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 mitochondrial inner membrane (TIM23) complex, inhibiting mitochondrial protein import and altering the mitochondrial proteome. The HTT N-terminal 17-amino acids sequence (N17) acts as a regulatory domain in HD pathogenesis; phosphomimetic modification of serine 13 and 16 of the N17 domain impacts subcellular localization, 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 to mice expressing mHTT. Consistent with reduced toxicity in vivo, we found that the amount of full-length mHTT in brain mitochondria of BACHD-SD transgenic mice is less when the mHTT has two phosphomimetic mutations. To complement the relevance of the phosphomimic HTT findings, endogenous N17 phospho mHTT is less likely to translocate to the mitochondria compared to non-phosphorylated 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. Significance Statement We establish the first 17 amino acids of mHTT as a mitochondrial targeting sequence driving mHTT accumulation in mitochondria and show that N-terminal phosphomimetic modifications reduce mitochondrial accumulation and toxicity. Utilizing super-resolution live imaging and expansion microscopy of isolated mitochondria and full-length mHTT immunoprecipitation from human cells, we demonstrate HTT intramitochondrial localization and interaction with the mitochondrial protein importing complex. mHTT N17 domain phosphomimetic mutations reduce binding affinity for the TIM23 subunit, preventing mHTT mitochondrial accumulation. This lessens mHTT inhibitory effect on mitochondrial protein import. Our study elucidates the molecular mechanism responsible for the BACHD-SD mouse ameliorated phenotype and underscores a critical need to identify enzymes responsible for regulating HTT phosphorylation to exploit their potential as therapeutic HD targets.

Regulation of mutant huntingtin mitochondrial toxicity by phosphomimetic mutations within its N-terminal region.