Src tyrosine kinase signaling regulates microglial protection of neurons from excitotoxicity

Abstract

Microglial activation is thought to be important in Alzheimer's disease (AD). However, signal transduction pathways mediating microglia activation are incompletely understood. Src tyrosine kinase is important in the regulation of a variety of cellular functions, including inflammation. We previously found that overexpressing the M-CSF receptor (M-CSFR; encoded by c-fms) on microglia leads to rescue of neurons from NMDA-induced excitotoxicity. We sought to determine if Src signaling mediates M-CSFR-induced neuroprotection. We examined the effects of M-CSFR overexpression on Src kinase levels and activity. We also tested whether LPS, a potent inflammatory stimulus, activated Src. To determine the specificity of M-CSFR activation of Src, we quantified the activity of the Src-family kinase Lyn. A mutagenesis approach was used to block M-CSFR-induced Src activation, and the effects on microglial neuroprotection were examined. We also tested whether a Src dominant negative (DN) construct would block M-CSFR-induced neuroprotection. Western analysis showed that microglial M-CSFR activation resulted in a greater than 2 fold increase in Src expression, whereas an enzymatic assay showed a greater than 3.5 fold increase in Src kinase activity. Microglia treatment with LPS did affect Src activity. M-CSFR activation had no effect on Lyn activity. In the M-CSFR, tyrosine position c-fmsY559 binds Src and induces activation by phosphorylation. Position c-fmsY807, the global c-fms activation site, indirectly affects Src activation. Using co-cultures consisting of microglia overexpressing the M-CSFR and rat organotypic hippocampal cultures subjected to NMDA-induced excitotoxicity, overexpression of the Y559F or Y807F c-fms mutant constructs in microglia resulted in a loss of neurprotection. Similarly, microglial expression of a Src DN form resulted in loss of M-CSFR-induced neuroprotection, and also inhibited microglial proliferation. Increasing the number of microglia expressing the Src DN form in co-culture only partially restored neuroprotection. Adding exogenous M-CSF also partially restored neuroprotection by Src DN-treated microglia in co-cultures. Our results show that the Src signal transduction pathway is important in microglial neuroprotection. Identification of downstream Src kinase targets in microglia could lead to new pharmacological treatments for AD (Funding: Alzheimer's Association and NIH MH57833).