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Title: Analysis of mutations in superoxide dismutase-1 and the protective effect of heat shock proteins against mutant-SOD1 toxicity
Author: Patel, Yogesh Jayendra Kumar
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2006
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Genetic studies have revealed over 100 mutations in the gene encoding superoxide dismutase type-1 (SOD1) that cause Familial Amyotrophic Lateral Sclerosis (FALS). For the purpose of this thesis an in vitro model has been developed by stably over-expressing wild type (wt), G93A or G93R mutant SOD1 in neuronally derived ND7 cell line. It was found that wt-SOD1 could provide protection against a range of cell stresses including serum removal (plus retinoic acid) IFN-gamma, staurosporine camptothecin ischemia/reoxygenation glutamate and hydrogen peroxide (H2O2). In contrast, both mutant forms of SOD1 enhanced cell death with the G93R mutation being the more severe of the two mutations tested. Hence, the disease-associated mutations convert wt-SOD1 from a protective anti-apoptotic protein to a pro-apoptotic form. Most of the above stresses are FALS relevant stresses, which primarily induce apoptotic cell death in ND7 cells, implicated in FALS pathology. The neuroprotective effect of various heat shock proteins (Hsps) in the above system was studied, utilising a Herpes Simplex Virus (HSV) - based gene delivery system. For the first time, it was demonstrated that in an in vitro model of mutant-SOD1-induced toxicity, the exogenous expression of Hsp27 and/or Hsp70 protects both G93A and G93R-SOD1-mutant expressing cells, under all the stresses tested, and the dual expression of Hsp27 and Hsp70 is more effective in protecting against mutant-SOD1 cytotoxicity than either Hsp individually as assessed by trypan blue and TUNEL analysis. In addition, G93A and G93R- SOD1 mutant expressing cells were markedly protected by caspase-8 and caspase-9 inhibition. However no additive protective effect of Hsp and caspase inhibitor was observed. To further investigate the protective effect of wt-SOD1 and damaging effect of the mutant, an HSV-based gene delivery system was utilised. Primary cultures of dorsal root ganglia (DRGs) from postnatal rats, wild-type mice, transgenic Hsp27 and transgenic Hsp70 mice were infected with SOD1 viruses for wt-SOD1 and G93R-mutant SOD1 and subjected to stresses of NGF withdrawal, IFN-gamma and staurosporine treatment. Finally, the DRG experiments were repeated with additional delivery of Hsps via HSV vectors to further investigate the protective effect of the Hsps. These experiments provided further evidence on the protective role of Hsp27 and/or Hsp70 against mutant-SOD1 induced toxicity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available