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Induction of Autophagy Promotes Clearance of RHOP23H Aggregates and Protects From Retinal Degeneration

ABSTRACT: Autophagy is a critical metabolic process that acts as a major self-digestion and recycling pathway contributing to maintain cellular homeostasis. An emerging field of research supports the therapeutic modulation of autophagy for treating human neurodegenerative disorders, in which toxic aggregates are accumulated in neurons. Our previous study identified Ezrin protein as an inhibitor of autophagy and lysosomal functions in the retina; thus, in turn, identifying it as a potential pharmacological target for increasing retinal cell clearance to treat inherited retinal dystrophies in which misfolded proteins have accumulated. This study aimed to verify the therapeutic inhibition of Ezrin to induce clearance of toxic aggregates in a mouse model for a dominant form of retinitis pigmentosa (i.e., RHOP23H/+). We found that daily inhibition of Ezrin significantly decreased the accumulation of misfolded RHOP23H aggregates. Remarkably, induction of autophagy, by a drug-mediated pulsatile inhibition of Ezrin, promoted the lysosomal clearance of disease-linked RHOP23H aggregates. This was accompanied with a reduction of endoplasmic reticulum (ER)-stress, robust decrease of photoreceptors' cell death, amelioration in both retinal morphology and function culminating in a better preservation of vision. Our study opens new perspectives for a pulsatile pharmacological induction of autophagy as a mutation-independent therapy paving the way toward a more effective therapeutic strategy to treat these devastating retinal disorders due to an accumulation of intracellular toxic aggregates.

SUBMITTER: Intartaglia D

PROVIDER: S-EPMC9281868 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We previously reported autophagic structures in rod photoreceptors expressing a misfolding RHO (rhodopsin) mutant (RHOP23H), suggesting that autophagy may play a role in degrading the mutant RHO and/or be involved in photoreceptor cell death. To further examine autophagy in normal and diseased rods, we generated transgenic Xenopus laevis tadpoles expressing the dually fluorescent autophagy marker mRFP-eGFP-LC3 in rods, which changes from green to yellow and finally red as autophagic structures develop and mature. Using transgenic lines with constitutive and inducible expression, we determined the time-course of autophagy in rod photoreceptors: autophagosomes last for 6 to 8 hours before fusing with lysosomes, and acidified autolysosomes last for about 28 hours before being degraded. Autophagy was diurnally regulated in normal rods, with more autophagic structures generated during periods of light, and this regulation was non-circadian. We also found that more autophagosomes were produced in rods expressing the misfolding RHOP23H mutant. The RHO chromophore absorbs photons to initiate phototransduction, and is consumed in this process; it also promotes RHO folding. To determine whether increased autophagy in light-exposed normal rods is caused by increased RHO misfolding or phototransduction, we used CRISPR/Cas9 to knock out the RPE65 and GNAT1 genes, which are essential for chromophore biosynthesis and phototransduction respectively. Both knockouts suppressed light-induced autophagy, indicating that although light and misfolded rhodopsin can both induce autophagy in rods, light-induced autophagy is not due to misfolding of RHO, but rather due to phototransduction. Abbreviations: CYCS: cytochrome c; bRHOP23H: bovine RHOP23H; Cas9: CRISPR associated protein 9; dpf: days post-fertilization; eGFP: enhanced green fluorescent protein; GNAT1: guanine nucleotide-binding protein G(t) subunit alpha-1 aka rod alpha-transducin; HSPA1A/hsp70: heat shock protein of 70 kilodaltons; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1A/1B light chain 3; mRFP: monomeric red fluorescent protein; RHO: rhodopsin; RP: retinitis pigmentosa; RPE65: retinal pigment epithelium-specific 65 kDa protein: sfGFP: superfolding GFP; sgRNA: single guide RNA; WGA: wheat germ agglutinin; RHOp: the Xenopus laevis RHO.2.L promoter.

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Induction of Autophagy Promotes Clearance of RHOP23H Aggregates and Protects From Retinal Degeneration

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