The Mouse Homolog of the Human LCA3 Gene, Spata7, Plays a Critical Role in Cilium Structural Maintenance and Protein Trafficking in Photoreceptor Cells

Abstract

 

Purpose

 

LCA is a severe, early onset congenital retinal disease. Our group has identified SPATA7 as a novel disease gene at the LCA3 locus. In this study, the molecular function of mouse Spata7 in the retina was investigated to offer clues about the disease mechanism, which will form the basis for developing treatment of human patients.

 

Methods

 

Spata7 knock-out mice were generated to create an LCA3 disease model. A combination of histology, immunohistochemistry, physiology, and molecular and cell biology approaches were used to characterize the Spata7 mutant phenotype in detail. Proteins that interact with SPATA7 were identified using yeast two hybrid, BiFC, and IP-Mass spectrometer sequencing. Genetic interactions between Spata7 and Rho were also tested.

 

Results

 

Spata7 knock-out mice present with a phenotype similar to human patients, with progressive degeneration of photoreceptor cells and a rapid decrease of response to light as measured by ERG. Immunohistochemistry indicates that SPATA7 protein specifically localizes to the transition zone of the photoreceptor connecting cilium. Correspondingly, structural defects have been observed at the connecting cilium of photoreceptor cells using electron tomography in Spata7 mutant retinas. In parallel, protein interaction studies reveal that SPATA7 directly interacts with members of multiple transition zone protein complexes that play important roles in protein trafficking between photoreceptor inner and outer segments. Strikingly, mislocalization of transition zone proteins is observed in the Spata7 mutant retina. Consistently, accumulation of Rhodopsin is detected in the inner segments and cell bodies of photoreceptors, which likely triggers photoreceptor cell death. Indeed, reduced expression of rod opsin suppresses photoreceptor apoptosis and partially rescues the retinal degeneration phenotype observed in Spata7 mutants.

 

Conclusions

 

Functioning as a novel component of the photoreceptor connecting cilium, SPATA7 is critical for maintaining connecting cilium structural integrity, proper transition zone protein complex localization, and protein transport.

 

 

Keywords: 695 retinal degenerations: cell biology • 660 proteins encoded by disease genes • 663 proteomics