Kirk Mykytyn

Bardet–Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia

Primary cilia are ubiquitous cellular appendages that provide important yet not well understood sensory and signaling functions. Ciliary dysfunction underlies numerous human genetic disorders. However, the precise defects in cilia function and the basis of disease pathophysiology remain unclear. Here, we report that the proteins disrupted in the human ciliary disorder Bardet–Biedl syndrome (BBS) are required for the localization of G protein-coupled receptors to primary cilia on central neurons. We demonstrate a lack of ciliary localization of somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1) in neurons from mice lacking the Bbs2 or Bbs4 gene. Because Mchr1 is involved in the regulation of feeding behavior and BBS is associated with hyperphagia-induced obesity, our results suggest that altered signaling caused by mislocalization of ciliary signaling proteins underlies the BBS phenotypes. Our results also provide a potential molecular mechanism to link cilia defects with obesity.

Identification of the gene that, when mutated, causes the human obesity syndrome BBS4.

Bardet–Biedl syndrome (BBS, MIM 209900) is a heterogeneous autosomal recessive disorder characterized by obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation, and hypogenitalism. The disorder is also associated with diabetes mellitus, hypertension, and congenital heart disease. Six distinct BBS loci map to 11q13 (BBS1), 16q21 (BBS2), 3p13–p12 (BBS3), 15q22.3–q23 (BBS4), 2q31 (BBS5), and 20p12 (BBS6). Although BBS is rare in the general population (<1/100,000), there is considerable interest in identifying the genes causing BBS because components of the phenotype, such as obesity and diabetes, are common. We and others have demonstrated that BBS6 is caused by mutations in the gene MKKS (refs. 12,13), mutation of which also causes McKusick–Kaufman syndrome (hydrometrocolpos, post-axial polydactyly, and congenital heart defects). MKKS has sequence homology to the alpha subunit of a prokaryotic chaperonin in the thermosome Thermoplasma acidophilum. We recently identified a novel gene that causes BBS2. The BBS2 protein has no significant similarity to other chaperonins or known proteins. Here we report the positional cloning and identification of mutations in BBS patients in a novel gene designated BBS4.

Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-coupled Receptors

Primary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling proteins localized to the ciliary membrane. Certain G protein-coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis we expressed chimeric receptors containing fragments of Sstr3 and Htr6 in the nonciliary receptors Sstr5 and Htr7, respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is sufficient for ciliary localization. Comparison of these loops revealed a loose consensus sequence. To determine whether this consensus sequence predicts ciliary localization of other GPCRs, we compared it with the third intracellular loop of all human GPCRs. We identified the consensus sequence in melanin-concentrating hormone receptor 1 (Mchr1) and confirmed Mchr1 localizes to primary cilia in vitro and in vivo. Thus, we have identified a putative GPCR ciliary localization sequence and used this sequence to identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our results implicate ciliary signaling in the regulation of body weight.

Type III adenylyl cyclase localizes to primary cilia throughout the adult mouse brain

Solitary primary cilia project from nearly every cell type in the human body. These organelles are considered to have important sensory and signaling functions. Although primary cilia have been detected throughout the mammalian brain, their functions are unknown. The study of primary cilia in the brain is constrained by the scarcity of specific markers for these organelles. We previously demonstrated that type III adenylyl cyclase (ACIII) is a marker for primary cilia on neonatal hippocampal neurons in vivo and in vitro. We further showed that ACIII localizes to cilia on cultured glial cells. Here, we report that ACIII is a marker for primary cilia throughout many regions of the adult mouse brain. Furthermore, we report that ACIII localizes to primary cilia on choroid plexus cells and some astrocytes in the brain, which to our knowledge is the first report of a marker for visualizing cilia on glia in vivo. Overall, our data indicate that ACIII is a prominent marker of primary cilia in the brain and will provide an important tool to facilitate further investigations into the functions of these organelles. J. Comp. Neurol. 505:562–571, 2007.

Dopamine Receptor 1 Localizes to Neuronal Cilia in a Dynamic Process that Requires the Bardet-Biedl Syndrome Proteins

Primary cilia are nearly ubiquitous cellular appendages that provide important sensory and signaling functions. Ciliary dysfunction underlies numerous human diseases, collectively termed ciliopathies. Primary cilia have distinct functions on different cell types and these functions are defined by the signaling proteins that localize to the ciliary membrane. Neurons throughout the mammalian brain possess primary cilia upon which certain G protein-coupled receptors localize. Yet, the precise signaling proteins present on the vast majority of neuronal cilia are unknown. Here, we report that dopamine receptor 1 (D1) localizes to cilia on mouse central neurons, thereby implicating neuronal cilia in dopamine signaling. Interestingly, ciliary localization of D1 is dynamic, and the receptor rapidly translocates to and from cilia in response to environmental cues. Notably, the translocation of D1 from cilia requires proteins mutated in the ciliopathy Bardet-Biedl syndrome (BBS), and we find that one of the BBS proteins, Bbs5, specifically interacts with D1.

Hippocampal neurons possess primary cilia in culture

Primary cilia are cellular appendages that provide important sensory functions and defects in primary ciliary signaling have been implicated in the pathophysiology of human diseases and developmental abnormalities. Almost all human cell types possess a primary cilium. Neurons throughout the brain possess primary cilia on which certain receptors localize, suggesting that neurons possess cilia‐mediated signaling. However, the functional significance of neuronal cilia is unknown. Although there is a great deal of interest in understanding the functions of neuronal cilia, their study is hampered by the lack of an in vitro model system. We report that the majority of hippocampal neurons cultured from postnatal mice possess primary cilia in vitro. Further, we describe cilia proteins that can be labeled to readily visualize neuronal primary cilia in culture. These findings are the first characterization of neuronal primary cilia in vitro and should greatly facilitate further investigations into the function of these organelles.

Neuronal ciliary signaling in homeostasis and disease

Primary cilia are a class of cilia that are typically solitary, immotile appendages present on nearly every mammalian cell type. Primary cilia are believed to perform specialized sensory and signaling functions that are important for normal development and cellular homeostasis. Indeed, primary cilia dysfunction is now linked to numerous human diseases and genetic disorders. Collectively, primary cilia disorders are termed as ciliopathies and present with a wide range of clinical features, including cystic kidney disease, retinal degeneration, obesity, polydactyly, anosmia, intellectual disability, and brain malformations. Although significant progress has been made in elucidating the functions of primary cilia on some cell types, the precise functions of most primary cilia remain unknown. This is particularly true for primary cilia on neurons throughout the mammalian brain. This review will introduce primary cilia and ciliary signaling pathways with a focus on neuronal cilia and their putative functions and roles in human diseases.

Arborization of dendrites by developing neocortical neurons is dependent on primary cilia and type 3 adenylyl cyclase.

The formation of primary cilia is a highly choreographed process that can be disrupted in developing neurons by overexpressing neuromodulatory G-protein-coupled receptors GPCRs or by blocking intraflagellar transport. Here, we examined the effects of overexpressing the ciliary GPCRs, 5HT6 and SSTR3, on cilia structure and the differentiation of neocortical neurons. Neuronal overexpression of 5HT6 and SSTR3 was achieved by electroporating mouse embryo cortex in utero with vectors encoding these receptors. We found that overexpression of ciliary GPCRs in cortical neurons, especially 5HT6, induced the formation of long (>30 μm) and often forked cilia. These changes were associated with increased levels of intraflagellar transport proteins and accelerated ciliogenesis in neonatal neocortex, the induction of which required Kif3a, an anterograde motor critical for cilia protein trafficking and growth. GPCR overexpression also altered the complement of signaling molecules within the cilia. We found that SSTR3 and type III adenylyl cyclase (ACIII), proteins normally enriched in neuronal cilia, were rarely detected in 5HT6-elongated cilia. Intriguingly, the changes in cilia structure were accompanied by changes in neuronal morphology. Specifically, disruption of normal ciliogenesis in developing neocortical neurons, either by overexpressing cilia GPCRs or a dominant-negative form of Kif3a, significantly impaired dendrite outgrowth. Remarkably, coexpression of ACIII with 5HT6 restored ACIII to cilia, normalized cilia structure, and restored dendrite outgrowth, effects that were not observed in neurons coexpressing ACIII and dominant-negative form of Kif3a. Collectively, our data suggest the formation of neuronal dendrites in developing neocortex requires structurally normal cilia enriched with ACIII.

Clinical evidence of decreased olfaction in Bardet–Biedl syndrome caused by a deletion in the BBS4 Gene

Recent discoveries have lead to the hypothesis that ciliary dysfunction is a mechanism underlying the pathogenesis of Bardet–Biedl syndrome (BBS). Here, we describe two individuals with decreased olfaction who are members of an extended family affected with BBS caused by a homozygous deletion (c.77‐220del) in the BBS4 gene. These findings correlate with the evidence that several BBS proteins, including BBS4, are expressed in the olfactory epithelium (OE). Although the prevalence and the spectrum of impaired olfaction in BBS are not known, the causal relationship of the BBS4 deletion in this family and the decreased olfaction is corroborated by evidence that Bbs2 and Bbs4 knockout mice have severe olfaction deficits and that also patients with BBS caused by mutations in other BBS genes can have impaired olfaction. This finding broadens the spectrum of clinical manifestations associated with BBS, confirms the role of BBS4 in olfaction, and lends support to the hypothesis that ciliary dysfunction is an important aspect of BBS pathogenesis.

Differences in renal tubule primary cilia length in a mouse model of Bardet-Biedl syndrome.

Background: Bardet-Biedl syndrome (BBS) is a heterogeneous genetic disorder that comprises numerous features, including renal cystic disease. Twelve BBS genes have been identified (BBS1–12). Although the exact functions of the BBS proteins are unknown, evidence suggests that they are involved in cilia assembly, maintenance and/or function. Renal primary cilia dysfunction can lead to cystic kidney disease. To test whether lacking Bbs4 affects cilia assembly and structure, we analyzed primary cilia in Bbs4-null (Bbs4–/–) mice. Methods: Renal tubule cultures from wild-type (Bbs4+/+) and Bbs4–/– mice were examined by immunocytochemistry and scanning and transmission electron microscopy. Results: Our culture conditions generated ciliated epithelial cells that were mostly of collecting duct origin. The microtubule ultrastructure of cilia and basal bodies did not appear disrupted in Bbs4–/– cells. In control cells, cilia length was maximal at 7 days in culture. In cells cultured from Bbs4–/– mice, cilia were shorter initially, but surpassed the length of control cilia by 10 days. Renal primary cilia were also longer in Bbs4–/– kidneys. Conclusions: Lacking Bbs4 does not lead to aberrant cilia or basal body structure. However, the dynamics of cilia assembly is altered in Bbs4–/– cells, suggesting a role for Bbs4 in the regulation of ciliary assembly.