MicroRNA-129-5p-mediated translational repression of microglial ROCK1 leads to enhanced phagocytosis.

ROCK1 plays an important role in phagocytosis by inducing cytoskeletal rearrangement. Although the transcriptional regulation of ROCK1 is known but its post-transcriptional regulation is underexplored. We intended to find a mechanism of microglial phagocytosis through possible post-transcriptional regulation of ROCK1. The study identified miR-129-5p as a regulator of microglial phagocytosis following exposure to an environmental stressor, arsenic, combining in silico analysis, mutational analysis, in vitro experiments, and validation in BALB/c mouse. The in silico analysis and in vitro studies with mouse primary neonatal microglia, BV2 microglia, ex vivo microglia, and human microglial cell line CHME3 revealed that arsenic exposure increases microglial phagocytosis. Arsenic exposure was also observed to increase the level of miR-129-5p and consequently decrease the level of ROCK1 protein. In vitro experiments and mutational analysis confirmed the in silico predicted binding site of miR-129-5p on the 3'UTR of ROCK1 and also confirmed the shuttling of ROCK1 mRNA into the cytoplasmic-processing body (p-body) in mouse microglia. Downstream to ROCK1, Rac1 has also been studied to pinpoint the partners in the signaling axis. The role of miR-129-5p in microglial phagocytosis was studied in vitro and validated in vivo in BALB/c mouse by stereotactically injecting anti-miR-129-5p and assessing the phagocytosis in ex vivo microglia and colocalization of Iba1 and PSD95 in brain cryosection. Finally, experiments with arsenic, anti-miR-129-5p, ROCK1 & Rac1 siRNA in various combinations confirmed the miR-129-5p→ROCK1→Rac1→Phagocytosis signaling axis. Overall, the study revealed miR-129-5p as an important regulator of microglial phagocytosis with potential implication in synaptic plasticity and neurodegenerative complications.