Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are severe hereditary diseases that causes visual impairment in infants and children. SPATA7 has recently been identified as the LCA3 and juvenile RP gene in humans, whose function in the retina remains elusive. Here, we show that SPATA7 localizes at the primary cilium of cells and at the connecting cilium (CC) of photoreceptor cells, indicating that SPATA7 is a ciliary protein. In addition, SPATA7 directly interacts with the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1), a key connecting cilium protein that has also been linked to LCA. In the retina of Spata7 null mutant mice, a substantial reduction of RPGRIP1 levels at the CC of photoreceptor cells is observed, suggesting that SPATA7 is required for the stable assembly and localization of the ciliary RPGRIP1 protein complex. Furthermore, our results pinpoint a role of this complex in protein trafficking across the CC to the outer segments, as we identified that rhodopsin accumulates in the inner segments and around the nucleus of photoreceptors. This accumulation then likely triggers the apoptosis of rod photoreceptors that was observed. Loss of Spata7 function in mice indeed results in a juvenile RP-like phenotype, characterized by progressive degeneration of photoreceptor cells and a strongly decreased light response. Together, these results indicate that SPATA7 functions as a key member of a retinal ciliopathy-associated protein complex, and that apoptosis of rod photoreceptor cells triggered by protein mislocalization is likely the mechanism of disease progression in LCA3/ juvenile RP patients.

P23H-hRho-GFP mice provide a valuable tool for evaluating the efficacy of potential therapies for ADRP that influence the levels or localization of P23H-rhodopsin.

Patients with Bardet-Biedl syndrome (BBS) experience severe retinal degeneration as a result of impaired photoreceptor transport processes that are not yet fully understood. To date, there is no effective treatment for BBS-associated retinal degeneration, and blindness is imminent by the second decade of life. Here we report the development of an adeno-associated viral (AAV) vector that rescues rhodopsin mislocalization, maintains nearly normalappearing rod outer segments, and prevents photoreceptor death in the Bbs4-null mouse model. Analysis of the electroretinogram awave indicates that rescued rod cells are functionally indistinguishable from wild-type rods. These results demonstrate that gene therapy can prevent retinal degeneration in a mammalian BBS model. ciliopathies | intra-flagellar transport | electroretinography B ardet-Biedl syndrome (BBS) is clinically diagnosed by the presence of at least four of the following signs: retinal dystrophy, polydactyly, obesity, learning disabilities, male hypogonadism, and renal anomalies (1). Of these, the visual phenotype is particularly devastating: the average BBS patient will progress to legal blindness before his or her 16th birthday. There are currently 15 genetic loci (Online Mendelian Inheritance in Man #209900) that are known to be associated with BBS, and although inheritance of this disease was once thought to follow a classic autosomal recessive pattern, recent evidence suggests that a more complex pattern, termed "triallelic inheritance," is involved at certain BBS loci (2). Only within the last decade has evidence emerged that BBS proteins play a role in ciliary function (3). More specifically, Bbs4 and the other BBSome components seem to be involved in both recruitment of cargo toward the ciliary basal body (4, 5) and in intraflagellar transport along the cilium (5-7). Several reports have elucidated the mechanisms by which disruption of BBS proteins give rise to the individual phenotypes seen in this highly pleiotropic syndrome (8-11).Retinal degeneration is a central feature of all BBS mouse models generated to date (8,(12)(13)(14)(15)(16). In rod and cone photoreceptors, the connecting cilium is a highly specialized ciliary structure that serves as the sole conduit from the inner segment to the outer segment. Because protein synthesis occurs proximal to the outer segment, rhodopsin and other visual proteins must be trafficked through the connecting cilium to reach their site of action in the outer segment. Abd-El-Barr et al. (11) showed that when Bbs4 was deleted in mice, rhodopsin and cone opsins became grossly mislocalized in rod and cone photoreceptors, respectively. This was followed by apoptotic photoreceptor death and deterioration of the electroretinogram (ERG) a-and b-waves (14). Ultrastructural analysis of rods from young animals revealed normal-appearing connecting cilia and basal body structures. This latter finding has important implications for gene therapy: because the structural transport apparatus seems to develop normally in Bbs4-nu...

Mutation of the polarity gene Crumbs homolog 1 (CRB1) is responsible for >10% of Leber congenital amaurosis (LCA) cases worldwide; LCA is characterized by early-onset degenerative retinal dystrophy. The role of CRB1 in LCA8 pathogenesis remains elusive since Crb1 mouse mutants, including a null allele, have failed to mimic the early-onset of LCA, most likely due to functional compensation by closely related genes encoding Crb2 and Crb3. Crb proteins form an evolutionarily conserved, apical polarity complex with the scaffolding protein associated with lin-seven 1 (Pals1), also known as MAGUK p55 subfamily member 5 (MPP5). Pals1 and Crbs are functionally inter-dependent in establishing and maintaining epithelial polarity. Pals1 is a single gene in the mouse and human genomes; therefore, we ablated Pals1 to establish a mouse genetic model mimicking human LCA. In our study, the deletion of Pals1 leads to the disruption of the apical localization of Crb proteins in retinal progenitors and the adult retina, validating their mutual interaction. Remarkably, the Pals1 mutant mouse exhibits the critical features of LCA such as early visual impairment as assessed by electroretinogram, disorganization of lamination and apical junctions and retinal degeneration. Our data uncover the indispensible role of Pals1 in retinal development, likely involving the maintenance of retinal polarity and survival of retinal neurons, thus providing the basis for the pathologic mechanisms of LCA8.

Extensive studies have identified many growth factors and intracellular pathways that can promote neuronal survival after retinal injury, but the intrinsic survival mechanisms in the normal retina are poorly understood. Here we report that genetic ablation of Shp2 (Ptpn11)