Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Suzhou, 215006, Jiangsu, China.
Department of Neurosurgery, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, China.
Mol Brain. 2022 Apr 23;15(1):35. doi: 10.1186/s13041-022-00919-6.
RUN and FYVE domain-containing 3 (Rufy3) is a well-known adapter protein of a small GTPase protein family and is bound to the activated Ras family protein to maintain neuronal polarity. However, in experimental subarachnoid hemorrhage (SAH), the role of Rufy3 has not been investigated. Consequently, we aimed to investigate the potential role of Rufy3 in an in vivo model of SAH-induced early brain injury (EBI). In addition, we investigated the relevant brain-protective mechanisms. Oxyhemoglobin (OxyHb) stimulation of cultured primary neurons simulated vitro SAH condition. The SAH rat model was induced by infusing autologous blood into the optic chiasma pool and treating the rats with lentivirus-negative control 1 (LV-NC1), lentivirus-Rufy3 shRNA (LV-shRNA), lentivirus-negative control 2 (LV-NC2), lentivirus-Rufy3 (LV-Rufy3), or 8-pCPT-2'-O-Me-cAMP (8p-CPT) (Rap1 agonist). In experiment one, we found that the protein level of Rufy3 decreased and neuronal axon injury in the injured neurons but was rectified by LV-Rufy3 treatment. In experiment two, mRNA and protein levels of Rufy3 were downregulated in brain tissue and reached the lowest level at 24 h after SAH. In addition, the expression of Myelin Basic Protein was downregulated and that of anti-hypophosphorylated neurofilament H (N52) was upregulated after SAH. In experiments three and four, Rufy3 overexpression (LV-Rufy3) increased the interactions between Rufy3 and Rap1, the level of Rap1-GTP, and the ratio of Rap1-GTP/total GTP. In addition, LV-Rufy3 treatment inhibited axon injury and accelerated axon repair by activating the Rap1/Arap3/Rho/Fascin signaling pathway accompanied by upregulated protein expression levels of ARAP3, Rho, Fascin, and Facin. LV-Rufy3 also enhanced synaptic plasticity by activating the Rap1/MEK/ERK/synapsin I signaling pathway accompanied by upregulated protein expression levels of ERK1, p-ERK1, MEK1, p-MEK1, synaspin I, and p-synaspin I. Moreover, LV-Rufy3 also alleviated brain damage indicators, including cortical neuronal cell apoptosis and degeneration, brain edema, and cognitive impairment after SAH. However, the downregulation of Rufy3 had the opposite effect and aggravated EBI induced by SAH. Notably, the combined application of LV-Rufy3 and 8p-CPT showed a significant synergistic effect on the aforementioned parameters. Our findings suggest that enhanced Rufy3 expression may reduce EBI by inhibiting axon injury and promoting neuronal axon repair and synaptic plasticity after SAH.
RUN 和 FYVE 结构域包含蛋白 3（Rufy3）是一种已知的小 GTPase 蛋白家族的衔接蛋白，与激活的 Ras 家族蛋白结合以维持神经元极性。然而，在实验性蛛网膜下腔出血（SAH）中，Rufy3 的作用尚未得到研究。因此，我们旨在研究 Rufy3 在 SAH 诱导的早期脑损伤（EBI）体内模型中的潜在作用。此外，我们还研究了相关的脑保护机制。培养的原代神经元的氧合血红蛋白（OxyHb）刺激模拟了体外 SAH 条件。通过将自体血液注入视交叉池来诱导 SAH 大鼠模型，并使用慢病毒阴性对照 1（LV-NC1）、慢病毒-Rufy3 shRNA（LV-shRNA）、慢病毒阴性对照 2（LV-NC2）、慢病毒-Rufy3（LV-Rufy3）或 8-pCPT-2'-O-Me-cAMP（8p-CPT）（Rap1 激动剂）处理大鼠。在实验一中，我们发现 Rufy3 蛋白水平降低，损伤神经元中的轴突损伤，但 LV-Rufy3 治疗可纠正。在实验二中，脑内 Rufy3 的 mRNA 和蛋白水平下调，SAH 后 24 小时达到最低水平。此外，SAH 后髓鞘碱性蛋白表达下调，抗低磷酸化神经丝 H（N52）表达上调。在实验三和实验四中，Rufy3 过表达（LV-Rufy3）增加了 Rufy3 与 Rap1、Rap1-GTP 水平和 Rap1-GTP/总 GTP 比值之间的相互作用。此外，LV-Rufy3 治疗通过激活 Rap1/Arap3/Rho/Fascin 信号通路抑制轴突损伤并加速轴突修复，同时上调 ARAP3、Rho、Fascin 和 Fascin 的蛋白表达水平。LV-Rufy3 还通过激活 Rap1/MEK/ERK/synapsin I 信号通路增强突触可塑性，同时上调 ERK1、p-ERK1、MEK1、p-MEK1、synapsin I 和 p-synapsin I 的蛋白表达水平。此外，LV-Rufy3 还减轻了 SAH 后皮质神经元细胞凋亡和变性、脑水肿和认知障碍等脑损伤标志物。然而，下调 Rufy3 则产生相反的效果，并加重了 SAH 引起的 EBI。值得注意的是，LV-Rufy3 和 8p-CPT 的联合应用对上述参数具有显著的协同作用。我们的研究结果表明，增强 Rufy3 的表达可能通过抑制轴突损伤和促进神经元轴突修复和突触可塑性来减轻 SAH 后的 EBI。
