Structural Changes in the Primary Visual Cortex of Alzheimer’s Disease Model Mice As A Correlate to

Diminished Plasticity

Kiana Brown

May 2015

 

 

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Structural Changes in the Primary Visual Cortex of Alzheimer’s Disease Model Mice As A Correlate to

Diminished Plasticity

An  Honors  Thesis  Submitted  to  the  Department  of  Biology  

in  partial  fulfillment  of  the  Honors  Program  STANFORD  UNIVERSITY  

By Kiana Brown

May 2015

 

 

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Acknowledgments

I would like to thank my graduate student mentor George Vidal for teaching me the techniques used in my research, as well as for his support and assistance throughout my past year in the lab. I would also like to thank him for reading through many drafts of my thesis. I could not have completed this project without him. I would also like to thank Richie Sapp, who has been an outstanding peer and support through my time in the lab, and especially helpful in writing this thesis. I would like to thank Maja Djurisic for teaching me confocal microscopy, which has been central to imaging and obtaining results, as well as for general help throughout my time in the lab. I would like to thank Taeho Kim for his intellectual contributions that have prefaced my current work. I would also like to thank Dr. Carla Shatz, Principal Investigator, for the opportunity to conduct research in her lab and for her feedback and contributions to my research techniques and experimental design. She has been an amazing mentor and taught me a great deal about research in general. I would also like to thank the other members of the Shatz lab, Barbara Brott, Hanmi Lee, Kylie Chew, and Michael Birnbaum, for their support and help throughout my time in the lab. I would like to thank Thomas Joseph for his support in editing my thesis, as well as formatting figures. Nora Sotelo-Kury has been an invaluable resource in logistical and organizational support and I cannot thank her enough for that. Finally, I am grateful to Peggy Kemper and Christina Chechelski for their hard work on animal breeding and animal transfers of all the mice used in this study.

 

 

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Table of Contents

Abstract…………………………………………………………………………………………...6 Introduction………………………………………………………………………………………7 Neural Plasticity…………………………………………………………………………...8 Molecular Controls of Plasticity…………………………………………………………..9

Structural Changes and Correlates of Plasticity………………………………………….11 Alzheimer’s Disease……………………………………………………………………..13

Materials and Methods………………………...……………………………………………….15 Mice……………………………………………………………………………………...15 Perfusions………………………………………………………………………………...15 Immunostaining………………………………………………………………………….15

Dendritic Spine Analysis………………………………………………………………...16 Results…………………………………………………………………………………………...16 Dendritic Spine Density of Juvenile Transgenic Mice Is Not Decreased………………..16 Dendritic Spine Density of Adult Transgenic Mice Is Not Decreased…………………..17 Discussion/Conclusions…………………………………………………………………………17 References……………………………………………………………………………………….21 Figures…………………………………………………………………………………………...23

 

 

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List of Figures

Figure A Schematic of Mouse Visual System

Figure B Schematic of APP/PS1;YFP-H+ Mouse Breeding

Figure C Western Blot Analysis of APP and Aβ Expression

Figure 1 Dendritic spine density remains unaltered in P30 APP/PS1 mice

Figure 2 P30 APP/PS1 mice show similar dendritic spine morphology as compared to wildtype

Figure 3 Dendritic spine density elevated in P74 APP/PS1 mice

 

 

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Abstract

Amyloid-β (Aβ) accumulation has been implicated in Alzheimer’s disease pathogenesis.

Mice with transgenic expression of mutant amyloid precursor protein (APP) and presenilin-1

(PS1) accumulate Aβ and have therefore been used as models of the disease. Previous research

has shown that critical period ocular dominance (OD) plasticity is defective in these mice, which

indicate an early effect of Alzheimer’s on neural plasticity, even before the typical Aβ plaques

accumulate. In an attempt to determine the mechanism responsible for this defect, I investigated

layer 5 apical dendritic spine density and morphology in the primary visual cortex of juvenile

(P30) and adult (P74) APP/PS1 mice. We found that wildtype and APP/PS1 transgenic juvenile

and adult mice both displayed comparable spine densities and initially concluded that the critical

period ocular dominance plasticity defect observed in APP/PS1 transgenic mice is not associated

with a decrease in spine density in layer 5 apical dendrites. However, recent discoveries within

the lab have led us to believe that the APP/PS1;YFP-H+ mouse line used in these experiments

may have epigenetic or other modifications that would inhibit transcription of YFP and/or

APP/PS1. Further experiments should be conducted to verify the presence of Aβ accumulation in

this mouse line before continuing research.

 

 

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Introduction

Neural plasticity refers to the brain’s ability to rewire pathways and synapses in response

to changes in behavior and environment, and to injury. The Shatz lab recently discovered that

specific molecules such as Major Histocompatibility Complex Class I (MHCI) (Corriveau et al,

1998) and its receptor, Paired Immunoglobulin-like receptor B (PirB) (Syken et al, 2006) appear

to act as “brakes” on forms of synaptic plasticity in the visual system, and may do so by

regulating synaptic density on pyramidal neurons of the cerebral cortex (Djurisic et al, 2013).

These molecules could therefore play a crucial role in understanding and treating Alzheimer’s

and other diseases that are characterized by loss of neural plasticity. Current mouse models for

Alzheimer’s disease express a transgenic, mutant form of amyloid precursor protein (APPswe)

and presenillin (PS1dE9), a subcomponent of the gamma secretase that cuts APP into its mature

form, amyloid-β (Aβ). The fibrillar form of this Aβ protein causes the amyloid plaques that are a

signature of Alzheimer’s pathology (Cerasoli et al, 2015). PirB is a high affinity receptor for Aβ

(Kim et al, 2013). Studies have already shown that APP/PS1 mice show an early form of

synaptic plasticity defect (William et al, 2012) that could possibly be restored. However, while

synaptic loss is an important characteristic of Alzheimer’s pathology, it is unknown whether

APP/PS1 mice exhibit an early loss of synapses, and whether blocking PirB function would

restore synapse number to normal levels. Therefore, monitoring structural and functional

synaptic changes in APP/PS1 mice is a crucial first step. If there is measurable synapse loss, next

steps are to determine whether PirB level modulation could act to restrict synaptic density early

on. If there is a structural correlate for the early plasticity deficits found in APP/PS1, and if this

correlate can be restored when blocking or removing PirB, then blocking the PirB-Aβ interaction

could become an extremely important early therapeutic.

 

 

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Neural Plasticity

A crucial aspect of the brain is its ability to adapt according to sensory input and learning

(Feldman, 2009). Neural plasticity, the term used to describe these changes, is one of the major

fields of study within the field of neuroscience and is crucial for normal development of learning

and memory, the development of sensory maps, and recovery following injury. By uncovering

the mechanisms of neural plasticity, we can discover therapeutic techniques for disease such as

Alzheimer’s, which is neurodegenerative in nature and restricts various forms of neural plasticity

(William et al, 2012).

One method to study neural plasticity is through experience-driven or training-induced

changes in primary sensory cortex topographical maps. In the primary visual cortex, current

research focuses on ocular dominance plasticity and synaptic formation and loss (Bear &

Rittenhouse, 1999; Hensch, 2005; Levelt & Hübener, 2012). By studying the mouse visual

system through these methods, we have access to a highly adaptable model of neural plasticity,

as wiring is refined during critical periods of plasticity by interactions involving neural activity

and structural remodeling.

For example, wiring from the retina to the thalamic lateral geniculate nucleus (LGN)

occurs as spontaneous patterns of neural activity are sent to the brain from the eye, and is refined

by this activity. By blocking activity from retinal ganglion cells with injections of tetrodotoxin (a

voltage-gated sodium channel blocker) in utero or after birth through the critical period, normal

wiring is disrupted. Specifically, blocking activity from one retina induces projections from the

unmanipulated eye to expand into LGN territory normally belonging to the blocked eye (Penn, et

al, 1998). In the case of alterations to the synaptic plasticity of mice, these patterns get altered

and are often abolished or attenuated.

 

 

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Another example of plasticity can be found

in the visual cortex. The visual cortex of normally

reared adult mice receives functional input from

both eyes. The monocular zone, where neurons are

driven solely by input from the contralateral eye,

encompasses a majority of the visual cortex. A

much smaller binocular zone also exists, receiving

input from both the ipsilateral and contralateral

eye. During development, larger areas of the visual cortex initially receive input bilaterally, but

ipsilateral eye input becomes restricted in an activity-dependent manner by the postnatal week

four, known as the critical period (Gordon & Styker, 1996). In the adult visual cortex, signals

from the eye are relayed mainly to the contralateral visual cortex, as well as a smaller binocular

zone of the ipsilateral cortex. This is called ocular dominance and can be altered in mice if the

relative ratio of activity being driven by both eyes is changed. Closing or removing one eye

during the critical period has been shown to shift ocular dominance towards the open functioning

eye (Antonini & Styker, 1993).

Molecular Controls of Plasticity Neural plasticity is under the control of many factors, including a number of molecules,

such as MHC I and its receptor, PirB. Originally thought to be located only in the immune

system, the Shatz lab has since discovered these molecules also regulate plasticity in the central

nervous system (CNS). In the immune system, MHCI molecules present antigens to cytotoxic T-

cells. There are over 50 different MHCI genes that encode MHCI proteins, which are cell-surface

ligands and must act through a receptor-mediated mechanism (Ploegh   et   al,   1981). In the

Figure A: Schematic of Mouse Visual system. Binocular Zone (BZ) receives input from both eyes (Datwani et al, 2009)  

 

 

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nervous system, an unbiased screen for activity-regulated genes in the LGN was performed

during layer-specific eye formation. This led to the discovery that MHCI mRNA is

downregulated in correlation with activity blockade (Corriveau  et  al,  1998). Subsequent studies

have shown that synaptic plasticity is dependent on cell surface expression of MHC I (Huh,  et  al  

2000) and in mice lacking two specific MHCIs, H2-Kb and H2-Kb, ocular dominance plasticity

during the critical period and adulthood is enhanced. Together, these results suggest that

neuronal MHCI negatively regulates ocular dominance plasticity and is involved in the

regression and/or elimination of inappropriate synaptic connections. Known receptors for

immune MHCI ligands were not found to be expressed in the CNS, (Syken  &  Shatz,  2003) so a

screen to find a novel receptor expressed in the cortex was performed. This study led to the

identification of PirB as a MHCI receptor.

Separate studies have shown that MHCI is a physiological ligand for PirB, meaning

MHCI can bind and signal through PirB (Takai, 2005) The PirB molecule consists of six

extracellular immunoglobulin (IG)–like domains, a transmembrane domain, and four

intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) domains. Found in a

number of immune cells, including mast cells, B-cells, and macrophages, PirB aids in anti-

inflammation and recruiting phosphatases to decrease signaling in other pathways that

antagonize integrin and MAP Kinase cascade pathways (Maeda et al, 1999; Takaix, 2005). In

neurons, when ligands bind PirB, the ITIM domains are phosphorylated and PirB recruits

members of the SHP family of phosphatases (SHP-1,2), which sends an inhibitory signal to other

cellular pathways (Syken et al 2006). The subsequent pathways are currently undetermined.

 

 

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Structural Changes and Correlates of Plasticity In addition to molecular controls of plasticity, structural changes are found to occur, both

through Arc mRNA induction and spine density measurements. Arc mRNA induction involves

exposing an eye to visual stimulation, which rapidly induces activity-regulated intermediate-

early gene Arc mRNA in visual cortex neurons driven by the stimulated eye. During monocular

enucleation (removal of one eye), inputs to the binocular zone driven by the remaining functional

eye expand. This is a measure of the strengthening of inputs from the open eye, which is a major

component of ocular dominance plasticity. By studying Arc mRNA induction in mice, it was

uncovered that mice lacking PirB display increased ocular dominance plasticity in the visual

cortex in both the critical period and adulthood as compared with wildtype (Syken et al, 2006).

A recently investigated mechanism for how ocular dominance plasticity occurs is by

identifying structural synapse changes that occur along with this shift in ocular dominance.

Inspired by Eric Knudsen’s experiments in barn owls, Hofer et al, 2009 studied dendritic spine

density. It was already known that temporary closure of one eye leads to ocular dominance

plasticity shifts that are reversed once normal vision is restored. When these same mice

experience a second monocular deprivation weeks later, the ocular dominance plasticity shifts

that occur are much more accelerated than the first, indicating that a structural remnant exists in

cortical circuits. By repeatedly imaging dendritic spine through the cycle of two monocular

deprivations, it was discovered that dendritic spine densities increase following the first

monocular deprivation, persist even after normal vision is restored, and allow for a more

accelerated ocular dominance shift when the mice undergo a second monocular deprivation.

Conclusions from the research noted that in the normal adult mouse visual cortex, monocular

deprivation will increase spine density and that this increase is stable and thought to be a

structural substrate for enhanced ocular dominance plasticity (Hofer et al, 2009).

 

 

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In PirB deficient mice, Djurisic et al (2013) studied changes in dendritic spine density as

well as miniature excitatory postsynaptic current (mEPSC) frequency to determine that the

spines are indeed functional synapses. Imaging of apical dendrites of layer 5 pyramidal neurons

from wildtype and PirB germline knockout mice showed that PirB -/- mice have increased spine

density. This increase is insensitive to the effects of visual deprivation suggesting that PirB -/-

mice have already reached a ceiling spine density level, which cannot be further increased even

following monocular deprivation.

Following this study, the Shatz lab created sPirB, a soluble form of PirB that lacks the

intracellular domains necessary for PirB signaling. sPirB may act as decoy to endogenous

ligands (Bochner et al, 2014). In effect, sPirB blocks the function of endogenous PirB. This

approach was chosen as a proof-of-concept to achieve rapid and local disruption of PirB as a

therapeutic reagent.

Minipump infusion of sPirB during the critical period resulted in a marked increase in

ocular dominance plasticity, as assessed by Arc mRNA induction. Layer 5 pyramidal neurons

also showed increased spine density compared to wildtype, even in the absence of visual

deprivation (Bochner et al. 2014). Taken together with the increase in mEPSC frequency, these

results readily support the theory that sPirB infusion causes an increase in synaptic connectivity,

bolstering the role of PirB as a negative regulator of neural plasticity in the cortex.

To study PirB function in the hippocampus, an area of the brain intimately associated

with memory formation, LTP in in vitro hippocampal slice physiology was examined for PirB

KO mice (Djurisic et al, 2011; Sapp, 2013). Increased excitatory LTP in CA1-CA3 synapses

were identified. Further behavioral studies indicated that PirB KO mice display increased

acquisition and increased extinction in a hippocampal contextual and fear-conditioning paradigm.

 

 

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Additionally, PirB KO mice showed improved learning in the Morris Water maze spatial

learning task (Djurisic et al, 2011). These results indicate a behavioral increase in learning speed

and strength.

Alzheimer’s Disease Although decreases in plasticity are important for stabilizing matured neural circuits by

studying ways to increase plasticity, we endeavor to find possible therapeutics that can be used in

treatments for neurodevelopmental and neurodegenerative diseases. PirB has been shown to be

an important regulator of neural connections and decreases in the protein, either through genetic

knockouts or decoy sPirB infusion, predictably lead to increased synaptic connectivity that

appears completely functional. Additionally, these physiological changes were correlated with

behavioral changes of increased speed and strength of learning.

Alzheimer’s Disease is a complex neurodegenerative disease that affects somewhere

between 3 and 4 million people and costs upwards of $1 billion annually in the United States

alone. Characterized mainly by dementia and memory loss symptoms, it is complex to treat

because of the array of molecular defects that can spur its pathology. Hallmarks of the disorder

include substantial cortical atrophy and upon silver stain examination, loss of neurons and

buildup of amyloid plaques (Selkoe & Lansbury, 1999). Amyloid β (Aβ) is a 36-43 amino acid

peptide formed from amyloid precursor protein (APP) being cleaved by specific enzymes.

Aggregation of misfolded Aβ leads to plaques, which are toxic to nerve cells. Soluble Aβ

oligomers are known to impair synaptic plasticity and cause the neuroplasticity loss prevalent in

Alzheimer’s.

An in vivo murine model of Alzheimer’s, which expressed mutant alleles of both amyloid

precursor protein (APPswe) and presenillin 1 (PSEN1ΔE9) showed very early loss of activity-

dependent plasticity. Aβ plaque deposition occurs at several months of age, long after the critical

 

 

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period of visual cortical plasticity. (Garcia-Alloza et al, 2006 & Jankowksy et al, 2003).

However, normal ocular dominance plasticity was disrupted early on. (William et al, 2012). This

follows the progression thought to occur in human Alzheimer’s patents, where those with Aβ

plaques have already reached a late stage of the disease. In another study, PirB was shown to

regulate plasticity in Alzheimer’s mouse models (Kim et al, 2013). Both PirB and LilrB2, the

human homolog to PirB, are receptors for Aβ oligomers. Using biotinylated synthetic human

Aβ1-42 (Aβ42) peptides as monomers and oligomers, Aβ42 binding to human embryonic kidney

cells expressing PirB or control were tested. Oligo-Aβ42 was bound PirB-expressing cells six

times more than monomeric Aβ42 and found to be constantly associated with PirB protein in

both coimmunoistaining and coprecipitation experiments. This binding is specific to PirB, as

Aβ42 did not bind cells expressing mouse PirA1, mouse PirA4 or an isoform of rat PirB to any

appreciable degree.

Once it was known that PirB binds Aβ42, a more direct contribution of PirB to

Alzheimer’s pathology was sought. APP/PS1 transgenic (Tg) mice were crossed with PirB-/-

mice to generate littermates that were either PirB+/-Tg or PirB -/-Tg. At 9mo of age, recognition

memory was evaluated through novel object and novel place behavioral tests. (Kim et al, 2013)

While both tests revealed the expected impaired behaviors in PirB+/-Tg mice, these learning and

memory deficits were not apparent in mice lacking PirB (PirB -/-Tg). In summary, these results

suggest that PirB may act by negatively regulating plasticity and limiting PirB could be a method

of alleviating the effects of Alzheimer’s.

In order to elucidate how PirB plays a role in Alzheimer’s and how effective sPirB would

be as a potential therapeutic, the details of synaptic alteration need to be examined in more depth.

Since it is known that PirB-/- mice show increased layer 5 pyramidal neuron spine densities as

 

 

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well as an increased area of Arc mRNA induction, the effect of Alzheimer’s on these structural

measures of plasticity should be examined. This project aims to identify and quantify changes in

APP /PS1 transgenic mouse spine density to characterize the development of Alzheimer’s.

Drawing from previous research that shows ocular dominance plasticity is defective in juvenile

transgenic mice, we hypothesize that APP/PS1 juvenile (P30) transgenic mice will also have

lower spine densities than wildtype littermates. Furthermore, this effect should become more

pronounced in adult (P74) mice as the disease has more time to develop.

Materials and Methods Mice APP/PS1 heterozygous transgenic mice (strain B6C3 Tg(APPswe,PSEN1dE9)85Dbo/J; Jan-kowsky et al., 2001) from The Jackson Laboratory were crossed into a PirB +/+ line, generating male mice in line 1 of the schematic below. All subsequent crosses were conducted following Figure B. Crosses generated APP/PS1 Tg+ and APP/PS1 Tg- mice, which were all YFP+ and were used to determine dendritic spine density at juvenile (P30) and adult (P74) ages. Juvenile females and males and adult males were used for experimentation. Perfusions Brains from APP/PS1 Tg+; (P30 and P74) were fixed via transcardial perfusion with 4% (wt/vol) paraformaldehyde in PBS. Coronal sections of 150 µm thickness were used for fluorescence microscopy of YFP+ neurons in P30 mice. Coronal sections of 50 µm were used for fluorescence miscropy of YFP+ neurons in P74 mice, to allow for immunostaining. All sections were cut on a vibratome (Leica VT1200S) and mounted on slides with Prolong Gold Antifade Reagent. Immunostaining Immunostaining was used to amplify the signal in P74 brains, but not P30 brains. All P74 brains analyzed were cut into 50 µm thick coronal sections, permeabilized in 0.2% Triton-X100, incubated in GFP primary antibody (NB600-308 from Novus Biologicals) at 1:500 concentration in 5% normal goat serum in 1x PBS, and then Alexa-488 conjugated goat anti-rabbit secondary antibody (A11008 from Life Technologies) at 1:500 concentration in 5% normal goat serum in 1x TBS.

B  

Figure B: Schematic of APP/PS1;YFP-H+ Mouse Breeding. (Schematic by Taeho Kim, Shatz Lab, 2013)  

 

 

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Dendritic Spine Analysis Distal apical tufts of layer 5 neurons labeled with YFP were imaged with a confocal microscope (Leica Microsystems SP8) using a 63× oil immersion objective. Neurons with cell bodies in layer 5 were anatomically selected from the binocular zone of the visual cortex. High-resolution images of apical tufts and the primary dendritic shaft immediately below the main branching of the dendrite (roughly in layer 2) were taken using confocal microscopy. Initial neuron localization was done using a 10x objective and imaging was done using a 60x oil-immersion objective and 4x optical zoom. Z-stacks were taken to capture as much of the neuron as possible in three dimensions within the section. Spine density analysis was done blind to the genotype using FIJI, a build of ImageJ software. Densities were calculated manually using data from Cell Counter and Simple Neurite Tracer plugins to count spines and measure dendrite length. Results Layer 5 apical dendrites of APP/PS1 transgenic mice were imaged at P30 and P74 in an

effort to identify structural changes occurring in the brain of Alzheimer’s models before the

onset of typical behavioral phenotypes. Previous research had identified an ocular dominance

plasticity defect as early as P28 (William et al, 2012) and measuring changes in dendritic spines,

namely changes in spine density, provide a structural substrate for rapid synaptic changes (Hofer

et al, 2009). I hypothesized that spine densities in both P30 and P74 APP/PS1 mice would be

decreased compared to wildtype, correlating with the ocular dominance plasticity defect.

Dendritic Spine Density of Juvenile Transgenic Mice Is Not Decreased

Unexpectedly, results in juvenile (P30) mice have significant difference in spine density

between APP/PS1 transgenic and wildtype mice (Fig. 1B). This indicates that the critical period

ocular dominance plasticity defect observed in APP/PS1 transgenic mice is not associated with a

decrease in spine density in layer 5 apical dendrites in juvenile mice. Additionally, there is not

measurable change in spine morphology that could account for the ocular dominance plasticity

defect (Fig. 2A and B). It is possible that while morphology has not yet changed, physiology has

already been altered, causing the ocular dominance plasticity defect.

Previous research has shown that APP/PS1 mice have measurable synaptic plasticity

defects when measured using Arc mRNA induction (William et al, 2012) Determination of spine

 

 

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density changes, which are a known structural correlate for rapid synaptic change, (Hofer et al,

2009) would illuminate early effects of Alzheimer’s on the brain and indicate sPirB as a potential

therapeutic. We hypothesized APP/PS1 mice would have decreased spine densities as compared

with wildtype as APP/PS1 mice display ocular dominance defects via Arc induction. While

wildtype mice demonstrated a normal expansion in the width of visual cortex responding to

ipsilateral eye input, APP/PS1 did not show this expansion (William et al, 2012).

Dendritic Spine Density of Adult Transgenic Mice Is Not Decreased

In order to gain a better understanding of Alzheimer’s disease progression, spine

densities were also measured in adulthood where structural changes were more likely to have

developed. P74 APP/PS1 transgenic mouse spine densities were compared with their wildtype

littermates as a control. Here as well, there was no significant difference in spine density

between the APP/PS1 mice and wildtype, although data did show an interesting trend not present

in juvenile mice (Fig. 3C). In general, this trend would indicate that individual spine densities for

P74 APP/PS1 mice were higher than those for P74 wildtype littermates. In summary, these

results are unexpected given our hypothesis that spine density would decrease and that this

would correlate to ocular dominance plasticity defects.

Discussion and Conclusions The spine densities of layer 5 pyramidal neurons were analyzed to identify structural

changes occurring in APP/PS1 mice in correlation with known ocular dominance plasticity

defects. As this defect is seen as early as the critical period, identification of structural changes

could provide a clue into Alzheimer’s disease progression and a target for early therapeutics. It

was hypothesized that spine densities measured would be lower in APP/PS1 mice compared to

wildtype, as this seems the most likely cause of plasticity defects. Results showed that neither

juvenile nor adult APP/PS1 mice displayed any significant change in spine density compared to

 

 

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wildtype. These results seem to indicate that the critical period ocular dominance plasticity

defect previously seen were not associated with any changes in spine density of layer 5 apical

dendrites. The ocular dominance plasticity defect may then be due to physiological changes that

have occurred and would not be detectable via spine density measurements. This conclusion

would be inconsistent with other experiments that have indicated dendritic spines to be a

structural substrate for rapid synaptic change (Hofer et al, 2009).

P74 mice did show an interesting trend, where APP/PS1 generally had higher spine

densities than wildtype mice. As only 7 cells were analyzed for P74 APP/PS1 mice and 6 cells

for P74 wildtype, it is possible that with a larger population sample, data would be more

conclusive. If larger samples were measured and the trend found to be significant, it could have

important implications about the mechanism of Alzheimer’s disease development.

Primarily, it would suggest that Aβ could be causing problems with the brain’s ability to

form connections and wire correctly. As a method of compensating for this, increasing numbers

of spines are formed in an attempt to form new connections. In vivo time course imaging of

spines as well as a developmental study of mEPSC frequency and amplitude would help

illuminate whether new spines are being formed at any appreciable rate, if spine turnover has

increased, and if the spines are correlated with functional synapases. In the case of this

compensation model, it would be likely that while new spines are being formed, they are not all

able to wire correctly and are therefore not functional.

This trend towards increased spine density is also interesting in light of a known

association between Alzheimer’s and an elevated risk for seizures present in both mice and

humans (Amantneik et al, 2006). Many mouse models for Alzheimer’s exhibit abnormal

electroencephalogram activity, suggesting fundamental cognitive dysfunction (Born et al, 2014;

 

 

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Palop & Mucke, 2010). If all spines counted are functional excitatory synapses, the high level of

neural excitation may be causing increased rates of epilepsy. Furthermore, when APP

overexpression was withheld to adulthood, epileptic activity was substantially delayed (Born et

al, 2014). If pruning defects during development are the cause of high spine density in adult

APP/PS1 mice, it would also explain this result, as APP overexpression after development would

not have the same effect on spine density.

In order to validate or disprove these hypotheses, further research would need to be

conducted. This would include in vivo imaging of spines over an extended time period in order to

determine how the “extra” spines occur and whether they are an artifact of pruning deficits or are

actually the appearance of new spines for another reason. Another next step would be to measure

mEPSCs from layer 5 pyramidal neurons in adult APP/PS1 mice and wildtype littermates as a

control (Djurisic et al, 2013). This would indicate whether the increase in spine density on the

apical dendrites is accompanied by an increase in functional excitatory synaptic inputs. If the

spines are functional excitatory synapses, further credence would be given to the theory that the

increased spine density in adult APP/PS1 mice could be responsible, at least in part, for the

increased risk for epilepsy associated with Alzheimer’s.

However, recent developments within the lab have indicated that the APP/PS1;YFP-H+

mouse line generated to measure spines in APP/PS1 mice may not have ocular dominance

plasticity defects at all (Richard W. Sapp, Shatz Lab, personal communication, April 28, 2015).

Recent measurements of ocular dominance plasticity in P30 APP/PS1;YFP-H+ mice appear to

match those of wildtype littermates. Since previous research has indicated that APP/PS1 mice

without the YFP transgene show ocular dominance plasticity defects, it is possible that the new

line generated for these sets of experiments has been epigenetically modified, and do not

 

 

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transcribe the mutant APP or PS1 protein at high levels. Furthermore, YFP expression was

noticeably low in this line, leading us to immunostain sections for analysis, and in retrospect

these low YFP levels may have been an indication to study the expression of all transgenes in the

line, including APP and PS1.

In order to determine the presence of APP

protein, a Western blot should be conducted similar to

that shown in Fig. C, where both APP and Aβ

expression were measured. Older APP/PS1;YFP-H+

mouse line brains can also be examined for the

presence of Aβ plaques, which would be a positive

indicator for APP/PS1 expression and reaffirm the line

as a valid model for Alzheimer’s disease.

In conclusion, current results indicate that ocular dominance plasticity defects previously

seen in Alzheimer’s model mice are not associated with changes in spine density as compared to

wildtype. Next steps are to first evaluate that our APP/PS1;YFP-H+ mouse line is a valid model

for Alzheimer’s disease or if APP and PS1 expression have been altered, changing Aβ

accumulation. In the case that these results are valid, research should extend to include more data

points, as well as time course measurements of spines and mEPSCs. Using this new information,

we could better characterize the early development of Alzheimer’s in mouse models, which may

provide new avenues for earlier and more targeted treatments to counteract the disease.

Fig. C Western Blot Analysis of APP and Aβ Expression (William et al, 2012)

C  

   

 

 

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Figures Figure 1: A) WT APP/PS1 B)

Figure 2: A) B)

Dendritic spine density remains unaltered in P30 APP/PS1 mice. 1A) Examples of apical tufts of YFP-labeled layer 5 pyramidal neurons in the binocular zone of the visual cortex. Scale bar represents 10 µm 1B) APP/PS1 transgene does not have an effect on dendritic spine densities in critical period-aged mice. N= 3/7, 3/11 animals/cells.

P30 APP/PS1 mice show similar dendritic spine morphology as compared to wildtypes. A) The distribution of dendritic spine length and width measurements for both wildtype and APP/PS1 critical period-age mice are strikingly similar. B) Histograms of frequency distribution illustrating that the proportion of dendritic spines of specific widths and lengths remain steady between wildtype and APP/PS1 P30 mice.

 

 

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Figure 3: A) WT APP/PS1 B)

Dendritic spine density not significantly different P74 APP/PS1 mice, but trends towards an increase. 1A) Examples of apical tufts of YFP-labeled layer 5 pyramidal neurons in the binocular zone of the visual cortex. Scale bar represents 10 µm 1B) APP/PS1 transgenic mice do not have significantly different spine densities compared with wildtype. However, the data shows a trend towards elevated spine density for APP/PS1 mice. (p=0.11) N= 3/7, 3/6 animals/cells.