Autophagy in <i>Xenopus laevis</i> rod photoreceptors is independently regulated by phototransduction and misfolded RHO^P23H

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 <i>Xenopus laevis</i> 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 <i>RPE65</i> and <i>GNAT1</i> 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. <b>Abbreviations</b>: CYCS: cytochrome c; bRHO^P23H: bovine RHO^P23H; 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; <i>RHO_p</i>: the <i>Xenopus laevis RHO.2.L</i> promoter.我们先前曾报道，在表达错误折叠视紫红质（rhodopsin, RHO）突变体（RHO^P23H）的视杆感光细胞中存在自噬结构，提示自噬可能参与突变型RHO的降解，或参与感光细胞的死亡进程。为进一步探究正常与病变视杆细胞中的自噬过程，我们构建了在视杆细胞中表达双荧光自噬标记物mRFP-eGFP-LC3（单体红色荧光蛋白-增强绿色荧光蛋白-微管相关蛋白1A/1B轻链3）的转基因非洲爪蟾（Xenopus laevis）蝌蚪。该标记物会随自噬结构的形成与成熟，从绿色变为黄色，最终转为红色。通过使用组成型表达与诱导型表达的转基因品系，我们明确了视杆感光细胞中自噬的时间进程：自噬体与溶酶体融合前可存活6至8小时，酸化后的自噬溶酶体在降解前可维持约28小时。正常视杆细胞中的自噬受昼夜光照调控：光照时段会产生更多自噬结构，且该调控不依赖生物钟。我们还发现，表达错误折叠RHO^P23H突变体的视杆细胞中会产生更多自噬体。视紫红质的生色团可吸收光子以启动光转导过程，并在该过程中被消耗；同时它还能促进视紫红质的折叠。为探究光照暴露后正常视杆细胞中自噬水平升高，是由RHO错误折叠增加还是光转导过程介导的，我们利用CRISPR/Cas9系统分别敲除了<i>RPE65</i>与<i>GNAT1</i>基因，二者分别为生色团生物合成与光转导过程所必需。两种基因的敲除均抑制了光照诱导的自噬，这表明：尽管光照与错误折叠的视紫红质均可诱导视杆细胞的自噬，但光照诱导的自噬并非由RHO错误折叠介导，而是源于光转导过程。 **缩写说明**： CYCS：细胞色素c； bRHO^P23H：牛源RHO^P23H； Cas9：CRISPR相关蛋白9（CRISPR associated protein 9）； dpf：受精后天数（days post-fertilization）； eGFP：增强型绿色荧光蛋白（enhanced green fluorescent protein）； GNAT1：鸟苷酸结合蛋白G(t)α亚基1（亦称为视杆α转导蛋白，guanine nucleotide-binding protein G(t) subunit alpha-1 aka rod alpha-transducin）； HSPA1A/hsp70：70 kDa热休克蛋白（heat shock protein of 70 kilodaltons）； LAMP1：溶酶体相关膜蛋白1（lysosomal-associated membrane protein 1）； LC3：微管相关蛋白1A/1B轻链3（microtubule-associated protein 1A/1B light chain 3）； mRFP：单体红色荧光蛋白（monomeric red fluorescent protein）； RHO：视紫红质（rhodopsin）； RP：色素性视网膜炎（retinitis pigmentosa）； RPE65：视网膜色素上皮特异性65 kDa蛋白（retinal pigment epithelium-specific 65 kDa protein）； sfGFP：超折叠绿色荧光蛋白（superfolding GFP）； sgRNA：单向导RNA（single guide RNA）； WGA：小麦凝集素（wheat germ agglutinin）； <i>RHO_p</i>：非洲爪蟾<i>RHO.2.L</i>基因启动子。