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Characterization of autophagy in retinal rod photoreceptor cells in Xenopus laevis Wen, Runxia

Abstract

Retinitis pigmentosa (RP) is a genetic neurodegenerative disorder that causes progressive cell death of the rod and cone photoreceptors, eventually leading to blindness. The light-sensitive protein in rods, rhodopsin, is composed of the chromophore 11-cis retinal and the protein rod opsin. Mutations in the rhodopsin gene are common causes of RP. Autophagy is a lysosomal-turnover pathway for degrading dysfunctional proteins, organelles or other cellular components that is necessary for maintaining cellular homeostasis. We observed an increase of autophagy structures in rods expressing the misfolding-prone rhodopsin mutant P23H (Bogéa et al. 2015). However, the role autophagy plays in RP is not clearly understood. To examine the role of autophagy in normal and diseased rods, I generated transgenic Xenopus laevis tadpoles expressing the autophagy reporter mRFP-eGFP-LC3. My results demonstrate that the autophagy process lasts for about 34 h in normal rods. Early autophagic structures persist for 6 to 8 h before fusing with lysosomes and acidification; acidified autolysosomes persist for about 28 h before complete digestion. Autophagy in normal rods is diurnally regulated, with more autophagic structures generated in light and fewer in darkness; this regulation is non-circadian. Autophagy also increased in rods co-expressing P23H rhodopsin. The rhodopsin chromophore, a pharmacological chaperone for rhodopsin, absorbs photons to initiate phototransduction, and is consumed in this process; it also promotes proper rhodopsin folding. To determine whether increased autophagy in light-exposed normal rods is caused by increased misfolding of wildtype rhodopsin due to lack of chromophore, I used CRISPR/Cas9 to knock out the gene RPE65, which is essential for chromophore biosynthesis. I observed that eliminating chromophore does not promote autophagy in dark-reared rods, but prevents induction of autophagy in light-exposed rods. This combination of outcomes suggests that, although rhodopsin misfolding can induce autophagy, light-induced autophagy is not due to misfolding of rhodopsin, but rather due to phototransduction. Further, I found that a group of compounds called histone deacetylase (HDAC) inhibitors, valproic acid (VPA), sodium butyrate (NaBu) and CI-994, consistently promote autophagy in rods; these compounds were previously demonstrated to ameliorate retinal degeneration associated with P23H rhodopsin (Vent-schmidt et al. 2017).

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