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In vitro and ex vivo exploration of knock-in mouse models harboring mutations linked to late onset Parkinson’s… Tatarnikov, Igor 2020

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IN VITRO AND EX VIVO EXPLORATION OF KNOCK-IN MOUSE MODELS HARBORING MUTATIONS LINKED TO LATE ONSET PARKINSON’S DISEASE  by  Igor Tatarnikov  B.Sc., The University of British Columbia, 2014  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  February 2020  © Igor Tatarnikov, 2020  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled:  In Vitro and Ex Vivo Exploration of Knock-In Mouse Models Harboring Mutations Linked to Late Onset Parkinson’s Disease.  Submitted by  Igor Tatarnikov      in partial fulfillment of the requirements for  the degree of  Master of Science   in   Neuroscience    Examining Committee:  Additional Supervisory Committee Members:  Dr. Matthew Farrer, Medical Genetics Supervisor  Dr. Lynn A. Raymond, Psychiatry Supervisory Committee Member  Dr. Doris J. Doudet, Neurology  Additional Examiner   Dr. Vesna Sossi, Physics and Astronomy Supervisory Committee Member iii  Abstract  Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting 2-3% of the population aged 65 or older1. Most PD cases are idiopathic i.e. without identifiable cause. Currently, therapeutic options for PD, which include dopamine replacement and deep brain stimulation, manage motor symptoms relatively well immediately following diagnosis. Unfortunately, neither therapeutic strategy modifies the progression of the disease or address the plethora of non-motor symptoms. Historically, PD was thought of as a disease of the environment with little to no genetic contribution. However, within the last two decades mutations in Alpha-synuclein (SNCA), leucine-rich repeat kinase (LRRK2), vacuolar protein sorting 35 (VPS35), and DnaJ heat shock protein family (Hsp40) member C13 (DNAJC13) have been linked to autosomal dominantly inherited parkinsonism that is clinically indistinguishable from idiopathic PD2.  In this study we began by comparing mature primary cortical cultures derived from knock-in mice harboring either the Lrrk2 p.R1441C (RKI), or the Dnajc13 p.N855S (DKI) mutation. Previously we have shown that cortical neurons derived from Lrrk2 p.G2019S (GKI) mice showed an increased frequency of miniature excitatory post-synaptic currents (mEPSCs) with no change in amplitude. RKI neurons showed a significantly higher frequency of mEPSCs with no change in amplitude. DKI neurons were indistinguishable from their WT counterparts in the frequency or amplitude of mEPSCs.   Next, we characterized the nigrostriatal dopamine system in 3-month-old DKI mice. By fast-scan cyclic voltammetry (FSCV) there were no significant changes in evoked release or clearance of dopamine. At a low dose, GBR-12909 acts as a potent and selective dopamine reuptake inhibitor. iv  When applied to brain slices, DKI mutant animals were less responsive to dopamine active transporter (DAT) inhibition, suggesting lower levels of DAT, as verified by western blotting.   Lastly, the effect of Lrrk2 kinase inhibition on the dopaminergic phenotypes previously described in Vps35 p.D620N (VKI) mice was examined. Lrrk2 kinase activity has been shown to be elevated in not only VKI mice but also in idiopathic PD. Previous work has shown changes in both DAT and VMAT2 levels along with increased evoked release and longer decays in 3-month-old VKI animals. Daily MLi-2 injections rescued the phenotypes previously observed3.    v  Lay Summary  Parkinson’s disease (PD) is the second most common neurodegenerative disorder, affecting 2-3% of the population aged 65 or older. Historically PD has been thought of to be a disease of the environment with no genetic contribution. However, within the last two decades many genes have been linked to familial PD. Current treatments manage the motor symptoms, yet are ineffective at slowing down the progression of the disease or at addressing the non-motor symptoms. The biggest roadblock to developing new treatments has been the lack of appropriate models replicating the early stages of the disease. This study compares the early changes present in several genetic models of mutations linked to late-onset PD.  The changes observed are similar across multiple models, and were reversed using a drug targeting one of the genes. This work highlights the similarities between genetic models of late-onset PD, suggesting a potential common mechanism underlying disease ontology. vi  Preface  The work contained within this thesis was performed at the University of British Columbia, chiefly at the Djavad Mowafaghian Centre for Brain Health and the Centre for Disease Modelling. All projects were approved by the University of British Columbia’s Research Ethics Board and procedures were carried out in strict accordance with the Canadian Council on Animal Care guidelines: • Animal care protocols: A15-0105 and A16-0088 • Personal animal care and safety certificates: Canadian Council on Animal Care (7224-15); Introduction to Working with Rodents in Research, Rodent Restraint and Subcutaneous/Intraperitoneal Injections (RBH-50-15); Introduction to Rodent Anesthesia - Inhalational and Injectable (RA-36-15); Biological Safety (2018-ZXO99); Chemical Safety (2018-2Ijjv).   This thesis is based on work conducted at the Centre for Applied Neurogenetics by Igor Tatarnikov, Dr. Jordan Follet, Li-Ping Cao, Cindy Wong, and Brittney Smaila, working under the supervision of Professor Matthew J. Farrer.. I was responsible for all electrophysiological experiments and a portion of the biochemical findings presented in chapter 4. Dr. Jordan Follet was responsible for the western blots in chapter 3 and 4, along with an initial draft of the methods section on protein biochemistry. Li-Ping Cao was responsible for the creation and maintenance of cultured cortical neurons for chapter 2. Cindy Wong and Brittney Smaila were responsible for all genotyping of transgenic animals. Professor Matthew J. Farrer and Igor Tatarnikov were responsible for experimental design. vii  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Figures ............................................................................................................................... xi List of Symbols ............................................................................................................................ xii List of Abbreviations ................................................................................................................. xiv Acknowledgements ................................................................................................................... xvii Dedication ................................................................................................................................. xviii Chapter 1: Introduction ................................................................................................................1 1.1 Epidemiology .................................................................................................................. 1 1.2 Idiopathic PD .................................................................................................................. 1 1.2.1 Modeling ..................................................................................................................... 2 1.3 Toxin related ................................................................................................................... 2 1.3.1 Modeling ..................................................................................................................... 3 1.4 PD linked genes .............................................................................................................. 3 1.4.1 Juvenile and early onset .............................................................................................. 4 1.4.1.1 Modeling ............................................................................................................. 5 1.4.2 SNCA .......................................................................................................................... 5 1.4.2.1 Modeling ............................................................................................................. 5 1.4.3 LRRK2 ........................................................................................................................ 6 viii  1.4.3.1 Modeling ............................................................................................................. 7 1.4.4 VPS35 ......................................................................................................................... 7 1.4.4.1 Modeling ............................................................................................................. 7 1.4.5 DNAJC13 .................................................................................................................... 8 1.5 Summary ......................................................................................................................... 8 1.6 Aims ................................................................................................................................ 9 Chapter 2: In-vitro comparison of gene mutations lined to late-onset Parkinson’s disease .10 2.1 Introduction ................................................................................................................... 10 2.2 Materials and methods .................................................................................................. 11 2.2.1 Transgenic mice handling and culture preparation ................................................... 11 2.2.2 Electrophysiology ..................................................................................................... 12 2.3 Results ........................................................................................................................... 13 2.3.1 In vitro synaptic transmission in R1441C LRRK2 cortical neurones ....................... 13 2.3.2 In vitro synaptic transmission in N855S Dnajc13 cortical neurones ........................ 13 2.4 Discussion ..................................................................................................................... 14 Chapter 3: Ex vivo characterization of the nigrostriatal dopaminergic system of N855S Dnajc13 mice. ...............................................................................................................................18 3.1 Introduction ................................................................................................................... 18 3.2 Materials and Methods .................................................................................................. 18 3.2.1 Dnajc13 N855S knock-in mice ................................................................................. 18 3.2.2 Protein analysis ......................................................................................................... 19 3.2.3 Fast-scan cyclic voltammetry (FSCV) ...................................................................... 20 3.2.4 Statistics and data reporting ...................................................................................... 21 ix  3.3 Results ........................................................................................................................... 22 3.3.1 Striatal dopaminergic markers in N855S Dnajc13 knock-in mice ........................... 22 3.3.2 Striatal dopaminergic release in N855S Dnajc13 knock-in mice ............................. 22 3.4 Discussion ..................................................................................................................... 23 Chapter 4: LRRK2 inhibition as a therapeutic target in Parkinson’s disease .......................27 4.1 Introduction: .................................................................................................................. 27 4.2 Materials and methods: ................................................................................................. 27 4.2.1 Vps35 D620N knock-in mice ................................................................................... 27 4.2.2 Fast-scan cyclic voltammetry (FSCV) ...................................................................... 28 4.2.3 MLi-2 injection ......................................................................................................... 29 4.2.4 Protein analysis ......................................................................................................... 29 4.2.5 Statistics and data reporting ...................................................................................... 30 4.3 Results: .......................................................................................................................... 31 4.3.1 Striatal dopamine release in Vps35 D620N knock-in mice ...................................... 31 4.3.2 Inhibiting Lrrk2 activity Vps35 D620N knock-in mice at 3 months of age ............. 31 4.4 Discussion: .................................................................................................................... 33 Chapter 5: Conclusion .................................................................................................................40 5.1 Summary ....................................................................................................................... 40 5.2 Significance................................................................................................................... 41 5.3 Limitations .................................................................................................................... 41 5.3.1 Chapter 2 ................................................................................................................... 41 5.3.2 Chapter 3 and 4 ......................................................................................................... 42 5.4 Future Directions .......................................................................................................... 42 x  5.4.1 Chapter 2 ................................................................................................................... 42 5.4.2 Chapter 3 ................................................................................................................... 43 5.4.3 Chapter 4 ................................................................................................................... 43 References .....................................................................................................................................45   xi  List of Figures  Figure 2.1 Whole-cell patch-clamp recordings of cortical neurons in DIV21 CTX cultures from RKI mice. ...................................................................................................................................... 16 Figure 2.2 Whole-cell patch-clamp recordings of cortical neurons in DIV21 CTX cultures from DKI mice. ...................................................................................................................................... 17 Figure 3.1 Western blotting for DAT in microdissected striatal tissue from 3 month old. .......... 25 Figure 3.2 Nigrostriatal dopamine transmission in 3 month old DKIs. ........................................ 26 Figure 4.1 Nigrostriatal dopamine transmission in 1 month old VKIs. ........................................ 36 Figure 4.2 Western blotting for Lrrk2 and pLrrk2 in microdissected striatal tissue from 3 month old VKIs treated with 7 daily injections of either vehicle or MLi-2. ........................................... 37 Figure 4.3 Western blotting of dopaminergic markers in microdissected striatal tissue from 3 mo. old VKIs treated with 7 daily injections of either vehicle or MLi-2. ........................................... 38 Figure 4.4 Nigrostriatal dopamine transmission in 3 month old VKIs treated with vehicle or MLi-2..................................................................................................................................................... 39  xii  List of Symbols  °  Degree a.u.   Arbitrary units Cm  Membrane capacitance  g  Gram Hz  Hertz mg  Milligram min  Minute mL  Milliliter mM  Millimole Mohm  Megaohm mOsm   Milliosmole ms  Millisecond N  Number pA  Picoampere pF  Picofarads pg  Picogram Rp  Pipette resistance  Rs  Series resistance  s  Second v  Version Δ  Delta xiii  μA  Microampere μM  Micromolar xiv  List of Abbreviations  6-OHDA 6-hydroxydopamine ACSF  Artificial cerebrospinal fluid BCA  Bicinchoninic acid assay BoNT/C Botulinum neurotoxin C DA  Dopamine DAT  Dopamine transporter DBS  Deep brain stimulation DIV  Days in vitro DKI  Dnajc13 p.N855S knock-in mice DN  D620N DNAJC13 DnaJ heat shock protein family (Hsp40) member C13 ECS  extracellular solution  fEPSP  field excitatory postsynaptic potentials  FSCV  Fast-scan cyclic voltammetry GAPDH Glyceraldehyde 3-phosphate dehydrogenase GBR-12909  1-[2-[Bis-(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine dihydrochloride GKI  Lrrk2 G2019S knock-in mice HBSS  Hank's Balanced Salt Solution  HET  Heterozygous HRP  Horseradish peroxidase  xv  IP  Intraperitoneal L-DOPA Levodopa LRRK2 Leucine-rich repeat kinase 2 mEPSC Miniature excitatory post-synaptic currents  mGluR5 Metabotropic glutamate receptor 5 MLi-2  Merck LRRK2 inhibitor-2 MPTP  1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MSNs  Medium spiny neurons PD  Parkinson's disease PET  Positron emission tomography  PM  Plating medium  PSD95  Post-synaptic density protein 95  PTX  Picrotoxin RKI  Lrrk2 R1441C knock-in mice RME8  Receptor-mediated endocytosis 8 RT  Room temperature SEP  Superecliptic pHlourin SNpc  Substantia nigra pars compacta SNX1  Sorting nexin 1 Syn1A  Syntaxin 1A TBST  Tris-buffered saline containing 0.1% Tween 20  TH  Tyrosine Hydroxylase TTX  Tetrodotoxin xvi  VGluT1 Vesicular glutamate transporter-1 VKI  Vps35 D620N knock-in mice VMAT2 Vesicular mono-amine transporter 2 VPS35  Vacuolar protein sorting 35  WB  Western blot xvii  Acknowledgements  I thank Dr. Matthew J. Farrer for providing the space and occasion for me to grow as a scientist and as a person. The opportunities he gave me have forever changed my life.   I would also like to thank Dr. Austen J. Milnerwood for encouraging me to explore and question everything I am told. I will forever appreciate the time he took to pass on his technical and scientific knowledge.  Further, I would like to thank Drs. Dayne A. Beccano-Kelly, Mattia Volta and Lise Munsie for training me in the multitude of techniques I’ve had the pleasure of learning throughout my degree and for encouraging my passion for learning.  I would also like to thank my graduating committee, Drs. Lynn Raymond and Vesna Sossi for guiding me through this journey.   Finally, I would like to thank my family who have supported me throughout this entire degree. I would like to especially thank my partner for her constant words of encouragement and support.  This work would not have been possible without the support of funding agencies such as Canadian Institutes for Health Research (CIHR), Weston Brain Institute, Parkinson’s Canada, Parkinson’s BC, and the Michael J. Fox Foundation.  xviii  Dedication I dedicate this thesis to everyone that believed in me and guided me to becoming the person I am today. I especially want to thank my partner and best friend. Her contributions to my success cannot be understated, without her none of this would have been possible. 1  Chapter 1: Introduction  1.1 Epidemiology Parkinson’s disease (PD) is associated with an insidious but progressive loss of midbrain dopaminergic neurons within the substantia nigra pars compacta (SNpc)1. Classical motor symptoms present when >50% of the SNpc is lost, reducing striatal dopaminergic innervation by >80%4. While dopamine-replacement therapies and/or deep brain stimulation (DBS) can provide effective symptomatic relief, such treatments fail to modify the disease progression and, over time, greater dosing often leads to disabling dyskinesia, wearing off effects, and dose failure. Most patients with late-onset PD are idiopathic, ascribed to a multifactorial etiology, albeit with a relatively modest but well defined genetic component.  1.2 Idiopathic PD The majority of PD cases are idiopathic in nature, suggesting a complex interaction between the genetics and environment that is not yet fully understood. The disease is classically late-onset affecting approximately 2-3% of the population aged 655. The progression is slow but insidious: mortality is unchanged in the first decade following diagnosis but eventually doubling6. Men are twice as likely to be affected by PD in most populations7,8 suggesting a role for the protective effect of female sex hormones, sex-specific differences in exposure to environmental risk factors, and sex-associated genetic mechanisms. Intriguingly, one study reports no difference or even an increased likelihood of female cases9. Health care quality and willingness to seek out treatment may also play a role in the observed sex differences10. Several environmental factors have also been associated with the incidence of PD. Organic pesticides and traumatic brain injury are 2  associated with an increased incidence of PD, while smoking and caffeine consumption are associated with a lower incidence11. Pathologically idiopathic PD is defined by the loss of neurons within the SNpc and the presence of Lewy bodies. These two findings are required for a definitive diagnosis of idiopathic PD10. Idiopathic PD is the standard when comparing other forms of PD with identified causes.   1.2.1 Modeling Modeling idiopathic PD has proven difficult as, by definition, the cause is unknown and impossible to replicate. Recently, advances in the reprogramming of cells into iPSCs and their derivation into relevant cell types has allowed some replication of the unique complex combination of the environmental and genetic factors resulting in idiopathic PD.  Neurons derived from patients with PD showed increased apoptosis12, lower dopamine (DA) levels and impaired network activity13, impaired mitophagy14 and autophagy15. Interestingly, these phenotypes mirror those observed in iPSCs derived from patients harboring mutations in genes linked to late-onset PD (LRRK2, SNCA), suggesting that there is a common etiological mechanism between idiopathic and late-onset gene linked PD16.    1.3 Toxin related The use of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to induce nigral cell death was identified as a potential model for PD following a string of hospitalizations of heroin users with irreversible PD symptoms17. A batch of contaminated synthetic heroin containing MPTP was identified as being the cause. MPTP when administered can cross the blood brain barrier and is then converted to MPP+ in astrocytes and taken up by dopaminergic neurons via the dopamine 3  transporter (DAT). Once imported, MPP+ targets mitochondrial complex I resulting in cell death18. Exposure to certain chemicals used in farming increased PD risk substantially. Paraquat, an analogue of MPP+ and a commonly used herbicide, is also associated with PD risk in farming communities19. Rotenone, an organic pesticide, has also been found to cause oxidative damage and also inhibits mitochondrial complex I, leading to cell death20.   1.3.1 Modeling Toxin administration has proven valuable to model the end state of disease. MPTP, rotenone, and paraquat can induce dopaminergic cell loss, with limited progression, and aggregation of α-synuclein depending on the schedule of administration21–25. Furthermore, 6-hydroxydopamine (6-OHDA), administered by stereotaxic injection into both the striatum and the SNpc, has been used to selectively lesion SNpc dopaminergic neurons26. The loss of dopaminergic neurons induced by these toxins leads to a parkinsonian state in animals and facilitated the development of therapeutic interventions aimed at improving symptoms and the quality of life for patients. However, since the cell death is induced in an artificial manner, the etiology of idiopathic disease has remained unknown27.  1.4 PD genetics The majority of PD is idiopathic in nature; however, several genes have been linked to rare familial cases. Parkinson’s disease can be separated into categories based on the age at onset. Juvenile (≤20 years at diagnosis), early-onset (≤50 years at diagnosis) and typical, late-onset disease (>50 years at diagnosis)1. Examples of recessively inherited genes linked to juvenile and early-onset parkinsonism include DnaJ (Hsp40) homolog, subfamily C, member 6 (DNAJC6)28,29, DnaJ 4  (Hsp40) homolog, subfamily C, member 12 (DNAJC12)30, Synaptojanin 1 (SYNJ1)31  PINK1, parkin (also known as PARK2) and DJ-1 (also known as PARK7)2. Genes linked with dominantly inherited late onset PD include α-synuclein (SNCA), coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2)32, GTP cyclohydrolase I (GCH1)33, leucine-rich repeat kinase (LRRK2), vacuolar protein sorting 35 (VPS35), and DnaJ heat shock protein family (Hsp40) member C13 (DNAJC13), also known as required for receptor-mediated endocytosis 8 (RME8)2. Further, heterozygous mutations in the enzyme glucocerebrosidase (GBA) have been found to be the largest genetic risk factor contributing to the development of PD, enhancing the risk five-fold34. The implicated genes contribute to many important molecular pathways, suggesting a strong link between PD etiology and mitochondrial, lysosomal, and endosomal function, as well as protein trafficking and recycling.  1.4.1 Juvenile and early onset PINK1, parkin and DJ-1 are the main genes associated with juvenile and early onset PD. PINK1 and parkin play an important role in mitophagy. PINK1 is a cytoplasmic protein kinase associated with the mitochondria acting upstream of the E3 ubiquitin ligase Parkin. Together these protein promote the degradation of damaged mitochondria35. DJ-1 is a redox reactive protein which responds to cellular stress. DJ-1 may become active in the presence of reactive oxygen species and under oxidative stress36. Together with the toxin models of PD the recessively associated genes suggest a crucial role for mitochondrial dysfunction and oxidative stress in the death of SNpc neurons.   5  1.4.1.1 Modeling The nature of mutations inherited in a recessive manner tends to be loss of function. This fact makes knock-out models ideal for studying genes related to juvenile and early onset PD. Interestingly, while some minor behavioral deficits and alterations in striatal dopamine signaling were reported, no apparent neurodegeneration was observed in DJ-1-/-, Pink1-/-, or parkin-/- mice37–39. Interestingly, intestinal infection with gram negative bacteria in Pink1-/- mice created mitochondria specific T cells in the periphery and the brain. These animals showed a decrease in the density of dopaminergic axonal varicosities in the striatum and levodopa (L-DOPA) sensitive motor impairment40. Rats seem to be more sensitive to the genetic knock-out of these genes.       DJ1-/-, and Pink1-/- rats showed a progressive motor phenotype accompanied by substantial dopaminergic cell death within the SNpc41.  1.4.2 SNCA α-synuclein is the main component in Lewy bodies and neurites42. Several point mutations (p.A30P, p.E46K, p.H50Q, p.G51D, and p.A53T)43–48 along with locus duplication and triplication have been linked to dominantly inherited familial PD49–51. α-synuclein is expressed broadly throughout development and enriched in the brain in adulthood52. Currently, α-synuclein is thought to be involved in neurotransmitter release by binding and regulating SNARE complex assembly53,54, and by regulating synaptic vesicle endocytosis55.  1.4.2.1 Modeling Knock-out models of α-synuclein show mild alteration in synaptic vesicle dynamics with no overt behavioral phenotypes56,57. However, there are two additional homologues in the synuclein family 6  β-, and γ-synuclein. A triple knock-out presents with a hyperactive phenotype in novel environments combined with an increase in evoked dopamine and lower tissue dopamine content58. Several mouse models overexpressing wild-type or mutant α-synuclein under several different promoters (PDGβ, PrP, Thy1) have been created to model PD. These models are able to induce cell death, early pre-synaptic alterations, and PD like behavioral phenotypes59–61. Stereotaxic delivery of recombinant adeno-associated virus inducing expression of WT and mutant α-synuclein has also been used to induce cell death and pathology in rats62. Overall, these models are fantastic at replicating the end stage of the disease with rapid progression and some Lewy-like pathology, akin to the toxin models. Nevertheless, while these models replicate the synucleinopathy, they may not necessarily replicate the underlying etiological mechanisms leading to the diseased state.   1.4.3 LRRK2 Dominantly-inherited mutations in leucine-rich repeat kinase 2 (LRRK2 p.N1437H, p.R1441C/G/H, p.Y1699C, p.G2019S, and p.I2020T)63–66 are linked to a syndrome that is clinically, and often pathologically, indistinguishable from idiopathic PD2. Further, polymorphic variants in LRRK2 have been associated with both higher and lower risk of PD67. Brain imaging in asymptomatic LRRK2 heterozygotes clearly reveals physiological differences in dopaminergic and serotonergic systems68. Pathogenic LRRK2 mutations constitutively activate the kinase directly69 or indirectly 70–72. However, post-mortem examination of SNpc from patients with idiopathic PD also shows elevated LRRK2 kinase activity73.   7  1.4.3.1 Modeling Several knock-in models of Lrrk2 associated mutations have been characterized in full. The R1441C mutation does not cause any overt behavioral alterations or changes in steady state dopamine levels in mice up to 22 months of age74. Lrkk2 p.G2019S knock-in mice show hyperactivity, along with an increased probability of glutamatergic release at corticostriatal synapses, and slower clearance of dopamine at a young age75. Older animals lose the behavioral phenotypes and synaptic hyperactivity observed75, reversing to display a reduction in striatal dopamine release76. The phenotypes observed in the knock-in mice models of Lrrk2 mutations model the early dysfunction and progression of the disease. The models are congruous with the early hyperdopaminergia observed in asymptomatic LRRK2 G2019S carriers.   1.4.4 VPS35 VPS35 p.D620N is linked to a late-onset familial form of PD reminiscent of idiopathic PD with a mean age at onset of ~51 years of age77,78 . VPS35 is responsible for the retrieval of transmembrane proteins from endosomes to the trans-Golgi network79. VPS35 is a key component of the retromer complex, along with VPS26, and VPS29. The mutation has no effect on the expression of the retromer complex, or the interaction between subunits80.  1.4.4.1 Modeling Our lab has shown that Vps35 plays an important role at the synapse, interacting with GluR1 and GluR2 when overexpressed in mature murine cultured cortical neurons. The trafficking of GluR1 was inhibited by the overexpression of VPS35 WT with p.D620N (DN) presenting as a loss of function81. Recently, we showed that Vps35 p.D620N knock-in mice (VKI) have early alterations 8  within the dopaminergic system, and in the absence of any overt motor phenotypes3. Fast-scan cyclic voltammetry (FSCV) revealed increased dopamine release and slower clearance in VKI striatal slices. Biochemistry and immunohistochemistry supported observations by FSCV and showed decreased dopamine transporter (DAT) levels and increased vesicular monoamine transporter 2 (VMAT2) levels. These mice have also been shown to have tau pathology and progressive neurodegeneration at 13+ months of age82 However, an independently generated Vps35 p.D620N model shows no loss of tyrosine hydroxylase (TH) positive cells, or α-Synuclein positive inclusions up to 70 weeks of age80. .  1.4.5 DNAJC13 Recently, a mutation in DNAJC13 (p.N855S) has been identified in a German-Russian Mennonite kindred83. Dopaminergic positron emission tomography (PET) imaging of symptomatic mutations carriers revealed rostrocaudal striatal deficits similar to idiopathic PD84. Dnajc13 p.N855S knock-in mice were found to have no change in expression of Dnajc13 in mature cultured cortical neurons but were shown to have increased formation of sorting nexin-1 (SNX1) enriched tubules85.  1.5 Summary PD is a complex syndrome with a currently unidentified etiology. Current therapeutic options address the motor symptoms, but their efficacy diminishes with use and disease progression, and increased dosage may lead to disabling dyskinesia. Historically, PD was thought to have no genetic component. However, several mutations in multiple genes have been linked to juvenile, early, and late-onset PD in the previous two decades. In addition, 187 genes in 90 loci have been modestly associated with risk through meta-analyses of genome-wide association studies86. Mouse models 9  of PD cannot replicate the full condition. Toxin and certain overexpression models can induce cell death and pathology but achieve it in an extremely acute manner which does not represent the slowly progressive ontology of disease. However, genetic knock-in models of mutations linked to late-onset PD may provide the best opportunity to recapitulate pathophysiology, and study early changes of specific gene dysfunction. Synergy across such models may identify common mechanisms, enable disease modifying therapies and new treatment strategies. Importantly, LRRK2 activity, as assayed by the levels of autophosphorylation at serine 910, 935, and 1292, has been shown to be elevated across several models of late-onset mutations linked to PD as well as post-mortem idiopathic PD samples 69,73. Taken together, we propose that there exist common phenotypes across knock-in models of late-onset PD-linked mutations, with increased LRRK2 activity being key to pathogenesis.  1.6 Aims The specific aims of this thesis are 1) to compare the effects of several mutations (Lrrk2 p.R1441C and Dnajc13 p.N855S) linked to late-onset PD in-vitro, 2) to examine the effects of Dnajc13 p.N855S mutation on dopamine release within the dorsolateral striatum, and 3) to examine the effect of inhibiting the kinase activity of Lrrk2 on dopaminergic phenotypes previously observed in mice harboring the Vps35 p.D620N mutation.     10  Chapter 2: In-vitro comparison of gene mutations linked to late-onset Parkinson’s disease  2.1 Introduction Published work suggests many genes linked to PD play an important role in the normal function of the synapse. Studies have shown that Lrrk, the homologue of human LRRK1 and LRRK2, plays a role at the neuromuscular junction of Drosophila melanogaster. Flies lacking Lrrk showed an increased number of synaptic boutons with overexpression of both LRRK and human LRRK2 having the opposite effect87. LRRK2 was also shown to phosphorylate EndophilinA, inhibiting membrane association and decreasing synaptic vesicle endocytosis in Drosophila melanogaster88. Mammalian models also suggest that Lrrk2 has a role at the synapse. RNA mediated silencing of LRRK2 expression induced a redistribution of vesicles within the synaptic bouton, altered recycling dynamics, and increased vesicle kinetics in cortical neurons89. Previously, our lab has shown elevated spontaneous release of glutamate in cultures derived from Lrrk2 p.G2109S (GKI) mice compared to their wild-type (WT) counterparts90. This was accompanied by alterations in Synapsin-1 phosphorylation with no changes in synaptic density. Interestingly neither Lrrk2 knock-out (KO) nor overexpressing (OE) cells showed any alteration in the probability of spontaneous release in cortical culture, arguing a unique gain of function caused by the increased kinase activity resulting from the p.G2019S mutation. Further, we have examined the effect of overexpression of both VPS35 WT and p.D620N (DN) in mature murine cortical neurons. Overexpression of VPS35 WT and DN caused a marked decrease in the frequency of miniature excitatory post-synaptic currents (mEPSCs) and a corresponding decrease in synaptic density in 11  cultured cortical neurons 81. VPS35 DN was shown to alter the trafficking of GluR1 as evidenced by quicker recovery of fluorescence following photobleaching of SEP-GluR1 and an increase in the amplitude of mEPSCs81. DnaJC13 has been shown to be expressed in cortical neurons, and the p.N855S mutations was shown to alter SNX1 membrane dynamics91. Altogether these findings suggest key synaptic roles for multiple PD-linked genes. DnaJC13, and Lrrk2, and Vps35 have also been shown to physically interact with each other92, suggesting that mutations found in these genes are disrupting the same function. In this chapter, the effect of Lrrk2 p.R1441C (RKI) and Dnajc13 p.N855S (DKI) on synaptic transmission in cortical cultured neurons was examined. We hypothesize that the probability of release will be altered in cortical neurons derived from RKI and DKI mice when compared to their WT counterparts similar to the phenotype found in GKI neurons.  2.2 Materials and methods 2.2.1 Transgenic mice handling and culture preparation All breeding, housing, and experimental procedures were performed according to Canadian Council on Animal Care regulations with appropriate ethical approvals (UBC RISe Ethics approvals A15-0105 & A16-0088; PI Matt Farrer). Lrrk2 p.R1441C knock-in (RKI), and Dnajc13 p.N855S knock-in (DKI) mice were maintained for >10 generations on a C57Bl/6J background and kept on a reverse cycle (light on from 7pm to 7am). Heterozygous (HET) KI × HET KI breeds yielded homozygous, heterozygous KI and WT littermates. Primary neuronal cultures were prepared from timed pregnancy dams from background strain C57BL/6J mice with the aforementioned transgenic crosses. Tails from each embryo were genotyped during the single-pup neuronal isolation, before cells were pooled by genotype and plated. Only WT and heterozygous knock-in mice were included and are herein referred to as WT and KI. Cultures were prepared as 12  previously described93; briefly, cortical neurons were isolated from pups at E16.5, brains were removed and placed on ice in Hank's Balanced Salt Solution (HBSS, GIBCO). 24-well plates were seeded at 115 k cells/well in 1 ml plating medium (PM, 2% B27+1/100 penicillin/streptomycin, Invitrogen; 0.5 mM α-glutamine; neurobasal medium, GIBCO). From DIV4, 10% of media was added every 3–5 days. All experiments, data processing and analysis was conducted in a blinded manner.  2.2.2 Electrophysiology Whole-cell patch-clamp recordings were performed on cortical cells at DIV21–26 in voltage clamp at Vh −70 mV and the membrane test function was used to determine intrinsic membrane properties ~1 min after obtaining whole-cell configuration, as described previously93–96. Briefly, neurons were perfused at room temperature with extracellular solution (ECS) containing (in mM unless stated): 167 NaCl, 2.4 KCl, 1 MgCl2, 10 glucose, 10 HEPES, 2 CaCl2, pH 7.4, 300 mOsm. Tetrodotoxin (TTX, 0.2 μM), and picrotoxin (PTX, 100 μM, except when analyzing GABA currents) were added before use. Pipette resistance (Rp) was 3–6 MΩ when filled with (in mM): 130 Cs methanesulfonate, 5 CsCl, 4 NaCl, 1 MgCl2, 5 EGTA, 10 HEPES, 5 QX-314, 0.5 GTP, 10 Na2-phosphocreatine, and 5 MgATP, 0.1 spermine, pH 7.2, 290 mOsm. Data were acquired by Multiclamp 700 B amplifier and signals were filtered at 2 kHz, digitized at 10 kHz, and analyzed in Clampfit10 (Molecular Devices). Tolerance for series resistance (Rs) was <25 MΩ and uncompensated; ΔRs tolerance cut-off was <10%. Miniature excitatory post-synaptic currents (mEPSCs) were analyzed with experimenter blind to genotype using Clampfit10 (threshold 5pA mEPSC); all events were checked by eye and monophasic events were used for amplitude and decay kinetics, while others were suppressed but included in frequency counts93–96. Data are 13  presented as mean ±SEM. where n is cells from a minimum of 3 separate cultures (culture n in brackets).  2.3 Results 2.3.1 In vitro synaptic transmission in LRRK2 p.R1441C cortical neurones The intrinsic cell properties of LRRK2 p.R1441C (RKI) cortical neurons were electrophysiologically similar between WT and mutant genotypes. Analysis of mEPSCs demonstrated no change in the mean amplitude of events (Figure 2.1 A, n=15(4) WT, n=19(4) RKI, p<0.279, two-tailed unpaired t-test, df=31), however similar to previously published work97 there was significant elevation in the mean frequency of mEPSCs onto RKI cells compared to WT (Figure 2.1 B, n=15(4) WT, n=19(4) RKI, *p<0.019, two-tailed unpaired t-test, df=31). While no significant changes in membrane capacitance, or resistance were observed (Figure 2.1 C., D., n=15(4) WT, n=19(4) RKI, p<0.087, two-tailed Mann-Whitney test; n=15(4) WT, n=19(4) RKI, p<0.642, two-tailed unpaired t-test, df=31, respectively) there was a strong trend towards a higher membrane capacitance in the mutant neurons. These results are suggestive of an increase in the probability of glutamatergic release, the number of active synapses or an alteration in both parameters in cortical neurons derived from Lrrk2 p.R1441C knock-in mice.   2.3.2 In vitro synaptic transmission in Dnajc13 p.N855S cortical neurones Analysis of mEPSCs revealed no significant alterations in either mean amplitude or frequency of the events between WT and heterozygous mutant genotypes (Figure 2.2 A., B., n=21(4) WT, n=19(4) DKI, p>0.999, two-tailed Mann-Whitney test; n=21(4) WT, n=19(4) DKI, p<0.226, two-tailed Mann-Whitney test, respectively). Similarly, to the previous experiment genotype had no 14  significant effect on the membrane capacitance, or resistance (Figure 2.2 C., D, n=21(4) WT, n=19(4) DKI, p<0.537, two-tailed Mann-Whitney test; n=21(4) WT, n=19(4) DKI, p<0.937, two-tailed unpaired t-test, df=38). These results suggest no differences in spontaneous excitatory neurotransmission in this model.    2.4 Discussion This chapter highlights electrophysiological phenotypes observed in knock-in models of mutations linked to late-onset PD. The RKIs showed a propensity towards increased spontaneous glutamatergic release with no change in baseline cell characteristics. Previously, we have shown a similar increase in mEPSC frequency with no significant change in synapse density in GKI-derived cortical neurons versus wildtype controls, as measured by synapsin-1 and post-synaptic density protein 95 (PSD95) colocalization97. Thus, we did not assess synapse number in RKI derived cortical neurons. Curiously, DKI cortical cultures have half as many synapses but equal frequency of mEPSCs when compared to their WT counterparts (personal communication, Dr. Jordan Follet). Taken together, these data suggest that even though the mean frequency of mEPSCs in DKI cultures was unchanged the probability of release at each individual synapse is increased. This hypothesis can be tested by expressing VGluT1 or synaptophysin tagged with a pH sensitive fluorophore (pHlourin) and measuring spontaneous release frequency on the synapse level using live cell microscopy98. This study did not examine the underlying mechanism of the observed changes. Future work should focus on characterizing the expression and secondary modifications of proteins associated with release probability, and those shown to be reliant on LRRK2 kinase activity. Previous work has suggested that members of the Rab family of proteins are 15  phosphorylated in a LRRK2 dependent manner99. Further, LRRK2 has also been shown to interact with endophilinA88, auxillin (DNAJC6)100 and clathrin-light chains101. These interactions should be explored in both RKIs and DKIs. It would also be interesting to examine the effect of acute silencing of Lrrk2, or Dnajc13 pharmacologically or through siRNA-mediated knockdown on phenotypes described in this study. Overall, the work outlined in this chapter examined the effect of late-onset PD linked mutations on cultured cortical neurons. Further examining the phenotypes observed may provide new targets for developing disease modifying therapies.   16   Figure 2.1 Whole-cell patch-clamp recordings of cortical neurons in DIV21 CTX cultures from RKI mice.  Quantification of mean mEPSC amplitude (A.) and frequency (B.) showed no significant difference in amplitude (n=15 (4) WT, n=19 (4) RKI p<0.279), while a significantly higher frequency of mEPSCs in RKI cultures was observed (n=15 (4) WT, n=19 (4) RKI *p<0.019). C.,D.) Intrinsic cell properties of patched neurons showed no effect of genotype on membrane capacitance (n=15 (4) WT, n=19 (4) RKI, p<0.087) and resistance (n=15 (4) WT, n=19 (4) RKI p<0.615). Comparisons by unpaired t-test for all except membrane capacitance which was analyzed Mann-Whitney, results shown are mean ±SEM.    17   Figure 2.2 Whole-cell patch-clamp recordings of cortical neurons in DIV21 CTX cultures from DKI mice.  Quantification of mean mEPSC amplitude (A. n=21(4) WT, n=19(4) DKI, p>0.999, two-tailed Mann-Whitney test) and frequency (B. n=21(4) WT, n=19(4) DKI, p<0.226, two-tailed Mann-Whitney test) showed no significant difference in DKI cultures. C.,D.) Intrinsic cell properties of patched neurons showed no effect of genotype on membrane capacitance (n=21(4) WT, n=19(4) DKI, p<0.537, two-tailed Mann-Whitney test) and resistance (n=21(4) WT, n=19(4) DKI, p<0.937, two-tailed unpaired t-test, df=38). Comparisons by Mann-Whitney for all except membrane resistance which was analyzed with an unpaired t-test, results shown are mean ±SEM. 18  Chapter 3: Ex vivo characterization of the nigrostriatal dopaminergic system of Dnajc13 p.N855S mice.  3.1  Introduction The nigrostriatal dopaminergic system is of utmost interest in the study of PD, because of the cell loss observed at autopsy1. Previous work has shown alterations in the dorsolateral striatum of GKI and VKI mice, where activity of the dopaminergic system was examined by fast-scan cyclic voltammetry (FSCV) to assay DA release on a millisecond timescale. GKI mice were found to have elevated sustained release upon repeat stimulation, increased D2 autoreceptor activity, and decreased DAT activity at 3 months of age75. The observed phenotypes reverted by 12+ months of age75. Similarly, 3-month-old VKI mice were found to have significantly elevated release, increased D2 autoreceptor activity, and decreased DAT activity. Further, VKI mice showed significantly higher levels of VMAT2 along with lower levels of DAT mirroring the results obtained by FSCV. We hypothesized that DKI mice would have alterations in nigrostriatal dopamine clearance as it is a shared phenotype observed in both GKI and VKI mice.  3.2 Materials and Methods 3.2.1 Dnajc13 p.N855S knock-in mice  All breeding, housing, and experimental procedures were performed according to Canadian Council on Animal Care regulations with appropriate ethical approvals (UBC RISe Ethics approvals A15-0105 & A16-0088; PI Matt Farrer). Dnajc13 p.N855S knock-in (DKI) mice were maintained for >10 generations on a C57Bl/6J background and kept on a reverse cycle (light on 19  from 7pm to 7am) and single-sex group-housed in enrichment cages after weaning at post-natal day 21. For all experiments, 3 month old male animals were used as human data has shown that PD is more likely to occur in males102. Post-mortem ear notches were taken for post-hoc genotyping. All experiments, data processing and analysis were conducted in a blinded manner.  3.2.2 Protein analysis  For western blot (WB) analysis, dissected striatal brain tissue was lysed in HEPES buffer (20 mM HEPES, pH 7.2, 50mM KAc, 200 mM Sorbitol, 2 mM EDTA, 0.1% Triton X-100, 0.5% NP-40) containing protease and phosphatase inhibitors (Roche), homogenized, and incubated (on ice, 45 min, gentle agitation every 15 min). Lysates were cleared by centrifugation at 4000×g for 12 min at 4 °C and supernatant was quantified by BCA assay (Pierce). Lysates were denatured in 1× LDS sample buffer (Thermo Fisher Scientific) and heated for 10 min at 70 °C and 10–15 µg of protein was resolved by SDS-PAGE as previously described103, or using NuPage 4–12% Bis–Tris gel (Thermo Fisher Scientific) and transferred to PVDF membranes (EMD Millipore). Membranes blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) were probed overnight at 4 °C with primary antibodies diluted in 3% BSA (Sigma-Aldrich) in PBS. The following day, membranes were washed in TBST (3 × 5 min) at room temperature (RT) followed by incubation for 1 h at RT with horseradish peroxidase-conjugated anti-mouse, rabbit, or Rat IgG (Santa-Cruz Biotechnology). Blots were washed in TBST (3 × 5 min) at RT, and developed using enhanced chemiluminescence plus reagent (Thermo Scientific). Subsequent imaging was performed with a Chemi-Doc imaging system (Cell-Bio), and images were analyzed for band intensity with Image J software. The following primary antibodies were used for WB: Mouse anti-VPS35 (Abnova, H00055737-M02); anti-GAPDH (Cell Signaling, 2118); anti-PSD95 (6G6- 1C9, 20  Thermofisher Scientific, MA1-045); anti-TH (Sigma-Aldrich, T2928); Rabbit anti-mouse VMAT2 (Phoenix Pharmaceuticals, Inc); and Rat anti-DAT (EMD Millipore, MAB369). The detection of primary antibodies was achieved by probing membranes with Donkey anti-Rabbit or mouse IgG conjugated horseradish peroxidase (HRP) (Santa-Cruz Biotechnology, sc-2318, sc-2313, respectively), or Rabbit anti-Rat IgG HRP (Abcam, ab6734).   3.2.3 Fast-scan cyclic voltammetry (FSCV) FSCV was conducted in the dorsolateral striatum in 300 μm thick coronal slices from 3 months old male mice, as previous. Slices were perfused at RT with artificial cerebrospinal fluid [ACSF—containing in mM: 130 NaCl, 10 glucose, 26 NaHCO3, 3 KCl, 1 MgCl2·6H20, 1.25 NaH2PO4 (monobasic monohydrate), 2 CaCl2; pH 7.2–7.4, mOsm 290–310] and oxygenated (95% O2, 5% CO2) at room temperature for >1 h prior to experiments. Individual slices were then placed in a recording chamber at temperature 26–27 °C, and perfused at 1–2 mL/min with ACSF. Stimuli (150 μs duration) were delivered by nickel-chromium bipolar electrodes (made in house) placed in the dorsolateral striatum, optically isolated (A365, World Precision Instruments, USA) and controlled/sequenced with ClampEx software. Voltammetric responses were recorded, standardized and analyzed with an Invilog Voltammetry system and software (Invilog Research Ltd., Finland). Carbon fiber electrodes (diameter: 32 μm, length: 300 μm, sensitivity: >20 nA/μM) were purchased prefabricated (Invilog) and placed within 100–200 μm of the stimulating electrode in the dorsolateral striatum. Triangular waveforms (ramp from −400 mV to 1200 mV to −400 mV, 10 ms duration at 10 Hz) were used to detect the oxidation and redox peaks for dopamine between 700 and 800 mV. Striatal field excitatory postsynaptic potentials (fEPSPs) were also recorded to ensure slice viability during the duration of each experiment. The input/output paradigm consisted 21  of single pulses of increasing intensities (100–700 μA, delivered every 2 min/ 0.0083 Hz) to determine the input required to evoke ~70% of the maximum response, which was used for the rest of the experiment. Five single pulses were delivered at 0.0083 Hz to calculate an average dopamine transient to be used for a more accurate representation of the decay characteristics.  At the end of each recording session, a three-point calibration of each carbon fiber electrode was conducted (final concentrations 0.5, 1.0, 2.0 μM dopamine in ACSF). 1-[2-[Bis-(4-fluorophenyl) methoxy]ethyl]-4-(3-phenylpropyl)piperazine dihydrochloride (GBR-12909 dihydrochloride, Tocris 0421) was perfused at a concentration of 1 μM in ACSF for 20 minutes. Five single pulses were then delivered at 0.0083 Hz to calculate an average dopamine transient following DAT inhibition.   3.2.4 Statistics and data reporting  Data are presented throughout as mean ± SEM where “n” equals the number of animals for western blot experiments, or shown as per slice “n” with the total number of animals indicated in parentheses for experiments involving FSCV. Sample sizes estimates used the resource equation method104 but were refined using G power software105 with pilot data (n = 3) on effect size (mean/standard deviation within groups) for beta = 0.8 and alpha = 0.05. Throughout the study, comparisons were conducted by 1-, or 2-way ANOVA with appropriate post-hoc tests, as detailed in the text, using Prism 5.0 or 8.2 (GraphPad, San Diego, CA, USA).  22  3.3 Results 3.3.1 Striatal dopaminergic markers in Dnajc13 pN855S knock-in mice The levels of DAT were examined in the striatum of 3 month old DKIs. There was significant loss of DAT levels (normalized to GAPDH) in the DKI mice when compared to their WT counterparts (Figure 3.1 A., B., n=4 WT, n=4 DKI, **p<0.007, two-tailed unpaired t-test, df=6)  3.3.2 Striatal dopaminergic release in Dnajc13 p.N855S knock-in mice Nigrostriatal dopamine release was assessed on a millisecond timescale using FSCV. Single stimuli of increasing intensity evoked increasing concentrations of dopamine and were used to construct an input-output relationship. The genotype had no effect on the input/output relationship (Figure 3.2 A. n=16(4) WT, n=18(5) DKI, stimulation intensity F1.780,56.95=61.97 p<0.0001, genotype F1,32=0.1229 p<0.728, stimulation intensity × genotype F4,128=0.2355 p<0.918, two-way RM ANOVA with Bonferroni’s correction). Since DAT levels were found to be significantly lower in the DKIs, GBR-12909 (GBR), a potent and selective dopamine reuptake inhibitor, was used to further examine DAT activity. GBR had a significant effect on the concentration of evoked dopamine with no genotype effect (Figure 3.2 B., n=16(4) WT, n=18(5) DKI, GBR F1,32=52.94 p<0.0001, genotype F1,32=0.6514 p<0.426, GBR × genotype F1,32=1.604 p<0.215 two-way RM ANOVA; Bonferroni’s correction WT Baseline vs. DKI baseline p>0.999, WT GBR vs. DKI GBR p<0.492). Further the effectiveness of DAT inhibition was examined by comparing decay time constants (Tau). There was a significant effect of GBR, genotype, and an interaction between the two, with DKI mice having a significantly faster decay following GBR application (Figure 3.2 C., n=16(4) WT, n=18(5) DKI, GBR F1,32=69.54 p<0.0001, genotype F1,32=5.784 p<0.022, GBR × 23  genotype F1,32=5.916 p<0.021 two-way RM ANOVA; Bonferroni’s correction WT baseline vs. DKI baseline p>0.9999, WT GBR vs. DKI GBR p<0.003).   3.4 Discussion This chapter examined the nigrostriatal dopamine system of DKI mice.  No baseline differences were observed in dopamine release within the dorsolateral striatum; further, the amplitude and decay characteristics were not significantly different from WT animals. Intriguingly, there was a lower effect of DAT inhibition using GBR-12909. Together these data indicate a subtle physiological perturbance in nigrostriatal neurotransmission that seems to be fully compensated at baseline. This compensation can be explained in a multitude of ways. There could be more DAT at the cell surface, an increase in the activity of DAT, an increase in the activity of DA metabolic enzymes or some combination of these factors. To probe further, measuring the surface level of DAT using a biotynilation assay on acute slices would be a promising approach. Previous work has shown that Syntaxin1A (syn1a) regulates DAT activity, and surface expression106. It would also be interesting to apply the syn1a protease Botulinum Neurotoxin C (BoNT/C) to acute slices and measure DA clearance using FSCV. If  the compensation in DKIs relies on syn1a then BoNT/C should have no effect on the mutant and lower the decay times for WT slices. Metabotropic glutamate receptor 5 (mGluR5) agonism decreased DAT capacity and efficiency with antagonism having the opposite effect107. Inhibitors of protein kinase C and calcium calmodulin-dependent kinase II blocked the effect of mGluR5 agonism107. The effects of mGluR5 agonism and antagonism should also be explored in acute DKI slices. If the compensation is acting through this system, then antagonism would have no effect, while agonism would slow clearance substantially. Lastly, isolating DAT from the striata of both WT and DKI animals followed by mass spectrometry 24  would facilitate the comparison of post-translational modifications across genotypes. Altogether these results, while not identical to those of VKI and GKI studies, are reminiscent of previous findings. Mutations linked to autosomal dominant late-onset PD affect DAT expression and activity, providing an attractive target for early therapeutic intervention.    25   Figure 3.1 Western blotting for DAT in microdissected striatal tissue from 3 month old.  A.) Representative images of DAT, and GAPDH in the synaptic fraction following synaptosome isolation. B.) Quantification of DAT levels (normalized to GAPDH) within the synaptic fraction showed a significant loss in DKI animals (n=4 WT, n=4 DKI, **p<0.007, two-tailed unpaired t-test, df=6). Comparison by unpaired t-test, results shown are mean ±SEM.   26   Figure 3.2 Nigrostriatal dopamine transmission in 3 month old DKIs.  Electrically evoked DA transients were detected by ex-vivo fast-scan cyclic voltammetry (FSCV) in the dorsolateral striatum of 3 month old DKIs and their WT littermates. A.) There was a significant effect of stimulation intensity (****p<0.0001) with no genotype effect (p<0.728) on the concentration of dopamine evoked over a range of stimulus intensities. Comparison by ANOVA with Bonferroni correction. B.) There was a significant effect of GBR-12909 (****p<0.0001) on the averaged concentration of dopamine evoked at 50-70% of the maximum with no genotype (p<0.426) or interaction effect (p<0.215). C.) Dopamine transient decay times calculated from the evoked averages showed a significant effect of GBR-12909 (****p<0.0001), genotype (*p<0.022), and GBR-12909 × genotype (p<0.021) (post test: WT GRB-12909 vs. DKI GBR-12909 **p<0.003). Comparisons by ANOVA with Bonferroni correction, results shown are mean ±SEM.   27  Chapter 4: LRRK2 inhibition as a therapeutic target in Parkinson’s disease  4.1 Introduction: Prior investigations of Lrrk2 and Vps35 knock-in mouse models focused on striatal function have revealed subtle synaptic differences between mutant and wildtype littermates. Briefly, Vps35 p.D620N knock-in mice (VKI) showed increased release, and slower clearance of dopamine at 3 months of age. The expression of DAT was substantially lower in mutant animals, while VMAT2 was increased3. Lrrk2 p.G2019S knock-in (GKI) mice also have slower dopamine clearance at 3 months of age108. Mutations in LRRK2, especially p.G2019S, have been found to increase kinase activity, as assayed by levels of autophosphorylation at serine 1292, 910 and 93569,73. Interestingly, endogenous Lrrk2 was found to be activated in VKI mouse brain109, and in nigrostriatal neurons of idiopathic PD patients73. Here, we examine dopaminergic phenotypes of VKI mice at 1 month of age, and subsequently explore the effects of MLi-2 administration, a well-characterized LRRK2 kinase inhibitor110, at 3 months of age. We hypothesized that the phenotypes observed in the nigrostriatal dopamine system of VKI mice would be age-dependent, with no observed differences at 1 month of age, and that inhibiting LRRK2 activity would rescue the phenotypes observed in 3 month old VKIs.  4.2 Materials and methods: 4.2.1 Vps35 p.D620N knock-in mice  All breeding, housing, and experimental procedures were performed according to Canadian Council on Animal Care regulations with appropriate ethical approvals (UBC RISe Ethics approvals A15-0105 & A16-0088; PI Matt Farrer). VKI mice were maintained for >10 generations 28  on a C57Bl/6J background and kept on a reverse cycle (light on from 7pm to 7am) and single-sex group-housed in enrichment cages after weaning at post-natal day 21. For all experiments, 1 and 3 month old male animals were used. Post-mortem ear notches were taken for post-hoc genotyping. All experiments, data processing and analysis was conducted in a blinded manner.  4.2.2 Fast-scan cyclic voltammetry (FSCV) FSCV was conducted in the dorsolateral striatum in 300 μm thick coronal slices from 3 months old male mice, as previous. Slices were perfused at RT with artificial cerebrospinal fluid [ACSF—containing in mM: 130 NaCl, 10 glucose, 26 NaHCO3, 3 KCl, 1 MgCl2·6H20, 1.25 NaH2PO4 (monobasic monohydrate), 2 CaCl2; pH 7.2–7.4, mOsm 290–310] and oxygenated (95% O2, 5% CO2) at room temperature for >1 h prior to experiments. Individual slices were then placed in a recording chamber at temperature 26–27 °C, and perfused at 1–2 mL/min with ACSF. Stimuli (150 μs duration) were delivered by nickel-chromium bipolar electrodes (made in house) placed in the dorsolateral striatum, optically isolated (A365, World Precision Instruments, USA) and controlled/sequenced with ClampEx software. Voltammetric responses were recorded, standardized and analyzed with an Invilog Voltammetry system and software (Invilog Research Ltd., Finland). Carbon fiber electrodes (diameter: 32 μm, length: 300 μm, sensitivity: >20 nA/μM) were purchased prefabricated (Invilog) and placed within 100–200 μm of the stimulating electrode in the dorsolateral striatum. Triangular waveforms (ramp from −400 mV to 1200 mV to −400 mV, 10 ms duration at 10 Hz) were used to detect the oxidation and redox peaks for dopamine between 700 and 800 mV. Striatal field excitatory postsynaptic potentials (fEPSPs) were also recorded to assess slice viability during the duration of each experiment. The input/output paradigm consisted of single pulses of increasing intensities (100–700 μA, delivered every 2 min/ 0.0083 Hz) to 29  determine the input required to evoke ~70% of the maximum response, which was used for the rest of the experiment. Five single pulses were delivered at 0.0083 Hz to calculate an average dopamine transient to be used for a more accurate representation of the decay characteristics.  At the end of each recording session, a three-point calibration of each carbon fiber electrode was conducted (final concentrations 0.5, 1.0, 2.0 μM dopamine in ACSF).   4.2.3 MLi-2 injection Due to the low solubility of MLi-2 in water, 45% Captisol (https://www.captisol.com/) in sterile PBS was used to dilute and deliver the compound. A solution of 0.5 mg/mL MLi-2 in 45% Captisol was prepared for injection along with a vehicle control of 45% Captisol alone. Both solutions were filter sterilized prior to injection. The mice were weighed and injected intraperitoneally (IP) with 10 μL per gram of either MLi-2 (0.5 mg/mL) or control every morning for 7 days. The final dosage of MLi-2 was 5 mg/kg. The brains were collected following decapitation 1 hour after the final injection. Extracted brains and allowed to rest in ice cold, oxygenated ACSF for 30 seconds prior to microdissection.   4.2.4 Protein analysis  For western blot (WB) analysis, dissected striatal brain tissue was lysed in HEPES buffer (20 mM HEPES, pH 7.2, 50mM KAc, 200 mM Sorbitol, 2 mM EDTA, 0.1% Triton X-100, 0.5% NP-40) containing protease and phosphatase inhibitors (Roche), homogenized, and incubated (on ice, 45 min, gentle agitation every 15 min). Lysates were cleared by centrifugation at 4000×g for 12 min at 4 °C and supernatant was quantified by BCA assay (Pierce). Lysates were denatured in 1× LDS sample buffer (Thermo Fisher Scientific) and heated for 10 min at 70 °C and 10–15 µg of protein 30  was resolved by SDS-PAGE as previously described103, or using NuPage 4–12% Bis–Tris gel (Thermo Fisher Scientific) and transferred to PVDF membranes (EMD Millipore). Membranes blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) were probed overnight at 4 °C with primary antibodies diluted in 3% BSA (Sigma-Aldrich) in PBS. The following day, membranes were washed in TBST (3 × 5 min) at room temperature (RT) followed by incubation for 1 h at RT with horseradish peroxidase-conjugated anti-mouse, rabbit, or Rat IgG (Santa-Cruz Biotechnology). Blots were washed in TBST (3 × 5 min) at RT, and developed using enhanced chemiluminescence plus reagent (Thermo Scientific). Subsequent imaging was performed with a Chemi-Doc imaging system (Cell-Bio), and images were analyzed for band intensity with Image J software. The following primary antibodies were used for WB: Mouse anti-VPS35 (Abnova, H00055737-M02); anti-GAPDH (Cell Signaling, 2118); anti-PSD95 (6G6- 1C9, Thermofisher Scientific, MA1-045); anti-TH (Sigma-Aldrich, T2928); Rabbit anti-mouse VMAT2 (Phoenix Pharmaceuticals, Inc), Rat anti-DAT (EMD Millipore, MAB369). The detection of primary antibodies was achieved by probing membranes with Donkey anti-Rabbit or mouse IgG conjugated horseradish peroxidase (HRP) (Santa-Cruz Biotechnology, sc-2318, sc-2313, respectively), or Rabbit anti-Rat IgG HRP (Abcam, ab6734).   4.2.5 Statistics and data reporting  Data are presented throughout as mean ± SEM where “n” equals the number of animals, or else are shown as per slice “n” for the total number of animals (indicated in parentheses). Sample sizes estimates used the resource equation method104 but were refined using G power software105 with pilot data (n = 3) on effect size (mean/standard deviation within groups) for beta = 0.8 and alpha = 0.05. Throughout the study, comparisons were conducted by 1-, 2- or 3-way ANOVA with 31  appropriate post-hoc tests, as detailed in the text, using Prism 8.0 (GraphPad, San Diego, CA, USA).  4.3 Results: 4.3.1 Striatal dopamine release in Vps35 p.D620N knock-in mice Fast-scan cyclic voltammetry (FSCV) was performed on striatal brain slices of VKI animals and their wild type littermates. Single pulses evoking dopamine release at increasing stimulation intensities showed no differences in overall concentration of dopamine release at 1 month of age (Figure 4.1 A. n=16(4) WT, n=15(4) VKI, stimulation intensity F1.217,28.95=32.47 p<0.0001, genotype F1,30=0.01940 p<0.890, stimulation intensity × genotype F5,119=0.3578 p<0.8763 two-way RM ANOVA). Dopamine clearance was calculated from the average of 5 stimuli evoked at 70% of the maximum. There were no differences in either the concentration (Figure 4.1 B., n=16(4) WT, n=15(4) VKI, p<0.7405, two-tailed Mann-Whitney test) or decay constant (Tau) (Figure 4.1 C., n=16(4) WT, n=15(4) VKI, p<0.9789, two-tailed unpaired t-test, df=29) of the averaged dopamine transients.   4.3.2 Inhibiting Lrrk2 activity Vps35 p.D620N knock-in mice at 3 months of age Lrrk2 kinase inhibitor MLi-2110 was administered daily for seven days by intraperitoneal (IP) injection to heterozygous VKI and wild type littermate mice. We achieved a ~75% reduction in pS935 Lrrk2 levels (Figure 4.2 B., n=3 WT Vehicle, n=3 WT MLi-2, n=3 VKI Vehicle, n=3 VKI MLi-2, MLi-2 F1,8=24.89 p<0.001, genotype F1,8=0.8554 p<0.382, MLi-2 × genotype F1,8=2.947 p<0.124 two-way ANOVA; Tukey’s post-test WT MLi-2 vs. VKI vehicle p<0.013, VKI vehicle vs. VKI MLi-2 p<0.006, DF=8), and a ~75% decrease in the ratio of pLrrk2/Lrrk2 (Figure 4.2 C., 32  n=3 WT Vehicle, n=3 WT MLi-2, n=3 VKI Vehicle, n=3 VKI MLi-2, MLi-2 F1,8=94.92 p<0.0001, genotype F1,8=0.005887 p<0.9407, MLi-2 × genotype F1,8=2.179 p<0.9407; Tukey’s post-test WT vehicle vs. WT MLi-2 p<0.002, WT vehicle vs. VKI MLi-2 p<0.001, WT MLi-2 vs. VKI vehicle p<0.001, VKI vehicle vs VKI MLi-2 p<0.001, DF=8).   We next examined protein levels of the dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) in whole striatal lysates and in synaptosome preparations. There was a significant effect of genotype (F1,12=5.815  p<0.03), and a significant interaction between genotype × MLi-2 treatment (F1,12=6.388 p<0.03) associated with a recovery in DAT levels (Figure 4.3 B., n=4 for all groups, 2-way ANOVA followed by Tukey’s post-hoc comparisons: WT Vehicle vs. VKI Vehicle *p<0.03, VKI Vehicle vs. WT MLi-2 *p<0.05, VKI Vehicle vs. VKI MLi-2 *p<0.04). VKI genotype had a significant effect on VMAT2 levels (F1,12=27.40 p<0.0002), along with a significant MLi-2 effect (F1,12=4.962 p<0.05) and a significant interaction between genotype × treatment  indicating that MLi-2 rescued the genotype dependent increase in VMAT2 levels (F1,12=5.961 p<0.03) (Figure 4.3 C., n=4 for all groups, 2-way ANOVA followed by Tukey’s post-hoc comparisons: WT vehicle vs. VKI vehicle ***p<0.001, VKI vehicle vs. WT MLi-2 ***p<0.002, VKI vehicle vs. VKI MLi-2 *p<0.03). These results suggest that lowering Lrrk2 activity for 7 days rescued the levels of both VMAT2 and DAT.  FSCV was subsequently assessed in striatal brain slices, ex vivo, from VKI animals treated with MLi-2 or vehicle. As previous, single pulses of increasing stimulation intensity were used to construct an input/output relationship (Figure 4.4 A.). There was a significant effect of stimulation intensity (F2.094, 104.7=55.66 p<0.001) along with significant interactions between stimulation 33  intensity × genotype (F7,350=2.398 p<0.03), genotype × treatment (F1,75=6.106 p<0.01), and stimulation intensity × genotype × treatment (F7,350=2.697 p<0.01) on the input/output relationship (n=19(5) WT Vehicle, n=20(5) WT MLi-2, n=20(5) VKI Vehicle, n=20(5) VKI MLi-2, 3-way ANOVA followed by Tukey’s post-hoc comparisons: 400 μA WT Vehicle vs. VKI Vehicle **p<0.005). Dopamine release kinetics were calculated from the average of 5 pulses evoked at 70% of the maximum. Genotype (F1,75=5.776 p<0.02) and the interaction between genotype × MLi-2 treatment (F1,75=5.506 p<0.02) significantly affected the average dopamine concentration of single pulses (Figure 4.4 B., n=19(5) WT Vehicle, n=20(5) WT MLi-2, n=20(5) VKI Vehicle, n=20(5) VKI MLi-2, 2-way ANOVA followed by Tukey’s post-hoc comparisons: WT Vehicle vs. VKI Vehicle **p<0.008, VKI Vehicle vs. WT MLi-2 *p<0.02, VKI Vehicle vs. VKI MLi-2 *p<0.02). The dopamine decay constant (tau) showed a similar pattern with a significant effect of genotype (F1,75=7.702  p<0.01) and an interaction between genotype × treatment (F1,75=5.347 p<0.02). The decay constant was significantly slower in slices from vehicle treated VKI mice, compared to both vehicle-and MLi-2-treated WT (Tukey’s multiple comparisons test **p<0.004, *p<0.03 respectively) with no significant changes for MLi-2 treated VKIs (Figure 4.4 C n=19(5) WT Vehicle, n=20(5) WT MLi-2, n=20(5) VKI Vehicle, n=20(5) VKI MLi-2, 2-way ANOVA; followed by Tukey’s post-hoc comparisons).    4.4 Discussion: Dopaminergic phenotypes in Vps35 D620N knock in (VKI) mice3 are age-dependent, rather than developmental, as mutant and wildtype animals are comparable at one month of age. In mutant animals, pathophysiological alterations in DAT and VMAT2 levels are associated with increased 34  Lrrk2 kinase activity, which is restored by short-term (1 week) administration of a Lrrk2 kinase inhibitor, MLi-2. Hence, VKI mice may provide a biologically and clinically-relevant assay to titrate such drugs, to identify the lowest possible dose with lasting efficacy on disease-relevant endpoints, to potentially avoid side-effects. In VKI mice, it will be important to assess whether normalizing Lrrk2 activity and related dopaminergic phenotypes can ameliorate neurodegeneration and neuropathology at later time-points. Nevertheless, with this model we can compare different classes of Lrrk2 inhibitors regardless of whether their effects are mediated through Lrrk2 kinase inhibition, the Roc GTPase (GTP-GDP binding and guanine nucleotide exchange factors), dimerization and/or by a reduction in Lrrk2 protein levels. Alternative treatments must be considered for long-term administration, to mitigate potential side effects.   VPS35 and LRRK2 parkinsonism are clinically indistinguishable from idiopathic PD; most patients appear late-onset and follow an insidious but progressive course. However, while VPS35 p.D620N parkinsonism is rare, increased Lrrk2 kinase activity mediated by LRRK2 mutations and/or LRRK2 polymorphic variability arguably has become a focal point in the etiology and pathophysiology of both autosomal dominant gene-linked and idiopathic PD. Pre-symptomatic imaging in human LRRK2 heterozygotes demonstrates significant alterations in dopamine metabolism many decades before motor symptoms arise, also suggesting an opportunity for meaningful intervention. Nevertheless, prolonged exposure to high doses of Lrrk2 kinase inhibitors can lead to loss of Lrrk2 levels and type II pneumocyte lung pathology110. Lrrk2 KO mice also develop age-associated lung and kidney phenotypes 111. Behavioral and synaptic phenotypes in Lrrk2 p.G2019S knock in mice (GKI) recapitulate aspects of the insidiously progressive pathophysiology observed in human LRRK2 p.G2019S heterozygotes. Relative to wild-type littermates, young GKI mice exhibit more 35  vertical exploration, have elevations in striatal glutamate and dopamine transmission and altered synaptic responses to the D2-dopamine receptor agonist quinpirole. These phenomena decline significantly with age in mutant mice but are stable in littermates. In young GKI brain slices, evoked nigrostriatal dopamine release is significantly slower and the extracellular lifetime of single release events is increased compared to wild-type controls. While the physiological effects of mutant expression are reproducible, they are subtle and challenging to quantify.   Our results highlight the value of Vps35 p.D620N knock-in mice as an orthogonal model for the pathophysiology of VPS35 and LRRK2 parkinsonism, and potentially sporadic PD.  A corollary of our study in mice is that PET imaging of DAT (11C-methylphenidate) and VMAT2 binding (11C-dihydro-tetrabenezine) might provide a clinically-relevant assay of Lrrk2 kinase inhibition in human VPS35 p.D620N parkinsonism; with such an assay it may be possible to guide the minimal effective, but long-term, therapeutic dose required in brain to achieve functional recovery (disease modification), lessening the potential for unwanted side-effects. Recent studies in blood leucocytes show LRRK2 activity is elevated in VPS35 parkinsonism and idiopathic PD 73,109, and may provide a peripheral measure of Lrrk2 kinase inhibition, and now it is clear how such measures might be extrapolated in clinical trials.    36   Figure 4.1 Nigrostriatal dopamine transmission in 1 month old VKIs.  Electrically evoked DA transients were detected by ex-vivo fast-scan cyclic voltammetry (FSCV) in the dorsolateral striatum of 1 month old VKIs and their WT littermates. A.) There was a significant effect of stimulation intensity (****p<0.0001) with no genotype effect (p<0.890) on the concentration of dopamine evoked over a range of stimulus intensities. Comparison by two-way ANOVA with Bonferroni correction. B.,C.) There was also no significant effect of genotype on the concentration (p<0.741) or the transient decay time calculated from the average of 5 pulses evoked at 50-70% of the maximum (p<0.979). Comparisons by Mann-Whitney and unpaired t-test respectively, results shown are mean ±SEM.   37   Figure 4.2 Western blotting for Lrrk2 and pLrrk2 in microdissected striatal tissue from 3 month old VKIs treated with 7 daily injections of either vehicle or MLi-2.  A.) Representative images of Lrrk2, Lrrk2 pSer935 and their respective GAPDH loading controls. B.) Quantification of Lrrk2 levels (normalized to GAPDH) showed a significant effect of treatment (*p<0.038) indicating a loss of Lrrk2 levels following MLi-2 treatment, with no effect of genotype (p<0.597). C.) Quantification of Lrrk2 pSer935 levels (normalized to GAPDH) showed a significant effect of treatment (**p<0.001), with no effect of genotype (p<0.382) (Tukey’s post-test: WT MLi-2 vs. VKI vehicle *p<0.013, VKI vehicle vs. VKI MLi-2 **p<0.006). D.) Ratio of Lrrk2 pSer935 and Lrrk2 showed a highly significant effect of treatment (****p<0.0001)  with no effect of genotype (p<0.9407) (Tukey’s post test: WT Vehicle vs. WT MLi-2 **p<0.002, WT Vehicle vs. VKI MLi-2 ***p<0.001, WT MLi-2 vs. VKI Vehicle ***p<0.001, VKI Vehicle vs. VKI MLi-2 ***p<0.001). Comparisons by two-way ANOVA with Tukey’s post test shown on graph, results shown are mean ±SEM. 38   Figure 4.3 Western blotting of dopaminergic markers in microdissected striatal tissue from 3 mo. old VKIs treated with 7 daily injections of either vehicle or MLi-2.  A.) Representative images of VMAT2, DAT, and Gapdh in the synaptic fraction following synaptosome isolation. B.) Quantification of DAT levels (normalized to Gapdh) within the synaptic fraction showed a significant effect of genotype (*p<0.033) and a significant interaction between genotype × treatment (*p<0.027) (post-test: WT Vehicle vs. VKI Vehicle *p<0.020, VKI Vehicle vs. MLi-2 *p<0.035, VKI Vehicle vs. VKI MLi-2 *p<0.030). C.) Quantification of VMAT2 levels (normalized to Gapdh) within the synaptic fraction showed a significant effect of genotype (***p<0.001), treatment (*p<0.046), and a significant interaction between  genotype × treatment (p<0.031) (post-test: WT vehicle vs. VKI vehicle ***p<0.001, VKI vehicle vs. WT MLi-2 ***p<0.001, VKI vehicle vs. VKI MLi-2 *p<0.028). Comparisons by two-way ANOVA with Tukey’s post test shown on graph, results shown are mean ±SEM. 39   Figure 4.4 Nigrostriatal dopamine transmission in 3 month old VKIs treated with vehicle or MLi-2.  Electrically evoked DA transients were detected by ex-vivo fast-scan cyclic voltammetry (FSCV) in the dorsolateral striatum of 3 month old VKIs and their WT littermates treated with 7 daily injections of either vehicle (45% Captisol®) or MLi-2 (5 mg/kg). A.) There was a significant effect of stimulation intensity (****p<0.0001) effect along with a significant interaction between stimulation intensity × genotype (*p<0.024), genotype × treatment (*p<0.040), and stimulation intensity × genotype × treatment (**p<0.007) on peak dopamine release evoked over a range of stimulus intensities (post-test: 400 μA WT Vehicle n=19 (5) vs. VKI Vehicle n=20 (5)**p<0.058). B.) The average amplitude calculated from 5 pulses at 70% of the maximum showed a significant effect of genotype (*p<0.022) and a significant interaction between genotype × treatment (*p<0.019) (post-test: WT Vehicle vs. VKI Vehicle **p< 0.0071, VKI Vehicle vs. WT MLi-2 *p<0.0145, VKI Vehicle vs. VKI MLi-2 *p<0.0162) C.) There was a significant effect of genotype (**p<0.007) and a significant interaction between genotype × treatment (*p<0.024) in dopamine transient decay times (post-test: WT Vehicle vs. VKI Vehicle **p<0.003, VKI Vehicle vs WT MLi-2 *p<0.026). Comparisons by three and two-way ANOVA with Tukey’s post test shown on graph, results shown are mean ±SEM.   40  Chapter 5: Conclusion 5.1 Summary Overall, this work has identified the similarities and differences in genetic models of late-onset PD linked mutations. In vitro, the cortical neurons derived from RKI mice showed elevated glutamatergic release, similar to our previously published work in GKIs97. DKI neurons were found to have a similar frequency of mEPSCs with drastically lower synapse number.  Ex vivo, DKI mice showed a subtle deficit in dopamine release and clearance as measured by FSCV. Interestingly, even though the levels of both DAT are drastically altered in mutant animals, when compared to their WT littermates, baseline release characteristics are identical by FSCV. However, when the DAT inhibitor GBR-12909 was introduced, slices derived from the mutant animals showed a much lower response to DAT inhibition when compared to their WT counterparts. Together, these data suggest that while baseline the DKI mutant mice have somehow compensated for the lack of DAT, there are fewer DAT molecules available for inhibition, leading to a lowered effect of GBR-12909. The compensation observed may be due to higher levels of DAT present at the cell surface in DKIs, post-translational modifications, such as phosphorylation of DAT, or both. Overall, while subtle, this phenotype is similar to those observed in both the GKI108 and VKI3. All models show early changes to DAT levels and/or activity. The GKIs showed lower DAT activity as observed by the longer decay constants without any alterations to DAT levels themselves, whereas VKI animals showed a drastic reduction in DAT levels along with a corresponding decrease in dopamine decay constants.  41  Lastly, we used the kinase inhibitor MLi-2110 to explore the relationship between Lrrk2 kinase activity and DAT levels and activity in the VKIs. The phenotypes observed were extremely sensitive to Lrrk2 kinase inhibition and after 7 daily injections, leading to a complete rescue physiologically and biochemically.  5.2 Significance Overall this work highlights a potential nexus to focus on when studying the pathogenesis of the disease. Mutations linked to autosomal dominant PD produce strikingly similar early synaptic phenotypes both in vitro and ex vivo. Specifically, multiple genes linked to PD have been shown to not only affect synaptic transmission, but also autophagy and protein clearance. LRRK2 kinase activity has been shown to disrupt both synaptic vesicle recycling and autophagy in Drosophila Melanogester112, while one of the key roles of VPS35 is directing cargo from the endosomal compartment back to the trans-golgi or the cell surface113. Findings presented in this thesis suggest that the interplay between protein endocytosis, recycling, and degradation lies at the heart of PD pathogenesis113, LRRK2 kinase activity plays an important role in this nexus, and highlights the therapeutic potential of LRRK2 kinase inhibitors as a disease modifying therapy for both gene-linked and idiopathic late-onset PD.  5.3 Limitations and alternative approaches 5.3.1 Chapter 2 The core limitation of this chapter is the model chosen. While cortical neurons are easy to grow and mature, they are not the cells most uniquely affected nor are they classically related to the pathogenesis of PD. Further, the effects of these mutations could be unique to the cultured 42  environment as the stressors are vastly different from in vivo conditions, with the vast network of support cells missing from this model. Ideally these experiments should be repeated using a co-culture of cortical and medium spiny neurons (MSNs), or even a tri-culture of SNpc, cortical and MSNs.   5.3.2 Chapter 3 and 4 The limitations of this chapter once again lie in the chosen model. Ex vivo slicing exposes the samples to unique stressors which, while minimized through experimental design, can never be fully eliminated. Further, FSCV is an indirect measure of dopamine release, relying on the consistency of the calibration, electrode manufacturing, and solution temperature and pH. Supplementing these results with HPLC and other methods of monitoring DAT trafficking would be beneficial.   5.4 Future Directions 5.4.1 Chapter 2 The results of this chapter should be verified in iPSC cultures derived from human carriers of the mutation compared to their isogenic controls. Several lines of patient derived cells and their isogenic controls should be created from each mutation to avoid other underlying genetic variation affecting the results. It would be interesting to see if the results are similar in human cortical neurons.  Further, live cell technology can be used to monitor synaptic vesicle release and clearance. Examining the characteristics of synaptic vesicle release and clearance using pHlourin tagged constructs98 would allow assaying release on a synaptic scale, independent of the number of cells and synapses on the coverslip. These methods can then be matched with targeted 43  pharmacological and genetic manipulation of Lrrk2, Dnajc13 and any other genes that are found to play a role in the observed phenotypes. This would further our understanding into how these mutations affect the probability of release and potentially identify new therapeutic targets by illuminating signaling pathways altered by the mutations.  5.4.2 Chapter 3 The results of this chapter can supplemented by examining the phenotypes at different ages and through different techniques. Firstly, investigating DKI mice at younger and older ages will be fruitful to fully describing the effects of the mutation. Next, determination of steady state dopamine level within the dorsolateral striatum of these animals by HPLC, both in awake behaving animals and from tissue punches at multiple time points. We may see a more severe phenotype at a younger age, or a failing of compensation at older ages. Lastly, it would be of great interest to monitor DAT trafficking using live cell. Briefly, superecliptic pHluorin (SEP) tagged DAT constructs can be packed into AAV viruses. These viruses can be stereotaxically  introduced into the nigra. Several weeks later, acute slices can be prepared, allowing visualization of surface DAT. Baseline rates of trafficking and rates following stimulation and pharmacological intervention can be examined. DAT trafficking has quickly become the focus across multiple models and identifying the specific interaction between DAT and the many PD associated genes will highlight new therapeutic targets.  5.4.3 Chapter 4 This chapter can be furthered by exploring the effects of long term MLi-2 administration, along with investigating the specific mechanisms connecting DAT trafficking and Lrrk2 kinase activity. Previous studies have shown that long-term Lrrk2 kinase inhibition can lead to loss of Lrrk2 levels 44  itself and damage to both kidneys and lungs. It would be interesting to examine the effects of long term (3 month+) MLi-2 administration with respect to kidney and lung pathology, along with outcomes associated with late-onset tau deposition observed by 82,110. One could also probe the connection between DAT trafficking and Lrrk2 kinase activity. One way this can be approached is by examining the effect of MLi-2 on activity dependent trafficking of DAT, using SEP-tagged DAT introduced virally into the substantia nigra and monitored by live cell. Further, one could examine the relationship between Lrrk2 activity and DAT binding by comparing interaction partners of DAT or Lrrk2 in the presence or absence of MLi-2 in order to identify a pathway linking them. The pattern of post-translational modifications of DAT in mutant (VKI, GKI, DKI) animals can be compared to their corresponding WT counterparts. This may be achieved by isolating DAT through immunoprecipitation, followed by mass spectrometry to identify any alteration in baseline modifications of DAT. 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