Benn, Susanna Clare; (2002) Neuroprotection by heat shock protein 27 in sensory and motor neurons. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The ability of the small heat shock protein 27 (Hsp27) to prevent apoptotic neuronal death has been examined in primary sensory and motor neurons. The first part of the thesis investigates whether there is a correlation between the expression of Hsp27 and the survival of adult and neonatal sensory and motor neurons. Peripheral nerve injury is well known to induce a substantial death of neonatally injured, but not of adult injured sensory and motor neurons. In the adult, sciatic nerve axotomy results in a rapid upregulation of Hsp27 expression in all injured sensory and motor neurons. In contrast, sciatic nerve injury performed at birth induces Hsp27 upregulation only in a very small proportion (~5%) of injured neurons. Those neonatal neurons that express Hsp27 after nerve injury survive, and those that do not undergo apoptosis. The hypothesis that Hsp27 functions as an intrinsic neuronal survival factor is directly tested. Employing viral vectors to induce human Hsp27 expression in neonatal sensory neurons in vitro and neonatal motor neurons in vivo, Hsp27 is demonstrated to rescue both sensory and motor neurons from nerve injury induced cell death. Delivery of an antisense human Hsp27 construct with a Herpes-Simplex viral vector to knockdown endogenous Hsp27 is shown to induce apoptotic cell death in adult primary sensory neurons. These findings suggest that Hsp27 is both necessary and sufficient for cell survival in these neurons. Immunohistochemical analysis of the apoptotic pathway reveals that Hsp27 inhibits apoptosis in sensory neurons by acting downstream of cytochrome-c release from the mitochondria but upstream of caspase-3 activation. The contribution of post-translational changes in conferring a survival role for Hsp27 is studied by examining the ability of a non-phosphorylatable mutant of Hsp27 to protect neonatally injured motor neurons from death in vivo, and by investigating changes in the phosphorylation of Hsp27 on Seri5 after nerve injury. These findings suggest phosphorylation is important in the neuroprotective role of Hsp27. These findings that all injured adult sensory and motor neurons upregulate Hsp27 and survive and that only a minority of neonatal sensory and motor neurons upregulate Hsp27 and most die, together with the demonstration that Hsp27 sense expression can rescue neonatal cells while antisense constructs induce adult neuronal death, indicate that Hsp27 is indeed an intrinsic neuronal survival factor. That the Hsp27 in all adult injured sensory and motor neurons is phosphorylated, while constitutively expressed Hsp27 is not, as well as the failure of a non-phosphorylatable mutant Hsp27 to rescue cells, suggests that post-translational as well as transcriptional regulation of Hsp27 is important for promoting neuronal survival. The survival of sensory and motor neurons depends on the presence of intrinsic factors that include Hsp27, preventing full activation the apoptotic pathway.

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