Disturbed Ca<sup>2+</sup> 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 (Ca) signaling and apoptosis of HD medium spiny neurons (MSN). Repetitive application of glutamate elevates cytosolic Ca 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 Ca overload and can be prevented by the mitochondrial Ca 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 Ca signaling and degeneration of MSN in the caudate nucleus in HD. These findings also suggest that Ca and MPTP blockers may have a therapeutic potential for treatment of HD.
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 Htt 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 Htt facilitates activity of the NR2B subtype of NMDA receptors (NMDARs) (6–8) and the type 1 inositol 1,4,5-trisphosphate receptors (InsP3R1) (9). A connection between disturbed Ca signaling and neuronal apoptosis is well established (10, 11), and we therefore proposed that Htt-induced Ca overload results in degeneration of MSN in HD (12). To test this hypothesis, we analyzed Ca signals and apoptotic cell death in primary cultures of MSN from the YAC128 mice. Our results provide further support to the hypothesis that disturbed Ca underlies neuronal cell death in HD (12) and allowed us to identify a number of potential therapeutic targets for HD treatment.
The BKA concentration is 10 μM, and the concentration of all of the other putative MPTP blockers is 2 μM. , P<0.05 compared with control experiment. Promethazine was toxic.
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 Ca 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.
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