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Xenopus laevis P23H rhodopsin transgene causes rod photoreceptor degeneration that is more severe in the ventral retina and is modulated by light.

ABSTRACT: Rhodopsin transgenes carrying mutations that cause autosomal dominant retinitis pigmentosa in humans have been used to study rod photoreceptor degeneration in various model organisms including Xenopus laevis. To date, the only transgenes shown to cause rod photoreceptor degeneration in Xenopus laevis have been either mammalian rhodopsins or chimeric versions of rhodopsin based mainly on Xenopus laevis rhodopsin sequences but with a mammalian C-terminus. Since the C-terminal sequence of rhodopsin is highly conserved in mammals and divergent in Xenopus laevis, and mammalian and epitope-tagged rhodopsins may have unexpected properties as transgenes, we decided to test whether a Xenopus laevis rhodopsin transgene carrying only the P23H mutation could also cause rod photoreceptor degeneration. Xenopus laevis tadpoles expressing these transgenes indeed had shortened outer segments and, in severely affected animals, the loss of rod photoreceptors but not the loss of cone photoreceptors. RT-PCR analyses showed that less than 10% of mutant transgenic rhodopsin relative to wild-type endogenous rhodopsin mRNA was sufficient to produce severe rod photoreceptor degeneration. As observed in other animal models as well as humans carrying this particular rhodopsin mutation, the rod photoreceptor degeneration was most severe in the ventral retina and was modified by light. Thus, the rod photoreceptor degeneration produced in Xenopus laevis by the P23H mutation in an otherwise untagged Xenopus laevis rhodopsin is generally similar to that seen with mammalian rhodopsins and epitope-tagged versions of Xenopus laevis rhodopsin, though some differences remain to be explained.

SUBMITTER: Zhang R

PROVIDER: S-EPMC4127875 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Xenopus laevis P23H rhodopsin transgene causes rod photoreceptor degeneration that is more severe in the ventral retina and is modulated by light.

Zhang Rui R Oglesby Ericka E Marsh-Armstrong Nicholas N

Experimental eye research 20080112 4

Rhodopsin transgenes carrying mutations that cause autosomal dominant retinitis pigmentosa in humans have been used to study rod photoreceptor degeneration in various model organisms including Xenopus laevis. To date, the only transgenes shown to cause rod photoreceptor degeneration in Xenopus laevis have been either mammalian rhodopsins or chimeric versions of rhodopsin based mainly on Xenopus laevis rhodopsin sequences but with a mammalian C-terminus. Since the C-terminal sequence of rhodopsin ...[more]

PMID: 18291367

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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.

Project description:PurposeTo characterize the optical coherence tomography (OCT) appearances of photoreceptor degeneration in the rhodopsin P23H transgenic rat (line 2) in relation to the histological, ultrastructural, and electroretinography (ERG) findings.Materials and methodsHomozygous rhodopsin P23H transgenic albino rats (line 2, very-slow degeneration model) were employed. Using OCT (Micron IV®; Phoenix Research Labs, Pleasanton, CA, USA), the natural course of photoreceptor degeneration was recorded from postnatal day (P) 15 to P 287. The OCT images were qualitatively observed by comparing them to histological and ultrastructural findings at P 62 and P 169. In addition, each retinal layer was quantitatively analyzed longitudinally during degeneration, compared it to that observed in wild type Sprague-Dawley (SD) rats. The relationships between the ERG (full-field combined rod-cone response, 3.0 cds/m2 stimulation) findings and OCT images were also analyzed.ResultsIn the qualitative study, the two layers presumably corresponding to the photoreceptor inner segment ellipsoid zone (EZ) and interdigitation zone (IZ) were identified in the P23H rat until PN day 32. However, the photoreceptor inner and outer segment (IS/OS) layer became diffusely hyperreflective on OCT after P 46, and the EZ and IZ zones could no longer be identified on OCT. In contrast, in the SD rats, the EZ and IZ were clearly distinguished until at least P 247. The ultrastructural study showed partial disarrangements of the photoreceptor outer segment discs in the P23H rats at P 62, although a light-microscopic histological study detected almost no abnormality in the outer segment. In the quantitative study, the outer retinal layer including the outer plexiform layer (OPL) and the outer nuclear layer (ONL) became significantly thinner in the P23H rats than in the SD rats after P 71. The thickness of the IS/OS layer was maintained in the P23H rats until P 130, and it became statistically thinner than in the SD rats at P 237. The longitudinal attenuation in the amplitude of the a- and b-waves of ERG was significantly correlated with the thickness of the combined OPL and ONL but not with that of the IS/OS layer.ConclusionOCT showed the degenerated photoreceptor IS/OS layer in rhodopsin P23H transgenic rats (line 2) as a diffuse hyperreflective zone, even in the early stage, with the partially disarranged and destabilized OS discs recognizable by ultrastructural assessment but not by a histological study. The amplitude of the a- and b-waves mainly depends on the thickness of the OPL and ONL layer rather than the thickness of the photoreceptor IS/OS layer in P23H rats.

Dataset Information

Xenopus laevis P23H rhodopsin transgene causes rod photoreceptor degeneration that is more severe in the ventral retina and is modulated by light.

Publications

Xenopus laevis P23H rhodopsin transgene causes rod photoreceptor degeneration that is more severe in the ventral retina and is modulated by light.

Similar Datasets

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