Zourlidou, Alexandra; (2005) Analysis of mutations in alpha-synuclein and the protective effect of heat shock proteins in a model of alpha-synuclein-induced toxicity. Doctoral thesis , University of London.

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Abstract

Genetic studies have revealed three mutations (A30P, A53T and E46K) in alpha-synuclein (alpha-Syn) that cause Parkinson's disease (PD) in a small number of pedigrees with autosomal dominant inheritance. For the purpose of this thesis an in vitro model has been developed by stably over-expressing wild type (wt), A30P or A53T mutant alpha-Syn in ND7 neuronal cells. Wt alpha-Syn can enhance cell death in response to ischaemia/reoxygenation or staurosporine treatment whilst protecting against serum removal and dopamine-induced cell death in this system. In contrast, both mutant forms of alpha-Syn enhance cell death. The above stresses were used to induce primarily apoptotic cell death, implicated in PD pathology. Hence, the PD-associated mutations convert alpha-Syn from a protein which could modulate cell death differently in different circumstances to forms which are deleterious in response to various stresses. Subsequently, the neuroprotective effect of various heat shock proteins (hsps) in the above system was studied, utilising a Herpes Simplex Virus-based gene delivery system. For the first time, it was demonstrated that in an in vitro mammalian model of alpha-Syn-induced toxicity over-expression of hsp27 protects, under all the stresses tested, both wt and mutant alpha-Syn expressing cells, as assessed by multiple apoptotic/necrotic death assays. Interestingly, A30P alpha-Syn expressing cells were markedly protected by caspase-8 and caspase-9 inhibition as well as by hsp27 over-expression. No synergy between hsp27 and the caspase inhibitors was observed. In addition, hsp70 conferred protection only to wt alpha-Syn expressing cells exposed to ischaemia whereas hsp56 had no protective role in this system. Hence, hsp27 was neuroprotective by interfering with the enhanced caspase-dependent cell death resulting from mutant A30P alpha-Syn over-expression. Finally, studies of the mitochondrial status in this system were performed to further explore the site of action of hsp27. Hsp27 reduced significantly the mitochondrial membrane potential loss in stressed A30P mutant alpha-Syn cells and this correlates well with their enhanced cell survival. These findings suggest that hsp27 has a novel neuroprotective role against mutant alpha-Syn toxicity and this is achieved by interfering with the caspase cascade and mechanisms modulating the mitochondrial membrane potential.

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