Qihong Zhang

A Core Complex of BBS Proteins Cooperates with the GTPase Rab8 to Promote Ciliary Membrane Biogenesis

Primary cilium dysfunction underlies the pathogenesis of Bardet-Biedl syndrome (BBS), a genetic disorder whose symptoms include obesity, retinal degeneration, and nephropathy. However, despite the identification of 12 BBS genes, the molecular basis of BBS remains elusive. Here we identify a complex composed of seven highly conserved BBS proteins. This complex, the BBSome, localizes to nonmembranous centriolar satellites in the cytoplasm but also to the membrane of the cilium. Interestingly, the BBSome is required for ciliogenesis but is dispensable for centriolar satellite function. This ciliogenic function is mediated in part by the Rab8 GDP/GTP exchange factor, which localizes to the basal body and contacts the BBSome. Strikingly, Rab8(GTP) enters the primary cilium and promotes extension of the ciliary membrane. Conversely, preventing Rab8(GTP) production blocks ciliation in cells and yields characteristic BBS phenotypes in zebrafish. Our data reveal that BBS may be caused by defects in vesicular transport to the cilium.

A BBSome Subunit Links Ciliogenesis, Microtubule Stability, and Acetylation

Primary cilium dysfunction affects the development and homeostasis of many organs in Bardet-Biedl syndrome (BBS). We recently showed that seven highly conserved BBS proteins form a stable complex, the BBSome, that functions in membrane trafficking to and inside the primary cilium. We have now discovered a BBSome subunit that we named BBIP10. Similar to other BBSome subunits, BBIP10 localizes to the primary cilium, BBIP10 is present exclusively in ciliated organisms, and depletion of BBIP10 yields characteristic BBS phenotypes in zebrafish. Unexpectedly, BBIP10 is required for cytoplasmic microtubule polymerization and acetylation, two functions not shared with any other BBSome subunits. Strikingly, inhibition of the tubulin deacetylase HDAC6 restores microtubule acetylation in BBIP10-depleted cells, and BBIP10 physically interacts with HDAC6. BBSome-bound BBIP10 may therefore function to couple acetylation of axonemal microtubules and ciliary membrane growth.

BBS6, BBS10, and BBS12 form a complex with CCT/TRiC family chaperonins and mediate BBSome assembly

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.

A novel protein LZTFL1 regulates ciliary trafficking of the BBSome and Smoothened.

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.

Intrinsic Protein-Protein Interaction-mediated and Chaperonin-assisted Sequential Assembly of Stable Bardet-Biedl Syndrome Protein Complex, the BBSome

Background: Bardet-Biedl syndrome proteins form a complex known as the BBSome. The details of BBSome assembly are unknown. Results: The BBSome is assembled via intrinsic protein-protein interactions, some of which involve cytoplasmic chaperonins. Conclusion: BBSome assembly is a regulated stepwise process. Significance: Understanding the assembly process of the BBSome might help to better understand the molecular mechanisms involved in cilia-related diseases. The pleiotropic features of obesity, retinal degeneration, polydactyly, kidney abnormalities, cognitive impairment, hypertension, and diabetes found in Bardet-Biedl syndrome (BBS) make this disorder an important model disorder for identifying molecular mechanisms involved in common human diseases. To date, 16 BBS genes have been reported, seven of which (BBS1, 2, 4, 5, 7, 8, and 9) code for proteins that form a complex known as the BBSome. The function of the BBSome involves ciliary membrane biogenesis. Three additional BBS genes (BBS6, BBS10, and BBS12) have homology to type II chaperonins and interact with CCT/TRiC proteins and BBS7 to form a complex termed the BBS-chaperonin complex. This complex is required for BBSome assembly. Little is known about the process and the regulation of BBSome formation. We utilized point mutations and null alleles of BBS proteins to disrupt assembly of the BBSome leading to the accumulation of BBSome assembly intermediates. By characterizing BBSome assembly intermediates, we show that the BBS-chaperonin complex plays a role in BBS7 stability. BBS7 interacts with BBS2 and becomes part of a BBS7-BBS2-BBS9 assembly intermediate referred to as the BBSome core complex because it forms the core of the BBSome. BBS1, BBS5, BBS8, and finally BBS4 are added to the BBSome core to form the complete BBSome.

BBS proteins interact genetically with the IFT pathway to influence SHH-related phenotypes

There are numerous genes for which loss-of-function mutations do not produce apparent phenotypes even though statistically significant quantitative changes to biological pathways are observed. To evaluate the biological meaning of small effects is challenging. Bardet–Biedl syndrome (BBS) is a heterogeneous autosomal recessive disorder characterized by obesity, retinopathy, polydactyly, renal malformations, learning disabilities and hypogenitalism, as well as secondary phenotypes including diabetes and hypertension. BBS knockout mice recapitulate most human phenotypes including obesity, retinal degeneration and male infertility. However, BBS knockout mice do not develop polydacyly. Here we showed that the loss of BBS genes in mice result in accumulation of Smoothened and Patched 1 in cilia and have a decreased Shh response. Knockout of Bbs7 combined with a hypomorphic Ift88 allele (orpk as a model for Shh dysfuction) results in embryonic lethality with e12.5 embryos having exencephaly, pericardial edema, cleft palate and abnormal limb development, phenotypes not observed in Bbs7−/− mice. Our results indicate that BBS genes modulate Shh pathway activity and interact genetically with the intraflagellar transport (IFT) pathway to play a role in mammalian development. This study illustrates an effective approach to appreciate the biological significance of a small effect.

Bardet-Biedl syndrome 3 (Bbs3) knockout mouse model reveals common BBS-associated phenotypes and Bbs3 unique phenotypes.

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.

Abnormal development of NG2 + PDGFR-α + neural progenitor cells leads to neonatal hydrocephalus in a ciliopathy mouse model

Hydrocephalus is a common neurological disorder that leads to expansion of the cerebral ventricles and is associated with a high rate of morbidity and mortality. Most neonatal cases are of unknown etiology and are likely to have complex inheritance involving multiple genes and environmental factors. Identifying molecular mechanisms for neonatal hydrocephalus and developing noninvasive treatment modalities are high priorities. Here we use a hydrocephalic mouse model of the human ciliopathy Bardet-Biedl Syndrome (BBS) and identify a role for neural progenitors in the pathogenesis of neonatal hydrocephalus. We found that hydrocephalus in this mouse model is caused by aberrant platelet-derived growth factor receptor α (PDGFR-α) signaling, resulting in increased apoptosis and impaired proliferation of chondroitin sulfate proteoglycan 4 (also known as neuron-glial antigen 2 or NG2)+PDGFR-α+ neural progenitors. Targeting this pathway with lithium treatment rescued NG2+PDGFR-α+ progenitor cell proliferation in BBS mutant mice, reducing their ventricular volume. Our findings demonstrate that neural progenitors are crucial in the pathogenesis of neonatal hydrocephalus, and we identify new therapeutic targets for this common neurological disorder.

BBS7 is required for BBSome formation and its absence in mice results in Bardet-Biedl syndrome phenotypes and selective abnormalities in membrane protein trafficking.

Summary Bardet-Biedl Syndrome (BBS) is a pleiotropic and genetically heterozygous disorder caused independently by numerous genes (BBS1–BBS17). Seven highly conserved BBS proteins (BBS1, 2, 4, 5, 7, 8 and 9) form a complex known as the BBSome, which functions in ciliary membrane biogenesis. BBS7 is both a unique subunit of the BBSome and displays direct physical interaction with a second BBS complex, the BBS chaperonin complex. To examine the in vivo function of BBS7, we generated Bbs7 knockout mice. Bbs7−/− mice show similar phenotypes to other BBS gene mutant mice including retinal degeneration, obesity, ventriculomegaly and male infertility characterized by abnormal spermatozoa flagellar axonemes. Using tissues from Bbs7−/− mice, we show that BBS7 is required for BBSome formation, and that BBS7 and BBS2 depend on each other for protein stability. Although the BBSome serves as a coat complex for ciliary membrane proteins, BBS7 is not required for the localization of ciliary membrane proteins polycystin-1, polycystin-2, or bitter taste receptors, but absence of BBS7 leads to abnormal accumulation of the dopamine D1 receptor to the ciliary membrane, indicating that BBS7 is involved in specific membrane protein localization to cilia.

BBS mutations modify phenotypic expression of CEP290-related ciliopathies

Ciliopathies are a group of heterogeneous disorders associated with ciliary dysfunction. Diseases in this group display considerable phenotypic variation within individual syndromes and overlapping phenotypes among clinically distinct disorders. Particularly, mutations in CEP290 cause phenotypically diverse ciliopathies ranging from isolated retinal degeneration, nephronophthisis and Joubert syndrome, to the neonatal lethal Meckel-Gruber syndrome. However, the underlying mechanisms of the variable expressivity in ciliopathies are not well understood. Here, we show that components of the BBSome, a protein complex composed of seven Bardet-Biedl syndrome (BBS) proteins, physically and genetically interact with CEP290 and modulate the expression of disease phenotypes caused by CEP290 mutations. The BBSome binds to the N-terminal region of CEP290 through BBS4 and co-localizes with CEP290 to the transition zone (TZ) of primary cilia and centriolar satellites in ciliated cells, as well as to the connecting cilium in photoreceptor cells. Although CEP290 still localizes to the TZ and connecting cilium in BBSome-depleted cells, its localization to centriolar satellites is disrupted and CEP290 appears to disperse throughout the cytoplasm in BBSome-depleted cells. Genetic interactions were tested using Cep290(rd16)- and Bbs4-null mutant mouse lines. Additional loss of Bbs4 alleles in Cep290(rd16/rd16) mice results in increased body weight and accelerated photoreceptor degeneration compared with mice without Bbs4 mutations. Furthermore, double-heterozygous mice (Cep290(+/rd16);Bbs4(+/-)) have increased body weight compared with single-heterozygous animals. Our data indicate that genetic interactions between BBSome components and CEP290 could underlie the variable expression and overlapping phenotypes of ciliopathies caused by CEP290 mutations.