Veronika E. Neubrand
Over-activated microglia, the resident immune cells of the brain, play a central role during neuroinflammation, leading to neuronal cell death and neurodegeneration, for example in Alzheimer´s and Parkinson´s disease. Reversion of these over-activated microglia to a neuroprotective phenotype could regenerate a healthy Central Nervous System (CNS)-supporting microglial environment. Our aim was to identify a dataset of intracellular molecules in primary microglia that play a role in the transition of a neurotoxic phenotype to a ramified, neuroprotective one. To do this, we exploited the anti-inflammatory and neuroprotective properties of conditioned medium of adipose-derived mesenchymal stem cells (CM) as a tool to generate the neuroprotective phenotype of microglia in vitro, and we set up a microscopy-based siRNA screen to identify its hits by cell morphology. We assayed an siRNA array targeting more than 150 genes that codify proteins of cytoskeleton and inflammatory pathways in microglia. From them, siRNA-downregulation of more than 40 genes significantly inhibited the CM-induced transition from a neurotoxic to a neuroprotective microglia phenotype, and 50 siRNA-downregulated genes had the opposite effect. As a proof-of-concept, seven of these targets were validated by downregulation of protein expression with individual siRNAs. They were assayed in functional screens that revealed that the atypical RhoGTPase RhoE/Rnd3 is necessary for BDNF expression and plays an essential role in controlling microglial migration. Besides the identification of RhoE/Rnd3 as a novel inducer of a neuroprotective phenotype in microglia, we propose a list of potential targets to be further confirmed with selective activators or inhibitors. Acknowledgements: This work was supported by a Michael J Fox Foundation research grant.
