Kingham, Paul; (2000) Microglial-neuronal interactions. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Recent evidence suggests that microglia, the resident macrophages of the CNS, play a role in neurodegeneration. In the Alzheimer's brain there are increased levels of both activated and apoptotic microglia. Chromogranin A (CGA), is a peptide upregulated in Alzheimer's disease and may be a physiological activator of microglia. The signalling pathways involved in the response to CGA were investigated in cell cultures of microglia as well as the subsequent effect of secreted factors on neuronal cultures. Following exposure to CGA, cultured microglia displayed apoptotic changes - pyknotic nuclei and DNA fragmentation. Activated microglia produced nitric oxide prior to a collapse in mitochondrial membrane potential (ΔΨm) which could be blocked with cyclosporin A suggesting the involvement of the mitochondrial permeability transition. Nitric oxide synthase inhibitors prevented both the fall in ΔΨm and downstream apoptosis whereas the caspase inhibitor, YVAD-CHO, only prevented the latter. The translocation of cytochrome c from the inner mitochondrial membrane to cytosol was not necessary for caspase activation. CGA activated microglia also released glutamate, attenuation of which prevented the microglial death as did the metabotropic glutamate receptor antagonist, MSPG. Cultured neurones treated with conditioned medium from CGA activated microglia underwent apoptosis which was blocked by z-DEVD-fmk, a different class of caspase inhibitor. Neuronal death was in part mediated by activation of ionotropic glutamate receptors and also by proteins released from activated microglia. Exposure of neuronal cultures to cathepsin B, which was secreted by CGA activated microglia, resulted in neuronal apoptosis which could also be blocked with z-DEVD-fmk. Single cell fluorescence imaging of intracellular calcium ([Ca2+]i) in neurones revealed a small elevation in [Ca2+]i following the addition of conditioned medium which may constitute an early step in the apoptotic pathway. The discovery of these distinct signalling pathways could allow for new therapeutic strategies in the treatment of disorders such as Alzheimer's disease.

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