Identifying molecular drivers of pathology provides potential therapeutic targets. Differentiating between drivers and coincidental molecular alterations presents a major challenge. Variation unrelated to pathology further complicates transcriptomic, proteomic and metabolomic studies which measure large numbers of individual molecules. To overcome these challenges towards the goal of determining drivers of Huntington's disease (HD), we generated an allelic series of HD knock-in mice with graded levels of phenotypic severity for comparison with molecular alterations. RNA-sequencing analysis of this series reveals high numbers of transcripts with level alterations that do not correlate with phenotypic severity. These discorrelated molecular changes are unlikely to be drivers of pathology allowing an exclusion-based strategy to provide a short list of driver candidates. Further analysis of the data shows that a majority of transcript level changes in HD knock-in mice involve alteration of the rate of mRNA processing and/or degradation rather than solely being due to alteration of transcription rate. The overall strategy described can be applied to assess the influence of any molecular change on pathology for diseases where different mutations cause graded phenotypic severity.

Chronic ethanol consumption increases sensitivity of the mitochondrial permeability transition (MPT) pore induction in liver. Ca(2+) promotes MPT pore opening, and genetic ablation of cyclophilin D (CypD) increases the Ca(2+) threshold for the MPT. We used wild-type (WT) and CypD-null (CypD(-/-)) mice fed a control or an ethanol-containing diet to investigate the role of the MPT in ethanol-mediated liver injury. Ca(2+)-mediated induction of the MPT and mitochondrial respiration were measured in isolated liver mitochondria. Steatosis was present in WT and CypD(-/-) mice fed ethanol and accompanied by increased terminal deoxynucleotidyl transferase dUTP-mediated nick-end label-positive nuclei. Autophagy was increased in ethanol-fed WT mice compared with ethanol-fed CypD(-/-) mice, as reflected by an increase in the ratio of microtubule protein 1 light chain 3B II to microtubule protein 1 light chain 3B I. Higher levels of p62 were measured in CypD(-/-) than WT mice. Ethanol decreased mitochondrial respiratory control ratios and select complex activities in WT and CypD(-/-) mice. Ethanol also increased CypD protein in liver of WT mice. Mitochondria from control- and ethanol-fed WT mice were more sensitive to Ca(2+)-mediated MPT pore induction than mitochondria from their CypD(-/-) counterparts. Mitochondria from ethanol-fed CypD(-/-) mice were also more sensitive to Ca(2+)-induced swelling than mitochondria from control-fed CypD(-/-) mice but were less sensitive than mitochondria from ethanol-fed WT mice. In summary, CypD deficiency was associated with impaired autophagy and did not prevent ethanol-mediated steatosis. Furthermore, increased MPT sensitivity was observed in mitochondria from ethanol-fed WT and CypD(-/-) mice. We conclude that chronic ethanol consumption likely lowers the threshold for CypD-regulated and -independent characteristics of the ethanol-mediated MPT pore in liver mitochondria.

Recent evidence suggests that interneurons are involved in the pathophysiology of Huntington Disease (HD). Abnormalities in the function of interneurons expressing the calcium buffer parvalbumin (PV) have been observed in multiple mouse models of HD, although it is not clear how PV-positive interneuron dysfunction contributes to behavioral and synaptic deficits. Here, we use the cre-lox system to drive expression of mutant huntingtin (mthtt) in parvalbumin (PV)-positive neurons and find that mutant mice exhibit diffuse mthtt immunoreactivity in PV-rich areas at 10months of age and mthtt aggregates in PV-positive processes at 24months of age. At midlife, mutant mice are hyperactive and display impaired GABA release in the motor cortex, characterized by reduced miniature inhibitory events and severely blunted responses to gamma frequency stimulation, without a loss of PV-positive interneurons. In contrast, 24month-old mutant mice show normalized behavior and responses to gamma frequency stimulation, possibly due to compensatory changes in pyramidal neurons or the formation of inclusions with age. These data indicate that mthtt expression in PV-positive neurons is sufficient to drive a hyperactive phenotype and suggest that mthtt-mediated dysfunction in PV-positive neuronal populations could be a key factor in the hyperkinetic behavior observed in HD. Further clarification of the roles for specific PV-positive populations in this phenotype is warranted to definitively identify cellular targets for intervention.

Huntington's disease (HD) causes preferential loss of a subset of neurons in the brain although the huntingtin protein is expressed broadly in various neural cell types, including astrocytes. Glutamate-mediated excitotoxicity is thought to cause selective neuronal injury, and brain astrocytes have a central role in regulating extracellular glutamate. To determine whether full-length mutant huntingtin expression causes a cell-autonomous phenotype and perturbs astrocyte gliotransmitter release, we studied cultured cortical astrocytes from BACHD mice. Here, we report augmented glutamate release through Ca(2+)-dependent exocytosis from BACHD astrocytes. Although such release is usually dependent on cytosolic Ca(2+) levels, surprisingly, we found that BACHD astrocytes displayed Ca(2+) dynamics comparable to those in wild type astrocytes. These results point to a possible involvement of other factors in regulating Ca(2+)-dependent/vesicular release of glutamate from astrocytes. We found a biochemical footprint that would lead to increased availability of cytosolic glutamate in BACHD astrocytes: i) augmented de novo glutamate synthesis due to an increase in the level of the astrocyte specific mitochondrial enzyme pyruvate carboxylase; and ii) unaltered conversion of glutamate to glutamine, as there were no changes in the expression level of the astrocyte specific enzyme glutamine synthetase. This work identifies a new mechanism in astrocytes that could lead to increased levels of extracellular glutamate in HD and thus may contribute to excitotoxicity in this devastating disease.

Huntington's disease (HD) is a devastating autosomal-dominant neurodegenerative disorder initiated by an abnormally expanded polyglutamine in the huntingtin protein. Determining the contribution of specific factors to the pathogenesis of HD should provide rational targets for therapeutic intervention. One suggested contributor is the type 2 transglutaminase (TG2), a multifunctional calcium dependent enzyme. A role for TG2 in HD has been suggested because a polypeptide-bound glutamine is a rate-limiting factor for a TG2-catalyzed reaction, and TG2 can cross-link mutant huntingtin in vitro. Further, TG2 is up regulated in brain areas affected in HD. The objective of this study was to further examine the contribution of TG2 as a potential modifier of HD pathogenesis and its validity as a therapeutic target in HD. In particular our goal was to determine whether an increase in TG2 level, as documented in human HD brains, modulates the well-characterized phenotype of the R6/2 HD mouse model. To accomplish this objective a genetic cross was performed between R6/2 mice and an established transgenic mouse line that constitutively expresses human TG2 (hTG2) under control of the prion promoter. Constitutive expression of hTG2 did not affect the onset and progression of the behavioral and neuropathological HD phenotype of R6/2 mice. We found no alterations in body weight changes, rotarod performances, grip strength, overall activity, and no significant effect on the neuropathological features of R6/2 mice. Overall the results of this study suggest that an increase in hTG2 expression does not significantly modify the pathology of HD.

Huntington Disease (HD) is an autosomal dominant neurological disorder characterized by motor, psychiatric and cognitive disturbances. Recent evidence indicates that the viability and function of cerebellar Purkinje cells (PCs) are compromised in an aggressive mouse model of HD. Here we investigate whether this is also the case in the HdhQ200 knock-in mouse model of HD. Using quantitative-real time-PCR and immunofluorescence, we observed a loss of the PC marker and calcium buffer calbindin in 50week-old symptomatic mice. Reductions were also observed in parvalbumin and glutamic acid decarboxylase protein expression, most markedly in the molecular cell layer. Stereological analysis revealed an overall reduction in the PC population in HdhQ200/Q200 mice by nearly 40%, and loose patch electrophysiology of remaining PCs indicated a reduction in firing rate in HD mice compared to control littermates. Taken together, these data demonstrate that PC survival and function are compromised in a mouse model of adult-onset HD and suggest that further experiments should investigate the contribution of PC death and dysfunction to HD-associated motor impairment.

Huntington Disease (HD) is a devastating neurological disorder characterized by progressive deterioration of psychiatric, motor, and cognitive function. Purkinje cells (PCs), the output neurons of the cerebellar cortex, have been found to be vulnerable in multiple CAG repeat disorders, but little is known about the involvement of PC dysfunction in HD. To investigate possible PC abnormalities, we performed quantitative real time PCR, Western blot analysis, and immunohistochemistry experiments to explore the changes in PC markers in the R6/2 mouse model of severe HD. There were reductions in the transcript and protein levels of the calcium-binding proteins parvalbumin and calbindin, as well as the enzyme glutamic acid decarboxylase 67. Immunohistochemistry supported these results, with the most substantial changes occurring in the PC layer. To determine whether the reductions in PC marker expression were due to cell loss, we performed stereology on both presymptomatic and end-stage R6/2 mice. Stereological counts indicated a significant reduction in PC number by end-stage but no change in presymptomatic animals (4 weeks of age). To assess cellular function prior to cell loss and symptom onset, we measured spontaneous firing in PCs from 4-week old animals and found a striking deficit in PC firing as indicated by a 57% decrease in spike rate. Interestingly, huntingtin inclusions were not widely observed in PCs until 12 weeks of age, indicating that soluble huntingtin and/or abnormalities in other cell types may contribute to PC dysfunction. Considering the roles for PCs in motor control, these data suggest that early PC dysfunction potentially contributes to motor impairment in this model of HD.

Immunophilins are receptors for immunosuppressive drugs such as the macrolides cyclosporin A (CsA) and FK506; correspondingly these immunophilins are referred to as cyclophilins and FK506-binding proteins (FKBPs). In particular, CsA targets cyclophilin D (CypD), which can modulate mitochondrial Ca(2+) dynamics. Since mitochondria have been implicated in the regulation of astrocytic cytosolic Ca(2+) (Ca(cyt)(2+)) dynamics and consequential Ca(2+)-dependent exocytotic release of glutamate, we investigated the role of CypD in this process. Cortical astrocytes isolated from CypD deficient mice Ppif(-/-) displayed reduced mechanically induced Ca(cyt)(2+) increases, even though these cells showed augmented exocytotic release of glutamate, when compared to responses obtained from astrocytes isolated from wild-type mice. Furthermore, acute treatment with CsA to inhibit CypD modulation of mitochondrial Ca(2+) buffering, or with FK506 to inhibit FKBP12 interaction with inositol-trisphosphate receptor of the endoplasmic reticulum, led to similar reductive effects on astrocytic Ca(cyt)(2+) dynamics, but also to an enhanced Ca(2+)-dependent exocytotic release of glutamate in wild-type astrocytes. These findings point to a possible role of immunophilin signal transduction pathways in astrocytic modulation of neuronal activity at the tripartite synapse.

Disruption of 14-3-3 function by alpha-synuclein has been implicated in Parkinson's disease. As 14-3-3s are important regulators of cell death pathways, disruption of 14-3-3s could result in the release of pro-apoptotic factors, such as Bax. We have previously shown that overexpression of 14-3-3θ reduces cell loss in response to rotenone and MPP(+) in dopaminergic cell culture and reduces cell loss in transgenic C. elegans that overexpress alpha-synuclein. In this study, we investigate the mechanism for 14-3-3θ's neuroprotection against rotenone toxicity. While 14-3-3s can inhibit many pro-apoptotic factors, we demonstrate that inhibition of one factor in particular, Bax, is important to 14-3-3s' protection against rotenone toxicity in dopaminergic cells. We found that 14-3-3θ overexpression reduced Bax activation and downstream signaling events, including cytochrome C release and caspase 3 activation. Pharmacological inhibition or shRNA knockdown of Bax provided protection against rotenone, comparable to 14-3-3θ's neuroprotective effects. A 14-3-3θ mutant incapable of binding Bax failed to protect against rotenone. These data suggest that 14-3-3θ's neuroprotective effects against rotenone are at least partially mediated by Bax inhibition and point to a potential therapeutic role of 14-3-3s in Parkinson's disease.

Huntington's disease (HD) is an incurable autosomal-dominant neurodegenerative disorder initiated by an abnormally expanded polyglutamine domain in the huntingtin protein. It is proposed that abnormal mitochondrial Ca2+ capacity results in an increased susceptibility to mitochondrial permeability transition (MPT) induction that may contribute significantly to HD pathogenesis. The in vivo contribution of these hypothesized defects remains to be elucidated. In this proof-of-principle study, we examined whether increasing mitochondrial Ca2+ capacity could ameliorate the well-characterized phenotype of the R6/2 transgenic mouse model. Mouse models lacking cyclophilin D demonstrate convincingly that cyclophilin D is an essential component and a key regulator of MPT induction. Mitochondria of cyclophilin D knockout mice are particularly resistant to Ca2+ overload. We generated R6/2 mice with normal, reduced or absent cyclophilin D expression and examined the effect of increasing mitochondrial Ca2+ capacity on the behavioral and neuropathological features of the R6/2 model. A predicted outcome of this approach was the finding that cyclophilin D deletion enhanced the R6/2 brain mitochondria Ca2+ capacity significantly. Increased neuronal mitochondrial Ca2+ capacity failed to ameliorate either the behavioral and neuropathological features of R6/2 mice. We found no alterations in body weight changes, lifespan, RotaRod performances, grip strength, overall activity and no significant effect on the neuropathological features of R6/2 mice. The results of this study demonstrate that increasing neuronal mitochondrial Ca2+-buffering capacity is not beneficial in the R6/2 mouse model of HD.

Huntington's disease (HD) is a progressive autosomal dominant neurodegenerative disorder caused by an abnormally expanded CAG repeat in the huntingtin gene. Expansion of the repeat above a critical length results in severe neurodegeneration primarily of the medium spiny neurons in the striatum. A consistent neuropathological hallmark of HD, and others neurodegenerative disorders caused by a pathological expansion of a glutamine track, is the presence of neuronal inclusion bodies or aggregates composed primarily of the mutated proteins but that also sequester various proteins such as components of the 19S and 20S proteasomes. Given that most of the inclusion bodies contain proteins that are post-translationally conjugated with ubiquitin residues, a canonical marker for protein degradation by the ubiquitin proteasome system, a connection between the formation of aggregates and the ubiquitin proteasome system can be postulated. In support of this hypothesis, the presence of mutant protein aggregates is enhanced in mouse and cellular models of HD in response to inhibition of the proteasome core catalytic activities. Further, several reports provide evidence that the ubiquitin proteasome system is impaired in postmortem HD tissues and in HD cellular models overexpressing mutant huntingtin. The recent development of specific reporters designed to examine specific steps of the ubiquitin proteasome system in HD cellular models has made it possible to identify specific steps of the ubiquitin proteasome system altered in HD. Further, in vitro enzymatic studies have revealed that specific fibrillar conformational assemblies of the mutant huntingtin inhibit the catalytic activity of the 26S proteasome. Because expression of the mutant huntingtin is associated with impairment of several cellular processes that may indirectly affect the ubiquitin proteasome function, the exact contribution of ubiquitin proteasome system dysfunction to the etiology of HD remains to be elucidated. In this chapter we will examine the current literature relevant to the role of the ubiquitin proteasome system in HD and other polyglutamine disorders.

Chronic ethanol consumption increases mitochondrial oxidative stress and sensitivity to form the mitochondrial permeability transition pore (MPTP). The mechanism responsible for increased MPTP sensitivity in ethanol-exposed mitochondria and its relation to mitochondrial Ca2+handling is unknown. Herein, we investigated whether increased sensitivity to MPTP induction in liver mitochondria from ethanol-fed rats compared with controls is related to an ethanol-dependent change in mitochondrial Ca2+accumulation. Liver mitochondria were isolated from control and ethanol-fed rats, and Ca2+-mediated induction of the MPTP and mitochondrial Ca2+retention capacity were measured. Levels of proposed MPTP proteins as well as select pro- and antiapoptotic proteins were measured along with gene expression. We observed increased steatosis and TUNEL-stained nuclei in liver of ethanol-fed rats compared with controls. Liver mitochondria from ethanol-fed rats had increased levels of proapoptotic Bax protein and reduced Ca2+retention capacity compared with control mitochondria. We observed increased cyclophilin D (Cyp D) gene expression in liver and protein in mitochondria from ethanol-fed animals compared with controls, whereas there was no change in the adenine nucleotide translocase and voltage-dependent anion channel. Together, these results suggest that enhanced sensitivity to Ca2+-mediated MPTP induction may be due, in part, to higher Cyp D levels in liver mitochondria from ethanol-fed rats. Therefore, therapeutic strategies aimed at normalizing Cyp D levels may be beneficial in preventing ethanol-dependent mitochondrial dysfunction and liver injury.

The presence of aggregates of abnormally expanded polyglutamine (polyQ)-containing proteins are a pathological hallmark of a number of neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxia-3 (SCA3). Previous studies in cellular, Drosophila, and mouse models of HD and SCA have shown that neurodegeneration can be prevented by manipulations that inhibit polyQ aggregation. We have shown that the UL97 kinase of the human cytomegalovirus (HCMV) prevents aggregation of the pp71 and pp65 viral tegument proteins. To explore whether UL97 may act as a general antiaggregation factor, we examined whether UL97 prevents aggregation of cellular non-polyQ and polyQ proteins. We report that UL97 prevents the deposition of aggregates of two non-polyQ proteins: a protein chimera (GFP170*) composed of the green fluorescent protein and a fragment of the Golgi Complex protein (GCP-170) and a chimera composed of the red fluorescent protein (RFP) fused to the Werner syndrome protein (WRN), a RecQ helicase and exonuclease involved in DNA repair. Furthermore, we show that UL97 inhibits aggregate deposition in cellular models of HD and SCA3. UL97 prevents the deposition of aggregates of the mutant huntingtin exon 1 containing 82 glutamine repeats (HttExon1-Q82) or full length ataxin-3 containing a 72 polyQ track (AT3-72Q). The kinase activity of UL97 appears critical, as the kinase-dead UL97 mutant (K335M) fails to prevent aggregate formation. We further show that UL97 disrupts nuclear PML bodies and decreases p53-mediated transcription. The universality of the antiaggregation effect of UL97 suggests that UL97 targets a key cellular factor that regulates cellular aggregation mechanisms. Our results identify UL97 as a novel means to modulate polyQ aggregation and suggest that UL97 can serve as a novel tool to probe the cellular mechanisms that contribute to the formation of aggregates in polyglutamine disorders.

The aggregation of mutant polyglutamine (polyQ) proteins has sparked interest in the role of protein quality-control pathways in Huntington's disease (HD) and related polyQ disorders. Employing a novel knock-in HD mouse model, we provide in vivo evidence of early, sustained alterations of autophagy in response to mutant huntingtin (mhtt). The HdhQ200 knock-in model, derived from the selective breeding of HdhQ150 knock-in mice, manifests an accelerated and more robust phenotype than the parent line. Heterozygous HdhQ200 mice accumulate htt aggregates as cytoplasmic aggregation foci (AF) as early as 9 weeks of age and striatal neuronal intranuclear inclusions (NIIs) by 20 weeks. By 40 weeks, striatal AF are perinuclear and immunoreactive for ubiquitin and the autophagosome marker LC3. Striatal NIIs accumulate earlier in HdhQ200 mice than in HdhQ150 mice. The earlier appearance of aggregate pathology in HdhQ200 mice is paralleled by earlier and more rapidly progressive motor deficits: progressive imbalance and decreased motor coordination by 50 weeks, gait deficits by 60 weeks and gross motor impairment by 80 weeks of age. At 80 weeks, heterozygous HdhQ200 mice exhibit striatal and cortical astrogliosis and a approximately 50% reduction in striatal dopamine receptor binding. Increased LC3-II protein expression, which is noted early and sustained throughout the disease course, is paralleled by increased expression of the autophagy-related protein, p62. Early and sustained expression of autophagy-related proteins in this genetically precise mouse model of HD suggests that the alteration of autophagic flux is an important and early component of the neuronal response to mhtt.

One of the most notable developments in research concerning Alzheimer's disease is the solid evidence directly linking glycogen synthase kinase-3 (GSK-3) to most, if not all, of the major mechanisms known to contribute to the neuropathology of the disease. GSK-3 interacts with multiple components of the plaque-producing amyloid system associated with Alzheimer's disease. GSK-3 participates in phosphorylating the microtubule-binding protein tau, which may contribute to the formation of neurofibrillary tangles in Alzheimer's disease. GSK-3 interacts with presenilin and other Alzheimer's disease-associated proteins. GSK-3 has a central role in neuronal plasticity and memory. GSK-3 promotes both inflammation and apoptosis. These many links between GSK-3 and Alzheimer's disease are discussed in this chapter.

Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disease caused by an abnormally expanded CAG repeat in the HD gene. Ubiquitylated aggregates containing mutant huntingtin protein in neurons are hallmarks of HD. Misfolded mutant huntingtin monomers, oligomers, or aggregates may be a result of, and cause, ubiquitin‐ proteasome dysfunction. To investigate the ubiquitin‐proteasome system we designed a series of firefly luciferase reporters targeted selectively to different points along this pathway. These reporters were used to monitor ubiquitin‐proteasome system function in a striatal cell culture model of HD. Ubiquitylation processes were not reduced in mutant huntingtin cells but recognition or degradation of ubiquitylated substrates was decreased. We also found mutant huntingtin expressing cells had slight but significant decreases in chymotrypsin‐like and caspase‐like activities, and an unexpected increase in trypsin‐like activity of the proteasome core. General proteasome core inhibitors, as well as selective caspase‐like activity inhibitors, were less effective in mutant cells. Finally, treatment with 3‐nitropropionic acid, a succinate dehydrogenase inhibitor, had opposite effects on the ubiquitin‐proteasome system with activation in wild‐type and decreased activity in mutant huntingtin expressing cells. The results of these experiments show clearly selective disruption of the ubiquitin‐proteasome system in this cell culture model of HD. The high throughput tools that we have designed and optimized will also be useful in identifying compounds that alter ubiquitin‐proteasome system function and to investigate other neurodegenerative diseases such Alzheimer's disease and Parkinson's disease. © 2007 Wiley‐Liss, Inc.

Abstract Cystamine significantly improved motor deficits and extended survival in mouse models of Huntington's disease (HD); however, the precise mechanism(s) by which cystamine and the related compound cysteamine are beneficial remain to be elucidated. Using clonal striatal cell lines from wild-type (STHdhQ7/HdhQ7) and mutant huntingtin knock-in (STHdhQ111/HdhQ111) mice, we have tested the hypothesis that cystamine and cysteamine could be beneficial by preventing the depolarization of mitochondria in cell cultures. Treatment with 3-nitroproprionic acid (3-NP), a mitochondrial complex II inhibitor, induces mitochondrial depolarization and cell death of mutant HD striatal cells but not of wild-type cells. The 3-NP-mediated decrease in the mitochondrial membrane potential was attenuated by 50 microm cystamine and completely inhibited by 250 microm cystamine. Similar results were obtained using cysteamine (50-500 microm). In addition, both cystamine and cysteamine significantly attenuated the 3-NP-induced cell death. Treatment of mutant HD striatal cells with 3-NP resulted in a robust decrease in the cellular and mitochondrial levels of glutathione (GSH) compared with cells exposed to the vehicle alone. Pre-treatment of the cells with cystamine and cysteamine completely prevented the 3-NP-mediated decrease in cellular and mitochondrial GSH levels. Incubation with L-buthionine (S,R) sulfoximine (BSO) 250 microm in combination with cystamine (250 microm) or cysteamine (250 microm) prior to being treated with 3-NP completely prevented the beneficial effects of cystamine and cysteamine on the 3-NP-mediated mitochondrial depolarization. These results demonstrate that cystamine and cysteamine prevent the 3-NP-induced mitochondrial depolarization of HD striatal cell cultures.

The inheritance of a long CAG repeat causes several late onset neurological disorders including Huntington's disease (HD). Longer CAG repeats correlate with earlier onset of HD suggesting an increased toxicity for the products of long repeat alleles. PCR based data has been used to show that HD CAG repeat expansion beyond the inherited length occurs in affected tissues indicating a possible role for somatic instability in the disease process. PCR, however, is prone to artifacts resulting from expansion of repeat sequences during amplification. We describe a method to distinguish between CAG repeat expansions that exist in vivo and those that potentially occur during PCR. The method involves size fractionation of genomic restriction fragments containing the expanded repeats followed by PCR amplification. The application of this method confirms the presence of somatic expansions in the brains of a knock-in mouse model of HD.

Huntington's disease (HD) is a progressive neurodegenerative disease characterized by a severe neuronal loss that occurs primarily in the neostriatum. It has been postulated that mitochondria dysfunction and oxidative stress may play significant roles in the etiology of the disease. Indeed, markers of oxidative stress damage have been detected in the brains of HD patients and in mouse models of HD. In this study, we evaluate the changes in the levels of the potent, endogenous antioxidant glutathione and enzymes involved in its metabolism or recycling in the cortex and striatum of an extensively studied HD mouse model (R6/2). In both cortex and striatum, the levels of cellular glutathione were not significantly different in the R6/2 mice when compared with littermate wild type controls. Remarkably, the levels of glutathione were significantly increased in mitochondria isolated from the cortex and striatum of R6/2 mice when compared with wild type control mice. This specific increase in the levels of glutathione in mitochondria suggests that a compensatory mechanism is induced in the R6/2 mice to protect against an increase in oxidative stress in mitochondria.

The 'expanded' HD CAG repeat that causes Huntington's disease (HD) encodes a polyglutamine tract in huntingtin, which first targets the death of medium-sized spiny striatal neurons. Mitochondrial energetics, related to N-methyl-d-aspartate (NMDA) Ca2+-signaling, has long been implicated in this neuronal specificity, implying an integral role for huntingtin in mitochondrial energy metabolism. As a genetic test of this hypothesis, we have looked for a relationship between the length of the HD CAG repeat, expressed in endogenous huntingtin, and mitochondrial ATP production. In STHdhQ111 knock-in striatal cells, a juvenile onset HD CAG repeat was associated with low mitochondrial ATP and decreased mitochondrial ADP-uptake. This metabolic inhibition was associated with enhanced Ca2+-influx through NMDA receptors, which when blocked resulted in increased cellular [ATP/ADP]. We then evaluated [ATP/ADP] in 40 human lymphoblastoid cell lines, bearing non-HD CAG lengths (9-34 units) or HD-causing alleles (35-70 units). This analysis revealed an inverse association with the longer of the two allelic HD CAG repeats in both the non-HD and HD ranges. Thus, the polyglutamine tract in huntingtin appears to regulate mitochondrial ADP-phosphorylation in a Ca2+-dependent process that fulfills the genetic criteria for the HD trigger of pathogenesis, and it thereby determines a fundamental biological parameter--cellular energy status, which may contribute to the exquisite vulnerability of striatal neurons in HD. Moreover, the evidence that this polymorphism can determine energy status in the non-HD range suggests that it should be tested as a potential physiological modifier in both health and disease.