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Photoreceptors possess ribbon synapses distinct from the conventional synapses in the brain. Little is known about the function of the BBSome, a complex integral in ciliary and intracellular trafficking, in ribbon synaptic formation. We performed immunohistochemistry using retinas from Bardet-Biedl Syndrome (BBS) mouse models and found that BBS mutant animals have significantly fewer ribbon synapses in the outer plexiform layer and increased ectopic synapses in the outer nuclear layer compared to controls. Many ectopic synapses in BBS mutant retinas are associated with horizontal cell axonal processes that aberrantly intrude into the outer nuclear layer. To determine whether this horizontal cell phenotype is a consequence of retinal degeneration, we examined this phenotype in mice with photoreceptor-specific inactivation of the BBSome induced by Cre recombinase driven by the rhodopsin promoter. At three months of age, despite retinal degeneration, Bbs8 floxed/floxed ; Rho-Cre + mice lack the aberrant intrusion of horizontal cell processes. At 6 months, some horizontal cell processes intrude into the outer nuclear layer in Bbs8 floxed/floxed ; Rho-Cre + mice, but the phenotype does not recapitulate the phenotypic severity observed in young congenital BBS mutant mice. Therefore, the lack of BBSome function negatively impacts retinal synaptogenesis, and causes horizontal cell defects in a potentially cell-autonomous fashion.

Nephrocystin (NPHP1) is a ciliary transition zone protein and its ablation causes nephronophthisis (NPHP) with partially penetrant retinal dystrophy. However, the precise requirements of NPHP1 in photoreceptors are not well understood. Here, we characterize retinal degeneration in a mouse model of NPHP1 and show that NPHP1 is required to prevent infiltration of inner segment plasma membrane proteins into the outer segment during the photoreceptor maturation. We demonstrate that Nphp1 gene-trap mutant mice, which were previously described as null, are likely hypomorphs due to the production of a small quantity of functional mRNAs derived from nonsense-associated altered splicing and skipping of two exons including the one harboring the gene-trap. In homozygous mutant animals, inner segment plasma membrane proteins such as syntaxin-3 (STX3), synaptosomal-associated protein 25 (SNAP25), and interphotoreceptor matrix proteoglycan 2 (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. Consistent with this, some photoreceptor cell loss (~30%) occurs during the photoreceptor maturation period but it stops afterward. 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 mutant mice. These findings demonstrate that NPHP1 is required for the confinement of inner segment plasma membrane proteins 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 retinopathy in human NPHP1 patients.

Mutations affecting ciliary components cause a series of related genetic disorders in humans, including nephronophthisis (NPHP), Joubert syndrome (JBTS), Meckel-Gruber syndrome (MKS), and Bardet-Biedl syndrome (BBS), which are collectively termed “ciliopathies.” Recent protein–protein interaction studies combined with genetic analyses revealed that ciliopathy-related proteins form several functional networks/modules that build and maintain the primary cilium. However, the precise function of many ciliopathy-related proteins and the mechanisms by which these proteins are targeted to primary cilia are still not well understood. Here, we describe a protein–protein interaction network of inositol polyphosphate-5-phosphatase E (INPP5E), a prenylated protein associated with JBTS, and its ciliary targeting mechanisms. INPP5E is targeted to the primary cilium through a motif near the C terminus and prenyl-binding protein phosphodiesterase 6D (PDE6D)-dependent mechanisms. Ciliary targeting of INPP5E is facilitated by another JBTS protein, ADP-ribosylation factor-like 13B (ARL13B), but not by ARL2 or ARL3. ARL13B missense mutations that cause JBTS in humans disrupt the ARL13B–INPP5E interaction. We further demonstrate interactions of INPP5E with several ciliary and centrosomal proteins, including a recently identified ciliopathy protein centrosomal protein 164 (CEP164). These findings indicate that ARL13B, INPP5E, PDE6D, and CEP164 form a distinct functional network that is involved in JBTS and NPHP but independent of the ones previously defined by NPHP and MKS proteins.

Bardet-Biedl syndrome (BBS) is a human genetic disorder resulting in obesity, retinal degeneration, polydactyly, and nephropathy. Recent studies indicate that trafficking defects to the ciliary membrane are involved in this syndrome. Here, we show that a novel complex composed of three chaperonin-like BBS proteins (BBS6, BBS10, and BBS12) and CCT/TRiC family chaperonins mediates BBSome assembly, which transports vesicles to the cilia. Chaperonin-like BBS proteins interact with a subset of BBSome subunits and promote their association with CCT chaperonins. CCT activity is essential for BBSome assembly, and knockdown of CCT chaperonins in zebrafish results in BBS phenotypes. Many disease-causing mutations found in BBS6, BBS10, and BBS12 disrupt interactions among these BBS proteins. Our data demonstrate that BBS6, BBS10, and BBS12 are necessary for BBSome assembly, and that impaired BBSome assembly contributes to the etiology of BBS phenotypes associated with the loss of function of these three BBS genes.

Many signaling proteins including G protein-coupled receptors localize to primary cilia, regulating cellular processes including differentiation, proliferation, organogenesis, and tumorigenesis. Bardet-Biedl Syndrome (BBS) proteins are involved in maintaining ciliary function by mediating protein trafficking to the cilia. However, the mechanisms governing ciliary trafficking by BBS proteins are not well understood. Here, we show that a novel protein, Leucine-zipper transcription factor-like 1 (LZTFL1), interacts with a BBS protein complex known as the BBSome and regulates ciliary trafficking of this complex. We also show that all BBSome subunits and BBS3 (also known as ARL6) are required for BBSome ciliary entry and that reduction of LZTFL1 restores BBSome trafficking to cilia in BBS3 and BBS5 depleted cells. Finally, we found that BBS proteins and LZTFL1 regulate ciliary trafficking of hedgehog signal transducer, Smoothened. Our findings suggest that LZTFL1 is an important regulator of BBSome ciliary trafficking and hedgehog signaling.

Genetic mutations disrupting the structure and function of primary cilia cause various inherited retinal diseases in humans. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic ciliopathy characterized by retinal degeneration, obesity, postaxial polydactyly, intellectual disability, and genital and renal abnormalities. To gain insight into the mechanisms of retinal degeneration in BBS, we developed a congenital knockout mouse of Bbs8, as well as conditional mouse models in which function of the BBSome (a protein complex that mediates ciliary trafficking) can be temporally inactivated or restored. We demonstrate that BBS mutant mice have defects in retinal outer segment morphogenesis. We further demonstrate that removal of Bbs8 in adult mice affects photoreceptor function and disrupts the structural integrity of the outer segment. Notably, using a mouse model in which a gene trap inhibiting Bbs8 gene expression can be removed by an inducible FLP recombinase, we show that when BBS8 is restored in immature retinas with malformed outer segments, outer segment extension can resume normally and malformed outer segment discs are displaced distally by normal outer segment structures. Over time, the retinas of the rescued mice become morphologically and functionally normal, indicating that there is a window of plasticity when initial retinal outer segment morphogenesis defects can be ameliorated.

Bardet-Biedl syndrome (BBS) is a heterogeneous disorder characterized by obesity, retinopathy, polydactyly, and congenital anomalies. The incidence of hypertension and diabetes are also increased in BBS patients. Mutation of 16 genes independently causes BBS, and seven BBS proteins form the BBSome that promotes ciliary membrane elongation. BBS3 (ARL6), an ADP ribosylation factor-like small GTPase, is not part of the BBSome complex. The in vivo function of BBS3 is largely unknown. Here we developed a Bbs3 knockout model and demonstrate that Bbs3–/– mice develop BBS-associated phenotypes, including retinal degeneration, male infertility, and increased body fat. Interestingly, Bbs3–/– mice develop some unique phenotypes not seen in other BBS knockout models: no overt obesity, severe hydrocephalus, and elevated blood pressure (shared by some but not all BBS gene knockout mice). We found that endogenous BBS3 and the BBSome physically interact and depend on each other for their ciliary localization. This finding explains the phenotypic similarity between Bbs3–/– mice and BBSome subunit knockout mice. Loss of Bbs3 does not affect BBSome formation but disrupts normal localization of melanin concentrating hormone receptor 1 to ciliary membranes and affects retrograde transport of Smoothened inside cilia. We also show that the endogenous BBSome and BBS3 associate with membranes and the membrane association of the BBSome and BBS3 are not interdependent. Differences between BBS mouse models suggest nonoverlapping functions to individual BBS protein.

Bardet-Biedl syndrome (BBS) is a heterogeneous genetic disorder characterized by many features, including obesity and cardiovascular disease. We previously developed knockout mouse models of 3 BBS genes: BBS2, BBS4, and BBS6. To dissect the mechanisms involved in the metabolic disorders associated with BBS, we assessed the development of obesity in these mouse models and found that BBS-null mice were hyperphagic, had low locomotor activity, and had elevated circulating levels of the hormone leptin. The effect of exogenous leptin on body weight and food intake was attenuated in BBS mice, which suggests that leptin resistance may contribute to hyperleptinemia. In other mouse models of obesity, leptin resistance may be selective rather than systemic; although mice became resistant to leptin's anorectic effects, the ability to increase renal sympathetic nerve activity (SNA) was preserved. Although all 3 of the BBS mouse models were similarly resistant to leptin, the sensitivity of renal SNA to leptin was maintained in Bbs4 -/- and Bbs6 -/- mice, but not in Bbs2 -/- mice. Consequently, Bbs4 -/- and Bbs6 -/- mice had higher baseline renal SNA and arterial pressure and a greater reduction in arterial pressure in response to ganglionic blockade. Furthermore, we found that BBS mice had a decreased hypothalamic expression of proopiomelanocortin, which suggests that BBS genes play an important role in maintaining leptin sensitivity in proopiomelanocortin neurons.

RPGR (retinitis pigmentosa GTPase regulator) is a ciliary protein associated with several forms of inherited retinal degenerative diseases. PDE6D is a ubiquitously expressed prenyl-binding protein and involved in ciliary targeting of prenylated proteins. The current working model for the RPGR function depicts that RPGR acts as a scaffold protein to recruit cargo-loaded PDE6D to primary cilia. Here, we present evidence demonstrating an alternative relationship between RPGR and PDE6D, in which RPGR is a cargo of PDE6D for ciliary targeting. We found that the constitutive isoform of RPGR, which is prenylated, requires prenylation for its ciliary localization. We also found that there are at least two independent ciliary targeting signals in RPGR: one within the N-terminal region that contains the RCC1-like domain and the other near the prenylation site at the C-terminus. Ablation of PDE6D blocked ciliary targeting of RPGR. Our study indicates that prenylated RPGR is one of the cargos of PDE6D for ciliary trafficking and provides insight into the mechanisms by which RPGR is targeted to cilia.

Nephronophthisis-related ciliopathies (NPHP-RC) are a group of disorders that present with end-stage renal failure in childhood/adolescence, kidney cysts, retinal degeneration, and cerebellar hypoplasia. One disorder that shares clinical features with NPHP-RC is Bardet-Biedl Syndrome (BBS). Serologically defined colon cancer antigen 8 (SDCCAG8; also known as NPHP10 and BBS16) is an NPHP gene that is also associated with BBS. To better understand the patho-mechanisms of NPHP and BBS caused by loss of SDCCAG8 function, we characterized an SDCCAG8 mouse model (Sdccag8Tn(sb-Tyr)2161B.CA1C2Ove) generated by Sleeping Beauty Transposon (SBT)-mediated insertion mutagenesis. Consistent with the previously reported, independent SDCCAG8 mouse models, our mutant mice display pre-axial polydactyly in their hind limbs. In addition, we report patterning defects in the secondary palate, brain abnormalities, as well as neonatal lethality associated with developmental defects in the lung in our mouse model. The neonatal lethality phenotype is genetic background dependent and rescued by introducing 129S6/SvEvTac background. Genetic modifier(s) responsible for this effect were mapped to a region between SNPs rs3714172 and rs3141832 on chromosome 11. While determining the precise genetic lesion in our mouse model, we found that SBT insertion resulted in a deletion of multiple exons from both Sdccag8 and its neighboring gene Akt3. We ascribe the patterning defects in the limb and the secondary palate as well as lung abnormalities to loss of SDCCAG8, while the developmental defects in the brain are likely due to the loss of AKT3. This mouse model may be useful to study features not observed in other SDCCAG8 models but cautions are needed in interpreting data.