The atypical RhoGTPase RhoE/Rnd3 is a key molecule to acquire a neuroprotective phenotype in microglia Neubrand, Veronika Elisabeth Forte-Lago, Irene Caro, Marta Delgado, Mario Microglia Neuroinflammation siRNA screen Cell morphology Michael J Fox Foundation (MJFF grant ID 9404 to VEN and MD) and an Excellence Grant of the Andalusian Government (to MD). Both funding bodies supported the collection, analysis, and interpretation of data. Availability of data and materials The datasets analyzed during the current study are all available on the PD map webpage of the University of Luxembourg, https://pdmap.uni.lu/ minerva/. In detail, we refer to the Parkinson’s UK Gene Ontology Project (Foulger et al., 2016), the PD map tool of the University of Luxembourg (Fujita et al., 2014), and the differential transcriptome expression data from post mortem tissue of the substantia nigra (Glaab &Schneider, 2015), as referenced here: Foulger RE, Denny P, Hardy J, Martin MJ, Sawford T, Lovering RC. Using the Gene Ontology to Annotate Key Players in Parkinson’s Disease. Neuroinformatics. 2016;14(3):297–304. doi:https://doi.org/10.1007/s12021-015-9293-2 Fujita KA, Ostaszewski M, Matsuoka Y, Ghosh S, Glaab E, Trefois C, et al. Integrating pathways of Parkinson’s disease in a molecular interaction map. Molecular Neurobiology. 2014;49(1):88–102. doi:1007/s12035-013-8489-4 Glaab E, Schneider R. Comparative pathway and network analysis of brain transcriptome changes during adult aging and in Parkinson’s disease. Neurobiology of Disease. 2015;74:1–13. doi:https://doi.org/10.1016/j.nbd.2014.11.002 Background: Over-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. Methods: 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. Results: We 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. Conclusions: Besides 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. 2024-01-02T12:27:51Z 2024-01-02T12:27:51Z 2018 journal article Neubrand, V.E., Forte-Lago, I., Caro, M. et al. The atypical RhoGTPase RhoE/Rnd3 is a key molecule to acquire a neuroprotective phenotype in microglia. J Neuroinflammation 15, 343 (2018). [https://doi.org/10.1186/s12974-018-1386-z] https://hdl.handle.net/10481/86490 10.1186/s12974-019-1416-5 10.1186/s12974-018-1386-z eng http://creativecommons.org/licenses/by-nc-nd/3.0/ open access Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License Springer Nature