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. The genetic context of these models however differs from that of HD patients.
This article employed human pluripotent stem cells (hiPSCs), which express endogenous full-length mHTT. Using genome editing, isogenic hiPSC lines were generated in which the S421 site in mHTT has been mutated into a phospho-mimetic aspartic acid (S421D) or phosphoresistant alanine (S421A). It was observed that S421D, rather than S421A, confers neuroprotection in hiPSC-derived neural cells. Although no effect of S421D on mHTT clearance or axonal transport was observed, two aspects previously reported to be impacted by phosphorylation of mHTT at S421, 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.
The 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.
Genome-editing strategy to generate isogenic S421A/D-mHTT hiPSC lines. A) TALEN-binding sequences flanking the S421 codon in HTTexon 9. B) TALEN cleavage activity as assessed by Surveyor assay. C) ssODN donor DNA with BglI restriction enzyme site for screening. D) Verification of targeting and screening strategy in K562 cells. E) Expected restriction fragment pattern with BglI screening. F) HTT Exon 9 PCR followed by BglI screening identifies a number of targeted CAG180 HD hiPSC clones (black arrows); example shown is for S421(A) targeting; red arrows represent mixed clones. G) Summary of targeting efficiency. H) Phasing of targeted allele by allele-specific PCR followed by Sanger sequencing.
Results show that PTM at S421 may modulate the toxicity of the full-length mHTT protein at least in part by affecting HD-associated mitochondrial phenotypes. These findings provide further support that targeting phosphorylation of mHTT at S421 site can be considered for further development as a therapeutic strategy for HD.
The full article can be accessed here.
1Department of Neurology and Stroke Center, Clinical Neuroscience Institute, The First Affiliated Hospital, Jinan University, 613 Huangpu Avenue West, Guangzhou, Guangdong 510632, China. 2Translational Laboratory in Genetic Medicine, 8A Biomedical Grove, Immunos, Level 5, Singapore 138648, Singapore. 3Institute of Medical Biology, 8A Biomedical Grove, Immunos, Level 5, Singapore 138648, Singapore. 4Immuneering Corporation, 157 Columbus Avenue, Suite 537, New York, NY 10023, USA. 5Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver V5Z4H4, Canada. 6Division of Neurobiology, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 7Department of Medicine, National University of Singapore, Singapore 117597, Singapore. 8Department of Physiology, National University of Singapore, Singapore 117597, Singapore.
