The atypical RhoGTPase RhoE/Rnd3 is a key molecule to acquire a neuroprotective phenotype in microglia

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.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1386-z) contains supplementary material, which is available to authorized users.

Switching Roles: Beneficial Effects of Adipose Tissue-Derived Mesenchymal Stem Cells on Microglia and Their Implication in Neurodegenerative Diseases

“…After 60 min, Rac1 activation and lamellipodia formation completely diminish (Figure 3). In line with these results, Rac1 downregulation by siRNA reduces ASC-CM-induced microglial ramification, indicating an essential role of Rac1 in this process [54]. domains [59] that can bind to PIP3 generated by PI3K and might take part in the PI3K/Akt pathway described above.…”

“…[ 56 , 57 ], there might also be other signaling molecules and pathways involved in the switch to a neuroprotective microglial phenotype, leading to synergistic effects with the pathway described above. In order to shed light on genes implicated in these pathways, Neubrand and colleagues performed an siRNA screen of primary microglia stimulated with ASC-CM to induce microglial ramification [ 54 ]. The siRNA-downregulated expression of certain genes significantly inhibits microglial ramification.…”

Inhibition of microRNA-128-3p alleviates liver ischaemia–reperfusion injury in mice through repressing the Rnd3/NF‐κB axis

“…Meanwhile, in the inflammatory area, Rnd3 was reported as a new fine-tuning factor regulating myocardial infarction-induced inflammation and promoted cardiac cell recovery. 15 Neubrand et al 33 showed that Rnd3 might be an underlying neuroprotective phenotype in microglia, while Sun et al 34 further showed that Rnd3 could inhibit NF-jB signalling to induce glioblastoma multiforme cell apoptosis by binding p65 and promoting its ubiquitination, resulting in decreased p65.…”

Long-termin vivoimaging of mouse spinal cord through an optically cleared intervertebral window

“…Unlike the immunostaining method that is only applicable to postmortem study, the morphological analysis of microglia combined with highresolution in vivo imaging techniques can serve as a versatile and sensitive means to detect subtle inflammatory activity in living animals, which is indispensable for in vivo longitudinal study of immune responses to different pathological situations. Among the morphological parameters, the ramification index (RI) 32,[44][45][46][47] and the number of process endpoints (NPE) 33,36,[48][49][50] are widely used to describe the ramification of microglial cells quantitatively. RI is calculated as the ratio of the cell's perimeter to its area normalized to that of a circle with the same area 32 , while NPE counts the total number of microglial cell processes 33 .…”