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N-terminal mutant huntingtin associates with mitochondria and impairs mitochondrial trafficking.

ABSTRACT: Huntington's disease (HD) is caused by polyglutamine (polyQ) expansion in huntingtin (htt), a large (350 kDa) protein that localizes predominantly to the cytoplasm. Proteolytic cleavage of mutant htt yields polyQ-containing N-terminal fragments that are prone to misfolding and aggregation. Disease progression in HD transgenic models correlates with age-related accumulation of soluble and aggregated forms of N-terminal mutant htt fragments, suggesting that multiple forms of mutant htt are involved in the selective neurodegeneration in HD. Although mitochondrial dysfunction is implicated in the pathogenesis of HD, it remains unclear which forms of cytoplasmic mutant htt associate with mitochondria to affect their function. Here we demonstrate that specific N-terminal mutant htt fragments associate with mitochondria in Hdh(CAG)150 knock-in mouse brain and that this association increases with age. The interaction between soluble N-terminal mutant htt and mitochondria interferes with the in vitro association of microtubule-based transport proteins with mitochondria. Mutant htt reduces the distribution and transport rate of mitochondria in the processes of cultured neuronal cells. Reduced ATP level was also found in the synaptosomal fraction isolated from Hdh(CAG)150 knock-in mouse brain. These findings suggest that specific N-terminal mutant htt fragments, before the formation of aggregates, can impair mitochondrial function directly and that this interaction may be a novel target for therapeutic strategies in HD.

SUBMITTER: Orr AL

PROVIDER: S-EPMC2652473 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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N-terminal mutant huntingtin associates with mitochondria and impairs mitochondrial trafficking.

Orr Adam L AL Li Shihua S Wang Chuan-En CE Li He H Wang Jianjun J Rong Juan J Xu Xingshun X Mastroberardino Pier Giorgio PG Greenamyre J Timothy JT Li Xiao-Jiang XJ

The Journal of neuroscience : the official journal of the Society for Neuroscience 20080301 11

Huntington's disease (HD) is caused by polyglutamine (polyQ) expansion in huntingtin (htt), a large (350 kDa) protein that localizes predominantly to the cytoplasm. Proteolytic cleavage of mutant htt yields polyQ-containing N-terminal fragments that are prone to misfolding and aggregation. Disease progression in HD transgenic models correlates with age-related accumulation of soluble and aggregated forms of N-terminal mutant htt fragments, suggesting that multiple forms of mutant htt are involve ...[more]

PMID: 18337408

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Project description:Although the mechanisms by which mutant huntingtin (mHtt) results in Huntington's disease (HD) remain unclear, mHtt-induced mitochondrial defects were implicated in HD pathogenesis. The effect of mHtt could be mediated by transcriptional alterations, by direct interaction with mitochondria, or by both. In the present study, we tested a hypothesis that mHtt directly damages mitochondria. To test this hypothesis, we applied brain cytosolic fraction from YAC128 mice, containing mHtt, to brain non-synaptic and synaptic mitochondria from wild-type mice and assessed mitochondrial respiration with a Clark-type oxygen electrode, membrane potential and Ca2+ uptake capacity with tetraphenylphosphonium (TPP+ )- and Ca2+ -sensitive electrodes, respectively, and, reactive oxygen species production with Amplex Red assay. The amount of mHtt bound to mitochondria following incubation with mHtt-containing cytosolic fraction was greater than the amount of mHtt bound to brain mitochondria isolated from YAC128 mice. Despite mHtt binding to wild-type mitochondria, no abnormalities in mitochondrial functions were detected. This is consistent with our previous results demonstrating the lack of defects in brain mitochondria isolated from R6/2 and YAC128 mice. This, however, could be because of partial loss of mitochondrially bound mHtt during the isolation procedure. Consequently, we increased the amount of mitochondrially bound mHtt by incubating brain non-synaptic and synaptic mitochondria isolated from YAC128 mice with mHtt-containing cytosolic fraction. Despite the enrichment of YAC128 brain mitochondria with mHtt, mitochondrial functions (respiration, membrane potential, reactive oxygen species production, Ca2+ uptake capacity) remained unchanged. Overall, our results suggest that mHtt does not directly impair mitochondrial functions, arguing against the involvement of this mechanism in HD pathogenesis. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Project description:The precise degradation of dysfunctional mitochondria by mitophagy is essential for maintaining neuronal homeostasis. HTT (huntingtin) can interact with numerous other proteins and thereby perform multiple biological functions within the cell. In this study, we investigated the role of HTT during mitophagy and analyzed the impact of the expansion of its polyglutamine (polyQ) tract. HTT is involved in different mitophagy steps, promoting the physical proximity of different protein complexes during the initiation of mitophagy and recruiting mitophagy receptors essential for promoting the interaction between damaged mitochondria and the nascent autophagosome. The presence of the polyQ tract in mutant HTT affects the formation of these protein complexes and determines the negative consequences of mutant HTT on mitophagy, leading to the accumulation of damaged mitochondria and an increase in oxidative stress. These outcomes contribute to general mitochondrial dysfunction and neurodegeneration in Huntington disease.Abbreviations: AMPK: AMP-activated protein kinase; ATG13: autophagy related 13; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chlorophenyl hydrazone; DMEM: Dulbecco's modified eagle medium; EDTA: ethylene-diamine-tetra-acetic acid; EGFP: enhanced green fluorescent protein; EGTA: ethylene glycol bis(2-aminoethyl ether)tetraacetic acid; FUNDC1: FUN14 domain containing 1; HD: Huntington disease; HRP: horseradish peroxidase; HTT: huntingtin; LC3-II: lipidated form of MAP1LC3/LC3; mtDNA: mitochondrial deoxyribonucleic acid; MTDR: MitoTracker Deep Red; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1, autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; OCR: oxygen consumption rate; OPTN: optineurin; OXPHOS: oxidative phosphorylation; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PINK1: PTEN induced putative kinase 1; PLA: proximity ligation assay; PMSF: phenylmethylsulfonyl fluoride; polyQ: polyglutamine; PtdIns3K: phosphatidylinositol 3-kinase; ROS: reactive oxygen species; Rot: rotenone; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TMRM: tetramethylrhodamine methyl ester; UB: ubiquitin; ULK1: unc-51 like kinase 1.

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N-terminal mutant huntingtin associates with mitochondria and impairs mitochondrial trafficking.

Publications

N-terminal mutant huntingtin associates with mitochondria and impairs mitochondrial trafficking.

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