Regulation of Mutant Huntingtin Mitochondrial Toxicity by Phosphomimetic Mutations within Its N-Terminal Region

Huntington's disease (HD), a neurodegenerative disease, affects approximately 30,000 people in the United States, with 200,000 more at risk. Mitochondrial dysfunction caused by mutant huntingtin (mHTT) drives early HD pathophysiology. mHTT binds the translocase of the mitochondrial inner membrane (TIM23) complex, inhibiting mitochondrial protein import and altering the mitochondrial proteome. The 17 aa HTT N-terminal sequence (N17) acts as a regulatory domain in HD pathogenesis; phosphomimetic modification of serines 13 and 16 of the N17 domain impacts subcellular localization and degradation and ameliorates toxicity in mouse and cell models of HD. Using cellular and mouse (either sex) HD models, we investigated the mechanisms by which HTT phosphorylation affects intracellular localization. We demonstrate that introducing phosphomimetic mutations within the mHTT fragment N17 domain decreased TIM23 binding affinity and reduced inhibition of mHTT-mediated mitochondrial protein import. BACHD-SD mice expressing full-length mHTT harboring the same two N17 phosphomimetic mutations have an ameliorated HD-like phenotype as compared with mice expressing mHTT. Consistent with reduced toxicity in vivo, we found that the amount of full-length mHTT in the brain mitochondria of BACHD-SD transgenic mice is less when the mHTT has two phosphomimetic mutations. To complement the relevance of the phosphomimetic HTT findings, endogenous N17 phospho-mHTT is less likely to translocate to the mitochondria compared with nonphosphorylated mHTT. We demonstrate that phosphorylation of mHTT at serines 13 and 16 is critical for negatively regulating mHTT mitochondrial targeting and that reducing mHTT mitochondrial localization and binding to TIM23 results in amelioration of mHTT-induced mitochondrial and neuronal toxicity.