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Fas Receptor Mediates seizure induced neuronal death and Epileptogenesis

Final Activity Report Summary - FARMINDE (Fas Receptor Mediates seizure induced neuronal death and Epileptogenesis)

Epilepsy is a significant health problem in the EU, affecting 1-3% of the population. Understanding the molecular mechanisms underlying brain damage after prolonged seizures may help optimise treatment of epilepsy, the brain injuries which cause it and the brain injury caused by seizures themselves. Work leading up to this project had identified the molecular pathways of apoptosis (programmed cell death) as important. Of particular interest, the role of a group of receptors present on neurons that belong to the tumour necrosis factor (TNF) family. The aim of the work was to improve our understanding of death receptor activity after seizures and investigate longer-term roles: the Fas receptor has been implicated in determining neuronal shape and so our hypothesis was that Fas might also be active during brain re-modelling as epilepsy develops. The following objectives were proposed and results obtained:
Project Objective 1: Characterise the role of Fas receptor signalling during seizure-induced neuronal death in mice.

Working on our first objective we followed the time course of Fas and related gene expression levels in the hippocampus of mice subject to prolonged (damaging) seizures. Our results showed regulation of some components of the Fas pathway. We also used subcellular fractionation to detect changes in the levels of Fas signalling proteins within specific compartments of the cell after seizures. These findings were supported by fluorescence microscopy evidence of Fas overexpression within neurons of the damaged hippocampus 7 days after the prolonged seizure, at a time when epilepsy begins to develop.

Next, we undertook experiments to manipulate the Fas signalling pathway using two different antibody approaches: a neutralizing antibody and a Fas-activating antibody. Note: studies on knockout mice were aborted before commencement because of published reports of abnormalities in neuron shape that would preclude our use of them. These experiments demonstrated that modulating the Fas pathway by this approach did not significantly affect seizure-induced cell death.

To learn whether these mouse results were clinically relevant we examined death receptor signalling in human hippocampus, comparing findings between controls and patients with intractable temporal lobe epilepsy. This work, published in the journal Experimental Neurology revealed levels of the signalling adaptor TRADD and cleaved caspase-8 were higher in epilepsy samples than controls. Within intracellular compartments patients had higher levels of cytoplasmic TNFR1, TRADD and FADD and microsomal levels of TRADD and cleaved caspase-8. Levels of the Fas target ASK1 (apoptosis signal regulating kinase 1) were also higher in epilepsy patient brain. Immunoprecipitation studies detected TRADD-FADD binding in TLE hippocampus and fluorescence microscopy revealed TRADD co-localization with FADD. These data showed for the first time that death receptor signalling is engaged in the hippocampus of patients with refractory temporal lobe epilepsy and may be a potential target for adjunctive neuroprotective therapy.

Project Objective 2:
Characterise Fas receptor signalling during epileptogenesis and the epilepsy phenotype of Fas receptor-deficient mice.

Studies in our second aim set about defining the significance of Fas for development of epilepsy in our mouse model. Mice were subject to prolonged seizures and then EEG was recorded intermittently thereafter. Our findings show for the first time that status epilepticus in this model triggers development of epilepsy. Mice became epileptic, exhibiting epileptic seizures characterised by variable periods of high amplitude high frequency seizure activity that began as early as 4 days after the initial insult.