Dey Rajib Kumar, Kumari Ranjana, Patra Roni, Soni Dharmendra Kumar, Biswas Roopa, Patnaik Satyakam, Ghosh Debabrata
Immunotoxicology Laboratory, Systems Toxicology Group, FEST Division, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow, Uttar Pradesh, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India.
Department of Anatomy, Physiology and Genetics, School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.
J Biol Chem. 2025 May 24;301(7):110293. doi: 10.1016/j.jbc.2025.110293.
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.
ROCK1通过诱导细胞骨架重排在吞噬作用中发挥重要作用。虽然ROCK1的转录调控已为人所知，但其转录后调控仍未得到充分研究。我们旨在通过ROCK1可能的转录后调控来寻找小胶质细胞吞噬作用的机制。该研究通过计算机分析、突变分析、体外实验以及在BALB/c小鼠中的验证，确定miR-129-5p是暴露于环境应激源砷后小胶质细胞吞噬作用的调节因子。对小鼠原代新生小胶质细胞、BV2小胶质细胞、离体小胶质细胞和人小胶质细胞系CHME3进行的计算机分析和体外研究表明，砷暴露会增加小胶质细胞的吞噬作用。还观察到砷暴露会增加miR-129-5p的水平，从而降低ROCK1蛋白的水平。体外实验和突变分析证实了miR-129-5p在ROCK1的3'UTR上的计算机预测结合位点，也证实了ROCK1 mRNA在小鼠小胶质细胞中穿梭进入细胞质加工体（p小体）。在ROCK1的下游，还对Rac1进行了研究，以确定信号轴中的伙伴。通过立体定向注射抗miR-129-5p并评估离体小胶质细胞中的吞噬作用以及脑冰冻切片中Iba1和PSD95的共定位，在体外研究了miR-129-5p在小胶质细胞吞噬作用中的作用，并在BALB/c小鼠体内进行了验证。最后，用砷、抗miR-129-5p、ROCK-1和Rac1 siRNA进行的各种组合实验证实了miR-129-5p→ROCK1→Rac1→吞噬作用信号轴。总体而言，该研究揭示了miR-129-5p是小胶质细胞吞噬作用的重要调节因子，对突触可塑性和神经退行性并发症具有潜在影响。
