Spinal Cord and Brain Injury Research Group, Stark Neurosciences Research Institute, Indiana University School of Medicine, 320 W. 15th Street, NB 500E, Indianapolis, IN, 46202, USA.
Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, 46202, USA.
Mol Neurobiol. 2017 Nov;54(9):6885-6895. doi: 10.1007/s12035-016-0187-6. Epub 2016 Oct 22.
Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A (cPLA), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA. Inhibition of RhoA, Rho kinase and cPLA significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA. The immunofluorescence staining showed that ROCK or ROCK, two isoforms of Rho kinase, was co-localized with cPLA in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK or ROCK bonded directly with cPLA and phospho-cPLA. When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA activation.
RhoA/Rho 激酶的激活导致生长锥塌陷和轴突回缩。虽然 RhoA/Rho 激酶抑制已被证明可以改善轴突再生、髓鞘再生和功能恢复，但它在神经元细胞死亡中的作用尚不清楚。为了确定 RhoA/Rho 激酶在损伤后神经元死亡中是否起作用，我们使用体外神经元死亡模型和第十胸椎（T10）水平的创伤性脊髓损伤模型，研究了 RhoA/Rho 激酶与细胞质磷脂酶 A（cPLA）之间的关系，cPLA 是一种介导炎症和细胞死亡的脂肪酶。我们发现，TNF-α 和谷氨酸共同给药诱导脊髓神经元死亡，并激活 RhoA、Rho 激酶和 cPLA。RhoA、Rho 激酶和 cPLA 的抑制分别显著降低 TNF-α/谷氨酸诱导的细胞死亡 33%、52%和 43%（p<0.001）。RhoA 和 Rho 激酶的抑制也分别显著下调 cPLA 激活 66%和 60%（p<0.01）。此外，RhoA 和 Rho 激酶的抑制还降低了 cPLA 下游底物花生四烯酸的释放。免疫荧光染色显示，ROCK 或 ROCK，即 Rho 激酶的两种同工型，与神经元细胞质中的 cPLA 共定位。有趣的是，共免疫沉淀（Co-IP）实验表明，ROCK 或 ROCK 与 cPLA 和磷酸化 cPLA 直接结合。当 Rho 激酶抑制剂 Y27632 应用于 T10 挫伤损伤的小鼠时，它显著降低了 cPLA 的激活和表达，并减少了损伤诱导的在损伤部位及其附近的细胞凋亡。总之，我们的结果揭示了 RhoA/Rho 激酶通过调节 cPLA 激活介导神经元死亡的新机制。
