Abstract
Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre‐clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non‐human or non‐neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra‐nuclear inclusions in a polyQ length‐dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction.
Highlights
Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder, typically of adult onset, with irreversible progression of motor, cognitive, and psychiatric symptoms over 10-15 years.[1]
Cells were washed with Hank's balanced salt solution (HBSS) and trypsinized using TrypzeanEDTA [Lonza] that was added in sufficient volume to coat the surface of the flask and incubated at 37°C; 5% of CO2 for 5 minutes
In contrast to some other cells lines that express mutant exon 1 huntingtin protein (HTT), these lines do not show any overt cell death or toxicity at up to six weeks in culture, though there is a possible subtle deficit in neuronal differentiation in the 122 CAG line. They do show, CAG-length dependent, “pre-pathological” changes in terms of inclusion bodies (IB) formation, increased nuclear size, and mitochondrial dysfunction. These cellular changes may reflect some of the earliest changes that occur in HD pathogenesis prior to disease onset
Summary
Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder, typically of adult onset, with irreversible progression of motor, cognitive, and psychiatric symptoms over 10-15 years.[1]. The causative mutation is an expanded CAG repeat in exon 1 of the gene encoding the huntingtin protein (HTT), which leads to an elongated polyglutamine (polyQ) stretch within its N-terminal domain. The length of the CAG repeat is critical in determining disease status; inheritance of 36-39 CAG repeats leads to a reduced penetrance form of HD, whereas 40 or more CAGs causes the fully penetrant form. Longer repeat lengths are associated with an earlier age of onset and faster disease progression, with the inheritance of more than 55 CAGs leading to juvenile HD, in which age of onset occurs before 20 years of age.[2]. This incompletely spliced transcript is present in all knock-in HD mouse models[7] and HD post-mortem brains and fibroblasts,[8] supporting a role in HD pathogenesis
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