Autophagy has a fundamental role in maintaining cell homeostasis. Although autophagy has been implicated in glaucomatous pathology, how it regulates retinal ganglion cell (RGC) injury is largely unknown. In the present work, we found that biphasic autophagy in RGCs occurred in a mouse model of chronic ocular hypertension (COH), accompanied by activation of Rac1, a member of the Rho family. Rac1 conditional knockout (Rac1 cKO) in RGCs attenuated RGC apoptosis, in addition to blocking the increase in the number of autophagosomes and the expression of autophagy-related proteins (Beclin1, LC3-II/I, and p62) in COH retinas. Electron micrograph and double immunostaining of LAMP1 and LC3B showed that Rac1 cKO accelerated autolysosome fusion in RGC axons of COH mice. Inhibiting the first autophagic peak with 3-methyladenine or Atg13 siRNA reduced RGC apoptosis, whereas inhibiting the second autophagic peak with 3-MA or blocking autophagic flux by chloroquine increased RGC apoptosis. Furthermore, Rac1 cKO reduced the number of autophagosomes and apoptotic RGCs induced by rapamycin injected intravitreally, which suggests that Rac1 negatively regulates mTOR activity. Moreover, Rac1 deletion decreased Bak expression and did not interfere with the interaction of Beclin1 and Bcl-2 or Bak in COH retinas. In conclusion, autophagy promotes RGC apoptosis in the early stages of glaucoma and results in autophagic cell death in later stages. Rac1 deletion alleviates RGC damage by regulating the cross talk between autophagy and apoptosis through mTOR/Beclin1-Bak. Interfering with the Rac1/mTOR signaling pathway may provide a new strategy for treating glaucoma.

自噬在维持细胞稳态方面具有重要作用。尽管自噬与青光眼病理有关，但它如何调节视网膜神经节细胞 (RGC) 损伤在很大程度上是未知的。在目前的工作中，我们发现 RGC 中的双相自噬发生在慢性高眼压 (COH) 小鼠模型中，伴随着 Rho 家族成员 Rac1 的激活。RGC 中的 Rac1 条件性敲除 (Rac1 cKO) 减弱了 RGC 细胞凋亡，此外还阻止了自噬体数量的增加和自噬相关蛋白（Beclin1、LC3-II/I 和 p62）在 COH 视网膜中的表达。LAMP1 和 LC3B 的电子显微照片和双重免疫染色表明，Rac1 cKO 加速了 COH 小鼠 RGC 轴突中的自溶酶体融合。用 3-甲基腺嘌呤或 Atg13 siRNA 抑制第一个自噬峰可减少 RGC 细胞凋亡，而用 3-MA 抑制第二个自噬峰或通过氯喹阻断自噬通量可增加 RGC 细胞凋亡。此外，Rac1 cKO 减少了玻璃体内注射雷帕霉素诱导的自噬体和凋亡 RGC 的数量，这表明 Rac1 负向调节 mTOR 活性。此外，Rac1 缺失降​​低了 Bak 的表达并且不干扰 Beclin1 和 Bcl-2 或 Bak 在 COH 视网膜中的相互作用。总之，自噬在青光眼早期促进 RGC 细胞凋亡，并在后期导致自噬细胞死亡。Rac1 缺失通过 mTOR/Beclin1-Bak 调节自噬和细胞凋亡之间的串扰来减轻 RGC 损伤。