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Mutant Huntingtin-mediated alterations of N-Methyl-D-Aspartate receptor currents and downstream events… Fernandes, Herman Brian 2007

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MUTANT JLUNTINGTIN-MEDIATED ALTERATIONS OF N-METHYL-D-^ S P A R T A T E RECEPTOR CURRENTS AND DOWNSTREAM EVENTS UNDERLYING ENHANCED EXCITOTOXICITY IN HUNTINGTON'S DISEASE by H E R M A N BRIAN FERNANDES B.Sc, McMaster University, 1997 M.Sc., University of British Columbia, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Neuroscience) THE UNIVERSITY OF BRITISH COLUMBIA August 2007 © Herman Brian Fernandes, 2007 A B S T R A C T Expansion of a C A G repeat in the Huntington disease (HD) gene results in progressive neuronal loss, particularly of medium-sized spiny neurons of the striatum (MSNs). Previous studies suggest that increased activity of N-methyl-D-aspartate (NMDA) receptors (NMDARs) and altered mitochondrial function contribute to selective neuronal degeneration. Here I utilized MSNs obtained from transgenic mice expressing human huntingtin (htt) on a yeast artificial chromosome (YAC) to study alterations in NMDAR function and possible downstream consequences, particularly in regards to altered calcium (Ca ) handling and mitochondrial function. I examined the relationship between the length of the polyglutamine (polyQ) repeat in mutant htt (mhtt) and N M D A R currents in MSNs in vitro. I found that NMDAR current density was significantly enhanced in YAC72 MSNs compared to wild type (WT), Y A C 18, YAC46 and Y A C 128 MSNs, and that there was a trend toward larger N M D A R current density with increasing polyQ length for Y A C 18, YAC46 and YAC72 MSNs. This increase was selective for NMDARs and occurred in a neuronal. type-specific fashion. The mechanism for increased N M D A R current in YAC72 MSNs appears due to a mhtt-mediated increase in trafficking of NMDARs to the plasma membrane. While NMDA-induced apoptosis is enhanced in Y A C 128 MSNs, I found the initial steps in the intrinsic apoptotic pathway, including N M D A R current and cytosolic 9-1-Ca loading, are similar to those observed in WT MSNs. In contrast, the NMDAR-mediated Ca load triggered a significantly greater depolarization of mitochondria in Y A C 128 MSNs, suggesting that N M D A R signaling via the mitochondrial apoptotic pathway is altered. Y A C 128 MSNs demonstrated impaired cytosolic C a 2 + clearance following N M D A application, a difference that was not apparent following depolarization-evoked C a 2 + entry. Inhibitors of the mitochondrial permeability transition 9+ (mPT) reduced peak cytosolic Ca and mitochondrial depolarization evoked by N M D A in Y A C 128, but not WT, MSNs. Hence, in contrast to Y A C models with moderate C A G expansions, enhanced NMDA-induced apoptosis in Y A C 128 MSNs is largely due to augmented mitochondrial sensitivity to Ca2+-induced activation of the mPT. These findings suggest that the C A G repeat length can influence the mechanism by which mhtt enhances NMDAR-mediated excitotoxicity. However, as signaling pathways activated by strong N M D A R stimulation converge on mitochondria in all Y A C HD models, bolstering mitochondrial function and/or inhibiting the mPT represent possible therapeutic targets. iii TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables :.. ix List of Figures x List of Abbreviations xiii Acknowledgements xvii Dedication . xviii Chapter 1 Introduction 1 1.1 Huntington's Disease 1 1.1.1 Clinical Features of HD 1 1.1.2 Neuropathology in HD 2 1.1.3 Huntingtin Distribution, Cellular Roles and Function 4 1.1.4 Model Systems for the Study of HD....... 6 1.1.4.1 Heterologous Expression Systems 6 1.1.4.2 Mouse Models of HD 7 1.1.5 Mechanisms of Cellular Pathology inHD 10 1.1.5.1 The.Toxic Fragment Hypothesis 10 1.1.5.2 Mhtt Aggregates and Inclusions Contain N-Terminal Fragments 12 1.1.5.3 Mhtt Expression is Associated with Transcriptional Deficiencies 15 1.1.5.4 Mhtt May Alter Trafficking of Several Cellular Components 16 1.1.6 Summary 18 1.2 The Excitotoxicity Hypothesis in HD Pathogenesis 18 1.2.1 Excitotoxicity Defined 19 1.2.2 General Properties of the N M D A R 19 1.2.3 The Basis for the Excitotoxicity Hypothesis in HD 22 1.2.3.1 Evidence from Chemical Models of HD Support the Excitotoxic Hypothesis 23 1.2.4 Alterations of N M D A R Function in HD Models 24 1.2.4.1 N M D A R Currents in HD Models 24 1.2.4.2 The NR2B-Selective Hypothesis of Mhtt-Mediated Enhancement of N M D A R Function 26 1.2.4.3 Neurotoxicity as an Indicator of PolyQ Length-Dependent Alterations in NMDAR Function by Mhtt 27 1.2.5 Downstream Consequences of N M D A R Activation in Cells Expressing Mhtt 28 1.2.5.1 C a 2 + Homeostasis is Affected by Mhtt 28 1.2.6 Mitochondrial and Bioenergetic Impairment in HD 31 1.3 Research Hypotheses and Goals 34 v Chapter 2 Materials and Methods 37 2.1 Animals 37 2.2 Primary Neuronal Cultures 37 2.2.1 Preparation of Primary Striatal Neurons 37 2.2.2 Preparation of Primary Cortical Neurons 38 2.2.3 Preparation of Primary Cerebellar Granule Neurons 39 2.3 Electrophysiological Experiments. 40 2.3.1 Measurement of Current Density, Steady-State to Peak Ratio, Ifenprodil Sensitivity 41 2.3.2 Kainate Stimulation 42 2.3.3 Electrophysiological Measurement of N M D A R Insertion to the Plasma Membrane 42 2.4 Visualization of Relative Changes in Cytosolic Free Ca and Mitochondrial Membrane Potential (A^Fm) 43 2.4.1 Determination of Resting A T m 46 2.4.2 Calculation of CCCP/NMDA (C/N) Ratio to Determine Efficiency of Mitochondrial C a 2 + Uptake 47 2.4.3 Quantifying the Temporal Relationship Between Increases in C a 2 + and Loss of A^m 47 2.5 Data Analysis 48 2.6 Materials 49 Chapter 3 N M D A R Function in the Y A C Mouse Models of HD 50 vi 3.1 N M D A R Current Density in Neuronal Primary Cultures 51 3.2NMDARDesensitization 53 3.3 NRl/NR2B-Containing NMDARs 53 3.4 Shift of NMD'AR Expression in YAC72 MSNs to the Surface of the Plasma Membrane 54 3.5 Summary 56 Chapter 4 Aberrant Cytosolic C a 2 + Handling in the YAC128 Mouse Model of HD 57 4.1 Considerations Regarding N M D A Concentrations Used for These Studies 58 4.2 Resting and NMDA-Induced Changes in Cytosolic C a 2 + in YAC128 MSNs 59 4.3 Altered Cytosolic C a 2 + Clearance Following N M D A Stimulation in YAC128 MSNs 60 4.4 Effect of Route of C a 2 + Entry on Recovery from an Induced Cytosolic Ca 2 + Load in YAC128 MSNs 61 4.5 Summary 62 Chapter 5 Mitochondrial Dysfunction in Y A C 128 MSNs 64 5.1 Equivalent Cytosolic C a 2 + Loads Induce Significantly Different Changes in A ^ in YAC128 MSNs Compared with WT MSNs 65 5.2 Comparison of Resting A^Fm 66 vii 5.3 Assessing the Ability of Y A C 128 MSNs to Act as a C a 2 + Sink During N M D A R Activity 66 5.4 Potential mPT Involvement in Mitochondrial Depolarization in YAC128 MSNs.. . . . . 68 5.5 Summary..... 69 Chapter 6 General Discussion 70 6.1 Synopsis of Findings 70 6.2 Dissociation Between NMDAR-Mediated Current and Downstream Changes in Cytosolic C a 2 + and A T m in Y A C 128 MSNs 73 6.3 Acute NMDA-Evoked mPT Activation in YAC128 MSNs 75 6.4 Mhtt-Induced Impairment of C a 2 + Homeostasis in Y A C 128 MSNs 78 6.5 Selective Vulnerability of MSNs in HD 83 6.6 Limitations of This Study and Suggested Further Experiments... 85 6.7 Conclusions 88 Tables and Figures 90 References 141 LIST OF TABLES Table 1 A Summary of Relevant Full Length and N-Terminal Fragment HD Models : 90 ix LIST OF FIGURES Figure 1 Representative whole-cell current response of a voltage-clamped (V H = -60 mV) WT MSN to a 4 s application of 1 mM N M D A 91 Figure 2 Peak N M D A R current density varies with htt polyQ length in cultured MSNs . 93 Figure 3 PolyQ length does not affect kainate receptor current density......... 95 Figure 4 Peak N M D A R current density is not influenced by htt polyQ length in cultured CPNs 97 Figure 5 Desensitization of whole-cell NMDAR current in MSNs is not governed in a polyQ length-dependent fashion 99 Figure 6 Desensitization of whole-cell N M D A R current in CPNs is not altered by polyQ length. 101 Figure 7 IFN blocks NR 1 /NR2B-containing NMDARs 103 Figure 8 Neither overexpression of htt or expression of mhtt affects the proportion of NMDARs containing NR1/NR2B in cultured MSNs... 105 Figure 9 Neither overexpression of htt or expression of mhtt affects the proportion of NMDARs containing NR1/NR2B in cultured CPNs... 107 Figure 10 Electrophysiological silencing of surface NMDARs allows for detection of naive N M D A R insertion to the plasma membrane 109 Figure 11 The rate of whole-cell current recovery in YAC72 MSNs following MK-801 binding is significantly faster than in WT MSNs : I l l Figure 12 Example of cytosolic Ca imaging experiment using the x fluorescent Ca indicator Fura-2 113 Figure 13 Representative cytosolic C a 2 + responses of WT and Y A C 128 MSNs to 500 pM N M D A 115 94-Figure 14 WT and Y A C 128 MSNs have similar cytosolic Ca responses to N M D A . . . 117 Figure 15 Recovery from an NMDA-induced C a 2 + load in WT and Y A C 128 MSNs 119 Figure 16 Contrasting responses of WT and Y A C 128 MSNs to different stimuli that increase cytosolic C a 2 + to similar peak levels 121 Figure 17 The impairment in recovery of Y A C 128 MSNs from a cytosolic C a 2 + load is selective for NMDA-induced C a 2 + loads 123 Figure 18 Simultaneous monitoring of NMDA-induced changes in cytosolic C a 2 + and A T m in WT. and YAC128 MSNs 125 Figure 19 Y A C 128 MSNs undergo a more dramatic loss of A ^ m as a consequence of N M D A challenge than WT MSNs, despite similar cytosolic C a 2 + loads 127 Figure 20 Characteristics of resting mitochondria in WT and Y A C 128 MSNs.. 129 Figure 21 Mitochondria in both WT and Y A C 128 MSNs have a similar capability to buffer transient cytosolic C a 2 + increases produced by short application of N M D A 131 Figure 22 Recovery of Y A C 128 MSNs from a cytosolic Ca 2 + load produced by a short application of N M D A is similar in timecourse to that of WTMSNs 133 xi Figure 23 The effect of the mPT inhibitor CsA on simultaneously measured peak cytosolic C a 2 + and A ¥ m in WT and Y A C 128 MSNs 135 Figure 24 The effect of the mPT inhibitor BkA on simultaneously measured peak cytosolic C a 2 + and in WT and YAC128 MSNs 137 Figure 25 A general overview of the main excitotoxic events in the Y A C HD mouse models of varying htt polyQ length 139 LIST OF ABBREVIATIONS 3-NP, 3-nitropropionic acid ADP, adenosine diphosphate AM, acetoxymethyl AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid AMPAR, AMPA receptor ' ANOVA, analysis of variance ANT, adenine nucleotide translocase APV, 2-amino-5-phosphonovalerate ATP, adenosine 5'-triphosphate AUC, area under the curve BAPTA, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid BDNF, brain-derived neurotrophic factor BkA, bongkrekic acid BSA, bovine serum albumin BSS, balanced salt solution C 6 R , caspase-6 resistant CaBP, calbindin cAMP, cyclic adenosine 5'-monophosphate CBP, CREB binding protein CCCP, carbonyl cyanide 3-chloro-phenylhydrazone CGN, cerebellar granule neuron CHO, Chinese hamster ovary C/N ratio, CCCP/NMDA ratio . CNS, central nervous system ; COS, Simian fibroblasts (CV-1 cells) transformed by SV40 CPN, cortical pyramidal neuron CREB, cAMP response element binding protein CsA, cyclosporin A DARPP-32, dopamine- and cAMP-regulated phosphoprotein 32 kDa A T m , mitochondrial membrane potential DNA, deoxyribonucleic acid DRPLA, dentatorubral pallidoluysian atrophy d.i.v., days in vitro DMSO, dimethyl sulfoxide EAA, excitatory amino acid EC50, effective concentration to achieve 50% of maximal response EPSC, excitatory post-synaptic current ER, endoplasmic reticulum FPL, FPL64176 GABA, y-aminobutyric acid GAD-65, glutamic acid decarboxylase GluR, glutamate receptor HAP-1, huntingtin-associated protein-1 HBSS, Hank's balanced salt solution HD, Huntington's disease HEK293 cells, human embryonic kidney 293 cells HEPES, (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) HIP-1, huntingtin-interacting protein-1 htt, huntingtin IFN, ifenprodil iGluR, ionotropic glutamate receptor Ipeak, peak current IP3, inositol 1,4,5-trisphosphate IP3R, IP3 receptor Iss, steady-state current Kj, dissociation constant mGluR, metabotropic glutamate receptor mhtt, mutant htt MK-801, dizocilpine or (5R, 10S)-(+)-5-methyl-10,l l-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate mPT, mitochondrial permeability transition mRNA, messenger RNA MSN, medium-sized spiny neuron of the striatum NCX3, Na + /Ca 2 + exchanger isoform 3 N F - K B , nuclear factor kappa-B Nil, neuronal intranuclear inclusion NMDA, N-methyl-D-aspartate NMDAR, N M D A receptor NR1, N M D A R subunit-1 NR2, N M D A R subunit 2 P, postnatal day polyQ, polyglutamine PSD-95, post-synaptic density protein 95 kDa REST/NRSF, repressor element 1 (REl)-silencing transcription factor (REST)/] restrictive silencer factor (NRSF) Rhod-123, rhodamine-123 RNA, ribonucleic acid ROI, region of interest SBMA, spinal bulbar muscular atrophy SCA, spinocerebellar ataxia SEM, standard error of the mean TTX, tetrodotoxin UV, ultraviolet VDAC, voltage-dependent anion channel V G C C , voltage-gated calcium channel V H , membrane holding potential WT, wild type Y A C , yeast artificial chromosome ACKNOWLEDGEMENTS I would like to thank Dr. Lynn Raymond for many things, including her knowledge, insight, dedication, and for. her supportive yet 'hands-off style of supervision that allowed me to explore and expand my limits and abilities as a scientist. I would also like to thank the other members of my supervisory committee, including Drs. Ken Baimbridge, John Church, and Blair Leavitt for their guidance, ideas and help carrying out the various aspects of my research. I wish to particularly thank Drs. Church and Baimbridge for the use of their equipment and lab resources to carry out all of the imaging experiments presented here. A number of other people in the lab have also provided help and support along the way. Bo Li gave me valuable advice while learning the patch-clamp technique, and Nansheng Chen shared with me the secrets of the theta-tube. Jacqueline Shehadeh, Mannie Fan, Tao Luo, Lily Zhang and Esther Yu all helped me with cell culture as well as other day-to-day matters too numerous to mention. Drs. Timothy Murphy, Cath Cowan, Austen Milnerwood, and Andy Shih, as well as Lavan Sornarajah, whiled away many an hour with me discussing ideas, enriching the quality of my work and work experience by their contributions. I would also like to thank our collaborators, Drs. Michael Hayden, Blair Leavitt, and Roha Graham for providing and replenishing the transgenic mice which were essential to my project. Dr. Gordon Rintoul provided both analysis tools and valuable input regarding my work, and Drs. Claire Sheldon and Tony Kelly provided technical assistance. Lastly, I would like to thank the other members of the Kinsmen Labs for good times... good times. xvii D E D I C A T I O N This work is dedicated to my former girlfriend and now wife Amanda, for her unconditional and endless patience and support throughout my graduate degrees. I would also like to thank my family and friends for their support and sense of humour while I slowly but surely progressed towards finally finishing my education - for now.... xviii Chapter 1 Introduction 1.1 Huntington's Disease Huntington's disease (HD) is an inherited, progressive neurodegenerative disorder, with a prevalence of -5-10 per 100 000 people (Vonsattel and DiFiglia, 1998). This genetic, autosomal dominant disease is caused by a mutation in exon 1 of the IT15 gene, resulting in the expansion of a C A G repeat (HDCRG, 1993) encoding a polyglutamine (polyQ) region in the protein huntingtin. Normal individuals have 35 or fewer C A G repeats (Kremer et al., 1994), while 36 or more C A G repeats results in the eventual development of the disease (Rubinsztein et al., 1996). HD is one of nine currently identified neurodegenerative diseases resulting from the expansion of a C A G tract within the coding region of nine different genes reviewed in (Tobin and Signer, 2000; Ross, 2002), which include spinocerebellar ataxia (SCA) 1, 2, 3, 6, 7, 17, spinal bulbar muscular atrophy (SBMA), and dentatorubral pallidoluysian atrophy (DRPLA). 1.1.1 Clinical Features of HD The clinical presentation of HD includes a range of motor, cognitive and mood (emotional) changes. The age of onset generally occurs between the ages of 35-50,-progressing over 15-20 years until death (Hayden, 1981). Motor symptoms include choreiform involuntary movements, postural imbalance, uncoordinated voluntary movements and dysphagia, followed at later stages by akinesia and rigidity. Depression is a common symptom, and patients often display personality changes such as apathy and 1 flashes of temper. Generally, emotional and cognitive changes precede the motor symptoms (Harper, 1996; Paulsen et al., 2001). Cases of juvenile-onset HD also occur and these cases have a somewhat different clinical presentation, with symptoms including bradykinesia, rigidity, dystonia, and often the presence of epileptic seizures (Vonsattel and DiFiglia, 1998). In contrast to the adult-onset form of HD, juvenile-onset patients often have little or no chorea (Brandt et al., 1996). Expanded C A G regions show increased instability, particularly when passed via the paternal germline, with expansions in C A G length occurring more often than reductions. This produces the phenomenon known as anticipation, where the C A G repeat number tends to increase in subsequent family generations (Myers et al., 1982; Duyao et al., 1993). The age of onset of symptoms generally correlates inversely with the length of the C A G expansion (Andrew et al., 1993; Brinkman et al., 1997; Stine et al., 1993). Most adult-onset cases have C A G repeat lengths of 40-50, whereas juvenile-onset cases have somewhat longer C A G expansions (>60) (Brandt et al., 1996). 1.1.2 Neuropathology in H D The pattern of neurodegeneration in HD is particularly selective for the medium-sized spiny neurons (MSNs) of the striatum, which normally project to other areas of the basal ganglia, specifically the substantia nigra and globus pallidus (Graveland et al., 1985), and make up approximately 95% of neurons in the striatum (Surmeier et al., 1988). In an extensive study characterizing postmortem HD brains, the highest degree of degeneration was found in the caudate and putamen (Vonsattel et al., 1985). The authors of this study used a five-point grading system (0-4) to describe striatal neuropathology in 2 ascending order of severity, correlating closely with the extent of clinical disability. For example in grade 0 HD brains no discernable neuropathological changes could be seen; in grade 1 brains there are only microscopic neuropathological changes (50% loss of neurons in the caudate nucleus) evident. The more severe cases showed increasing neuronal loss within the caudate as well as other striatal regions; for example, grade 4 brains show >95% neuronal loss in the caudate, as well as extensive neuronal losses in the putamen, globus pallidus and nucleus accumbens, accompanied by increasing numbers of astrocytes through grades 2-4 (Vonsattel et al., 1985). • MSNs are the first population of neurons to die, and generally show the greatest losses in numbers (Vonsattel and DiFiglia, 1998). In the early stages of HD, the subpopulation of MSNs expressing enkephalin and projecting to the external segment of the globus pallidus (the indirect basal ganglia pathway) die first, followed by the substance P-expressing MSNs (Richfield et al., 1995) that project to the internal segment of the globus pallidus (the direct basal ganglia pathway). Notably, the large aspiny cholinergic and nitric oxide synthase-containing interneurons are relatively spared (Ferrante et al., 1985; Ferrante et al., 1987). In later stage HD cases, a number of brain regions display atrophy, or a loss in cross-sectional area. The caudate and putamen show approximately 60% area loss whereas other brain regions (e.g. substantia nigra, globus pallidus, thalamus, hippocampus) display lesser atrophy of 20-30% in later stages (de la Monte et al., 1988; Cowan and Raymond, 2006). Hence, the striatum,undergoes the greatest extent of neuronal loss, and in the most severe cases (grade 4) this results in a significant decrease in neuronal density despite the concurrent regional atrophy. 3 A loss of cortical neurons and volume also occurs in more advanced cases of HD, particularly loss of the large pyramidal neurons in layers III, V and VI which project directly to the striatum (Cudkowicz and Kowall, 1990; Hedreen et al., 1991). 1.1.3 Huntingtin: Distribution, Cellular Roles and Function The protein huntingtin (htt) is widely distributed throughout many tissues of the body (Strong et al., 1993) and throughout most brain regions (Landwehrmeyer et al., 1995b) although there is no particular enrichment of the protein in the striatum (Sharp et al., 1995). Expansion of the C A G repeat region of the HD gene to produce polyglutamine expanded, mutant htt (mhtt) does not appear to alter the tissue distribution of the protein (Aronin et al., 1995). Htt is a 350 kDa cytosolic protein and can be found in the soma and throughout the dendrites as well as in synaptic terminals (Aronin et al., 1995). To this point the function of htt is unknown. It has been found associated with membrane-bound organelles including mitochondria (Choo et al., 2004) and vesicular membranes (DiFiglia et al., 1995; Velier et al., 1998). Htt is also found in the nucleus, and this has implications for a possible role in gene transcription for both the normal and mutant forms (Kegel et al., 2002). Htt expression is essential for normal development, as disruption of the endogenous htt protein results in embryonic lethality (Leavitt et al., 2001; Nasir et al., 1995; Zeitlin et al., 1995). However, mhtt retains at least some of the developmentally required functionality of the wild type protein, as expression of mhtt can rescue the lethal suppression of endogenous htt expression (Leavitt et al., 2001). 4 In the context of the yeast artificial chromosome (YAC) mouse model, it could also be said that a function of htt is to prevent neuronal death, as overexpression of human htt with 18 glutamine repeats is neuroprotective against a variety of toxic insults in primary MSN cultures (Leavitt et al., 2006; Shehadeh et al., 2006), and endogenous murine htt counters the pro-apoptotic effects of human mhtt in YAC46 mice (Leavitt et al., 2001). Additionally, normal htt was found to have an anti-apoptotic effect against a number of pro-apoptotic stimuli in cloned striatal cells, acting upstream of caspase-3 activation (Rigamonti et al., 2000). One controversy in the HD field revolves around the functions of htt and mhtt, specifically whether HD represents a gain of function of the mutant protein, or a loss of function of the normal protein. Several lines of evidence exist to support both sides of this argument. Conditional inactivation of endogenous htt in the brain of mice produced a progressively severe limb-clasping behaviour when picked up by the tail, a behaviour commonly observed in HD mouse models; this was accompanied by motor deficits progressing to hypoactivity and death by 13 months, with obvious striatal degeneration (Dragatsis et al., 2000). Additionally, mhtt retains at least some required activity of htt, as it can rescue the embryonic lethality of endogenous htt disruption in mice (Leavitt et al., 2001). These observations support the idea that the loss of htt function is sufficient to cause HD-like pathology. However, the case can also be made that this last observation "supports a toxic gain of function in mhtt; since the mutant protein has been shown to retain some semblance of physiological function, an additional toxic function could be conferred by the mutation and would be necessary to produce the pathological changes associated with HD. Furthermore, HD shows a pattern of dominant inheritance, 5 suggesting that one copy of the mutant allele is sufficient; but again, this does not exclude a 'loss of htt function' component to the disease. Several clinical studies (Wexler et al., 1987; Myers et al., 1989; Durr et al., 1999) suggest that homozygous HD appears clinically identical to heterozygous HD, and as the normal allele does not improve disease phenotype, this suggests that normal htt function does not play a role in the development or progression of HD. However, methodological weaknesses in these studies render this evidence inconclusive (Cattaneo et al., 2001), and another study suggests that phenotype and disease progression are altered in HD patients with two mutant alleles compared to heterozygous HD patients (Squitieri et al., 2003). Overall, it is likely that both gain and loss of htt function contribute to the overall development and progression of HD. 1.1.4 Model Systems for the Study of HD 1.1.4.1 Heterologous Expression Systems The simplicity and degree of control possible in heterologous expression systems made them an ideal starting point for examining the basis of mhtt-mediated toxicity. For example, the HEK293 cell line has been used extensively to study ionotropic glutamate receptor-mediated toxicity and function in the absence of htt (Monyer et al., 1992; Cik et al., 1994; Anegawa et al., 1995; Anegawa et al., 2000; Raymond et al , 1996), as well as the influence of overexpression of both htt and mhtt on N-methyl-D-aspartate receptor (NMDAR) function and excitotoxicity (Chen et al., 1999b; Zeron et al., 2001). Other cell lines such as COS and CHO cells have been used to also study N M D A R function and 6 toxicity (Boeckman and Aizenman, 1996; Anegawa et al., 2000; Rameau et al., 2000). The finding that expression of mhtt along with NMDARs composed of NR1 and NR2B subunits enhanced N M D A R current amplitude (Chen et al., 1999b), similar to subsequent observations in acutely.dissociated and cultured MSNs (Zeron et al., 2002; Zeron et al., 2004), was the genesis for studies of NMDAR-mediated excitotoxicity in our laboratory (Zeron et al , 2001; Zeron et al , 2002; Zeron et al., 2004; Shehadeh et al , 2006) and indeed for the work presented here. 1.1.4.2 Mouse Models of HD A number of different mouse models have been established as tools to provide insights into the pre-symptomatic changes, pathogenic mechanisms, progression of disease pathology, and possible areas of therapeutic intervention in HD. A summary of the relevant mouse models discussed here can be found in Table 1. A comprehensive summary of these and other models can be found courtesy of the Hereditary Disease Foundation (www.hdfoundation.org/PDF/hdmicetable.pdf). Generally speaking, the most commonly used models fall into one of three categories: transgenic mice expressing an N-terminal fragment of human htt containing an expanded C A G region (N-terminal fragment models); transgenic mice that express full length human htt in addition to their own endogenous murine htt (full length models); and knock-in mouse models where an expanded C A G repeat region has been inserted into the endogenous Hdh gene. The most commonly used N-terminal fragment models are the R6/1 and R6/2 lines, expressing exon 1 of the human HD gene containing 116 and 144 C A G repeats, respectively (Mangiarini et al., 1996), with the R6/2 model showing the more aggressive 7 disease phenotype. Generally these models both feature early death (approximately 12 months for R6/1, 4 months for R6/2) preceded by an early onset of motor symptoms (as early as 1 month in R6/2) and overall brain atrophy (-20%, Davies et al., 1999)-which is apparent prior to neuronal loss (Mangiarini et al., 1996). Another well characterized fragment model expresses an N-terminal fragment of 171 residues, which contains a C A G repeat length of 82 and also dies prematurely, having a lifespan between 2.5 to 11 months (Schilling et al., 1999). In a study comparing striatal gene expression changes mediated by a polyglutamine repeat expansion in the R6/2 and N171-82Q models, both models produce similar changes to striatal gene expression (Luthi-Carter et al., 2000), indicating common pathological changes. Interestingly, both of these models also display striatal resistance to excitotoxins after HD-like symptoms have developed (Hansson et al., 2001; MacGibbon et al., 2002; Jarabek et al., 2004), but they also lack frank striatal neuronal loss, which is inconsistent with the notable striatal degeneration in human postmortem HD brains. Hence one key criticism qf these models may be that expression of the full-length htt gene, with all critical regulatory sequences, is required to most accurately reproduce the human disease. However, the aggressive pathology and shortened lifespan make toxic fragment models ideal for therapeutic testing in one sense, as beneficial effects of intervention will be more obvious than in a more discrete phenotype. Knock-in HD mouse models generally exhibit a late onset of motor symptoms, with a relatively mild disease phenotype and little to no neuropathology evident, aside from predominantly striatal aggregate formation, despite longer C A G repeat lengths ranging from 72 up to 150 (Shelbourne et al., 1999; Wheeler et al., 2000; Lin et al., 2001; 8 Menalled et al., 2002; Menalled et al., 2003). One particular advantage of these models is the elimination of a key confounding factor in interpreting results from other HD models, that of htt (mutant or otherwise) overexpression. As well, the long lifespan make these models ideal for studying behavioural changes as a proxy for neuronal dysfunction prior to neuronal loss. Full length models, such as the Y A C mouse model (Hodgson et al., 1999; Slow et al, 2003) or the C M V promoter model (Reddy et al., 1998) recapitulate the pattern of selective striatal neuronal loss seen in human HD patients, making them ideal for studying changes in neuronal function that could underlie human disease. Therefore, the work presented here utilizes Y A C mouse models of HD, which were developed at UBC in the Hayden lab. Mice in these models generally have a later onset (2-7 months, depending on the htt polyQ repeat length) of the HD motor phenotype, and have a relatively normal length lifespan (Hodgson et al., 1999; Slow et al., 2003; Van Raamsdonk et al., 2005). For this project, we made use of several different lengths of C A G repeat expansions to represent a range of human phenotypes, from non-pathogenic repeat lengths of 18 to sizes correlating to adult (46 repeats) and juvenile (72 or 128 repeats) HD (Hodgson et al., 1999; Slow et al., 2003). In this model, the full length human htt gene with a specific C A G repeat length and all regulatory domains is integrated into a Y A C , leaving expression under control of the endogenous human HD promoter. Using this method, expression of transgenic htt is regulated in a developmentally and anatomically appropriate fashion. The expression of transgenic htt in the Y A C lines used (YAC 18 - line 212, YAC46 - line 668, YAC72 - line 2511, Y A C 128 - line 55) is approximately one third to one half the endogenous amount and 9 equivalent across genotypes (Hodgson et al., 1999; Slow et al , 2003); these lines were selected for use to minimize mhtt expression level-related phenomena, specifically differences in phenotype that could be attributed to the amount of transgenic protein expressed (Hickey and Chesselet, 2003; Graham et al., 2006a). 1.1.5 Mechanisms of Cellular Pathology in HD There are several different mechanisms which have been proposed as the underlying cause of neurodegeneration in HD, based on evidence from a variety of sources including observations gleaned from examination of human brain tissue, study of HD model organisms, in vitro experiments in cellular preparations of both neuronal and non-neuronal origin, and in vitro experiments using isolated proteins and/or organelles. The work here focuses exclusively on one particular mechanism, the excitotoxicity hypothesis, which will be discussed in depth in a later section. Here I will describe several of the competing hypotheses, several (or all) of which may well act in concert to produce neuronal dysfunction and death in HD. 1.1.5.1 The Toxic Fragment Hypothesis Both htt and mhtt can be proteolytically cleaved, producing N-terminal fragments which contain the polyQ domain. There is substantial evidence in the literature suggesting that htt is proteolytically targeted by the family of cysteine proteases known as caspases. There are both caspase-3 cleavage sites (residues 513 and 552) and a caspase-6 cleavage site (residue 586), which have been shown to be targeted in vitro (Wellington et al , 1998; Wellington et al , 2000). Mutation of these caspase cleavage 10 sites resulted in reduced apoptosis (both in neurons and non-neuronal cell lines) and protein aggregate formation (Wellington et al., 2000). The identification of htt as being cleaved by apopain (a cysteine protease, also known as caspase-3) led to the hypothesis that HD pathogenesis involved conditions where inappropriate apoptosis occurred, and that the cleavage fragments of mhtt could be the toxic agent (Goldberg et al., 1996). Expression of N-terminal htt fragments containing expanded polyQ regions has been demonstrated to be toxic by a number of studies (Martindale et al., 1998; Hackam et al., 1998; Cooper et al., 1998; Saudou et al., 1998), and it was proposed that proteolytic cleavage of mhtt produces toxic fragments containing expanded polyQ regions which are neurotoxic themselves, and in addition may also stimulate further proteolytic activity (Wellington and Hayden, 1997). This idea gained further support with the demonstration that htt was proteolytically cleaved by caspase-3 in vivo in brains from both HD mouse models (Hodgson et al., 1999; Wellington et al., 2002) and human patients (Kim et al., 2001; Wellington et al., 2002). These N-terminal fragments derived from mhtt have been proposed to be more toxic than full-length mhtt (Wellington et al., 2000), and there may be a tissue-specific component to htt cleavage activity, as both N- and C-terminal htt fragments were found to be enhanced in the striatum of HD versus control brains (Mende-Mueller et al., 2001). This could be a result of enhanced cleavage of htt and/or impaired degradation of htt proteolysis products. Evidence for an increased rate of htt cleavage with increasing polyQ length (Goldberg et al., 1996) supports the idea that mhtt cleavage is enhanced, although other reports dispute this notion, suggesting either that that normal htt is more susceptible to cleavage than mhtt (Dyer and McMurray, 2001) or 11 that polyQ repeat length does not affect the likelihood of cleavage (Wellington et al., 1998). It has been reported that htt from both human and mouse brain can also be cleaved by another family of cysteine proteases known as calpains (Kim et al., 2001). Gafni and Ellerby demonstrated that N-terminal htt cleavage fragments found in the caudate of HD patient post-mortem brain were products of calpain cleavage, and confirmed this finding in vitro; they did not find similar cleavage fragments in brain tissue from controls (Gafni and Ellerby, 2002). Studies using a chemical model of HD in rats found activated calpain selectively in the striatum, which was associated with the appearance of calpain cleavage products of htt (Bizat et al., 2003); another recent study using the same model found neuroprotection with memantine that coincided with reduced calpain activity (Lee et al., 2006). Further evidence of a role for calpain-mediated cleavage of htt in HD pathology was provided by a study demonstrating increased striatal and cortical calpain activity in a knock-in mouse model of HD, and that mutation of calpain-cleavage sites to produce a cleavage-resistant form of mhtt reduced toxicity in a cell line (Gafni et al., 2004). Since it is established that (m)htt is a target for proteolytic cleavage, the question becomes how do these fragments of mhtt differ from cleavage products of htt to produce toxic effects? 1.1.5.2 Mhtt Aggregates and Inclusions Contain N-terminal Fragments It was noted in the R6/2 model of HD that the appearance of neuronal intranuclear inclusions (Nils) correlates with the onset of symptoms, and these Nils contain both N-12 terminal htt and ubiquitin (Davies et al., 1997). Similar Nils have also been noted in the striatum and cortex of human brain tissue from HD patients, and N-terminal mhtt fragments were found in dystrophic neurites (Sapp et al., 1999). It appears that smaller htt fragments containing an expanded polyQ region preferentially form Nils compared to larger fragments, and that the expanded polyQ region increases the susceptibility of cells to toxic stimuli (Hackam et al., 1998; Martindale et al., 1998), further implicating cleavage fragments of mhtt as potential toxic mediators underlying neuronal dysfunction in HD. It is notable that aggregates of mhtt are also ubiquitinated, suggesting that mhtt fragments are targeted for proteasomal degradation but cannot be processed and impair cellular function leading to degeneration (DiFiglia et al., 1997). This hypothesis is supported by the observation that the proteasomal degradation of another protein containing an expanded polyQ region, ataxin-1, is impaired, underlying aggregate formation and appearance of ubiquitinated nuclear inclusions in SCA-1 (Cummings et al., 1999). Hence the presence of aggregates may reflect an inability to process expanded polyQ repeats, leading to cell dysfunction and eventual death (Bence et al , 2001). Recent evidence from HD patient brains supports this deficiency as a factor in human disease development, as decreased proteasomal activity and association of ubiquitin with inclusions is noted particularly in the caudate and putamen in late stages (Seo et al., 2004). Aside from possible effects on protein degradation, Nils containing mhtt fragments have also been implicated in interference with transcriptional activity (Kegel et al., 2002), most notably binding to the transcriptional co-activator CREB binding protein (CBP) in models of HD (Nucifora et al , 2001; Steffan et al., 2000) and SBMA and SCA3 13 (McCampbell et al., 2000). Overexpression of CBP (McCampbell et al , 2000; Nucifora et al., 2001) or the application of H D A C inhibitors (McCampbell et al., 2001) successfully reduced toxicity in these models, implying that transcriptional repression is in part responsible for the toxicity in these disease models (Nucifora et al., 2001; Steffan etal., 2000). Another possible mechanism for mhtt fragments to mediate toxicity would be through documented interactions with organelle structures, possibly impairing subcellular function. This localization characteristic may be a feature of the expanded polyQ repeat itself (Monoi et al., 2000). Caspase-3 cleavage fragments of mhtt have been shown to preferentially interact with membrane-bound structures (Kim et al., 2001). It has also been reported that fragments of mhtt, but not endogenous or non-pathogenic length htt, become lodged in the membranes of mitochondria from YAC72 mice, possibly compromising their bioenergetic functions (Panov et al., 2002). Another recent study demonstrated that the first 17 amino acids in the N-terminus of mhtt fragments alter association with the ER and Golgi as well as mitochondria, and cause acute dysfunction in isolated mitochondria (Rockabrand et al., 2006). The altered association of htt with these organelles caused by the polyQ expansion may also adversely affect organelle morphology and function (Hilditch-Maguire et al., 2000). Recent studies concerning variants of the Y A C 128 HD mouse model provide further controversy regarding the toxic fragment hypothesis. The shortstop mouse (Slow et al., 2005) has a truncated form of htt expressed with an intact polyQ region of 128 repeats, and yet lacks any behavioural symptoms and shows no signs of neurodegeneration, seemingly contradicting the hypothesis that N-terminal mhtt 14 fragments are toxic on their own. Another interesting finding was that Y A C 128 mice expressing full length caspase-6 resistant (C6R) mhtt showed a remarkable lack of HD symptoms or neuropathological changes, whereas Y A C 128 mice expressing full length caspase-3 resistant mhtt resembled Y A C 128 mice in terms of behaviour, neuropathology, and neuronal toxicity (Graham et al., 2006b). Hence the toxic fragment hypothesis remains a controversial basis for explaining disease development and progression in HD. 1.1.5.3 Mhtt Expression is Associated with Transcriptional Deficiencies A particularly interesting property of polyQ tracts is their tendency to fold into a p-sheet configuration (Chen et al., 2002), allowing both self-association and association with other proteins containing polyQ stretches through a polar zipper interaction (Perutz, 1994). This tendency underlies aggregation of polyQ-containing proteins, and occurs in proportion to polyQ length (Chen et al., 2001b). As briefly mentioned earlier (see Section 1.1.5.1), mhtt interacts with a number of transcription factors and nuclear proteins including CBP, Spl , p53, N F - K B , and several others, with the overall effect generally trending towards reduced transcriptional activity (Sugars and Rubinsztein, 2003). A number of striatal transcriptional and signaling pathways are altered by mhtt in two different HD mouse models, again the overall effect being a reduction in expression of striatal genes required for normal neuronal function (Luthi-Carter et al., 2000); this effect was found to be particularly evident with N-terminal fragments of mhtt (Chan et al., 2002). A recent study found that mRNA profiles in HD patient brains exhibited the greatest changes in the caudate 15 nucleus, followed by motor cortex and cerebellum, showing a parallel between molecular expression events and neuropathology in the human disease (Hodges et al., 2006). One particularly well-studied consequence of mhtt-mediated transcriptional repression is the effect on brain-derived neurotrophic factor (BDNF) production, where mhtt expression decreases cortical production of BDNF at the transcriptional level, resulting in poor trophic support of MSNs (Zuccato et al., 2001). This result was noted in both human HD brain tissue as well as transgenic mouse HD models (R6/2 and N171-82Q), and the levels of cortical BDNF mRNA correlated well with HD disease progression in these mice (Zuccato et al., 2005). Normal htt is known to interact with the transcriptional repressor REST/NRSF and retain it in the cytosol, inhibiting its function and thus allowing transcription of BDNF, among other genes (Zuccato et al., 2003). However, mhtt loses this ability to restrict REST/NRSF translocation to the nucleus, resulting in suppression of BDNF transcription (Zuccato et al., 2003). Hence, transcriptional alterations may produce sub-optimal conditions for neuronal survival and promote dysfunction and premature death in HD. 1.1.5.4 Mhtt May Alter Trafficking of Several Cellular Components As discussed earlier, htt is associated with vesicular membranes, providing possible clues to a physiological role. This is supported by the apparent functions of some its interacting proteins, particularly huntingtin interacting protein 1 (HIP-1) and huntingtin associated protein 1 (HAP-1). HIP-1 is associated with the clathrin-mediated endocytosis pathway and is enriched on clathrin-coated vesicles (Metzler et al., 2001). HIP-1 is also a homologue of 16 the yeast protein Sla2p (Wanker et al., 1997), which is essential for appropriate cytoskeletal function (Kalchman et al., 1997). The strength of interaction between htt and HIP-1 is inversely proportional to the polyQ length, hence mhtt may indirectly, through its altered interaction with HIP-1, perturb membrane-cytoskeleton interactions (Kalchman et al., 1997). For example, HIP-1 has been strongly implicated in the normal endocytosis and trafficking of a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs; Metzler et al., 2003). Another possible manifestation of the aberrant mhtt-HIP-1 interaction is altered neurotransmitter release, as demonstrated in corticostriatal terminals in symptomatic R6/2 mice (Cepeda et al., 2003), and a reduction in the presynaptic vesicular pool in several transgenic HD mouse models. The finding of a reduced presynaptic vesicle pool may also be attributed to the effect of mhtt expression on the association of HAP-1 with synaptic vesicles (Li et al., 2003b). HAP-1 also interacts with the cytoskeletal microtubule network via an association with dynactin, and is likely involved in intracellular vesicular transport (Engelender et al., 1997). In an interesting parallel to mhtt-mediated transcriptional inhibition of BDNF production, the transport of BDNF in corticostriatal axons and MSNs is impaired by mhtt, resulting in neurotoxicity (Gauthier et al., 2004; del Toro et al., 2006). Mhtt also appears to interfere with the trafficking of organelles other than vesicles. A recent report noted that mhtt effectively limited the mobility of mitochondria along cortical neuronal processes, an event attributed to the enhanced ability of full length mhtt to form cytosolic aggregates (Chang et al., 2006). This impairment could reduce the ability of neurons to remove injured mitochondria, provide adequate energy 17 production at a given site, or prevent adequate and timely buffering of intracellular Ca (Chang et al., 2006). Therefore mhtt can impair essential trafficking functions through several mechanisms, each of which may contribute to the underlying neuronal dysfunction in H D . 1.1.6 Summary These are several of the main hypotheses for the underlying causes of neuronal dysfunction and eventual death in H D . There are several other hypotheses to explain the neuropathological changes seen in both the human condition and the changes in animal models (genetic and chemical models of HD) with substantial supporting evidence. The mechanisms of neuropathology are largely thought to be postsynaptic, and here I have grouped them under the umbrella of the excitotoxicity hypothesis of H D pathogenesis. This categorization best represents the underlying rationale for the research performed in support of this thesis project. 1.2 The Excitotoxicity Hypothesis in HD Pathogenesis There are many lines of evidence which both directly and indirectly support the role of excitotoxicity in H D , from alterations of N M D A R function to bioenergetic impairment. Here I w i l l provide an overview of evidence supporting the excitotoxicity hypothesis from human patients and in vivo and in vitro experiments using animal and cellular models. In doing so we wi l l arrive-at the hypotheses that we attempt to answer with this body of work. 18 1.2.1 Excitotoxicity Defined Excitotoxicity in neurons is a toxic consequence of the actions of excitatory amino acids (EAAs), whether endogenous or exogenous (in the case of some chemical models in animals or in vitro). Within the CNS, since glutamate is the major excitatory neurotransmitter, excitotoxicity is generally considered to result from neurons being exposed to glutamate, either for prolonged periods or in excessive concentrations (Lucas and Newhouse, 1957; Olney, 1969). This can result in a number of pathological changes including ion influx, osmotic dysregulation, energy depletion and biochemical changes, eventually leading to cellular death (Sattler and Tymianski, 2001; Mattson, 2003). Glutamate activates two classes of receptors in neurons: 1) metabotropic glutamate receptors (mGluRs), which exert their effects via coupling to G-proteins; and 2) ionotropic glutamate receptors (iGluRs), which upon binding of the appropriate ligands allow passage of cations through a channel pore formed by the receptor subunits (Dingledine et al., 1999). The most intensely studied of the iGluRs in excitotoxicity is the N M D A R , a subclass of receptor with several key features of relevance to neuronal death, i.e. a relatively high permeability to C a 2 + and relatively slow activation and deactivation kinetics. 1.2.2 General Properties of the NMDAR N M D A R s are heteromeric protein complexes, composed of two NR1 subunits and at least two NR2 subunits (Monyer et a l , 1994; Mcllhinney et al., 1998; Laube et a l , 1998), most likely in a tetrameric configuration (Clements and Westbrook, 1991; Laube et al., 1998). Functional N M D A R s require a combination of both NR1 and NR2 subunits 19 (Monyer et al., 1992). There are four different genes that encode the NR2 (NR2A, NR2B, NR2C, NR2D) subunits, and their expression is both developmentally and spatially regulated, on a cellular level as well as a brain-regional level (Ishii et al., 1993; Monyer et al., 1994; Akazawa et al., 1994; Laurie et al., 1997). There are eight possible splice variants of the NR1 subunit, generated at the mRNA level (Sugihara et al., 1992; Hollmann et al., 1993). The combination of NR1 splice variants with different NR2 subunits alters ion channel characteristics (Monyer et al., 1992; Ishii et al., 1993; Flint et al , 1997; Misra et al., 2000; Monyer et al.,1994; Chen et al., 1999a), providing significant potential for functional diversity. Activation of the N M D A R ion channel complex requires concurrent events: the binding of the co-agonists glutamate and glycine, and depolarization of the plasma membrane. The binding site for glutamate is located on NR2 subunits (Anson et al., 1998; Anson et al., 2000), whereas glycine binds to the NR1 subunit (Kuryatov et al., 1994). Binding of the two co-agonists has the property of negative co-operativity (Regalado et al., 2001), thus ensuring that sufficient concentrations of both co-agonists must be present to activate the channel. Mg physically blocks the N M D A R channel pore in a voltage-dependent fashion, requiring depolarization of the plasma membrane to allow channel function (Mayer et al., 1984; Nowak et al., 1984); this is physiologically achieved by the activation of postsynaptic AMPARs by synaptically-released glutamate. Activation of the N M D A R channel under physiological conditions allows for the conductance of cations, predominantly Na + and C a 2 + influx accompanied by K + efflux, with the Ca component of the current being -10-18% (Burnashev et al., 1995; Schneggenburger, 1996). Hence NMDARs are a major route for Ca influx. 20 As mentioned above, NR2 subunit expression is both developmentally and spatially regulated. In the mammalian brain, certain brain regions characteristically express particular NR2 subunits (Akazawa et al., 1994; Monyer et al., 1994). In the adult forebrain, the main NR2 subunits expressed are NR2A and NR2B, indicating that the majority of NMDARs in these regions are diheteromers composed of either NR1/NR2A or NR1/NR2B, or are in a triheteromeric configuration of NR1/NR2A/NR2B (Sheng et al., 1994; Li et al., 1998; Chapman et al , 2003). These subunit combinations produce NMDARs that are similar in certain channel properties such as permeability to Ca , single channel conductance, and sensitivity to voltage-dependent M g 2 + block. However, there are key differences between NR2A and NR2B subunits, both in terms of the functional properties they convey to channels, and how they are distributed on a subcellular level. NR2A and NR2B subunits have differential sensitivity to both agonists and antagonists (Buller et al , 1994; Vicini et al., 1998; Dingledine et al., 1999; Christie et al., 2000), and channel gating properties are altered in a subunit-dependent fashion (Monyer et al., 1994; Vicini et al , 1998). The NR2A receptor is generally expressed within synaptic NMDARs, and this subcellular expression pattern is considered to be a consequence of developmental regulation and synaptic maturation (Sheng et al., 1994; Li et al., 1998; Tovar and Westbrook, 1999; Chapman et al., 2003). In contrast, NR2B receptors appear to predominate at extrasynaptic sites (Li et al., 1998; Stocca and Vicini, 1998; Tovar and Wesbrook, 1999; Barria and Malinow, 2002) and the specificity of this spatial distinction between NR2A and NR2B expression patterns may reflect differential roles in determining cell survival and cell death (Hardingham et al., 2002; Hardingham and Bading, 2003; Liu et al., 2004; Massey et al , 2004). Interestingly the striatum 21 appears to express higher levels of NR2B relative to other NR2 subunits, compared to other regions of the brain; this pattern of expression is observed in several species, including man (Landwehrmeyer et al , 1995a; Rigby et al., 1996; Ghasemzadeh et al., 1996; Standaert et al., 1999; Kuppenbender et al., 1999; Li et al., 2003a). 1.2.3 The Basis for the Excitotoxicity Hypothesis in HD Critical evidence to support the idea of excitotoxicity being involved in HD pathogenesis came from studies of post-mortem HD brains, showing a loss of striatal NMDAR binding sites (Young et al., 1988; Albin et al., 1990a; Albin et al., 1990b). These observations even extended to brains from individuals who were pre-symptomatic, indicating that NMDAR-expressing MSNs were at particular risk and loss occurred very early in disease progression, possibly underlying further symptoms. These observations correlate with the selective loss of MSNs in HD patient brains (Graveland et al, 1985). A survey of transgenic HD mouse models also reveals changes in N M D A R composition and subunit expression. In the R6/2 mouse model, immunostaining of striatal NR2A and NR2B are both decreased in symptomatic mice, although NR1 immunostaining is enhanced (Cepeda et al., 2001b). Other studies in the same model showed that NR1 protein levels in brain homogenates were unchanged at 12 weeks, and striatal N M D A R binding had not changed (Cha et al., 1999). However, in another N-terminal fragment model (N171-82Q, see Section 1.1.4.2 and Table 1), no significant changes in NR1 or NR2B expression relative to controls were found (Jarabek et al., 2004). In the Y A C mouse models, striatal NR1 and NR2B expression were similar in WT, YAC46 and YAC72 mice prior to any onset of symptoms (2 mos; Li et al., 2003a), 22 whereas in the more aggressive Y A C 128 model, striatal atrophy and neuronal loss were highly correlated with motor dysfunction (Slow et al., 2003). Therefore, while there is an inconsistency between the human disease and transgenic mouse models in terms of early-stage loss of MSNs, it is likely that HD produces a decrease in the overall number of functional NMDARs within the striatum. This may reflect a deficit in neuronal function, which can precede neuronal death (Van Raamsdonk et al., 2005). Changes in NMDAR function will be addressed in a later section. 1.2.3.1 Evidence From Chemical Models of HD Support the Excitotoxic Hypothesis Several studies have demonstrated that intrastriatal injection of N M D A R agonists results in the selective loss of MSNs while sparing interneurons, reproducing many behavioural and neuropathological characteristics of HD (Schwarcz et al., 1984; Sanberg et al., 1989; Hantraye et al., 1990; Beal et al., 1986; Beal et al., 1991). Additionally, striatal MSNs show increased sensitivity to NMDA-induced swelling (a correlate of current and toxicity) compared with large-sized striatal interneurons, whereas kainate produced similar swelling in both neuronal populations (Cepeda et al., 2001a). Moreover, N M D A R agonists are more effective than other GluR agonists for inducing striatal neuronal excitotoxicity (DiFiglia, 1990). A different means of chemically inducing HD-like symptoms and neuropathology involves the use of inhibitors of mitochondrial complex II. Both malonate and 3-nitropropionic acid (3-NP) can be either injected intrastriatally, or interestingly enough, given systemically to rodents to produce selective degeneration of striatal MSNs (Beal et al., 1993b; Greene et al., 1993; Brouillet et al, 1995; Bogdanov et al., 1998), which can be blocked by N M D A R antagonists 23 (Greene et al., 1993; Bogdanov et al., 1998). While these models effectively replicate many aspects of advanced HD, they cannot be used to study early presymptomatic changes, which may be critical to understanding disease pathogenesis and dysfunction prior to neuronal death. However, these models do provide further evidence for the excitotoxic involvement of NMDARs in HD pathogenesis, and in the case of mitochondrial complex II inhibitors, implicate mitochondrial dysfunction as a predisposing factor in excitotoxicity in HD. 1.2.4 Alterations of NMDAR Function in HD models Given the likely involvement of NMDARs in HD pathology, whether causative or not, there has been a great deal of interest addressing the question of whether N M D A R function in HD models is altered, and if so, how? Studies in intact HD animal models, in vitro preparations derived from HD model animals, and heterologous systems have been performed to answer this question. The most direct method to look for potential HD-associated changes in N M D A R function would be observation of N M D A R function through electrophysiological means. A number of studies have demonstrated enhancement of N M D A R currents, in several different transgenic HD mouse models. 1.2.4.1 NMDAR Currents in HD Models Enhancement of N M D A R currents was observed in MSNs acutely dissociated from striata of both pre-symptomatic and symptomatic R6/2 mice (Starling et al., 2005). The same study also observed decreased sensitivity of N M D A R currents to M g 2 + block in 24 a subpopulation of MSNs which had significantly larger N M D A R currents, possibly reflecting an alteration of N M D A R subunit composition. This observation extended to MSNs from pre-symptomatic mice, indicating altered N M D A R function prior to any apparent onset of symptoms. NMDA-evoked currents in striatal slices taken from both pre-symptomatic and symptomatic R6/2 mice are increased over WT, whereas in the same mice AMPA-evoked currents are actually smaller, indicating a process selective for NMDARs (Cepeda et al., 2001b). Later studies in this same model demonstrated that this enhancement of N M D A R current is specific for the striatum, as cortical NMDAR-mediated currents were not enhanced (Andre et al., 2006). These enhanced striatal NMDAR currents also explain an earlier observation of enhanced swelling of MSNs in R6/2 striatal slices compared to slices from WT animals in response to exogenous N M D A application, an observation which was also confirmed with MSNs in slices from a knock-in HD mouse model with a polyQ length of 94 (Levine et al., 1999). Similar observations were also documented in another N-terminal fragment model of HD (with a polyQ length of 100 contained within the N-terminal third of the human htt gene), showing significant enhancement of N M D A R peak currents and current density in MSNs from striatal slices (Laforet et al., 2001). Our lab has reported similar changes in N M D A R function in the Y A C mouse models of HD. Acutely dissociated MSNs from 6-11 week old YAC72 animals had increased N M D A R peak current densities relative to those in MSNs from WT animals (Zeron et al., 2002). This age corresponds to the pre-symptomatic period in these animals, indicating that aberrant N M D A R activity may be present prior to, and possibly play a causative role in, any noticeable behavioural changes associated with HD. As 25 these data were obtained using exogenously applied N M D A and stimulating presumably extrasynaptic NMDARs, subsequent studies focused on possible changes in synaptic NMDAR properties produced by mhtt. Synaptic N M D A R currents recorded from MSNs in corticostriatal slices from YAC72 mice were also found to be enhanced compared to those recorded from WT mice, and this enhancement was found to reflect a postsynaptic NMDAR-selective mechanism (Li et al., 2003a; Li et al., 2004), suggesting that mhtt preferentially modulates N M D A R function. 1.2.4.2 The NR2B-Selective Hypothesis of Mhtt-Mediated Enhancement of NMDAR Function The first demonstration of an effect of mhtt on N M D A R function was in a study where NMDARs, composed of either NR1/NR2A or NR1/NR2B, were expressed in conjunction with full length human htt containing either 15 or 138 polyQ repeats (Chen et al., 1999b). The authors observed that mhtt enhanced the responses of NMDARs composed of NR1/NR2B, whereas NR1/NR2A NMDARs were not differentially affected by the presence of htt or mhtt (Chen et al., 1999b). These results were underscored by a later finding that mhtt selectively enhanced apoptotic cell death in H E K cells co-transfected with NR1/NR2B (Zeron et al., 2001), indicating a possible preferential modulation of NR2B-containing NMDARs by mhtt (the NR2B-selective hypothesis). The possibility that the NR2B subunit could be necessary for selective modulation by mhtt is not surprising, considering that the adult striatum is enriched in NR2B (as mentioned previously). In the same paper that demonstrated enhanced N M D A R currents in acutely dissociated MSNs from YAC72 mice, the authors found that greater than 50% 26 of the N M D A R current was mediated by NR1/NR2B NMDARs (Zeron et al., 2002). This work provided several other supporting lines of evidence for a specialized role for NR2B in mhtt-mediated changes in NMDAR function. In conjunction with enhanced NMDAR currents in YAC72 MSNs NMDA-induced apoptosis was enhanced in YAC72 vs. WT MSNs, but there was no differential effect observed in cerebellar granule neurons (CGNs), which did not express NR2B under the culture conditions used. The NR1/NR2B selective antagonist ifenprodil (IFN) effectively reduced apoptosis in both WT and YAC72 MSNs (Zeron et al., 2002), providing further evidence of the important role that this particular N M D A R subunit combination may play in mediating mhtt-enhanced excitotoxic cell death in MSNs. 1.2.4.3 Neurotoxicity as an Indicator of PolyQ Length-Dependent Alterations in NMDAR Function by Mhtt Building on the aforementioned studies in support of the NR2B-selective hypothesis (Chen et al., 1999b; Zeron et al., 2001; Zeron et al., 2002), the influence of the polyQ length in mhtt on neurotoxicity, and in particular apoptosis, was further examined. Cultured YAC72 MSNs were more susceptible to NMDA-induced toxicity than WT MSNs, although no difference was observed between the two genotypes in response to AMPA application (Zeron et al., 2002), supporting observations of selective enhancement of N M D A R currents in corticostriatal slices (Li et al., 2003a; Li et al., 2004) and correlating electrophysiological observations with neurotoxicity (Zeron et al., 2002). Additionally, it was found that the enhancement of apoptosis by mhtt in YAC46 and YAC72 MSNs was proportional to the length of the polyQ repeat (Zeron et al., 27 2002). This observation was further bolstered by a subsequent study demonstrating that rates of NMDA-induced apoptosis induction were maximal and equivalent in YAC72 and YAC128 MSNs when saturating concentrations of N M D A were used, with YAC128 MSNs showing enhanced sensitivity to apoptosis at subsaturating concentrations of N M D A (Shehadeh et al., 2006). An IFN-sensitive enhancement of glutamate-induced apoptosis in Y A C 128 relative to WT MSNs was also reported by another group (Tang et al.,2005). 1.2.5 Downstream Consequences of NMDAR Activation in Cells Expressing Mhtt A recent paper by our lab illustrated how polyQ length influences the induction of apoptosis in MSNs (from Y A C HD mice) in response to a number of noxious stimuli that increase intracellular C a 2 + , and found that Y A C 128 MSNs are generally more sensitive to most toxic stimuli; however, the most effective inducer of neuronal apoptosis relative to control Y A C 18 MSNs was N M D A R activation (Shehadeh et al., 2006). This finding illustrates how the presence of mhtt may preferentially affect the relationship between intense N M D A R stimulation and the resultant intracellular C a 2 + elevations in a manner that results in neuronal death. 1.2.5.1 Ca 2 + Homeostasis is Affected by Mhtt A number of damaging events can ensue from an excessive amount of free cytosolic Ca 2 + , which is a downstream consequence of N M D A R activation in MSNs in the presence of mhtt (Zeron et al., 2004; Tang et al., 2005; Shehadeh et al., 2006). These could include inappropriate enzyme activation (i.e. calpains, calcineurin, other Ca -28 regulated enzymes) and mitochondrial dysfunction (elaborated on below) which in turn has a number of toxic consequences. There is evidence from several HD models that the regulation of intracellular Ca is altered in several ways, although not always predictably. For example, resting Ca levels are elevated in MSNs from R6/2 mice (Hansson et al., 2001), hippocampal neurons in YAC72 mice and an immortalized cell line derived from striatal neurons from a knock-in HD mouse model (Seong et al., 2005). On the other hand, in studies using primary MSN cultures from Y A C mice resting Ca levels were equivalent between WT, YAC46 and YAC72 MSNs (Zeron et al , 2004) and WT, Y A C 18 and Y A C 128 MSNs (Tang et al., 2005). The former study found that stimulation of NMDARs in mhtt-expressing MSNs resulted in elevated C a 2 + levels relative to controls. Additionally, the latter study by Tang et al., (2005) in Y A C 128 MSNs found increased cytosolic C a 2 + levels in Y A C 128 MSNs following glutamate 2+ stimulation that were attributed in part to enhanced IP3 receptor-mediated Ca release from the ER, downstream of mGluRl/5 activation, as mhtt was found to sensitize the IP3 receptor (IP3R) (Tang et al., 2003). Hence not only does mhtt appear to enhance NMDAR activity, and subsequently C a 2 + influx via NMDARs (Zeron et al., 2004), but it may also enhance the probability of intracellular C a 2 + release. Similar findings of mhtt-enhanced NMDAR-mediated C a 2 + entry in MSNs have been found in N-terminal fragment HD models, including the R6/2 (Cepeda et al., 2001b) and tgHDIOO mice (Laforet et al., 2001). Importantly, these findings of altered C a 2 + regulation in Y A C HD mice have been observed in MSNs obtained from early postnatal mice (Zeron et al., 2004; Tang et al., 2005), implying that these changes are present at birth and may over time increase the risk of neuronal dysfunction and death. 29 Shehadeh et al., (2006) compared the toxic effects of Ca influx via both NMDAR and non-NMDAR routes, and the findings raised two particularly important points to consider: 1) the route of C a 2 + entry could play a significant role in determining the extent of toxicity; 2) The level of toxicity produced by N M D A R activation in the presence of mhtt may be proportional to cytosolic C a 2 + elevations. Addressing the first point, previous work suggests that mitochondria preferentially buffer Ca entering via NMDARs, the so-called privileged access hypothesis (Peng and Greenamyre, 1998; Sattler et al., 1998). As N M D A R activation and consequent Ca entry are increased in MSNs expressing mhtt, the possible consequences of this preferential pathway include enhanced mitochondrial stress, free radical generation, loss of adenosine 5'-triphosphate (ATP)-generating capability, and generalized mitochondrial dysfunction, which could result in the eventual activation of the intrinsic apoptotic pathway. The finding that N M D A application was a more potent inducer of apoptosis than other means to raise intracellular C a 2 + , such as treatment with C a 2 + ionophores or membrane depolarization, is consistent with this hypothesis. As to the second point, evidence from our lab has shown that N M D A R currents (Zeron et al., 2002; Zeron et al., 2004) and C a 2 + influx downstream of N M D A R activation (Zeron et al , 2004) are both enhanced in YAC72 compared to WT MSNs, correlating with enhanced apoptosis levels in YAC72 MSNs compared to controls (Zeron et al., 2002; Shehadeh et al., 2006). This difference in apoptosis was abolished by using the competitive N M D A R antagonist 2-amino-5-phosphonovalerate (APV) to reduce NMDAR-mediated C a 2 + influx in YAC72 MSNs down to a level equivalent to that seen in WT MSNs. This indicated that the enhanced level of neurotoxicity produced by 30 N M D A R activation in the presence of mhtt is due to the augmentation of N M D A R function by mhtt in Y A C 7 2 M S N s (Shehadeh et al., 2006). A relationship where increased cytosolic C a 2 + corresponded to increased neuronal death following stimulation with glutamate was also found with Y A C 128 MSNs (Tang et al., 2005). 1.2.6 Mitochondrial and Bioenergetic Impairment in HD Given the influence that mhtt exerts on both N M D A R function and subsequent C a 2 + entry and regulation, it follows that by virtue of these changes mitochondrial responses will also be affected. While there is direct evidence of this, there is also evidence that mhtt may independently impair mitochondrial function via different mechanisms. Mitochondria have an electrochemical gradient across their inner membrane established by the active extrusion of H + ions, in order to couple re-entry of protons to oxidative phosphorylation in the generation of A T P . Mitochondria may also utilize this 9 + gradient to drive the uptake of Ca from the cytosol in situations where local concentrations exceed ~ 1 u M (Nicholls and Ward, 2000). During this process the mitochondrial membrane potential (ATm) is dissipated; under normal conditions it is re-established through continued activity of active H + transport, but excessive mitochondrial depolarization is associated with increased risk of apoptotic neuronal death (Schinder et al., 1996). N M D A R activation in Y A C 4 6 and Y A C 7 2 M S N s has been shown to produce enhanced mitochondrial depolarization, associated with increased cytosolic C a levels and apoptosis as noted earlier (Zeron et al., 2004; Shehadeh et al., 2006). Compounding this is the observation that mitochondria isolated from H D patients, as well as from 31 YAC72 mice, tend to have a reduced resting A^Fm and depolarize to a greater extent when stressed (Sawa et al., 1999; Panov et al., 2002), and that expression of mhtt may reduce the ability of mitochondria to re-establish baseline A^Fm (Oliveira et al., 2006). These observations demonstrate that mhtt-mediated changes in N M D A R function can have negative consequences to neuronal health beyond altered activity of the receptor itself. Ca cycling between the mitochondria and cytosol occurs normally as a consequence of physiological activity, such as during synaptic activity. However, under conditions where excessive concentrations of C a 2 + are achieved, such as during an excitotoxic stimulus, mitochondrial C a 2 + uptake leads to pathological activation of a conductance known as the mitochondrial permeability transition (mPT; White and Reynolds, 1996; Crompton, 1999; Dubinsky and Levi, 1998), which is associated with apoptotic neuronal death processes ((Marchetti et al., 1996; Nicholls and Budd, 1998; Brustovetsky et al., 2002). The mPT is formed by the association of several mitochondrial membrane proteins, including the voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and cyclophilin D to create a pore with a conductance of-1.5 kDa, allowing the movement of ions and small proteins out of the mitochondria (Bernardi et al., 1994; Petit et al., 1996; Green and Reed, 1998). Activation of the mPT short-circuits A^Fm, preventing ATP generation and allowing the release of C a 2 + and apoptotic factors into the cytosol (Green and Reed, 1998; Crompton, 1999; Duchen, 2004; Orrenius, 2004). NMDA-induced mPT activity has been previously observed in murine MSNs (Alano et al., 2002), and inhibitors of mPT formation have been shown to prevent NMDA-induced apoptosis in Y A C HD MSN culture preparations including YAC46 and Y A C 128 (Zeron et al., 2004; Tang et al., 2005). The release of 32 cytochrome c from mitochondria has been shown to occur following N M D A R stimulation in primary MSN cultures from YAG128 mice (Tang et al., 2005), indicating induction of the mPT and intrinsic apoptotic pathway. A number of observations, from alterations in energy status indicators to direct physical interactions, in both human HD patients and HD models, have indicated that mhtt may directly impair mitochondrial function. Reduced levels of cAMP have been reported in the cerebrospinal fluid, brain tissues and lymphoblasts of HD patients (Cramer et al., 1984; Gines et al., 2003), and striata of HD knock-in mice (Wheeler et al., 2000) while lactate levels seem to be increased (Jenkins et al., 1993; Koroshetz et al., 1997), possibly indicating a shift to greater reliance on glycolytic ATP generation. Consistent with this idea are observations of reduced cAMP generation and decreased ATP:ADP (Gines et al., 2003; Seong et al., 2005) accompanied by reduced mitochondrial ATP levels (Seong et al., 2005) in a cell line (STHdfP^) derived from MSNs from a knock-in HD mouse model. Other studies of human HD brain tissue have noted decreased enzyme activity in a number of electron transport chain components, including complexes II, III and IV (Brennan et al., 1985; Beal, 1995; Gu et al., 1996; Browne et al , 1997) as well as other enzymes (i.e. aconitase) involved in mitochondrial ATP generation (Tabrizi et al., 1999). Studies on mitochondria isolated from both human and Y A C HD mice are more depolarized at rest, and are more sensitive to imposed C a 2 + loads, which may be due to direct interactions between mhtt (or polyOJ-containing protein fragments and mitochondria (Panov et al , 2002; Choo et al., 2004). Other studies which have focused on mitochondrial function, by the use of either mitochondrial toxins (Sawa et al., 1999; Shehadeh et al., 2006) or conditions where mitochondria are solely relied upon for 33 energy production (Oliveira et al., 2006), indicate that mitochondrial function is impaired by expression of mhtt. Indeed, defects in mitochondrial complex II have been noted in HD patient brains and MSNs expressing HttN171-82Q (Benchoua et al., 2006). Lastly, the aforementioned studies using systemic inhibitors of mitochondrial complex II to mimic HD pathology in animal models (Beal et al., 1993a; Brouillet et al., 1993; Palfi et al., 1996) broadly illustrate that mitochondrial dysfunction, either alone or in conjunction with upstream alterations in N M D A R function and C a 2 + handling changes, may impact neuronal survival in the context of HD. 1.3 Research Hypotheses and Goals The results of this thesis project have been divided into three chapters, each representative of a different phase of the project, correspondingly driven by changing hypotheses along the way. Our initial goal was to characterize the electrophysiological properties of NMDARs in primary cultures of MSNs from Y A C HD mice, with several hypotheses inherent in the objectives, as described in Chapter 3:1) We wanted to correlate N M D A R current amplitudes within primary M S N cultures to the observed NMDA-induced apoptosis, and hypothesized that the amplitude of N M D A R currents would correlate with increasing polyQ lengths in the Y A C HD mice, thus explaining enhanced neurotoxicity with increasing polyQ length. Characterization of N M D A R current in the Y A C HD mouse model had not been done in cultured primary MSNs, which was the preparation used for many of the toxicity studies (Zeron et al., 2002; Graham et al., 2006b; Shehadeh et al., 2006). We started with WT, Y A C 18, YAC46 and YAC72 mice (Hodgson et al., 34 1999), with the later addition of Y A C 128 mice as that model became available (Slow et al , 2003) for testing. 2) We hypothesized that any enhancement of N M D A R activity that could be correlated to increased apoptosis would be selective for NMDARs and not other glutamate receptors, and that there would be neuronal-type specificity to explain selective striatal loss in HD. 3) Given the possibility of an NR2B-related component to augmentation of N M D A R function by mhtt, we hypothesized that mhtt might alter the subunit composition of NMDARs in a striatal-specific fashion. 4) Having examined the relationship between polyQ length and NMDAR current density in the various Y A C HD mouse lines and finding a significant difference, we then attempted to address the underlying mechanisms for this observation. Given the findings regarding the characterization of N M D A R properties in MSNs from HD mice described in Chapter 3, our overall objective for the experiments outlined in Chapters 4 and 5 was to explain the enhanced sensitivity to NMDAR-mediated apoptosis in. Y A C 128 MSNs in the absence of enhanced N M D A R function. Using fluorescent imaging techniques, the hypotheses investigated were as follows: 1) Ca 2_|_ homeostasis was altered in Y A C 128 MSNs, and our hypothesis was that resting Ca 94- • levels or responses to NMDAR-mediated Ca entry would be augmented in a toxic 2+ fashion. 2) Although we did not find any genotypic differences in the magnitude of Ca responses, we did observe a difference in recovery rate from an intracellular C a 2 + load. We hypothesized that Y A C 128 MSNs could have a generalized defect in C a 2 + handling, and designed experiments to address this possibility. 3) Having observed mitochondrial 2+ membrane potential changes downstream of N M D A R and cytosolic Ca changes in YAC46 and YAC72 MSNs (Zeron et al., 2004), we hypothesized that a similar event in 35 Y A C 128 MSNs could perhaps explain their enhanced rates of NMDAR-mediated apoptosis. To address this question, we simultaneously monitored changes in cytosolic C a 2 + and A^m, and attempted to define the relationship between the two parameters. This thesis describes a transition of hypotheses that were initially focused on describing broad trends to provide a unifying theory describing neurotoxicity in Y A C HD mouse models. Subsequently my research became focused on neurotoxic events within one particular HD mouse model, the Y A C 128 model, which exemplified enhanced neuronal apoptosis via a mechanism contrary to our initial hypotheses. 36 Chapter 2 Materials and Methods 2.1 Animals Primary striatal neuronal cultures were prepared from offspring of breedings between two homozygous yeast artificial chromosome (YAC) transgenic mice on a pure FVB/N strain background (Hodgson et al , 1999; Slow et al, 2003). YAC46 (line 668), YAC72 (line 2511) and YAC 128 (line 55) mice expressed full length human mhtt containing 46, 72 or 128 glutamine repeats. These mice were compared to both'FVB/N WT mice and in some experiments compared against YAC 18 (212 line) mice, which express full length human htt but contain a non-pathogenic number (18) of glutamine repeats. All mice were bred and maintained according to Canadian Council on Animal Care (CCAC) and UBC Animal Care guidelines. 2.2 Primary Neuronal Cultures 2.2.1 Preparation of Primary Striatal Neurons Primary MSN cultures were prepared as previously described (Zeron et al., 2004; Shehadeh et al., 2006). Briefly, sterile poly-D-lysine (low molecular weight, MW 30-70K, 50 ug/ml) coated glass coverslips (12 mm; Paul Marienfeld GmbH & Co. KG, Lauda-Kdnigshofen, Germany) were placed in 24-well, plates, and allowed to dry prior to culturing. Striata were dissected from postnatal day 0-1 (P0-P1) mice in Hank's Balanced Salt Solution (HBSS) on ice and then digested in warmed (37°C) papain solution (HBSS, 37 40 U/ml papain) for ten minutes. Cells were pelleted by centrifugation, then further dissociated using a series of reducing bore-size Pasteur pipettes in warmed trypsin inhibitor solution (containing Neurobasal media, 0.25% bovine serum albumin, 0.25% trypsin inhibitor, 20 pg/ml DNAse) and subsequently transferred to warmed serum-free plating media containing Neurobasal medium with 2% B27 without supplements, 2 mM L-glutamine, and 1% penicillin/streptomycin. Neurons were plated at a density of approximately 5.0 x 105 cells/well (1.0 x 106 cells/ml), and incubated (37°C, 5% CO2) for 9-12 days. In order to maintain the cells, every 4-5 days half of the medium was replaced with fresh medium. Approximately 80-90%> of cells cultured in this serum-free medium have the characteristics of medium-sized spiny neurons (MSNs), as evidenced by morphology, DARPP-32 staining, and/or staining for the GABAergic neuronal marker glutamic acid decarboxylase-65 (Hansson et al., 1999; Kovacs et al., 2001; Shehadeh et al., 2006). In all experiments measurements were taken only from cells with morphological features consistent with MSNs, including an ovoid soma with a short diameter of approximately 10 um and 2-4 short projecting processes (Shi and Rayport, 1994). 2.2.2 Preparation of Primary Cortical Neurons Primary cortical neuronal cultures were occasionally prepared in parallel from the same animals used for MSN cultures, and in largely similar fashion with a few differences. Cells obtained from cortical tissue were plated in Neurobasal medium with a composition identical to that used for MSN cultures, except for a lower concentration of L-glutamine (0.5 mM) used in the plating medium. Cortical neurons were plated at a 38 density of 3.0 x 105 cells/well (5.0 x 105 cells/ml), and incubated (37°C, 5% C0 2 ) for 4-21 days. In order to maintain the cells, every 4-5 days half of the medium was replaced with fresh medium. In all experiments measurements were taken only from cells with morphological features consistent with cortical pyramidal neurons (CPNs), including a general pyramidal (triangular) somatic structure with prominent neurites (de Lima et al., 1997). 2.2.3 Preparation of Primary Cerebellar Granule Neurons Primary cultures of cerebellar granule neurons (CGNs) were prepared to provide a control for the selectivity of IFN for NRl/NR2B-containing NMDARs. CGNs prepared in the manner described here express NMDARs that lack NR2B by 9 d.i.v. (Vallano et al., 1996), and thus N M D A R currents in these cells should not be sensitive to IFN antagonism (see Results, Figure 7B). Coverslips (12 mm) were prepared as described for MSN cultures. Cerebella were dissected from P7-P9 mice in HBSS on ice, and then digested for 15 min in a warmed (37°C) papain solution (HBSS, 60 U/ml papain). Cells were pelleted and resuspended in trypsin inhibitor solution (same composition as that used for MSN cultures), and passed through either polished Pasteur pipettes or a 5 ml plastic culture pipette with a standard yellow 100 ul plastic tip on the end. After several passes, the cell suspension was placed on ice for 2-3 minutes, during which time clumps of non-dissociated tissue settled to the bottom. The supernatant (containing single cells) was carefully removed with a pipette, DNAse (final concentration 20 u.g/ml) was added, and the suspension was centrifuged to produce a pellet. After the cells were resuspended in ice-cold HBSS they were centrifuged at 4°C for 15 min @ 2000 RPM through 40.5% 39 Percoll. The resulting cell pellet was resuspended in cerebellar plating media (Basal Medium Eagle with Earle's salts from Invitrogen, containing 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, 2 mM L-glutamine, 25 mM KC1, 17 mM D -glucose), re-spun for 1 min, and the resulting pellet resuspended in warmed (37°C) cerebellar plating medium. CGNs were plated at a density of 5.0 x 105 cells/well (1.0 x 106 cells/ml), and incubated (37°C, 5% C0 2 ) for 8-10 days. 2.3 Electrophysiological Experiments All MSNs were used for electrophysiological analysis at 9-11 days in vitro (d.i.v.); CPNs were used at 4-7 d.i.v., whereas CGNs were recorded from between 8-10 d.i.v. Coverslips with cultured neurons were placed in the recording chamber on the stage of an inverted microscope (Axiovert 100, Carl Zeiss, Thornburg, NY). Intracellular recording solution contained: 115 mM CsMeS0 3 , 10 mM HEPES, 10 mM BAPTA, 4 mM Mg-ATP, 20 mM K2-creatine phosphate, and 50 U/ml creatine phosphokinase, titrated to pH 7.25 using CsOH and with an osmolarity of 309-310 mosm. Extracellular recording solution contained: 167 mM NaCl, 2.4 mM KC1, 1.8 mM CaCl 2 , 10 mM HEPES, 10 mM D-(+) glucose, titrated to pH 7.3 using NaOH and with an osmolarity of 325 mosm. Both intracellular and extracellular solutions were filtered prior to use. In all experiments, 50 uM glycine and 0.3 pM tetrodotoxin (TTX; to block voltage-gated sodium currents) were added to all extracellular solutions. Superfusion of neurons with different agonist solutions was achieved using a gravity-fed solenoid system attached to a theta tube. Currents were sampled at 5 kHz and acquired and analyzed using pCLAMP 40 software and an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Current amplitude measurement and kinetics fittings were performed using Clampfit software. 2.3.1 Measurement of Current Density, Steady-State to Peak Ratio, Ifenprodil Sensitivity Primary neuronal cultures were prepared as described, and whole-cell currents were recorded under voltage clamp (VH = -60 mV; except where otherwise noted). All currents were leak-subtracted prior to analysis. N M D A R currents were elicited by a 4s application of 1 mM NMDA. This was repeated twice at 60s intervals, for a total of 3 separate trials. Peak current density was calculated by dividing the peak current amplitude by the cell capacitance, thus normalizing current to cell surface area (allowing comparisons between cells with potentially different membrane surface areas). NMDAR steady-state to peak current ratios (Iss/IPeak) were calculated by averaging N M D A R currents over the last 200 ms prior to cessation of N M D A application (to get a measure of steady-state current) and then dividing by the peak current amplitude and expressing the ratio as a percentage. This was done for each cell using the first and second trials (to minimize any effect of rundown), so that the Iss/Ipeak expressed for each cell is an average of two responses. The N M D A stimulation paradigm was then repeated in the presence of 3 uM ifenprodil (IFN), for a total of 3 trials. The percentage of current inhibited by IFN was calculated by dividing the average peak NMDAR current of the last two responses in the presence of IFN by the peak N M D A R current prior to IFN application. IFN was washed 41 out, and the stimulation paradigm repeated to monitor recovery of N M D A R current from IFN block. 2.3.2 Kainate Stimulation In some experiments, 1 mM kainate was used instead of 1 mM N M D A to assess possible changes in AMPA/kainate receptor current density. Kainate was used because it activates AMPARs in a non-desensitizing fashion (Hollmann and Heinemann, 1994), making it easier to accurately measure peak current. Measurements of IFN sensitivity or Iss/Ipeak were not carried out in these experiments. 2.3.3 Electrophysiological Measurement of NMDAR Insertion to the Plasma Membrane Whole-cell patch clamp recordings were performed under voltage clamp (VH = -80 mV, to minimize any possibility of MK-801 dissociation) on cultured striatal MSNs as described above. NMDAR-mediated current was evoked by bath application of saturating (1 mM) concentrations of NMDA. To irreversibly block all surface NMDARs, 5 uM MK-801 (a use-dependent, open-channel blocker) was co-applied with 1 mM N M D A for 9 s immediately following a 1 s application of N M D A alone. During this period whole-cell N M D A current could be observed to decay to pre-NMDA application levels (see Results, Figure 10A). Thirty seconds following initial blockade, this protocol was repeated to ensure complete block of all surface N M D A receptors. After extensive washout of MK-801, subsequent recovery of NMDAR-mediated current was measured for as long as possible at 5 min intervals by stimulating three times with a 500 ms 42 application of 1 mM NMDA, 30 seconds apart. The three responses at each 5 min interval were recorded and averaged, and expressed as an average Area Under the Curve (AUC) for the NMDA-evoked current, normalized to cell capacitance (pF) to obtain a measure of integrated current density, which over time could be interpreted as integrated current recovery (or density of NMDARs) per unit of cell membrane. The integrated NMDA-evoked current over 500 ms was used in order to circumvent the effect of noise and difficulty in accurately discriminating peak amplitudes of these relatively small whole-cell currents. 2.4 Visualization of Relative Changes in Cytosolic Free C a 2 + and Mitochondrial Membrane Potential (A^ Fm) Measurements were taken at room temperature (20-21°C) from primary cultures of age-matched WT or Y A C 128 MSNs between 9 and 11 d.i.v. Neurons were loaded with the acetoxymethyl (AM) esters of Fura-2, Fura-FF or MagFura-2 by incubation at 32°C for 45 min in 6.7 pM of indicator dye in balanced salt solution (BSS, containing 139 mM NaCl, 3.5 mM KC1, 2 mM NaHC0 3 , 10 mM HEPES, 3 mM Na 2 HP0 4 -7H 2 0, 1.8 mM CaCl 2 , 11 mM D-glucose, 50 uM glycine, pH adjusted to 7.35) containing 0.05% bovine serum albumin. The coverslips were then transferred to BSS alone for 15 min before each experiment to ensure hydrolysis of the fluorophore. Experiments were performed using a Zeiss Attofluor digital fluorescence imaging system as previously described (Abdel-Hamid and Baimbridge, 1997). Neurons were superfused continuously with BSS at 2.4 ml/min. Regions of interest (ROIs) were sized and placed over the soma of cells that morphologically resembled MSNs. Cells were stimulated with varying concentrations.(100 or 500 uM) of 43 N M D A or 50 mM KC1 + 5 uM FPL 64176 (FPL), with 50 uM glycine in all experiments; other compounds were added where indicated. Exposure of cells to 50 mM KC1 was achieved by replacing BSS (during the stimulation period) with a high KC1 solution containing: 70 mM NaCl, 50 mM KC1, 2 mM NaHC0 3 , 10 mM HEPES, 3 mM Na 2 HP0 4 -7H 2 0, 1.8 mM CaCl 2 , 11 mM D-glucose, 50 uM glycine, pH adjusted to 7.35. Cells that morphologically resembled MSNs and responded to N M D A or KC1/FPL application with an increase in cytosolic C a 2 + were used in the analysis. Data conversion calculations and analysis were performed using Microsoft Excel macros created in Microsoft Visual Basic by Dr. Gordon Rintoul. For estimation of relative changes in cytosolic Ca , the mean peak F 3 3 4 / F 3 8 0 response for each cell was calculated by averaging over a 30s period around the maximal peak response during application of N M D A or KC1/FPL, after subtraction of mean baseline F 3 3 4 / F 3 8 0 for a 30s period before stimulus application. The resulting values from each cell were then used to calculate an overall mean value for that experiment; mean and standard error for each treatment group were calculated from the means of each experiment for that treatment group (i.e., n is the number of experiments). Estimates of cytosolic calcium recovery were made by expressing the mean F334 /F380 value every 5 min following N M D A or KC1/FPL washout as a percentage of the maximal peak F334 /F380 response. Changes in AT™ were assessed by single wavelength imaging of rhodamine-123 (rhod-123) fluorescence. This dye is preferentially sequestered in mitochondria (on the basis of their negative transmembrane potential relative to the cytosol) where its fluorescence is quenched due to dye stacking. A loss of A T m results in the redistribution of rhod-123 back into the cytosol, producing an increase in overall cell fluorescence. The 44 absolute magnitude, of the fluorescence change is dependent on several factors (including variability in dye-loading and de-esterification times, and changes in the intensity of the UV light source over time) that vary from experiment to experiment, not the least of which are the health of the cells and consequent mitochondrial polarization that day, and which other dyes, if any, are present. Hence the maximum fluorescence measured using rhod-123 should ideally be compared in experiments performed on the same day under the same conditions. Importantly, maximum mitochondrial depolarization is only achieved using protonophores or other measures to directly dissipate A^m. When other stimuli are used that result in loss of A'Fm, unless they are compared within the same experiment to a stimulus that produces maximum depolarization, it is not possible to accurately assess the relative mitochondrial depolarization achieved using different stimuli between different experiments. This caveat can also be extended to data sets from experiments performed on different days; hence, genotoype-paired experiments were performed wherever possible (the notable exception being those experiments where mPT inhibitors were used, as paired cultures were not reliably available). In some experiments the initial loading of C a 2 + indicator (either Fura-2 or Fura-FF) was followed immediately by an addition of 26.3 mM rhod-123 to the loading buffer to achieve a final concentration of 5.0 uM rhod-123. Fifteen minutes after the addition of rhod-123, coverslips were transferred to BSS alone, at room temperature. Dual imaging experiments were then performed where both cytosolic Ca and A ^ n , were monitored simultaneously under the same conditions and in the same age-matched and paired (except for experiments using mPT inhibitors) WT and Y A C 128 MSNs. The same chamber and imaging system were used as described above, and the same stimulation and 45 measurement paradigm with the addition of an excitation (488 nm) and emission (>510 nm) cycle for rhod-123. Data for A ^ were expressed as the background-corrected change in fluorescence compared with baseline (AF/Fo). The mean peak AF/Fn during NMDA application (calculated by averaging over a 30 s period around the peak response) was calculated from measurements for all responding neurons during stimulus (NMDA or KC1/FPL) application in each experiment. The resulting means from each experiment were then used to calculate an overall mean and standard error (based on n's reflecting number of experiments) for each treatment group. 2.4.1 Determination of Resting A ^ m To get an approximate measurement of resting A^Fm in intact MSNs, MSNs were loaded with rhod-123 and visualized as described above. In some experiments, MSNs were co-loaded with Fura-FF for two reasons: 1) to help visualize neuronal soma, and 2) to allow for monitoring of C a 2 + release from mitochondria. The mitochondrial protonophore CCCP (5 uM) was applied, resulting in maximal depolarization of A^m and release of accumulated rhod-123 (Toescu and Verkhratsky, 2000). The relative fold-increase in rhod-123 fluorescence following application of CCCP provides a rough indication of the degree of depolarization achieved, and in doing so allows indirect measurement of the relative resting A^m prior to depolarization. 46 2.4.2 Calculation of CCCP/NMDA (C/N) Ratio to Determine Efficiency of Mitochondrial Ca 2 + Uptake For some experiments, mitochondrial Ca content was measured following N M D A application by depolarizing mitochondria using 5 pM CCCP, causing mitochondria to release accumulated free C a 2 + into the cytosol. In order to isolate this source of C a 2 + influx to the cytosol (and exclude C a 2 + entry from the extracellular space), neurons were superfused with a Ca2+-free BSS (same composition as normal BSS above, except 1.8 mM CaC^ is replaced by 0.9 mM MgC^) for 2 min prior to the addition of CCCP, during the washout of NMDA. We used previously established methods to determine the efficiency of mitochondrial C a 2 + uptake (Brocard et al., 2001). The area under the curve (AUC) of C a 2 + responses (during stimulus application) was measured, in serial fashion, in response to a 1-min exposure to 500 pM N M D A (N) followed 2 min later by a 1-min application of CCCP (C; to release free Ca accumulated by mitochondria). The regions where A U C measurements were performed were limited to periods during N M D A or CCCP application, i.e. a 60 s region in each case. The C/N ratio was then calculated. 2.4.3 Quantifying the Temporal Relationship Between Increases in Ca 2 + and Loss of To calculate the lag time between initial changes in cytosolic C a 2 + and A T m , 2*1* experiments using the dye pair of Fura-2 (due to high sensitivity at low [Ca ]) and rhodamine-123 were analyzed. The time bases for both variables were aligned and the initiation of fluorescence changes for each variable were recorded. The start time for a 47 fluorescence change was defined as the initial data point in a series of at least five data points that successively increased in a positive direction, following N M D A application. Increases in Ca were observed to always be initiated prior to changes in A^m. 2.5 Data Analysis All data are presented as means ± standard error of the mean (SEM), unless otherwise noted. T-tests, one-way, two-way and Repeated Measure ANOVAs followed by Bonferroni post-test were performed using GraphPad Prizm statistical analysis software. For imaging experiments, ANOVAS were used where experiments using WT and, Y A C 128 MSNs could be age-matched and performed on the same days (the majority of experiments), allowing for variability in dye-loading and de-esterification times, and changes in the intensity of the UV light source over time, to be accounted for equally in all genotype and treatment groups within and across culture batches. Where experiments for both genotypes could not be performed on the same day (those using mPT inhibitors), unpaired t-tests were used to analyze data within genotype only, in order to assess the effects of treatment. All C a 2 + imaging data were baseline-subtracted prior to analysis. For statistical purposes, the number of experiments (n) was considered to be the number of cells tested for electrophysiological experiments, and the number of individual coverslips used for imaging experiments. For all data sets, cells from at least 3 different litters of pups (culture batches) were used, so n is theoretically representative of results from a number of different mice. The number of experiments carried out was not dependent on achieving a given result; in fact, most significant differences found were 48 apparent with low n's. Rather, the numbers of experiments performed were largely determined by the birth rate of pups of the relevant genotypes, using the minimum standard of the results of at least 3 separate litters per genotype being incorporated into the overall data set. In certain data sets, for example as represented in Figures 8 and 9, this approach limited the size of n as these data were obtained towards the end of experimental protocols where cells died prematurely. Hence, in these cases, it may be desirable for n to be increased. 2.6 Materials Acetoxymethyl esters of Fura-2, Fura-FF, and MagFura-2, as well as rhodamine-123 were purchased from Invitrogen Canada Inc. (Burlington, ON). T T X was purchased from Alomone labs (Israel). Neurobasal medium, B27 with no added supplements, HBSS, and penicillin/streptomycin were purchased from Invitrogen Canada Inc. (Burlington, ON). All other chemicals were purchased from Sigma Aldrich Canada (Oakville, ON). 49 Chapter 3 NMDAR Function in the YAC Mouse Models of HD Our laboratory previously reported that co-expression of NR1/NR2B-containing NMDARs with mhtt (httl38, 138 polyQ) in HEK293 cells led to an enhancement of NMDAR-mediated current density (Chen et al., 1999b). Quantification of NMDA-induced apoptosis in primary MSN cultures from WT, YAC46 and YAC72 MSNs showed that the presence of full-length mhtt significantly increased NMDAR-mediated apoptosis in a polyQ length-dependent fashion (Zeron et al., 2002). In this paper the authors noted that YAC72 MSNS acutely dissociated from the striata of 6-11 week old YAC72 mice had enhanced N M D A R peak current densities, a phenomenon similar to that observed in the studies using transfected HEK cells as noted above. Since these results were obtained in two distinct neuronal preparations, our first question here was to determine whether the same enhancement of N M D A R current occurred in the primary MSN culture preparation, and whether these changes were specific to N M D A R activity or a symptom of a broader alteration in neuronal function which also affected other ionotropic glutamate receptors. Secondly, we wondered whether any changes that were observed could be correlated to the length of the polyQ repeat region in htt. A parallel question would be whether any changes in N M D A R function seen would be specific to the population of neurons most at risk in HD, the MSN, or would different neuronal subtypes also be affected in a similar fashion? And finally, we set out to determine what mechanism(s) underlie any functional changes observed. 50 3.1 NMDAR Current Density in Neuronal Primary Cultures In order to investigate whether any relationship existed between htt polyQ length and NMDAR-mediated current, we established primary cultures of striatal MSNs from early postnatal mice on a FVB/N strain background (WT) or from Y A C transgenic mice expressing full-length human htt containing various polyQ lengths: YAC18 (18Q, line 212), YAC46 (46Q, line 668), YAC72 (72Q, line 2511), and YAC128 (128Q, line 55). The generation and characterization of these different Y A C transgenic mouse lines have been previously described (Hodgson et al., 1999; Slow et al, 2003; Leavitt et al., 2006). The first set of experiments sought to establish whether cultured early postnatal MSNs from Y A C mice expressing mhtt with various polyQ lengths showed" increased NMDA-evoked current. Figure 1 illustrates a typical MSN (in this case WT) whole-cell current evoked by the application of a saturating (1 mM) concentration of N M D A while under voltage clamp (V H = -60 mV). Note that upon application of N M D A the current rapidly reaches peak amplitude, and then proceeds to decay toward a steady-state plateau in the continued presence of agonist. The measurements of both peak current amplitude (Ipeak) and steady-state current (Iss) are demonstrated here. Comparing N M D A R peak current density (Ipeak normalized to the cell capacitance) across genotypes, we found that NMDAR peak current density generally increased with longer htt polyQ length, up to 72Q (YAC 18 vs. YAC72, **P<0.01; YAC46 vs. YAC72, *P<0.05; One-way A N O V A , Bonferroni post-test, Figure 2). Similar to previously reported results with acutely dissociated MSNs from ~2-month old mice (Zeron et al., 2002), YAC72 MSNs exhibited significantly increased N M D A R peak current density vs. WT MSNs (*P<0.05, One-way A N O V A , Bonferroni post-test, Figure 2). This enhancement of current was specific to 51 N M D A receptors, as activation of the other classes of non-NMDA ionotropic glutamate receptors by bath application of kainate (an agonist at both A M P A and kainate receptors) showed no significant difference in current density between WT and YAC72 MSNs (Figure 3). It should be noted that the mean cell capacitance was not significantly different across all genotypes of cultured MSNs studied (12.6 ± 1 . 8 pF, n=42 for WT; 15.5 ± 2 . 3 pF,n=13 for YAC18; 15.1 ± 1.2 pF,n=ll for YAC46; 12.1 ± 0.9 pF, n=16 for 72Q; and 13.2 ± 0.9 pF, n=20 cells for Y A C 128). Interestingly, N M D A R peak current density was not increased in Y A C 128 MSNs vs. WT (Figure 2), despite the fact that primary cultures of Y A C 128 MSNs exhibit enhanced sensitivity to NMDA-induced apoptosis (Shehadeh et al., 2006). This observation formed the basis for Chapters 4 and 5, which describe our attempts to resolve this paradox. To determine whether other neuronal populations expressing mhtt would also have enhanced N M D A R current, we assessed N M D A R current density in primary cultures of CPNs taken from the same mice used to prepare our MSN cultures. Since there were no significant differences in N M D A R current density between YAC46 and WT or Y A C 18 MSNs, we chose not to pursue these recordings in YAC46 CPNs. As Figure 4 illustrates, we found similar NMDA-evoked peak current densities in cultured CPNs from WT, Y A C 18, YAC72 and Y A C 128 mice. This observation suggests that neither the overexpression of non-pathogenic htt nor (m)htt polyQ length serve to regulate N M D A R activity in CPNs. Hence, htt polyQ length-dependent potentiation of NMDAR current is neuronal-type specific and may contribute to enhanced excitotoxic 52 striatal vulnerability in the YAC72 mice as well as in human HD expressing htt with moderate repeat lengths. 3.2 NMDAR Desensitization The level of desensitization of NMDAR current could play a significant role in determining the total charge transfer and C a 2 + influx that occurs during N M D A R activation, possibly influencing the vulnerability of MSNs to excitotoxicity. To determine whether mhtt, in addition to increasing peak N M D A R current density, decreased desensitization (i.e. resulted in a higher I s s ) of NMDARs as an alternate mechanism to enhance excitotoxicity, we examined I s s/Ipeak ratios across genotypes in both MSNs (Figure 5) and CPNs (Figure 6). We found that desensitization of N M D A R currents recorded from Y A C transgenic MSNs was not significantly different regardless of mhtt polyQ length; however, NMDAR currents in Y A C 18 MSNs desensitized significantly less than those in WT MSNs (WT vs. Y A C 18, **P<0.01; One-way ANOVA, Bonferroni post-test). No significant differences in N M D A R desensitization . were found between genotypes in CPNs. 3.3 NRl/NR2B-Containing NMDARs NMDARs are composed of both NR1 and NR2 subunits, and it is the NR2 subunits that define N M D A R channel properties including peak open probability, single-channel conductance, and desensitization (reviewed in Cull-Candy et al., 2001). As well, since mhtt significantly enhanced N M D A R currents and NMDA-induced apoptosis in NRl/NR2B-expressing H E K cells (but not in HEK cells expressing NR1/NR2A), we 53 hypothesized that an mhtt-influenced alteration of N M D A R subunit composition could play a role in enhanced excitotoxicity in YAC72 MSNs (Zeron et al., 2002). To probe N M D A R subunit composition, we conducted our whole-cell patch clamp recordings in both MSNs and CPNs in the absence and presence of 3 uM IFN, an NRl/NR2B-selective antagonist of NMDARs (Williams, 1993). As illustrated in Figure 7, IFN blocked a significant proportion of NMDAR current in MSNs (Figure 7A), but had no effect on N M D A R current in cultured CGNs (Figure 7B), which do not express NR2B under the culture conditions used (see Methods). As summarized in Figure 8, NRl/NR2B-containing NMDARs mediated the majority of current in MSNs, and this proportion was similar across genotypes. Similar observations were made regarding NRl/NR2B-containing NMDARs in CPNs (Figure 9), with no significant difference between genotypes. Together, these results indicate that mhtt likely does not alter the subunit composition of NMDARs expressed at the plasma membrane. It should be noted that the proportion of current mediated by NR2B-containing NMDARs was generally less in CPNs compared with MSNs of the same genotype, significantly so for both WT and YAC72 (**P< 0.01 for wt and *P<0.05 for YAC72 by unpaired t-test, percentage of IFN-sensitive current in CPNs vs. MSNs within genotypes). 3.4 Shift of NMDAR Expression in YAC72 MSNs to the Surface of the Plasma Membrane Although we found an increase in N M D A R current density in YAC72 MSNs, the overall level of protein expression of NR1 and NR2B subunits in striatal tissue of YAC72 54 mice is not increased above that in striatal tissue from WT mice (Li et al., 2003a). Therefore, it is unlikely that the increase in N M D A R current density in YAC72 MSNs is simply a function of greater amounts of N M D A R proteins. However, in other experiments we found that the expression pattern of NR1 and NR2B was shifted in YAC72 MSNs such that a greater proportion (approximately 50% increase in surface:internal NR1 expression, *P<0.01 by one-way A N O V A , Bonferroni post-test and approximately 30% increase in surface:internal NR2B expression, T O . 0 5 by one-way A N O V A , Bonferroni post-test) was expressed at the plasma membrane than in WT or Y A C 18 MSNs, while the total amount of NR1 expressed is similar across genotypes (Fan et al., 2007). Hence, a greater number of surface NMDARs could explain the enhanced current in YAC72 MSNs. One possible mechanism to explain the shift in N M D A R expression pattern towards the plasma membrane is that the rate of forward trafficking (towards the plasma membrane) of NMDARs is enhanced. To determine whether the insertion of NMDARs into the plasma membrane occurred at a faster rate in YAC72 MSNs vs. WT MSNs, I. utilized an electrophysiological approach similar to that described previously (Lan et al., 2001; Tovar and Westbrook, 2002; Mu et al., 2003; Lin et al , 2004), monitoring the recovery of NMDAR-mediated current following functional blockade of surface receptors by the use-dependent, irreversible N M D A R antagonist MK-801 (electrophysiological silencing). All surface NMDARs were first fully stimulated and then blocked by application of a saturating (1 mM) concentration N M D A , first alone and then in conjunction with MK-801 (see Methods, Figure 10A). Once 'silenced', surface NMDARs remain inactivated for the duration of the experiment as MK-801 binding to 55 the NMDAR channel is expected to be irreversible under the voltage-clamp conditions j used (Huettner and Bean, 1988). Therefore, any subsequent NMDA-evoked current reflects the insertion of new N M D A receptors (or ones that were not on the surface during'silencing') to the plasma membrane. Following N M D A R block by MK-801, NMDA-evoked current was monitored at 5 min intervals (as in Figure 10B), and quantified as described (see Methods). The recovery of N M D A R current was ~3-, 3-, and 2-fold higher in YAC72 compared with WT MSNs at 5, 10 and 15 min following MK-801 washout, respectively (Figure 11), a difference that was significant at the 10- and 15-minute time points (*P< 0.05, **p< 0.01 respectively by two-way A N O V A followed by Bonferroni post-test). This difference in the rate of N M D A R current recovery indicates a faster rate of insertion of NMDARs to the plasma membrane from membrane proximal intracellular pools in YAC72 MSNs. 3.5 Summary Taken together, these observations suggest that N M D A R current density is enhanced in early postnatal cultured MSNs expressing mhtt with a polyQ length up to a certain ceiling, beyond which other competing mechanisms may prevent the enhancement of N M D A R current. Since these cells are obtained from very early postnatal mice, this would suggest that mhtt-mediated mechanisms that result in the potentiation of N M D A R activity are present at birth, and are selectively expressed in MSNs (and not other neuronal types). Hence this functional alteration may serve as an ongoing source of neuronal stress, possibly underlying a cumulative insult, which renders MSNs vulnerable in HD. 56 Chapter 4 Aberrant Cytosolic Ca 2 + Handling in the YAC128 Mouse Model of HD Y A C 128 mice, carrying a more extreme C A G expansion than the other Y A C HD mouse models described earlier, show an accelerated phenotype and significant striatal neurodegeneration by 6-12 months (Slow et al., 2003). Here we wanted to examine the events downstream of N M D A R activation, specifically regulation of cytosolic Ca 2 + , preceding the previously reported (Graham et al., 2006b) enhanced NMDA-induced apoptosis in YAC128 compared with WT MSNs. As shown in Chapter 3, the N M D A R current density of MSNs from Y A C transgenic mice containing full-length human (m)htt generally correlates to the length of the polyQ repeat region in the human (m)htt transgene. However, this relationship is clearly not intact for polyQ lengths of 128, despite MSNs from this line of mice (YAC 128 line 55) showing enhanced NMDA-mediated excitotoxicity to the same maximal level as that seen in YAC72 MSNs (Shehadeh et al , 2006). Having previously documented events downstream of enhanced NMDAR activation in YAC72 MSNs which predictably lead to an apoptotic endpoint, namely enhanced peak cytosolic C a 2 + and mitochondrial membrane depolarization (Zeron et al., 2004), we followed the same line of investigation with YAC128 MSNs to resolve the discrepancy between normal NMDAR currents and enhanced N M D A -induced neurotoxicity. Therefore, the goal was to explain the apparent paradox: mhtt expression in Y A C 128 MSNs does not increase the N M D A R peak current density as it does in YAC72 MSNs or affect desensitization (and hence the integrated N M D A R current) or NMDAR 57 subunit composition, yet N M D A exposure induces apoptosis more efficiently in Y A C 128 than in WT MSNs (Graham et al., 2006b), and indeed produces the largest enhancement of apoptosis in Y A C 128 MSNs relative to Y A C 18 controls of any stimulus (Shehadeh et al, 2006). 4.1 Considerations Regarding NMDA Concentration Used for These Studies Given the results presented in Chapter 3, enhanced excitotoxicity in Y A C 128 MSNs in the absence of increased N M D A R current could result from impaired C a 2 + homeostasis and/or mitochondrial dysfunction. Therefore, I began to investigate these events by comparing cytosolic Ca changes as a predictable consequence of N M D A R activation. For several reasons we decided that 500 uM N M D A was an appropriate agonist concentration for maximally stimulating NMDARs in the majority of these experiments. The EC50 for sustained glutamate stimulation of recombinant NR1/NR2B NMDARs in H E K cells was found to be -20 pM (Chen etal., 2001a), and N M D A is a less efficacious N M D A R agonist with a higher EC50 than glutamate (Sather et al., 1992; Banke and Traynelis, 2003). Furthermore, in a series of experiments measuring cytosolic C a 2 + changes in cultured rat MSNs, Alano et al (2002) found that N M D A concentrations 94-on the order of 500 pM - 1 mM were required to induce maximal Ca responses. Lastly, our own previous results confirmed that 500 uM N M D A was sufficient to achieve maximal apoptosis induction in cultured MSNs (Zeron et al., 2004), while 100 uM NMDA was insufficient (Shehadeh et al., 2006). As the goal of these experiments was to examine changes in cytosolic C a 2 + (and also mitochondrial function, as described later in Chapter 5) that would explain enhanced apoptosis in Y A C 128 MSNs, we thought it most 58 appropriate to use an N M D A concentration proven to most efficiently induce such an endpoint. 4.2 Resting and NMDA-Induced Changes in Cytosolic Ca 2 + in YAC128 MSNs We initially hypothesized that Y A C 128 MSNs would have a higher resting C a 2 + level, which could possibly indicate a stress-induced change (Hansson et al., 2001) or pre-existing mitochondrial impairment (Xiong et al., 2002) leaving cells more vulnerable to further insult. Alternatively, an equivalent concentration of N M D A under the aforementioned conditions could produce a larger increase in cytosolic C a i T in Y A C 128 than seen in WT MSNs. To address this question, I performed fluorescence imaging of cytosolic free C a 2 + levels in MSNs using the high-affinity calcium dye Fura-2. An example of a typical experiment is shown in Figure 12, depicting how and when measurements for resting (basal) C a 2 + levels and peak cytosolic C a 2 + were performed. Representative mean responses (one experiment each for WT and Y A C 128) to a 5 min application of 500 uM N M D A are illustrated in Figure 13. Also shown in this experiment (Figure .12) is the application of the C a 2 + ionophore 4Br-A23187, to illustrate the maximal fluorescence signal in this experiment and demonstrate that our responses were not saturated by NMDA-induced Ca influx. / Contrary to our hypothesis, we found similar basal cytosolic Ca levels, as reflected in similar F 3 3 4 / F 3 8 0 ratios (P>0.05 by paired t-test) for WT and Y A C 128 MSNs 2"i" (Figure 14A). Furthermore, the maximal peak cytosolic Ca evoked as a consequence of the application of either 100 uM N M D A or 500 uM N M D A was not significantly different (P>0.05 by paired t-test) between WT and Y A C 128 MSNs (Figure 14A), as 59 expected from the similar whole-cell current densities evoked by 1 mM N M D A (Figure 94-2). As the relatively high affinity of Fura-2 for free Ca (Kd = 145 nM) is ideal for estimating resting Ca levels but not necessarily optimal for discerning peak amplitudes 94-of cytosolic Ca (Hyrc et al , 1997; Stout and Reynolds, 1999), we used the lower affinity C a 2 + indicator MagFura-2 (Kd = 25 pM) to confirm the lack of difference in peak NMDAR-evoked C a 2 + increases. In response to a 5 min application of 500 pM NMDA, peak F334 /F380 ratios using MagFura-2 (Figure 14B) for WT vs. Y A C 128 MSNs were 0.54 ± 0 . 1 6 vs. 0.37 ± 0 . 1 0 , respectively (P>0.05 by paired t-test), ruling out the possibility that indicator dye saturation artifactually equalized the C a 2 + responses. We 94-concluded that neither changes in resting Ca levels nor enhancement of peak cytosolic C a 2 + responses, as seen in YAC46 and YAC72 MSNs (Zeron et al., 2004), explained the enhanced NMDA-induced apoptosis characteristic of Y A C 128 MSNs. 4.3 Altered Cytosolic C a 2 + Clearance Following NMDA Stimulation in YAC128 MSNs As illustrated in representative experiments (Figure 13), the recovery of cytosolic C a 2 + toward baseline levels following washout of N M D A appeared slower in Y A C 128 MSNs. To investigate this further a subset of experiments, in which C a 2 + changes were monitored .over longer periods, were performed and cytosolic Ca recovery was quantified as a percentage of the peak NMDA-induced change remaining over time following washout of NMDA. These experiments found a significantly slower rate of recovery of C a 2 + towards pre-stimulus (500 pM NMDA) levels in YAC128 vs. WT MSNs as early as 15 min (repeated measures A N O V A followed by Bonferroni post-test, 60 *P<0.05 at 15 min, *P<0.05 at 20 min, **P<0.01 at 25 min) following N M D A washout (Figure 15). These data demonstrated a prolonged elevation of cytosolic C a 2 + in Y A C 128 MSNs that may be toxic in and of itself and/or reflect pathological changes in neuronal function, such as the failure of calcium extrusion processes or sequestration into intracellular stores within organelles. 4.4 Effect of Route of Ca 2 + Entry on Recovery from an Induced Cytosolic Ca 2 + Load in YAC128 MSNs 2+ Having identified an unexpected genotypic difference in the return of Ca 9 + towards pre-stimulus levels, we wondered whether the impaired cytosolic Ca recovery reflected a general defect in C a 2 + handling in Y A C 128 MSNs, or if it was specific for NMDAR-mediated C a 2 + accumulation. To stimulate an alternate pathway of calcium influx from the extracellular compartment without activating NMDARs, I used 50 mM 9 + KC1 in combination with the L-type Ca channel modulator FPL 64176 (5 uM; FPL), a combination that optimizes depolarization-induced C a 2 + influx (Hardingham et al., 1999; Shehadeh et al, 2006) via voltage-gated C a 2 + channels (VGCCs). I then compared the cytosolic C a 2 + responses to those induced by 100 uM N M D A (representative responses to 2*F both stimuli shown in Figure 16A). Quantified in Figure 16B, the maximal peak Ca response was similar for these two different stimuli (area under the curve analysis, calculating integrated C a 2 + responses during stimulus application, also confirmed no significant differences for genotype or treatment at 1 and 5 min; data not shown), and there was no significant difference in peak cytosolic Ca for either stimulus between genotypes (two-way A N O V A ; no effect of treatment or genotype). Using the same 61 method to quantify cytosolic Ca clearance as in section 4.3, we found that recovery 94-from a Ca load induced by a 5 min application of 100 uM N M D A was significantly slower in Y A C 128 compared with WT MSNs (two-way A N O V A followed by Bonferroni post-test, *P<0.05 at 5 min, **P<0.01 at 10 min, ***P<0.01 at 15 min). These results are similar to those found using 500 u.M N M D A as the incident stimulus (Section 4.3, Figure 15). In stark contrast, recovery from a C a 2 + load induced by membrane depolarization and V G C C activation with KC1 and FPL was relatively rapid, nearly complete by 15 min post-stimulus washout (-10% above pre-stimulus levels), and proceeded at equivalent rates in both genotypes (two way A N O V A ; no effect of genotype) (Figure 17). Thus, we concluded that the defect in recovering from an imposed C a 2 + load was relatively selective for C a 2 + influx that occurred via N M D A R activation, and specific to Y A C 128 MSNs. 4.5 Summary Contrary to our hypothesis, we found that Y A C 128 MSNs did not have a 2_|_ significantly higher basal cytosolic Ca level, nor did they respond to N M D A R 94-stimulation with higher peak cytosolic Ca than observed in WT MSNs. However, we found an interesting dichotomy in the ability of Y A C 128 MSNs to recover from an imposed cytosolic Ca 2 +load. Whereas WT and Y A C 128 MSNs recovered from 94-depolarization-mediated Ca influx at similar rates, Y A C 128 MSNs were slower than 94-WT MSNs to recover from Ca influx via N M D A R activation. Hence it appears that 94-Y A C 128 MSNs are impaired in their ability to regulate cytosolic Ca , particularly after 62 intense N M D A R activity, which may increase their vulnerability to excitotoxic damage in the context of HD. 63 Chapter 5 Mitochondrial Dysfunction in YAC128 MSNs Previous work from our lab documents that mitochondrial membrane depolarization downstream of strong NMDAR stimulation in both YAC46 and YAC72 cultured MSNs is significantly greater than in cultured WT MSNs (Zeron et al., 2004). This enhanced depolarization of mitochondria in mhtt-expressing MSNs correlated with 9 + enhanced Ca entry resulting from N M D A R activation, which was characterized in parallel experiments. Several studies have demonstrated that NMDAR-mediated excitotoxicity is likely dependent on mitochondrial calcium uptake and overload (Castilho et al., 1998; Stout et al , 1998), and that the loss of mitochondrial membrane potential induced by mitochondrial uptake of free Ca from the cytosol is one indicator of the risk for subsequent apoptotic death (Ankarcrona et al., 1995; Ankarcrona et al., 1996; Schinder et al., 1996). In this series of experiments, our goal was to understand how mitochondria in Y A C 128 MSNs behaved in response to an NMDA-induced cytosolic Ca load (as documented in Chapter 4), and whether any differences existed between the responses of these mitochondria and those in WT MSNs which could explain the enhanced apoptosis of Y A C 128 MSNs as previously described. In order to accomplish this we combined the monitoring of both parameters, namely cytosolic C a 2 + and mitochondrial membrane potential (A^Fm) in an attempt to understand how the two factors interrelate and affect each other, and whether a loss of mitochondrial function similar to that seen in less 64 extreme polyQ expansions could occur in the absence of a correspondingly larger Ca load. 5.1 Equivalent Cytosolic Ca Loads Induce Significantly Different Changes in ATm in YAC128 Compared with WT MSNs The initial step was to establish our parameters for imaging both C a 2 + and A T m together using two dyes - Fura-2 and rhod-123 - simultaneously. Figure 18 shows two 94-typical experiments monitoring cytosolic Ca and A T m at the same time in the same cells in WT (top panel) and Y A C 128 (bottom panel) MSNs. This method allowed one particular advantage over monitoring each parameter in sister experiments, specifically the ability to directly assess the temporal relationship between changes in cytosolic Ca and loss of A^Frn- We predicted that following N M D A R activity, an increase in cytosolic C a 2 + would precede a change in A H r V as suggested by the literature. Indeed, we found that the increase in cytosolic C a 2 + indicated by an increase in Fura-2 ratio preceded any change in rhod-123 fluorescence (mean lag time between initial changes in Fura-2 and rhod-123 signals: 4.39 ± 0.98 s for WT, 5.89 ± 1.2 s for YAC128 MSN, P>0.05 by paired t-test; n= 8 paired experiments per genotype; total of 131 and 147 neurons for WT and YAC128, respectively, from 4 different culture batches); one example is illustrated in the 2"b inset in Figure 18. These data are consistent with an increase in cytosolic Ca triggering uptake of free C a 2 + by mitochondria and, consequently, a reduction in A ^ V We expected under our dual-imaging conditions that our previous findings, that WT and Y A C 128 MSNs have similar basal and NMDA-induced peak C a 2 + levels, would still hold true. As expected, there was no difference between WT and Y A C 128 MSNs 65 (P>0.05 by paired t-test) with regard to resting Ca or peak Ca in response to a 5 min application of 500 U.M N M D A (Figure 19, top panel). Surprisingly, however, the simultaneously monitored changes in A^Fm were markedly different with a greater loss of A^Frn in Y A C 128 MSNs as indicated by an approximately 3-fold greater increase in rhod-123 fluorescence compared with WT MSNs (Figure 19, bottom panel; *P<0.05 by paired t-test). 5.2 Comparison of Resting A ^ m There are several possible reasons for the larger NMDA-induced loss of A^Fm in Y A C 128 MSNs. This finding could reflect altered resting mitochondrial function and/or enhanced mitochondrial access to the NMDAR-induced Ca influx leading to increased uptake and depolarization. We attempted to address the first possibility by comparing the relative resting A ^ m in WT and Y A C 128 MSNs, as this would provide an indication as to the potential for resting mitochondria of either genotype to act as a C a 2 + buffering reservoir. We utilized 5 uM CCCP (a mitochondrial protonophore which dissipates A ^ F m ; Toescu and Verkhratsky, 2000) to depolarize the mitochondria of MSNs loaded with both Fura-FF and rhod-123 (resulting in the release of accumulated rhod-123 to the cytosol). By comparing the maximum fluorescence responses, we determined that resting mitochondria in Y A C 128 MSNs had an average polarization state similar as those in WT MSNs (P>0.05 by paired t-test; Figure 20A). In some experiments neurons were co-loaded with the C a 2 + indicator Fura-FF, allowing us to visualize the release of free C a 2 + from the mitochondria during CCCP-induced mitochondrial depolarization. As illustrated in Figure 20B very little free C a 2 + was released from resting mitochondria. J 66 5.3 Assessing the Ability of YAC128 MSNs to Act as a Ca 2 + Sink During NMDAR Activity To address the possibility that mitochondria in Y A C 128 MSNs had privileged access to the Ca influx pathway activated by NMDA'application, we used previously established methods (Brocard et al., 2001) to compare the efficiency of mitochondrial C a 2 + uptake between WT and YAC128 MSNs. Illustrated in Figure 21 A, we measured the area under the curve (AUC) of C a 2 + responses during a 1-min exposure to 500 uM N M D A (N) followed 2 min later by a 1-min application of CCCP (C; to release free Ca accumulated by mitochondria) to calculate the C/N ratio (as described in the Methods). We found similar C/N ratios (Figure 2IB, P>0.05, unpaired t-test), indicating that mitochondria in WT and Y A C 128 MSNs are equally capable of initially buffering Ca entering via NMDARs. We also noted similar, rapid rates of recovery of cytosolic C a 2 + towards baseline following N M D A application for WT and Y A C 128 MSNs (see Figure 21A as an example) that were not significantly different (Figure 22; P>0.05 by unpaired t-test), suggesting that the C a 2 + handling defect documented in Chapter 4 (and again demonstrated in Figure 18) is only observed after prolonged N M D A R activity. Based on these experiments, we conclude that mitochondria rapidly and efficiently buffer the Ca influx through NMDARs in both WT and Y A C 128 MSNs, implying that an enhanced mitochondrial C a 2 + uptake cannot explain the enhanced depolarization of mitochondria in Y A C 128 MSNs. 67 5.4 Potential mPT Involvement in Mitochondrial Depolarization in YAC128 MSNs Another possible explanation for the dramatically enhanced mitochondrial depolarization found in Y A C 128 MSNs during 5-min N M D A applications is that.the mPT might be acutely activated in this paradigm. We therefore decided to test the effect of mPT inhibition on changes in AT™, maximal peak C a 2 + and the subsequent recovery of cytosolic C a 2 + towards baseline. Note that for MSNs used in these experiments, the inhibitors of the mPT were present 1 hour prior to N M D A exposure and during the entire experiment, in all solutions used. Cyclosporin A (CsA), which binds the mPT modulator cyclophilin D, had no effect (P>0.05 by paired t-test) on NMDA-induced peak cytosolic C a 2 + in WT MSNs but was found to significantly (*P<0.05 by unpaired t-test) reduce peak C a 2 + in YAC128 MSNs (Figure 23A). There was also a trend toward CsA reducing simultaneously monitored NMDA-induced mitochondrial depolarization in Y A C 128 (but not WT) MSNs, although this effect was not significant (Figure 23B; P>0.05 by unpaired t-test). Since CsA is known to inhibit protein phosphatase 2B (calcineurin) in addition to exerting an inhibitory effect on mPT formation, we elected to test another mPT inhibitor, bongkrekic acid (BkA) which binds the ANT, an integral mPT component protein. BkA (5 pM) was found to significantly reduce both peak Ca (Figure 24A; *P<0.05, unpaired t-test) and mitochondrial depolarization (Figure 24B; *P<0.05, unpaired t-test) in Y A C 128 MSNs, thus having similar effects to CsA. Interestingly, neither parameter was affected by BkA treatment (P>0.05, unpaired t-test) in WT MSNs (Figures 24A and 24B). These results suggest that mPT activation occurs within 5 min of N M D A R activity in Y A C 128 MSNs, contributing to both peak C a 2 + and mitochondrial 68 depolarization. Neither of the mPT inhibitors had an effect on the rate of Ca recovery over a 15 minute period following N M D A application (data not shown). 5.5 Summary We found that mitochondria in Y A C 128 MSNs have a similar relative resting membrane potential as those in WT MSNs. This allows mitochondria in Y A C 128 MSNs to efficiently buffer cytosolic C a 2 + elevations that result from transient (60 s) N M D A R stimulation. However, when NMDARs are activated for a longer time period, in this case 5 min, mitochondria in Y A C 128 MSNs depolarize to a greater extent than those in WT MSNs. We found that the greater relative depolarization of mitochondria in Y A C 128 MSNs is due, at least in part, to activation of the mPT, and this event contributes to the peak cytosolic C a 2 + elevation. Interestingly mPT inhibitors had no effect on NMDA-induced changes in peak cytosolic Ca or AT™ in WT MSNs, indicating that mPT formation is not acutely induced. Based on the results presented here we conclude that a greater tendency for mPT activation, a common step in induction of apoptosis, is responsible for enhanced apoptosis in Y A C 128 MSNs following N M D A R activation. 69 Chapter 6 General Discussion 6.1 Synopsis of Findings In this thesis I have examined, for the first time, the relationship between the length of the polyQ repeat in mhtt and NMDAR currents in MSNs in vitro. I found that NMDAR current density was significantly enhanced in YAC72 MSNs compared to WT, Y A C 18, and YAC46, and that there was a trend toward larger N M D A R current density with increasing polyQ length for Y A C 18, YAC46 and YAC72 MSNs. Furthermore, this increase was selective for NMDARs as kainate-evoked current densities were equivalent between WT and YAC72 MSNs. This finding is consistent with previous work from our lab showing N M D A R currents enhanced by the expression of mhtt in H E K cells (Chen et al., 1999b) and acutely dissociated MSNs from YAC72 mice (Zeron et al., 2002). The increasing N M D A R current correlated with polyQ length for Y A C 18, YAC46 and YAC72 MSNs is consistent with the idea that increased N M D A R currents can act as an initiating event, producing elevated cytosolic Ca 2 + , mitochondrial membrane depolarization, mPT and caspase activation to enhance apoptosis in cultured MSNs expressing mhtt (Zeron et al., 2002; Zeron et al., 2004; Shehadeh et al., 2006), and increasing sensitivity to excitotoxic neuronal death in vivo (Zeron et al., 2002). The lack of NMDAR current augmentation in Y A C 128 MSNs will be discussed in the next section. In this thesis I also showed that the NMDAR current enhancement in YAC72 compared with WT MSNs was dependent upon neuronal subtype, as it was not observed 70 in GPNs derived from the same mice. Previous findings published by our laboratory showed that NMDAR-mediated apoptosis was not enhanced in cultured CGNs from Y A C HD mice (Zeron et al. 2002), consistent with the idea that mhtt increases N M D A R currents and/or cell death signaling in a neuronal type-specific fashion, a finding also confirmed in R6/2 mice (Cepeda et al., 2001b; Andre et al., 2006). As CGNs, under the culture conditions used, did not express NR2B at the time of experimentation (Zeron et al., 2002), it is tempting to suggest that the lack of increased apoptosis in mhtt-expressing CGNs was due to the absence of the NR2B subunit. The demonstration that NR1/NR2B N M D A R currents are selectively enhanced by mhtt (Chen et al., 1999b) supports this hypothesis. Additionally, CPNs did not show mhtt-enhanced N M D A R current and also expressed a significantly lower proportion of NRl/NR2B-type NMDARs.than MSNs. On the other hand, -45-55% of N M D A R current recorded from CPNs was mediated by NR1/NR2B, suggesting that the expression of the NR2B subunit may be required, but is not sufficient, for the mhtt-mediated enhancement of N M D A R activity. Furthermore, I found that neither the proportion of N M D A R current in MSNs sensitive to IFN nor NMDAR desensitization varied significantly with the expression of mhtt containing different polyQ lengths, indicating that a mhtt-mediated shift in N M D A R subunit expression was unlikely. Since YAC72 MSNs exhibited an increased N M D A R current density without any differences in neuronal capacitance, I attempted to discern the mechanism underlying the increase. It was initially observed that YAC72 MSNs express a greater proportion of their pool of NR1 at the cell surface, relative to WT and Y A C 18 MSNs (which had similar NR1 surface:internal ratios), while the total amounts of NR1 were similar across 71 genotypes (Fan et al., 2007). Biotinylation experiments subsequently confirmed that YAC72 MSNs express higher levels of both NR1 and NR2B at the plasma membrane than WT MSNs, but GluRl expression at the surface was similar for both genotypes (Fan et al., 2007). Previous work from our laboratory established that single-channel properties of NMDARs are not altered by mhtt (Chen et al., 1999b), it follows that this redistribution of NMDARs to the plasma membrane explains the enhancement of peak current density in YAC72 MSNs. Subsequently, seeking an explanation for the altered NMDAR distribution in YAC72 MSNs, our laboratory demonstrated elevated rates for both forward trafficking to the plasma membrane as well as removal and degradation of NMDARs from the surface (Fan et al., 2007); the former is consistent with the finding reported here of a faster rate of N M D A R insertion to the plasma membrane, as assessed by electrophysiological experiments. The net difference in rates favours an accumulation of NMDARs at the plasma membrane, thus explaining the redistribution of NMDARs in YAC72 compared with WT MSNs. Importantly, this enhanced trafficking effect was not noted for GluRl, indicating a change that would selectively affect NMDARs (Fan et al., 2007). For the first time, I recorded N M D A R currents in Y A C 128 MSNs, and surprisingly found current densities that were not significantly increased compared to WT or Y A C 18 MSNs, despite an increased sensitivity to NMDA-induced apoptosis with a maximum ceiling equivalent to YAC72 MSNs (Graham et al., 2006b; Shehadeh et al., 2006). I compared a number of factors with respect to cytosolic Ca handling and how changes in ATm are impacted downstream of N M D A R activation. To summarize, I found that while Y A C 128 MSNs have a resting level of cytosolic C a 2 + similar to WT 72 MSNs and reach a similar peak cytosolic Ca after prolonged (5 min) N M D A application, recovery of Ca towards baseline following washout of N M D A is significantly impaired in Y A C 128 MSNs. This genotypic difference in C a 2 + recovery appears to be dependent on the route of C a 2 + entry, as the rate of recovery from V G C C -mediated Ca 2 +entry was similar in both WT and Y A C 128 MSNs. I also found mitochondria in Y A C 128 MSNs depolarized to a greater extent than those in WT MSNs, downstream of prolonged N M D A application. Inhibitors of mPT formation were able to reduce both peak C a 2 + and the extent of mitochondrial depolarization in Y A C 128 MSNs but had no effect in WT MSNs, implying that activation of the mPT accounts for some of the differences in these factors between genotypes. Hence mhtt appears to impair mitochondrial responses in Y A C 128 MSNs downstream of N M D A R activation, resulting in the initiation of mPT formation. This sequence of events can account for the previously reported enhanced neuronal apoptosis in YAC128 MSNs (Tang et al., 2005; Graham et al., 2006b; Shehadeh et al., 2006). 6.2 Dissociation Between NMDAR-Mediated Current and Downstream Changes in Cytosolic Ca 2 + and A*Fm in YAC128 MSNs I have shown that N M D A R peak current density increases with the length of the htt polyQ repeat in MSNs obtained from YAC18, YAC46, and YAC72 mice, and yet YAC128 MSNs exhibit current densities similar to WT and YAC18. Thus, the observed correlation between htt polyQ length and N M D A R current density in MSNs does not extend to more extreme repeat sizes in the Y A C transgenic mouse model. 73 The increased N M D A R current density correlates well with increased N M D A R surface expression in YAC72 MSNs compared with WT or Y A C 18 MSNs in culture (Fan et al., 2007). Preliminary data from our lab also indicates that the distribution of NMDARs between surface and internal pools is similar for Y A C 128, WT and Y A C 18 MSNs (L. Zhang and L A - Raymond, unpublished results), which would be consistent with similar current densities between these genotypes. It is possible that mechanisms underlying altered N M D A R trafficking in YAC72 MSNs (Fan et al., 2007) are also active in Y A C 128 MSNs but that additional mechanisms, arising from the further expansion of the polyQ region that result in the down-regulation of N M D A R surface expression, are recruited in Y A C 128 MSNs. These pathways could include aberrant activation of proteases, or protein phosphatases and kinases, particularly if stimulated by prolonged elevation of C a 2 + levels following sustained N M D A R activity. For example, increased activation of casein kinase 2, which is a calcium-activated protein kinase that phosphorylates NR2B, reduces N M D A R surface expression in hippocampal neurons 9 + (Chung et al., 2004). Another candidate for N M D A R downregulation is Ca -activated calpain activity, which can cause cleavage of the C-terminus of surface NR2B subunits (Guttmann et al , 2002; Simpkins et al., 2003; Dong et al., 2006) and promote NMDAR internalization (Scott et al., 2004). In further contrast to our previously published observations in MSN cultures from YAC46 and YAC72 mice (Zeron et al., 2004) but consistent with the results of the N M D A R peak current density experiments, there was no significant difference in NMDA-induced maximal peak cytosolic C a 2 + between WT and Y A C 128 MSNs. This 9 + lack of difference in NMDA-induced peak Ca was consistently observed using three 74 different fluorescent Ca indicators (Fura-2, Fura-FF and MagFura-2) with differing Kd values, and with two concentrations (100 and 500 \xM) of NMDA. Our results in WT and Y A C 128 MSNs using the combination of KC1/FPL to induce C a 2 + influx suggest that VGCCs behave similarly in both genotypes with regards to their contribution to peak Ca 2 + , and the similar recovery of cytosolic C a 2 + following depolarization argues against differential regulation of V G C C function by mhtt in Y A C 128 MSNs. Since Y A C 128 MSNs exhibited a dramatically enhanced loss df A T m 9 4 -downstream of N M D A R activation, indicative of mitochondrial Ca uptake and overload (Castilho et al., 1998; Stout et al., 1998; Pivovarova et al., 2004), and the extent of loss of 9 4 -A ^ m induced by Ca uptake is one indicator of the risk for subsequent excitotoxic or apoptotic death (White and Reynolds, 1996; Schinder et al., 1996), our results help explain the heightened sensitivity of Y A C 128 MSNs to NMDA-induced apoptosis. 6.3 Acute NMDA-Evoked mPT Activation in YAC128 MSNs My results indicate that both mPT inhibitors CsA and BkA, which interact with different components of the mPT, significantly reduced peak C a 2 + induced by a 5 min application of 500 pM N M D A in YAC128 MSNs. The change in A^¥m was also 9 4 -attenuated, whereas the rate of recovery of the Ca response towards pre-stimulus (resting) levels was not significantly altered by either inhibitor. Another study using cultured WT rat MSNs observed activation of the mPT during a prolonged (>20.min) exposure to 1 mM N M D A (Alano et al., 2002). It was therefore surprising to us that the mPT is activated as a consequence of a 5 min application of 500 pM N M D A in YAC128 MSNs, which suggests that very early induction of the apoptotic pathway may occur. 75 There was no indication of mPT activity during or following a 5 min application of 500 pM N M D A in my experiments with WT MSNs. Others have also reported mPT activation in paradigms of glutamate excitotoxicity in cultured forebrain neurons (White and Reynolds, 1996) and in hippocampal neurons (Schinder et al., 1996; Vergun et al., 1999). The acute NMDA-induced mPT activation observed in Y A C 128 MSNs occurred in response to similar levels of cytosolic Ca as in WT MSNs. Others have reported significant protection against glutamate-induced cell death using putative mPT inhibitors in Y A C 128 MSNs from a higher mhtt-expressing line (line 53) of Y A C transgenic mice (Tang et al., 2005), further supporting our hypothesis that mPT induction contributes to the enhanced NMDA-induced apoptosis in Y A C 128 MSNs. Interestingly, the relative changes in cytosolic C a 2 + and A'Pm in Y A C 128 MSNs following prolonged N M D A R stimulation contrast with data obtained in YAC72 MSNs, where N M D A R current, cytosolic Ca levels and mitochondrial membrane depolarization induced by N M D A application are all increased compared with WT MSNs and correlate with enhanced mPT activation and apoptosis (Zeron et al., 2002; Zeron et al., 2004). In this regard, it is important to note that once NMDAR-mediated cytosolic C a 2 + levels in YAC72 MSNs are reduced to those observed in WT MSNs, the amount of NMDA-induced apoptosis is also reduced accordingly, becoming equivalent to levels seen in WT (Zeron et al., 2004; Shehadeh et al., 2006). Taken together, these results suggest that the mechanism by which mhtt enhances NMDAR-mediated apoptosis is different, depending on the length of the C A G repeat (Figure 25). Previous studies conducted on isolated mitochondria from human lymphoblasts demonstrated that mitochondria from HD patients depolarized more readily than those 76 from controls when incubated with Ca (Panov et al., 2002; Panov et al., 2005), and that this depolarization caused the release of C a 2 + from the mitochondria, an event which could be delayed by inhibition of the mPT (Panov et al., 2002). Importantly, the authors observed that mitochondria from juvenile-onset HD patients were even more sensitive to stepwise additions of C a 2 + than those from adult-onset patients. These data are consistent with the idea that C A G repeat length can modulate the mechanisms underlying MSN sensitivity to apoptosis. I have demonstrated that mitochondria in Y A C 128 MSNs can efficiently buffer C a 2 + during a 60s application of 500 pM N M D A , and that a longer duration of N M D A application results in mPT activation. A leak of free Ca from compromised mitochondria in Y A C 128 MSNs may contribute to the difference in intracellular C a 2 + recovery rates following 5 min N M D A R activation, although other mitochondrial C a 2 + transport mechanisms including a mitochondrial Na + /Ca 2 + exchanger (White and Reynolds, 1997; Gunter et al., 2000) or the mitochondrial Ca uniporter (Kirichok et al., 2004) could also play a role in this difference. Further experiments would be required to determine the possible relative contributions of these different mechanisms to mitochondrial failure to regulate cytosolic Ca . Extrapolating these observations together with my data to the human condition, raises the possibility that HD patients with more extreme C A G repeat expansions may be best served by targeting therapies at the level of the mitochondria, for example to better resist mPT induction. A recent study by Oliveira et al., (2006) found mhtt-induced impairment of cytosolic C a 2 + and A T m responses to a 10 min application of 100 uM N M D A in YAC128 MSNs. However, this study was aimed at delineating events which occur during 77 neuronal dysfunction, and thus were achieved under conditions of glycolytic inhibition and ATP depletion. In contrast, my results were obtained in Y A C 128 MSNs in their normal metabolic state, in order to elucidate events that could lead specifically to apoptosis, as characterized by our lab (Shehadeh et al., 2006). This difference in methodology makes a direct comparison of results difficult to interpret. However, their findings also support the idea of a deficit in mitochondrial function that is present, as in our experiments, from an early age during development and that underlies neuronal dysfunction preceding death in HD (Oliveira et al., 2006). 6.4 Mhtt-Induced Impairment of Ca 2 + Homeostasis in YAC128 MSNs Consistent with a previous report (Tang et al., 2005), we found similar resting Ca2 +levels in WT and Y A C 128 MSNs. As steady-state resting Ca 2 + i s determined primarily by the balance between C a 2 + entry and efflux/sequestration mechanisms (Xiong et al., 2002), these processes are likely to be normally functional during resting (i.e. unstressed) conditions within Y A C 128 MSNs, at least at the early time-point of our assessment. Further evidence of this comes from our results showing that cytosolic calcium recovery in Y A C 128 MSNs occurs normally (i.e. shows a rate of recovery similar to WT MSNs) following a brief (60s) stimulation of NMDARs, or even prolonged (5 min) stimulation of VGCCs using KC1 with FPL (the L-type Ca2+channel modulator). These data suggest that V G C C activation is also similar between WT and Y A C 128 MSNs, and that differences in intracellular handling of Ca 2 + , such as mitochondrial buffering, only become apparent during prolonged (5-min) NMDAR-induced stress, a finding supported by previous research (White and Reynolds, 1995). This requirement of 78 a stressful stimulus to uncover underlying functional deficits in mitochondria is not surprising, given findings that active glycolysis in the cytosol can mask mhtt-induced mitochondrial deficiencies in C a 2 + handling in STHdhQni cells that become apparent when glycolysis is inhibited (Oliveira et al., 2006). While the experiments presented here have largely focused on mitochondrial regulation of cytosolic Ca 2 + , mPT inhibition had no effect on the rate of C a 2 + recovery towards baseline following an NMDA-induced C a 2 + load. This observation suggests that other intracellular C a 2 + stores and extrusion/sequestration or buffering mechanisms may contribute to impaired calcium homeostasis. Indeed, an interaction between mhtt, htt-associated protein 1A (HAP-1A) and the IP3 receptor (IP3R) has been shown, resulting in increased sensitivity of the IP 3 receptor to IP3 (Tang et al., 2003). In turn, this abnormal interaction appears to enhance the toxicity of glutamate-mediated IP 3 R activation in YAC128 MSNs, facilitating C a 2 + release from the ER in YAC128 MSNs in response to the selective mGluRl/5 agonist 3 ,5 - DHPG (Tang et al., 2005). This mechanism was not likely to be activated in our paradigm using N M D A , although Tang et al. (2005) also reported enhanced sensitivity of Y A C 128 MSNs to NMDAR-mediated apoptosis. Consistent with our results demonstrating impaired Ca recovery following N M D A R activation in Y A C 128 MSNs, repetitive glutamate stimulation produced a gradual elevation in cytosolic C a 2 + in YAC128 MSNs, but not WT or Y A C 18 MSNs over a similar timeframe (Tang et al., 2005). One interesting possibility that could contribute to impaired extrusion of cytosolic C a 2 + is dysfunction of the Na + /Ca 2 + exchanger isoform 3 (NCX3). A recent report (Bano et al., 2005) demonstrated that NCX3 is cleaved by calpains in striatal tissue following 79 focal ischemia and in cerebellar granule neurons under excitotoxic conditions. Overexpression of calpastatin, an endogenous inhibitor of calpains, in CGNs prevented secondary elevations in cytosolic C a 2 + following glutamate application and protected neurons from glutamate-mediated excitotoxicity (Bano et al., 2005). Others have reported that plasmalemmal Na /Ca exchange activity plays a role in buffering glutamate-mediated C a 2 + increases in forebrain neurons (White and Reynolds, 1995). If activation of p- or m-calpains in Y A C 128 MSNs following N M D A R activity is enhanced (Nicotera et al., 1986), another important homeostatic C a 2 + clearance route would be impaired. If this increased calpain activity is Ca2+-dependent (Gafni and Ellerby, 2000; Gafni et al., 2004), then it could result in a feed-forward mechanism that slowly amplifies to a maximum over time, resulting in the gradual development of more severe C a 2 + -handling deficits. 9-f-Another interesting explanation for impaired Ca clearance in Y A C 128 MSNs following prolonged N M D A application is a possible difference in cytosolic Ca buffering factors, for example differential expression of C a 2 + buffering proteins such as calbindin (CaBP), or other cytosolic buffering factors in Y A C 128 compared to WT 9 + MSNS. CaBP overexpression did not alter peak Ca downstream of N M D A R activation 94-but did prolong the recovery of Ca towards baseline values (Rintoul et al., 2001), reminiscent of our observations in Y A C 128 MSNs. Evidence from human HD patient brains indicates increasing CaBP expression, with increases in expression in MSN dendrites and spines associated with more advanced (grades 3 and 4) cases (Huang et al., 1995). Following intrastriatal injection of quinolinic acid, surviving MSNs expressed increased levels of CaBP; the upregulated expression 80 was found to be dependent upon N M D A R activity, suggesting that this expression change was a excitotoxicity-driven attempt to buffer elevated C a 2 + levels (Huang et al., 1995). Similar observations have been made in the striatum of R6/2 mice. A pattern of increased CaBP expression was observed in the dorsolateral striatum, an area normally associated with low CaBP expression; this increase was viewed as an attempt to buffer C a 2 + downstream of an excitotoxic process (Sun et al., 2005), and occurred at an age associated with resistance to excitotoxicity in R6/2 mice (Hansson et al., 2001). However, our data in Y A C 128 MSNs do not support the idea of enhanced N M D A R activity driving a compensatory upregulation in CaBP expression. CaBP overexpression was also found to reduce mitochondrial depolarization in response to ionophore-mediated Ca elevations, and enhance the 24h survival rate following N M D A incubation (Rintoul et al., 2001), findings which are inconsistent with our observations of increased apoptosis in Y A C 128 MSNs 24h following N M D A exposure (Graham et al., 2006b; Shehadeh et al., 2006;) and enhanced mitochondrial depolarization in Y A C 128 MSNs following NMDARactivity. There is however an interesting similarity between results obtained in my study and a previous study using artificial C a 2 + buffers in an excitotoxicity paradigm in hippocampal neurons. In that study, following glutamate exposure, the presence of buffers did not have an impact on peak cytosolic Ca but significantly slowed the recovery of C a 2 + towards baseline levels and enhanced, rather than reduced, neuronal death (Abdel-Hamid and Baimbridge, 1997). These are similar observations to what we report here in Y A C 128 MSNs. The authors argued that the effect of artificial C a 2 + -buffering actually enhanced overall C a 2 + entry into the neurons, possibly by inhibiting 81 normal Ca -dependent feedback mechanisms on N M D A R function (Abdel-Hamid & Baimbridge, 1997), and that excitotoxicity better correlates with total Ca influx rather than peak free cytosolic C a 2 + (Michaels and Rothman, 1990; Hartley et al., 1993; Abdel-Hamid and Baimbridge, 1997). This last point is an issue of controversy, as other work has suggested that the C a 2 + influx pathway (Peng and Greenamyre, 1998; Sattler et al., 1998;) is the most important factor determining neuronal excitotoxicity, while a different set of studies showed that peak C a 2 + is in fact predictive of excitotoxicity under the right conditions (Cheng et al., 1999; Stout and Reynolds, 1999). The findings regarding Y A C 128 MSNs presented here, along with data on NMDA-induced apoptosis in the same cells (Graham et al., 2006b; Shehadeh et al., 2006) indicate that peak C a 2 + was not predictive of excitotoxicity even when low-affinity C a 2 + indicators were used. It could be that the presence of mhtt, which affects mitochondrial function and Ca handling as mentioned 94-earlier, alters the predictive value of Ca changes on excitotoxic outcomes. Also, there is no evidence presented here to support the idea that total Ca influx is greater in Y A C 128 MSNs, or that the C a 2 + buffering potential of the cytosol in Y A C 128 MSNs is in any way different from WT. As suggested earlier (Section 6.2), it may be that 9 + mitochondrial depolarization and Ca uptake are the best predictors for neuronal viability in HD models. Further experiments will be required to address these questions. 94-Overall, these observations suggest that a strategy of using Ca buffers as a therapeutic intervention to spare MSNs in HD would be difficult at best. The finding that Y A C 128 MSNs exhibit significantly longer delays in recovery of cytosolic C a 2 + following N M D A challenge than WT MSNs may be important with regard 82 to downstream toxic consequences, particularly those mediated by Ca -activated pathways, as in calpain activation. Indeed, in excitotoxicity-resistant R6/2 mice, it is 94-speculated that elevated resting Ca levels in MSNs may be a stress-induced alteration which could cause neuronal dysfunction underlying behavioural deficits preceding neuronal death (Hansson et al., 2001). A similar phenomenon, stemming from a cumulative dysfunction downstream of N M D A R activity, may manifest itself over time in Y A C 128 mice, which start showing behavioural changes prior to any apparent neuronal loss (Slow et al., 2003). The changes in Y A C 128 M S N function that we have characterized here are present from early development, and thus are consistent with a progressively developing condition arising from cumulative insult. 6.5 Selective Vulnerability of MSNs in HD There are a number of observations that could provide an explanation for the selective vulnerability of MSNs to neurodegeneration in HD. A natural starting place would be the distribution of htt; however, studies of htt distribution in rat striatal and cortical neurons showed no correlation between neuronal subtypes that express htt and those that preferentially degenerate in HD (Fusco et al., 1999). In fact, mRNA evidence suggests that several regions of the brain in humans, including hippocampus, cerebellum, and cerebral cortex, exhibit higher levels of htt than the striatum (Landwehrmeyer et al., 1995b). These observations suggest that the expression pattern of htt does not explain selective neurodegeneration. Mitochondria Isolated from rat striatal tissue were found to depolarize to a greater extent, have reduced ATP:ADP ratios, and be more susceptible to permeability 83 transition than those isolated from cortical tissue, when challenged with Ca . This inherent difference in brain mitochondria was attributed to an increased expression of the mPT component cyclophilin D in striatal mitochondria, suggesting that striatal mitochondria are more prone to mPT activation than mitochondria from other brain regions (Brustovetsky et al., 2003). My findings of enhanced mitochondrial depolarization in the absence of increased N M D A R current in Y A C 128 MSNs, and the effects of mPT inhibitors on peak Ca and mitochondrial depolarization support the idea of a mhtt-mediated enhancement of mPT formation. However, in this study I have not addressed the issue of what molecular mechanisms underlie the enhanced sensitivity to Ca2+-induced mPT formation (e.g., whether cyclophilin D expression is altered in mitochondria from Y A C 128 versus WT MSNs) nor whether enhanced mPT activation occurs selectively in mhtt-expressing MSNs versus CPNs. Further experiments would be required to investigate these questions. Another strong possible candidate as a factor explaining enhanced MSN vulnerability in HD would be the expression pattern of NMDARs and in particular the specific N M D A R subunits expressed within neuronal subtypes which may underlie selective neurodegeneration of MSNs. Whereas MSNs express predominantly NR1-1A, NR2A and NR2B (Landwehrmeyer et al., 1995a; Kuppenbender et al., 1999; Chapman et al., 2003), striatal interneurons express reduced levels of NR2B and preferentially express NR2D (Landwehrmeyer et al , 1995a). NMDA-evoked currents were much larger in MSNs vs. larger interneurons, as were EPSCs, whereas kainate-evoked currents showed the opposite pattern (Cepeda et al., 2001a). Notably, MSNs swelled in response to exogenous N M D A application, whereas striatal interneurons responded with very little 84 change, indicating that MSNs are at a greater risk, even in the absence of mhtt, for excitotoxic damage (Cepeda et al., 2001a). Perhaps the context of impaired mitochondrial function in MSNs provides the background conditions required for the NR2B-selective hypothesis (see Section 1.2.4.2) to be realized, i.e. the combination of NR2B-containing NMDARs on a cellular background with mitochondria more likely to transition to mPT formation is a key factor in MSN vulnerability in HD. Our findings of selective enhancement of N M D A R (but not AMPA/kainate receptor) currents in MSNs (and not CPNs) support this concept, but further experiments with respect to mitochondrial function would be required to determine whether this hypothesis is valid. 6.6 Limitations of This Study and Suggested Further Experiments The main goals of this study were to examine the relationship between polyQ length and modulation of N M D A R function, and explain the mechanism(s) underlying enhanced NMDA-induced apoptosis in Y A C 128 MSNs. The opportunity to look back with hindsight allows the subsequent identification of limitations on interpretation of results, either because of a deficit in experimental design or execution, or because further experimentation is required to answer new questions which arise from our findings and the recent findings of others. Our finding of an enhanced rate of NMDAR insertion into the plasma membrane over a span of minutes (Chapter 3) in YAC72 MSNs relative to WT MSNs may be attributable to the effect of mhtt on overall rates of N M D A R trafficking towards the plasma membrane (Fan et al , 2007). However, considering that YAC72 MSNs express a greater total amount of htt, mutant or otherwise, than WT MSNsjaises the possibility that 85 the difference in insertion rates was due to an effect related to the total amount of htt (including mhtt) rather than the polyQ expansion in mhtt, Although the finding that surface internal NR1 ratios are equivalent in WT and Y A C 18 MSNs argues against this possibility, the inclusion of data from Y A C 18 MSNs on insertion rate to the plasma membrane (as determined electrophysiologically) would provide a more definitive answer to this question. The lack of such a finding would not disprove the hypothesis being true in MSNs, but rather indicate a striatal-specific trafficking mechanism that may in turn help to explain striatal vulnerability in HD. Furthermore, if the enhanced rates of NMDAR trafficking are due to the expanded polyQ region in YAC72 MSNs, then a similar alteration in trafficking would be expected in Y A C 128 MSNs. A confirmation of increased N M D A R forward trafficking in Y A C 128 MSNs would necessitate the existence of an opposing mechanism in order to maintain a steady-state distribution of NMDARs similar to that seen in WT MSNs. Hence it would be useful to conduct these experiments in genotypes other than what I have assessed thus far. All of the experiments in this thesis monitoring C a 2 + dynamics and A^Fm changes involved a comparison between WT and Y A C 128 MSNs. This choice of comparison is subject to the same criticism as above in that our control expresses less total htt, endogenous and mhtt combined, than the experimental group, a possible confounding factor. Our choice of WT as the control was driven by observations that YAC18 mice, and cultured MSNs derived from them, show a resistance to noxious stimuli including excitotoxins, C a 2 + ionophores, and even general apoptotic inducers (Leavitt et al., 2006; Shehadeh et al., 2006;). This resistance is ascribed to a possible neuroprotective role of normal htt (Leavitt et al., 2001; Leavitt et al., 2006). In our mind, the use of a control 86 that is inherently resistant to excitotoxicity, the induction of which we are attempting to characterize in order to differentiate alterations caused by mhtt expression, would not be an appropriate representation of normal events. However, having obtained data from the 'normal' situation (WT MSNs) it would be of great interest to see if the neuroprotection afforded by htt overexpression is due to neuroprotective modulation of C a 2 + handling and A ^ m maintenance. This would be of further interest when compared in parallel with MSNs from Y A C 128 mice expressing a caspase-6 resistant (C6R) form of mhtt. These mice do not develop HD and appear to have a WT phenotype, and MSN cultures derived from these mice show no enhanced apoptosis or neurotoxicity in response to excitotoxins (Graham et al., 2006b). A particularly interesting comparison of C a 2 + handling and mitochondrial function would be between Y A C 18, C6R-YAC128, and YAC128 to see if neuroprotection in the first two models correlates with reduced sensitivity of mitochondria to Ca2+-induced mPT formation. If so, it would support the ideas that mhtt retains at least some normal function of htt (arguing against the loss of function hypothesis) and that cleavage of mhtt is an initiating toxic event. As the correlative relationship between htt polyQ length and N M D A R current density in MSNs is intact up to YAC72 but does not extend beyond to Y A C 128 MSNs, the idea that altered N M D A R function is solely responsible for the selective vulnerability of MSNs must be questioned. Our results suggest that mhtt containing a polyQ expansion greater than a certain threshold number may modulate neurotoxicity in a different manner than shorter pathogenic polyQ expansions. If the impairment of mitochondrial function in Y A C 128 MSNs is also apparent in other neuronal populations, 87 it would support the argument that different lengths of pathogenic polyQ expansions in htt are toxic by different mechanisms. Lastly, the work presented here has identified an abnormality in Y A C 128 MSN responses to N M D A that can explain enhanced excitotoxicity in these cells; however, the reason for the particular sensitivity of mitochondria in Y A C 128 MSNs still needs to be elucidated. One possibility may be that the physical localization of mitochondria within Y A C 128 MSNs makes them more vulnerable to sudden Ca changes, for example if a disproportionate number of mitochondria are near extrasynaptic NMDARs, the activation of which are associated with neuronal death (Hardingham et al., 2002; Liu et al., 2004). Another related possibility is that movement of mitochondria, shown to be impaired by mutant htt (Chang et al., 2006), is restricted in Y A C 128 MSNs such that they are unable to respond appropriately to localized energy (or other functional) demands within the cell. Combined with the greater predisposition of striatal mitochondria towards mPT formation (Brustovetsky et al., 2003), such circumstances could not only explain enhanced sensitivity to N M D A R activation in YAC128 MSNs (Shehadeh et al., 2006) but also possibly explain the enhanced vulnerability of MSNs in HD. 6.7 Conclusions Our findings using primary MSN cultures from Y A C mice transgenic for human htt suggest a critical role for NMDARs in initiating MSN dysfunction and death in HD. Our research suggests that N M D A R activity can be selectively enhanced in a neuronal type-specific manner, through a mechanism that increases the expression of NMDARs on the plasma membrane (Fan et al., 2007). This redistribution results in an increased 88 sensitivity to NMDAR-mediated excitotoxicity. Our findings also support a mhtt-mediated mechanism whereby sustained N M D A R activation reveals an inability to regulate cytosolic C a 2 + in MSNs, in the absence of increased N M D A R peak current. Diverging from Y A C models with shorter polyQ expansions, the enhanced N M D A -induced apoptosis in Y A C 128 MSNs appears largely determined by increased susceptibility to Ca2+-induced mitochondrial depolarization, acute mitochondrial dysfunction, and mPT activation. These observations may help to refine the choice of therapeutic strategies to test in Y A C HD mouse models with different C A G sizes. Importantly, signalling pathways activated by strong N M D A R stimulation converge on the mitochondria in all Y A C HD models, indicating that intervention downstream of NMDAR activity, such as bolstering mitochondrial function and/or mPT inhibition, may represent viable targets for ameliorating excitotoxicity-driven apoptosis in HD. 89 Table 1. A Summary of Relevant Full-Length and N-Terminal Fragment H D Models R6/1. R6/2 YAC46. YAC72, YAC 128 N171 CAG80 CAG94 HdhQ)2, HdhQ\ 11 tgHDIOO References Promoter Transcript # C A G repeals Striatal N M D A R protein expression Electrophysiological properties Hippocampal and/or striatal LTP/LTD Striatal NMDAR-current Striatal N M D A R - C a 1 * or cell swelling response Striatal N M D A R -niitochondrial membrane depolarization Cellular energy metabolism Striatal N M D A R toxicity Mangiarini etal . (1996) Human Exon I 120. 150 NR1 unchanged or increased, NR2A/NR2B decreased Altered Altered Increased Increased N.R. N R . No change early, resistant late Hodgson et al. (1999), Sbw et aL (2003) Human A l l 67 exoas 46, 72, 128 NR1/NR2B unchanged in YAC46 or YAC72 Altered Altered Increased Increased Increased N R . Increased; remains susceptible Schilling et al. (1999) Shclbourne et al. (1999) Levine et al. (1999) Wheeler et al. (2000) Laforet et al. (2001) Prion Murine Murine Murine Rat NSE cDNA for C A G knock-in C A G knock-in C A G knock-in cDNA for N-terminal 17laa N-tcrniinal 3 kb 82 80 NR2A transiently N.R. decreased N.R. N.R. N.R. N.R. N.R. N.R. Resistant late N.R. Altered N R . N.R. N.R. N.R. N.R. 94 N.R. Altered N.R. N.R. Increased N.R. N.R. N.R. 92, 111 N.R. Altered N.R^ N.R. •' N.R. N.R. Impaired N.R. 100 N.R. Altered N.R. Increased Increased N.R. N.R. Not different from wt N . R . = not reported N S E = neuron specific enolase Table taken directly from (Fan and Raymond, 2007) FIGURES Figure 1. Representative whole-cell current response of a voltage-clamped ( V H = -60 mV) WT MSN to a 4 s application of 1 mM NMDA. The peak current amplitude (Ipeak) is measured as indicated, and the 200 ms region which is averaged to derive the value for steady-state current (Iss) is identified near the end of the NMDA application period. 9 1 NMDA Figure 2. Peak N M D A R current density varies with htt polyQ length in cultured MSNs. Whole-cell patch clamp recording was performed on cultured MSNs at 9-10 d.i.v. stimulated with 1 mM NMDA. Bars represent mean peak current density (peak current normalized to cell capacitance) ± SEM. n = 42 (WT), 13 (YAC 18), 11 (YAC46), 16 (YAC72) or 20 (YAC 128) cells total. Significant difference between genotypes by one-way A N O V A , effect of genotype F(4,97) = 5.113, PO.001; * P< 0.05, ** P< 0.01 by Bonferroni post-test. 93 WT YAC 18 YAC46 YAC72 YAC128 94 Figure 3. PolyQ length does not affect kainate receptor current density. A) Representative whole-cell current response of a voltage-clamped ( V H = -60 mV) MSN to a 4 s application of 1 mM kainate. B) Mean peak current densities of WT and YAC72 MSNs in response to exogenous application of 1 mM kainate as in A). Whole-cell patch clamp recordings were performed on cultured MSNs at 9-10 d.i.v. as described in Methods. n=l 1 (WT) or 10 (YAC72) cells total. Bars represent mean peak current density (peak current normalized to cell capacitance) ± SEM. No significant difference between genotypes by unpaired t-test. 95 Kainate 400 pA 1000 ms 100 LU CO i c 80 n=11 Figure 4. Peak N M D A R current density is not influenced by htt polyQ length in cultured CPNs. Whole-cell current density was measured under voltage clamp (V H = -60 mV) in cultured CPNs (4-7 d.i.v.) from WT, Y A C 18, YAC72 and YAC128 mice, stimulated with 1 mM NMDA. n=20 (WT), 11 (YAC 18), 19 (YAC72) or 17 (YAC 128) cells total. Bars represent mean peak current density (peak current normalized to cell capacitance) ± SEM. No significant difference between genotypes by one-way A N O V A . 97 Figure 5. Desensitization of whole-cell NMDAR current in MSNs is not governed in a polyQ length-dependent fashion. Steady-state (Iss) to peak (I p e ak) current was calculated and expressed as a percentage as described in Methods, from MSNs responding to exogenous application of 1 mM N M D A while held under voltage clamp ( V H = -60 mV). Bars represent mean peak current density (peak current normalized to cell capacitance) ± SEM. n=42 (WT), 13 (YAC 18), 11 (YAC46), 16 (YAC72) or 20 (YAC 128) cells total. Significant difference between genotypes by one-way A N O V A , F(4,96) - 4.935, PO.01; *P< 0.05 Y A C 18 vs. WT, **P<0.01 Y A C 18 vs. YAC46 or Y A C 18 vs. Y A C 128 by Bonferroni post-test. 99 Figure 6. Desensitization of whole-cell NMDAR current in CPNs is not altered by polyQ length. Steady-state (I s s) to Peak (I p e ak) current was calculated and expressed as a percentage as described in Methods, from CPNs responding to exogenous application of 1 mM N M D A while held under voltage clamp ( V H = -60 mV). Bars represent mean peak current density (peak current normalized to cell capacitance) ± SEM. n=20 (WT), 11 (YAC 18), 19 (YAC72) or 16 (YAC 128) cells total. No significant difference between genotypes by one-way A N O V A . 101 Figure 7. IFN blocks NRl/NPv2B-containing NMDARs. A) Representative whole-cell current responses of a voltage-clamped (V ' H = -60 mV) YAC72 MSN to a 4 s application of 1 mM NMDA, in the absence (black trace) and subsequent presence (grey trace) of 3 uM IFN. The difference between the two responses represents the fraction of current conducted by NRl/NR2B-containing NMDARs. Note that co-application of IFN with NMDA blocks the majority of N M D A R current. B) Representative whole-cell current responses of a voltage-clamped (V H = -60 mV) WT C G N to a 4 s application of 1 mM NMDA, in the absence (black trace) and subsequent presence (grey trace) of 3 uM IFN. In contrast to the effect of IFN in MSNs, note that co-application of IFN with N M D A in CGNs had no effect on N M D A R Ipeak or I s s, indicating its selectivity for NR1/NR2B-containing NMDARs. 103 Figure 8. Neither overexpression of htt or expression of mhtt affects the proportion of NMDARs containing NR1/NR2B in cultured MSNs. The percentage of current mediated by NRl/NR2B-containing NMDARs (those blocked by IFN) was calculated as described in Methods, from cells responding to exogenous application of 1 mM N M D A (± 3 pM IFN) while held under voltage clamp ( V H = -60 mV). Bars represent mean peak current density (peak current normalized to cell capacitance) ± SEM. n=18 (WT), 5 (YAC18), 5 (YAC46), 5 (YAC72) or 10 (YAC 128) cells total. No significant difference between genotypes by one-way A N O V A . 105 Figure 9. Neither overexpression of htt or expression of mhtt affects the proportion of NMDARs containing NR1/NR2B in cultured CPNs. The percentage of current mediated by NR1/NR2B-containing NMDARs (those blocked by IFN) was calculated as described in Methods, from cells responding to exogenous application of 1 mM N M D A (± 3 uM IFN) while held under voltage clamp (V H = -60 mV). Bars represent mean peak current density (peak current normalized to cell capacitance) ± SEM. n=9 (WT), 6 (YAC 18), 10 (YAC72) or 7 (YAC128) cells total. No significant difference between genotypes by one-way A N O V A . 107 Figure 10. Electrophysiological silencing of surface NMDARs allows for detection of naive NMDAR insertion to the plasma membrane. A) Example of method used for electrophysiological silencing of cell surface NMDARs in an MSN using whole-cell patch clamp. Note clear N M D A R Ip e ak and desensitization of current during Pre-Block (pre-MK-801) phase, and decay of current towards zero during the Post-Block (Post-MK-801) phase. Initial stimulation used 1 mM N M D A (Pre-Block), followed by blockade of surface NMDARs by co-application of 1 mM N M D A and 5 pM MK-801 (Post-Block). Application of N M D A + MK-801 continued for approximately 4 s beyond what is depicted in figure. This paradigm was repeated to ensure full block of all surface NMDARs. B) Example of N M D A R current recovery following block by MK-801. A representative series of recordings from a YAC72 MSN showing NMDA-evoked current recovery following electrophysiological silencing of surface NMDARs; each current trace represents average of 3 consecutive responses (every 5 min) to 500 ms pulses of 1 mM NMDA. 109 0 500 1000 1500 2000 Time (ms) n o Figure 11. The rate of whole-cell current recovery in YAC72 MSNs following MK-801 binding is significantly faster than in WT MSNs. Plot of recovery of NMDA-evoked Uuc (integrated current, normalized to C m ) over time shows that N M D A R current recovery is significantly faster in YAC72 vs. WT MSNs by 10 min post-block. Each data point represents mean ± SEM from n = 15, 10, 6 (WT) and 8, 6, 5 (YAC72) cells for 5, 10 and 15 min time points, respectively, post-block by MK-801. Significant difference in recovery of current, WT vs. YAC72 by two-way A N O V A ; effect of genotype F(l,44) = 18.39, PO.001; effect of time F(2,44) = 11.00, PO.001; *P< 0.05, **P<0.01 by Bonferroni post-test. I l l 1400 1200 § 1000-< 800-8) 600J g> 400 -I < 200 0 0 - • - W T - Q - Y A C 7 2 10 15 20 Time (min) following NMDAR Block Figure 12. Example of cytosolic Ca imaging experiment using the fluorescent Ca indicator Fura-2. During the initial monitoring phase, the resting C a 2 + level is determined. A rise in cytosolic Ca imaging is produced by a 5 min application of 500 9+ pM NMDA, and the NMDA-induced Ca peak is calculated accordingly. This example also shows a subsequent rise in cytosolic C a 2 + following the application of 2 pM 4Br-A23187, a calcium ionophore, demonstrating that our responses were not saturated by NMDA-induced Ca influx. The response shown is a mean of 13 MSNs, and in this example is not baseline-subtracted. 113 1.4 1.2 1.0 o CO n LL 0.8 CO CO LL 0.6 0.4 0.2 0.0 4Br-A23187 NMDA NMDA-induced C a 2 + peak Resting Ca 2 * level 600 1200 1800 Time (s) n=13 cells 2400 3000 114 Figure 13. Representative cytosolic Ca z responses of WT and Y A C 128 MSNs to 500 u.M NMDA. Intracellular C a 2 + was monitored using Fura-2. Responses shown are mean responses of one experiment each from WT (solid line, n=27 cells) and Y A C 128 (dashed line, n=21 cells) MSNs prior to, during, and following a 5 min application of 500 pM N M D A as indicated. 115 116 Figure 14. WT and Y A C 128 MSNs have similar cytosolic Ca responses to NMDA. A) Basal (n=10 experiments per genotype, total of 168 and 163 neurons for WT and Y A C 128, respectively, from 6 different batches of cultured neurons) and peak cytosolic C a 2 + responses to 100 uM N M D A (n=4 experiments per genotype, total of 73 and 68 neurons for WT and Y A C 128, respectively, from 2 different culture batches) or 500 uM N M D A (n=10 experiments per genotype, total of 168 and 163 neurons for WT and Y A C 128, respectively, from 6 different culture batches) in WT (black bars) or Y A C 128 (open bars) MSNs. Not significant between genotypes by paired t-test for basal cytosolic Ca 2 + , or responses to 100 or 500 pM NMDA. Data expressed as mean ± SEM. B) Peak cytosolic C a 2 + responses of WT (black bar) and Y A C 128 (open bar) MSNs to 500 uM N M D A using the low affinity C a 2 + indicator MagFura-2. n=4 experiments per genotype, 74 and 80 neurons total for WT and Y A C 128, respectively, from 2 different culture batches. No significant difference between genotypes by paired t-test. 117 A 0.6 n NMDA, jaM • WT • YAC128 B _ 0.81 MagFura-2 data, 4 exp'ts per genotype, 80 cells for Y128 and 74 cells for WT. Data from Dec-05 XL spreadsheet 118 Figure 15. Recovery from an NMDA-induced Ca z load in WT and Y A C 128 MSNs. Recovery of cytosolic Ca (imaged using Fura-2) towards basal levels following the application of 500 pM N M D A in WT (black squares, n=5 experiments, total of 91 neurons from 3 different culture batches) or Y A C 128 (open circles, n=5 experiments, total of 90 neurons, from 3 different culture batches) MSNs. Repeated measures ANOVA, effect of genotype F(l,20) = 33.78, PO.0001; effect of time F(4,20) = 4.04, *P<0.05; *P<0.05 at 15 min, *P<0.05 at 20 min, **P<0.01 at 25 min by Bonferroni post-test. 119 Figure 16. Contrasting responses of WT and Y A C 128 MSNs to different stimuli that increase cytosolic C a 2 + to similar peak levels. A) Representative mean responses of WT MSNs to 100 pM N M D A (solid line, n=14 cells) or 50 mM KC1 + 5 uM FPL64176 9+ (dotted line, n=23 cells) as indicated. Note that the peak amplitude of the cytosolic Ca response to either stimulus is similar. Each trace is representative of a single experiment. B) Peak cytosolic C a 2 + responses to 100 pM N M D A (n=6 experiments for WT, n=5 for YAC128; total of 108 and 86 neurons for WT and YAC128, respectively, from 3 different culture batches) or 50 mM KC1 + 5 pM FPL64176 (n=6 experiments per genotype, total of 107 and 106 neurons for WT and Y A C 128, respectively, from 3 different culture batches) in WT (black bars) or Y A C 128 (open bars) MSNs. No significant difference by two-way A N O V A ; no effect of treatment F(l , 19) = 1.99, P>0.05 or genotype (F(l,19)= 0.01, P>0,05. Data expressed as mean ± SEM. Intracellular C a 2 + was monitored using Fura-FF in these experiments. 121 03 o < o 00 CO LL CO CO 0.8 0.6 0.4 0.2 KCI+FPL NMDA 0 300 600 900 1200 1500 Time (s) r - 1.6 CM 03 O < 1.2 WT • YAC128 Figure 17. The impairment in recovery of Y A C 128 MSNs from a cytosolic Ca load is selective for NMDA-induced C a 2 + loads. Recovery of cytosolic C a 2 + towards pre-stimulus levels following application of 100 pM N M D A (WT: black squares; Y A C 128: open circles; n=5 experiments per genotype, total of 97 and 86 neurons for WT and Y A C 128, respectively, from 5 different culture batches) or 50 mM KC1 + 5 uM FPL64176 (WT: black triangles; Y A C 128: open triangles; n=6 experiments per genotype, total of 107 and 106 neurons for WT and Y A C 128, respectively, from 3 different culture batches). Significant difference in recovery of cytosolic Ca 2 + , WT vs. YAC128 (100 uM NMDA): two-way A N O V A ; effect of genotype F(l,21) = 36.76, PO.0001; *PO.05, **PO.01, ***PO.01 by Bonferroni post-test; effect of time F(2,21) = 2.23, P>0.05. Significant difference in recovery of cytosolic Ca 2 + , Y A C 128 (NMDA vs. KC1 + FPL64176): two-way A N O V A ; effect of treatment F(l,24) = 136.34, PO.0001; effect of time F(2,24) = 14.98, PO.0001; effect of interaction F(2,24) = 8.94, PO.01; # PO.05, # # # PO.001 by Bonferroni post-test. Significant difference in recovery of cytosolic Ca 2 + , WT (NMDA vs. KC1 + FPL64176): two-way A N O V A ; effect of treatment F(l,27) = 31,46, PO.0001; effect of time F(2,27) = 10.64, PO.001; @ @ @ P O . 0 0 1 by Bonferroni post-test. No significant difference in recovery of cytosolic Ca , WT vs. Y A C 128 (50 mM KC1 + 5 pM FPL64176): two way A N O V A ; effect of genotype F(l,39) = 0,20, P>0.05; effect of time F(3,39) = 30.94, PO.0001. 123 100i 0-i . • . • r -5 10 15 Time (min) Figure 18. Simultaneous monitoring of NMDA-induced changes in cytosolic Ca and A ^ m in WT and Y A C 128 MSNs. A) Representative mean cytosolic C a 2 + (solid line, Fura-2) and mitochondrial A^Fm (dashed line, rhodamine-123) responses of WT (top, n=20 cells) and Y A C 128 (bottom, n=15 cells) MSNs prior to, during, and following 5 min application of 500 uM N M D A as indicated. Note the relatively rapid recovery of C a 2 + in WT MSNs, compared to that seen in Y A C 128 MSNs, as well as the relative changes in A T m in each genotype. Expanded region of Y A C 128 timescale (bottom) illustrates the temporal order of changes in Ca (solid line) preceding changes in AT™ (dashed line) during the initial phase of response to N M D A (arrow indicates start of N M D A application). 125 + CM 2.5n 2.CH CO < 1 - 5 i o 00 CO LL? 0.5 0.0 WT NMDA > > 2 0 0 300 600 900 1200 1500 Time (s) + CM 03 o < 2.5 2.0 1.5 o r LO 11? 0.5H 0.0 YAC128 NMDA C a 2 * 0 300 600 900 1200 1500 Time (s) 1 2 l Nco 0.9 O s 0.6 0.3 0.0 he > 4 ^ 3 2 0 > T I — C a 2 * - - AH' T 320 340 360 Time (s) Figure 19. Y A C 128 MSNs undergo a more dramatic loss of A^ Fm as a consequence of N M D A challenge than WT MSNs, despite similar cytosolic C a 2 + loads. Top panel: basal and peak cytosolic Ca responses (measured using Fura-2) to 500 pM N M D A in WT (black bars) or Y A C 128 (open bars) MSNs. No significant difference between genotypes by paired t-test; data expressed as mean ± SEM. Bottom panel: Concurrent changes in A T m (AF/F) in WT (black bar) or Y A C 128 (open bar) MSNs, n= 8 paired experiments per genotype; total of 131 and 147 neurons for WT and YAC128, respectively, from 4 different culture batches. Significant difference between genotypes T O . 0 5 by paired t-test; data expressed as mean ± SEM. 127 Figure 20. Characteristics of resting mitochondria in WT and Y A C 128 MSNs. A) Determination of resting mitochondrial polarization (ATm) status (i.e. prior to any stimulation). Mitochondria in previously unstimulated WT and Y128 MSNs were maximally depolarized by the addition of 5 pM CCCP (see Methods), and the mean fluorescent signal for each genotype was quantified. n=6 paired experiments per genotype; total of 121 and 113 neurons for WT (black bar) and Y A C 128 (open bar), respectively, from 3 different culture batches. No significant difference between genotypes by paired t-test; data expressed as mean ± SEM. B) Depolarization of resting mitochondria by 5 pM CCCP in previously unstimulated Y A C 128 MSNs results in the release of very little free C a 2 + from the mitochondria into the cytosol. Mean cytosolic C a 2 + (solid line, Fura-FF) and A ^ m (dashed line, rhodamine-123) responses of 13 MSNs. 129 • W T • YAC128 0.6 CM 03 O 0.4 < o 00 C O C O C O 0.2] 0.0-CCCP Ca 2 + m r4 2 3 > > 0 400 600 800 1000 Time (s) 130 Figure 21. Mitochondria in both WT and Y A C 128 MSNs have a similar capability to buffer transient cytosolic Ca increases produced by short application of NMDA. A) A 94-representative example of a mitochondrial Ca uptake experiment, showing the mean cytosolic C a 2 + responses (± SEM) of 13 WT MSNs, to a 60 s application of 500 pJvl N M D A followed 2 min later by a 60 s application of 5 uM CCCP (in Ca2+-free buffer). Fura-FF was used as the C a 2 + indicator for these experiments. The hatched areas, corresponding to the stimulus duration, were used in A U C calculations to determine C/N ratios as described in the Methods. B) C/N ratios for WT (black bar, n=8 experiments) and Y A C 128 (open bar, n=6 experiments) MSNs (total of 92 and 105 neurons for WT and Y A C 128, respectively, from 3 - 4 different culture batches). No significant difference by unpaired t-test; data expressed as mean ± SEM. 131 • WT • YAC128 132 Figure 22. Recovery of Y A C 128 MSNs from a cytosolic Ca load produced by a short application Of N M D A is similar in timecourse to that of WT MSNs. The recovery of WT and Y A C 128 MSNs from peak Ca towards prestimulus levels following a 60 s application of 500 uM N M D A (as performed to calculate the C/N ratios) was quantified by calculating the mean time of recovery to 50% and 25% of the peak. n=8 experiments and 92 cells total for WT; n=6 experiments and 105 cells total for Y A C 128. No significant difference between genotypes by unpaired t-test. 133 • WT • YAC128 Figure 23. The effect of the mPT inhibitor CsA on simultaneously measured peak cytosolic C a 2 + and A ^ in WT and YAC128 MSNs. A) Peak cytosolic C a 2 + in WT MSNs (black bars; with 10 pJVl CsA: n= 6 experiments, 81 neurons total; without 10 p.M CsA: n=6 experiments, 109 neurons total; from 3-4 different culture batches) and Y A C 128 MSNs (open bars; with 10 u.M CsA: n=5 experiments, 106 neurons total; without 10 pM CsA: n=7 experiments, 88 neurons total; from 3 - 4 different culture batches) following a 5 min application of 500 uM NMDA, with or without 10 pM CsA present in all solutions as indicated. *P<0.05 by unpaired t-test comparing treatments within genotype; data expressed as mean ± SEM. Intracellular calcium was monitored using Fura-FF in these experiments. B) Simultaneously observed changes in (AF/F). No significant difference by unpaired t-test within genotypes; data expressed as mean ± SEM. 135 136 Figure 24. The effect of the mPT inhibitor BkA on simultaneously measured peak cytosolic C a 2 + and A ¥ m in WT and Y A C 128 MSNs. A) Peak cytosolic C a 2 + in WT MSNs (black bars; with BkA: n=9 experiments, 135 cells total; without BkA: n=10 experiments, 129 cells total; from 5 different culture batches) and Y A C 128 MSNs (open bars; with 5 uM BkA: n=10 experiments, 145 cells total; without 5 uM BkA: n=9 experiments, 148 cells total; from 5 different culture batches) following a 5 min application of 500 uM N M D A , with or without 5 uM BkA present in all solutions as indicated. No significant difference by unpaired t-test within genotypes; data expressed as mean ± SEM. B) Simultaneously observed changes in A ^ m (AF/F). T O . 0 5 by unpaired t-test comparing treatments within genotype; data expressed as mean ± SEM. 137 138 Figure 25. A general overview of the main excitotoxic events in the Y A C HD mouse models of varying htt polyQ length. Previous research established that enhanced NMDAR activation (step 1) leads to elevated peak cytosolic C a 2 + (step 2) and subsequent mitochondrial depolarization (step 3). Consequently, activation of caspases (step 4) and apoptosis (step 5) in YAC46 and YAC72 MSNs occurs. In the Y A C 128 mouse model, steps 1 and 2 do not appear to play a role in the enhancement of apoptosis for Y A C 128 versus WT MSNs, as the work presented here indicates that neither the N M D A R peak current nor the resultant peak in cytosolic C a 2 + is any different from that seen in WT MSNs. The key excitotoxic step augmented in Y A C 128 MSNs appears to occur at the level of the mitochondria (step 3, mitochondrial depolarization), and yet is still dependent on NMDAR activation. The release of free C a 2 + from the mitochondria back into the cytosol (step 3 a, recently taken up downstream of N M D A R activation) follows mitochondrial depolarization. Thus, the increased sensitivity to NMDA-induced toxicity occurs at a later point in the sequence of events leading to apoptotic death in Y A C 128 compared with YAC72 MSNs. 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