Pittler, S. J. | Search bioRxiv

bioRxiv2025

Selective Knockout of Murine Glutamic Acid-rich Protein 2 (GARP2) Significantly Alters Cellular Dark Noise in Rod Photoreceptors

GARP2, a glutamic-acid-rich protein found exclusively in rod photoreceptors, has been suggested to function as a structural protein, a modulator of the cGMP phosphodiesterase enzyme (PDE6), and a gating inhibitor of the rod cGMP-gated cation channel. GARP2 is a splice variant of the Cngb1 gene, which in the rods encodes the {beta}-subunit of the cyclic nucleotide-gated cation channel. Mutations in Cngb1 cause retinitis pigmentosa (RP45), and {beta}-subunit knockout mice are being studied as models of this disease. In this study, using ZFN-mediated gene editing, we have selectively eliminated GARP2 expression, while not affecting expression of the cyclic nucleotide gated cation channel {beta} -subunit, to determine its essential functions in mouse rods. The absence of GARP2 caused no consistent perturbations of retinal structure. Transiently, rod outer segment length was regionally greater than WT and infrequently misaligned, appearing parallel to the retinal pigment epithelium. Electroretinography of the knockout mice did reveal consistent functional alterations over time, seen as a reduction in the ERG response amplitudes in older mice, albeit with no significant alterations in sensitivity to light. Interestingly, single-cell patch-clamp recordings showed a significant reduction in rod photoreceptor dark noise consistent with a previously proposed role for GARP2 in binding to PDE6 and affecting its basal activity. Our results suggest a role for the GARP2-PDE6 interaction in stabilizing the PDE6 enzyme and controlling the turnover rate of cGMP in darkness, adjusting the level of dark noise and implicating an influence on the signal and noise properties of rod photoreceptors. Key PointsO_LIGlutamic acid-rich protein 2 (GARP2), an alternatively spliced variant of the Cngb1 gene, is exclusively expressed in rod photoreceptors, but the in vivo role of GARP2 remains unestablished. C_LIO_LIWe used precision gene editing technology to selectively knockout soluble GARP2 expression in rods, to determine its essential functions in structure and function of the retina. C_LIO_LIWe show that GARP2 has a minor role in maintaining the structural integrity of the rod outer segments as a function of age. C_LIO_LIOur study indicates that GARP2 has an important role in regulating rod photoreceptor dark noise, likely through stabilization of PDE6 basal activity to maintain the appropriate cGMP turnover rate. C_LIO_LIThis regulation is critical to facilitate the single-photon sensitivity and function of the rod photoreceptors. C_LI

neuroscience↗

bioRxiv2025

Modeling Retinitis Pigmentosa 59: Dhdds T206A and Dhdds K42E knock-in mutant mice are phenotypically similar.

Dehydrodolichyl diphosphate synthase (DHDDS) is an essential enzyme required for several forms of protein glycosylation in all eukaryotic cells. Surprisingly, three mutant alleles, (DhddsK42E/K42E (K42E/K42E), DhddsT206A/K42E (T206A/K42E), and found in only one patient, DhddsR98W/K42E (R98W/K42E) have been reported that cause non-syndromic retinitis pigmentosa (RP59), an inherited retinal degeneration (IRD). Because T206A was only observed heterozygously with the K42E allele in RP59 patients, we used CRISPR/CAS9 technology to generate T206A/T206A, and subsequently T206A/K42E alleles in mice to assess the contribution of the T206A allele to the disease phenotype, to model the human disease, and to compare resulting phenotypes to our homozygous K42E mouse model. By postnatal (PN) 12-mo, T206A/K42E mice exhibit significant reduction of inner nuclear layer thickness as was observed in K42E/K42E mice. No change in outer nuclear layer thickness is observed in all mutant phenotypes up to PN 12 mo. Electroretinography (ERG) showed a significantly reduced b-wave without a-wave decrement and by PN 3-mo, ERG c- and d-wave responses were significantly attenuated in all phenotypes. Consistent with a reduction in inner nuclear layer thickness seen by OCT and cell loss observed by histology, bipolar and amacrine cell densities were reduced in all Dhdds mutant phenotypes compared to PN 8-12 mo age-matched controls. These results indicate that the DHDDS T206A allele causes retinal disease independent of the K42E allele, and that there likely is a common disease mechanism involving RP59-associated DHDDS mutations. We conclude that the physiological basis of retinal dysfunction in RP59 involves defective signaling in the inner retina resulting in bipolar/amacrine cell degeneration.

cell biology↗