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Proc Natl Acad Sci U S A. Feb 15, 2005; 102(7): 2602–2607.
Published online Feb 3, 2005. doi:  10.1073/pnas.0409402102
PMCID: PMC548984
Neuroscience

Disturbed Ca2+ signaling and apoptosis of medium spiny neurons in Huntington's disease

Abstract

Huntington's disease (HD) is caused by polyglutamine expansion (exp) in huntingtin. Here, we used a yeast artificial chromosome (YAC) transgenic mouse model of HD to investigate the connection between disturbed calcium (Ca2+) signaling and apoptosis of HD medium spiny neurons (MSN). Repetitive application of glutamate elevates cytosolic Ca2+ levels in MSN from the YAC128 mouse but not in MSN from the wild-type or control YAC18 mouse. Application of glutamate results in apoptosis of YAC128 MSN but not wild-type or YAC18 MSN. Analysis of glutamate-induced apoptosis of the YAC128 MSN revealed that (i) actions of glutamate are mediated by mGluR1/5 and NR2B glutamate receptors; (ii) membrane-permeable inositol 1,4,5-trisphosphate receptor blockers 2-APB and Enoxaparin (Lovenox) are neuroprotective; (iii) apoptosis involves the intrinsic pathway mediated by release of mitochondrial cytochrome c and activation of caspases 9 and 3; (iv) apoptosis requires mitochondrial Ca2+ overload and can be prevented by the mitochondrial Ca2+ uniporter blocker Ruthenium 360; and (v) apoptosis involves opening of mitochondrial permeability transition pore (MPTP) and can be prevented by MPTP blockers such as bongkrekic acid, Nortriptyline, Desipramine, Trifluoperazine, and Maprotiline. These findings describe a pathway directly linking disturbed Ca2+ signaling and degeneration of MSN in the caudate nucleus in HD. These findings also suggest that Ca2+ and MPTP blockers may have a therapeutic potential for treatment of HD.

Keywords: Enoxaparin, neurodegeneration, transgenic mouse, mitochondria, Lovenox

Huntington's disease (HD) has onset usually between 35 and 50 years with chorea and psychiatric disturbances and gradual but inexorable intellectual decline to death after 15–20 years (1). Neuropathological analysis reveals selective and progressive neuronal loss in the striatum (1), particularly affecting the GABAergic medium spiny neurons (MSN). At the molecular level, the cause of HD is a polyglutamine expansion (exp) in the amino terminus of huntingtin (Htt), a 350-kDa ubiquitously expressed cytoplasmic protein (2). Despite significant progress, cellular mechanisms that link the Httexp mutation with the disease are poorly understood (3).

A number of transgenic HD mouse models have been generated that reproduce many HD-like features (4). In the yeast artificial chromosome (YAC128) mouse model, the full-length human Htt protein with polyglutamine exp (128Q) is expressed under the control of its endogenous promoter and regulatory elements (5). The onset of a motor deficit before striatal neuronal loss in the YAC128 mouse model accurately recapitulates the progression of HD (5). Thus, the YAC128 mouse model is ideal for understanding the cellular mechanisms that lead to neurodegeneration in HD, as well as for validating potential therapeutic agents.

Previous studies demonstrated that Httexp facilitates activity of the NR2B subtype of NMDA receptors (NMDARs) (68) and the type 1 inositol 1,4,5-trisphosphate receptors (InsP3R1) (9). A connection between disturbed Ca2+ signaling and neuronal apoptosis is well established (10, 11), and we therefore proposed that Httexp-induced Ca2+ overload results in degeneration of MSN in HD (12). To test this hypothesis, we analyzed Ca2+ signals and apoptotic cell death in primary cultures of MSN from the YAC128 mice. Our results provide further support to the hypothesis that disturbed Ca2+ underlies neuronal cell death in HD (12) and allowed us to identify a number of potential therapeutic targets for HD treatment.

Materials and Methods

Primary Neuronal Cultures. Generation and breeding of YAC18 and YAC128 transgenic mice (FVBN/NJ background strain) are described in refs. 5 and 13. Heterozygous male YAC128 or YAC18 mice were crossed with the wild-type (WT) female mice and resulting litters were collected at postnatal days 1–2. The pups were genotyped by PCR with primers specific for exons 44 and 45 of human Htt gene and the medium spiny neuronal (MSN) or hippocampal neuronal (HN) cultures of WT, YAC18, and YAC128 mice were established and maintained as described in ref. 9.

Ca2+ Imaging Experiments. Fura-2 Ca2+ imaging experiments with 14- to 16-DIV (days in vitro) MSN cultures were performed as described in ref. 9, using a DeltaRAM illuminator, an IC-300 camera, and imagemaster pro software (all from PTI, South Brunswick, NJ). The cells were maintained in artificial cerebrospinal fluid (aCSF) (140 mM NaCl/5 mM KCl/1 mM MgCl2/2 mM CaCl2/10 mM Hepes, pH 7.3) at 37°C during measurements (PH1 heater, Warner Instruments, Hamden, CT). Fura-2 340/380 ratio images were collected every 6 sec for the duration of the experiment. Baseline (1–3 min) measurements were obtained before first pulse of glutamate. The 20 μM glutamate solution was dissolved in aCSF and 1-min pulses of 37°C glutamate solution (SH-27B in-line solution heater, Warner Instruments) were applied by using a valve controller (VC-6, Warner Instruments) driven by a square-pulse electrical waveform generator (Model 148A, Wavetek, San Diego). DHPG (3,5-dihydroxyphenylglycine)-induced Ca2+ responses have been measured in Ca2+-free aCSF (omitted CaCl2 from aCSF and supplemented with 100 μM EGTA). For the Enoxaparin experiments, MSN were preincubated in medium containing 200 μg/ml Enoxaparin for 3 h. Enoxaparin was included at 200 μg/ml in all solutions during Fura-2 loading and Ca2+ imaging steps.

TUNEL Staining Experiments. The 14-DIV MSN or HN were exposed for 8 h to a range of glutamate concentrations added to the culture medium. During exposure to glutamate, the cells were maintained in a cell culture incubator (humidified 5% CO2, 37°C). (+)MK-801, ifenprodil, MPEP [2-methyl-6-(phenylethynyl)pyridine hydrochloride], CPCCOEt {7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester}, and Ruthenium (Ru) 360 were added to cell culture media 30 min before the addition of glutamate. 2-APB, Enoxaparin, Z-VAD-FMK, Z-FA-FMK, Z-DEVD-FMK, Z-LEHD-FMK, Z-IETD-FMK, bongkrekic acid, Nortriptyline, Pirenzepine, Promethazine, Desipramine, Trifluoperazine, Thiothixene, and Maprotiline were added 2–3 h before glutamate exposure. Immediately after exposure to glutamate, neurons were fixed for 30 min in 4% paraformaldehyde plus 4% sucrose in PBS (pH 7.4), permeabilized for 5 min in 0.25% Triton X-100, and stained by using the DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer's instructions. Nuclei were counterstained with 5 μM propidium iodide (PI) (Molecular Probes). Coverslips were extensively washed with PBS and mounted in Mowiol 4-88 (Polysciences). FITC- and PI-fluorescent images were collected with an Olympus IX70 microscope with ×40 objectives, using a Cascade:650 camera (Roper Scientific) and metafluor software (Universal Imaging, Downingtown, PA). Four to six randomly chosen microscopic fields containing 200–300 MSN were captured, and the number of TUNEL-positive neuronal nuclei was calculated as a fraction of PI-positive neuronal nuclei in each microscopic field by an observer blinded to the nature of the sample. Nuclei of glial cells, identified by large size and weak PI staining, were not counted in the analysis. The fractions of TUNEL-positive nuclei determined for each microscopic field were averaged, and the results are presented as means ± SD (n = number of fields counted).

Planar Lipid Bilayer Experiments. Single-channel recordings of rat cerebellar InsP3R1 were performed as described in ref. 14. Briefly, rat cerebellar microsomes were isolated by differential centrifugation and fused to planar lipid bilayers. The activity of InsP3-gated channels was recorded at a 0-mV transmembrane potential by using 50 mM Ba2+ (trans) dissolved in Hepes (pH 7.35) as a charge carrier. The cis (cytosolic) chamber contained 110 mM Tris dissolved in Hepes (pH 7.35), 0.5 mM Na2ATP, and pCa 6.7 (0.2 mM EGTA plus 0.14 mM CaCl2). Cerebellar InsP3R1 was activated by addition of 2 μM InsP3 (Alexis) to the cis chamber. Different concentrations of Enoxaparin were added to the cis chamber. The InsP3-gated currents were amplified (OC-725, Warner Instruments), filtered at 1 kHz by low-pass 8-pole Bessel filter, digitized at 5 kHz (Digidata 1200, Axon Instruments), and stored on a computer hard drive and optical discs. For off-line computer analysis (pclamp 6, Axon Instruments), currents were filtered digitally at 500 Hz. For presentation of the current traces, data were filtered at 200 Hz.

Cytochrome c Release Assay. MSN cultures at 12- to 14-DIV were exposed to glutamate for 5 h, washed once with PBS, scraped off in the homogenization buffer (0.32 M sucrose/25 mM Hepes, pH 8.0/1 mM EDTA/1 mM DTT/protease inhibitors), homogenized, and clarified by a 60-min spin at 100,000 × g. The supernatant was collected as the soluble fraction, and the protein concentration of each sample was determined by using a BioRad protein assay kit. Both pellet and supernatant were lysed in SDS-loading buffer and boiled. Samples were resolved by SDS/PAGE, analyzed by Western blotting with anti-cytochrome c monoclonal antibody (BD Pharmingen), and quantified by densitometry.

Drugs. Ru360 and 2-APB were from Calbiochem. Glutamate, MPEP, CPCCOEt, (+)-MK801 maleate, and ifenprodil were purchased from Tocris. Z-VAD-FMK, Z-FA-FMK, Z-DEVD-FMK, Z-LEHD-FMK, and Z-IETD-FMK were from R & D Systems. Bongkrekic acid, Nortriptyline, Pirenzepine, Promethazine, Desipramine, Trifluoperazine, Thiothixene, and Maprotiline were from Sigma-Aldrich.

Results

Disturbed Ca2+ Signals in YAC128 MSN. Previous findings (69) suggested that glutamate should induce supranormal Ca2+ responses in HD MSN. To test this hypothesis, we compared glutamate-induced Ca2+ signals in MSN primary cultures established from the WT, YAC18 (13), and YAC128 (5) mice. To mimic physiological conditions more closely, we applied repetitive pulses of 20 μM glutamate 1 min in duration, followed by a 1-min washout (Fig. 1). The intracellular Ca2+ concentration in these experiments was continuously monitored by Fura-2 imaging, and the data were presented as 340/380 ratios (Fig. 1). On average, basal Ca2+ levels before glutamate application were not significantly different from each other for all three groups of MSN (Fig. 1D). Thus, increase in basal rat and mouse MSN Ca2+ levels as a result of Htt overexpression observed in our previous experiments (9, 15) most likely results from high levels of Htt expression in transfected MSN. Repetitive pulses of glutamate caused large elevation of Ca2+ levels in the YAC128 MSN (Fig. 1C), versus much smaller Ca2+ increases in the WT (Fig. 1 A) and YAC18 (Fig. 1B) MSN. On average, Ca2+ levels after 20 pulses of glutamate were not significantly different between WT and YAC18 MSN but were significantly (P < 0.05) higher in YAC128 MSN when compared with either WT or YAC18 MSN (Fig. 1D).

Fig. 1.
Deranged Ca2+ signaling in YAC128 MSN. (A–C) Repetitive application of 20 μM glutamate induces Ca2+ signals in MSN from the WT (A), YAC18 (B), and YAC128 (C) mice. Cytosolic Ca2+ levels are presented as a 340/380 Fura-2 ratio. Each pulse ...

In Vitro HD Model. Next, we established an “in vitro HD” model that reproduces Httexp-dependent degeneration of MSN. These experiments were performed with primary cultures of WT, YAC18, and YAC128 MSN. At 14 DIV, all three groups of MSN were challenged by an 8-h application of glutamate (from 0 to 250 μM) to mimic physiological stimulation. After exposure to glutamate, MSN were fixed, permeabilized, and scored for apoptotic cell death by using TUNEL staining. We determined that, in basal conditions (no glutamate added), ≈10% of MSN in all three experimental groups were apoptotic (TUNEL-positive) (Figs. 2 A and B). Addition of 25 or 50 μM glutamate increased the number of apoptotic cells to 15–20% in all three experimental groups (Figs. 2 A and B). Addition of 100 or 250 μM glutamate increased apoptotic death to 60–70% for YAC128 MSN (Figs. 2 A and B) but only to 25–30% for WT and YAC18 MSN (Figs. 2 A and B). Thus, we reasoned that exposure to glutamate concentrations in the 100–250 μM range leads to selective apoptosis of YAC128 MSN. As an additional control for specificity of observed degeneration, we compared glutamate-induced apoptosis in primary cultures of WT and YAC128 hippocampal neurons (HN), which are spared in HD (1). No significant differences in apoptosis of HN from WT and YAC128 mice in the 0–500 μM range of glutamate concentrations (8-h exposure) were apparent (data not shown).

Fig. 2.
In vitro HD assay. (A) Fourteen-DIV MSN from WT, YAC18, and YAC128 mice were exposed to a range of glutamate concentrations for 8 h, fixed, permeabilized, and analyzed by TUNEL staining (green) and PI counterstaining. (B) The fraction of TUNEL-positive ...

Disturbed Ca2+ Signaling and Degeneration of MSN in HD. To test the connection between disturbed Ca2+ signaling (Fig. 1) and glutamate-induced degeneration of YAC128 MSN (Fig. 2), we assessed the previously described “in vitro HD” assay in the presence of Ca2+ signaling blockers. We found that inhibition of mGluR1/5 receptors (by a mixture of MPEP and CPCCOEt) reduced the glutamate-induced apoptosis of YAC128 MSN to WT MSN levels (Fig. 3A). Inhibition of the NMDAR by (+)MK801 had a similar neuroprotective effect (data not shown). The NR2B-specific antagonist ifenprodil also decreased glutamate-induced apoptosis of YAC128 MSN to WT MSN levels (Fig. 3B). A combination of mGluR1/5 and NMDAR blockers [MPEP, CPCCOEt, and (+)MK801] completely eliminated glutamate-dependent apoptotic cell death in both WT and YAC128 MSN (Fig. 3C). In a previous study, we demonstrated that InsP3R1-mediated Ca2+ release is potentiated in MSN transfected with Httexp expression plasmids (9). Consistent with direct involvement of InsP3R1, preincubation of the MSN cultures with a membrane-permeable InsP3R blocker 2-APB (16) protected YAC128 MSN from glutamate-induced apoptosis (Fig. 3D).

Fig. 3.
Ca2+ blockers prevent apoptosis of YAC128 MSN. Fourteen-DIV MSN were exposed to a range of glutamate concentrations for 8 h, fixed, permeabilized, and analyzed by TUNEL staining as described in the legend of Fig. 2. The fraction of TUNEL-positive MSN ...

Because InsP3R1 appears to be involved in the glutamate-induced apoptosis of YAC128 MSN (Fig. 3D), we hypothesized that InsP3R1 might be a potential drug target for HD treatment. Bath application of a low-molecular-weight heparin sulfate Enoxaparin (Lovenox) prevents glutamate-induced Ca2+ release in cerebellar slices, presumably by blocking InsP3R1 activity (17). To directly test the ability of Enoxaparin to block InsP3R1, we performed a series of planar lipid bilayer reconstitution experiments with rat cerebellar microsomes. Addition of Enoxaparin efficiently and reversibly inhibited InsP3R1 in planar lipid bilayers (Fig. 4A), similar to actions of a high-molecular-weight heparin. Quantitative analysis of the inhibitory effects of Enoxaparin on InsP3R1 yielded an IC50 of 50 ng/ml and a Hill coefficient of 3.4 (Fig. 4B), consistent with Enoxaparin binding to each subunit of the InsP3R1 tetramer. To establish that Enoxaparin is able to block InsP3R1 in striatal MSN, we performed a series of Ca2+ imaging experiments with Fura-2. As described in ref. 9, application of 25 μM DHPG induced Ca2+ release in cultured MSN in Ca2+-free medium (Fig. 4C). Preincubation with 200 μg/ml Enoxaparin for 3 h prevented DHPG-induced Ca2+ release in cultured MSN (Fig. 4D), consistent with the ability of Enoxaparin to permeate cell membranes (17) and block InsP3R1 activity (Figs. 4 A and B). Using the “in vitro HD” assay, we found that 200 μg/ml Enoxaparin protected YAC128 MSN from glutamate-induced apoptosis (Fig. 4E), consistent with 2-APB data (Fig. 3D).

Fig. 4.
Enoxaparin protects YAC128 MSN from apoptosis. (A) Enoxaparin reversibly inhibits the activity of cerebellar InsP3R1 in planar lipid bilayers. Each current trace corresponds to 2 sec of current recording from the same experiment. InsP3-gated channels ...

Intrinsic Apoptotic Pathway, Mitochondria, and HD MSN Degeneration. Which apoptotic pathway is involved in glutamate-induced degeneration of HD MSN? Preincubation with the pan-caspase membrane-permeable inhibitor (Z-VAD-FMK), but not with the control peptide (Z-FA-FMK), reduced glutamate-induced apoptosis of YAC128 MSN to WT levels (Fig. 5A). Thus, apoptosis of YAC128 MSN occurs via a caspase-dependent pathway. Membrane-permeable inhibitors of caspase-3 (Z-DEVD-FMK) and caspase-9 (Z-LEHD-FMK) also protected YAC128 MSN from glutamate-induced apoptosis (Figs. 5 B and C). In contrast, a membrane-permeable inhibitor of caspase-8 (Z-IETD-FMK) was ineffective (Fig. 5D). These results are consistent with glutamate-induced apoptosis of YAC128 MSN occurring via the intrinsic (mitochondria and caspase-9-mediated) and not via the extrinsic (death receptor and caspase-8-mediated) pathway.

Fig. 5.
Apoptosis of YAC128 MSN is mediated by intrinsic pathway. (A–D) Membrane-permeable caspase inhibitors were added to 14-DIV WT and YAC128 (YAC) MSN 3 h before the application of glutamate. The results of TUNEL staining were quantified and presented ...

Activation of an intrinsic apoptotic pathway requires release of cytochrome c from the intramitochondrial space into the cytoplasm (10, 11). When WT MSN were incubated with 100 or 250 μM of glutamate for 5 h, a small elevation in cytosolic cytochrome c levels was observed (Fig. 5E). In contrast, a 5-h incubation of YAC128 MSN with 250 μM glutamate resulted in a much larger increase of cytosolic cytochrome c signal (Fig. 5E). These data are consistent with the activation of the intrinsic apoptotic pathway in YAC128 MSN but not in WT MSN, as a result of exposure to 250 μM glutamate in our experiments (Figs. 2 A and B).

Whether disturbed cytosolic Ca2+ signaling in YAC128 MSN (Figs. 1 C and D) is linked to the release of cytochrome c from the innermitochondrial space (Fig. 5E) is unknown. It is well established that excessive cytosolic Ca2+ is efficiently taken up by the mitochondria via activity of the mitochondrial Ca2+ uniporter/channel (MCU) located in the mitochondria inner membrane (18). Consistent with involvement of MCU in our experiments, preincubation of MSN with the membrane-permeable MCU blocker Ru360 (18, 19) reduced the glutamate-induced apoptotic cell death of YAC128 MSN to WT MSN levels (Fig. 5F).

It has been proposed that mitochondrial Ca2+ overload leads to release of cytochrome c due to opening of the mitochondrial permeability transition pore (MPTP) (10, 11). Consistent with the role of MPTP, we demonstrated that preincubation with the 10 μM MPTP inhibitor bongkrekic acid (BKA) (20) reduced glutamate-induced apoptosis of YAC128 MSN to WT levels (Table 1). Recently, a number of biologically active Food and Drug Administration-approved heterocyclic, tricyclic, and phenothiazine-derived compounds have been identified as putative MPTP blockers (21). To determine the potential usefulness of these compounds for HD, we performed “in vitro HD” assay experiments with compounds from this class. We found that preincubation with 2 μM Nortriptyline, Desipramine, Trifluoperazine, or Maprotiline protected YAC128 MSN from glutamate-induced apoptosis (Table 1), consistent with their putative MPTP-blocking activity (21). As a control for specificity of observed effects, we demonstrated that Pirenzepine and Thiothixene were not protective (Table 1), despite similarity in chemical structure.

Table 1.
Effects of putative MPTP blockers on glutamate-induced apoptosis in WT and YAC128 MSN

Discussion

Pathway Leading to Apoptosis of HD MSN. How polyglutamine exp in Httexp leads to neuronal death of MSN in the striatum is a central question in understanding the pathogenesis of HD (3). Our results are consistent with a model that links Httexp-induced disturbance of neuronal Ca2+ signaling with apoptosis of MSN in HD (Fig. 6). Specifically, we propose that glutamate released from corticostriatal projection neurons stimulates NR1/NR2B NMDAR and mGluR5 receptors, both of which are abundantly expressed in the striatal MSN (22, 23). Activation of NR1/NR2B NMDAR leads to Ca2+ influx and activation of mGluR5 receptors, leading to production of InsP3 and Ca2+ release via InsP3R1 (Fig. 6). Httexp affects Ca2+ signaling in HD MSN by sensitizing InsP3R1 to activation by InsP3 (9), stimulating NR1/NR2B NMDAR activity (68), and directly destabilizing mitochondrial Ca2+ handling (24, 25). As a result, stimulation of glutamate receptors results in supranormal Ca2+ responses in HD MSN, leading to cytosolic Ca2+ overload (Fig. 6). Excessive cytosolic Ca2+ is taken into the mitochondria via activity of the MCU (Fig. 6). With time, mitochondrial Ca2+ storage capacity is exceeded, leading to an opening of MPTP, release of cytochrome c into the cytosol, and activation of the caspase-mediated intrinsic apoptotic program (Fig. 6). These observations suggest that delayed onset of MSN degeneration in HD is at least in part a result of significant mitochondrial Ca2+ storage capacity that takes a long time to exceed, as we previously speculated (12). Consistent with mitochondrial involvement is the observation of dysfunctional mitochondria in HD mouse models and in HD patients (24, 25). The abnormal cytosolic and mitochondrial Ca2+ levels may contribute to neurological abnormalities observed in early-grade HD patients and in HD mouse models before onset of neurodegeneration.

Fig. 6.
Proposed mechanisms linking disturbed Ca2+ signaling and apoptosis of HD MSN. Glutamate released from corticostriatal projection neurons stimulates NR1A/NR2B NMDAR and mGluR5 receptors abundantly expressed in striatal MSN (22, 23). Httexp affects Ca2+ ...

Potential Therapeutic Targets for Treatment of HD. The proposed model (Fig. 6) may have implications for the treatment of HD. Our results suggest that NR2B NMDAR (Fig. 3B) and mGluR5 (Fig. 3A) can be considered potential drug targets for HD treatment. We have also demonstrated that inhibition of InsP3R1 by 2-APB (Fig. 3D), inhibition of MCU by Ru360 (Fig. 5F), and inhibition of MPTP by bongkrekic acid (Table 1) reduced glutamate-induced apoptosis of YAC128 MSN to WT MSN levels. Thus, InsP3R1 and MPTP would appear to constitute therapeutic targets for HD. In our experiments, it was demonstrated that low-molecular-weight heparin Enoxaparin (Lovenox) exerts a neuroprotective effect on YAC128 MSN (Fig. 4E), apparently by directly inhibiting InsP3R1 function (Figs. 4 A–D). Intravenous injections of Enoxaparin are neuroprotective in animal models of stroke and neurodegenerative diseases (2628), and our results indicate that Enoxaparin may also be worthy of assessment as HD therapeutics. Recently, a number of biologically active Food and Drug Administration-approved heterocyclic, tricyclic, and phenothiazine-derived compounds have been identified as putative MPTP blockers (21). In our experiments, it is demonstrated that several members of this class of compounds (Nortriptyline, Desipramine, Trifluoperazine, and Maprotiline) protect YAC128 MSN from glutamate-induced apoptosis (Table 1). Future studies with animal models of HD or human clinical trials will be required to test the utility of putative MPTP blockers as potential therapeutics for HD. The potential side effects of these compounds, such as bleeding potential for Enoxaparin and anticholinergic actions of Nortriptyline and Desipramine, must be considered in the clinical setting.

Acknowledgments

We thank Tianhua Lei for help with maintaining the YAC mouse colony and genotyping, Linda Patterson for administrative assistance, Ethan Signer for facilitating our collaboration on MPTP blockers, and Xiaodong Wang for advice on cytochrome c release experiments. I.B. is supported by the Robert A. Welch Foundation, the Huntington's Disease Society of America, the Hereditary Disease Foundation, the High Q Foundation, and National Institute of Neurological Disorders and Stroke (NINDS) Grant R01 NS38082. M.R.H. is supported by the Canadian Institutes of Health Research, the Hereditary Disease Foundation, the Huntington's Disease Society of America, and the High Q Foundation, and holds a Canada Research Chair in Human Genetics. B.S.K. is supported by the Hereditary Disease Foundation and the High Q Foundation. R.L. is supported by NINDS Grant NINDS-NS13742.

Notes

Author contributions: R.L., B.S.K., M.R.H., and I.B. designed research; T.-S.T., E.S., V.L., I.G.S., and M.S. performed research; T.-S.T., E.S., V.L., I.G.S., M.S., R.L., M.R.H., and I.B. analyzed data; and B.S.K., M.R.H., and I.B. wrote the paper.

Abbreviations: CPCCOEt, 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester; DIV, days in vitro; exp, expansion; HD, Huntington's disease; Htt, huntingtin; InsP3, inositol 1,4,5-trisphosphate; InsP3R1, type 1 InsP3 receptor; MCU, mitochondrial Ca2+ uniporter/channel; MPEP, 2-methyl-6-(phenylethynyl)pyridine hydrochloride; MPTP, mitochondrial permeability transition pore; MSN, medium spiny neurons; NMDAR, NMDA receptor; PI, propidium iodide; Ru360, Ruthenium 360.

References

1. Vonsattel, J. P. & DiFiglia, M. (1998) J. Neuropathol. Exp. Neurol. 57, 369-384. [PubMed]
2. The Huntington's Disease Collaborative Research Group (1993) Cell 72, 971-983. [PubMed]
3. Tobin, A. J. & Signer, E. R. (2000) Trends Cell Biol. 10, 531-536. [PubMed]
4. Rubinsztein, D. C. (2002) Trends Genet. 18, 202-209. [PubMed]
5. Slow, E. J., van Raamsdonk, J., Rogers, D., Coleman, S. H., Graham, R. K., Deng, Y., Oh, R., Bissada, N., Hossain, S. M., Yang, Y. Z., et al. (2003) Hum. Mol. Genet. 12, 1555-1567. [PubMed]
6. Chen, N., Luo, T., Wellington, C., Metzler, M., McCutcheon, K., Hayden, M. R. & Raymond, L. A. (1999) J. Neurochem. 72, 1890-1898. [PubMed]
7. Sun, Y., Savanenin, A., Reddy, P. H. & Liu, Y. F. (2001) J. Biol. Chem. 276, 24713-24718. [PubMed]
8. Zeron, M. M., Hansson, O., Chen, N., Wellington, C. L., Leavitt, B. R., Brundin, P., Hayden, M. R. & Raymond, L. A. (2002) Neuron 33, 849-860. [PubMed]
9. Tang, T.-S., Tu, H., Chan, E. Y., Maximov, A., Wang, Z., Wellington, C. L., Hayden, M. R. & Bezprozvanny, I. (2003) Neuron 39, 227-239. [PMC free article] [PubMed]
10. Orrenius, S., Zhivotovsky, B. & Nicotera, P. (2003) Nat. Rev. Mol. Cell Biol. 4, 552-565. [PubMed]
11. Hajnoczky, G., Davies, E. & Madesh, M. (2003) Biochem. Biophys. Res. Commun. 304, 445-454. [PubMed]
12. Bezprozvanny, I. & Hayden, M. R. (2004) Biochem. Biophys. Res. Commun. 322, 1310-1317. [PubMed]
13. Hodgson, J. G., Agopyan, N., Gutekunst, C. A., Leavitt, B. R., LePiane, F., Singaraja, R., Smith, D. J., Bissada, N., McCutcheon, K., Nasir, J., et al. (1999) Neuron 23, 181-192. [PubMed]
14. Lupu, V. D., Kaznacheyeva, E., Krishna, U. M., Falck, J. R. & Bezprozvanny, I. (1998) J. Biol. Chem. 273, 14067-14070. [PubMed]
15. Tang, T. S., Tu, H., Orban, P. C., Chan, E. Y., Hayden, M. R. & Bezprozvanny, I. (2004) Eur. J. Neurosci. 20, 1779-1787. [PubMed]
16. Maruyama, T., Kanaji, T., Nakade, S., Kanno, T. & Mikoshiba, K. (1997) J. Biochem. (Tokyo) 122, 498-505. [PubMed]
17. Jonas, S., Sugimori, M. & Llinás, R. (1997) Ann. N.Y. Acad. Sci. 825, 389-393. [PubMed]
18. Kirichok, Y., Krapivinsky, G. & Clapham, D. E. (2004) Nature 427, 360-364. [PubMed]
19. Ying, W. L., Emerson, J., Clarke, M. J. & Sanadi, D. R. (1991) Biochemistry 30, 4949-4952. [PubMed]
20. Halestrap, A. P., McStay, G. P. & Clarke, S. J. (2002) Biochimie 84, 153-166. [PubMed]
21. Stavrovskaya, I. G., Narayanan, M. V., Zhang, W., Krasnikov, B. F., Heemskerk, J., Young, S. S., Blass, J. P., Brown, A. M., Beal, M. F., Friedlander, R. M. & Kristal, B. S. (2004) J. Exp. Med. 200, 211-222. [PMC free article] [PubMed]
22. Landwehrmeyer, G. B., Standaert, D. G., Testa, C. M., Penney, J. B., Jr., & Young, A. B. (1995) J. Neurosci. 15, 5297-5307. [PubMed]
23. Testa, C. M., Standaert, D. G., Landwehrmeyer, G. B., Penney, J. B., Jr., & Young, A. B. (1995) J. Comp. Neurol. 354, 241-252. [PubMed]
24. Panov, A. V., Gutekunst, C. A., Leavitt, B. R., Hayden, M. R., Burke, J. R., Strittmatter, W. J. & Greenamyre, J. T. (2002) Nat. Neurosci. 5, 731-736. [PubMed]
25. Choo, Y. S., Johnson, G. V., MacDonald, M., Detloff, P. J. & Lesort, M. (2004) Hum. Mol. Genet. 13, 1407-1420. [PubMed]
26. Stutzmann, J. M., Mary, V., Wahl, F., Grosjean-Piot, O., Uzan, A. & Pratt, J. (2002) CNS Drug Rev. 8, 1-30. [PubMed]
27. Mary, V., Wahl, F., Uzan, A. & Stutzmann, J. M. (2001) Stroke 32, 993-999. [PubMed]
28. Bergamaschini, L., Rossi, E., Storini, C., Pizzimenti, S., Distaso, M., Perego, C., De Luigi, A., Vergani, C. & De Simoni, M. G. (2004) J. Neurosci. 24, 4181-4186. [PubMed]

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