Science, Irving K. Barber Faculty of (Okanagan)
DSpace
UBCO
Lu, Xueying
Master of Science - MSc
University of British Columbia
Developmental axon remodeling is an essential process that ensures the proper development and functioning of the nervous system. Dysregulation of this process is associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Tropomyosin receptor kinase A (TrkA) has shown characteristics of a dependence receptor for NGF. However, how TrkA initiates degeneration and the downstream players remain unclear. This thesis aims to determine if PLCγ1 acts downstream of TrkA-dependent axonal degeneration in dorsal root ganglion (DRG) sensory neurons. It is hypothesized that in the absence of NGF, TrkA induced axon degeneration by activating PLCγ1, thereby inducing cytotoxicity. The first chapter of this thesis investigates the role of PLCγ1 in the axonal degeneration of nerve growth factor (NGF) dependent DRG through drug and in the second chapter, a genetic approach is utilized. First, a drug inhibitor, U73122, is employed to inhibit PLC activities. Unexpectedly, U73122 is proven to be an unsuitable inhibitor of PLCγ1 in DRGs. Our results reveal that U73122 induced changes in axon morphology, increases in Ca²⁺ mobilization, disruption of mitochondria outer membrane potential (MOMP), and a significant increase in PLCγ1 phosphorylation in DRGs. Next, a PLCγ1 Cre-Lox mouse model is used to study DRG degeneration in the absence of PLCγ1. Our results determined that PLCγ1 fl/fl DRGs show similar levels of NGF-dependent growth and degeneration as wild-type DRGs, but in the absence of PLCγ1, DRGs show significantly reduced axonal-induced Ca²⁺ mobilization after NGF deprivation. In Chapter 5, TrkAY⁷⁸⁵F knock-in DRGs were generated and characterized. Phosphorylation of TrkA at Y785 allows PLCγ1 to interact, become phosphorylated, and activated. They were shown to possess normal NGF-dependent growth and degeneration. Moreover, they were shown to have defective TrkA Y785 phosphorylation and show reduced Ca²⁺ mobilization following NGF deprivation compared to wild-type DRGs. Together these results suggest PLCγ1 may play an important role in TrkA dependent degenerative pathway in DRG sensory neurons. A better understanding of this degenerative pathway could advance therapeutic drug development in the treatment of neurodegenerative disorders by targeting specific components of TrkA-PLC degenerative pathway.
The Investigation of the Role of PLCgamma1 in the TrkA Dependent Degenerative Pathway within Dorsal Root Ganglion Sensory Neurons by Xueying Lu B.Sc., The University of British Columbia, 2021 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERISTY OF BRITISH COLUMBIA (Okanagan) August 2024 © Xueying Lu 2024 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis entitled: The Investigation of the Role of PLCgamma1 in TrkA Dependent Degenerative Pathway in Dorsal Root Ganglion submitted by Xueying Lu in partial fulfillment of the requirements of the degree of Master of Science in Biochemistry and Molecular Biology Dr. Phil A. Barker; Faculty of Science Co-supervisor Dr. Julien Gibon; Faculty of Science Co-supervisor Dr. David Kaplan; Faculty of Medicine Supervisory Committee Member Dr. Sepideh Pakpour; Faculty of Applied Science Supervisory Committee Member Dr. Sean Lawrence; Faculty of Creative and Critical Studies, Language and World Literatures University Examiner Dr. Paul Van Donkelaar; School of Health and Exercise Sciences External Examiner iii Abstract Developmental axon remodeling is an essential process that ensures the proper development and functioning of the nervous system. Dysregulation of this process is associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Tropomyosin receptor kinase A (TrkA) has shown characteristics of a dependence receptor for NGF. However, how TrkA initiates degeneration and the downstream players remain unclear. This thesis aims to determine if PLCγ1 acts downstream of TrkA-dependent axonal degeneration in dorsal root ganglion (DRG) sensory neurons. It is hypothesized that in the absence of NGF, TrkA induced axon degeneration by activating PLCγ1, thereby inducing cytotoxicity. The first chapter of this thesis investigates the role of PLCγ1 in the axonal degeneration of nerve growth factor (NGF) dependent DRG through drug and in the second chapter, a genetic approach is utilized. First, a drug inhibitor, U73122, is employed to inhibit PLC activities. Unexpectedly, U73122 is proven to be an unsuitable inhibitor of PLCγ1 in DRGs. Our results reveal that U73122 induced changes in axon morphology, increases in Ca2+ mobilization, disruption of mitochondria outer membrane potential (MOMP), and a significant increase in PLCγ1 phosphorylation in DRGs. Next, a PLCγ1 Cre-Lox mouse model is used to study DRG degeneration in the absence of PLCγ1. Our results determined that PLCγ1 fl/fl DRGs show similar levels of NGF-dependent growth and degeneration as wild-type DRGs, but in the absence of PLCγ1, DRGs show significantly reduced axonal-induced Ca2+ mobilization after NGF deprivation. In Chapter 5, TrkAY785F knock-in DRGs were generated and characterized. Phosphorylation of TrkA at Y785 allows PLCγ1 to interact, become phosphorylated, and activated. They were shown to possess normal NGF-dependent growth and degeneration. Moreover, they were shown to have defective TrkA Y785 phosphorylation and show reduced Ca2+ mobilization following NGF deprivation compared to wild-type DRGs. Together these results suggest PLCγ1 may play an important role in TrkA dependent degenerative pathway in DRG sensory neurons. A better understanding of this degenerative pathway could advance therapeutic drug development in the treatment of neurodegenerative disorders by targeting specific components of TrkA-PLC degenerative pathway. iv Lay Summary Malfunctioning of our nervous system may cause various diseases such as Alzheimer’s and Parkinson’s disease. These diseases often involve the loss of communication between cells leading to their eventual death. There is currently no cure for these brain disorders. The overarching goal of my master’s thesis project is to provide more insights into the biological processes of the mechanism of brain cell death. To achieve this, I have studied how peripheral sensory neurons eliminate excessive or damaged cells during early development, with the eventual goal of linking pathways utilized in peripheral neurons to those that may become active in degenerative brains diseases. With greater knowledge of how neurons are dying, we may reveal new therapeutic strategies to better treat neurological diseases and increase the quality of life for patients and their families. v Preface Xueying Lu planned, performed, and analyzed all experiments presented in this thesis. Additionally, Xueying Lu wrote the thesis. Generative AI was not used in any way in this thesis. The research conducted in this thesis was under the approval of the UBC animal care committee breeding protocol, A24-0101 and experimental protocol, A24-0103. Dr. Andres de Leon contributed to Figure 2B. Dr. Sepideh Pakpour and Dr. David Kaplan made suggestions on the thesis. Dr. Julien Gibon contributed to the design of experiments and edited the thesis. Dr. Philip A. Barker directed experiments, contributed to data presentation, and was involved in the preparation and editing of the thesis. vi Table of Contents Abstract ............................................................................................................................ iii Lay Summary ................................................................................................................... iv Preface.............................................................................................................................. v Table of Contents ............................................................................................................ vi List of Tables ................................................................................................................. viii List of Figures .................................................................................................................. ix Abbreviations .................................................................................................................... x Acknowledgements .......................................................................................................... xi Chapter 1: Introductory Literature Review ....................................................................... 1 1.1 Neurotrophins and neurotrophic receptors ............................................................. 1 1.2 The neurotrophic theory ......................................................................................... 1 1.3 TrkA dependent survival and apoptosis ................................................................. 2 1.4 PLCγ1 protein function ........................................................................................... 4 1.5 Research Objective and Hypothesis .......................................................................... 5 Chapter 2: Materials and Methods ................................................................................... 7 2.1 Animals................................................................................................................... 7 2.1.1 PLCγ1 conditional knockout mice .................................................................... 7 2.1.2 TrkAY785F knock-in mice ............................................................................... 7 2.2 DRG neuron culture ............................................................................................... 8 2.3 Immunoblotting and Quantifications ....................................................................... 8 2.4 DRG Immunocytochemistry and Quantifications ................................................... 9 2.5 Wheat Germ-Agglutinin Pull Down ......................................................................... 9 2.6 Live Ca2+ Imaging with Fluo-4 and Quantifications .............................................. 10 2.7 Statistical Analysis ............................................................................................... 10 Chapter 3: The effect of U73122 on axonal morphology in NGF dependent DRG sensory neurons ............................................................................................................ 11 3.1 Introduction .......................................................................................................... 11 3.2 Results ................................................................................................................. 11 3.3 Chapter Summary ................................................................................................ 15 vii Chapter 4: Investigating the role of PLCγ1 in NGF-dependent axonal degeneration in DRG sensory neurons ................................................................................................... 17 4.1 Introduction .......................................................................................................... 17 4.2 Results ................................................................................................................. 17 4.3 Chapter Summary ................................................................................................ 18 Chapter 5: Determining if PLCγ1 is involved in TrkA-dependent NGF dependent degeneration in DRG sensory neurons ......................................................................... 23 5.1 Introduction .......................................................................................................... 23 5.2 Results ................................................................................................................. 23 5.3 Chapter Summary ................................................................................................ 24 Chapter 6: Discussion .................................................................................................... 27 Chapter 7: Conclusion ................................................................................................... 31 Bibliography ................................................................................................................... 32 Appendix ........................................................................................................................ 38 viii List of Tables SI. Table 1. Statistical Analysis ...................................................................................... 38 ix List of Figures Figure 1. PLCγ inhibitor, U73122, alters DRG axon morphology in vitro ....................... 13 Figure 2. PLCγ inhibitor disrupts mitochondrial membrane potential and induces axonal Ca2+ influx ...................................................................................................................... 14 Figure 3. U73122 induces PLCγ1 Y783 phosphorylation in DRG sensory neurons in a dose and time dependent manner ................................................................................. 16 Figure 4. PLCγ1 fl/fl DRG explants show normal growth and degeneration and PLCγ1 fl/fl::AdvCre DRGs show significant reduction in PLCγ1 protein level ........................... 20 Figure 5. PLCγ1 fl/fl::AdvCre significantly reduced Ca2+ mobilization induced by NGF deprivation ..................................................................................................................... 21 Figure 6. TrkAY785F double knock-in DRGs have defective phosphorylation at Y785 25 Figure 7. TrkAY785F double knock-in DRGs show attenuation of in Ca2+ mobilization induced by NGF deprivation .......................................................................................... 25 x Abbreviations BDNF Brain-derived neurotrophic factor DRG Dorsal root ganglia ESC Embryonic stem cell MMP Mitochondrial membrane permeabilization MOMP Mitochondrial outer membrane potential NGF Nerve growth factor NT3 Neurotrophin-3 NT4 Neurotrophic-4 PLCγ1 Phospholipase C gamma 1 PKC Protein kinase C ROS Reactive oxygen species SCG Superior cervical ganglion TrkA Tropomyosin receptor kinase A TRPC3 Transient receptor potential canonical subfamily 3 xi Acknowledgements I would first like to thank my mentors Dr. Philip A. Barker and Dr. Julien Gibon for accepting me into their laboratories and providing me with an extraordinary learning environment, training, and resources to excel in my master’s thesis program. Thank you for granting me the freedom to express my thoughts and ideas and to discuss and execute them. It’s been a real pleasure and honour to be part of such an incredible lab. I want to thank the members of my advisory committee for providing guidance and support over the years: Dr. David Kaplan and Dr. Sepideh Pakpour. They provided me with invaluable advice for the development of my project and the kind words they spoke about my work have been truly validating. I want to thank Dr. Andres de Leon for all of the advice, laboratory training, and support over the years. He inspired me to learn from his meticulous approaches to tackling scientific problems and experimental setups. I like to thank all my colleagues, including Svetlana Simtchouk, for all of her hard work in keeping the laboratory in working order and doing an amazing job in organizing the animal colonies. Noah Zurfluh, for working alongside me and keeping me in check. I would also like to thank Mahdi Abbasian, Abbie Zhou, and Filip Tomas for their teachings and support. 1 Chapter 1: Introductory Literature Review 1.1 Neurotrophins and neurotrophic receptors During development, axons and dendrites extend toward target sites to form complex neuronal networks, allowing neighboring neurons to properly communicate with one another (Silbereis et al., 2016). Neurotrophins are a group of closely related mammalian trophic factors known to regulate neuronal survival and death, as well as synaptic plasticity, learning, and memory (Gibon & Barker, 2017; B. Hempstead, 2006). There are four distinct trophic factors in the neurotrophin family including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4, also referred to as NT5) (Gibon & Barker, 2017). Neurotrophins bind to pan-neurotrophin receptor p75 (p75NTR) and tropomyosin kinase receptors (Trk) A, B and C to exert their cellular functions. NGF preferentially binds to TrkA (Harrington et al., 2011), brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) bind and activate TrkB selectively and neurotrophin-3 (NT3) specifically binds to TrkC (Chao, 2003; Squinto et al., 1991). All of the above mentioned neurotrophins associate with p75NTR with similar affinity (Chao, 1994). Additionally, p75NTR may also act as a co-receptor that increases neurotrophin binding affinity to its receptors (Bibel, 1999; Chao, 2003; B. L. Hempstead et al., 1991; Lee et al., 2001). Specifically, coexpression of p75 with TrkA increases binding affinity of NGF by 2 magnitude compared to when either receptors is expressed independently (B. L. Hempstead et al., 1991). Neurotrophic receptors may function independently, however, they may also interact with one other affecting their affinities towards neurotrophic factor (Barker & Shooter, 1994). 1.2 The neurotrophic theory NGF was first discovered and isolated through the work of Rita Levi-Montalcini, Victor Hamburger, and Stanley Cohen in the 1950s (Cohen, 1960; Levi-Montalcini & Cohen, 1956; Levi-Montalcini & Hamburger, 1951). These pioneering studies helped to form the neurotrophic hypothesis which proposes that neurons are initially produced in excess. During development, neurons extend axons and dendrites that compete for the limiting 2 amounts of trophic factor synthesized endogenously at target tissues (Levi-Montalcini, 1987; Miguel-Aliaga & Thor, 2009; Oppenheim, 1989). Axons that receive sufficient amounts of trophic support will survive while those that do not will be eliminated by the process of degeneration. Utilizing the neuro-protective characteristics of NGF on degenerating neurons in the peripheral nervous system has allowed significant advancement in our understanding of the molecular mechanisms underlying neuron degeneration triggered by insufficient trophic support (Levi-Montalcini, 1987; Levi-Montalcini et al., 1994). 1.3 TrkA dependent survival and apoptosis NGF promotes survival and growth by binding and activating TrkA cell surface receptor. TrkA has a single transmembrane and a cytoplasmic domain containing a tyrosine kinase domain (Huang & Reichardt, 2003). The tyrosine residues in the receptor’s kinase domain undergo autophosphorylation induced upon ligand-receptor interaction and subsequent receptor dimerization (Jing et al., 1992; Ullrich & Schlessinger, 1990). NGF not only induces homodimerization of TrkA receptor and intermolecular phosphorylation but also drives rapid internalization of the ligand-receptor complex (Jing et al., 1992). The tyrosine residues in TrkA that are autophosphorylated upon NGF binding are in the kinase domain (specifically Y670, Y674, and Y675) whereas tyrosine residues outside of the kinase domain that become phosphorylated by receptor dimerization are Y490 and Y785. These phosphorylated tyrosines, and their flanking sequences, function as docking sites for adaptor proteins and enzymes that mediate intracellular signaling cascade downstream of TrkA receptor activation (Huang & Reichardt, 2003). While TrkA is well recognized for its role in promoting neuronal survival, several studies have shown that it shows characteristics of a dependence receptor (Feinberg et al., 2017; Mehlen & Bredesen, 2004). The idea of dependence receptors arose from the observation that cells depend on the presence of various stimulus for survival (Mehlen & Bredesen, 2011). These receptors promote cell survival when bound to their respective ligand, but mediate apoptosis in the absence of their ligand (Mehlen & Bredesen, 2011). Dependence receptors are active in both the presence and absence of their ligand. In the presence of NGF, it is certain that the TrkA receptor promotes neuronal survival, but 3 emerging evidence suggests that when NGF is withdrawn, TrkA switches to mediate pro-death signaling. The Barde lab has shown that expression of TrkA in embryonic stem cells (ESC) derived neuronal cultures leads to cell death within a week in the absence of NGF. Interestingly, the death of these neurons can be prevented with the addition of NGF verifying that TrkA is a dependence receptor (Nikoletopoulou et al., 2010). Moreover, similar dependence on NGF was observed in sympathetic superior cervical ganglion neurons (SCG). SCG neurons lacking TrkA survived in the absence of NGF, while control neurons containing TrkA were rapidly eliminated (Nikoletopoulou et al., 2010). Additionally, the Kaplan lab has shown that TrkA maintains a low level of phosphorylation in the absence of its ligand, NGF, and this basal activity leads to degeneration of sympathetic and sensory neurons (Feinberg et al., 2017). Pan-kinase inhibitors including foretinib and lestaurtinib blocked TrkA tyrosine phosphorylation and protected neurons from NGF deprivation induced apoptosis and axon degeneration (Feinberg et al., 2017). Moreover, it has been suggested that foretinib could rescue axon degeneration via an anterograde signaling process as inhibition of TrkA in the cell body compartment alone was sufficient to suppress degeneration in the axon compartment (Feinberg et al., 2017). Furthermore, it has been demonstrated that specific TrkA inhibition rescued axonal degeneration partially by suppressing signaling pathways underlying mitochondria integrity (Feinberg et al., 2017). Ras-MAPK signaling, and PI3K signaling are well studied pathways initiated through TrkA activation and both are initiated by phosphorylation of TrkA at Y490. This phosphorylated residue recruits Shc which in turn recruits the Grb2 adaptor protein leading to the activation of guanine nucleotide exchange factor (GEF), SOS (Nimnual et al., 1998). GEFs regulate the activation and inactivation of Ras by switching between GTP-bound and GDP-bound status respectively (Nimnual et al., 1998). Activation of Ras is involved in activating PI3K and in activating the MAPK kinase signaling cascade (Huang & Reichardt, 2003; Nimnual et al., 1998). Ras activation mediates mitogen-activated protein kinase (MEK) and extracellular signal-related kinase (ERK) activation by activating Raf 4 (English et al., 1999). MEK-ERK can activate the cAMP response element binding protein (CREB) which plays a crucial role in the regulation of genes essential for neuronal survival (Gao et al. 2004). The PI3K signaling pathway promotes neuronal survival by inhibiting proteins involved in apoptosis (Huang & Reichardt, 2003). PI3K and phosphatidylinositde protein kinase (PDK-1) generate phosphatidylinositides that activate Akt (Huang & Reichardt, 2003). One of the main functions of Akt in this context is to mediate the phosphorylation of Bad which blocks BAD-induced neuronal death (Datta et al., 1997). PLCγ1 signaling also lies downstream of TrkA activation and directly associates with TrkA at its Y785 residue. Unlike the important roles Ras-MAPK and PI3K pathways play in promoting neuronal survival, the role of PLCγ1 in neuronal survival and death has been largely unexplored and remain enigmatic. However, one paper published in 2010 showed that inhibition of TrkA by kinase inhibitors, K-252a and CEP-2563, promoted hippocampal neuronal survival in the absence of NGF (Matrone et al., 2009). In agreement with previous studies, silencing TrkA by RNAi protected hippocampal neurons from NGF deprivation induced apoptosis. Notably, the Calissano lab reported that following NGF deprivation, PLCγ1 phosphorylation levels immediately drop and returns to basal level 24 hours later. Moreover, silencing of TrkA prevented the increase of PLCγ1 phosphorylation (Matrone et al., 2009). This publication was the first to suggest a possible role for PLCγ1 in TrkA-mediated neuronal degeneration (Matrone et al., 2009). 1.4 PLCγ1 protein function PLCγ1 is a membrane associated enzyme involved in ligand-mediated signal transduction that is recruited to the TrkA receptor following phosphorylation of TrkA at Y785 (Inagaki et al., 1995). PLCγ1 is autoinhibited until the enzyme undergoes a conformational change induced by phosphorylation at Y783 (Hajicek et al., 2019). This activates the enzymes lipase activity, allowing it to cleave phosphatidylinositol-4,5-bisphosphate (PIP2) to produce inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG) (Chuang et al., 2001; Hajicek et al., 2019). IP3 induces Ca2+ release from endoplasmic reticulum (ER) stores into the cytosol through binding to IP3 receptor (Chuang et al., 2001) whereas DAG, activates several members of the protein kinase C (PKC) family to induce an array of downstream events (Chuang et al., 2001; Johnstone et al., 2019). 5 PLCγ has been described several cellular processes induced by BDNF including synapse plasticity, neurotransmitter release, and dendritic spine plasticity. PLCγ has been reported to become phosphorylated by NGF-mediated endogenous TrkA activation as well as BDNF-induced activation of TrkB. It has been demonstrated that deletion of the PLCγ binding site in TrkB has little effect on sympathetic SCG axon outgrowth mediated by BDNF (Atwal et al., 2000; Sciarretta et al., 2010). Moreover, TrkB-PLCγ signaling was required for the normal calyx formation around hair cells, where afferent sensory neuron envelopes the hair cell to formation an enlarged synaptic terminal (Sciarretta et al., 2010). This suggests an essential role for the PLCγ interaction site in structural synaptic plasticity in the peripheral nervous system. In cortical neurons, TrkB-PLCγ was thought to be facilitated by TrkB glucocorticoid receptor interaction which led to the release of neurotransmitter, glutamate (Numakawa et al., 2009). Furthermore, BDNF has been described to sustain membrane permeabilization through transient receptor potential canonical subfamily 3 (TRPC3) by mediating IP3 and DAG formation through PLCγ (Amaral & Pozzo-Miller, 2007). A recent publication from the Barker lab showed that PKC activation which lies downstream of PLCγ1 signaling leads to NOX activation resulting in generation of reactive oxygen species (ROS) following NGF deprivation. This drives increases in axoplasmic Ca2+ via the TRPV1 Ca2+ channel in sensory neurons causing axon degeneration (Johnstone et al., 2019). It has been shown that applications of PKC inhibitors, NOX complex inhibitor, ROS scavenger, and EDTA were all sufficient to protect axons from degeneration (Johnstone et al., 2019). While it has been assumed that PLCγ1 activation induced by TrkA plays a pro-growth or pro-survival role, the goal of my thesis project was to test the hypothesis that the TrkA-PLCγ1 signaling complex plays a critical role in DRG sensory neuron degeneration induced by NGF withdrawal. 1.5 Research Objective and Hypothesis This master’s thesis project aims to investigate the role of PLCγ1 in TrkA-dependent DRG sensory neuron degeneration. I first investigated if PLCγ1 is involved in DRG sensory neuron axonal degeneration. To examine DRG degeneration induced by NGF deprivation in the absence of PLCγ1, a known PLCγ inhibitor, U73122 was used. Additionally, a 6 genetic deletion of PLCγ1 specifically in DRG sensory neurons using the Cre-Lox system was used to overcome nonspecific targeting of the U73122. Axon morphology, axonal Ca2+ levels and mitochondria membrane permeabilization (MMP) was compared between DRGs with and without PLCγ1 activity. I hypothesized that in the absence of PLCγ1 activity, DRG axons will be protected from degeneration indicated by healthy axons with low axonal Ca2+ level and healthy mitochondrial outer membrane potential (MOMP). In my second objective, I determined if the role of PLCγ1 in DRG axon degeneration is dependent on TrkA signaling. We generated a mouse model with a TrkAY785F knock in mutation that I anticipated would ablate the interaction of PLCγ1 with TrkA. Quantification of axon morphology, axonal Ca2+ levels, MMP, PLCγ1 phosphorylation and total PLCγ1 protein levels in wild-type DRGs was compared with DRGs containing the TrkAY785F mutation. It was hypothesized that if PLCγ1 is unable to interact with TrkA, DRG axon degeneration would be reduced. TrkAY785F knock in DRG axons were hypothesized to undergo reduced or delayed degeneration following NGF withdrawal compared to wild-type DRGs. The goal of this thesis project was to provide a better understanding of the molecular mechanisms underlying developmental degenerative diseases such as Alzheimer’s disease and Parkinson’s disease. This approach may elucidate potential therapeutic drug candidates targeting the signaling molecules involved in neuron degeneration in aid of their treatment. 7 Chapter 2: Materials and Methods 2.1 Animals 2.1.1 PLCγ1 conditional knockout mice Plcg floxed mice were provided by Dr. Florian Heidel at the Friedrich-Schiller-University in Jena Germany. These mice have flox sites flanking exons 2-4 (Schnoeder et al., 2016). The Plcg1-floxed mice were genotyped using primer set 1, forward (5’ ACCTCAGGCTCGTGTACG-3’) and reverse (5’-CTAGGTCAGAGCAGGTCACT -3’). To generate sensory neuron specific Plcg1 deletion, Plcg1-floxed mice were crossed with Advillin-Cre transgenic mice obtained from Jackson Laboratory. Plcg1-floxed mice were crossed with Plcg1-floxed::Advillin-Cre mice to generate Plcg1-floxed embryos that either lacks Advillin-Cre or are heterozygous for Advillin-Cre. Wild-type littermates were used as experimental controls. Tails of embryos were used for genotyping prior to experimentations. Primer set 1 was used to determine presence of recombination and a second primer set was used to determine if the Advillin-Cre allele was present. Primer set 2 includes forward (5’- AATGGCTCCCTGTTCACTGT-3’), wild-type reverse (5’- TGACTAGGTAGAGGTGCAAATGTC-3’), and mutant reverse primer (5’-AGGCAAATTTTGGTGTACGG-3’). 2.1.2 TrkAY785F knock-in mice To determine if TrkA-dependent PLCγ activation plays a role in NGF withdrawal induced sensory neuron degeneration, we created a TrkAY785F knock-in mouse model using CRISPR-mediated ES cell mutagenesis, in collaboration with Mitra Cowan (MICAM, University of McGill). The TrkAY785F mice expresses a mutant form of TrkA in which tyrosine 785 is replaced with a phenylalanine residue. The expectation was that this mutation will prevent normal Y785 phosphorylation and thereby reduce the ability of PLCγ to bind to the activated TrkA receptor. Genotyping of TrkAY785F knock-in mice and embryos were verified by sequencing genomic DNA produced using the following primers, forward (5’–AGGCGATCGAGTGTATCACG–3’) and reverse (5’-ACCCAGAGGGACCAAAATGG-3’) respectively. Animals bearing the sequence (5’- 8 CCCAGTTACCTA-3’) were wild-type and those containing (5’-CCCAGTTTCCTA-3’) contained the TrkAY785F mutation. All animal experimental procedures were approved by the University of British Columbia animal care committee and in compliance with the Canadian Council on Animal Care guidelines. 2.2 DRG neuron culture E13 DRG explants were dissected from either PLCγ fl/fl::AdvCre or TrkAY785F pregnant female mice. DRG explants were seeded in either 12 multi-well (MW) plastic bottom plates (Grenier), or glass-bottom dish (CellVis). Plates were coated with 0.5 g/L poly-D-lysine (Sigma-Aldrich) for glass-bottom dishes and 5 mg/L poly-D-lysine for plastic dishes, followed by 10 μg/ml laminin-entactin complex (Corning), and 0.1 mg/ml PurCol bovine collagen (Advanced Biomatrix). DRG explants were grown in complete neurobasal media (Invitrogen) containing 2% B27 serum-free supplement (Invitrogen), 1% L-glutamine (Wisent), 1% penicillin/ streptomycin (Wisent), 10 μM 5-fluoro-2’deoxyuridine (FDU, Sigma-Aldrich) and 12.5 ng/ml nerve growth factor (NGF, CedarLee) at 37˚C with 5% CO2. DRG explants were allowed 2 days of growth before experimentations. 2.3 Immunoblotting and Quantifications DRG explants were seeded in a 12 well multi-well plastic plates (Corning). DRG explants were washed with 1x PBS once and scraped into 50 μL of 2x sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M Tris-HCl, pH ~6-8). Samples were boiled for 5 min, centrifuged, and stored at -80˚C. Samples were separated using 12% SDS-polyacrylamide gel electrophoresis at 60 V for stacking gel followed by 160 V in the running gel. Proteins were transferred from gel to nitrocellulose membranes in 1x Towbin buffer at 0.3 A for 1h. The nitrocellulose membranes were blocked with 5% milk for 1 hour at room temperature followed by overnight incubation with primary antibodies phos-PLCγ1 (1:1000, Cell Signaling), C-terminal PLCγ1 (1:5000, NEB), actin (1:1000, Fisher Scientific), neurofilament (1:1000, Millipore), and βIII-tubulin (1:100,000, Millipore). The blots were washed 6 times for 10 min at room temperature followed by 1 h incubation with goat anti-mouse (1:3000) and goat anti-rabbit (1:2000) 9 secondary antibodies. Densitometric analyses were performed using Bio-Rad ImageLabTM software. 2.4 DRG Immunocytochemistry and Quantifications Dissociated DRG neuronal cultures were grown on acid-treated glass coverslips and fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature, washed once with 1x phosphate buffered saline (PBS), then blocked for 1 h with 5% dry milk powder (BioShop) and 0.3% Triton X100 (Millipore) in Tris Buffered Saline with Tween (TBST, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween-20) at room temperature. Neurons were incubated with mouse monoclonal antibody against βIII-tubulin (1:5000, Millipore) and rabbit monoclonal antibody against PLCγ1 (1:100, ThermoFisher) overnight at 4˚C. Primary and secondary antibodies were diluted in blocking solution. Neurons were washed 3 times with 1x PBS, and further incubated with goat anti-mouse secondary antibody conjugated to Alexa Fluor 488 (1:2000, Jackson ImmunoResearch) and goat anti-rabbit secondary antibody conjugated to Alexa Fluor 546 (1:2000, ThermoFisher) for 3 h at room temperature. Neurons were washed once with 1x PBS, stained with 1:1000 4′,6-diamidino-2-phenylindole (DAPI) diluted in PBS for 5 minutes at room temperature, and washed once with 1x PBS. Coverslips were mounted onto microscope slides with Fluoroshield (Millipore). DRG neurons images were captured in Leica DMi8 confocal microscope laser in 1 μm increments with 63x oil immersed objective. National Institutes of Health (NIH) ImageJ software Z stacks were visualized through maximal intensity projection image. Mean PLCγ intensity values per neuron area were quantified using in NIH ImageJ. 2.5 Wheat Germ-Agglutinin Pull Down TrkAY785F DRG explants and wild-type controls were seeded in 12 multi-well plastic dishes. DRG explants were deprived of NGF for 3 h (with no anti-NGF added) followed by 15 min 12.5 ng/ml NGF stimulation. DRG explants were washed once with PBS and scaped in 200 μL NP40 cell lysis buffer. 40 μL of cell lysate were isolated, mixed with 2x sample buffer, boiled for 5 minutes, centrifuged, and stored at -80˚C. 50 μL of agarose bound wheat germ-agglutinin (WGA) were added to the remaining cell lysate and incubated overnight at 4˚C with gentle shaking. Cell lysate containing the WGA suspended in NP40 10 lysis buffer were centrifuged at 1000 RPM for 30 secs and supernatant were removed, and this process was repeated 3 times. The mixture was spun at 13200 RPM for 15 sec liquid was removed and samples were mixed with 2x sample buffer, boiled for 5 min, and stored at -80˚C. 2.6 Live Ca2+ Imaging with Fluo-4 and Quantifications DRG explants were grown on glass-bottom dishes (CellVis). Explants were deprived of NGF for 16 hours by removing NGF and provided 1.5 μg/ml anti-NGF to sequester NGF residuals remaining in media. To assess Ca2+ mobilization in the axons, DRG explants were treated with 5 μM Fluo-4 AM (Invitrogen) in neurobasal media for 20 min at 37˚C, washed once with neurobasal media, and incubated in neurobasal for an additional 20 min at 37˚C. DRG explants were washed once with HBSS, and switched to clear HBSS-based complete media supplemented with 20mM HEPES (final concentration) to maintain pH at 7.4. DRG explants were tile-imaged using Zeiss Observer Z.1 inverted epifluorescence microscope with an automated motorized stage at 20x magnification. NIH ImageJ software was used to crop the soma and Schwann-cell rich area of each DRG explant. Then, binary images were generated with the remaining axons and the mean pixel intensity corrected by background pixel intensity was quantified. All control DRG explants were pooled and averaged to generate basal Ca2+ level. Measurements were normalized to the mean basal Ca2+ level and expressed as fold change from control DRG explants. 2.7 Statistical Analysis All visualization and statistical analyses were conducted using GraphPad Prism version 9.4.1 (Dotmatics, Boston, MA, USA). Normality tests were conducted on experiments with larger sample size (n > 5). Experiments with smaller sample size were analyzed with Kruskal Wallis test. Depending on normality of data, either Kruskal Wallis or one-way ANOVA was chosen. Appropriate post hoc test was conducted for statistically significant data. Dun’s test was used with Kruskal Wallis and Tukey’s post hoc test was used with ANOVA. 11 Chapter 3: The effect of U73122 on axonal morphology in NGF dependent DRG sensory neurons 3.1 Introduction U73122 is a synthetic compound initially discovered as an inhibitor for platelet activation and later found to inhibit PLC function (Bleasdale et al., 1989; Smith et al., 1990). Since then, the U73122 has been widely used as a PLC inhibitor with over 3500 citations. Although a few reports have reported nonspecific and cytotoxic effects of this chemical, most studies do not control for side effects, nor do they directly measure PLC inhibition or Ca2+ mobilization. This lack of rigor may lead to potentially biased or incorrect interpretation of experimental findings. In this thesis, U73122 was initially used to investigate axon degeneration in the absence of PLCγ activity. However, we have revealed that U73122 is profoundly cytotoxic to DRG sensory neurons. In this chapter, the effects of U73122 on DRG morphology, PLCγ1 phosphorylation and Ca2+ mobilization was examined. 3.2 Results Figure 1A shows DRG explants extracted from CD1 E13 mouse embryos and cultured with or without NGF in the presence and absence of 10 μM U73122; quantification of the axonal area of these DRGs is provided in Figure 1B. Typical U73122 concentrations applied in in vitro experiments is between 0.1 μM and 10 μM. U73122 is known to be unstable in solution, therefore a higher concentration of 10 μM was chosen to account for the loss of U73122 potency over the course of an experiment. When NGF is withdrawn for 24 hours, DRG explants undergo severe axonal degeneration characterized by blebbing, fragmentation of axons, and reduced axonal area. The addition of 10 μM U73122 increases the DRG axonal area covered in regions close to the soma (0 – 1500 μm) and U73122-treated DRGs showed higher levels of β-tubulin III at regions proximal to the soma, indicating an accumulation of β-tubulin III fragments (Figure 1A), Notably, the U73122-treated DRG explants showed reduced NGF dependent growth, and axon 12 fragmentation and blebbing (Figure 1A and 1B). Together, these data indicate that U73122 is compromising axonal function and producing a degenerative phenotype even in the presence of NGF. To further investigate the effect of U73122 on DRGs, in vivo imaging of dissociated DRG cultures was used to assess axonal morphology, Ca2+ levels and mitochondrial potential. Dissociated E13 DRG culture were infected with a lentivirus expressing GFP (lenti-gfp) to assess morphological changes in live neurons exposed to 10 μM U73122 (Figure 2A). To our surprise, U73122 treatment induced rapid degeneration of distal axons, with axonal fragmentation starting within 40 minutes of U73122 application, and a complete disappearance of the axonal GFP signal at 3 hours of treatment (Figure 2A). This rapid axonal loss contrasts with NGF-deprivation induced degeneration in which axonal blebbing was observed only after 12 hours of deprivation and axons remained intact and remained GFP positive even at 24 hours of NGF deprivation (Figure 2A). To further characterize the effects of U73122, DRG explants were stained with Fluo-4 and TMRE to detect intracellular Ca2+ and membrane outer membrane potential (MOMP) within axons. Figure 2B shows that DRG explants treated with U73122 in the presence of NGF showed drastically increased axonal Ca2+ mobilization and a complete loss of MOMP after only 20 minutes following treatment (Figure 2B). Taken together, these data demonstrate that U73122 exerts a profoundly toxic effect on DRG axons. This cytotoxicity may reflect off-target effects, effects on PLCγ1 activity, or a combination of both factors. 13 Figure 1. PLCγ inhibitor, U73122, alters DRG axon morphology in vitro. 13-day DRG sensory neuron explants were grown on poly-D-Lysine/laminin/collagen coated plates in neurobasal media containing NGF for 2 days. (A) β-tubulin III staining of DRG explants either maintained in the presence or absence of 10 μM PLCγ inhibitor, U73122. PLCγ inhibitor treated DRGs show growth reduction, axon fragmentation, swellings of neurite end. (B) Axon density as a function of distance from the soma was quantified by Axoquant 2.0 (Johnstone et al., 2018) and plotted in 500 μm intervals. Differences in the relative axon area between control DRGs and DRGs treated with U73122 at different time points were analyzed by two-way ANOVA and Tukey’s post hoc comparison and plotted with mean and SEM (n = 36 DRG explants per condition from 3 independent experiments). *p<0.05, **p<0.01, ***p<0.001. Scale bar indicate 1000 μm. 14 Figure 2. PLCγ inhibitor disrupts mitochondrial membrane potential and induces axonal Ca2+ influx. 13-day DRGs were grown on poly-D-Lysine/laminin/collagen coated plates in neurobasal media containing 12.5 ng/ml NGF for 1 day. (A) Representative frame capture of in vivo videos of lenti-gfp infected dissociated DRG neurons following 10 μM U73122 treatment in the presence and absence of NGF. 4 Independent experiments were performed. (B) Cultures were stained with Fluo-4 and TMRE and images were captured 20 minutes after treatment using 10 μM U73122 in the presence or absence of 12.5 g/ml NGF. Representative images show that the treatment with U73122 results in a massive loss of active mitochondria 20 minutes following treatment along with large increases in axonal Ca2+ level. 4 Independent experiments were performed. B A 15 To determine the effect of U73122 on PLCγ1, phosphorylation of PLCγ1 at Y783 was examined. As noted above, phosphorylation at this site relieves autoinhibition of PLCγ1 and is crucial for its activation. Figure 3A – 3D shows that treatment with U73122 results in a drastic increase in PLCγ1 Y783 phosphorylation in a time and dose dependent manner. Taken together with data above, these finding suggest that U73122 is highly toxic and that, at least in sensory DRG neurons, it may act as a PLCγ1 activator rather than an inhibitor. 3.3 Chapter Summary In this chapter, the effects of U73122 on DRG sensory neurons was examined. Although U73122 has been widely used as a PLC inhibitor, we discovered that it is highly toxic to DRG sensory neurons. Figures 1 and 2 revealed a substantial increase in axonal Ca2+ level and rapid MMP eventually leading to axon degeneration in DRGs. Surprisingly, U73122 induced a time and dose dependent increase in PLCγ1 phosphorylation suggesting that it may act as a PLCγ1 activator (Figure 3). It remains unclear whether U73122 activates PLCγ1 directly or through an alternative pathway induced by other U73122 off target effects. While a lower dosage could have been used to minimize or to avoid the cytotoxicity induced by U73122, the drug is unstable in solution, so a lower dosage may result in a loss of drug potency over the course of the experiment. 16 Figure 3. U73122 induces PLCγ1 Y783 phosphorylation in DRG sensory neurons in a dose and time dependent manner. 13-day DRG sensory neurons were maintained on poly-D-Lysine/laminin/collagen coated plates in neurobasal media containing 12.5 ng/mL NGF for 2 days. (A) DRG explant cultures treated with 10 μM U73122 for 3 and 6 hours. (B) Western blot quantification by densitometry of the corresponding bands for total PLCγ1 were normalized to βIII-tubulin. Differences in relative phos-PLCγ1to total PLCγ1 levels were analyzed by Kruskal-Wallis test (n = 4 independent experiment) and Dunn’s post hoc test. (C) dissociated DRG culture were exposed to 0.3 μM, 1 μM, 3 μM, and 10 μM U73122 for 6 hours. (D) western blot quantification of 3 independent experiments. Quantification by densitometry of the corresponding bands for total PLCγ1 were normalized to βIII-tubulin. Differences in the relative phos-PLCγ1 to total PLCγ1 levels were analyzed with Kruskal-Wallis test (n = 3 independent experiment) and Dunn’s post hoc test. *p < 0.05, **p < 0.01. A C B D 17 Chapter 4: Investigating the role of PLCγ1 in NGF-dependent axonal degeneration in DRG sensory neurons 4.1 Introduction In Chapter 3, U73122 was utilized to determine if PLCγ1 was involved in axonal degeneration in DRG sensory neurons. However, it is now clear that use of U73122 is problematic. A genetic approach is an alternative method to achieve my experimental goal. In Chapter 4, a PLCγ1 Cre-Lox mouse model was utilized to determine the role of PLCγ1 in axonal degeneration in DRG sensory neurons. PLCγ1 fl/fl mice have flox sites embedded in the Plcg gene that should not affect the normal expression of PLCγ1 but allow excision of the PLCγ1 locus in the presence of the cre recombinase. To excise PLCγ1 specifically in DRG sensory neurons, we crossed the PLCγ1 mice with those expressing a sensory neuron specific cre line, Advillin-cre with the expectation that PLCγ1 fl/fl::AdvCre mice will lack PLCγ1 expression only in sensory neurons. 4.2 Results Although results from Chapter 3 did not directly demonstrate that PLCγ1 plays a role in facilitating axonal degeneration in DRGs, western blot analysis reveal a gradual increase in PLCγ1 levels following NGF deprivation treatment (Figure 4A-4B). This increase was observed in 4 distinct experiments (Figure 4C). but when data pooled from these, the combined data approached but did not reach statistical significance. To confirm that PLCγ1 fl/fl control DRGs display NGF-induced growth and NGF deprivation induced degeneration, axonal areas of PLCγ1 fl/fl DRG explants were compared to control CD1 DRG explants in both the presence and absence of NGF. Figure 4C and 4D show that there was no difference in DRG explant size between the CD1 DRG explants controls and PLCγ1 fl/fl explants. Next, the ability of PLCγ1 fl/fl::AdvCre to delete PLCγ1 in DRG sensory neurons was assessed. The level of PLCγ1 in PLCγ1 fl/fl versus PLCγ1 fl/fl::AdvCre DRG explants was quantified through western blot analysis. Figure 4E-4G show a significant reduction but not a complete elimination of total PLCγ1 protein level in PLCγ1 fl/fl::AdvCre explants compared to PLCγ1 fl/fl littermate counterparts. Importantly, 18 PLCγ1 levels were reduced but levels of neurofilament, actin and βIII-tubulin were the same in the PLCγ1 fl/fl::AdvCre neurons versus PLCγ1 fl/fl littermate counterparts (Figure 5C-5E). Interestingly, the volume of PLCγ1 found in DRGs varies considerably between individual embryos. Immunocytochemistry was performed to further validate the reduction of PLCγ1 in PLCγ1 fl/fl::AdvCre DRGs. It was hypothesized that the differences in PLCγ1 levels observed across embryos may be the result of differential expression of PLCγ1 in individual sensory neuron due to varying penetrance of advillin-cre. Unfortunately, immunocytochemistry did not reveal significant differences in PLCγ1 expression between PLCγ1 fl/fl control and PLCγ1 fl/fl::AdvCre dissociated neuron cultures (data not shown). This may be caused by an oversaturation or unspecific binding of the rabbit monoclonal primary PLCγ1 antibody that was used. To determine if NGF-induced axonal growth or NGF deprivation-induced axonal loss differed between PLCγ1 fl/fl::AdvCre and PLCγ1 fl/fl DRGs explants, littermate explants with these genotypes were maintained in the presence of NGF or were grown in NGF and then deprived for 24 hours. Figure 4A and 4B show that NGF responses do not differ between PLCγ1 fl/fl::AdvCre and PLCγ1 fl/fl DRGs in these assays. I next compared intra-axonal Ca2+ mobilization and caspase 3 cleavage 16 hours following NGF deprivation in PLCγ1 fl/fl::AdvCre and PLCγ1 fl/fl DRGs. Interestingly, PLCγ1 fl/fl::AdvCre DRGs showed a significant attenuation of Ca2+ entry compared to DRGs derived from PLCγ1 fl/fl mice (Figure 5A and 5B). In contrast, caspase 3 cleavage observed in PLCγ1 fl/fl::AdvCre and PLCγ1 fl/fl DRGs following 16 hours NGF deprivation did not differ significantly (Figure 5C and 5G). 4.3 Chapter Summary The role of PLCγ1 in axonal degeneration was investigated using the PLCγ1 fl/fl::AdvCre mouse model in Chapter 4. First, I determined that PLCγ1 fl/fl::AdvCre expressed significantly lower PLCγ1 protein levels with normal responses to NGF (Figure 4). In Figure 5, I showed that DRG sensory neurons with reduced PLCγ1 level experienced 19 reduced Ca2+ mobilization. This finding suggests that PLCγ1 may play a protective role in DRG axon degeneration. However, PLCγ1 fl/fl::AdvCre DRGs showed no difference in cleaved caspase 3 levels. actin 40 t-PLCγ1 155 Advillin Cre -/- +/- +/- β-tubulin III 55 E F G D A B C 20 Figure 4. PLCγ1 fl/fl DRG explants show normal growth and degeneration and PLCγ1 fl/fl::AdvCre DRGs show significant reduction in PLCγ1 protein level. DRG explants from E13 mice were maintained in 12.5 ng/mL NGF for 2 days. (A) Western blot analysis of time dependent PLCγ1 phosphorylation in response to NGF deprivation treatment. (B) Quantification by densitometry of the corresponding bands for total PLCγ1 were normalized to βIII-tubulin. Relative phos-PLCγ1 level were analyzed with Kruskal-Wallis test (n = 4 independent experiment). (C) βIII tubulin staining of DRG explants either maintained in the presence or absence of NGF. CD1, PLCγ1 fl/fl, and PLCγ1 fl/fl::AdvCre show similar levels of growth and degeneration. (D) Axon density as a function of distance from the soma was quantified by Axoquant 2.0 (Johnstone et al., 2018) and plotted in 500 μm intervals. Differences in the relative axon area between CD1, PLCγ1 fl/fl, and PLCγ1 fl/fl::AdvCre DRGs treated with NGF deprivation at different time points were analyzed by two-way ANOVA and Tukey’s post hoc comparison and plotted with mean and SEM (n = 36 DRG explants per condition from 3 independent experiments). (E) Western blot analysis of PLCγ1 protein level in PLCγ1 fl/fl and PLCγ1 fl/fl::AdvCre DRG explants. (F) Quantification by densitometry of the corresponding bands for total PLCγ1 were normalized to βIII-tubulin. Differences in the total PLCγ1 level and (G) βIII tubulin level between PLCγ1 fl/fl and PLCγ1 fl/fl::AdvCre DRGs were analyzed with unpaired t-test, t = 4.156, df= 19, two-tailed, p < 0.001 (n = 9 PLCγ1 fl/fl embryos and n = 12 PLCγ1 fl/fl::AdvCre embryos collected from 4 independent experiment). 21 Figure 5. PLCγ1 fl/fl::AdvCre significantly reduced Ca2+ mobilization induced by NGF deprivation. E13 DRG explants from PLCγ1 fl/fl and PLCγ1 fl/fl::AdvCre embryos were isolated and maintained in poly-D-Lysine/laminin/collagen coated plates in C A E B D F G H 22 neurobasal media containing 12.5 ng/mL NGF for 2 days. (A) DRGs were stained with Fluo-4 after 16 h of NGF deprivation. PLCγ1 fl/fl::AdvCre DRGs show reduced Ca2+ mobilization compared to PLCγ1 fl/fl control explants. (B) Quantification of the mean pixel intensity corrected by background pixel intensity with National Institutes of Health (NIH) ImageJ Software. Fluo-4 intensities values were normalized to the mean intensity of PLCγ1 fl/fl NGF control DRG explants. Relative fluo-4 levels were analyzed using two-way ANOVA and Tukey’s post hoc comparison (n = 24 PLCγ1 fl/fl and n = 32 PLCγ1 fl/fl::AdvCre DRG explants from 4 independent experiments). *p < 0.01. (C) Western blot analysis of total PLCγ1, neurofilament, βIII tubulin, caspase 3 and cleaved caspase 3 following 16 h NGF deprivation in PLCγ1 fl/fl and PLCγ1 fl/fl::AdvCre DRG explants. Quantifications by densitometry of protein volumes were normalized by βIII-tubulin reveals (D) significant reductions of PLCγ1 were observed in in PLCγ1 fl/fl::AdvCre DRGs. (E) there was no significant differences in neurofilament levels, (F) caspase 3 levels, (G) cleaved caspase 3 levels, and (H) βIII tubulin levels between PLCγ1 fl/fl and PLCγ1 fl/fl::AdvCre DRG neurons. Results were analyzed with two-way ANOVA and Tukey’s post hoc comparison (n = 9 PLCγ1 fl/fl and n = 12 PLCγ1 fl/fl::AdvCre embryos collected from 4 independent experiments). ****p < 0.0001. 23 Chapter 5: Determining if PLCγ1 is involved in TrkA-dependent NGF dependent degeneration in DRG sensory neurons 5.1 Introduction In the previous chapter, I demonstrated that Ca2+ influx induced by NGF withdrawal from DRG sensory neurons was compromised when PLCγ1 levels were reduced. In chapter 5, I investigated if PLCγ1 functions downstream of a TrkA-dependent degenerative pathway. To achieve this, we generated TrkA mouse in which PLCγ1 tyrosine 785 was mutated to a phenylalanine, using CRISPR Cas9 mediated ESC mutagenesis. Phosphorylation of TrkA at tyrosine 785 is required for the interaction of PLCγ1 with the receptor and I anticipated that the Y785F mutation introduced at this site would prevent PLCγ1 from binding TrkA in vivo. 5.2 Results These novel TrkAY785F knock in mice are the only known mouse model bearing a mutation that interferes the PLCγ1-TrkA interaction by preventing phosphorylation at the tyrosine 785 residue. Fortunately, both heterozygous and homozygous crossings of these mice can generate viable and fertile offspring with expected proportions of sexes. While both TrkAY785F heterozygous and homozygous mice are generally normal sized offspring, a few offspring are smaller in size. Further, a significant proportion of this mice line show a barbering and over-grooming phenotype, however, there isn’t a preference for homozygous wild-type, heterozygous, or homozygous knock-ins that bear this unusual phenotype. In this series of experiments, TrkAY785F homozygotes crossings with wild-type homozygotes crosses of the same genetic background used as controls. This was necessary because the only method to distinguish TrkA homozygotes from heterozygotes is through sequencing as this induced mutation only differs from wild-type animals by a single nucleotide. In some experiments heterozygotes crossings were performed to control minor differences in the age of embryos. 24 To begin the biochemical characterization of these animals, NGF-induced phosphorylation of TrkA was examined in TrkAY785F DRGs and from wild-type littermate counterparts (Figure 6A). For this, wheat germ agglutinin pull downs were performed to extract glycosylated cell surface proteins, including TrkA, from the treated DRGs and these were analyzed by immunoblot. Figure 6A confirms that DRGs carrying two knock-in mutant alleles did not display NGF-dependent phosphorylation at Y785 while phosphorylation at Y490 phosphorylation remained normal. Additionally, phosphorylation of PLCγ1, Akt, and Erk were examined in both TrkAY785F homozygotes and wild-type DRGs. Figure 6B shows that the TrkAY785F mutation did not change phosphorylation levels of TrkA dependent signaling pathways. We then turned to examining Ca2+ mobilization in axons 16 hours following NGF deprivation. Figure 6B demonstrates that DRGs that carried two knock-in mutant alleles have significantly reduced Ca2+ mobilization after NGF deprivation control to wild-type control explants. 5.3 Chapter Summary This chapter explores if the protective role of PLCγ1 observed in chapter 4 is dependent on PLCγ1-TrkA interaction. I first determined that the TrkAY785F double knock-in mice can produce viable and fertile offspring. Additionally, the Y785F mutation only disrupted TrkA tyrosine 785 phosphorylation (Figure 6A). Moreover, TrkAY785F knock-in DRGs reduced Ca2+ mobilization induced by NGF deprivation (Figure 7A and 7B). Together these data suggest a possible TrkA-PLCγ1 dependent degenerative pathway. 25 Figure 6. TrkAY785F double knock-in DRGs have defective phosphorylation at Y785. E13 DRG explants from TrkAY785 and wild-type embryos were isolated and maintained in poly-D-Lysine/laminin/collagen coated plates in neurobasal media containing 12.5 ng/mL NGF for 2 days. (A) western blot of TrkA phopshorylations at Y490, Y785, total TrkA, and βIII tubulin from wheat germ agglutinin pull down of TrkA. (B) Western blot analysis of PLCγ1 Y783, Akt, and Erk phosphorylation in homozygous TrkAY785F and wild-type DRGs of 1 independent experiment. Figure 7. TrkAY785F double knock-in DRGs show attenuation of in Ca2+ mobilization induced by NGF deprivation. E13 DRG explants from TrkAY785F and A B A B 26 wild-type embryos were isolated and maintained in poly-D-Lysine/laminin/collagen coated plates in neurobasal media containing 12.5 ng/mL NGF for 2 days and subject to NGF deprivation treatment. (A) DRGs were stained with Fluo-4 after 16 h of NGF deprivation. TrkA785F and wild-type DRGs show reduced Ca2+ mobilization compared to wild-type control explants. (B) Quantification of the mean pixel intensity corrected by background pixel intensity with National Institutes of Health (NIH) ImageJ Software. Fluo-4 intensities values were normalized to the mean intensity of WT NGF control DRG explants. Relative fluo-4 levels were analyzed using two-way ANOVA and Tukey’s post hoc comparison (n = 28 WT and n = 32 TrkAY785F DRG explants from 4 independent experiments). ****p < 0.0001. 27 Chapter 6: Discussion Developmental neuronal axonal degeneration is crucial for the normal development and functioning of the neuronal networks. TrkA is important in the studying of neurodegenerative pathways due to its characteristics of a dependence receptor. However, the signaling pathway for TrkA-dependent axonal degeneration is not well characterized. In fact, there hasn’t been any mechanistic linkages between TrkA and its downstream targets in the degenerative pathway. This thesis project attempts to shed light on downstream proteins that may be involved in facilitating axon degeneration in DRGs. This project was divided into two main parts. In the first part, I determined if PLCγ1 plays a role in sensory neuron axonal degeneration. Then, I investigated if PLCγ1 functions downstream of TrkA dependent degenerative pathway. In determining the degenerative role of PLCγ1, the U73122 drug and a genetically modified mice were employed to inhibit and to nullify PLCγ1 expression respectively. While the U73122 has been a very widely used for inhibiting PLCγ expression (Bleasdale et al., 1989; Smith et al., 1990), its specificity for PLCγ1 remains somewhat murky. Despite the few publications reporting the nonspecific effects of the U73122 (Chen et al., 2006; Klose et al., 2008; Pulcinelli et al., 1998). It has been widely accepted that U73122 was responsible to inhibit PLC activity and limit Ca2+ mobilization, and that its close analogue, U73343, does not inhibit PLC (Heemskerk et al., 1997). However, little attention has been given to these significant findings and the science community remain to take little notice of the importance to include proper controls to ensure that the U73122 was achieving the desired experimental effect. In this project, U73122 was utilized to determine whether inhibition of PLC activity would rescue axonal degeneration induced by NGF deprivation. It was quite surprising to discover the substantial amount of degeneration and potential activation of PLCγ1 the U73122 application has on DRG sensory neurons (Figure 2 and 3 respectively). While it remains unclear if these findings are specific to sensory neurons, U73122 is clearly unsuitable for determining the role of PLCγ1 in sensory neurons. Additionally, it is important for all future research utilizing this drug compound to have proper controls in place to avoid biases in interpretations. One of the limitations of the project was that the 28 PLCγ1 activity was measured indirectly assessing phosphorylation levels at Y783 residue. While the phosphorylation of the tyrosine 783 residue is required for PLCγ1 enzymatic activity (Hajicek et al., 2019), it was an indirect way of measuring PLCγ1 activity. Performing an IP1 activity assay would allow a direct and accurate measurement of PLCγ1 enzymatic activity through quantifications of the hydrolyzed product IP1 (Liu et al., 2020). Additionally, an unexpected discovery I made was the instability of the drug even in its powder form. It is well known that the U73122 is unstable in DMSO (Wilsher et al., 2007). U73122 from a newly purchased bottle was significantly more potent compared to an old bottle. The results shown in Figure 1 was obtained using an old U73122, while results from Figures 2 and 3 were obtained using new drug. Overall, the same conclusion can be reached regardless of whether new or old drug was used. However, a delay in the raise of Ca2+ and disruption of MOMP was observed compared to that shown in Figure 2. More specifically, when using the old U73122 drug, MOMP was completely disrupted by 40 min and maximal Ca2+ mobilization was achieved between 1 h and 2 h following drug treatment. Moreover, a 10-fold reduction in U732122 concentration needed to be done to prevent DRG explants from completely detaching and lifting off the plastic culture dishes and to allow cell lysate collection and quantification of PLCγ1 Y783 phosphorylation. It was worth noting that the new and old drugs was stored in their respective stable powder forms and only made into stock solutions close to the experimentation times. This suggests that the U73122 is unstable even in its powder form. The rate of decay falls outside the scope of this project. Perhaps a drug activity assay could be performed if the potency of U73122 becomes important in an experimental set up. Despite the limitations in utilizing the U73122 to determine the role of PLCγ1 in axonal degeneration. It remains hopeful that PLCγ1 may potentially be involved in this degenerative pathway. PLCγ1 Cre-Lox mouse model was used to determine if PLCγ1 plays a role in axonal degeneration induced by NGF deprivation. The advantage of this mouse model is the ability to examine neuronal responses in DRGs with significantly reduced PLCγ1 levels as PLCγ1 null mouse embryos are known to be nonviable. I have determined that a significant reduction of PLCγ1 protein level was achieved in PLCγ1 fl/fl::AdvCre sensory neurons and the level of reduction in PLCγ1 was sufficient to achieve a significant 29 attenuation of Ca2+ mobilization induced by NGF deprivation (Figure 5A – 5B). This data suggests that PLCγ1 may play a role in facilitating axon degeneration in DRG sensory neurons. It is worth noting that these PLCγ1 fl/fl::AdvCre mice does not achieve a complete knock-out of PLCγ1 in sensory neurons (Figure 4E – 4G) and that PLCγ1 expression in PLCγ1 fl/fl::AdvCre vary among individual embryos. More work may be done in the future to optimize the reduction of PLCγ1 protein levels. One way to further reduce PLCγ1 levels in sensory neurons is to increase the Cre recombinase penetration by crossing PLCγ1 fl/fl mice with different Cre mouse line perhaps under a stronger promoter. Alternatively, a greater Cre penetrance may be achieved through the use of the PLCγ1 fl/fl::AdvCre mice in conjunction with an alternative method for Cre delivery such as viral infections with Lenti-Cre or AAV-Cre virus, or transfections of Cre-containing plasmids through delivery methods such as nucleofection or magnetofection. One potential pitfall for the use of viral infections is that it may take too long for the virus to penetrate the DRG axons with a MOI that doesn’t overwhelm and kill the DRGs. As previously mentioned, that there was a significant decrease in Ca2+ mobilization observed in PLCγ1 fl/fl::AdvCre sensory neurons. Moreover, a binary distribution in the Ca2+ mobilization induced by NGF deprivation in the PLCγ1 fl/fl DRG explants was shown in Figure 5B. Quantifications represent four independent experiments where two experiments show a significant reduction in the amount of Ca2+ mobilization induced by NGF deprivation in PLCγ1 fl/fl::AdvCre explants when compared to PLCγ1 fl/fl explants. The other two experiments show no difference in the amount Ca2+ mobilization induced by NGF deprivation. In the experiments where no difference in Ca2+ mobilization was observed, the PLCγ1 fl/fl control DRG explants did not seem to show significant increases in NGF deprivation induced Ca2+ mobilization like one would expect to see on wild-type control DRG explants. It is unclear why there are two distinct populations of PLCγ1 fl/fl DRG explants were observed in the absence of NGF. This may likely be due to natural biological variation where certain PLCγ1 fl/fl DRG explants did not show as strong of an intense Ca2+ mobilization induced by NGF deprivation as other PLCγ1 fl/fl explants. Performing more replicates of this experiment will hopefully reflect that the population of PLCγ1 fl/fl accounts for only a small proportion of the overall population. Additionally, quantifications based on embryos rather than DRG explants would have given the data 30 more confidence. However, more repeats of the experiment will be necessary for that. None the less, the quantifications in Figure 5B with each data point representing a DRG explant was significant with p < 0.05. Finally, the TrkAY785F double knock-in mice was utilized to determine if the protective role of PLCγ1 observed in 4 was dependent on TrkA. They are the first mice to be generated that bears a mutation to prevent TrkA-PLCγ1 interaction. Fortunately, these animals are fertile and can generate viable offspring. There are several behavioural differences observed in these animals such as excessive barbering and grooming, as well as occasional smaller animals were found. Further investigations are required to determine if this is as result of the TrkAY785F mutation since these phenotypes were found in wild-type, heterozygotes, and homozygote knock-in animals of the same lineage. Additionally, I determined that DRGs bearing two alleles of the Y785F mutation disrupted tyrosine 785 phosphorylation (Figure 6A) while phosphorylation of other TrkA dependent proteins remain the same (Figure 6B). Additionally, Ca2+ mobilization was significantly reduced 16 h after NGF deprivation in TrkAY785F double knock-in DRGs. This confirms that PLCγ1 may play a role in NGF deprivation induced axon degeneration. More importantly, interaction of PLCγ1 with TrkA is required to dampen Ca2+ mobilization induced by NGF deprivation in DRGs. 31 Chapter 7: Conclusion One of the significances of studying axon degeneration is due to the irreplaceable nature of post-mitotic neurons. This means that once a patient has been diagnosed with neurodegenerative disorders, there is no way to regenerate new neurons to replace the lost cells making treatments for these diseases extremely challenging. There are many factors that may trigger axonal degeneration whether it is part of a normal pruning or pathological degenerating process. This master’s thesis project aims to explore the TrkA-PLCγ1 degenerative pathway. The thesis first examines if PLCγ1 play a role in NGF-dependent DRG axonal degeneration. It was hypothesized that in the absence of PLCγ1, axons will be rescued from NGF deprivation induced degeneration. Both inhibitor and genetic approach were employed to answer this research question. It was confirmed that the genetic knock down of PLCγ1 reduces NGF deprivation induced Ca2+ mobilization confirming a role of PLCγ1 in DRG axon degeneration. Interestingly, the use of U73122 to inhibit PLCγ activity did not achieve the desired inhibition despite U73122 is established as the only specific inhibitor for PLCγ. Furthermore, the thesis explores whether PLCγ1 functions in a TrkA-dependent manner. It was believed that if PLCγ1 is unable to interact with TrkA, then it will be unable to carry out its role in facilitating axon degeneration. By utilizing TrkAY785F knock-in mice, it has been demonstrated that the absence of TrkA-PLCγ1 interaction also reduces NGF deprivation induced Ca2+ mobilization. The thesis project reveals novel sights to the degenerative pathway facilitated by TrkA-PLCγ1interaction. It has been shown that both PLCγ1 and PLCγ1-TrkA interaction is both necessary and sufficient to significantly reduce Ca2+ mobilization induced by NGF deprivation. Further research is required to provide a more wholistic understanding of the TrkA-PLCγ1 degenerative signaling. 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Statistical Analysis Figure 1B Comparison p-value Normality Test used Post-hoc used 1000-1500 μm CTRL vs. 24h NGF DEP 0.0365 N Two-way ANOVA TukeyHSD 1000-1500 μm 24h NGF DEP vs. PLCγ CTRL 0.0334 1500-2000 μm CTRL vs. 24h NGF DEP <0.0001 1500-2000 μm 24h NGF DEP vs. PLCγ CTRL 0.0004 2000-2500 μm CTRL vs. 24h NGF DEP <0.0001 2000-2500 μm CTRL vs. PLCγ 24h NGF DEP <0.0001 2000-2500 μm 24h NGF DEP vs. PLCγ CTRL 0.0004 2500-3000 μm CTRL vs. 24h NGF DEP <0.0001 2500-3000 μm CTRL vs. PLCγ CTRL 0.0014 2500-3000 μm CTRL vs. PLCγ 24h NGF DEP <0.0001 3000-3500 μm CTRL vs. 24h NGF DEP <0.0001 3000-3500 μm CTRL vs. PLCγ CTRL <0.0001 3000-3500 μm CTRL vs. PLCγ 24h NGF DEP <0.0001 39 3500-4000 μm CTRL vs. 24h NGF DEP 0.0008 3500-4000 μm CTRL vs. PLCγ CTRL 0.0005 3500-4000 μm CTRL vs. PLCγ 24h NGF DEP 0.0001 Figure 3B Comparison p-value Normality Test used Post-hoc used 0h vs. 6h treatment 0.0211 N Kruskal-Wallis Dunn’s Test Figure 3D Comparison p-value Normality Test used Post-hoc used 0μM vs. 10μM 0.0097 N Kruskal-Wallis Dunn’s Test Figure 4B Comparison p-value Normality Test used Post-hoc used ns N Kruskal-Wallis Dunn’s Test Figure 4D Comparison p-value Normality Test used Post-hoc used 40 1000-1500 μm CD1 control vs. CD1 24h dep 0.0032 N Two-way ANOVA TukeyHSD 1000-1500 μm CD1 control vs. PLCγ fl/fl::AdvCre 24h dep 0.0031 1000-1500 μm CD1 24h dep vs. PLCγ fl/fl::AdvCre control 0.0009 1000-1500 μm CD1 24h dep vs. PLCγ fl/fl control 0.0023 1000-1500 μm PLCγ fl/fl::AdvCre control vs. PLCγ fl/fl::AdvCre 24h dep 0.0008 1000-1500 μm PLCγ fl/fl::AdvCre control vs. PLCγ fl/fl 24h dep 0.0411 1000-1500 μm PLCγ fl/fl::AdvCre 24h dep vs. PLCγ fl/fl control 0.0023 1500-2000 μm CD1 control vs. CD1 24h dep <0.0001 1500-2000 μm CD1 control vs. PLCγ fl/fl::AdvCre 24h dep <0.0001 1500-2000 μm CD1 control vs. PLCγ fl/fl 24h dep <0.0001 1500-2000 μm CD1 24h dep vs. PLCγ fl/fl::AdvCre control <0.0001 1500-2000 μm CD1 24h dep vs. PLCγ fl/fl control <0.0001 1500-2000 μm PLCγ fl/fl::AdvCre control vs. PLCγ fl/fl::AdvCre 24h dep <0.0001 1500-2000 μm PLCγ fl/fl::AdvCre control vs. PLCγ fl/fl 24h dep <0.0001 41 1500-2000 μm PLCγ fl/fl::AdvCre 24h dep vs. PLCγ fl/fl control <0.0001 1500-2000 μm PLCγ fl/fl control vs. PLCγ fl/fl 24h dep <0.0001 2000-2500 μm CD1 control vs. CD1 24h dep <0.0001 2000-2500 μm CD1 control vs. PLCγ fl/fl::AdvCre 24h dep <0.0001 2000-2500 μm CD1 control vs. PLCγ fl/fl 24h dep <0.0001 2000-2500 μm CD1 24h dep vs. PLCγ fl/fl::AdvCre control <0.0001 2000-2500 μm CD1 24h dep vs. PLCγ fl/fl control <0.0001 2000-2500 μm PLCγ fl/fl::AdvCre control vs. PLCγ fl/fl::AdvCre 24h dep <0.0001 2000-2500 μm PLCγ fl/fl::AdvCre control vs. PLCγ fl/fl 24h dep <0.0001 2000-2500 μm PLCγ fl/fl::AdvCre 24h dep vs. PLCγ fl/fl control <0.0001 2000-2500 μm PLCγ fl/fl control vs. PLCγ fl/fl 24h dep <0.0001 2500-3000 μm CD1 control vs. CD1 24h dep 0.0288 2500-3000 μm CD1 control vs. PLCγ fl/fl::AdvCre 24h dep 0.0321 2500-3000 μm CD1 control vs. PLCγ fl/fl 24h dep 0.0329 42 2500-3000 μm CD1 24h dep vs. PLCγ fl/fl::AdvCre control 0.0396 2500-3000 μm PLCγ fl/fl::AdvCre control vs. PLCγ fl/fl::AdvCre 24h dep 0.0440 2500-3000 μm PLCγ fl/fl::AdvCre control vs. PLCγ fl/fl 24h dep 0.0450 Figure 4F Comparison p-value Normality Test used Post-hoc used PLCγ1 level in PLCγ1 fl/fl vs. PLCγ1 fl/fl::AdvCre 0.0005 N Unpaired t-test N/A Figure 4G Comparison p-value Normality Test used Post-hoc used βIII-tubulin in PLCγ1 fl/fl vs. PLCγ1 fl/fl::AdvCre ns N Unpaired t-test N/A Figure 5B Comparison p-value Normality Test used Post-hoc used PLCγ1 fl/fl:NGF Control vs. PLCγ1 fl/fl:16 h NGF dep. <0.0001 Y Two-way ANOVA TukeyHSD PLCγ1 fl/fl:NGF Control vs. PLCγ1 fl/fl::CRE :16 h NGF dep. <0.0001 PLCγ1 fl/fl:16 h NGF dep. vs. PLCγ1 fl/fl::CRE :NGF Control <0.0001 43 PLCγ1 fl/fl:16 h NGF dep. vs. PLCγ1 fl/fl::CRE :16 h NGF dep. 0.0094 PLCγ1 fl/fl::CRE :NGF Control vs. PLCγ1 fl/fl::CRE :16 h NGF dep. <0.0001 Figure 5D Comparison p-value Normality Test used Post-hoc used PLCγ1 fl/fl:NGF Control vs. PLCγ1 fl/fl:16h NGF dep. <0.0001 Y Two-way ANOVA TukeyHSD PLCγ1 fl/fl:NGF Control vs. PLCγ1 fl/fl AdvCre:NGF Control <0.0001 PLCγ1 fl/fl:NGF Control vs. PLCγ1 fl/fl AdvCre:16h NGF dep. <0.0001 PLCγ1 fl/fl:16h NGF dep. vs. PLCγ1 fl/fl AdvCre:NGF Control 0.0020 PLCγ1 fl/fl:16h NGF dep. vs. PLCγ1 fl/fl AdvCre:16h NGF dep. ns PLCγ1 fl/fl AdvCre:NGF Control vs. PLCγ1 fl/fl AdvCre:16h NGF dep. <0.0001 Figure 5E Comparison p-value Normality Test used Post-hoc used PLCγ1 fl/fl:NGF Control vs. PLCγ1 fl/fl:16h NGF dep. <0.0001 Y Two-way ANOVA TukeyHSD PLCγ1 fl/fl:NGF Control vs. PLCγ1 fl/fl AdvCre:NGF Control ns 44 PLCγ1 fl/fl:NGF Control vs. PLCγ1 fl/fl AdvCre:16h NGF dep. <0.0001 PLCγ1 fl/fl:16h NGF dep. vs. PLCγ1 fl/fl AdvCre:NGF Control <0.0001 PLCγ1 fl/fl:16h NGF dep. vs. PLCγ1 fl/fl AdvCre:16h NGF dep. ns PLCγ1 fl/fl AdvCre:NGF Control vs. PLCγ1 fl/fl AdvCre:16h NGF dep. <0.0001 Figure 5F Comparison p-value Normality Test used Post-hoc used PLCγ1 fl/fl:NGF control vs. PLCγ1 fl/fl:16h NGF dep. 0.0005 Y Two-way ANOVA TukeyHSD PLCγ1 fl/fl:NGF control vs. PLCγ1 fl/fl AdvCre:NGF control ns PLCγ1 fl/fl:NGF control vs. PLCγ1 fl/fl AdvCre:16h NGF dep. <0.0001 PLCγ1 fl/fl:16h NGF dep. vs. PLCγ1 fl/fl AdvCre:NGF control 0.0015 PLCγ1 fl/fl:16h NGF dep. vs. PLCγ1 fl/fl AdvCre:16h NGF dep. ns PLCγ1 fl/fl AdvCre:NGF control vs. PLCγ1 fl/fl AdvCre:16h NGF dep. <0.0001 Figure 5G Comparison p-value Normality Test used Post-hoc used 45 PLCγ1 fl/fl:NGF control vs. PLCγ1 fl/fl:16h NGF dep. 0.0173 Y Two-way ANOVA TukeyHSD PLCγ1 fl/fl:NGF control vs. PLCγ1 fl/fl AdvCre:NGF control ns PLCγ1 fl/fl:NGF control vs. PLCγ1 fl/fl AdvCre:16h NGF dep. 0.0119 PLCγ1 fl/fl:16h NGF dep. vs. PLCγ1 fl/fl AdvCre:NGF control 0.0028 PLCγ1 fl/fl:16h NGF dep. vs. PLCγ1 fl/fl AdvCre:16h NGF dep. ns PLCγ1 fl/fl AdvCre:NGF control vs. PLCγ1 fl/fl AdvCre:16h NGF dep. 0.0015 Figure 5H Comparison p-value Normality Test used Post-hoc used PLCγ1 fl/fl:NGF Control vs. PLCγ1 fl/fl:16h NGF dep. ns Y Two-way ANOVA TukeyHSD PLCγ1 fl/fl:NGF Control vs. PLCγ1 fl/fl AdvCre:NGF Control ns PLCγ1 fl/fl:NGF Control vs. PLCγ1 fl/fl AdvCre:16h NGF dep. ns PLCγ1 fl/fl:16h NGF dep. vs. PLCγ1 fl/fl AdvCre:NGF Control ns PLCγ1 fl/fl:16h NGF dep. vs. PLCγ1 fl/fl AdvCre:16h NGF dep. ns PLCγ1 fl/fl AdvCre:NGF Control vs. PLCγ1 fl/fl AdvCre:16h NGF dep. ns 46 Figure 7B Comparison p-value Normality Test used Post-hoc used WT:NGF control vs. WT:dep. 16h <0.0001 Y Two-way ANOVA TukeyHSD WT:NGF control vs. TrkA Y785F:dep. 16h <0.0001 WT:dep. 16h vs. TrkA Y785F:NGF control <0.0001 WT:dep. 16h vs. TrkA Y785F:dep. 16h <0.0001 TrkA Y785F:NGF control vs. TrkA Y785F:dep. 16h <0.0001
Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Barker, Philip A.
Gibon, Julien
The investigation of the role of PLCgamma1 in the TrkA dependent degenerative pathway within dorsal root ganglion sensory neurons
Text