Autophagy inXenopus laevisrod photoreceptors is independently regulated by phototransduction and misfolded RHO^P23H

Abstract

We previously reported autophagic structures in rod photoreceptors expressing a misfolding RHO (rhodopsin) mutant (RHO P23H ), 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 RHO P23H 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.

Subcellular localization of mutant P23H rhodopsin in an RFP fusion knock-in mouse model of retinitis pigmentosa

“…8 A), indicating an autophagy component to the P23H-RFP pathology. Similar double-membrane vesiculations were observed in transgenic bovine P23H-Rho Xenopus tadpoles that were exposed to light ( Bogea et al, 2015 ), and, more recently, LC3-positive autophagosomes were localized adjacent to P23H-Rho protein in the ISs of these same Xenopus rods expressing bovine P23H-Rho ( Wen et al, 2019 ).…”

Subcellular localization of mutant P23H rhodopsin in an RFP fusion knockin mouse model of retinitis pigmentosa

“…Another study supported a key role for protein degradation by demonstrating that P23H-Rho photoreceptors were preserved by genetically over-activating the proteasome by crossing the untagged P23H-Rho knockin mouse with mice constitutively overexpressing either the PA28α or PSMD11 proteasomal cap protein (57). Therefore, the ER membrane expansion (23), and more recently LC3-positive autophagosomes were localized adjacent to P23H-Rho protein in the inner segments of these same Xenopus rods expressing bovine-P23H-Rho (58).…”

How to increase precision in Xenopus gene editing and base editing experiments

“…Some did use next generation sequencing (NGS) [12][13][14] or Sanger sequencing of cloned genomic isolates from the embryos [14][15][16][17][18][19]. Others merely monitored the breakdown of Sanger traces from directly amplified DNA at the target site, combined with a deconvolution algorithm to predict likely repairs [20,21]. Indels that cause reading frame-shifts are a frequent outcome, so analyses with basic DNA genotyping have instead emphasized the depletion of the targeted protein measured with specific antibody staining [21][22][23].…”

“…Others merely monitored the breakdown of Sanger traces from directly amplified DNA at the target site, combined with a deconvolution algorithm to predict likely repairs [20,21]. Indels that cause reading frame-shifts are a frequent outcome, so analyses with basic DNA genotyping have instead emphasized the depletion of the targeted protein measured with specific antibody staining [21][22][23]. Reported mutation efficiencies have varied, which probably reflects differing Cas9-gRNA reagent activity.…”