Huntington’s disease (HD) is an autosomal dominant inherited neurodegenerative disease that specifically affects the striatum of the human brain. HD is characterized by a chorea-like movement disorder, cognitive decline, and psychiatric symptoms. In Europe, it has a relatively high prevalence of about 2.17-7.33 per 100,000 people compared with other continents. By far, there is no cure for HD. The mean survival time of patients after the diagnosis of HD is 15 to 20 years. Although the mutant form of the Huntingtin (HTT) as the cause of HD has been confirmed for decades, the exact pathogenesis of HD is still elusive. More recently, large global genome-wide association studies (GWAS) and several other studies provided new insights for HD mechanism, by highlighting several genes involved in DNA damage repair mechanisms as modifiers of age at onset and disease severity in HD. Thus, this project focused on the investigation of DNA damage response and repair in HD in vitro models. Fused in sarcoma (FUS) was the protein of our interest, as it has been confirmed to participate in DNA damage response and repair in multiple ways. Furthermore, FUS protein was implicated to have a relationship with neurodegenerative diseases, as it was found to play a role in the pathogenesis of subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. FUS was also found to co-localize with mutant huntingtin protein in intracellular aggregates in HD mice models. In this project, donor/patient-specific induced pluripotent cells (iPSCs) and its derived striatal neurons were the main materials. By immunofluorescence staining approach of γH2AX and 53BP1, DNA double-strand breaks (DSBs) damage was investigated on iPSCs-derived in vitro striatal neurons. HD neurons showed an obvious and excessive accumulation of DNA DSBs damage. Then, in order to visualize FUS protein during DNA damage response procedure, eGFP tagged endogenous wild-type FUS iPSCs were generated, and later were differentiated into striatal neurons. UVA laser micro-irradiation was applied onto both hiPSCs and their differentiated striatal neurons in vitro models, simultaneously conducting with live-cell imaging approach. FUS was found to recruit to the DNA damage site induced by laser irradiation. For studying the kinetics of wild-type FUS protein during the response to laser irradiation, a novel and robust workflow was generated. By this workflow, the kinetics of FUS protein was characterized into four phases and a real-time scale of the kinetics was offered. After comparisons, a prominent change of FUS kinetics in HD at neuron-stage but not iPSC-stage was found. Furthermore, an intriguing different performance of FUS protein was found in different types of in vitro cellular models. In iPSCs, not all the laser-irradiated cells recruited FUS at the DNA damage site. The kinetics of the FUS protein also differed in different models. In conclusion, first, our in vitro striatal neuron model recapitulated the impaired DNA damage repair phenotype that published by other models. Second, new evidence was offered that wild-type FUS was involved in the pathogenesis of HD. Third, depending on cell-type, FUS performed differently during the response to the laser irradiation-induced DNA damage. Thus, these results suggest that the impaired DNA damage response and repair would be crucial to the mechanism of HD. Furthermore, the role of FUS protein playing especially the functional part in DNA damage response and repair might be a potential target for further investigation of neurodegenerative diseases including HD.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:74543 |
| Date | 23 April 2021 |
| Creators | Niu, Yu |
| Contributors | Hermann, Andreas, Ader, Marius, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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