Spadoni, Cesare Giuseppe; (2000) Identification of neuronal caspases and involvement of death domain proteins in neuronal apoptosis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Apoptosis is implicated in both developmental and disease related neuronal loss. The characteristic morphological changes that define apoptosis appear to result from the activation of members of a class of cysteine proteases known as caspases. While identification of the relevant caspases and their means of regulation have been investigated in many cell types, the corresponding pathways in neurones are still unclear. One activating mechanism in non neuronal cell types depends upon caspase-8 autocatalytic activation upon aggregation induced by interactions with protein domains referred to as death domains (DD) and death effector domains (DED). The aim of this study was the identification of neuronal caspases and potential DD/DED upstream regulators. These were studied in a model of developmental neuronal apoptosis, rat P1 superior cervical ganglion (SCG) neurones which are dependent upon nerve growth factor (NGF) for survival. The screening of a rat brain library for DD/DED encoding cDNAs resulted in the cloning of the rat homologue of FADD. Wild type (WT) FADD and a dominant negative (DN) FADD mutant were overexpressed by microinjection in SCG neurones. Overexpression of FADD WT did not induce cell death in neurones in the presence of NGF, whereas both the WT and DN rescued neurones from NGF withdrawal induced apoptosis, suggesting that DDs are involved in neuronal apoptosis. The expression of several caspases and DD-related proteins was assessed in SCG neurones with an optimised RT-PCR protocol. While caspases-2, -3, -6, -7 and -9 were found to be normally expressed, caspase-8, a known partner of FADD in non neuronal systems, was absent. Furthermore, the expression of caspase-8 and DD-related mRNAs such as FADD, Fas and Fas ligand were not induced after NGF withdrawal. These findings suggest a regulatory role for DD proteins in SCG neuronal apoptosis, which is different from the Fas-FADD-caspase-8 pathway described for non neuronal cells.

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