Abstract
BackgroundOver-activated microglia play a central role during neuroinflammation, leading to neuronal cell death and neurodegeneration. Reversion of over-activated to neuroprotective microglia phenotype could regenerate a healthy CNS-supporting microglia environment. Our aim was to identify a dataset of intracellular molecules in primary microglia that play a role in the transition of microglia to a ramified, neuroprotective phenotype.MethodsWe 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.ResultsWe initially assayed an array of 157 siRNAs against genes that codify proteins and factors of cytoskeleton and activation/inflammatory pathways in microglia. From them, 45 siRNAs significantly inhibited the CM-induced transition from a neurotoxic to a neuroprotective phenotype of microglia, and 50 siRNAs had the opposite effect. As a proof-of-concept, ten of these targets were validated with individual siRNAs and by downregulation of protein expression. This validation step resulted essential, because three of the potential targets were false positives. The seven validated targets were assayed in a functional screen that revealed that the atypical RhoGTPase RhoE/Rnd3 is necessary for BDNF expression and plays an essential role in controlling microglial migration.ConclusionsBesides the identification of RhoE/Rnd3 as a novel inducer of a potential neuroprotective phenotype in microglia, we propose a list of potential targets to be further confirmed with selective activators or inhibitors.
Highlights
Over-activated microglia play a central role during neuroinflammation, leading to neuronal cell death and neurodegeneration
Since a dominant-negative mutant of the small RhoGTPase Rac1 inhibited the Conditioned medium of ASCs (CM)-induced conversion to an elongated neuroprotective microglia [7], we expected the same phenotype for its small interference RNA (siRNA) knock down
The quantification shows that CM-treated microglia that were previously transfected with three individual siRNAs targeting Rac1 or a pool of them have a higher value for circularity, corresponding to a rounder cell shape, compared to control CM-treated microglia that were not transfected or transfected with scrambled siRNAs (Fig. 1c)
Summary
Over-activated microglia play a central role during neuroinflammation, leading to neuronal cell death and neurodegeneration. Our aim was to identify a dataset of intracellular molecules in primary microglia that play a role in the transition of microglia to a ramified, neuroprotective phenotype. The underlying neuroinflammation in these neurodegenerative diseases are characterized by microglia activation [2, 3] During this inflammatory process, microglia acquire an amoeboid cell shape [4], and amongst other mechanisms, they decrease the release of neurotrophic factors, such as the brain-derived neurotrophic factor (BDNF), and secrete cytotoxic substances, which lead to neuronal death [5]. Neubrand et al Journal of Neuroinflammation (2018) 15:343 underwent a dramatic cell shape change into a highly elongated morphology in vitro [7], similar to the phenotype of microglia observed in a healthy brain [8]. We previously identified that the small RhoGTPases Rac and Cdc, which are important regulators of the actin cytoskeleton [9, 10], play major roles in this phenotypic transition [7]
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