Contribution of TRPM2 to Memory Loss in an Alzheimer's ...

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Contribution of TRPM2 to Memory Loss in an Alzheimer's Mouse Contribution of TRPM2 to Memory Loss in an Alzheimer's Mouse

Model Model

Megan M. Chen, The University of Western Ontario

Supervisor: Dr. Marco Prado, The University of Western Ontario

Joint Supervisor: Dr. John MacDonald, The University of Western Ontario

A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in

Physiology

© Megan M. Chen 2013

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CONTRIBUTION OF TRPM2 TO MEMORY LOSS IN AN ALZHEIMER’S MOUSE

MODEL

(Thesis format: Monograph)

by

Megan Meng Chen

Graduate Program in Physiology and Pharmacology

A thesis submitted in partial fulfillment of the requirements for the degree of

Master of Science

The School of Graduate and Postdoctoral Studies Western University Canada London, Ontario, Canada

© Megan Chen 2013

ii

ABSTRACT

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the progressive

deterioration of memory and other intellectual abilities. Accumulation of amyloid-β (Aβ) peptide, the

major contributor to the senile plaques central to AD, is thought to mediate neurotoxicity by inducing

oxidative stress and calcium dysregulation. Transient Receptor Potential Melastatin type 2 (TRPM2)

is a calcium permeable, non-selective cation channel activated under oxidative stress and ultimately

induces cell death. The APPSWE/PSEN1∆E9 double transgenic mouse model carries the human

APPswe (Swedish mutations K594N/M595L) and PS1 mutations with a deletion in exon 9 (PS1-

dE9), and is one of the most commonly used AD model. Both mutations have been shown to be

linked to Familial (early onset) AD as well as an increased production in Aβ peptides. We propose

that TRPM2 contributes to pathological deficits in AD. To test this, we crossed TRPM2 knock-out

with APPSWE/PSEN1∆E9 mice to generate four mouse genotypes (WT, TRPM2 knock-out,

Alzheimer’s and Alzheimer’s TRPM2 knock-out). We hypothesize that Alzheimer’s TRPM2 knock-

out mice will have improved memory performance when compared to Alzheimer’s mice. To test this

hypothesis, anxiety, motor activity, recognition memory, as well as spatial learning and memory were

tested in these mutant mice at 3, 6, 9, 12, and 15 months of age. For these assessments we use open

field locomotor activity (OFT), elevated plus maze (Elev+), object recognition (OR), Barnes (BM)

and Morris water maze (MWM) respectively. Our data showed that no genotype difference was

observed in OFT, Elev+ and OR in terms of hyperactivity, anxiety, and recognition memory. On the

other hand, our data indicate that Alzheimer’s mice show cognitive impairment (spatial memory

deficit) compared to WT controls while Alzheimer’s mice without TRPM2 do not, suggesting that

elimination of TRPM2 activity prevents the spatial learning and memory deficits observed in

APPSWE/PSEN1∆E9 double transgenic mice.

Key words: Alzheimer’s disease, TRPM2, memory, cognitive impairment, elevated plus maze, open

field, object recognition, Barnes maze, Morris water maze

iii

ACKNOWLEGEMENTS

First of all, I would like to thank my supervisor Dr. John MacDonald for giving me

the opportunity to be a part of his lab and for helping me grow for the past two years. I would

also like to thank my co-supervisor Dr. Michael Jackson for his patience and guidance and

for always giving me advice throughout my journey as a Master student. Thank you to

everyone in the MacDonald/Jackson lab for all your help and support, especially Dr. Jillian

Belrose for her friendship and always answering my questions no matter how busy she is

with work/school. A special thank you goes to Dr. Amanda Martyn who has provided me

with intensive animal behaviour training and for always being patient with all my endless

questions.

I would like to sincerely thank the members of my advisory committee: Dr. Marco

Prado, Dr. Vania Prado, and Dr. Susanne Schmid for all the times they have spent reading

and discussing my work and for all their suggestions and recommendations throughout this

project. I would also like to thank Dr. Lina Dagnino for being a part of my advisory

committee after such short notice and for all her help and advice.

Finally, thank you to all my family (Cathy [mom], Charlie [dad], Tori [sister] and my

boyfriend Yuri) for their continued motivation and support; without whom this thesis would

not be possible.

Thank you all once again, I greatly appreciate it!

Megan Chen

iv

TABLE OF CONTENTS

Abstract and Key Words……………………………………............................................ii

Acknowledgements…………………………………….....................................................iii

Table of Contents……………………………………………………………………….iv-v

List of Figures…………………………………………………………………….…...vi-vii

List of Abbreviations………………………………………………………….……...viii-ix

Section 1 – Introduction………………………………………………………………….1

1.1 Alzheimer’s disease……………………………………………………….....2-6

1.2 TRP channels and TRPM2……………………………………………...…...7-8

1.3 Mouse models of Alzheimer’s disease…………………………………........8-9

1.4 APPSWE/PSEN1∆E9 double transgenic mouse model………………..…10-13

1.5 Cell death by oxidative stress and TRPM2-KO mice…………………..…13-15

1.6 Hypothesis and Objectives……………………………………………..……..15

Section 2 – Materials and Methods………………………………………………….....16 2.1 Animals……………………………………………………………………….17

2.2 General materials and methods……………………………………….…..17-18

2.3 Open field locomotor activity test…………………………………..…….18-20

2.4 Elevated plus maze……………………………………………………......21-22

2.5 Object recognition test………………………………………………….....23-24

2.6 Barnes maze………………………………………………………….…....25-27

2.7 Morris water maze and reversal…………………………………………...28-30

2.8 Statistical analysis…………………………………………………………….31

Section 3 – Results………………………………………………………………...…….32 3.1 Open field locomotor activity………………………………………….....33-39

3.2 Elevated plus maze………………………………………………………..40-43

3.3 Object recognition………………………………………………………...44-46

3.4 Barnes maze……………………………………………………………….47-61

3.5 Morris water maze hidden-platform test……………………………...…..62-67

3.6 Morris water maze reversal learning………………………………….......68-73

Section 4 – Discussion…………………………………………………………………..74 4.1 Summary of Key Findings…………………………………………………...75

4.2 Open field locomotor activity test…………………………………..…....75-77

4.3 Elevated plus maze…………………………………………………….....77-79

4.4 Object recognition…………………………………………………….......79-80

4.5 Barnes maze…………………………………………………………...….80-82

4.6 Morris water maze hidden-platform test………………………………....82-83

4.7 Morris water maze reversal learning……………………………………..83-86

4.8 Future directions…………………………………………………..….…..86-87

v

4.9 Overall conclusions…………………………………………………………..88

Section 5 – References…………………………………………………………......89-102

Curriculum Vitae………………………………………………………………......103-104

vi

LIST OF FIGURES

Figure 1.1. APP processing……………………………………………………….…….....5

Figure 2.1. Automated locomotor activity chambers……………………………………..20

Figure 2.2. Standard elevated plus maze………………………………………………....22

Figure 2.3. Homecage used during Object recognition……………………………..........24

Figure 2.4. Barnes maze setup…………………………………………………………...27

Figure 2.5. Morris water maze setup……………………………………………………..30

Figure 3.1. Locomotor activity at 3 months of age……………………………………....35



Figure 3.4. Locomotor activity at 12 months of age……………………………….........38

Figure 3.5. Locomotor activity at 15 months of age……………………………….........39

Figure 3.6. Assessment of anxiety at 3, 6, 9, 12, and 15 months of age……………...42-43

Figure 3.7. Assessment of recognition memory at 9 and 12 months of age……………..46

Figure 3.8. Barnes maze assessment of spatial memory at 3 months of age…………52-53

Figure 3.9. Barnes maze assessment of spatial memory at 6 months of age…………54-55

Figure 3.10. Barnes maze assessment of spatial memory at 9 months of age………..56-57

Figure 3.11. Barnes maze assessment of spatial memory at 12 months of age………58-59

Figure 3.12. Barnes maze assessment of spatial memory at 15 months of age………60-61

Figure 3.13. MWM assessment of spatial memory at 12 months of age……………..64-65

vii

Figure 3.14. MWM assessment of spatial memory at 15 months of age……………66-67

Figure 3.15. MWM assessment of reversal learning at 12 months of age…………..70-71

Figure 3.16. MWM assessment of reversal learning at 15 months of age…………..72-73

viii

LIST OF ABBREVIATIONS

α-secretase alpha secretase

β-secretase beta secretase

β-cells beta cells

γ-secretase gamma secretase

[Ca2+

]i intracellular calcium concentration

Aβ amyloid beta

AD Alzheimer’s disease

ADPR adenosine diphosphate ribose

AMPA α-Amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid

APP amyloid precursor protein

APP+ amyloid precursor protein positive

APP- amyloid precursor protein negative

APPSWE/PSEN1∆E9 Alzheimer’s transgenic double mutant:

transgenic mouse model expressing the Swedish

mutation of K594N/M595L and presenilin-1

mutations with an exon-9 deletion

BM Barnes maze

Ca2+

calcium

cADPR cyclic adenosine diphosphate ribose

CNS central nervous system

CTFα C-terminal fragment

Elev+ Elevated plus maze

FAD Familial Alzheimer’s disease

H2O2 hydrogen peroxide

LTD/LTP long-term depression/potentiation

ix

Mg2+

magnesium

MWM Morris water maze

NMDA/NMDAR N-methyl-D-aspartate receptor

OFT Open field locomotor test

OR Object recognition test

PrP prion protein promoter

PS1 presenilin 1

PS2 presenilin 2

RNS reactive nitrogen species

ROS reactive oxygen species

sAPPα soluble N-terminal fragment

TRP Transient Receptor Potential Channel

TRPA Transient Receptor Potential Ankyrin Channel

TRPC Transient Receptor Potential Canonical Channel

TRPM Transient Receptor Potential Melastatin Channel

TRPM2 Transient Receptor Potential Melastatin Type 2

Channel

TRPM2-/-

TRPM2 homozygous single mutant

TRPM2+/-

TRPM2 heterozygote

TRPM2-KO TRPM2 knock-out

TRPM2-KO-APPSWE/PSEN1∆E9 APPSWE/PSEN1∆E9 triple mutants

TRPML Transient Receptor Potential Mucolipin Channel

TRPP Transient Receptor Potential Polycystin Channel

TRPV Transient Receptor Potential Vanilloid Channel

WT wildtype

1

Section 1

INTRODUCTION

2

1.1 Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive, degenerative and fatal disease

characterized by deterioration of cognitive functions such as memory and the ability to

use judgment and reasoning (Coyle, Price, & Delong, 1983; Cummings, 2004). AD is the

most common neurodegenerative disease worldwide and is the leading cause of dementia

(Billings, Oddo, Green, McGaugh, & LaFeria, 2005; Cvetkovic-dozic, Skender-gazibara,

& Dozic, 2001; Goedert & Spillantini, 2006). At present, AD is prevalent in

approximately 500,000 Canadians over the age of 65, representing 1.5% of the total

population in Canada (Alzheimer Society of Canada, 2010). A deterioration of the

hippocampus and prefrontal cortex, entorhinal cortex, and the basal forebrain cholinergic

neurons are found to be present in individuals affected by AD (Coyle et al., 1983;

Gomez-Isla et al., 1996) and the degree of deterioration and the loss of synaptic function

is shown to be strongly correlated with the severity of dementia (Scheff & Price, 2003).

There is currently no cure for AD; however medications such as memantine, donepezil,

galantamine, and rivastigmine can be used to temporarily alleviate the symptoms of

dementia (Cummings, 2004).

The main pathological hallmark of AD is amyloid-beta (Aβ) plaques (Cvetkovic-

dozic et al., 2001; Yankner, Lu, & Loerch, 2008). Aβ plaques are extracellular and are

composed of Aβ peptides that are 39 to 42 amino acids in length, with Aβ40 and Aβ42

being the two most common lengths of Aβ found in the brain of AD patients (Oddo et al.,

2003; Selkoe, 2008). These filaments are created by the proteolytic cleavage of the

transmembrane glycoprotein, amyloid precursor protein (APP). There are two proteolytic

pathways under which APP is processed: the amyloidogenic pathway which leads to

3

amyloid plaque production and the non-amyloidogenic pathway which does not. In the

non-amyloidogenic pathway, APP is cleaved by α-secretase and γ-secretase within the

Aβ domain yielding a soluble N-terminal fragment (sAPPα) and a C-terminal fragment

(CTFα). These fragments do not accumulate in plaques. However, in the amyloidogenic

pathway, the cleavage of APP by β-secretase and γ-secretase results in the generation of

aggregation-prone Aβ peptides, eventually leading to amyloid plaque buildup (Figure

1.1) (Selcoe, 2001; Selkoe & Schenk, 2003; Xiong, 2011). Mutations in the APP gene

were found to influence Aβ peptide levels by altering the normal processing of the

protein, resulting in an elevation in both Aβ40 and 42 peptides (Citron et al., 1992;

Haass, Hung, Selkoe, & Teplow, 1994; Suzuki et al., 1994) with the latter more likely to

accumulate in the brain increasing the ratio of Aβ42/Aβ40 (Burdick et al., 1992; Haass &

Selkoe, 2007; Jarrett & Lansbury, 1993). Presenilin 1 (PS1) and 2 (PS2) were also found

to play a role in Aβ peptide production. Mutations in these genes were believed to further

increase Aβ42/ Aβ40 ratio as a result from a reduced γ-secretase activity, which in turn is

a critical protein involved in the within domain cleavage of the APP gene (Goedert &

Spillantini, 2006; Hardy & Higgins, 1992; Jankowsky et al., 2004; Xiong et al., 2011).

Brains affected by AD contain both insoluble and soluble oligomeric assemblies

(Hardy & Selkoe, 2002; Yu et al., 2009). Aβ peptides have the ability to self-aggregate

from smaller monomers into readily diffusible oligomers including, dimers, trimers,

higher order oligomers, as well as fibrils that ultimately accumulate into Aβ plaques. This

ability to self-aggregate was demonstrated by Pike, Walencewicz, Glabe, & Cotman

(1991) who identified aggregation in cultured cells that were pre-incubated by the Aβ

peptide solution for 2-4 days. In addition, Aβ peptides of different lengths were noted to

4

have different physical and biological properties (Haass & Selkoe, 2007; Selkoe, 2008;

Shankar et al., 2008; Townsend et al., 2006). Various studies have examined the

relationship between Aβ peptide length and its toxicity to cells, with the common

consensus that soluble forms of oligomers; including dimers and trimers, can cause long-

term potentiation (LTP) deficit and memory impairments in rodents while soluble

monomers and fibrils cannot (Cleary et al., 2005; Demuro et al., 2005; Shankar et al.,

2008). Dimers however, were found to be more damaging to the cells than trimers

(Selkoe, 2008; Townsend, Shankar, Mehta, Walsh, & Selkoe, 2006). In keeping with this,

it is the soluble form of Aβ present in the individual with AD that is most toxic and

correlate strongly with the disease (Hardy & Selkoe, 2002; Yu et al., 2009). Numerous

studies have examined the relationship between insoluble Aβ, memory impairment and

dementia associated with AD. These studies suggest that insoluble Aβ deposition results

in only minimal neuronal loss (Irizarry, McNamara, Fedorchak, Hsiao, & Hyman, 1997a;

Irizarry et al., 1997b) and has a weak correlation with memory impairment (Dickson et

al., 1995; Terry et al., 1991). The latter observation was further confirmed by the

existence of non-cognitively impaired individuals with high amounts of insoluble Aβ

deposits in the brain (Delaere et al., 1990; Dickson et al., 1995; Katzman et al., 1988). In

contrast, soluble Aβ oligomers induce a number of AD-related phenotypes including a

decrease in dendritic spine density and the disruption of LTP in the hippocampus of

rodents. These phenomenon correlates to learning and memory as well as impairing

memory in rats as measured by behavioural tests (Shankar et al., 2008; Townsend et al,

2006).

5

Figure 1.1. APP processing under amyloidogenic and non-amyloidogenic pathways.

α-secretase and γ-secretase goes under the non-amyloidogenic pathway to produce

non-plaque forming Aβ, while β-secretase and γ-secretase goes under the

amyloidogenic pathway to produce amyloid plaque forming Aβ [Image source:

http://www.ebi.ac.uk/interpro/potm/2006_7/Page2.htm].

6

The accumulation of Aβ peptides has been found by numerous studies to mediate

neurotoxicity by inducing oxidative stress in the brain (Mattson, 1998; Takahashi, Kozai,

Kobayashi, Ebert, & Mori, 2011). This oxidative stress is formed when an imbalance is

present between reactive oxygen species (ROS) such as hydrogen peroxide (H2O2),

reactive nitrogen species (RNS), and antioxidants (Butterfield, 2003; Mattson, 1998;

Takahashi et al., 2011). This relationship between Aβ peptide accumulation and oxidative

stress was demonstrated by Misonou, Morishima-Kawashima, & Ihara (2000) using

human neuroblastoma SH-SY5Y cells. These scientists used H2O2 to induce oxidative

stress into neuroblastoma cells and observed an increased level of intracellular Aβ

peptide. In addition, Aβ deposition was also found to take place in the striatum (Klunk et

al., 2004; Wolfer, Mohajeri, Lipp, & Thal et al., 1998) where oxidative stress may act as

the key factor of cell death (Lipton, 1999) under states of ischaemia and reperfusion

(Hawker & Lang, 1990). However, the relationship between Aβ, oxidative stress, and the

striatum is not well understood.

In recent studies, there has been evidence supporting that TRPM2 channels are

important in the pathogenesis of AD. TRPM2 is a ROS/RNS gated channel that has been

implicated in cell death. ROS/RNS is found to promote TRPM2 activation, leading to a

disruption in Ca2+

homeostasis and a toxic influx of Ca2+

, ultimately inducing cell death

(Goedert & Spillantini, 2006; Mattson, 2007; Takahashi et al., 2011). Activation by

ROS/RNS has been attributed to the generation of ADP-ribose (ADPR) which plays a

key role in the gating of TRPM2 (Perraud et al., 2001).

7

1.2 TRP channels and TRPM2

Transient receptor potential (TRP) channels are a superfamily of ion channels that are

non-selective cation channels, most of which have substantial permeability to Ca2+

(Nilius & Owsianik, 2011). The first TRP gene described was from the fruit fly

Drosophila melanogaster where TRP is used for phototransduction purposes (Montell &

Rubin, 1989). To present day, 28 mammalian TRP channels have been discovered and

can be subdivided into six main subfamilies including TRPC (canonical), TRPV

(vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA

(ankyrin). TRP channels are expressed in almost all cell types in most tissues of the

human body. These channels are usually localized in the plasma membrane where they

aid in the transport of Ca2+

, Mg2+

and other ions. TRP channels play a critical role in

regulating various cell functions including immune response and vasomotor functions,

cold and thermal pain sensing, organ development, and oxidative stress sensing in various

cells of the body (Nilius, Owsianik, Voets, & Peters, 2007; Nilius & Owsianik, 2011).

TRPM1 (melastatin) was the first member of the TRPM subfamily and was first

discovered from melanomas where it served as a tumor suppressor (Duncan et al., 1998).

There are currently eight family members (TRPM1-TRPM8) present in the TRPM

subfamily (Jiang, Yang, Zou, & Beech, 2010; Kraft & Harteneck, 2005; Nilius &

Owsianik, 2011). TRPM may induce cell death under certain conditions given that they

mediate cellular responses to a range of physiological stimuli and within this family, two

members (TRPM2 and TRPM7) have been identified to play key roles in cell death

pathways (McNulty & Fonfria, 2005).

8

Transient Receptor Potential Melastatin type 2 (TRPM2) is a member of the TRPM

subfamily and is a calcium permeable, non-selective cation channel. It is highly

expressed in the CNS including the hippocampus, cerebral cortex, thalamus and the

midbrain (Fonfria et al., 2005; Fonfria et al., 2006; Hara et al., 2002; Kraft et al., 2004;

Nagamine et al., 1998; Olah et al., 2009; Perraud et al., 2001) and in many tissues outside

of the CNS including pancreatic β-cells (Qian et al., 2002), bone marrow, spleen, heart,

as well as in immune cells of the monocytic lineage including monocytes and neutrophils

(Hara et al., 2002; Perraud et al., 2001; Sano et al., 2001).

1.3 Mouse models of Alzheimer’s disease

In order to investigate neurodegenerative diseases such as AD, animal models

have become a common tool used for the better understanding of the pathology,

underlying mechanisms, and investigation into potential treatments of the disease. While

invertebrate and primate models are used in some research, rodents are the most common

and widely used animal models. Due to the fact that the mouse genome can be easily

manipulated at relatively lower costs, the use of genetically modified mice strains to

model neurodegenerative diseases such as AD has become extensive (Ghorayeb, Page,

Gaillard, & Jaber, 2011).

Pharmacological models and transgenic mouse models have been used for AD.

Pharmacological models are generated by the direct injection of Aβ peptide into the brain

of the rodent and can be particularly useful for the understanding of amyloid toxicity.

However, not all neuropathology are observed in these mouse models of AD, including

9

the loss of neurons and synapses in the cortex and hippocampus, which leads to cognitive

impairments (Ghorayeb et al., 2011). Unlike pharmacological models, transgenic mouse

models of AD do not involve injections of Aβ peptides. Instead, they are made from the

expression of different variants of the APP gene (located on human chromosome 21

(Xiong et al., 2011)), PS1 or PS2, tau or apolipoprotein E (Ashe, 2001). One of the

mouse models most commonly used to identify potential therapeutic interventions for AD

is the APPSWE/PSEN1∆E9 double transgenic mouse line. These mice show both Aβ

deposition and memory loss (Ashe, 2001; Esh et al., 2005).

A number of other mouse transgenic lines have been generated using variants of

the APP and PS1 genes including, the NSE: β-APP751, homozygous for the transgene

array expressing wild-type human APP751 (Moran, Higgins, Cordell, & Moser, 1995);

PDAPP, carrying the transgene APP717 expressing the Indiana mutation V717F with a

portion of APP introns 6-8 (Chen et al., 2000; Duyckaerts, Potier, & Delatour, 2008; Esh

et al., 2005); TgCRND8, carrying the transgene APP695 expressing both Swedish double

mutation and the Indiana mutation K670N/M671L and V717F (Duyckaerts et al., 2008;

Janus et al., 2000); Tg2576, carries the transgene APP695 that expresses the Swedish

mutation K670N/M671L (Chapman et al., 1999; Duyckaerts et al., 2008; Hsiao et al.,

1996; Kawarabayashi et al., 2001) and APPSWE/PSEN1∆E9, expressing the Swedish

mutation of K594N/M595L and presenilin-1 mutations with an exon-9 deletion (Bonardi,

Pulford, Jennings, & Pardon, 2011; Duyckaerts et al., 2008; Malm, Koistinaho, &

Kanninen, 2011). All of which produce many biochemical, physiological, pathological

and behavioural characteristics that simulate AD (Ashe, 2001).

10

1.4 APPSWE/PSEN1∆E9 double transgenic mouse model

The APPSWE/PSEN1∆E9 double transgenic mouse model carries the human

APPswe (Swedish mutations K594N/M595L) and PS1 mutations with a deletion in exon

9 (PS1-dE9) (Bonardi et al., 2011; Duyckaerts et al., 2008; Jackson Laboratory, 2013;

Malm et al., 2011). It was created by coinjection, where each vector containing the

specific transgene has its own promoter component. In this case, the two specific

transgenes were Mo/HuAPP695swe and PS1-dE9. Each of these transgenes was then

inserted into a plasmid at a single locus and each of these plasmids was designed to be

controlled by an independent mouse prion protein (PrP) promoter. The

Mo/HuAPP695swe transgene expresses a “humanized” mouse amyloid beta (A4)

precursor protein gene modified to contain the K595N/M596L mutations that are linked

to familial Alzheimer’s disease (FAD). The PS1-dE9 transgene expresses the human

presenilin 1 mutant with the exon-9-deleted also associated with FAD. These two

plasmids were then coinjected into the B6C3HF2 pronuclei and have co-integrated in the

genome. Founder line 85 was obtained and by crossing transgenic mice to B6C3F1/J, the

resulting colony maintained as hemizygotes. Transgenic mice were then backcrossed to

the background strain C57BL/6J for at least 8 generations (Garcia-Alloza et al., 2006;

Jackson Laboratory, 2013; Jankowsky et al., 2001).

APPSWE/PSEN1∆E9 double transgenic mice have been noted to be the best

characterized AD model to date (Bonardi et al., 2011); both mutations have been shown

to be linked to FAD, as well as increased production of Aβ40 and Aβ42 peptides (Garcia-

Alloza et al., 2006; Malm et al., 2011; O’Leary and Brown, 2009; Reiserer, Harrison,

Syverud, & McDonald, 2007; Savonenko et al., 2005; Zhang et al., 2011); Increased

11

production of Aβ42 is one of the triggers of FAD (Savonenko et al., 2005). This increase

in Aβ42 subsequently results in large senile plaque buildup in the hippocampus, cortex

and subcortical nuclei (Duyckaerts et al., 2008). Moreover, various transgenic studies

have shown a direct correlation between the level of Aβ42 and the age at which amyloid

plaques appear (Jankowsky et al., 2002), with some studies presenting early development

of amyloid plaques in the hippocampus and cortex observed as early as 4-6 months of age

(Garcia-Alloza et al., 2006; Ghorayeb et al., 2011; Jackson Laboratory, 2013; Malm et

al., 2011; Minkeviciene et al., 2008; O’Leary and Brown, 2009; Savonenko et al., 2005).

Jankowsky et al. (2004), Jardanhazi-Kurutz et al. (2010) and Lalonde, Kim, & Fukuchi

(2004) have also observed a rapid accumulation of plaques within the cortex and

hippocampus of these mice at 6 months of age and even greater amounts at 9 (Lalonde et

al., 2004) and 12 months (Jardanhazi-Kurutz et al., 2010). Likewise, Reiserer et al.

(2007) found that Aβ plaques start to develop at 6-7 months of age.

APPSWE/PSEN1∆E9 bigenic mice have been used extensively for the study of

cognitive deficits, such as memory impairment observed in humans with AD. A variety

of tests including open field test (OFT), Elevated plus maze (Elev+ maze), Object

Recognition (OR), Barnes maze (BM) and Morris Water Maze (MWM) have been

conducted in these mice to measure their exploratory tendencies, anxiety and memory

ability. Park et al. (2010) compared the locomotor activity of aged WT with

APPSWE/PSEN1∆E9 mice and found no difference between the two genotypes. The

specific age of the mice were not indicated. Likewise, Bonardi et al. (2011) also did not

see a difference in locomotor activity in 4 months old WT and APPSWE/PSEN1∆E9

12

mice suggesting that at least at young age these mutants do not show changes in

locomotion behavior.

Elev+ maze is used to examine greater or lesser anxiety experienced by mice

through assessing an individual’s exploratory tendencies in walled vs. open arms (Cryan

& Holmes, 2005; File, 2001). Studies with 7-month-old APPSWE/PSEN1∆E9 mice

showed that these mutants spend less time in the closed arms than WT mice, suggesting

that the bigenic mice might be less anxious than WT controls (Lalonde et al., 2004;

Reiserer et al., 2007).

To test recognition memory, Bonardi et al. (2011) conducted an object

recognition test on 4-month-old mice and found no difference between WT and

APPSWE/PSEN1∆E9 mice. Similar results were observed in a study by Jardanhazi-

Kurutz et al. (2010) using mice at 4 and 6 months of age. Interestingly,

APPSWE/PSEN1∆E9 mice were found to show severe object recognition memory

impairment at the age of 9-13 months (Donkin et al., 2010; Yoshiike et al., 2008).

Spatial learning and memory in rodents were examined in the BM (Sunyer, Patil,

Hoger, & Lubec, 2007). Studies using 6-7 month old APPSWE/PSEN1∆E9 mice showed

cognitive impairment in the BM (Reiserer et al., 2007). O’Leary and Brown (2009) also

observed spatial learning and memory deficits in 16-month-old APPSWE/PSEN1∆E9

mice. MWM is another test that measures spatial learning and memory. BM and MWM

are similar because both tests measures spatial learning and memory, however BM is

conducted on land and MWM in water. Some studies have observed no cognitive deficit

in APPSWE/PSEN1∆E9 mice at 6 months of age as measured by MWM (Minkeviciene

13

et al., 2008; Savonenko et al., 2005). However, cognitive deficit was observed at 8-12

months of age (Butovsky et al., 2006; Cao, Lu, Lewis, & Li, 2007; Lalonde et al., 2004;

Minkeviciene et al., 2008; Oksman et al., 2006), 12-13 months of age (Malm et al., 2011;

Zhang et al., 2011) and 16-18 months of age (Savonenko et al., 2005). Thus, although

there is still controversy about when spatial deficits are first observed in this mouse line,

a number of studies seem to indicate that by 12 months of age APPSWE/PSEN1∆E9

mice show spatial learning and memory deficits.

1.5 Cell death by oxidative stress and TRPM2-KO mice

Calcium is an intracellular messenger that mediates many physiological responses

of neurons. The concentration of intracellular Ca2+

([Ca2+

]i) fluctuates with time under

normal physiological conditions and do not result in adverse effects for neurons.

However, in pathological conditions, the concentration of intracellular Ca2+

may rise

significantly as H2O2 and Cyclic ADP-ribose (ADPR) activates TRPM2, compromising

calcium homeostasis in neurons (Mattson, 2007). A loss of calcium homeostasis leads to

sequential alterations in neuronal function, including disrupting the structure and function

of synapses, impairment of synaptic plasticity and culminating with cell death. TRPM2

channels have been shown to play a critical role in cell death (Hara et al., 2002). When

activated by ADPR and oxidative stress such as H2O2, high levels of Ca2+

will move

through the channel into the cell resulting in a continued increase in [Ca2+

]i, ultimately

inducing cell death (Fonfria et al., 2004; Hara et al., 2002; Wehage et al., 2002). This link

between H2O2-induced cell death and TRPM2 channels were investigated by Kaneko et

al. (2006), who demonstrated that a severe deterioration of primary cultured neurons was

14

present after H2O2 exposure. Similarly, Zhang et al. (2006) demonstrated that activation

of TRPM2 by oxidative stress triggers apoptotic cell death in hematopoietic cells. Recent

work by Fonfria et al. (2005) has shown that the inhibition of TRPM2 function reduces

the increase in [Ca2+

]i and toxicity induced by Aβ, and therefore provides concrete

evidence that TRPM2 may contribute to neuronal cell death under circumstances in

which oxidative stress are generated.

Synaptic plasticity is the ability of synapses to either strengthen or weaken with

time, in response to increases or decreases in their activity and has been established to be

Ca2+

-dependent (Cavazzini, Bliss, & Emptage, 2005). NMDA (N-methyl-D-aspartate)

and AMPA (α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) glutamate

receptors are the two main receptors for the excitatory transmitter, glutamate. The

opening of NMDA receptors leads to an influx of Ca2+

leading to long-term potentiation

(LTP) while a more modest influx of Ca2+

into the post-synaptic neuron leads to long-

term depression (LTD). LTP and LTD are thought to represent the cellular basis for

learning and memory (Bear & Malenka, 1994; Cavazzini et al., 2005). Using

hippocampal cultures and slices from TRPM2 knockout mice, a recent study by Xie et

al., (2011) demonstrated that the loss of TRPM2 channels impairs LTD while having no

effect on LTP. These findings suggest that TRPM2 contributes to synaptic plasticity.

Moreover, as TRPM2 is oxidative stress responsive, it is anticipated that oxidative stress

associated with elevated Aβ in Alzheimer’s mouse models will provoke aberrant TRPM2

channel activation leading to altered synaptic plasticity. Therefore, eliminating TRPM2

may be beneficial to learning and memory in mice.

15

Our hypothesis is that when TRPM2 expression is genetically ablated, there will

be reduced [Ca2+]i elevation in response to Aβ and therefore reduced toxicity. We believe

that the loss of TRPM2 expression will better preserve neuronal function and cognition in

mice. To test that, we will investigate whether knocking out the TRPM2 gene influences

behavioral deficits observed in the APPSWE/PSEN1∆E9 mouse model of AD. We will

also investigate whether TRPM2 knockout (TRPM2-KO) mice show any behavioral

changes. This thesis focuses on 4 genotypes of mice: wild-type mice (WT), TRPM2-KO

(TRPM2-/-

), Alzheimer transgenic (APPSWE/PSEN1∆E9+) and APPSWE/PSEN1∆E9

TRPM2 knock-out (TRPM2-KO-APPSWE/PSEN1∆E9) mice.

1.6 Hypothesis and Objectives

TRPM2 is a calcium permeable, non-selective cation channel activated by ADPR and

oxidants such as hydrogen peroxide (H2O2). Activated TRPM2 causes a continued

increase in the intracellular Ca2+

concentration and ultimately disrupts neuronal function.

Given the fact that oxidative stress can promote TRPM2 activation and calcium

dysregulation, we propose that abnormal TRPM2 activation may contribute to the Aβ-

induced neurotoxicity with consequences for cognitive dysfunction.

Therefore, we propose that TRPM2 contributes to memory deficits in Alzheimer’s

disease. To test the hypothesis, we crossed the TRPM2-KO mice with

APPSWE/PSEN1∆E9 mice in order to conduct cognitive behavioral testing.

16

Section 2

MATERIALS AND METHODS

17

2.1 Animals

All animal experiments were conducted in accordance to the policies indicated on

the animal use protocol at Western University. TRPM2 knockout (TRPM2-KO) mice

were provided by Dr. Yasuo Mori from Kyoto University, Kyoto, Japan.

APPSWE/PSEN1∆E9 double transgenic mice were provided by Dr. Marco Prado at

Western University. All mice were housed in the animal facility at Robarts Research

Institute, Western University. Both mouse strains were on a mixed C57Bl/6J-C3H/HeJ

background.

TRPM2 homozygous (TRPM2-/-

) mice were crossed with the Alzheimer’s

transgenic (APPSWE/PSEN1∆E9) bigenic mice to produce TRPM2+/-

;APP+ and

TRPM2+/-

;APP-. Heterozygous littermates were then interbred (TRPM2

+/-;APP

+ x

TRPM2+/-

;APP-) to generate the four genotypes in our study: Wild-type (WT); TRPM2

knock-out (TRPM2-KO) mice; APPSWE/PSEN1∆E9, and TRPM2 and

APPSWE/PSEN1∆E9 triple mutants (TRPM2-KO-APPSWE/PSEN1∆E9 ).

Due to the fact that this is a longitudinal study and that death is common at an

older age, we added another group of mice to the original cohort at the 6 month time

point and continued experimentation as to make sure the N value will not be too low.

2.2 General materials and methods

This study was done longitudinally using the same cohort of mice. Only male

mice were used. We did not use females since their estrous cycle may interfere with our

experiments.

18

Anxiety, motor activity, recognition memory, spatial learning and memory were

tested in mice at 3, 6, 9, 12, and 15 month time points, as measured by the open field

locomotor activity (OFT), elevated plus (Elev+), object recognition(OR), Barnes (BM)

and Morris water maze (MWM) respectively.

For all behaviour tests, 70% ethanol was used prior to each mouse trial to

obliterate any odour cues left from the previous individual, thus preventing bias in our

results. In order to reduce the level of stress experienced by these mice so that

behavioural responses will not be influenced, all individuals were placed in the test room

before experimentation was to begin and were given a thirty minute habituation session.

In order to minimize any alteration in these rodents’ behaviour while the observer

is in the room, all experimental apparatus were placed at the back of the testing room

with a curtain hanging from the ceiling, separating the area where the maze and observer

are located (Vorhees & Williams, 2006; Walf & Frye, 2007). All tests were conducted in

the same animal facility located at Robarts Research Institute between 0900 and 1700

hours.

2.3 Open field locomotor activity

Locomotor activity was measured using an automated system (Figure 2.1)

(Versamax animal activity monitor; Accuscan Instruments, Inc., Columbus, OH, USA).

Vertical light beam holes are positioned at the base of the monitors to record activities of

mice. When mice move about the activity chamber, these light beams are interrupted, and

these light beam breaks are then converted to distance (cm) for simple data analysis of

activity (Sotnikova et al., 2004; de Castro et al., 2009a; de Castro et al., 2009b).

19

Mice were placed into several 20 cm x 20 cm locomotor activity chambers made

out of plexiglass with 30.5 cm high walls and left alone for two hours, while the

automated activity monitor recorded the discrete movements of mice including total

distance travelled during exploration, total number of rear movements, and the amount of

time spent in the center which is a measure of anxiety. All activities were measured in

blocks of five minute intervals.

20

Figure 2.1. Automated Locomotor activity chamber used to measure locomotor

activity. [Image source: http://phenome.jax.org].

21

2.4 Elevated plus maze

A standard elevated plus maze (Med Associates Inc., St. Albans, VT, USA) was

used in this test, as seen in Figure 2.2. This maze is composed of opaque white acrylic

material and consists of four arms, each being 38 cm long and 6 cm wide. Two of the

arms are open without walls and the other two arms were enclosed by black

polypropylene walls of 20 cm in height. The maze is elevated 75 cm off the floor with

metal legs supporting each arm. A camera mounted overhead on the ceiling is used to

record the animals’ movements on the maze, while this behaviour data are automatically

collected with the ANY-maze video-tracking system (Stoelting Co., Wood Dale, IL,

USA).

Each mouse is placed in the center of the maze facing the open arm and is then

allowed to explore the maze for 5 minutes while the video tracking system automatically

records the animals’ movements. During this time, different variables were recorded

including the total distance travelled, latency to enter each arm, the number of entries into

and the amount of time individual mice spent in each arm.

22

Figure 2.2. A standard elevated plus maze used to measure anxiety responses of

rodents. [Image source:

http://iobs.fudan.edu.cn/En/techs_view.asp?id=27&EnBigclassname=Animal%20Model

%20and%20Behavior%20Facility&EnSmallclassname=Equipments].

23

2.5 Object recognition

Object recognition test was performed over the course of 2d and consisted of a 1d

learning trial and a 1d testing trial. Each mouse was placed in a shoebox cage (homecage)

(Figure 2.3) in the test room and given a 30 minute habituation session before the test

was to begin. Training was conducted immediately following habituation on day 1, where

two identical objects were positioned on the opposite ends of the homecage at a fixed

distance. The mouse was then left to explore its surroundings for 5 minutes. Each mouse

underwent three trials on training day. After a latency of 24 hours, mice were presented

with one familiar object and one novel object in the same homecage. Exploration was

again recorded for 5 minutes. Exploration was defined as either sniffing or touching the

object with the nose and/or forepaw (de Lima et al. 2005). All objects had similar

colours, textures, and sizes, but distinctive shapes.

A counterbalance measure was created prior to experimentation in order to

randomly assign the mouse with their familiar and novel object. The ANY-maze video

system (Stoelting Co., Wood Dale, IL, USA) was used to record the animals’ movements.

While the video records, the observer uses stopwatches to manually record the

time each mouse spends exploring either the familiar or novel object.

24

Figure 2.3. Homecage used during training and test days for object recognition. Red

circles represent familiar objects, while purple square represent the novel object.

[Image adapted from

http://www.nature.com/ncomms/journal/v1/n6/images_article/ncomms1064-f1.jpg].

25

2.6 Barnes maze

Barnes maze is made up of a white opaque circular platform (91.5cm in diameter)

made of a plastic material, elevated 105cm above the floor, with 20 holes equally spaced

around the perimeter (5cm in diameter; 7.5cm between holes) (Figure 2.4) (Sandiego

Instruments, San Diego, CA, USA). An escape box or target hole (5cm x 11cm x 5cm)

acts as positive reinforcement since they provide the mice with an escape from the

brightly lit and exposed platform. The location of the escape box depends on the

counterbalance made prior to experimentation. All holes need to look identical to avoid

any potential behavioural bias that could arise.

In order to motivate mice so they will escape from the platform and into the

escape box, a weak aversive stimulus (sound of a buzzer) is applied; the same parameters

of the sound were used throughout the experiment. Visual cues of different shapes and

colors were placed on the walls surrounding the maze. These cues act as the animal’s

reference points for locating the target hole (Sunyer et al., 2007).

The Barnes maze test was performed over the course of 6d and consisted of a 4d

acquisition trial (4 trials per day) and a 2d (1 trial per day) testing trial. For the adaptation

period on the first trial of the first day of training, the mouse was placed in a cylindrical

white start chamber at the center of the maze. After 10 seconds, the chamber was lifted

and the mouse guided to the escape box, again with the sound of a buzzer acting as a

weak aversive stimulus to provide increased motivation for the mouse to escape from the

platform and into the escape box.

Spatial acquisition follows the adaptation period, where each mouse was given 3

minutes on the maze to locate the escape box. A camera mounted overhead on the ceiling

is used to record the animals’ movements on the maze, while this behaviour data are

26

automatically collected with the ANY-maze video-tracking system (Stoelting Co., Wood

Dale, IL, USA). The variables collected include the primary and total latency to get to

target, primary and total number of errors, as well as total distance travelled.

To test reference memory, either short or long-term retention in these mice, two

probe trials are conducted after training, with the first trial performed on day 5 and the

second trial on day 12. In these trials, the number of times a mouse pokes its nose into or

hovers its head above the escape box are recorded, also with the automated video-

tracking system.

27

Figure 2.4. Barnes maze used to measure spatial learning and memory, including

distal visual cues used during experimentation.

28

2.7 Morris Water maze and reversal

The Morris water maze (MWM) (Med Associates Inc., St. Albans, VT, USA) is

made up of a smooth white opaque plastic pool 120cm in diameter and 40cm in height for

the walls. The pool was filled approximately half way with clear water kept at

temperatures ranging from 24-26°C with a circular escape platform 10cm in diameter

submerged 1cm below the surface of the water. Two axes were designated for the maze,

creating an imaginary “+” in the pool leading to the formation of four equal quadrants

numbered 1-4 with the platform positioned in the middle of one quadrant. The location of

the quadrant depends on the counterbalance made prior to experimentation where at least

one-quarter of the animals are tested with the platform in each individual quadrants.

This behaviour data were automatically collected with the ANY-maze video-

tracking system (Stoelting Co., Wood Dale, IL, USA) (Figure 2.5).

Visual cues of different colours and shapes (gift wrap bows, large decorative

flower, and plastic Hawaiian leis) were used as a guide to the escape box and were placed

on the walls surrounding the maze. These cues act as the animal’s reference points for

locating the target quadrant (Vorhees & Williams, 2006).

The MWM requires 4d of acquisition (4 trials per day) and 1d of testing (1 trial

per day). In the acquisition phase (days 1-4), the mouse was released into the water at the

desired start position of the maze, facing the pool wall. The mouse was then required to

locate the hidden platform with the guidance of visual cues on the walls surrounding the

maze. Once the mouse reaches the platform, the video-tracking system is stopped by the

experimenter and variables such as the total distance travelled, the escape latency, and

speed are recorded. In order to assess the retention of spatial memory, specifically short-

term memory, a probe trial was performed 24 hours after the last training trial. The

29

platform was removed in the test trial and mice were allowed to swim freely for 60

seconds. During this session, the amount of time spent in each of the quadrants was

measured (Faivre, Hamilton, & Holscher, 2012; Vorhees & Williams, 2006).

MWM reversal was conducted the week after MWM, under identical conditions

and methodology with the only difference being the location of the platform. In this

reversal learning period, the platform was relocated to the quadrant opposite of its

previous location.

30

Figure 2.5. Morris water maze used to measure spatial learning and memory,

including distal visual cues used during experimentation.

31

2.8 Statistical Analysis

All data are expressed as mean ± SEM. Statistical analyses were carried out using

SigmaStat® (Aspire Software International, Ashburn, VA). Graphs were constructed

using GraphPad Prism® (GraphPad Software, San Diego, CA). Repeated measures one-

way and two-way ANOVA followed by the Holm-Sidak post hoc comparison test were

used (where appropriate) to compare multiple groups. Results were considered significant

when p < 0.05.

32

Section 3

RESULTS

33

3.1 Open field locomotor activity

An open field activity test was conducted to measure locomotor activity in mice

of all genotypes. In addition to measuring locomotion, the open field test can also

measure anxiety levels in rodents. Mice have a natural tendency to avoid open spaces by

travelling along the walls of enclosures as a means of protection and easy escape

(Lalonde et al., 2004). Therefore, the amount of time individuals spent in the center area

of the open field test provides a measure of anxiety.

No statistical differences in locomotor activity were observed between genotypes

at three months (Fig.3.1A,B; F(3,920) = 1.105, p = 0.358; there was no interaction

between genotype x time - Fig.3.1 A,B; F(69,920) = 0.669, p = 0.982; and habituation to the

novel environment was found to occur for mice from all four genotypes, indicated by a

continued decrease in distance with time - Fig.3.1A; F(69,920) = 0.669, p = 0.982); six

months (Fig.3.2A,B; F(3,1081) = 0.369, p = 0.775; there was no interaction between

genotype x time - Fig.3.2A,B; F(69,1081) = 0.850, p = 0.803; and habituation to the novel

environment was found to occur for mice from all four genotypes - Fig. 3.2A; F(23,1081) =

51.910, p < 0.001); or nine months (Fig.3.3A,B; F(3,897) = 0.701, p = 0.557; there was no

interaction between genotype x time - Fig.3.3A,B; F(69,897) = 1.031, p = 0.412; and

habituation to the novel environment was found to occur for mice from all four genotypes

- Fig.3.3A; F(23,897) = 44.401, p < 0.001). Furthermore, no change was observed in terms

of the amount of time spent in the center of the chamber at these different time points

(three months: Fig.3.1C; F(3,920) = 2.016, p = 0.127; six months: Fig.3.2C; F(3,1081) =

0.755, p = 0.525; and nine months: Fig.3.3C; F(3,897) = 0.370, p = 0.775).

34

At twelve months, no difference in locomotor activity was observed between

TRPM-KO and WT mice. However, APPSWE/PSEN1∆E9 and TRPM2-KO-

APPSWE/PSEN1∆E9 mice showed significantly decreased horizontal activity

(Fig.3.4A,B; F(3,1035) = 3.745, p = 0.017; post hoc analysis show p<0.05). In addition,

APPSWE/PSEN1∆E9 and TRPM2-KO-APPSWE/PSEN1∆E9 mice spent less time in

the center than the control mice (Fig.3.4C; p < 0.01 and p < 0.01 respectively, in the

Holm-Sidak post-hoc test). However, this difference in center time might reflect the fact

that these mutant mice are hypoactive rather than any difference in anxiety.

Unfortunately, for the fifteen months analysis we had a smaller number of mice

due to death or fight injury. Our analysis did not show any significant difference in

locomotor activity between genotypes (Fig.3.5A,B; F(3,374) = 1.622, p = 0.202). A

statistical significance in center time was observed between genotypes as measured by

two-way repeated measures ANOVA. Post hoc analysis indicates that

APPSWE/PSEN1∆E9 mice spent significantly less time in the center than WT, TRPM2-

KO and TRPM2-KO-APPSWE/PSEN1∆E9 mice (Fig.3.5C).

35

Figure 3.1. Locomotor activity at 3 months of age

(A) Horizontal activity in an open field for WT, TRPM2-KO, APPSWE/PSEN1∆E9

and TRPM2-KO-APPSWE/PSEN1∆E9 mice was measured over time (F(23,920) = 61.052,

p < 0.001 in a two-way repeated measures ANOVA) and (B) cumulatively over 2 h and

(C) amount of time spent in the center. All data were plotted as Mean ± SEM.

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 1001051101151200

200

400

600 WT N=13

TRPM2-KO N=13

APPSWE/PSEN1∆E9 N=8

TRPM2-KO-APPSWE/PSEN1∆E9 N=10

Time (min)

To

tal D

ista

nce

(cm

/5m

in)

0

2000

4000

6000

Day 1

To

tal D

ista

nc

e

(cm

/2h

r)

n.s.

0

1000

2000

3000

Day 1

n.s.

WT N=13

TRPM2-KO N=13



To

tal C

en

ter

Tim

e(s

)

A

B C

36






0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 1200

100

200

300

400



WT N=13

TRPM2-KO N=12

Time (min)

To

tal D

ista

nce

(cm

/5m

in)

0

1000

2000

3000

Day 1

To

tal D

ista

nce

(cm

/2h

r)

n.s.

0

1000

2000

3000

Day 1

WT N=13

TRPM2-KO N=12



n.s.

To

tal

Ce

nte

r T

ime

(s)

A

B C

37






0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 1200

100

200

300

400

500

WT N=13

TRPM2-KO N=11



Time (min)

To

tal D

ista

nce

(cm

/5m

in)

0

500

1000

1500

2000

2500

3000

3500

Day 1

To

tal D

ista

nce

(cm

/2h

r)

n.s.

0

1000

2000

3000

Day 1

WT N=13

TRPM2-KO N=11


TRPM2-KO-APPSWE/PSEN1∆E9 N=14n.s.

To

tal C

en

ter

Tim

e(s

)

A

B C

38





(C) amount of time spent in the center. All data were plotted as Mean ± SEM. *p = 0.05

in a Holm-Sidak post hoc test; **p < 0.01 in a Holm-Sidak post hoc test.

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 1200

50

100

150

200

250

300

350

400

450

WT N=12

TRPM2-KO N=11



***

Time (min)

To

tal D

ista

nce

(cm

/5m

in)

0

1000

2000

3000

4000

**

Day 1

To

tal D

ista

nce

(cm

/2h

r) *

0

500

1000

1500

2000

2500

3000

3500

Day 1

WT N=12

TRPM2-KO N=11



****

To

tal

Ce

nte

r T

ime

(s)

A

B C

39





(C) amount of time spent in the center. All data were plotted as Mean ± SEM. **p < 0.01

in a Holm-Sidak post hoc test; ***p < 0.001 in a Holm-Sidak post hoc test.

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 1200

50

100

150

200

250

300

350

400

450WT N=8

TRPM2-KO N=7



Time (min)

To

tal D

ista

nce

(cm

/5m

in)

0

1000

2000

3000

4000

5000

Day 1

To

tal D

ista

nce

(cm

/2h

r)

n.s.

0

500

1000

1500

2000

2500

3000

Day 1

**,***

WT N=8

TRPM2-KO N=7



To

tal

Ce

nte

r T

ime

(s)

A

B C

40

3.2 Elevated Plus maze

The elevated plus maze task was used to examine whether anxiety levels differed

between the four genotypes of mice (WT, TRPM2-KO, APPSWE/PSEN1∆E9, and

TRPM2-KO-APPSWE/PSEN1∆E9) at five age points (3, 6, 9, 12, and 15 months).

No statistical differences in anxiety were observed between genotypes at three

months (Fig.3.6A; F(3,28) = 0.152, p = 0.928 in terms of the percentage of time spent in

open arms; there was no statistical difference in terms of the number of arm entries to the

open arms – Fig.3.6B; F(3,28) = 0.121, p = 0.947); nine months (Fig.3.6E; F(3,32) = 0.551,

p = 0.651 in terms of the percentage of time spent in open arms; there was no statistical

difference in terms of the number of arm entries to the open arms – Fig.3.6F; F(3,32) =

1.248, p = 0.309); or fifteen months (Fig.3.6I; F(3,30) = 1.241, p = 0.312 in terms of the

percentage of time spent in open arms; there was no statistical difference in terms of the

number of arm entries to the open arms – Fig.3.6J; F(3,30) = 1.296, p = 0.294).

At six months, while TRPM2-KO and APPSWE/PSEN1∆E9 mice did not differ

significantly from WT in the time spent in the open arms; TRPM2-KO-

APPSWE/PSEN1∆E9 mice spent significantly longer time in the open arms than the

control mice (Fig.3.6C; F(3,33) = 3.756, p = 0.020; post hoc analysis show p<0.05 and

p<0.01). However, no statistical difference was observed between genotypes in terms of

the number of arm entries into the open arms (Fig.3.6D; F(3,33) = 2.023, p = 0.130). These

results might suggest that TRPM2-KO-APPSWE/PSEN1∆E9 mice were less anxious

than WT, TRPM2-KO and APPSWE/PSEN1∆E9 at this age.

No statistical difference was observed in the percentage of time spent in the open

arms at twelve months of age (Fig.3.6G; F(3,31) = 0.417, p = 0.742). On the other hand,

41

APPSWE/PSEN1∆E9 and TRPM2-KO-APPSWE/PSEN1∆E9 mice visited the open

arms significantly fewer times than the control mice (Fig.3.6H; F(3,31) = 5.799, p = 0.003;

post hoc analysis show p<0.05 and p<0.01), suggesting that at this age these mutant mice

might be more anxious than WT and TRPM2-KO. However, this may also be a

consequence of their reduced activity when aged.

42

0

10

20

30

40

n.s.%

op

en

arm

s

0

5

10

15

20

25n.s.

WT N=13

TRPM2-KO N=13



# e

ntr

ies

to o

pen

arm

0

10

20

30*,**

% o

pen

arm

s

0

5

10

15

WT N=13

TRPM2-KO N=11


TRPM2-KO-APPSWE/PSEN1∆E9 N=14n.s.

# e

ntr

ies

to o

pen

arm

0

5

10

15

% o

pen

arm

s

n.s.

0

5

10

15

n.s.

WT N=13

TRPM2-KO N=11



# e

ntr

ies

to o

pen

arm

0

5

10

15

20

25 n.s.

% o

pen

arm

s

0

5

10

15

*,****

WT N=12

TRPM2-KO N=11



# e

ntr

ies

to o

pen

arm

0

10

20

30

% o

pen

arm

s

n.s.

0

5

10

15

n.s.

WT N=12

TRPM2-KO N=10



# e

ntr

ies

to o

pen

arm

A B

C D

E F

G H

I J

43

Figure 3.6. Assessment of anxiety at 3, 6, 9, 12, and 15 months of age

(A) Percentage of time WT, TRPM2-KO, APPSWE/PSEN1∆E9, and TRPM2-KO-

APPSWE/PSEN1∆E9 mice spent in the open arms measured at 3 months (B) Number of

arm entries measured at 3 months and (C) Percentage of time spent in open arms

measured at 6 months (F(3,33) = 3.756, p < 0.05 in a one-way repeated measures ANOVA)

(D) Number of arm entries measured at 6 months and (E) Percentage of time spent in

open arms measured at 9 months and (F) Number of arm entries measured at 9 months

(G) Percentage of time spent in open arms measured at 12 months and (H) Number of

arm entries measured at 12 months (F(3,31) = 5.799, p < 0.01 in a one-way repeated

measures ANOVA) (I) Percentage of time spent in open arms measured at 15 months of

age and (J) Number of arm entries measured at 15 months of age. All data were plotted as

Mean ± SEM. *p < 0.05 in a Holm-Sidak post hoc test; **p < 0.01 in a Holm-Sidak post

hoc test.

44


To test for recognition memory, which is the ability of mice to discriminate

between familiar and novel objects, we conducted an object recognition test at 9 and 12

months. Mice have a spontaneous tendency to spend more time exploring a novel object

than a familiar one. Therefore, the choice to explore the novel object will reflect the use

of learning and recognition memory (Otalora et al., 2012; Prado et al., 2006). This task

requires that the individuals be able to distinguish between the two familiar objects on

training day and recognize the novel object on test day where both familiar and novel

objects are present. Although we are showing only the 9 and 12 months data here, we did

conduct this test at 3 and 6 months of age. However, our protocol was flawed and

therefore we could not use those results. The protocol was modified in order to conduct

the test at 9 and 12 months of age. We decided not to conduct object recognition test at

15 months of age because we assumed no deficit will be seen; that mice of all genotypes

will be able to discriminate between the familiar and novel objects, just like the data

shown at 9 and 12 months.

No significant difference was observed in terms of the percentage of time spent

exploring the familiar (F) and novel (N) objects. Mice from all genotypes preferred the

novel object on testing day, as shown by an increased exploration of N at nine months

(Fig.3.7B; WT - F(1,5) = 33.235, p = 0.002; post hoc analysis show p<0.01; TRPM2-KO -

F(1,5) = 17.506, p = 0.009; post hoc analysis show p<0.01; APPSWE/PSEN1∆E9 - F(1,9) =

7.625, p = 0.022; post hoc analysis show p<0.01; TRPM2-KO-APPSWE/PSEN1∆E9

- F(1,11) = 12.696, p = 0.004; post hoc analysis show p<0.01) and twelve months

(Fig.3.7D; WT - F(1,7) = 10.469, p = 0.014; post hoc analysis show p<0.01; TRPM2-KO -

45

F(1,7) = 27.963, p = 0.001; post hoc analysis show p<0.01; APPSWE/PSEN1∆E9- F(1,11) =

6.621, p = 0.026; post hoc analysis show p<0.05; TRPM2-KO-APPSWE/PSEN1∆E9

- F(1,13) = 20.757, p < 0.001; post hoc analysis show p<0.01).

46

Figure 3.7. Assessment of recognition memory at 9 and 12 months of age

(A) The time that WT, TRPM2-KO, APPSWE/PSEN1∆E9 and TRPM2-KO-

APPSWE/PSEN1∆E9 mice spent exploring the homecage on training trials 1-3 and test

trial 4 was measured at 9 months and (C) 12 months. (B) Percentage of time mice spent

exploring the familiar (F) and novel (N) objects on both training and test days were

measured at 9 months and (D) 12 months. All data were plotted as Mean ± SEM. *p <

0.05 in a Holm-Sidak post hoc test; **p < 0.01 in a Holm-Sidak post hoc test.

1 2 3 40

25

50

75

100

125



WT N=9

TRPM2-KO N=8

Training Test

Trial

To

tal

Exp

lora

tio

n (

sec)

0

10

20

30

40

50

60

70

80

90

TRPM2-KO-

APPSWE/PSEN1∆E9

APPSWE/

PSEN1∆E9

N=12

NF F N

N=14

Training

Test

F FF F

WT TRPM2-KO

N=9 N=8

NF F F NF F F

**** ** **

% E

xp

lora

tio

n

1 2 3 40

20

40

60

80

100

WT N=8

TRPM2-KO N=8



Training Test

Trial

To

tal E

xp

lora

tio

n (

sec)

0

10

20

30

40

50

60

70

80

90

N=12

NF F N

N=14

Training

Test

F FF F

WT TRPM2-KO

N=8 N=8

NF F F NF F F

** ***

**

% E

xp

lora

tio

n

APPSWE/

PSEN1∆E9

TRPM2-KO-

APPSWE/PSEN1∆E9

A B

C D

47

3.4 Barnes maze

Spatial learning and memory were examined in the Barnes maze (Sunyer et al.,

2007) for all four groups of mice at 3, 6, 9, 12, and 15 months of age. Learning was

observed for all ages as measured by the primary latency to find the escape box, indicated

by the reduced time needed for the mice to find the escape box. Primary number of

errors, the number of errors made by the mice in order to reach the target hole was also

measured. Memory, or the ability to remember the location of the target hole with the

guidance of visual cues, was assessed on test/probe days 5 and 12. Here, the number of

times the mouse pokes its nose into, or hover its head above the escape box was

measured.

No statistical differences in spatial memory was observed between genotypes at

three months (there was no statistical difference between genotypes in terms of primary

latency to target hole for training days 1 to 4 as well as testing days 5 and 12 - Fig.3.8A

(left); F(3,195) = 1.128, p = 0.350; there was no statistical difference between genotypes in

terms of primary latency to target hole when only looking at training days – Fig.3.8A

(middle); F(3,117) = 0.861, p = 0.469; and there was no statistical difference between

genotypes in terms of primary number of errors to target hole on training days – Fig.3.8A

(right); F(3,117) = 1.167, p = 0.335. There was no statistical difference between genotypes

on test day 5 (Fig.3.8B (left); F(3,117) = 1.213, p = 0.318) or day 12 (Fig.3.8C (left); F(3,117)

= 2.807, p = 0.052) in terms of number of times the mouse pokes its nose into the escape

box.

At six months, APPSWE/PSEN1∆E9 and TRPM2-KO-APPSWE/PSEN1∆E9

mice showed longer primary latency to the target hole than control mice - Fig.3.9A (left);

48

F(3,230) = 4.051, p < 0.05; post hoc analysis show p≤0.01 and p<0.05;

APPSWE/PSEN1∆E9 and TRPM2-KO-APPSWE/PSEN1∆E9 mice also showed longer

primary latency to the target hole than control mice over the 4 days of training - Fig.3.9A

(middle); F(3,138) = 4.125, p < 0.05; post hoc analysis show p<0.01 and p<0.05; and

APPSWE/PSEN1∆E9 mice made more errors compared to the others throughout the 4

training days – Fig.3.9A (right); F(3,138) = 2.970, p <0.05; post hoc analysis show p<0.05.

On probe trial day 5, latency to find the target hole was no different between genotypes

(Fig 3.9A (left)) and time spent investigating the target quadrant was significantly longer

compared with the other quadrants for all genotypes (Fig.3.9B (left); F(3,138) = 3.176, p =

0.033; post hoc analysis show p<0.05; all mice visited the target hole location more

regardless of genotypes as indicated by the percentage of time spent in each quadrant -

Fig.3.9B (right); F(3,138) = 91.008, p < 0.001; post hoc analysis show p<0.001. On probe

trial day 12, even though APPSWE/PSEN1∆E9 and TRPM2-KO-APPSWE/PSEN1∆E9

mice visited the target hole significantly less than controls - Fig.3.9C (left); F(3,138) =

4.512, p = 0.007; post hoc analysis show p<0.05 and p<0.01, the results indicate that

these mutants do remember the position of the target hole.

At nine months, there was no statistical difference between genotypes in terms of

primary latency to target hole for training days 1 to 4 as well as testing days 5 and 12 -

Fig.3.10A (left); F(3,230) = 2.269, p = 0.093; there was no statistical difference between

genotypes in terms of primary latency to target hole when only looking at training days –

Fig.3.10A (middle); F(3,138) = 1.631, p = 0.195; and there was no statistical difference

between genotypes in terms of primary number of errors to target hole on training days –

Fig.3.10A (right); F(3,138) = 0.506, p = 0.680. On probe trial day 5, APPSWE/PSEN1∆E9

49

and TRPM2-KO-APPSWE/PSEN1∆E9 mice visited the target hole location significantly

less than controls - Fig.3.10B (left); F(9,138) = 3.223, p = 0.001; post hoc analysis show

p≤0.001 and p<0.05; TRPM2-KO-APPSWE/PSEN1∆E9 mice also visited the left hole

location significantly less than controls - Fig.3.10B (left); F(9,138) = 3.223, p = 0.001; post

hoc analysis show p<0.05; all mice visited the target hole location more regardless of

genotypes on this day as indicated by the percentage of time spent in each quadrant -

Fig.3.10B (right); F(3,138) = 103.229, p < 0.001; post hoc analysis show p<0.001; and

there was no statistical difference between genotypes on test day 12 – Fig.3.10C (left);

F(9,138) = 1.407, p = 0.191); again, all mice visited the target hole location more regardless

of genotypes as indicated by the percentage of time spent in each quadrant - Fig.3.10C

(right); F(3,138) = 68.221, p < 0.001; post hoc analysis show p<0.001 and p < 0.01.

At twelve months, there was no statistical difference between genotypes in terms

of primary latency to target hole for training days 1 to 4 as well as testing days 5 and 12

(Fig.3.11A (left); F(3,185) = 0.856, p = 0.472); there was no statistical difference between

genotypes in terms of primary latency to target hole when only looking at training days –

Fig.3.11A (middle); F(3,111) = 0.575, p = 0.635; and there was no statistical difference

between genotypes in terms of primary number of errors to target hole on training days –

Fig.3.11A (right); F(3,111) = 1.129, p = 0.350. On probe trial days 5 and 12, while target

hole location visits of TRPM2-KO and TRPM2-KO-APPSWE/PSEN1∆E9 mice did not

differ from that of WT, APPSWE/PSEN1∆E9 mice visited the target hole location

significantly less than control and TRPM2-KO-APPSWE/PSEN1∆E9 mice - Fig.3.11B

(left); F(3,111) = 2.284, p = 0.035; post hoc analysis show p<0.01 and p<0.05; and

Fig.3.11C (left); F(3,111) = 1.789, p = 0.05; post hoc analysis show p<0.05. All mice

50

visited the target hole location more regardless of genotypes on both day 5 (Fig.3.11B

(middle); F(3,111) = 59.827, p < 0.001; post hoc analysis show p<0.001 and p<0.01) and

day 12 (Fig.3.11C (middle); F(3,111) = 54.250, p < 0.001; post hoc analysis show p<0.001

and p<0.01) as indicated by the percentage of time spent in each quadrant.

At fifteen months, APPSWE/PSEN1∆E9 and TRPM2-KO-

APPSWE/PSEN1∆E9 mice had longer primary latency to target hole than control mice

(Fig.3.12A (left); F(3,200) = 7.198, p < 0.001; post hoc analysis show p<0.001 and

p<0.01); APPSWE/PSEN1∆E9 and TRPM2-KO-APPSWE/PSEN1∆E9 mice also had

longer primary latency to target hole than control mice when only looking at the first 4

training days (Fig. 3.12A (middle); F(3,120) = 4.763, p < 0.01; post hoc analysis show

p<0.01 and p<0.05); and there was no statistical difference between genotypes in terms of

primary number of errors to target hole on training days (Fig.3.12A (right); F(3,120) =

0.803, p = 0.499). On probe trial day 5, APPSWE/PSEN1∆E9 mice visited the target hole

location significantly less than control and TRPM2-KO-APPSWE/PSEN1∆E9 mice

(Fig.3.12B (left); F(9,120) = 4.344, p < 0.001; post hoc analysis show p<0.001 and p<0.05).

On probe trial day 12, APPSWE/PSEN1∆E9 mice visited the target hole location

significantly less than control and TRPM2-KO-APPSWE/PSEN1∆E9 mice (Fig.3.12C

(left); F(9,120) = 2.006, p = 0.044; post hoc analysis show p<0.01). Thus,

APPSWE/PSEN1∆E9 mice visited less and spent significantly less time in the target

quadrant on both probe days compared to TRPM2-KO-APPSWE/PSEN1∆E9 mice.

These results indicate that TRPM2-KO-APPSWE/PSEN1∆E9 mice perform significantly

better than APPSWE/PSEN1∆E9 mice.

51

Taking all of the above findings into consideration, results from BM indicate that

APPSWE/PSEN1∆E9 mice have impaired spatial memory and that AD mouse model

that lack TRPM2 (TRPM2-KO-APPSWE/PSEN1∆E9) had their performance rescued in

the Barnes maze.

52

Figure 3.8. Barnes Maze assessment of spatial memory at 3 months of age.

1 2 3 4 5 120

15

30

45

60

75

90

Day

Pri

mary

Late

ncy (

sec)

1 2 3 40

15

30

45

60

75

90

Day

Pri

mary

Late

ncy (

sec)

1 2 3 40

5

10

15

20

25

Day

Pri

mary

# o

f E

rro

rs

Target Opposite Left Right0

5

10

15

20

25n.s.

Quadrant

# o

f P

okes

T O L R T O L R T O L R T O L R0

2

4

6

8

Quadrant

% N

ose P

okes

**

***'

**

*** ***

***


5

10

15

20

25 n.s.

Quadrant

# o

f P

okes


2

4

6

8

Quadrant

% N

ose P

okes

*** ***

*** ***

A

B

C

Probe Day 5

Probe Day 12

WT

TRPM2-KO

APPSWE/PSE

N1∆E9

TRPM2-KO- APPSWE/PSEN1∆E9

WT

TRPM2-KO

APPSWE/PSE

N1∆E9


53

(A (left)) Primary latency to target hole on acquisition days 1-4 (the average values of

four 3min trials per day are plotted) and test days 5 and 12 was measured for WT,

TRPM2-KO, APPSWE/PSEN1∆E9, and TRPM2-KO-APPSWE/PSEN1∆E9 mice at 3

months. (A (middle)) Primary latency to target hole on acquisition days 1-4 (the average

values of four 3min trials per day are plotted) was measured for WT, TRPM2-KO,

APPSWE/PSEN1∆E9 , and TRPM2-KO-APPSWE/PSEN1∆E9 mice at 3 months. (A

(right)) Primary number of error to target hole on acquisition days 1-4 was measured for

all genotypes at 3 months. (B (left)) Number of nose pokes per quadrant measured on test

day 5 in a 90s probe trial. (B (middle)) Percentage of nose pokes per quadrant separated

by genotypes on day 5. (B (right)) Representative path traces on day 5 comparing WT,

TRPM2-KO, APPSWE/PSEN1∆E9, and TRPM2-KO-APPSWE/PSEN1∆E9 mice.

Target hole indicated by black dot. (C (left)) Number of nose pokes per quadrant

measured on test day 12 in a 90s probe trial. (C (middle)) Percentage of nose pokes per

quadrant separated by genotypes on day 12. (C (right)) Representative path traces on day

12 comparing WT, TRPM2-KO, APPSWE/PSEN1∆E9, and TRPM2-KO-

APPSWE/PSEN1∆E9 mice. All data were plotted as Mean ± SEM. *p < 0.05 in a Holm-

Sidak post hoc test; **p < 0.01 in a Holm-Sidak post hoc test; ***p < 0.001 in a Holm-

Sidak post hoc test; ***’p = 0.001 in a Holm-Sidak post hoc test. L, left; O, opposite; R,

right; T, target.

54

Figure 3.9. Barnes Maze assessment of spatial memory at 6 months of age.

(A (left)) Primary latency to target hole on aquisition days 1-4 (the average values of four

3min trials per day are plotted) and test days 5 and 12 was measured for WT, TRPM2-

KO, APPSWE/PSEN1∆E9, and TRPM2-KO-APPSWE/PSEN1∆E9 mice at 6 months

(F(3,230) = 4.051, p < 0.05 in a two-way repeated measures ANOVA). (A (middle))

1 2 3 4 5 120

10

20

30

40

50

60

*,**

*,**'

Day

Pri

mary

Late

ncy (

sec)

1 2 3 40

10

20

30

40

50

60

Day

Pri

mary

Late

ncy (

sec)

*,**

*,**

1 2 3 40

5

10

15

20

Day

Pri

mary

# o

f E

rro

rs

*,**'


5

10

15

20

25

30

35

** *

Quadrant

# o

f P

okes


2

4

6

Quadrant

% N

ose P

okes

*** *** ******


5

10

15

20

25

30

35

*,** **

*

*,***

Quadrant

# o

f P

okes


1

2

3

4

5

Quadrant

% N

ose P

okes

****** ***

***

A

B

C

Probe Day 5

Probe Day 12

A

B

C

55

Primary latency to target hole on acquisition days 1-4 (the average values of four 3min

trials per day are plotted) was measured for WT, TRPM2-KO, APPSWE/PSEN1∆E9,

and TRPM2-KO-APPSWE/PSEN1∆E9 mice at 6 months (F(3,138) = 4.125, p < 0.05 in a

two-way repeated measures ANOVA). (A (right)) Primary number of error to target hole

on acquisition days 1-4 was measured for all genotypes at 6 months (F(3,138) = 2.970, p <

0.05 in a two-way repeated measures ANOVA). (B (left)) Number of nose pokes per

quadrant measured on test day 5 in a 90s probe trial (F(3,138) = 3.176, p < 0.05 in a two-

way repeated measures ANOVA). (B (right)) Percentage of nose pokes per quadrant

separated by genotypes on day 5 (F(3,138) = 91.008, p < 0.001 in a two-way repeated

measures ANOVA). (C (left)) Number of nose pokes per quadrant measured on test day

12 in a 90s probe trial (F(3,138) = 4.512, p < 0.01 in a two-way repeated measures

ANOVA). (C (right)) Percentage of nose pokes per quadrant separated by genotypes on

day 12 (F(3,138) = 80.773, p < 0.001 in a two-way repeated measures ANOVA). All data

were plotted as Mean ± SEM. *p < 0.05 in a Holm-Sidak post hoc test; **p < 0.01 in a

Holm-Sidak post hoc test; **’p = 0.01 in a Holm-Sidak post hoc test; *** p < 0.001 in a

Holm-Sidak post hoc test. L, left; O, opposite; R, right; T, target.

56

Figure 3.10. Barnes Maze assessment of spatial memory at 9 months of age






1 2 3 4 5 120

10

20

30

40

50

Day

Pri

mary

Late

ncy (

sec)

1 2 3 40

10

20

30

40

50

Day

Pri

mary

Late

ncy (

sec)

1 2 3 40

5

10

15

20

Day

Pri

mary

# o

f E

rro

rs


10

20

30

40

***,***'

*,***

*

Quadrant

# o

f P

okes


1

2

3

4

5

***

Quadrant

% N

ose P

okes

*** ***

***


10

20

30

Quadrant

# o

f P

okes


1

2

3

4

5

**

Quadrant

% N

ose P

okes

****** ***

***

A

B

C

Probe Day 5

Probe Day 12

A

B

57

APPSWE/PSEN1∆E9, and TRPM2-KO-APPSWE/PSEN1∆E9 mice at 9 months. (A


all genotypes at 9 months. (B (left)) The number of nose pokes per quadrant were

measured on test day 5 in a 90s probe trial (F(9,138) = 3.223, p = 0.001 in a two-way

repeated measures ANOVA). (B (right)) Percentage of nose pokes per quadrant separated

by genotypes on day 5 (F(9,138) = 103.229, p < 0.001 in a two-way repeated measures

ANOVA). (C (left)) The number of nose pokes per quadrant were measured on test day

12 in a 90s probe trial. (C (right)) Percentage of nose pokes per quadrant separated by

genotypes on day 12 (F(9,138) = 68.221, p < 0.001 in a two-way repeated measures

ANOVA). All data were plotted as Mean ± SEM. *p < 0.05 in a Holm-Sidak post hoc

test; **p < 0.01 in a Holm-Sidak post hoc test; ***p < 0.001 in a Holm-Sidak post hoc

test; ***’p = 0.001 in a Holm-Sidak post hoc test. L, left; O, opposite; R, right; T, target.

58




1 2 3 4 5 120

20

40

60

Day

Pri

mary

Late

ncy (

sec)

1 2 3 40

20

40

60

Day

Pri

mary

Late

ncy (

sec)

1 2 3 40

5

10

15

20

Day

Pri

mary

# o

f E

rro

rs


5

10

15

20

25

30

*,**

Quadrant

# o

f P

okes


2

4

6

8

**

***

Quadrant

% N

ose P

okes

***

******


5

10

15

20

25

30

**

Quadrant

# o

f P

okes


2

4

6

8

*******

Quadrant

% N

ose P

okes

*** ***

***

A

B

C

Probe Day 5

Probe Day 12

WT

TRPM2-KO

APPSWE/PS

EN1∆E9

TRPM2-KO-

APPSWE/PS

EN1∆E9

WT

TRPM2-KO

APPSWE/P

SEN1∆E9

TRPM2-KO-

APPSWE/PS

EN1∆E9

A

B

C

59




APPSWE/PSEN1∆E9 , and TRPM2-KO-APPSWE/PSEN1∆E9 mice at 12 months. (A


all genotypes at 12 months. (B (left)) The number of nose pokes per quadrant were

measured on test day 5 in a 90s probe trial (F(3,111) = 2.284, p = 0.035 in a two-way

repeated measures ANOVA). (B (middle)) Percentage of nose pokes per quadrant


measures ANOVA). (B (right)) Representative path traces on day 5 comparing WT,

TRPM2-KO, APPSWE/PSEN1∆E9, and TRPM2-KO-APPSWE/PSEN1∆E9 mice.

Target hole indicated by black dot. (C (left)) The number of nose pokes per quadrant

were measured on test day 12 in a 90s probe trial (F(3,111) = 1.789, p = 0.05 in a two-way

repeated measures ANOVA). (C (middle)) Percentage of nose pokes per quadrant


measures ANOVA). (C (right)) Representative path traces on day 12 comparing WT,

TRPM2-KO, APPSWE/PSEN1∆E9, and TRPM2-KO-APPSWE/PSEN1∆E9 mice. All

data were plotted as Mean ± SEM. *p < 0.05 in a Holm-Sidak post hoc test; **p < 0.01 in

a Holm-Sidak post hoc test; ***p < 0.001 in a Holm-Sidak post hoc test. L, left; O,

opposite; R, right; T, target.

60





1 2 3 4 5 120

20

40

60

80

100

**,***

**

Day

Pri

mary

Late

nc

y (

sec)

1 2 3 40

20

40

60

80

100

***

Day

Pri

mary

Late

ncy (

sec)

1 2 3 40

5

10

15

20

Day

Pri

mary

# o

f E

rro

rs


10

20

30

***

*,***

** *

**,***

*,**

*,***,***'

Quadrant

# o

f P

okes


1

2

3

4

5

6

***

***

Quadrant

% N

ose P

okes

*****


10

20

30

**

Quadrant

# o

f P

okes


1

2

3

4

5

**

***

Quadrant

% N

ose P

okes

***

***

***

A

B

C

Probe Day 5

Probe Day 12

A

B

C

61

months (F(3,200) = 7.198, p < 0.001 in a two-way repeated measures ANOVA). (A

(middle)) Primary latency to target hole on acquisition days 1-4 (the average values of

four 3min trials per day are plotted) was measured for WT, TRPM2-KO,

APPSWE/PSEN1∆E9 , and TRPM2-KO-APPSWE/PSEN1∆E9 mice at 15 months

(F(3,120) = 4.763, p < 0.05 in a two-way repeated measures ANOVA). (A (right)) Primary

number of error to target hole on acquisition days 1-4 was measured for all genotypes at

15 months. (B (left)) The number of nose pokes per quadrant were measured on test day

5 in a 90s probe trial (F(9,120) = 4.344, p < 0.001 in a two-way repeated measures

ANOVA). (B (right)) Percentage of nose pokes per quadrant separated by genotypes on

day 5 (F(9,120) = 22.974, p < 0.001 in a two-way repeated measures ANOVA). (C (left))

The number of nose pokes per quadrant were measured on test day 12 in a 90s probe trial

(F(9,120) = 2.006, p < 0.05 in a two-way repeated measures ANOVA). (C (right))

Percentage of nose pokes per quadrant separated by genotypes on day 12 (F(9,120) =

14.823, p < 0.001 in a two-way repeated measures ANOVA). All data were plotted as

Mean ± SEM. *p < 0.05 in a Holm-Sidak post hoc test; **p < 0.01 in a Holm-Sidak post

hoc test; ***p < 0.001 in a Holm-Sidak post hoc test; ***’p = 0.001 in a Holm-Sidak post

hoc test. L, left; O, opposite; R, right; T, target.

62

3.5 Morris water maze hidden-platform test

Spatial learning and memory were examined by Morris water maze (MWM) at

twelve and fifteen months of age. In acquisition trials, mice were trained to locate a

hidden platform placed in 1 of 4 quadrants in the maze. On day 5 of the probe trial, the

platform was removed and mice were allowed to swim freely for 60 seconds. During this

session, the amount of time spent in each of the quadrants was measured.

No statistical differences in spatial memory were observed between WT, TRPM2-

KO, APPSWE/PSEN1∆E9 and TRPM2-KO-APPSWE/PSEN1∆E9 mice at twelve

months. There was no statistical difference between genotypes in terms of the latency to

find the escape platform in the 4d training period, and all mice improved in the time it

took to find the platform over training trials (Fig.3.13A; F(3,105) = 1.279, p = 0.297). No

statistical difference between genotypes were observed in terms of distance required to

find the platform (Fig.3.13B; F(3,105) = 1.636, p = 0.199). When spatial memory was

investigated on probe trial day 5, the time WT, TRPM2-KO, APPSWE/PSEN1∆E9, and

TRPM2-KO-APPSWE/PSEN1∆E9 mice spent investigating the target quadrant was

significantly longer than all other quadrants (Fig.3.13C; F(3,105) = 39.134, p<0.001; post

hoc analysis show p<0.05, p<0.01, p=0.001, and p<0.001), indicating that all mice have

learned the location of the target quadrant. Closer examination of representative path

traces indicate that mice of all genotype are spending more time in the target quadrant

and thus are learning, as well as remembering the locations of the platform (Fig.3.13D).

At fifteen months, APPSWE/PSEN1∆E9 and TRPM2-KO-

APPSWE/PSEN1∆E9 mice had a longer latency to find the escape platform in the 4d

training period than control mice (Fig.3.14A; F(3,117) = 4.167, p < 0.05; post hoc analysis

63

show p<0.05 and p<0.01). In addition, the distance required for APPSWE/PSEN1∆E9

and TRPM2-KO-APPSWE/PSEN1∆E9 mice to find the platform is significantly longer

than controls (Fig.3.14B; F(3,117) = 9.619, p < 0.001; post hoc analysis show p<0.01 and

p<0.001). When spatial memory was investigated on probe trial day 5, the time WT,

TRPM2-KO, and TRPM2-KO-APPSWE/PSEN1∆E9 mice spent investigating the target

quadrant was significantly longer than all other quadrants (Fig.3.14C; F(3,117) = 30.070,

p<0.001; post hoc analysis show p<0.05, p<0.01, and p<0.001), suggesting that these

mice remembered where the platform was located. However, APPSWE/PSEN1∆E9

mice did not remember the location of the platform since these mice did not spend a

significantly longer time investigating the target quadrant. Representative path traces

further indicate that APPSWE/PSEN1∆E9 mice, contrary to WT, TRPM2-KO and

TRPM2-KO-APPSWE/PSEN1∆E9 mice, do not remember the position of the platform

and their swim paths are in a random pattern around the pool instead of only focusing on

the target quadrant (Fig.3.14D).

64

Figure 3.13. MWM assessment of spatial memory at 12 months of age

(A) WT, TRPM2-KO, APPSWE/PSEN1∆E9, and TRPM2-KO-APPSWE/PSEN1∆E9

mice were subject to the MWM paradigm. Latency to find the platform on acquisition

days 1-4 (the average of four 90s trials per day) is plotted (B) Total distance traveled to

the platform on acquisition days 1-4 (the average of four 90s trials per day) is plotted (C)

Percentage of time spent in each quadrant was measured on day 5 in a 60s probe trial

1 2 3 40

20

40

60

80

Day

Es

ca

pe

L

ate

nc

y (

se

c)

1 2 3 40

5

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10

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Quadrant

******

*,***'

***

****,***

***'**

*********%

Tim

e

A B

C D


APPSWE/PS

EN1∆E9

TRPM2-KO

WT

65

with the platform removed (F(3,105) = 39.134, p < 0.001 in a two-way repeated measures

ANOVA). (D) Representative path traces on probe day 5 comparing WT, TRPM2-KO,

APPSWE/PSEN1∆E9 , and TRPM2-KO-APPSWE/PSEN1∆E9 mice. All data were

plotted as Mean ± SEM. *p < 0.05 in a Holm-Sidak post hoc test; **p < 0.01 in a Holm-

Sidak post hoc test; ***p < 0.001 in a Holm-Sidak post hoc test; ***’p = 0.001 in a

Holm-Sidak post hoc test. L, left; O, opposite; R, right; T, target.

66

Figure 3.14. MWM assessment of spatial memory at 15 months of age


mice were subject to the MWM paradigm. Latency to find the platform on acquisition

days 1-4 (the average of four 90s trials per day) is plotted (F(3,117) = 4.167, p < 0.05 in a

two-way repeated measures ANOVA) (B) Total distance traveled to the platform on

acquisition days 1-4 (the average of four 90s trials per day) is plotted (F(3,117) = 9.619, p <

1 2 3 40

20

40

60

*,***

Day

Es

ca

pe

L

ate

nc

y (

se

c)

1 2 3 40

5

10

15

***

**

Day

To

tal D

ista

nc

e (

m)


10

20

30

40

50

60

Quadrant

******

*,**

*** ***

*

*,***

*** ***

***

% T

ime

B

B A

C D

WT

TRPM2-KO

APPSWE/P

SEN1∆E9


67

0.001 in a two-way repeated measures ANOVA) (C) Percentage of time spent in each

quadrant was measured on day 5 in a 60s probe trial with the platform removed (F(3,117) =

16.466, p < 0.001 and F(3,117) = 30.070, p < 0.001 in a two-way repeated measures

ANOVA). (D) Representative path traces on probe day 5 comparing WT, TRPM2-KO,

APPSWE/PSEN1∆E9, and TRPM2-KO-APPSWE/PSEN1∆E9 mice. All data were

plotted as Mean ± SEM. *p < 0.05 in a Holm-Sidak post hoc test; **p < 0.01 in a Holm-

Sidak post hoc test; ***p < 0.001 in a Holm-Sidak post hoc test. L, left; O, opposite; R,

right; T, target.

68

3.6 Morris water maze reversal learning

Reversal learning is another set of experiment conducted one week after Morris

water maze, again requiring 4d of acquisition (4 trials per day) and 1d of testing (1 trial

per day). In this test however, the platform is relocated to the quadrant opposite of its

former location. This reversal learning reveals whether or not animals can extinguish

their previously learnt platform location and acquire a direct path to the new location

(Faivre et al., 2012; Vorhees & Williams, 2006). In order to re-learn the location of this

newly placed platform, cognitive flexibility needs to be present in these animals

(Rosczyk, Sparkman, & Johnson, 2008).

At 12 months of age, reversal learning experiments show no statistical difference

between genotypes in terms of the latency to find the escape platform in the 4d training

period, with all mice improved in the time it took to find the platform over training

(Fig.3.15A; F(3,105) = 1.936, p = 0.142). No statistical difference between genotypes were

observed in terms of distance required to find the platform (Fig.3.15B; F(3,105) = 2.891, p

= 0.059). When spatial memory was investigated on probe trial day 5, the time WT,

APPSWE/PSEN1∆E9, and TRPM2-KO-APPSWE/PSEN1∆E9 mice spent investigating

the target quadrant was significantly longer than all other quadrants (Fig.3.15C; F(3,105) =

39.134, p<0.001; post hoc analysis show p<0.05, p<0.01, and p<0.001). However,

TRPM2-KO mice did not spend more time investigating the target quadrant. Instead,

these mice spent similar amounts of time investigating all quadrants. This suggests that

TRPM2-KO mice show impaired reversal learning. The representative path traces

indicate that TRPM2-KO mice do not remember the position of the platform and their

swim paths are in a random pattern around the pool instead of only focusing on the target

69

quadrant. On the other hand, WT, APPSWE/PSEN1∆E9 and TRPM2-KO-

APPSWE/PSEN1∆E9 mice are spending more time in the target quadrant and thus are

learning, as well as remembering the locations of the platform (Fig.3.15D).

At 15 months of age, no statistical differences was observed between genotypes

in terms of the latency to find the escape platform in the 4d training period, and all mice

improved in the time it took to find the platform over training (Fig.3.16A; F(3,117) = 2.017,

p=0.127). The distance required for APPSWE/PSEN1∆E9 and TRPM2-KO-

APPSWE/PSEN1∆E9 mice to find the platform is significantly longer than controls

(Fig.3.16B; F(3,117) = 6.069, p < 0.01; post hoc analysis show p<0.05, p<0.01, and

p<0.001). When spatial memory was investigated on probe trial day 5, the time WT and

TRPM2-KO-APPSWE/PSEN1∆E9 mice spent investigating the target quadrant was

significantly longer than all other quadrants (Fig.3.16C; F(3,117) = 29.914, p<0.001; post

hoc analysis show p<0.05, p<0.01, and p<0.001). Both TRPM2-KO and

APPSWE/PSEN1∆E9 mice did not learn the location of the target quadrant since these

mice did not spend a significantly longer time investigating the target quadrant,

indicating that at this age both TRPM2-KO and APPSWE/PSEN1∆E9 mice show

impaired reversal learning. The representative path traces indicate that both TRPM2-KO

and APPSWE/PSEN1∆E9 mice show swim paths that are random around the pool

instead of only focusing on the target quadrant (Fig.3.16D).

70

Figure 3.15. MWM assessment of reversal learning at 12 months of age


mice were subject to reversal learning in MWM. Latency to find the platform on

acquisition days 1-4 (the average of four 90s trials per day) is plotted (B) Total distance

traveled to the platform on acquisition days 1-4 (the average of four 90s trials per day) is

plotted (C) Percentage of time spent in each quadrant was measured on day 5 in a 60s

1 2 3 40

20

40

60

Day

Es

ca

pe

L

ate

nc

y (

se

c)

1 2 3 40

5

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tal D

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m)


10

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50

60

70

Quadrant

*** ***

*,**

******

*,**

***

******%

Tim

e

A B

C D WT

TRPM2-KO

APPSWE/P

SEN1∆E9


71

probe trial with the platform removed (F(3,105) = 31.318, p < 0.001 in a two-way repeated

measures ANOVA). (D) Representative path traces on probe day 5 of reversal comparing

WT, TRPM2-KO, APPSWE/PSEN1∆E9, and TRPM2-KO-APPSWE/PSEN1∆E9 mice.

All data were plotted as Mean ± SEM. *p < 0.05 in a Holm-Sidak post hoc test; **p <

0.01 in a Holm-Sidak post hoc test; ***p < 0.001 in a Holm-Sidak post hoc test. L, left;

O, opposite; R, right; T, target.

72

Figure 3.16. MWM assessment of reversal learning at 15 months of age


mice were subject to reversal learning in MWM. Latency to find the platform on

acquisition days 1-4 (the average of four 90s trials per day) is plotted (B) Total distance

traveled to the platform on acquisition days 1-4 (the average of four 90s trials per day) is

1 2 3 40

10

20

30

40

50

Day

Es

ca

pe

L

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nc

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se

c)

1 2 3 40

5

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*,***

*,**

Day

To

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m)


10

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50

60

Quadrant

***

***

*,**

*,***

******

***

% T

ime

A B

C D


APPSWE/PS

EN1∆E9

TRPM2-KO

WT

73

plotted (F(3,117) = 6.069, p < 0.01 in a two-way repeated measures ANOVA) (C)

Percentage of time spent in each quadrant was measured on day 5 in a 60s probe trial

with the platform removed (F(3,117) = 11.375, p < 0.001 and F(3,117) = 29.914, p < 0.001 in

a two-way repeated measures ANOVA). (D) Representative path traces on probe day 5 of

reversal comparing WT, TRPM2-KO, APPSWE/PSEN1∆E9, and TRPM2-KO-

APPSWE/PSEN1∆E9 mice. All data were plotted as Mean ± SEM. *p < 0.05 in a Holm-

Sidak post hoc test; **p < 0.01 in a Holm-Sidak post hoc test; ***p < 0.001 in a Holm-

Sidak post hoc test. L, left; O, opposite; R, right; T, target.

74

Section 4

DISCUSSION

75

4.1 Summary of Key Findings

The main purpose of this thesis was to explore the relationship between TRPM2

channels and cognitive impairment observed in an Alzheimer’s mouse model. I tested the

hypothesis that genetic ablation of TRPM2 would prevent cognitive deficits in

APPSWE/PSEN1∆E9 transgenic mice. Behavioral analysis suggested that:

1) TRPM2 channels are important for reversal learning in aged mice, as TRPM2-KO

mice at 12 month and older showed impaired performance in the MWM reversal

learning task.

2) Genetic ablation of TRPM2 improves cognition in APPSWE/PSEN1∆E9

transgenic mice, as TRPM2-KO-APPSWE/PSEN1∆E9 mice showed improved

performance on spatial memory, measured by BM and MWM tests and reversal

learning.

4.2 Open field locomotor activity test

The primary reason of conducting an open field locomotor activity test was to

test whether mice were hyperactive, which is a common characteristic associated with

several neurodegenerative disease models (Arendash et al., 2001). It is expected that mice

will spend more time exploring their environment at the beginning because of the novel

environment; however they will gradually decrease their exploration with time, since they

will become familiar with the environment, otherwise known as habituation. This change

in exploratory tendency was observed in our study; WT, TRPM2-KO,

APPSWE/PSEN1∆E9 and TRPM2-KO-APPSWE/PSEN1∆E9 mice at all ages showed a

continued decrease in distance travelled during the two hour test period. Likewise,

76

Daenen et al. (2001) and Bolivar et al. (2000) also observed habituation in their cohort of

male APPSWE/PSEN1∆E9 mice.

Anxiety level in these rodents can also be recorded, by measuring the amount of

time spent in the center of the open field activity chamber. Mice have a natural tendency

to avoid open spaces by travelling along the walls of enclosures as a means of protection

and easy escape (Lalonde et al., 2004). Therefore, the amount of time individuals spent in

the center area of the open field test provides a measure of anxiety.

No difference between WT, TRPM2-KO, APPSWE/PSEN1∆E9, and TRPM2-

KO-APPSWE/PSEN1∆E9 mice was observed in terms of total distance travelled and

total time spent in the center of the chamber at 3, 6, and 9 months of age. However, after

12 months of age, while no difference was seen between control mice (WT and TRPM-

KO), APPSWE/PSEN1∆E9 and TRPM2-KO-APPSWE/PSEN1∆E9 mice showed

significantly decreased horizontal activity. These mice also spent less time in the center

of the chamber than controls. A number of studies in the literature have investigated

locomotor activity in APPSWE/PSEN1∆E9 transgenic mice. Consistent with our results,

no difference in locomotor activity was observed when APPSWE/PSEN1∆E9

transgenic mice were compared to controls at different age points, such as at 4 months

(Bonardi et al., 2011), 5-7 months (Lalonde et al., 2004), and 8 months (Park et al., 2010)

of age.

As mentioned above, no difference was seen between WT and TRPM2-KO mice

while hypoactivity was observed in APPSWE/PSEN1∆E9 and TRPM2-KO-

APPSWE/PSEN1∆E9 mice. Because hypoactivity was only observed in Alzheimer’s

mice and not in the control mice, regardless of the presence of TRPM2, we can conclude

77

that TRPM2 did not play a role in causing a decrease in horizontal activity in the

Alzheimer’s mice. The behaviour of TRPM2-KO-APPSWE/PSEN1∆E9 mice was not

improved when compared to the behaviour of APPSWE/PSEN1∆E9 mice, suggesting

that knocking out TRPM2 did not interfere with locomotor activity in mice.

4.3 Elevated plus maze

The elevated plus maze is used to measure anxiety responses of rodents, where

mice choose to either enter the open or closed arms. Mice were placed in the maze and

their activities were measured for 5 minutes. This length of recording is based on an early

study by Montgomery, (1955) who demonstrated that strongest avoidance behaviour for

the open arms was seen in rats in the first 5 minutes after placement. If mice were left in

the maze for a longer period of time, such as 10 or 15 minutes, these individuals would

no longer experience fear and avoidance toward the open arms since they would

habituate to the environment.

The anxiety levels of WT, TRPM2-KO, APPSWE/PSEN1∆E9 and TRPM2-KO-

APPSWE/PSEN1∆E9 mice did not differ at 3, 9, and 15 months of age. All mice

performed equally well when placed on the elevated plus maze in terms of number of

entries onto the open arm and the time spent on the open arms. To date, there have been

controversial results regarding whether anxiety occurs or are absent in the

APPSWE/PSEN1∆E9 transgenic mice. Consistent with these results, Arendash et al.

(2001) studied the Tg2576 transgenic mice and found no difference between transgenic

and control mice at 5-7 and 15-17 months of age in terms of performance in closed and

open arm entries as well as time spent in open arms. On the other hand, there have been

reports on APPSWE/PSEN1∆E9 mice which had results opposite of those found in this

78

current study. Lalonde et al. (2004) and Reiserer et al. (2007) found that 7 months old

transgenic mice display reduced anxiety compared with wild type controls as measured

by the elevated plus maze. Lalonde et al., (2004) also observed an increase in open arm

entries as well as more time spent in the open arms in 7-month-old transgenic mice,

suggesting that APPSWE/PSEN1∆E9 mice were less anxious than the control mice. The

reason why results from our study and previous literatures did not match could be due to

many factors such as temperature, noise levels, social interaction, the presence of

enrichment and diet, which can impact anxiety levels and influence outcomes of the

study. Light/dark cycle was found to be a significant factor in anxiety-like behaviour

measured with the elevated plus maze (Clénet et al., 2006; Gomes et al., 2011). These

scientists demonstrated that mice in the dark cycle spent more time in the open arm of the

elevated plus maze, thus less anxious than mice in the light cycles. Elevated plus maze in

our study was conducted when mice were in their light cycles which could be the reason

why we are not seeing results consistent with others. In addition, housing conditions of

mice has also been found to influence anxiety levels, as demonstrated by da Silva et al.

(1996) where mice were either housed individually or in groups. They observed that mice

housed individually had a reduced number of entries and spent less time in the open arms

compared to mice housed in groups, coming to the conclusion that mice housed

individually were more anxious than grouped mice, and vice versa. Although our mice

were housed in groups and therefore should be less anxious, we did not see this result in

mice at 3, 9, and 15 months of age.

At 6 months however, TRPM2-KO-APPSWE/PSEN1∆E9 mice spent

significantly longer time in the open arms than the control mice, suggesting that these

79

mice may be less anxious than WT, TRPM2-KO and APPSWE/PSEN1∆E9 mice. In

contrast, at 12 months of age, APPSWE/PSEN1∆E9 and TRPM2-KO-

APPSWE/PSEN1∆E9 mice visited the open arms significantly fewer times than control

mice, suggesting that these mice are more anxious than controls. Consistent with this,

Filali, Lalonde, and Rivest (2011) found that APPSWE/PSEN1∆E9 transgenic mice spent

more time in the closed arms and less time in the open arms and therefore were more

anxious than controls. Overall, our results suggest that APPSWE/PSEN1∆E9 and

TRPM2-KO-APPSWE/PSEN1∆E9 mice do not differ in anxiety levels, in terms of

hyperactivity when compared to control mice. Because there were no consistent changes

in anxiety in all mice, this suggests that elimination of TRPM2 does not affect anxiety

levels in the elevated plus maze.


The object recognition test, which is based on the ability of mice to discriminate

novel objects, was used to evaluate the performance of WT, TRPM2-KO,

APPSWE/PSEN1∆E9 , and TRPM2-KO-APPSWE/PSEN1∆E9 mice. Mice have a

natural tendency to explore a novel object, which can reflect the individual’s use of

discriminatory short- or long-term memory (Otalora et al., 2012; Prado et al., 2006).

Short-term memory would involve a test being given to the mice shortly (a few hours)

after training, whereas long-term memory involves a test being given 24 hours after

training. Our study tested the long-term memory of WT, TRPM2-KO,

APPSWE/PSEN1∆E9 and TRPM2-KO-APPSWE/PSEN1∆E9 mice.

80

Our results demonstrated no genotype difference in terms of the percentage of

time spent exploring the familiar and novel objects. All mice preferred to explore the

novel object on testing day at both 9 and 12 months of age. A number of researchers have

studied the APPSWE/PSEN1∆E9 transgenic mouse model using this test and

controversial results have been found regarding the age at which discriminatory memory

deficits occur. Consistent with our results, Bonardi et al. (2011) and Jardanhazi-Kurutz et

al. (2010) found no memory deficit in the same APPSWE/PSEN1∆E9 transgenic mice at

earlier ages (4 and 6 months). However, findings from Donkin et al. (2010) and Yoshiike

et al. (2008) on the APPSWE/PSEN1∆E9 mice were contradictory to our results since

both cohorts of mice demonstrated a severe object recognition memory deficit in

APPSWE/PSEN1∆E9 mice at 9-13 months of age. Overall, our results suggest that

discriminatory memory of APPSWE/PSEN1∆E9 and TRPM2-KO-APPSWE/PSEN1∆E9

mice do not differ from control mice. Because all mice learnt well in this test, this

allowed me to demonstrate that knocking out TRPM2 does not affect mice’s

discriminatory memory in object recognition.

4.5 Barnes maze

One of the methods to assess spatial learning and memory in mice is with the

Barnes maze, consisting of an open, exposed platform elevated off the ground with an

escape box located somewhere along the perimeter of the platform.

At 3, 6, and 9 months of age, no cognitive impairment was present in

APPSWE/PSEN1∆E9 mice since all mice remembered the location of the escape box

with the guidance of visual cues surrounding the maze. At 12 months however, when

81

testing memory retention on day 5, APPSWE/PSEN1∆E9 mice visited the target hole

location significantly less than control indicating spatial memory deficit in these mice.

Day 12 of the probe trial also indicated a spatial memory deficit in APPSWE/PSEN1∆E9

mice. TRPM2-KO-APPSWE/PSEN1∆E9 mice had improved performance in the BM on

both test days 5 and 12. Therefore, elimination of TRPM2 alleviates spatial memory

deficit seen in mice expressing the APPSWE/PSEN1∆E9 transgene. In addition,

APPSWE/PSEN1∆E9 mice at 15 months of age also showed spatial memory deficit as

measured on probe day 5 and 12, as well as an improvement in performance when the

TRPM2 gene is eliminated. These results suggest that spatial memory deficits, as

measured by BM, only occur in aged APPSWE/PSEN1∆E9 mice, at 12 and 15 months of

age. Our data also support the notion that elimination of TRPM2 can reverse or prevent

these alterations in spatial memory.

APPSWE/PSEN1∆E9 transgenic mice have been shown to exhibit age-dependent

spatial learning and memory impairment in the Barnes maze. To date, there have been

controversial results regarding whether spatial memory deficit occurs at an earlier age or

a later age. For instance, Reiserer et al. (2007) has demonstrated that 6-7 months old

APPSWE/PSEN1∆E9 mice shows cognitive impairment in BM. However, O’Leary &

Brown, (2009) have shown results consistent with our current results; spatial memory

deficit only occurs in aged mice. O’Leary & Brown, (2009) assessed spatial memory of

APPSWE/PSEN1∆E9 transgenic mice at 16 months of age using the Barnes maze and

observed that transgenic mice took a longer time to locate the target hole than WT mice,

as well as made more errors during acquisition trials. The above observations therefore

82

demonstrate the presence of spatial learning and memory impairment in

APPSWE/PSEN1∆E9 mice as measured by Barnes maze.

4.6 Morris water maze hidden-platform test

Morris water maze (MWM) is a test used for spatial learning and memory

(Vorhees & Williams, 2006). Since mice have a natural fear of water, this test uses this

fear as a motivation for these individuals to find the hidden platform with the aid of

visual cues. Similarly to the Barnes maze, WT mice are expected to utilize spatial

learning and memory in order to locate the hidden platform. Because of the potential

spatial memory deficits we observed in the BM, we decided to determine if a second task

would allow us to confirm these results.

At 12 months of age, all mice learned and retained the memory of the location of

the target platform, as seen by the large amount of time spent in the target quadrant.

Opposite from our findings, Zhang et al. (2011) demonstrated the presence of spatial

memory deficit in the APPSWE/PSEN1∆E9 mice at 12 months of age.

At 15 months of age, APPSWE/PSEN1∆E9 and TRPM2-KO-

APPSWE/PSEN1∆E9 mice took a longer period of time to find the target location

(Fig.3.14A) as well as travelled a longer distance (Fig.3.14B) than control mice but they

learnt the test. It is interesting to note that, likely, WT and TRPM-KO mice remember

that they have done this experiment multiple times and already in the first day of training

they promptly find the platform, while APPSWE/PSEN1∆E9 and TRPM2-KO-

APPSWE/PSEN1∆E9 mice behave as if they were doing the test for the first time.

However, we observe a spatial memory deficit in APPSWE/PSEN1∆E9 mice, since these

83

mice do not remember the location of the target platform on the probe trial. TRPM2-KO-

APPSWE/PSEN1∆E9 mice were found to have improved performance in this test since

they remember the location of the target platform and therefore spent more time in that

quadrant. This demonstrates that knocking out the expression of TRPM2 led to an

improved cognitive function in APPSWE/PSEN1∆E9 mice.

APPSWE/PSEN1∆E9 transgenic mice have been shown to exhibit age-dependent

spatial learning and memory impairment in the MWM. To date, just like BM, there have

been controversial results regarding whether spatial memory deficit occurs at an earlier

age or a later age in the MWM. Some studies have observed a lack of spatial memory

impairment in APPSWE/PSEN1∆E9 mice at 6 months (Holcomb et al., 1999;

Minkeviciene et al., 2008; Savonenko et al., 2005) and 9 months (Holcomb et al., 1999)

of age, while memory deficit have been found to present itself at 8-12 (Butovsky et al.,

2006; Cao et al., 2007; Lalonde et al., 2004; Minkeviciene et al., 2008; Oksman et al.,

2006), 12-13 months of age (Malm et al., 2011; Zhang et al., 2011), 14 months of age

(Liu et al., 2003), and 16-18 months of age (Malm et al., 2007; Minkeviciene et al., 2008;

Savonenko et al., 2005). The results from our study are consistent with numerous reports

that indicate APPSWE/PSEN1∆E9 mice show spatial learning and memory deficits at 12

and 15 months of age. Overall, our results demonstrated that elimination of TRPM2

allows for improved performance compared to APPSWE/PSEN1∆E9 mice in MWM.

4.7 Morris water maze reversal learning

MWM reversal, or reversal learning, tests an animal’s ability to extinguish their

memory of the previously learnt platform position and essentially to “re-learn” the new

84

platform position (Vorhees & Williams, 2010). This test was conducted a week after the

original MWM, using the same materials but a slight modification to the methodology,

with the location of the hidden platform now being placed in the opposite quadrant of

where it used to be. In order to re-learn the location of this newly placed platform,

cognitive flexibility needs to be present in these animals.

An unexpected result arose when observing MWM reversal learning. At 12

months of age, cognitive flexibility was present in WT, APPSWE/PSEN1∆E9 and

TRPM2-KO-APPSWE/PSEN1∆E9 mice since they found the new location of the hidden

platform quickly and accurately. However, TRPM2-KO mice did not find the new hidden

platform location and therefore presented a deficit in cognitive flexibility. At 15 months

of age, WT and TRPM2-KO-APPSWE/PSEN1∆E9 mice were observed to remember

the new location of the platform while TRPM2-KO and APPSWE/PSEN1∆E9 mice did

not; indicating a deficit in cognitive flexibility in these mice as well as a possible

impairment in LTP in the APPSWE/PSEN1∆E9 mice since these mice had trouble

remembering the new location of the platform. The fact that we observe cognitive

flexibility deficits in mice with elimination of TRPM2 (TRPM2-KO) at 12 months of age

but no deficits in Alzheimer’s mice, were interesting yet puzzling. Moreover, 15 months

results showed that mice with elimination of TRPM2 (TRPM-KO) as well as

APPSWE/PSEN1∆E9 mice had a deficit in cognitive flexibility, again suggesting a

possible LTP impairment in these mice. However, TRPM2-KO-APPSWE/PSEN1∆E9

mice had no deficit in cognitive flexibility at 12 and 15 months of age, instead they had

normal performance at both age points, suggesting that at both time points, the

elimination of TRPM2 channels may be beneficial and may rescue the LTP impairment.

85

There is no explanation for our results at this time; future in vivo research should further

investigate TRPM2-KO mice and reversal learning as well as the role that TRPM2 has in

these behaviour tests. For example, because there are numerous mouse models of AD as

noted in the introduction, longitudinal research could be conducted in those mouse

models to investigate whether or not knocking out TRPM2 played a role in performance.

TRPM2-KO mice and reversal learning can also be further investigated using other

mouse models of AD to see if the same results are obtained compared to the unexpected

results we saw in reversal learning. These results observed in the MWM reversal tests in

our study could be correlated to an in vitro experiment conducted by Xie et al., (2011),

described below.

TRPM2 channels are highly expressed in the CNS and have previously been

found to be expressed in the CA1 pyramidal neurons in the hippocampus (Olah et al.,

2009). Based on this finding, an in vitro study by Xie et al., (2011) used hippocampal

cultures and slices from TRPM2 knockout mice and demonstrated that a loss of TRPM2

channels selectively impairs NMDAR-dependent LTD while sparing LTP. LTD is an

activity-dependent reduction in the efficacy of excitatory synaptic transmission while

LTP is the opposite; an enhancement in the efficacy of transmission. In order to further

explore the loss of NMDAR-dependent LTD in TRPM2 knockout mice, these scientists

examined the activity of GSK-3β (glycogen synthase kinase-3β), which has been widely

implicated in AD and is required in the formation of this LTD (Jo et al., 2011). Genetic

deletion of TRPM2 was to be associated with decreased GSK-3β activity. However, it

was noted that a rescue of LTD was observed in TRPM2 knockout mice when GSK-3β

86

activity was restored. Therefore, TRPM2 channels have been demonstrated to play a key

role in hippocampal synaptic plasticity.

Recall that Aβ plaques are the main pathological hallmark of AD (Cvetkovic-

dozic et al., 2001; Yankner, Lu, & Loerch, 2008) and that Aβ40 and Aβ42 are the two

most common species of Aβ (Oddo et al., 2003; Selkoe, 2008). An increased production

of oligomeric Aβ42 is one of the triggers of FAD (Savonenko et al., 2005), resulting in

the accumulation of large senile plaque in the hippocampus, cortex and subcortical nuclei

(Duyckaerts et al., 2008). Oligomeric Aβ42 has been shown by numerous studies to

inhibit LTP, important in memory formation (Shankar et al., 2008; Walsh et al., 2002) as

well as increase LTD, contributing in memory loss (Hsieh et al., 2006; Li et al., 2009;

Shankar et al., 2008).

Given that the loss of TRPM2 expression leads to deficits in LTD induction, an

increase in TRPM2 channel function, for example as a result of oxidative stress, may

favor LTD. Accordingly, we propose that the increased LTD associated with AD is

partially contributed by augmented TRPM2 function, via Aβ42 initiated oxidative stress.

And therefore, it can be concluded that a loss of TRPM2 channel activity may play a

beneficial role in learning and memory in AD.

4.8 Future directions

As noted in the introduction, soluble oligomers have been found by numerous

studies to mediate neurotoxicity by inducing oxidative stress in the brain which promote

TRPM2 activation, leading to a disruption in Ca2+

homeostasis and a toxic influx of

Ca2+

, ultimately inducing cell death (Goedert & Spillantini, 2006; Mattson, 2007;

87

Takahashi et al., 2011). An improvement in memory in the TRPM2-KO-

APPSWE/PSEN1∆E9 mice has been observed in our study and therefore knocking out

TRPM2 has been found to be protective. Cognition was found to be mildly impaired in

TRPM2-KO mice, as shown by impaired reversal learning. Future in vitro research could

explore Aβ load in these TRPM2-KO individuals and see whether or not the load has

decreased in these individuals since there was an improvement in behaviour. Presently,

there are no specific antagonists of TRPM2. However, a number of pharmacological

agents are able to inhibit the channel, although only non-selectively. These agents include

flufenamic acid, econazole, clotrimazole, and N-(p-amycinnamoyl) (Eisfeld & Luckhoff,

2007; Olah et al., 2009). Thus, these potential TRPM2 antagonists should also be

investigated in order to examine whether TRPM2 represents a target for the treatment of

AD. Given our findings, we predict that a TRPM2 antagonist could potentially be

effective in treating AD.

My study focuses on mice with TRPM2 absent from birth, which is different than

humans who develop AD with age. To test the effectiveness of a TRPM2 blocker in

patients already suffering the symptoms, longitudinal studies in which treatment is

initiated at various time points (e.g. 3, 6, 9 months etc.) should be performed on mouse

models of AD. In addition, biomarkers of AD may soon allow us to identify humans at

risk of developing AD; treatment in these patients with a TRPM2 blocker could be

considered.

88

4.9 Overall conclusions

Although the precise mechanism through which Aβ causes AD pathogenesis is

not known at present, this mechanism can be speculated taking into consideration the

large amount of research on this topic. To date, it is known that the accumulation of Aβ is

the main pathological hallmarks of AD, and that Aβ induces oxidative stress and a loss of

Ca2+

homeostasis in the brain. According to the amyloid cascade hypothesis, the

accumulation of soluble Aβ peptides causes progressive synaptic and neuritic injury by

reducing glutamatergic synaptic transmission strength and plasticity (Hardy, & Selkoe,

2002; Palop, & Mucke, 2010). Similarly, a loss of Ca2+

homeostasis is known to disrupt

the structure and function of synapses, impair synaptic plasticity, and ultimately cause

cell death. Although the precise mechanisms through which oxidative stress causes a loss

of Ca2+

homeostasis is not known, we do know that oxidative stress promotes the

activation of TRPM2 channels and that this activation causes a toxic influx of Ca2+

and

ultimately induce cell death (Takahashi et al., 2011). Collectively, these findings led us to

consider TRPM2 channels as a likely contributor to the neurotoxic cascades initiated by

elevated Aβ levels.

This thesis was the first to explore the relationship between TRPM2 and cognitive

deficits associated with a well characterized mouse model of Alzheimer’s disease. Our

results suggest that knocking out the TRPM2 transgene improves the spatial learning and

memory abilities of the APPSWE/PSEN1∆E9 double transgenic mice, suggesting that

TRPM2 might represent a potential target for the treatment of AD.

89

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Curriculum Vitae

EDUCATION

Master of Science (Physiology) 2011-2013 Western University, London, Canada

Bachelor of Science (Honours Specialization Biology) 2006-2011 Western University, London, Canada

LABORATORY AND RESEARCH EXPERIENCE

Master’s Thesis Research – Dr. John MacDonald’s Laboratory 2011-2013

Department of Physiology, Robarts Research Institute, Western University Thesis: Contributions of TRPM2 to memory deficiency in an Alzheimer’s mouse model

Summer Student Research Assistant – Dr. John MacDonald’s Laboratory 2011

Department of Physiology, Robarts Research Institute, Western University

Investigated the role of TRPM2 on the life span and memory deficit of Alzheimer’s mice

by conducting behaviour experiments using elevated plus maze, object recognition,

locomotor, Barnes maze and Morris water maze.

Summer Student Research Assistant – Dr. Subrata Chakrabarti’s Laboratory

Department of Pathology, Western University 2004, 2007-2009 Investigated the pathogenic mechanisms and treatment of heart failure in diabetes

patients using RT-PCR, Western blot and ELISA techniques; assisted other

research team members with their experiments

TEACHING EXPERIENCE Western University Graduate Teaching Assistantship, London, Canada

Physiology 3130Y – 3rd

year undergraduate lab course 2011-2013 Assisting professors with the teaching of course materials; assisting students with

various experiments; evaluating lab reports and presentations; proctoring exams

PUBLICATIONS AND PRESENTATIONS

Journal articles: Coauthored three articles in the following journals:

• Investigative Ophthalmology & Visual Science 53(13): 1-11 2012

• Journal of Diabetes Investigation 2(2): 123–131 2011

• American Journal of Physiology –

Endocrinology and Metabolism 298: E127-E137 2009

Presentations (with Abstracts):

Participated in group presentations at the following events and venues:

• 38th J.A.F. Stevenson Memorial Lecture at the Department of Physiology and

Pharmacology Research Day, London, Canada, November 6, 2012 2012

• Canadian Association for Neuroscience, Vancouver, Canada, May 20-23, 2012 2012

• Annual Meeting of the Southern Ontario Neuroscience Association, Toronto,

Canada, April 30, 2012 2012

• Canadian Diabetes Association, Toronto, Canada, October 26-29, 2011 2011

• American Transplant Congress, Toronto, Canada, May 30-June 4, 2008 2008

104

VOLUNTEER AND EXTRACURRICULAR ACTIVITIES Alzheimer’s Society, London, Canada

Volunteer Companion 2012-2013 Providing relief for caregivers by offering companionship and social stimulation for

persons in the early stages of Alzheimer’s disease and related dementias

Participation House, London, Canada

Friendship/Buddy Volunteer 2012-2013 Assisting physically and mentally disabled individuals with handcrafts, gardening,

walks, swimming, yoga, manicures, music, and sensory room activities

Western Serves, Western University, London, Canada

Participation House Volunteer 2012 Assisted at the annual cookout, bonfire, and games; provided support in preparing for and

running games; assisted individuals with developmental disabilities and complex physical

needs in engaging in conversation and participating in games

Indian Cultural Connection Camp, London, Canada

Camp Counselor 2012

Assisted in organizing a number of activities during camp week; supervised and assisted

children in doing various activities such as sports and crafts

Chinese Students Association, Western University, London, Canada

Executive and Director in the Cultural and Academics Department 2007-2009 Provided information about Asian culture to the general public; organized review

sessions to help students with exam preparation; organized various club activities

University Hospital, London Health Sciences Center, London, Canada

Outpatient Clinic and Dietary Department 2007-2009

Recorded patient data, organized charts, and kept track of patients for the Blood Taking

Clinic; assisted patients in making meal choices, retrieved menus from patients, and

delivered cards and flowers to patients while working in the Dietary Department

AWARDS AND ACHIEVEMENTS

• Western University Graduate Student Teaching Award Nominee 2012

• Western Graduate Research Scholarships,

Western University, London, Canada 2011-2013

• Dean’s Honour List, Western University, London, Canada 2010

• Orchard Park Public School ESL Award, London, Canada 2002

SKILLS

• Language: Fluent in English and Mandarin (Chinese)

• Computer: Proficient in word processing and the use of quantitative data analysis

software; conversant with the entire range of Internet functions and applications

• Interpersonal: excellent verbal and written communication skills; social skills; leadership

skills; problem-solving and conflict resolution

Contribution of TRPM2 to Memory Loss in an Alzheimer's ...

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