Loyola University ChicagoLoyola eCommons

Dissertations Theses and Dissertations

2009

Regulation of Transglutaminase by 5-HT2AReceptor Signaling and CalmodulinYing DaiLoyola University Chicago

This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion inDissertations by an authorized administrator of Loyola eCommons. For more information, please contact ecommons@luc.edu.

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.Copyright © 2009 Ying Dai

Recommended CitationDai, Ying, "Regulation of Transglutaminase by 5-HT2A Receptor Signaling and Calmodulin" (2009). Dissertations. Paper 86.http://ecommons.luc.edu/luc_diss/86

LOYOLA UNIVERSITY CHICAGO

REGULATION OF TRANSGLUTAMINASE BY

5-HT2A RECEPTOR SIGNALING AND CALMODULIN

A DISSERTATION SUBMITTED TO

THE FACULTY OF THE GRADUATE SCHOOL

IN CANDIDACY FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

PROGRAM IN NEUROSCIENCE

BY

YING DAI

CHICAGO, IL

DECEMBER 2009

Copyright by Ying Dai, 2009 All rights reserved.

iii

ACKNOWLEDGEMENTS

Over five years of study at two great Universities make it impossible for me to

extend my gratitude to all of the people who have assisted me throughout my doctoral

education. First of all, I wish to express my gratitude to all the members of my

dissertation committee: Dr. George Battaglia, Dr. Thackery Gray, Dr. Nancy Muma,

Dr. Edward J. Neafsey and Dr. Karie Scrogin. Their kind encouragement, perceptive

criticism, and willing assistance made this dissertation possible. I sincerely thank my

advisor, Dr. Nancy Muma, who has supervised my doctoral research, overseen the

planning of the project and given my work minute consideration for the last five years.

Without her mentoring and prodding, I would never have made it this far.

I appreciate the help of many KU faculties who have supported me during my

dissertation studies. I would especially like to extend my gratitude to Dr. Qian Li and

Dr. Stephen Fowler, for their invaluable guidance and friendship during this process.

I would also like to thank the faculty and staff of Neuroscience Program and my

laboratory colleagues who have provided me with tremendous support in my graduate

studies at Loyola University Chicago. In particular, I would like to thank Dr. Lydia

DonCarlos, Peggy Richied, Nichole Dudek, Ju Shi, Cuihong Jia, Bozena Zemaitaitis

and Fran Garcia.

Lastly, I want to take this opportunity to thank my loving parents, Yiquan Dai and

iv

Zhiyang Song, who always support me and believe in me. Even though they are

thousands of miles away, their constant love has given me much needed

encouragement and inspiration throughout all of my work in graduate school.

I am grateful to the financial support from High-Q/CHDI Foundation and NIH

(Grant# MH068612) that made the research in this dissertation possible.

v

Dedicated to my parents—

Your love and support have made me the person I am today.

vi

TABLE OF CONTENTS

ACKNOWLEDGEMENTS iii LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xi ABSTRACT xiv CHAPTER ONE: BACKGROUND 1

INTRODUCTION 1

REVIEW OF RELEVANT LITERATURE 7 Transglutaminases (TGases), a family of enzymes 7 Physiological functions of TGases 8 Regulatory mechanisms and substrates of TGases 9 5-HT2A receptor signaling and small G protein transamidation 11 Small G proteins 18 Calmodulin (CaM) 23 Pathological role of TGases in neurodegenerative diseases 25 Alzheimer’s disease (AD) and TGase 25 Parkinson’s disease (PD) and TGase 26 Huntington's disease (HD) and TGase 27

CHAPTER TWO: TRANSGLUTAMINASE-CATALYZED TRANSAMIDATION: A NOVEL MECHANISM FOR RAC1 ACTIVATION BY 5-HT2A RECEPTOR STIMULATION 34

ABSTRACT 34

INTRODUCTION 35

MATERIALS AND METHODS 37

RESULTS 44

DISCUSSION 66 CHAPTER THREE: PHOSPHOLIPASE C, CALCIUM AND CALMODULIN SIGNALING ARE REQUIRED FOR 5-HT2A RECEPTOR-MEDIATED TRANSAMIDATION OF RAC1 BY TRANSGLUTAMINASE 73

vii

ABSTRACT 73

INTRODUCTION 74

MATERIALS AND METHODS 76

RESULTS 80

DISCUSSION 97

CHAPTER FOUR: STRIATAL EXPRESSION OF A CALMODULIN FRAGMENT IMPROVED MOTOR FUNCTION, WEIGHT LOSS AND NEUROPATHOLOGY IN THE R6/2 MOUSE MODEL OF HUNTINGTON’S DISEASE 104

ABSTRACT 104

INTRODUCTION 105

MATERIALS AND METHODS 107

RESULTS 116

DISCUSSION 134 CHAPTER FIVE: GENERAL CONCLUSION 141

REVIEW OF RESULTS AND SIGNIFICANCE 141

LIMITATIONS OF THE PRESENT STUDIES 152

FUTURE STUDIES: DISRUPTING CAM-HTT INTERACTION AS A

POTENTIAL THERAPEUTIC STRATEGY IN HUMANS 149

CONCLUSIONS 151 REFERENCES LIST 155 VITA 179

viii

LIST OF TABLES

Table Page

1. Quantitative analysis of neuropathology 133

ix

LIST OF FIGURES Figure Page

1. Molecular switch of the small G-proteins 19

2. Proposed involvement of TGase and CaM in the formation of htt- containing aggregates 33

3. Serotonin-induced increase of Rac1 transamidation in A1A1v cells 52

4. Serotonin receptor specificity on induction of Rac1 transamidation in A1A1v cells 53

5. The effects of cell differentiation on Rac1 transamidation 55

6. Rac1 activity was transiently increased after serotonin treatment 57

7. Dose-dependent effects of cystamine on Rac1 transamidation 58

8. Rac1 activity is inhibited by cystamine 59

9. Knockdown of TGase2 by siRNAs prevents Rac1 transamidation 61

10. Transamidation of serotonin to Rac1 is mediated by TGase 63

11. DOI stimulates transamidation of Rac1 to serotonin to a similar extent as those treated with serotonin 65

12. Effect of PLC inhibition on DOI-induced Ca2+ signals and TGase- modified Rac1 in A1A1v cells 86

13. Pre-incubation with a Ca2+ chelator attenuated the DOI-mediated elevation of cytosolic Ca2+ and TGase-modified Rac1 88

14. The Ca2+ ionophore mimiced the DOI-induced increase in cytosolic Ca2+ and TGase-catalyzed transamidation of Rac1 90

15. The CaM inhibitor W-7 caused a dose-dependent reduction in DOI- stimulated TGase-modified Rac1 92

x

16. Rac1 activity-related domains and potential TGase-targeted amino acid residues 94

17. Schematic representation showing 5-HT2A receptor signaling-mediated transamidation of Rac1 by TGase 96

18. Effect of CaM-fragment on body weight and survival 124

19. Video-based gait analysis on the treadmill 125

20. Expression of CaM-fragment enhanced the locomotor activity in R6/2 mice 126

21. CaM-fragment expression delayed the onset of the rotarod defects in R6/2 mice 127

22. TGase-modified htt in R6/2 mice striatum was reduced by CaM- fragment expression 128

23. Histological evaluation of neuropathology 129

24. Expression of CaM-fragment in mouse striatum did not significantly affect the activity CaM kinase II or TGase 131

25. Regulation of TGase by 5-HT2A receptor signaling and CaM 153

xi

LIST OF ABBREVIATIONS 5-HT serotonin

5-HT2A R serotonin 2A receptor

AA arachidonic acid

AAV adeno-associated virus

ANOVA analysis of variance

BDNF brain-derived neurotrophic factor

BSA bovine serum albumin

Ca2+ calcium

CaM Calmodulin

CaMKII Ca2+-calmodulin dependent kinase type II

DAG diacylglycerol

DAPI 4',6-diamidino-2-phenylindole

DOI (-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane HC1

ECL enhanced chemiluminescence

EDTA ethylenediaminetetraacetic acid

EGTA ethylene glycol tetraacetic acid

ELISA enzyme-linked immunosorbent assay

ER endoplasmic reticulum

xii

FBS fetal bovine serum

G protein guanine nucleotide-binding protein

GDP guanosine diphosphate

GFP green fluorescent protein

GPCR G protein-coupled receptor

GTP guanosine triphosphate

GTPyS Guanosine-5'-O-(thiotriphosphate)

HD Huntington's disease

HEK 293 human embryonic kidney cells

HRP horseradish peroxidase

htt huntingtin protein

IOD integrated optical density

IP3 inositol 1,4,5-triphosphate

PBS phosphate-buffered saline

PIP2 phophatidyl inositol-4,5-bisphosphate

PKA cAMP-dependent protein kinase

PKC protein kinase C

PLC phospholipase C

PMSF phenylmethylsulphonyl fluoride

SEM standard error of the mean

SSRI selective serotonin reuptake inhibitor

TBS Tris-buffered saline

xiii

TGase transglutaminase

WT wild type

xiv

ABSTRACT

Transglutaminase (TGase), nature’s biological glue, catalyzes the

post-translational modification of proteins by formation of intra- and intermolecular

protein cross-links or by primary amine incorporation. TGase has various

physiological functions, such as skin-barrier formation and blood clot stabilization,

whereas increasing evidence indicates they may also involved in neurodegenerative

diseases including Huntington’s disease (HD), Alzheimer disease’s, and progressive

supranuclear palsy. Mutant huntingtin (htt) and small G proteins (e.g. Rac 1) are

potential substrates of TGases. The purpose of this dissertation was to characterize the

mechanisms by which 5-HT2A receptor signaling and calmodulin (CaM) regulate

TGase-catalyzed transamidation of Rac1 and htt in cultured neuronal cells and HD

transgenic animals, respectively.

5-HT2A receptors are G-protein coupled receptors which are widely expressed in

the brain, peripheral vasculature, platelets and skeletal muscle. They are involved in

diverse physiological functions, from platelet aggregation to neuroendocrine release.

In A1A1v cells, a rat cortical cell line, stimulation of 5-HT2A receptor induces small G

protein Rac1 transamidation and activation. An inhibitory agent or knockdown of

TGase by siRNA revealed that TGase is responsible for transamidation and activation

of Rac1. Moreover, serotonin was identified as an amine that becomes transamidated

xv

to Rac1 by TGase. The classical signal transduction pathway of 5-HT2A receptors is

Gq/11-coupled activation of phospholipase C (PLC), which hydrolyzes

phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and

diacyglycerol. IP3 mobilizes Ca2+ from the endoplasmic reticulum and thereby

increases intracellular Ca2+ and consequently, it may enhance TGases enzymatic

activity. Inhibition of PLC or manipulation of intracellular Ca2+ by a chelating agent

suppressed 5-HT2A receptor-mediated Rac1 transamidation, whereas an elevation in

intracellular Ca2+ via an ionophore can mimic 5-HT2A receptor-induced cytosolic Ca2+

increases and is sufficient to induce TGase-catalyzed Rac1 transamidation. Moreover,

a CaM inhibitor decreased 5-HT2A receptor-stimulated Rac1 modification by TGase in

a dose-dependent manner. These results suggest that PLC, Ca2+ and CaM signaling

are required for 5-HT2A receptor-mediated transamidation of Rac1 by TGase.

In the next study, we propose that interrupting CaM interactions with htt is

therapeutically beneficial in HD. This hypothesis was based on the observations that

CaM regulates TGase-modification of mutant htt in cells and co-localizes with TGase

and htt in intranuclear inclusions in HD cortex. Furthermore the association of CaM

with mutant htt was demonstrated by affinity purification and coimmunoprecipitation

approaches. Our previous studies demonstrated that in HEK293 and SH-SY5Y cells,

expression of a CaM-fragment, consisting of amino acids 76-121 of CaM, decreased

binding of CaM to mutant htt, decreased TGase-modified htt, decreased cytotoxicity

associated with mutant htt and normalized intracellular calcium release. In this study,

xvi

an adeno-associated virus (AAV) that expresses the CaM-fragment was injected into

the striatum of HD transgenic R6/2 mice and littermate control mice. The HD mice

with CaM-fragment expression had significantly reduced body weight loss and

improved motor function compare to HD control mice. Without affecting the activity

of CaM-dependent enzymes such as CaM-dependent kinase II, CaM-fragment

specifically reduced TGase-modified htt, the percentage of htt-positive nuclei and the

size of intranuclear htt aggregates in HD mouse striatum. Thus, disrupting CaM-htt

interaction with CaM-fragment may provide a new therapeutic strategy for HD

patients.

The data presented here support our hypothesis that stimulation of 5-HT2A

receptors induces TGase-catalyzed Rac1 transamidation and activation by PLC and

Ca2+/CaM signaling, whereas disruption of CaM-htt interaction inhibits

TGase-catalyzed modification of htt and provides neuronal protection in HD. These

results further suggest that CaM regulates the TGase transamidation reaction and

TGase modification of htt is involved in the formation and stabilization of

htt-aggregates and may play a role in the pathogenesis of HD.

1

CHAPTER ONE

BACKGROUND

INTRODUCTION

Transglutaminases (TGases, EC 2.3.2.13) are a family of calcium

(Ca2+)-dependent enzymes that catalyze the formation of a covalent bond between

peptide-bound glutamine residues and either peptide-bound lysine residues or mono-

or polyamines (Folk and Finlayson, 1977, Folk et al., 1980, Walther et al., 2003).

TGases are widely distributed in the human body with various physiological functions,

such as extracellular matrix organization, skin-barrier formation and blood

coagulation (Griffin et al., 2002;Lorand and Graham, 2003). TGases are subject to

transcriptional regulation by retinoic acid and steroid hormones (Fujimoto et al.,

1996;Ou et al., 2000), and require the binding of Ca2+ for their activity (Burgoyne and

Weiss, 2001).

Serotonin signaling may also regulate TGases activity possibly by increasing

cytosolic Ca2+ (Walther et al., 2003, Guilluy et al., 2007). Stimulation of 5-HT2A

receptors activate phospholipase C (PLC) through Gq/11, leading to an accumulation

of inositol 1,4,5-trisphosphate (IP3) and consequent release of Ca2+ from the

endoplasmic reticulum. In platelets, activation of 5-HT2A receptors stimulate

TGase-catalyzed transamidation of serotonin to small G proteins, RhoA and Rab4,

2

making them constitutively active (Walther et al., 2003). TGase-dependent RhoA

transamidation and activation by serotonin stimulation was also observed in vascular

smooth muscle cells (Guilluy et al., 2007). Neuronal differentiation of SH-SY5Y cells

induced by retinoic acid increases the expression and activation of TGases, resulting

in transamination of putrescine to RhoA and activation of RhoA (Singh et al., 2003).

Small G proteins are a family of monomeric 20~30 kDa GTP-binding proteins,

which are homologous to the alpha subunit of heterotrimeric G-proteins. Small G

proteins regulate a wide variety of processes in the cell, including growth, cellular

differentiation, cell movement and lipid vesicle transport (Matozaki et al., 2000).

Rac1 belongs to the Rho family of small G proteins, a subgroup of the Ras

superfamily. Members of the Rho family are involved primarily in the regulation of

cytoskeletal organization (Etienne-Manneville and Hall, 2002). Bearing 5 glutamine

residues (Gln 2,61,74,141,162) and 17 lysine residues in the amino acid sequence

(Matos et al., 2000), Rac1 might serve as a suitable substrate of TGases.

TGase-catalyzed transamination of small G proteins may result in constitutive

activation of the proteins (Walther et al., 2003). Investigating the regulatory

mechanisms of TGase-modification of small G proteins and the functional

consequences in a neuronal cell line may further explore the role of TGase in neuronal

differentiation and neurite outgrowth.

Calmodulin (CaM), another Ca2+-binding protein, has also been shown to play

a role in regulating TGase. CaM is a 17 kDa protein that activates a host of enzymes

3

during Ca2+ binding (Cheung, 1982). CaM coimmunoprecipitated with TGase in

transfected cells (Zainelli et al., 2004) and increased TGase activity in human

erythrocyte (Billett and Puszkin, 1991), platelets and chicken gizzard (Plank et al.,

1983). Recently, a series of studies showed that the activity of TGase is critical for the

pathology of a number of neurodegenerative diseases, such as Huntington's disease

(HD)(Karpuj and Steinman, 2004;Violante et al., 2001). CaM may also be involved in

HD based on its interaction with both TGase and huntingtin (htt) (Bao et al., 1996,

Zainelli et al., 2004).

HD is an autosomal dominant neurodegenerative disorder with progressive

neurological symptoms and mental decline such as chorea, rigidity, gait disturbance,

abnormal posturing, seizure, depression and dementia (Harper, 1991). HD occurs in

individuals whose HTT gene has more than 36 CAG repeats, resulting in a mutant

protein with abnormal expansions of the polyglutamine domain in the N-terminus.

The pathological hallmark of HD is intranuclear inclusions and cytoplasmic

aggregates composed of mutant htt protein (DiFiglia et al., 1997;Ross et al., 1998). In

vitro and in cell culture, mutant htt is an excellent substrate for TGases (Gentile et al.,

1998;Kahlem et al., 1998;Karpuj et al., 1999). The mRNA, protein, and enzymatic

activity of TGases are elevated in HD brain (Karpuj et al., 1999;Lesort et al.,

1999;Zainelli et al., 2003). Increasing evidence indicates that TGases may contribute

to the formation of mutant htt aggregates in HD (Cooper et al., 1999). Htt protein

colocalizes with TGase2 and its product, -( -glutamyl) lysine covalent bond in

4

intranuclear inclusions in the frontal cortex of HD (Zainelli et al., 2004). On the other

hand, our lab and others reported CaM associates with mutant htt as demonstrated by

coimmunoprecipitation (Zainelli et al., 2004) and affinity purification (Bao et al.,

1996). Furthermore, CaM colocalizes with TGase2 and with htt in HD intranuclear

inclusions (Zainelli et al., 2004). Taken together, these studies suggest that mutant htt

may bind to CaM resulting in increases TGase-modification to htt. This modification

may stabilize htt monomers or polymers and contribute to htt-containing aggregate

formation and cytotoxicity.

Both TGases and CaM have been explored as therapeutic targets for HD.

Administration of the TGase inhibitor, cystamine, extended survival, improved motor

performance and increased neuroprotective gene transcription in HD transgenic mice

(Dedeoglu et al., 2002, Karpuj et al., 2002a). TGases2 ablation in HD transgenic mice

results in a drastic reduction in -( -glutamyl) lysine bond levels and neuronal death

in the cortex and striatum (Mastroberardino et al., 2002). CaM inhibitor, W5, can

decrease TGases-catalyzed cross-linking of htt in cells transfected with TGase2 and

mutant htt (Zainelli et al., 2004). However, direct inhibition of CaM or TGase may not

be the best therapeutic approach, since both enzymes have a lot of other biological

functions and long-term inhibition might cause untoward effects. A new strategy is to

identify the htt-binding domain in CaM, and express this CaM fragment in HD

models. It might disrupt CaM-htt interaction by competing with endogenous CaM,

and thereby decrease TGase-modified htt, htt-aggregates and cell death. This

5

therapeutic strategy has been tested in cell models of HD and will next be tested in an

HD transgenic mouse model.

We hypothesize that stimulation of 5-HT2A receptors induces

TGase-catalyzed Rac1 transamidation and activation by increasing intracellular

Ca2+, whereas disruption of CaM-htt interaction inhibits TGase-catalyzed

modification of htt and provides neuronal protection in HD. Three specific aims

are proposed to test this hypothesis.

Specific Aim 1: To determine if the activity and transamidation of Rac1 by

TGase are increased by 5-HT2A receptor signaling.

The first specific aim is to determine if the activity and transamidation of Rac1

are increased by 5-HT2A receptor signaling in a rat cortical cell line, A1A1v cells. We

will use either a TGase inhibitor or siRNA targeting TGase to determine if Rac1

transamidation and activation are mediated TGase. In addition, we will determine if

serotonin is incorporated into Rac1 by TGase following 5-HT2A receptor stimulation.

Lastly, we will test if DOI, a 5-HT2A/2c receptor agonist, selectively increases

TGase-catalyzed transamination of Rac1 in vivo.

Specific Aim 2: To determine if 5-HT2A receptor-regulated transamination of

Rac1 by TGase is dependent on an increase of intracellular Ca2+ mediated by PLC

signaling, and if calmodulin regulate TGase-modification of Rac1.

The second specific aim designed is to explore the downstream mechanistic

pathways in 5-HT2A receptor-mediated Rac1 transamidation. We will determine if

6

inhibition of PLC prevents the increase of intracellular Ca2+ and transamination of

Rac1 in response to activation of 5-HT2A receptors. A Ca2+ chelator or Ca2+ ionophore

will be used to manipulate intracellular Ca2+ and determine if the increase in

intracellular Ca2+ is necessary and sufficient to induce Rac1 transamination. We will

also determine if inhibiting calmodulin prevents 5-HT2A receptor-stimulated

transamidation of Rac1.

Specific Aim 3: To determine if disrupting the interaction of calmodulin and

huntingtin decreases TGase-modified huntingtin and huntingtin-aggregates, improves

motor function and increases survival of HD transgenic mice.

We will use a viral vector to deliver a calmodulin-fragment, which can

interrupt calmodulin and mutant huntingtin binding, to the striatum of R6/2 HD

transgenic mice, then monitor survival and body weight, evaluate motor performance

by behavioral tests such as DigiGait, Actometer and Rotarod. We will also measure

neuropathological changes such as intranuclear htt-aggregates and striatal atrophy by

immunofluorescence and Nissl staining. The specificity of calmodulin-fragment will

be tested by measuring calmodulin-dependent protein kinase II (CaMKII) and TGase

activity.

7

REVIEW OF RELEVANT LITERATURE

In the literature review, the family of TGases will be introduced first,

followed by their physiological functions. The next section will focus on regulatory

mechanisms and substrates of TGases. The regulation of TGases-mediated

transamidation of small G protein by 5-HT2A receptor signaling will be highlighted.

Rac1, as a potential substrate of TGases, will be presented. Regulation of TGases by

calmodulin will also be discussed in this section. Lastly, the pathological role of

TGases in neurodegenerative diseases including Alzheimer disease, Parkinson disease

and especially Huntington’s disease will be reviewed.

Transglutaminases (TGases), a family of enzymes

TGases (EC 2.3.2.13) are a family of Ca2+-dependent enzymes. Once activated,

TGases catalyze the cross-linking of proteins via the γ-carboxamide group of

peptide-bound glutamine and the є-amino group of peptide-bound lysine, forming a

inter- or intramolecular isodipeptide bond (Griffin et al., 2002). The enzyme can also

covalently link biogenic amines and polyamines, such as spermine or serotonin

(5-Hydroxytryptamine; 5-HT), to a peptide-bound glutamine residue (Dale et al.,

2002;Folk et al., 1980).

To date, nine isoforms of transglutaminase have been identified, each encoded

by different but structurally and functionally related related gene. Family members

include keratinocyte transglutaminase (TGase1), tissue transglutaminase (TGase2),

epidermal transglutaminase (TGase3), prostate transglutaminase (TGase4),

8

transglutaminase X (TGase5), transglutaminase Y (TGase6), transglutaminase Z

(TGase7), factor XIII-A subunit (fibrin-stabilizing factor) and ATP-binding

erythrocyte membrane protein band 4.2 (B4.2) (Lorand and Graham, 2003).

At least 4 isoforms, TGase1, 2, 3 and TGase7, are expressed in normal human

brain tissue (Kim et al., 1999, Grenard et al., 2001) and mutant huntingtin protein is

identified as a substrate of the first three TGase isoforms (Zainelli et al., 2005).

However, of the nine known human isozymes, tissue TGase (TGase2) is ubiquitously

expressed and most well characterized.

Physiological functions of TGases

TGases are widely distributed in various tissues and are involved in various

physiological functions such as blood clot formation, wound healing, skinbarrier

formation, extracellular matrix assembly, cell death, and cell differentiation (Griffin et

al., 2002, Lorand and Graham, 2003).

For example, following exposure to thrombin and Ca2+, fibrin stabilizing

factor (factor XIII) is activated and converted to factor XIII-A containing only the A

subunit that, in turn, catalyzes the formation of Nε(γ-glutamyl)lysine

protein-to-protein side chain bridges within the clot network. Introduction of these

covalent crosslinks greatly stabilize the fibrin and augments its resistance to

fibrinolytic enzymes (Lorand, 2001).

In addition to cytoplasmic and nuclear localization, a significant part of the

TGase2 protein pool is present on the cell surface. Cell surface TGase2 acts as a

9

coreceptor of integrins, coordinating binding of extracellular matrix (ECM) proteins

(Zemskov et al., 2006). It also crosslinks the fibronectin (as well as other ECM

proteins) to improve cell adhesion (Aeschlimann and Thomazy, 2000). TGase2

functions to stabilize the ECM by forming large polymeric structures that are resistant

to proteolytic/chemical degradation and mechanical stresses (Griffin et al., 2002).

The homozygous null TGase2 mice are viable and have normal size and

weight with no severe phenotype (De Laurenzi and Melino, 2001, Nanda et al., 2001),

suggesting that other TGase isoforms may compensate for the lack of TGase2 to some

extent. However, TGase2-/- mice do have altered fibroblast function, defective wound

healing (Nanda et al., 2001), decreased phagocytic clearance of apoptotic cells

(Szondy et al., 2003, Rose et al., 2006), mild glucose intolerance and hyperglycemia

likely due to reduced insulin secretion (Bernassola et al., 2002). Therefore, it is

plausible that under disease or stress, TGase2 play an essential role in tissure repair or

healing and that further testing of these mice will reveal how TGase2 is critical in

other physiologic states.

Regulatory mechanisms and substrates of TGases

TGases are Ca2+-dependent enzymes containing a cysteine in their active site

that is unmasked only in the presence of Ca2+ (Hand et al., 1993) and Ca2+ activation

of TGase is further regulated by other signal modulators such as GTP (Monsonego et

al., 1998), phospholipids (Griffin et al., 2002, Beninati and Piacentini, 2004), tumor

necrosis factor alpha (Chen et al., 2000), nitric oxide (Bernassola et al., 1999) and

10

CaM (Puszkin and Raghuraman, 1985, Zainelli et al., 2004).

An elevation in intracellular Ca2+ is the required element for activation of

TGases. Elevations in intracellular Ca2+ needed to activate TGases may arise via

extracellular influx, release from intracellular stores, or decreased levels of other Ca2+

binding proteins (Lorand and Conrad, 1984). The binding of Ca2+ to TGases induces

conformational changes and exposes the active site to substrate proteins (Hand et al.,

1993, Casadio et al., 1999).

TGase2 has been reported to be a GTP-binding protein with GTPase activity

(Achyuthan and Greenberg, 1987). GTP binding to the enzyme can inhibit its

transamidation activity at physiological Ca2+ levels (Bergamini et al., 1987). Binding

of GTP to TGase keeps the enzyme in a conformation such that the C-terminus blocks

the enzyme's catalytic core (Monsonego et al., 1998, Casadio et al., 1999).

Interestingly, an elevation in cytosolic Ca2+ will overcome GTP inhibition and activate

TGase (Bergamini, 1988).

Other regulatory effects may also occur through the interaction of TGase with

phospholipids, nitric oxide and CaM. When phospholipid vesicles are in the

crystalline-liquid transition state, they can interact with and inhibit TGase activity.

The inhibition may be due to the hydrophobic environment provided by the

phospholipid hydrocarbon chain when the enzyme is inserted into the bilayer (Fesus

et al., 1983). Exposure to tumor necrosis factor alpha causes fibronectin

multimerization in lung endothelial matrix by increasing TGase activity (Chen et al.,

11

2000). Nitric oxide has an inhibitory effect on both TGase2 and coagulation factor

XIII, probably via S-nitrosylation of their crucial thiol groups (Bernassola et al.,

1999). CaM increased TGase activity in human erythrocyte (Billett and Puszkin,

1991), platelets and chicken gizzard (Plank et al., 1983). A CaM inhibitor can

decrease TGases-catalyzed cross-linking of htt in cells transfected with TGase2 and

mutant htt (Zainelli et al., 2004).

Small G proteins have emerged as the new substrates of TGase. It has

been reported that neuronal differentiation of SH-SY5Y cells induced by retinoic

acid increases the expression and activation of TGases, resulting in transamidation of

putrescine to RhoA and activation of RhoA (Singh et al., 2003). In platelets and aortic

smooth muscle cells, TGases can catalyze the incorporation of serotonin into

small GTPases ("transamidation"), such as RhoA and Rab4. This process of

transamidation renders these small GTPases constitutively active (Walther et al.,

2003, Guilluy et al., 2007). Activation of 5-HT2A receptors, which increases Ca2+

availability, induces serotonin transamidation to small GTPases (Walther et al.,

2003). Therefore, the next section will introduce the 5-HT2A receptor, including

its structure, signal transduction, distribution and pathophysiological roles.

5-HT2A receptor signaling and small G protein transamidation

Overview and classification of 5-HT receptors

As one of the most complex families of neurotransmitter receptors, 5-HT

receptors have at least 16 distinct members cloned, identified and classified into 7

12

different subfamilies according to their ligand recognition profiles, signal transduction

mechanisms, and structural characteristics (Humphrey et al., 1993, Hoyer et al., 1994,

Hoyer et al., 2002). Except for the 5-HT3 receptor, which is a ligand-gated ion

channel, the rest of 5-HT receptors belong to the G protein-coupled receptor (GPCR)

superfamily.

The 5-HT1 receptor class includes 5 members, 5-HT1A, 5-HT1B, 5-HT1D,

5-HT1F and 5-HT1F, which are classically coupled to the Gi/o proteins that

negatively regulate adenylyl cyclase and induces the opening of K+ channels. They

are expressed both pre and post-synaptically and involved in the regulation of

serotonin release (Hoyer et al., 2002).

The 5-HT2 family consists of 5-HT2A, 5-HT2B and 5-HT2C receptors that

primarily activate phospholipase C (PLC) via coupling with Gq/11. Activation of

5-HT2A receptors also mediates neuronal depolarization, a result of the closing of K+

channels (Aghajanian and Marek, 1999, Lambe and Aghajanian, 2001).

The 5-HT3 receptor is unique among the currently known subtypes not only

because it is one of the first 5-HT receptors identified, corresponding to the M

receptor (Gaddum and Picarelli, 1957), but also because it belongs to the ligand-gated

channel superfamily instead of coupling to G proteins or second messenger systems.

The 5-HT3 receptor mediates the rapid excitatory electrophysiological response due

to a transient inward current, subsequent to the opening of nonselective cation

channels (Na+, Ca2+ influx, K+ efflux) (Peters et al., 1992, Hoyer et al., 2002).

13

The 5-HT4, 5-HT6 and 5-HT7 receptors are single members in different families

that mainly stimulate adenylyl cyclase through the activation of Gαs proteins

(Raymond et al., 2001).

The 5-HT5 family (5-HT5A and 5-HT5B receptors) is neither coupled to adenylyl

cyclase nor PI-PLC. The effector systems of this family remain unclear, but some

evidence indicates 5-HT5A receptor is implicated in the action of LSD (Grailhe et al.,

1999, Nelson, 2004).

5-HT2A receptor distribution and pathophysiological roles

5-HT2A receptors are widely distributed in central and peripheral tissues. In the

central nervous system (CNS), these receptors have been found mainly in the cortical

areas (neocortex, entorhinal and pyriform cortex, claustrum), caudate nucleus, nucleus

accumbens, olfactory tubercle and hippocampus (Hoyer et al., 1986, Pazos et al.,

1987). In the cortex the great majority of labeled cells were pyramidal neurons

(Wright et al., 1995, Willins et al., 1997). In the periphery, 5-HT2A receptors are

located in platelets (de Chaffoy de Courcelles et al., 1987), vascular smooth muscle

(Cohen et al., 1981), and uterine smooth muscle (Wilcox et al., 1992), inducing

vasoconstriction and platelet aggregation. Studies that have investigated the

subcellular location of the 5-HT2A receptor indicate that the majority of 5-HT2A

receptors in the human cerebellum and rat CNS are in cytosol, rather than the cellular

membrane (Cornea-Hebert et al., 1999, Eastwood et al., 2001).

The 5HT2A receptor has been shown to be involved in a number of different

14

processes including endocrine modulation (Van de Kar et al., 2001), pain modulation

(Sommer, 2004), thermoregulation (Ootsuka and Blessing, 2006), cognitive function

and memory (Meneses, 2002, Williams et al., 2002). In addition, 5-HT2A receptor

signaling is implicated in a number of disorders including hypertension,

atherosclerosis, and different psychiatric diseases such as anxiety, stress, suicide

behavior and schizophrenia (Roth, 1994, Weisstaub et al., 2006).

Serotonin is one of many neurotransmitters that influence the activation of

hypothalamic-pituitary-adrenal (HPA) axis and stimulate secretion of oxytocin,

adrenocorticotrophic hormone (ACTH), corticosterone (Van de Kar et al., 2001,

Hemrick-Luecke and Evans, 2002, Zhang et al., 2002). Serotonergic terminals from

the medial raphe nucleus make synaptic interaction with the corticotrophin releasing

factor (CRF) synthesizing neurons in the paraventricular nucleus of rat hypothalamus

(Liposits et al., 1987). Moreover, 5-HT2A receptors are found in hypothalamic

paraventricular nucleus and in both CRF synthesizing neurons and

oxytocin-containing neurons (Van de Kar et al., 2001, Zhang et al., 2002). When CRF

is released, it induces ACTH secretion from the anterior pituitary gland, which in turn

stimulates the release of corticosterone from the adrenal cortex. Administration of

DOI, a 5-HT2A/2C receptor agonist increases plasma levels of oxytocin, ACTH,

corticosterone in rats. The 5-HT2A receptor selective antagonist, MDL 100,907, but

not the 5-HT2C receptor antagonist SB242084 can dose-dependently inhibit the

neuroendocrine effects of DOI (Van de Kar et al., 2001, Zhang et al., 2002).

15

5-HT2A receptors are the target of some therapeutic agents for a variety of

psychiatric disorders. Atypical antipsychotic drugs such as clozapine display potent

5-HT2A receptors antagonism with relatively weak blockade at D2 dopamine (DA)

receptors, various effects at D1 receptors (Meltzer et al., 1989, Arora and Meltzer,

1994). Moreover, the clinical response to the selective serotonin reuptake inhibitors

(SSRI) in treatment-resistant patients is augmented by atypical antipsychotic drugs

and some antidepressants (Ostroff and Nelson, 1999, Carpenter et al., 2002,

Marangell et al., 2002). The common feature of these agents is that they are able to

occupy 5-HT2 receptors and to block the responses mediated by those receptors, in

particular by 5-HT2A receptors (Marek et al., 2003). These reports support a role for

5-HT2A receptors in antipsychotic drug action.

5-HT2A receptors not only mediated the effects of some antipsychotic drugs, but

also are involved in pathophysiology of psychiatric disorders. Abnormalities in

5-HT2A receptors have been proposed to be implicated in the pathology of

schizophrenia (Woolley and Shaw, 1954, Dean, 2003). Direct evidence supporting this

hypothesis came from radioligand binding studies in post-mortem schizophrenia

patients, which suggest there is a decrease in the density of cortical 5-HT2A receptors

in those patients (Arora and Meltzer, 1991, Dean and Hayes, 1996). The levels of

mRNA encoding the 5-HT2A receptors were also reduced in the superior temporal

gyrus of schizophrenic subjects (Hernandez and Sokolov, 2000). In depressed patients

and suicide victims, the postmortem studies indicated an elevated level of 5-HT2A

16

receptor in the cortex, amygdala and hippocampus (Arango et al., 1990, Hrdina et al.,

1993, Pandey et al., 2002). However, positron emission tomography (PET) and

radioligand binding studies detected a significant decrease in 5-HT2A receptor density

in hippocampus of both young and old depressed patients (Mintun et al., 2004,

Sheline et al., 2004). Furthermore, 5-HT2A receptors are also involved in modulating

anxiety-related behaviors in humans and rodents. For example, global disruption of

5-HT2A receptor signaling in htr2a–/– mice reduces the inhibition in conflict anxiety

paradigms, whereas selective restoration of 5-HT2A receptor signaling to the cortex

normalized conflict anxiety behaviors (Weisstaub et al., 2006).

5-HT2A receptor structure and signal transduction

The serotonin 5-HT2A receptor gene is located on human chromosome 13q14-q21

and comprises 471 amino acid in rats, mice, and humans (Hoyer et al., 2002). It

contains three exons, separated by two introns, with the coding region of the gene

spanning 1.4 kb (Sanders-Bush et al., 2003).

The 5-HT2A receptor is coupled via the Gq/11 proteins to the phospholipase С

(PLC) signaling cascade. PLC hydrolyzes phosphatidyl-inositol 4,5-bisphosphate

(PIP2) to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 mobilizes

Ca2+ from the endoplasmic reticulum and thereby leads to an increase of cytosolic

Ca2+ concentration (Roth et al., 1998).

In addition to the major pathways mentioned above, extensive evidence suggests

that 5-HT2A receptors couple to other effector pathways. 5-HT2A receptor stimulation

17

also induces the activation of phospholipase A2 and the release of the second

messenger arachidonic acid, which is involving Gβγ-mediated ERK1/2 activation and

Gα12/13-coupled, Rho-mediated p38 activation (Felder et al., 1990, Kurrasch-Orbaugh

et al., 2003). Through interaction with ADP-ribosylation factor 1 (ARF1) via its

C-terminal domain, 5-HT2A receptor is able to signal through the phospholipase D

(PLD) pathway (Mitchell et al., 1998, Robertson et al., 2003).

5-HT2A receptor is known to trigger MAPK activation via PKC/Raf-1 pathway

(Hershenson et al., 1995, Watts, 1996). The JAK/STAT signaling pathway is activated

by a number of G protein coupled receptors including 5-HT2A receptors

(Guillet-Deniau et al., 1997, Singh et al., 2009).

Depending on the cell types, 5-HT2A receptor is able to increase or diminish

cyclic adenosine monophosphate (cAMP) accumulation. It increases cAMP in a cell

line derived from embryonic rat cortex (A1A1 cells) by protein kinase C-dependent

and calcium/calmodulin-dependent mechanisms (Berg et al., 1994a) and in FRTL-5

thyroid cells through a pertussis toxin-sensitive mechanism (Tamir et al., 1992).

However, in rat renal mesangial cells, 5-HT2A receptor activation can inhibit adenylyl

cyclase activity and forskolin-stimulated cAMP accumulation (Garnovskaya et al.,

1995).

Because transamidation of small G proteins (e.g. RhoA and Rab4) mediated by

5-HT2A receptor signaling leads to platelet α-granule release and aggregation (Walther

et al., 2003), and TGase-dependent Rho A transamidation also inhibits contraction of

18

vascular smooth muscle cells, a brief introduction of small G proteins especially Rac1

whose transamidation is extensively studied in this dissertation will be presented in

the next section.

Small G proteins

Small G-proteins, short for small guanine nucleotide-binding proteins, are

monomeric G proteins with molecular masses of 20–30 kDa. They are homologous to

the alpha subunit of heterotrimeric G-proteins, but they can function on their own by

cycling between an inactive GDP-bound state and an active GTP-bound state. The

GTP/GDP cycling is controlled by many regulator molecules such as

GTPase-activating proteins (GAPs), guanine nucleotide exchange factors (GEFs) and

GDP dissociation inhibitors (GDIs) (Hakoshima et al., 2003). GTP hydrolysis is

accelerated by GAPs, resulting in inactivation of the cognate G-protein.

GAP-dependent signaling is antagonized by GEF, which activate small G proteins by

promoting the nucleotide exchange of GTP for GDP. Furthermore, small G proteins

are regulated by GDIs, which maintain small G proteins in their inactive GDP-bound

form, in addition to preventing their association to the plasma membrane (Figure 1).

19

Figure 1. Molecular switch of the small G-proteins. The activation of small G protein

is mediated by three types of regulatory proteins: GDI (guanine nucleotide

dissociation inhibitor), GAP (GTPase activating protein), and GEF (guanine

nucleotide exchange factors).

20

Biological functions of Rac1

Rac1, short for Ras-related C3 botulinum toxin substrate 1, belongs to the Rho

family of small G proteins, a subgroup of the Ras superfamily. Binding to a variety of

downstream target proteins (effectors), members of the Rho family (e.g. RhoA, Rac1,

Cdc42) are associated with a wide array of cellular processes such as cytoskeletal

organization, vesicular transport, cell cycle progression, cell adhesion and migration,

neuronal differentiation and a variety of enzymatic activities (Etienne-Manneville and

Hall, 2002, Burridge and Wennerberg, 2004).

Rac1 is best known as a regulator for the assembly of the actin cytoskeleton,

thereby playing a role in neurite outgrowth and neuronal differentiation. Expression of

a constitutively active mutant of Rac1 in neuroblastoma cells leads to the formation of

neurites (Leeuwen et al., 1997), and production of filopodia and lamellipodia in the

developing growth cone (Kozma et al., 1997). In contrast, over-expression of

dominant negative Rac1 in SH-SY5Y cells blocks retinoic acid (RA)-induced neurite

outgrowth and expression of neuronal markers, suggesting that activation of Rac1

regulates neuronal differentiation (Pan et al., 2005). Rac1 play an essential role in

fibroblasts cell growth in response to mitogenic stimulation since microinjection of

Rac1 stimulated cell cycle progression through G1 and subsequent DNA synthesis.

This effect can be blocked by microinjection of dominant negative forms of Rac1

(Olson et al., 1995). In growth factor-stimulated fibroblasts and neuronal cells, Rac1

was transiently activated in a broad area of the plasma membrane, followed by a

21

localized activation at nascent lamellipodia (Kurokawa et al., 2005). Consistent with

this finding, Rac1 is required to stimulate the formation of lamellipodia and

membrane ruffles.

Rac1 is also involved in multiple cell death pathways. Depending on the

interaction with other signaling molecules and on the type of cells, it becomes

pro-apoptotic (Embade et al., 2000, Aznar and Lacal, 2001, Harrington et al., 2002) or

anti-apoptotic (Nishida et al., 1999, Deshpande et al., 2000, Murga et al., 2002). For

example, neurotrophin receptor p75 activates Rac1, which in turn activates c-Jun

N-terminal kinase (JNK) and causes apoptosis in neuronal cells (Harrington et al.,

2002). On the other hand, Rac1 protects endothelial cells from tumor necrosis

factor-alpha-induced apoptosis (Deshpande et al., 2000) and promotes COS7 cell

survival by the activation of phosphatidylinositol 3-kinase (PI3K) and Akt (Murga et

al., 2002).

Structure of Rac1

The Rho family are monomeric globular proteins consisting of less than 300

amino acids (Wennerberg and Der, 2004). Like all members of the Rho family, Rac1

functions as a conformational switch by cycling active GTP and inactive GDP-bound

forms. Rho GTPases share a common GTPase domain, which carries out the basic

function of nucleotide binding and hydrolysis. The GTPase domain consists of a

six-stranded β-sheet surrounded by α-helices (Vetter and Wittinghofer, 2001). Most

typical Rho proteins consist only of the GTPase domain and short N- and C-terminal

22

extensions. C-terminal post-translational modifications such as isoprenoid addition

facilitate specific subcellular location and association with specific membranes, which

is crucial for their function (Wennerberg and Der, 2004). The differences between the

GDP- and GTP- bound structural forms of RhoA are confined primarily to two

segments, referred to as switch I and switch II, and this feature is probably shared by

all Rho GTPases (Ihara et al., 1998, Vetter and Wittinghofer, 2001).

Bearing five glutamine residues in the amino acid sequence (Matos et al., 2000),

Rac1 might serve as a suitable substrate of TGases. The transamidation of primary

amines to RhoA and Rab4 by TGases renders these small G proteins constitutively

active (Walther et al., 2003, Guilluy et al., 2007). Similarly, post-translational

modifications, such as transamidation and phosphorylation of Rac1 may affect its

ability to interact with regulatory proteins (e.g. GAPs, GEFs and GDIs) or to convert

GTP back to GDP, resulting in increased activation of Rac1 and stimulation of its

signaling cascade in the cells.

The Conserved Domain Database (CDD) (http://www.ncbi.nlm.nih.gov/

entrez/query.fcgi? db=cdd) provides an interactive tool to identify conserved domains

present in protein sequences. The residues, which regulate Rac1 activity after

post-translational modifications, are most likely to be located at GTP/Mg2+ binding

sites, and GAPs, GEFs and GDIs interaction sites in the Rac1 sequence. Two

glutamine residues (Q61, Q74) and three lysine residues (K5, K16, and K116),

potential tagets of TGase-catalyzed modification, are identified within these

23

activity-related domains using CDD search of the Rac1 sequence (PSSM-Id: 57957).

In line with this finding, it has been reported that site-specific deamidation of a Gln

residue in the Rho family of G proteins (Q61 in Rac and Cdc42, Q63 in RhoA) by

CNF-1 inhibits both intrinsic and GAPs-stimulated GTP hydrolysis activity, resulting

in constitutive activation of these proteins (Flatau et al., 1997, Schmidt et al., 1997).

CNF-1 has also been shown to possess in vitro TGase activity. In the presence of

primary amines, RhoA is transamidated in vitro at Q63 by CNF-1 and at positions 52,

63 and 136 by guinea pig liver TGase (Schmidt et al., 1998). Similarly, in addition to

Q61 which is critical in regulating Rac1 activity, the other four glutamine residues in

Rac1 may also be modified by TGase.

Calmodulin (CaM)

The other regulator molecule of TGase that will be focused on this dissertation is

CaM. It is a 17 kDa, dumbbell shaped, Ca2+ binding protein that is ubiquitously

expressed in cells (Babu et al., 1985). There are two EF hand motifs at each end of its

dumbbell-like structure, separated by a helical linker region. The C-terminal EF hand

motifs have three to five fold higher affinity for Ca2+ than those in the N-terminal

(Chin and Means, 2000). Upon Ca2+ binding to the EF hand motifs, both N-terminal

and C-terminal domains adopt an open confirmation, exposing hydrophobic surfaces,

which then interact with a variety of proteins triggering events such as the release of

autoinhibitory domains (for CaM-dependent kinases and calcineurin), active site

remodeling (anthrax adenylyl cyclase), and CaM-induced dimerization of membrane

24

proteins (Bachs et al., 1994, Hoeflich and Ikura, 2002). However, CaM can interact

with target proteins in both Ca2+ dependent as well as independent manners. In the

absence of Ca2+, the N-terminal domain adopts a closed confirmation whereas the

C-terminal domain is in a semi-open state with partial exposure of a hydrophobic

patch. Thus the C-terminal domain of CaM could interact with target proteins even in

the absence of Ca2+ (Swindells and Ikura, 1996).

The temporal and spatial associations between changes in Ca2+ and the function of

CaM have been identified in numerous studies. Changes in intracellular Ca2+

concentration induce dynamic compartmentalization and mobilization of CaM

including translocation from the cytosol to the nucleus (Luby-Phelps et al., 1995,

Deisseroth et al., 1998, Craske et al., 1999). CaM activation is coupled to elevation in

intracellular Ca2+ and also correlates with the spatial pattern of increased Ca2+ (Hahn et

al., 1992).

CaM not only acts as a ubiquitous transducer of intracellular Ca2+ signals but also

modulates intracellular Ca2+ concentrations. CaM can act as intracellular Ca2+ buffer,

regulate the plasma membrane Ca2+ ATPase, ryanodine receptors, IP3 receptors and

cyclic nucleotide gated Ca2+ channels (Baimbridge et al., 1992, Liu et al., 1994, Patel et

al., 1997, Balshaw et al., 2001).

Various downstream substrates of CaM have been studied extensively and the

studies presented here focus on delineating the role of Ca2+/CaM in regulation of

TGase-catalyzed transamidation activity. CaM increased TGase activity in human

25

erythrocyte (Billett and Puszkin, 1991), platelets and chicken gizzard (Plank et al.,

1983). In the presence of CaM, TGases are activated at lower Ca2+ concentrations than

the concentration that is normally needed for activation (Billett and Puszkin, 1991). In

cells transfected with TGase2 and mutant htt, CaM co-immunoprecipitates with both

TGase2 and mutant htt. A CaM inhibitor can decrease TGases-catalyzed cross-linking

of htt (Zainelli et al., 2004). Furthermore, fluorescence confocal microscopy

demonstrated that CaM colocalizes with TGase2 and with htt in HD intranuclear

inclusions (Zainelli et al., 2004), suggesting that CaM may play a pivotal role in HD.

Pathological role of TGases in neurodegenerative diseases

Protein aggregation and inclusion bodies are the common features of several

neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and

Huntington’s disease. Selkoe and colleagues were the first to propose the role for

TGases in neurodegeneration. They demonstrated that TGases could catalyze the

formation of insoluble polymers composed of human neurofilament proteins by

forming covalently crosslink (Selkoe et al., 1982). Increasing evidence suggests

TGases may be involved in protein aggregation in those neurodegenerative diseases

(Ross and Poirier, 2004, Muma, 2007).

Alzheimer’s disease (AD) and TGase

Neurofibrillary tangles (NFTs), deposits of tau filaments in the neuronal cell

body, are one of the most important pathologic characteristics of AD. Numerous

groups have shown that tau is cross-linked by TGases in vitro, forming insoluble

26

filamentous polymers (Dudek and Johnson, 1993, Appelt and Balin, 1997, Murthy et

al., 1998, Norlund et al., 1999). In post-mortem studies, it has been reported that

TGase2 colocalizes with paired helical filaments (PHFs), which are composed of tau,

in AD brain (Appelt et al., 1996). Our laboratory and others further demonstrated that

PHF-tau from AD brain contains TGase-catalyzed gamma-glutamyl-lysine

isodipeptide bonds (Norlund et al., 1999), and there is extensive co-localization of

TGase-catalyzed isodipeptide bonds and PHF-tau (Citron et al., 2002, Singer et al.,

2002), providing circumstantial evidence for the involvement of TGases in AD

pathology. Moreover, PHF-tau co-localizes with TGase-catalyzed cross-links in brain

regions devoid of NFT in stage II AD, which would be expected to contain them in

stage III, suggesting TGase as a contributing factor in NFT formation induces tau

cross-links in an early stage of AD (Singer et al., 2002).

Furthermore, TGase protein expression and cross-linking activity are

increased in AD brain and cerebrospinal fluid (CSF) as compared to controls (Kim et

al., 1999, Bonelli et al., 2002). Interestingly, TGase activity are only increased in the

brain regions with abundant neurofibrillary pathology, but not in the cerebellum, a

region spared of NFT pathology (Johnson et al., 1997).

Parkinson's disease (PD) and TGases

Multiple lines of evidence suggest that TGase-catalyzed bonds are associated

with Lewy bodies, the major inclusion body in PD. In vitro and in cell culture, TGase

is also capable of crosslinking a-synuclein, a major component of Lewy bodies in PD,

27

to form intramolecular cross-links and high molecular weight polymers (Jensen et al.,

1995, Junn et al., 2003). Moreover, Glu79 and Lys80 in a-synuclein have been

identified as the TGase-reactive residues and Lys80 residue is located in a conserved

sequence in the synuclein gene family (Jensen et al., 1995). Immunohistochemistry

studies demonstrate that TGase-catalyzed cross-links are elevated in dopaminergic

neurons in PD substantia nigra, and co-localize with α-synuclein in Lewy bodies

(Junn et al., 2003, Andringa et al., 2004). On Western blots, the TGase-catalyzed

cross-link bond comigrates with α-synuclein from PD substantia nigra (Citron et al.,

2002, Andringa et al., 2004).

Huntington's disease (HD) and TGase

HD is an autosomal dominant neurodegenerative disorder characterized by

cognitive and behavioral disturbance, involuntary movements (chorea), striatal and

cortical neurodegeneration and neuronal inclusions (Gusella and MacDonald, 2000).

HD is caused by expansion of CAG repeats in exon1 of HTT gene, which is translated

into an expanded polyglutamine (polyQ) stretch at the N-terminus of the huntingtin

protein (htt) (Huntington’s Disease Collaborative Research Group, 1993).

Consequently, N-terminal proteolytic fragments of mutant htt form intranuclear and

cytoplasmic aggregates in neurons, the prominent neuropathologic hallmarks of HD.

This CAG “triplet” is normally repeated about 20 times, but an increase in the number

of repeats to 40 or more results in the expression of the disease (Duyao et al., 1993,

Huntington’s Disease Collaborative Research Group, 1993). Additionally, there is an

28

inverse correlation between age of onset of HD symptoms and the number of CAG

repeats (Duyao et al., 1993).

Several lines of evidence suggest that TGase may be involved in the

pathophysiology of HD. It was reported firstly by Cariello and colleagues (Cariello et

al., 1996) that TGase activity is above maximum control levels in some HD patients

and is correlated with the CAG repeat length. In vitro and in cell culture, htt is an

excellent substrate for TGase (Gentile et al., 1998, Kahlem et al., 1998, Karpuj et al.,

1999). The rate of the cross-linking reaction increases over an order of magnitude

with repeat lengths of over 40 glutamines. Additionally, TGase-catalyzed

cross-linking of mutant htt resulted in the formation of htt polymers (Kahlem et al.,

1998). TGase mRNA, protein levels, and activity are selectively elevated in HD

affected brain regions (Karpuj et al., 1999, Lesort et al., 1999, Lesort et al., 2002,

Zainelli et al., 2003).

TGase has become a therapeutic target for HD in both preclinical experiments

and clinical trials. Knocking-out TGase2 in an HD transgenic mouse model, R6/1

mice, results in improved motor performance and increases survival (Mastroberardino

et al., 2002). R6/2 mice treated with cystamine, a TGase inhibitor, showed extended

survival, improved body weight and motor performance and delayed

neuropathological sequela (Dedeoglu et al., 2002, Karpuj et al., 2002a). In the other

mouse model of HD, the YAC 128 mouse, treatment with cystamine ameliorates

striatal volume loss and decreases striatal neuronal atrophy (Van Raamsdonk et al.,

29

2005). Cysteamine, the dimer of cystamine, is an orphan drug approved for the

treatment of nephropathic cystinosis in humans. In 2008, researchers at Raptor

Pharmaceuticals Corp began with Phase II clinical trials of cysteamine for HD.

Distribution and functions htt protein

As a 350 kDa protein composed of 3,144 amino acids, htt is expressed

ubiquitously throughout the body. The highest concentrations are found in the brain

and testes, with moderate amounts in the liver, heart, and lungs (Walker, 2007). In the

brain, htt has the highest expression in the neocortex, cerebellar cortex, striatum, and

hippocampus (Fusco et al., 1999). Although htt is found primarily in the cytoplasm,

smaller amounts have also be detected in other cellular compartments, including the

nucleus (Hoogeveen et al., 1993, Kegel et al., 2002)

Several studies have revealed the role of wildtype htt in development. Complete

knockout of the mouse Htt gene is embryonic lethal between embryonic days 7.5 and

8.5 (Duyao et al., 1995, Nasir et al., 1995, Zeitlin et al., 1995). It was later found

that htt contributes to the formation of the nervous system. Mice carrying reduced

levels of wild-type htt display profound malformations of the cortex and striatum

(White et al., 1997). Several lines of evidence indicate that wild-type htt facilitates

neuronal survival as well. For example, overexpression of wildtype htt exhibits

neuroprotection against ischemic injury, excitotoxicity, and caspase activation

(Rigamonti et al., 2000, Zhang et al., 2003, Leavitt et al., 2006), where as disruption

of the mouse homologue of HTT gene results in a progressive degenerative neuronal

30

phenotype and sterility (O'Kusky et al., 1999, Dragatsis et al., 2000). Moreover, htt

may also participate in the regulation of apoptosis, control of BDNF production,

vesicular and mitochondrial transport, neuronal gene transcription, and synaptic

transmission (Cattaneo et al., 2005).

Similar to wildtype htt, mutant htt with expanded polyglutamine stretch is found

in the cytoplasm as well as the nucleus. In HD, cleavage of the full-length mutant htt

is critical for the formation of htt-containing aggregates which are a prominent

pathological feature in neurons in the cortex and striatum (DiFiglia et al., 1997, Sapp

et al., 1997). Htt has been defined as a caspase substrate with numerous sites for

specific caspase activity, such as amino acids 552 (caspase 2), amino acids 513, 552,

530, and 589 (caspase 3), and amino acid 586 (caspase 6) (Wellington et al., 1998,

Wellington et al., 2000). In HD and in transgenic mouse models, the activity of

caspases 3 and 9 is increased (Wellington et al., 2000, Zeron et al., 2004) and

inhibition of caspase 3, 6 or 9 activity decreases toxicity in models of HD (Wellington

et al., 2000, Tang et al., 2005).

While mutant htt is primarly cytosolic, the cleavage products of mutant htt,

N-terminal fragment with the polyglutamine stretch, can translocate into the nucleus

and mediate toxic functions of htt in HD (Hodgson et al., 1999, Graham et al., 2006).

Mutant htt can induce neurodegeneration by an apoptotic mechanism in a cell model

(Saudou et al., 1998). Proteasomal activity is decreased and the proteasome is unable

to process the polyglutamine repeat in htt-transfected cells and HD knock-in mouse

31

brains (Zhou et al., 2003, Venkatraman et al., 2004), suggesting that mutant htt may

disturb normal proteasome function. Also, mutant htt appears to interfere with other

cellular mechanisms such as gene transcription. It has been reported that mutant htt

causes decreased transcription of the brain-derived neurotrophic factor (BDNF) gene

(Zuccato et al., 2001), which is is necessary for survival of striatal neurons. cAMP

response element (CRE)-mediated transcription, which promotes the expression of

several pro-survival genes, are also impaired by mutant htt (Steffan et al., 2000,

Wyttenbach et al., 2001).

Although some evidence suggests that htt-associated toxicity is linked to soluble

htt and its protein–protein interactions (Petersen et al., 1999), it may also be correlated

with the formation of toxic aggregate species, which are found in the neuronal

intranuclear inclusions (NIIs) and cytoplasmic aggregates in the cortex and striatum

of HD brains (Davies et al., 1997, DiFiglia et al., 1997, Sapp et al., 1997).

Interestingly, htt-containing aggregates are predominately nuclear in juvenile HD

cases, while they are mainly localized to the neural processes in adult-onset cases

(DiFiglia et al., 1997).

CaM-regulated TGase-modification of htt

We previously reported that CaM associates with htt and TGase2 as

demonstrated by coimmunoprecipitation in transfeced cells and fluorescence

colocalization in intranuclear inclusions in HD brains (Zainelli et al., 2004). CaM has

been shown to increase TGase activity in different cells and tissues (Puszkin and

32

Raghuraman, 1985, Billett and Puszkin, 1991). Inhibition of CaM results in decreased

TGase-catalyzed modifications of htt in cells transfected with mutant htt and TGase2.

Using affinity purification, mutant htt with an expanded glutamine repeat binds to

CaM with a higher affinity than wild-type htt, suggesting in vivo interaction between

CaM and htt as well (Bao et al., 1996). These data, along with the observation that

TGase mRNA, protein, and activity are elevated in HD (Karpuj et al., 1999;Lesort et

al., 1999;Zainelli et al., 2003), and TGase2 colocalizes with both htt protein and

TGase-catalyzed cross-links in HD intranuclear inclusions (Zainelli et al., 2003),

implicate CaM and TGase in htt-aggregate formation in HD (Figure 2).

Hypothetically, interaction of mutant htt with both CaM and TGase results in an

increase in TGase-modified htt and consequent htt stabilization and aggregate

formation.

33

Figure 2. Proposed involvement of TGase and CaM in the formation of htt-containing

aggregates. When there are more than 35 glutamines (Gln) repeat in mutant htt,

protein conformation is changed. Some proteases such as caspase 3 and calpain can

cleave mutant htt and generate a toxic N-terminal htt fragment, which can be

transported to nucleus and neurites. We propose that N-terminal htt associated with

CaM at a specific binding domain, and htt were modified by TGase. This modification

may stabilize htt monomers or polymers and contribute to cytotoxicity and formation

of htt-containing aggregates.

34

CHAPTER TWO

TRANSGLUTAMINASE-CATALYZED TRANSAMIDATION: A NOVEL MECHANISM FOR RAC1 ACTIVATION BY 5-HT2A RECEPTOR

STIMULATION

(Published in J Pharmacol Exp Ther. 2008; 326(1):153-62)

ABSTRACT

Transglutaminase (TGase)-induced activation of small G proteins via 5-HT2A

receptor signaling leads to platelet aggregation (Walther et al., 2003). We hypothesize

that stimulation of 5-HT2A receptors in neurons activates TGase, resulting in

transamidation of serotonin to a small G protein, Rac1, thereby constitutively

activating Rac1. Using immunoprecipitation and immunoblotting, we show that in a

rat cortical cell line, A1A1v cells, serotonin increases TGase-catalyzed transamidation

of Rac1. This transamidation occurs in both undifferentiated and differentiated cells.

Treatment with a 5-HT2A/2C receptor agonist, 2,5-dimethoxy-4-iodoamphetamine

(DOI), but not the 5-HT1A receptor agonist, 5-hydroxy-2-dipropylamino tetralin

(DPAT), increases transamidation of Rac1 by TGase. In A1A1v cells, 5-HT2A

receptors mediate the transamidation reaction since expression of 5-HT2C receptors

was not detectable and the selective 5-HT2A receptor antagonist blocked

transamidation. Time course studies demonstrate that transamidation of Rac1 is

significantly elevated after 5 and 15 minutes of serotonin treatment, but returns to

35

control levels after 30 minutes. The activity of Rac1 is also transiently increased

following serotonin stimulation. Inhibition of TGase by cystamine or siRNA reduces

TGase-modification of Rac1 and cystamine also prevents Rac1 activation. Serotonin

itself is bound to Rac1 by TGase following 5-HT2A receptor stimulation as

demonstrated by co-immunoprecipitation experiments and a dose-dependent decrease

of serotonin-associated Rac1 by cystamine. These data support the hypothesis that

Rac1 activity is transiently increased due to TGase-catalyzed transamidation of

serotonin to Rac1 via stimulation of 5-HT2A receptors. Activation of Rac1 via TGase

is a novel effector and second messenger of the 5-HT2A receptor signaling cascade in

neurons.

INTRODUCTION

5-HT2A receptors are G-protein coupled receptors which are widely expressed

in the brain, peripheral vasculature, platelets and skeletal muscle. 5-HT2A receptors

are involved in diverse physiological functions, from platelet aggregation to

neuroendocrine release (Van de Kar et al., 2001, Walther et al., 2003).

Pathophysiologically, 5-HT2A receptor signaling is implicated in a number of

disorders including hypertension, atherosclerosis, anxiety and depression (Roth, 1994,

Weisstaub et al., 2006). The classical signal transduction pathway of 5-HT2A receptors

is Gq/11-coupled activation of phospholipase C (PLC) (Sanders-Bush et al., 2003),

which hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol

36

1,4,5-trisphosphate (IP3) and diacyglycerol. IP3 mobilizes calcium from the

endoplasmic reticulum and thereby increases intracellular Ca2+ (Julius, 1991).

TGases (EC 2.3.2.13) are a family of Ca2+-dependent enzymes. Once activated,

TGases can catalyze the cross-linking of proteins via the γ-carboxamide group of

peptide-bound glutamine and the є-amino group of peptide-bound lysine, forming an

inter- or intramolecular isodipeptide bond (Griffin et al., 2002). TGases can also

covalently link biogenic amines and polyamines, such as serotonin or spermine, to a

peptide-bound glutamine residue in a transamidation reaction (Folk et al., 1980, Dale

et al., 2002). In platelets, activation of 5-HT2A receptors stimulates TGase-catalyzed

transamidation of serotonin to small G proteins, such as RhoA and Rab4, rendering

them constitutively active (Walther et al., 2003). Furthermore, neuronal differentiation

of SH-SY5Y cells induced by retinoic acid increases the expression/activation of

TGases, resulting in transamidation of putrescine to RhoA and activation of RhoA

(Singh et al., 2003).

Rac1 belongs to the Rho family of small G proteins, a subgroup of the Ras

superfamily. Members of the Rho family (e.g. RhoA, Rac1, Cdc42) are associated

with a wide array of cellular processes such as cytoskeletal organization, vesicular

transport, cell cycle progression, cell adhesion and migration, neuronal differentiation

and a variety of enzymatic activities (Etienne-Manneville and Hall, 2002, Burridge

and Wennerberg, 2004). Like other small G proteins, Rac1 functions as a molecular

switch which cycles between two conformational states: a GTP-bound, active state

37

and a GDP bound, inactive state. The GTP/GDP cycling is controlled by many

regulator molecules such as GTPase-activating proteins (GAPs), guanine nucleotide

exchange factors (GEFs) and GDP dissociation inhibitors (GDIs) (Hakoshima et al.,

2003). Post-translation modification of Rac1 in the regulator-interaction sites or in the

GTP-hydrolyzing domain may have an enormous effect on its activation cycle.

Bearing five glutamine residues in the amino acid sequence (Matos et al., 2000), Rac1

might serve as a suitable substrate of TGases resulting in the transamidation of

primary amines to Rac1 and rendering this small G protein constitutively active.

A1A1v cells derived from embryonic rat cortex (Scalzitti et al., 1998) provide

an excellent experimental model to study 5-HT2A receptor signaling. In the present

study, we use A1A1v cells to test whether 5-HT2A receptor stimulation increases

TGase-catalyzed transamidation and activation of Rac1 since they endogenously

express 5-HT2A/1A receptors, Gαq/11, PLC β, TGase 2, the serotonin transporter, and

small G proteins including Rac1, RhoA and Cdc42.

MATERIALS AND METHODS

Cell Culture

A1A1v cells, a rat cortical cell line, were grown on 100 mm2 plates coated with

poly-L-ornithine (Sigma, St Louis, MO) and maintained in 5% CO2 at 33°C, in

Dulbecco’s modified Eagle medium (DMEM) (Fisher Scientific, Pittsburgh, PA)

containing 10% fetal bovine serum (FBS) (Fisher Scientific, Pittsburgh, PA). Cells

38

were induced to differentiate by incubation at 37°C for 4 days. Before each

experiment, cells were maintained in DMEM with 10% charcoal-treated FBS for 48

hours. Charcoal adsorption removes most but not all serotonin in the medium. The

maximal final concentration of serotonin in the medium is approximately 3 nM

(Unsworth and Molinoff, 1992). Cells from passages 8-15 were used for all

experiments.

Drugs

The following drugs were used in this study: (-)-1-(2,5-dimethoxy-4-

lodophenyl)-2-aminopropane HCl (DOI) and serotonin (Sigma, St Louis, MO).

(R)-(+)-8-hydroxy-2-dipropylamino tetralin hydrobromide (DPAT) (Tocris, Ellisville,

MO). 2-aminoethyl disulfide dihydrochloride (cystamine) (MP biomedicals, Costa

Mesa, CA). α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidine

methanol (MDL 100907) (a gift from Hoechst Marion Roussel Research Institute,

Cincinnati, OH). Serotonin was dissolved in 10 µM HCl and MDL 100907 was

dissolved in a minimal volume of Dimethyl sulfoxide (DMSO) then diluted with

saline. The remaining drugs were dissolved in purified water. All compounds were

further diluted (at least 1:100) in cell culture media before they were applied to the

cells and washed away prior to lysing the cells.

Immunoprecipitation of TGase-modified Protein

A1A1v cells were harvested and lysed using lysis buffer A (25 mM Tris-HCl, pH 7.5,

250 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 1:1000 protease inhibitor cocktail

39

(Sigma, St Louis, MO) containing 104 µM AEBSF, 0.08 µM aprotinin, 2 µM

leupeptin, 4 µM bestatin, 1.5 µM pepstatin A and 1.4 µM E-64). Protein

concentration was determined using the BCA Protein Assay kit (Pierce, Rockford, IL).

Immunopurification of proteins containing TGase-catalyzed bonds was performed

using 81D4 mAb (mouse IgM) prebound to Sepharose beads (Covalab, Lyon, France)

using a protocol developed by Covalab and as described previously (Norlund et al.,

1999, Zainelli et al., 2005). The 81D4 antibody is well characterized and has been

previously shown to be specific for the N epsilon-(gamma-L-glutamy)-L-lysine

isopeptide and N epsilon-(gamma-L-glutamy)-L-lysine isopeptide cross-link

generated by TGase (Sarvari et al., 2002, Thomas et al., 2004). The 81D4 antibody

has been extensively used to demonstrate increases in TGase-catalyzed bonds (Citron

et al., 2002, Junn et al., 2003, Andringa et al., 2004). The use of the 81D4 antibody in

a competitive ELISA assay was shown to result in isodipeptide cross-link

measurements that correlate well to those measured by HPLC analysis but provide

more sensitivity than the HPLC approach (Sarvari et al., 2002). Transfection of cells

to over-express TGases, including TGase 1, 2 and 3, and a substrate protein such as

mutant huntingtin protein results in increased presence of the isodipeptide cross-link

in mutant huntingtin protein as demonstrated with immunoprecipitation with the 81D4

antibody (Zainelli et al., 2005).

Briefly, 20µl of sepharose-81D4 beads were washed three times in TBS/0.1%

Tween 20 with gentle shaking for 15 minutes, followed by adding 200µg cell lysate (1

40

μg/μl) to the washed beads and incubating for 2 hours at 37°C. After incubation, the

pellets were washed four times in TBS/0.1% Tween 20 for 15 minutes. Then 20µl of

loading buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, 10%

glycerol and 5% β-mercaptoethanol) was added to the washed pellets followed by 5

minutes incubation at 90°C. The samples were then centrifuged at 9,000 × g for 2

minutes and the supernatant was transferred and stored at -80°C until immunoblot

analysis.

Immunoprecipitation of Rac1

200 µg of protein from each sample was brought up to a total volume of 100 µl with

IP buffer (50 mM Tris-HCl, pH 7.4, 10 mM EGTA, 100 mM NaCl, 0.5% Triton-X

100 and 0.1% protease inhibitor cocktail), then the samples were precleared using

10µl of recombinant protein G (rProtein G) agarose (Invitrogen, Carlsbad, CA) for 1

hour. The samples were centrifuged at 9,000 × g for 10 minutes and the supernatant

was incubated for 1.5 hour at 4ºC with either 2 µg of Rac1 antibody (Upstate, Lake

Placid, NY) or 2 µg of normal mouse IgG (Santa Cruz, Santa Cruz, CA) as a control

for non-specific binding. The immuno-complexes were precipitated using 20 µl of

rProtein G agarose at 4ºC for 1 hour. The agarose-immuno complexes were washed

three times in IP buffer and centrifuged after each wash at 100 × g for 3 minutes. After

the last wash, bound proteins were eluted by adding 2× PAGE sample buffer and

heating for 5 minutes at 90ºC. The samples were centrifuged at 13,000 × g for 5

minutes and the supernatant was transferred and stored at -80°C until immunoblot

41

analysis.

Immunoblot

Immunoaffinity purified proteins and cell lysates were separated on 12%

SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose

membranes. Membranes were then incubated in blocking buffer (5% non-fat dry milk,

0.1% Tween 20, 1× TBS) for 1 hour at room temperature. Membranes were incubated

overnight at 4°C with primary antibodies on a shaker. Primary antibodies (Upstate

Biotechnology, NY: anti-Rac1, mouse IgG, 1:700; anti-Na+/K+ ATPase, mouse IgG, 1:

10000; Abcam, Cambridge, MA: anti-serotonin, rabbit IgG, 1:1000; BD Pharmingen,

San Jose, CA: anti-5-HT2C receptor, mouse IgG, 1:300; Cell Signaling, Danvers, MA:

anti-phosphorylated ERK1/2 at residues Thr202/Tyr204, mouse IgG, 1:1,000;

anti-ERK1/2, rabbit IgG, 1:1,000; MP Biomedicals, Aurora, OH: anti-actin, mouse

IgG, 1:20,000; CovalAb, Lyon, France: 81D4, mouse IgM, 1: 500; NeoMarkers,

Fremont, CA: TG100, mouse IgG, 1:100) were diluted in antibody buffer (1% non-fat

dry milk, 0.1% Tween 20, 1× TBS). The next day, membranes were washed with

TBS/0.1% Tween 20 and then incubated with goat-anti-mouse or goat-anti-rabbit

secondary antibody conjugated to HRP (Jackson ImmunoResearch, West Grove, PA)

diluted in antibody buffer. Membranes were washed and signal was detected using

enhanced chemiluminescence (ECL) western blotting detection reagents (Amersham

Biosciences, Piscataway, NJ). Using Scion Image for Windows (Scion, Frederick,

MD), immunoblots were quantified by calculating the integrated optical density (IOD)

42

of each protein band on the film.

Expression and Purification of Glutathione S-Transferase (GST)-PAK1

The pGEX-2T plasmid was used to express Rac1 interactive domain of human PAK1

(residues 51–135) fused to GST. The BL21 (DE3) E. coli (Invitrogen life technologies,

Carlsbad, CA) were transformed with plasmid expressing GST-PAK1 and grown

overnight at 37°C on LB-ampicillin plates. Then a single colony was used to inoculate

25ml LB-ampicillin (100 µg/ml) which was shaken overnight at 37°C. 10 ml of the

overnight culture was used to inoculate 500ml LB-ampicillin which was grown for 3

h at 37°C while shaking. After induction with 0.4 mM isopropyl

β-D-1-thiogalactopyranoside (IPTG) for 2 h at 30-32ºC, bacteria were lysed in lysis

buffer B (50 mM Hepes, pH 7.6, 1% Triton X-100, 100 mM NaCl, 5 mM MgCl2, 10%

glycerol, 1 mM PMSF and 0.1% protease inhibitor cocktail). The lysates were

sonicated and centrifuged for 15 minutes at 9,000 × g at 4ºC. Then the supernatant

was added to glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ) and

the mixture was incubated at 4ºC for 2 hours. The beads were then centrifuged at 4ºC

at 2,000 × g and were washed four times with lysis buffer B. The beads were

resuspended in lysis buffer B and were stored at -80ºC for at most two weeks.

Rac1 Activity Assay

A1A1v cells were treated for 5 or 15 minutes with 14 µM serotonin or vehicle (10 µM

HCl). The cells were harvested in lysis buffer C (50 mM Tris–HCl, pH 7.4, 100 mM

NaCl, 10 mM MgCl2, 1% Triton X-100, 0.1% SDS, 1 mM PMSF and 0.1% protease

43

inhibitor cocktail). Cell lysates were centrifuged at 14,500 × g for 20 minutes at 4°C

and 200 µl of the supernatant was incubated with 20 µl of GST-PAK1-Sepharose

beads for 40 minutes at 4°C under constant agitation. The beads were washed three

times with lysis buffer C, and 2× Laemmli sample buffer was added to the washed

beads followed by 5 minutes incubation at 90°C. The samples were then centrifuged

at 9,000 × g for 2 minutes and equivalent amounts of proteins in the supernatants

were loaded on 12% SDS-PAGE as well as 20 µg cell lysates (in order to detect total

Rac1 protein levels), followed by immunoblot analysis as described above.

Small Interfering RNA

To induce TGase2 gene silencing, two siRNA duplexes to target the coding sequence

of rat TGase2 mRNA were designed and synthesized by QIAGEN (Germantown,

MD). The target sequence is 5’-AAGAGCGAGATGATCTGGAAT-3’ for siRNA1

and is 5’- AGAGCCAACCACCTGAACAAA-3’ for siRNA2. The sense strand of

each siRNA was labeled at 3' end with Alexa Fluor 488 to monitor transfection

efficiency. At 30~50% confluence, A1A1v cells were transfected with siRNA at a

final concentration of 90 nM using Lipofectamine™ 2000 (Qiagen, Germantown, MD)

according to the manufacturer’s instructions. 24 hours after transfection, siRNA-lipid

complexes were removed by changing medium. Transfection efficiency was assessed

based on the percentage of fluorescent cells observed under Nikon Eclipse TE 2000U

microscopy. 48 hours after transfection, cells were stimulated with DOI or vehicle for

5 minutes, and then cell lysates were collected to measure TGase2 expression and

44

TGase-modified Rac1 by immunoblot and immunoprecipitation. Cells incubated with

Lipofectamine™ 2000 alone were used as the nontransfected control.

Statistical Analyses

All data are presented as group mean ± the standard error of the mean (SEM) and

analyzed by one-way or two-way ANOVA. Post hoc tests were conducted using

Newman-Keuls Multiple Comparison Test. SYSTAT 11 (Systat Software, Inc., San

Jose, CA) and GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA) were

used for all statistical analyses. A probability level of p<0.05 was considered to be

statistically significant for all statistical tests.

RESULTS

Serotonin treatment induces Rac1 transamidation in A1A1v cells

In order to determine whether TGase-catalyzed transamidation of Rac1 is increased

after serotonin treatment, we treated A1A1v cells with 14 µM serotonin for 5, 15 or

30 minutes. The transamidation of Rac1 is significantly elevated after 5 or 15 minutes

of serotonin treatment by approximately 2~2.5 fold as compared to vehicle

(HCl)-treated cells (Fig. 3). However, there is no significant difference in the amount

of TGase-modified Rac1 in cells treated with serotonin for 30 minutes as compared to

vehicle-treated cells (Fig. 3A, B).

5-HT2A receptor stimulation increases the transamidation of Rac1

In order to explore the serotonin receptor specificity for induction of TGase-catalyzed

45

transamidation, A1A1v cells were stimulated with 14 µM serotonin, 3 µM DOI

(5-HT2A/2C receptor agonist), 10 nM DPAT (5-HT1A receptor agonist) or 10 µM HCl

(vehicle) for 15 minutes. Treatment with serotonin or DOI significantly (p<0.05)

increases the amount of TGase-modified Rac1 2-fold as compared to vehicle-treated

cells, whereas treatment with DPAT has no effect on the amount of TGase-modified

Rac1 compared to vehicle-treated cells (Fig. 4A, B). Expression of 5-HT2A receptors

in A1A1v cells has been previously confirmed using anti-sense oligodeoxynucleotide

strategies and radioligand binding assays (Scalzitti et al., 1998). Here, 5-HT2C

receptor expression in undifferentiated and differentiated A1A1v cells was examined

by immunoblot analysis. Choroid plexus tissue from the fourth ventricle of a rat was

used as a positive control and HEK 293 cell lysates were used as a negative control.

We found that 5-HT2C receptors are expressed in rat choroid plexus, but 5-HT2C

receptor expression was not detected in either HEK293 cells or undifferentiated or

differentiated A1A1v cells (Fig. 4C). To exclude the possibility that the lack of

involvement of the 5-HT1A receptors in Rac1 transamidation is not due to insufficient

concentration of DPAT, we treated cells with 14 µM serotonin and increasing

concentrations of DPAT (1 nM, 10 nM, and 100 nM) for 5 minutes, and then detected

ERK phosphorylation by immunoblot. Treatment with 14 µM serotonin and 10 nM or

100 nM DPAT significantly increased phosphorylation of ERK (Fig. 4D, E),

indicating 10 nM DPAT is enough to activate 5-HT1A receptors in A1A1v cells. To

further confirm that the effect of DOI on Rac1 transamidation in A1A1v cells is due

46

to stimulation of the 5-HT2A receptors, cells were pretreated with 100 nM MDL

100907 (a selective 5-HT2A receptor antagonist) for 15 minutes, and then stimulated

with DOI for 5 minutes. We found the pretreatment of MDL 100907 significantly

reduced DOI-induced Rac1 transamidation without itself having any effect on Rac1

transamidation (Fig. 4F).

DOI increases Rac1 transamidation in both undifferentiated and differentiated

A1A1v cells

After differentiation of A1A1v cells, the cytoskeleton undergoes a dramatic

reorganization and the cells acquire a neuronal-like cell shape with long processes

similar to axons and dendrites compared to undifferentiated cells (Fig. 5A). Rho

family GTPases are critical regulators of the actin cytoskeleton organization

(Etienne-Manneville and Hall, 2002, Burridge and Wennerberg, 2004). This prompted

us to explore the effects of cell differentiation on Rac1 transamidation stimulated by

5-HT2A receptor activation. Stimulation of 5-HT2A receptors with DOI increased

TGase-catalyzed transamidation of Rac1 in both undifferentiated and differentiated

cells (Fig. 5B). Treatment with DOI in A1A1v cells significantly increased the amount

of TGase-modified Rac1 by approximately 2~2.5 fold as compared to vehicle-treated

cells (p<0.01). In vehicle-treated groups, the amount of TGase-modified Rac1 in

differentiated cells was similar to the amount in undifferentiated cells, indicating that

cell differentiation has no effect on basal level of Rac1 transamidation. Although the

amount of TGase-modified Rac1 in DOI-treated differentiated cells was increased

47

compared to DOI-treated undifferentiated cells, this increase was not significant,

suggesting that cell differentiation also does not have a significant effect on

DOI-induced transamidation of Rac1 (Fig. 5C).

The activity of Rac1 is transiently increased following serotonin stimulation

Racl is GDP-bound in the inactive state and GTP-bound when activated. Activated

Rac1 binds to its downstream effectors such as PAK1, therefore a GST-PAK1 fusion

protein purified from E. coli was used to determine if serotonin stimulation increases

the activated from of Rac1. A1A1v cells were treated with serotonin for 5 or 15

minutes, the cells were harvested and cell lysates were incubated with GST-PAK1

prebound to glutathione-Sepharose beads. Then equivalent amounts of the purified

proteins were resolved by SDS-PAGE. Immunoblot analysis was performed using an

antibody against Rac1. As shown in Fig. 6, the activity of Rac1 is increased by 80

percent after 5 minutes of serotonin treatment, but returns back to baseline after 15

minutes in the presence of serotonin, indicating that Rac1 becomes transiently

activated after serotonin treatment in A1A1v cells. In addition, there is no significant

change in the total amount of Rac1 in the cell lysate after serotonin treatment (Fig.

6A), suggesting that the activity increase is not due to synthesis of new Rac1.

TGase inhibition reduces Rac1 transamidation and activity

The activity of Rac1 is under the direct control of a large set of regulatory proteins. To

exam whether the serotonin-stimulated increase of Rac1 activity is due to

TGase-catalyzed transamidation, A1A1v cells were treated with increasing

48

concentrations (0 µM, 100 µM, 500 µM and 1000 µM) of the TGase inhibitor,

cystamine, for 1 h, followed by treatment with 14 µM serotonin for 5 minutes.

Precipitation of TGase-modified proteins and the activated Rac1 were performed as in

previous experiments. Then immunopurified proteins and activated Rac1 were

examined on immunoblots using anti-Rac1 and 81D4 antibodies. We found that

pretreatment with cystamine significantly decreases Rac1 transamidation (Fig. 7) and

activity (Fig. 8) in a dose-dependent manner. Treatment with 100 µM, 500 µM or

1000 µM of cystamine decreases the amount of TGase-modified Rac1 by

approximately 50%, 70% or 80%, respectively, as compared to untreated cells (Fig.

7A, C). A similar dose-dependent decrease in TGase-modified Rac1 was also

observed when cells were treated with serotonin for 15 minutes following cystamine

inhibition (data not shown). Treatment with 500 µM or 1000 µM of cystamine

decreases the amount of activated Rac1 by approximately 20% or 40%, respectively,

as compared to untreated cells (Fig. 8A, C). These results indicate that

TGase-catalyzed transamidation of Rac1 contributes to the increase in Rac1 activity

upon serotonin stimulation. The ubiquitously expressed Na+/K+ ATPase is a

well-established plasma membrane marker and its function underlies essentially all of

mammalian cell physiology (Kaplan, 2002). In order to verify that the dose-dependent

decrease of Rac1 transamidation and activation was not due to cystamine-induced

total Rac1 reduction or cellular toxicity, cell lysates from the same experiment were

examined on western blots with antibodies for Rac1 or Na+/K+ ATPase. There is no

49

significant difference in total Rac1 or Na+/K+ ATPase levels between

cystamine-treated and untreated cells, indicating that the concentrations of cystamine

were not toxic to the cells (Fig. 7B, 8B).

Next, a second and more specific approach was used to determine the

significance of TGase in Rac1 transamidation; we designed two siRNA duplexes to

inhibit endogenous TGase expression. Several isoenzymes of TGase are found in the

brain, of which TGase2 is the most abundant, therefore siRNAs were developed to

silence rat TGase2 gene. At 24 hours post-transfection, transfection efficiency of both

siRNA reached about 90%. 48 hours after transfection, siRNA1 and siRNA2

transfection of A1A1v cells resulted in 95% and 65% down-regulation of TGase2

protein expression, respectively (data not shown). Reprobing of membrane with

anti-Rac1 antibody revealed that neither siRNA changed Rac1 protein level,

indicating no off-target effect on Rac1 (Fig. 9A). DOI induced TGase-modification of

Rac1 was significantly reduced by knocking down TGase2 expression with siRNA1

or siRNA2. As compared to DOI-stimulated nontransfected cells, siRNA1 and

siRNA2 transfection caused 70% and 30% decreases in TGase-modified Rac1

responding to DOI, respectively (Fig. 9B, C). These results confirm that 5-HT2A

receptor-mediated Rac1 transamidation is dependent on TGase2 expression in A1A1v

cells.

TGase-mediated transamidation of serotonin to Rac1

TGase-modified Rac1 did not show a significant upward shift on immunoblots

50

compared to native Rac1 in cell lysates (data not shown), indicating that the molecular

weight of Rac1 does not significantly increase after modification by TGase. Therefore,

a small amine such as serotonin is most likely incorporated into Rac1 upon

stimulation of 5-HT2A receptors. To test this hypothesis, we stimulated A1A1v cells

with serotonin or vehicle for 5 minutes then cells were harvested and cell lysates were

used to immunoprecipitate Rac1 with an anti-Rac1 antibody, or immunoprecipitate

TGase-modified proteins with the 81D4 antibody. The immunopurified proteins were

examined on immunoblots using antibodies directed against serotonin.

Immunoprecipitation of Rac1 and probing for serotonin reveals an association

between Rac1 and serotonin in A1A1v cells (Fig. 10A). However we were unable to

detect serotonin in TGase-modified proteins (Fig. 10A). This may be due to a

compromise of the serotonin antibody epitope during the immunoprecipitation of

TGase-modified proteins, since this procedure uses higher temperatures and longer

incubation times compared to the Rac1 immunoprecipitation procedure. Therefore in

order to confirm that serotonin is associated with Rac1 by a TGase-catalyzed covalent

bond, we treated cells with increasing concentrations (0 μM, 100 μM, 500 μM, 1000

μM) of cystamine for 1 hour followed by stimulating the cells with serotonin for 5

minutes. Then we immunoprecipitated Rac1 and detected associated serotonin by

immunoblot, as described above. We found that treatment with cystamine decreases

the serotonin-associated Rac1 in a dose-dependent manner (Fig. 10B, D), thus

supporting the hypothesis that serotonin is incorporated into Rac1 by a

51

TGase-catalyzed covalent bond. To compare the effects of different agonists on the

incorporated serotonin, cells were exposed to either serotonin or DOI for 5 min. Then

Rac1 was immunoprecipitated and the incorporated serotonin was detected by

immunoblot. There was no significant difference in serotonin incorporation between

serotonin and DOI-treated cells. Pretreatment with cystamine also inhibited

DOI-induced serotonin incorporation (Fig. 11).

52

Fig.3 Serotonin-induced increase of Rac1 transamidation in A1A1v cells. (A) After 5,

15 or 30 minutes of 14 µM serotonin treatment, TGase-modified proteins were

immunoprecipitated with 81D4 antibody coupled to sepharose. TGase-modified Rac1

was then detected on immunoblots with anti-Rac1 antibody. (B) Quantitation of

immunoblots for 3 separate experiments. Data shown are the mean IOD ± SEM and

normalized to vehicle-treated control levels. One-way ANOVA (F(3,8)=7.31, p<0.05)

indicates a significant difference among groups. Newman-Keuls Multiple Comparison

Test indicates p<0.05* as compared to vehicle treatment.

53

Fig. 4 Serotonin receptor specificity on induction of Rac1 transamidation in A1A1v

cells. (A) Cells were stimulated with 14 µM serotonin, 3 µM DOI (5-HT2A/2C receptor

agonist), 10 nM DPAT (5-HT1A receptor agonist) or vehicle for 15 minutes.

TGase-modified Rac1 was detected by immunoprecipitation and immunoblot analysis.

(B) Quantitation of immunoblots for 3 separate experiments. Data shown are the

mean IOD ± SEM and normalized to vehicle-treated control levels. One-way ANOVA

(F(3,8)=10.05, p<0.01) indicates a significant difference among various treatments.

Newman-Keuls Multiple Comparison Test indicates p<0.05* as compared to vehicle

treatment. (C) Immunoblot analysis of 5-HT2C receptor expression in undifferentiated

(UD) and differentiated (DI) A1A1v cells. Rat choroid plexus was used as a positive

54

control and HEK 293 cell was used as a negative control. (D) A1A1v cells were

treated with vehicle, 14 µM serotonin or DPAT (1 nM to 100 nM) for 5 minutes. Cell

lysates were then resolved by SDS-PAGE, followed by immunoblotting with an

anti-phosphorylated ERK antibody. Equal loading was verified by reprobing the same

membrane with antibodies to total ERK and actin. (E) Quantitation of immunoblots

for 3 separate experiments. Data shown are the mean IOD ± SEM and normalized to

serotonin-treated control levels. Each phospho-ERK value was normalized using the

corresponding total ERK value to control for the possibility of differential loading.

One-way ANOVA (F(4,10)=18.12, p<0.001) indicates a significant difference among

various treatments. Newman-Keuls Multiple Comparison Test *indicates p<0.05 as

compared to vehicle treatment and #indicates p<0.05 as compared to serotonin

treatment. (F) Upper, cells were stimulated with vehicle, 14 µM serotonin, 3 µM DOI.

Some cells were pretreated with 100 nM MDL 100907 prior to treatment with DOI or

treated with MDL100907 alone. TGase-modified Rac1 was detected by

immunoprecipitation and immunoblot analysis. Lower, quantitation of immunoblots

for 3 separate experiments. Data shown are the mean IOD ± SEM and normalized to

vehicle-treated control levels. One-way ANOVA (F(4,10)=38.96, p<0.001) indicates a

significant difference among various treatments. Newman-Keuls Multiple

Comparison Test indicates p<0.05* as compared to vehicle treatment and p<0.05# as

compared to DOI treatment.

55

Fig. 5 The effects of cell differentiation on Rac1 transamidation. (A) A1A1v cells

were incubated at 37°C for 4 days to induce differentiation. Compared to

undifferentiated cells (left), differentiated cells (right) develop a neuron-like cell

shape with long neurites. (B) Cells were treated with 3 µM DOI for 15 minutes,

followed by immunoprecipitation and immunoblot analysis. Stimulation of 5-HT2A

receptors by DOI increased the TGase-catalyzed transamidation of Rac1 in both

undifferentiated and differentiated cells. (C) Quantitation of immunoblots for 3

separate experiments. Data shown are the mean IOD ± SEM and normalized to

vehicle-treated control levels in undifferentiated cells. Two-way ANOVA indicates a

56

significant main effect of DOI treatment (F(1,8)=49.48, p<0.01). However, there was

no significant main effect of cell differentiation on TGase-modified Rac1 (F(1,8)=4.07,

p=0.08). The interaction between DOI treatment and cell differentiation was also not

significant for TGase-modified Rac1 (F(1,8)=0.34, p=0.58). Newman-Keuls

Multiple Comparison Test indicates p<0.01* as compared to vehicle treatment in

undifferentiated cells, p<0.01# as compared to vehicle treatment in differentiated cells.

Scale bar, 105 µm.

57

Fig. 6 Rac1 activity was transiently increased after serotonin treatment. (A) Top ,

activated Rac1 was purified using GST-PAK1 coupled to glutathione-Sepharose beads

and detected on immunoblot with an antibody against Rac1. Middle, the total amount

of Rac1 in the cell lysates used for pull-down of activated Rac1 was monitored by

immunoblot. Bottom, Ponceau staining of GST-PAK1 documents equal amounts of

protein loading in each lane. (B) Quantitation of immunoblot for 3 separate

experiments. Data shown are the mean IOD ± SEM and normalized to vehicle-treated

control levels. The two-way ANOVA reveals a significant (F(1,8)=8.64, p<0.05) main

effect for serotonin treatment, and a significant (F(1,8)=6.32, p<0.05) interaction

between serotonin treatment and time course. Newman-Keuls Multiple Comparison

Test indicates p<0.05* as compared to 5 minutes of serotonin treatment.

58

Fig.7 Dose-dependent effects of cystamine on Rac1 transamidation. (A)

Immunoprecipitation and immunoblot analyses reveal that cystamine causes a

dose-dependent (100 to 1000 µM) inhibition of the transamidation of Rac1 as

indicated by less intense bands. (B) Cells lysates from the same experiment were

examined on immunoblots with antibodies for Rac1 and Na+/K+ ATPase, which

indicate that cystamine has no effect of on total Rac1 and cell viability, respectively.

(C) Quantitation of effects of cystamine on Rac1 transamidation in 3 separate

experiments. Data shown are the mean IOD ± SEM and normalized to untreated

control levels. One-way ANOVA (F(3,8)=19.41, p<0.001) indicates a significant effect

of cystamine on Rac1 transamidation. Newman-Keuls Multiple Comparison Test

indicates p<0.05* as compared to untreated cells.

59

Fig.8 Rac1 activity is inhibited by cystamine. (A) Top , pull-down and immunoblot

analyses reveal that cystamine causes a dose-dependent (100 to 1000 µM) inhibition

of the activation of Rac1. Middle, the same membrane was reprobed with antibody

81D4, directed against TGase-catalyzed covalent bonds. Bottom, Ponceau staining of

GST-PAK1 (~35 kDa) documents equal amounts of protein loading in each lane. (B)

Cell lysates from the same experiment were examined on immunoblots with an

antibody for Na+/K+ ATPase. (C) Quantitation of the effects of cystamine on Rac1

activation in 3 separate experiments. Data shown are the mean IOD ± SEM and

normalized to untreated control levels. One-way ANOVA (F(3,8)=20.94, p<0.001)

60

indicates a significant effect of cystamine on Rac1 activity. Newman-Keuls Multiple

Comparison Test indicates p<0.05* as compared to untreated cells, p<0.001** as

compared to untreated cells.

61

Fig.9 Knockdown of TGase2 by siRNAs prevents Rac1 transamidation. (A) Cells

were incubated with 90nM of TGase2-specific siRNAs for 24 hours, and then treated

with 3 µM of DOI or vehicle for 5 minutes at 48 hours post-transfection. Immunoblot

analyses confirmed that transfection of siRNA1 or siRNA2 significantly inhibited

TGase2 protein expression compared to nontransfected control (NT). The membranes

were stripped and reprobed with anti-Rac1 and anti-actin antibodies. (B)

TGase-modified Rac1 was detected by immunoprecipitation and immunoblot analysis.

DOI induced TGase-modification of Rac1 were significantly decreased in

siRNAs-transfected cells compared to NT cells. (C) Quantitation of effects of siRNAs

62

on Rac1 transamidation in 3 separate experiments. Data shown are the mean IOD ±

SEM and normalized to DOI-stimulated nontransfected cells. Two-way ANOVA

indicates a significant main effect of transfection (F(2,12)=24.68, p<0.0001), a

significant main effect of DOI treatment (F(1,12)=63.23, p<0.0001) and a significant

interaction between transfection and DOI treatment (F(2,12)=9.42, p<0.01) on

TGase-modified Rac1. Newman-Keuls Multiple Comparison Test indicates p<0.01*

or p<0.001** as compared to vehicle treatment in nontransfected cells, p<0.01# or

p<0.001## as compared to DOI treatment in nontransfected cells.

63

Fig.10 Transamidation of serotonin to Rac1 is mediated by TGase. (A)

Immunoprecipitation of Rac1, but not TGase-modified proteins, reveals an

association between Rac1 and serotonin in A1A1v cells upon 5 minutes of 14 µM

serotonin stimulation. (B) Immunoprecipitation and immunoblot analysis reveal that

cystamine causes a dose-dependent (100 to 1000 µM) reduction of the

serotonin-associated Rac1. Normal mouse IgG is used as a negative control (NC) for

immunoprecipitation. (C) Cell lysates from the same experiment were examined on

immunoblots with an antibody for Na+/K+ ATPase, which indicates cystamine has no

effect of on cell viability. (D) Quantitation of the effects of cystamine on

64

serotonin-induced transamidation of Rac1. Data shown are the mean IOD ± SEM and

normalized to untreated control levels. One-way ANOVA (F(3,8) = 7.15, p<0.05)

indicate a significant difference in serotonin-associated Rac1 among cells treated with

different concentration of cystamine. Newman-Keuls Multiple Comparison Test

indicates p<0.05* as compared to untreated cells.

65

Fig.11 DOI stimulates transamidation of Rac1 to serotonin to a similar extent as those

treated with serotonin. (A) Cells were stimulated with vehicle, 14 µM 5-HT or 3 µM

DOI for 5 minutes. Some cells were pretreated with 500 µM cystamine (Cys) for 1

hour prior to stimulation with DOI. Transamidation of serotonin to Rac1 was detected

by immunoprecipitation and immunoblot analysis. (B) Quantitation of immunoblot

for 3 separate experiements. Data shown are the mean IOD ± SEM and normalized to

serotonin-treated levels. One-way ANOVA (F(3,8)=19.80, p<0.001) indicates a

significant difference in Rac1-incorporated serotonin among groups. Newman-Keuls

Multiple Comparison Test * indicates p<0.05 as compared to vehicle-treated cells and

# indicates p<0.05 as compared to serotonin-treated cells.

66

DISCUSSION

In platelets, 5-HT2A receptor activation causes TGase-catalyzed

transamidation of RhoA and Rab4, leading to activation of these proteins and platelet

aggregation (Walther et al., 2003). In aplysia ganglia, serotonin-treatment induces the

activation of Cdc42 and its downstream effector PAK to regulate the actin

cytoskeleton (Udo et al., 2005). In the present study, we extend these findings to a rat

cortical cell model and find that 5-HT2A receptor stimulation increased

TGase-catalyzed transamidation of Rac1 and Rac1 activity, both of which can be

suppressed by TGase inhibition. In elucidating the mechanism further, we identify

serotonin as an amine that becomes transamidated to Rac1 by TGase. Activation of

Rac1 via TGase is a novel effector and second messenger of the 5-HT2A receptor

signaling pathway in neurons.

To exam the serotonin receptor specificity on induction of Rac1

transamidation, we treated cells with different serotonin receptor agonists and found

that the 5-HT2A/2C receptor agonist DOI, but not the 5-HT1A receptor agonist DPAT

induces a significant increase in TGase-modified Rac1 with a similar magnitude to

that observed in serotonin-treated cells. Since there is no 5-HT2C receptor expression

in A1A1v cells and MDL 100907 reversed the effect of DOI on Rac1 transamidation

(Fig. 4), serotonin or DOI-induced Rac1 transamidation is selectively mediated by

activation of 5-HT2A receptors in A1A1v cells.

Previous studies demonstrated that TGase-mediated transamidation of the Rho

67

family of small G proteins blocked the GTP-hydrolyzing activity of these proteins,

rendering these small GTPases constitutively active for their respective signaling

pathways (Masuda et al., 2000, Walther et al., 2003). Based on these results, we

hypothesized that 5-HT2A receptor-stimulated transamidation of Rac1 by TGase will

result in prolonged activation of Rac1. Unexpectedly, we found a transient increase in

both Rac1 activity and TGase-catalyzed transamidation which returned to baseline

levels following continuous exposure to serotonin (Figs. 3 and 6). The transient

response may due to selective degradation of transamidated Rac1. Mammalian cells

contain multiple proteolytic systems for degradation of various classes of intracellular

proteins. Most abnormal proteins (mutant or misfolded) are degraded in the

ubiquitin–proteasome pathway. Rac1 is normally a stable protein, but activation by

cytotoxic necrotizing factor-1 (CNF-1) in rat bladder carcinoma cells (804G) and

human umbilical vein endothelial cells resulted in increased sensitization to

ubiquitination and proteasomal degradation (Doye et al., 2002, Munro et al., 2004).

Further, dominant-positive forms of Rac1 appeared more susceptible to

ubiquitin-mediated proteasomal degradation compared to both dominant-negative and

wild-type Rac1(Doye et al., 2002). Activated and transamidated Rac1 may be targeted

for proteasomal degradation, resulting in transient activation of Rac1. However, there

is no significant change in total Rac1 protein 15 min after serotonin stimulation (Fig.

4A), suggesting that activated or transamidated Rac1 may only account for small

fraction of total Rac1.

68

In this study, we found differences in the time course for Rac1 transamidation

and for its increased activity. After serotonin stimulation for 15 minutes, Rac1 activity

is reduced to baseline (Fig. 6) while TGase-catalyzed transamidation of Rac1 still

remains at a relatively high level (Fig. 3). These results suggest that transamidation

may not only occur at the GTPase activity-related residues, but also at residues which

do not have an effect on Rac1 activity. Essentially, transamidation of Rac1 at some

sites may induce activation of the small G proteins, while transamidation at other sites

may not affect the GTPase activity of Rac1. If activated Rac1 is more sensitive to

proteasomal degradation, the activated Rac1 may be degraded earlier than

non-activated Rac1 that is transamidated at sites not involved in activation.

Cystamine has been shown to decrease TGase activity and TGase-catalyzed

є-(γ-glutamyl) lysine bonds in cultured cells and animal models of neurodegenerative

diseases (Karpuj et al., 2002a, Ientile et al., 2003, Zainelli et al., 2005). As a primary

amine, cystamine is a potential substrate for TGase, and acts as a competitive

inhibitor for TGase by blocking access to the active site of the enzyme for the

glutamine residues in proteins which would otherwise participate in forming

є-(γ-glutamyl) lysine bonds (Lorand et al., 1979). In our study, pretreatment with

cystamine reduced both serotonin-stimulated Rac1 transamidation (Fig. 7A, C) and

activation (Fig. 8A, C) in a dose-dependent manner. Those observations were further

supported by the use of TGase2-specific siRNAs, which significantly suppressed DOI

induced TGase-modification of Rac1 (Fig. 9B, C). These results suggest that

69

cystamine prevented Rac1 transamidation through TGase inhibition, and that

transamidation of Rac1 by TGase may contribute to the increase in Rac1 activity.

Post-translational modifications, such as transamidation and phosphorylation

of Rac1 may affect its ability to interact with regulatory proteins (e.g. GAPs, GEFs

and GDIs) or to convert GTP back to GDP, resulting in increased activation of Rac1

and stimulation of its signaling cascade in the cells. The Conserved Domain Database

(CDD) (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd) provides an

interactive tool to identify conserved domains present in protein sequences. We

searched the CDD for GTP/Mg2+ binding sites, and GAPs, GEFs and GDIs interaction

sites in the Rac1 sequence (PSSM-Id: 57957) because they will be most likely to

modify Rac1 activity after TGase-catalyzed transamidation at these sites. We

identified two glutamine residues (Gln61, Gln74) located within these domains. It has

been reported that site-specific deamidation of a Gln residue in the Rho family G

proteins (Gln61 in Rac and Cdc42, Gln63 in RhoA) by CNF-1 inhibits both intrinsic

and GAPs-stimulated GTP hydrolysis activity, resulting in constitutive activation of

these proteins (Flatau et al., 1997, Schmidt et al., 1997). CNF-1 has also been shown

to possess in vitro TGase activity. In the presence of primary amines, RhoA is

transamidated in vitro at Gln63 by CNF-1 and at positions 52, 63 and 136 by guinea

pig liver TGase (Schmidt et al., 1998). Similarly, in addition to Gln61 which is critical

in regulating Rac1 activity, the other four glutamine residues in Rac1 may also be

modified by TGase. It is not surprising, therefore, that the same dosage of cystamine

70

prevents Rac1 transamidation and its activation to different extents (Fig. 7C, 8C).

Walther et al. found that in platelets TGase covalently cross-links serotonin to

RhoA or Rab4 at a position in the phosphate-binding site that is conserved in the

sequences of all Ras-related small GTPases (Walther et al., 2003). It has also been

shown that polyamines such as putrescine, spermidine and spermine could be

incorporated into Rac1 by bacterial TGase in vitro (Masuda et al., 2000). In our study,

co-immunoprecipitation assays demonstrated that serotonin is associated with Rac1 in

A1A1v cells after serotonin stimulation (Fig. 10A). To explore the association

mechanism further, we inhibited TGase by cystamine and found a reduction of

serotonin-associated Rac1 with increasing concentrations of cystamine (Fig. 10B, D),

indicating that serotonin is incorporated into Rac1 by a TGase-catalyzed bond. Since

both serotonin and DOI stimulation can lead to serotonin incorporation to similar

levels (Fig. 11), the serotonin bound to 5-HT2A receptors is not likely the source of

Rac1-incorporated serotonin; the serotonin bound to Rac1 may originate from

endogenously synthesized serotonin or serotonin transported into the cell from serum

in the cell culture media.

The first signal transduction mechanism identified for the 5-HT2A receptor was

Gq/11 mediated activation of PLC, leading to increased accumulation of IP3 and an

increase in intracellular Ca2+ (Boess and Martin, 1994). In addition to activation of

PLC, extensive evidence suggests that 5-HT2A receptors couple to other effector

pathways, such as phospholipase A2/arachidonic acid cascade (Berg et al., 1994b),

71

Mitogen-Activated Protein Kinase signaling (Watts, 1998) and phospholipase

D/protein kinase C pathway (Mitchell et al., 1998). On the other hand, TGases are

subjected to transcriptional regulation by retinoic acid and steroid hormones

(Fujimoto et al., 1996, Ou et al., 2000), and require the binding of Ca2+ for their

activity (Burgoyne and Weiss, 2001). Evidence indicates that TGase activity can be

significantly enhanced in response to increased intracellular Ca2+ through IP3

generation (Zhang et al., 1998). In aplysia, serotonin mediated activation of Cdc42

was dependent on PLC and phosphatidylinositol 3-kinase (PI3K) (Udo et al., 2005).

In RA-induced neuronal differentiation of SH-SY5Y cells, TGase-catalyzed

transamidation is required for activation of RhoA (Singh et al., 2003), whereas

activation of Rac1 is mediated by PI3K in a transamidation-independent manner (Pan

et al., 2005). Further studies are needed to elucidate the underlying molecular

mechanisms by which the 5-HT2A receptor signaling regulates TGase-catalyzed

activation of Rac1.

The present study provides the first evidence in neurons that stimulation of

5-HT2A receptors induces an increase in TGase-catalyzed transamidation of serotonin

into Rac1 and constitutive activation of Rac1. Further studies using alternative

approaches to measure Rac1 transamidation, such as mass spectroscopy, are needed to

confirm this hypothesis. Rac1 activation via 5-HT2A receptor stimulation may have an

important functional impact given the plethora of pathways that utilize this small G

protein. For example, Rac1 is best known as a regulator for the assembly of the actin

72

cytoskeleton, thereby playing a role in neurite outgrowth and neuronal differentiation.

We demonstrate that stimulation of 5-HT2A receptor can increase the transamidation

of Rac1 in both undifferentiated and differentiated A1A1v cells, suggesting that

transamidation and constitutive activation of this signal transducer are not altered by

neuronal differentiation of cells. However, the effect of 5-HT2A receptor-mediated

activation of Rac1 on cytoskeleton organization, cell cycle progression,

transcriptional activation or other crucial cellular functions in neurons has yet to be

explored.

34

CHAPTER THREE:

PHOSPHOLIPASE C, CALCIUM AND CALMODULIN SIGNALING ARE

REQUIRED FOR 5-HT2A RECEPTOR-MEDIATED TRANSAMIDATION OF

RAC1 BY TRANSGLUTAMINASE

ABSTRACT

Increasing evidence indicates that small G proteins undergo transglutaminase

(TGase)-catalyzed transamindation and activation in neurons, platelets, and smooth

muscle cells. We have previously reported that 5-HT2A receptor stimulation increased

TGase-catalyzed transamidation and activation of Rac1 small G protein in A1A1v

cells, a rat embryonic cortical cell line. In this study, we explore the signaling

pathways involved in 5-HT2A receptor-mediated Rac1 transamidation. In cells

pretreated with phospholipase C (PLC) inhibitor U73122, the 5-HT2A receptor

specific agonist, DOI-stimulated increase in intracellular Ca2+ concentration and

TGase-modified Rac1 were significantly attenuated as compared to those pretreated

with U73343, an inactive analog. Addition of the membrane-permeant Ca2+ chelator,

BAPTA-AM strongly reduced TGase-catalyzed Rac1 transamindation upon DOI

stimulation. Conversely, when cells were treated with Ca2+ ionophore, ionomycin at a

concentration that produced an elevation of cytosolic Ca2+ to a level comparable to

cells treated with DOI, ionomycin mediated an increase in TGase-modified Rac1

73

74

without 5-HT2A receptor activation. Moreover, calmodulin (CaM) inhibitor W-7,

significantly decreased Rac1 transamindation in a dose-dependent manner in

DOI-treated cells. To identify potential TGase-targeting residues that could render

Rac1 constitutively active upon transamidation, a search in NCBI Conserved Domain

Database was conducted, which revealed that two glutamine residues (Q61, Q74) and

three lysine residues (K5, K16, and K116) are located within activity-related domains.

Taken together, these results indicated that 5-HT2A receptor-coupled PLC activation

and subsequent Ca2+/CaM signaling are necessary in TGase-catalyzed Rac1

transamidation, and an increase in intracellular Ca2+ is sufficient to induce Rac1

transamination in A1A1 cells.

INTRODUCTION

Rac1 (Ras-related C3 botulinum toxin substrate 1), one of the most extensively

characterized members in the Rho family of small G proteins, was initially discovered

as a substrate for ADP-ribosylation induced by the C3 component of botulinum toxin

(Didsbury et al., 1989). Subsequently, Rac1 and its downstream effectors were

identified as key signaling molecules in various cell functions, such as cytoskeleton

reorganization, cell transformation, axonal guidance, and cell migration

(Etienne-Manneville and Hall, 2002, Bosco et al., 2009).

Like all members of the Rho superfamily, Rac1 functions as a molecular switch,

cycling between an inactive GDP-bound state and an active GTP-bound state. Rho

75

small G proteins can be activated by different signal transduction pathways initiated

by extracellular factors such as platelet-derived growth factor, insulin or epidermal

growth factor (Bishop and Hall, 2000). Serotonin has also been shown to induce

activation via transglutaminase (TGase)-catalyzed transamidation of small G proteins

in neurons, platelets, and aortic smooth muscle cells (Walther et al., 2003, Guilluy et

al., 2007, Dai et al., 2008). TGases catalyze the transamidation of protein-bound

glutamines and primary amines such as serotonin in a Ca2+-dependent manner

(Ahvazi et al., 2002). We previously reported that the effect of serotonin on Rac1

transamidation in A1A1v cells, a rat cortical cell line, is due to stimulation of the

5-HT2A receptors (Dai et al., 2008). However, the underlying molecular mechanisms

by which the 5-HT2A receptor signaling regulates TGase-catalyzed transamidation and

activation of Rac1 are still unclear.

5-HT2A receptor activation can increase IP3 via Gq/11-mediated activation of

phospholipase C (PLC) (Conn and Sanders-Bush, 1984, de Chaffoy de Courcelles et

al., 1985). PLC plays an important role in intracellular signal transduction by

hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid,

and thereby generating two second messengers: inositol trisphosphate (IP3) which

diffuses through the cytosol and releases Ca2+ from intracellular endoplasmic

reticulum stores and diacylglycerol (DAG) (Boess and Martin, 1994). It has been

reported that TGase activity can be significantly enhanced in response to increased

intracellular Ca2+ through inositol 1,4,5-trisphosphate (IP3) generation (Zhang et al.,

76

1998). Moreover, serotonin-mediated activation of small G protein cdc42 was

dependent on PLC signaling pathways (Udo et al., 2005).

Calmodulin (CaM) has been shown to increase TGase enzymatic activity. In the

presence of CaM, a membrane-associated erythrocyte TGase is activated at

physiological and lower than physiological Ca2+ concentrations (Billett and Puszkin,

1991). Moreover, in the presence of Ca2+, CaM enhanced TGase activity 3-fold in

human platelets and the chicken gizzard (Puszkin and Raghuraman, 1985). A CaM

inhibitor prevented TGase-catalyzed cross-linking of huntingtin in cells co-transfected

with mutant huntingtin and TGase (Zainelli et al., 2004). Taken together, we

hypothesize that TGase-catalyzed Rac1 modification upon 5-HT2A receptor

stimulation is dependent on PLC-mediated increases in intracellular Ca2+ and is

regulated by CaM. To test this hypothesis, we examined the effects of PLC inhibition,

CaM inhibition, and manipulation of intracellular Ca2+ by means of a Ca2+ ionophore

or a chelating agent on TGase-modified Rac1 in response to 5-HT2A receptor

activation in A1A1v cells.

MATERIALS AND METHODS

Cell Culture

A1A1v cells, a rat cortical cell line, were grown on 100 mm2 plates coated with

poly-L-ornithine (Sigma, St Louis, MO) and maintained in 5% CO2 at 33°C, in

Dulbecco’s modified Eagle medium (DMEM) (Fisher Scientific, Pittsburgh, PA)

77

containing 10% fetal bovine serum (FBS) (Fisher Scientific, Pittsburgh, PA). Before

each experiment, cells were maintained in DMEM with 10% charcoal-treated FBS for

48 hours. Charcoal adsorption removes most but not all serotonin in the medium. The

maximal final concentration of serotonin in the medium is approximately 3 nM

(Unsworth and Molinoff, 1992). Cells from passages 8-15 were used for all

experiments.

Drugs

The following drugs were used in this study: (-)-1-(2,5-dimethoxy-4-lodophenyl)-

2-aminopropane HCl (DOI) (Sigma, St Louis, MO), U73122 (Tocris Bioscience

Ellisville, MO), U73343 (Sigma, St Louis, MO), BAPTA AM (Invitrogen, Carlsbad,

CA), ionomycin (Invitrogen, Carlsbad, CA), and W-7 hydrochloride (Tocris

Bioscience Ellisville, MO).

Measurement of intracellular Ca2+ concentration

Cells were grown to 90% confluence in black-sided 96-well plates (Fisher Scientific,

Pittsburgh, PA). Cells were washed twice with modified Kreb's medium (135 mM

NaCl, 5.9 mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 11.5 mM D-glucose, 11.6 mM

Hepes, pH 7.3) and then incubated in the same medium with 5 µM Fura-2 AM

(Molecular Probes, Carlsbad, CA), 0.1% bovine serum albumin, and 0.02% Pluronic

F127 detergent (Poenie et al., 1986) for 60 min at 33°C incubator in the dark. After

loading, the cells were washed twice and incubated in the dark in modified Kreb's

78

medium or pretreated with drugs for 30 min. Following 1 min of equilibration, the

cells were stimulated with a single injection of 3 µM DOI, and the response was

recorded until a plateau was reached (about 4 min). Fura-2 fluorescence (at 510 nm)

was measured with a BioTek fluorescence plate reader. After background fluorescence

was subtracted, the ratio of fluorescence at 340 nm excitation to that at 380 nm

excitation was calculated and used as an index of the intracellular Ca2+ concentration

(the 340/380 nm fluorescence ratio is positively correlated with the absolute values of

intracellular Ca2+ concentration).

Immunoprecipitation of TGase-modified Protein

A1A1v cells were harvested and lysed using lysis buffer A (25 mM Tris-HCl, pH 7.5,

250 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 1:1000 protease inhibitor cocktail

(Sigma, St Louis, MO) containing 104 µM AEBSF, 0.08 µM aprotinin, 2 µM

leupeptin, 4 µM bestatin, 1.5 µM pepstatin A and 1.4 µM E-64). Protein

concentration was determined using the BCA Protein Assay kit (Pierce, Rockford, IL).

Immunopurification of proteins containing TGase-catalyzed bonds was performed

using 81D4 mAb (mouse IgM) prebound to Sepharose beads (Covalab, Lyon, France)

using a protocol developed by Covalab and as described previously (Norlund et al.,

1999, Zainelli et al., 2005). The 81D4 antibody has been extensively used to

demonstrate increases in TGase-catalyzed bonds (Citron et al., 2002, Junn et al., 2003,

Andringa et al., 2004). The use of the 81D4 antibody in a competitive ELISA assay

was shown to result in isodipeptide cross-link measurements that correlate well with

79

those measured by HPLC analysis but provide more sensitivity than the HPLC

approach (Sarvari et al., 2002). Transfection of cells to over-express TGases,

including TGase 1, 2 and 3, and a substrate protein such as mutant huntingtin protein

results in increased presence of the isodipeptide cross-link in mutant huntingtin

protein as demonstrated with immunoprecipitation with the 81D4 antibody (Zainelli et

al., 2005).

Briefly, 20µl of sepharose-81D4 beads were washed three times in TBS/0.1%

Tween 20 with gentle shaking for 15 minutes, followed by adding 200µg cell lysate (1

μg/μl) to the washed beads and incubating for 2 hours at 37°C. After incubation, the

pellets were washed four times in TBS/0.1% Tween 20 for 15 minutes. Then 20µl of

loading buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, 10%

glycerol and 5% β-mercaptoethanol) was added to the washed pellets followed by 5

minutes incubation at 90°C. The samples were then centrifuged at 9,000 × g for 2

minutes and the supernatant was transferred and stored at -80°C until immunoblot

analysis.

Immunoblot

Immunoaffinity purified proteins and cell lysates were separated on 12%

SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose

membranes. Membranes were then incubated in blocking buffer (5% non-fat dry milk,

0.1% Tween 20, 1× TBS) for 1 hour at room temperature. Membranes were incubated

overnight at 4°C with primary antibodies on a shaker. Primary antibodies (Upstate

80

Biotechnology, NY: anti-Rac1, mouse IgG, 1:700; anti-Na+/K+ ATPase, mouse IgG, 1:

10000) were diluted in antibody buffer (1% non-fat dry milk, 0.1% Tween 20, 1×

TBS). The next day, membranes were washed with TBS/0.1% Tween 20 and then

incubated with goat-anti-mouse or goat-anti-rabbit secondary antibody conjugated to

HRP (Jackson ImmunoResearch, West Grove, PA) diluted in antibody buffer.

Membranes were washed and signal was detected using enhanced chemiluminescence

(ECL) western blotting detection reagents (Amersham Biosciences, Piscataway, NJ).

Using Scion Image for Windows (Scion, Frederick, MD), immunoblots were

quantified by calculating the integrated optical density (IOD) of each protein band on

the film.

Statistical Analyses

Data are presented as mean ± the standard error of the mean (SEM) and analyzed by

one- or two-way ANOVA. Post hoc tests were conducted using Bonferroni's multiple

comparison tests. GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA) was

used for all statistical analyses. A probability level of p < 0.05 was considered to be

statistically significant for all statistical tests.

RESULTS

PLC inhibitor decreases the response of intracellular Ca2+ and TGase-modified

Rac1 to 5-HT2A receptor activation

We previously reported that 5-HT2A receptor stimulation induced

81

TGase-catalyzed Rac1 transamidation and activation in A1A1v cells (Dai et al., 2008).

To test the dependence of the intracellular Ca2+ response and Rac1 transamidation on

PLC activation, cells were pretreated with 5 µM of the PLC inhibitor, U73122, or an

inactive analog, U73343, for 30 min prior to the stimulation of 5-HT2A receptors by 3

µM DOI. U73122 inhibits both PLC-mediated hydrolysis of PIP2 to IP3 and the

coupling of G protein-PLC activation (Bleasdale et al., 1990). Changes in intracellular

Ca2+ were monitored by the 340/380 nm fluorescence ratio of the Fura-2 loaded cells.

DOI-stimulation induced a robust increase (by at least 200%) in the intracellular Ca2+

concentration within cells pretreated with U73343, where as PLC inhibition by

U73122 reduced the intracellular Ca2+ response to DOI (Fig. 12A). TGase-modified

Rac1 was evaluated by immunoprecipitation using the 81D4 antibody directed against

the TGase-catalyzed covalent bond and immunoblots in parallel experiments. There

were significant increases in TGase-modified Rac1 within DOI-stimulated cells, but

pretreatment with U73122 significantly attenuated DOI-induced Rac1 modification by

TGase (Fig. 12B, C). These results suggest a specific role of PLC upstream of Rac1

transamidation in A1A1v cells. Two-way ANOVA indicates a significant main effect

of DOI [F(1,8) =108.3; p<0.001] and U73122 [F(1,8) =29.99; p<0.001] on

TGase-modified Rac1. There was also a significant interaction between DOI and

U73122 [F(1,8) =17.40; p<0.01]. The ubiquitously expressed Na+/K+ ATPase is a well

established plasma membrane marker, and its function underlies essentially all of

mammalian cell physiology (Kaplan, 2002). To verify that the PLC inhibitor-mediated

82

the decrease in Rac1-transamidation by TGase was not due to U73122-induced

cellular toxicity, cell lysates from the same experiment were examined on immunoblot

with antibodies for Na+/K+ ATPase. There are no significant differences in Na+/K+

ATPase levels among cells with different treatments (Fig. 12B). Similar

measurements were used in all of the following experiments.

Ca2+ chelator inhibits DOI-stimulated modification of Rac1 by TGase

In order to examine whether TGase-modified Rac1 specifically depends on an

increase in intracellular Ca2+ upon 5-HT2A receptor activation, A1A1v cells were

pretreated with increasing concentrations of the membrane-permeant Ca2+ chelator,

BAPTA-AM (0, 5, 10, 20 µM) for 30 min, then the cells were challenged by DOI.

Dose-dependent reductions in the DOI-mediated Ca2+ response were observed by

measuring Fura-2 fluorescence (Fig. 13A). Since 20 µM BAPTA caused the most

significant inhibition in Ca2+ increase, it was used in the parallel experiments that

evaluated TGase-modified Rac1. DOI-stimulation in cells without

BAPTA-pretreatment significantly increases the amount of TGase-modified Rac1

about 2-fold compared with vehicle-stimulated cells, whereas pretreatment with

BAPTA has a notable inhibitory effect on DOI-induced Rac1 modification by TGase

(Fig. 13B, C), suggesting that an increase in intracellular Ca2+ is required for

DOI-mediated Rac1 transamidation. Two-way ANOVA indicates a significant main

effect of DOI [F(1,8) =37.09; p<0.001] and BAPTA [F(1,8) =9.85; p<0.05] on

TGase-modified Rac1. There was also a significant interaction between DOI and

83

BAPTA [F(1,8) =9.50; p<0.05].

Ca2+ ionophore induces TGase-modified Rac1 without 5-HT2A receptor

activation

To determine if an increase in intracellular Ca2+ is sufficient to enhance Rac1

modification by TGase, a Ca2+ ionophore, ionomycin was used to produce an

elevation of cytosolic Ca2+ concentration without 5-HT2A receptor activation.

Ionomycin is an effective and specific Ca2+ carrier, so it has been used in studies of

Ca2+ flux across plasma and ER membranes. To determine the concentration of

ionomycin that raises the levels of cytosolic Ca2+ to levels comparable to 3µM DOI

(the concentration of DOI used to induce TGase-modified Rac1), serial dilutions of

ionomycin (from 1000 nM to 12.5 nM) were tested for their ability to induce

increases in intracellular Ca2+. As shown in Fig. 14A, 25 nM ionomycin was able to

produce a comparable increase in cytosolic Ca2+ in comparison to 3 µM DOI, so this

concentration was used in the parallel experiments to measure the effects on

TGase-modified Rac1. After cells were treated with either 3 µM DOI or 25 nM

ionomycin for 15 min, ionomycin mediated an increase in TGase-modified Rac1 to

levels similar to those induced by DOI. One-way ANOVA indicated there is no

significant difference in TGase-modified Rac1 between DOI- and ionomycin-treated

cells. Taken together, these data suggest an increase in intracellular Ca2+ via an

ionophore can mimic 5-HT2A receptor-induced cytosolic Ca2+ increases and is

sufficient to induce TGase-catalyzed Rac1 transamidation.

84

TGase-modified Rac1 is reduced by CaM inhibition in a dose-depend manner

TGase is not only a Ca2+-dependent enzyme, but its activity is also positively

regulated by CaM. Moreover, Ca2+ signaling in cells is mainly mediated by CaM, a

potent Ca2+-binding protein. The CaM inhibitor, W-7, binds with high affinity to CaM

in the presence of Ca2+ and thereby inhibits the activation of CaM-dependent enzymes

(Asano and Hidaka, 1984). To test whether CaM modulates TGase-catalyzed

transamidation of Rac1, cells were pre-incubated for 1 h with 0, 0.5 or 5 µM W-7

hydrochloride, and then stimulated with 3 µM DOI or vehicle for 15 min. We found

that in DOI-stimulated cells, the levels of TGase-modified Rac1 were significantly

higher than in vehicle-stimulated cells, whereas pretreatment with W-7 decreased

DOI-stimulated Rac1 modification by TGase in a dose-dependent manner (Fig. 15).

Two-way ANOVA indicates a significant main effect of DOI [F(1,12) =144.6;

p<0.001] and W-7 [F(2,12) =8.152; p<0.01] on TGase-modified Rac1. There was also

a significant interaction between DOI and W-7 [F(2,12) =5.01; p<0.05].

Searching potential TGase-targeting residues at the activity-related domains in

Rac1

We have previously reported that TGase-catalyzed transamidation of Rac1 was

able to transiently increase Rac1 activity (Dai et al., 2008). Like other small G

proteins, Rac1 acts as a molecular switch and cycles between the GDP-bound inactive

and GTP-bound active forms. There are at least three types of regulators for small G

proteins: GTPase activating protein (GAP) which accelerates GTP hydrolysis,

85

resulting in inactivation of small G proteins; guanine nucleotide exchange factors

(GEF) which stimulates conversion from the GDP-bound to the GTP-bound form; and

GDP dissociation inhibitor (GDI) which inhibits this reaction. Post-translational

modifications, such as TGase-catalyzed transamidation, of Rac1 at the conserved

domains that interact with these regulators may affect its ability to convert GTP to

GDP, resulting in increased activation. The Conserved Domain Database (available on

http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) provides an interactive tool to

identify conserved domains present in protein sequences. We searched the Conserved

Domain Database for GTP/Mg2+ binding sites, and GAP, GEF, and GDI interaction

sites in the Rac1 sequence (accession numbers: NP_008839) since TGase-catalyzed

modifications at theses sites would more likely lead to functional changes that would

impact on Rac1 activity (Fig. 16A). TGase-catalyzed transamidation may not only

incorporate an amine into the glutamine residue of the acceptor protein, but also form

intra-molecular cross-links between glutamine and lysine residues within the protein

(Hand et al., 1993). Two glutamine residues (Q61, Q74) and three lysine residues (K5,

K16, and K116) are located at the activity-related domains and are highlighted in

Rac1 structure (Fig. 16B).

86

Figure 12. Effect of PLC inhibition on DOI-induced Ca2+ signals and TGase-modified

Rac1 in A1A1v cells. (A) After pretreatment with 5 µM U73122 or U73343 (negative

control) for 30 min, and stimulation with either 3 µM DOI or vehicle (Ve), changes in

the fluorescence ratio (340/380 nm) were recorded from cells loaded with Fura-2. A

representative example from three independent experiments conducted in triplicate is

shown. Arrows indicate the time of drug application. (B) Upper,

immunoprecipitation (IP) and immunoblot (IB) analyses revealed that U73122

87

pretreatment inhibited the increase in TGase-modified Rac1 upon DOI stimulation.

Lower, cells lysates from the same experiment were examined on IB with Na+/K+

ATPase antibody, which indicated that U73122 had no effect of on cell viability. (C)

Quantitation of effects of U73122 and DOI on TGase-modified Rac1 in three separate

experiments. Data shown were the mean IOD±S.E.M., and they were normalized to

U73343- and Ve- treated cells. Two-way ANOVA followed by Bonferroni test: * and

*** indicated p<0.05 and 0.001 as compared to U73343- and Ve- treated cells,

respectively; # indicated p<0.001 as compared to U73343- and DOI- treated cells.

88

Figure 13. Pre-incubation with a Ca2+ chelator attenuated the DOI-mediated elevation

of cytosolic Ca2+ and TGase-modified Rac1. (A) Dynamic changes in fluorescence

ratio (340/380 nm) in Fura-2 loaded A1A1v cells. The Ca2+ response to exposure to 3

µM DOI was gradually reduced with the increasing concentrations of BAPTA. A

representative example of three independent experiments conducted in triplicate is

shown. (B) Upper, cells were pretreated with 20 µM BAPTA or the vehicle for

BAPTA (Ve1) for 30 mins, and then stimulated with 3 µM DOI or the vehicle for DOI

(Ve2) for 15 mins. TGase-modified Rac1 was detected by IP and IB analysis. BAPTA

inhibited the increase in TGase-modified Rac1in DOI- stimulated cells. Lower, cells

89

lysates from the same experiment were examined on IB with Na+/K+ ATPase

antibody. There was no significant difference among various treatments. (C)

Quantitation of effects of BAPTA and DOI on TGase-modified Rac1 in three separate

experiments. Data shown were the mean IOD±S.E.M., and they were normalized to

Ve1- and Ve2- treated cells. Two-way ANOVA followed by Bonferroni test: *

indicated p<0.01 as compared to Ve1- and Ve2- treated cells; # and ## indicated

p<0.05 and p<0.01 as compared to Ve1- and DOI- treated cells, respectively.

90

Figure 14. The Ca2+ ionophore mimiced the DOI-induced increase in cytosolic Ca2+

and TGase-catalyzed transamidation of Rac1. (A) Fura-2 loaded cells were stimulated

with either 3µM DOI or different concentrations of ionomycin. The changes in the

fluorescence ratio (340/380 nm) were recorded. A representative example from three

independent experiments conducted in triplicate is shown. (B) Upper, cells were

treated for 15 mins with 3µM DOI, 25 nM ionomycin or vehicles (Ve). Ionomycin

caused an increase in TGase-modified Rac1 comparble to that induced by DOI. Lower,

cell viability, as indicated by Na+/K+ ATPase levels was not significantly different

91

among the various treatments. (C) Quantitation of effects of ionomycin and DOI on

TGase-modified Rac1 in three separate experiments. Data shown were the mean

IOD±S.E.M., and were normalized to Ve-1-treated cells. One-way ANOVA followed

by Bonferroni test: * indicates p<0.01 as compared to Ve-1-treated cells; # indicates

p<0.01 as compared DOI-treated cells.

92

Figure 15. The CaM inhibitor W-7 caused a dose-dependent reduction in

DOI-stimulated TGase-modified Rac1. (A) Cells were pretreated with increasing

concentrations of W-7 for 1 h and then stimulated with either 3 µM DOI or vehicle

(Ve). TGase-modified Rac1 and Na+/K+ ATPase were measured by IP and/or IB. W-7

decreased the levels of TGase-modified Rac1 upon DOI stimulation, but did not

significantly affect Na+/K+ ATPase at the concentrations applied. (B) Quantitation of

effects of W-7 on TGase-modified Rac1 in three separate experiments. Data shown

were the mean IOD±S.E.M., and were normalized to Ve-stimulated cells without W-7

pretreatment. Two-way ANOVA followed by Bonferroni test: * and ***indicate

93

p<0.05 and p<0.001 as compared to Ve-stimulated cells without W-7 pretreatment,

respectively; # and ## indicate p<0.01 and p<0.001 as compared to DOI-stimulated

cells without W-7 pretreatment, respectively.

94

Figure 16. Rac1 activity-related domains and potential TGase-targeted amino acid

residues. (A) Amino acid sequence alignment of human Rac1. Conserved

activity-related sites were identified by searching NCBI Conserved Domain Database

(CDD). # indicates a GTP/Mg2+ binding site; * indicates GAP interaction sites; +

indicates GEF interaction sites; & indicated GDI interaction sites. Two glutamine

residues (Q61, Q74) and three lysine residues (K5, K16, and K116), highlighted in

gray, are directly localized in these potential activity-related domains. (B) Rac1

structure adapted from http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?

uid=56843. The potential TGase-targets, glutamine and lysine residues at Rac1

95

activity-related domains are highlighted in yellow.

96

Figure 17. Schematic representation showing 5-HT2A receptor signaling-mediated

transamidation of Rac1 by TGase. DOI stimulates Gq/11 proteins-coupled 5-HT2A

receptors, leading to the activation of PLC, which in turn hydrolyses PIP2 into IP3. IP3

releases Ca2+ from ER and results in the activation of Ca2+-dependent proteins, such

as CaM and TGase. Positively regulated by both Ca2+ and CaM, TGase catalyzes

transamidation of Rac1 to bioamines, such as serotonin transported into the cytoplasm

by SERT. Abbreviations: 5-HT2AR, 5-HT2A receptor; CaM, calmodulin; ER,

endoplasmic reticulum; PLC, phospholipase C; PIP2, phophatidyl

inositol-1,4-bisphosphate; IP3, inositol-1,4,5-triphosphate; SERT, serotonin

transporter; TGase, transglutaminase; α, β, γ, G protein α, β, γ subunits

97

DISCUSSION

Several lines of evidence indicate Gq/11-protein-coupled receptors, such as

bradykinin or endothelin-1 receptor, are also able to stimulate the activation of Rac1

(van Leeuwen et al., 1999, Clerk et al., 2001), but the mechanisms by which Rac1

becomes activated through these receptors remains poorly characterized. Nevertheless,

it is worth noting that the activation of Rac1 is highly regulated by physiological

stimuli, and aberrant activation occurs under many pathological conditions such as

Salmonella invasion, tumor cell migration, and retinal degeneration (Bourguignon et

al., 2000, Belmonte et al., 2006, Brown et al., 2007). Therefore, further studies that

delineate signaling pathways involved in Rac1 activation may uncover new potential

therapeutic targets for these diseases. We have previously demonstrated that the

5-HT2A receptor, another Gq/11-protein-coupled receptor, is responsible for

TGase-catalyzed transamidation and activation of Rac1 (Dai et al., 2008). In the

present study, we investigated the mechanisms involved in Rac1 transamidation in

response to 5-HT2A receptor stimulation. Our experimental results identified that

PLC-mediated increase in intracellular Ca2+ and subsequent CaM activation are

required for 5-HT2A receptor-mediated Rac1 transamidation. 5-HT2A receptor

stimulation activates PLC which catalyzes the hydrolysis of PIP2 into IP3. IP3

mobilizes Ca2+ from ER stores and thereby increases the intracellular Ca2+ and leads

to the activation of CaM. Our data suggest that both Ca2+ and CaM activate TGase,

which catalyzes transamidation of Rac1 to bioamines, such as serotonin (Fig. 17).

98

Except for the 5-HT3 receptor, which is a ligand-gated ion channel, the other

5-HT receptors belong to the G protein-coupled receptor (GPCR) superfamily. G

proteins such as Gα12 and Gα13, have been shown to transduce signals from GPCR to

activate Rho small G proteins (Suzuki et al., 2003). One of the GEFs for Rho,

p115RhoGEF, has served as a direct link between Gα12/Gα13 and Rho (Hart et al.,

1998, Kozasa et al., 1998). However, the signaling pathways between 5-HT2A

receptors and Rac1 transamidation and activation are not well understood. 5-HT2A

receptor is primarily coupled to Gq/11 and activates various isoforms of PLC (Conn

and Sanders-Bush, 1984, de Chaffoy de Courcelles et al., 1985). It also induces the

activation of phospholipase A2 (PLA2) and the release of arachidonic acid (Felder et

al., 1990, Kurrasch-Orbaugh et al., 2003). Moveover, through interaction with

ADP-ribosylation factor 1 (ARF1) via its C-terminal domain, 5-HT2A receptor is able

to signal through the phospholipase D (PLD) pathway (Mitchell et al., 1998,

Robertson et al., 2003). Our results suggest that 5-HT2A receptor activation triggers

TGase-catalyzed Rac1 transamidation at least predominantly through a pathway

involving PLC (Fig.12).

Although it has been reported that PLC activates Rac in a manner dependent on

Iba1, a Ca2+-binding protein specifically expressed in macrophage/microglia

(Kanazawa et al., 2002), Rac1 transamidation and activation in A1A1v cells, a

neuronal cell line, could be mediated by other signaling pathways. Cleavage of PIP2

by PLC yields two important second messengers, IP3 and DAG, to initiate a variety of

99

cellular functions. Since Ca2+ chelation reduces DOI-stimulated Rac1 transamidation

to basal levels (Fig.13), IP3-induced Ca2+ release from ER rather than DAG signaling

plays a pivotal role in TGase-catalyzed Rac1 transamidation. These results are in

agreement with an earlier study suggesting that in thrombin- and collagen-stimulated

human blood platelets, PLC activity and an increase in intracellular Ca2+ concentration

were required for Rac activation, whereas PKC inhibition had no effect (Soulet et al.,

2001). Increases in intracellular Ca2+ concentration upon 5-HT2A receptor stimulation

is achieved by releasing intracellular Ca2+ stores and/or by activating Ca2+ channels,

depending on the cell of interest. 5-HT2A receptor-induced contraction of rat aorta

may require the concerted action of voltage-gated Ca2+ channels and phosphoinositide

turnover (Nakaki et al., 1985, Roth et al., 1986, Watts, 1998). More interestingly,

stimulation of 5-HT2A receptors on astrocytes caused Ca2+ influx through voltage

independent Ca2+ channels which were dependent of the IP3 production and

subsequent Ca2+ release from internal stores (Eberle-Wang et al., 1994, Hagberg et al.,

1998). Therefore, both Ca2+ mobilization from internal stores and influx of

extracellular Ca2+ could contribute to the PLC/IP3-mediated intracellular Ca2+

increase.

CaM and TGase are Ca2+ dependent proteins. CaM activation is coupled to

intracellular Ca2+ elevation and also correlates with the spatial pattern of increased

Ca2+ (Hahn et al., 1992). Upon Ca2+ binding to the EF hand motifs of CaM, both N-

and C-terminal domains adopt an open confirmation, exposing hydrophobic surfaces,

100

which then interact with a variety of target proteins including TGase (Puszkin and

Raghuraman, 1985, Zainelli et al., 2004). TGase contains a cysteine in their active site

that is unmasked only in the presence of Ca2+ (Hand et al., 1993) and Ca2+ activation

of TGase is further regulated by other signal modulators such as GTP (Monsonego et

al., 1998) and CaM (Puszkin and Raghuraman, 1985, Zainelli et al., 2004). In order

for TGase to become receptive to the glutamine substrate, Ca2+ first must bind to

TGase, inducing its conformational change which permits the glutamine substrate

binding to the catalytic center (Folk, 1983, Greenberg et al., 1991). A rise of Ca2+

concentration increases TGase activity in a dose dependent manner in vitro and in

cells (Siefring et al., 1978, Shin et al., 2004, Li et al., 2009). Consistent with these

results, the elevation of intracellular Ca2+ induced via a Ca2+ ionophore lead to

increased TGase-catalyzed Rac1 transamidation (Fig. 14).

In addition to Ca2+, CaM also interacts with TGase and regulates its

cross-linking activity. CaM has been shown to coimmunoprecipitate with TGase2 in

transfected cells (Zainelli et al., 2004). CaM increased TGase activity in human

erythrocyte (Billett and Puszkin, 1991), platelets, chicken gizzard (Puszkin and

Raghuraman, 1985) and rat liver (Hand et al., 1985). Interestingly, exogenous CaM

with a binding site on the α-subunits of the hexadecameric phosphorylase kinase

molecule (αβγδ)4, stimulates TGase-catalyzed incorporation of putrescine into the β-

and γ-subunits, but inhibits incorporation of amines into the α-subunit (Nadeau and

Carlson, 1994). Some of the CaM antagonists, such as dansylcadaverine, inhibit TGase

101

(Lorand et al., 1979) and its protein cross-linking ability. We have previously reported

that a CaM antagonist or inhibitory CaM fragments, attenuated TGase-modified mutant

huntingtin in cell and animal models of Huntington's disease (Zainelli et al., 2004, Dai

et al., 2009, Dudek et al., 2009). CaM antagonists and Ca2+-chelators also have been

shown to depress the motile progression of sperm cells possibly mediated by

TGase-catalyzed cross-linking of the contractile proteins (Cariello and Nelson, 1985).

Our present results revealed that CaM inhibitor, W-7, reduced TGase-modified-Rac1 in

dose-dependent fashion (Fig. 15), indicating that TGase-catalyzed Rac1 transamidation,

at least in part, was positively regulated by CaM. Moreover, in the presence of 5-HT2A

receptor agonist, CaM has been shown to interact with 5-HT2A receptor at the sites

that are important for G protein coupling, suggesting a putative role for CaM in

regulating 5-HT2A receptor function (Turner and Raymond, 2005). Thus, one could

not exclude the possibility that the effect of CaM inhibition is mediated by alterations

in 5-HT2A receptor signaling.

The significance of TGase in Rac1 transamidation was confirmed by TGase

siRNA and inhibitor in our previous studies (Dai et al., 2008). In addition to small G

proteins such as Rac1 and RhoA (Walther et al., 2003, Dai et al., 2008) that could

undergo TGase-catalyzed transamidation to serotonin (serotonylation), more recently,

arterial delete cytoskeletal proteins including actin, myosin heavy chain and filamin A

have been identified as serotonylated proteins (Watts et al., 2009). However, serotonin

may not be the only amine that could be incorporated in these proteins. Neuronal

102

differentiation of SH-SY5Y cells induced by retinoic acid increases the

expression/activation of TGases, resulting in transamidation of putrescine to RhoA and

activation of RhoA (Singh et al., 2003). Moreover, spermidine and putrescine are

suitable substrates for transglutamination of RhoA in vitro (Schmidt et al., 2001). In

addition to amine incorporation, another important TGase-catalyzed modification of

small G proteins is deamidation of glutamine residue. The site-specific deamidation of

Gln53 and Gln61 of RhoA and Ras, respectively, by the bacterial toxin TGases

inhibits the GTPase activity and renders small G proteins constitutively active (Lorand

and Graham, 2003).

In addition to TGase-catalyzed reactions, other Ca2+/CaM dependent pathways

may also contribute to Rac1 activation. For example, platelet-derived growth factor

(PDGF) stimulates phosphorylation of Tiam1, the Rac1-specific GEF, and this

phosphorylation was significantly reduced by Ca2+ chelation, inhibition of Ca2+/CaM

dependent kinase II (CaMKII), or disruption of PLC-γ expression in Swiss 3T3

fibroblasts (Fleming et al., 1998). Targeted CaMKII knockdown using siRNA resulted

in the inhibition of wound-induced Rac activation and vascular smooth muscle cell

migration (Mercure et al., 2008).

In conclusion, our study provides important new insights into the signaling

mechanisms underlying 5-HT2A receptor-induced Rac1 transamidation and activation

by TGase in A1A1v cells, and highlights the necessary role of PLC-mediated Ca2+

mobilization and CaM in TGase-catalyzed Rac1 transamidation. As an activating

103

signal for both TGase and CaM, an elevation of intracellular Ca2+ is sufficient to

induce Rac1 transamidation. Accumulating evidence suggests that Rho family of

small G proteins is subject to TGase-mediated modification and activation. The

essential role of Rac1 in cytoskeletal organization is well characterized, while the

biological functions of Rac1 transamidation by TGase of in neurons have yet to be

explored.

104

CHAPTER FOUR

STRIATAL EXPRESSION OF A CALMODULIN FRAGMENT IMPROVED MOTOR FUNCTION, WEIGHT LOSS AND NEUROPATHOLOGY IN THE R6/2

MOUSE MODEL OF HUNTINGTON’S DISEASE

(Published in J Neurosci. 2009; 29(37):11550-9)

ABSTRACT

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder,

caused by a polyglutamine expansion in the huntingtin protein (htt). Increasing

evidence suggests that transglutaminase (TGase) plays a critical role in the

pathophysiology of HD possibly by stabilizing monomeric, polymeric and aggregated

htt. We previously reported that in HEK293 and SH-SY5Y cells expression of a

calmodulin (CaM)-fragment, consisting of amino acids 76-121 of CaM, decreased

binding of CaM to mutant htt, TGase-modified htt and cytotoxicity associated with

mutant htt and normalized intracellular calcium release. In this study, an

adeno-associated virus (AAV) that expresses the CaM-fragment was injected into the

striatum of HD transgenic R6/2 mice. The CaM-fragment significantly reduced body

weight loss and improved motor function as indicated by improved rotarod

performance, longer stride length, lower stride frequency, fewer low mobility bouts

and longer travel distance than HD controls. A small but insignificant increase in

survival was observed in R6/2 mice with CaM-fragment expression.

104

105

Immunoprecipitation studies show that expression of the CaM-fragment reduced

TGase-modified htt in the striatum of R6/2 mice. The percentage of htt-positive nuclei

and the size of intranuclear htt aggregates were reduced by the CaM-fragment without

striatal volume changes. The effects of CaM-fragment appear to be selective, as

activity of another CaM-dependent enzyme, CaM-dependent kinase II, was not altered.

Moreover, inhibition of TGase-modified htt was substrate-specific since overall

TGase activity in the striatum was not altered by treatment with the CaM-fragment.

Taken together, these results suggest that disrupting CaM-htt interaction may provide

a new therapeutic strategy for HD.

INTRODUCTION

Patients with Huntington’s disease (HD) present symptoms such as chorea,

irregular gait, reduced motor coordination and weight loss (Harper, 1991). HD is a

progressive and fatal neurological disorder caused by expansion of CAG repeats in

HTT gene, conferring a toxic gain of function to the huntingtin (htt) protein

(Huntington’s Disease Collaborative Research Group, 1993). It is characterized

neuropathologically by intranuclear inclusions and cytoplasmic aggregates composed

of htt with an expanded polyglutamine domain (DiFiglia et al., 1997, Ross et al.,

1998). Currently, there are no effective treatments to prevent or slow the progression

of the disease.

Increasing evidence indicates that TGases may contribute to the

106

pathophysiology of HD (Cooper et al., 1999, Karpuj et al., 2002b). TGases, a family

of Ca2+-dependent enzymes, catalyze a covalent bond between peptide-bound

glutamine residues and either lysine-bound peptide residues or mono- or polyamines

(Folk et al., 1980, Griffin et al., 2002). In cell culture, mutant htt is an excellent

substrate for TGases (Gentile et al., 1998, Kahlem et al., 1998, Zainelli et al., 2005).

The mRNA, protein and enzymatic activity of TGases are elevated in HD brain

(Karpuj et al., 1999, Lesort et al., 1999, Zainelli et al., 2003). TGase 2 ablation in HD

transgenic mice resulted in a drastic reduction in -(γ-glutamyl) lysine bond levels

and neuronal death in the cortex and striatum (Mastroberardino et al., 2002).

Administration of the TGase inhibitor, cystamine, extended survival and improved

motor performance in HD transgenic mice (Karpuj et al., 2002a).

Calmodulin (CaM) increased TGase activity in human erythrocyte (Billett and

Puszkin, 1991), platelets and chicken gizzard (Puszkin and Raghuraman, 1985). CaM

activates a host of enzymes upon Ca2+ binding (Cheung, 1982) and associates with

mutant htt as demonstrated by affinity purification (Bao et al., 1996) and

immunoprecipitation (Zainelli et al., 2004). Moreover, CaM colocalizes with htt and

TGase 2 in HD intranuclear inclusions (Zainelli et al., 2004). Since a CaM inhibitor

decreased TGase-catalyzed cross-linking of htt in cells (Zainelli et al., 2004), we

tested the ability of fragments of CaM to interrupt the interaction of CaM and mutant

htt and decrease the deleterious effects of TGase in HEK-293 cells (Dudek et al.,

2008). A CaM-fragment, containing amino acids 76-121, reduced binding of CaM to

107

mutant htt in vitro and in HEK-293 cells. AAV-mediated expression of the

CaM-fragment significantly decreased TGase-modified htt, and mutant htt-associated

cytotoxicity in differentiated SH-SY5Y cells, a neuroblastoma cell line, which stably

express mutant htt. Importantly, CaM-fragment did not alter the total activity of

TGase or another CaM-dependent enzyme, CaM kinase II, suggesting that

CaM-fragment has specific effects on the CaM-htt interaction (Dudek et al., 2009). In

the present study, we assessed the therapeutic potential of CaM-fragment in the R6/2

mouse model of HD. R6/2 transgenic mice express exon 1 of the human HD gene

with an increased CAG repeat. Consequently, they develop a progressive neurological

phenotype and pathological changes that resemble many features of HD (Mangiarini

et al., 1996, Li et al., 2000). Survival, body weight, motor performance and

neuropathological features were monitored to determine whether AAV-mediated

expression of the CaM-fragment has beneficial effects on the course of HD in R6/2

mouse model.

MATERIALS AND METHODS

Animals. Male R6/2 transgenic mice (with 100-115 CAG repeats in the

transgene) and wild-type littermate mice were purchased from Jackson Laboratories

(Bar Harbor, ME) at 6 weeks of age. The mice had free access to food and water in an

environment controlled for temperature and humidity and a 12 h light/dark cycle. The

behavioral tests were conducted in the light part of the cycle. All procedures were

108

conducted in accordance with the National Institutes of Health Guide for the Care and

Use of Laboratory Animals as approved by the University of Kansas Institutional

Animal Care and Use Committee.

Adenoassociated Virus (AAV) Injections. Recombinant AAV serotype 2 vectors

were generated as described in our previous studies (Dudek et al., 2009). Three

resultant vectors expressed each of the following: GFP and the CaM-fragment

(containing amino acids 76-121 of calmodulin), GFP and a scrambled peptide

(containing the same amino acids of the CaM-fragment but in a randomly scrambled

sequence), and GFP alone. The titers of final AAV products were ranged from 2~5 ×

1014 vector genomes per ml. Bilateral striatal injections of GFP-expressing AAV were

performed in 7-week-old mice (1 µl per each side with 200 nl/min infusion rate at

coordinates of anterior/posterior 0.5 mm, lateral/medial ±2 mm and dorsal/ventral -3

mm relative to bregma (Franklin et al., 1997)). Mice were randomly divided into five

groups (11~14 mice for each group) with a defined genotype and treatment: CaM-HD

(R6/2 mice injected with AAV expressing the CaM-fragment), Vec-HD (R6/2 mice

injected with empty vector AAV expressing GFP alone), Scr-HD (R6/2 mice injected

with AAV expressing a scrambled version of the CaM-fragment), CaM-WT

(wild-type mice injected with AAV expressing CaM-fragment) and Vec-WT

(wild-type mice injected with empty vector AAV).

Body weight and survival. Body weights were measured twice a week.

Survival was checked twice daily at 8 h intervals. Brains were removed after death

109

and snap frozen in 2-methylbutane/dry ice bath and stored at -80°C.

Behavioral tests. Three behavioral tests were performed between 10 and 14

weeks of age at weekly intervals. Rotarod performance, gait dynamics and

locomotion were examined every Wednesday, Friday and Sunday, respectively.

Gait dynamics. The DigiGait imaging system (Mouse Specifics, Inc., Boston,

MA) was utilized for gait analyses as previously described (Hampton et al., 2004).

Briefly, mice were placed on a motorized transparent treadmill belt moving at a speed

of 14 cm/s. Digital images of paw placement were recorded at 150 Hz through a video

camera mounted below the animal. The proprietary software analyzed the resulting

digital images to generate a set of periodic waveforms that described the paw area and

movement of each limb relative to the treadmill belt through consecutive strides. Gait

data were pooled from all four paws and used to determine numerous gait dynamic

measures including stride length and frequency, variability of stance width and paw

area at peak stance.

Locomotion. To accurately measure locomotor behaviors including the total

distance traveled and the number of low mobility bouts (defined as remaining

continuously in a virtual circle of 15 mm radius for 10 sec), a force-plate actometer

(constructed in Dr. Fowler’s laboratory, University of Kansas) was used as described

previously (Fowler et al., 2001). Mice were placed in a 28 cm × 28 cm force-plate

actometer for a 30-min recording once a week. When the animal moves on the plate,

its movements are sensed by four supporting force transducers positioned at the

110

corners of the plate. The signals are processed by specialized computer algorithms

written in house to yield measurements of travel distance and bouts of low mobility.

Rotarod performance. A rotarod apparatus (MED-Associates, St. Albans, VT)

was used to measure fore- and hindlimb motor coordination and balance. The mice

were given a training session at 9 weeks of age (four trials per day for 3 consecutive

days) to acclimate them to the rotarod apparatus. During the test period, each mouse

was placed on the rotarod with increasing speed, from 4 rpm to 40 rpm in 300 seconds.

The latency to fall off the rotarod within this time period was recorded. Mice were

tested on the rotorod once a week. Each mouse received two consecutive trials and the

mean latency to fall was used in the analysis.

Immunoprecipitation (IP). Mouse brains were bisected mid-saggitally. One

half was used in IP, CaM Kinase II Activity and TGase assays. The other half was

used for immunohistochemistry assays. To prepare striatal homogenates for IP, a half

brain was cut into 300-μm coronal sections with a cryostat (Leica, Nussloch,

Germany). Striatum was punched out from five consecutive sections and

homogenized in lysate buffer (10 mM Tris-HCl, pH 7.5, 0.14 M NaCl, 1 mM EDTA,

and 1:1000 protease inhibitor mixture) in a Tissue Tek homogenizer. Protein

concentration was determined using the BCA Protein Assay kit (Pierce Chemical,

Rockford, IL) and tissue homogenates containing 150 µg of protein were centrifuge at

12,000 × g for 5 min at 4°C to separate the insoluble fraction. After removal of the

supernatant, the insoluble fraction was resuspended in 60 µl of 95% formic acid and

111

incubated in a shaking water bath at 37°C for 40 min. The formic acid was then

evaporated using a CentriVap Speed Vacuum (Labconco, Kansas City, MO) for 1.5 h

at 45°C. The pellets were resuspended in 150 µl of IP wash buffer (10 mM Tris-HCl,

pH 7.5, 0.14 M NaCl, and 0.1% Tween 20) and sonicated on ice at 10 x 5 sec pulse.

Immunopurification of proteins containing TGase-catalyzed bonds was performed

using 81D4 monoclonal antibody prebound to Sepharose beads (Covalab, Lyon,

France) using a protocol developed by Covalab and as described previously (Norlund

et al., 1999, Zainelli et al., 2005).

Immunoblot. Immunoaffinity-purified proteins were separated on 10%

SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose

membranes. Membranes were then incubated in blocking buffer (5% nonfat dry milk,

0.1% Tween 20, and 1× TBS) for 1 h at room temperature. Membranes were

incubated overnight at 4°C with primary antibody on a shaker. Primary antibody

(Chemicon International, Temecula, CA: anti-Huntingtin amino acids 1-82, mouse

IgG, 1:500) was diluted in antibody buffer (1% nonfat dry milk, 0.1% Tween 20, and

1× TBS). The next day, membranes were washed with TBS/0.1% Tween 20, and then

they were incubated with goat anti-mouse secondary antibody conjugated to

horseradish peroxidase (Jackson ImmunoResearch Laboratories Inc., West Grove, PA)

diluted in antibody buffer. Membranes were washed, and signal was detected using

enhanced chemiluminescence Western blotting detection reagents (GE Healthcare,

Chalfont, St.Giles, UK). Using Scion Image for Windows (Scion Corporation,

112

Frederick, MD), immunoblots were quantified by calculating the integrated optical

density (IOD) of each protein band on the film.

Evaluation of htt aggregates by immunofluorescence. Sections of frozen

mouse brain tissue (20 µm thick) were mounted on glass slides, and then fixed in 4%

paraformaldehyde. After fixation, the sections were washed in PBS (pH 7.4), and then

nonspecific binding was blocked with 5% normal goat serum (NGS). Sections were

incubated overnight with primary antibody: MAB5374 (mouse IgG, 1:100, directed

against human huntingtin amino acids 1~256) in 1% NGS + 1X PBS. Next, sections

were washed and incubated for 1h with secondary antibody: goat anti-mouse IgG, Fcγ

fragment specific, conjugated to DyLight 649 (1:400) (Jackson ImmunoResearch,

West Grove, PA). Next, coverslips were mounted on tissue sections with Prolong

Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA). As a control for

nonspecific labeling with secondary antibody, omission of the primary antibody was

also performed on a slide from each case. Htt-aggregate positive nuclei and size of

htt-aggregates were measured in each of ten 215 ×215 µm microscope fields by

Olympus IX-81 microscope (Olympus, Japan), in each of five rostrocaudally spaced

sections in the striatum of 5 mice from the three groups of R6/2 mice (wild-type

littermates did not show htt immunoreactivity). At least 450 nuclei per mouse were

obtained and the percentage of nuclei containing htt-aggregates and area of

htt-aggregates were calculated by CellProfiler software (Whitehead Institute,

Cambridge, MA) and a mean value was obtained for each group.

113

Measurement of striatal volume by Nissl staining. Cryostat sections (20 µm

thick) were postfixed in 4% paraformaldehyde, then dehydrated in graded alcohols.

After delipidated in 1:1 alcohol/chloroform for 1.5 h, sections were rehydrated

through graded alcohols and stained with 0.2% aqueous solution of cresyl violet

(Sigma, St. Louis, MO) for 5 min, followed by a brief rinse with water and

dehydration in graded alcohols. Sections were cleared in xylene (two changes, 5

min each) and cover-slipped with Permount (Fisher Scientific, Fair Lawn, NJ). The

volume of the striatum was measured according to the principle of Cavalieri (Cyr et

al., 2005) (volume=s1d1+s2d2…sndn, where s=surface area and d=distance between

two sections). We considered 15 coronal levels of the striatal sections (from Bregma

1.7 mm, with an interval of 200 μm between the sections) for the volumetric

measurement study. Image capturing was performed by using an inverted microscope

(Olympus IX-81) coupled to a digital camera (Hamamatsu EMCCD, Japan). The

montage image of a coronal brain section was constructed by Slidebook (Intelligent

Imaging Innovations, Denver, CO). The surface area of striatum in each section was

measured using NIH Image J [http://rsbweb.nih.gov/ij/]. Data were expressed as the

average Cavalieri volume ±SEM (mm3) of 4~5 mice per group.

CaM Kinase II Activity Assay. CaM kinaseII enzyme activity was analyzed

using the SignaTECT Calcium/Calmodulin-Dependent Protein Kinase Assay System

(Promega, Madison, WI), according to the manufacturer's protocol. Mouse striatum

was homogenized in the extraction buffer (20mM Tris-HCl pH 8.0, 2mM EDTA,

114

2mM EGTA, 2mM DTT, 1mM PMSF and 1:1000 Protease Inhibitor Cocktail (Sigma,

St Louis, MO)). The homogenate was centrifuged at 350 × g for 5 min at 4°C and the

supernatant was mixed with [γ-32P]ATP (at 3000Ci/mmol, 10mCi/ml), 50 µM

biotinylated CaM kinase II substrate, reaction buffer containing 50mM Tris-HCl (pH

7.5), 10mM MgCl2 and 0.5mM DTT, with or without control buffer containing 1mM

EGTA. After incubation at 30°C for 2 min, the reaction was terminated by adding 7.5

M guanidine hydrochloride and spotted to a streptavidin-impregnated membrane.

Membranes were washed and the retained radioactive substrate of CaM kinase II was

quantitated by liquid scintillation counting (Beckman, Fullerton, CA). Radioactive

counts were used as an index of endogenous CaM kinase II activity in the sample.

TGase II Activity Assay. TGase activity was measured by detecting the

incorporation of a biotinylated TGase amine substrate, 5-(biotinamido)pentylamine

(Pierce, Rockford, IL) into N,N’-dimethylcasein (Calbiochem, San Diego, CA) as

previously described (Dudek et al., 2009). 96-well Immulon 4 HBX plates (Dynatech,

Franklin, MA) were coated with 100 µl of N,N’-dimethylcasein (20 mg/ml) in 0.05M

sodium bicarbonate, and stored at 4°C overnight. After the unbound

N,N’-dimethylcasein was discarded, the plate was washed with PBS and blocked with

nonfat dry milk (2% milk in PBS) for 1 hour at 37°C, followed by 3 washes with PBS.

Striatum from the various mice were homogenized in 0.1 M Tris-HCl pH 8.3, 1 mM

EDTA, 1 mM PMSF, and protease inhibitor cocktail. Striatal homogenates (15 µg of

protein/sample) were added to the plate with 0.1 M Tris-HCl pH 8.5, 0.15 M NaCl, 5

115

mM DTT, 0.5 mM biotin-labeled pentylamine, and 5 mM CaCl2. The mixture was

incubated for 1 hour at 37°C. The plate was washed with TBS and 0.001% Tween

followed by incubation with streptavidin conjugated to HRP (Jackson

ImmunoResearch, West Grove, PA) for 1 hour at room temperature. After five washes,

1 x TMB substrate solution (eBioscience, SanDiego, CA) was added and color was

allowed to develop for 3 min. Then the reaction was stopped with 3N sulfuric acid

and the absorbance was read at 450nm. For GTP-inhibited TGase enzymatic activity,

the assay was performed in the presence of 500 µM GTPγS (Sigma-Aldrich, St Louis,

MO). Negative controls were run in the absence of pentylamine or dimethylcasein.

Each assay also included a standard curve of varying amounts of guinea pig TGase

(Sigma, St. Louis, MO). Each sample was measured in triplicate. TGase activity was

converted to a percentage control based on the mean value for the Vec-WT mice.

Statistics. All data are presented as mean ± S.E.M. and analyzed by one- or

two-way ANOVA with repeated measures. Post hoc tests were conducted using

Bonferroni's multiple comparison tests. There were one or two mice dead after week

13 in each HD group, so the last observed value was used for the subsequent missing

values. Survival time was demonstrated by Kaplan-Meier survival curves and

analyzed by a log-rank test. GB-STAT 10.0 (Dynamic Microsystems, Inc., Silver

Spring, MD) and GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA) were

used for all statistical analyses. A probability level of p < 0.05 was considered to be

statistically significant for all statistical tests.

116

RESULTS

AAV-mediated delivery of CaM-fragment in R6/2 mice attenuated body weight

loss

Body weight was monitored from 7 weeks of age onwards. All groups of mice were of

similar initial body weight (22.9±0.3 g, n＝10~14 mice in each group). Changes in

body weight were expressed as a percentage of body weight measured at 7 weeks of

age (Fig. 18a). Body weight in all groups slowly increased or remained unchanged

until week 10. Thereafter, mice in the two wild-type (WT) groups continued gaining

body weight. The Vec-HD (R6/2 mice injected with empty vector AAV expressing

GFP alone) and Scr-HD mice (R6/2 mice injected with AAV expressing a scrambled

version of the CaM-fragment) had profound weight loss over the duration of the

observation period, whereas CaM-HD mice (R6/2 mice injected with AAV expressing

the CaM-fragment) maintained their body weight, or slightly increased their body

weight (Fig. 18a). One-way ANOVA [F(4,50)=82.48; p<0.0001] followed by

Bonferroni's multiple comparison test indicated that mice in CaM-HD group had less

change in body weight than those in the Vec-HD group at 12-16 weeks of age (p <

0.05), and there was no significant difference in body weight change between Scr-HD

and Vec-HD at each time point. Similarly, body weight change in the CaM-WT (WT

mice injected with AAV expressing the CaM-fragment) was not significantly different

from the Vec-WT (WT mice injected with empty vector AAV expressing GFP alone).

CaM-fragment expression did not significantly increase R6/2 mice survival

117

Although the first death took place on day 88, 78 and 67 and mean survival time was

112, 102.9, 99.7 days in CaM-HD, Vec-HD and Scr-HD mice, respectively,

suggesting a small (about 10%) increase in life span of CaM-HD mice, Kaplan-Meier

survival curves demonstrated no statistically significant effect of CaM-fragment

expression on the mortality in R6/2 mice (Fig. 18b). By log-rank comparison, the

three groups of HD mice did not significantly differ from each other (p > 0.05). None

of the mice in WT groups died during the observation period (from 6 to 27 weeks of

age).

CaM-fragment expression increases stride length and lowers stride frequency in

R6/2 mice

Gait dynamics were characterized using the DigiGait Imaging System. Generally,

there is a clear difference between WT mice and Vec-HD or Scr-HD mice starting

from week 11 in all the parameters measured, whereas the difference between WT

mice and CaM-HD mice is not significant in stride length and frequency. At a speed

of 14 cm/s, WT mice maintained a regular alternating gait at a consistent stride length

of 5.8±0.3 cm and stride frequency of 2.5±0.1 steps/sec, whereas Vec-HD and Scr-HD

mice walked at a gradually reduced stride length and higher stride frequency. For

stride length (Fig. 19a), the two-way ANOVA with repeated measures indicated a

significant main effect of group [F(4,126)= 35.77; p<0.0001] and week [F(3,126)= 12.12;

p<0.0001]. There was also a significant interaction between group and week [F(12,126)=

5.71; p<0.0001]. The post hoc Bonferroni test indicated that stride length of CaM-HD

118

mice is indistinguishable from the two HD control groups at 10 weeks of age, but

CaM-HD mice displayed a longer stride length as compared to Vec-HD or Scr-HD

mice at week 11~13. Stride length of CaM-HD mice was not significantly shorter than

WT mice until at week 13. For stride frequency (Fig. 19b), two-way ANOVA

indicated significant differences among groups [F(4,126)=11.99; p<0.0001], but there is

no significant effect of week [F(3,126)= 0.202; p=0.895] or interaction between group

and week [F(12,126)= 1.368; P=0.190]. The Bonferroni test indicated that CaM-HD

mice had a significantly lower stride frequency than Vec-HD at week 11-13. Although

Vec-HD and Scr-HD were statistically indistinguishable at any time point, there was a

statistically significant difference between CaM-HD and Scr-HD at week 12. There

was no significant difference between CaM-HD and WT mice in stride frequency.

Analysis of some other gait dynamics revealed differences between genotypes. For

example, the measure of stance width variation between steps was greater (Fig. 19c)

and paw area at peak stance was smaller (Fig. 19d) in HD mice than in WT mice, but

the CaM-fragment had no beneficial effects on these gait indices. For stance width

variation, two-way ANOVA followed by Bonferroni test indicated significant

differences between HD and WT groups [F(4,126)=13.61; p<0.0001], but no significant

differences among HD groups or WT groups (P>0.05). There is no significant effect

of week [F(3,126)= 1.21; p=0.31] although interaction between group and week is

significant [F(12,126)= 1.97; P=0.03]. Similar results were obtained in the analysis of

paw area at peak stance [Group factor: F(4,126)=10.42; p<0.0001], whereas week effect

119

is significant [F(3,126)= 13.68; p<0.0001] and interaction between group and week is

not [F(12,126)= 1.55; P=0.11].

CaM-HD mice had fewer low mobility bouts and longer travel distance than

Scr-HD or Vec-HD mice

Force-plate actometers were used to measure the locomotor activity of R6/2 and WT

mice. The number of low mobility bouts (LMB, defined as remaining continuously in

a virtual circle of 15 mm radius for 10 sec) is presented in Fig. 20a. Two-way ANOVA

with repeated measures indicated a significant effect of group [F(4,144) =27.75, p <

0.001] and a significant effect of week [F(3, 144) = 3.179, p < 0.05], but the interaction

between group and week was not significant. The number of LMB gradually

increased in HD mice without CaM-fragment expression, whereas CaM-HD mice,

similar to WT mice, maintained LMB at a relatively lower level. At week 12 and 13,

the Bonferroni post hoc test detected significantly fewer LMB in CaM-HD mice as

compared to either Scr-HD or Vec-HD mice. Total travel distance in a 30 min session

provided another index of locomotor activity of HD mice (Fig. 20b). Two-way

ANOVA with repeated measures indicated a significant effect of group [F(4,138) = 3.40,

p = 0.02], but the effect of week [F(3,138) = 0.17, p=0.92] and interaction between

group and week [F(12,138) =1.12, p=0.35] were not significant. Consistent with reduced

LMB in HD mice treated with CaM-fragment, CaM-fragment expression also

increased the locomotor activity in HD mice as indicated by a significantly greater

travel distance in CaM-HD mice than in either Scr-HD (at week 12,13) or Vec-HD

120

mice (at week 12). There was no significant difference between CaM-HD and WT

mice in distance traveled in 30 min.

CaM-fragment improved performance on the rotarod in R6/2 mice

Rotarod was used to measure fore- and hindlimb motor coordination and balance

starting at 10 weeks of age (i.e., three weeks after the AAV injections). At ten weeks

of age, the two groups of WT mice were able to stay on the rod during the 300 sec

time period measured, whereas none of the three groups of HD mice could finish the

test without falling in 300 sec. Even at this early time point after injection of the AAV,

the mean latency to fall was significantly differed among the HD groups, 245.9±32.0,

150.2±29.5, 98.1±19.8 sec in CaM-HD, Vec-HD and Scr-HD mice, respectively. After

10 weeks of age, there was a progressive decline in performance of all HD mice, but

the CaM-HD mice still achieved better performance than the other two HD groups

(Fig 21). Using analysis using two-way ANOVA with repeated measurements, we

found a significant main effect of group [F(4,188) = 49.35, p < 0.0001], a significant

main effect of week [F(4,188) = 18.86, p < 0.0001] and a significant interaction between

group and week [F(16,188) = 4.714, p < 0.0001] on latency to fall off the rotarod.

Bonferroni post hoc test indicated that CaM-HD mice had longer latency to fall as

compared to Scr-HD mice at 10-14 weeks of age and Vec-HD mice at 10-11 weeks of

age (p < 0.05).

CaM-fragment expression reduced TGase-modified htt in R6/2 mice striatum

Cell culture studies indicated that AAV-mediated CaM-fragment expression can

121

significantly inhibit the formation of TGase-modified htt (Dudek et al., 2009). Here

we used striatal homogenates from HD or WT mice to detect TGase-catalyzed

cross-linking of htt. Immunopurification of proteins containing є-(γ-glutamyl) lysine

bonds was performed using 81D4 mAb prebound to Sepharose beads.

TGase-modified htt was then detected on immunoblots as shown in the Fig. 22a.

TGase-modified htt was only detected in HD but not WT mice. One-way ANOVA

[F(2,15) =18.44, p<0.0001] followed by Bonferroni's comparison test found there was a

significant reduction (by approximate 50%) in TGase-modified htt in the striatum of

CaM-HD mice as compared to Scr-HD or Vec-HD mice (Fig. 22b).

CaM-fragment expression decreases the percentage of htt-positive nuclei and the

size of htt aggregates in the striatum of R6/2 mice

Striatal intranuclear and neuropil inclusions containing mutant htt are prominent

neuropathological hallmarks of HD and may play an important role in disease

progression. In R6/2 mice, intranuclear aggregates are much larger than neuropil

aggregates and amenable to quantification, so we examined the effect of

CaM-fragment on intranuclear htt aggregates in the striatum of R6/2 mice. In

CaM-HD mice, the percentage of htt-positive nuclei was lower and the size of

intranuclear aggregates appeared smaller than in the control virus-treated HD mice

(Fig. 23a). These observations were confirmed by quantification of nuclear aggregates

(Table 1). We found a significant reduction in the percentage of striatal nuclei

containing htt-aggregates (approximately 18%) and the nuclear aggregate size

122

(approximately 35%) in CaM-HD mice as compared with either Vec- or Scr-HD mice.

GFP fluorescence was observed in coronal brain sections from AAV-injected mice,

indicating a widespread infection and expression of CaM-fragment in the striatum

(Fig. 23a). Both human HD and R6/2 mouse brains are characterized by atrophy of

the striatum. To exam whether the CaM-fragment delivery decreases gross striatal

atrophy, we used Nissl staining and the Cavalieri principle to estimate striatal volumes.

There was no statistically significant difference in mean striatal volumes between

CaM-HD and the two groups of control HD mice. However, all three groups of HD

mice had significantly smaller striatal volumes as compared with WT littermates (Fig.

23b, Table 1). As expected, striatal neuronal atrophy was observed in HD mice in

high-power images.

CaM Kinase II activity was not affected by CaM-fragment expression

To test if expression of the CaM-fragment would interfere with other CaM-dependent

enzymes, we measured the activity of calmodulin-dependent protein kinase II (CaM

kinase II) in mouse striatal homogenates. We found no significant differences in CaM

kinase II activity among the various HD or WT mouse groups in either the presence or

absence of EGTA, a calcium chelator (Fig. 24a). However, EGTA caused a general

decrease in CaM kinase II activity in all mouse groups. Two-way ANOVA indicated a

significant main effect of EGTA [F(1,50)=72.12; p<0.0001, but there was no significant

effect of mouse group [F(4,50)=0.543; p=0.705] or an interaction between EGTA and

group [F(4,50)=0.966; p=0.435].

123

CaM-fragment did not change total TGase activity

In order to explore whether the reduced TGase-modified htt in CaM-HD mice is due

to a substrate-dependent inhibition or a general decrease in TGase enzymatic activity,

we compared total TGase activity in the striatum among the different groups of mice.

There is a significant increase in TGase activity in the three groups of HD mice as

compared to CaM-WT or Vec-WT mice (p<0.01). Expression of the CaM-fragment

did not have an effect on TGase activity in either HD or WT mice. In the presence of

GTPγS, which specifically inhibits TGase activity, TGase activity is dramatically

reduced (at least 60%) in all groups (Fig. 24b). Two-way ANOVA indicated a

significant main effect of GTPγS [F(1,50)=428.149; p<0.0001] and mouse group

[F(4,50)= 5.535; p<0.01], and there was a significant interaction between GTPγS and

mouse group [F(4,50)= 5.032; p<0.01]. The activity of guinea pig liver TGase was

measured in each assay in the range from 0.01 to 0.5 milliunits/ml and resulted in a

linear correlation with an R-square value from 0.95 to 0.99. The activity of TGase in

the striatal samples fell within this linear range for guinea pig liver TGase.

124

Fig.18 Effect of CaM-fragment on body weight and survival. (a) Changes in body

weight were expressed as a percentage of body weight measured at 7 week of age.

One-way ANOVA and Bonferroni's test found that the change in body weight was

significantly smaller in CaM-HD than in Vec-HD (p< 0.05) starting from week 12,

and there is no significant difference between Scr-HD and Vec-HD. * indicates p<0.05

(CaM-HD vs. Vec-HD). (b) Kaplan-Meier survival curves showed the first death was

at day 88, 78 and 67 in CaM-HD, Vec-HD and Scr-HD mice, respectively. By

log-rank comparison, three groups of HD mice did not differ from each other (p>0.05).

(n＝9~14 in each group)

125

Fig.19 Video-based gait analysis on the treadmill. Mice were placed on a treadmill

belt moving at a speed of 14 cm/s. Gait data were pooled from all four paws.

CaM-HD exhibited a significantly greater stride length (a), and a lower stride

frequency (b). There is no significant difference between CaM-HD and Vec-HD or

Scr-HD in stance width variability (c) and paw area at peak stance (d). Two-way

ANOVA with repeated measures followed by Bonferroni test. * indicates p<0.05

(CaM-HD vs. Vec-HD), # indicates p<0.05 (CaM-HD vs. Scr-HD), n=6-14 in each

group

126

Fig.20 Expression of CaM-fragment enhanced the locomotor activity in R6/2 mice.

Spontaneous locomotor activities of R6/2 and WT control mice were recorded in a

force-plate actometer apparatus for 30 min. (a) CaM-HD mice displayed a

significantly fewer number of low mobility bouts than the two HD control groups at

week 12-13. (b) Distance traveled by CaM-HD mice was significantly longer than

Scr-HD at week 12-13 and Vec-HD at week 12. Two-way ANOVA with repeated

measures followed by Bonferroni test. * indicates p<0.05 (CaM-HD vs. Vec-HD), #

indicates p<0.05 (CaM-HD vs. Scr-HD), n=9-14 in each group

127

Fig.21 CaM-fragment expression delayed the onset of the rotarod defects in R6/2

mice. Mice were placed on a rotating rod with increasing speed, from 4 rpm to 40 rpm

in 300 seconds. The latency to fall off the rotarod within this time period was

recorded. Two-way ANOVA with repeated measures followed by Bonferroni test

found that CaM-HD mice had significantly longer latency to fall than Scr-HD starting

from week 10, and than Vec-HD at week 10-11. * indicates p<0.05 (CaM-HD vs.

Vec-HD), # indicates p<0.05 (CaM-HD vs. Scr-HD), n=9-14 in each group

128

Fig.22 TGase-modified htt in R6/2 mice striatum was reduced by CaM-fragment

expression. (a) The insoluble fraction from mouse striatal homogenates was dissolved

with formic acid. Immunopurification (IP) of proteins containing є-(γ-glutamyl)

lysine bonds was performed using 81D4 mAb prebound to Sepharose beads.

Immunopurified proteins were then examine on immunoblots (IB) using an antibody

against htt. (b) Quantitation of immunoblots. Data shown are the mean IOD± S.E.M.,

and they are normalized to CaM-HD mice. * indicates p<0.05 as compared to

CaM-HD (n=5 in each group at 13-14 weeks of age).

129

Fig. 23 Histological evaluation of neuropathology (a) Top and middle,

immunofluorescent labeling of striatum in R6/2 mice at 14 weeks of age.

Htt-aggregates were labeled with htt antibody MAB5374 (red), the nuclei were

labeled with DAPI (blue). The composite images show that the percentage of

htt-positive nuclei and the size of nuclear htt-aggregates were decreased in CaM-HD

as compared to the control-HD mice. Scale bar=30µm. Bottom, photomicrographs of

130

GFP protein distribution in the brain of representative AAV-injected animals indicated

that the same vector-derived CaM-fragment was expressed in the striatum. Scale

bar=1 mm. (b) Top , montage images of Nissl-stained brain coronal sections from

CaM-HD, Vec-HD and CaM-WT mice at the level when the corpus callosum starts to

merge in the middle. Scale bar=1 mm. Bottom, high magnification of micrograph of

the dorsomedial aspect of the striatum from the sections above. There is marked

neuronal atrophy with small angulated neurons in Vec-HD mouse, with relative

preservation of neuronal size in CaM-HD mouse. Scale bar=50 µm.

131

Fig. 24 Expression of CaM-fragment in mouse striatum did not significantly affect the

activity CaM kinase II or TGase. (a) Mouse striatal homogenates were used to

measure CaM kinase II activity. Two-way ANOVA indicates a significant main effect

of EGTA. However, there was no significant main effect of mouse group. The

interaction between EGTA and group was also not significant. Bonferroni posttest

indicates no significant differences in CaM kinase II activity among the various

groups in either the presence or absence of EGTA. (b) The presence of the

132

CaM-fragment did not significantly change the levels of TGase activity in either HD

or WT mice. However, a significant elevation of TGase activity in three groups of HD

mice was observed as compared with WT mice. When 500 µM GTPγS was added,

there is a dramatic reduction in all mouse groups. Two-way ANOVA indicates a

significant main effect of GTPγS and mouse group, and there is a significant

interaction between GTPγS and group. Bonferroni posttest *indicates p<0.01 as

compared to Vec-WT; # indicates p<0.01 as compared to CaM-HD in the absence of

GTPγS. Each column represents the mean ± SEM. (n=5 in each group at 13-14 weeks

of age).

133

134

DISCUSSION

Although there have been enormous strides in understanding the molecular

and mechanistic pathways that mediate the progression of HD, an effective treatment

to slow or prevent the disease-associated physical and mental decline has yet to be

discovered. A number of reports in cultured cells have shown that interfering peptides

can selectively inhibit pathological interactions between mutant htt and other proteins,

and therefore are a potential therapeutic strategy for HD (Nagai et al., 2000, Dudek et

al., 2008, 2009). However, only a few studies have been performed to verify the

efficacy of these peptides in vivo (Kazantsev et al., 2002, Tang et al., 2009). We

previously found that plasmid or AAV-mediated delivery of CaM-fragment

significantly attenuated TGase-modified htt, htt-associated cytotoxicity and

intracellular Ca2+ disturbances without interfering with the activity of other

CaM-dependent enzymes in HD cell models (Dudek et al., 2008, 2009). We now

report that expression of the CaM-fragment in the striatum ameliorated body weight

loss, improved motor performance of R6/2 mice, reduced TGase-modified htt and

decreased the number and size of nuclear htt-aggregates in the striatum.

Striatal delivery of CaM-fragment significantly delayed the onset of

movement abnormalities and improved motor function as measured by analysis of

gait, rotarod performance and locomotor behavior. It has been reported that gait

deficiencies in R6/2 mice became apparent from 8 weeks of age. As visualized in the

footprint analysis, R6/2 mice displayed a significantly shorter stride length, a

135

staggering movement and a gait that lacked a uniform step pattern compared to WT

littermates (Carter et al., 1999). Striatal injection of AAV-CaM-fragment conferred

significant improvements in stride length and frequency measurements, and a mild,

insignificant increase in paw area at peak stance in R6/2 mice. Decreased motor

function on the rotarod is the most commonly reported measure among animal models

of HD (Schilling et al., 1999, von Horsten et al., 2003). Three weeks after

CaM-fragment expression, R6/2 mice started to show significantly improved rotarod

performance and this beneficial effect was maintained from 10 weeks of age onward.

Although motor abnormalities such as bradykinesia (abnormally slow movements)

and pronounced hypoactivity were frequently observed in HD animal models

(Mangiarini et al., 1996, Keene et al., 2002), there have been relatively few

quantitative studies on spontaneous locomotor activity of R6/2 mice. In the present

experiments using a force-plate actometer, progressive locomotor deterioration

(assessed by number of low mobility bouts and travel distance) was observed in R6/2

mice injected with control AAV, whereas delivery of AAV expressing CaM-fragment

to R6/2 mice striatum significantly improved their locomotor deficits.

In comparison to the TGase inhibitor cystamine, which prolonged the life span

of R6/2 mice by 12%~19% (Dedeoglu et al., 2002, Karpuj et al., 2002a), there was a

non-significant increase in the mean survival time by approximately 10% in the

CaM-HD mice. Differences in other mechanisms of action could possibly account for

the different outcomes. In addition to inhibiting TGase, cystamine increased

136

transcription of neuroprotective genes, increased glutathione levels, and ameliorated

apoptosis (Dedeoglu et al., 2002, Karpuj et al., 2002a). In contrast, that

CaM-fragment decreased TGase-modified htt, normalized intracellular calcium

release, and reduced mutant htt-associated cytotoxicity but had no effect on TGase

activity (Dudek et al., 2008, 2009). Secondly, the different outcomes could be due to

the different brain regions treated. The CaM-fragment was expressed in the striatum

only while the cystamine-treatment was systemically delivered and could affect other

brain regions that display neuropathology in HD such as the frontal cortex. Rather

than unspecific inhibition of TGase by cystamine, disrupting the CaM-htt interaction,

as with the CaM-fragment, is a promising new approach for the treatment HD.

As a result of its expanded N-terminal polyglutamine region, mutant htt is

processed and deposited as a component of insoluble protein aggregates that persist in

neuronal nuclei, perikarya, and processes (DiFiglia et al., 1997). Although some

evidence suggests that htt-associated toxicity is linked to soluble htt and its

protein–protein interactions (Petersen et al., 1999), htt aggregates act as a phenotypic

readout that can reflect pathologically relevant processes such as htt cleavage,

misfolding, and sequestration (Chopra et al., 2007). A number of compounds which

are neuroprotective in R6/2 mice, such as the TGase inhibitor cystamine (Dedeoglu et

al., 2002), the antioxidant coenzyme Q10 (Ferrante et al., 2002) and the energy buffer

creatine (Ferrante et al., 2000), significantly suppress htt aggregates. Our present

results revealed that CaM-fragment significantly reduced TGase-modified htt in the

137

insoluble fraction, the percentage of htt-positive nuclei and intranuclear htt-aggregate

size in the striatum, which may contribute to the benefits of CaM-fragment. These

findings support the hypothesis that CaM-regulated TGase modification of htt

contributes to the formation and stabilization of htt-aggregates and may play a role in

the pathogenesis of HD. However, it is difficult to speculate whether there would be

an associated increase in soluble or oligomeric htt in the R6/2 mouse striatum with

CaM-fragment expression. There is evidence that TGase modifications stabilize

proteins (Tucholski et al., 1999), so inhibiting TGase modifications to htt may

increase clearance of the htt protein.

R6/2 mice developed significant striatal atrophy as compared to WT

littermates, a pathological characteristic associated with the gradual

neurodegeneration occurring in the mice. Although there is no significant difference

in striatal volume between CaM-HD and control-HD mice by light microscopy, we

could not exclude the possibility that expression of CaM-fragment would reduce the

striatal atrophy if a more sensitive technology (MR scanning) was applied. Moreover,

if striatal injections of AAV were performed at an earlier age, there might be more

notable beneficial results including increases in survival and striatal volume.

Similar to increases in TGase protein level and enzymatic activity in human

HD brain (Karpuj et al., 1999, Lesort et al., 1999), our results show that TGase

activity is elevated in R6/2 mice compared with WT littermates. Interestingly, striatal

delivery of CaM-fragment did not change the overall activity of TGase in either R6/2

138

mice or WT mice. These results are consistent with our previous studies in cell culture

(Dudek et al., 2009), suggesting that CaM-fragment may specifically affect the

CaM-mutant htt interaction and selectively alter TGase-catalyzed modifications of

mutant htt without interfering with the activity of TGase on other substrate proteins.

Furthermore, CaM-fragment expression did not affect CaM kinase II activity,

indicating that CaM-fragment had no effect on the activity of other CaM-dependent

enzymes. Most importantly, expression of CaM-fragment in WT mice had no effect

on any of the behavioral tests nor striatal histopathology, suggesting there is minimal,

if any, deleterious effect of long-term expression of CaM-fragment.

A prominent hypothesis on the pathogenesis of HD is that the aberrant

interactions of the extended polyglutamine repeats (polyQs) of mutant htt, either with

other proteins or with each other result in htt-aggregate formation (Wanker, 2000).

Glutamine repeats may function as polar zippers and expansion of polyQs may

increase the affinity of htt for its protein binding partners (Perutz et al., 1994). The

principal proteins that are known to interact with htt are: htt associated protein 1 (Li et

al., 1995), htt interacting protein (Kalchman et al., 1997), type 1 inositol

1,4,5-trisphosphate receptor (InsP3R1) (Tang et al., 2003) and calmodulin (Bao et al.,

1996, Zainelli et al., 2004). As revealed by our previous in vitro assay (Dudek et al.,

2009), the neuroprotective effects of CaM-fragment may depend on its ability to

competitively bind to mutant htt thereby disrupting the CaM-mutant htt interaction.

First, this disruption may result in the CaM and mutant htt no longer in close

139

proximity to each other, thus the TGase activity in htt modification and aggregate

formation may be reduced. Second, the association between mutant htt and

CaM-fragment may prevent the further interaction of mutant htt with other proteins,

such as InsP3R1, an intracellular Ca2+ release channel. Since mutant htt selectively

associates with and activates InsP3R1(Tang et al., 2003), disrupting their interaction

should stabilize neuronal Ca2+ homeostasis. This hypothesis is supported by in vitro

experiments that indicated the CaM-fragment (Dudek et al., 2008) or

InsP3R-fragment (Tang et al., 2009) attenuates mutant htt-associated intracellular

Ca2+ disturbances.

Although it will be advantageous to confirm the results from the current study

using a different HD animal model, special care should be taken to choose a different

strain other than R6/2 mice. Instead of body weight loss, YAC128 mice have

significantly (27%) increased body weight at 12 months of age (Van Raamsdonk et al.,

2006, Van Raamsdonk et al., 2007), which in turn can cause bias in most behavioral

tests. Obvious or diffuse nuclear htt accumulation is not detectable in the striatum or

cortices of BAC HD mice even in 18-month-old mice (Gray et al., 2008).

Intergenerational instability and somatic instability of the HD CAG repeat have been

observed in HdhQ111 knock-in mice in all three background strains: C57BL/6, FVB/N

and 129Sv (Lloret et al., 2006). The phenotype of N171-82Q mice is more variable

than that of R6/2 mice, and therefore a much larger number of mice are necessary to

provide adequate power (Hersch and Ferrante, 2004).

140

Many therapeutic targets for HD today are based on the pathogenesis

hypotheses, from mitochondrial dysfunction to transcriptional perturbation to TGase

hyperactivity (Beal and Ferrante, 2004). However, since clinical trials of

mechanism-based therapies for HD have been limited by insufficient statistical power

and insignificant improvement (Handley et al., 2006), current clinical treatments are

symptomatic. The major neurological symptoms associated with HD include

disordered voluntary movements: uncoordinated, arrhythmic, and slow fine motor

movements; rigidity; and gait disturbances (Harper, 1991). Our in vivo results

demonstrated that viral delivery of CaM-fragment to the striatum of R6/2 mice

significantly improved motor coordination and balance, enhance locomotor activity

and alleviated gait disturbances, suggesting the possibility of a better quality of life

for HD patients if this new therapeutic strategy is successfully translated to the

clinical trials.

The present studies provide, for the first time, in vivo evidence that

CaM-fragment has significant efficacy in improving the behavioral deficits and

neuropathological phenotype in the HD animal model. The positive effects of

CaM-fragment in R6/2 mice provide further evidence that CaM-regulated TGase

modification of htt may contribute to HD pathogenesis. More importantly, these

studies have identified a novel therapeutic strategy for treating HD patients.

141

141

CHAPTER FIVE

GENERAL CONCLUSION

REVIEW OF RESULTS AND SIGNIFICANCE

In chapter two, we identified a novel effector mechanism attributed to 5-HT2A

receptor signaling. This chapter described the effect of 5-HT2A agonists (5-HT or DOI)

on transamidation and activation of Rac1 in a cell line derived from rat embryonic

cortex. The transamidation is mediated by TGase, as suggested by a TGase inhibitor,

cystamine or knockdown of TGase with siRNA. The transamidation of Rac1

contributed to its transient activation, since TGase inhibition significantly reduced

both transamidation and activation. Moreover, serotonin was identified as an amine

that becomes transamidated to Rac1 by TGase.

TGase-catalyzed transamidation of small G proteins not only occurred in

platelets, vascular smooth muscle cells and cells derived from cervical cancer (Singh

et al., 2001, Walther et al., 2003, Guilluy et al., 2007), but also occurred in neuronal

cell lines, such as SH-SY5Y neuroblastoma cell line (Singh et al., 2003) as well as

A1A1v cell line derived from rat embryonic cortex (Dai et al., 2008). One of the

consequences of small G protein transamidation was constitutive activation. Members

of the Rho family, particularly Rac1, are associated with a wide array of cellular

142

processes such as cytoskeletal organization, vesicular transport, cell cycle progression,

cell adhesion and migration (Etienne-Manneville and Hall, 2002, Burridge and

Wennerberg, 2004) and pathological conditions such as Salmonella invasion, tumor

cell migration, and retinal degeneration (Bourguignon et al., 2000, Belmonte et al.,

2006, Brown et al., 2007). The physiological functions of TGase-catalyzed

transamidation and activation of small G proteins in the nervous system remains

poorly understood. Recent studies have demonstrated that activation of TGase and

transamidation of RhoA to putrescine induced by retinoic acid in SH-SY5Y cells,

results in activation of ERK1/2, JNK1, and p38 MAP kinases, which may stimulate

transcription factors and induce gene expression for neuronal differentiation, as

indicated by neurite outgrowth and neuronal marker expression (Singh et al., 2003).

In addition to small G proteins, cytoskeletal proteins such as intermediate filaments in

neurons, have also been shown to undergo TGase-catalyzed transamidation in vitro

(Selkoe et al., 1982, Miller and Anderton, 1986) and in human cerebral cortex

(Norlund et al., 1999). We demonstrate that stimulation of 5-HT2A receptors can

increase the transamidation of Rac1 in both undifferentiated and differentiated A1A1v

cells, suggesting that transamidation and constitutive activation of this signal

transducer are not altered by neuronal differentiation of cells. The functional

consequences of transamidation of Rac1 by TGase especially in neurons needs to be

further explored.

The signaling pathways involved in Rac1 transamidation have been

143

investigated in chapter three. The different components in the potential signaling

pathways have been targeted by the antagonists and /or agonists. We found inhibition

PLC decreased intracellular Ca2+ concentration as well as TGase-modified Rac1 upon

5-HT2A receptor activation. The crucial role of Ca2+ signaling in Rac1 transamidation

was confirmed by manipulation of intracellular Ca2+ by means of a Ca2+ chelating

agent or an ionophore. When cells were pretreated with a Ca2+ chelator,

TGase-catalyzed Rac1 transamidation was significantly reduced. Conversely,

treatment with a Ca2+ ionophore was sufficient to induce an increase in Rac1

transamidation by TGase. Moreover, the transamidation reaction was also regulated

by CaM, a Ca2+ binding protein, as demonstrated by a CaM antagonist which

depresses the stimulatory effect of 5-HT2A receptor activation on Rac1 transamidation.

Searching the Conserved Domain Database revealed that two glutamine residues (Q61,

Q74) and three lysine residues (K5, K16, and K116) are located at the activity-related

domains. These results provided new insights into the signaling mechanisms

underlying 5-HT2A receptor-induced Rac1 transamidation and activation by TGase in

A1A1v cells. The present study indicated that 5-HT2A receptor-coupled PLC

activation and subsequent Ca2+/CaM signaling are necessary in TGase-catalyzed Rac1

transamidation, and identified potential TGase-targeting residues that could render

Rac1 constitutively active upon transamidation.

In the studies presented in chapter four, we shifted our focus from Rac1 to

mutant htt as another substrate of TGase-catalyzed transamidation reaction. Our

144

previous studies in the cell models of HD screened various CaM fragments based on

their ability to decrease TGase-modified htt and cytotoxicity, as well as normalize

intracellular Ca2+ release. In this study, an AAV that expresses a CaM fragment was

delivered into the striatum of HD transgenic R6/2 mice. Ameliorated body weight loss

and improved motor performance were observed in those mice as compared to control

virus-injected HD mice. CaM-fragment expression also specifically reduced

TGase-modified htt, the percentage of htt-positive nuclei and the size of intranuclear

htt aggregates in the striatum.

Although the life span of CaM-fragment treated HD mice was not

significantly prolonged (there was a non-significant increase in the mean survival

time by ~10%), our results demonstrated that CaM-fragment expression in the

striatum of HD mice significantly improved motor coordination and balance, enhance

locomotor activity and alleviated gait disturbances. The gene mutation that causes

HD was identified in 1993 (Huntington’s Disease Collaborative Research Group,

1993), but there is currently no effective therapy and the disease inevitably leads to

death within 10 to 15 years of symptom onset (Bates, 2003). Medications are

available to help manage the signs and symptoms associated with HD, but will not

stop the progressive physical and mental decline. AAV-mediated gene therapy for HD

by preventing mutant htt expression or interupting its interaction with other proteins

has emerged as an attractive new strategy. The AAV-mediated knock-down of htt via

bilateral injections into the striatum of HD transgenic mice resulted in a reduction in

145

neuronal intranuclear inclusions, and improvements on behavioral phenotypes and

histological deficits (Harper et al., 2005, Rodriguez-Lebron et al., 2005). Disruption

interaction mutant htt with either CaM (Dai et al., 2009) or type 1 IP3 receptor (Tang

et al., 2009) by AAV expressing competitive fragments alleviated motor deficits and

improved neuropathological abnormalities. Our results demonstrate the importance of

CaM-htt association for HD pathogenesis and suggested that CaM fragment is a

potential therapeutic agent for HD.

According to our in vivo studies, wild-type mice injected with the AAV

expressing CaM-fragment did not differ from those injected with AAV vector in terms

of body weight, any of the behavioral tests, striatal histopathology, nor CaM KII or

TGase enzyme activity up to 29 weeks old, suggesting there are minimal, if any,

deleterious effects of long term expression of CaM-fragment. The CaM-fragment

might not be able to disrupt the binding of CaM to other proteins to inhibit other

biologic functions. The CaM target database

(http://calcium.uhnres.utoronto.ca/ctdb/flash.htm) provides a program that searches

for possible CaM-binding motifs in a protein sequence (Yap et al., 2000). The

database currently includes the CaM-binding sites identified in over 300 proteins. No

putative binding sites were located in the sequence of CaM-fragment suggesting that

the interruption of binding of CaM to mutant huntingtin protein is selective.

LIMITATIONS OF THE PRESENT STUDIES

There are certain limitations that should be noted when interpreting the data of

146

present studies.

We did not have direct evidence to establish a causal relationship between

Rac1 transamidation and activation. Based on the facts that inhibition of TGase

caused decreases in both Rac1 transamidation and activation, and that two glutamine

residues (Gln61, Gln74) located in Rac1 activity-related domain are potential targets

of transglutaminase, we are only able to conclude that 5-HT2A receptor stimulation

increased TGase-catalyzed transamidation of Rac1, which may contribute to the

increase in Rac1 activity. Although retinoic acid promoted activation of RhoA by

TGase-catalyzed transamidation in neuroblastoma SH-SY5Y cells (Singh et al., 2003),

transamidation-independent activation of Rac1 was observed in SH-SY5Y cells

stimulated with retinoic acid (Pan et al., 2005). Further experiments could be carried

out to explore if other mechanisms are also involved in 5-HT2A receptor-mediated

Rac1 activation in A1A1v cells.

Although the TGase inhibitor cystamine decreased the serotonin-associated

Rac1 in co-immunoprecipitation experiments, we could not confirm a direct

association between Rac1 and serotonin via a TGase-catalyzed covalent bond.

Another common binding partner could link serotonin to Rac1. However, this

possibility is undermined by the fact that serotonin-associated Rac1 did not show a

significant upward shift on immunoblots compared with native Rac1 in cell lysates,

indicating that the molecular weight of serotonin-associated Rac1 does not

significantly increase. Therefore, the binding partner, if it exists, should also be a

147

small molecule likely an amine.

The attempt to replicate 5-HT2A receptor-mediated Rac1 transamidation in rat

cortex failed. In Sprague Dawley rats that were given subcutaneously injections of

DOI (2.5 mg/kg) and sacrificed 5 or 15 minutes later, there is no significant increase

in TGase-modified Rac1 in the frontal cortex compared to saline-treated rats. The

discrepancy between cell-based and in vivo studies could be attributed to the

following possibilities. The DOI dosage and route of administration in rats may not be

able to mimic the circumstances under which 5-HT2A receptor stimulation induces

Rac1 transamidation in A1A1v cells. Moreover, the time course of reactions for

DOI-mediated Rac1 transamidation may be different between cultured cells and

whole animals. Lastly, the cultured cells grow in an environment that lack

intercellular communication or neurotransmitter cross-talk, which differs from the

normal surrounding milieu presenting in the brain.

In our studies that evaluated protective effects of CaM-fragment in R6/2

mice, immunofluorescence staining demonstrated that the percentage of htt-positive

nuclei and the size of intranuclear htt aggregates in the striatum were reduced by

CaM-fragment expression. Although TGase-modified htt in the striatal homogenates

was decreased in CaM-expressing HD mice as compared to the other two groups of

control HD mice (as indicated in immunoprecipitation and immunoblotting

experiments), we were unable to confirm that the reduction of htt aggregates was due

to decreased TGase-modification of htt. Our original experimental design that aimed

148

to measure the colocalization of htt aggregates with TGase-modified covalent bonds

could help to test this hypothesis. However, the only commercially available antibody

directed against TGase-modified covalent bonds (81D4) is raised in mouse. High

levels of background staining were observed with the 81D4 antibody in R6/2 mouse

tissues, likely due to the binding of secondary anti-mouse antibody to endogenous

mouse tissue Igs and other components. This technical difficulty compromised our

ability to explore the role of TGase-modified covalent bonds in the formation of htt

aggregation in HD mice.

Our previous in vitro experiment indicated that CaM-fragment can directly

inhibit the binding of mutant htt and CaM (Dudek et al., 2009). In this in vivo study,

since the striatum of age matched HD mice were used in immunofluorescence

staining, Nissil staining, immunoprecipitation, TGase and CaM activity assay, we did

not have enough striatal homogenates to confirm the hypothesis that striatal

CaM-fragment expression disrupted mutant htt-CaM association in vivo. The other

experiment that could support the hypothesis that the CaM-fragment can directly

disrupt the interaction between mutant htt and CaM is co-immunoprecipitation to

demonstrate the binding of CaM-fragment to mutant htt. However, we have not been

able to find a specific antibody directed against this CaM-fragment. Moreover, due to

the small size of the CaM-fragment (consisting of 46 amino acids and weighing

5,158.6 daltons), detection of the proteins on immunoblots was difficult.

As for the timing of the delivery of AAV expressing CaM-fragment, our

149

original plan was to inject AAV to R6/2 mice at a much younger age, but the youngest

mice we could purchase from Jackson Laboratories were at 6 weeks of age due to the

genotyping process. Therefore, we chose the new R6/2 strain

(B6CBA-Tg(HDexon1)62Gpb/3J) which has fewer CAG repeats in exon 1 (100-115

CAG repeats) over the previous strain (B6CBA-Tg(HDexon1)62Gpb/ 1J) with

154-159 CAG repeats. The new strain exhibited a concomitant decrease in severity

and delayed onset in the expected neurological phenotype (Jackson Laboratories,

http://jaxmice.jax.org/strain/002810.html), which actually gave CaM fragment more

time to perform its function. However, if striatal injection of AAV was performed at a

younger age, we might have more beneficial results including significant increases in

survival and striatal volume.

FUTURE STUDIES: DISRUPTING CAM-HTT INTERACTION AS A

POTENTIAL THERAPEUTIC STRATEGY IN HUMANS

Currently there is no cure for HD. On August 15, 2008 the U.S. Food and

Drug Administration (FDA) approved the use of tetrabenazine to treat Huntington’s

chorea (the involuntary writhing movements), making it the first drug approved for

use in the United States to treat the disease. Tetrabenazine works mainly as a

Vesicular Monoamine Transporter (VMAT)-inhibitor (Zheng et al., 2006) and thereby

increases the early metabolic degradation of dopamine. Other agents targeting

dopamine system have also been used to ameliorate chorea and treat irritability,

hallucinations and delusions. Those agents include dopamine antagonists such as

150

haloperidol, fluphenazine and pimozide (Caine and Shoulson, 1983, Girotti et al.,

1984, Barr et al., 1988).

Our in vivo studies demonstrated that AAV-mediated striatal expression of

CaM-fragment improved motor function, weight loss, and neuropathology in HD

mice. In the long run, to extend the application of CaM-fragment to HD patients, a

less invasive method has to be developed since AAV-mediated delivery of

CaM-fragment by stereotaxic surgery could cause brain injury. The less invasive

approaches include intravenous administration or systemic injection. The delivery of

CaM-fragment to the brain by the intravenous route could be efficient and widespread

to all parts of the brain once the blood-brain barrier (BBB) is traversed. Certain

endogenous large-molecule neuropeptides such as insulin, transferrin, or leptin are

transported into the brain from blood via receptor mediated transport (RMT) across

the BBB (Pardridge, 2003). These transporters are regulated by ligand-specific

receptors such as insulin receptors and transferring receptors, which are highly

expressed on the capillary endothelium of brain (Rip et al., 2009). Molecular Trojan

horses (MTH) are certain monoclonal antibodies (MAbs) or peptide ligands that target

RMT systems (e.g., receptor-binding sequences of insulin) that bind to BBB receptors

and induce receptor mediated transcytosis through the BBB (Pardridge, 2002).

Therefore, the construction of fusion proteins between the CaM-fragment and the

MTH may enable the CaM-fragment to cross the BBB in vivo. It has been reported

that a fusion protein between the insulin receptor mAb and brain-derived neurotrophic

151

factor (BDNF) enables rapid brain penetration of the neurotrophin in animal models,

including nonhuman primates and is intended for treatment of stoke and

neurodegenerative diseases (Boado et al., 2007, Boado et al., 2008, Boado and

Pardridge, 2009).

On the other hand, the identification of the sites on mutant htt that binds to

CaM may also provide an alternative approach to disrupt CaM-htt interaction. A yeast

two-hybrid assay or affinity purification could be used to rapidly screen the sequential

deletions of htt to identify binding domain, and then test their ability to disrupt the

binding of CaM to mutant huntingtin. Next, similar to CaM-fragment (Dudek et al.,

2008, 2009), we could test the ability of these fragments from mutant htt for their

ability to inhibit TGase-catalyzed modifications to mutant htt and for their ability to

increase cell viability in HD cell models. Once the beneficial effects were observed,

we could deliver these fragments to the animal models of HD via different approaches

depending on their molecular size and charge character. The binding domain in

mutant htt could be located at the amino acid sequences upstream or downstream of

the expanded polyglutamine tract or in the middle of the polyglutamine tract. If the

polyglutamine domain is responsible for binding to CaM, the therapeutic strategy that

targets CaM-htt interaction could be applied to the other eight polyglutamine repeat

diseases as well.

CONCLUSIONS

TGase-catalyzed transamidation and activation of the small G protein Rac1 in

152

A1A1v cells is regulated by 5-HT2A receptor signaling and Ca2+/CaM (Figure 25).

Active Rac1 has various physiological functions in neuronal cells, including neurite

outgrowth and neuronal differentiation, via modulating the activity of diverse effector

proteins (Leeuwen et al., 1997, Pan et al., 2005). Pathologically, TGase-modification

of htt is regulated by the association of htt with CaM, which may be involved in the

formation and stabilization of htt-aggregates and play a role in the pathogenesis of

HD. Direct inhibition of TGase or CaM may be able to suppress TGase- and

htt-mediated neuropathological perturbations, but also will interrupt the normal

physiological functions of TGase and CaM because both enzymes regulate a variety

of effectors including kinases and cytoskeletal proteins. Alternatively, specific

disruption of the interaction of CaM with mutant htt, by a competitive

CaM-fragement, is a useful tool to determine the pathogenic mechanisms involved in

HD and can be used to develop new therapeutic strategies for HD (Figure 25). Our in

vivo study demonstrated that striatal expression of the CaM-fragment significantly

improved motor function and neuropathological perturbations in HD mice. Moreover,

TGase and CaM activity were not altered by the CaM-fragment in either HD or WT

mice, suggesting that disrupting CaM-htt interaction is an important new therapeutic

strategy for HD.

153

Figure 25. Regulation of TGase by 5-HT2A receptor signaling and CaM. In A1A1v

cells, upon stimulation of Gq/11 protein-coupled 5-HT2A receptor, the phosphatidyl

inositol pathway was activated, resulting in PLC-mediated hydrolysis of PIP2 to IP3,

which in turn releases Ca2+ from ER stores. A rise of cytoplasmic Ca2+ is sufficient to

activate TGase, which catalyzed the transamidation of Rac1 to bioamines, such as

serotonin transported into the cytoplasm by SERT. CaM, another Ca2+-dependent

enzyme, also contributed to Rac1 transamidation by positive regulation of TGase. In

HD cells, mutant htt associated with CaM at a specific binding domain, and htt were

modified by TGase. This modification may stabilize htt monomers or polymers and

contribute to cytotoxicity and formation of htt-containing aggregates. CaM-fragment

may disrupt CaM-htt interaction by competing with endogenous CaM, resulting in

154

decreased TGase-modified htt and aggregates. Abbreviations: 5-HT2AR, 5-HT2A

receptor; CaM, calmodulin; ER, endoplasmic reticulum; PLC, phospholipase C; PIP2,

phophatidyl inositol-1,4-bisphosphate; IP3, inositol-1,4,5-triphosphate; SERT,

serotonin transporter; TGase, transglutaminase; αq/11, , Gq/11 protein α subunits

155

155

REFERENCES LIST

Achyuthan KE, Greenberg CS (Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase. Role of GTP and calcium ions in modulating activity. J Biol Chem 262:1901-1906.1987).

Aeschlimann D, Thomazy V (Protein crosslinking in assembly and remodelling of extracellular matrices: the role of transglutaminases. Connect Tissue Res 41:1-27.2000).

Aghajanian GK, Marek GJ (Serotonin, via 5-HT2A receptors, increases EPSCs in layer V pyramidal cells of prefrontal cortex by an asynchronous mode of glutamate release. Brain Res 825:161-171.1999).

Andringa G, Lam KY, Chegary M, Wang X, Chase TN, Bennett MC (Tissue transglutaminase catalyzes the formation of alpha-synuclein crosslinks in Parkinson's disease. Faseb J 18:932-934.2004).

Appelt DM, Balin BJ (The association of tissue transglutaminase with human recombinant tau results in the formation of insoluble filamentous structures. Brain Res 745:21-31.1997).

Appelt DM, Kopen GC, Boyne LJ, Balin BJ (Localization of transglutaminase in hippocampal neurons: implications for Alzheimer's disease. J Histochem Cytochem 44:1421-1427.1996).

Arango V, Ernsberger P, Marzuk PM, Chen JS, Tierney H, Stanley M, Reis DJ, Mann JJ (Autoradiographic demonstration of increased serotonin 5-HT2 and beta-adrenergic receptor binding sites in the brain of suicide victims. Arch Gen Psychiatry 47:1038-1047.1990).

Arora RC, Meltzer HY (Serotonin2 (5-HT2) receptor binding in the frontal cortex of schizophrenic patients. J Neural Transm Gen Sect 85:19-29.1991).

Arora RC, Meltzer HY (Effect of clozapine treatment on serotonin-2-receptor binding in the blood platelets of schizophrenic patients. Neuropsychopharmacology 10:109-114.1994).

Asano M, Hidaka H (1984) Biopharmacological properties of naphthalenesulfonamides as potent calmodulin antagonists. In: Calcium and Cell Function, vol. 5 (Cheung, W. Y., ed), pp 123–164 New York: Academic Press.

Aznar S, Lacal JC (Rho signals to cell growth and apoptosis. Cancer Lett 165:1-10.2001).

Babu YS, Sack JS, Greenhough TJ, Bugg CE, Means AR, Cook WJ (Three-dimensional structure of calmodulin. Nature 315:37-40.1985).

156

Bachs O, Agell N, Carafoli E (Calmodulin and calmodulin-binding proteins in the nucleus. Cell Calcium 16:289-296.1994).

Baimbridge KG, Celio MR, Rogers JH (Calcium-binding proteins in the nervous system. Trends Neurosci 15:303-308.1992).

Balshaw DM, Xu L, Yamaguchi N, Pasek DA, Meissner G (Calmodulin binding and inhibition of cardiac muscle calcium release channel (ryanodine receptor). J Biol Chem 276:20144-20153.2001).

Bao J, Sharp AH, Wagster MV, Becher M, Schilling G, Ross CA, Dawson VL, Dawson TM (Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin. Proc Natl Acad Sci U S A 93:5037-5042.1996).

Barr AN, Fischer JH, Koller WC, Spunt AL, Singhal A (Serum haloperidol concentration and choreiform movements in Huntington's disease. Neurology 38:84-88.1988).

Bates G (Huntingtin aggregation and toxicity in Huntington's disease. Lancet 361:1642-1644.2003).

Beal MF, Ferrante RJ (Experimental therapeutics in transgenic mouse models of Huntington's disease. Nat Rev Neurosci 5:373-384.2004).

Belmonte MA, Santos MF, Kihara AH, Yan CY, Hamassaki DE (Light-Induced photoreceptor degeneration in the mouse involves activation of the small GTPase Rac1. Invest Ophthalmol Vis Sci 47:1193-1200.2006).

Beninati S, Piacentini M (The transglutaminase family: an overview: minireview article. Amino Acids 26:367-372.2004).

Berg KA, Clarke WP, Chen Y, Ebersole BJ, McKay RD, Maayani S (5-Hydroxytryptamine type 2A receptors regulate cyclic AMP accumulation in a neuronal cell line by protein kinase C-dependent and calcium/calmodulin-dependent mechanisms. Mol Pharmacol 45:826-836.1994a).

Berg KA, Clarke WP, Sailstad C, Saltzman A, Maayani S (Signal transduction differences between 5-hydroxytryptamine type 2A and type 2C receptor systems. Mol Pharmacol 46:477-484.1994b).

Bergamini CM (GTP modulates calcium binding and cation-induced conformational changes in erythrocyte transglutaminase. FEBS Lett 239:255-258.1988).

Bergamini CM, Signorini M, Poltronieri L (Inhibition of erythrocyte transglutaminase by GTP. Biochim Biophys Acta 916:149-151.1987).

Bernassola F, Federici M, Corazzari M, Terrinoni A, Hribal ML, De Laurenzi V, Ranalli M, Massa O,

157

Sesti G, McLean WH, Citro G, Barbetti F, Melino G (Role of transglutaminase 2 in glucose tolerance: knockout mice studies and a putative mutation in a MODY patient. FASEB J 16:1371-1378.2002).

Bernassola F, Rossi A, Melino G (Regulation of transglutaminases by nitric oxide. Ann N Y Acad Sci 887:83-91.1999).

Billett HH, Puszkin EG (The red cell membrane contains calmodulin-regulated crosslinking and proteolytic activity. Hematol Pathol 5:185-193.1991).

Bishop AL, Hall A (Rho GTPases and their effector proteins. Biochem J 348 Pt 2:241-255.2000).

Boado RJ, Pardridge WM (Comparison of Blood-Brain Barrier Transport of GDNF and an IgG-GDNF Fusion Protein in the Rhesus Monkey. Drug Metab Dispos.2009).

Boado RJ, Zhang Y, Pardridge WM (Genetic engineering, expression, and activity of a fusion protein of a human neurotrophin and a molecular Trojan horse for delivery across the human blood-brain barrier. Biotechnol Bioeng 97:1376-1386.2007).

Boado RJ, Zhang Y, Wang Y, Pardridge WM (GDNF fusion protein for targeted-drug delivery across the human blood-brain barrier. Biotechnol Bioeng 100:387-396.2008).

Boess FG, Martin IL (Molecular biology of 5-HT receptors. Neuropharmacology 33:275-317.1994).

Bonelli RM, Aschoff A, Niederwieser G, Heuberger C, Jirikowski G (Cerebrospinal fluid tissue transglutaminase as a biochemical marker for Alzheimer's disease. Neurobiol Dis 11:106-110.2002).

Bosco EE, Mulloy JC, Zheng Y (Rac1 GTPase: a "Rac" of all trades. Cell Mol Life Sci 66:370-374.2009).

Bourguignon LY, Zhu H, Shao L, Chen YW (CD44 interaction with tiam1 promotes Rac1 signaling and hyaluronic acid-mediated breast tumor cell migration. J Biol Chem 275:1829-1838.2000).

Brown MD, Bry L, Li Z, Sacks DB (IQGAP1 regulates Salmonella invasion through interactions with actin, Rac1, and Cdc42. J Biol Chem 282:30265-30272.2007).

Burgoyne RD, Weiss JL (The neuronal calcium sensor family of Ca2+-binding proteins. Biochem J 353:1-12.2001).

Burridge K, Wennerberg K (Rho and Rac take center stage. Cell 116:167-179.2004).

Caine ED, Shoulson I (Psychiatric syndromes in Huntington's disease. Am J Psychiatry 140:728-733.1983).

158

Cariello L, de Cristofaro T, Zanetti L, Cuomo T, Di Maio L, Campanella G, Rinaldi S, Zanetti P, Di Lauro R, Varrone S (Transglutaminase activity is related to CAG repeat length in patients with Huntington's disease. Hum Genet 98:633-635.1996).

Cariello L, Nelson L (Transglutaminase inhibitors, calmodulin antagonists, and calcium channel blockers: Influence on Arbacia sperm motility. Gamete Research 11:261-270.1985).

Carpenter LL, Yasmin S, Price LH (A double-blind, placebo-controlled study of antidepressant augmentation with mirtazapine. Biol Psychiatry 51:183-188.2002).

Carter RJ, Lione LA, Humby T, Mangiarini L, Mahal A, Bates GP, Dunnett SB, Morton AJ (Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation. J Neurosci 19:3248-3257.1999).

Casadio R, Polverini E, Mariani P, Spinozzi F, Carsughi F, Fontana A, Polverino de Laureto P, Matteucci G, Bergamini CM (The structural basis for the regulation of tissue transglutaminase by calcium ions. Eur J Biochem 262:672-679.1999).

Cattaneo E, Zuccato C, Tartari M (Normal huntingtin function: an alternative approach to Huntington's disease. Nat Rev Neurosci 6:919-930.2005).

Chen R, Gao B, Huang C, Olsen B, Rotundo RF, Blumenstock F, Saba TM (Transglutaminase-mediated fibronectin multimerization in lung endothelial matrix in response to TNF-alpha. Am J Physiol Lung Cell Mol Physiol 279:L161-174.2000).

Cheung WY (Role of calmodulin in brain function. Prog Brain Res 56:237-253.1982).

Chin D, Means AR (Calmodulin: a prototypical calcium sensor. Trends Cell Biol 10:322-328.2000).

Chopra V, Fox JH, Lieberman G, Dorsey K, Matson W, Waldmeier P, Housman DE, Kazantsev A, Young AB, Hersch S (A small-molecule therapeutic lead for Huntington's disease: preclinical pharmacology and efficacy of C2-8 in the R6/2 transgenic mouse. Proc Natl Acad Sci U S A 104:16685-16689.2007).

Citron BA, Suo Z, SantaCruz K, Davies PJ, Qin F, Festoff BW (Protein crosslinking, tissue transglutaminase, alternative splicing and neurodegeneration. Neurochemistry international 40:69-78.2002).

Clerk A, Pham FH, Fuller SJ, Sahai E, Aktories K, Marais R, Marshall C, Sugden PH (Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1. Mol Cell Biol 21:1173-1184.2001).

Cohen ML, Fuller RW, Wiley KS (Evidence for 5-HT2 receptors mediating contraction in vascular smooth muscle. J Pharmacol Exp Ther 218:421-425.1981).

159

Conn PJ, Sanders-Bush E (Selective 5HT-2 antagonists inhibit serotonin stimulated phosphatidylinositol metabolism in cerebral cortex. Neuropharmacology 23:993-996.1984).

Cooper AJ, Sheu KF, Burke JR, Strittmatter WJ, Gentile V, Peluso G, Blass JP (Pathogenesis of inclusion bodies in (CAG)n/Qn-expansion diseases with special reference to the role of tissue transglutaminase and to selective vulnerability. J Neurochem 72:889-899.1999).

Cornea-Hebert V, Riad M, Wu C, Singh SK, Descarries L (Cellular and subcellular distribution of the serotonin 5-HT2A receptor in the central nervous system of adult rat. J Comp Neurol 409:187-209.1999).

Craske M, Takeo T, Gerasimenko O, Vaillant C, Torok K, Petersen OH, Tepikin AV (Hormone-induced secretory and nuclear translocation of calmodulin: oscillations of calmodulin concentration with the nucleus as an integrator. Proc Natl Acad Sci U S A 96:4426-4431.1999).

Cyr M, Caron MG, Johnson GA, Laakso A (Magnetic resonance imaging at microscopic resolution reveals subtle morphological changes in a mouse model of dopaminergic hyperfunction. Neuroimage 26:83-90.2005).

Dai Y, Dudek NL, Li Q, Fowler SC, Muma NA (Striatal expression of a calmodulin fragment improved motor function, weight loss, and neuropathology in the R6/2 mouse model of Huntington's disease. J Neurosci 29:11550-11559.2009).

Dai Y, Dudek NL, Patel TB, Muma NA (Transglutaminase-catalyzed transamidation: a novel mechanism for Rac1 activation by 5-hydroxytryptamine2A receptor stimulation. J Pharmacol Exp Ther 326:153-162.2008).

Dale GL, Friese P, Batar P, Hamilton SF, Reed GL, Jackson KW, Clemetson KJ, Alberio L (Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface. Nature 415:175-179.2002).

Davies SW, Turmaine M, Cozens BA, DiFiglia M, Sharp AH, Ross CA, Scherzinger E, Wanker EE, Mangiarini L, Bates GP (Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell 90:537-548.1997).

de Chaffoy de Courcelles D, Leysen JE, De Clerck F, Van Belle H, Janssen PA (Evidence that phospholipid turnover is the signal transducing system coupled to serotonin-S2 receptor sites. J Biol Chem 260:7603-7608.1985).

de Chaffoy de Courcelles D, Roevens P, Wynants J, Van Belle H (Serotonin-induced alterations in inositol phospholipid metabolism in human platelets. Biochim Biophys Acta 927:291-302.1987).

De Laurenzi V, Melino G (Gene disruption of tissue transglutaminase. Mol Cell Biol

160

21:148-155.2001).

Dean B (The cortical serotonin2A receptor and the pathology of schizophrenia: a likely accomplice. J Neurochem 85:1-13.2003).

Dean B, Hayes W (Decreased frontal cortical serotonin2A receptors in schizophrenia. Schizophr Res 21:133-139.1996).

Dedeoglu A, Kubilus JK, Jeitner TM, Matson SA, Bogdanov M, Kowall NW, Matson WR, Cooper AJ, Ratan RR, Beal MF, Hersch SM, Ferrante RJ (Therapeutic effects of cystamine in a murine model of Huntington's disease. J Neurosci 22:8942-8950.2002).

Deisseroth K, Heist EK, Tsien RW (Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons. Nature 392:198-202.1998).

Deshpande SS, Angkeow P, Huang J, Ozaki M, Irani K (Rac1 inhibits TNF-alpha-induced endothelial cell apoptosis: dual regulation by reactive oxygen species. FASEB J 14:1705-1714.2000).

Didsbury J, Weber RF, Bokoch GM, Evans T, Snyderman R (rac, a novel ras-related family of proteins that are botulinum toxin substrates. J Biol Chem 264:16378-16382.1989).

DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, Aronin N (Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science 277:1990-1993.1997).

Doye A, Mettouchi A, Bossis G, Clement R, Buisson-Touati C, Flatau G, Gagnoux L, Piechaczyk M, Boquet P, Lemichez E (CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion. Cell 111:553-564.2002).

Dragatsis I, Levine MS, Zeitlin S (Inactivation of Hdh in the brain and testis results in progressive neurodegeneration and sterility in mice. Nat Genet 26:300-306.2000).

Dudek NL, Dai Y, Muma NA (Protective effects of interrupting the binding of calmodulin to mutant huntingtin. J Neuropathol Exp Neurol 67:355-365.2008).

Dudek NL, Dai Y, Muma NA (Neuroprotective Effects of Calmodulin Peptide 76-121aa: Disruption of Calmodulin Binding to Mutant Huntingtin. Brain Pathol.2009).

Dudek SM, Johnson GV (Transglutaminase catalyzes the formation of sodium dodecyl sulfate-insoluble, Alz-50-reactive polymers of tau. J Neurochem 61:1159-1162.1993).

Duyao M, Ambrose C, Myers R, Novelletto A, Persichetti F, Frontali M, Folstein S, Ross C, Franz M, Abbott M, et al. (Trinucleotide repeat length instability and age of onset in Huntington's disease. Nat Genet 4:387-392.1993).

161

Duyao MP, Auerbach AB, Ryan A, Persichetti F, Barnes GT, McNeil SM, Ge P, Vonsattel JP, Gusella JF, Joyner AL, et al. (Inactivation of the mouse Huntington's disease gene homolog Hdh. Science 269:407-410.1995).

Eastwood SL, Burnet PW, Gittins R, Baker K, Harrison PJ (Expression of serotonin 5-HT(2A) receptors in the human cerebellum and alterations in schizophrenia. Synapse 42:104-114.2001).

Eberle-Wang K, Braun BT, Simansky KJ (Serotonin contracts the isolated rat pylorus via a 5-HT2-like receptor. Am J Physiol 266:R284-291.1994).

Embade N, Valeron PF, Aznar S, Lopez-Collazo E, Lacal JC (Apoptosis induced by Rac GTPase correlates with induction of FasL and ceramides production. Mol Biol Cell 11:4347-4358.2000).

Etienne-Manneville S, Hall A (Rho GTPases in cell biology. Nature 420:629-635.2002).

Felder CC, Kanterman RY, Ma AL, Axelrod J (Serotonin stimulates phospholipase A2 and the release of arachidonic acid in hippocampal neurons by a type 2 serotonin receptor that is independent of inositolphospholipid hydrolysis. Proc Natl Acad Sci U S A 87:2187-2191.1990).

Ferrante RJ, Andreassen OA, Dedeoglu A, Ferrante KL, Jenkins BG, Hersch SM, Beal MF (Therapeutic effects of coenzyme Q10 and remacemide in transgenic mouse models of Huntington's disease. J Neurosci 22:1592-1599.2002).

Ferrante RJ, Andreassen OA, Jenkins BG, Dedeoglu A, Kuemmerle S, Kubilus JK, Kaddurah-Daouk R, Hersch SM, Beal MF (Neuroprotective effects of creatine in a transgenic mouse model of Huntington's disease. J Neurosci 20:4389-4397.2000).

Fesus L, Horvath A, Harsfalvi J (Interaction between tissue transglutaminase and phospholipid vesicles. FEBS Lett 155:1-5.1983).

Flatau G, Lemichez E, Gauthier M, Chardin P, Paris S, Fiorentini C, Boquet P (Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature 387:729-733.1997).

Fleming IN, Elliott CM, Exton JH (Phospholipase C-gamma, protein kinase C and Ca2+/calmodulin-dependent protein kinase II are involved in platelet-derived growth factor-induced phosphorylation of Tiam1. FEBS Lett 429:229-233.1998).

Folk JE (Mechanism and basis for specificity of transglutaminase-catalyzed epsilon-(gamma-glutamyl) lysine bond formation. Adv Enzymol Relat Areas Mol Biol 54:1-56.1983).

Folk JE, Finlayson JS (The epsilon-(gamma-glutamyl)lysine crosslink and the catalytic role of transglutaminases. Adv Protein Chem 31:1-133.1977).

162

Folk JE, Park MH, Chung SI, Schrode J, Lester EP, Cooper HL (Polyamines as physiological substrates for transglutaminases. J Biol Chem 255:3695-3700.1980).

Fowler SC, Birkestrand BR, Chen R, Moss SJ, Vorontsova E, Wang G, Zarcone TJ (A force-plate actometer for quantitating rodent behaviors: illustrative data on locomotion, rotation, spatial patterning, stereotypies, and tremor. J Neurosci Methods 107:107-124.2001).

Fujimoto M, Kanzaki H, Nakayama H, Higuchi T, Hatayama H, Iwai M, Kaneko Y, Mori T, Fujita J (Requirement for transglutaminase in progesterone-induced decidualization of human endometrial stromal cells. Endocrinology 137:1096-1101.1996).

Fusco FR, Chen Q, Lamoreaux WJ, Figueredo-Cardenas G, Jiao Y, Coffman JA, Surmeier DJ, Honig MG, Carlock LR, Reiner A (Cellular localization of huntingtin in striatal and cortical neurons in rats: lack of correlation with neuronal vulnerability in Huntington's disease. J Neurosci 19:1189-1202.1999).

Gaddum JH, Picarelli ZP (Two kinds of tryptamine receptor. Br J Pharmacol Chemother 12:323-328.1957).

Garnovskaya MN, Nebigil CG, Arthur JM, Spurney RF, Raymond JR (5-Hydroxytryptamine2A receptors expressed in rat renal mesangial cells inhibit cyclic AMP accumulation. Mol Pharmacol 48:230-237.1995).

Gentile V, Sepe C, Calvani M, Melone MA, Cotrufo R, Cooper AJ, Blass JP, Peluso G (Tissue transglutaminase-catalyzed formation of high-molecular-weight aggregates in vitro is favored with long polyglutamine domains: a possible mechanism contributing to CAG-triplet diseases. Arch Biochem Biophys 352:314-321.1998).

Girotti F, Carella F, Scigliano G, Grassi MP, Soliveri P, Giovannini P, Parati E, Caraceni T (Effect of neuroleptic treatment on involuntary movements and motor performances in Huntington's disease. J Neurol Neurosurg Psychiatry 47:848-852.1984).

Graham RK, Deng Y, Slow EJ, Haigh B, Bissada N, Lu G, Pearson J, Shehadeh J, Bertram L, Murphy Z, Warby SC, Doty CN, Roy S, Wellington CL, Leavitt BR, Raymond LA, Nicholson DW, Hayden MR (Cleavage at the caspase-6 site is required for neuronal dysfunction and degeneration due to mutant huntingtin. Cell 125:1179-1191.2006).

Grailhe R, Waeber C, Dulawa SC, Hornung JP, Zhuang X, Brunner D, Geyer MA, Hen R (Increased exploratory activity and altered response to LSD in mice lacking the 5-HT(5A) receptor. Neuron 22:581-591.1999).

Gray M, Shirasaki DI, Cepeda C, Andre VM, Wilburn B, Lu XH, Tao J, Yamazaki I, Li SH, Sun YE, Li XJ, Levine MS, Yang XW (Full-length human mutant huntingtin with a stable polyglutamine repeat can elicit progressive and selective neuropathogenesis in BACHD mice. J Neurosci

163

28:6182-6195.2008).

Greenberg CS, Birckbichler PJ, Rice RH (Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J 5:3071-3077.1991).

Grenard P, Bates MK, Aeschlimann D (Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15. Structure of the gene encoding transglutaminase X and a novel gene family member, transglutaminase Z. J Biol Chem 276:33066-33078.2001).

Griffin M, Casadio R, Bergamini CM (Transglutaminases: nature's biological glues. Biochem J 368:377-396.2002).

Guillet-Deniau I, Burnol AF, Girard J (Identification and localization of a skeletal muscle secrotonin 5-HT2A receptor coupled to the Jak/STAT pathway. J Biol Chem 272:14825-14829.1997).

Guilluy C, Rolli-Derkinderen M, Tharaux PL, Melino G, Pacaud P, Loirand G (Transglutaminase-dependent RhoA activation and depletion by serotonin in vascular smooth muscle cells. J Biol Chem 282:2918-2928.2007).

Gusella JF, MacDonald ME (Molecular genetics: unmasking polyglutamine triggers in neurodegenerative disease. Nat Rev Neurosci 1:109-115.2000).

Hagberg GB, Blomstrand F, Nilsson M, Tamir H, Hansson E (Stimulation of 5-HT2A receptors on astrocytes in primary culture opens voltage-independent Ca2+ channels. Neurochemistry international 32:153-162.1998).

Hahn K, DeBiasio R, Taylor DL (Patterns of elevated free calcium and calmodulin activation in living cells. Nature 359:736-738.1992).

Hakoshima T, Shimizu T, Maesaki R (Structural basis of the Rho GTPase signaling. J Biochem (Tokyo) 134:327-331.2003).

Hampton TG, Stasko MR, Kale A, Amende I, Costa AC (Gait dynamics in trisomic mice: quantitative neurological traits of Down syndrome. Physiol Behav 82:381-389.2004).

Hand D, Bungay PJ, Elliott BM, Griffin M (Activation of transglutaminase at calcium levels consistent with a role for this enzyme as a calcium receptor protein. Biosci Rep 5:1079-1086.1985).

Hand D, Perry MJ, Haynes LW (Cellular transglutaminases in neural development. Int J Dev Neurosci 11:709-720.1993).

Handley OJ, Naji JJ, Dunnett SB, Rosser AE (Pharmaceutical, cellular and genetic therapies for Huntington's disease. Clin Sci (Lond) 110:73-88.2006).

164

Harper PS (1991) Huntington’s Disease London: W.B. Saunders.

Harper SQ, Staber PD, He X, Eliason SL, Martins IH, Mao Q, Yang L, Kotin RM, Paulson HL, Davidson BL (RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model. Proc Natl Acad Sci U S A 102:5820-5825.2005).

Harrington AW, Kim JY, Yoon SO (Activation of Rac GTPase by p75 is necessary for c-jun N-terminal kinase-mediated apoptosis. J Neurosci 22:156-166.2002).

Hart MJ, Jiang X, Kozasa T, Roscoe W, Singer WD, Gilman AG, Sternweis PC, Bollag G (Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13. Science 280:2112-2114.1998).

Hemrick-Luecke SK, Evans DC (Comparison of the potency of MDL 100,907 and SB 242084 in blocking the serotonin (5-HT)(2) receptor agonist-induced increases in rat serum corticosterone concentrations: evidence for 5-HT(2A) receptor mediation of the HPA axis. Neuropharmacology 42:162-169.2002).

Hernandez I, Sokolov BP (Abnormalities in 5-HT2A receptor mRNA expression in frontal cortex of chronic elderly schizophrenics with varying histories of neuroleptic treatment. J Neurosci Res 59:218-225.2000).

Hersch SM, Ferrante RJ (Translating therapies for Huntington's disease from genetic animal models to clinical trials. NeuroRx 1:298-306.2004).

Hershenson MB, Chao TS, Abe MK, Gomes I, Kelleher MD, Solway J, Rosner MR (Histamine antagonizes serotonin and growth factor-induced mitogen-activated protein kinase activation in bovine tracheal smooth muscle cells. J Biol Chem 270:19908-19913.1995).

Hodgson JG, Agopyan N, Gutekunst CA, Leavitt BR, LePiane F, Singaraja R, Smith DJ, Bissada N, McCutcheon K, Nasir J, Jamot L, Li XJ, Stevens ME, Rosemond E, Roder JC, Phillips AG, Rubin EM, Hersch SM, Hayden MR (A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration. Neuron 23:181-192.1999).

Hoeflich KP, Ikura M (Calmodulin in action: diversity in target recognition and activation mechanisms. Cell 108:739-742.2002).

Hoogeveen AT, Willemsen R, Meyer N, de Rooij KE, Roos RA, van Ommen GJ, Galjaard H (Characterization and localization of the Huntington disease gene product. Hum Mol Genet 2:2069-2073.1993).

Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR, Humphrey PP (International Union of Pharmacology classification of receptors for 5-hydroxytryptamine

165

(Serotonin). Pharmacol Rev 46:157-203.1994).

Hoyer D, Hannon JP, Martin GR (Molecular, pharmacological and functional diversity of 5-HT receptors. Pharmacol Biochem Behav 71:533-554.2002).

Hoyer D, Pazos A, Probst A, Palacios JM (Serotonin receptors in the human brain. II. Characterization and autoradiographic localization of 5-HT1C and 5-HT2 recognition sites. Brain Res 376:97-107.1986).

Hrdina PD, Demeter E, Vu TB, Sotonyi P, Palkovits M (5-HT uptake sites and 5-HT2 receptors in brain of antidepressant-free suicide victims/depressives: increase in 5-HT2 sites in cortex and amygdala. Brain Res 614:37-44.1993).

Humphrey PP, Hartig P, Hoyer D (A proposed new nomenclature for 5-HT receptors. Trends Pharmacol Sci 14:233-236.1993).

Huntington’s Disease Collaborative Research Group (A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell 72:971-983.1993).

Ientile R, Campisi A, Raciti G, Caccamo D, Curro M, Cannavo G, Li Volti G, Macaione S, Vanella A (Cystamine inhibits transglutaminase and caspase-3 cleavage in glutamate-exposed astroglial cells. J Neurosci Res 74:52-59.2003).

Ihara K, Muraguchi S, Kato M, Shimizu T, Shirakawa M, Kuroda S, Kaibuchi K, Hakoshima T (Crystal structure of human RhoA in a dominantly active form complexed with a GTP analogue. J Biol Chem 273:9656-9666.1998).

Jensen PH, Sorensen ES, Petersen TE, Gliemann J, Rasmussen LK (Residues in the synuclein consensus motif of the alpha-synuclein fragment, NAC, participate in transglutaminase-catalysed cross-linking to Alzheimer-disease amyloid beta A4 peptide. Biochem J 310 ( Pt 1):91-94.1995).

Johnson GV, Cox TM, Lockhart JP, Zinnerman MD, Miller ML, Powers RE (Transglutaminase activity is increased in Alzheimer's disease brain. Brain Res 751:323-329.1997).

Julius D (Molecular biology of serotonin receptors. Annu Rev Neurosci 14:335-360.1991).

Junn E, Ronchetti RD, Quezado MM, Kim SY, Mouradian MM (Tissue transglutaminase-induced aggregation of alpha-synuclein: Implications for Lewy body formation in Parkinson's disease and dementia with Lewy bodies. Proc Natl Acad Sci U S A 100:2047-2052.2003).

Kahlem P, Green H, Djian P (Transglutaminase action imitates Huntington's disease: selective polymerization of Huntingtin containing expanded polyglutamine. Mol Cell 1:595-601.1998).

166

Kalchman MA, Koide HB, McCutcheon K, Graham RK, Nichol K, Nishiyama K, Kazemi-Esfarjani P, Lynn FC, Wellington C, Metzler M, Goldberg YP, Kanazawa I, Gietz RD, Hayden MR (HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nat Genet 16:44-53.1997).

Kanazawa H, Ohsawa K, Sasaki Y, Kohsaka S, Imai Y (Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma -dependent pathway. J Biol Chem 277:20026-20032.2002).

Kaplan JH (Biochemistry of Na,K-ATPase. Annu Rev Biochem 71:511-535.2002).

Karpuj MV, Becher MW, Springer JE, Chabas D, Youssef S, Pedotti R, Mitchell D, Steinman L (Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine. Nat Med 8:143-149.2002a).

Karpuj MV, Becher MW, Steinman L (Evidence for a role for transglutaminase in Huntington's disease and the potential therapeutic implications. Neurochem Int 40:31-36.2002b).

Karpuj MV, Garren H, Slunt H, Price DL, Gusella J, Becher MW, Steinman L (Transglutaminase aggregates huntingtin into nonamyloidogenic polymers, and its enzymatic activity increases in Huntington's disease brain nuclei. Proc Natl Acad Sci U S A 96:7388-7393.1999).

Kazantsev A, Walker HA, Slepko N, Bear JE, Preisinger E, Steffan JS, Zhu YZ, Gertler FB, Housman DE, Marsh JL, Thompson LM (A bivalent Huntingtin binding peptide suppresses polyglutamine aggregation and pathogenesis in Drosophila. Nat Genet 30:367-376.2002).

Keene CD, Rodrigues CM, Eich T, Chhabra MS, Steer CJ, Low WC (Tauroursodeoxycholic acid, a bile acid, is neuroprotective in a transgenic animal model of Huntington's disease. Proc Natl Acad Sci U S A 99:10671-10676.2002).

Kegel KB, Meloni AR, Yi Y, Kim YJ, Doyle E, Cuiffo BG, Sapp E, Wang Y, Qin ZH, Chen JD, Nevins JR, Aronin N, DiFiglia M (Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription. J Biol Chem 277:7466-7476.2002).

Kim SY, Grant P, Lee JH, Pant HC, Steinert PM (Differential expression of multiple transglutaminases in human brain. Increased expression and cross-linking by transglutaminases 1 and 2 in Alzheimer's disease. J Biol Chem 274:30715-30721.1999).

Kozasa T, Jiang X, Hart MJ, Sternweis PM, Singer WD, Gilman AG, Bollag G, Sternweis PC (p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13. Science 280:2109-2111.1998).

167

Kozma R, Sarner S, Ahmed S, Lim L (Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid. Mol Cell Biol 17:1201-1211.1997).

Kurokawa K, Nakamura T, Aoki K, Matsuda M (Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells. Biochem Soc Trans 33:631-634.2005).

Kurrasch-Orbaugh DM, Parrish JC, Watts VJ, Nichols DE (A complex signaling cascade links the serotonin2A receptor to phospholipase A2 activation: the involvement of MAP kinases. J Neurochem 86:980-991.2003).

Lambe EK, Aghajanian GK (The role of Kv1.2-containing potassium channels in serotonin-induced glutamate release from thalamocortical terminals in rat frontal cortex. J Neurosci 21:9955-9963.2001).

Leavitt BR, van Raamsdonk JM, Shehadeh J, Fernandes H, Murphy Z, Graham RK, Wellington CL, Raymond LA, Hayden MR (Wild-type huntingtin protects neurons from excitotoxicity. J Neurochem 96:1121-1129.2006).

Leeuwen FN, Kain HE, Kammen RA, Michiels F, Kranenburg OW, Collard JG (The guanine nucleotide exchange factor Tiam1 affects neuronal morphology; opposing roles for the small GTPases Rac and Rho. J Cell Biol 139:797-807.1997).

Lesort M, Chun W, Johnson GV, Ferrante RJ (Tissue transglutaminase is increased in Huntington's disease brain. J Neurochem 73:2018-2027.1999).

Lesort M, Chun W, Tucholski J, Johnson GV (Does tissue transglutaminase play a role in Huntington's disease? Neurochemistry international 40:37-52.2002).

Li H, Li SH, Johnston H, Shelbourne PF, Li XJ (Amino-terminal fragments of mutant huntingtin show selective accumulation in striatal neurons and synaptic toxicity. Nat Genet 25:385-389.2000).

Li X, Wei XL, Meng LL, Chi MG, Yan JQ, Ma XY, Jia YS, Liang L, Yan HT, Zheng JQ (Involvement of tissue transglutaminase in endothelin 1-induced hypertrophy in cultured neonatal rat cardiomyocytes. Hypertension 54:839-844.2009).

Li XJ, Li SH, Sharp AH, Nucifora FC, Jr., Schilling G, Lanahan A, Worley P, Snyder SH, Ross CA (A huntingtin-associated protein enriched in brain with implications for pathology. Nature 378:398-402.1995).

Liposits Z, Phelix C, Paull WK (Synaptic interaction of serotonergic axons and corticotropin releasing factor (CRF) synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A light and electron microscopic immunocytochemical study. Histochemistry 86:541-549.1987).

168

Liu M, Chen TY, Ahamed B, Li J, Yau KW (Calcium-calmodulin modulation of the olfactory cyclic nucleotide-gated cation channel. Science 266:1348-1354.1994).

Lloret A, Dragileva E, Teed A, Espinola J, Fossale E, Gillis T, Lopez E, Myers RH, MacDonald ME, Wheeler VC (Genetic background modifies nuclear mutant huntingtin accumulation and HD CAG repeat instability in Huntington's disease knock-in mice. Hum Mol Genet 15:2015-2024.2006).

Lorand L (Factor XIII: structure, activation, and interactions with fibrinogen and fibrin. Ann N Y Acad Sci 936:291-311.2001).

Lorand L, Conrad SM (Transglutaminases. Mol Cell Biochem 58:9-35.1984).

Lorand L, Graham RM (Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 4:140-156.2003).

Lorand L, Parameswaran KN, Stenberg P, Tong YS, Velasco PT, Jonsson NA, Mikiver L, Moses P (Specificity of guinea pig liver transglutaminase for amine substrates. Biochemistry 18:1756-1765.1979).

Luby-Phelps K, Hori M, Phelps JM, Won D (Ca(2+)-regulated dynamic compartmentalization of calmodulin in living smooth muscle cells. J Biol Chem 270:21532-21538.1995).

Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, Hetherington C, Lawton M, Trottier Y, Lehrach H, Davies SW, Bates GP (Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell 87:493-506.1996).

Marangell LB, Johnson CR, Kertz B, Zboyan HA, Martinez JM (Olanzapine in the treatment of apathy in previously depressed participants maintained with selective serotonin reuptake inhibitors: an open-label, flexible-dose study. J Clin Psychiatry 63:391-395.2002).

Marek GJ, Carpenter LL, McDougle CJ, Price LH (Synergistic action of 5-HT2A antagonists and selective serotonin reuptake inhibitors in neuropsychiatric disorders. Neuropsychopharmacology 28:402-412.2003).

Mastroberardino PG, Iannicola C, Nardacci R, Bernassola F, De Laurenzi V, Melino G, Moreno S, Pavone F, Oliverio S, Fesus L, Piacentini M ('Tissue' transglutaminase ablation reduces neuronal death and prolongs survival in a mouse model of Huntington's disease. Cell Death Differ 9:873-880.2002).

Masuda M, Betancourt L, Matsuzawa T, Kashimoto T, Takao T, Shimonishi Y, Horiguchi Y (Activation of rho through a cross-link with polyamines catalyzed by Bordetella dermonecrotizing toxin. Embo J 19:521-530.2000).

169

Matos P, Skaug J, Marques B, Beck S, Verissimo F, Gespach C, Boavida MG, Scherer SW, Jordan P (Small GTPase Rac1: structure, localization, and expression of the human gene. Biochem Biophys Res Commun 277:741-751.2000).

Matozaki T, Nakanishi H, Takai Y (Small G-protein networks: their crosstalk and signal cascades. Cell Signal 12:515-524.2000).

Meltzer HY, Matsubara S, Lee JC (The ratios of serotonin2 and dopamine2 affinities differentiate atypical and typical antipsychotic drugs. Psychopharmacol Bull 25:390-392.1989).

Meneses A (Involvement of 5-HT(2A/2B/2C) receptors on memory formation: simple agonism, antagonism, or inverse agonism? Cell Mol Neurobiol 22:675-688.2002).

Mercure MZ, Ginnan R, Singer HA (CaM kinase II delta2-dependent regulation of vascular smooth muscle cell polarization and migration. Am J Physiol Cell Physiol 294:C1465-1475.2008).

Miller CC, Anderton BH (Transglutaminase and the neuronal cytoskeleton in Alzheimer's disease. J Neurochem 46:1912-1922.1986).

Mintun MA, Sheline YI, Moerlein SM, Vlassenko AG, Huang Y, Snyder AZ (Decreased hippocampal 5-HT2A receptor binding in major depressive disorder: in vivo measurement with [18F]altanserin positron emission tomography. Biol Psychiatry 55:217-224.2004).

Mitchell R, McCulloch D, Lutz E, Johnson M, MacKenzie C, Fennell M, Fink G, Zhou W, Sealfon SC (Rhodopsin-family receptors associate with small G proteins to activate phospholipase D. Nature 392:411-414.1998).

Monsonego A, Friedmann I, Shani Y, Eisenstein M, Schwartz M (GTP-dependent conformational changes associated with the functional switch between Galpha and cross-linking activities in brain-derived tissue transglutaminase. J Mol Biol 282:713-720.1998).

Muma NA (Transglutaminase is linked to neurodegenerative diseases. J Neuropathol Exp Neurol 66:258-263.2007).

Munro P, Flatau G, Doye A, Boyer L, Oregioni O, Mege JL, Landraud L, Lemichez E (Activation and proteasomal degradation of rho GTPases by cytotoxic necrotizing factor-1 elicit a controlled inflammatory response. J Biol Chem 279:35849-35857.2004).

Murga C, Zohar M, Teramoto H, Gutkind JS (Rac1 and RhoG promote cell survival by the activation of PI3K and Akt, independently of their ability to stimulate JNK and NF-kappaB. Oncogene 21:207-216.2002).

Murthy SN, Wilson JH, Lukas TJ, Kuret J, Lorand L (Cross-linking sites of the human tau protein, probed by reactions with human transglutaminase. J Neurochem 71:2607-2614.1998).

170

Nadeau OW, Carlson GM (Zero length conformation-dependent cross-linking of phosphorylase kinase subunits by transglutaminase. J Biol Chem 269:29670-29676.1994).

Nagai Y, Tucker T, Ren H, Kenan DJ, Henderson BS, Keene JD, Strittmatter WJ, Burke JR (Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening. J Biol Chem 275:10437-10442.2000).

Nakaki T, Roth BL, Chuang DM, Costa E (Phasic and tonic components in 5-HT2 receptor-mediated rat aorta contraction: participation of Ca++ channels and phospholipase C. J Pharmacol Exp Ther 234:442-446.1985).

Nanda N, Iismaa SE, Owens WA, Husain A, Mackay F, Graham RM (Targeted inactivation of Gh/tissue transglutaminase II. J Biol Chem 276:20673-20678.2001).

Nasir J, Floresco SB, O'Kusky JR, Diewert VM, Richman JM, Zeisler J, Borowski A, Marth JD, Phillips AG, Hayden MR (Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell 81:811-823.1995).

Nelson DL (5-HT5 receptors. Curr Drug Targets CNS Neurol Disord 3:53-58.2004).

Nishida K, Kaziro Y, Satoh T (Anti-apoptotic function of Rac in hematopoietic cells. Oncogene 18:407-415.1999).

Norlund MA, Lee JM, Zainelli GM, Muma NA (Elevated transglutaminase-induced bonds in PHF tau in Alzheimer's disease. Brain Res 851:154-163.1999).

O'Kusky JR, Nasir J, Cicchetti F, Parent A, Hayden MR (Neuronal degeneration in the basal ganglia and loss of pallido-subthalamic synapses in mice with targeted disruption of the Huntington's disease gene. Brain Res 818:468-479.1999).

Olson MF, Ashworth A, Hall A (An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science 269:1270-1272.1995).

Ootsuka Y, Blessing WW (Thermogenesis in brown adipose tissue: increase by 5-HT2A receptor activation and decrease by 5-HT1A receptor activation in conscious rats. Neurosci Lett 395:170-174.2006).

Ostroff RB, Nelson JC (Risperidone augmentation of selective serotonin reuptake inhibitors in major depression. J Clin Psychiatry 60:256-259.1999).

Ou H, Haendeler J, Aebly MR, Kelly LA, Cholewa BC, Koike G, Kwitek-Black A, Jacob HJ, Berk BC, Miano JM (Retinoic acid-induced tissue transglutaminase and apoptosis in vascular smooth muscle cells. Circ Res 87:881-887.2000).

171

Pan J, Kao YL, Joshi S, Jeetendran S, Dipette D, Singh US (Activation of Rac1 by phosphatidylinositol 3-kinase in vivo: role in activation of mitogen-activated protein kinase (MAPK) pathways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells. J Neurochem 93:571-583.2005).

Pandey GN, Dwivedi Y, Rizavi HS, Ren X, Pandey SC, Pesold C, Roberts RC, Conley RR, Tamminga CA (Higher expression of serotonin 5-HT(2A) receptors in the postmortem brains of teenage suicide victims. Am J Psychiatry 159:419-429.2002).

Pardridge WM (Drug and gene targeting to the brain with molecular Trojan horses. Nat Rev Drug Discov 1:131-139.2002).

Pardridge WM (Blood-brain barrier drug targeting: the future of brain drug development. Mol Interv 3:90-105, 151.2003).

Patel S, Morris SA, Adkins CE, O'Beirne G, Taylor CW (Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: redistribution of calmodulin as a possible means of regulating Ca2+ mobilization. Proc Natl Acad Sci U S A 94:11627-11632.1997).

Pazos A, Probst A, Palacios JM (Serotonin receptors in the human brain--IV. Autoradiographic mapping of serotonin-2 receptors. Neuroscience 21:123-139.1987).

Perutz MF, Johnson T, Suzuki M, Finch JT (Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases. Proc Natl Acad Sci U S A 91:5355-5358.1994).

Peters JA, Malone HM, Lambert JJ (Recent advances in the electrophysiological characterization of 5-HT3 receptors. Trends Pharmacol Sci 13:391-397.1992).

Petersen A, Mani K, Brundin P (Recent advances on the pathogenesis of Huntington's disease. Exp Neurol 157:1-18.1999).

Poenie M, Alderton J, Steinhardt R, Tsien R (Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science 233:886-889.1986).

Puszkin EG, Raghuraman V (Catalytic properties of a calmodulin-regulated transglutaminase from human platelet and chicken gizzard. J Biol Chem 260:16012-16020.1985).

Raymond JR, Mukhin YV, Gelasco A, Turner J, Collinsworth G, Gettys TW, Grewal JS, Garnovskaya MN (Multiplicity of mechanisms of serotonin receptor signal transduction. Pharmacol Ther 92:179-212.2001).

Rigamonti D, Bauer JH, De-Fraja C, Conti L, Sipione S, Sciorati C, Clementi E, Hackam A, Hayden MR, Li Y, Cooper JK, Ross CA, Govoni S, Vincenz C, Cattaneo E (Wild-type huntingtin protects from apoptosis upstream of caspase-3. J Neurosci 20:3705-3713.2000).

172

Rip J, Schenk GJ, de Boer AG (Differential receptor-mediated drug targeting to the diseased brain. Expert Opin Drug Deliv 6:227-237.2009).

Robertson DN, Johnson MS, Moggach LO, Holland PJ, Lutz EM, Mitchell R (Selective interaction of ARF1 with the carboxy-terminal tail domain of the 5-HT2A receptor. Mol Pharmacol 64:1239-1250.2003).

Rodriguez-Lebron E, Denovan-Wright EM, Nash K, Lewin AS, Mandel RJ (Intrastriatal rAAV-mediated delivery of anti-huntingtin shRNAs induces partial reversal of disease progression in R6/1 Huntington's disease transgenic mice. Mol Ther 12:618-633.2005).

Rose DM, Sydlaske AD, Agha-Babakhani A, Johnson K, Terkeltaub R (Transglutaminase 2 limits murine peritoneal acute gout-like inflammation by regulating macrophage clearance of apoptotic neutrophils. Arthritis Rheum 54:3363-3371.2006).

Ross CA, Margolis RL, Becher MW, Wood JD, Engelender S, Cooper JK, Sharp AH (Pathogenesis of neurodegenerative diseases associated with expanded glutamine repeats: new answers, new questions. Prog Brain Res 117:397-419.1998).

Ross CA, Poirier MA (Protein aggregation and neurodegenerative disease. Nat Med 10 Suppl:S10-17.2004).

Roth BL (Multiple serotonin receptors: clinical and experimental aspects. Ann Clin Psychiatry 6:67-78.1994).

Roth BL, Nakaki T, Chuang DM, Costa E (5-Hydroxytryptamine2 receptors coupled to phospholipase C in rat aorta: modulation of phosphoinositide turnover by phorbol ester. J Pharmacol Exp Ther 238:480-485.1986).

Roth BL, Willins DL, Kristiansen K, Kroeze WK (5-Hydroxytryptamine2-family receptors (5-hydroxytryptamine2A, 5-hydroxytryptamine2B, 5-hydroxytryptamine2C): where structure meets function. Pharmacol Ther 79:231-257.1998).

Sanders-Bush E, Fentress H, Hazelwood L (Serotonin 5-ht2 receptors: molecular and genomic diversity. Mol Interv 3:319-330.2003).

Sapp E, Schwarz C, Chase K, Bhide PG, Young AB, Penney J, Vonsattel JP, Aronin N, DiFiglia M (Huntingtin localization in brains of normal and Huntington's disease patients. Ann Neurol 42:604-612.1997).

Sarvari M, Karpati L, Fesus L, Deli L, Muszbek L, Nemes Z (Competitive enzyme-linked immonosorbent assay for N epsilon gamma-glutamyl lysine. Analytical biochemistry 311:187-190.2002).

173

Saudou F, Finkbeiner S, Devys D, Greenberg ME (Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell 95:55-66.1998).

Scalzitti JM, Berg KA, Kratowicz SA, Hensler JG (Regulation of serotonin2A receptor expression by an antisense oligodeoxynucleotide. J Neurochem 71:1457-1463.1998).

Schilling G, Becher MW, Sharp AH, Jinnah HA, Duan K, Kotzuk JA, Slunt HH, Ratovitski T, Cooper JK, Jenkins NA, Copeland NG, Price DL, Ross CA, Borchelt DR (Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum Mol Genet 8:397-407.1999).

Schmidt G, Goehring UM, Schirmer J, Uttenweiler-Joseph S, Wilm M, Lohmann M, Giese A, Schmalzing G, Aktories K (Lysine and polyamines are substrates for transglutamination of Rho by the Bordetella dermonecrotic toxin. Infect Immun 69:7663-7670.2001).

Schmidt G, Sehr P, Wilm M, Selzer J, Mann M, Aktories K (Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature 387:725-729.1997).

Schmidt G, Selzer J, Lerm M, Aktories K (The Rho-deamidating cytotoxic necrotizing factor 1 from Escherichia coli possesses transglutaminase activity. Cysteine 866 and histidine 881 are essential for enzyme activity. J Biol Chem 273:13669-13674.1998).

Selkoe DJ, Abraham C, Ihara Y (Brain transglutaminase: in vitro crosslinking of human neurofilament proteins into insoluble polymers. Proc Natl Acad Sci U S A 79:6070-6074.1982).

Sheline YI, Mintun MA, Barch DM, Wilkins C, Snyder AZ, Moerlein SM (Decreased hippocampal 5-HT(2A) receptor binding in older depressed patients using [18F]altanserin positron emission tomography. Neuropsychopharmacology 29:2235-2241.2004).

Shin DM, Jeon JH, Kim CW, Cho SY, Kwon JC, Lee HJ, Choi KH, Park SC, Kim IG (Cell type-specific activation of intracellular transglutaminase 2 by oxidative stress or ultraviolet irradiation: implications of transglutaminase 2 in age-related cataractogenesis. J Biol Chem 279:15032-15039.2004).

Siefring GE, Jr., Apostol AB, Velasco PT, Lorand L (Enzymatic basis for the Ca2+-induced cross-linking of membrane proteins in intact human erythrocytes. Biochemistry 17:2598-2604.1978).

Singer SM, Zainelli GM, Norlund MA, Lee JM, Muma NA (Transglutaminase bonds in neurofibrillary tangles and paired helical filament tau early in Alzheimer's disease. Neurochem Int 40:17-30.2002).

Singh RK, Dai Y, Staudinger JL, Muma NA (Activation of the JAK-STAT pathway is necessary for

174

desensitization of 5-HT2A receptor-stimulated phospholipase C signalling by olanzapine, clozapine and MDL 100907. Int J Neuropsychopharmacol 12:651-665.2009).

Singh US, Kunar MT, Kao YL, Baker KM (Role of transglutaminase II in retinoic acid-induced activation of RhoA-associated kinase-2. Embo J 20:2413-2423.2001).

Singh US, Pan J, Kao YL, Joshi S, Young KL, Baker KM (Tissue transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells. J Biol Chem 278:391-399.2003).

Sommer C (Serotonin in pain and analgesia: actions in the periphery. Mol Neurobiol 30:117-125.2004).

Soulet C, Gendreau S, Missy K, Benard V, Plantavid M, Payrastre B (Characterisation of Rac activation in thrombin- and collagen-stimulated human blood platelets. FEBS Lett 507:253-258.2001).

Steffan JS, Kazantsev A, Spasic-Boskovic O, Greenwald M, Zhu YZ, Gohler H, Wanker EE, Bates GP, Housman DE, Thompson LM (The Huntington's disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc Natl Acad Sci U S A 97:6763-6768.2000).

Suzuki N, Nakamura S, Mano H, Kozasa T (Galpha 12 activates Rho GTPase through tyrosine-phosphorylated leukemia-associated RhoGEF. Proc Natl Acad Sci U S A 100:733-738.2003).

Swindells MB, Ikura M (Pre-formation of the semi-open conformation by the apo-calmodulin C-terminal domain and implications binding IQ-motifs. Nat Struct Biol 3:501-504.1996).

Szondy Z, Sarang Z, Molnar P, Nemeth T, Piacentini M, Mastroberardino PG, Falasca L, Aeschlimann D, Kovacs J, Kiss I, Szegezdi E, Lakos G, Rajnavolgyi E, Birckbichler PJ, Melino G, Fesus L (Transglutaminase 2-/- mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells. Proc Natl Acad Sci U S A 100:7812-7817.2003).

Tamir H, Hsiung SC, Yu PY, Liu KP, Adlersberg M, Nunez EA, Gershon MD (Serotonergic signalling between thyroid cells: protein kinase C and 5-HT2 receptors in the secretion and action of serotonin. Synapse 12:155-168.1992).

Tang TS, Guo C, Wang H, Chen X, Bezprozvanny I (Neuroprotective effects of inositol 1,4,5-trisphosphate receptor C-terminal fragment in a Huntington's disease mouse model. J Neurosci 29:1257-1266.2009).

Tang TS, Slow E, Lupu V, Stavrovskaya IG, Sugimori M, Llinas R, Kristal BS, Hayden MR, Bezprozvanny I (Disturbed Ca2+ signaling and apoptosis of medium spiny neurons in Huntington's disease. Proc Natl Acad Sci U S A 102:2602-2607.2005).

175

Tang TS, Tu H, Chan EY, Maximov A, Wang Z, Wellington CL, Hayden MR, Bezprozvanny I (Huntingtin and huntingtin-associated protein 1 influence neuronal calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1. Neuron 39:227-239.2003).

Thomas V, Fournet G, Simonet F, Roch AM, Ceylan I, El Alaouia S, Quash G (Definition of the fine specificity of the monoclonal antibody 81D4: its reactivity with lysine and polyamine isopeptide cross-links. Journal of immunological methods 292:83-95.2004).

Tucholski J, Kuret J, Johnson GV (Tau is modified by tissue transglutaminase in situ: possible functional and metabolic effects of polyamination. J Neurochem 73:1871-1880.1999).

Turner JH, Raymond JR (Interaction of calmodulin with the serotonin 5-hydroxytryptamine2A receptor. A putative regulator of G protein coupling and receptor phosphorylation by protein kinase C. J Biol Chem 280:30741-30750.2005).

Udo H, Jin I, Kim JH, Li HL, Youn T, Hawkins RD, Kandel ER, Bailey CH (Serotonin-induced regulation of the actin network for learning-related synaptic growth requires Cdc42, N-WASP, and PAK in Aplysia sensory neurons. Neuron 45:887-901.2005).

Unsworth CD, Molinoff PB (Regulation of the 5-hydroxytryptamine1B receptor in opossum kidney cells after exposure to agonists. Mol Pharmacol 42:464-470.1992).

Van de Kar LD, Javed A, Zhang Y, Serres F, Raap DK, Gray TS (5-HT2A receptors stimulate ACTH, corticosterone, oxytocin, renin, and prolactin release and activate hypothalamic CRF and oxytocin-expressing cells. J Neurosci 21:3572-3579.2001).

van Leeuwen FN, van Delft S, Kain HE, van der Kammen RA, Collard JG (Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading. Nat Cell Biol 1:242-248.1999).

Van Raamsdonk JM, Gibson WT, Pearson J, Murphy Z, Lu G, Leavitt BR, Hayden MR (Body weight is modulated by levels of full-length huntingtin. Hum Mol Genet 15:1513-1523.2006).

Van Raamsdonk JM, Metzler M, Slow E, Pearson J, Schwab C, Carroll J, Graham RK, Leavitt BR, Hayden MR (Phenotypic abnormalities in the YAC128 mouse model of Huntington disease are penetrant on multiple genetic backgrounds and modulated by strain. Neurobiol Dis 26:189-200.2007).

Van Raamsdonk JM, Pearson J, Bailey CD, Rogers DA, Johnson GV, Hayden MR, Leavitt BR (Cystamine treatment is neuroprotective in the YAC128 mouse model of Huntington disease. J Neurochem 95:210-220.2005).

Venkatraman P, Wetzel R, Tanaka M, Nukina N, Goldberg AL (Eukaryotic proteasomes cannot digest polyglutamine sequences and release them during degradation of polyglutamine-containing

176

proteins. Mol Cell 14:95-104.2004).

Vetter IR, Wittinghofer A (The guanine nucleotide-binding switch in three dimensions. Science 294:1299-1304.2001).

von Horsten S, Schmitt I, Nguyen HP, Holzmann C, Schmidt T, Walther T, Bader M, Pabst R, Kobbe P, Krotova J, Stiller D, Kask A, Vaarmann A, Rathke-Hartlieb S, Schulz JB, Grasshoff U, Bauer I, Vieira-Saecker AM, Paul M, Jones L, Lindenberg KS, Landwehrmeyer B, Bauer A, Li XJ, Riess O (Transgenic rat model of Huntington's disease. Hum Mol Genet 12:617-624.2003).

Walker FO (Huntington's disease. Lancet 369:218-228.2007).

Walther DJ, Peter JU, Winter S, Holtje M, Paulmann N, Grohmann M, Vowinckel J, Alamo-Bethencourt V, Wilhelm CS, Ahnert-Hilger G, Bader M (Serotonylation of small GTPases is a signal transduction pathway that triggers platelet alpha-granule release. Cell 115:851-862.2003).

Wanker EE (Protein aggregation and pathogenesis of Huntington's disease: mechanisms and correlations. Biol Chem 381:937-942.2000).

Watts SW (Serotonin activates the mitogen-activated protein kinase pathway in vascular smooth muscle: use of the mitogen-activated protein kinase kinase inhibitor PD098059. J Pharmacol Exp Ther 279:1541-1550.1996).

Watts SW (Activation of the mitogen-activated protein kinase pathway via the 5-HT2A receptor. Ann N Y Acad Sci 861:162-168.1998).

Watts SW, Priestley JR, Thompson JM (Serotonylation of vascular proteins important to contraction. PLoS One 4:e5682.2009).

Weisstaub NV, Zhou M, Lira A, Lambe E, Gonzalez-Maeso J, Hornung JP, Sibille E, Underwood M, Itohara S, Dauer WT, Ansorge MS, Morelli E, Mann JJ, Toth M, Aghajanian G, Sealfon SC, Hen R, Gingrich JA (Cortical 5-HT2A receptor signaling modulates anxiety-like behaviors in mice. Science 313:536-540.2006).

Wellington CL, Ellerby LM, Hackam AS, Margolis RL, Trifiro MA, Singaraja R, McCutcheon K, Salvesen GS, Propp SS, Bromm M, Rowland KJ, Zhang T, Rasper D, Roy S, Thornberry N, Pinsky L, Kakizuka A, Ross CA, Nicholson DW, Bredesen DE, Hayden MR (Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract. J Biol Chem 273:9158-9167.1998).

Wellington CL, Singaraja R, Ellerby L, Savill J, Roy S, Leavitt B, Cattaneo E, Hackam A, Sharp A, Thornberry N, Nicholson DW, Bredesen DE, Hayden MR (Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells. J Biol

177

Chem 275:19831-19838.2000).

Wennerberg K, Der CJ (Rho-family GTPases: it's not only Rac and Rho (and I like it). J Cell Sci 117:1301-1312.2004).

White JK, Auerbach W, Duyao MP, Vonsattel JP, Gusella JF, Joyner AL, MacDonald ME (Huntingtin is required for neurogenesis and is not impaired by the Huntington's disease CAG expansion. Nat Genet 17:404-410.1997).

Wilcox BD, Rydelek-Fitzgerald L, Jeffrey JJ (Regulation of collagenase gene expression by serotonin and progesterone in rat uterine smooth muscle cells. J Biol Chem 267:20752-20757.1992).

Williams GV, Rao SG, Goldman-Rakic PS (The physiological role of 5-HT2A receptors in working memory. J Neurosci 22:2843-2854.2002).

Willins DL, Deutch AY, Roth BL (Serotonin 5-HT2A receptors are expressed on pyramidal cells and interneurons in the rat cortex. Synapse 27:79-82.1997).

Woolley DW, Shaw E (Some neurophysiological aspects of serotonin. Br Med J 2:122-126.1954).

Wright DE, Seroogy KB, Lundgren KH, Davis BM, Jennes L (Comparative localization of serotonin1A, 1C, and 2 receptor subtype mRNAs in rat brain. J Comp Neurol 351:357-373.1995).

Wyttenbach A, Swartz J, Kita H, Thykjaer T, Carmichael J, Bradley J, Brown R, Maxwell M, Schapira A, Orntoft TF, Kato K, Rubinsztein DC (Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington's disease. Hum Mol Genet 10:1829-1845.2001).

Yap KL, Kim J, Truong K, Sherman M, Yuan T, Ikura M (Calmodulin target database. J Struct Funct Genomics 1:8-14.2000).

Zainelli GM, Dudek NL, Ross CA, Kim SY, Muma NA (Mutant huntingtin protein: a substrate for transglutaminase 1, 2, and 3. J Neuropathol Exp Neurol 64:58-65.2005).

Zainelli GM, Ross CA, Troncoso JC, Fitzgerald JK, Muma NA (Calmodulin regulates transglutaminase 2 cross-linking of huntingtin. J Neurosci 24:1954-1961.2004).

Zainelli GM, Ross CA, Troncoso JC, Muma NA (Transglutaminase cross-links in intranuclear inclusions in Huntington disease. J Neuropathol Exp Neurol 62:14-24.2003).

Zeitlin S, Liu JP, Chapman DL, Papaioannou VE, Efstratiadis A (Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue. Nat Genet 11:155-163.1995).

Zemskov EA, Janiak A, Hang J, Waghray A, Belkin AM (The role of tissue transglutaminase in

178

cell-matrix interactions. Front Biosci 11:1057-1076.2006).

Zeron MM, Fernandes HB, Krebs C, Shehadeh J, Wellington CL, Leavitt BR, Baimbridge KG, Hayden MR, Raymond LA (Potentiation of NMDA receptor-mediated excitotoxicity linked with intrinsic apoptotic pathway in YAC transgenic mouse model of Huntington's disease. Mol Cell Neurosci 25:469-479.2004).

Zhang J, Lesort M, Guttmann RP, Johnson GV (Modulation of the in situ activity of tissue transglutaminase by calcium and GTP. J Biol Chem 273:2288-2295.1998).

Zhang Y, Damjanoska KJ, Carrasco GA, Dudas B, D'Souza DN, Tetzlaff J, Garcia F, Hanley NR, Scripathirathan K, Petersen BR, Gray TS, Battaglia G, Muma NA, Van de Kar LD (Evidence that 5-HT2A receptors in the hypothalamic paraventricular nucleus mediate neuroendocrine responses to (-)DOI. J Neurosci 22:9635-9642.2002).

Zhang Y, Li M, Drozda M, Chen M, Ren S, Mejia Sanchez RO, Leavitt BR, Cattaneo E, Ferrante RJ, Hayden MR, Friedlander RM (Depletion of wild-type huntingtin in mouse models of neurologic diseases. J Neurochem 87:101-106.2003).

Zheng G, Dwoskin LP, Crooks PA (Vesicular monoamine transporter 2: role as a novel target for drug development. AAPS J 8:E682-692.2006).

Zhou H, Cao F, Wang Z, Yu ZX, Nguyen HP, Evans J, Li SH, Li XJ (Huntingtin forms toxic NH2-terminal fragment complexes that are promoted by the age-dependent decrease in proteasome activity. J Cell Biol 163:109-118.2003).

Zuccato C, Ciammola A, Rigamonti D, Leavitt BR, Goffredo D, Conti L, MacDonald ME, Friedlander RM, Silani V, Hayden MR, Timmusk T, Sipione S, Cattaneo E (Loss of huntingtin-mediated BDNF gene transcription in Huntington's disease. Science 293:493-498.2001).

179

179

VITA

Ying Dai was born on July 3, 1978 in Hunan, China to Yiquan Dai and

Zhiyang Song. He attended Nanhua University, China, where he earned his

Bachelor of Medicine degree, with highest distinction in 2001. After graduation, Ying

entered the Master’s Program in the Department of Anatomy and Histology at Jinan

University, China, and joined the laboratory of Dr. Yunquan Liu. His project focused

on cerebral ischemia and reperfusion injury. He completed his Master of Medicine in

July, 2004.

In August of 2004, Ying began to pursue a doctorate of philosophy in

Neuroscience Program at Loyola University Chicago. In 2005, Ying joined the

laboratory of Dr. Nancy A. Muma in the Department of Pharmacology and

Experimental Therapeutics, to study the regulation of transglutaminases by 5-HT2A

receptor signaling and calmodulin. In January of 2007, Ying moved to University of

Kansas with his advisor Dr. Muma, and continued his research at the Department of

Pharmacology and Toxicology.

Following the completion of the doctoral program in Neuroscience at Loyola

University Chicago, Ying will continue to pursue his interest in development of new

therapeutic strategies for neurodegenerative diseases.

180

DISSERTATION APPROVAL SHEET

The dissertation submitted by Ying Dai has been read and approved by the following committee: Nancy A. Muma, Ph.D., Director Professor and Chair Department of Pharmacology and Toxicology, University of Kansas George Battaglia, Ph.D. Professor of Pharmacology Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago Thackery Gray, Ph.D. Professor of Anatomy and Associate Dean School of Medicine, American University of the Caribbean Edward J. Neafsey, Ph.D. Professor and Program Director, Neuroscience Program, Loyola University Chicago Karie Scrogin Associate Professor of Pharmacology Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago

The final copies have been examined by the director of the dissertation and the signature which appears below verifies the fact that any necessary changes have been incorporated and that the dissertation is now given final approval by the committee with reference to content and form.

The dissertation is therefore accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

__________________ ____________________________________

Date Director’s Signatur