Rac1 conditional deletion attenuates retinal ganglion cell apoptosis by accelerating autophagic flux in a mouse model of chronic ocular hypertension

Abstract

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.

Interplay between Müller cells and microglia aggravates retinal inflammatory response in experimental glaucoma

“…We first examined the dynamic changes of Müller cell and microglia activation in the mouse model of COH that was produced by injecting micro-magnetic beads into the anterior chamber [ 12 , 13 ]. Changes of IOP in COH mice are shown in Additional file 1 : Fig.…”

“…Mouse COH model was produced by injection of the micro-magnetic beads (2 μL, diameter ≈ 10 μm, BioMag ® Superparamagnetic Iron Oxide, Bangs Laboratories, Ins, USA) into the anterior chamber of the right eyes, referring to the procedure previously described in detail [ 12 , 13 ]. In brief, the mice were anesthetized by 2% pentobarbital sodium (40 mg/kg, i.p.)…”

Rapamycin and Resveratrol Modulate the Gliotic and Pro-Angiogenic Response in Müller Glial Cells Under Hypoxia

“…Thereby, it would be predictable that the autophagic flux have a relevant role during initial stages of the stimulus, until the cell reach a stable metabolic state. Prolonged exposition frequently show a different autophagy flux ( Zhang et al, 2020 ). By Western blot and immunofluorescence assays we demonstrated that autophagic flux returned to normoxic levels when incubated in 1% O 2 during 24 h. This result indicates that the activation of the flux is transient and takes place immediately after the exposition to hypoxic environment.…”

The interplay of autophagy and oxidative stress in the pathogenesis and therapy of retinal degenerative diseases

“…Chloroquine (CQ) and hydroxychloroquine (HCQ) are autophagic inhibitors popularly used as antitumor agents [ 241 ]. Both CQ and HCQ have been reported to cause RGC damage [ 242 , 243 ]. In the clinic, the mean values of quantified fundus autofluorescence (QAF, an indirect approach to measuring lipofuscin in the RPE in vivo) were significantly higher in patients receiving CQ/HCQ than in healthy controls, indicating that CQ/HCQ treatment leads to retinal damage [ 244 ].…”