Huntington's Disease (HD) is an inherited, neurodegenerative disorder associated with the abnormal expansion of CAG triplet that encodes a polyglutamine domain in huntingtin, a 350 kDa protein expressed in various tissues. A mechanistic link between Htt gene mutation and neuronal loss leading to neurological abnormalities in HD has not yet been determined, but mitochondrial dysfunction has emerged as a causal factor involved in HD pathogenesis. Despite extensive studies, the mechanisms of mitochondrial dysfunction in HD remain unclear. The overall objectives of the proposed study are to clarify the role of mitochondrial porin, also known as voltage-dependent anion channel (VDAC), in mutant huntingtin (mHtt)-induced mitochondrial dysfunction and abnormal mitochondrial fragmentation in mHtt-expressing neurons. In the proposed study, we will test a novel hypothesis that mHtt binds to VDAC and inhibits metabolite transport across the OMM, leading to mitochondrial dysfunction, Ca2+ handling defects, mitochondrial oxidative stress, and augmented mitochondrial fission. We will address the following questions: (1) Does mHtt diminish VDAC transport activity by binding to the channel? (2) Is VDAC inhibition accountable for respiratory suppression, depolarization, and accumulation of superoxide anion O2? - in mitochondria exposed to mHtt? (3) Does mHtt result in increased susceptibility to mitochondrial Ca2+-induced injury and decreased Ca2+ uptake capacity by inhibiting VDAC? (4) Does VDAC inhibition lead to mitochondrial oxidative stress and augmented mitochondrial fission in cultured neurons expressing mHtt? To answer these questions we will use VDAC-reconstituted giant proteoliposomes in conjunction with electrophysiological patch-clamp technique and glutathione-S-transferase (GST)-polyQ fusion proteins. We will use synaptic and non-synaptic purified brain mitochondria isolated from wild-type mice and transgenic and knock-in HD mouse models in combination with modern pharmacological, biochemical, and bioenergetic methodologies. To analyze mitochondrial dynamics, we will use live-cell, laser spinning-disk confocal microscopy followed by sophisticated image processing and quantitative 3D image rendering applied to cultured striatal and cortical neurons derived from wild-type and HD mice with mitochondria visualized by mitochondrially targeted fluorescent proteins. At the conclusion of this research program, we will establish the role of VDAC inhibition in mitochondrial dysfunction, Ca2+ handling defects, mitochondrial oxidative stress, and augmented fission in mitochondria exposed to mHtt. Thus, our study will provide novel, vital knowledge about molecular mechanisms of mitochondrial dysfunction in HD and build a platform for future HD research. This will lay a solid foundation for creating treatments aimed at improving mitochondrial functioning and neuronal survival in HD. Most importantly, this will immensely help in the development of new therapeutic strategies to alleviate neurological deficits in HD and significantly diminish suffering of HD patients, improve quality of their life, and lessen the emotional and financial burden on the family and the whole society.

Public Health Relevance

The proposed research is aimed at elucidating the molecular mechanisms of mitochondrial dysfunction that might contribute to development of Huntington Disease (HD), one of the most devastating neurodegenerations. The proposed research will significantly advance our knowledge about molecular mechanisms involved in mitochondrial injury and brain damage in HD and will lead to the design of more effective therapeutic strategies directed at protecting mitochondria and neurons thus diminishing neurological abnormalities in HD.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS078008-02
Application #
8416946
Study Section
Neural Oxidative Metabolism and Death Study Section (NOMD)
Program Officer
Sutherland, Margaret L
Project Start
2012-02-15
Project End
2016-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
2
Fiscal Year
2013
Total Cost
$326,819
Indirect Cost
$115,725
Name
Indiana University-Purdue University at Indianapolis
Department
Pharmacology
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Hamilton, James; Pellman, Jessica J; Brustovetsky, Tatiana et al. (2016) Oxidative metabolism and Ca2+ handling in isolated brain mitochondria and striatal neurons from R6/2 mice, a model of Huntington's disease. Hum Mol Genet 25:2762-2775
Lakhter, Alexander J; Hamilton, James; Konger, Raymond L et al. (2016) Glucose-independent Acetate Metabolism Promotes Melanoma Cell Survival and Tumor Growth. J Biol Chem 291:21869-21879
Brustovetsky, Nickolay (2016) Mutant Huntingtin and Elusive Defects in Oxidative Metabolism and Mitochondrial Calcium Handling. Mol Neurobiol 53:2944-53
Pellman, Jessica J; Hamilton, James; Brustovetsky, Tatiana et al. (2015) Ca(2+) handling in isolated brain mitochondria and cultured neurons derived from the YAC128 mouse model of Huntington's disease. J Neurochem 134:652-67
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Hamilton, James; Pellman, Jessica J; Brustovetsky, Tatiana et al. (2015) Oxidative metabolism in YAC128 mouse model of Huntington's disease. Hum Mol Genet 24:4862-78
Brustovetsky, Tatiana; Pellman, Jessica J; Yang, Xiao-Fang et al. (2014) Collapsin response mediator protein 2 (CRMP2) interacts with N-methyl-D-aspartate (NMDA) receptor and Na+/Ca2+ exchanger and regulates their functional activity. J Biol Chem 289:7470-82
Ashpole, Nicole M; Chawla, Aarti R; Martin, Matthew P et al. (2013) Loss of calcium/calmodulin-dependent protein kinase II activity in cortical astrocytes decreases glutamate uptake and induces neurotoxic release of ATP. J Biol Chem 288:14599-611
Brittain, Joel M; Pan, Rui; You, Haitao et al. (2012) Disruption of NMDAR-CRMP-2 signaling protects against focal cerebral ischemic damage in the rat middle cerebral artery occlusion model. Channels (Austin) 6:52-9
Ashpole, Nicole M; Song, Weihua; Brustovetsky, Tatiana et al. (2012) Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibition induces neurotoxicity via dysregulation of glutamate/calcium signaling and hyperexcitability. J Biol Chem 287:8495-506

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