Abstract
The glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for several neuronal populations in different brain regions, including the hippocampus. However, no information is available on the: (1) hippocampal subregions involved in the GDNF-neuroprotective actions upon excitotoxicity, (2) identity of GDNF-responsive hippocampal cells, (3) transduction pathways involved in the GDNF-mediated neuroprotection in the hippocampus. We addressed these questions in organotypic hippocampal slices exposed to GDNF in presence of N-methyl-D-aspartate (NMDA) by immunoblotting, immunohistochemistry, and confocal analysis. In hippocampal slices GDNF acts through the activation of the tyrosine kinase receptor, Ret, without involving the NCAM-mediated pathway. Both Ret and ERK phosphorylation mainly occurred in the CA3 region where the two activated proteins co-localized. GDNF protected in a greater extent CA3 rather than CA1 following NMDA exposure. This neuroprotective effect targeted preferentially neurons, as assessed by NeuN staining. GDNF neuroprotection was associated with a significant increase of Ret phosphorylation in both CA3 and CA1. Interestingly, confocal images revealed that upon NMDA exposure, Ret activation occurred in microglial cells in the CA3 and CA1 following GDNF exposure. Collectively, this study shows that CA3 and CA1 hippocampal regions are highly responsive to GDNF-induced Ret activation and neuroprotection, and suggest that, upon excitotoxicity, such neuroprotection involves a GDNF modulation of microglial cell activity.
Highlights
Considerable interest has been devoted to neurotrophins as candidate neuroprotective agents for several neurodegenerative disorders since they promote neuronal survival, neuritic growth, and differentiation of several, but selective, neuronal populations
With the aim to investigate the intracellular signalling mechanisms triggered by glial cell line-derived neurotrophic factor (GDNF) stimulation of rat organotypic hippocampal slices, we first tested the effect on ERK phosphorylation in slice cultures at day of culture in vitro (DIV) 2, 7, and 14 following dissection
In order to determine whether the stimulation of phosphorylated ERK levels by GDNF treatment was mediated by a Ret-dependent or by a p140NCAM-dependent mechanism, we stimulated hippocampal slices at 7 and 14 DIV with 200 ng/ml GDNF for 30 minutes and looked for Ret and FAK tyrosine phosphorylation, the latter being an upstream effector of neural cell adhesion molecule (NCAM)-dependent GDNF signalling [4]
Summary
Considerable interest has been devoted to neurotrophins as candidate neuroprotective agents for several neurodegenerative disorders since they promote neuronal survival, neuritic growth, and differentiation of several, but selective, neuronal populations. One such candidate is glial cell line-derived neurotrophic factor (GDNF) given its spectrum of demonstrated activities which includes, but is not limited to, potent trophic actions on a wide variety of neuronal populations of the central and peripheral nervous systems [1]. A Ret-independent pathway of GDNF signalling that involves the association of GFRa-1 with the p140NCAM isoform of the neural cell adhesion molecule (NCAM) and subsequent activation of Fyn and FAK kinases, has been as well demonstrated to take place in primary glial cells and neurons [4,5]
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