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Investigating the role of Alzheimer's disease-associated presenilin mutations in olfactory deficits Parvand, Mahraz 2018

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INVESTIGATING THE ROLE OF ALZHEIMER’S DISEASE-ASSOCIATED PRESENILIN MUTATIONS IN OLFACTORY DEFICITS  by  Mahraz Parvand  BSc., The University of British Columbia, 2016  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA Vancouver  July 2018  © Mahraz Parvand, 2018                                                                                                                                          ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled:  Investigating the role of Alzheimer’s disease-associated presenilin mutations in olfactory deficits  submitted by Mahraz Parvand in partial fulfillment of the requirements for the degree of Master of Science  in Experimental Medicine   Examining Committee: Dr. Catharine Rankin Supervisor  Dr. Weihong Song Supervisory Committee Member  Dr. Haakon Nygaard Supervisory Committee Member Dr. Neil Cashman  Additional Examiner   Additional Supervisory Committee Members:  Supervisory Committee Member  Supervisory Committee Member                                                                                                                                            iii  Abstract Although olfactory dysfunction is one of the hallmark symptoms in Alzheimer’s disease (AD) prior to the onset of cognitive impairments, little is known about the causes of this dysfunction. The nematode Caenorhabditis elegans is an ideal model for system-level genetic understanding of sensory neural circuits and behavior. Many cases of familial AD are linked to mutations of the presenilin (PS) genes. These genes are homologues with sel-12 genes in C. elegans. The purpose of this project was to examine the association between PS1 and olfactory deficits in order to investigate the cellular mechanism of these dementia-linked deficits. To gain a better understanding of the relationship between presenilin 1 (PS1) mutations in AD and olfactory deficits, chemotaxis experiments (with the attractant diacetyl, and the aversive octanol) were conducted on worms with a mutation in sel-12. I found that adult sel-12 mutant worms had a significantly decreased sensitivity to both odorants compared to wild-type worms. Extrachromosomal array expression of human wild-type PS1 into C. elegans rescued olfactory defects, confirming functional homology between the C. elegans and human gene.  However, a PS1 mutant from an Alzheimer’s family was unable to rescue olfactory deficits. Moreover, C. elegans sel-12 mutant worms presented olfactory deficits throughout their lifespan, and the deficit increased with age, similar to the neurodegenerative progression of AD. Based on these data, I concluded that a mutation in the C. elegans homologue of PS1 is associated with decreased olfactory function, and this deficit was rescued by wild-type human PS1 gene. I suggest that altered functioning of the Notch pathway may be involved in these chemosensory deficits. Additionally, to localize the neuron(s) where wild-type sel-12 function is required for normal olfaction, sel-12 and PS1 rescues were conducted in specific sensory neurons, namely the ASH and the AWA neurons responsible for detecting octanol and diacetyl, respectively. Further, an examination of                                                                                                                                          iv  the morphology of the ASH neurons showed increased neurodegeneration over time in sel-12 mutant worms, demonstrating an association with the observed behavioral deficits.                                                                                                                                                               v  Lay Summary Currently, the underlying causes of Alzheimer’s disease (AD) are unknown, and despite its growing prevalence, no effective treatments exist. In this thesis, I have studied AD from a novel perspective. Olfactory deficits are one of the primary symptoms of AD that tend to appear prior to cognitive deficits. However, the cause of these deficits is unknown. Most cases of familial Alzheimer’s disease are linked to mutations in the presenilin 1 gene. This gene is similar to the sel-12 gene in Caenorhabditis elegans, a transparent roundworm. Chemotaxis experiments were conducted on worms with a mutation in sel-12 to gain a better understanding of the relationship between presenilin 1 and olfactory deficits. Worms with sel-12 mutations showed chemotaxis deficits as well as degeneration in their sensory neurons. Understanding the role of presenilin 1 in these deficits may lead to opportunities for new therapeutic targets.                                                                                                                                                        vi  Preface This thesis was conducted in Dr. Catharine Rankin’s lab at the Djavad Mowafaghian Centre for Brain Health at UBC Hospital. I was responsible for the identification and design of the research project, conducting the experiments, and the analysis of research data. Under my supervision, some of the data included were collected by an undergraduate research assistant (Born, D).                                                                                                                                                            vii  Table of Contents  Abstract  ........................................................................................................................................ iii Lay Summary  ................................................................................................................................v Preface  .......................................................................................................................................... vi Table of Contents  ....................................................................................................................... vii List of Tables  ............................................................................................................................... xi List of Figures  ............................................................................................................................. xii List of Abbreviations  ................................................................................................................ xvi Acknowledgements  ................................................................................................................... xix Dedication .....................................................................................................................................xx Chapter 1: Introduction ................................................................................................................1  1.1 Alzheimer’s Disease and Related Dementias  ...............................................................1  1.2 Amyloid-beta Precursor Protein (APP) and Presenilin  .................................................4   1.2.1 The role of Amyloid-beta Precursor Protein (APP)  .......................................4   1.2.2 Alzheimer’s Disease and APP Processing  .....................................................5   1.2.3 The g-Secretase Complex ................................................................................8   1.2.4 Presenilin  ........................................................................................................9   1.2.5 The Amyloid Hypothesis  .............................................................................12  1.3 Notch Signaling and Alzheimer’s Disease  .................................................................14 1.4 Olfactory Deficits in Alzheimer’s Disease  .................................................................15  1.4.1 The Olfactory System  ..................................................................................15  1.4.2 Alzheimer’s Disease and Olfactory Impairments  ........................................17 1.5 Use of Caenorhabditis elegans (C. elegans) to Understand Disease Mechanisms  ....23                                                                                                                                          viii   1.5.1 C. elegans as a Genetic Tool to Study Human Disease  ...............................23  1.5.2 Use of C. elegans in Studying AD  ...............................................................24  1.5.3 Alzheimer’s Disease-Associated Genes in C. elegans .................................25  1.5.4 C. elegans Olfactory System  ........................................................................26 1.6 Project Objectives  .......................................................................................................27 Chapter 2: sel-12 mutant C. elegans display olfactory impairments from hatch and these impairments increase over time  .................................................................................................29  2.1 Introduction ..................................................................................................................29  2.2 Methods .......................................................................................................................34   2.2.1 Generation of transgenic lines and strain maintenance  ................................34   2.2.2 Chemotaxis Assay  ........................................................................................35   2.2.3 Multi-worm Tracker (MWT)  .......................................................................37   2.2.4 Statistical Analysis  .......................................................................................38  2.3 Results  .........................................................................................................................39 2.3.1 Worms with a sel-12 mutation had more severe chemotaxis deficits compared to wild-type worms than hop-1 mutant worms ......................................................39 2.3.2 sel-12 mutant worms had chemotaxis deficits from hatching that increased with age ..................................................................................................................40 2.3.3 Locomotion speed was not a confounding variable in sel-12 mutant chemotaxis  ............................................................................................................43 2.3.4 Both the chemotaxis deficit and the slow speed could be rescued at all ages by expression of PS1 driven by the sel-12 promoter .............................................45  2.4 Discussion ....................................................................................................................49                                                                                                                                          ix  Chapter 3: Cell-specific expression of wild-type PS1 and PS1Δs169, but not PS1C410Y, rescued chemotaxis impairments in sel-12 mutant worms  ....................................................................50  3.1 Introduction ..................................................................................................................50  3.2 Methods .......................................................................................................................56   3.2.1 Generation of transgenic lines and strain maintenance  ................................56   3.2.2 Chemotaxis Assay  ........................................................................................58   3.2.3 Statistical Analysis  .......................................................................................58   3.2.3 Mutations of both Notch receptors via heat shock and developmental  stage synchronization .............................................................................................58  3.3 Results  .........................................................................................................................59   3.3.1 Nervous system expression of wildtype sel-12, wildtype PS1, and PS1Δs169,  but not PS1C410Y, rescued chemotaxis deficits in sel-12 mutant worms .................59 3.3.2 The chemotaxis deficit was independent of any motor deficit due to the sel-12 egl phenotype ....................................................................................................64 3.3.3 ASH neuron expression of wildtype sel-12, wildtype PS1, and PS1Δs169,  but not PS1C410Y, rescued chemotaxis deficits in sel-12 mutant worms  ................68 3.3.4 Wildtype PS1 rescue lines in the ASH neuron had a cell-specific effect on chemotaxis .............................................................................................................72 3.3.5 C. elegans with mutations in Notch receptors do not show increased chemotaxis deficits over time ................................................................................75  3.4 Discussion ....................................................................................................................78 Chapter 4: sel-12 mutant C. elegans demonstrate ASH neuron morphological abnormalities that are rescued by wildtype PS1 and PS1Δs169, but not PS1C410Y ............................................82                                                                                                                                          x   4.1 Introduction ..................................................................................................................82  4.2 Methods........................................................................................................................84   4.2.1 Generation of transgenic lines and strain maintenance .................................84   4.2.2 ASH neuron imaging ....................................................................................85   4.2.3 C. elegans Dye-filling staining assay ............................................................87   4.2.4 Statistical Analysis ....................................................................................... 88  4.3 Results ..........................................................................................................................88 4.3.1 Strains in which ASH expressed extrachromosomal wildtype PS1 rescues had normal octanol chemotaxis ....................................................................................88 4.3.2 sel-12 mutant worms had abnormal ASH neurons which were rescued by wildtype Psra6::PS1 and Psra-6::PS1Δs169, but not Psra-6::PS1C410Y ..................90  4.4 Discussion ....................................................................................................................95 Chapter 5: General discussion ....................................................................................................98   5.1 Conclusion  ..................................................................................................................99  5.2 Thesis limitations .......................................................................................................100  5.3 Future directions ........................................................................................................101 Bibliography ...............................................................................................................................104 Appendices ..................................................................................................................................133  Appendix A  .....................................................................................................................133   A.1 sel-12 and hop-1 mutant worms’ chemotaxis in response to diacetyl ..........133   A.2 Nervous system rescues in response to diacetyl ...........................................134   A.3 Neuron specific rescues in response to diacetyl  ...........................................135                                                                                                                                           xi  List of Tables  Table 1.1 Alzheimer’s disease-associated genetic risk factors ........................................................4 Table 1.2 Recent articles assessing Alzheimer’s disease-related olfactory deficits ......................21 Table 3.1 Orthologues of the core components of the LIN-12/Notch pathway among C. elegans and mammals .................................................................................................................................53 Table 4.1 ASH neuron morphology chi-square test results  ..........................................................90                                                                                                                                                           xii  List of Figures  Figure 1.1 APP trafficking in neurons .............................................................................................6 Figure 1.2 Sequential cleavage of the amyloid precursor protein (APP) occurs by two pathways .7 Figure 1.3 The four γ-secretase complex subunits ...........................................................................9 Figure 1.4 Presenilin 1 structure ....................................................................................................11 Figure 1.5 Alzheimer’s disease amyloid cascade hypothesis ........................................................13 Figure 1.6 Olfactory functioning ...................................................................................................16 Figure 1.7 Structure of chemosensory organs ................................................................................27 Figure 2.1 Inferred Topology of SEL-12 .......................................................................................31 Figure 2.2 sel-12 mutant C. elegans chemotaxis assay .................................................................32 Figure 2.3 C. elegans life cycle .....................................................................................................33 Figure 2.4 The chemotaxis assay layout ........................................................................................37 Figure 2.5 The Multi-Worm Tracker .............................................................................................38 Figure 2.6 Chemotaxis assay in sel-12 mutant, hop-1 mutant, and sel-12/hop-1 double mutant worms .............................................................................................................................................40 Figure 2.7 Chemotaxis assay in wild-type and sel-12 mutant worms from L1-adult stage ...........41 Figure 2.8 Chemotaxis assay in wild-type and sel-12 mutant worms from 78-108 hours old ......42 Figure 2.9 Average forward movement speed of 68-108 hour old wild-type and sel-12 mutant worms .............................................................................................................................................44 Figure 2.10 78 hour old wild-type and sel-12 mutant worm tracks on a plate over 250 seconds .44 Figure 2.11 Ubiquitous expression of wild-type presenilin 1 (PS1) in worms with a sel-12 mutation chemotaxis assay over time ............................................................................................46                                                                                                                                          xiii  Figure 2.12 Average forward movement speed groups of 88, 98, and 108 hour old wild-type, sel-12 mutant, and ubiquitous PS1 rescue worms during 250 seconds of tracking .............................47 Figure 2.13 88, 98, and 108 hour old wild-type (A), sel-12 mutant (B), and ubiquitous PS1 rescue (C) worm tracks on a plate over 250 seconds .....................................................................48 Figure 3.1 Expression of Psel-12::PS1 in sel-12 mutant Caenorhabditis elegans chemotaxis assay ...............................................................................................................................................53 Figure 3.2 Expression of Psel-12::PS1C410Y in sel-12 mutant Caenorhabditis elegans chemotaxis assay  ..............................................................................................................................................54 Figure 3.3 Expression of Psel-12::PS1Δs169  in sel-12 mutant Caenorhabditis elegans chemotaxis assay  ..............................................................................................................................................55 Figure 3.4 Expression of Ptag-168::sel-12 chemotaxis assay .......................................................62 Figure 3.5 Expression of Ptag-168::PS1 chemotaxis assay  .........................................................62 Figure 3.6 Expression of Ptag::PS1C410Y chemotaxis assay  .........................................................63 Figure 3.7 Expression of Ptag::PS1Δs169 chemotaxis assay  ..........................................................63 Figure 3.8 Nervous system expression of wild-type PS1 in worms with a sel-12 mutation chemotaxis assay over time ...........................................................................................................65 Figure 3.9 Average forward movement speed groups of 88-108 hour old wild-type, sel-12 mutant, and nervous system PS1 rescues during 250 seconds of tracking ....................................66 Figure 3.10 88-108 hour old wild-type (A), sel-12 mutant (B), and nervous system PS1 rescue (C) worm tracks on a plate over 250 seconds ................................................................................67  Figure 3.11 Expression of Psra-6::sel-12 in sel-12 mutant Caenorhabditis elegans chemotaxis assay ...............................................................................................................................................70 Figure 3.12 Expression of Psra-6::PS1 in sel-12 mutant Caenorhabditis elegans chemotaxis assay ...............................................................................................................................................70 Figure 3.13 Expression of Psra-6::PS1C410Y  in sel-12 mutant Caenorhabditis elegans chemotaxis assay ...............................................................................................................................................71                                                                                                                                          xiv  Figure 3.14 Expression of Psra-6::PS1 Δs169  in sel-12 mutant Caenorhabditis elegans chemotaxis assay ...............................................................................................................................................71 Figure 3.15 Expression of Pod-10::PS1 in sel-12 mutant Caenorhabditis elegans chemotaxis assay ...............................................................................................................................................73 Figure 3.16 Expression of Podr-10::PS1 in sel-12 mutant Caenorhabditis elegans chemotaxis assay ...............................................................................................................................................74 Figure 3.17 Expression of Psra-6::PS1 in sel-12 mutant Caenorhabditis elegans chemotaxis assay ...............................................................................................................................................74 Figure 3.18. RNAi knockdown of glp-1 in a lin-12 null background chemotaxis assay ...............76 Figure 3.19 Chemotaxis experiments on worms with RNAi knockdown of glp-1 in a lin-12 null background .....................................................................................................................................77 Figure 3.20 Amino acid conservation of PS1Δs169 and PS1C410Y between C. elegans’ sel-12 and human PS1 .....................................................................................................................................81 Figure 4.1 ASH sensory neuron morphology in worms ................................................................86 Figure 4.2 ASH sensory neuron morphology categories ...............................................................87 Figure 4.3 Psra-6::PS1 octanol chemotaxis assay ........................................................................89 Figure 4.4 Psra-6::PS1 diacetyl chemotaxis assay ........................................................................89 Figure 4.5 ASH neuron morphology of five strains (n=100 per strain per time point) over time (78, 98, and 108 hour old worms)  .................................................................................................92 Figure 4.6 Neurodegeneration in the wild-type worms’ ASH neurons .........................................93 Figure 4.7 Neurodegeneration in the sel-12 mutant worms’ ASH neurons ...................................93 Figure 4.8 DiI dye fill (in red) and GFP (in green) used for ASH neurons’ confocal imaging .....94 Figure A.1 Chemotaxis assay in sel-12 mutant, hop-1 mutant, and sel-12/hop-1 double mutant worms ...........................................................................................................................................133 Figure A.2 Pan neuronal expression of wild-type sel-12 chemotaxis assay ................................134                                                                                                                                          xv  Figure A.3 Pan neuronal expression of wild-type PS1 chemotaxis assay ...................................134 Figure A.4 Pan neuronal expression of PS1C410Y chemotaxis assay ............................................135 Figure A.5 Pan neuronal expression of PS1Δs169 chemotaxis assay .............................................135 Figure A.6 Wild-type sel-12 expressed under an AWA-specific promoter (odr-10) chemotaxis assay .............................................................................................................................................136 Figure A.7 Wild-type PS1 expressed under an AWA-specific promoter (odr-10) chemotaxis assay .............................................................................................................................................137 Figure A.8 PS1C410Y expressed under an AWA-specific promoter (odr-10) chemotaxis assay ...138 Figure A.9 PS1Δs169 expressed under an AWA-specific promoter (odr-10) chemotaxis assay ...138                                                                                                                                                       xvi  List of Abbreviations AD: Alzheimer’s disease  ADAM 10: a disintegrin and metalloproteinase domain-containing protein 10 AICD: amyloid precursor protein intracellular domain Aph1: anterior pharynx defective 1  APLP1: amyloid precursor like protein 1 APLP2: amyloid precursor like protein 2  APOE: apolipoprotein E APP: amyloid precursor protein Aß: amyloid beta  BACE1: beta secretase 1  C. elegans: Caenorhabditis elegans  CGC: Caenorhabditis Genetics Center ChR2: channelrhodopsin CI: chemotaxis index  CNS: central nervous system  CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats CSF: cerebrospinal fluid  CTF: C-terminal fragment  CVD: cerebrovascular disease  DCC: deleted in colorectal carcinoma DR6: death receptor 6 DSL: Delta, Serrate, or Lag2                                                                                                                                          xvii  E. coli: Escherichia coli ER: endoplasmic reticulum  FAD: familial Alzheimer’s disease  FUdR: 5’-fluorodeoxyuridine GPCR: G protein-coupled receptor HNR: Heinz Nixdorf Recall LDLR: low density lipoprotein receptors  LRP:  lipoprotein-related protein-1  MEMRI: manganese-enhanced magnetic resonance imaging miRNAs: micro RNA MMSE: Mini-mental state examinations MRI: magnetic resonance imaging  MWT: multi-worm tracker  NGM: nematode growth medium NICD: Notch intracellular ectodomain NTF: N-terminal fragment  NTF: neurofibrillary tangles  OB: olfactory bulb OE: olfactory epithelium  OR: olfactory receptors  ORN: olfactory receptor neurons  Pen-2: presenilin enhancer 2  PET: positron emission tomography                                                                                                                                          xviii  PS1: presenilin 1 PS2: presenilin 2  ptl-1: protein with tau-like repeats RNAi: RNA interference  sAPPb: soluble APP extracellular domain SEM: standard error of the mean  SNB-1: synaptobrevin TGN: trans Golgi network  TRPV: transient receptor potential vanilloid UPSIT: university of Pennsylvania smell identification test  US: United States                                                                                                                                            xix  Acknowledgements I would like to express my sincere gratitude to my supervisor, Dr. Catharine Rankin, for providing me with an amazing learning opportunity by accepting me to become a part of her research team. I would like to thank Dr. Rankin for her patience in responding to my many questions, motivation, and her openness to share her immense knowledge. Her guidance has not only helped me become a better scientist, but has opened my eyes in recognizing the value of creativity and perseverance in all of my endeavors. Besides my supervisor, I would like to thank the rest of my thesis committee:  Dr. Weihong Song, Dr. Haakon Nygaard, and Dr. Neil Cashman, for their continuous support and insightful feedback throughout this journey.  I would also like to acknowledge our amazing lab manager, Alvaro Luna, for his hard work in teaching me a variety of techniques and for his kind words and positive attitude. I would further like to thank my lab-mates, Troy, Joseph, Aram, and Alex for the stimulating discussions and advice, and for creating a collaborative environment in the lab. Moreover, my sincere thanks goes to the Canadian Federation of University Women for providing me with the Ecole Polytechnique Commemorative Award.  Last but not least, this journey would not have been possible without the support of my parents and friends. Thank you for encouraging me to pursuit what inspired me the most in life and for coping with my busy schedule during these last couple of months.                                                                                                                                              xx   To my parents, whose courage instilled in me the value of perseverance and self-development.                                                                                                                                            1  Chapter 1: Introduction 1.1 Alzheimer’s Disease and Related Dementias  There are a variety of disorders that lead to impaired memory and cognition among older adults, of which Alzheimer’s disease (AD) is the most common cause, accounting for 60% of all dementias (Qiu, Kivipelto, & von Strauss, 2009), followed by vascular dementia, frontotemporal dementia, Lewy body dementias, and prion disease (Elahi & Miller, 2017; Montine et al., 2014). According to the 2011 report from the National Institute on Aging, dementia is defined as deficits in at least two cognitive or neuropsychiatric domains that cannot be further explained by other psychiatric or non-degenerative disorders (McKhann et al., 2011). As the majority of AD and related dementias are chronic illnesses, they appear in stages, and patients’ symptoms worsen with time prior to a complete clinical manifestation (Montine et al., 2014). This leads to substantial decrements in the affected individual’s daily activities, social relationships, and their overall quality of life. Patients who are newly diagnosed with AD have a median survival time of 3-6 years (Helzner et al., 2008). Age is the most common risk factor for dementia and the majority of cases appear after the age of 65 years, with an overrepresentation in females over the age of 75 years (Ruitenberg, Ott, van Swieten, Hofman, & Breteler, 2001). A meta-analysis in 2015 reported that worldwide approximately 46.8 million individuals suffer from dementia, and that this number is expected to reach 131.5 million by the year 2050 (World Alzheimer Report 2015: The Global Impact of Dementia., 2015). This rapid increase in number of persons with dementia will have a tremendous impact on the economy and society. In 2010, the annual cost of care of patients with AD in the United States (US) was estimated to be more than $172 billion and this number is expected to rise to a trillion dollars by 2050 (Brookmeyer et al., 2011). Despite the increase in AD                                                                                                                                             2  prevalence as the result of the aging population, the underlying molecular and cellular mechanisms of this disease remain unclear.  One of the early signs of AD is deficits in episodic memory as a result of neurodegeneration of the limbic system and the medial temporal lobe (Elahi & Miller, 2017). Definitive diagnosis of AD is based on an underlying neuropathology of protein plaques as discovered by autopsies or biopsies (Elahi & Miller, 2017). These aggregates are followed by neuronal death, loss of synaptic connections, and gliosis, causing disruptions in cognition, personality and behavior, and sensorimotor functioning (Elahi & Miller, 2017). However, it is unclear whether these proteins act as biomarkers of the disease or initiate neuron toxicity themselves. The dual proteinopathy of AD encompasses extracellular aggregates of amyloid beta42 (Ab42) and to a lesser extent Ab40 fibrils leading to neuritic plaques, as well as the existence of intracellular hyper-phosphorylated tau aggregates leading to neurofibrillary tangles (NFTs) (Braak & Braak, 1991). However, the progression of AD-related cognitive deficits is better associated with the gradual spread of NFTs than amyloid burden (Jack et al., 2013; Thal, Rub, Orantes, & Braak, 2002). The transentorhinal cortex of the medial temporal lobes, the hippocampal formation, and the neocortex are among the first brain regions affected by tau aggregations (Elahi & Miller, 2017). Neurons that are affected by this proteinopathy undergo premature apoptosis and thus a decrease in grey matter, leading to symptom progression.   Most forms of AD are sporadic AD where genetic risk factors are inherited in a non-Mendelian fashion, or familial AD (FAD) where genetic risk factors are autosomal dominant (Lista et al., 2015). Sporadic AD, characterized by complex genetic and environmental risk factors, accounts for more than 95% of all cases, and occurs after the age of 65 years (late-onset), presenting with a heterogeneous risk factor profile and neuropathology. The genetics of sporadic                                                                                                                                             3  AD is complex and its single known monogenic risk factor is the apolipoprotein E ε4 allele (APOE4). Apolipoprotein E, which is produced by the liver and macrophages in peripheral tissues and by astrocytes in the central nervous system (CNS), plays a role in lipid homeostasis by transporting lipids between different tissues and cell types (Giau, Bagyinszky, An, & Kim, 2015). Receptors of APOE are members of the low-density lipoprotein receptors (LDLR) and aid in lipid transport by APOE among neurons (Bu, 2009). In humans, the APOE gene consists of three polymorphic alleles, the ε2 (8.4% frequency), ε3 (77.9% frequency), and the ε4 (13.7% frequency) (Farrer et al., 1997). The occurrence of the ε4 allele leads to higher AD susceptibility and an earlier age on onset, but its presence is neither necessary nor sufficient for an AD prognosis (Qiu et al., 2009). Individuals who are homozygous for this allele have up to a 15 fold increased risk of AD, and heterozygous individuals experience a threefold increase (Farrer et al., 1997). Other sporadic AD risk factors include age, traumatic brain injury, family history, hypertension, diabetes, and a history of cerebrovascular disease (CVD) (Baumgart et al., 2015). Familial AD, although much less prevalent, has more identified genetic risk factors and presents earlier than the sixth or seventh life decade (Table 1.1). Mutations in three genes, each acting in an autosomal dominant fashion, lead to FAD: amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2) (Bertram & Tanzi, 2012). Mutations in any one of these genes lead to an increased production of Ab42 and inflammation (Bateman et al., 2012).   Alzheimer’s disease pathology is believed to begin years prior to symptom manifestation (Jack et al., 2010). Various biomarkers are used to evaluate AD diagnosis. These include magnetic resonance imaging (MRI) to investigate differential patterns of atrophy, cerebrospinal fluid (CSF) to evaluate Ab42/40 ratio for Ab deposition as well as CSF total tau and hyper-phosphorylated tau to assess neurofibrillary tangles and neuron loss, and genetic testing (Huynh & Mohan, 2017). In                                                                                                                                             4  recent years, it has been noted that olfactory impairments are commonly seen in AD patients prior to clinical symptoms. This thesis explores the role of PS1 in FAD-associated olfactory impairments.  Table 1.1 Alzheimer’s disease-associated genetic risk factors Gene  Chromosome Disease Pattern of Inheritance and prevalence Outcome APP  21q21.3 Familial AD Autosomal dominant (10-15% of FAD) ­ in Ab42  PS1 14q24.2 Familial AD Autosomal dominant (30-70% of FAD) ­ in Ab42  PS2 1q42.13 Familial AD Autosomal dominant (<5% of FAD) ­ in Ab42  ApoE4 19q13.32 Sporadic AD Irregular-sporadic ­ in Ab42  APP: amyloid precursor protein; PS1: presenilin1; PS2: presenilin2; ApoE4: apolipoprotein E4; Ab42: amyloid beta 42  1.2 Amyloid-beta Precursor Protein (APP) and Presenilin  1.2.1 The Role of Amyloid-beta Precursor Protein (APP) Amyloid beta plaques found in the brains of AD patients are encoded by APP, a gene localized on chromosome 21 (Y. W. Zhang, Thompson, Zhang, & Xu, 2011). Interestingly, Trisomy 21, more commonly known as Down’s syndrome, in which an individual has 3 copies of chromosome 21, leads to increased production of APP and thus AD neuropathology (Y. W. Zhang et al., 2011). In mammals, APP is a member of the amyloid precursor-like proteins (made up of APP, APLP1 and APLP2) which are single-pass transmembrane proteins consisting of large extracellular domains (O'Brien & Wong, 2011). However, only APP generates the AD-associated amyloidogenic fragment; APLP1 and APLP2 do not contain the Aß sequence (Wasco et al., 1992). Members of the APP family have essential and redundant functions in brain development, and these functions do not require Aß (Weyer et al., 2011). Many studies have been conducted to                                                                                                                                             5  determine the role of APP. Studies using mice reported that wild-type APP overexpression leads to improved cell health and enlarged neurons (Oh et al., 2009). Other studies using cell culture have demonstrated a role for APP in cell motility, neurite outgrowth, and cell survival. These roles were further highlighted in an in vivo study reporting abnormal neuronal migration as a result of APP RNA interference (RNAi) injection in embryonic rodents (Young-Pearse et al., 2007). In mice, after APP ectodomain intracerebral injections, cognitive functioning and synaptic density was reported to improve (Meziane et al., 1998). The two heparin binding domains of APP were found to be most responsible for its bioactivity, one of which is also the site of F-spondin binding, the only known APP ligand (Ho & Sudhof, 2004). Note that though F-spondin has a role in neuronal development, it is not known whether APP is necessary for its functioning. In more recent studies, it was shown that the APP ectodomain cleaved by beta secretase 1 (BACE1) acts as a ligand for death receptor (DR6), activating caspases 6 and 3, leading to neuron degeneration (Nikolaev, McLaughlin, O'Leary, & Tessier-Lavigne, 2009). The Ab itself has also been shown to play a role in synaptic scaling regulation and synaptic vesicle release (Abramov et al., 2009). Thus it is possible that APP ectodomains cleaved by different secretases have different properties. Despite these findings, APP knockouts in mice demonstrate no deleterious phenotypes. However, cortical neuronal migration abnormalities are seen in triple knockouts of APP, APLP1 and APLP2 (Herms et al., 2004). To date, the precise role of APP in brain function remains elusive.  1.2.2 Alzheimer’s Disease and APP Processing  Neurons produce large quantities of APP which are then metabolized quickly. From the trans Golgi network (TGN), APP is either transported directly to an endosome compartment or to the cell surface (O'Brien & Wong, 2011) (Figure 1.1). If APP is proteolyzed directly on the cell surface by a-secretase prior to g-secretase, no Ab is produced.  If, however, APP is cleaved by                                                                                                                                             6  BACE1 and g-secretase sequentially, Ab toxic peptides, soluble APP extracellular domain (sAPPb), and APP intracellular domain (AICD) are generated (Sachse et al., 2013) (Figure 1.2). After the production of sAPPb by BACEI cleavage, the C-terminal of APP is cleaved via g-secretase  at sites that vary from +40 to +44 and generate Ab peptides (O'Brien & Wong, 2011). These Ab peptides then aggregate and form neurotoxic oligomeric structures and senile plaques seen in AD.  The first APP missense mutation was reported in 1991 in a family with FAD. The majority of APP mutations are located near sites that are normally cleaved by the a, b, or g-secretase, highlighting the importance APP cleavage in amyloid deposition.   Figure 1.1 APP trafficking in neurons. Newly synthesized APP (purple) is transported from the Golgi down the axon (1) or into a cell body endosomal compartment (2). After insertion into the cell surface, some APP is cleaved by α-secretase (6) generating the sAPPα fragment, which diffuses away (green), and some is reinternalized into endosomes (3), where Aβ is generated (blue). Following proteolysis, the endosome recycles to the cell surface (4), releasing Aβ (blue) and sAPPβ. Transport from the endosomes to the Golgi prior to APP cleavage can also occur, mediated by retromers (5). Figure adopted from O’Brien and Wong, Annual Reviews of Neuroscience. Permission obtained.                                                                                                                                              7    Figure 1.2 Sequential cleavage of the amyloid precursor protein (APP) occurs by two pathways. (a) The APP family of proteins has large, biologically active, N-terminal ectodomains as well as a shorter C-terminus that contains a crucial Tyrosine–Glutamic Acid-Asparagine-Proline-Threonine-Tyrosine (YENPTY) protein-sorting domain to which the adaptor proteins X11 and Fe65 bind. The Aβ peptide starts within the ectodomain and continues into the transmembrane region (red ). (b) Nonamyloidogenic processing of APP involving α-secretase followed by γ-secretase is shown. (c) Amyloidogenic processing of APP involving BACE1 followed by γ-secretase is shown. Both processes generate soluble ectodomains (sAPPα and sAPPβ) and identical intracellular C-terminal fragments (AICD). Figure adopted from O’Brien and Wong, Annual Reviews of Neuroscience. Permission obtained.                                                                                                                                                8  1.2.3    g-Secretase Complex  The g-secretase complex consists of four different integral membrane proteins (De Strooper, Iwatsubo, & Wolfe, 2012). The presenilins alongside nicastrin, presenilin enhancer 2 (Pen-2), and anterior pharynx defective 1 (Aph1) make up the catalytic core of g-secretase (Figure 1.3) (De Strooper, 2003). These four proteins are both necessary and sufficient for g-secretase processing (De Strooper et al., 2012). As there are two different presenilins (PS1 and PS2) and Aph1 genes (Aph1a, Aph1b) in the human genome, at least four different g-secretase complexes exist (De Strooper, 2003). In addition to APP and Notch, g-secretase is responsible for the proteolysis of over 80 additional proteins including adhesion molecules N-cadherin and E-cadherin (Struhl & Adachi, 2000), deleted in colorectal carcinoma (DCC) axon guidance molecule (Taniguchi, Kim, & Sisodia, 2003), and neuregulin which plays a role in myelin formation (Dejaegere et al., 2008). However, studies in mice suggest some specificity in substrates that are cleaved by different g-secretase complexes. In studies in which different subunits of g-secretase have been knocked out, different phenotypes are observed. For example, in mice when the Aph1b was knocked out, no Notch defects were observed, but this was not true when Aph1a was knocked out as this leads to adverse embryonic phenotypes (Serneels et al., 2005). The two substrates of g-secretase that have been most implicated in AD are APP and the Notch receptor (Bergmans & De Strooper, 2010). The cleavage of APP by g-secretase primarily happens in the TGN, whereas Notch is often cleaved at the plasma membrane (Tarassishin, Yin, Bassit, & Li, 2004). Since g-secretase is able to cleave APP at various sites, several Ab of different amino acid sizes are produced. Of these, Ab40 is the most abundant type, and the hydrophobic Ab42 occurs at around one tenth the level of Ab40 (Bergmans & De Strooper, 2010).                                                                                                                                               9    Figure 1.3 The four γ-secretase complex subunits. Presenilin consists of 2 fragments known as the amino terminal fragment (NTF) and the carboxyl terminal fragment (CTF). Black circles represent the two highly conserved catalytic aspartate residues.  1.2.4 Presenilin (PS) The presenilins nine-transmembrane domain proteins are the catalytic subunit of g-secretase, consisting of two highly conserved aspartate residues that are critical for g-secretase functioning (Wolfe et al., 1999) (Figure 1.4). These proteins are expressed ubiquitously but are found abundantly in the endoplasmic reticulum (ER) and the TGN, and undergo autoendoproteolysis on the cytoplasmic side to generate a ~25kDa N-terminal fragment (NTF) and a 19 kDa C-terminal fragment (CTF) (Thinakaran et al., 1996; S. Zhang, Zhang, Cai, & Song, 2013). In the early 1990s, multiple laboratories reported that mutations in the PS1 gene lead to FAD, where patients displayed symptoms generally in their fifth decade of life (Mullan et al., 1992; Schellenberg et al., 1992; Sherrington et al., 1995). To date, there are over 180 PS1 mutations reported (Bertram & Tanzi, 2012). A second protein known as presenilin 2 (PS2) which has a significant homology to PS1 at both the gene (67% identical sequence) and protein level has                                                                                                                                             10  also been noted as a cause of FAD (Levy-Lahad et al., 1995). These two presenilins are functionally redundant and highly conserved (Levitan & Greenwald, 1995). Mutations in PS2 account for the smallest percentage of FAD cases and lead to a later age of onset compared to PS1 and APP mutations (Bertram & Tanzi, 2012). The majority of presenilin mutations are missense substitutions in the transmembrane domains and act in an autosomal dominant manner and increase the ratio of Aß42 to Aß40, the well-known biomarker of AD (Bergmans & De Strooper, 2010). Importantly, most AD-associated presenilin mutations also lead to a reduction in the overall proteolytic activity (Song et al., 1999). None of these mutations lead to protein truncation or loss, and assemble with the rest of the components to make a full g-secretase complex (De Strooper et al., 2012). There may be additional as yet undiscovered causative genes as several families with FAD do not display mutations in APP, PS1, or PS2 (De Strooper et al., 2012). No new major monogenic loci that is associated with FAD has been discovered since the mid 1990s, suggesting that other families with inherited FAD have a more complex oligogenic etiology (Bergmans & De Strooper, 2010).     Presenilins also display a number of g-secretase independent functions (S. Zhang et al., 2013). Conditional presenilin knockout studies in mice demonstrate neurodegeneration irrespective of age (Wines-Samuelson et al., 2010), though it is unclear whether this is due to impairments in Notch signaling. However, presenilin knockout mice show more severe somite phenotypes than mice with Notch impairments (Huppert, Ilagan, De Strooper, & Kopan, 2005), supporting a g-secretase independent function of  presenilin (S. Zhang et al., 2013). More recently, it has been noted that presenilins function as passive ER leak channels (Tu et al., 2006). Presenilin mutations that lead to AD disturb these channels and cause an increased accumulation of calcium ions in the ER (Nelson et al., 2007). Knockout of both presenilins in mice demonstrated striking                                                                                                                                             11  neurodegeneration with age, though it was not clear whether this was due to disturbances in g-secretase functioning, imbalances of calcium ion homeostasis, or both (Saura et al., 2004). Thus the role of these calcium imbalances as a result of presenilin mutations remains unclear. Other g-secretase independent roles of PS1 include negative regulation of ß-catenin, a cell adhesion molecule as well as a signal transducer protein in Wnt signaling (H. Huang & He, 2008; Murayama et al., 1998). Lastly, there is an association between presenilin deficiency and abnormality of autophagic vacuoles, though this relationship is not well understood in AD-associated presenilin mutations (S. Zhang et al., 2013).   Figure 1.4 Presenilin 1 structure. This diagram shows the amino acid sequence of PS1 and the distribution of the FAD-associated mutations. Blue circles represent the FAD-associated mutations and red circles indicate the two catalytic active aspartates. Figure adopted from Zhang et al., Journal of Translational Neurodegeneration. No permission required as figure was available from creative commons.                                                                                                                                               12  1.2.5   The Amyloid Hypothesis “Whenever a theory appears to you as the only possible one, take this as a sign that you have neither understood the theory nor the problem which it was intended to solve.” Karl Popper  In the past several decades, as a result of the accumulating evidence that mutations in PS1 and PS2 lead to enhanced APP processing via g-secretase cleavage and an increased production of Ab causing an imbalance of Ab production and its clearance, the amyloid hypothesis has been one of the major foci of AD research (Figure 1.5). Mutations in genes encoding tau protein lead to neurofibrillary tangles and Parkinson’s disease-associated frontotemporal dementia, but do not cause amyloid plaques, further supporting this theory (Goedert & Jakes, 2005). Further, in APP and tau double mutant mice, tau neurofibrillary tangles are increased compared to mutations in tau alone with the same number of amyloid plaques, suggesting that altered APP metabolism occurs prior to tau pathogenesis  (Lewis et al., 2001). The type of Aß instead of the amount of Aß seems to be more crucial in AD pathology (Bergmans & De Strooper, 2010). In their oligomeric state, Aß seems to be more toxic than single Aß peptides, though increasing the amount of Aß peptides can also lead to AD (Bergmans & De Strooper, 2010). Despite the correlation between Aß and pathology, the amyloid hypothesis remains controversial.   There is evidence countering the amyloid hypothesis. Firstly, approximately 40% of individuals without dementia develop some level of AD neuropathology containing Aß and tau (Price et al., 2009). Some of these individuals have no cognitive deficits while imaging shows that they have plaque burdens equivalent to patients that do have dementia (Rentz et al., 2010). However, a study by Mormino et al. (2009) offers an explanation for this observation (Mormino                                                                                                                                             13  et al., 2009). Mormino et al. used positron emission tomography (PET) imaging to measure the levels of Aß deposition in the brains of patients with AD and older non-demented individuals and compared this to hippocampal volume and episodic memory. They found that Aß deposition was followed by hippocampal atrophy and then episodic memory loss, suggesting that high Aß deposition in healthy subjects may indicate early AD stages. Secondly, it is important to note that almost all AD mouse models over-express APP, however there is little evidence of APP overexpression in AD patients (Robakis, 2011). Most of these mouse models do not demonstrate neuronal loss in the presence of large Aß deposition (Bryan, Lee, Perry, Smith, & Casadesus, 2009), and unlike humans with AD who show synaptic loss, mouse models show a variety of presentations (Morris, Clark, & Vissel, 2014). Thirdly, some FAD-causing PS1 mutations do not change the Aß42:40 ratio and family members with the same mutations often show differences in their clinical and neuropathology phenotypes (Gomez-Isla et al., 1999; Shioi et al., 2007). Therefore, over time the AD field has become more open to alternative ideas.   Figure 1.5 Alzheimer’s disease amyloid cascade hypothesis: sequence of pathogenic events leading to increased amyloid beta 42 (Aβ42) production and AD symptomology.                                                                                                                                              14  1.3 Notch Signaling and Alzheimer’s disease  The Notch protein is a type I transmembrane cell surface receptor that plays a role in cell fate decisions of both vertebrates and invertebrates (Artavanis-Tsakonas, Matsuno, & Fortini, 1995; Kopan, Schroeter, Weintraub, & Nye, 1996). Binding to a member of the Delta, Serrate, or Lag2 (DSL) family of ligands triggers Notch proteolysis, leading to the cleavage of Notch by many of the same secretases that cleave APP (Louvi & Artavanis-Tsakonas, 2006). After binding to its ligand, Notch is initially cleaved by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM 10) at its extracellular domain, which in turn triggers cleavage of its transmembrane domain by g-secretase (De Strooper et al., 1999). This leads to the release of the soluble intracellular Notch ectodomain (NICD) which translocates to the nucleus and regulates transcription of genes that play a role in cell differentiation and development (Kopan & Goate, 2000). Impairments of the Notch pathway lead not only to embryogenesis disturbances, but also to disturbances in many oncogenic signaling pathways (Kopan & Goate, 2000; Palomero & Ferrando, 2008). The Notch pathway may also play a role in the adult brain’s neurogenesis (Sestan, Artavanis-Tsakonas, & Rakic, 1999), neural stem cell maintenance (Hitoshi et al., 2002), and long term memory (Poirazi & Mel, 2001). There have also been reports about the involvement of Notch signaling pathway in synaptic plasticity and cognition (Salama-Cohen, Arevalo, Grantyn, & Rodriguez-Tebar, 2006; Sestan et al., 1999).  It has been suggested that alterations in Notch proteolysis by g-secretase may be involved in AD pathogenesis. In the adult brain, Notch is expressed in neurons at especially high levels in the hippocampus (Berezovska, Xia, & Hyman, 1998). Mice with Notch mutations demonstrate long-term spatial memory deficits suggesting that Notch-dependent transcription is important for spatial learning (Costa, Honjo, & Silva, 2003; Woo, Park, Gwon, Arumugam, & Jo, 2009). Further,                                                                                                                                             15  in animals with presenilin deficiencies, Notch phenotypes are observed. One of the greatest concerns with using g-secretase inhibitors to treat AD is their interference with the Notch signaling pathway (De Strooper et al., 2012). Knocking out presenilins in mice abolishes Notch cleavage via g-secretase as well as inhibiting NICD release (De Strooper et al., 1999). Further, mice with FAD-associated presenilin mutations have impairments in their NICD (Song et al., 1999). Studies have also reported that the Notch signaling pathway may contribute to neurodegeneration. Notch mRNA and NICD in nuclei are increased in mice with prion infections that result in dendritic atrophy (Ishikura et al., 2005). However, whether or not Notch signaling plays a role in AD pathogenesis remains unclear (S. Zhang et al., 2013).  1.4  Olfactory Deficits in Alzheimer’s Disease  1.4.1 The Olfactory System  Our sense of smell is essential for recognizing environmental hazards, and it has a considerable impact on our appetite as well as flavor detection. The human olfactory system begins at a pseudostratified columnar epithelium in the nasal cavity known as the mucosa where odorants arrive via nasal airflow and are projected to the olfactory bulb (OB) (Misiak et al., 2017). The olfactory receptor neurons (ORN) of the nasal mucosa consist of cilia that house olfactory receptors (OR) which odorants come in contact with (Misiak et al., 2017). Each ORN innervates a maximum of two OB glomeruli per OB, and each ORN has only one type of OR. Once the odorant binds to its receptor, a G-coupled protein-mediated signaling cascade is activated leading to an action potential. In younger individuals, there are thousands of glomeruli, but as we age, this number decreases (R. L. Doty & Kamath, 2014). Currently, the University of Pennsylvania smell identification test (UPSIT) is the most frequently used test to assess olfactory functioning (R. L. Doty, Shaman, Kimmelman, & Dann, 1984). This test consists of 40 scratch and sniff items, and                                                                                                                                             16  participants are required to identify the detected odorant via a multiple choice questionnaire format.  Olfactory functioning, as measured by the ability to discriminate, identify, and detect odorants at different thresholds decreases with age as does odor memory (Figure 1.6) (Jimbo, Inoue, Taniguchi, & Urakami, 2011; Mobley, Rodriguez-Gil, Imamura, & Greer, 2014). Most people over the age of 65 years present with hyposmia (decreased olfactory ability) or anosmia (complete olfactory loss) (C. Murphy et al., 2002). Olfactory deficits in old age have been associated with depressive symptoms and a loss of self-esteem (Croy, Nordin, & Hummel, 2014; Kollndorfer, Reichert, Bruckler, Hinterleitner, & Schopf, 2017). Intact olfactory functioning relies on normal cellular regeneration of the OB, neuroepithelium, and the hippocampus (Pagano et al., 2000; Schwob, 2002). The human olfactory system is dynamic with continuous sensory and inhibitory neuron neurogenesis (Tsai & Barnea, 2014). This continuous plasticity of the olfactory system persists throughout adulthood.  Figure 1.6 Olfactory functioning: assessed by measuring four odor-related tasks: identification, discrimination, threshold, and memory.                                                                                                                                             17  1.4.2 Alzheimer’s Disease and Olfactory Impairments  Olfactory impairments may be a sign of preclinical neurological disease such as AD, Parkinson’s disease, Huntington’s disease, or vascular dementia (Richard L. Doty, 2017).  Olfactory losses in AD are often drastic and are likely primarily the result of central olfactory system degeneration (Vasavada et al., 2017). In a study of 318 Japanese outpatients at a psychiatric unit, participants and their relatives underwent open essence olfactory testing (Okutani, Hirose, Kobayashi, Kaba, & Hyodo, 2013; Ryo et al., 2017). This test is used to assess odor identification abilities via 12 odorants that participants were asked to identify. Those with AD had more severe olfactory deficits compared to participants with other neuropsychiatric disorders (Ryo et al., 2017). Moreover, in AD, odorant recognition and identification are believed to be more impaired than memory of visual stimuli (Gilbert & Murphy, 2004; Liang et al., 2016; Woodward et al., 2017). Olfactory functioning, especially odorant recall and recognition, involves many complex processes and is associated with episodic and semantic activation of odorant memories. (Kollndorfer, Reichert, Braunsteiner, & Schopf, 2017; Schubert et al., 2008).  The relationships between APOE, olfactory deficits, and dementia are complex. The APOE ɛ4 allele has been demonstrated to be strongly correlated with sporadic AD (Corder et al., 1993; Kanekiyo, Xu, & Bu, 2014). APOE is associated with cholesterol transportation and is expressed in the OB, the olfactory epithelium (OE), as well as the rest of the CNS (Hof & Mobbs, 2010; Y. Huang & Mahley, 2014). In a study of 76 participants, elderly healthy men who were carriers of the APOE4 allele had more impairments in olfactory recognition tasks than did men with AD (Sundermann, Gilbert, & Murphy, 2007). In addition, women carriers of the APOE4 allele had more severe memory impairments than women who were negative for the APOE4 allele. In a later study, ɛ4 carriers aged 70-80 years showed olfactory deficits, even after controlling for their                                                                                                                                             18  cognitive status, suggesting that the ɛ4 allele has a negative impact on the olfactory functioning of older cohorts (Olofsson et al., 2010). An association between dementia and olfactory deficits was also observed, yet dementia did not mediate the effect of ɛ4 and age, suggesting that non-demented ɛ4 carriers with olfactory deficits may not necessarily develop cognitive decline in the future. In summary, APOE4 may increase the risk of toxic neuropathology in the olfactory system and impair olfaction without leading to a diagnosis of dementia.   Overall, the olfactory deficits in AD patients with or without APOE4 tends to be highly correlated with cognitive state. Individuals with a rapid rate of olfactory progression develop more cognitive deficits, lower independence, and in general more severe AD symptoms. In the Heinz Nixdorf Recall (HNR) study, 2,640 randomly sampled participants aged 45-75 years underwent cognitive assessment and olfactory testing every 5 years (Tebrugge et al., 2018). Anosmic and normosmic participants had the worst and the best cognitive performance, respectively. Moreover, in a study of 57 individuals with sporadic AD and 24 older individuals without dementia, UPSIT scores at baseline were correlated with Mini-Mental Sate Examinations (MMSE, assessing cognitive ability) (Velayudhan, Pritchard, Powell, Proitsi, & Lovestone, 2013). Thus evidence is accumulating that in AD, olfactory function correlates with cognitive function, with both declining at a similar rate (Hidalgo, Chopard, Galmiche, Jacquot, & Brand, 2011; Sohrabi et al., 2009). The deposition of Aß in the brain’s olfactory circuit can have an adverse impact on olfaction. J.Y. Kim et al. (2018) demonstrated altered APP processing machinery in AD patients in which higher levels of g-secretase were observed in the OE compared to other brain regions and an increased expression of PS2 in the OE (J. Y. Kim et al., 2018). Examinations of post-mortem brains of AD patients show evidence of Aß deposition and tau neurofibrillary tangles (M. P. Murphy & LeVine, 2010), inflammation as a result of microglia activation after recognition of                                                                                                                                             19  amyloid plaques, (Wyss-Coray & Rogers, 2012), cerebral hypoperfusion in both regional and global regions (Austin et al., 2011), hypometabolism in brain areas with Aß plaques (Klupp et al., 2014), and oxidative stress (W. J. Huang, Zhang, & Chen, 2016). Using MRI, a volumetric analysis of an AD mice model with double mutations affecting APP was conducted before and after Aß accumulation (Badea, Johnson, & Jankowsky, 2010). Volumetric losses were identified in the cortex, hippocampus, pons, and the substantia nigra preceding Aß deposits. These findings further support the hypothesis that physical changes in the brain lead to pre-clinical symptoms such as depression and olfactory deficits prior to cognitive impairments. Kim et al. used manganese-enhanced magnetic resonance imaging (MEMRI) to demonstrate that axonal transport of the OB neurons was compromised in an AD mice model in which mutant APP was overexpressed (J. Kim, Choi, Michaelis, & Lee, 2011). A second study was able to reverse this deficit using a selective g-secretase inhibitor that also significantly decreased both soluble and insoluble Aß (Wang et al., 2012). In a study of the anterior piriform cortex of AD mice models, the presence of Aß led to deficits in odor habituation, but basic odor discrimination was intact (Xu et al., 2014). Odor habituation involves pathways upstream of the primary olfactory pathway, suggesting that odor identification deficits in AD may be the result of impairments in pairing the respective odorant to its associated label in cortical areas upstream of the olfactory circuit.  Various hypotheses have been proposed regarding the etiology of olfactory deficits in AD. It has been suggested that Aß may be behaving in a prion-like manner, spreading from the OB to other connected regions of the brain (Stohr et al., 2012). In a recent in vivo study, human insoluble Aß monomers and oligomers were injected into the mouse OB while being tracked for potential spreading to other areas (He et al., 2017). As expected, both monomers and oligomers of Aß quickly spread to other connected brain regions and led to neuronal apoptosis. However,                                                                                                                                             20  oligomeric Aß induced more damage than monomeric Aß. Previous in vitro studies have also demonstrated the same pattern of Aß propagation (Morales, Callegari, & Soto, 2015; Poon et al., 2013). The role of MicroRNAs (miRNAs) in olfaction has also been examined. MicroRNA, a form of single stranded RNA, plays a role in guiding molecules during post-transcriptional genetic processes (V. N. Kim, Han, & Siomi, 2009). Once the miRNA is bound to the mRNA, most often the mRNA is degraded and translation is repressed. One such miRNA, miRNA-206, is known for its role of regulating muscle development and tumor suppressor functions in cancer (McCarthy, 2008; Mitchelson & Qin, 2015). The effects of expression level of miRNA-206 in the OE of early AD participants was investigated (Moon et al., 2016). It was found that miRNA-206 overexpression in the OE of patients was highly associated with their cognitive assessments, suggesting that miRNA-206 levels might be a biomarker for early AD diagnosis.  In conjunction with other AD biomarkers such as the level of Aß42, olfactory testing can be used to predict disease progression in AD patients. Odorant identification deficits in AD are observed prior to deficits in odorant detection and discrimination. Further, it has been demonstrated that APOE4 increases the risk of neuropathology accumulation in the olfactory system without impairing cognition. However, AD-related olfactory deficits are correlated with cognitive state. Despite the advancements in this area in the past decade (Table 1.2), the underlying mechanisms leading to olfactory deficits in AD remain unclear.                                                                                                                                                   21  Table 1.2: Recent articles assessing Alzheimer’s disease-related olfactory deficits Study Subjects Design and Participant Number Major Findings (Peng, Mathews, Levy, & Wilson, 2017) Mice - Human APOE e4 or APOE e3 was expressed in mice, and olfactory functioning was examined at 6 or 12 months (n=14-24).  - Young (6 months old) APOE e4 mice exhibited olfactory deficits and hyperactive LFP response. - Middle aged mice (12 months old) had reduced spontaneous LFP activity.  (Ryo et al., 2017) Human - 318 participants with varying psychiatric diagnoses and their relatives underwent olfactory testing using open essence olfactory discrimination test.  - Participants with AD had more severe olfactory deficits. compared to those with other psychiatric disorders.  (Woodward et al., 2017) Human - Measured smell identification of 262 AD, 110 aMCI, and 194 control participants using UPSIT, and conducted interim analyses after one year. - Cognitive status was highly correlated with odor identification.  - 36.4% hyposmotic and 17.3% normosmotic participants converted from aMCI to AD after one year. (He et al., 2017) Mice - Monomeric and oligomeric amyloid ß was injected in vivo in the OB of mice to investigate spreading pattern.  - Both monomeric and oligomeric amyloid ß spread from the OB to other interconnected areas and induced apoptosis. - Oligomeric amyloid ß spread more efficiently and lead to more apoptosis than monomeric forms. (Kohl et al., 2017) Human - Characterized OB microglial proliferation and activation in 10 AD patients and 6 healthy participants via Iba1 expressing microglia.  - Protein aggregation of the OB lead to microgliosis as a result of increased microglial activation, but not proliferation.  (Hu, Geng, & Hou, 2017) Rat - Examined the effect of oligomeric amyloid ß on mitral cells (neurons of the OB) using 4-6 rats.  - Amyloid ß oligomers decreased OB’s signal to noise ratio, and increased mitral cells firing activities.                                                                                                                                              22  - Amyloid ß oligomers lead to deficits in the olfactory system’s inhibitory circuits.  (Moon et al., 2016) Human - Intranasal biopsies were conducted on the OE of 24 participants with early signs of dementia and 9 control participants. - Elevated microRNA-206 levels in the OE was highly correlated with cognitive impairments.  (Olofsson et al., 2016) Human - longitudinally (10-20 years) investigated the association between APOE e4, olfactory deficits, and episodic memory impairments in 1087 participants (324= APOE e4 carriers). - In APOE e4 carriers, episodic memory was associated with olfactory deficits.  (Kjelvik et al., 2014) Human - Investigated the relationship between brain structure volumes, cognitive functioning, and olfactory deficits. - Study consisted of 12 aMCI patients, 6 early AD patients, and 30 controls.  - Hippocampal volume reduction in early AD patients is associated with the loss of olfactory ability, but not a loss in cognitive functioning.  (Xu et al., 2014) Mice - Studied odor coding in the APC of transgenic and wild-type mice at 3, 6, and 12 months using olfactory habituation tasks and electrophysiology. - AD mice had odor habituation impairments, but odor identification remained intact.  - AD odor deficits may be due to disruptions of linkage between the odorant and upstream primary olfactory pathways.  APOE: apolipoprotein E; LFP: local field potentials; AD: Alzheimer’s Disease; aMCI: amnestic mild cognitive impairment; UPSIT: University of Pennsylvania Smell Identification Test; OB: olfactory bulb; Iba1: ionized calcium binding adaptor molecule 1; OE: Olfactory Epithelium; APC: anterior piriform cortex.                                                                                                                                                  23  1.5 Use of Caenorhabditis elegans (C. elegans) to Understand Disease Mechanisms In the 1960s, Sydney Brenner introduced the use of the roundworm C. elegans for the study of developmental biology and neurobiology. This nematode is non-parasitic and has a life cycle of 3.5 days at 20°C and a lifespan of approximately 2-3 weeks (Markaki & Tavernarakis, 2010). The worm consists of a total of 959 somatic cells, 302 of which are neurons (Corsi, Wightman, & Chalfie, 2015). Adult worms are around 1mm long and, in the laboratory, feed on Escherichia coli (E. coli) on agar plates. C. elegans have 6 pairs of chromosomes: five pairs of autosomes along with one pair of sex chromosomes. They display two sexes, either hermaphrodites or males, with the ratio of sex chromosomes to autosomes determining their sex (Markaki & Tavernarakis, 2010). Their genome is comprised of 100,000,000 base pairs and an estimate of 60-80% of their proteome have human homologous genes, of which roughly 42% are orthologues, meaning that they are corresponding genes in different lineages that result from speciation (Harris et al., 2004; Vahdati Nia, Kang, Tran, Lee, & Murakami, 2017). Thus C. elegans has been used as a powerful experimental system to study the underlying molecular and cellular processes of human diseases in vivo.  1.5.1 C. elegans as a Genetic Tool to Study Human Disease  There are a number of advantages to using C. elegans as a model for studying neurological diseases as it has a short life span, a sequenced genome, and a defined nervous system that is well characterized with a known connectome (Alexander, Marfil, & Li, 2014). Basic cellular neurobiological functions are well conserved, with C. elegans having basic cell biology and biochemistry similar to mammals. C. elegans nervous system shows both genetic and functional similarity having many of the basic mammalian neurotransmitters (ex. dopamine, serotonin, GABA, glutamate, acetylcholine and their receptor subtypes) and many classes of ion channels                                                                                                                                             24  (ex. sodium, calcium, and potassium channels) (Hobert, 2013). Many insights into nervous system and development have come from studies in C. elegans, for example the first axon guidance molecule, netrin (unc-6) was first identified in the worm (Merz, Zheng, Killeen, Krizus, & Culotti, 2001), several of the known components of vesicle release were first identified in C. elegans (unc-19; unc-13) (Maduro & Pilgrim, 1995; Richmond, Davis, & Jorgensen, 1999), and their homologues were later found in mammals (Munc-19; Munc13). Further, since C. elegans are self-fertilizing hermaphrodites, large clonal populations of genetically indistinguishable individuals can be studied.  1.5.2 Use of C. elegans in Studying AD Studies in C. elegans were critical for understanding the biological function of presenilins. Mutations in one of the C. elegans presenilin homologues, spe-4, lead to disruption of Golgi protein trafficking and thus impairments in spermatogenesis (L'Hernault & Arduengo, 1992; Markaki & Tavernarakis, 2010). Moreover, the original observation that presenilin cleaves Notch came from work using C. elegans (Levitan & Greenwald, 1995). Levitan et al. showed that in C. elegans, mutations in sel-12, a homologue of human PS1, lead to an egg-laying deficit because of the failure of sel-12 to cleave the C. elegans Notch protein that is critical for development of the vulva (Levitan & Greenwald, 1995).  Both Levitan et al. and Baumeister et al. found that they could rescue mutations in sel-12 with the C. elegans wild-type gene or with human wildtype PS1, however introduction of human PS1 carrying FAD mutations did not rescue egg laying. These data indicated that there is functional homology between C. elegans sel-12 and human PS1 and was the first demonstration of a role for presenilin in cleavage of Notch (Baumeister et al., 1997; Levitan et al., 1996). Lastly, it is interesting to note that the majority of presenilin mutations leading to                                                                                                                                             25  FAD do not occur at random locations in the PS1 gene, rather they generally occur in residues that are conserved between PS1 and sel-12 (Hardy, 1997).  1.5.3  Alzheimer’s disease-associated genes in C. elegans  C. elegans has orthologs of some of the genes implicated in AD, and not others. For example, C. elegans does not have an APOE gene and has only one APP-related gene known as apl-1, which contains a large extracellular domain that shares a 46-49% sequence similarity to human APP and a cytosolic domain which has a 71% sequence similarity to human APP (Daigle & Li, 1993). Similar to APLP1 and APLP2, APL-1 does not have an Aß sequence and is expressed in neurons, supporting cells, head muscles, and vulva cells (Daigle & Li, 1993).  In C. elegans, Notch proteins undergo three proteolytic cleavages at three distinct sites. Site 1 cleavage occurs when the protein is transported to the surface, generating a heterodimer between N and C terminals (Jarriault & Greenwald, 2005). Once a ligand binds to the Notch receptor, cleavage at site 2 occurs, creating the substrate for presenilin cleavage at site 3 (Jarriault & Greenwald, 2005). C. elegans has two α-secretase proteins known as SUP-17 and ADM-4 which work redundantly in cleaving C. elegans Notch homologues, LIN-12 and GLP-1, at site 2 (Jarriault & Greenwald, 2005). It is unclear in C. elegans whether these two proteins also cleave APL-1. As no BACE or other ß-secretase homologue has been identified in C.elegans, it is suggested that APL-1 is cleaved directly by α/γ-secretase activity (Link, 2006). First, α-secretase cleaves APL-1 which releases sAPL-1, the extracellular fragment, and next APL-1-CTFα is cleaved by γ-secretase to release the intracellular domain (AICD) (Alexander et al., 2014). Further, as discussed previously, the C. elegans presenilin gene family includes spec-4 (expressed only in male gonad), sel-12, and hop-1. In the LIN-12 and GLP-1 Notch pathways, sel-12 and hop-1 have redundant functions (Westlund, Parry, Clover, Basson, & Johnson, 1999).                                                                                                                                              26   Finally, C. elegans has one tau homologue named “protein with tau-like repeats” (ptl-1), consisting of two isoforms which have a high level of sequence homology with mammalian tau and promote microtubule assembly (Goedert et al., 1996). PTL-1 is expressed in the embryonic epidermis where microtubules aid in distributing the force generated during elongation, and are also found in mechanosensory neurons where microtubules are needed to respond to touch (Goedert et al., 1996).  1.5.4 C. elegans Olfactory System More than 5% of the C. elegans genes are responsible for the recognition of environmental chemicals such as volatile (olfaction) and water-soluble (gustation) compounds (Bargmann, 2006). C. elegans use their chemosensation ability to avoid unpleasant stimuli, increase or decrease their speed, mate, and enter or exit dauer during development (Bargmann, 2006). The amphid (head) region of the worm contains 11 of the 32 pairs of chemosensory neurons, each consisting of candidate G protein-coupled receptors (GPCRs) responsible for detecting multiple odorants (Bargmann, 2006). The rest of the chemosensory neurons are housed in the phasmid (tail) region or the inner or outer labia (mouth) region (Figure 1.7). Two signal transduction pathways have been described in C. elegans chemosensation, one that uses cyclic guanosine monophosphate (cGMP)-gated channels and the other that uses transient receptor potential vanilloid (TRPV) channels (Bargmann, 2006). Each neuron pair is structurally similar and pairs are recognized by their specific cilium and axon morphology as well as their synaptic targets (White, Southgate, Thomson, & Brenner, 1986). The cilia of these neurons are either directly or indirectly exposed to the environment via pores made by glial cells. The chemosensory neurons send information to premotor interneurons that drive motor neurons to direct the forward or backward movement of worms (Chalfie et al., 1985).                                                                                                                                              27   Figure 1.7 Structure of chemosensory organs. Disposition of chemosensory neurons in the animal. Each of the two amphids contains 12 associated chemosensory or thermosensory neurons. Each of the two phasmids contains 2 chemosensory neurons, PHA and PHB. There are six inner labial organs, each of which contains one IL2 chemosensory and one IL1 mechanosensory neuron. There are two URX neurons, one AQR neuron, and one PQR neuron; the endings of these neurons are within the animal, and not exposed. Figure adopted from Bargmann, Wormbook. No permission required as figure is available from creative commons.  1.6  Project Objectives  Alzheimer’s disease is a fatal disease and is the most common form of neurodegenerative disorder in the elderly, impacting all aspects of patients’ lives. To date, no cure exists for AD. Three autosomal dominant causative genes have been associated in FAD (APP, PS1, and PS2), and one genetic risk factor has been identified for sporadic AD (APOE4). The PS1 gene is the most common cause of FAD. Presenilins are a part of the catalytic subunit of γ-secretase, an enzyme that cleaves many proteins including APP and Notch that are implicated in AD. A great deal of research on AD has focused on the abnormal cleavage of APP. The majority of FAD mutations in PS1 disrupt both APP and Notch processing, yet the role of Notch in AD remains unclear. A recent study identified a new PS1 serine deletion mutation at locus 169 (PS1∆S169 ) in a Chinese family with FAD (Guo et al., 2010). A study of PS1∆S169  by Dr. Weihong Song’s lab at the University of British Columbia (UBC) demonstrated that this mutation impairs APP processing                                                                                                                                             28  but does not impair Notch processing, a unique phenomenon observed in FAD-causing presenilin mutations (S. Zhang et al., 2018). Although many patients with AD experience olfactory impairments before manifestation of other clinical symptoms, the underlying molecular and cellular etiology of these impairments are poorly understood. C. elegans is a suitable animal in which to investigate the role of PS1 in AD-associated olfactory deficits. This approach allows an investigation of the function of PS1 in chemosensory processing in individual neurons-something not possible in other in vivo systems. Understanding the gene pathway underlying the olfactory deficit in AD may identify new genes for more intensive studies in mammals. Ultimately, understanding the role of presenilin in olfaction may lead to the development of novel ways to diagnose and possibly treat AD in humans.  Our lab has previously demonstrated that mutations in sel-12, the homologue of human PS1, lead to chemotaxis deficits in worms. This deficit could be rescued by ubiquitous expression of both human PS1 and PS1∆S169. The objectives of my research are therefore:  1) To investigate the role of PS1 in AD-associated olfactory deficits using nervous system and neuron-specific rescues.  2) To briefly examine the role of Notch signaling in AD-associated olfactory deficits. 3) To determine whether the observed olfactory deficits are correlated with neuronal degeneration.                                                                                                                                                 29  Chapter 2: sel-12 mutant C. elegans display olfactory impairments from hatch and these impairments increase over time. 2.1 Introduction Currently, a definitive diagnosis of AD requires brain autopsy after death. Therefore, there is a critical need for biological and behavioral markers for AD classification in living patients (Wilson et al., 2009). This is driven by a belief that therapeutic advancements in AD would be most effective if patients could be enrolled in clinical trials as early as possible in the course of the disease, prior to the formation of AD-related neuropathology (Frank et al., 2003). Deficits in the identification of odorants has been associated with later development of mild cognitive impairment, a precursor to AD-related dementia, and is robustly correlated with the amount of AD neuropathology examined post-mortem (Wilson et al., 2007). Although the underlying mechanism is unclear, it has been suggested that, in general, sensory systems may be more susceptible to AD pathology compared to the rest of the CNS (Yoo et al., 2017). Changes in the brains of those with AD follow a pattern that leads to worsening of clinical symptoms such as decreased memory over time (Thies & Bleiler, 2013). The pattern that has been observed begins with increased levels of Aß in the brain and increased levels of tau in the CSF, eventually decreasing the brain’s ability to use glucose as an energy source (Thies & Bleiler, 2013).  Interestingly, the rate of progression of olfactory impairments in AD patients is similar to their rate of cognitive decline (Hidalgo et al., 2011). Although mutations in PS1 are the most common cause of FAD, the role of PS1 is AD-associated olfactory deficits remains unknown.  An organism with tractable genetics and a small nervous system such as C. elegans can be used to investigate the role of PS1 in AD-associated olfactory deficits. C. elegans have three homologues of presenilins, sel-12 (PS1), hop-1 (PS1), and spe-4 (PS1) (Smialowska &                                                                                                                                             30  Baumeister, 2006). Similar to human presenilins, sel-12 is expressed in a variety of cell types throughout development (X. Li & Greenwald, 1997). Though sel-12 and hop-1 have redundancies with regards to their enzymatic activities, only mutations in sel-12 lead to a strong egg-laying deficits and additional phenotypes (Smialowska & Baumeister, 2006). Further, hop-1 displays a much lower sequence identity to human presenilins (31%) compared to sel-12 (50%) (X. Li & Greenwald, 1997) (Figure 2.1). However, a recent article reported that the maintenance of adult stem cells in C. elegans relies exclusively on Notch signaling mediated by HOP-1 (Agarwal et al., 2018).    Our lab has previously demonstrated that C. elegans with a mutation in sel-12 has chemotaxis deficits to octanol, an aversive odorant (Figure 2.2). In this chapter, I extended this work by testing the chemotaxis abilities of a sel-12 mutant, a hop-1 mutant, and sel-12/hop-1 double mutant worms. As sel-12 mutant worms had a more severe chemotaxis impairment than hop-1, I explored whether the sel-12 olfactory deficits increased with age, similar to AD-associated symptoms. I further tested whether, as a result of their egg-laying phenotype, speed was a confounding variable in sel-12 mutants.   Embryogenesis takes around 9 hours at 25°C in C. elegans (Corsi et al., 2015). The parent lays an impermeable egg after approximately 24-cell divisions, that hatches about 8-9 hours later and the larva grows independent of the parent. C. elegans have four larval stages (L1-L4); the L1 stages lasts 16 hours and the rest last about 12 hours at 25℃ (Figure 2.3) (Corsi et al., 2015). Prior to the completion of each stage, the worms go through a sleep-like period known as lethargus during which they make a new cuticle  (Raizen et al., 2008). Twelve hours after the final L4 mold, adult hermaphrodites are capable of using their self-produced sperm to lay eggs for 2-3 days, after which they live for a couple more weeks (Corsi et al., 2015).                                                                                                                                              31   Figure 2.1 Inferred Topology of SEL-12: Hydrophobic regions are indicated by Arabic numerals, and transmembrane domains deduced from this study are indicated by Roman numerals. As detailed in the Discussion, the eight transmembrane domains may associate with each other to form a β barrel, and the seventh and tenth hydrophobic regions may be associated with the membrane (represented by a double-headed arrow), with other domains of SEL-12, or with other proteins. The deduced cleavage site is likely to be within amino acids G235 to D284; for simplicity in this diagram, the uncleaved form is shown. Cleavage does not appear to be absolutely required for PS1 function in a C. elegans functional assay. Figure adopted from Li & Greenwald, Neuron. Permission obtained.                                                                                                                                                   32   Figure 2.2 sel-12 mutant C. elegans chemotaxis assay: Chemotactic indices generated from assays performed on wild-type and sel-12 mutant worms using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=30-60 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference (p<0.05) from wild-type worms. Figure adapted from T. Bozorgmehr, University of British Columbia Thesis. Permission obtained.                                                                                                                                             33   Figure 2.3 C. elegans life cycle. C. elegans larval development proceeds through 4 larval stages (L1 through L4). L4 larvae molt into young adults which then develop into reproductive adults that survive for approximately 3 weeks under normal laboratory conditions. If L1s are starved, crowded and experience elevated temperatures, they may select an alternative developmental pathway developing into L2d and then dauer larvae. Dauer larvae are adapted for survival in the absence of food by dispersing into new environments. When suitable environmental conditions return, dauers may reenter reproductive development by molting into L4 larvae. Figure adapted from WormAtlas, Altun, Z.F., Herndon, L.A., Wolkow, C.A., Crocker, C., Lints, R. and Hall, D.H. (ed.s) 2002-2018. No permission required as figure is available from creative commons.                                                                                                                                                  34  2.2 Methods  2.2.1 Generation of transgenic lines and strain maintenance  C.elegans wild-type (N2) and sel-12 mutant (RB1672), hop-1 mutant (GS2447), and sel-12/hop-1 double mutant (CE1239) strains were provided by Caenorhabditis Genetics Center (CGC), and worms were cultured on Escherichia coli (E. coli) seeded nematode growth medium (NGM) (Brenner, 1974). All experiments were conducted in 6cm Petri plates that were filled with agar a maximum of two weeks prior to use. Worms were stored in a 20℃ incubator and all experiments were conducted in a room with controlled humidity (40±	5	%	RH)	and temperature (20±1℃).  Transgenic lines were constructed by DNA microinjection into young adult C. elegans germ line of sel-12 mutant worms at a concentration of 10 ng/μl along with a co-injection GFP marker (Evans, 2006). This DNA mixture was injected into the distal arm of the worm’s gonad, which contains a central cytoplasm core that is shared by germ cell nuclei (Evans, 2006). Worms were injected under an inverted Zeiss DIC microscope equipped with a 40X Nomarski objective. The plasmid pBY140 containing the wild-type PS1 coding region expressed by the sel-12 promoter was provided by Dr. Baumeister’s lab (Albert-Ludwig University in Freiburg/Breisgau, Germany) and was modified with different promoter sequences used to generate the plasmids used. Lines with >50% GFP transmissions were selected. As this DNA microinjection created an extra-chromosomal array, there was inherent variability of expression levels because there were probably different copy numbers of plasmids incorporated into the array and the possibility of different cells having different genotypes (mosaic expression) (Yochem & Herman, 2005). To overcome this, three independent lines for each construct were used (Levitan & Greenwald, 1995).                                                                                                                                              35  Strains:  RB1762; sel-12(ok2078) X; description: deletion Size: 1525 bp. Deletion left flank: TCTGGTTGTTTTTACGATGAACACGATTAC. Deletion right flank: TCAGCTGAATAT ATTTTGTTCATTTAAAGT. GS2447; hop-1 (ar179) I; description: deletion size: 718bp. Has break points within codon Ser-57 and codon Tyr-218.  CE1239; hop-1 (ep369) I; sel-12 (ep6) spr-3(ep17) X; ep369 is a weak allele. ep6 is a deletion allele. The following strains were created:   VG546 sel-12 (ok2078); Psel-12::PS1 wt Punc-122:: GFP  VG547 sel-12 (ok2078); Psel-12::PS1 wt Punc-122:: GFP  VG548 sel-12 (ok2078); Psel-12::PS1 wt Punc-122:: GFP  2.2.2 Chemotaxis Assay:  The chemotaxis procedure used was adapted from a study by (Margie, Palmer, & Chin-Sang, 2013). To ensure that all animals tested were the same developmental age, adult worms (20-30) were placed in a droplet of 4µl of bleach solution (1:1 ratio of 100% bleach and 1M NaCl). The worms’ bodies disintegrated but their eggs survived. All eggs hatched at the same time, and larvae were allowed to grow for three days on agar seeded with E. coli in a 20℃ incubator. On day three, 72-hour-old worms were collected using 700µl of liquid M9 buffer into 1.5mL centrifuge tubes. To remove OP50 E. coli from the bodies of the worms, worms were centrifuged for a minute at 1000rcf and the supernatant was removed. Another 700ul of M9 buffer was added to the tubes, and the worms were centrifuged again (done 3 time). After the third time, the supernatant was removed and the pellet of the worms remained in the tube.                                                                                                                                              36  Eight NGM plates (4 control, 4 test) per strain were divided into four equal quadrants, with a circle of diameter 1cm that was measured to be 2.3cm away from the center of the plate (Figure 2.4). For test plates, each quadrant was marked as control or test. Note that the 2 control quadrants and the 2 test quadrants were on opposite sides. For control plates, all 4 quadrants were labelled as control. The octanol solution was prepared by mixing of a 1:1 ratio of 100% octanol and 1M-sodium azide (NaAz) that was used to immobilize worms. The diacetyl solution was prepared by diluting it to 0.5% diacetyl using 99.5µl of M9 and 5µl of diacetyl. A 1:1 ratio of this 0.5% diacetyl solution was then added to NaAz. Control solution was prepared by mixing equal volumes of M9 and NaAz. Washed worms (50-100) were pipetted onto the center of the plates. Then 2µl of the volatile odorants octanol or diacetyl were placed on test sites, and 2µl of the control solution was placed on control sites. Plate lids were kept open until the odorants soaked into the agar, and the worms were allowed to move around undisturbed for one hour in a 20°C	room. Plates were then transferred into a 4°𝐶	fridge for two hours to immobilize worms, and were later taken out for counting worms. Worms were counted by a researcher blind to the condition/strain/the study objectives as plates were relabeled (and a key generated) by another student prior to counting, and a chemotaxis index (CI) was calculated (Figure 2.4b).  Use of 5’-fluorodeoxyuridine (FUdR) in aging studies:  Because of the egg-laying deficit in sel-12 mutant worms, eggs buildup inside the gonad, and then the young worms hatch inside the adult and kill it at a young age.  This makes studies of older worms very difficult. To stop the hatching of eggs inside adults so the adults lived to an older age, I used 5’-fluorodeoxyuridine (FUdr). FUdr is an inhibitor of DNA synthesis that is used in C. elegans lifespan studies to preserve a synchronized aging population (Anderson et al., 2016). In                                                                                                                                             37  my research, two days prior to adding E. coli to NGM plates, 250µl of 100µM FUdR was spread on plates. Two days after the addition of E. coli, L4 stage animals were transferred to NGM plates with FUdr. For aging studies, adult worms were tested from 78 to 108 hours at 10 hour intervals.          Figure 2.4 a. The chemotaxis assay layout. 2ul of octanol/M9 buffer was placed on black circles. 50-100 worms were placed in the middle circle. b. Chemotaxis index calculation per plate.  2.2.3 Multi-worm tracker (MWT) Behavioral experiments on different-aged worms were used to determine speed and the locomotory ability of worms on plates. The MWT is an automated machine vision system that can record various components of worm’s behavior such as their probability of reversal due to a stimulus, their speed, and the duration of movements (Figure 2.5) (Swierczek, Giles, Rankin, & Kerr, 2011). To assess locomotion, approximately 50 worms of each age were handpicked onto a plate without FUdR or E. coli (n=3 plates), placed on the MWT, and the lid of each plate was lifted briefly to provide an air-puff stimulus to arouse worms so they were moving and could be recognized by the MWT. Worms were tracked for 250 seconds.                                                                                                                                                38   Figure 2.5 The Multi-Worm Tracker. (Top) The Multi-Worm Tracker allows for high-throughput, high-resolution behavioral analysis of Caenorhabditis elegans. The Multi-worm tracker delivers mechnosensory stimuli and preforms image acquisition, object selection and parameter selection in real time while choreography software extracts detailed phenotypic information offline. (Bottom) (A) A petri plate of C. elegans, (B) a petri plate of C. elegans selected for analysis by the Multi-Worm Tracker, (C) a Multi-worm tracker digital representation showing the degree of phenotypic detail. The C. elegans response to a mechanosensory tap to the side of the petri plate is brief backward locomotion (from C to D). This habituates (decreases in probability, duration and speed) with repeated taps. Scale bars are 1, 1, 0.25 and 0.25 mm from left to right. Figure adopted from McDiarmid et al., Genes, Brain, and Behavior. Permission obtained.  2.2.4 Statistical Analysis  Data are reported as means ±  standard error of the mean (SEM) of three or four independent plates with 50-100 worms on each for each strain tested. Presenilin rescue strains are                                                                                                                                             39  reported as the average of three extrachromosomal lines. Data analyses were conducted using the statistical program SPSS version 22.0. Data were checked for normal distribution and parametric tests were used when appropriate. When comparing three or more groups, a one-way ANOVA was conducted with a Tukey’s Honest Significant Difference test for post-hoc analyses. P values of less than 0.05 were considered significant.  2.3 Results  2.3.1 Worms with a sel-12 mutation had more severe chemotaxis deficits compared to wild-type worms than hop-1 mutant worms. Because previous studies reported that sel-12 and hop-1 have redundant functions, we investigated mutations in both to determine whether there was a difference in their chemotaxis abilities. Seventy-two hour old wildtype worms had a normal CI	in response to octanol. All mutant strains had significantly lower CIs indicating a lower ability to chemotax away from octanol compared to wild-type [F (3, 12) = 13.89, p<0.01] (Figure 2.6). Since sel-12 has a higher sequence homology to PS1 compared to hop-1 (50% vs 31%), all future experiments in this thesis will use sel-12(ok2078) for chemotaxis assays.                                                                                                                                                40   Figure 2.6 Chemotaxis assay in sel-12 mutant, hop-1 mutant, and sel-12/hop-1 double mutant worms: Chemotactic indices generated from assays using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference from wild-type (p<0.05). 2.3.2 sel-12 mutant worms had chemotaxis deficits from hatching that increased with age.   In patients with AD, olfactory functioning declines at a similar rate to cognitive functioning, and these symptoms worsen over time (Hidalgo et al., 2011). As C. elegans have 4 larval stages, I tested their chemotaxis response to octanol from L1-L4 as well as at 56 hours old (adult worms). Compared to wildtype worms, C. elegans with a sel-12 mutation had significant chemotaxis deficits from L1 that persists into L2, L3, L4, and adulthood [F (9, 30) = 65.60, p <0.01] (Figure 2.7).  To determine whether these deficits increased with time, 78-108 hour old worms were tested on chemotaxis assays at 10 hour intervals (Figure 2.8). There was a statistically significant difference between groups [F (7, 24) = 60.11, p <0.01]. The results showed that there was a mild but non-significant pattern of decrease in chemotaxis over time in wild-type worms (CI of 78 hour                                                                                                                                             41  old worms -0.54±0.04	vs CI of 108 hour old worms -0.41±0.02, p=0.06). This pattern was much more rapid and significant in sel-12 mutant worms (CI of 78 hour old worms -0.16±0.02 vs CI of 108 hour old worms -0.03±0.01,	p=0.03). Overall, wild-type and sel-12 mutant worms had a chemotaxis decrement of 15% and 81% from 78 to 108-hours old, respectively.   Figure 2.7 Chemotaxis assay in wild-type and sel-12 mutant worms from L1-adult stage: Chemotactic indices generated from assays using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant difference from wild-type of same age (p<0.05).                                                                                                                                                  42    Figure 2.8 Chemotaxis assay in wild-type and sel-12 mutant worms from 78-108 hours old: Chemotactic indices generated from assays using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Single asterisks show significant differences from wild-type worms of same age and double asterisk shows significant differences from 78 hour old sel-12 mutant worms (p<0.05).                                                                                                                                                      43  2.3.3 Locomotion speed was not a confounding variable in sel-12 mutant chemotaxis.   Because sel-12 mutant worms have an egg-laying phenotype in which the parent is unable to lay eggs and becomes bloated, it is possible that this could affect their ability to chemotax properly or within the allotted time. To determine if this was the case I conducted experiments to examine locomotor speed as a function of age on the MWT. Although sel-12 mutant worms appeared a bit slower than wild-type at the outset of the experiment, forward speed was not significantly different (wild-type speed 0.16±0.01mm/s vs sel-12 speed 0.17±	0.03mm/s, p=0.08) (Figure 2.9). As worms aged, wild-type worms’ forward speed peaked at 88 hours (0.25±0.01mm/s) and slowed down at 108 hours old (0.11±0.01mm/s). However, sel-12 mutant worms did not get faster with age and their speed slowed down at a younger age than wild-type mutant worms, being significantly slower at 88 and 98-hour old compared to wild-type worms (p<0.01).  Using the MWT, I could also determine whether 78 hour old sel-12 mutant worms could navigate around the entire test plate within the 1-hour chemotaxis test period as this is near the age (72 hours old) when they are usually tested for chemotaxis. At this age, sel-12 mutant worms traversed the entire distance of the petri plate in only approximately 5 minutes (Figure 2.10), suggesting that slow speed is not a confounding variable for chemotaxis as the worms have one hour to reach the test circle with the chemical cue of interest.                                                                                                                                                   44   Figure 2.9 Average forward movement speed of 68-108 hour old wild-type and sel-12 mutant worms. Blue bars and red bars represent wild-type (N2) and sel-12 mutant worms respectively. Each bar represents an average from 3 independent plates (n=50 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant difference from wild-type of same age (p<0.05).   Figure 2.10 78 hour old wild-type and sel-12 mutant worm tracks on a plate over 250 seconds. Different colors indicate various tracking time points after a brief air-puff. Wild-type and sel-12 mutant worms are both capable of navigating the entire plate (n=50 worms per plate).                                                                                                                                               45  2.3.4 Both the chemotaxis deficit and the slow speed could be rescued at all ages by expression of PS1 driven by the sel-12 promoter.  Previously, our lab showed that expression of sel-12 and wildtype PS1 driven by the sel-12 promoter (Psel-12::sel-12 and Psel-12::PS1) in sel-12 mutant worms rescued the worms’ octanol chemotaxis deficits and their egg laying impairment (Bozorgmehr, 2015). In this chapter, I replicated the rescue of the chemotaxis deficits by Psel-12::PS1 and extended the findings across three different ages. Chemotaxis tests were conducted on wild-type worms, sel-12 mutant worms, and rescue lines of Psel-12::PS1 at 88, 98, and 108 hours of age (Figure 2.11). For each age, all three Psel-12::PS1 rescue lines restored chemotaxis deficits in sel-12 mutant worms and had CI values significantly different from sel-12 mutant worms (88-hour old F(4, 15) = 9.20, p<0.01; 98-hour old F(4,15) = 31.41, p<0.01; 108-hour old F(4,15) = 15.65, p<0.01).  I also tested whether the slowed locomotion observed in sel-12 mutant worms could be rescued by Psel-12::PS1 as worms aged. Tracking experiments were conducted to determine whether speed differed among sel-12 mutant worms, and Psel-12::PS1 (rescues egg laying behavior) as worms aged. Figure 2.12 shows the forward speed of wildtype worms (black and grey lines), sel-12 mutant worms (red lines), and ubiquitous PS1 rescue lines (blue lines). The overlap of the black and blue lines suggests the slowed locomotion phenotype of sel-12 mutant worms was rescued by the expression of Psel-12::PS1 in the whole body. Figure 2.13 shows the tracks around the plate of the same worms over time and shows that sel-12 mutant worms moved around much less (panel B) than wildtype and ubiquitous PS1 rescue lines (panels A and C). These data suggest that expression of Psel-12::PS1 in sel-12 mutant worms rescued speed across all ages (p<0.01, Figure 2.12) as well as dispersal defects (Figure 2.13).                                                                                                                                              46    Figure 2.11 Ubiquitous expression of wild-type PS1 in worms with a sel-12 mutation chemotaxis assay over time: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Psel-12::PS1 worms using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant difference (p<0.05) from wild-type of same age.                                                                                                                                                47   Figure 2.12 Average forward movement speed groups of 88, 98, and 108 hour old wild-type, sel-12 mutant, and ubiquitous PS1 rescue worms during 250 seconds of tracking. Values are averages from 3 independent plates (n=50 worms per plate).                                                                                                                                                      48   Figure 2.13 88, 98, and 108 hour old wild-type (A), sel-12 mutant (B), and ubiquitous PS1 rescue (C) worm tracks on a plate over 250 seconds. Different colors indicate various tracking time points after a brief air-puff. (n=50 worms per plate). Note that in the rescue panels (C), only one of the three extrachromosomal rescue lines is shown as all followed the same trend.                                                                                                                                                    49  2.4 Discussion  In this chapter, I showed that both sel-12 and hop-1 mutant worms had olfactory deficits. This underscores that both SEL-12 and HOP-1 play roles in C. elegans olfaction. Because SEL-12 has a higher sequence homology to human PS1 than HOP-1, I chose to test SEL-12 in the remainder of my experiments.  C. elegans with a mutation in sel-12 have olfactory deficits that are present from hatching and increase with time/age, similar to symptoms of AD. This may indicate that wildtype sel-12 is important during the development of sensory neurons, interneurons, or synapses responsible for detecting octanol and controlling the worm’s behavior in response to aversive stimuli. This question is further explored in chapter 4 of this thesis. As in normal aging, I see a pattern of a decrease in olfactory ability in wildtype worms, though this pattern is not observed in the extrachromosomal Psel-12::PS1 rescue lines. Note that as these are extrachromosomal arrays, the expression levels among different lines are unpredictable, and the expression pattern may not be the same as the endogenous gene (Evans, 2006). Therefore, overexpression of Psel-12::PS1 in sel-12 mutant worms may mask the pattern observed in wild-type worms over time. However, since chemotaxis is rescued at all time points, a functional homology in octanol chemotaxis among sel-12 and PS1 is demonstrated.  The MWT was used to show that sel-12 mutant worms display no locomotion or coordination deficits severe enough to alter chemotaxis in this assay. Although at all ages, sel-12 mutant worms move much slower than wildtype worms, they were able to navigate around the entire petri plate within approximately 5 minutes, leading to the conclusion that slow speed was not a confounding variable in the chemotaxis tests.                                                                                                                                              50  Chapter 3: Cell-specific expression of wild-type PS1 and PS1Δs169, but not PS1C410Y, rescued chemotaxis impairments in sel-12 mutant worms. 3.1 Introduction The presenilin hypothesis of AD was introduced in 2007, stating that the loss of normal presenilin functions may explain AD-related dementia and neurodegeneration better than the amyloid hypothesis, and that the change in Aß42:Aß40 may arise secondarily to the loss of presenilin functions (Shen & Kelleher, 2007). Presenilins are a part of the catalytic subunit of the γ-secretase complex, alongside nicasterin, Pen-2, and Aph1, and are responsible for the proteolytic cleavage of many type I transmembrane proteins such as APP, Notch, and lipoprotein-related protein-1 (LRP) (Francis et al., 2002). To date, more than 180 PS1 mutations have been identified that lead to FAD (Cacquevel, Aeschbach, Houacine, & Fraering, 2012). Clinical PS1 mutations do lead to an increase in the relative amount of Aß42 compared to Aß40 (De Strooper, 2007); however, PS1 mutations also lead to impairments in several other pathways such as Notch signaling (Song et al., 1999) and calcium signaling (Mattson, Chan, & Camandola, 2001). The role of impaired Notch signaling associated with FAD causing PS1 mutations remains unclear.  Unlike the majority of FAD-causing APP mutations which are clustered around the Aß domain, PS1 mutations are scattered throughout the entire presenilin gene in amino acid sequences that are well conserved across species (De Strooper, 2007). This leads to difficulties in understanding how these mutations lead to similar neuropathological consequences. One of the earliest PS1 mutations found is PS1C410Y, discovered in 1995, which leads to FAD age of onset ranging from 48-56 years (Poorkaj et al., 1998; Sherrington et al., 1995). Similar to other PS1 mutations, PS1C410Y increases the Aß42:Aß40 ratio and impairs Notch-1 cleavage in mouse cells (Nakajima, Shimizu, & Shirasawa, 2000) as well as Notch signaling in C. elegans (Baumeister et                                                                                                                                             51  al., 1997).  In 2010, a novel PS1 deletion mutation, PS1∆S169, was discovered in a Chinese family with FAD in their early 40s (Guo et al., 2010). Later, Dr. Weihong Song’s lab at UBC discovered that this mutation impairs APP processing thus increasing Aß42:Aß40 ratio and leads to amyloid plaque formation in mouse brains, but does not affect Notch signaling (S. Zhang et al., 2018). Based on this data, I have assumed that PS1∆S169 impacts the same Notch pathway, as no experiments were conducted to directly measure C.elegans’ Notch levels.  Notch signaling is well known for its role in development and cell fate specification. Many studies report that Notch signaling plays non-developmental roles in the adult nervous system of many species including mammals, C. elegans, and Drosophila. Impairments in Notch signaling impacts spatial learning, memory, and long term potentiation (Chao, Larkins-Ford, Tucey, & Hart, 2005; Ge et al., 2004). Notch receptors are activated by binding ligands that contain DSL domains, leading to their cleavage by a-secretase and the translocation of the NICD to the nucleus. Next, the NICD works with a transcription factor known as LAG-1 in C. elegans to activate target genes (Fortini, 2009). The two Notch receptors in C. elegans are known as LIN-12 and GLP-1. These two receptors play both unique and redundant roles in the development of C. elegans (Greenwald, 2005). For example, LIN-12 is important in the development of somatic gonad and GLP-1 is important for germ cell proliferation (Singh et al., 2011). Both receptors are type I transmembrane proteins containing multiple epidermal growth factor (EGF)-like motifs (Greenwald, 2005). Ligands for both of these receptors are a part of the DSL family, also containing EGF motifs. There are 10 genes in C. elegans that encode members of the DSL ligand family, including osm-7 and osm-11 (Table 3.1) (Chen & Greenwald, 2004).  In her Master’s Thesis Tahereh Bozorgmehr (2015) showed that Psel-12::PS1 rescued the sel-12 chemotaxis deficits (Figure 2.2). I replicated that chemotaxis result and extended it to show                                                                                                                                             52  that Psel-12::PS1 also rescued the locomotion and dispersal phenotype. Bozorgmehr (2015) also tested whether 2 mutations found in FAD families (Psel-12::PS1∆S169 and Psel-12::PS1C410Y) would rescue the chemotaxis deficits of a mutation in sel-12. She found that Psel-12::PS1, and Psel-12::PS1∆S169 but not Psel-12::PS 1 C410Y, were able to rescue chemotaxis impairments in sel-12 mutant worms (Figures 3.1-3.3) suggesting a possible role for Notch in the chemotaxis deficits we observed.  The aim in this chapter was to identify in which cells the expression of sel-12 is required for proper octanol chemotaxis. One of the advantages of C. elegans is the ability to use specific promoters to express target genes in only the nervous system or only in neurons of interest.  In C. elegans, volatile repellants such as octanol are detected by the paired ASH neurons (Hart & Chao, 2010), therefore I first showed that nervous system expression would rescue the chemotaxis deficits in sel-12 worms and then did a series of experiments in which I rescued PS1 in the ASH neurons only. To provide evidence that restored olfaction was a result of cell-specific expression of PS1 and not just normal PS1 anywhere in the animal, another strain was generated in which PS1 was expressed in another pair of chemosensory neurons, the AWA neurons, responsible for detecting the attractive odorant diacetyl. Finally, the role of potential interactions between PS1 and Notch in the chemotaxis deficits was briefly explored. In C. elegans, there are 2 Notch receptors, glp-1 and lin-12. Worms with mutations in either one of them are viable, however the double mutation is lethal.  Thus in order to study chemotaxis in the absence of Notch, heat shock promoter driven RNAi was used to knock down glp-1 in adult worms with a lin-12(null) mutation as originally described in Singh et al. (2011). 			                                                                                                                                            53  Table 3.1 Orthologues of the core components of the LIN-12/Notch pathway among C. elegans and mammals. Table adopted from Greenwald, Wormbook. No permission required as figure is available from creative commons.   Role C. elegans Mammals Ligand OSM-11, OSM-7, LAG-2, APX-1, DSL-1 and 5 others Jagged, Delta-like, 4 others Receptor LIN-12, GLP-1 Notch 1-4 Site 2 cleavage SUP-17, ADM-4 (Adam-10) TACE Site 3 cleavage  SEL-12, HOP-1, APH-1, APH-2, PEN-2 Presenilin 1, 2 APH-1 Nicastrin PEN-2 Nuclear complex LAG-1, SEL-8 CBF1, RBP-J Mastermind-like   Figure 3.1 Expression of Psel-12::PS1 in sel-12 mutant Caenorhabditis elegans chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, Psel-12::PS1 rescue worms using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=30-60 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference (p<0.05) from wild-type worms. Figure adapted from T. Bozorgmehr, University of British Columbia Thesis. Permission obtained.                                                                                                                                             54   Figure 3.2 Expression of Psel-12::PS1C410Y in sel-12 mutant Caenorhabditis elegans chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Psel-12::PS1C410Y rescue worms using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=30-60 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant difference (p<0.05) from wild-type worms. Figure adapted from T. Bozorgmehr, University of British Columbia Thesis. Permission obtained.                                                                                                                                                 55   Figure 3.3 Expression of Psel-12::PS1Δs169 in sel-12 mutant Caenorhabditis elegans chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Psel-12::PS1Δs169 rescue worms using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=30-60 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference (p<0.05) from wild-type worms. Figure adapted from T. Bozorgmehr, University of British Columbia Thesis. Permission obtained.                                                                                                                                                     56  3.2 Methods  3.2.1 Generation of transgenic lines and strain maintenance  Please refer to Chapter 2, section 2.2.  Plasmids containing PS1Δs169 and PS1C410Y were provided by Dr. Weihong Song’s lab at UBC. The HA1712 strain containing osm-11(null) and a glp-1 RNAi activated by a heat shock promoter was provided by Dr. Anne Hart’s lab at Brown University. Three independent lines for each construct were generated.  Strains: Ptag-168 is a nervous system specific promoter (Saheki & Bargmann, 2009) , Psra-6 is an ASH neuron promoter (Troemel, Chou, Dwyer, Colbert, & Bargmann, 1995), and Podr-10 is an AWA neuron specific promoter (Sengupta, Chou, & Bargmann, 1996). Note that for the purpose of imaging the injection marker was changed from GFP (green fluorescent marker) to mCherry (red fluorescent marker) in strains that had GFP expressed in their ASH neurons (further explored in chapter 4).  The following strains were created: VG559 sel-12 (ok2078); Ptag-168::sel-12 wt Punc-122:: GFP  VG560 sel-12 (ok2078); Ptag-168::sel-12 wt Punc-122:: GFP VG561 sel-12 (ok2078); Ptag-168::sel-12 wt Punc-122:: GFP VG601 sel-12 (ok2078); Ptag-168::PS1 wt Punc-122:: GFP VG602 sel-12 (ok2078); Ptag-168::PS1 wt Punc-122:: GFP VG603 sel-12 (ok2078); Ptag-168::PS1 wt Punc-122:: GFP VG734 sel-12 (ok2078); Ptag-168::PS1Δs169 Punc-122:: GFP VG735 sel-12 (ok2078); Ptag-168::PS1Δs169 Punc-122:: GFP                                                                                                                                             57  VG736 sel-12 (ok2078); Ptag-168::PS1Δs169 Punc-122:: GFP VG697 sel-12 (ok2078); Ptag-168::PS1C410Y  Punc-122:: GFP VG698 sel-12 (ok2078); Ptag-168::PS1C410Y  Punc-122:: GFP VG699 sel-12 (ok2078); Ptag-168::PS1C410Y  Punc-122:: GFP VG669 sel-12 (ok2078); Psra-6::sel-12 wt Punc-122:: GFP VG670 sel-12 (ok2078); Psra-6::sel-12 wt Punc-122:: GFP VG671 sel-12 (ok2078); Psra-6::sel-12 wt Punc-122:: GFP VG680 sel-12 (ok2078); Podr-10::sel-12 wt Punc-122:: GFP VG681 sel-12 (ok2078); Podr-10::sel-12 wt Punc-122:: GFP VG682 sel-12 (ok2078); Podr-10::sel-12 wt Punc-122:: GFP VG675 sel-12 (ok2078); Psra-6::PS1 wt Punc-122:: GFP VG676 sel-12 (ok2078); Psra-6::PS1 wt Punc-122:: GFP VG677 sel-12 (ok2078); Psra-6::PS1 wt Punc-122:: GFP VG691 sel-12 (ok2078); Podr-10::PS1 wt Punc-122:: GFP VG692 sel-12 (ok2078); Podr-10::PS1 wt Punc-122:: GFP VG693 sel-12 (ok2078); Podr-10::PS1 wt Punc-122:: GFP VG822 sel-12 (ok2078); Psra-6:: PS1Δs169 Pmyo-3:: mCherry VG823 sel-12 (ok2078); Psra-6:: PS1Δs169 Pmyo-3:: mCherry VG824 sel-12 (ok2078); Psra-6:: PS1Δs169 Pmyo-3:: mCherry VG843 sel-12 (ok2078); Podr-10:: PS1Δs169 Pmyo-3:: mCherry VG844 sel-12 (ok2078); Podr-10:: PS1Δs169  Pmyo-3:: mCherry  VG845 sel-12 (ok2078); Podr-10:: PS1Δs169 Pmyo-3:: mCherry                                                                                                                                              58  VG819 sel-12 (ok2078); Psra-6:: PS1C410Y  Pmyo-3:: mCherry VG820 sel-12 (ok2078); Psra-6:: PS1C410Y Pmyo-3:: mCherry VG821 sel-12 (ok2078); Psra-6:: PS1C410Y Pmyo-3:: mCherry VG840 sel-12 (ok2078); Podr-10:: PS1C410Y Pmyo-3:: mCherry VG841 sel-12 (ok2078); Podr-10:: PS1C410Y Pmyo-3:: mCherry VG842 sel-12 (ok2078); Podr-10:: PS1C410Y Pmyo-3:: mCherry 3.2.2 Chemotaxis Assay  FUdR was used in these aging experiments to maintain synchronous cultures of aged animals. For details on FUdR, please refer to section 2.2.2. For details of the chemotaxis assay protocol, please refer to chapter 2, section 2.2. Note that in this chapter, to test for cell-specificity in neuron-specific rescue worms, ASH and AWA rescue strains were tested on both the repellent odorant octanol and the attractant odorant diacetyl. The diacetyl solution was prepared by diluting it to 0.5% diacetyl using 99.5µl of M9 and 5µl of diacetyl. A 1:1 ratio of this 0.5% diacetyl solution was then added to NaAz. Control solution was prepared by mixing equal volumes of M9 and NaAz.   3.2.3 Statistical Analysis  Please refer to section 2.2.4.  3.2.3 Mutations of both Notch receptors via heat shock and developmental stage synchronization:  Heat shock promoter driven RNAi was used to knock down glp-1 in adult worms with a lin-12(null) mutation (strain name: HA1712). These animals with a heat shock promoter driving                                                                                                                                             59  glp-1 RNAi were raised at 15°C, the permissive temperature, and moved to 33°C, the restrictive temperature, for 2 hours as described in Singh et al. (Singh et al., 2011). After this, animals were allowed to recover for 3 hours in a 20°C incubator prior to chemotaxis experiments that were conducted in a 20°C behavior room.  HA1712: lin-12 (n941) null allele and glp-1(q231) temperature sensitive loss of function (tslf).  Note that Notch mutant worms have developmental delays compared to wildtype. To test worms at similar stages in development, wild-type and lin-12/glp-1 mutant worms were closely observed every 4 hours to determine when these strains reached young adulthood (defined as containing at least 4 eggs) (Chao et al., 2005). Compared to wild-type worms, worms with a mutation in the Notch receptor had a 6-hour lag in development (reached young adults at 68 hours in 20°C).  3.3 Results 3.3.1 Nervous system expression of wildtype sel-12, wildtype PS1, and PS1Δs169, but not PS1C410Y, rescued chemotaxis deficits in sel-12 mutant worms.  Throughout the life cycle of a worm sel-12 is expressed in all cells. We previously showed that ubiquitous expression of Psel-12::PS1 and Psel-12::PS1Δs169 rescued chemotaxis deficits in worms with a sel-12 mutation (Bozorgmehr, 2015). However, ubiquitous expression of Psel-12::PS1C410Y did not rescue chemotaxis deficits. In order to understand the role of PS1 in octanol chemotaxis, the experiments described here were designed to test whether  the expression of either sel-12 or PS1 in only the nervous system was sufficient for normal chemotaxis.  Three extrachromosomal lines were generated in which wildtype sel-12 was expressed under the control of a pan-neuronal promoter (tag-168) in sel-12 mutant worms (Figure 3.4). Strains tested were wild-type, sel-12 mutant, and 3 lines of sel-12 mutant worms expressing Ptag-168::sel-12 in only the neurons. There was a statistically significant difference between groups as                                                                                                                                             60  determined by one-way ANOVA [F (4,15) = 18.70, p<0.01]. Chemotaxis results indicated that seventy-two hour old sel-12 mutant animals showed a significantly decreased response to octanol compared to wildtype animals (p<0.01). The three Ptag-168::sel-12 rescue lines had CI indices that were not significantly different from wildtype worms in response to octanol (p values=1.00, 0.98, 0.97), and had a significantly increased chemotaxis ability compared to sel-12 mutant worms (p<0.01). Three extrachromosomal lines were generated in which wildtype human PS1 was expressed under the control of a pan-neuronal promoter (Ptag-168::PS1). Strains tested were wild-type, sel-12 mutant, and 3 lines of sel-12 mutant worms expressing Ptag-168::PS1  in only the neurons (Figure 3.5) . A one way ANOVA indicated a statistically significant difference between groups [F (4,15) = 14.70, p<0.01]. Chemotaxis results indicated that two out of three Ptag-168::PS1 lines rescued chemotaxis deficits in sel-12 mutant worms (Figure 3.5). Replicating previous results, sel-12 mutant worms had a significantly decreased chemotaxis to octanol compared to wildtype worms (p<0.01). Two Ptag-168::PS1 rescue lines were not significantly different from wildtype (p values = 0.18, 0.08). However, one Ptag-168::PS1 rescue line  showed a significant difference compared to both wildtype (p=0.03) and sel-12 mutant worms (p=0.01) and therefore I concluded that this line showed only a partial rescue for octanol chemotaxis deficits in sel-12 mutant worms. I next expressed a canonical FAD PS1 gene mutation (PS1C410Y) in the nervous system of sel-12 mutant worms to investigate its effect on chemotaxis. Three extrachromosomal lines were generated with Ptag-168::PS1 C410Y which did not rescue chemotaxis deficits in sel-12 mutant worms (Figure 3.6). Strains tested were wild-type, sel-12  mutant, and 3 lines of Ptag-168::PS1C410Y. A one way ANOVA indicated a statistically significant difference between groups                                                                                                                                             61  [F (4,15) = 21.33, p<0.01]. Out of the three Ptag-168::PS1 C410Y  rescue lines, all showed CI values that were not significantly different from CI values of sel-12 mutant worms (p values 0.13, 0.10, 0.10), but were significantly difference compared to wild-type worms (p<0.01).  Finally, I tested whether the novel FAD mutation, PS1Δs169, had an effect on worm’s chemotaxis. Three extrachromosomal lines Ptag-168::PS1 Δs169 were also generated which rescued chemotaxis deficits in sel-12 mutant worms (Figure 3.7). Strains tested were wild-type, sel-12 mutant, and 3 lines of sel-12 mutant worms expressing Ptag-168::PS1 Δs169. A one way ANOVA indicated a statistically significant difference between groups  [F (4,15) = 40.44, p<0.01]. The results of this assay was that, as expected, sel-12 mutant worms had significant chemotaxis deficits compared to wildtype (p<0.01) and that two out of three extrachromosomal Ptag-168::PS1 Δs169 rescue lines demonstrated no significant differences compared to wildtype worms (p values = 0.85, 0.97), but were significantly different from sel-12 mutant worms (p <0.01). The third Ptag-168::PS1 Δs169  rescue line had a CI that showed significant differences compared to both wildtype (p=0.04) and sel-12 mutant worms (p<0.01).                                                                                                                                                    62   Figure 3.4 Expression of Ptag-168::sel-12 chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Ptag-168::sel-12 using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference (p<0.05) from wild-type and rescue lines.  Figure 3.5 Expression of Ptag-168::PS1 chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Ptag-168::PS1 using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Single asterisk shows significant difference from wild-type and rescue lines, double asterisk shows significant difference from wild-type and sel-12 mutant (p<0.05).                                                                                                                                              63   Figure 3.6 Expression of Ptag-168::PS1C410Y  chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Ptag-168::PS1C410Y using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Single asterisks show significant difference from wild-type  (p<0.05).   Figure 3.7 Expression of Ptag-168::PS1Δs169 chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Ptag-168::PS1Δs169 using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Single asterisk shows significant difference from wild-type and rescue lines, double asterisk shows significant difference from wild-type and sel-12 mutant (p<0.05).                                                                                                                                             64  3.3.2 The chemotaxis deficit was independent of any motor deficits due to the sel-12 egl phenotype.   One possibility is that the chemotaxis deficits observed in the sel-12 mutant worms were the result of locomotor effects of the buildup of eggs inside the worms, however, when PS1 is expressed in only the nervous system, the worms egg laying deficit is not rescued, and their crawling speed is slower than wild-type worms. Therefore, I next tested sel-12 mutant worms with wildtype PS1 expression under the control of Ptag-168, a pan neuronal promoter (Figure 3.8). At all ages, all three wildtype PS1 nervous system rescue lines were able to restore chemotaxis deficits in sel-12 mutant worms (88-hour old worms F (4, 15) = 23.53, p<0.01; 98-hour old worms F (4,15) = 12.24, p<0.01; 108-hour old worms F (4,15) = 23.50, p<0.01).  Figure 3.9 shows the forward speed of wildtype worms (black and grey lines), sel-12 mutant worms (red lines), and nervous system PS1 rescue lines (green lines). This figure shows that the black and green lines do not overlap, and only one of the three nervous system specific wildtype PS1 lines partially rescues locomotion speed. All three rescue lines were able to fully restore dispersal defects at 88 hours, but only partially at 98 and 108 hours (Figure 3.10), indicating that the egg-laying deficit was not responsible for the chemotaxis deficit observed in sel-12 mutant worms.                                                                                                                                                    65   Figure 3.8 Nervous system expression of wild-type PS1 in worms with a sel-12 mutation chemotaxis assay over time: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Ptag-168::PS1 using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant difference (p<0.05) from wild-type of same age.                                                                                                                                                       66   Figure 3.9 Average forward movement speed groups of 88-108 hour old wild-type, sel-12 mutant, and nervous system PS1 rescues during 250 seconds of tracking. Values are averages from 3 independent plates (n=50 worms per plate).                                                                                                                                                        67   Figure 3.10 88-108 hour old wild-type (A), sel-12 mutant (B), and nervous system PS1 rescue (C) worm tracks on a plate over 250 seconds. Different colors indicate various tracking time points after a brief air-puff. (n=50 worms per plate). Note that in the rescue panels (C), only one of the three extrachromosomal rescue lines is shown as all followed the same trend.                                                                                                                                                  68  3.3.3 ASH neuron expression of wildtype sel-12, wildtype PS1, and PS1 Δs169, but not PS1C410Y, rescued chemotaxis deficits in sel-12 mutant worms.  In C. elegans, the ASH neurons are  considered polymodal nociceptors that detect a number of noxious and potentially toxic stimuli including octanol (Bargmann, 2006). C. elegans shows an avoidance response to octanol mediated by ASH activation (Bargmann, 2006). To determine whether presenilin played a role in chemotaxis by acting in just the sensory neurons, I expressed sel-12 and wild-type PS1, PS1Δs169 and PS1C410Y using the sra-6, a promoter that is strongly expressed in the ASH neurons, and weakly expressed in the ASI neurons and PVQ interneurons (Troemel et al., 1995).    Three extrachromosomal lines were generated in which wildtype sel-12 was expressed under the control of the sra-6 promoter (Figure 3.11). Strains tested were wild-type, sel-12 mutant, and 3 lines of sel-12 mutant worms expressing Psra-6::sel-12. Worms with a mutation in sel-12 showed chemotaxis impairments in response to octanol compared to wildtype [F (4,15) = 17.30, p<0.01]. Two out of the three Psra-6::sel-12 lines restored chemotaxis impairments in sel-12 mutant worms (p values compared to wildtype = 0.11, 0.38) and had chemotaxis indices that were significantly different from sel-12 mutant worms (p values <0.01) (Figure 3.11). However, one Psra-6::sel-12 line had a CI that was significantly different from wild-type (p<0.01) but not sel-12 mutant worms (p = 0.27).  Next, three extrachromosomal lines were generated in which wildtype human PS1 was expressed under the control of the sra-6 promoter (Figure 3.12). Strains tested were wild-type, sel-12 mutant, and 3 lines of sel-12 mutant worms expressing Psra-6::PS1. All three Psra-6::PS1 lines restored the chemotaxis impairments observed in sel-12 mutant worms [F(4,15) = 12.67, p<0.01] (Figure 3.12). Wild-type and sel-12 mutant worms had significantly different CIs (p<0.01).                                                                                                                                             69  The three wildtype PS1 rescue lines had similar CIs to wildtype worms (p values 0.33, 0.15, 0.45) and were significantly different from sel-12 mutant worms (p values <0.01).   I then expressed a canonical FAD PS1 gene mutation (PS1C410Y) in the ASH neurons [F (4,15) = 15.57, p<0.01] (Figure 3.13). Strains tested were wild-type, sel-12 mutant, and 3 lines of sel-12 mutant worms expressing Psra-6:: PS1C410Y. None of the Psra-6: PS1C410Y lines restored chemotaxis deficits in sel-12 mutant worms (p values 0.07, 0.08, 0.07)  and all rescue lines had a significantly lower CI than wildtype (p<0.01).  Finally, I expressed PS1Δs169 in the ASH neurons. Strains tested were wild-type, sel-12 mutant, and 3 lines of sel-12 mutant worms expressing Psra-6:: PS1Δs169. In contrast to Psra-6: PS1C410Y , Psra-6:: PS1Δs169 lines restored chemotaxis deficits in sel-12 mutant worms [F (4,15) = 13.27, p value <0.01] (Figure 3.14). All rescue lines had comparable CIs to wildtype worms  (p values 0.10, 0.84, 0.10) and were significantly different from sel-12 mutant worms (p values <0.01).                                                                                                                                                   70   Figure 3.11 Expression of Psra-6::sel-12  in sel-12 mutant Caenorhabditis elegans chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Psra-6::sel-12 using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Single asterisk shows significant difference from wild-type and rescue lines, double asterisk shows significant difference from wild-type, and remaining rescue lines (p<0.05).   Figure 3.12 Expression of Psra-6::PS1 in sel-12 mutant Caenorhabditis elegans chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Psra-6::PS1 using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference from wild-type and rescue lines (p<0.05).                                                                                                                                              71   Figure 3.13 Expression of Psra-6::PS1C410Y in sel-12 mutant Caenorhabditis elegans chemotaxis assay:: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Psra-6::PS1C410Y using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant difference from wild-type (p<0.05).   Figure 3.14 Expression of Psra-6::PS1Δs169 in sel-12 mutant Caenorhabditis elegans chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Psra-6::PS1 Δs169 using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference from wild-type and rescue lines (p<0.05).                                                                                                                                              72  3.3.4 Wildtype PS1 rescue lines in the ASH neuron had a cell-specific effect on chemotaxis.    Although wildtype PS1 in ASH was sufficient to rescue octanol chemotaxis, it is possible that the rescue was not cell-autonomous, but that expression of PS1 anywhere in the animal would rescue the phenotype. To rule out this hypothesis, I expressed wildtype PS1 driven by an odr-10 promoter in another pair of chemosensory neurons, the AWA neurons, that are responsible for chemotaxis towards that attractant diacetyl. I then tested chemotaxis away from octanol in worms with wildtype PS1 in the AWA neurons of sel-12 mutant worms. This then allowed me to also test  the effect of expressing wildtype PS1 in ASH and AWA in a diacetyl chemotaxis assay.   Three extrachromosomal lines were generated in which wildtype PS1 was expressed in the AWA neurons under the control of the odr-10 promoter. Strains tested were wild-type, sel-12 mutant, and 3 lines of sel-12 mutant worms expressing odr-10:: PS1. As shown in figure 3.15 [F (4,15) = 13.00, p<0.01], expression of wildtype PS1 in the AWA neurons did not rescue chemotaxis impairments towards octanol. Moreover, all PS1 rescue lines in AWA had similar CIs compared to sel-12 mutant worms in response to octanol (p values 1.00, 0.39, 0.98), but were significantly different from wildtype (p values <0.01).  Expression of wildtype PS1 in the AWA neurons rescued the chemotaxis deficit in response to diacetyl in sel-12 mutant worms [F (4,15) = 22.19, p<0.01] (Figure 3.16). Strains tested were wild-type, sel-12 mutant, and 3 lines of sel-12 mutant worms expressing odr-10:: PS1. All odr-10:PS1 lines had significantly different CIs compared to sel-12 mutant worms in response to diacetyl (p values <0.01), but were similar to wildtype worms (p values 0.84, 0.80, 0.37).  Lastly, expression of wildtype PS1 in the ASH neurons did not rescue sel-12 mutant worm’s chemotaxis response towards diacetyl [F (4,15) = 9.14, p<0.01] (Figure 3.17). Strains tested were wild-type, sel-12 mutant, and 3 lines of sel-12 mutant worms expressing sra-6:: PS1.                                                                                                                                             73  All sra-6:: PS1 lines had a similar chemotaxis response to diacetyl compared to sel-12 mutant worms (p values 1.00) and were significantly different from wildtype (p values <0.01). These data indicate that expression of PS1 in ASH rescued chemotaxis for octanol sensed by ASH, but not for diacetyl sensed by different sensory neurons; at the same time expression of PS1 in the AWA sensory neurons did not rescue chemotaxis to octanol, but did rescue chemotaxis to diacetyl.    Figure 3.15 Expression of Pod-10::PS1 in sel-12 mutant Caenorhabditis elegans chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Podr-10::PS1 using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant differences from wild-type (p<0.05).                                                                                                                                              74   Figure 3.16 Expression of Podr-10::PS1 in sel-12 mutant Caenorhabditis elegans chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Podr-10::PS1 using either diacetyl (attractive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference from wild-type (p<0.05).   Figure 3.17 Expression of Psra-6::PS1 in sel-12 mutant Caenorhabditis elegans chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, Psra-6::PS1 rescues in ASH using either diacetyl (attractive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant differences from wild-type (p<0.05).                                                                                                                                              75  3.3.5 C. elegans with mutations in Notch receptors do not show increased chemotaxis deficits over time.  Like APP, Notch receptors are cleaved and activated by the presenilins of g-secretase. In the past, Singh et al. reported that the two Notch receptors, lin-12 and glp-1, regulate C. elegans chemosensory avoidance behavior in response to octanol (Singh et al., 2011). They found that a knockdown of glp-1 in a lin-12 null background led to behavioral impairments in response to octanol. I investigated whether the octanol chemosensory deficit observed in Notch mutants showed the same characteristics as the sel-12/PS1 mutation. As single allele mutations did not lead to chemotaxis impairments, to study this, I used the same Notch mutant strain used by Singh et al. in which heat shock promoter driven glp-1 was knocked down in adult lin-12 mutants, and tested the chemotaxis to octanol of this strain over time to determine whether these deficits increased in the same way as did chemotaxis in the sel-12 mutant worms.  Strains tested were wildtype worms, sel-12 mutant worms, and a strain with a heat shock promoter driven glp-1 knockout in lin-12 mutants (Figure 3.18). Both sel-12 mutant worms and lin-12/glp-1 mutant worms had a significantly lower CI compared to wildtype worms (p values <0.01). Figure 3.19 shows that compared to age-matched wild-type worms, C. elegans with lin-12/glp-1 reduction of function did not show an increase in their chemotaxis impairments towards octanol over time (p values = 1.00). This is in contrast with sel-12 mutant worms that demonstrated an increased chemotaxis deficit with time (p values <0.01). These findings suggest that Notch associated chemotaxis deficits do not phenocopy all of the characteristics of mutations in sel-1.                                                                                                                                                76   Figure 3.18. RNAi knockdown of glp-1 in a lin-12 null background chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and lin-12/glp-1 knockout using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant differences from wild-type (p<0.05).                                                                                                                                               77   Figure 3.19 Chemotaxis experiments on worms with RNAi knockdown of glp-1 in a lin-12 null background: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and lin-12/glp-1 knockout worms using either octanol (attractive odorant) as a repellant or M9 (odorless buffer) as control. Values are averages from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant differences from 78 hour old sel-12 mutant worms (p<0.05).                                                                                                                                                        78  3.4 Discussion In this chapter, I showed that nervous system and neuron specific rescues of wildtype sel-12, wildtype PS1, and the atypical  PS1Δs169 mutation restored chemotaxis impairments to octanol in sel-12 mutant worms. However, expression of the canonical PS1C410Y mutation did not rescue these impairments. To my knowledge, PS1Δs169 is the first known mutation in PS1 that does not impact Notch signaling. The observation that PS1Δs169 did rescue the chemotaxis deficit suggests a role for PS1 cleavage of  Notch in the sel-12 chemotaxis deficit. I investigated this by testing chemotaxis away from octanol in worms with reduction of Notch signaling across aging.  Like sel-12 mutant worms, worms with reduced Notch functioning showed deficits in octanol chemotaxis (p values <0.01, Figure 3.18). Unlike sel-12 mutations, Notch mutant worms did not show an increased chemotaxis deficits with time (Figure 3.19). This suggested the possibility of a Notch independent role of sel-12 in chemotaxis deficits.  Because of the impacts that PS1Δs169 and PS1C410Y had on the worm’s nervous system and thus chemotaxis, I compared the sequences of these mutant alleles to sel-12 to determine whether they were in regions of the gene conserved from C. elegans to humans. PS1Δs169 leads to a serine deletion, and serines are critical in the biosynthesis of both purines and pyrimidines. Likewise, PS1C410Y leads to a point mutation in a cysteine residue, and cysteines are critical amino acids that help stabilize proteins’ secondary structures via hydrogen bonding. Cysteines tend to be highly conserved across species. As shown in Figure 3.16, both of these presenilin mutations are in residues conserved in C. elegans sel-12.  When I examined the locomotion of worms expressing Ptag-168::PS1 pan neuronally (nervous system only) in sel-12 mutant worms, I found that this was sufficient to rescue chemotaxis impairments over time, but not speed of locomotion, and thus concluded that the chemotaxis deficit                                                                                                                                             79  was unrelated to speed. If C. elegans expressing PS1 in their nervous system move as slowly as sel-12 mutants at 98 hours and 108 hours old, and they can still navigate away from the aversive odorant octanol, this suggests that sel-12 mutants would avoid the odorant if they could detect it.  In some of my experiments, there was variability in the transgenic lines and all three sel-12/PS1 rescue lines did not restore the chemotaxis deficits in sel-12 mutant worms (Figures 3.5, 3.6, 3.8). This is probably due to the variable expression pattern of the extrachromosomal arrays. These arrays usually have a high expression pattern (overexpression) with some mosaicism, and it is very likely that each rescue line expressed  sel-12/PS1 at different levels (Chao, Komatsu, Fukuto, Dionne, & Hart, 2004). Although I have shown functional conservation in sel-12/PS1 role in octanol chemotaxis, this type of rescue is a limitation in this study, as it may reflect the importance of the level of PS1 expression in the worms to rescue chemotaxis impairments. This is not surprising as Levitan et al. showed that level of PS1 expression was important in rescuing the egg laying deficit (Levitan et al., 1996). One solution to the overexpression issue would be to use the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system to replace PS1 in a single copy at the exact correct position of se1-12 in worms.     My experiments suggested that sel-12/PS1 cell specific expression in C. elegans’ sensory neurons rescued the worm’s chemotaxis to an odorant detected by that neuron. By expressing sel-12/PSI in either the octanol detecting neuron (ASH) or diacetyl detecting neuron (AWA), my experiments demonstrated that sel-12/PS1 rescue chemotaxis deficits in a cell-specific manner.   I also tested the chemotaxis abilities of worms with nervous system and neuron specific PS1Δs169 and PS1C410Y  rescues and showed that, similar to wild-type PS1, PS1Δs169 also rescued chemotaxis deficits in worms. In contrast, PS1C410Y did not rescue the chemotaxis deficits. One of the major functional difference between these two mutations is the fact that PS1Δs169 is the only                                                                                                                                             80  known presenilin mutation that does not impact Notch signaling (Zhang, 2013). Therefore, I tested a C. elegans strain with decreased Notch signaling for changes in chemotaxis ability over time. In the future, I will test neuron specific rescues of both PS1Δs169 and PS1C410Y over time to determine if PS1Δs169 shows a similar pattern to wildtype worms and whether PS1C410Y shows a similar pattern to sel-12 mutant worms.  To activate LIN-12 and GLP-1 Notch receptors in C. elegans, 10 different ligands function in conjunction with the DSL ligand LAG-2 and regulates avoidance to octanol. The LIN-12 Notch receptor is essential for the proper spontaneous backward crawling of the worm through the RIG interneurons (Chao et al., 2005). From the data reported here, it is clear that both sel-12 and Notch play important roles in worm olfaction, but differences in the increased impairment over time suggested that mutations in sel-12 may also lead to olfactory impairments via a Notch independent pathway.  In conclusion, although PS1 is the most commonly mutated FAD-causing gene, its role in AD-associated olfactory deficits, one of the first pre-clinical symptoms of AD, remains unknown. In this chapter, I presented compelling evidence that C. elegans can be used to study non-Aß cell-specific roles of presenilins in olfaction and neuronal function. Investigating the cellular mechanisms of this chapter’s results further could lead to potential therapeutic targets.                                                                                                                                                 81   Figure 3.20 Amino acid conservation of PS1Δs169 and PS1C410Y between C. elegans’ sel-12 and human PS1.                                                                                                                                                       82  Chapter 4: sel-12 mutant C. elegans demonstrate ASH neuron morphological abnormalities that are rescued by wildtype PS1 and PS1Δs169, but not PS1C410Y. 4.1 Introduction  One of the key pathological changes in the brains of patients with AD is the gradual degeneration of neurons and the loss of synaptic connections. In this chapter I will investigate whether a sel-12 mutation lead to neuronal degeneration in C. elegans. Though the precise mechanisms that lead to neuronal degeneration in AD remain unknown, many studies have focused on the role of APP products. The amyloid hypothesis suggests that Aß aggregates are toxic to neurons and thus Aß is considered to play a central role in neuronal cell death (Niikura, Tajima, & Kita, 2006). But how does Aß kill neurons? It has been suggested that hydrophobic Aß oligomers bind to cellular membranes and trigger cytotoxic pathways that involve an increase in intracellular calcium levels, oxidative stress, and receptor mediated activation of apoptosis (LaFerla, 2002; Niikura et al., 2006). Moreover, Aß alters many kinases’ activities and leads to tau protein hyperphosphorylation, which in turn causes neurofibrillary tangle formation (Niikura et al., 2006). Together, these toxicities are thought to be Aß initiated, leading to neuronal death. A caveat for this interpretation is that the concentration of Aß required to kill cells in experimental assays are much higher than physiological levels (Vickers et al., 2000). Further, concentration of soluble Aß, but not Aß plaques, are what distinguishes AD brains from control brains of those without dementia but with high levels of Aß deposition, supporting the hypothesis that it may be soluble Aß that plays a role in neurodegeneration (Lue et al., 1999).  The first aggregates of Aß and neurofibrillary tangles are found in the hippocampal formation and the olfactory bulb (OB), which is thought to correlate to memory and olfactory impairments (De la Rosa-Prieto, Saiz-Sanchez, Ubeda-Banon, Flores-Cuadrado, & Martinez-                                                                                                                                            83  Marcos, 2016). Further, mutations in proteins such as APP and presenilins which are highly implicated in AD commonly lead to neurogenic development (De la Rosa-Prieto et al., 2016). In our lab, we have found that C. elegans with mutations in sel-12, the homologue of human PS1, have chemotaxis deficits in response to the aversive odorant octanol and the attractive odorant diacetyl and that expression of PS1 in a single pair of neurons can rescue the deficit. The focus of this chapter is to determine whether or not there is an association between the morphology of C. elegans’ neurons responsible for the detection of these two odorants and chemotaxis deficits.  C. elegans use their sense of smell to navigate their environment. From the 302 neurons in C. elegans, 16 pairs (32 neurons in total) of anatomically bilaterally symmetric neurons have been identified to be chemosensory (Hart & Chao, 2010). These neurons synapse either directly (such as the ASH neuron) or indirectly to command interneurons which in turn controls the forward (in cases of attractive stimuli) or backwards (in cases of aversive stimuli) movement of the worm (Hart & Chao, 2010). There are four chemosensory organs in C. elegans, known as the amphid, phasmid, inner labial, and outer labial organs. Some of these neurons have cilia that is exposed to the environment that come in contact with the odorant (such as ASH neurons), and some have embedded cilia that work via particle diffusion (such as AWA neurons). Ablation studies have suggested that specific sensory neurons in C. elegans are required for the detection of either attractive or aversive stimuli, but not both (Bargmann & Avery, 1995). Generally, the worms ASE neurons detect soluble attractants, the AWA and AWC neurons detect volatile attractants, and the ASH, ADL, and AWB neurons detect volatile repellants (Hart & Chao, 2010). Specifically, the AWA neurons detect diacetyl and the ASH neurons detect octanol, the two odorants tested in my thesis.                                                                                                                                             84  In chapter 2, I demonstrated that chemotaxis deficits in sel-12 mutant worms worsen with time, similar to AD symptoms. In chapter 3, I showed that the expression of human PS1 in just the ASH neurons was able to rescue chemotaxis deficits to octanol in sel-12 mutant worms. These findings provided me with a target to look more closely at the morphology of the ASH neurons in wildtype worms, sel-12 mutant worms, and ASH specific PS1 rescued worms over time.   4.2 Methods  4.2.1 Generation of transgenic lines and strain maintenance For generation of transgenic lines and strain maintenance, please refer to section 2.2.1. For details of the chemotaxis assay, please refer to section 2.2.2. The following strains were created and tested in this chapter. To make these strains, a strain (HA3; [osm-10::GFP + lin-15(+)]) carrying integrated GFP in its ASH neurons and ASI interneurons was crossed with our wild-type and sel-12 mutant strains prior to injection.   VG789 sel-12 (ok2078); Posm-10::GFP Psra-6:: PS1 wt, Pmyo-3:: mCherry  VG790 sel-12 (ok2078); Posm-10::GFP Psra-6:: PS1 wt Pmyo-3:: mCherry VG791 sel-12 (ok2078); Posm-10::GFP Psra-6:: PS1 wt Pmyo-3:: mCherry VG822 sel-12 (ok2078); Posm-10::GFP Psra-6:: PS1Δs169 Pmyo-3:: mCherry VG823 sel-12 (ok2078); Posm-10::GFP Psra-6:: PS1Δs169 Pmyo-3:: mCherry VG824 sel-12 (ok2078); Posm-10::GFP Psra-6:: PS1Δs169 Pmyo-3:: mCherry VG819 sel-12 (ok2078); Posm-10::GFP Psra-6:: PS1C410Y  Pmyo-3:: mCherry VG820 sel-12 (ok2078); Posm-10::GFP Psra-6:: PS1C410Y Pmyo-3:: mCherry VG821 sel-12 (ok2078); Posm-10::GFP Psra-6:: PS1C410Y Pmyo-3:: mCherry The above strains were made by extrachromosomal injections, where mCherry was used as a marker, into a sel-12 mutant strain with an integrated GFP in the ASH neurons.                                                                                                                                              85  4.2.2 ASH neuron imaging  FUdR was used in these aging experiments to maintain synchronous cultures of aged animals. For details on FUdR, please refer to section 2.2.2.  The osm-10::GFP expressing ASH neurons that detect the octanol odorant were imaged in both wildtype and sel-12 mutant worms over time (78, 98, and 108 hour old worms). For all extrachromosomal rescue strains, the line that had the closest chemotaxis index to wildtype was chosen for imaging. Worms were placed on 2% agar pads on sterile glass microscope slides and immersed in 15uL of 50mM sodium azide which was used to paralyze worms. Worms were given approximately one minute to become paralyzed prior to being covered with a 1.5mm thick coverslip. Images were obtained using a Leica SP8 white light confocal microscope. To excite GFP, a 488nm wavelength laser was used and the emitted light was collected by passing through a 510-550nm bandpass filter. Depending on the thickness of the worm and the brightness of its GFP, optical sections were collected at various intervals using a 63X oil immersion lens and summed into a single Z projected image. A graduate student who was blinded to the nature and hypotheses of the experiment was asked to image 50 wildtype worms, 50 sel-12 mutant worms, and 50 rescue worms (wild-type PS1 rescue in ASH) to confirm my findings.  One hundred worms were imaged per strain per time point (a total of 300 images per strain). After careful observation, the ASH neurons containing GFP driven by osm-10 promoter were placed into three categories: normal, gapped/missing arm, and bleb (Figure 4.1). If after increasing power and performing z-stacks, there were still gaps present in the arms or the nerve ring of the ASH neuron, then they were placed in the gapped arm category (Figure 4.2). The presence of circular structures in the nerve ring or the arms of the ASH neuron lead to their categorization as blebs (Figure 4.2). If gaps and blebs were both present, the neuron would be categorized as blebs.                                                                                                                                              86   In order to determine if the ASH neurons were undergoing degeneration, I imaged 10 ASH neuron pairs of wild-type and sel-12 mutant worms over time from 68-108 hours old. Each worm was placed on a separate agar plate with food and assigned a number. However, because using sodium azide for worm paralysis would delay the post imaging by many hours, I used 10mM levamisole which is a weaker anesthetic agent from which worms recover more quickly. Under these conditions, worms were given 3-5 minutes to become paralyzed prior to imaging.    Figure 4.1 ASH sensory neuron morphology in worms: A. a 72-hour old adult worm, white circle shows the head where the ASH neuron resides. B. A close up image of the worm’s amphid with the green fluorescent depicting the ASH neuron. C. A wild-type worm’s ASH neuron pair. The cilia of the ASH neuron protrude outwards and come in contact with the odorant, send signals through the dendrite to the cell body and axon and eventually the nerve ring. The nerve ring is a horseshoe shaped axon bundle that makes synaptic connections with other neurons.                                                                                                                                                 87   Figure 4.2 ASH sensory neuron morphology categories: A. an ASH neuron pair with normal morphology. B. an ASH neuron pair with the presence of gaps in one of its dendrites and axons. C. an ASH neuron pair with the presence of both gaps and circular blebs in its dendrites and axons (in the case of both blebs and gaps, the neuron was categorized as blebs).  4.2.3 C. elegans Dye-filling staining assay   To confirm the GFP observations, a second imaging technique (DiI Dye filling assay) was used to also determine whether there were abnormalities in the cilia of the ASH neurons. The DiI is a red fluorescent dye that is taken up by the cilia of the ASH neurons that stains the entire sensory neuron. Worms were transferred from agar plates into 1.5mL tubes using M9 buffer and centrifuged for 30 seconds at 2600rpm. The supernatant was removed, 1mL of M9 buffer was added to the tube, and worms were centrifuged again (repeated 3 times). All but 100ul of M9 was removed. A mixture of 0.5ul of DiI (Vybrant DiI Cell-Labeling Solution, catalogue number V-22885) plus 500ul of M9 was added to the tube containing the worms. The tube was vortexed quickly for 1 minute and stored in the dark for 30 minutes (at the 15-minute time point, the tube was vortexed again for 1 minute). After this, the worms were centrifuged for 30 seconds at                                                                                                                                             88  2600rpm and the supernatant was removed. Worms were pipetted onto seeded plates and left to eat for 30 minutes to remove gut bacteria that were also stained with DiI.  4.2.4 Statistical Analysis  For statistical analysis of all chemotaxis assays, please refer to section 2.2.4. The percentage of normal versus abnormal ASH morphology of each strain was compared to other strains using aggregated Pearson's chi-squared test on SPSS version 22.0. The average percentages of normal and abnormal (blebs and gaps) ASH neurons were used in the analyses across time for each strain, as there were no differences in the proportion of normal vs abnormal processes with age seen in strain groups (p<0.05).    4.3 Results 4.3.1 Strains in which ASH expressed extrachromosomal wildtype PS1 rescues had normal octanol chemotaxis.  To image the ASH neurons, I made new rescue lines in which the ASH neurons were marked with GFP driven by osm-10 promoter (as a marker I used red fluorescent mCherry for my extrachromosomal rescue lines in this chapter). Because these were new transgenic lines, I replicated the behavioral experiments on chemotaxis and tested wildtype, sel-12 mutant, and ASH-specific PS1 rescue worms for octanol chemotaxis.  As expected, there was a significant difference between the sel-12 mutant worms and the three PS1 rescue lines in response to octanol [F (4,15) =43.78, p<0.001] (Figure 4.3), but not in response to diacetyl (p>0.05) (Figure 4.4). This confirmed a cell-specific rescue in response to octanol chemotaxis in these strains.                                                                                                                                                89   Figure 4.3 Psra-6::PS1 octanol chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Psra-6::PS1 using either octanol (aversive odorant) as a repellant or M9 (odorless buffer) as control. Values are averages from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference from wild-type and rescue lines (p<0.05).   Figure 4.4 Psra-6::PS1 diacetyl chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and Psra-6::PS1 using either diacetyl (attractive odorant) as a repellant or M9 (odorless buffer) as control. Values are averages from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant differences from wild-type (p<0.05).                                                                                                                                               90  4.3.2 sel-12 mutant worms had abnormal ASH neurons which were rescued by Psra-6::PS1 and Psra-6::PS1Δs169, but not Psra-6::PS1C410Y.  To address the question of whether the mutation in sel-12 altered the morphology of the ASH neurons, I imaged the ASH neurons of wildtype, sel-12 mutant, Psra-6::PS1, Psra-6::PS1Δs169, and Psra-6::PS1C410Y worms over time. Within the same strain, no significant differences in the number of abnormal ASH neurons were observed over time (p value of all >0.05, Figure 4.5B). A chi-square test showed that worms with a sel-12 mutation had a significantly smaller number of normal ASH neurons compared to wildtype worms across all time points, X2 (1, N=200) = 16.33, p<0.01 (Figure 4.5 A&B). However, ASH neuron-specific expression of wildtype PS1 and PS1Δs169, but not PS1C410Y, significantly increased the number of normal ASH neurons in sel-12 mutant worms (Figure 4.5). Statistical evaluation of this data is shown in table 4.1.  Table 4.1: ASH neuron morphology chi-square test results   Compared groups  Chi-square test results Wildtype and ASH-specific expression of PS1C410Y X2 (1, N=200) = 14.21, p<0.001 ASH specific expression of wildtype PS1 and sel-12 mutant worms  X2 (1, N=200) = 11.82, p<0.001 ASH specific expression of wildtype PS1 and PS1C410Y X2 (1, N=200) = 10.00, p<0.001 ASH specific expression of wildtype PS1Δs169 and sel-12 mutant worms X2 (1, N=200) = 9.84, p=0.002 ASH specific expression of wildtype PS1Δs169 and PS1C410Y X2 (1, N=200) = 8.21, p=0.004  Although within a strain the proportion of normal and abnormal ASH neurons stayed fairly constant over time, the severity of the defect as measured by the number of “blebs” increased over time in certain strains (Figure 4.5A). Wildtype worms did not have blebs at any time point and ASH specific Psra-6::PS1 transgenic worms had a low number of blebs (5-8) present over time                                                                                                                                             91  (Figure 4.5A). In sel-12 mutant worms, the occurrence of blebs significantly increased from 78-108 hours (0 at 78h; 9 at 98h; 31 at 108h), X2 (1, N=137) = 22.31, p<0.01. This pattern of increased bleb occurrence over time was also present in ASH specific PS1C410Y rescue worms (5 at 78h; 15 at 98h; 26 at 108 h), X2 (1, N=138) = 9.91, p<0.01, and to a lesser degree in Psra-6::PS1Δs169 transgenic worms (3 at 78h; 6 at 98h; 12 at 98h), X2 (1, N=76) = 2.97, p = 0.69 (Figure 4.5A).  As a result of the increased number of blebs in sel-12 mutant worms, I imaged 10 single wildtype and sel-12 mutant worms every 10 hours over time (68-108 hour old worms) to determine if this pattern demonstrated ASH degeneration. As shown in Figure 4.6, ASH neurons in wildtype worms had normal morphology at 68 hours old, though small gaps could appear at 98 hours, and at 108 hours some ASH neurons looked quite abnormal. In contrast, the majority of sel-12 mutant worms looked abnormal at 68 hours and the ASH neurons degenerated much more rapidly than wildtype worms (Figure 4.7).  In order to assay the structural integrity of the ASH cilia and confirm my GFP imaging results, I tested their ability to take up a red fluorescent dye. This process happens through the exposed ciliated endings of the ASH neurons. As a GFP marker was present in the ASH neurons, the overlap between the green GFP and the red dye would indicate proper uptake of the dye. As shown in Figure 4.8, the yellow overlap is present in normal as well as abnormal ASH neurons, suggesting intact cilia structure and confirming the phenotypes observed using GFP.                                                                                                                                                   92   Figure 4.5 ASH neuron morphology of five strains (n=100 per strain per time point) over time (78, 98, and 108 hour old worms): A. The ASH neurons of wildtype, sel-12 mutant, ASH-specific wildtype PS1 rescues, ASH-specific PS1Δs169 rescues, and ASH-specific PS1C410Y rescues were imaged and categorized as either normal or abnormal (gapped arm or blebs). B. Number of abnormal (gapped arm or blebs) ASH neurons over time. Within strains, there were no significant differences in the number of abnormal ASH neurons over time. Asterisk shows significant differences between wildtype/wildtype PS1/PS1Δs169  and sel-12 mutant/ PS1C410Y  average number of abnormal ASH neurons.                                                                                                                                                93   Figure 4.6 Neurodegeneration in the wild-type worms’ ASH neurons: single worms (n=10) were paralyzed using 10mM levamisole and their ASH neurons were imaged over time (from 68-108 hours old). The images in each column represent the same ASH neuron over time.   Figure 4.7 Neurodegeneration in the sel-12 mutant worms’ ASH neurons: single worms (n=10) were paralyzed using 10mM levamisole and their ASH neurons were imaged over time (from 68-108 hours old). The images in each column represent the same ASH neuron over time.                                                                                                                                              94   Figure 4.8 DiI dye fill (in red) and GFP (in green) used for ASH neurons’ confocal imaging: In ASH neurons with normal (A), gapped (B), and blebs (C), there is an overlap between GFP and dye fill shown in yellow.                                                                                                                                                      95  4.4 Discussion  In this chapter, I showed that sel-12 mutant worms had more severe and significantly higher number of morphologically abnormal ASH neurons compared to wildtype. These abnormalities were rescued by ASH-specific expression of wildtype PS1 and PS1Δs169, but not PS1C410Y. I further demonstrated that the cilia of these neurons are structurally intact, and used single worm imaging over time to demonstrate a more rapid ASH degeneration in sel-12 mutant worms compared to wildtype worms.   These data show that in C. elegans, mutations in the homologue of PS1 lead to neuronal degeneration. In humans, one of the described roles of FAD-associated presenilin mutations in degeneration is an increased production of Aß42 (N. Li et al., 2016). The neurodegenerative process in AD begins with synaptic impairments followed by neuronal loss (Crews & Masliah, 2010). Many hypotheses regarding the pathogenesis of AD associated neurodegeneration has been proposed; these include apoptosis, formation of free radicals, oxidative stress and mitochondrial damage, inflammation, and environmental pollution (Crews & Masliah, 2010; Yankner, 1996). This pathogenesis is thought to be the result of abnormal accumulation of Aß oligomers (Sisodia & Price, 1995). In FAD, mutations lead to an increased production of Aß oligomers, but in in sporadic AD it is thought that Aß clearance mechanisms including both lysosomal and non-lysosomal pathways may play a role in neuronal death (Crews & Masliah, 2010; Marambaud, Zhao, & Davies, 2005). As C. elegans lack an Aß sequence in their APP homologue, the rapid degeneration of sel-12 mutant worms’ sensory neurons cannot be explained by Aß accumulation, suggesting that a sel-12 mutation in C. elegans leads to neurodegeneration in an Aß independent manner.                                                                                                                                              96  Another possible role for presenilin in cell death comes from in vitro studies that have reported that presenilin mutations sensitize cells to apoptotic stimuli (Popescu & Ankarcrona, 2004).  Both in vivo and in vitro assays have suggested that apoptosis (programmed cell death), necroptosis (non-apoptotic cell death), and autophagy play a role in neurodegenerative processes (Fan, Dawson, & Dawson, 2017). These cell death mechanisms can be triggered by excitotoxicity, oxidative stress, cytosolic calcium overload, and aggregated misfolded proteins (Fan et al., 2017). Moreover, it has also been reported that many presenilin mutations impair calcium homeostasis by interacting with a number of calcium-related proteins, and thus it has been proposed that disruptions of calcium signaling may play a role in AD-related neuron death (Mattson et al., 2001).   The pattern of neurodegeneration demonstrated in this chapter is very similar to findings of a recent article by Ghose et al. (2018) who found that with age, some C. elegans neurons develop gaps in their processes to signal to and attract certain specialized proteins involved in phagocytosis (Ghose et al., 2018). Nass et al. (2002) reported a similar morphology of dopamine neuron degeneration in C. elegans’ model of Parkinson’s disease (Nass, Hall, Miller, & Blakely, 2002).  The neuronal changes observed here are consistent with other neuronal degeneration seen in C. elegans.  With this cell specific degeneration, we may be able to determine the mechanism by which presenilin kills neurons in the absence of Aß.  As wildtype PS1 and PS1Δs169 rescued ASH morphological impairments in sel-12 mutant worms, the role of Notch signaling in chemotaxis deficits and ASH neuron degeneration needs to be investigated. Although worms with mutations in one of their Notch ligands, osm-11, have chemotaxis deficits to octanol, no morphological impairments were observed in their ASH neurons (Singh et al., 2011). Thus far, the role of Notch in AD-associated neurodegeneration remains elusive. However, this is not true in the case of ischemic stroke. A recent report demonstrated that                                                                                                                                             97  in the case of  cerebral ischemia and hypoxia, the Notch signaling pathway as well as other gene transcription regulators converge on a DNA-associated nuclear multi-protein complex and upregulate genes that trigger neuronal death (Arumugam et al., 2018). Therefore, it has been suggested that the blockage of Notch signaling via g-secretase is neuroprotective against stroke (Balaganapathy et al., 2017). Although in this chapter my data suggests that Notch plays a role in neurodegeneration, and though Notch is expressed throughout the adult brain and has been implicated in apoptosis in stroke cases, whether or not the interaction between PS1 and Notch played a critical role in the ASH degeneration of sel-12 mutant worms remains unknown and requires further investigation.                                                                                                                                                            98  Chapter 5: General discussion  The focus of my thesis was to investigate the role of human PS1 in AD-associated olfactory deficits using sel-12, the C. elegans homologue of PS1. Worms with sel-12 mutations had chemotaxis deficits from hatching which increased with age, similar to olfactory deficits in AD patients, confirming that these deficits are not a circuit problem. This is not surprising since we know that the worms’ sensory neurons are functional at the time they hatch as these neurons play a critical role in controlling the entry into and exit from the alternative dauer larva stage (Golden & Riddle, 1982). The ASH neurons that are responsible for detection of the octanol odorant showed morphological abnormalities and a more rapid degeneration in sel-12 mutant worms compared to wild-type worms. The expression of wildtype PS1 and PS1Δs169, but not PS1C410Y,  in only the ASH neurons was able to rescue both chemotaxis deficits and ASH morphological abnormalities in sel-12 mutant worms. As PS1Δs169 does not affect Notch signaling but PS1C410Y does, I questioned whether or not the interaction between PS1 and Notch played a role in these chemotaxis deficits. A previous study reported that worms with mutations in the two Notch receptors had octanol chemotaxis deficits (Singh et al., 2011). I showed that chemotaxis deficits in this Notch mutant strain do not increase with time. This suggests that although Notch seems to be important in octanol chemotaxis, PS1 may be functioning in chemotaxis in a Notch-independent manner. However, further studies are required to confirm this conclusion.           In my thesis, I demonstrated that wildtype PS1 and PS1Δs169 rescued neuron morphology, suggesting that human and worm presenilins and Notch have conserved functions in post-mitotic neurons. In Drosophila Notch signaling is involved in the guidance of neuronal outgrowth (Giniger, Jan, & Jan, 1993). This may also be the case in vertebrates where presenilins may play a role in controlling neuron morphology in adult cells. Moreover, Wittenburg et al. showed that sel-12                                                                                                                                             99  mutant worms have morphological defects in two interneurons involved in thermotaxis (Wittenburg et al., 2000). Thus, it is possible that impairments in sel-12 activity impacts a subset of neurons owing to its broad expression in the worm’s nervous system. My locomotion data suggests that not all neurons are affected in sel-12 mutant worms as the motor neurons continue to drive movement even in the oldest worms I tested.   Human and mouse studies suggest that AD-associated olfactory deficits may be the result of soluble Aß aggregated in the olfactory system (Wesson, Levy, Nixon, & Wilson, 2010; Wu, Rao, Gao, Wang, & Xu, 2013). Moreover, the accumulation of Aß has been suggested to lead to apoptosis in AD (Rohn, 2010). As C. elegans do not have an Aß sequence, my study shows that sel-12 mutations can cause chemotaxis impairments and neurodegeneration in an Aß independent manner. Therefore, C. elegans provides a unique system in which  the role of presenilin in olfactory dysfunction and neuron death can be investigated using single cell rescues. As presenilins are substrates for different caspase proteins, the interaction between PS1 and caspases can be further explored.  This use of single cell resolution phenotyping allows the characterization of even subtle and pleotropic phenotypes, providing potential novel therapeutic opportunities.  5.1 Conclusion In this thesis, I developed a model in which to investigate non Aß impacts of PS1 in a behavioral phenotype that occurs as a symptom of AD. I found a correlation between behavioral and morphological assays to further understand the role presenilins play in olfactory deficits that occur pre-clinically in many AD patients. This improves our knowledge with regards to presenilins’ role in the nervous system, and could help understand the abnormalities in neuronal pattern of AD in the hopes of finding new therapeutics prior to the manifestation of cognitive decline in patients.                                                                                                                                               100  5.2 Thesis limitations   One of the major advantages of using C. elegans to understand gene function is the ability to study comparatively simple phenotypes in mutant worms. Despite this, there are several disadvantages for using worms to study human disease. It is important to keep in mind that not all aspects of AD can be studied using C. elegans, as these worms do not have an adaptive immune system or a circulatory system. In my study, I used transgenes that were presented as extrachromosomal arrays and were thus not integrated into the genome. It is not possible to know the copy number of the transgenes in these arrays (can be few copies or several hundred copies), and the expression level could have been much higher than what is found in vivo. Newer techniques such as the Mos10 mediated Single Copy Insertion (MosSCI) (Frokjaer-Jensen et al., 2008) and the CRISPR-Cas9 system are now developed and can be used in C. elegans (Dickinson & Goldstein, 2016). In MosSCI, the mobilization of a transposon creates DNA double strand breaks in the non-coding region, which gets repaired by using the extrachromosomal template to copy DNA into a chromosomal site (Frokjaer-Jensen et al., 2008). In CRISPR-Cas9, a spacer sequence is transcribed into an RNA sequence that can then locate matching DNA sequences. When this is done, the Cas9 enzyme can either activate or deactivate gene expression (Dickinson & Goldstein, 2016).   In patients with AD, it has been reported that the severity of olfactory deficits is associated with cognitive decline. Unfortunately, because of the sensory deficits observed in sel-12 mutant worms it is difficult to test memory. Tap habituation is used to test long-term memory in C. elegans (Ardiel & Rankin, 2010), however sel-12 mutant worms have deficits in responding to tap, likely due to impairments in their mechanosensory neurons (personal observation). In other C. elegans’ memory tests, odorants or tastes are associated with food or used for chemosensory habituation                                                                                                                                             101  (Colbert & Bargmann, 1995). This cannot be done in sel-12 mutant worms as they display olfactory and taste (personal observation) impairments.   Because the chemotaxis defects I observed in sel-12 mutant worms were more severe than ASH morphological defects (43-51% normal ASH in sel-12 worms), it may be that sel-12 mutations may have further subtle morphological or synaptic defects that cannot be visualized with the GFP construct used here (Hedgecock, Culotti, Thomson, & Perkins, 1985) and further examination with synaptic markers might be needed.  5.3 Future directions  There are several future experiments that would enhance our understanding of the role of PS1 in chemotaxis. I plan to conduct experiments to explore the role of Notch in sel-12 mutant worms’ chemotaxis. A previous study reported that mutations in one of the worm’s Notch receptors, lin-12, lead to morphological abnormalities in two of interneurons in the nerve ring (Wittenburg et al., 2000). I will image the ASH neurons of worms with mutations in both Notch receptors to determine if they show the same pattern of abnormality as in the sel-12 mutant worms. The release of the Notch intracellular domain leads to its translocation to the nucleus and interaction with DNA binding proteins, activating transcription. I plan to use a heat shock promoter driven a human NICD expression (ubiquitously, nervous system, and neuron specific) to test the involvement of canonical Notch signaling in olfaction. In order to avoid developmental defects, I will express this NICD specifically in adult stages.  I hypothesize that the AWA neurons will show a similar pattern to ASH and so I will make and image sel-12 mutant strains that express GFP in the worm’s diacetyl detecting neurons. The interneurons that ASH and AWA synapse onto such as AIB and AVA will also be imaged to determine which, if any, of the neurons in the chemosensory pathway are affected by a presenilin                                                                                                                                             102  mutation. To further determine whether PS1 impacts interneuron functioning, I will specifically express PS1 in different interneurons of the chemosensory circuit and test the worms’ chemotaxis abilities. I have already generated three lines with transgenic expression of PS1 driven by heat shock promoters which I will express at different larval and adult life stages in sel-12 mutant worms to find at what time point(s) PS1 expression is necessary for normal chemotaxis. This will provide more information with regards to the role of PS1 in chemotaxis.  Another route to understanding the effects of sel-12 mutations on neuron function involves bypassing transduction and depolarizing the neurons directly to determine whether the neurons can still drive locomotion.  To do this, I will use optogenetics in which I will express channelrhodopsin-2 (ChR2), a light gated cation channel, in the worm’s ASH neurons (Nagel et al., 2005). Through this, I attempt to activate ASH neurons in sel-12 mutant and wildtype worms to observe whether the worms crawl backwards. If ASH neuron activation leads to proper phenotype (backward crawling) in sel-12 mutant worms, then I will visualize ASH synapses in living worms using the vesicle protein synaptobrevin (SNB-1) tagged with GFP. The SNB-1 protein is an integral membrane protein found in synaptic vesicles (Jin, 2005).   Based on the results found in this thesis, a new project can explore the use of intragenic (suppressor lies in same gene as starting mutation) and extragenic suppression screening to learn more about presenilin function and to understand the molecular basis of how PS1 produces neuronal degeneration without the present of Aß (Hodgkin, 2005). As C. elegans are self-fertilizing diploids, both dominant and recessive suppressors may be conducted (Hodgkin, 2005). Moreover, high-throughput molecule screening could be conducted to search for molecules that rescue chemotaxis and neuron morphology in sel-12 mutant worms (O'Reilly, Luke, Perlmutter,                                                                                                                                             103  Silverman, & Pak, 2014). These molecules would somehow bypass the necessity of presenilins or activate pathways compensating for the lack of presenilin function. This would provide potential new drug and therapeutic targets for treating AD.                                                                                                                                                                  104  Bibliography  Abramov, E., Dolev, I., Fogel, H., Ciccotosto, G. D., Ruff, E., & Slutsky, I. (2009). Amyloid-beta as a positive endogenous regulator of release probability at hippocampal synapses. 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Biological function of Presenilin and its role in AD pathogenesis. Transl Neurodegener, 2(1), 15. doi:10.1186/2047-9158-2-15 Zhang, Y. W., Thompson, R., Zhang, H., & Xu, H. (2011). APP processing in Alzheimer's disease. Mol Brain, 4, 3. doi:10.1186/1756-6606-4-3                                                                                                                                                133  Appendix A A.1 sel-12 and hop-1 mutant worms’ chemotaxis in response to diacetyl   Figure A.1 Chemotaxis assay in sel-12 mutant, hop-1 mutant, and sel-12/hop-1 double mutant worms: Chemotactic indices generated from assays using either diacetyl (attractive odorant) as an attractant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference from wild-type (p<0.05).                                                                                                                                                      134  A.2 Nervous system rescues in response to diacetyl  Figure A.2 Pan neuronal expression of wild-type sel-12 chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and wild-type sel-12 nervous system rescues using either diacetyl (attractive odorant) as an attractant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference (p<0.05) from wild-type and rescue lines.   Figure A.3 Pan neuronal expression of wild-type PS1 chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and wild-type PS1 nervous system rescues using either diacetyl (attractive odorant) as an attractant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference from wild-type and rescue lines (p<0.05).                                                                                                                                              135   Figure A.4 Pan neuronal expression of PS1C410Y chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and PS1C410Y nervous system rescues using either diacetyl (attractive odorant) as an attractant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant difference from wild-type (p<0.05).   Figure A.5 Pan neuronal expression of PS1Δs169 chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and PS1Δs169 nervous system rescues using either diacetyl (attractive odorant) as an attractant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference from wild-type and rescue lines (p<0.05).                                                                                                                                              136  A.3 Neuron specific rescues in response to diacetyl  Figure A.6 Wild-type sel-12 expressed under an AWA-specific promoter (odr-10) chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and wild-type sel-12 rescues in AWA using either diacetyl (attractive odorant) as an attractant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Single asterisk shows significant difference from wild-type and rescue lines, double asterisk shows significant difference from wild-type, and remaining rescue lines (p<0.05).                                                                                                                                                   137   Figure A.7 Wild-type PS1 expressed under an AWA-specific promoter (odr-10) chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and wild-type PS1 rescues in AWA using either diacetyl (attractive odorant) as an attractant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference from wild-type and rescue lines (p<0.05).                                                                                                                                                        138   Figure A.8 PS1C410Y expressed under an AWA-specific promoter (odr-10) chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and PS1C410Y rescues in AWA using either diacetyl (attractive odorant) as an attractant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisks show significant difference from wild-type (p<0.05).   Figure A.9 PS1Δs169 expressed under an AWA-specific promoter (odr-10) chemotaxis assay: Chemotactic indices generated from assays performed on wild-type, sel-12 mutant, and PS1Δs169 rescues in ASH using either diacetyl (attractive odorant) as an attractant or M9 (odorless buffer) as control. Each bar represents an average from 4 independent plates (n=50-100 worms per plate). Error bars reflect the standard error of the mean. Asterisk shows significant difference from wild-type and rescue lines (p<0.05).  

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