CELL VIABILITY, PROLIFERATION RATE, AND EXTENSION …

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CANNABINOID RECEPTOR SIGNALING IN HUMAN SH-SY5Y NEUROBLASTOMA

CELL VIABILITY, PROLIFERATION RATE, AND EXTENSION LENGTH

BY

ERICA LYNN LYONS (NEE WIESER)

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Molecular Medicine and Translational Science

May 2019

Winston-Salem, North Carolina

Approved by:

Allyn Howlett, Ph.D., Advisor

Graca Almeida-Porada, Ph.D., Chair

John Parks, Ph.D.

Rong Chen, Ph.D.

Brian Thomas, Ph.D.

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ACKNOWLEDGEMENTS

I would like to begin by thanking my mentor, Dr. Allyn Howlett, for her years of

guidance and support. She was absolutely critical to the design and completion of my work. Her

experience and knowledge and ready availability to discuss everything from technical aspects of

experimental design to big picture ideas about the roles of cannabinoid systems in physiological

systems make her a fantastic teacher. The entire Howlett lab provided me with help and support

and I am so grateful for their constant encouragement. Sandra Kabler, Dr. Khalil Eldeeb, Dr.

Lawrence Blume, Alex Ilyasov, Carolina Burgos-Aguilar, and everyone contributed to a positive

lab environment that encouraged creative thinking and open discussion.

Thank you to my committee members, the people of Molecular and Cellular Biosciences,

Molecular Medicine and Translational Science, Molecular Pathology, Lipid Sciences, and

everyone at the Wake Forest School of Medicine. Your encouragement to advance my studies

has supported me to progress forward. I enjoyed learning from all of you not only in classes and

journal club, but also in a wonderful community of scientist friends who excitedly chat about

cutting edge techniques and experimental design as we pursue our work across multiple campuses.

Everyone was always so open to discuss technical details of protocols or how to best design an

experiment to address a hypothesis, and so willing to collaborate and share equipment. Thank

you to the FACS Core, the Diabetes Center, and the Lipids Sciences group for collaborating with

me on this project.

Finally, thank you to my family and friends for your constant love and encouragement

throughout this experience. I greatly appreciate everyone’s time and effort to support me for

these past years.

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TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENTS…………………………………………………...…………………2

TABLE OF CONTENTS……………………………………………………...……………...….3

LIST OF ABBREVIATIONS………………………………………………………...………….4

LIST OF TABLES………………………………………………………….………...…………11

LIST OF FIGURES………………………………………………………...………....………...12

ABSTRACT………………………………………………………………………...……………15

CHAPTER

I. INTRODUCTION…………………………………………………………………...18

II. EXPERIMENTAL DESIGN AND METHODS……………...…………………..77

III. HUMAN NEUROBLASTOMA SH-SY5Y CELL LINES......………………….87

IV. CANNABINOID RECEPTORS, VIABILITY, AND PROLIFERATION …..109

V. CANNABINOID RECEPTORS AND NEURONAL EXTENSIONS……..…...121

VI. DISCUSSION AND FUTURE PERSPECTIVES…………...………………....136

REFERENCES………………………………………………………………………...157

CURRICULUM VITAE………………………………………………………………178

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LIST OF ABBREVIATIONS

AG arachidonoyl glycerol

2-AG 2-arachidonoyl glycerol

12-HAEA 12-hydroxy-N-arachidonoylethanolamine

12-HETE 12- hydroxyeicosatetraenoic acid

15-HAEA 15-hydroxy-N-arachidonoylethanolamine

15-HETE 15- hydroxyeicosatetraenoic acid

ACEA arachidonyl-2'-chloroethylamide, selective CB1 agonist

ABHD6 alpha/beta-hydrolase domain containing 6

ABHD12 alpha/beta-hydrolase domain containing 12

AC adenylyl cyclase

ADP adenosine diphosphate

AEA anandamide or N-arachidonoylethanolamine

AKT protein kinase B

AM251 N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-

1H-pyrazole-3-carboxamide, a CB1 receptor selective antagonist

AM630 1-[2-(morpholin-4-yl)ethyl]-2-methyl-3-(4-methoxybenzoyl)-6-

iodoindole or 6-Iodopravadoline, CB2 receptor selective antagonist

AMPK 5' adenosine monophosphate-activated protein kinase

ANOVA analysis of variance

β3Tub beta 3 tubulin, also known as Tuj1

BDNF brain-derived neurotrophic factor

BRCA1 breast cancer type 1 susceptibility protein

BrdU bromodeoxyuridine

BSA bovine serum albumin

cAMP cyclic adenosine monophosphate

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CB1R CB1 cannabinoid receptor

CB1XS CB1 cannabinoid receptor stably expressing SH-SY5Y clonal cell line

CB2R CB2 cannabinoid receptor

CB2XS CB2 cannabinoid receptor stably overexpressing SH-SY5Y cell line

cDNA complementary deoxyribonucleic acid

cfos Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog C

CNPase 2',3'-cyclic-nucleotide 3'-phosphodiesterase

CNS central nervous system

COX2 cyclooxygenase 2

CP 55,940 (-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-

hydroxypropyl)cyclohexanol, a dual cannabinoid receptor agonist

CRE cre recombinase enzyme

CRAC cholesterol recognition/interaction amino acid consensus sequence

CRIP1a cannabinoid receptor interacting protein 1a

Ctip2 COUP TF1-interacting protein 2, also known as Bcl11b

DAGL diacylglycerol lipase

DAPI 4',6-diamidino-2-phenylindole

DIAPH1 diaphanous homolog, a rhoA effector molecule

DMEM Dulbecco’s modified Eagle medium

DNA deoxyribonucleic acid

EC50 half maximal (50%) effective concentration

ECS endogenous cannabinoid system

EDTA ethylenediaminetetraacetic acid

EGF epidermal growth factor

ERK extracellular signal regulated kinase

FAAH fatty acid amide hydrolase

6

FACS fluorescence activated cell sorting

FAK focal adhesion kinase

FDA Food and Drug Administration

FTY720 fingolimod

G protein guanine nucleotide binding protein

G418 geneticin

GABA gamma-aminobutyric acid

GAP GTPase activating protein

GAP1m Ras GTPase-activating protein 2 or 1m

GAP43 growth cone associated protein 43

GEF guanine nucleotide exchange factor

GIRK G protein-coupled inwardly-rectifying potassium channels

GDI guanosine nucleotide dissociation inhibitor

GDP guanosine diphosphate

GFAP glial fibrillary acidic protein

GPCR G protein coupled receptor

GPR55 G protein coupled receptor 55

GRK G protein coupled receptor kinase

GTP guanosine triphosphate

GTPase guanosine triphosphate hydrolase

H8 helix 8

HeLa human cervical cancer cell line derived from Henrietta Lacks in 1951

HEK293 293rd human embryonic kidney cell line made by Frank Graham in 1973

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HHT hydroxyheptadecatrienoic acid

HU210 Hebrew University compound 210, dual cannabinoid receptor agonist

7

HU308 Hebrew University compound 308, selective CB2 receptor agonist

ICL intracellular loop

ITGA1 integrin alpha 1, also known as CD49a

JNK c-Jun N-terminal kinase

JTE907 a quinolinecarboxamide compound, CB2 receptor specific antagonist

JWH015 John Huffman compound 015, selective CB2 receptor agonist



JZL184 4-nitrophenyl-4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-

1-carboxylate, an irreversible inhibitor of MAGL

Ki67 antigen KI-67, from Kiel, Germany clone 67

Lox locus of X-over P1, flanks DNA to be cut with CRE

LPA lysophosphatidic acid

LPL-C lysophospholipase C

LY294002 2-morpholin-4-yl-8-phenylchromen-4-one

MAPK mitogen activated protein kinase

MGL monoacylglycerol lipase

MAGL monoacylglycerol lipase

mAEA methanandamide, a stable anandamide analog

Met-F-AEA 2-methyl-2'-F-anandamide, a stable anandamide analog

mRNA messenger ribonucleic acid

MTORC1 mammalian or mechanistic target of rapamycin complex 1

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

N18TG2 mouse neuroblastoma cell line

NAPEPLD N-acyl phosphatidylethanolamine phospholipase D

NCBI National Center for Biotechnology Information

8

NCAM neural cell adhesion molecule

NGF nerve growth factor

NUCCORE NCBI nucleotide reference database

O2050 (6aR,10aR)-1-Hydroxy-3-(1-Methanesulfonylamino-4-hexyn-6-yl)-

6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran, a CB1

receptor specific antagonist

OxoM oxotremorine M

P115 RhoGEF 115 kDa guanine nucleotide exchange factor or rho GEF 1

PAGE polyacrylamide gel electrophoresis

PARP poly (ADP-ribose) polymerase

PBS phosphate buffered saline

PCR polymerase chain reaction

PD98059 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one

PDZ-RhoGEF rho guanine nucleotide exchange factor 11

PGD2 prostaglandin D2

PGE2 prostaglandin E2

PGH2 prostaglandin hydroxy-endoperoxide

PLC phospholipase C

PPAR peroxisome proliferator-activated receptor

Pen/Strep penicillin/streptomycin

PTV piccolo-transport vesicles

PTX pertussis toxin

qPCR quantitative real-time polymerase chain reaction

RA all trans retinoic acid

Rac ras-related C3 botulinum toxin substrate 1

Ral ras related protein ral

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Rap1 ras-proximate-1 or ras-related protein 1

Rap1GAPII RAP1 GTPase activating protein 2

REST relative expression software tool

RhoA ras homolog gene family member A, a small GTPase

RhoGEF rho guanine nucleotide exchange factor

ROCK1 rho-associated coiled-coil containing protein kinase1

RGS regulator of G-protein signaling

S1P sphingosine-1-phosphate

Satb2 special AT-rich sequence-binding protein 2

SCG10 neuron-specific isoform of stathmin 10

SDS sodium dodecyl sulfate

SH-SY5Y clonal human neuroblastoma cell line

siRNA small interfering or silencing ribonucleic acid

SRC proto-oncogene tyrosine-protein kinase

SR141716 5-(4-Chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-

yl)-1H-pyrazole-3-carboxamide, CB1 receptor selective antagonist

SR144528 5-(4-Chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-

[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-1H-pyrazole-3-

carboxamide, CB2 receptor selective antagonist

Stat3 signal transducer and activator of transcription 3

ST8SIA2 ST8 Alpha-N-Acetyl-Neuraminide Alpha-2,8-Sialyltransferase 2 or

Alpha-2,8-sialyltransferase 8B

SYT synaptotagmin 1

THC (−)-trans-Δ⁹-tetrahydrocannabinol

THL tetrahydrolipstatin

TRPV1 transient receptor potential cation channel subfamily V member 1

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TR-WGA Texas red conjugated wheat germ agglutinin

URB597 [3-(3-Carbamoylphenyl)phenyl] N-cyclohexylcarbamate, inhibits fatty

acid amide hydrolase

VAChT vesicular acetylcholine transporter

WB Western blot

WIN 55,212-2 (11R)-2-methyl-11-[(morpholin-4-yl)methyl]-3-(naphthalene-1-

carbonyl)-9-oxa-1-azatricyclo[6.3.1.0⁴,¹²]dodeca-2,4(12),5,7-tetraene,

dual cannabinoid receptor agonist

ZIF 268 zinc finger protein 268 or 225, also known as early growth response

protein 1, nerve growth factor-induced protein A, Krox-24, TIS8, and

ZENK.

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LIST OF TABLES

PAGE

CHAPTER I

CHAPTER II

Table 2.4.1 Primers Designed For This Investigation……………………………...…...…...81

CHAPTER III

CHAPTER IV

CHAPTER V

CHAPTER VI

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LIST OF FIGURES

PAGE

CHAPTER I

Figure 1.3.1 Chemical Structures during Synthesis of Endogenous Cannabinoids…….…….34

Figure 1.3.2 Concept Map of Endogenous Cannabinoid Ligand Production....……………...35

Figure 1.3.3 Literature Values of the Endogenous Cannabinoids 2-AG, AEA in Brain.….....39

Figure 1.6.3 Concept Map of Gα12/13 and Gαi/o Mediated Cannabinoid Signaling ….……63

Figure 1.6.4 Concept Map of Gβγ Mediated Cannabinoid Signaling ….………………....….67

CHAPTER II

CHAPTER III

Figure 3.1 Characterization of CB1 or CB2 Receptor-Expressing Human SH-SY5Y

Neuroblastoma Cell Lines……………………………………………...…...…..92

Figure 3.2 Quantification of Endogenous Cannabinoid Ligands 2-AG and AEA in Parental

SH-SY5Y Cells…………………...………………………..…………........……93

Figure 3.3 Identification of mRNA Targets Responding to Retinoic Acid or Cannabinoid

Receptor Expression.………..……………………………..…………........……94

Figure 3.4 Effect of Cannabinoid Receptor Overexpression on mRNA of Endogenous

Cannabinoid System Component Enzymes…...…………..…………........……95

Figure 3.5 Effect of Cannabinoid Receptor Overexpression on mRNA of Transcription

Factors……….……………………………………………..…………........……96

Figure 3.6 Effect of 7 Day of 20 μM Retinoic Acid (RA) Every Other Day on mRNA of

Endogenous Cannabinoid System Component Enzymes….…………........……97

Figure 3.7 Effect of 7 Days of 20 μM Retinoic Acid (RA) Every Other Day on mRNA of

Proteins Involved in Neurotransmitter Regulation……..…………….......…….98


Proteins Involved in Cytoskeletal Regulation……..……….………........……..99

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Proteins Involved in Cellular Adhesion…………..……….………........…...…100


Transcription Factors……………………………..……….………........…...…101

Figure 3.11 mRNA Targets Not Altered by CB1 Receptor Stable Expression….….........…102

Figure 3.12 mRNA Targets Not Altered by CB2 Receptor Stable Expression….….........…103

Figure 3.13 Feedback Inhibition and Desensitization is Evidence of a Functional Signaling

Pathway……….……………………………………………..…………........…104

Figure 3.14 mRNA Abundance in CB1XS and CB2XS Cell Lines Treated for 7 Days with 10

nM of Dual Cannabinoid Agonist CP-55,940….……………..………….....…106

Figure 3.15 Representative Western Blot of CB1 Receptor Abundance….……………...…107

Figure 3.16 Representative Western Blot of CB2 Receptor Abundance….……………...…108

CHAPTER IV

Figure 4.1 Stable Expression of Cannabinoid Receptors Does not Alter SH-SY5Y Viability

or Promote Apoptosis………………………..………….……………....……..113

Figure 4.2 Stable Expression of Cannabinoid Receptors Does not Alter the Proliferation

Rate of Human SH-SY5Y Neuroblastoma Cells…..…………………………..114

Figure 4.3 Stable Expression of Cannabinoid Receptors Does Not Alter Apoptosis in

Human SH-SY5Y Neuroblastoma Cells……………….…..…………………..116

Figure 4.4 Application of mAEA onto Cells Stably Expressing Cannabinoid Receptors Does

Not Alter Apoptosis in Human SH-SY5Y Neuroblastoma Cells....…………..118

Figure 4.5 Application of mAEA onto Cells Stably Expressing Cannabinoid Receptors Does

Not Alter Cell Number of Human SH-SY5Y Neuroblastoma Cells…………..119

Figure 4.6 Application of 2-AG Ether onto Cells Stably Expressing Cannabinoid Receptors

Does Not Alter Cell Number of Human SH-SY5Y Neuroblastoma Cells...…..120

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CHAPTER V

Figure 5.1 Stable Expression of Cannabinoid Receptors Is Associated With Neurite

Development in SH-SY5Y Cells…….……………...…………………………126

Figure 5.2 Histochemistry of SH-SY5Y Stably Transfected Cell Lines...………………...127

Figure 5.3 Endocannabinoid System Factors Affecting Neurite Extension in CB1XS SH-

SY5Y Cells.……………………...............………………………………….....128

Figure 5.4 Histochemistry of CB1XS SH-SY5Y Stably Transfected Cell Lines.………...129

Figure 5.5 Intracellular Signaling Targets of CB1 Receptor Activation…………...……...130

Figure 5.6 Histochemistry of CB1XS SH-SY5Y Stably Transfected Cell Lines.………...131

Figure 5.7 Histochemistry of CB1XS SH-SY5Y Stably Transfected Cell Lines ………...132

Figure 5.8 Effect of Cannabinoid Receptor Expression on mRNA of Proteins Involved in

Cytoskeletal Regulation………………………………………………...……...133


Neurotransmitter Regulation.…………………………………………...……...134


Cellular Adhesion…….………………………………………………...……...135

CHAPTER VI

Figure 6.1 Cannabinoid Signaling Pathways Affecting Neuronal Extension Length……..152

Figure 6.2 Effect of CB1 and CB2 Cannabinoid Receptors Extension Length, Apoptosis,

Proliferation Rate, and Gene Expression. …………………...……...….……..156

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ABSTRACT

Erica L. Lyons

CANNABINOID RECEPTOR OVEREXPRESSION IN HUMAN SH-SY5Y

NEUROBLASTOMA CELLS

Dissertation under the guidance of Allyn C. Howlett, PhD

Professor, Department of Physiology and Pharmacology, and Asst Dean, WFU Graduate School

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We created clonal cell lines that stably overexpress either CB1 (CB1XS) or CB2 receptor

(CB2XS) in human neuroblastoma SH-SY5Y as a model to investigate the role of cannabinoid

signaling in neurite extension, proliferation, apoptosis, and cell viability. The mRNA of the

enzymatic components of the endogenous cannabinoid system diacylglycerol lipase,

monoacylglycerol lipase, alpha/beta-hydrolase domain containing 6, alpha/beta-hydrolase domain

containing 12, N-acyl phosphatidylethanolamine phospholipase D, and fatty acid amide hydrolase

are present in SH-SY5Y cells. Activity of these enzymes produced endocannabinoid ligands,

resulting in a steady state concentration of 2-arachidonoylglycerol of 135 pmol per gram of

protein and anandamide of 0.82 pmol per gram protein. Application of tetrahydrolipstatin, an

inhibitor of diacylglycerol lipase activity, decreased 2-arachidonoylglycerol concentration to 15

pmol per gram of protein while anandamide remained at 0.79 pmol per gram of protein.

Oxotremorine M, an agonist of Gq coupled muscarinic acetylcholine receptors, mobilized

calcium from intracellular stores to stimulate diacylglycerol lipase, increasing 2-

arachidonoylglycerol concentration to 564 pmol per gram of protein. A 2-arachidonoylglycerol

hydrolysis enzyme inhibitor cocktail increased 2-arachidonoylglycerol to 2,140 pmol per gram

protein. Transfection resulted in a stable increase of CB1 receptor mRNA by 11 fold increase in

abundance in the CB1XS cell line and of CB2 receptor mRNA by 14 fold increase in abundance in

the CB2XS cell line. Cannabinoid receptor overexpression did not alter the mRNA abundance of

the enzyme components of the endogenous cannabinoid system in SH-SY5Y cells. Parental SH-

SY5Y cells had an average neurite length per nuclei of 0.7 μm per nuclei. Stable increased

expression of CB2 receptor in three clonal cell lines did not consistently change extension length,

and resulted in a maximal neurite length per nuclei increase to 1.69 μm. Stable increased

expression of CB1 receptor increased the abundance of one soma long neuritic extensions from

0.7 to 6.0 micrometers per nuclei per field. This CB1 stimulated increase in neurite length was not

constitutive receptor activity as it was halved by tetrahydrolipstatin inhibition of diacylglycerol

lipase enzymatic production of the endogenous cannabinoid ligand 2-arachidonoylglycerol.

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Neurite extension length was halved with a p of 0.07 by gallein inhibition of G protein beta

gamma dependent signaling. Stable increased expression of CB1 receptor increased the mRNA

abundance of growth cone associated protein 43 and ST8 Alpha-N-Acetyl-Neuraminide Alpha-

2,8-Sialyltransferase 2 mRNA and decreased integrin alpha 1 mRNA. Stable increased

expression of CB2 receptor increased neural cell adhesion molecule and synaptotagmin 1 mRNA.

We identified the endogenous cannabinoid enzyme monoacylglycerol lipase as a novel target of

retinoic acid mRNA transcription in the SH-SY5Y neuroblastoma model of neuron differentiation

and neurite extension. Overexpression of cannabinoid receptors did not alter the viability,

apoptosis rate, or proliferation rate of cells relative to parental or empty vector SH-SY5Y.

This research investigated the gaps in knowledge of cannabinoid signaling in neuronal

functional development. There had never been a direct side by side comparison of the impact of

CB1 versus CB2 cannabinoid receptors on extension length. We have addressed the role of ligand

stimulated CB1 and CB2 cannabinoid receptor signaling in human β-3-tubulin positive neuronal

cells. We identified novel mRNA targets of cannabinoid receptor signaling and provided

evidence that CB1 and CB2 cannabinoid receptors play different roles in neurite extension during

synapse development. We demonstrate cannabinoid system mediated changes to neuronal

extensions independent of cell viability, apoptosis rate, or proliferation rate. We demonstrated

that CB1 receptor, more than CB2 receptor, stimulates a 2-arachidonoylglycerol dependent

increase in human neurite length without altering cell viability, apoptosis, or proliferation rate.

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CHAPTER I

INTRODUCTION

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1.1 DISCOVERY OF AND PHSYICAL AND TEMPORAL LOCALIZATION OF THE

ENDOGENOUS CANNABINOID SYSTEM

1.1.1: The history of cannabis sativa, marijuana, or hashish

Cannabis sativa is a plant which originated in Asia (Li 1974). Cannabis was used in

China for thousands of years as a textile fiber, consumable grain, and for production of paper

until it was replaced in the past five hundred years by cotton, rice, and other crops (Li 1974).

Cannabis also has a long history of use for its psychoactive properties. There is evidence that

cannabis was used medicinally in India in 1,000 B.C. to facilitate meditation by the Tantric

Buddhists of the Himalayas, as incense by the Assyrians in the ninth century B.C, and for

euphoric purposes at a Scythian funeral in 450 B.C. (Zuardi 2006). Cannabis was introduced to

Africa in the 15th century, to South American in the 16

th century, and to Europe in the 19

th century

(Zuardi 2006). Recreational use of cannabis was practiced in Northeast Brazil in the 16th century

and by the early 20th century had expanded north to Mexico and the United States (Zuardi 2006).

1.1.2: The discovery of the cannabinoid receptors

Many decades of chemical investigation were required to first identify Δ9-THC as the

constituent of cannabis responsible for central nervous system action, and then identify

cannabinoid receptors as the mechanism of action. In the 1800’s, T. and H. Smith first

determined that the alkali-insoluble, high boiling fraction of hemp resin contained the active

constituents, and that it was not an alkaloid (Todd 1946). In 1896, Wood, Spivey, and Easterfield

isolated a red oil from Indian charas (Wood et al., 1899). Acetylated purified crystallines from

this time period did not have the activity of hashish in rabbits and the active component of the

resin remained unknown (Todd 1946). By 1960, the status of the field was that isolates named

cannabidiol, cannabinol and tetrahydrocannabinol (Korte et al., 1960) had been separated

chromatographically, but the exact structure of the euphorically active isolate

tetrahydrocannabinol had not yet been described. In 1964, Mechoulam and Gaoni reported the

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isolation, structure, and partial synthesis of what they termed Δ1-3,4-trans-tetrahydrocannabinol

(Gaoni et al., 1964), later called Δ9-THC, and showed using the ataxia test in dogs that this was

the active component of marijuana.

There was some uncertainty over whether or not a receptor was required as the lipid

soluble Δ9-THC can pass freely through the plasma membrane and might take effect on any

number of intracellular components. It was speculated that perhaps like ether and other lipophilic

general anaesthetics, the lipophilic Δ9-THC might also exert its analgesic effect by acting upon

the plasma membrane of neurons.

The structural determination of the phytocannabinoid Δ9-THC sparked investigation into

the creation of architecturally related compounds that were hoped to have a similar analgesic

effect. It was clear that Δ9-THC was not an opiate because its analgesic action was not

antagonized by naloxone (Razdan 1986). Pharmacologists were interested in cannabinoids as

analgesics because unlike opiates, cannabinoids did not induce respiratory depression (Razdan

1986). By 1986, hundreds of synthetic cannabinoids had been generated based on Δ9-THC

(Razdan 1986), although design was hampered by a lack of understanding of the cellular

mechanism of action of these compounds.

In 1984 it was discovered that Δ9-THC inhibited adenylate cyclase in neuroblastoma cells

(Howlett 1984) and membranes (Howlett et al., 1984; Howlett 1985) in a concentration

dependent manner and in the nanomolar range. In 1988, tritium-labelling of the synthetic

cannabinoid CP-55,940 allowed measurement of cannabinoid binding to rat brain membranes

(Devane et al., 1988). Binding was found to be rapid, reversible, and specific. The tritiated CP-

55,940 could be displaced by 1 μM Δ9-THC, and this specific binding could be eradicated by

heating the membrane to 60°C. Tritiated CP-55,940 showed for the first time that cannabinoid

binding was saturable (Devane et al., 1988). Saturation of radioligand binding upon a finite

number of binding sites rules out passive diffusion into the cell and indicates the presence of a

receptor. In 1990 an orphan receptor which bound CP-55,940 was cloned from rat cerebral

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cortex cDNA, the CB1 receptor (Matsuda et al., 1990), and in 1993 a second CP-55,940 binding

receptor was cloned from a human promyelocytic leukemic line (Munro et al., 1993), the CB2

receptor.

1.1.3: The structure of the cannabinoid receptors

Receptor theory is the idea that the binding of a extracellular ligand to a receptor in the

plasma membrane leads to manyfold amplification and a signaling cascade that changes the

behavior of the cell. The receptor is assumed to exist in an inactive state that, when activated by

the binding of the extracellular ligand, either changes receptor conformation to activate an already

coupled signaling complex of proteins or moves the receptor laterally in the cell membrane to

couple to and activate a signaling complex of proteins. Alternatively, a receptor may achieve its

active state spontaneously without a ligand, which is known as constitutive activity. In this case,

the binding of a molecule that inhibits spontaneous activity is called inverse agonism. Finally, it

is possible that a receptor might have multiple different potential active conformation states. In

this case, the identity of either the ligand or the tissue specific auxiliary coupling proteins are

what determines the receptor’s active state and its resulting multi unit signaling complex

(Kenakin 2004).

There are many different types of receptors including ligand gated ion channels, voltage

gated ion channels, enzyme linked receptors such as receptor tyrosine kinases, and G protein

coupled receptors (GPCRs) to name only a few. GPCRs are the most pharmacologically relevant

receptor class. From the more than 20,000 FDA approved products as of 2006, the most common

drug target was Class A rhodopsin-like GPCRs, accounting for 27% of all drug targets

(Overington et al., 2006). The GPCR superfamily consists of more than 800 unique sequences in

the human genome, of which more than 300 are nonolfactory (Fredriksson et al., 2003). GPCRs

are integral membrane proteins with seven transmembrane alpha helices, three intracellular loops

(ICL), a generally extracellular N terminus and a generally intracellular C terminus. Conserved

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GPCR structure generally has a six amino acid long alpha helical region that is called ICL1, a one

or two turn alpha helical or unstructured ICL2, and a large amount of variation in ICL3 and the C

terminal tail (Venkatakrishnan et al., 2013). Many GPCRs have alpha helicity in the C terminal

region named helix 8 (H8), which because of its amphipathic nature leads to physical proximity

to the plasma membrane and creates a pseudo ICL4, a region implicated in contact with a G

protein (Venkatakrishnan et al., 2013). Unlike other Class A rhodopsin-like GPCRs, the sub

group of receptors that binds lipid-derived endogenous ligands do not have readily water soluble

ligands and are unlikely to share the same ligand binding pocket on the extracellular side of the

transmembrane bundle (Hurst et al., 2013; Venkatakrishnan et al., 2013). Instead, lipid binding

GPCRs such as cannabinoid CB1 and CB2 receptors, S1P1-5 receptors, and rhodopsin itself which

binds the lipophilic 11-cis-retinal, likely experience ligand approach via the plasma membrane

through an opening between transmembrane helices (Hurst et al., 2013).

There are two cannabinoid receptors, CB1 anad CB2, which share many structural

similarities but also some differences. Both receptors bind Δ9-THC with nanomolar affinities.

The affinity of Δ9-THC to displace [

3H]CP-55,940 from its binding site was a Ki of 41 +/- 2 nM

from CB1 receptor in rat brain membranes and a Ki of 36 +/- 10 nM from membranes of CHO

cells transfected to express CB2 receptor (Huffman et al., 2006). The crystal structure of CB1

receptor has been solved (Hua et al., 2016; Shao et al., 2016). The receptors share an overall

sequence homology of 44% (Munro et al., 1993), with important differences occuring in the N-

terminal, extracellular loop 2, the C-terminal of transmembrane helix VII, and the C-terminal

(Montero et al., 2005). The CB1 receptor is the only Class A GPCR with a 70 residue long N-

terminal (Montero et al., 2005), more than twice as long as the N-terminal of CB2 receptor. CB1

receptor’s gene sequence is found on human chromosome 6 (Hoehe et al., 1991) while the

sequence for CB2 receptor is found on chromosome 1 (Valk et al., 1997). As of May 2018, the

NCBI nucleotide reference database NUCCORE lists five confirmed human mRNA transcript

variants for CB1 receptor. Variants of the CB1 receptor may impact its function; for example the

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TAG haplotype of CB1 receptor was significantly associated with the polysubstance abuse

compared with the non-substance-abuse patients in three difference ancestry populations of a

human study (Zhang et al., 2004). The NUCCORE lists one mRNA splicing possibility for CB2

receptor. Although CB1 receptor possesses a CRAC domain in transmembrane helix 7 and its

adenylyl cyclase (AC) signaling is acutely affected by methyl-β-cyclodextrin depletion of plasma

membrane cholestrol, CB2 receptor was completely insensitive to cholesterol depletion by

methyl-β-cyclodextrin of DAUDI cells (Bari et al., 2006), likely due to the lack of a completely

consensus CRAC domain. CB2 receptor appears to not reside in lipid rafts due to the 304th

residue of the otherwise consensus CRAC domain being G instead of K, which when point

mutated as K402G in the CB1 receptor reduced residence in cholesterol-rich membrane regions

and induced insensitivity to changes in membrane cholesterol (Oddi et al., 2011). In rat C6

glioma cells, CB1 receptor colocalizes with caveolin-1 and resides in caveolae, a specialized type

of lipid raft, in the plasma membrane (Bari et al., 2008).

The N-terminus of the CB1 receptor contains three distinct consensus sequences for N-

linked glycosylation, (N-X-S/T), and two of the sites are glycosylated in the rat brain (Song et al.,

1995). After translation of mRNA to protein, palmitoylation of Cys415

of CB1 receptor is required

for recruitment to plasma membrane and lipid rafts (Oddi et al., 2012).

1.1.4: Physical location of expression of the cannabinoid receptors

Cannabinoid receptor mRNA and protein are expressed at distinct phases and tissues in

development and throughout life. Cannabinoid receptor signaling is involved in implantation of

the early blastocyst. Knocking out either CB1 or CB2 receptor induces asynchronicity in

implantation of the blastocyst in the uterus of mice (Paria et al., 2001).

The tissue that expresses the most CB1 receptor is the brain (Onaivi et al., 2006). In-situ

hybridization mRNA probes for CB1 receptor in chicken brains found it to increase from

relatively low abundance at midgestation stage 10 to great abundance by embryonic stage 15

24

(Begbie et al., 2004). CB1 receptor then remains one of the most abundant GPCRs in the brain

from birth throughout adulthood. Autoradiography for dual CB1 and CB2 receptor agonist CP-

55,940 binding in rats shows strong specific staining in brain regions in embryonic day 21 which

is shortly before birth (Fernandez-Ruiz et al., 2000; Romero et al., 1997) and in the adult brain

(Herkenham et al., 1990). In-situ hybridization using an antisense riboprobe for CB1 receptor

mRNA on a whole brain slice from a 20 week (approximately embryonic stage 10) human fetal

brain and an adult human both resulted in regions of high specific staining (Wang et al., 2003).

The CB2 receptor is not as abundant as CB1 in the midgestate or adult brain, and initially

was thought to be entirely absent from the brain (Munro et al., 1993). An antisense probe for CB2

receptor yields only diffuse nonspecific staining in both 20 week and adult human brain (Wang et

al., 2003). Although much lower abundance than CB1, CB2 receptor is present in certain brain

regions and on specific cell types. CB2 receptor is present in the adult mouse brain subventricular

zone (Goncalves et al., 2008). Onaivi et al. were able to detect CB2 receptor mRNA in adult rat

brainstem and hypothalamus at 100x lower abundance than its presence in the spleen, and more

than 100x lower abundant than hypothalamic and brainstem CB1 receptor (Onaivi et al., 2006).

CB2 receptor mRNA is also present in the adult rat spinal cord and dorsal root ganglia (Hsieh et

al., 2011). Mouse postnatal day 7 retinal ganglion nerve cells express both CB1 and CB2

receptors on growth cones (Duff et al., 2013).

Cannabinoid receptors are also present outside of the central nervous system. Liver

hepatocytes express CB1 receptor (Osei-Hyiaman et al., 2005) and CB2 receptor (Gertsch et al.,

2008). CB1 receptor abundance in the liver is increased after chronic alcohol (Jeong et al., 2008))

and high fat diet (Mukhopadhyay et al., 2010). Visceral and subcutaneous mouse adipose tissue

stain positive for CB1 and CB2 receptor proteins (Starowicz et al., 2008). CB1 receptor is found in

rat brown adipose tissue but not white adipose tissue, and has been proposed as a protein marker

of brown adipose tissue (Eriksson et al., 2015). Brown adipose tissue can be found in both

visceral and subcutaneous depots (Sacks et al., 2013). When human visceral and subcutaneous

25

adipose tissue were examined for CB1 receptor, the abundance of CB1 receptor mRNA and

protein was lower in subcutaneous abdominal adipose tissue from obese subjects and higher in

lean subjects (Bennetzen et al., 2010). There is very low or unmeasurable CB1 or CB2 mRNA in

mouse intestine, lung, or muscle (Onaivi et al., 2006). There are functional CB1 receptors in

human vascular endothelial cells that are capable of activating p42/44 MAP kinase (Liu et al.,

2000).

The tissue that expresses the most CB2 receptor is the spleen (Munro et al., 1993; Onaivi

et al., 2006), and the spleen is regarded as the best tissue from which to make a positive control

that contains abundant CB2 receptor (Marchalant et al., 2014). The mRNA abundance of CB2

receptor in tonsils and spleen is as abundant as the mRNA of CB1 receptor in the brain (Galiegue

et al., 1995). The abundance of CB2 receptor mRNA in the spleen is due to its immune cell

content. CB2 receptor mRNA abundance in leukocytes can be ranked as followed: B-cells >

natural killer cells S monocytes > polymorphonuclear neutrophil cells > T8 cells > T4 cells

(Galiegue et al., 1995). CB2 receptor mRNA in the spleen is 10 to 100 times more abundant that

CB1 receptor (Galiegue et al., 1995).

1.2 CANNABINOID RECEPTOR SIGNALING PARTNERS, TARGETS, AND

RECYCLING

1.2.1: Cannabinoid Receptor Coupled G Proteins

A receptor’s resting state may be solitary, with the receptor only coupling to effector

molecules after binding to an agonist (Kenakin 2004). For GPCRs, the agonist receptor

complexes are a larger molecular weight than antagonist receptor complexes (Limbird et al.,

1978). This can be interpreted to mean that the receptor requires a coupling partner in order to

signal when bound by the ligand, and that it does not couple to this partner until also bound to a

ligand. Binding data from the β-adrenergic receptor, a GPCR, supports the ternary complex

model more than the unimolecular reaction model (De Lean et al., 1980). Receptor activation

26

requires not only the ligand and the receptor, but also an additional component(s) in the

membrane that upon ligand binding must then join together to form a ternary complex in order to

activate effector molecules.

GPCRs may couple with a variety of different partners. This includes, among others, 21

different Gα, 6 unique Gβ, and 12 distinct Gγ subunits (Moreira 2014) which can lead to a large

number of possible αβγ heterotrimer combinations. Binding of the agonist to the receptor leads to

a physical exchange of GDP for GTP in the Gα and a dissociation of Gα and Gβγ from the

receptor. This frees the Gα and Gβγ to perform a number of activities in the cell. The Gα family

has inherent GTPase activity that is increased by the regulator of G-protein signaling (RGS)

protein family (Kach et al., 2012). The Gβγ can inhibit ACI, activate AC II, IV, and VII, activate

phospholipase-C-β, activate GIRK1-4, activate phosphatidylinositol-3-kinase-β, and inhibit

voltage dependent calcium channels (Wettschureck et al., 2005). Gallein is a compound which

can inhibit Gβγ activity (Lehmann et al., 2008).

There are four families of Gα: Gα s which stimulate AC, Gα i/o which inhibit AC, Gα

q/11 which stimulate phospholipase-C-β 1-4, and Gα 12/13 which affect PDZ-RhoGEF, Bruton

tyrosine kinase, GAP1m, cadherin, p115RHOGEF, radixin, etc (Wettschureck et al., 2005).

Cannabinoid receptors signal through the Gα i/o family which inhibit AC to decrease the

accumulation of cAMP (Eldeeb et al., 2017). The Gα i/o family members include Gαi1, Gαi2,

Gαi3, Gαo, Gαz, Gαgust, Gαt-r, and Gαt-c (Wettschureck et al., 2005). The members that may be

expected to be found in the central nervous system are Gαi1, Gαi2, Gαi3, Gαo, and Gαz (Casey et

al., 1990; Wettschureck et al., 2005). Pertussis toxin (PTX) is often used as a tool to study the

Gαi/o family because it ADP ribosylates most of the Gαi/o family near the carboxylic acid

termini and prevents the G protein from interacting with the receptor (Wettschureck et al., 2005).

There are exceptions to this generalization; three Gα i/o are insensitive to PTX . Gαo mediates

most of its effects through its Gβγ complex (Wettschureck et al., 2005). Gαz and G16 are PTX

insensitive (Chan et al., 1998; Wettschureck et al., 2005). The SH-SY5Y human neuroblastoma

27

cell line used in the following study is known to express Gαz (Chan et al., 1998). Also, although

there are some situations in which CB1 receptor and Gαz interaction has not been observed (data

not shown in (Mukhopadhyay et al., 2000)), CB1 receptor can interact with Gαz (Garzon et al.,

2009) and mediates long lasting CB1 desensitization in the mouse brain . The presence of Gαz can

make receptors that are PTX sensitive in other tissues that lack Gαz become PTX insensitive in

tissues that have Gαz. Gαz abundance can direct receptor signaling to new targets such as ion

channels (Jeong et al., 1998). A yeast two hybrid screen found that Gαz interacts with Rap1GAP,

a protein involved in GTP hydrolysis of Rap1 and possible cytoskeletal regulator (Meng et al.,

1999). Gαz also reduced levels of Rock2, RhoA, and RhoGAP and increases expression of Rnd3

in C2C12 myoblasts, suppressing myotubule formation (Mei et al., 2011).

1.2.2: Cannabinoid Receptor Activated Intracellular Signaling Pathways

The cannabinoid receptors can act upon a variety of different intracellular signaling

pathways. Which signaling pathway occurs depends upon the abundance of a variety of signaling

molecules present in the cell of a given type responding to the environmental cues around it; for

example COX2 is not normally expressed in neurons or glia but can be induced by

lipopolysaccharide in microglia cells (Schlachetzki et al., 2010). The first cannabinoid signaling

discovered was Δ9-THC inhibition of adenylate cyclase in neuroblastoma cells (Howlett 1984) by

receptor coupling to the Gα i/o family of G proteins. CB1 receptors activate inwardly rectifying

potassium channels and inhibit voltage dependent calcium channels (Mackie et al., 1995). CB1

receptors also inhibit acid sensing ion channels in rat dorsal ganglion (Liu et al., 2012). CB1

receptor activation can also lead to focal adhesion kinase (FAK) phosphorylation (Dalton et al.,

2013; Derkinderen et al., 1996). Activation of CB1 receptors can stimulate MAPK (Bouaboula et

al., 1995). Intracellular ceramide levels can be elevated after CB1 receptor activation (Sanchez et

al., 1998). The c-Jun N-Terminal Kinase (JNK) can be activated by CB1 receptors (Rueda et al.,

2000). Stimulation of CHO-CB1 cells with 1 μM THC induced JNK activity that is maximal at

28

10 minutes and returns near basal after 30 minutes. The JNK activation was also duplicated with

15 μM 2-AG and 15 μM anandamide. The stimulation of JNK was prevented by coapplication of

1 μM of CB1 receptor antagonist SR141716 with the 1 μM THC (Rueda et al., 2000).

Stimulation of CB1 receptor caused release of nitric oxide (Prevot et al., 1998). There is great

diversity of signaling pathways cannabinoid receptors can activate.

Cannabinoid signaling has an important role in preventing excitotoxicity and seizures.

The most abundant excitatory neurotransmitter is L-glutamate. There are two major types of

glutamatergic receptors: ionotropic and metabotropic glutamate receptors. The ionotropic

glutamate receptors are ligand gated cation channels which when bound by glutamte affect the

concentration of Ca2+

and/or Na+, while the metabotropic glutamate receptors affect Gq/G11

(groups 1 and 2) or Gi/Go (group 3) (Kew et al., 2005). Kainic acid is an excitotoxin that causes

seizures by binding to an ionotropic glutamatic receptor. In 2003, Marsicano et al. hypothesized

that it might be possible to identify the signaling molecules that dampen excitotoxicity by

inducing excitotoxic seizures in mice and then measuring the molecules whose abundance

increases in response. Twenty minutes after dosing with kainic acid, the endogenous cannabinoid

agonist anandamide had tripled relative to basal concentration. Whole body knockout mice

lacking CB1 receptor had significantly more intense seizures than wild type mice after both were

dosed with kainic acid. Heterozygote whole body CB1 receptor knockouts could be induced to

the full magnitude of homozygote mouse seizure intensity by the application of SR141716, a CB1

receptor antagonist (Marsicano et al., 2003). A tissue specific knockout model of CB1 receptor

was created by using mice whose CB1 receptor genetic sequence was fragmented by a cre-lox

system targeting all but a 2.5 kilobase region of the CB1 receptor and whose Cre was promoted

with the Ca2+

/calmodulin-dependent kinase IIα gene. The resultant CB1f/f;CaMKIIαCre

knockout

mouse lacked CB1 receptor solely in principal neurons of the forebrain but not any other cell type.

Expression of CB1 receptor was maintained in cortical and hippocampal GABAergic interneurons

and in cerebellar neurons. These principal forebrain neuron specific knockout mice experienced

29

the full magnitude of seizure intensity experienced by whole body CB1 receptor knockout mice.

Seizures were not further exacerbated by application of SR141716, demonstrating that CB1

receptors located in places other than principal forebrain neurons were not contributing to the

effect. Dark field micrographs of protein markers of neuronal activity including c-fos, zif268,

and BDNF showed that the CB1f/f;CaMKIIαCre

knockout mice that experienced significantly less

neuronal activation after being dosed with kainic acid than their floxed controls. Interruption of

the signaling pathway of anandamide activating CB1 receptors in principal forebrain neurons

dramatically reduced recovery from excitation and increased excitotoxic seizure intensity

(Marsicano et al., 2003). This work showed that CB1 receptor activation of principal forebrain

neurons is critical for regulating excitatory neuron activation and decreasing susceptibility for

seizures. The CB1 receptor’s ability to inhibit glutamate receptor induced excitotoxicity is likely

via Giβγ signaling. Postsynaptic cannabinoid ligand binds to presynaptic CB1 receptor,

stimulating Giβγ to inhibit voltage-gated calcium channels which suppresses neurotransmitter

release (Katona et al., 2008).

1.2.3: Cannabinoid Receptor Endocytosis, Desensitization, Trafficking, and Recycling

Within seconds of ligand binding, GPCRs are phosphorylated by a G protein receptor

kinase (GRK), bound by arrestin, and uncoupled from G proteins. Within minutes of ligand

binding, GPCRs are internalized in an endosome. Arrestin dissociates during receptor

internalization (Wolfe et al., 2007). The endocytosed GPCR is then either dephosphorylated and

returned to the plasma membrane in preparation for future activation or trafficked to a lysosome

for degradation (Wolfe et al., 2007).

There are multiple different endocytosis mechanisms. Clathrin coated pits bud within 20

to 90 seconds and may endocytose a large quantity of diverse cargo (Wolfe et al., 2007). The

GTPase dynamin and the actin cytoskeleton regulate clathrin-coated pit dissociation from the

plasma membrane (Wolfe et al., 2007). Caveolae are a type of pit that do not have clathrin but

30

have the protein caveolin (Silva et al., 1999) that both spatially organizes proteins in lipid rafts on

the plasma membrane (Song et al., 1996; Xiang et al., 2002b) and endocytoses them to form

internal caveolae vesicles (Corrotte et al., 2013).

Endocytosis of the receptor to intracellular vesicles was first identified in connection with

agonist induced desensitization (Waldo et al., 1983) that decreased the number of receptors

present at the plasma membrane and reduced specific binding of the receptor to its ligand and G

proteins. Internalization was viewed for years only as a means of silencing the receptor. A small

fraction of endocytosed receptors would receive a phosphorylation status reset and, their ligand

receptivity restored, return to the plasma membrane. Endocytosed receptors might also be sent to

a lysosome for degradation (Wolfe et al., 2007). However, the traditional view that the only

roles of endocytosis are to reset or degrade a receptor has recently been challenged. It is possible

for endocytosis to prolong a receptor’s G protein mediated signaling intracellularly (Irannejad et

al., 2014). One example of endocytosis not leading to receptor silencing is the mechanism of

action of FTY720, a clinically approved treatment for multiple sclerosis. One of the cell-level

effects of FTY720 is to tighten the endothelial barrier to prevent lymphocytes from

transmigrating through and attacking myelin sheaths. FTY720 leads to S1P receptor

internalization and does eventually lead to ubiquitination and degradation. The compound was

therefore interpreted to act as a functional antagonist that achieved efficacy by decreasing the

receptor’s plasma membrane abundance. However, when other S1P receptor selective antagonist

compounds were designed and tested they not only failed to reduce lymphocyte transmigration,

but even enhanced vascular leakage, worsening symptoms. This is because the mechanism of

action of FTY720 was to serve not as an antagonist of the S1P receptor but as a long term agonist.

Endocytosis of FTY720 induces long-lasting S1P receptor initiated Gαi signaling. This long term

Gαi signaling inhibits AC and phosphorylates ERK for hours, increasing endothelial cell

migration and barrier function (Mullershausen et al., 2009). This was some of the first evidence

that endocytosis may not always terminate receptor initiated G protein signaling. The lipid

31

signaling S1P receptors can still signal through G proteins from inside the endosome.

Furthermore, sequestration and protection of the ligand bound receptor in the endosomal pocket

promoted a novel time scale of hours-long G protein signaling that was distinct from the minutes

time scale of plasma membrane localized G protein activation. Further investigation of endocytic

regulation of cAMP has found endocytosed G protein coupled receptors to be capable of acute

and rapidly reversible Gαs signaling (Tsvetanova et al., 2015). Interaction with the retromer

complex, perhaps via the retromer subunit VPS26, has been proposed as a mechanism of

termination of sustained endocytic receptor signaling (Tsvetanova et al., 2015).

Cannabinoid receptors are capable of responding differently to the binding of different

agonists. Δ9-THC, a partial agonist, does not cause internalization even at 3 μM, while full

agonists CP-55,940, WIN 55,212-2, and HU 210 cause rapid internalization (Hsieh et al., 1999).

Cannabinoid agonist WIN 55,212-2 reduced cell surface CB1 receptor immunoreactivity by up to

84% (Coutts et al., 2001). The last 14 residues of the C terminus were required for CB1 receptor

internalization (Hsieh et al., 1999), a region later discovered to be the interaction site of

cannabinoid receptor interacting protein1a (CRIP1a) (Blume et al., 2017). This protein competes

with β-arrestins for interaction with the CB1 receptor C terminus and affects agonist initiated

receptor internalization (Blume et al., 2017). It was thought that CB1 receptor internalization was

via clathrin coated pits based upon internalization being inhibited by hypertonic sucrose (Hsieh et

al., 1999), however hypertonic sucrose also inhibits caveolae as well (Page et al., 1998). In C6

glioma cells which endogenously express the protein, CB1 receptor colocalizes almost entirely

with caveolin-1(Bari et al., 2008). In HeLa cells transfected to express the receptor, CB1 binds to

β-arrestin2 but not β-arrestin1 and its internalization is decreased by 30% by siRNA knockdown

of clathrin (Gyombolai et al., 2013). Approximately 85% of CB1 receptor is located in

intracellular vesicles in CB1 receptor transfected HEK-293 cells (Leterrier et al., 2004).

32

1.3 STIMULATED SYNTHESIS AND BIOTRANSFORMATION OF ENDOGENOUS

LIGANDS

1.3.1: Discovery of the Enzymes that Produce and Hydrolyze Endogenous Ligands of the

Cannabinoid Receptors

In 1992, screens for compounds that activate a cannabinoid receptor revealed the first

endogenous agonist: anandamide also known as arachidonylethanolamide (AEA) (Devane et al.,

1992). In 1994, Di Marzo et al proposed a pathway by which neural membrane depolarization

leads to increased intracellular calcium concentration, which activates the enzyme NAPE-PLD

(Di Marzo et al., 1994). NAPE-PLD is located in adult presynaptic membranes, postsynaptic

membranes, in dendrites, and in smooth endoplasmic reticulum (Reguero et al., 2014). This

enzyme converts N-arachidonoyl phosphatidylethanolamine (NAPE) in the plasma membrane to

anandamide, which is then released extracellularly (Di Marzo et al., 1994). In the adult brain,

anandamide travels across the synapse to bind presynaptic CB1 receptor and decrease

neurotransmitter release (Howlett et al., 2004). Fatty acid amide hydrolase (FAAH) is a

membrane bound serine hydrolase that has a high activity with oleamide, arachidonamide, and

other lipid signaling molecules. In 1996, Cravatt et al proposed that anandamide was a substrate

for FAAH (Cravatt et al., 1996). FAAH converts anandamide into arachidonic acid and

ethanolamine (Cravatt et al., 1996). FAAH is highly expressed in liver, small intestine, brain, and

other organs, and is associated with mitochondrial and microsomal membrane fractions (Deutsch

et al., 2002). Although a transporter has not yet been identified (Chicca et al., 2012), anandamide

is also localized in intracellular membranes, with 85% of anandamide localized to the

endoplasmic reticulum and 15% in the plasma membrane (Dainese et al., 2014). Lipoxygenases

have also been proposed to oxidize anandamide. In human platelets, 12-lipoxygenase and 15-

lipoxygenase convert anandamide to 12(S)- and 15(S)-hydroxy-arachidonylethanolamide,

respectively (Edgemond et al., 1998). The enzyme cyclooxygenase 2 also acts on anandamide,

33

converting it to prostaglandin E2 ethanolamide (Yu et al., 1997) in a biologically relevant amount

in the brain (Glaser et al., 2010).

A lipid signaling molecule, 2-arachidonoylglycerol (2-AG), was identified to be a second

endogenous cannabinoid agonist in the late 1990’s (Stella et al., 1997; Sugiura et al., 1995). 2-

AG is present in the brain at approximately one thousand times higher concentration than

anandamide (Zoerner et al., 2011). It is also present in plasma, adipose tissue, and liver (Zoerner

et al., 2011). The mechanism for the synthesis of 2-AG was proposed to initiated by an increase

in intracellular calcium leading to the activation of the synthetic enzyme, in this case

diacylglycerol lipase-α (DAGLα) (Sugiura et al., 1995). DAGLα and β catalyze the hydrolysis of

membrane diacylglycerol into 2-AG (Bisogno et al., 2003). DALGα is localized around the

postsynaptic spine, placing it in a good position for interacting with presynaptic cannabinoid

receptors (Yoshida et al., 2006). Alternatively, lysophospholipase-C is present in the brain and

could synthesize 2-AG as well (Lambert et al., 2005; Sugiura et al., 1995). The biotransformation

of 2-AG into arachidonic acid and glycerol can be mediated by a number of different enzymes.

In 2007, Blankman et al. found that in the adult mouse brain, the majority of hydrolytic activity

can be attributed to monoacyl glycerol lipase (MAGL, 85%), ABHD12 (9%), ABHD6 (4%), and

FAAH (2%) (Blankman et al., 2007). Alpar et al. showed with an ex-vivo activity based protein

profile of brain derived serine hydrolases that in the embryonic 18.5 day mouse cortex, ABHD6

is more active than MAGL (Alpar et al., 2014). As with anandamide, COX2 can also convert 2-

AG to prostaglandins. The conversion products 12-hydroxyeicosatetraenoic acid glycerol ester,

15- hydroxyeicosatetraenoic acid glycerol ester, and prostaglandin H2 glycerol ester can be

measured using mass spectrometry (Kozak et al., 2000). This enzyme activity is shown in Figure

1.3.2 below (Ueda 2002), (Lambert et al., 2005), (Di Marzo et al., 1994), (Yu et al., 1997),

(Cravatt et al., 1996), (Edgemond et al., 1998), (Kozak et al., 2000), (Stella et al., 1997),

(Blankman et al., 2007).

34

Figure 1.3.1: Chemical structures during the steps to synthesize endogenous cannabinoids

anandamide and 2-AG from plasma membrane components. The endogenous cannabinoids are

rapidly hydrolyzed into arachidonic acid and either ethanolamine or glycerol.

35

Fig

ure

1.3

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36

1.3.2: Subcellular Localization of the Endogenous Cannabinoid System Enzymes

The endogenous cannabinoid ligand anandamide can be synthesized by NAPE-PLD and

hydrolyzed to arachidonic acid and ethanolamine by the enzyme FAAH. Analysis of enzyme

activity and abundance show that NAPE-PLD is present in the mouse brain as a membrane, not

cytosolic, protein (Morishita et al., 2005). FAAH is an integral membrane protein (Patricelli et al.,

1998). Both NAPEPLD and FAAH are present in the mouse at birth and triple in abundance

during the first postnatal month (Morishita et al., 2005). In the mouse hypothalamus, NAPE-PLD

protein can be detected in both pre and post-synaptic membranes, with a significantly greater

abundance in dendrites than synaptic terminals (Reguero et al., 2014). FAAH is primarily located

on intracellular membranes. In the mouse hippocampus, 50% of FAAH is in smooth endoplasmic

reticulum (primarily on the cytoplasmic surface), 24% is present in the cytoplasm, 16% is in

mitochondrial membrane (primarily on the cytoplasmic side of the outer membrane), and 11% of

FAAH is in surface membrane (Gulyas et al., 2004). In the mouse cerebellum, 47% of FAAH is

in smooth endoplasmic reticulum, 37% is in mitochondrial membrane (primarily on the

cytoplasmic side of the outer membrane), 12% is in the cytoplasm, 2% is in the stacked lamellae

of endoplasmic reticulum, and 2% is in surface membrane (Gulyas et al., 2004). FAAH protein

was detected in hippocampal pyramidal cell dendritic shafts and spines, but not in axon terminals

or on glial processes (Gulyas et al., 2004).

The ability of FAAH to hydrolyze 2-AG is only 2% of the 2-AG hydrolysis occuring in

the adult mouse brain (Blankman et al., 2007) and FAAH knockout mice do not have an

increased abundance of 2-AG in the brain (Lichtman et al., 2002), while the same FAAH

knockout mice had a 15 fold increase in anandamide abundance (Lichtman et al., 2002).

The endogenous cannabinoid ligand 2-AG can be synthesized from DAG by DAGL or

from 2-AG lysophospholipid by lysophospholipase C, and hydrolyzed to arachidonic acid and

glycerol by MAGL, ABHD12, and ABDH6. This is shown in detail in Figure 1.3.2. DAGLα,

DAGLβ, and MAGL mRNA are present in the mouse embryo from the earliest time point

37

measured, embryonic day 7, and with increasing abundance as gestation progresses and near birth

(Psychoyos et al., 2012). In humans, DAGLα is more abundant in the human brain than it is in

any other tissue except for the pancreas (Bisogno et al., 2003). DAGLα is enriched in the tips of

mouse hippocampal CA1 pyramidal cell dendritic spines (Katona et al., 2006). In mouse

prelimbic prefrontal cortical sections, 56% of postsynaptic dendritic spines had DAGLα

immunoreactivity, and 64% of presynaptic terminals had DAGLα immunoreactivity (Lafourcade

et al., 2007). In the dorsomedial region of the ventromedial nucleus of the hypothalamus, there is

no difference in DAGLα protein abundance between dendrites and dendritic spines (Reguero et

al., 2014). In mouse Purkinje cells, there was far more DAGLα in extensions than in the soma;

14 DAGLα metal particles per micrometer in spiny branchlets, 6 in spines, 3 in proximal

dendrites, yet only 0.5 in soma (Yoshida et al., 2006). Yoshida also like Katona found that

DAGLα is enriched in the hippocampal CA1 pyramidal dendritic spine tip and base, relative to

the region in between (Yoshida et al., 2006). Both DAGL α and β are present in the adult mouse

brain, and in neurofilament positive axonal tracts (Bisogno et al., 2003). Both DAGL α and β are

present in the embryonic day 14 rodent cortical pyramidal cell axonal growth cone (Mulder et al.,

2008). Comparing DAGL α and β, they are both very abundant in the mouse brain, but DAGLα

has approximately as much abundance in the thalamus, cortex, hippocampus, and cerebellum,

while DAGLβ is less abundant in the the thalamus than it is in the cortex, hippocampus, or

cerebellum (Yoshida et al., 2006). DAGLα and MAGL have a visually indistinguishable mRNA

in situ hybridization expression pattern in the mouse and rat hippocampus (Dinh et al., 2002;

Katona et al., 2006). There is abundant MAGL mRNA in the rat cortex, hippocampus,

cerebellum, and thalamus (Dinh et al., 2002). In the mouse prefrontal cortex, electron

micrographs of ABHD6 antibodies exhibit an 8-fold increased abundance in postsynaptic rather

than presynaptic side of the synapse (16 postsynaptic gold particles per square micrometer

compared with 2 presynaptically) (Marrs et al., 2010). Alpar et al. showed with an ex-vivo

38

activity based protein profile of brain derived serine hydrolases that in the embryonic 18.5 day

mouse cortex, ABHD6 is more active than MAGL (Alpar et al., 2014).

39

Fig

ure

1.3

.3

Lit

erat

ure

Val

ues

of

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1.4 ROLE OF ENDOGENOUS CANNABINOID SIGNALING IN REGULATION OF

CELLULAR PROLIFERATION

Both CB1 and CB2 cannabinoid receptors have been implicated in altering proliferation

rate of neuronal and neuronal precursor cells. These observations have been made in a variety of

models, ranging from neurospheres to two dimensional cell culture to animal models.

Cellular proliferation rate may be measured using several different types of assays. In

general these assays can be divided into two types: identifying a protein that is present only

during certain phases of the cell cycle, or applying a tag to the cell that is only incorporated

during certain phases of the cell cycle. The advantage to the first strategy is that immunolabeling

of proteins present only during certain stages of the cell cycle can easily be performed both in

vitro and in vivo, while the advantage to the second strategy is that pulse labeling an incorporated

tag for a brief amount of time can quantify the difference in proliferation rates between two

proliferating populations. The proteins known to be present only during certain phases of the cell

cycle are as follows: the 57 kDa protein statin is present only in G0, while proliferating cell

nuclear antigen, Ki67, p53, p105, and DNA polymerase are only present during G1, S, G2, and M

phases (Dolbeare 1995). Histone H3 is phosphorylated during mitosis (Hans et al., 2001). The

labeling of these proteins only identifies the phase of the cell cycle and not the rate at which it is

progressing. In contrast, pulse labeling of a tagged DNA base analog can compare the speed of

two different proliferating populations. A short pulse of a tagged DNA base analog labels only

the cells that were incorporating free bases into polymerized DNA in their S phase during the

time of the pulse. Examples of tagged DNA base analogs include radiodetection of thymidine

([3H]dThd), or immunohistochemical detection of bromodeoxyuridine (BrdU) (Dolbeare 1995).

These thymidine-like molecules which are incorporated into the DNA of the S phase cell can be

used to compare two proliferating populations or a change in the rate of proliferation after a

treatment, as the percentage of cells that incorporated the tagged thymidine analog may increase

or decrease during the same pulse time period.

41

1.4.1 Proliferation in Cortical Brain Derived Neurosphere Cell Models

The neurosphere model was first established in 1992 by Reynolds et al. (Reynolds et al.,

1992). Cells harvested from prenatal day 14 mouse striatal primordia and grown at low density in

serum free DMEM/F12 with epidermal growth factor (EGF) formed spheres that were positive

for nestin (Reynolds et al., 1992). Nestin is an intermediate filament that is expressed in

proliferating central nervous system progenitor cells and whose mRNA expression rapidly

decreases as the cell transitions from a proliferating to a post-mitotic state (Dahlstrand et al.,

1995). The neurosphere is therefore regarded as an example of proliferating mixed progenitor

cells that model the cells that would in vivo differentiate into central nervous system tissue.

However, the heterogeneous cell population with its differences in survival and proliferation rate

between cell types yields neurospheres in a range of compositions based upon methodological

procedure (Jensen et al., 2006).

Molina-Holgado et al. investigated the effect of cannabinoid receptor signaling on mouse

neural stem cell proliferation (Molina-Holgado et al., 2007). Cultures of neural cells were

prepared as neurospheres from the cortex of NIH Swiss White mouse embryos at embryonic day

15-16 (75% of the 20 day gestation). The neurospheres were plated at 20 cells per microliter and

fixed after seven days. Stains for CB1 and CB2 receptors showed that the neurospheres were

positive for both cannabinoid receptors in a highly overlapping manner. The cells that expressed

one receptor tended to also express the other. Molina-Holgado et al. (Molina-Holgado et al.,

2007) then investigated the requirement of functional cannabinoid signaling for proliferation of

the neurosphere. The percentage of cells that were positive for BrdU out of total DAPI stained

cell significantly increased from 50% to 75% after application of HU210 dual receptor agonist.

Co-application with the HU210 of either the CB1 receptor antagonist SR141716 or CB2 receptor

antagonist SR144528 abrogated the effect of HU210 alone (Molina-Holgado et al., 2007).

Antagonism of either cannabinoid receptor was enough to fully prevent the increase in

42

proliferation caused by the HU210 dual cannabinoid receptor agonist. This can be interpreted to

mean that both cannabinoid receptors were important for the proliferation of the cells in the

neurosphere derived from the embryonic day 15-16 Swiss White mouse cortices.

Palazuelos et al. investigated the effect of CB2 cannabinoid receptor signaling on

neurosphere proliferation rate (Palazuelos et al., 2006). Neurospheres were derived from

embryonic day 17.5 (90% of the 20 day gestation) mouse cortex. Application for seven days of

nanomolar concentrations of selective CB2 agonists HU308 or JWH133 significantly increased

the percentage of BrdU positive cells from 40% in control neurospheres to 70% and 50%,

respectively. This CB2 agonist-induced increase in BrdU positive proliferating cells was receptor

specific because application of CB2 receptor specific antagonist SR144528 thirty minutes before

the injection of CB2 agonist did not result in an increase of BrdU positive proliferating cells

relative to control. The injection of SR144528 alone did not change the percentage of BrdU

positive proliferating cells relative to control (Palazuelos et al., 2006). The downstream signaling

pathway after HU308 CB2 receptor stimulation that resulted in an increase in proliferation rate

was found to be dependent upon phosphoinositide 3-kinase and MEK/ERK because

phosphoinositide 3-kinase inhibitor LY294002 and MEK/ERK inhibitor PD98059 each had the

same effect as CB2 antagonist SR144528; preventing a significant increase above basal when

coapplied with HU308 and not altering basal proliferation rate when applied alone (Palazuelos et

al., 2006).

Aguado et al. investigated the effect of CB1 cannabinoid receptor signaling on

proliferation rate of rat-derived neurospheres (Aguado et al., 2005). Neurospheres derived from

embryonic day 17 (80% of 22 day gestation) rat cortices were positive for immunostaining of

CB1 receptor. The application of nanomolar concentrations of dual receptor agonist WIN 55,212-

2 increased the percent of BrdU positive cells 50% above basal (Aguado et al., 2005). The

individual applications of endogenous agonists anandamide and 2-AG both each increased the

BrdU positive proliferating population compared with control by 50%. This increase in

43

proliferation rate was CB1 receptor specific because coapplication of SR141716 selective CB1

receptor antagonist with WIN 55,212-2 did not significantly alter the percentage of BrdU positive

cells when compared with basal. The application of CB1 receptor antagonist SR141716 alone did

not alter proliferation rate when compared with control (Aguado et al., 2005). To summarize the

results of Molina-Holgado et al., 2007, Palazuelos et al. 2006, and Aguado et al. 2005, exogenous

stimulation of both CB1 and CB2 cannabinoid receptors increase the proliferation rate of

neurospheres derived from embryonic mouse and rat cortex in, for at least CB2 receptor, a

phosphoinositide 3-kinase and MEK/ERK dependent manner. However, the application of

cannabinoid receptor antagonists did not decrease proliferation rate relative to control treated

cells.

1.4.2 Proliferation in Subventricular Zone Rodent Brain Models

Mulder et al. employed CB1 receptor and FAAH knockout mice to investigate the role of

cannabinoid receptor signaling on proliferation rate in the embryonic rodent brain subventricular

zone (Mulder et al., 2008). Pregnant female mice were injected with BrdU when their embryos

were 14.5 days developed using a timed mating strategy. Two hours after injection, the embryos

were harvested and assessed. In CB1 receptor knockout mice, the percentage of BrdU

incorporating proliferating cells in the ventricular and subventricular brain regions was

significantly decreased from 40 to 30 percent of total Hoechst stained cells. In the FAAH

knockout mice, which would be anticipated to have 15-fold more anandamide than wild type

mice (Lichtman et al., 2002), the percentage of BrdU incorporating proliferating cells was

significantly increased from to 60% compared with 40% in the wild type mice (Mulder et al.,

2008). The FAAH mice data can be interpreted to mean that overabundance of anandamide can

at embryonic day 14.5 stimulate proliferation in the subventricular and ventricular zone brain

tissue. The CB1 receptor knockout mice data can be interpreted to mean that CB1 receptor is

contributing to the proliferation rate of embryonic day 14.5 day ventricular and subventricular

44

zone brain cells because decreased cannabinoid receptor activation impaired their proliferation

rate. However, Mulder et al. did not identify the specific type of cell that was proliferating at this

gestation stage, and it is not known whether or not the population of cells utilizing this

cannabinoid receptor-dependent proliferation pathway at embryonic day 14 was neurons,

astrocytes, or oligodendroglia.

Arevalo-Martin et al. investigated what cell types were affected in proliferation rate by

CB1 and CB2 cannabinoid receptor signaling in the postnatal day 15 Wistar rat subventricular

zone brain region (Arevalo-Martin et al., 2007). Postnatal day 15 Wistar rats were injected

subcutaneously in the neck every day from postnatal day 0 to 14 with either CB1 receptor specific

agonist ACEA or vehicle (1% bovine serum albumin and 0.2% ethanol in saline). On postnatal

day 15, the population of proliferating cells in the subventricular zone was identified by staining

for phospho-histone 3, which is phosphorylated during mitosis (Hans et al., 2001). The

monoclonal antibody 4A4 was used to label mitotic glia because it recognizes vimentin which has

been phosphorylated by a mitosis specific kinase, Cdc2 kinase, (Kamei et al., 1998). ACEA

injection did not change the overall number of proliferating cells as stained by phospho-histone

H3, but the 4A4 positive proliferating glia were increased in abundance by 50% above control

(Arevalo-Martin et al., 2007). This can be interpreted to mean that during the time window of

postnatal days 0 to 15, glial cells are proliferating in the rat subventricular zone, and their

proliferation rate or percentage that enter proliferation can be increased by selective agonist

stimulation of the CB1 cannabinoid receptor. It is interesting to note that the significant increase

in the proliferation rate of the mitotic glial population was not a large enough contributor to the

pool to change the total number of proliferating cells. This finding that total number of

proliferating cells in the postnatal days 0 to 15 subventricular zone was not increased by ACEA, a

selective CB1 receptor agonist, is similar to and agrees with the results found by Goncalves et al.

(Goncalves et al., 2008), who found that CB1 receptor antagonists did not decrease the Ki67

45

positive proliferation rate of total proliferating cells in the postnatal week 6 rodent subventricular

zone.

Goncalves et al. investigated the effect of CB1 and CB2 cannabinoid receptor signaling on

proliferation rate in the adult rodent brain subventricular zone (Goncalves et al., 2008). Young

adult six week old mice were intraperitoneal injected every day for ten days with a compound.

One day after drug cessation, viral vectors containing green fluorescent protein were injected

directly into the subventricular zone. The purpose of this labeling was to identify the neuronal

precursors that are known to migrate from their origin in the subventricular zone to their

destination in the olfactory bulb, which would appear as the only green fluorescent cells present

in the olfactory bulb. Injection of DAGL inhibitors RHC-80267 or tetrahydrolipstatin, which

decrease the abundance of cannabinoid ligand 2-AG, significantly decreased the green fluorescent

protein in the olfactory bulb from 1.0 to 0.4 area units (Goncalves et al., 2008). This can be

interpreted to mean that 2-AG is involved in a signaling process in the six week old mouse,

perhaps proliferation or migration, that increases the abundance of subventricular zone neurons in

the olfactory bulb. The ligand 2-AG can stimulate both cannabinoid receptors. In order to

investigate whether the activity of CB1 or CB2 receptors were responsible, antagonists of both

receptors were injected and the Ki67 positive proliferating cell population was assessed.

Injection of CB2 receptor specific antagonists JTE907 and AM630 significantly decreased the

Ki67 positive cell count in the subventricular zone from 60 to 20, while application of CB1

receptor specific antagonists SR141716 and LY320135 did not alter the subventricular zone Ki67

positive cell count (Goncalves et al., 2008). In the AM630 injected mouse olfactory bulb, the

green fluorescent protein area of migrated neurons compared with control was significantly

decreased from 30 to 20 area units. This can be interpreted to mean that activity of CB2 receptor

but not CB1 receptor is required for proliferation of the cells, including but possibly not limited to

neurons, in the subventricular zone of the six week mouse brain subventricular zone.

46

Goncalves et al. (Goncalves et al., 2008) also investigated the effect of CB2 receptor

signaling on proliferation of subventricular zone neuronal precursors in mouse models of aging.

Mice that were twenty months old and were treated with synthetic dual cannabinoid receptor

agonist WIN55,212-2 had 7 times more Ki67 positive proliferating cells in their subventricular

zones. Also, six month old mice were intraperitoneal injected every day for ten days with

selective CB2 agonist JWH133. One day after drug cessation, viral vectors containing green

fluorescent protein were injected directly into the subventricular zone. This labeling identifies the

neuronal precursors which migrate from their origin in the subventricular zone to their destination

in the olfactory bulb, appearing as the only green fluorescent cells in the olfactory bulb. Two

weeks later, the animals were sacrificed and the olfactory bulb was sagittally sectioned. The

JWH133 CB2-receptor stimulated six month old animals had a significantly increased green

fluorescent protein area, from 0.1 to 0.3 area units, and a significant increase in neuron count

from 100 to 300 neurons per 40 micrometer saggital section area (Goncalves et al., 2008).

Taken together, these results mean that stimulation of CB2 but not CB1 receptors in six

week old young adult and six month old and twenty month old aged adult subventricular zone

neuronal precursors increases the number of neurons that become present weeks later in the

olfactory bulb by a mechanism contributed to by increased proliferation in the subventricular

zone.

1.5 ROLE OF ENDOGENOUS CANNABINOID SIGNALING IN REGULATION OF

CELLULAR DEATH MECHANISMS

There are two major mechanisms of cell death: necrosis and apoptosis (Nikoletopoulou

et al., 2013). Necrosis involves the swelling of organelles and rupture of the plasma membrane,

while leaving the nucleus relatively intact. Necrosis involves inflammasome activation and a

strong inflammatory response. Apoptosis, or programmed cell death, involves the activation of

cysteine aspartyl proteases, or caspases. Apoptosis results in membrane permeabilization,

47

chromatin condensation, DNA fragmentation, and a relatively lower inflammatory response

(Nikoletopoulou et al., 2013). After commitment to apoptosis, the end result of caspase activity

is poly ADP ribose polymerase (PARP) cleavage. Cleaved PARP fragments are not present

unless a cell has become committed to and is currently undergoing apoptosis (Chaitanya et al.,

2010).

Cannabinoid receptor signaling has been shown to affect cell viability by triggering cell

death. Application of 24 to 48 hrs of 20 to 50 μg/mL of Δ9-THC (0.06 to 0.15 μM) in DMEM

triggered cell death in human U87-MG glioblastoma cells (Galanti et al., 2008). Cellular

abundance as measured by the MTT absorbance assay was below the absorbance seen at time

zero before application of the drug (Galanti et al., 2008). In human neuroblastoma cell lines, the

application of endogenous cannabinoid anandamide induced apoptosis in LAN-5 cells which

endogenously express CB1 receptor but not in SKNBE cells which do not endogenously express

CB1 receptor (Pasquariello et al., 2009). Co-application of SR141716 completely reversed the

anandamide induced apoptosis in LAN-5 cells (Pasquariello et al., 2009). Application of Δ9-THC

and CB2 receptor specific agonist JWH015 induced cell death in the human HCC hepatocellular

carcinoma cell line in an AMPK dependent manner (Vara et al., 2011). These studies

demonstrate that both cannabinoid receptors are capable of triggering cell death. It is therefore

important when working with cannabinoid receptor signaling to measure cell viability and

determine whether the effects witnessed are a side effect of cell death.

1.5.1 Cannabinoid Receptor, BDNF, and Recovery from Excitotoxicity

Brain Derived Neurotrophic Factor (BDNF) is an abundant secreted growth factor in the

central nervous system. The brain appears to require BDNF for synaptic plasticity, and

dysfunction in BDNF is present in many diseases including major depressive disorder,

schizophrenia, addiction, and more (Autry et al., 2012). The binding of mature BDNF to the

tropomyosin-related kinase B (TrkB) receptor can activate a variety of pathways including

48

stimulation of the mitogen-activated protein kinase (MAPK) or ERK pathway, phospholipase C γ

(PLCγ) activation of inositol trisphosphate (IP3) leading to release of intracellular calcium stores

and activation of calmodulin kinase and protein kinase C (PKC), and phosphatidylinositol 3-

kinase (PI3K) activation of AKT leading to increased survival (Autry et al., 2012). BDNF is

essential for axon growth and synaptic plasticity (Autry et al., 2012). BDNF knockout mice die

shortly after birth, likely due to respiratory suppression (Balkowiec et al., 1998).

Intrastriatal injection of quinolic acid is often employed as a model of induced

excitotoxicity. Quinolic acid injection decreases glutamate uptake and increases calcium uptake,

leading to astrogliosis after seven days and progressing to neuronal death after fourteen days

(Pierozan et al., 2014). The injected animals exhibit cognitive and motor deficits (Pierozan et al.,

2014).

De March et al. (De March et al., 2008) investigated the relationship between CB1

cannabinoid receptor and BDNF. Male Wistar rats were anesthetized and injected with 1 μL of

100 mM quinolic acid into the right striatum. One week later, the cortical BDNF immunostaining

intensity was significantly decreased and the CB1 receptor immunostaining intensity was

unchanged when compared with control. After six weeks, the BDNF remained significantly

decreased and the CB1 receptor immunofluorescent intensity was significantly increased when

compared with control (De March et al., 2008). The six week finding was found to be paralleled

using the ELISA assay to quantify BDNF protein abundance and [3H]CP-55,940 binding to

approximate CB1 receptor abundance (De March et al., 2008). De March et al. interpreted this to

mean that BDNF and CB1 receptor cortical protein abundance change after a lesion. They

investigated further with the goal of determining whether the early time point decrease in BDNF

protein abundance was the cause of the increased CB1 receptor abundance at later time points. In

a second publication by the group (De Chiara et al., 2010), male 8-10 week old C57/Bl6 mice

were sacrificed and coronal corticostriatal slices were patch clamped to measure postsynaptic

electrical current. In a control situation, the application of dual agonist HU210 decreased the

49

spontaneous inhibitory postsynaptic electrical current (sIPSC) from 100% at time zero down to a

maximal inhibition to 80% at 15 to 20 minutes, and returning to the basal 100% by 30 minutes

(De Chiara et al., 2010). The application of HU210 did not alter the sIPSC in slices taken from

mice that had been inject with BDNF 24 hours before sacrifice. Preapplication of BDNF also

prevented HU210-induced changes in the sIPSC frequency. The ability of BDNF to block the

drug induced decrease in sIPSC was specific to HU210; the response of the sIPSC to baclofen to

decrease from 100% to 40% of predrug frequency was not altered by pretreatment with BDNF

(De Chiara et al., 2010). The actions of HU210 and BDNF were likely CB1 receptor and Trk

receptor specific, respectively, because the effects were abrogated by CB1 receptor antagonist

AM251 and the broad spectrum tyrosine kinase receptor inhibitor lavendustin A. Because the

preapplication of BDNF did not affect the HU210-induced decrease in spontaneous excitatory

postsynaptic electrical current (sEPSC) frequency (De Chiara et al., 2010), we can interpret this

to mean that the BDNF is not affecting glutamate release itself and the BDNF inhibition of CB1

receptor is limited to CB1 receptors controlling GABA-mediated IPSCs. De Chiara et al. applied

methyl-β-cyclodextrin in order to investigate whether the decrease in sIPSC by HU210 was

cholesterol dependent, and found no different in HU210 response before or after application of

methyl-β-cyclodextrin, meaning that this particular CB1 cannabinoid receptor affect is cholesterol

independent (De Chiara et al., 2010), although the action of BDNF does depend on cholesterol

abundance. Taken together, these data can be interpreted to mean that striatal CB1 receptor

control of GABA-mediated IPSCs is inhibited by BDNF, without BDNF affecting glutamate

transmission or GABAB receptors.

Marsicano et al. (Marsicano et al., 2003) also investigated the roles of CB1 receptor and

BDNF in excitotoxicity. Their hypothesis was as follows. Abnormally high spiking activity can

damage neurons. A signaling system to protect neurons from the consequences of abnormal

discharge may exist. This signaling system that protects neurons might be identified by

triggering toxic levels of excitation and measuring whether or not suspected excitoprotectants had

50

increased. Kainic acid can be used to model excitotoxicity in neurons. Kainic acid stimulates

kainate receptors and mimics glutamate excitotoxicity (Zheng et al., 2011). Twenty minutes after

kainic acid injection, the abundance of anandamide in the C57/Bl6 mouse brain significantly

increased from 0.5 to 1.5 pmol/mg extract (Marsicano et al., 2003). This increased anandamide

abundance after kainic acid stress lead Marsicano et al. to investigate CB1 receptor knockout mice

response to excitotoxicity. Wild type or CB1 receptor knockout mice were injected with 30

mg/kg of kainic acid and the result was that the CB1 receptor knockout mice had a much more

severe reaction than the wild type mice. The behavioral score at fifteen minutes was 2 in the wild

type mice and 4 in the CB1 receptor knockout mice. More than 75% of the CB1 receptor

knockout mice died within one hour of kainic acid injection (Marsicano et al., 2003). The

behavioral score of the partial knockout CB1 receptor heterozygote mice could be increased from

2 to above 4 by functional depletion of the heterozygote abundance CB1 receptor by application

of the CB1 receptor antagonist SR141716 (Marsicano et al., 2003). Marsicano et al. then

investigated which type of neuron was responsible for this CB1 receptor effect by using a

Ca2+

/calmodulin-dependent kinase IIα (CaMKIIα Cre) gene specific CB1 receptor knockout

mouse that knocked out CB1 receptor only in principal forebrain neurons but not adjacent

inhibitory interneurons. After 15 minutes of 30 mg/kg kainic acid injection the CaMKIIα

principal forebrain neuron specific knockout mice had a significantly increased behavioral score

of 4 compared with the floxed control mice with a behavioral score of 2 (Marsicano et al., 2003).

Marsicano et al. also measured BDNF mRNA abundance in the kainic acid treated animals. In

the floxed control animals, kainic acid significantly increased the abundance of BDNF in the CA1,

CA2, and dentate gyrus regions of the hippocampus. However, in the principal forebrain neuron

specific CB1 receptor knockout CaMKIIα Cre mice, kainic acid failed to increase the mRNA

abundance of BDNF (Marsicano et al., 2003). Taken together, these data can be interpreted to

mean that ligand stimulated CB1 receptor activity in principal forebrain neurons protects the brain

51

from glutamate excitotoxicity, and that part of the mechanism of this protection from

excitotoxicity involves increasing the mRNA production of hippocampal BDNF.

1.6 ROLE OF ENDOGENOUS CANNABINOID SIGNALING IN NEURONAL

FUNCTIONAL DEVELOPMENT

The brain is composed of many connections between neuronal axons and dendrites called

synapses. For example, there are approximately 4 billion synapses per mm2 in the 5 week old rat

cortex (McAllister 2007). The formation of these synapses, or synaptogenesis, is only partially

understood and appears to be regulated by diverse mechanisms depending upon the specific type

of synapse, neuron, and brain region involved. In the hippocampus, accumulation of synaptic

vesicle precursors and piccolo-transport vesicles may lead to synapse formation in approximately

an hour (McAllister 2007). Also in the hippocampus, neuroligin scaffold complexes of the

proteins PSD-95, Shank, and guanylate kinase-associated protein may lead to synapse formation

in 2 hours (McAllister 2007). In cortical neurons, mobilization of synaptic vesicle precursors

and N-methyl-D-aspartate receptors can lead to synapse formation in 7 minutes (McAllister 2007).

Also in cortical neurons, filopodia from dendritic growth cones may define the site of synapse

stabilization and formation (McAllister 2007). The full variety of synapse formation mechanisms

has not yet been completely characterized and the proteins and signaling pathways involved are

still being discovered.

Both CB1 and CB2 cannabinoid receptors have been implicated in neuronal axon guidance

during synaptogenesis. CB1 receptors are located in the tip of Xenopus laevis spinal neuron

axonal growth cones, and bath application of the endogenous ligand anandamide was found to

disrupt the growth cone’s ability to turn in response to an electric field (Berghuis et al., 2007).

Both CB1 and CB2 receptors are present on the tip of the growth cone of mouse retinal ganglionic

neurons (Duff et al., 2013). Mouse retinal explant and primary neuron growth cone surface area

52

was decreased by CB2 receptor selective agonists JWH015 and JWH13, and increased by and

CB2 receptor selective antagonists AM630 and JTE907 (Duff et al., 2013).

In general, all studies of the effect of cannabinoid receptor stimulation on neurite length

performed in neuroblastoma cell lines has resulted in an increase in extension length. There are

two exception studies (Ishii et al., 2002; Zhou et al., 2001) in which cannabinoid receptor

stimulation did not increase extension length of neurites in neuroblastoma. The methods of these

two investigations (Ishii et al., 2002; Zhou et al., 2001) were unusual because both were

situations in which cell death was likely occurring. The cannabinoid receptors are known to

induce apoptosis through de novo synthesis of ceramide and activation of p38 MAPK (Fonseca et

al., 2013). One study (Zhou et al., 2001) involved pretreatment with 48 h of serum starvation,

enough time to cause cell death (Pasquariello et al., 2009), and one involved application of 1 μM

anandamide to virally infected cells grown on glass (Ishii et al., 2002).

Zhou et al. serum starved the murine N1E-115 neuroblastoma cells for 48 hours to induce

neurite outgrowth before then applying HU210 to measure the acute 15 minute change in pre-

formed neurite lengths (Zhou et al., 2001). Visually, after cannabinoid stimulation the cell nuclei

have gained punctae, a change that supports an apoptotic phenotype. As few as 24 hours of

serum starvation are capable of killing cultured cells (Braun et al., 2011). Both human and

murine neuroblastoma cells are generally cultured with 10% serum at all times to avoid growth

inhibition and cell death. The doubling time for N1E-115 is listed as 20 hours on the Swiss

Institute of Bioinformatics Expasy database (nearly three times faster than the 55 hour time listed

for the human SH-SY5Y cells used in our study). It is likely that 48 hours of serum starvation

was enough to introduce serum deprivation stress (Braun et al., 2011) on the 20 hour doubling

time N1E-115 murine neuroblastoma cells, predisposing the cells to a death response.

In the second study in which cannabinoid receptor stimulation induced neurite retraction,

Ishii et al. observed retraction of formed extensions after applying 1 μM anandamide to

retrovirally infected rat B103 cells (Ishii et al., 2002). This concentration of anandamide

53

approaches the concentration that is known activate cannabinoid receptor dependent apoptosis;

concentrations of 20 μM anandamide induced cannabinoid receptor antagonist-reversible

apoptosis in dendritic cells (Do et al., 2004) and 25 μM anandamide induced cannabinoid

receptor antagonist-reversible apoptosis in cytotrophoblasts (Costa et al., 2015).

In summary, these two reports in which HU210 or anandamide cannabinoid receptor

stimulation caused neurite retraction likely induced cell death which appeared similar to

retraction because dying cells can become more round, “balling up” in shape. Other than these

two studies, all other work with neuroblastoma cell lines has demonstrated that cannabinoid

receptor stimulation increased neurite length.

1.6.1 Cannabinoid Receptor Regulation of Oligodendrocyte Neuron Spatial Communication

Via Slit2/Robo1 Signaling

CB1 and CB2 cannabinoid receptors are expressed in multiple brain cell types including

neurons and oligodendrocytes. JZL184 causes an accumulation of 2-AG by inhibiting its

hydrolysis by MAGL. JZL184 significantly increased the abundance of 2-AG from 20 to 30

nanomol per gram of tissue in the cortex of embryonic mice whose mothers were intraperitoneal

injected with JZL184 from embryonic day 12.5-18.5. The abundance of anandamide, 2-linoleoyl

glycerol , or oleoylethanolamide was unaltered (Alpar et al., 2014). Alpar et al. showed that, in

pregnant mice intraperitoneal injected with JZL184 from embryonic day 12.5-18.5, the embryos

developed abnormal corticofugal axonal phenotypes and errors in axon fasciculation (Alpar et al.,

2014). The JZL184 had significantly increased the corpus callosum spread from 150 to 250

micrometers, and significantly increased the area of striatal fascicle from 200 to 500 micrometers

square when compared with control (Alpar et al., 2014). The altered morphology was very

similar to that seen in CB1 receptor knockout mice; the diameter of the individual fascicle was 5

micrometers in wild type animals, 15 micrometers in wild type animals treated with JZL184, and

15 micrometers in CB1 receptor knockout animals (Alpar et al., 2014). They interpreted this to

54

mean that in the time period from mouse embryonic days 12.5-18.5, either excessive

accumulation of cannabinoid ligand 2-AG by the application of JZL184 or knockout of CB1

receptor both alter neuron fasciculation to generate a similar morphology by altering how the

endogenous cannabinoid signaling pathway is guiding neuronal fasciculation and development in

a CB1 receptor and possibly also CB2 receptor dependent manner.

Slit 1, 2, and 3 are a family of secreted glycoproteins that bind Roundabout (Robo)

receptors (Hohenester 2008). Humans express three Slit family members, large homologously

structured glycoproteins that share a leucine rich repeat at the N terminus. This leucine rich

repeat, called D1-D4, is required for interaction with the extracellular domains of the Robo

receptors. Vertebrates express four Robo receptors in tissues including the brain, lung, kidney,

and mammary gland. Slit Robo signaling plays an important role in axon guidance during central

nervous system development (Hohenester 2008). Robo receptors span the plasma membrane and

exist both intracellularly and extracellularly. There are 500 intracellular Robo receptor amino

acid residues. The Robo receptor extracellular sequence contains multiple immunoglobulin like

domains and fibronectin type 3 repeats, which is similar to the structure of cell adhesion

molecules (Hohenester 2008). Slit activation of Robo is likely a result of a change in

oligomerization status, and may potentially be performed by one or both of two mechanisms.

First, Slit may bind to an inactive dimer and, with heparin sulfate stabilization, activate the Robo

as a monomer. Second, Slit binding to an inactive Robo monomer may induce dimerization, and

activation. Although it’s unclear which mechanisms is occuring in each situation of Slit Robo

signaling, the result of Slit glycoprotein binding to Robo receptors is generally chemorepulsion

for the cell (Hohenester 2008). It has recently been discovered that Robo2 inhibits the ability of

Slit to induce chemorepulsion via Robo1 (Evans et al., 2015), possibly by the extracellular

domain of Robo2 of one cell binding the extracellular domain of the Robo1 of another cell and

interfering with Slit binding to Robo1.

55

Alpar et al. observed that CB1 receptor and Robo1 colocalize in embryonic human and

mouse corticofugal axons (Alpar et al., 2014). In cultured embryonic day 16.5 C57/Bl6 mouse

cortical neurons, the application of the MAGL inhibitor JZL184 (which causes accumulation of

2-AG) led to a significant 50% increase in the ratio of Robo1 located in the growth cone tip

compared with the non-tip axon length (Alpar et al., 2014). This increased localization of Robo1

in the growth cone tip was CB1 receptor dependent because it was reversed upon coapplication of

CB1 receptor antagonist O2050. Robo1 accumulation in the growth cone tip also was not

observed in CB1 receptor knockout mouse derived neurons treated with JWL184. The ability of

CB1 receptor to enrich axonal growth cone localization was Robo1 specific as it was not observed

for Robo2 (Alpar et al., 2014).

CB1 receptor-induced Robo1 growth cone enrichment was dependent upon MEK1/MEK2

and cJun phosphorylation because the coapplication of U0126 or SP600125 prevented the

JZL184 effect (Alpar et al., 2014). Recalling that JZL184 and CB1 receptor knockout both

induced a significant increase in callosum spread in micrometers, it was found that JZL184 and

the environment of increased 2-AG and CB1 receptor signaling did not alter the corpus callosus

spread of Robo1 knockout mouse (Alpar et al., 2014). Based upon these data, Alpar et al.

concluded that in embryonic day 16.5 cortical neurons, stimulation of CB1 receptor leads

chemorepulsion via a MEK1/MEK2 and cJun phosphorylation mediated signaling pathway

leading to enrichment of Robo1 in the growth cone tip when compared with the axon length.

Because Robo1 is a chemorepulsive signaling receptor, this CB1 receptor stimulated enrichment

in Robo1 growth cone abundance increases chemorepulsion of the axon tip and steers the axon tip

away from Robo1 ligand stimuli. The Robo1 receptor stimuli was most likely to be its known

ligand Slit2, which Alpar et al. discovered is contained and excreted solely by oligodendrocytes,

but not astrocytes (Alpar et al., 2014).

Alpar et al. then investigated how increased cannabinoid signaling affects the Slit2

producing cells, oligodendrocytes. Application of JZL184 to increase 2-AG in cocultured

56

neurons and oligodendrocytes significantly increased the distance between the neuronal growth

cone and the oligodendrocytes from 10 to 15 micrometers in a manner that was not affected by

scrambled RNAi but was significantly reversed by Robo1 RNAi (Alpar et al., 2014). They

identified oligodendrocytes by labeling 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), an

enzyme required for oligodendrocyte creation of the myelin sheath and which is expressed

exclusively by fully differentiated myelinating oligodendrocytes. Oligodendrocytes are not

functional at myelination unless they express CNPase. Gaining expression of CNPase is a type of

functional differentiation for oligodendrocytes. Alpar et al. found that application of JZL184 to

increase 2-AG significantly increased the oligodendrocyte CNPase abundance, increasing from

0.5 to 4 CNPase positive oligodendrocytes per 100 micrometers square of area. The 2-AG

stimulated increase in functionally differentiated oligodendrocytes was not different in CB1

receptor knockout embryos (Alpar et al., 2014), and not dependent upon the presence of CB1

receptor. Alpar et al. interpreted this to mean that the oligodendrocyte differentiation was not

mediated by CB1 receptor, but perhaps by CB2 receptor. To test this, Alpar et al. measured the

ability of CB2 receptor antagonist AM630 to decrease Slit2 protein intensity in the

oligodendrocyte end feet. The protein abundance of Slit2 in the oligodendrocytes had increased

significantly from 1 to 1.4 intensity units in JZL184 treated oligodendrocytes. Coapplication of

AM630 abrogated the JZL184 effect on Slit2, meaning that the JZL184-induced increase in 2-AG

that resulted in increased Slit2 secretion and CNPase positive oligodendrocytes had signaled in a

CB2 receptor specific manner (Alpar et al., 2014).

Alpar et al. interpreted these data to mean that endocannabinoids simultaneously engage

cell-type-specific receptors to orchestrate chemoreplusion in-trans. CNPase positive

oligodendrocytes produce Slit2 chemorepellants that alter neuronal axon path via growth cone

located Robo1 signaling. Both CB2 cannabinoid receptor on oligodendrocyte precursors and CB1

cannabinoid receptor on neurons are involved in this axonal extension signaling pathway. CB2

receptor increases CNPase and Slit2 abundance in oligodendrocytes and CB1 receptor increases

57

Robo1 growth cone localization in neurons relative to the axon length, which can lead to Robo1

sensing of Slit2 and resultant chemorepulsion of the growing axon.

1.6.2 CB1 Receptor Regulation of RhoA GTPase G Proteins

There is a family of small GTPase or G proteins which have activity to hydrolyze GTP to

GDP. This is similar to the activity of the alpha G protein subunit, but independent of a G Protein

Coupled Receptor. The small G proteins are regulated by a variety of different mechanisms.

Guanine nucleotide exchange factors (GEFs) catalyze the physical exchange of GDP for GTP,

generally resulting in activation (Cook et al., 2014). Guanosine nucleotide dissociation inhibitors

(GDIs) bind to the cytosolic inactive form of the small G protein. GTPase activating proteins

(GAPs) accelerate the hydrolysis of GTP to GDP, terminating the small G protein’s active GTP

bound state (Huang et al., 2017). In general, the action of the G protein is as follows. An

extracellular signal leads to the GEF to exchange GTP for GDP, activating the membrane bound

small G protein. The GTP bound active form performs its action until either its inherent GTPase

activity or facilitation of hydrolysis by a GAP causes hydrolysis of the GTP into GDF. Inactive

GTPases may become dissociated from the plasma membrane, and a GDI may increase the

amount of time spent away from the plasma membrane and in the GDP bound form. Eventually

the GDI dissociates from the GTPase and with the GDI dissociated the small G protein is

available for a GEF to exchange its GDP for GTP and become active again (Kawano et al., 2014).

The RAS superfamily of small GTPases shares a common core G domain which has the

capacity for nucleotide exchange and is the location where GTP is hydrolyzed into GDP. There

are five families in the RAS superfamily: Ras which are generally involved in proliferation, Rho

which are generally involved in cellular morphology, Ran which are generally involved in nuclear

transport, and Rab and Arf which are generally involved in vesicular transport. Ras GTPases

coordinate multiple signal pathways with precise spatial control and are crucial for axonogenesis

(Hall et al., 2010). The axon’s unique actin, microtubule, and neurofilament skeleton serves as a

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metaphorical railroad for transporting proteins along the axon to the neuronal synapse (Kevenaar

et al., 2015). The RAS superfamily includes the actin regulating Rho family of GTPases, with

example members being Cdc42, Rac1, and RhoA. Myosin motors hydrolyze ATP to force

movement along the actin cytoskeleton. Rho GTPases control spine actin dynamics and

morphology, controlling where myosin directs protein traffic (Kneussel et al., 2013).

RhoA is a member of the Rho family of the RAS superfamily of GTPases. RhoA’s

downstream targets involve the actin cytoskeleton, cyclin D cell cycle regulation,

Serine/Threonine-Protein Kinase N2, regulation of apical junctions and cell-cell adhesion, Sox9

which is involved in developmental ontology, and transforming growth factor which is inolved in

tumorigenesis. In neurons, RhoA plays a role in altering cell extension, mobility, and migration.

GEFs phosphorylate RhoA to activate it. The C terminus of RhoA may receive a twenty carbon

geranylgeranylation prenylation to increase its lipophilicity and membrane anchor time. The

active form of RhoA is GTP bound and membrane localized, and results in increased activity of

effector molecules such as Rho-associated, coiled-coil containing protein kinase1 (ROCK1) and

diaphanous homolog (DIAPH1). G13 can directly interact with the Rho GEF domain of

p115RhoGEF (Chen et al., 2003). G12/13 is known to activate RhoA and ROCK (Bian et al.,

2006) to stimulate migration of human ovarian cancer cells. In CHO cells, G12/13 activation

stimulates RhoA activity (Sobel et al., 2015). Application of the ROCK inhibitor GSK269962

abrogated the ability of FTY720 to affect cell shape through the S1P1 receptor, as did knockdown

of Gα12 or Gα13 (Sobel et al., 2015). The application of ROCK-specific inhibitor Y27632

prevented LPA-induced filamentous actin polymerization in Swiss 3T3 fibroblasts, and prevented

LPA-induced neurite retraction in N1E-115 neuroblastoma (Narumiya et al., 2000).

Although the active form of RhoA is GTP bound and membrane localized, not all GTP

bound RhoA is active. RhoA activity can be regulated by phosphorylation at serine 188 and at

serine 26. The phosphorylation of serine 188 may be performed by protein kinase A (PKA) or

PKG. PKA phosphorylation of serine 188 decreased membrane localized GTP-bound active

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RhoA-GTP and through GDI activity increased the proportion of RhoA-GTP in its soluble

inactive form (Lang et al., 1996). This translocation of RhoA from the membrane into the cytosol

was repeated in YT natural killer cells with prostaglandin E2, isoproterenol, and the AC stimulant

forskolin, compounds known to lead to an increase intracellular cAMP (Lang et al., 1996).

Increasing intracellular cAMP and phosphorylation of serine 188 led to the GDI sequestration of

the GTP bound form of RhoA in the cytosol, despite other GTP bound forms of RhoA being the

membrane bound, active version (Lang et al., 1996). The phosphorylation of GTP bound RhoA

can separate it from its effectors independently of its GTP/GDP cycling. The PKA

phosphorylated GDI bound GTP-RhoA is no longer necessarily the active membrane bound form.

The location of the RhoA must therefore be considered and it should not be assumed that the GTP

bound RhoA is the active form. Cytosolic GDI bound RhoA-GTP is both GTP bound and

inactive. This affects the interpretation of a number of research results involving cannabinoid

receptors and RhoA not all of which quantified whether or not the RhoA-GTP was in the

membrane (active) or cytosolic (inactive, phosphorylated at S188, GDI bound) state. Some

researchers instead measure the GTPase activity of RhoA to measure RhoA activation (Berghuis

et al., 2007).

The effect of small G protein activation on neurons has become better understood

because of the investigations into the mechanism behind lysophosphatidic acid (LPA) and brain

derived neurotropic factor (BDNF) induced neuronal outgrowth. Decreased actin polymerization

and increased myosin activity results in filopodia retraction and repulsive turning (Yuan et al.,

2003). Rho family members Rac and Cdc42 promote neurite extension, while Rho inhibits or

collapses growth cones (Yuan et al., 2003). LPA induced chemorepulsion is known to be

mediated by Rho activated regulation of myosin (Frisca et al., 2013; Kim et al., 2011; Retzer et

al., 2000). Clostridium difficile Toxin B inhibits Rho GTPases by glucosylating Thr37,

preventing effector coupling (Aktories et al., 2000). Toxin B blocked both LPA and BDNF

induced growth cone turning (Yuan et al., 2003). The application of Y27632, an inhibitor of

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ROCK1, allowed for the LPA-induced increased extension length in the wild type and dominant

negative RhoA model of cultured Xenopus laevis spinal neurons, but not in the constitutively

active Cdc42 model (Yuan et al., 2003). These results were interpreted to mean that there is

crosstalk between the Cdc42 and RhoA pathways in myosin regulation. Cdc42 mediates BDNF

induced chemoattraction, while the BDNF induced repulsion observed in situations of low PKA

activity depended on both Cdc42 and RhoA (Yuan et al., 2003). The authors concluded that

BDNF may induce either chemoattraction or repulsion depending on intracellular calcium, PKA,

and cAMP concentrations (Yuan et al., 2003). This finding opens the possibility that BDNF and

cannabinoid receptor function in neuronal outgrowth may overlap. Indeed, seven years later the

De Chiara group found evidence that BDNF potently inhibits CB1 receptor function in the

striatum via a cholesterol dependent, lipid raft signaling mechanism (De Chiara et al., 2010).

BDNF, LPA, and CB1 cannabinoid receptors regulate neuronal outgrowth via small G protein

regulation of myosin and actin.

In primary cortical neurons, dual cannabinoid receptor agonist WIN 55,212-2 induced an

“increase in RhoA GTPase activity” (as measured by a Cell Biolabs Inc kit that precipitated the

GTP bound form) that was maximal between five and ten minutes and returned nearly to baseline

by fifteen minutes (Berghuis et al., 2007). This increase in RhoA activity by WIN 55,212-2 was

abrogated by pretreatment with CB1 receptor specific antagonist AM251 (Berghuis et al., 2007).

In J774A macrophages, the application of methanandamide increases the ratio of RhoA-GTP to

Total RhoA in a CB1 receptor-dependent manner, which was abrogated by CB1 specific

antagonist AM281 and by siCB1 but not siCB2 (Mai et al., 2015). It is noteworthy that the non-

lipid raft-localized CB2 receptor is not capable of increasing RhoA GTP binding (Mai et al.,

2015), and that RhoA GTP binding as a result of methanandamide stimulating the CB1 receptor is

pertussis toxin sensitive (Mai et al., 2015), implicating the non-Gαz members of the Gαi/o family

of G proteins as the transducers of CB1 receptor that lead to activation of RhoA. One caveat is

that the Berghuis and Mai groups (Berghuis et al., 2007; Mai et al., 2015) measured GTP

61

hydrolysis and GTP to GDP ratio, not membrane to cytosol translocation, and Lang et al. (Lang et

al., 1996) showed that PKA phosphorylation of RhoA on serine 188 may lead to GDI

sequestration of the GTP bound form of RhoA away from the membrane, in the cytosol, where it

does not have the same ability to activate signal transduction molecules.

The effect of cannabinoid receptors on membrane versus cytosolic GTP bound RhoA has

been measured in prostate cancer and breast cancer cells (Laezza et al., 2008; Nithipatikom et al.,

2012). In DU145 human prostate cancer cells, the application of dual cannabinoid receptor

agonist WIN 55,212-2 decreased the RhoA-GTP to total RhoA ratio, and the application of CB1

receptor antagonist AM251increased the ratio of GTP bound to total RhoA (Nithipatikom et al.,

2012). In PC3 human prostate cancer cells, the application of cannabinoid agonist WIN 55,212-2

increased the RhoA in the cytosol and decreased the RhoA in the membrane, and the application

of CB1 receptor antagonist AM251 increased the RhoA in the membrane and decreased the RhoA

in the cytosol. In human prostate cancer cells, cannabinoid receptor stimulation decreased RhoA

activity by leading to a decrease in the GTP to GDP bound ratio of RhoA and increasing

translocation to the cytosol (Nithipatikom et al., 2012). As in the prostate cancer cell lines,

MDA-MB-231 triple negative breast cancer cells treated with stable anandamide analog 2-

methyl-2’-F-anandamide also experienced a translocation of RhoA from the membrane to the

cytosol (Laezza et al., 2008). The result of dual cannabinoid receptor activation in primary

cortical neurons was an increase in GTP to GDP bound RhoA ratio (Berghuis et al., 2007), while

the result in breast and prostate cancer cells was a decrease in GTP to GDP bound RhoA ratio

(Laezza et al., 2008; Nithipatikom et al., 2012). Unlike neurons, in both prostate and breast

cancer cell lines, stimulation of the CB1 receptor inactivated RhoA by decreasing the GTP to

GDP bound ratio, and these two manuscripts did confirm a subsequent translocation from the

active site on the membrane to inactive sequestration in the cytosol. The SH-SY5Y

neuroblastoma cells have characteristics of both neurons and cancer cell lines. Whether the SH-

SY5Y neuroblastoma cells behaves like the primary neurons to increase the ratio of GTP to GDP

62

bound RhoA or the prostate and breast cancer cells to decrease the ratio of GTP to GDP bound

RhoA is unclear.

CB1 receptor can regulate small G proteins of the Rho family not only via activation of

Gα12/13 but also by activation of Gαi/o. Rap1 GTPases (Rap1a and Rap1b) are in the Ras-

related proteins family of small G proteins. Rap1 is present in neurons and regulates cell polarity

(Shah et al., 2017). The genetic knockout of Rap1 prevents formation of a synapse (Shah et al.,

2017). Anandamide decreases GTP-bound proportion of Rap1 and decreases B-Raf mediated

ERK phosphorylation in rat pheochromocytoma PC12 cells (Rueda et al., 2002). One mechanism

of action for CB1 activation of Rap1 is by Gαi/o induced ubiquitination and proteasomal

degradation of Rap1GAPII, an inhibitor of Rap1 (Jordan et al., 2005), which leads to neurite

outgrowth in the Neuro2A cells. In Neuro2A mouse neuroblastoma cells, CB1 receptor activity

through pertussis toxin sensitive Gαi/o activates Rap1 to induce Src and Stat3 phosphorylation

(He et al., 2005) and leads to neurite outgrowth (Bromberg et al., 2008a). CB1 receptor activation

of Gα12/13 decreases neurite extension, while CB1 activation of Gαi/o increases neurite extension.

63

Figure 1.6.3 Concept map of Gα12/13 and Gαi/o mediated cannabinoid signaling.

References: 1. (Diez-Alarcia et al., 2016), 2. (Siehler 2009), 3. (Garzon et al., 2009), 4. (Meng et

al., 1999), 5. (Jordan et al., 2005), 6. (Bromberg et al., 2008b), 7. (Gutkind 2000), 8. (Berghuis et

al., 2007), 9. (Mei et al., 2011), 10. (He et al., 2005), 11. (Yuan et al., 2003), 12. (Lawson et al.,

2014), 13. (Tortoriello et al., 2014), 14. (Alpar et al., 2014), 15. (Bromberg et al., 2008a), 16.

(Shin et al., 2012), 17. (Zhou et al., 2011), 18. (Keimpema et al., 2013)

64

1.6.4 Nerve Growth Factor Regulates CB1 Receptor Cytoskeletal Control Via BRCA1

Keimpema et al. investigated the role of localized degradation of cannabinoid ligands in

regulating the process of neurite extension (Keimpema et al., 2010). They quantified protein

abundance in the growth cone tip compared with the rest of the extension and found that DAGL,

MAGL, and CB1 receptor proteins are concentrated in specific motile regions on the growing

axon, suggesting functional activity of the cannabinoid system at these locations (Keimpema et

al., 2010). They hypothesized that in vivo, 2-AG concentration may be regulated by degradation

of MAGL at the protein level. Next, they utilized proteasome inhibition as a method of altering

protein abundance. Lactacystin is a molecule that was isolated from streptomyces lactacystinaeus

bacteria and found to increase neurite extension in Neuro2a mouse cells (Omura et al., 1991).

Lacacystin’s mechanism of action was discovered to be to prevent 20-S proteasomal degradation

of proteins (Fenteany et al., 1995). Keimpema et al. theorized that lactacystin’s mechanism of

action to increase neurite extension length was to inhibit proteasomal degradation of MAGL,

increasing MAGL’s abundance, and interfering with normal 2-AG concentration regulation.

Using a Western blot, Keimpema et al. showed that lactacystin increased total MAGL protein

abundance in primary cortical neurons isolated from E14.5-16.5 C57/Bl6 mice. This Western

blot data can be interpreted to mean that lactacystin’s inhibition of the proteasome had interfered

with normal MAGL protein degradation and function, increasing the net total amount of MAGL

protein, but in Figure 7 Keimpema showed that lactacystin actually decreased MAGL abundance

in the critical motile section of the extension (Keimpema et al., 2010). By interfering with

protein recycling, lactacystin had decreased MAGL abundance in the motile segment of the

neurite (Keimpema et al., 2010). Similar to how lactacystin decreases MAGL in the motile

segment in the tip of the neurite, JZL184 is also an inhibitor of MAGL. Application of JZL184 to

primary cortical neurons isolated from E14.5-16.5 C57/Bl6 mice also increased extension length,

and this increase in length could be prevented by a coapplication of either the 2-AG inhibitor

THL or the CB1 receptor specific antagonist O-2050. This work showed that the mechanism of

65

action of a known neurite extension increasing compound, lactacystin, was through decrease of

the protein abundance of cannabinoid system component MAGL locally in the motile section at

the tip of the growing extension (Keimpema et al., 2010). Lactacystin decreased motile tip

localized MAGL, decreased the hydrolysis of 2-AG, increased local 2-AG abundance, and

increased CB1 receptor activation specifically in the motile tip of the extension (Keimpema et al.,

2010).

Nerve Growth Factor (NGF) was the first discovered growth factor (Cohen et al., 1954;

Levi-Montalcini 1952). Depriving sensory and sympathetic neurons of NGF caused their death

(Levi-Montalcini et al., 1963) via a NEDD2/caspase2 dependent pathway (Park et al., 1998; Troy

et al., 1997). NGF binds to tyrosine kinase receptor TrkA (Kaplan et al., 1991). The TrkA

receptor can either be activated at the cell surface to signal via PI3 kinase / AKT and increase

survival, or the TrkA receptor can be endocytosed to signal from vesicles and increase neuronal

extension length (Zhang et al., 2000) if inactivation of Rab5 blocks endosomal fusion to establish

of a signaling endosome (Liu et al., 2007). RabGAP5 controls whether a signaling endosome

forms by inactivating Rab5 by promoting its GTP hydrolysis (Liu et al., 2007). NGF can increase

neuronal extension length via a JAK/Stat3 mediated signaling pathway (Quarta et al., 2014).

Nerve Growth Factor (NGF) is a trophic nutrient required for neuron survival. It binds to the

TrkA receptor and promotes neurite outgrowth. Due to the use of serum in cell culture, it is

possible that our SH-SY5Y cells are exposed to and stimulated by NGF.

Keimpema et al.’s discovery that exogenous lactacystin interfered with a MAGL

proteasome degradation pathway begged the question of which factors endogenously regulate

MAGL protein abundance in vivo. When BRCA1 and BARD1 heterodimerize, the resulting

complex functions as an E3 ubiquitin ligase (Wu et al., 2008). In cultured primary cortical

neurons isolated from E14.5-16.5 C57/Bl6 mice, NGF increased the total neurite length and the

number of vesicular acetylcholine transporter (VAChT) positive neurites in a manner that can be

completely inhibited by application of O2050, a CB1 receptor specific antagonist (Keimpema et

66

al., 2013). NGF induces spatial regulation of MAGL, affecting the “MAGL delay” or localized

protein abundance shift in the motile tip of the growth cone that was seen in lactacystin treated

cells. After three divisions, NGF treated cultured cortical neurons show an increase in BRCA1

mRNA expression (Keimpema et al., 2013). Cisplatin is a chemical inhibitor of BRCA1 activity

that decreases E3 ubiquitin ligase activity. The application of cisplatin alone decreased the

“MAGL delay”, or the distance between MAGL immunoreactivity (a quantified way to measure

MAGL in the motile tip of the extension), as cisplatin interfered with BRCA1 E3 ubiquitin ligase

activity and caused the accumulation of MAGL. Application of cisplatin plus NGF prevented

NGF from increasing neurite length (Keimpema et al., 2013). This means that in vivo, these

cholinergic neurons have a signaling system in which NGF increases the protein abundance of

BRCA1 to increase E3 ubiquitin ligase activity, decreasing the protein abundance of MAGL,

increasing the concentration of 2AG and increasing local CB1 receptor activation to drive

cytoskeletal changes that result in increased neurite extension. NGF regulates CB1 receptor

mediated cytoskeletal control via BRCA1 (Keimpema et al., 2013).

67

Figure 1.6.4 Concept map outlining G protein mediated cannabinoid signaling pathways.

References: 1. (Diez-Alarcia et al., 2016), 2. (Siehler 2009), 3. (Garzon et al., 2009), 4. (Meng et

al., 1999), 5. (Jordan et al., 2005), 6. (Bromberg et al., 2008b), 7. (Gutkind 2000), 8. (Berghuis et

al., 2007), 9. (Mei et al., 2011), 10. (He et al., 2005), 11. (Yuan et al., 2003), 12. (Lawson et al.,

2014), 13. (Tortoriello et al., 2014), 14. (Alpar et al., 2014), 15. (Bromberg et al., 2008a), 16.

(Shin et al., 2012), 17. (Zhou et al., 2011), 18. (Keimpema et al., 2013), 19. (Wang et al., 2018)

68

1.6.5 CB1 Receptor Regulation of Transcription Factors Involved in Neuronal Functional

Differentiation

The discovery of cannabinoid receptors in the nucleus (Glass et al., 1997; McIntosh et al.,

1998) raised the question: Is cannabinoid signaling affecting DNA production? There are a

variety of mechanisms through which this might be possible. For some nuclear receptors such as

the estrogen receptor, the lipophilic steroid molecule enters the plasma membrane of the cell and

either causes intracellular receptors to translocate to and become enriched in the nucleus (Parikh

et al., 1987) or activates receptor that are already present in the nucleus. Estrogen receptor

contains a DNA binding domain (Weinberg et al., 2007) and a nuclear localization sequence

(Casa et al., 2015) that allow for direct binding to DNA and an increase in transcription.

However, cannabinoid receptors don’t have known DNA binding domains. Without a DNA

binding domain it is unlikely that they are directly binding DNA, but they might be altering the

activity of or recruitment of nuclear transcription factors. A transcription factor could be defined

as any protein that can bind DNA and regulate its production. Transcription factors may be

regulated in a variety of manners including control of the transcription factor mRNA production

rate, mRNA and protein degradation rate, protein conformational activation, protein spatial

localization, etc. Cannabinoid ligands, estrogen, and testosterone all possess nonpolar

hydrophobic regions and great lipophilicity, and may not be confined by lipid bilayers. A

cannabinoid ligand might possibly enter the nucleus and bind to a nuclear cannabinoid receptor

that would signal through G proteins, resulting in an altered transcription factor

microenvironment. Cannabinoid receptors might then alter production of DNA by changing

transcription factor abundance or transcription factor conformational activation.

Retinoic acid activation of retinoic acid receptors RAR and RXR is an important

transcriptional master regulator of neuronal differentiation (Janesick et al., 2015). Chicken

ovalbumin upstream promoter transcription factor 1 (COUP-TF1) is an orphan receptor that can

competitively inhibit RAR and RXR to alter retinoic acid receptor interaction with DNA response

69

elements (Tran et al., 1992). Ctip2 is Coup-TF1 Interacting Protein 2. Ctip2 is a transcription

factor required for differentiation of medium spiny neurons in the striatum (Arlotta et al., 2008)

and corticospinal neurons (Diaz-Alonso et al., 2012). Diaz-Alonso et al. (Diaz-Alonso et al.,

2012) investigated cannabinoid mediated changes in transcription factor protein abundance.

Satb2 decreases the abundance of Ctip2 by binding to MAR promoter regions and sterically

inhibiting the transcription of Ctip2. Embryonic day 16.5 CB1 receptor knockout mice cortices

had less Ctip2+ cells and more Satb2+ cells than wild type controls (Diaz-Alonso et al., 2012). A

three day application of dual cannabinoid receptor agonist HU-210 to cortical slices increased the

number of Ctip2+ and decreased the number of Satb2+ cells. The co-application of CB1 receptor

antagonist SR141716 abrogated this effect, showing that CB1 receptor was required (Diaz-Alonso

et al., 2012). Diaz-Alonso created aHib5 cell line reporter model in which luciferasewas inserted

downstream of the MAR promoter region of Ctip2. This model directly quantified Satb2 steric

inhibition of the MAR site because when Satb2 is present, the MAR site is blocked and luciferase

and Ctip2 are not produced. Application of HU210 increased luciferase reporter activity in an

SR141716-dependent manner (Diaz-Alonso et al., 2012), meaning that CB1 receptor activation

uncouples Satb2 from the MAR sequence and allows for Ctip2 to be transcribed. This result was

confirmed in FAAH knockout mice which had significantly greater Ctip2 protein abundance in

the cortex at postnatal day 2 (Diaz-Alonso et al., 2012). Diaz-Alonso concluded that CB1

receptor activity increased Ctip2+ neurons by decreasing Satb2 inhibition of Ctip2 transcription

(Diaz-Alonso et al., 2012). Activation of cannabinoid receptor signaling may lead to changes in

Ctip2 inhibition of COUP interference in RAR-RXR interaction with certain of its DNA response

elements changing differentiation fate. Cannabinoid receptor-mediated regulation of COUP via

Ctip2 may be an important way in which the brain regulates neuronal differentiation in response

to RA.

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1.6.6 CB1 Receptor Regulation of SCG10

The survival of the axonal growth cone depends upon a number of maintenance proteins

that are under constant regulation. Deprivation of these proteins for several hours could collapse

the growth cone. One of these maintenance proteins is neuron-specific isoform of stathmin 10,

SCG10, a tubulin dimer binding protein that dynamically regulates the microtubule cytoskeleton.

The small G protein Rnd1 enhances the microtubule destabilizing activity of SCG10, inducing

axon formation (Li et al., 2009). SCG10 is an axonal maintenance factor required for survival of

the growth cone (Shin et al., 2012). Removing SCG10 by shRNA-mediated knockdown

accelerated the degeneration of severed axons, and maintaining SCG10 levels following axotomy

delayed axon breakdown (Shin et al., 2012). JNK phosphorylated SCG10, inactivating it and

causing rapid degradation within 10 minutes (Shin et al., 2012; Tortoriello et al., 2014). SCG10

is a rapidly degraded protein, and in normal physiological circumstances it is similarly rapidly

replenished by new protein synthesis and efficient transport along the axonal extension (Shin et

al., 2012). Overstimulating JNK phosphorylation of SCG10 and its proteasomal degradation

threatens the survival of the growth cone and may cause its collapse over the course of hours.

Maternal exposure to Δ9-THC activates fetal CB1 receptors, which stimulates JNK ,

which phosphorylate SCG10 leading to its degradation (Tortoriello et al., 2014). Maternal Δ9-

THC exposure decreased SCG10 abundance in both mouse and human fetal brains (Tortoriello et

al., 2014). Decreasing the abundance of SCG10 threatens the survival of axonal growth cones

and can alter the architecture of the developing brain. Maternally exposed fetuses have impaired

formation of perisomatic baskets in the mouse cortex, increased density of boutons in the statum

radiatum of the mouse hippocampal CA1 subfield, and diminished orthodromic stimulation-

evoked long term depression of CA1 pyramidal cells (Tortoriello et al., 2014). CB1 receptor

stimulated JNK phosphorylation of SCG10, leading to SCG10 protein degradation that if

sustained for hours may lead to microtubule instability and growth cone collapse. The

phosphorylation and degradation of SCG10 is one mechanism of action that can explain how

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hours of bath application of cannabinoid receptor agonists cause CB1 receptor dependent growth

cone collapse, as demonstrated by bath application of anandamide and WIN 55,212-2 by

Berghuis et al. (Berghuis et al., 2007).

1.7 THE SH-SY5Y HUMAN NEUROBLASTOMA CELL MODEL

The SK-N-SH cell line was first isolated from an aspiration biopsy in December 1970 of the

bone marrow of a four year old girl with metastatic neuroblastoma (Biedler et al., 1973). The

patient was not responding to chemotherapeutic agents or radiation therapy, and she died in

January 1971. The aspiration biopsy cells were grown in Eagle’s minimum essential medium

supplemented with 20% fetal bovine serum, penicillin, streptomycin, and fungizone (Biedler et al.,

1973). After two days the floating cells were washed off . After three months, the cells began to

be transferred to new cultures every two weeks by using three to five minute exposure to 0.125%

trypsin and 0.02% EDTA in calcium and magnesium free phosphate buffered salt solution to

obtain a cell suspension. Medium was replaced on days 5, 9, and 12. Inoculations of 107 isolated

cells induced tumors in cortisone induced immunocompromised hamsters. The doubling time

was 44 hours (Biedler et al., 1973). The SK-N-SH cells were of two morphologies: one with

short delicate processes that sometimes exceeded 100 μm in length, the other of larger size and

with epithelioid cell morphology (Biedler et al., 1973). The cells was maintained in vitro for two

years and as the culture aged the small “spiny” cells predominated. Microscopic observations of

these aged populations resembled dense mounds of the small spiny cells (Biedler et al., 1973).

Several clonal cell lines were derived from individual cells from the SK-N-SH cell line.

These clonal lines were assessed for their cholinergic and adrenergic enzyme activities and for

detection of neurotransmitters dopamine-β-hydroxylase, choline acetyltransferase,

acetylcholinesterase, and butyrylcholinesterase (Biedler et al., 1978). In general, the cultures

with a more neuronal “spiny” morphology had the greatest enzyme and neurotransmitter

abundance, while the epithelial morphology cell lines did not have enzyme activity or detectable

72

neurotransmitters (Biedler et al., 1978). SK-N-SH was the uncloned cell line cultured December

1970. SH-SY was a neuroblast-like clonal subline of SK-N-SH. SH-SY5 was subcloned from

SH-SY. SH-SY5Y was subcloned from SH-SY5. The thrice-cloned SH-SY5Y cell line was

homogenous and had the measured neurotransmitters (Biedler et al., 1978).

Later work (Jalava et al., 1988) found that the SH-SY5Y cells express abundant c-myc, an

oncogene transcription factor whose constitutive expression induces proliferation in a large

number of cancers (Dang 2012). A decline in c-myc expression, which can be induced by

tetradecanoylphorbol-13-acetate, was associated with morphological neuronal differentiation of

the SH-SY5Y cells (Jalava et al., 1988). Expression of c-myc can also be decreased by 3- to 5-

fold by 24 hour exposure to insulin-like growth factor 1, which led to morphological

differentiation of the SH-SY5Y cells and an increase in the neuronal differentiation marker

growth cone associated protein 43 (GAP43) (Sumantran et al., 1993).

We chose human SH-SY5Y neuroblastoma cells for this study because they are cells of a

human neuronal background that proliferate in culture and have relatively low basal cannabinoid

receptor mRNA and protein abundance. The SH-SY5Y cell line was chosen for its human

neuronal lineage and maintained expression of neuron specific proteins. SH-SY5Y cells have

been an important disease model for decades and are still currently being used to model

Parkinson’s disease (Su et al., 2016), Alzheimer’s disease (Song et al., 2015), and dopamine

production for functional differentiation during synapse development (Korecka et al., 2013). By

examining changes to the clonal cell lines after transfection and stable overexpression of either

CB1 or CB2 receptor we can more clearly distinguish effects between the two receptors than by

using an in vivo model.

1.8 RESEARCH OBJECTIVES

This research will investigate the gaps in knowledge of cannabinoid signaling in neuronal

functional development. There has never been a direct side by side comparison of the impact of

73

CB1 versus CB2 cannabinoid receptors on extension length. The relative contributions of CB1 and

CB2 cannabinoid receptors to axonal pathfinding, neurite elongation, and synaptic vesicle

formation and spatial regulation is not well understood.

There have been two longitudinal studies to examine the effect of prenatal marijuana

exposure upon the long term behavior of children. The Ottowa Prenatal Prospective Study

(OPPS) was begun in 1978 and followed approximately 145 children until up to 22 years of age.

At 13 to 16 years of age, the prenatally exposed children showed a decreased visual memory

because they had a significantly longer time to latency during the abstract design recognition test

(Fried et al., 2003). This effect was not seen in children who had been exposed prenatally to

cigarette smoking, and was likely not an effect of smoke itself (Fried et al., 2003). The second

longitudinal study, the Maternal Health Practices and Child Development Study (MHPCD) (Day

et al., 1994), similarly to the OPPS study found an increase in hyperactivity of the prenatally

marijuana exposed children at six years of age (McLemore et al., 2016). The OPPS also found

long term changes in prefrontal cortex activity during working memory FMRI tests in the

prenatally marijuana exposed children at 18-22 years of age (McLemore et al., 2016; Smith et al.,

2006).

The CB1 and CB2 cannabinoid receptors have been extensively studied in a variety of

different cell types and disease models. However, they are not often directly compared to one

another and the application of a dual agonist has sometimes confounds the interpretation

regarding whether there is activity at one of the receptors specifically. The discovery that both

cannabinoid receptors are capable of altering neuronal proliferation (Aguado et al., 2005),

initiating cell death (Galanti et al., 2008; Pasquariello et al., 2009), and that both are present in the

axonal growth cone (Berghuis et al., 2007; Bromberg et al., 2008b; Callen et al., 2012; Duff et al.,

2013; He et al., 2005; Jordan et al., 2005; Jung et al., 2011; Keimpema et al., 2013; Mulder et al.,

2008; Tortoriello et al., 2014) raises the question of the relative impact of one receptor over

another. This research will utilize a uniquely suited model of stable overexpression of either CB1

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receptor or CB2 receptor in an otherwise identical clonal human neuroblastoma cell line. The SH-

SY5Y cell line has a four decade history of being studied as a human disease model and is

regarded as being representative of human neurons. It has maintained expression of cellular

proteins such as β-3-tubulin that are specific to neurons and its other proteins have been

thoroughly characterized during the past forty years. For example, the G proteins present in the

SH-SY5Y cell line have already been documented (Chan et al., 1998).

Broadly, I hypothesize that cannabinoid receptors regulate neuronal differentiation and

functional development by decreasing proliferation rate and increasing extension length via a G

protein mediated signaling pathway. The relative contribution of CB1 versus CB2 cannabinoid

receptors to these effects will be investigated, and both will be quantitatively compared and

contrasted. Specific aims to test this hypothesis are:

CHAPTER III: AIM 1: CREATE MODELS OF INCREASED CB1 OR CB2

RECEPTOR SIGNALING IN A CELL LINE THAT ENDOGENOUSLY EXPRESSES

ENDOCANNABINOID ENZYMES, SYNTHESIZE ENDOCANNABINOID LIGANDS,

AND RETAINS CELLULAR ARCHITECTURE COMPONENTS OF A HUMAN

NEURON.

The unique and novel advantage of this investigation into the comparison of CB1 or CB2

cannabinoid receptors is the creation of two clonal and otherwise identical human neuronal type

cell lines that differ only in their expression of CB1 or CB2 cannabinoid receptors. To investigate

the effects of cannabinoid receptor signaling on neuronal proliferation, viability, and extension

length, the SH-SY5Y human neuroblastoma cell line will be stably transfected with either

cannabinoid receptor. A qPCR analysis will confirm transfection. Western blot will assess

protein quantification. An mRNA analysis of the endogenous cannabinoid system enzymatic

components will determine if cannabinoid receptor overexpression alters mRNA abundance. The

abundance of the endogenous ligands anandamide and 2-AG will be quantified using mass

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spectrometry. These cell lines will then be used to investigate the role of cannabinoid receptors

on cellular signaling pathways.

CHAPTER IV: AIM 2: DETERMINE THE CONSEQUENCES OF INCREASED

CB1 OR CB2 RECEPTOR SIGNALING ON CELLULAR PROLIFERATION RATE AND

VIABILITY.

The cannabinoid receptors have been shown to alter cellular proliferation rate and also

cellular viability via either increased necrosis or apoptosis. Changes in viability and proliferation

have known effects on neuronal functions such as altering extension length and must be

quantified in order to determine their contribution to the results. Aim 2 will investigate the

consequence of increased CB1 or CB2 cannabinoid receptor expression on proliferation rate using

a BrdU uptake assay, viability using a Trypan blue exclusion assay, and apoptosis using a PARP

cleavage assay. Data from these studies will determine if either or both cannabinoid receptors

regulate neuronal proliferation and viability, and if so which receptor has a larger impact.

CHAPTER V: AIM 3: DETERMINE THE CONSEQUENCES OF INCREASED

CB1 OR CB2 RECEPTOR SIGNALING ON NEURONAL EXTENSION LENGTH AS A

MODEL OF SYNAPTOGENESIS.

The presence of both cannabinoid receptors on the axonal growth cone of developing

neurons has raised the possibility that either or both cannabinoid receptors may be involved in

neuronal functional development. The focus of Aim 3 is to quantitatively compare the effect of

increased CB1 or CB2 cannabinoid receptor expression on the SH-SY5Y cell line. This aim will

use a novel technique created to quantify the extension length in neurons by staining the plasma

membrane using Texas Red conjugated Wheat Germ Agglutinin, tracing the cell outline, and

measuring extension length using the Neuron J plugin for Image J.

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1.9 DISSERTATION SUMMARY

The primary research objective of this research is to determine cannabinoid receptor

involvement in neuronal functional development. Specifically, by employing cannabinoid

receptor stable expression neuroblastoma cell line models that represent human neurons, the

direct effect of endogenously stimulated cannabinoid receptor activity on neuronal proliferation,

cell death, and extension length will be explored.

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CHAPTER II

EXPERIMENTAL DESIGN AND METHODS

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2.1. CELL CULTURE

SH-SY5Y clonal lines were grown in Dulbecco’s Modified Eagle’s Medium F12

supplemented with 2 mM L-alanyl-L-glutamine dipeptide as GlutaMAX, 100 IU/ml penicillin,

100 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and 10% heat

inactivated bovine serum (Gemini Bio Products, Sacramento, CA, USA). pcDNA3.1(+) plasmids

containing long isoform full length human CB1 receptor, CB2 receptor, or the neo gene (Missouri

S&T cDNA Research Center, St. Louis, MO, USA) all with the neo gene were transfected using

Lipofectamine (Invitrogen, Life Technologies, Carlsbad, CA, USA). The cells were selected for

plasmid expression using 450 mg/L geneticin (G418) (Life Technologies, Carlsbad, CA, USA)

and clonal cell lines were isolated.

2.1.1 Cell Abundance

Cell growth curves were created by plating 33,000 cells per well in six-well plates, with

treatment with compounds or vehicle beginning on day 3 and continuing every other day. Cells

were harvested from day 3 to day 9 by dissociation in 0.4 mL of sterile PBS EDTA (0.625 mM),

diluted 1:1 in 0.4% Trypan Blue in sterile 0.85% saline (Invitrogen, Life Technologies, Carlsbad,

CA, USA) and counted using a Reichert 0.1 mm hemocytometer.

2.1.2 Cell Viability

Cells were plated at 550,000 cells per well in a 96-well plate, and after one day the media

was changed in the presence or absence of 980 µM H2O2 (Walgreens, Deerfield, IL, USA). After

24 h, the media was removed and cells were dissociated with PBS/EDTA. The media was

combined with the dissociated cells, diluted (1:1) with 0.4% Trypan Blue and viable cells were

counted on a Countess Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA).

2.2 MASS SPECTROMETRY ANALYSIS

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2.2.1 Lipid Extraction of Endogenous Cannabinoids

Cells were harvested using dissociation by PBS/EDTA and sedimented at 1,000 x g at

4°C for 10 min. The pellet was resuspended in 150 μL of 50 mM Tris HCl, pH 7.4; 1 mM EDTA

and homogenized on ice with a Dounce glass-glass homogenizer. Lipid extraction followed the

procedure of Maccarrone and colleagues (Maccarrone et al., 2001). In a glass tube, 300 μL of

sample were combined with 600 μL of 2:1 (v/v) ice cold methanol:chloroform, and 180 μL ice

cold chloroform and vortexed. The suspension was centrifuged at 1,000 x g for 5 min at 4°C to

separate the aqueous (upper) and organic (lower) layers. The bottom organic layer was retrieved

using a glass pipette, taking care not to capture the protein residue at the interface, and dried to

approximately 30 μL of volume using N2 gas and stored frozen at -80°C.

Samples were analyzed by LC-MS/MS on an Acquity UPLC BEH C18 1.7 µm 2.1 x 50

mm column attached to a Sciex API 5000 ionizing via ESI. The mobile phases were 10 mM

ammonium acetate with 0.1% acetic acid in water (A) and 10 mM ammonium acetate with 0.1%

acetic acid in methanol (B) flowing at 0.3 mL/min. Starting conditions were 30% A ramping to

12% A over six min, followed by a ramp to 5% A over 1 min, 5% A held for 1.5 min, followed

by a re-equilibration to 30% A over one min and held for three min at 30% A for reconditioning.

Total run time was 12.5 min. Injection volume was 10 µL. Target column temperature was 30°C.

Target sample temperature was set to 4°C. Source parameters were set to 400°C, ion spray

voltage of 5500 volts. Ion source Gas 1 and 2 were set to 50 and 60, respectively. Curtain Gas

was ten and collision gas was five. Supplemental Tables 2 and 3 show detailed mass

spectrometry parameters (Schaich et al., 2015; Schaich et al., 2016).

2.3 MOLECULAR BIOLOGY STABLE TRANSFECTION TECHNIQUES

Plasmids containing CB1 receptor, CB2 receptor, or an empty vector (Missouri S&T

cDNA Research Center, St. Louis, MO, USA) all with the neo gene were transfected using

Lipofectamine (Invitrogen, Life Technologies, Carlsbad, CA, USA). The cells were selected for

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plasmid expression using geneticin G418 (Life Technologies, Carlsbad, CA, USA). Dilution to

approximately one cell per well was employed to isolate clonal cell lines. PCR confirmed

successful transfection and cannabinoid receptor mRNA abundance in the clonal cell lines. A

dose response curve determined that a concentration of 450 mg/L geneticin was sufficient to

prevent survival of the parental cell line and maintain stable overexpression.

2.4 REAL-TIME QUANTITATIVE PCR

Cells were harvested with 1 mL sterile PBS/EDTA (80 g NaCl or 1.37 M, 2 g KCl or

26.83 mM, 14.4 g Na2HPO4 or 101.43 mM, 2.4 g KH2PO4 or 17.64 mM, 25 mL of 250 mM

EDTA or 6.25 mM EDTA dissolved in 800 mL deionized H2O, adjusted to pH 7.4, and increased

to 1 L then filter sterilized to make a 10x stock. Final EDTA concentration is 0.625 mM in the 1x

PBS), sedimented at 2000 x g, 5 min in RNAse-free plastic tubes (Axygen, Corning Life Sciences,

Tewksbury, MA, USA), and pellets were stored at -80 for up to one week before extraction of

mRNA was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and a 15 min on-

column RNAse-free DNase digestion (Qiagen, Hilden, Germany). The mRNA was converted to

cDNA using a High Capacity cDNA Reverse Transcription Kit and Mutiscribe Reverse

Transcriptase (Applied Biosciences, Thermo Fisher Scientific, Waltham, MA, USA). The

concentration of cDNA was measured using a NanoDrop 2000 (Thermo Fisher Scientific,

Waltham, MA, USA) and qPCR was performed on 50 ng cDNA template per well plus All-In-

One PCR Mix (GeneCopoeia, Rockville, MD, USA) and 0.125 μM primers (listed in

Supplemental Table 1). The GeneMate 96-well plate (BioExpress, Kaysville, UT, USA) was

read by StepOnePlus Real Time PCR System (Applied Biosystems, Thermo Fisher Scientific,

Waltham, CA, USA). The threshold was manually adjusted to 0.5 for all wells, and cycle

threshold data were calculated using the 2^-ΔΔCt method (Livak et al., 2001) relative to ENO2

and GUSB as reference genes. Statistical analyses were performed using REST 2.0 with 2000

iterations (Pfaffl et al., 2002).

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2.4.1 Design and testing of primer sets

Gene Forward Primer Reverse Primer

ABHD6 ATG TCC GCA TCC CTC ATA AC CCT GGG TCT TTC CAT CAC TAC

ABHD12 CCT GGG TTT GAC TGG TTC TT CAG CTC AGG GCT CTT GTA AAT

CNR1 AGC AGA CCA GGT GAA CAT TAC TCA GGA CCA TGA AAC ACT CTA TG

CNR2 GAT TGG CAG CGT GAC TAT GA GAG CTT TGT AGG AAG GTG GAT AG

COX2 GTT CCA GAC AAG CAG GCT AAT A TAC CAG AAG GGC AGG ATA CA

DAGLα CTT CCT CTT TCT CCT GCA TAC C TGG CTT GAC CCT CCT CTA A.

DAGLβ TCA GCA TGA GAG GAA CGA TTT GGC TCT TGG TTG TTC CTG ATA

DDC CAG ACT TAA CGG GAG CCT TTA G CCA GAA TGA CTT CCA CAC AGA T

ENO2 CTG ATC CTT CCC GAT ACA TCA C CTG GTC AAA TGG GTC CTC AA

FAAH TCA GCT TTC CTC AGC AAC AT CCG CAG ACA CAA CTC TTC TT

GAP43 GCC AAG CTG AAG AGA ACA TAG A CAC ACG CAC CAG ATC AAA TAA C

GUSB AAG AGT GGT GCT GAG GAT TG GGA GGT GTC AGT CAG GTA TTG

ITGA1/CD49a GTG CCC GAA GAG GAG TTA AA CAT GAC CCA GTC CTG TGA ATA A

ITGB1/CD29 TGA TCC TGT GTC CCA TTG TAA G GTC AGT CCC TGG CAT GAA TTA

MAGL CTC ATT TCG CCT CTG GTT CT GAA GAC GGA GTT GGT GAC TT

MAP2 GAG AAT GGG ATC AAC GGA GAG GTG GAG AAG GAG GCA GAT TAG

NAPEPLD GCT GTG AGA ATG TGA TTG AGT TG CTG GGT CTA CAT GCT GGT ATT T

NCAM GGT GCC CAT CCT CAA ATA CA CAT CTG CCC TTC GAG CTT AG

NESTIN CGT TGG AAC AGA GGT TGG A CTC AGG ACT GGG AGC AAA G

PPARα AGT CTC CCA GTG GAG CAT TGA AAC GAA TCG CGT TGT GTG A

ST8SIA2 TAA TAA ACG GCT CCT CAT CAC C CAC AAG TCC CAA AGT GCT TAT TC

SYN1 CTC CTT GCA TTC TGT CTA CAA CT CTG GAC ACG CAC GTC ATA TT

SYN2 AGG TGG AAC AGG CAG AAT TT TCT CCT CCC AGT GTC TTA TAG ATA G

SYT CTG CTG GTA GGG ATC ATT CAG CAC GCC ATT CCT CAG TTA CA

β3TUB GAT CAG CGT CTA CTA CAA CGA G CAT CCG TGT TCT CCA CCA G

Table 2.4.1 Primers were generated using the IDT DNA PrimerQuest tool, modeled to detect

primer dimers using MFE 2.0, checked for hairpins using IDT DNA Oligo Analyzer tool,

referenced for alignment specificity using NCBI BLAST, and excluded if “expectation value” (E)

was less than three orders of magnitude away from the second closest target. PPARα was

generously gifted by Dr. John Parks.

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2.5 WESTERN IMMUNOBLOTTING PROCEDURES

Cells were harvested with PBS/EDTA, centrifuged at 2,000 g for 5 min, and resuspended on ice

with 200 μL of Pierce 87788 Lysis Buffer (25 mM TrisHCl pH 7.4, 150 mM NaCl, 1% NP-40, 1

mM EDTA, 5% glycerol, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with

1:100 six protease inhibitor cocktail #539134 with broad inhibition of aspartic, cysteine, and

serine proteases (Calbiochem, San Diego, CA, USA). For the CB1 receptor, RIPA buffer (25 mM

pH = 7.6 Trizma® Base, 150 mM NaCl, 1% by volume Triton X-100, 1% sodium deoxycholate,

0.1% sodium dodecyl sulfate) was used. All of the cell pellets were broken apart within less than

2 hours by pipetting up and down with a P1000 pipette and then a 28 gauge needle on ice.

Unsolubilized material was sedimented for 2 min at 2,000 g, and the supernatant was used. A

bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA, USA) was used to

determine protein concentration. A 40 μL volume (10 to 30 μg of protein) was loaded per lane

onto a 10% polyacrylamide gel and resolved in Tris/Glycine/SDS buffer on a BioRad Western

Blot electrophoresis chamber (BioRad Laboratories, Hercules, CA, USA) at 150 volts for one h.

The proteins were transferred onto nitrocellulose (Licor, Lincoln, NE, USA) using a BioRad

Transfer System at 85 volts on ice for 50 min. The blot was rinsed in PBS, blocked in Odyssey

Blocking Buffer (Licor, Lincoln, NE, USA) for one h, incubated with primary antibodies

overnight at 4°C and secondary antibodies were applied 1:20,000 for 2 h at room temperature,

and imaged on a Licor Odyssey (Licor, Lincoln, NE, USA). Primary antibodies include anti-CB1

receptor: sc10066 (Santa Cruz, Dallas, TX, USA); anti-CB2 receptor: sc25494 (Santa Cruz,

Dallas, TX, USA); and both cleaved and total poly (ADP-ribose) polymerase (PARP) #9542 (Cell

Signaling Technologies, Danvers, MA, USA). Secondary donkey and goat antibodies include

IRDye® 800CW and IRDye® 680RD (Licor, Lincoln, NE, USA).

83

2.6 IMMUNOFLUORESCENCE ANALYSIS

Clear plastic coverslips (Thermo Fisher Scientific, Waltham, MA, USA) were soaked in

ethanol for five minutes and then treated with ultraviolet light for twenty minutes to minimize

bacterial contamination. Cells were plated at a density of 1x105 cells per well in a six well plate.

After one week of growth, the cells were fixed for 10-20 minutes with 4% formaldehyde (Sigma

Aldrich, Missouri, USA), permeabilized for 10-20 minutes with 0.1% Triton X-100 in PBS

(Sigma Aldrich, St. Louis, MO, USA), and blocked for 30 minutes with 0.4% donkey serum in

PBS (Sigma Aldrich, St. Louis, MO, USA). The cells were stained for one hour with 1:100 goat

anti CB1 receptor antibody sc10066 (Santa Cruz, Dallas, TX, USA), CB2 receptor sc25494 (Santa

Cruz, Dallas, TX, USA) then rinsed with 0.1% Triton X-100 in PBS, incubated for 45 minutes

with 1:500 FITC channel donkey secondary antibodies (Abcam, Cambridge, United Kingdom),

and stained for 10 minutes with 1 μg/μL DAPI (Thermo Fisher Scientific, Waltham, MA, USA).

The coverslips were rinsed with PBS and then fixed onto SuperFrost Plus glass slides using

Prolong Gold antifade mountant (Thermo Fisher Scientific, Waltham, MA, USA). Images were

captured using CellSens software on an Olympus IX-71 inverted fluorescent microscope

(Olympus Microscopes, Tokyo, Japan).

2.6.1 Microscopy of PARP fragment abundance

Quantitative Immunocytochemical Determination of Apoptosis. Cells were settled on

glass coverslips at 20x10^4 cells per well in six well dishes. After one day, they were treated for

24 hours with 10 μM methanandamide (Cayman Chemicals, Ann Arbor, MI, USA) or 150 μM

H2O2, (Walgreens, Deerfield, IL, USA). One day after dosing, the slides were fixed for 10

minutes with 4% formaldehyde, permeabilized for 15 minutes with 0.1% Triton X-100, and then

blocked for 30 minutes with 4% donkey serum in PBS (Sigma Aldrich, St. Louis, MO, USA).

The cells were stained for one hour with 1:500 with mouse cleaved Parp antibody 19F4 (Cell

Signaling Technologies, Danvers, MA, USA) (Pasquariello et al., 2009) and 1:200 with Alexfluor

84

488 donkey anti mouse (Thermo Fisher Scientific, Waltham, MA, USA) for 30 minutes in the

dark. The cells were stained for 10 minutes with 1 μg/μL DAPI (Thermo Fisher Scientific,

California, USA), rinsed with PBS, mounted on a glass slide with Prolong Gold (Thermo Fisher

Scientific, Waltham, MA, USA), dried for one day, and then imaged on an Olympus IX71

microscope. The photon magnitude at one second of exposure (below saturation as per the

exposure compensation tool) was exported using CellSens software (Olympus Microscopes,

Tokyo, Japan) and divided by the number of DAPI positive nuclei as counted with the Cell

Counter plugin for ImageJ (National Institutes of Health, Rockville, MD, USA).

2.6.2 Microscopy of Bromodeoxyuridine uptake

Immunocytochemical Determination of Bromodeoxyuridine Uptake Rate- To quantitate

bromodeoxyuridine (BrdU) uptake, SH-SY5Y cells were plated at 100,000 cells per plastic

coverslip, and five days after plating, media was exchanged containing 15 μM BrdU or DMSO

control. After 48 h, the cells were fixed with 100% methanol for 5 min. PBS containing 2 M

HCl (Sasaki et al., 1988) and 0.1% Tween was applied for 30 min, followed by incubation with

PBS containing 10% serum, 0.3 M glycine and 0.1% Tween-20 for 1 h. The cells were then

incubated with rat anti- BrdU (1:200) (ab6326 Abcam, Cambridge, United Kingdom) in 4% goat

serum for 1 h, rinsed with 0.1% Triton X-100, and incubated with goat anti-rat Alexa Fluor 594

(1:1,000) in 4% goat serum (A11007 Thermo Fisher Scientific, Waltham, MA, USA) for 45 min.

The stained coverslips were then incubated with 1 μg/μL DAPI for 10 min, rinsed with 0.1%

Triton X-100, and mounted onto SuperFrost Plus glass slides using Prolong Gold. Images were

captured on an Olympus IX-71 inverted fluorescent microscope (Olympus Microscopes, Tokyo,

Japan). The nuclei were counted in the Texas Red (BrdU) channel (an excitatory beam at or near

596 nanometers and collects light at or near 615 nanometers) or DAPI (total nuclei) channel (an

excitatory beam at or near 350 nanometers and collects light at or near 470 nanometers) using the

85

Cell Counter plug-in for Image J, and the percent of BrdU-stained/DAPI-stained nuclei were

calculated.

2.6.3 Immunofluorescence of Texas Red Conjugated Wheat Germ Agglutinin stained extensions

SH-SY5Y human neuroblastoma cells were plated at a density of 100,000 cells per sterile

plastic coverslip, and treatments were applied as indicated. The RA treatment was applied every

other day while the other treatments were applied 24 h before fixation. On day 6, the cells were

fixed with 4% formaldehyde in PBS for 20 min, stained for 20 min with Texas Red conjugated

wheat germ agglutinin (Thermo Fisher Scientific, California, USA), and stained for 10 min with 1

μg/μL DAPI. The coverslips were rinsed with PBS and then mounted onto SuperFrost Plus glass

slides using Prolong Gold. Images were captured on an Olympus IX-71 inverted fluorescent

microscope (Olympus Microscopes, Tokyo, Japan) at 20x optical zoom at five fields per slide:

the four corners and the center. Image location was chosen in an unbiased manner based upon the

DAPI nuclei density, independent of the Texas Red channel’s view of cell shape. The nuclei

identified in the DAPI channel were counted using the Cell Counter plug-in for ImageJ (30 to 50

nuclei per field). All cell extensions per field visible in the Texas Red channel were traced using

the NeuronJ plug-in for Image J, recording the each individual tracing length and the longest

extension per field. Extensions that were longer than 30 micrometers were characterized as

“neurites”, whereas extensions that were less than 30 micrometers were considered to be

“filopodia” (Sainath et al., 2015). Data shown are the sum of all neurites or filopodia per field

normalized by dividing by the number of nuclei per field.

2.6.4 Cell Cycle Population Demographics Quantification Using FACS

Cell cycle was determined by dissociation of cells with PBS-EDTA, adjusting to

3,000,000 cells per sample, and sedimenting cells for 5 min at 2500 x g. The cells were

resuspended with 5 mL 70% ethanol at 4°C and incubated at 4°C for 2 h, sedimented, and the

86

pellet was resuspended in 900 μL PBS. Fluorescence Activated Cell Sorting (FACS) was

performed on Aria or Canto (BD Biosciences, California, USA) by treating the sample with DNA

binding reagent (100 μL; 0.036 M sodium citrate, 75 μM propidium iodide, 1 mg/mL RNAse A,

0.06% NP40) (Thermo Fisher Scientific, California, USA) for 10 min (Krishan 1975). The

samples were gated on forward and side scatter to exclude aggregates and debris. The two peaks

on the graph with FL2-A as the x axis were integrated to sum the area under each curve. The first

peak represents cells with single quanta DNA (cells in G0/G1 phase of the cell cycle) and the

second peak represents cells with double quanta DNA (cells in S/G2/M phase).

2.7 STATISTICAL ANALYSIS

Data were statistically analyzed and plotted with PRISM software version 6 or 7

(GraphPad, California, USA). Statistical differences were tested using the Student’s t-test, or, a

one way ANOVA was performed with either a Tukey’s post hoc test for multiple comparisons or

a Dunnett’s post hoc analysis to compare groups to the control. Differences were considered to be

significant at p < 0.05, and trends close to this level of significance are noted. Determinations of

significant difference in mRNA values were performed using Relative Expression Software Tool

(REST©) 2.0 with 2000 iterations (Pfaffl et al., 2002).

87

CHAPTER III

HUMAN NEUROBLASTOMA CELL MODEL OF NEURONAL CANNABINOID

SIGNALING

88

CHAPTER III: HUMAN NEUROBLASTOMA CELL MODEL OF NEURONAL

CANNABINOID SIGNALING

3.1 Creation of CB1 or CB2 receptor stably overexpressing SH-SY5Y neuroblastoma cell lines—

Clonal cell lines were created by lipofectamine plasmid transfection, geneticin selection

and maintenance of plasmid expression, and dilution to single cell founders of clonal cell lines.

Increased expression of CB1 receptors (the CB1XS cell line), CB2 receptors (the CB2XS cell lines),

or an empty vector that only expressed the neo geneticin resistance gene (the MT4 cell line) is

shown at the transcriptional and translational levels (Fig. 3.1). Transfection and selection of the

SH-SY5Y cells resulted in a stable increase of CB1 receptor mRNA by 10^11 abundance in the

CB1XS clonal cell line. Parental, MT4, and the CB2XS cell lines have less than 1% of the mRNA

abundance of CB1 receptor found in the CB1XS cell line (Fig. 3.1, ANOVA p < 0.0001,

Dunnett’s adjusted p for Par vs CB1XS = 0.0001, n = 3). Absolute cycle threshold (CT) value for

CB1 mRNA abundance was 34 for Parental SH-SY5Y, 33 for MT4, 23 for CB1XS, 36 for CB2XS

4-3, and 35 for the CB2XS 4-4 cell line (Fig. 3.1, n = 3). CB2 receptor mRNA increased by an

average of 10^12 abundance in the CB2XS clonal cell lines relative to the parental SH-SY5Y.

Parental, MT4, and the CB1XS cell line have less than 1% of the average mRNA abundance of

CB2 receptor found in the CB2XS cell lines (Fig. 3.1, ANOVA p < 0.0001, Dunnett’s adjusted p

for Par vs CB2XS 4-2 = 0.0001, for Par vs CB2XS 4-3 = 0.0001, for Par vs CB2XS 4-4 = 0.0001,

n = 3). Absolute CT for CB2 receptor mRNA abundance was 35 for Parental SH-SY5Y, 33 for

MT4, 34 for CB1XS, 24 for CB2XS 4-2, 22 for CB2XS 4-3, and 22 for the CB2XS 4-4 cell line

(Fig. 3.1, n = 3). Western blots were performed to quantify the protein abundance of cannabinoid

receptors in the parental SH-SY5Y, transfection control MT4, and stably transfected cell lines

CB1XS and CB2XS clones 4-2, 4-3, and 4-4 (Fig. 3.1, n = 3). Cannabinoid receptor protein

abundance was significantly increased in the XS cell lines (Fig. 3.1, n = 3).

SH-SY5Y cells express the mRNA of enzymatic components of the endogenous

cannabinoid system including DAGLα, DAGLβ, MAGL, ABHD6, ABHD12, NAPE-PLD, and

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FAAH (Fig. 3.2, n = 4). REST analysis found that increased expression of CB1 or CB2 receptors

did not alter mRNA abundance of the enzymatic components of the endogenous cannabinoid

system (Fig. 3.2, n = 4).

The SH-SY5Y human neuroblastoma cell line produces the endogenous cannabinoid

ligand 2-AG at a steady state concentration of 135 pmol per gram of protein (Fig. 3.2, n = 3).

Application of 2 h of 1 μM THL, an inhibitor of DAGL activity to produce 2-AG, did not

significantly alter 2-AG abundance but resulted in a concentration of 15 pmol per gram protein

(Fig. 3.2). Five min of 10 μM Oxotremorine M (an agonist of Gq coupled muscarinic

acetylcholine receptors that increases intracellular calcium (Billups et al., 2006), stimulating the

enzyme DAGL to produce 2-AG) did not significantly alter 2-AG abundance but resulted in a

concentration of 564 pmol per gram protein (Fig. 3.2). Inhibition of MAGL, ABHD6, and

ABHD12 was performed through a novel application of a combination of 1 μM of MAGL

inhibitor JZL184, 1 μM WWL70, and 10 μM ursolic acid 2 h before harvesting. These

compounds each individually inhibit one of the three major enzymes that hydrolyze 2-AG in-situ,

but have not previously been applied in combination. Inhibition of these hydrolytic enzymes

increased 2-AG abundance to 2,140 pmol per gram protein (Fig. 3.2, ANOVA p = 0.01, for Veh

vs J&W&U Dunnett’s adjusted p = 0.01, n = 3).

SH-SY5Y express endogenous cannabinoid ligand anandamide at a steady state

concentration of 0.82 pmol per gram of protein (Fig. 3.2). Application of 2 h of 1 μM THL did

not significantly alter anandamide concentration and resulted in 0.79 pmol per gram of protein

(Fig. 3.2). The novel cocktail of 2-AG hydrolysis inhibitors 1 μM of MAGL inhibitor JZL184, 1

μM WWL70, and 10 μM ursolic acid applied 2 h before cell harvesting increased anandamide

concentration, resulting in 2.4 pmol per gram of protein (Fig. 3.2, ANOVA p = 0.02, for Veh vs

J&W&U Dunnett’s adjusted p = 0.02, n = 3). Stimulation of muscarinic acetyl choline receptors

by five min of 10 μM OxoM (Billups et al., 2006) did not significantly change anandamide

concentration of 0.79 pmol per gram of protein (Fig. 3.2), likely due to the rapid hydrolytic

90

activity of FAAH (Cravatt et al., 1996) that rapidly biotransforms anandamide into arachidonic

acid and ethanolamine.

3.2 Cannabinoid Receptor Overexpression Alters Synaptogenic Protein mRNA Expression—

We designed primers, listed in Table 1, to detect mRNA of proteins known to be

involved in the endogenous cannabinoid system and neurite extension. Treatment with 10 to 100

μM all trans Retinoic Acid (RA) is known to increase SH-SY5Y extension length (Pahlman et al.,

1984) (reproduced here in Fig. 5.1) and to also alter the mRNA production of proteins that are

involved in increased neuronal extension length, neurotransmitter abundance, vesicle creation and

spatial regulation, and synapse formation (Korecka et al., 2013). The mRNA non-rate-limiting

catecholamine synthesis enzyme DOPA Decarboxylase (DDC) decreases after RA treatment

while dopamine abundance increases due to a greater increase in the mRNA enzymes that

degrade dopamine (Korecka et al., 2013). Replicating the work of Korecka et al., we also found

that treatment of the parental SH-SY5Y cells every other day for one week with 10 μM RA

decreased the mRNA of DDC (p=0.03, n = 3, Fig. 3.3). RA also decreased the mRNA of

endogenous cannabinoid enzyme MAGL (p=0.03, n = 3, Fig. 3.3) by approximately 80% relative

to untreated cells. Treatment with RA also decreased Synapsin 2 by approximately 40% relative

to vehicle control (p=0.03, n = 3, Fig. 3.3). Treatment of the parental SH-SY5Y cells every other

day for one week with 10 nM of the synthetic agonist CP-55,940 decreased the mRNA abundance

of ABHD6 (p = 0.02), DAGLβ (p = 0.049), ITGα1 (p = 0.02), ITGβ1 (p = 0.01), NAPE-PLD (p =

0.02), and PPARα (p = 0.03) to between 40 and 70% of the abundance of vehicle treated cells as

assessed by REST analysis (Fig 3.13, n = 4). Stable overexpression of cannabinoid receptors also

altered mRNA expression of proteins involved in neuronal functional development. GAP43 is a

protein that is enriched at the cytosolic surface of the growth cone membrane and which alters

neurite extension by modulating pertussis toxin-sensitive G-protein activity in the growth cone

(Strittmatter et al., 1994). ST8 Alpha-N-Acetyl-Neuraminide Alpha-2,8-Sialyltransferase 2

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(ST8SIA2) is a sialyl transferase that attaches polysialic acid to Neural Cell Adhesion Molecule,

NCAM, a major regulator of brain plasticity, to decrease its adhesive properties (Aonurm-Helm

et al., 2016; Schnaar et al., 2014; Szewczyk et al., 2017). The mRNA abundance of ST8SIA2 can

be increased by forskolin (Razmi et al., 2014), increasing rat memory. Stably increased

expression of CB1 receptor increased GAP43 (p = 0.01) and ST8SIA2 (p = 0.04) mRNA relative

to parental SH-SY5Y, and decreased Integrinα1 (p = 0.02, n = 4, Fig. 3.3). The mRNA targets

that were not altered by increased CB1 receptor abundance are listed in Fig. 3.11. Synaptotagmin

(SYT) is a calcium binding protein present in neurite growth cones and required for successful

neurite outgrowth in chick dorsal root ganglion neurons (Mikoshiba et al., 1999). Stably

increased expression of CB2 receptor increased NCAM and SYT mRNA abundance when REST

analysis compared them with parental SH-SY5Y cells (p < 0.001, p = 0.03, n = 4, Fig. 3.3). The

mRNA targets that were not altered by increased CB2 receptor abundance are listed in Fig. 3.12.

Stimulation of the cannabinoid receptors in the CB1XS and CB2XS cell lines altered the mRNA

of several proteins significantly versus CP-55,940 stimulated parental SH-SY5Y (Fig. 3.14). One

week of 1 nM CP-55,940 stimulation increased GAP43 (p = 0.04) and decreased Integrinα1 (p =

0.04) mRNA in CP treated CB1XS versus CP treated parental SH-SY5Y (n = 4). CP-55,940

stimulation of CB2XS increased GAP43 (p = 0.049) and decreased Dopa Decarboxylase (p =

0.04), ST8SIA2 (p = 0.01), and β-3-tubulin (p = 0.01) relative to CP stimulated parental cells (n =

4, Fig. 3.14). These findings show that cannabinoid receptor signaling can alter the mRNA

abundance of proteins involved in neuronal adhesion, extension length, and neurotransmitter

regulation.

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Figure 3.1 Characterization of CB1 or CB2 receptor-expressing human SH-SY5Y neuroblastoma

cell lines. PCR quantification of the steady state abundance of (A) hCB1 and (B) hCB2 receptor

mRNA in parental SH-SY5Y, empty neo vector MT4, and stably-transfected cannabinoid

receptor-expressing CB1XS and CB2XS cell lines. The average mRNA expression in the

transfected cell line is set at 100%. Statistical comparisons were made using ANOVA and then

Dunnett’s post hoc analysis, p for Par vs CB1XS = 0.0001, n = 3. Fig. 1 B, ANOVA p < 0.0001,

Dunnett’s p for Par vs CB2XS 4-2 = 0.0001, for Par vs CB2XS 4-3 = 0.0001, for Par vs CB2XS 4-

4 = 0.0001, n = 3. (C, D) Western blots of whole cell detergent lysates were performed as

described in the Methods section, and quantified for (C) CB1 receptors and (D) CB2 receptors in

parental SH-SY5Y, MT4, and stably transfected CB1XS and CB2XS cell lines. Data shown are

means ± SEM (C, n=5; D, n=3. Statistical analyses by ANOVA and Dunnett’s p value, **Par vs

CB2XS 4-4, p = 0.001)

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Figure 3.2 Quantification of endogenous cannabinoid ligands 2-AG and anandamide (AEA) in

parental SH-SY5Y cells. (A) Parental, CB1XS and CB2XS cells were grown in 10% heat-

inactivated bovine serum, harvested with PBS-EDTA, and mRNA was isolated and cDNA

prepared as described in the Methods. qPCR quantification of mRNA for the enzymes that

synthesize and biotransform endocannabinoid 2-AG and AEA was performed and ΔΔCt data are

reported relative to ENO2, X ± SEM (n= 4). Comparisons between cell lines were analyzed by

REST analysis (Livak et al., 2000) and found not to be significantly different for any of the genes

analyzed. (B, C) Parental SH-SY5Y cells were grown in media containing 10% heat-inactive

bovine serum, and media was changed to serum-free 12 h prior to addition of vehicle (serum-free

media containing 0.1% ethanol ) or indicated metabolic inhibitors: THL (1 µM, 2h); J&W&U: a

cocktail comprised of MAGL inhibitor JZL184 (1 μM), ABHD6 inhibitor WWL70 (1 μM), and

ABHD12 inhibitor ursolic acid (10 μM) (2h); or muscarinic agonist oxotremorine M (10 μM, 5

min). Cells were harvested, sedimented, and lipid extraction and mass spectroscopy was

performed as described in Methods. Data are shown for (B) 2-AG and (C) AEA as pmol/g

protein (X ± SEM, n = 3). Statistical differences from vehicle were determined by ANOVA and

Dunnett’s posthoc test , *p < 0.05.

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Figure 3.3 Identification of mRNA targets responding to retinoic acid or cannabinoid receptor

expression. (A) CB1XS and (B) CB2XS cell lines were grown in culture for 7 days, harvested and

mRNA extracted and subjected to real-time qPCR (n=4 experiments). (C) SH-SY5Y cells were

treated with RA (20 μM on days 2, 4, and 6 ), harvested on day 7, and RNA extracted and

subjected to real-time qPCR. (n=3 experiments). Primer sets shown on Supplemental Table 1.

ΔΔCT data were calculated relative to vehicle parental SH-SY5Y cells. Statistical differences

were identified using REST analyses (* = p < 0.05, *** = p < 0.001). Those mRNAs that were

tested and found not to be significantly different from parental SH-SY5Y cells are reported in Fig.

3.11 and 3.12.

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Figure 3.4: Effect of cannabinoid receptor overexpression on mRNA of endogenous

cannabinoid system component enzymes. Parental SH-SY5Y, CB1XS, and CB2XS mRNA are

shown. REST analysis of CB1XS compared with Parental and CB2XS compared with Parental

for 2,000 iterations did not find any significant differences. n = 4.

A B C

D E F

G H

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Figure 3.5: Effect of cannabinoid receptor overexpression on mRNA of transcription factors.

Parental SH-SY5Y, CB1XS, and CB2XS mRNA are shown. REST analysis of CB1XS

compared with Parental and CB2XS compared with Parental for 2,000 iterations did not find any

significant differences. n = 4.

A B

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Figure 3.6: Effect of 7 days of 20 uM Retinoic Acid (RA) every other day on mRNA of

endogenous cannabinoid system component enzymes. Parental SH-SY5Y vehicle and RA treated

mRNA are shown. REST analysis of RA treated compared with Vehicle for 2,000 iterations

identified MAGL as signficantly different (downregulated by a mean factor of 0.221, SE range

0.118 – 0.334, p = 0.031, n = 4).

A B C

D E F

G H

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Figure 3.7: Effect of 7 days of every other day application of 20 uM Retinoic Acid (RA) to SH-

SY5Y cells on the mRNA of proteins involved in neurotransmitter regulation. Vehicle and RA

treated parental SH-SY5Y mRNA are shown. REST analysis of RA treated compared with

Vehicle for 2,000 iterations identified DDC as signficantly different (downregulated by a mean

factor of 0.291, SE range 0.188 – 0.425, p = 0.029, n = 4). An analysis by the REST program run

for 2,000 iterations on the RA treated compared with Vehicle treated SH-SY5Y cells identified

SYN2 as signficantly different (downregulated by a mean factor of 0.574, SE range 0.466 – 0.637,

p = 0.032, n = 4).

A B

C D

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Figure 3.8: Effect of 7 days of 20 uM Retinoic Acid (RA) every other day on mRNA of proteins

involved in cytoskeletal regulation. Parental SH-SY5Y vehicle and RA treated mRNA are shown.

REST analysis of RA treated compared with Vehicle for 2,000 iterations did not find any

significant differences. N = 4.

A B

C D

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Figure 3.9: Effect of 7 days of 20 uM Retinoic Acid (RA) every other day on mRNA of proteins

involved in cellular adhesion. Parental SH-SY5Y vehicle and RA treated mRNA are shown.

REST analysis of RA treated compared with Vehicle for 2,000 iterations did not find any

significant differences. N = 4.

A B

C D

DD

Ct

% E

xp

ressio

n

Rela

tiv

e t

o E

NO

2

400

200

600

101

Figure 3.10: Effect of 7 days of 20 uM Retinoic Acid (RA) every other day on mRNA of

transcription factors. Parental SH-SY5Y vehicle and RA treated mRNA are shown. REST

analysis of RA treated compared with Vehicle for 2,000 iterations did not find any significant

differences. N = 4.

A B

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Figure 3.11: mRNA targets that were not altered by CB1 receptor stable expression as

determined by REST analysis, n = 4.

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Figure 3.12: mRNA targets that were not altered by CB2 receptor stable expression as

determined by REST analysis, n = 4.

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Figure 3.13: Feedback inhibition and desensitization is evidence of a functional signaling

pathway. One week of 10 nM of synthetic dual receptor cannabinoid agonist CP-55,940

decreases the mRNA abundance of endogenous cannabinoid system components ABHD6,

DAGLβ, NAPEPLD, and PPARα. n=3. (* denotes p < 0.05).

105

The functionality of the ECS in SH-SY5Y cells has been explored and corroborated by

others using radiolabeled displacement assays (Pasquariello et al., 2009). Previously in our lab

we have demonstrated that maximal CB1 receptor signaling requires integrins (Dalton, Peterson,

L. J., and Howlett, A. C. et al., 2013). PPARα has been suggested as a theoretical target of

cannabinoid signaling (O'Sullivan and Kendall, D. A. et al., 2010; Sun et al., 2006). We

demonstrated the functionality of the ECS enzymes by quantifying mRNA abundance regulatory

changes after one week of CP agonist treatment (Fig. 3.13). Chronic exogenous CP agonist

treatment decreased ABHD6, DAGLβ, NAPEPLD, and some proteins considered to be involved

with cannabinoid receptor signaling including Integrinα1, Integrinβ1, and PPARα to

approximately half of their steady state expression as if by feedback inhibition. Treatment with

CP-55,940 decreased ABHD6 (p=0.03), DAGLβ (p=0.04), Integrinα1 (p=0.03), Integrinβ1

(p=0.01), NAPEPLD (p=0.01), and PPARα (p=0.02) mRNA relative to vehicle treated SH-SY5Y

cells (Fig. 3.14). Our results therefore show that despite low receptor abundance, the internal

machinery required for endogenous cannabinoid signaling is intact in SH-SY5Y cells and that

desensitization can follow chronic exogenous agonist treatment. These data supports the

existence of the ECS in SH-SY5Y cells and suggests that there is endogenous production of

ligands by ECS enzymes.

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Figure 3.14: The mRNA abundance in CB1XS and CB2XS cell lines treated for 7 days with 10

nM of dual cannabinoid agonist CP-55,940, shown relative to the mRNA abundance in CP

stimulated parental SH-SY5Y. n = 4.

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Figure 3.15: Representative Western blot showing parental SH-SY5Y, MT4 SH-SY5Y, and three

CB2XS cell lines: 4-2, 4-3, and 4-4. The antibodies were Santa Cruz CB2 receptor rabbit sc-

25494 at 0.4 μg/mL and Santa Cruz GAPDH mouse sc-365062 at 0.4 μg/mL. Staining protocol

was as described in the methods, and image was captured and quantified using a Licor Odyssey

as described. Chameleon Duo ladder is shown.

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Figure 3.16: Representative Western blot showing parental SH-SY5Y, MT4 SH-SY5Y, and

CB1XS SH-SY5Y. The antibodies were Santa Cruz CB1 receptor goat sc-10066 at 0.4 μg/mL and

Santa Cruz GAPDH mouse sc-365062 at 0.4 μg/mL. Staining protocol was as described in the

methods, and image was captured and quantified using a Licor Odyssey as described. Chameleon

Duo ladder is shown.

109

CHAPTER IV

THE ROLE OF CANNABINOID SIGNALING IN CELL VIABILITY AND

PROLIFERATION

110

CHAPTER IV: THE ROLE OF CANNABINOID SIGNALING IN CELL VIABILITY

AND PROLIFERATION

Cannabinoid signaling has been discovered to alter the proliferation rate of cells in a

number of different neuronal systems ranging from early development of the embryonic brain to

sustained proliferation of progenitor populations in the adult brain. Both CB1 and CB2

cannabinoid receptor activation have been shown to be required for proliferation of neuronal

progenitors, with which is active depending upon signaling pathways that are possible in

different brain regions at different developmental stages.

4.1 Stable Overexpression of Cannabinoid Receptors Does Not Increase Apoptosis or Alter

Viability

Cannabinoid receptor activity has been associated with increased apoptosis (Pasquariello

et al., 2009) and decreased overall cell viability (Galanti et al., 2008). PARP cleavage is the end

result of caspase activity and is a mandatory step after commitment to apoptosis. Cleaved PARP

fragments are not present unless a cell has become committed to and is currently undergoing

apoptosis (Palazuelos et al., 2006). We investigated the capability of human neuroblastoma cells

to undergo cannabinoid stimulated apoptosis by quantifying cleaved PARP protein fragments.

Quantitative immunostaining of cleaved PARP found that stable increased expression of CB1 and

CB2 cannabinoid receptors did not increase the abundance of cleaved PARP fragments per nuclei

(ANOVA among vehicle groups Par, MT4, CB1XS and CB2XS p = 0.5, n = 6, Fig. 4.3).

Hydrogen peroxide (H2O2, 24 h of 500 μM) served as a positive control and significantly

increased cleaved PARP immunostaining per cell (p = 0.00002, n = 7, data not shown).

Treatment for 24 h with 10 μM methanandamide did not induce PARP cleavage in any of the cell

lines (ANOVA among methanandamide groups Par, MT4, CB1XS and CB2XS p = 0.2, n = 4, Fig.

4.4). A Western blot using an antibody that binds to both the 116 kDa holo PARP and the 89

kDa cleaved PARP fragment shows only a single 116 kDa protein band, demonstrating that

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PARP is present only in its uncleaved form (Fig. 4.1). A trypan viability assay that tests whether

the plasma membrane is intact was employed to detect the summation of all death mechanisms

that may alter overall viability. The increased expression of cannabinoid receptors did not alter

viability compared with parental or the MT4 transfection control, while the 979 μM H2O2 (24 h)

control had significantly reduced viability (Fig. 4.1, ANOVA p = 0.04, Dunnett’s adjusted p =

0.0001, n = 8). These data do not support a role for cannabinoid receptor signaling in apoptosis

or other mechanisms of decreased cell viability in SH-SY5Y human neuroblastoma cells.

4.2 Cannabinoid Receptors Do Not Alter Proliferation Rate in SHS5Y Human Neuroblastoma

We investigated whether cannabinoid receptor signaling could alter SH-SY5Y cellular

proliferation rate. Treatment of SH-SY5Y neuroblastoma cells with 20 μM RA every other day is

known to inhibit proliferation rate (Pahlman et al., 1984). This finding was reproduced here as a

positive control, showing significantly decreased percentage of proliferating cells compared with

vehicle. Treatment of SH-SY5Y neuroblastoma cells with 10 μM RA every other day

significantly decreased the number of cells on day nine versus vehicle treated cells (Fig. 4.2, p =

0.0005, n = 3). RA resulted in only 60% of the population incorporating the labeled nucleotide

bromodeoxyuridine (BrdU) over 48 h. (Fig. 4.2, Par RA vs Veh Dunnett’s adjusted p = 0.008, n =

4). Increased cannabinoid receptor expression did not significantly alter the rate of BrdU uptake

in SH-SY5Y cells (Fig. 4.2, n = 4). Approximately 85% of parental, empty vector MT4, and

cannabinoid receptor stably expressing CB1XS and CB2XS cell lines incorporated BrdU during

the 48 hours (Fig. 4.2, n = 4). The proliferation rate of the cell lines was also quantified by

determining the exponential growth rate and doubling time. When calculated using linear

interpolation, RA treatment increased the doubling time from 1.5 days to 2 days (n = 3, Fig. 4.2).

The doubling times calculated for Parental, CB1XS and CB2XS SH-SY5Y were 1.5, 1.5 and 1.5

days, respectively (Fig. 4.2, n = 3). Proliferation rate was also examined using fluorescence

activated cell sorting (FACS) of propidium iodide-stained cells, which quantified the percentage

112

of cells with single quanta DNA (cells in G0/G1 phase of the cell cycle) or doubled DNA (cells in

S/G2/M phase). The percentage of cells in S/G2/M phase versus total was not different between

parental SH-SY5Y and CB2XS, 31% versus 27% respectively (Fig. 4.2, ANOVA p = 0.3, n = 3).

These data does not support a role for cannabinoid receptor signaling in altering the proliferation

rate of SH-SY5Y human neuroblastoma cells.

Seven-day stimulation with methanandamide or 2-AG-ether, stable analogs of the

endogenous ligands anandamide and 2-AG, did not significantly alter the cell counts of parental

or CB1XS SH-SY5Y on day nine compared with vehicle (Fig. 4.2) over an extended

concentration range (Fig. 4.5, Fig 4.6), although application of 1 μM 2-AG-ether increased

CB2XS cell count (Fig 4.2, n = 4, p = 0.002). Cell counts relative to vehicle were not altered by

7-day application of CB1 receptor antagonist SR141716A (1 μM) to parental or CB1XS SH-SY5Y

cells, or CB2 receptor antagonists SR144528 (1 μM) or AM630 (0.5 μM) to parental or CB2XS

SH-SY5Y cells (Fig. 4.5).

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Figure 4.1 Stable expression of cannabinoid receptors does not alter SH-SY5Y viability or

promote apoptosis. (A) Cells were grown for 9 days, and where indicated subjected to 979 μM

H2O2 for 24 h. Total floating cells in media and attached cells were stained with trypan blue, and

assessed for viability on a Countess Automated Cell Counter. Data are means ± SEM (n=8), and

were analyzed using ANOVA with Dunnett’s test, ***, p<0.001. (B) Representative Western blot

of SH-SY5Y cell lines immunostained with a PARP antibody (whole PARP 116 kDa, cleaved

PARP 89 kDa, actin 42 kDa).

114

Figure 4.2 Stable expression of cannabinoid receptors did not alter the proliferation rate of

human SH-SY5Y neuroblastoma cells. (A) Cell clones were grown, and where indicated, all-

trans retinoic acid (RA; 1 μM) was applied to parental cells every other day. On day 5, cells were

incubated with BrdU for 48 h, immunostained, and nuclei were counted as described in Materials

and methods. BrdU positive nuclei are presented as a percent of total DAPI stained nuclei. Data

are means ± SEM (n=4), and statistical differences from parental vehicle was determined by

ANOVA with Dunnett’s test, ** p<0.01. (B) Cells from indicated lines were plated on day 0, and

counted every other day from days 3 to 9. Means ± SEM were determined from n =

4 experiments, and an exponential growth rate correlation curve was analyzed by linear

regression. (C) Indicated cell lines were counted on day 9, after 7 days of growth with vehicle, 1

μM methanandamide (n = 3) or 1 μM 2-AG-ether (n = 4). Data are shown as means ± SEM (n=3

experiments) normalized to the vehicle controls as 100%. Paired student’s two way t test of drug

compared with vehicle indicated a significant difference after Bonferroni correction between

CB2XS cells treated with 1 μM 2-AG-ether compared with vehicle (p = 0.002). (D) Cell lines

were plated and grown for 2 days, followed by 7 days of growth with or without 1 μM

SR141716A (n=5), 1 μM SR144528 (n=3), or 0.5 μM AM630 (n=5) and counted on day 9. Data

are means ± SEM. ANOVA of Parental SH-SY5Y indicated no significant differences between

SR141716, SR144528, and AM630 groups compared with vehicle SH-SY5Y determined as

100% (F (2, 10) = 0.03015; p = 0.97). Paired student’s two way t test of drug compared with

vehicle detected no significant difference between CB1XS vehicle and SR141716. ANOVA of

CB2XS SH-SY5Y indicated no significant differences between SR144528 and AM630 groups

115

compared with vehicle determined as 100% (F (2, 10) = 0.2134; p = 0.81). (E) Cell cycle phases

of SH-SY5Y cell lines was characterized using FACS of propidium iodide-stained cells as

described in Materials and methods. Data were normalized to the mean of the parental as 100%

(35.7% of total parental cells were in S+G2+M). Data are means ± SEM (n=3), and one-way

ANOVA indicated no significant differences between CB1XS or CB2XS compared with parental

SH-SY5Y.

116

Figure 4.3: Stable expression of cannabinoid receptors does not alter apoptosis in human SH-

SY5Y neuroblastoma cells. The fluorescent signal per cell from an antibody that detects only the

cleaved PARP fragment generated as the end result of caspase cleavage during apoptosis was not

altered by cannabinoid receptor overexpression, n=7. 24 hs of 500 μM H2O2 served as a positive

control known to induce apoptosis and reduce viability. (* denotes p < 0.05)

117

We investigated the susceptibility of the cells to undergo apoptosis by quantifying

cleaved PARP protein fragments. PARP cleavage is the end result of caspase activity and is a

mandatory step after commitment to apoptosis. Cleaved PARP fragments are not present unless a

cell has become committed to and is currently undergoing apoptosis (Palazuelos et al., 2006).

The stable increased expression of CB1 and CB2 cannabinoid receptors did not increase the

abundance of cleaved PARP fragments per nuclei as assessed by quantitative immunostaining of

cleaved PARP (ANOVA among vehicle groups Par, MT4, CB1XS and CB2XS p = 0.5, n = 6, Fig.

4.3). Hydrogen peroxide (H2O2, 24 h of 500 μM) served as a positive control and significantly

increased cleaved PARP immunostaining per cell (p = 0.00002, n = 7, Fig. 4.3).

118

Figure 4.4: Application of methanandamide (mAEA) onto cells stably expressing cannabinoid

receptors does not alter apoptosis in human SH-SY5Y neuroblastoma cells. The fluorescent

signal per cell from an antibody that detects only the cleaved PARP fragment generated as the

end result of caspase cleavage during apoptosis was not altered by cannabinoid receptor

overexpression or application of 24 hours of 10 μM mAEA , n=7. 24 hs of 500 μM H2O2 served

as a positive control known to induce apoptosis and reduce viability. (Dunnett’s post-ANOVA

comparison of parental vehicle vs CB2XS H2O2 p = 0.0001, ****).

119

Figure 4.5: Application of mAEA onto cells stably expressing cannabinoid receptors does not

alter cell number of human SH-SY5Y neuroblastoma cells. mAEA Dose Response Curve. Cells

were deposited in the same initial number, grown in media in an incubator for nine days, and then

counted. Every other day for seven days, doses were applied of 1 nM, 10 nM, 100 nM, 1μM, or

10 μM mAEA, a stable conjugate of the endogenous ligand AEA. Treatment did not alter the cell

counts compared with vehicle (ANOVA of Par doses p = 0.9, ANOVA of CB1XS doses p = 0.8,

ANOVA of CB2XS doses p = 0.9).

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Figure 4.6: Application of 2-AG ether onto cells stably expressing cannabinoid receptors does

not alter cell number of human SH-SY5Y neuroblastoma cells. 2-AG Ether Dose Response Curve.

Cells were deposited in the same initial number, grown in media in an incubator for nine days,

and then counted. Every other day for seven days, doses were applied of 1 nM, 10 nM, 100 nM,

1μM, or 10 μM 2-AG-ether, a stable conjugate of the endogenous ligand 2-AG. Treatment did

not alter the cell counts compared with vehicle (ANOVA of Par doses p = 0.9, ANOVA of

CB1XS doses p = 0.6, ANOVA of CB2XS doses p = 0.2).

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CHAPTER V

CANNABINOID RECEPTORS AND NEURONAL EXTENSIONS

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CHAPTER V: CANNABINOID RECEPTORS AND NEURONAL EXTENSIONS

5.1 CB1 Cannabinoid Receptor Overexpression Alters Neurite Outgrowth

SH-SY5Y cellular projections have been shown in the past to be lengthened by a variety

of signaling molecules, including 10 to 100 μM all trans retinoic acid (RA). Here, RA induction

of longer extensions in SH-SY5Y was used as a positive control to develop a Texas Red Wheat

Germ Agglutinin (TR-WGA) based Image J tracing assay that could numerically depict visual

cellular extension length. All cellular projections were traced. Extensions were summed per field

and divided by the number of nuclei per field. Neurites are defined as extensions longer than one

soma (at least 100 pixels or 30 micrometers) and filopodia are defined as extensions shorter than

one soma (less than 100 pixels or 30 micrometers). Treating SH-SY5Y cells for 5 days with 20

μM RA every other day increased the average neurite length. Vehicle treated parental SH-SY5Y

average neurite length was 0.72 μm, wheras SH-SY5Y cells treated for 7 days every other day

with 20 μM RA averaged 2.96 μm (Fig. 5.1, t test p = 0.004, n = 8). The assay was then applied

to quantify the extensions in the clonal cell lines. The average neurite length was 0.46 μm for the

MT4 neo empty vector control cell line (Fig. 5.1, n = 8), 6.02 μm for CB1XS (Fig. 5.1, ANOVA p

< 0.0001, Par vs CB1XS Dunnett’s adjusted p = 0.0001, n = 6), 0.65 μm for CB2XS 4-2 (Fig. 5.1,

n = 5), 2.65 μm for the CB2XS 4-3 (Fig. 5.1, ANOVA p < 0.0001, Par vs CB2XS 4-3 Dunnett’s

adjusted p = 0.002, n = 5), and 1.69 μm for the CB2XS 4-4 (Fig. 5.1, n = 8). The CB2XS 4-2 and

4-4 clonal cell lines were not significantly different versus parental SH-SY5Y while the CB2XS

4-3 cell was (Fig. 5.1). Application of 20 μM RA every other day did not further increase CB1XS

neurites compared with vehicle control (6.02 μm veh, 6.45 μm RA, p = 0.7, n = 6, Fig. 5.1).

5.2 CB1 Receptor Induced Cell Extensions Already Maximal with Endogenous 2-AG

Oxotremorine M is an agonist of M3 Gq coupled muscarinic acetylcholine receptors

(Billups et al., 2006), which leads via calcium mobilization from intracellular stores to

stimulation of DAGLα production of 2-AG. Mass spectrometry showed that 2-AG at five min

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after 10 μM OxoM treatment had not significantly changed but went from 135 to 564 pmol per

gram of protein (Fig. 3.2). We investigated whether OxoM application could induce a long term

increase in 2-AG abundance that would result in increased extension length. Application of 10

μM OxoM one day before fixation did not increase the neurite length compared with vehicle

CB1XS (5.94 μm, Fig. 5.3, ANOVA p = 0.7, n = 3).

5.3 CB1 Receptor Induced Cell Extension Maximal with Endogenous Receptor Stimulation

Stable overexpression of CB1 receptor in the CB1XS clonal cell line induced an increase

in neurites, defined as extensions that were at least one soma or 30 μm long. CB1 receptor

expression increased neurite length from an average of 0.72 μm per nuclei in the parental SH-

SY5Y to 6.02 μm in CB1XS cells (Fig. 5.1, p = 0.0001, n = 6). In order to determine whether the

increase in neurite length resulting from stimulation of the CB1 receptors by the 135 pmol per

gram 2-AG and 0.82 pmol per gram anandamide that is produced endogenously by SH-SY5Y

cells (Fig. 3.2) was maximal, we further stimulated the CB1XS cells with the exogenous

cannabinoid ligand CP-55,940. Application of 50 nM CP-55,940 on day 4, 24 hs before fixation

on day 5, did not increase CB1XS neurite length compared with CB1XS vehicle control (7.9 μm,

Fig. 5.3, ANOVA p = 0.7, n = 3).

5.4 CB1 Receptor Requires 2-AG Ligand Stimulation to Increase Extension Length

The increase in extension length in the CB1XS cell line could have been due to 2-AG

and anandamide endogenous stimulation of the CB1 receptor, or due to ligand independent

constitutive receptor activity. We investigated whether the receptor was ligand stimulated by

examining whether depletion of 2-AG would decrease extension length. THL is a hydrolase

inhibitor that impairs the activity of DAGLα and which decreased SH-SY5Y 2-AG concentration

from 135 to 15 pmol per gram of protein after application of 1 μM THL for 2 h (Fig. 5.3). The

application of 1 μM THL for 24 h significantly decreased neurite length in the CB1XS cell line

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compared with vehicle control by 42% (Fig. 5.3, p = 0.0002, n = 8), which can be interpreted to

mean that 2-AG ligand stimulation is required for CB1 receptor to increase neuronal extension

length. The experimental result of 42% reduction of neurite length was independent of passage

number as it was replicated at two distinct time points half a year apart with passage 50 and

passage 20 cells, at higher passage number by an average reduction of 43% (CB1XS vehicle

length of 6.02 μm, CB1XS THL treated length of 3.44 μm, n = 3) and at lower passage number

with an reduction of 42% (CB1XS vehicle length of 16.7 μm, CB1XS THL treated length of 9.61

μm, n = 5).

5.5 CB1 Receptor Mediated Cell Extension: Cellular Signaling Mechanisms

Cannabinoid receptors are coupled to multiple proteins that can convey signaling

messages including β-arrestin, Gαi, and the Gβγ complex (Howlett 2005). We used

pharmacological antagonists to identify which pathways were involved in transducing the signal

of the activated CB1 receptor to increase extensions. CB1XS cells were treated for 24 h before

fixation and staining with either 100 ng/mL of Gαi/o inactivator pertussis toxin (PTX), 100 μM of

β-arrestin inhibitor barbadin, or 10 μM of Gβγ inhibitor gallein. Filopodia were defined as short

extensions less than one soma (100 pixels, 30 micrometers) in length. CB1XS cells had

significantly more filopodia length per nuclei than parental SH-SY5Y (Fig. 5.5, Tukey’s adjusted

Par vs CB1XS Veh p < 0.0001, n = 5). Treating the CB1XS cells with 100 ng/mL of PTX for 24

hours significantly decreased the short filopodia length per nuclei compared with vehicle treated

CB1XS SHY5Y cells (Fig 5.5, Tukey’s adjusted CB1XS Veh vs PTX p = 0.02, n = 5). Neurites,

defined as extensions at least one cell body in length, were not affected by 24 hours of treatment

with 100 ng/mL of PTX (Fig 5.5, 14.5 μm, n = 5) or 100 μM barbadin (Fig. 5.5, 15.5 μm, n = 5

Fig.) compared with vehicle (Fig 5.5, 16.7 μm, n = 5). Treatment for 24 h with 10 μM of the Gβγ

inhibitor gallein did not significantly alter neurite length but at 37% length reduction had the most

impact of the three inhibitors (Fig. 5.5, 10.6 μm, Dunnett’s adjusted p = 0.07, n = 5).

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5.6 CB1 Receptor Induced Cell Extensions Increase After ROCK inhibition—

Y27632 selectively inhibits p160ROCK. One h of 50 μM Y27632 did not significantly

change neurite length (Fig. 5.5, 10.1 μm, n = 3). A 24-h application of 50 μm of Y27632

significantly increased the neurite length (Fig. 5.5, 14.0 μm, Dunnett’s adjusted p = 0.01, n = 3)

when compared with vehicle SH-SY5Y cells (Fig. 5.5, 6.02 μm, n = 6).

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Figure 5.1 Stable expression of cannabinoid receptors is associated with neurite development in

SH-SY5Y cells. (A) SH-SY5Y cells were grown for 5 days to 30% confluence on plastic

coverslips treated with RA (20 μM applied on days 2 and 4, fixed, stained and extensions

quantitated as described in Methods. Data are normalized to parental vehicle 0.72 +/- 0.21 as

100%, reported as mean ± SEM (n=8) and analyzed by unpaired two sided Student’s t-test. (B)

Parental SH-SY5Y, MT4, CB1XS and three different clones of CB2XS were grown for five days

to 30% confluence , fixed and stained with Texas red-conjugated wheat germ agglutinin and

DAPI, and traced using Image J as described in Methods. Data are normalized to parental as

100%, reported as mean ± SEM (n=5), and analyzed by ANOVA and Dunnett’s posthoc test. (C)

CB1XS SH-SY5Y cells were grown and treated with RA (20 μM added on days 2 and 4), and

fixed and stained as described in (A). Data are normalized to CB1XS vehicle and reported as

mean ± SEM (n=6), and analyzed by unpaired two sided Student’s t-test. (D, E) A neurite is

defined as an extension longer than 30 micrometers while a filopodia is an extension less than 30

micrometers. Statistically significant differences from control were: ** = p < 0.01; *** = p <

0.001.

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Figure 5.2: Histochemistry of SH-SY5Y stably transfected cell lines. Histochemistry of Texas

Red Wheat Germ Agglutinin and DAPI shows (a) parental SH-SY5Y, (b) parental treated every

other day with 20 μM RA, (c) MT4 neo gene transfection control cell line, (d) CB1XS, (e) CB2XS

4-2, (f) CB2XS 4-3, (g) CB2XS 4-4 which was used for further experiments, (h) CB1XS treated

with 20 μM RA every other day. Scale bar shows 50 micrometers.

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Figure 5.3 Endocannabinoid system factors affecting neurite extension in CB1XS SH-SY5Y cells.

CB1XS cells were grown for five days on plastic coverslips were stimulated on day 4 with CP-

55,940 (50 nM, 24 h) or oxotremorine M (10 μM, 24 h), fixed on day 5, and stained with Texas

Red-conjugated wheat germ agglutinin and DAPI. Extensions were traced in Image J and

quantitated as described in Methods. Data are normalized to vehicle as 100%, and reported as

X±SEM (n=3) and analyzed by ANOVA and Dunnett’s test. (B) CB1XS cells were treated with

vehicle or THL (1 μM, 24 h) before fixation and staining on day 5. Data are normalized to

CB1XS vehicle 6.02 +/- 0.52 as 100%, and reported as X±SEM (n=8) and analyzed by unpaired

two sided Student’s t-test (*** p < 0.001).

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Figure 5.4: Histochemistry of CB1XS SH-SY5Y stably transfected cell lines. Histochemistry of

Texas Red Wheat Germ Agglutinin and DAPI shows (a) CB1XS Veh grown on plastic coverslips

for one week, (b) CB1XS treated for 24 h with 50 nM CP-55,940 before fixation, (c) CB1XS

treated for 24 h with 10 uM of M3 Gq agonist OxoM which is shown to increase 2-AG, (d)

CB1XS treated for 24 h with 1 uM THL which was shown to decrease 2-AG from 135 to 15 pmol

per gram of protein. Scale bar shows 50 micrometers.

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Figure 5.5 Intracellular signaling targets of CB1 receptor stimulation. SH-SY5Y cell lines were

grown for five days on plastic coverslips, and treated with indicated compounds for 24 h prior to

fixation and staining with Texas red-wheat germ agglutinin as described in Methods. The effect

of GPCR activation on (A) neurite extensions greater than 30 micrometers in length or (B)

filopodia extensions less than 30 micrometers in length were determined after application of

pertussis toxin (PTX) (100 ng/ml for 24 h) ; gallein (10 μM), a Gβγ inhibitor or barbadin (100

μM), a β-arrestin inhibitor (n=5). (C) The effect of rho/rac pathways on neurite extensions (as

total length/cell) were determined by application of p160ROCK inhibitor Y27632 (50 μM) for 1

or 24 h prior to fixation and staining. Data are mean± SEM from n=3 experiments, and

normalized to CB1XS Veh 16.7 +/- 1.13. Statistical differences from vehicle control were

determined by ANOVA and Dunnett’s post-hoc test (* p < 0.05, **p < 0.01. ***p < 0.001, ****

p < 0.0001.)

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Figure 5.6 Histochemistry of CB1XS SH-SY5Ystably transfected cell lines. Histochemistry of

Texas Red Wheat Germ Agglutinin and DAPI shows (a) CB1XS SH-SY5Y grown for one week

on plastic coverslips and matching the passage plated for b-d, (b) CB1XS treated for the last 24 h

before the time of fixation with 100 ng/mL PTX, (c) CB1XS treated for the last 24 h before the

time of fixation with 100 μM of β-arrestin inhibitor Barbadin, (d) CB1XS treated for the last 24 h

before the time of fixation with 10 μM Gallein. Scale bar shows 50 micrometers.

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Figure 5.7: Histochemistry of CB1XS SH-SY5Y stably transfected cell lines. Histochemistry of

Texas Red Wheat Germ Agglutinin and DAPI shows (a) CB1XS SH-SY5Y grown for one week

on plastic coverslips and matching the passage plated for b-c, (b) CB1XS treated for the last 1 h

before the time of fixation with 50 μM Y27632, (c) CB1XS treated for the last 24 h before the

time of fixation with 50 μM Y27632, Scale bar shows 50 micrometers.

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Figure 5.8: Effect of cannabinoid receptor overexpression on mRNA of proteins involved in

cytoskeletal regulation. Parental SH-SY5Y, CB1XS, and CB2XS mRNA are shown. REST

analysis of CB1XS vs Par for 2,000 iterations identified GAP43 as significantly different

(upregulated by a mean factor of 4.395, SE range 2.273 – 7.428, p = 0.012, n = 4).

A B

C D

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neurotransmitter regulation. Parental SH-SY5Y, CB1XS, and CB2XS mRNA are shown. REST

analysis of CB2XS vs Par for 2,000 iterations identified SYT as significantly different

(upregulated by a mean factor of 1.749, SE range 1.369 – 2.307, p = 0.03, n = 4).

A B

C D

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cellular adhesion. Parental SH-SY5Y, CB1XS, and CB2XS mRNA are shown. REST analysis

of CB1XS vs Par for 2,000 iterations identified ITGA as significantly different (down regulated

by a mean factor of 0.445, SE range 0.325 – 0.583, p = 0.020, n = 4). CB1XS ST8SIA2 was also

significantly different versus parental (upregulated by a mean factor of 2.474, SE range 1.292 –

4.774, p = 0.038, n = 4). REST analysis of CB2XS vs Par identified NCAM as signficantly

different (upregulated by a mean factor of 2.043, SE range 1.456 – 2.889, p < 0.001, n = 4).

A B

C D

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CHAPTER VI

DISCUSSION AND FUTURE PERSPECTIVES

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6.1 IMPLICATIONS OF CURRENT RESEARCH ON CANNABINOID SIGNALING IN

HUMAN NEURITE EXTENSION

Multiple G protein coupled receptors (GPCRs) have been documented to increase or decrease

neurite extension length. Example GPCRs that increase neurite length include Pac1 (Guirland et

al., 2003), GPR3 (Tanaka et al., 2007), GPR6 (Tanaka et al., 2007), GPR12 (Tanaka et al., 2007),

GPR50 (Grunewald et al., 2009), melanopsin (Li et al., 2016), and CRHR1 (Inda et al., 2017).

Example GPCRs that decrease neurite length are CXCR4 (Xiang et al., 2002a), LPAR4 (Lee et

al., 2007), S1PR2 (Kempf et al., 2014), and muscarinic acetylcholine type 1 receptor (Sabbir et al.,

2018). The CB1 receptor has been documented to both increase length (Bromberg et al., 2008b;

He et al., 2005; Jordan et al., 2005; Jung et al., 2011; Keimpema et al., 2013) or decrease length

(Berghuis et al., 2007; Mulder et al., 2008; Tortoriello et al., 2014) in different test environments.

Recent evidence has also implicated the second cannabinoid receptor, CB2, in neurite outgrowth

to either shorten (Duff et al., 2013) or lengthen (Callen et al., 2012) neurites. These data suggests

a complicated role for cannabinoid receptors 1 and 2 to situationally either increase or decrease

neuritic extension length. The purpose of our investigation was to quantify the impact of CB1 and

CB2 receptor signaling on neurite outgrowth length in a purely neuronal human cell population.

There had never been a direct side by side comparison of the impact of CB1 versus CB2

cannabinoid receptors on extension length. We utilized transfection of CB1 or CB2 receptors to

create stably expressing SH-SY5Y clonal cell lines (CB1XS or CB2XS) to investigate how

cannabinoid receptors and their downstream signaling targets affect neurite extension.

6.2 OVERVIEW OF FINDINGS

In this study we investigated the role of cannabinoid receptors in neurite extension,

proliferation rate, apoptosis rate, and viability in a human SH-SY5Y neuroblastoma model of

increased CB1 or CB2 receptor cannabinoid signaling. The stably transfected exogenously

expressed cannabinoid receptors were maximally stimulated by the endogenously produced 2-AG

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and anandamide; the effect of the receptor on neurite length did not further increase with

exogenous stimulation by CP-55,940 agonist or Oxotremorine M induced calcium stimulated

production of endogenous agonist. Our work has clarified that although CB2 receptor has been

named as a potential initiator of neurite extension, it does not share the ability of CB1 receptor to

statistically significantly induce changes in neurite length, elongating only one CB2XS clone and

not at the magnitude achieved in the CB1XS cell line. The extensions induced by increased CB1

receptor was reversible when 2-AG concentrations were reduced by THL, implying that ligand

stimulation was required for receptor activity and that the CB1 was not constitutively active. The

inhibition of single receptor coupling complexes failed to reverse neurite length, but the greatest

inhibition of CB1 receptor induced neurite length was the application of gallein, p = 0.07, which

decreased extension length in the CB1XS cells. These results may identify the Gβγ subunit as the

greatest magnitude propagator of the extension length altering message, perhaps by coupling to

G12/13 or Gz or a combination therof. The application of all of the single inhibitors (PTX to

inhibit Gαi/o, barbadin to inhibit beta arrestin and possibly CRIP1a, and gallein to inhibit Gβγ,

and possibly its complexes with Gα12, Gα13, and the pertussis toxin insensitive Gαz) reduced

the length of filopodia, defined as extensions shorter than 30 micrometers.

The application of Retinoic Acid (RA) increased the length of neurites in parental SH-

SY5Y but not in CB1XS SH-SY5Y cells. At the mRNA level, MAGL, a cannabinoid receptor

system enzyme, was a mRNA target altered by RA induced neurite outgrowth, identifying a novel

junction point between the two signaling pathways.

We also identified a variety of mRNA targets that mediate cannabinoid receptor initiated

neurite outgrowth, neurotransmitter production, and vesicle regulation. The stable expression of

CB1 receptor increased ST8SIA2 and GAP43 mRNA and decreased ITGA1 mRNA. The stable

expression of CB2 receptor increased NCAM and SYT mRNA.

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Neither CB1 nor CB2 cannabinoid receptor signaling alters proliferation rate or viability

in the SH-SY5Y human neuroblastoma. This was tested by two different antibody assessments of

PARP cleavage to assess apoptosis rate, a trypan total viability stain, and cell counting with and

without methanandamide and 2-AG-ether, analogs of the endogenous agonists, and receptor

specific antagonists SR141716, SR144528, and AM630.

This study demonstrates a role for cannabinoid receptor signaling in neurite and filopodia

extension in a human neuroblastoma cell model that retains the enzymatic components of the

neuronal cannabinoid system and endogenously produces endocannabinoids. The increase in

extension length resulting from CB1 was of twice the magnitude of the maximal response

resulting from stimulating CB2. Of note, this CB1 receptor induced increase in neurite length was

dependent upon endogenous 2-AG production, showing that the receptor was not constitutively

active. Pertussis toxin inhibition of Gα i/o, barbadin inhibition of beta arrestin and possibly

CRIP1a, and gallein inhibition of the Gβγ complex all decreased filopodia that were less than one

soma in length but did not significnaly affect neurites that were at least one soma in length.

Inhibition by gallein of the Gβγ complex achieved a 40% reduction in length that reached p =

0.07, potentially implicating Gα12, Gα13, or Gαz coupling partners in the propagation of the

cannabinoid receptor signal to increase neurite length. Furthermore, the changes to extension

length occurred independent of any alteration in proliferation rate or decrease in cell viability.

6.3 PHYSIOLOGICAL RELEVANCE OF THE SHSH5Y CELL MODEL OF

NEURONAL CANNABINOID SIGNALING IN THE HUMAN BRAIN

The Endogenous Cannabinoid System (ECS) ECS is composed of two G protein coupled

receptors (CB1 and CB2 receptors), two ligands anandamide and 2-arachidonoylglycerol (2-AG)

capable of activating the receptors, and the enzymes that synthesize and biotransform the ligands.

Anandamide is synthesized from N-acyl lyso phosphatidyl ethanolamine by NAPE-PLD (Di

Marzo et al., 1994) and rapidly hydrolyzed to arachidonic acid and ethanolamine by FAAH

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(Cravatt et al., 1996). 2-AG is synthesized from diacylglycerol by DAGL (Stella et al., 1997) and

converted by MAGL, ABHD12, ABHD6, and FAAH to arachidonic acid and glycerol (Blankman

et al., 2007). The enzyme COX2 can also biotransform both anandamide and 2-AG into

prostaglandin products (Kozak et al., 2000; Yu et al., 1997). Anandamide and its metabolites can

also bind to receptor GPR55 and to vanilloid TRPV1 receptors.

Previous reports have published that the ECS is present and functional in SH-SY5Y cells

(Pasquariello et al., 2009) and that NAPEPLD, FAAH, DAGL, and MAGL mRNA are

measurable with PCR. We measured mRNA abundance of ECS components in SH-SY5Y cells

and were able to detect DAGLα, DAGLβ, MAGL, ABHD6, ABHD12, NAPEPLD, FAAH, and

COX2 and graphed these abundances all relative to ENO2 as per the Livak et al. method (Livak

et al., 2001). The presence of DAGL indicates that 2-AG can be synthesized from DAG. The

presence of MAGL, ABHD6, and ABHD12 indicates that 2-AG can be hydrolized to arachidonic

acid and glycerol. Because COX2 is one hundred times less abundant in mRNA than MAGL, 2-

AG is not likely to be being oxygenated to PGH2-G, 11-HETE-G, and 15-HETE-G. The

presence of NAPEPLD indicates that anandamide can be synthesized from N-acyl-

lysophosphatidylethanolamine. The presence of FAAH indicates that anandamide can be

hydrolized to arachidonic acid and ethanolamine. Because FAAH is ten times more abundant in

mRNA expression than COX2, hydrolysis of anandamide to arachidonic acid and ethanolamine is

more likely than oxygenation to PGE2. The mRNA for enzyme components of the endogenous

cannabinoid system were not altered by stable increased expression of CB1 or CB2 receptor.

The existence of an intact endocannabinoid system in parental SH-SY5Y cells was

supported by feedback inhibition of mRNA abundance of the enzymatic components in response

to one week of stimulation with 1 nM of the agonist CP-55,940. One week of treatment with dual

receptor agonist CP-55,940 led to feedback inhibition that halved mRNA abundance of the

enzymes ABHD6, DAGLβ, NAPE-PLD, and PPARα.Treatment with CP-55,940 decreased

ABHD6 (p=0.03), DAGLβ (p=0.04), Integrinα1 (p=0.03), Integrinβ1 (p=0.01), NAPEPLD

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(p=0.01), and PPARα (p=0.02) mRNA relative to vehicle treated SH-SY5Y cells. These data

supports the hypothesis that SH-SY5Y cells contain molecular machinery capable of producing

the ligands that stimulate cannabinoid activity.

Because the enzymes that create and then rapidly biotransform the endogenous ligands 2-AG

and anandamide are present, there was evidence to believe that although cannabinoid receptor

abundance was low in SH-SY5Y neuroblastoma, their neuronal cannabinoid system had remained

intact during the transition to a cancer cell. This was supported by lipid extraction and mass

spectrometry data finding that the steady state concentrations of 2-AG and anandamide. We

employed mass spectrometry to quantify the abundance of cannabinoid ligands 2-AG and

anandamide produced by the enzymes of the endogenous cannabinoid system, and determined the

steady state ligand concentrations. The steady state level of 2-AG in SH-SY5Y cells was 135 ±

47.0 pmol/g of protein. Steady state levels of anandamide in SH-SY5Y cells were 0.82 ± 0.10

pmol/g protein. We demonstrated the functionality of the ECS by applying THL, an inhibitor of

the synthetic enzyme DAGL, to deplete the abundance of 2-AG. Application of THL (1 μM), an

inhibitor of DAGL, for 2 h before cell harves resulted in a concentration of 15 ± 3.6 pmol/g

protein (p = 0.06). The application of THL for the last 24 hours of growth resulted in a 42%

decrease in the neurite extension length in the CB1XS cell line (p < 0.001). Our results therefore

show that despite low cannabinoid receptor abundance, the internal machinery required to

produce endogenous cannabinoid ligands is intact in SH-SY5Y cells. Endogenous 2-AG and

anandamide are produced at a magnitude capable of driving cannabinoid receptor activation and

signaling.

In the human brain 2-AG is hydrolyzed to arachidonic acid and glycerol by the actions of

enzymes MAGL, ABHD6, and ABHD12. Application of 2 h of MAGL inhibitor JZL184,

ABHD6 inhibitor WWL70, and ABHD12 inhibitor ursolic acid decreased the enzymatic

hydrolysis of 2-AG and significantly increased 2-AG concentration to 2,140 pmol per gram

protein. These data illustrates the active role played by the hydrolytic cannabinoid system

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enzymes in SH-SY5Y cells, suggesting that the enzymes involved in synthesis and degradation of

2-AG are similar in SH-SY5Y cells and the human brain. The SH-SY5Yhuman neuroblastoma

cell line was thus employed to model the signaling environment of the human brain, where

endogenous ligand production would stimulate receptors that we stably transfected and expressed

in great abundance. The CB1XS cell line has a greater abundance of CB1 receptor and therefore

greater CB1 receptor initiated signaling, and the CB2XS cell line has a greater abundance of CB2

and CB2 receptor signaling. These cell lines served as models of increased signaling from the

respective cannabinoid receptor.

The existence and functionality of low abundance cannabinoid receptors in SH-SY5Y has

been previously investigated. SH-SY5Y cells possess mRNA at an abundance of 35 cycle

thresholds, protein of both CB1 and CB2 receptors can be detected by Western blot and

immunofluorescence, and the radiolabeled agonist [3H] CP-55,940 can be displaced 50% by 0.1

μM of selective CB1 receptor agonist SR141716, or 0.1 μM of selective CB2 receptor agonist

SR144528 (Pasquariello et al., 2009). We created MT4, CB1XS, and CB2XS cell lines that

maintained expression of plasmid over time by co-culturing with geneticin in the media at a 450

mg/L concentration demonstrated to kill the parental cell line but not the transfected cell lines

(data not shown). We employed quantitative PCR to determine the mRNA abundance of CB1 and

CB2 receptors in the parental SH-SY5Y cell line, the empty vector geneticin resistant clone

(MT4), the CB1 receptor overexpressing clone (CB1XS) and the CB2 receptor overexpressing

clone (CB2XS clone 4-4) . The mRNA of CB1 receptor in the cells lines was stably maintained at

the following cycle thresholds: 34 in parental SH-SY5Y, 33 in the MT4 control, 23 in CB1XS, 36

in CB2XS 4-3, and 35 in CB2XS clone 4-4. The mRNA of CB2 receptor in the cells lines was

stably maintained at the following cycle thresholds: 35 in parental SH-SY5Y, 33 in the MT4

control, 34 in CB1XS, 24 in CB2XS clone 4-2, 22 in CB2XS clone 4-3, and 22 in CB2XS clone 4-

4. The mRNA expression of CB1 and CB2 receptors was maintained at greater abundance in their

respective stably transfected cell lines than in parental SH-SY5Y or the MT4 transfection control.

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We employed Western blot to determine the protein abundance of CB1 and CB2 receptors

in the parental SH-SY5Y cell line, the empty vector geneticin resistant clone (MT4), the CB1

receptor overexpressing clone (CB1XS) and the CB2 receptor overexpressing clone (CB2XS clone

4-4) . The protein of CB1 receptor in the cells lines was stably maintained at the following

abundance relative to parental SH-SY5Y set to 100%: 94% in the MT4 control and 2,611% in

CB1XS. The protein of CB2 receptor in the cells lines was stably maintained at the following

abundance relative to parental SH-SY5Y set to 100%: 65% in the MT4 control and 1,440% in

CB2XS clone 4-4. The protein expression of CB1 and CB2 receptors was maintained at greater

abundance in their respective stably transfected cell lines than in parental SH-SY5Y.

The effect of CB1 receptor on extension length was maximally attained by the steady

state concentrations of 2-AG and anandamide and not further increased by exogenous application

of 50 nM CP-55,940 24 hours before fixation.

These stably transfected human SH-SY5Y neuroblastoma cell lines may be seen as a

model of increased cannabinoid receptor signaling following receptor stimulation from

endogenously produced 2-AG and anandamide cannabinoid ligands. Our clonal SHSYSY cell

lines represent a human neuron derived cell population that contain no other brain cell types.

This model serves in contrast to models derived from isolation of primary cells from a rodent

brain which are rodent neurons and which may contain oligodendrocytes, etc variety of cells

present in the brain.

6.4 RETINOIC ACID DIFFERENTIATION OF SH-SY5Y CELLS AND CANNABINOID

RECEPTOR STABLY EXPRESSING SH-SY5Y CELL LINES

Retinoic Acid (RA) differentiation is frequently employed on SH-SY5Y cells to increase

neurite extension, decrease proliferation, and improve their usefulness as a model of human

disease such as Alzheimer’s disease (Martin et al., 1995). RA is a transcription factor whose

mechanism of action is mediated through heterodimerization of a family of nuclear retinoic acid

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receptors, RAR (Marill et al., 2003). One week of 1 μM RA increases SH-SY5Y mRNA of

neuronal associated proteins such as DDC, and increases protein abundance of dopamine

(Korecka et al., 2013). SH-SY5Y human neuroblastoma increase extension length upon

treatment with RA (Korecka et al., 2013). We employed RA treatment of SH-SY5Y as a known

positive control to design our neurite quantification assay, and confirmed that RA increased SH-

SY5Y neurite extension length.

We found that RA increased extension length in parental SH-SY5Y cells in line with

previous reports. Overexpression of CB1 but not CB2 receptor increased extension length versus

parental and empty vector controls. RA did not further increase the extension length of CB1

receptor stably expressing SH-SY5Y. It is possible that RA and CB1 receptor signaling pathways

converge or crosstalk. From the literature, RA increased CB1 receptor, DAGLα and β, and

ABHD6 mRNA in Neuro2A cells (Jung et al., 2011), and RA increased CB1 receptor expression

in the liver (Mukhopadhyay et al., 2010). Our own data found that amongst the targets of retinoic

acid, the endogenous cannabinoid enzyme MAGL had altered mRNA after one week of 10 μM

RA. The MAGL mRNA was increased at approximately the same magnitude as DDC. An

increase in MAGL activity would lead to a decrease in 2-AG, and could be predicted to reduce

the influence of 2-AG on neurite extension. Local alteration of MAGL abundance at the axonal

growth cone has been observed to be a mechanism of altering neurite extension length

(Keimpema et al., 2013). Another theoretical point of interaction between cannabinoid and

retinoic acid signaling is the COUP family of proteins, which regulates retinoic acid (Tran et al.,

1992) and whose member Ctip2 is regulated by cannabinoid receptor activity (Diaz-Alonso et al.,

2012).

6.5 COMPARISON OF CANNABINOID SIGNALING EFFECT ON PROLIFERATION

AND APOPTOSIS IN SH-SY5Y RELATIVE TO OTHER CELL TYPES

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Cannabinoid receptors have been implicated in altering apoptosis (Pasquariello et al., 2009),

proliferation (Galanti et al., 2008), and autophagy (Vara et al., 2011). PARP cleavage marks

apoptosis (Chaitanya et al., 2010). We assessed cleaved PARP per cell using a quantitative

immunostain and performed a Western blot to assess whole versus cleaved fragments. The

Western blots revealed only whole PARP in the Parental, CB1XS, and CB2XS cell lines, with no

second band for cleaved PARP present. The whole PARP protein was detectable but the cleavage

fragment was not, signifying that the parental, CB1XS, and CB2XS are not undergoing apoptosis

at any appreciable rate. A second, distinct antibody based immunofluorescent quantification assay

also revealed no difference in apoptosis rate in the cannabinoid receptor stimulated SH-SY5Y

cells. The cleaved PARP per nuclei assay revealed no difference between cell types with vehicle

(anova p = 0.5). The positive control of treatment with hydrogen peroxide did increase cleaved

PARP in this assay. Treatment with anandamide, an endogenous cannabinoid receptor agonist,

has been recognized to initiate apoptosis in a CB1 receptor reversible manner (Pasquariello et al.,

2009). The Par, CB1XS, and CB2XS cell lines did not experience PARP when treated with 24

hours of 10 μM methanandamide.

Cannabinoid receptor stable increased expression also did not alter survival in a Trypan assay

of total cell viability (ANOVA p = 0.7) that would have reflected any change in apoptosis,

autophagy necroptosis, pyroptosis, while the positive control of hydrogen peroxide treated cells

experienced reduced viability. This Trypan sum viability assay can be interpreted to mean that

the sum effect of cannabinoid signaling on SH-SY5Y apoptotic and non apoptotic death

mechanisms such as autophagy, necroptosis, and pyroptosis is insignificant. Stable expression of

cannabinoid receptors does not alter the viability of SH-SY5Y human neuroblastoma cells.

Both CB1 and CB2 receptors have had a documented impact upon proliferation in some

cell types. Aguado et al. showed that adult mouse cortex derived neurospheres treated with dual

synthetic agonist WIN-55,212-2, anandamide, and 2-AG increased their expression of

proliferation markers BrdU, Ki67, and nestin. The increase in proliferation was abrogated by

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coapplication of CB1 receptor antagonist SR141716 (Aguado et al., 2005). The mechanism of

CB1 receptor stimulated proliferation may be via AKT/glycogen synthase kinase-3beta/beta-

catenin cyclin D1 signaling, increasing proliferation by decreasing cell cycle exit (Trazzi et al.,

2010). Embryonic rat hippocampus derived neurospheres treated with dual synthetic agonist

HU210 increase incorporation of proliferation marker BrdU (Jiang et al., 2005). In the dentate

gyrus of adult rats, ten day chronic but not acute treatment with dual agonist HU210 increased

proliferation as measured by BrdU uptake (Jiang et al., 2005). Neurospheres derived from the

embryonic cortex of either wild type or CB2 receptor knockout mice treated with selective CB2

receptor agonists HU308 and JWH133 increased in proliferation when quantified by number of

neurospheres and BrdU incorporation (Palazuelos et al., 2006). This proliferative effect was not

seen in the CB2 receptor knockout mouse derived neurospheres and was abrogated in the wildtype

mouse derived neurospheres by CB2 receptor antagonist SR144528. The mechanism of action for

CB2 receptor instigated proliferation may be via PI3K/Akt activated mTORC1 signaling

(Palazuelos et al., 2012). Goncalves et al. showed using Ki67 staining in adult wild type and

TRPV1 knockout mice that subventricular zone adult progenitor cell proliferation is increased by

application of CB2 receptor agonist JWH133 and in a manner abrogated by CB2 receptor

antagonists JTE907 and AM630. Proliferation was also increased by dual cannabinoid receptor

agonist WIN 55,212-2 and FAAH inhibitor URB597. The number of proliferating cells was not

affected by CB1 receptor agonist ACEA (Goncalves et al., 2008).

Based upon these previous studies, we anticipated that stably increased expression of either

cannabinoid receptor would alter the proliferation rate of SH-SY5Y cells. We employed a

Bromodeoxyuridine, BrdU, uptake assay, FACS propidium iodide cell cycle demographics, and

doubling time calculated cell counts over time to test whether cannabinoid receptor signaling in

SH-SY5Y cells would alter proliferation rate. We detected no difference in parental, MT4,

CB1XS or CB2XS cells. There was no difference in growth rate derived from counting cells and

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calculating doubling time, no difference in the percentage of cells in the S, G2, and M phases of

the cell cycle using propidium iodide stained FACS, and no difference in the BrdU uptake rates.

6.6 CANNABINOID RECEPTOR INDUCED CHANGES IN NEURITE AND FILOPODIA

EXTENSION

Present data provides convincing evidence that cannabinoid signaling is involved in

neuronal extension, axon guidance, and synapse formation (Harkany et al., 2008). However, the

conclusion about whether cannabinoid receptor stimulation increases or decreases extension

length appears to depend upon the model chosen. Investigations involving primary cell isolates

from embryonic days 14-18 rodent cortex (Berghuis et al., 2007; Mulder et al., 2008; Tortoriello

et al., 2014) or serum starved neuroblastoma (Zhou et al., 2001) resulted in the conclusion that

CB1 signaling decreases extension length. Investigations involving β-3-tubulin positive purely

neuronal cells such as mouse Neuro2A neuroblastoma (Bromberg et al., 2008b; He et al., 2005;

Jordan et al., 2005; Jung et al., 2011) or purified embryonic day 16.5 cholinergic neurons

(Keimpema et al., 2013) resulted in the conclusion that CB1 signaling increases extension length.

The two models, neuroblastoma and brain cell isolates, in general reach two different

conclusions about cannabinoid mediated neurite extension or repulsion. Neuroblastoma studies

conclude that CB1 receptor increases neurite length while brain primary cell isolates conclude that

CB1 receptor decreases neurite length. This difference in results was not influenced by

cannabinoid alteration of cell fate determination in a mixed neuronal precursor population, such

as when cannabinoid agonist increased BrdU positive proliferating cells to the detriment of the

acquisition of β-3-Tubulin and NeuN in E17 rat cortical progenitors and PC12 cells (Rueda et al.,

2002). A population that has less neurons may have less neurite extensions. However, the cells

in these studies were already β-3-Tubulin positive neurons. The difference in results may lie in

the potential for excreted signaling from a small but nonzero population of non-neuronal cells in

the brain primary isolate culture. The Keimpema et al. group employed embryonic day 16.5 cells,

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but futher isolated a purified cholinergic neuronal culture, and observed increased extension

length after CB1 receptor stimulation. It is therefore reasonable that the difference in results may

have been due to the presence of mixed cell types versus a monotypic cell culture. One example

of a pathway involving paracrine signaling between two cell types is the cannabinoid mediated

Slit2/Robo1 axon repulsion pathway discovered by Alpar et al. (Alpar et al., 2014). This

repulsion growth signaling requires both neurons and oligodendrocytes. Cannabinoid receptor

activity causes excretion of Slit2 by oligodendrocytes and mobilization of Robo1 receptor to the

axon growth cone tip in the neuron. Neuronal CB1 receptor mobilized Robo1 receptor in the

axon tip is activated by Slit2 that was excreted by local oligodendrocytes and the Robo1 signaling

results in axon repulsion (Alpar et al., 2014). This is one example of a paracrine cannabinoid

signaling pathway that cannot be observed in monoculture which lacks both oligodendrocytes and

neurons in the cell culture. This and other yet to be discovered paracrine signaling pathways may

explain why the pure neuron studies conclude that cannabinoids increase extension length while

the embryonic brain isolate studies conclude that cannabinoid signaling decreases extension

length.

CB2 receptor is localized on the growth cone of mouse retinal ganglion cells (Duff et al.,

2013). The effect of CB2 receptor has been documented to increase or to decrease neurite length

depending upon the model. CB2 receptor agonists JWH015 and JWH133 decrease mouse retinal

ganglion neurite surface area. CB2 receptor antagonists AM630 and JTE907 increase neurite

surface area (Duff et al., 2013). The Duff et al. work supports a role of CB2 receptor in

decreasing neurite length. Transient transfection of CB2 receptor into SH-SY5Y combined with

50 nM JWH133 agonist stimulation resulted in an unquantified image and claim that some of the

cells visually appeared to have longer neurites (Callen et al., 2012). The Callen et al. work

supports a role of CB2 receptor in increasing neurite length. Our own findings were performed in

three different human SH-SY5Y neuroblastoma cell lines that had been transfected, clonally

isolated, and stably expressed increased CB2 receptor. Two of the three CB2XS clonal cell lines

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did not experience a significant change in neurite length while one cell line had a statistically

significant increase in length that was of much smaller magnitude than the increase in length that

can be induced by RA or CB1 receptor expression.

In the present study, stably increased expression of cannabinoid receptors altered the mRNA

abundance of proteins that are involved in neurite extension and synapse functional development.

CB1 receptor stably increased expression (CB1XS) increased GAP43 mRNA versus parental SH-

SY5Y. GAP43 is a protein that is present in the axonal growth cone (Strittmatter et al., 1994) and

whose protein concentration is proportional to neurite length (Kim et al., 2012). Integrinα1

mRNA decreased in CB1XS SH-SY5Y, perhaps due to the involvement of integrinα1 in CB1

receptor stimulated FAK activation (Dalton et al., 2013). ST8SIA2, ST8 Alpha-N-Acetyl-

Neuraminide Alpha-2,8-Sialyltransferase 2, is a glycosyltransferase that regulates the activity of

Neural Cell Adhesion Molecule (NCAM). CB1XS increased ST8SIA2 mRNA compared with

parental SH-SY5Y.

CB2 receptor overexpression (CB2XS) increased NCAM and Synaptotagmin (SYT) mRNA.

NCAM and polysialylated NCAM (PSA-NCAM) regulate synaptogenesis and brain plasticity

(Aonurm-Helm et al., 2016). Synaptotagmin is a calcium and inositol polyphosphate binding

protein involved in neurotransmitter release and neurite outgrowth (Mikoshiba et al., 1999).

Stably increased CB2 receptor (CB2XS) increased NCAM and Synaptotagmin (SYT) mRNA.

Stable expression of CB1 receptor induced a significant increase in both neurite (defined as an

extension at least 30 micrometers in length) and filopodia (defined as an extension less than 30

micrometers in length) compared with parental SH-SY5Y cell. The change in extension length in

CB2XS SH-SY5Y was only significantly different in one clonal cell line, CB2XS 4-3, which

experienced an increase in neurite length of minor magnitude. Stable expression of CB2 receptor

did not consistently alter the extension length compared with parental SH-SY5Y.

Reversal of the CB1 receptor signaling effect in CB1XS cells was not significantly achieved

with 24 hour doses of 0.1, 1, or 100 nM of CB1 receptor antagonist SR141716. The application

150

of 50 nM of CB2 receptor ligands CP-55,940 and JWH133 24 hours before fixation did not

change the results of CB2 receptor stable exogenous expression. It is unclear whether exogenous

application of antagonists can reverse neurite increase caused by a greatly transfected protein.

The steady state abundance of cannabinoids appears to be producing a maximal CB1 receptor

induced length response in the CB1XS cell line, as application of oxotremorine M, which

activates the Gq-coupled M3 muscarinic acetylcholine receptors in SH-SY5Y cells to activate

PLC, generate IP3, and release intracellular Ca2+

to stimulate DAGL production of 2AG (Billups

et al., 2006) did not increase extension length in the CB1XS cells. The application of THL, an

inhibitor of the enzyme DAGL that decreased endogenous 2-AG from 135 to 15 pmol per gram

of protein, decreased extension length by 42% (p < 0.05) in CB1XS. Application of 24 hours of

gallein induced a 40% decrease in neurite length in the CB1XS SH-SY5Y (p = 0.07).

Cannabinoid receptors may couple to a variety of molecules, including Gα12, Gα13, Gαz,

Gαi, Gαo, and Gβγ. It is well documented that the family of G αi/o protein coupled receptors

may control the cytoskeleton and neurite extension through a Rap1 to SRK to STAT3 mediated

mechanism (Bromberg et al., 2008a). CB1 receptor signaling through Gα i/o leads to inhibition of

Rap1GAPII, inhibition of Rap1, an increase in Ral, increased Src activity, phosphorylation of two

Stat3 sites and an increase in its activity, resulting in an increase in neurite extension length (He

et al., 2005) (Jordan et al., 2005) (Bromberg et al., 2008b) (Zhou et al., 2011). Src may also

increase Rac activity to lead to Jnk inhibition of SCG10 and an increase of extension length via

that pathway (He et al., 2005) (Shin et al., 2012) (Tortoriello et al., 2014). Through Gα 12 or 13,

cannabinoid 1 receptor may alter RhoA activation of ROCK and myosin to decrease extension

length (Berghuis et al., 2007). Perhaps the CB1 to Gα 12/13 to RhoA signaling pathway requires

interaction with another cell type and cannot be observed in a purely neuronal population. This

idea is supported by the Slit2/Robo1 oligodendrocyte/neuron cannabinoid extension decrease

pathway documented by Alpar et al. (Alpar et al., 2014), where CB2 receptor dependent Slit2

151

excretion by oligodendrocytes causes a CB1 receptor dependent Robo1 mediated repulsion in

neuron growth cones.

Through Gβγ, cannabinoid 1 receptor may activate PI3K and AKT to inhibit BRCA1

inhibition of STAT3 and result in an increase in extension length (Bromberg et al., 2008b;

Keimpema et al., 2013). The Gβγ also allows cannabinoid 1 receptor to increase MAPK activity,

activating CREB and leading to increased neurite extension in that manner (Bromberg et al.,

2008b).

In the present study, we provide evidence that the CB1 receptor induced increase in the human

neuroblastoma cell neurites of at leat 30 micrometers in length was not significantly inhibited by

the application the inhibitor of any single receptor coupling complex. Neither pertussis toxin

(PTX), an inhibitor of Gα i/o but not the family member Gαz, not barbadin an inhibitor of beta

arrestin and possibly CRIP1a, nor gallein an inhibitor of the Gβγ complex significantly decreased

the length of neurites, although gallein did achieve a 40% reduction in magnitude similar to THL

and achieved a p value of 0.07. All three inhibitors significantly decrease the length of filopodia,

or extensions that were less than 30 micrometers in length.

In agreement with the Neuro2A model of purely neuronal population studies in which

cannabinoid receptors increased extension length (He et al., 2005; Jordan et al., 2005; Bromberg

et al., 2008b; Jung et al., 2011), we found that CB1 receptor activation lead to an increase in

neurite extension length. In agreement with the findings of Berghuis et al. where CB1 receptor

activation of RhoA lead to activation of ROCK and a decrease in neurite length, we also found

ROCK activation to have a negative impact on extension length that can be reversed by

application of ROCK1 inhibitor Y27632. Exogenous inhibition of ROCK1 through application

of Y27632 further increased the extensions in the already lengthened CB1XS cells. This further

increase to extension length by ROCK inhibition in the CB1XS cells is especially notable when

contrasted with the application of RA, which did not further increase the length of neurites in

CB1XS SH-SY5Y.

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Figure 6.1 Cannabinoid signaling pathways affecting neuronal extension length. Studies which

observed a RhoA to ROCK mediated neurite length decrease uniformly employed primary cells

isolated from rodent E 14-17 brain cortex, and may support the idea of a multi cell type

collaborative signaling pathway that could not be observed in purely neuronal culture such as our

SH-SY5Y human neuroblastoma. Studies using either a neuroblastoma cell line or further

isolation of primary cells to obtain a purified neuronal population consistently report that CB1

stimulation increases extension length. References: 1. (Diez-Alarcia et al., 2016), 2. (Siehler

2009), 3. (Garzon et al., 2009), 4. (Meng et al., 1999), 5. (Jordan et al., 2005), 6. (Bromberg et al.,

2008b), 7. (Gutkind 2000), 8. (Berghuis et al., 2007), 9. (Mei et al., 2011), 10. (He et al., 2005),

153

11. (Yuan et al., 2003), 12. (Lawson et al., 2014), 13. (Tortoriello et al., 2014), 14. (Alpar et al.,

2014), 15. (Bromberg et al., 2008a), 16. (Shin et al., 2012), 17. (Zhou et al., 2011), 18.

(Keimpema et al., 2013), 19. (Wang et al., 2018)

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6.7 CONCLUSIONS

We have described a new model for investigating the divergent effects of CB1 versus CB2

receptor stimulation in human disease. We created clonal cell lines that stably express either

increased CB1 (CB1XS) or CB2 (CB2XS) receptor in human neuroblastoma SH-SY5Y as a model

to investigate the role of cannabinoid signaling in neurite extension, proliferation, apoptosis, and

cell viability. The mRNA of the enzymatic components of the endogenous cannabinoid system

DAGL, MAGL, ABHD6, ABHD12, NAPE-PLD, and FAAH are present in SH-SY5Y cells.

Cannabinoid receptor stably increased expression did not alter the mRNA abundance of the

enzyme components of the endogenous cannabinoid system in SH-SY5Y cells. Activity of these

enzymes produced endocannabinoid ligands 2-AG and anandamide, resulting in a steady state

concentration of 135 pmol of 2-AG and 0.82 pmol of anandamide per gram of protein.

Application of THL, an inhibitor of DAGL activity, decreased 2-AG abundance to 15 pmol per

gram protein and as expected did not alter anandamide abundance at 0.79 pmol per gram of

protein. Oxotremorine M, an agonist of Gq coupled muscarinic acetylcholine receptors,

stimulated DAGL, increasing 2-AG abundance to 564 pmol per gram protein. Inhibition of

hydrolysis of 2AG by MAGL, ABHD6, and ABHD12 significantly increased 2-AG abundance to

2,140 pmol per gram protein. Transfection of SH-SY5Y cells resulted in a stable increase of CB1

receptor mRNA by 10^11 abundance in the CB1XS cell line and of CB2 receptor mRNA by

10^12 abundance in the CB2XS cell lines. Increased expression of CB1 but not CB2 receptor

increased neurite length from 0.7 to 6.0 micrometers per nuclei. This neurite length was halved

by THL inhibition of DAGL production of 2-AG. The filopodia, or extensions less than 30

micrometers in length, induced by CB1 receptor expression were signficantly inhibited by the

application of PTX, barbadin, or gallein. Increased expression of CB1 receptor increased the

mRNA abundance of GAP43 and ST8SIA2 mRNA and decreased ITGA1 mRNA. Stably

increased expression of CB2 receptor increased NCAM and SYT mRNA. MAGL mRNA was

significantly altered by RA treatment, identifying a component of the endogenous cannabinoid

155

system as a novel RA transcription target. Overexpression of cannabinoid receptors did not alter

the viability, apoptosis rate, or proliferation rate of cells relative to parental or empty vector SH-

SY5Y.

We contrasted the role of cannabinoid receptors CB1 and CB2 receptors in neurite extension,

proliferation rate, apoptosis rate, and total cell viability. Cannabinoid receptor CB1 greatly

increased extension outgrowth length in human SH-SY5Y neuroblastoma, while CB2 receptor did

not. Both receptors altered mRNA of different proteins involved in neurite extension,

neurotransmitter organization, and release. Neither altered proliferation rate or overall cell

viability. We provide evidence that CB1 and CB2 cannabinoid receptors play different roles in

neurons.

156

Figure 6.2: Effect of CB1 and CB2 cannabinoid receptors extension length, apoptosis,

proliferation rate, and gene expression. CB1 receptor greatly increased extension length, while

CB2 receptor did not. CB1 receptor increased GAP43 and ST8SIA2 mRNA and decreased

ITGA1 mRNA. CB2 receptor increased NCAM and SYT mRNA. Neither CB1 nor CB2 receptor

altered proliferation rate, apoptosis rate, or overall SH-SY5Y cell viability.

157

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CURRICULUM VITAE

179

ERICA L. LYONS

2327 Winston Ct. Apt C, Winston-Salem, NC 27103

(216) 978-1886. [email protected]

EDUCATION

Wake Forest School of Medicine Department of Internal Medicine - Molecular Medicine. Medical Center Boulevard, Winston-Salem, NC 27157. Ph.D. in Molecular Medicine and Translational Science, May 2019 (expected). GPA 3.50 Advanced to Ph.D. Candidacy April 21st, 2014. Case Western Reserve University Department of Chemical Engineering, 10900 Euclid Ave, Cleveland, OH 44106. B.S.E. in Chemical Engineering specializing in Biochemical Engineering, 2011. GPA 3.3 Minor: Japanese.

RESEARCH EXPERIENCE

Wake Forest School of Medicine

May 2013 - present

Ph.D. Candidate

Advisor: Dr. Allyn Howlett

Successfully proposed and defended a research plan to a committee, advancing to Ph.D.

candidacy. Collected data and presented two posters at competitive conferences.

Prepared a first author manuscript that is currently being finalized for publication.

Wake Forest School of Medicine

June 2012 - Apr 2013

Ph.D. Student

Advisor: Dr. Mary Sorci-Thomas

Contributed mouse compound injection, aortic embedding, cryosectioning,

immunohistochemistry, and quantification to a published manuscript.

Wake Forest Institute for Regenerative Medicine

Mar 2012 - May 2012

Rotation Student

Advisor: Dr. Bryon Petersen

Trained in the decellularization of porcine liver in order to create a scaffold for cell

seeding and organoid development.


Oct 2011 - Feb 2012

180

Rotation Student

Advisor: Dr. James Yoo

Analyzed the extracellular matrix of decellularized porcine kidney in order to optimize a

scaffold for cell seeding and organoid development.


Aug 2011 - Oct 2011

Rotation Student

Advisor: Dr. Shay Soker

Contributed immunohistochemical staining of muscle tissue samples in a rat

compartment syndrome model.

Polymer Diagnostic Inc.

Jun 2010 - Jan 2011

Intern Laboratory Technician

Trained in and executed more than 20 different ASTM standard laboratory testing

procedures. Operated over 20 different instruments.

Case Western Reserve University

Sep 2008 – May 2010

Advisor: Dr. Heidi Martin

Modified the surface of diamond films by attaching paraphenylene diamine to create a

selective biosensor diamond electrode. Fully trained in diamond growth using a hot

filament diamond reactor, cyclic voltammetry using a potentiostat, and near surface

microscopy using X-Ray photoelectron spectroscopy.

Case Western Reserve University

Jan 2008 – May 2008

Advisor: Dr. David Schiraldi

Tested the feasibility of carbon dioxide and calcium hydroxide reaction within PAA

montmorillonite composite to potentially sequester carbon dioxide gas in an industrial

setting to reduce atmospheric emissions.

HONORS / AWARDS

Case Western Reserve University SOURCE Summer Research Grant 2009

Case Western Reserve University Provost’s Scholarship 2007-2011

Center for Molecular Communication and Signaling Mini Grant 2016

181

PUBLICATIONS

Pollard RD, Blesso CN, Zabalawi M, Fulp B, Gerelus M, Zhu X, Lyons EW, Nuradin N,

Francone OL, Li XA, Sahoo D, Thomas MJ, Sorci-Thomas MG. (2015) Procollagen C-

endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by

Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein

(HDL)-Cholesteryl Ester Uptake. J Biol Chem. 290(25):15496-511.

PMID: 25947382. PMCID: PMC4505464.

Lawrence C. Blume, Sandra Leone-Kabler, Deborah J. Luessen, Glen S. Marrs, Erica

Lyons, Caroline E. Bass, Rong Chen, Dana E. Selley, and Allyn C. Howlett. (2016)

Cannabinoid Receptor Interacting Protein (CRIP1a) suppresses agonist-driven CB1

receptor internalization, and regulates receptor replenishment in an agonist-biased

manner. J Neurochem. 2016 Nov;139(3):396-407. doi: 10.1111/jnc.13767. Epub 2016

Sep 26.

Lyons, EL, Kabler SK, Howlett AC. (2018) CB1 and CB2 Cannabinoid Receptors

Increase Neurite Extensions in a Human SH-SY5Y Neuronal Model (pending)

ABSTRACTS TO MEETINGS

Lyons, EL, Kabler SK, Howlett AC. (2014) “Endogenous Cannabinoid Signaling in

Neuronal Development” presentation. 4th Annual T32 Symposium on Integrative Lipid

Sciences, Inflammation, and Chronic Diseases. Winston-Salem, NC.

Lyons, EL, Kabler SK, Howlett AC. (2014) “Endogenous Cannabinoid Signaling In a

CB1R or CB2R Transfected SH-SY5Y Human Neuroblastoma Cell Model” poster.

Carolina Cannabinoid Collaborative Conference. Winston-Salem, NC.

Lyons, EL, Kabler SK, Howlett AC. (2015) “Stable Overexpression of Cannabinoid

Receptors in SH-SY5Y Neuroblastoma” poster. Center for Molecular Communication

and Signaling Annual Retreat. Winston-Salem, NC.

Lyons, EL, Kabler SK, Howlett AC. (2015) “Stable Overexpression of Cannabinoid

Receptors in SH-SY5Y Neuroblastoma” poster. Carolina Cannabinoid Collaborative

Conference. NIAAA, Rockville, MD.

Lyons, EL, Kabler SK, Howlett AC. (2017) “Cannabinoid Receptor CB1 but not CB2

Increases Neurite Extension in Human Neuroblastoma” poster. Carolina Cannabinoid

Collaborative Conference. Raleigh, NC.

182


Increases Neurite Extension in Human Neuroblastoma” poster. Experimental Biology

Conference. San Diego, CA.


Increases Neurite Extension in Human Neuroblastoma” poster. Center for Molecular

Signaling Conference. Winston Salem, NC.


Increases Neurite Extension in Human Neuroblastoma” poster. Center for Research on

Substance Use and Addiction Retreat. Winston Salem, NC.


Increases Neurite Extension in Human Neuroblastoma” poster. Carolina Cannabinoid

Collaborative Conference. Raleigh, NC.

PROFESSONAL SOCIETIES

International Cannabinoid Research Society

American Society for Biochemistry and Molecular Biology

FUNDING

T32 HL091797 Parks (PI)

08/13/12-08/13/15

Integrative Lipid Metabolism, Inflammation, and Chronic Diseases

The goal of this T32 grant was to prepare predoctoral trainees for careers that have a

significant impact on the health related research needs of the nation.

Role: predoctoral student trainee

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