Differential requirement of NPHP1 for compartmentalized protein localization during photoreceptor outer segment development and maintenance

Abstract

Purpose : Nephrocystin (NPHP1) is a ciliary transition zone protein and its ablation causes nephronophthisis (NPHP) with partially penetrant retinal dystrophy. This study is to determine the roles of NPHP1 in photoreceptors and uncover the molecular/genetic basis of the incomplete penetrance of retinal dystrophy in NPHP1-associated nephronophthisis.

Methods : Nphp1 gene-trap (Nphp1^gt) and Cep290^fl mice were obtained from the Jax lab (Nphp1^tm1Jgg/J; #013169 and Cep290^tm1Jgg/J; #013701). Nphp1 expression from normal and Nphp1^gt/gt mice was examined by immunoblotting, immunohistochemistry, and RT-PCR. Localization of various inner segment and outer segment proteins was examined by immunohistochemistry. Retinal functions were examined by electroretinography (ERG). Physical interactions of NPHP1 mutant variants with other NPHP proteins were tested by immunoprecipitation.

Results : In Nphp1^gt/gt retinas, inner segment plasma membrane proteins including STX3, SNAP25, and IMPG2 accumulate in the outer segment when outer segments are actively elongating. This phenotype, however, is spontaneously ameliorated after the outer segment elongation is completed. Retinal degeneration also occurs temporarily during the photoreceptor maturation but stops afterward. We found that the Nphp1^gt allele, which was previously described as null, was hypomorphic due to the production of a small quantity of functional mRNAs derived from nonsense-associated altered splicing and the consequent production of near-full-length, internal deletion mutant proteins. The mutant protein appears to retain most, if not all, functions of full-length NPHP1 as determined by in vitro studies. We further show that Nphp1 genetically interacts with Cep290, another NPHP gene, and that a reduction of Cep290 gene dose results in retinal degeneration that continues until adulthood in Nphp1^gt/gt mice.

Conclusions : Our data show that NPHP1 is essential to prevent infiltration of inner segment plasma membrane proteins into the outer segment during the outer segment development, but its requirement diminishes as photoreceptors mature. Our study also suggests that additional mutations in other NPHP genes may influence the penetrance of retinal dystrophy in human NPHP1 patients.

This is a 2021 ARVO Annual Meeting abstract.