Innes, AE; (2012) Characterisation of novel mutations within Heat Shock Protein 27 causing motor neuropathies. Doctoral thesis , UCL (University College London).

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Abstract

Charcot-Marie-Tooth disease (CMT) 2F and distal Hereditary Motor Neuropathy (dHMN) are peripheral motor axonopathies with limited sensory involvement, which usually present during the first decade of life. They are caused by mutations in heat shock protein 27 (Hsp27)/HSPB1, a highly conserved, ubiquitously expressed molecular chaperone. Hsp27 has several cytoprotective functions including the inhibition of apoptosis, protection against oxidative stress and promotion of axonal growth. In this Thesis, the effects of several pathogenic Hsp27 mutations were examined to elucidate their cellular effects in vitro and map these effects to different regions of the gene. The effects of Hsp27 mutations were first investigated in neuronal-like SH-SY5Y cells in vitro Analysis of cell survival and cellular morphology revealed that all mutations were cytotoxic under basal conditions. However, mutations located in the α-crystallin protein domain of Hsp27 resulted in a significant increase in the vulnerability of cells to cytoskeletal stressors and decreased neurite outgrowth. Using immunocytochemistry, interactions between mutant Hsp27 and cytoskeletal components were also examined. Mutations located in the Hsp27 α-crystallin domain increased co-localisation of Hsp27 with cytoskeletal elements. Although the mutation within the N-terminus did not have this effect it did result in the formation of distinct nuclear aggregates containing mutant Hsp27. The functional effects of Hsp27 mutations were investigated using lentiviral delivery of mutant Hsp27 in primary motoneurons. Examination of mitochondrial function showed that none of the Hsp27 mutants had any effect on mitochondrial membrane potential. The results presented in this Thesis show that disease-causing Hsp27 mutations have differential effects upon protein function in vitro depending upon the gene position of the mutation. Therefore, although all Hsp27 mutations in CMT patients result in motoneuron degeneration, these results suggest that this process may be initiated by different pathological mechanisms and that normal Hsp27 function is essential for the maintenance of motor-axonal function.

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