Anti-apoptotic cFLIP proteins in human brain tissue

The relative importance of apoptotic cell death in brain injury is still debated.The cFLIP proteins are endogenous caspase inhibitors, with well-established anti-apoptosis, cell-protective actions.Two splice variants a and d are known, both of which compete with initiator pro-caspases (8 and 10) at the death inducing signalling complex (DISC), thereby inhibiting death receptor-mediated cell death.The recent literature and our own data suggest that apoptotic molecular pathways are active in human brain. We have shown that cFLIPs are present in cerebral cortex and we are in process of determining what functional role these may have. We are thus well-placed to formulate the following Aims.

Aims:

1. To determine the interaction partners of cFLIP proteins in post mortem human brain. This analysis of protein interactions is novel, and vital for any understanding of mechanism of action.

2. To test for immunohistochemical localisation of cFLIP proteins and markers of cellular neuropathology in human post mortem brain.

The abundance of cFLIP isoforms in homogenates of human brain will be assayed in Western blots. The composition of molecular complexes of cFLIP proteins with other brain proteins will be determined, using co-immunoprecipitation from cortical homogenates. In particular, we will test whether the composition and cFLIP-content of death receptor (e.g. TNF receptor, Fas) complexes differ between AD cases and controls. This is a highly relevant investigation, as the functional effects of cFLIPs depend on the biochemical complexes that they form with other proteins (e.g. caspase 8, FADD, Daxx). This will give valuable pointers to the role of cFLIPs in brain tissue. The cellular localisation of cFLIP proteins will be determined immuno-histochemically in fixed cortical slices.We will test for co-localisation with biochemical markers of neuropathology such as FluoroJade stain, nuclear TUNEL reaction, activated caspase-3.

Immunoprecipitation of cFLIP-a by anti-Fas antibody.The cleaved form of cFLIP-a (44 kDa) was detected in complexes from control cortex (C ) or 6 hrs post-traumatic brain injury, indicating the presence of a Fas-cFLIPa complex.Heart (H) and spleen (S) lysates were positive and negative controls, respectively. The precipitating antibody JO2 shows no false-positive immunoreactivity (lane 1).

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