pS421 huntingtin modulates mitochondrial phenotypes and confers neuroprotection in an HD hiPSC model

Abstract

Huntington disease (HD) is a hereditary neurodegenerative disorder caused by mutant huntingtin (mHTT). Phosphorylation at serine-421 (pS421) of mHTT has been shown to be neuroprotective in cellular and rodent models. However, the genetic context of these models differs from that of HD patients. Here we employed human pluripotent stem cells (hiPSCs), which express endogenous full-length mHTT. Using genome editing, we generated isogenic hiPSC lines in which the S421 site in mHTT has been mutated into a phospho-mimetic aspartic acid (S421D) or phospho-resistant alanine (S421A). We observed that S421D, rather than S421A, confers neuroprotection in hiPSC-derived neural cells. Although we observed no effect of S421D on mHTT clearance or axonal transport, two aspects previously reported to be impacted by phosphorylation of mHTT at S421, our analysis revealed modulation of several aspects of mitochondrial form and function. These include mitochondrial surface area, volume, and counts, as well as improved mitochondrial membrane potential and oxidative phosphorylation. Our study validates the protective role of pS421 on mHTT and highlights a facet of the relationship between mHTT and mitochondrial changes in the context of human physiology with potential relevance to the pathogenesis of HD.

Huntingtin S421 phosphorylation increases kinesin and dynein engagement on early endosomes and lysosomes

“…1G). In iPSC-derived neurons expressing polyglutamine-expanded huntingtin (180Q), S421Dhtt led to increased mitochondrial surface area while the number of mitochondria remained similar (Xu et al, 2020). Immunofluorescence of the mitochondria in HEK293T cells showed a decrease in the mitochondrial number and area upon S421D mutation (Fig.…”

“…Htt has 40 known phosphosites, many of which have unknown biological functions (reviewed by Saudou and Humbert, 2016). The phosphorylation state of serine 421 affects axonal transport direction and velocity of BDNF vesicles and autophagosomes (Colin et al, 2008; Wong and Holzbaur, 2014), while mitochondrial transport is unaffected by this phosphosite (Xu et al, 2020). Each of htt’s post-translational modifications contribute to its role in transport of both signaling and degradative cargoes.…”

Modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550

“…Ser421 phosphorylation by AKT, and dephosphorylation by calcinurin, recruits and releases kinesin, respectively, thereby determining the direction of vesicle transport (e.g. BDNF) (Colin et al, 2008; Scaramuzzino, Cuoc, Pla, Humbert, & Saudou, 2022), while phosphorylation of this site is also reported to decrease huntingtin-cleavage and fragment-toxicity (Warby et al, 2005) and to influence mitochondrial phenotypes and toxicity in HD neuronal cells (Xu et al, 2020). CDK5 phosphorylation of pSer434 was associated with decreased caspase cleavage of huntingtin (Luo, Vacher, Davies, & Rubinsztein, 2005), whereas CDK5 phosphorylation of pSer1181 and pSer1201was reported to mediate huntingtin toxicity in neuronal cultures (Anne, Saudou, & Humbert, 2007).…”

Kinases and Their Potential as Therapeutic Targets in Huntington’s Disease

“…A summary of all identified HTT phosphorylations and their effects on HTT-mediated toxicity can be found in Table 2. Overall HTT phosphorylation tend to be decreased in the presence of expanded polyQ, which has pathological -associated effects on HTT nuclear localization, aggregation, and cellular toxicity [82,[84][85][86][106][107][108][109][110][111][112]. Three highly studied phosphorylations on HTT are located within the N17 domain and correspond to T3, S13 and S16 [82,84,113].…”