Bardet–Biedl syndrome: Is it only cilia dysfunction?

FEBS Letters xxx (2015) xxx–xxx

journal homepage: www.FEBSLetters.org

Review

Bardet–Biedl syndrome: Is it only cilia dysfunction? Rossina Novas a, Magdalena Cardenas-Rodriguez a, Florencia Irigoín a,b, Jose L. Badano a,⇑ a b

Human Molecular Genetics Laboratory, Institut Pasteur de Montevideo, Mataojo 2020, Montevideo CP11400, Uruguay Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Gral. Flores 2125, Montevideo CP11800, Uruguay

a r t i c l e

i n f o

Article history: Received 2 July 2015 Revised 14 July 2015 Accepted 15 July 2015 Available online xxxx Edited by Wilhelm Just Keywords: Bardet–Biedl syndrome Cilia Ciliopathies

a b s t r a c t Bardet–Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic disorder, characterized by both congenital and late onset defects. From the analysis of the mutational burden in patients to the functional characterization of the BBS proteins, this syndrome has become a model for both understanding oligogenic patterns of inheritance and the biology of a particular cellular organelle: the primary cilium. Here we briefly review the genetics of BBS to then focus on the function of the BBS proteins, not only in the context of the cilium but also highlighting potential extra-ciliary roles that could be relevant to the etiology of the disorder. Finally, we provide an overview of how the study of this rare syndrome has contributed to the understanding of cilia biology and how this knowledge has informed on the cellular basis of different clinical manifestations that characterize BBS and the ciliopathies. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Bardet–Biedl syndrome (BBS; MIM 209900) is a genetic pleiotropic condition characterized by retinal degeneration, obesity, postaxial polydactyly, mental retardation, hypogonadism and renal malformations, although patients can present a number of additional clinical manifestations including asthma, craniofacial defects, anosmia, hearing loss and diabetes [1]. Studies in humans, animal models, cells, and bioinformatics have shown that the cellular basis of BBS is intimately linked to the dysfunction of a particular cellular organelle, the primary cilium. Cilia are evolutionary conserved, microtubule-based organelles that are organized from a modified centriole, the basal body, and that emanate from the plasma membrane of most cells in vertebrates. In a simplistic classification, two main types of cilia can be distinguished: motile and primary cilia whereby motile cilia are composed by an axoneme of 9 pairs of outer microtubule doublets surrounding a central pair (9 + 2) while primary cilia present a 9 + 0 structure. In addition, while cilia and flagella (same ultrastructure) are motile, primary cilia are generally immotile and present as a single structure on the apical surface of cells. A mechanism that is critical for building, maintaining and reabsorbing cilia is intraflagellar transport (IFT), the movement of material into and out of the cilium that is carried out by IFT particles linking their cargoes to microtubule based molecular motors. ⇑ Corresponding author. Fax: +598 522 4185. E-mail address: [email protected] (J.L. Badano).

IFT-B particles are responsible for anterograde transport (toward the ciliary tip) whereas IFT-A directs retrograde movement (toward the cell body) in association with kinesin and dynein motors respectively (Fig. 1; reviewed in [2,3]). A large body of work has shown that primary cilia participate in cell signaling, sensing and transducing a host of inputs ranging from mechanical cues to chemical and paracrine signaling including Hedgehog, Wnt and PDGF (Fig. 1; reviewed in [4–6]). Not surprisingly then, primary cilia play a critical role during development and in maintaining cellular and tissue homeostasis, a fact that is most clearly highlighted by the consequences of cilia dysfunction. While complete absence of cilia is incompatible with life, defective ciliary function has been associated with a number of clinical entities ranging from polycystic kidney disease (PKD) and nephronophthisis (NPHP) to pleiotropic conditions such as BBS, Alström syndrome (ALMS), Joubert syndrome (JBTS), and Meckel Gruber syndrome (MKS), all members of a novel human disease category: the ciliopathies [7,8]. The ciliopathies encompass a range of clinically distinct entities that share, to varying degrees, a common cellular basis. In this review, we will focus on a model ciliopathy, BBS, discussing the genetics of the syndrome and the known roles of the BBS proteins both in the context of the cilium but importantly also outside of the organelle. 2. The genetics of BBS BBS is a genetically heterogeneous condition for which 19 BBS genes have been identified to date: BBS1-12, BBS13/MKS1,

http://dx.doi.org/10.1016/j.febslet.2015.07.031 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: Novas, R., et al. Bardet–Biedl syndrome: Is it only cilia dysfunction?. FEBS Lett. (2015), http://dx.doi.org/10.1016/ j.febslet.2015.07.031

R. Novas et al. / FEBS Letters xxx (2015) xxx–xxx

A

Kinesin Dynein IFT complex A/B

B A B

ANTEROGRADE TRANSPORT

GLI

RETROGRADE TRANSPORT

GLI

A B

2

Canonical Wnt

PDGF PDGFRα

Wnt PCP

A B

A B

Other Wnt receptors?

GLI

Frizzled

A B

Smo

PC1/PC2 Complex Ca2+ FLUID FLOW

Ca2+ Ca2+ Ca2+

Basal Body (BBSs, NPHP)

A B

PDGF signaling

Hedgehog activation

Balance Canonical/PCP signaling

GLI

Shh

B A

A

TRANSITION ZONE WITH TRANSITION FIBRES (NPHP, MKS, CEP290)

B

Ptch1

A

Basal Body

GLI

Ca2+ signaling

Smo

Proliferation Differentiation Tissue homeostasis Organogenesis

Activation of Hh target genes (nucleus) Fig. 1. Schematic representation of the cilium and its role in signal transduction. The cilium is composed by a microtubule axoneme organized from a basal body. Alongside the microtubules, IFT particles and motors are in charge of transporting moieties in and out of the cilium. Different receptors localize to the ciliary membrane and rely on the cilium for signal transduction to control several biological processes (A and B). One of the best characterized is the Hedgehog signaling pathway (A). Upon binding of the activator (Shh) to its receptor Ptch1, the complex is translocated outside of the cilium and no longer inhibits the ciliary entry of Smo, which in turn facilitates Gli processing and activation. Activated Gli transcription factors (Gli-star) exit the cilium and enter the nucleus to drive gene transcription. (B) Basal body proteins, such as the BBSs and NPHPs, are involved in regulating Wnt signaling whereby depletion of BBSs results in increased canonical signaling and reduced PCP.

BBS14/CEP290, BBS15/WDPCP/FRITZ, BBS16/SDCCAG8, BBS17/LZTFL1, BBS18/BBIP10/BBIP1 and BBS19/IFT27; ([9–14] and references within). This heterogeneity was originally thought to be the cause of the significant inter-familial variability that characterizes the syndrome. However, establishing genotype–phenotype correlations has been a challenge, likely due to the significant functional overlap between the BBS proteins that we will discuss in the following sections. Having said that, thorough analyzes of different BBS patient cohorts and animal models are demonstrating subtle differences in disease presentation that are likely due to specific functions of different BBS proteins. For example, studies suggest that mutations in BBS2, BBS3 and BBS4 are associated with characteristic ocular phenotypes [15,16] and patients bearing mutations in BBS16/SDCCAG8 are characterized by highly penetrant renal disease but do not present with polydactyly [13]. Similarly, patients with mutations in BBS6, BBS10 or BBS12 present with a more severe renal phenotype [17]. Importantly, BBS6, BBS10 and BBS12 belong to a functionally distinguishable subgroup of BBS proteins, a fact that could provide a molecular explanation to this finding [18,19]. Studies in mice are also highlighting differences linked to the identity of the mutated gene. Bbs3 / mice develop common features of Bbs mutants, such as increased body weight and retinal degeneration, but also distinct manifestations, such as severe hydrocephalus and increased blood pressure [20]. Likewise, Bbs4 / and Bbs6 / mice present differences in blood pressure compared to Bbs2 / animals [21]. BBS is also characterized by significant intra-familial variability, a phenomenon not readily explained by genetic heterogeneity. Although BBS is largely inherited as an autosomal recessive trait, the screening of patient cohorts showed that in some families the disorder behaves as an oligogenic condition whereby mutant alleles in more than one BBS gene and other modifier loci interact to modulate the penetrance and expressivity of the syndrome

[11,22–30]. Thus, differences in the total mutational load across different BBS associated genes likely contribute to the characteristic phenotypic variability in this syndrome [31,32]. The functional characterization of the BBS proteins has provided critical information to understand both the overall lack of genotype-phenotype correlations and the genetic interaction between different BBS genes. These proteins share biological functions and even work forming multi-protein complexes. Therefore, mutations in different BBS genes are likely to affect the same biological processes (i.e. the formation/function of the cilium) and thus correlations between mutations in specific BBS genes and subsets of phenotypes would not be expected and rather would only be explained by specific functions of individual BBS proteins. Likewise, the observation of oligogenic inheritance in BBS is likely a consequence of mutating different components of the same protein complex, where different subunits can at least partially compensate for the lack of another, or affecting the same biological pathway at more than one step, models that have been used to explain this type of genetic interactions (reviewed in [22,33]). Interestingly, oligogenicity is not restricted to BBS but appears to be relatively common among ciliopathies as examples have been documented or postulated for NPHP, MKS and Acrocallosal (ACLS) syndrome [34–36]. The ciliopathies encompass a range of human conditions that although clinically distinct, share significant aspects of both their phenotypic presentation and their cellular basis. In other words, mutations in genes associated with different disorders can result in similar phenotypes because they affect the same organelle. Thus, if all the ciliopathies are caused by cilia dysfunction, why are these disorders distinct clinical entities? The main factor is the identity of the mutated gene and the specific function of its encoded protein in the cilium. It is not the same to impair cilia formation than to affect a given ciliary channel or receptor. In

Please cite this article in press as: Novas, R., et al. Bardet–Biedl syndrome: Is it only cilia dysfunction?. FEBS Lett. (2015), http://dx.doi.org/10.1016/ j.febslet.2015.07.031

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addition, accumulating evidence is showing that the type of mutation and the degree of residual protein function, together with the mutational load across both bona fide ciliopathy genes and modifier loci can determine both the clinical entity and its presentation [31,32]. Therefore, a given gene can be causally related to more than one ciliopathy (Table 1). For example BBS14/CEP290 mutations have been found associated not only with BBS but also with MKS, NPHP, JBTS and other ciliopathies, and mutations in MKS genes can cause BBS [4,11,37–39]. Lastly, one largely overlooked aspect of this equation is that specific cilia-associated proteins might play extra-ciliary roles that could also contribute to the pathogenesis and presentation of the ciliopathy, a factor that we will discuss for the BBS proteins in following sections. 3. The role of the BBS proteins... in the cilium The initial link between BBS and primary cilia came from the identification of a homozygous null mutation in BBS8 in a family presenting situs inversus, a reversal in the left–right (LR) configuration of the body plan [40]. Perturbations in the establishment of LR are caused by defects at specific embryonic structures, LR organizers, such as the node in the mouse and the Kupffer’s vesicle in

zebrafish, where cilia play a key role generating asymmetries [41]. The characterization of BBS8 then showed that the protein localizes to the centrosome and basal body of cilia [40], and subsequent studies have shown that the vast majority of BBS proteins share this localization pattern and can also enter the organelle (Table 1; [24,40,42–47]). 3.1. The BBS proteins in ciliogenesis: the BBSome The link between the BBS proteins and the cilium supported the initial expectation, raised by the genetic analysis of BBS cohorts, that this group of seemingly unrelated proteins could display significant similarities at the functional level. This notion was further reinforced by the realization that several of the BBS proteins physically interact to form a complex that has been dubbed the BBSome. The BBSome is a 438kDa complex composed by eight BBS proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 and BBIP10/BBS18) that functions as a coat for vesicles destined to the cilium [14,47,48]. Furthermore, it has been shown that the BBSome recognizes ciliary targeting signals in cargo proteins thus sorting and trafficking membrane proteins to the primary cilium

Table 1 BBS genes: their function, localization, and association to BBS and other ciliopathies. Association with other ciliopathies BBS1 BBS2

MKS

BBS3/ARL6 BBS4

MKS

BBS5

BBS6/MKKS

MKS, MKKS

BBS7 BBS8 BBS9 BBS10 BBS11/TRIM32 BBS12 BBS13/MKS1

MKS

BBS14/CEP290/ NPHP6

JBTS, MKS, SLS, NPHP, LCA MKS

BBS15/WDPCP/ FRITZ BBS16/SDCCG8/ NPHP10 BBS17/LZTFL1 BBS18/BBIP10/ BBIP1 BBS19/IFT27

SLS, NPHP

Ciliary role

Extra ciliary role?

Member of the BBSome Member of the BBSome (core)

Notch recycling. Regulate RNF2 levels Regulate RNF2 levels

Localization

Centrosome/basal body/cilia Centrosome/basal body/cilia/nucleus Small GTPase. BBSome assembly and traffic Intracellular trafficking? Cytoplasm/Cilia Member of the BBSome. Recruits the BBSome to the Notch recycling. Targets cargo to the pericentriolar Centrosome/basal body/ pericentriolar cilium region. Regulates actin polymerization. region/centriolar satellites Transcriptional regulation (RNF2) Member of the BBSome Centrosome/basal body/cilia. Direct contact with the ciliary membrane BBS-chaperonin complex that assist in BBSome Possible role in cell cycle. Regulates actin Centrosome/basal assembly polymerization body/cytoplasm Member of the BBSome (core) Transcriptional regulation (RNF2). Proteasome Centrosome/basal body/cilia/nucleus Member of the BBSome Regulates actin polymerization. Membrane Centrosome/basal localization of Vangl2 body/cilia/focal adhesions Member of the BBSome (core) Centrosome/basal body/cilia/focal adhesions BBS-chaperonin complex that assist in BBSome Centrosome/basal body assembly Similar to E3 ubiquitin ligase. Possible role in Predominantly present at proteasome degradation the cytoplasm/nucleus BBS-chaperonin complex that assist in BBSome Centrosome/basal assembly body/cytoplasm Transition zone organization Centrosome/basal body/transition zone Transition zone organization. Assembly and ciliary Centrosome/centriolar entry of the BBSome satellites/basal body/transition zone Recruitment of proteins essential for ciliogenesis PCP effector protein. Cell migration, cell polarity Transition zone/cytoplasm/ by modulation of the actin cytoskeleton actin cytoskeleton and focal adhesions. Regulates centrosomal accumulation of DNA damage response. Centrosome organization Transition zone/centrosome pericentriolar material during interphase and mitosis. associated protein. BBSome-interacting protein. Regulates ciliary Cytoplasmic protein. trafficking of BBSome components and Smo Member of the BBSome. Required for BBSome and Required for cytoplasmic microtubule Cilia primary cilium assembly polymerization and acetylation (MT stability) Cilia Component of the IFT-B complex required for anterograde transport of ciliary proteins. Links the BBSome to the IFT machinery

MKS: Meckel–Gruber syndrome; JBTS: Joubert Syndrome; NPHP: nephronophthisis; SLS: Senior–Løken syndrome; LCA: Leber congenital amaurosis; MKKS; McKusickKaufman syndrome.

Please cite this article in press as: Novas, R., et al. Bardet–Biedl syndrome: Is it only cilia dysfunction?. FEBS Lett. (2015), http://dx.doi.org/10.1016/ j.febslet.2015.07.031

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such as the G protein coupled receptors somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1) (Fig. 2A; [48,49]). Therefore, the BBSome plays a critical role regulating cilia composition. In addition, evidence is showing that the BBSome plays a role in IFT likely by coating the IFT particles (see below). The assembly of the BBSome coat onto membranes is triggered by the small Arf-like GTPase ARL6/BBS3, which is activated upon GTP binding [48]. Moreover, the BBSome interacts with the small GTPase Rab8 via Rabin8, its guanosyl exchange factor (GEF). Importantly, Rab8 plays a role in the docking and fusion of post-Golgi vesicles to the base of the cilium, and enters the cilium, suggesting that it is critical to mediate the targeting of the BBSome to the cilium [47,50]. In addition, it was demonstrated that the BBSome subunit BBIP10/BBS18 affects microtubule polymerization and acetylation, a post-translational modification highly prevalent in cilia microtubules, suggesting that the BBSome might participate in cilia formation/maintenance both by regulating cilia membrane composition and microtubule dynamics [51]. The role of the BBS proteins in the context of the cilium has been the main attraction of this group of proteins and the functional characterization of at least some of the ‘‘non-BBSome’’ BBS proteins has further reinforced this cilia-centric view. As mentioned above, BBS3/ARL6 plays a critical role in the function of the BBSome. BBS6, BBS10 and BBS12 compose the BBS-chaperonin complex, which mediates the assembly of the BBSome together with CCT/TRiC chaperonins [19]. Interestingly, the assembly of the BBSome occurs in several steps involving the formation of intermediate structures (Fig. 2B; [52]). The CCT/TRiC/BBS-chaperonin complex first binds and stabilizes BBS7 and its interaction with BBS2 and BBS9 to form a BBSome core to which then the rest of the proteins are incorporated in an orderly fashion: BBS1, BBS5, BBS8 and lastly BBS4, to which BBIP10 then binds [51,52]. Understanding the order in which BBS proteins are incorporated into the BBSome is likely to provide clues to dissect putative specific functions of individual BBS proteins. Interestingly, both BBS8 and BBS4, two tetratricopeptide repeat (TPR) containing proteins with significant similarity [40,43], are incorporated at the end of the assembly process. TPRs are typically involved in protein– protein interactions and thus these BBS proteins could serve as adaptors or provide target specificity to the BBSome. Interestingly, BBS4 has been shown to interact directly with a number of proteins that are relevant to the function of the BBSome and the pericentriolar region (see below). 3.2. Recruiting the BBSome to the cilium: the role of the pericentriolar region Besides the role of Rab8 mediating the cilia targeting of the BBSome, the pericentriolar region and centriolar satellites appear to play a major role in this process. BBS4 and the BBSome interact with PCM1, the main component of pericentriolar satellites [43,47]. BBS4, binding the p150Glued subunit of the dynein/dynactin molecular motor, is required to transport PCM1 toward the pericentriolar region (Fig. 2C; [43]). Importantly, recent evidence is showing that this interaction might coordinate the availability of BBSome subunits with ciliogenesis. During ciliogenesis BBS4 relocalizes from the centriolar satellites to the cilium in a process that is mediated by Cep72/Cep290: Cep72 binds PCM1 and in turn recruits Cep290 (BBS14) and this interaction has been shown to keep Cep72 and Cep290 in the satellites and away from the centrosome [53]. PCM1 appears to regulate the centrosomal/basal body availability of Cep72/Cep290 and consequently that of BBS4 (Fig. 2D). In addition, BBS8 ciliary localization is also reduced upon depletion of

Cep72/Cep290 suggesting that failure of BBS4 to re-localize to the cilium impairs recruitment of BBS8 and likely the BBSome [53]. BBS4 also interacts with AZI1, another component of centriolar satellites, and depletion of AZI1 results in the accumulation of the BBSome in the cilium [54], therefore reinforcing the notion that pericentriolar satellites regulate the availability of BBSome subunits, in particular BBS4, for the recruitment of the BBSome to the cilium. Cep290/BBS14 not only localizes to centriolar satellites but is also an integral part of the transition zone, the proximal region of the cilium that plays a critical role in mediating trafficking of different moieties into the ciliary compartment [55,56]. At this level, Cep290 and NPHP5 mediate both the assembly and ciliary entry of the BBSome [57]. Other BBS related proteins such as MKS1/BBS13 and SDCCAG8/BBS16/NPHP10 also localize to the transition zone reinforcing the importance of this region for the function of the BBSome [11,13]. In addition, the novel centriolar satellite SSX2IP targets CEP290 to the transition zone and SDCCAG8/BBS16, a protein that interacts with PCM1, regulates the recruitment of pericentriolar material to the centrosomal region, thus further highlighting the critical role of the pericentriolar region in BBSome function and cilia biology [58,59]. 3.3. Inside the cilium: the BBSome and IFT In Caenorhabditis elegans, mutations in bbs-7 and bbs-8 result in shortened cilia and both genes are required for the correct movement of the IFT components OSM-5/Polaris and CHE-11, but not CHE2, suggesting that the BBS proteins could participate in IFT and play a role facilitating transport of a subset of these proteins [46]. In the nematode, anterograde IFT is carried out by the action of two molecular motors, kinesin-II and OSM-3, and both bbs-7 and bbs-8 are required to coordinate their action and to stabilize the IFT/motor complex [60]. Interestingly, it was postulated that the BBSome could function as a coat adaptor for IFT [47]. Later work shows that the BBS proteins participate in the assembly and remodeling (disassembly and reassembly) of IFT complexes, both at the base of the cilium and the ciliary tip [61]. Wei and colleagues performed a whole-genome mutagenesis screen in C. elegans and identified mutations in dyf-2 (homolog of the IFT-A component IFT144) and bbs-1 in worms presenting normal anterograde IFT but defective IFT remodeling at the cilia tip. In dyf-2 mutants the BBSome subunits accumulate at the ciliary base despite unaffected anterograde IFT. The authors present a model in which the BBSome participates in the assembly of IFT particles at the ciliary base, it is then transported as IFT cargo to the tip of the cilium, and there, together with IFT-A, organize retrograde transport (Fig. 2E; [61,62]). However, the requirement of BBS proteins for IFT might depend on the organism and even cell types. Like in C. elegans, the BBSome has been recently shown to be part of IFT complexes in olfactory neurons in a 1:1 stoichiometry with IFT particles [63]. In contrast, BBS proteins are only found in a subset of IFT particles in Chlamydomonas reinhardtii, and mutations in key BBSome components do not affect IFT particle assembly and trafficking, albeit functional defects and accumulation of signaling proteins are observed [64]. 3.4. Exiting the cilium: the BBSome and IFT27/BBS19 Exiting the cilium is important for IFT regulation as well as cilia-mediated signaling. For example, the BBSome has been shown to mediate the ciliary exit of Patched-1 (Ptch1) and Smoothened (Smo), two components of the Hedgehog (Hh) pathway, and depletion of Bbs proteins in mice result in decreased Shh signaling [65,66]. The identification of IFT27 as BBS19 [9] provided a key

Please cite this article in press as: Novas, R., et al. Bardet–Biedl syndrome: Is it only cilia dysfunction?. FEBS Lett. (2015), http://dx.doi.org/10.1016/ j.febslet.2015.07.031

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R. Novas et al. / FEBS Letters xxx (2015) xxx–xxx

B

BBSome

Arl6

A IFT27

F

Arl6

BBSome BBSom A B

BBSome A B

Arl6 BBSome BBSome

E BBSome

BBSom BBSome

Ciliary membrane proteins (Sstr3, Mchr1, others)

B

A

A Arl6 BBSom BBSome BBSome

BBSome

EXIT (IFT27)

Basal Body

PERICENTRIOLAR REGION

(BBS, CEP290, NPHP5)

BBIP10

BBS4

PCM1

BBS4

D BBS4

BBS5 BBS2 BBS7 BBS1 BBS8 BBS9 BBS6 BBS10

GOLGI COMPLEX

CENTRIOLAR SATELLITES (PCM1, BBS4, CEP72/290, AZI1)

PCM1

BBS4

BBSome

RABIN8 RAB8 8

post-Golgi vesicle

A

BBS5 BBS2 BBS4 BBS7 BBS1 BBS8 BBS9

A Arl6

B

ENTRY (ARL6 CEP290 NPHP5 LZTFL1)

TRANSITION ZONE WITH TRANSITION FIBRES (NPHP, MKS, CEP290)

BBS12

BBS2 BBS5 BBS7 BBS1 BBS9

PCM1 BBS4

C

1 PCM 4 BBS lued G 0 p15

CCT/TRiC/ BBS-CHAPERONIN COMPLEX

B BBS2

BBS7 BBS1 BBS2 BBS9 BBS7 BBSome CORE BBS9

Fig. 2. Schematic representation of the roles reported for the BBS proteins. Summary of the proposed roles for the BBSome in the cilium. (A) The BBSome recognizes and coats vesicles with transmembrane proteins destined to the cilium. (B) BBSome assembly involves a series of intermediate complexes and is facilitated by the action of the CCT/ TRiC/BBS chaperonin complex (composed of BBS6, BBS10 and BBS12). (C) BBS4 is involved in transporting proteins toward the pericentriolar region in a microtubule and dynein dependent process. (D) The pericentriolar region appears to act as a reservoir of BBS4 thus controlling BBSome availability. (E) The BBSome participates in the formation of IFT particles and its transported to the ciliary tip. (F) Disassembly of the BBSome is thought to mediate IFT remodeling at the ciliary tip and the recruitment of cargo destined for ciliary exit to IFTA particles.

entry-point to start understanding this aspect of BBSome regulation [67–69]. IFT27 (also known as Rabl4) is a small G protein that heterodimerizes with IFT25 forming a complex (IFT25/27) that can exist both associated with IFT-B particles or free [70,71]. Eguether and colleagues have shown that Ift25 and Ift27 knockout mice present with typical hedgehog related structural defects, and at the cellular level show ciliary accumulation of Ptch1 and Smo [68]. In addition, they showed that different BBSome components readily accumulate in cilia of their Ift27 mutants suggesting that disruption of Ift25/Ift27 results in impaired BBSome exit. LZTFL1/BBS17 also regulates ciliary trafficking of the BBSome and Smo [65]. In Ift27 mutants, the BBSome, ARL6/BBS3 and LZTFL1/BBS17 are found enriched in the cilium suggesting that IFT27 likely acts removing these proteins from the cilium, and with

them, Ptch1 and Smo [68]. In contrast, depletion of Lztfl1 does not result in ciliary accumulation of Ift27 prompting the authors to postulate a model where Lztfl1 acts downstream of Ift27 linking the BBSome to the IFT machinery (IFT25/27) for ciliary export [68]. Liew and colleagues added mechanistic information demonstrating a direct biochemical interaction between IFT27 and ARL6/BBS3 [69]. Again, in both Ift27 knockdown cells and Ift27 / MEFS, BBSome subunits and components of the hedgehog pathway accumulate in the ciliary compartment. Importantly, IFT27 separates from the IFT-B complex to interact only with the nucleotide free ARL6/BBS3, thus potentially acting as a regulator of its activity, a GEF (guanine nucleotide exchange factor) that would promote ARL6/BBS3 activation [69]. Thus, the authors suggest that the remodeling of IFT particles at the ciliary tip is started by GTP hydrolysis of ARL6/BBS3, which leads to disassembly of the

Please cite this article in press as: Novas, R., et al. Bardet–Biedl syndrome: Is it only cilia dysfunction?. FEBS Lett. (2015), http://dx.doi.org/10.1016/ j.febslet.2015.07.031

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BBSome. Then, IFT-B particles release IFT25/27 (by unknown mechanism) and free IFT27 is able to participate in the GDP to GTP exchange of ARL6/BBS3, thus triggering the assembly of a BBSome coat linking cargoes destined for ciliary exit with retrograde IFT particles (Fig. 2F; [69]). 3.5. The BBS proteins: ciliogenesis and cilia length regulation Despite the critical role of the BBSome in transporting ciliary cargo, IFT regulation and overall ciliary function, individual BBS proteins do not appear to be absolutely required for cilia formation or cilia length regulation. As already mentioned, bbs-7 and bbs-8 mutations in C. elegans result in shortened cilia and compromised IFT but cilia are still formed [46]. Bbs7 / mice present fewer and shorter motile cilia in the ependymal cell layer but do not display overt cilia defects in the kidney and pancreas [72]. However, Bbs7 mutant animals present sperm with absent or abnormal flagella, similarly to what is observed in Bbs1M390R/M390R, Bbs2, Bbs4 and Bbs6 mutant mice, in which cilia do form but animals are sterile due to sperm flagella defects [72–76]. In cells, depletion of different BBSome components does not result in cilia shortening or absence [51]. Therefore, the requirement of individual BBS proteins for ciliogenesis and cilia length regulation appears to be highly variable. This variability could be explained by differences in the pattern of gene expression for different BBS genes in different cells and tissues and the requirement/dependency of different cell types on the function of the BBS proteins for IFT and ciliogenesis. In addition, functional redundancy among BBS proteins likely explains why targeting individual BBS genes might not be sufficient to have a major impact on ciliogenesis. We have shown that CCDC28B, a protein that interacts with a number of BBS proteins that are integral components of the BBSome, does play a role in cilia length regulation whereby its depletion in both cells and zebrafish results in significantly shortened cilia [77]. At least part of the role of CCDC28B in ciliogenesis is mediated by its interaction with SIN1, a member of the mTORC2, albeit independently of this complex. CCDC28B is a coiled-coil protein that in the context of mTORC2 plays a role mediating complex assembly [78]. Thus, it will be interesting to study whether this protein and SIN1 participate in the assembly or function of the BBSome as part of its role in ciliogenesis. 4. Other roles for the BBS proteins: going beyond the cilium In addition to their role in cilia, the BBS proteins may have other roles in cell physiology (Table 1). A first approximation to addressing this possibility is to analyze whether there are BBS homologous genes in non-ciliated organisms. Earlier studies evaluating this issue showed that at least the BBS genes identified at that time (BBS1, 2, 4, 5, 6, 7 and 8) were present only in ciliated organisms [45,74,79]. Accordingly, genes homologous to CCDC28B are present only in ciliated metazoans [77]. Interestingly however, some BBS genes are absent from organisms that possess ciliated cells, as it is the case for bbs2 and bbs7 in Drosophila melanogaster. Therefore, in certain organisms, some BBS proteins appear to be dispensable for ciliary function or, alternatively, other BBSs or BBS-unrelated proteins took their place. The chaperonin-like BBS proteins (BBS6, BBS10 and BBS12) were considered exclusive of vertebrates until a recent phylogenetic analysis discovered orthologous genes in very ancient eukaryotes, including non-ciliated ones [19,80]. However, the functionality of these genes in non-ciliated organisms has not been fully explored. Thus, while these data support a role for BBS genes in cilia function or/and some other specialized function common to ciliated organisms, we cannot discard that the BBS proteins could have been recruited

for other, cilia-unrelated functions. Importantly, this phenomenon has been described for core members of the IFT machinery that, although conserved only in ciliated organisms [45,79], have been shown to be expressed and to be functional in non-ciliated cells such as lymphocytes [81]. Here we will describe examples where the BBS proteins might be playing roles that, albeit could be linked to cilia, are not restricted to the formation and maintenance of the organelle. 4.1. Intracellular vesicular traffic The BBSome is involved in delivering membrane proteins to the cilium but its participation in microtubule dependent trafficking is likely not restricted to that on route to the organelle. Several BBSome components have been involved in retrograde, dynein-mediated, melanosome transport in zebrafish, a process that is not linked to cilia [82]. BBS1 and BBS4 recycle the Notch receptor to the plasma membrane [83]. This receptor is present both at the plasma and ciliary membrane, and it is internalized after activation. From early endosomes it can be either directed to multi vesicular bodies for degradation or recycled back to the plasma membrane [84]. When levels of BBS1 or BBS4 are reduced, recycling of Notch from early endosomes is diminished, suggesting that the BBS proteins participate in the trafficking from early endosomes to the cell surface (Fig. 3A; [83]). The ciliary pocket appears as a privileged site for vesicle docking [85], so it is possible that recycling to the plasma membrane occurs at this site and receptors are then distributed to the rest of the cell surface. However, it cannot be discarded that the BBS proteins and the BBSome mediate receptor recycling to other parts of the cell surface, independently of the cilium. Importantly, a more generalized role of cilia proteins in cell trafficking has also been described for bona fide IFT proteins. IFT20 traffics membrane proteins from the Golgi to the cilium and inside the ciliary compartment but also participates in recycling signaling molecules to the immune synapse in non-ciliated T-lymphocytes [81,86]. Moreover, IFT20 and BBS8 have been involved in membrane localization of Vangl2, a protein of the PCP pathway, in a process that is not affected by other ciliary proteins and therefore has been proposed as cilia-independent [87]. As mentioned above BBS3/ARL-6 is a member of the ARF-related small GTPases, a family of proteins that are associated with membrane trafficking. Some of these GTPases act by promoting the assembly of protein coats on donor compartments, the first step in vesicle formation and cargo selection, and BBS3 fulfills this role mediating BBSome association to membranes and traffic of the complex into and out of the cilium [48,88]. Consequently, the ciliary localization of the BBSome, as well as that of membrane proteins known to be transported by the BBSome (Mchr1 and Smo), is impaired in the absence of BBS3 [20]. As mentioned earlier Bbs3 / mice present characteristics that are not readily observed in other BBS mutants and thus could indicate a broader role for this protein in intracellular traffic [20]. For example, it was observed that canonical Wnt signaling seems to be inversely modulated by BBS3 and members of the BBSome whereby the pathway is activated when BBS3 is overexpressed [89] or when members of the BBSome, BBS1, BBS4 or BBS6 are down-regulated [90]. While these observations suggest that BBS3 may be involved in other cellular processes that are not related to the BBSome, further experiments should be performed to determine if these processes are cilia-independent. 4.2. Regulation of proteasome activity Proteasome-mediated proteolysis is important for maintaining appropriate cellular levels of proteins and represents a critical

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Notch

Ciliogenesis regulation

Notch

Notch Recycling

Early endosomes

BBS1, BBS4 BBSome?

A

Basal Body

?

Notch

BBS11 BBS proteins

Endosomal

C BBS4 BBS6 BBS8

?

B Lysosome

Actin dynamics

MVB

Proteasome

Degradation

Other BBSs??

RNF2 BBS7

BBS7

D

?

Degradation

?

BBS2

Cytoplasm-nuclear shuttling?

RNF2

Other BBSs??

BBS7 BBS7 BBS2

Signaling regulation

Transcriptional regulation

Nucleus

Fig. 3. Potentially extraciliary roles of the BBS proteins. Schematic representation of putative roles of the BBS proteins that are not necessarily linked to the formation and maintenance of the cilium. (A) BBS proteins participate in the regulation of endosomal trafficking whereby Notch receptor recycling to the plasma membrane is impaired upon depletion of BBS1 and BBS4. (B) Several BBS proteins interact with proteasomal subunits and regulate its activity thus potentially affecting signaling. (C) BBS proteins have been reported to affect actin dynamics, which in turn regulate ciliogenesis. (D) BBS7 and BBS2 (and possibly other BBSs) are able to enter the nucleus and interact with the chromatin remodeler RNF2 regulating its protein levels and thus the transcription of its target genes. BBSs proteins such as BBS7 might regulate RNF2 levels by affecting its availability for proteasome-mediated degradation, a possibility that needs to be tested.

strategy in the regulation of several signaling pathways including Hedgehog, canonical Wnt, Notch and NFjB. Interestingly, the centrosome/pericentriolar region is highly enriched in proteasome and associated regulatory proteins [91]. It was observed that several BBS proteins (1, 2, 4, 6, 7 and 8, but not 10 or 5) are able to interact with different proteasomal subunits [90,92]. Decreased levels of BBS1, BBS4 and oral-facial-digital syndrome-1 (OFD1, another centrosomal protein) alter proteasomal subunit composition and consequently inhibit its activity, as shown by increased levels of proteins that are normally processed by the complex (Gli2, Gli3, Notch) [92]. Interestingly, this role of the BBS proteins could be independent of the cilium since the NFjB pathway, which does not use the cilium but it is regulated by the proteasome, is affected by decreased levels of BBS4 and OFD-1 (Fig. 3B; [92]). BBS11 is an E3-ubiquitin ligase whose relationship with the cilium is still poorly understood. It was proposed that BBS2 instability when the protein is not part of the BBSome might be due to ubiquitination by BBS11 and proteasomal degradation [52]. More recently it was described that BBS11 interacts with NPHP7, a transcriptional repressor associated with NPHP. However, contrarily to what was expected BBS11 is responsible for the accumulation of polyubiquitylated NPHP7 species, a modification that prolonged the half-life of NPHP7 and also altered its sub-nuclear localization

and transcriptional activity. Thus, the authors propose that this interaction may help explain the phenotypic similarities between these two ciliopathies [93]. It was reported that BBS11/TRIM32 can ubiquitinate actin and downregulate its levels upon overexpression in Hek293 cells [94], findings that might be relevant to link this protein with ciliogenesis as we will discuss next. 4.3. Actin cytoskeleton dynamics Several studies have shown that ciliogenesis is critically dependent on actin cytoskeleton dynamics. In particular, actin polymerization inhibits cilia assembly [95]. Interestingly, BBS4, BBS6 and BBS8 regulate actin polymerization. When these proteins are absent or their levels diminished the actin cytoskeleton is reorganized, increasing the amount of focal adhesions where BBS8 and BBS9 colocalize with vinculin. These changes in actin organization do not involve cortical actin and were ascribed to changes in RhoA activity [96,97]. Importantly, inhibitors of RhoA activity recovered the number of ciliated cells and cilia length in BBS4 / and BBS6 / cells, and rescued several of the ciliopathy-associated phenotypes observed in zebrafish treated with morpholinos against bbs8 [96]. These results suggest that BBS proteins might be involved in ciliogenesis not only through the BBSome but also through regulation

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of RhoA and actin polymerization (Fig. 3C). This latter effect could also affect other cellular activities that depend on actin dynamics, such as cell migration or cytokinesis thus potentially expanding the range of cellular effects of disrupting the function of the BBS proteins. 4.4. Regulation of gene expression Primary cilia are essential for the transduction of several signaling pathways where the end result is changes in gene expression. We have reported that BBS proteins regulate gene transcription through a mechanism that could be independent of the cilium and that could also involve regulation of proteasome-mediated degradation (Fig. 3D; [98]). We observed that BBS1, 2, 4, 5, 6, 7, 8 and 10 interact with RNF2, a chromatin remodeler of the polycomb group. Knockdown of BBS1, BBS2, BBS4 or BBS7 result in increased RNF2 protein levels which, at least for BBS4 and BBS7 knockdowns, translate into changes in the expression levels of several RNF2-target genes [98]. RNF2 is a nuclear protein and importantly, the interaction with BBS7 may occur in this compartment as we showed that BBS7 is able to enter the nucleus from where it is actively exported via a nuclear export signal (NES) in its primary sequence. This active shuttling of BBS7 between the nucleus and cytoplasm might be linked to its capacity to regulate the proteasome-mediated processing of RNF2 and possibly other targets (Fig. 3D). Interestingly, NES motifs were also identified in the other BBS proteins, though their functionality was not evaluated. These results suggest that the capacity to enter the nucleus and to function in that compartment might be shared by other BBS proteins, a possibility that will need to be tested. 5. Understanding the cellular basis of the BBS phenotypes A large body of work has shown that cilia are hubs for sensing and transducing different signaling pathways. Coupled to this complex biological function, the presence of cilia in almost all cell types in the human body explains why ciliary dysfunction can have the pleiotropic effect that characterize the ciliopathies. Importantly, we now recognize a number of phenotypes as hallmarks of ciliary dysfunction that are present to varying degrees in the different ciliopathies including cystic kidney disease, retinal degeneration, obesity, polydactyly, central nervous system (CNS) malformations, left–right asymmetry defects and others [4,7,8,33]. Importantly, major advances have been achieved in understanding the role of primary cilia in the etiology of these phenotypes, a matter that has been reviewed extensively in the last years [4,5,8,33,99–101]. Therefore, just the realization that a disorder, in this case BBS, is a ciliopathy represents a critical entry-point to start understanding the cellular and molecular basis of the different phenotypes that characterize the syndrome. However, there can be substantial differences in the presentation of a given phenotype among different clinical entities highlighting the need to understand specific functions, both ciliary and extra-ciliary, of the different ciliopathy associated proteins. Here we will briefly review how defects in the BBS proteins are thought to explain BBS characteristic phenotypes such as renal disease, retinal dystrophy, polydactyly, CNS defects, and finally obesity where recent studies on BBS are providing important insights. 5.1. Renal disease A critical link between primary cilia and the pathogenesis of cystic diseases of the kidney (CDKs) was initially provided by the characterization of the Oak Ridge polycystic kidney (orpk) mouse, a model of autosomal recessive polycystic kidney disease

(ARPKD), caused by a mutation in Tg737, the gene encoding a core protein in intraflagellar transport: IFT88/Polaris ([102] and references within). Subsequent studies in a number of CDK models, including BBS, have led to the realization that cilia in the epithelium of the renal tubules play a critical role maintaining the homeostasis of the tissue, to the extent of now considering that likely all forms of CDKs are linked to cilia dysfunction [103,104]. Although the renal aspect of the disease has been difficult to reproduce in different BBS models, especially mice, the study of the BBS proteins has contributed to understanding the cellular basis of CDKs. The Wnt signaling pathway plays an important role for both the development and maintenance of the kidney. Depletion of BBS proteins results in Wnt signaling defects whereby canonical b-catenin dependent signaling is upregulated at the expense of a reduction in the non-canonical planar cell polarity (PCP) pathway [90,105]. Importantly, both upregulated canonical Wnt signaling and defective PCP have been shown to promote cystogenesis (reviewed in [106]). In addition, the BBS proteins have been shown to participate in Shh signaling transduction, a cascade that also plays a role in renal cystogenesis [12,66,107]. In addition to modulating signaling cascades relevant to the pathogenesis of CDKs, some BBS proteins interact with PC1 and PC2, the two major proteins affected in autosomal dominant polycystic kidney disease (ADPKD). PC1 and PC2 form a channel that localizes to the ciliary membrane and in the epithelium lining the renal tubules participate in sensing and transducing fluid flow shear stress into Ca2+ signaling to control cell proliferation and differentiation [108]. Four members of the BBSome (BBS1, BBS4, BBS5 and BBS8) physically interact with PC1 but depletion of only BBS1 and BBS3 result in defective ciliary targeting of PC1, data that further supports the notion that different BBS proteins have specific functions [109]. In line with these observations, depletion of BBS7 does not affect the ciliary localization of PC1 and PC2 but affects that of dopamine D1 receptor [72]. The BBSome, or at least some BBS proteins, appear to also play a role mediating the ciliary targeting of PC2 since expression of a dominant negative form of Rab8a results in mislocalization of PC2 [110]. Importantly, mutations in the Paramecium BBS orthologs also result in PKD2 loss from cilia [111]. Finally, it was reported recently that bbs-4 and bbs-5 act together to regulate the ciliary exit and lysosomal degradation of PC2 and other ciliary receptors in C. elegans [112]. Therefore, the renal phenotype in BBS could be caused by a combination of signaling defects involving canonical Wnt, PCP, Shh and PC1/PC2 mediated Ca2+ signaling. 5.2. Retinal degeneration Retinal degeneration has been associated with impaired IFT-mediated transport across the connecting cilium (CC), a highly modified cilium linking the photoreceptor inner and outer segments [113,114]. The BBS proteins play a role transporting rhodopsin to the base of the CC and defective IFT has been shown to cause retinal degeneration in several mutants [33,50]. Therefore, retinal degeneration in BBS is thought to involve a defect in transport of specific cargo proteins to the base of the CC and defects in IFT-mediated transport across the CC. However, while these mechanisms are likely relevant and possibly represent the main activity of the BBS proteins in the retina, restricting the activity of this group of proteins to a transport-based model might be an oversimplification since several of the ‘‘non-ciliary’’ activities of the BBS proteins could also contribute to photoreceptor degeneration. For example, the documented perturbations in actin dynamics, gene expression and proteasome activity resulting from BBS malfunction could also play a significant role in the development of this phenotype, a possibility that will have to be addressed.

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5.3. Polydactyly Hedgehog signaling in vertebrates is heavily dependent on the primary cilium whereby all the proteins that participate in the transduction of the pathway are localized to the cilium and their localization changes upon binding of Shh to its receptor Ptch1 (Fig. 1A). The effectors of the pathway, the Gli transcription factors, also enter the ciliary compartment and their processing and activation occurs inside the organelle [6,115]. Shh signaling controls patterning in the limbs and therefore defects in this signaling cascade can be reconciled with defects in digit number and identity [6]. The BBS proteins participate in Shh signal transduction and BBS1 directly interacts with Ptch1 and Smo [66]. In zebrafish, fin bud patterning is affected by misexpression of different bbs genes causing altered Shh signaling [116]. Interestingly, in BBS the polydactyly is postaxial which led to the suggestion that a combination of defects in Shh and other signaling pathways, primarily Wnt, could be contributing to this phenotype in this ciliopathy [33]. 5.4. Central nervous system BBS is characterized by learning difficulties, mental retardation, behavioral defects and other CNS defects [1,117]. The presence of cilia in different neuronal cell types has been known for decades but the importance of this organelle in different aspect of neuronal biology has been realized only recently and it is an area of intense research. The role of the cilium in the transduction of key signaling pathways such as Wnt and Shh is undoubtedly relevant to understand their function in neurons but cilia are being shown to play additional roles. For example, conditional ablation of cilia has been shown to result in disrupted dendritic arborization and the establishment of neuronal networks [118]. In addition, cilia are being shown to participate in diverse aspects of neuronal biology such as maintenance of progenitor pools, neurogenesis and neuronal migration, findings that will promote a better understanding of the cellular basis of the different CNS defects that characterize BBS and other ciliopathies (for thorough reviews on the role of cilia in the CNS see [119,120]). 5.5. Obesity in BBS Since the initial link between BBS protein function and cilia two major hypotheses have been considered to explain the obesity aspect of the phenotype. The first model was developed centered on a hypothalamic/neuro-endocrine dysfunction where cilia in specific areas of the brain play a critical role regulating signaling cascades that affect feeding and satiety. On the other hand, more recent data support a role for the BBS proteins and cilia in maintaining the homeostasis of peripheral tissues (revised in [121,122]). 5.5.1. A neuroendocrine origin for obesity in BBS Specific loss of cilia in hypothalamic POMC neurons causes obesity, hyperphagia and elevated serum insulin levels, glucose and leptin, an anorexigenic hormone critical in weight regulation [123]. Therefore, it was initially hypothesized that the obesity in BBS could be primarily due to defects in ciliary function in the CNS. Bbs2 / , Bbs4 / , Bbs6 / , and Bbs1M390R/M390R mice develop obesity associated with an accumulation of abdominal fat, decreased locomotion activity and hyperphagia [73–76]. These animals present increased serum leptin levels and leptin resistance and it was shown that the BBS proteins physically interact with the leptin receptor and mediate its traffic, thus potentially explaining the leptin phenotype [21,124].

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However, while leptin resistance is observed in different Bbs mouse models, it is not yet clear whether it is the main driver leading to obesity or a consequence of it. In the Bbs mice mentioned above, leptin resistance preceded the onset of obesity, suggesting that alterations in the leptin-signaling axis could drive the increased in body weight [21]. However, it has been proposed that leptin resistance could be a secondary consequence to the increase in body weight and also that cilia may not be directly involved in leptin responses [125]. Using an inducible system, Berbari and colleagues targeted Ift88 in 8 week old mice to then assess body weight gain and the response to leptin stimulation. Upon inducing the mutation, an initial phase of 3 weeks where knockout and wild type littermates maintained a normal body weight was followed by the development of obesity at 3 months in Ift88 / animals. Importantly, obese Ift88 / animals then were shown to respond to caloric restriction reducing their body weight to that of controls. Therefore, the authors were able to assess leptin response in animals lacking cilia in hypothalamic neurons and in three different body weight contexts: lean, obese, and lean after being obese. Leptin administration in pre-obese, but cilia deficient Ift88 / mice, resulted in activation of the leptin cascade as measured by a reduction in food intake and phosphorylation of STAT3 showing that in the absence of cilia animals can still respond to leptin. When Ift88 / mice were obese, leptin administration did not have any effect of feeding behavior or pathway activation. Finally, in Ift88 / mice that regained normal body weight values after diet, the authors observed a reduction in leptin levels and a response to leptin administration. Overall, these data show that at least in this experimental setup, leptin resistance is a consequence rather than the driver of obesity [125]. Functional differences between the BBS proteins and IFT88 could certainly explain these seemingly contradictory results. More difficult to reconcile however is the fact that in the Berbari et al. study, pre-obese Bbs4 / mice did not present increased leptin levels and responded to exogenous leptin administration [125]. Differences in the genetic background of mice or in the experimental setup could account for the observed discrepancies. Importantly, these results might be indicating that while leptin signaling defects do play an important role in the development of obesity in BBS and other ciliopathies, mechanisms that are still unknown might be important to initiate the process. Interestingly, the functional relationship between leptin and cilia appears to be even more complex as a recent work reports on leptin regulating cilia assembly and ciliary length by destabilizing actin filaments and increasing intraflagellar transport proteins through gene transcription regulation [126,127]. Therefore, leptin could modulate cilia length and the sensitivity of hypothalamic neuronal cilia to metabolic signals. Another important CNS defect in BBS is the mislocalization of G protein coupled receptors (GPCRs) involved in the regulation of feeding behavior. Both Sstr3 and Mhcr1 are receptors localized in neuronal cilia involved in feeding satiety, and their localization and function is altered upon disruption of BBS proteins [49,128]. In addition, a recent localization screen of GPCRs in neuronal cilia identified seven receptors including the neuropeptide Y receptor (NPY2R), another important anorexigenic pathway [129]. Depletion of the BBSome subunit BBIP10 in RPE cells result in decreased ciliary localization of NPY2R, Bbip10 / mice do not present NPY2R in hypothalamic neuronal cilia, and mutant animals do not respond to anorexigenic NPY2R-based stimuli [129]. In addition to GPCRs, the BBS proteins participate in the intracellular traffic of other receptors that are likely relevant to understand neuronal signaling and disease pathogenesis. BBS4 regulates the cilia localization of the brain derived neurotrophic factor (BDNF) receptor, TrkB, and its mislocalization upon depletion of BBS4 results in impaired BDNF mediated signaling, a pathway involved in controlling feeding behavior [130]. Therefore, mislocalization

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and defective signaling of GPCRs and other relevant signaling pathways are likely contributors to the altered feeding behavior and obesity in BBS. 5.5.2. Adipogenesis defects in the etiology of the BBS related obesity Besides a defect in hypothalamic ciliated neurons, adipocyte differentiation and misregulation of adipose tissue homeostasis are proving to be important aspects in the development of BBS-associated obesity. First it was realized that the BBS genes change their expression pattern during adipogenesis [131]. In addition, it was shown that while cilia are present in differentiating pre-adipocytes, cilia are lost during differentiation and mature adipocytes are non-ciliated cells [132]. Albeit transient, the presence of a primary cilium during adipocyte differentiation appears to be important since mesenchymal stem cells (MSCs) require the presence of primary cilia to differentiate toward the adipogenic lineage [133]. Moreover, Wnt and Hedgehog receptors, pathways known to regulate adipogenesis, are present in these transient cilia of differentiating pre-adipocytes [132]. Therefore cilia reabsorption or cilia dysfunction promoted by defects in the BBS proteins could affect the rate of adipogenesis. In this context, it was shown that blocking BBS10 and BBS12 leads to defective ciliogenesis and exacerbated adipogenesis promoted by the activation of the proadipogenic pathways GSK3 and PPARc [132]. More recently, BBS4 was shown also to affect proliferation and differentiation of adipocytes [134]. However, the effect on adipogenesis varies depending on the specific ciliary protein being affected. In contrast to BBS10 and BBS12, knockdown of ALMS1 (mutated in ALMS) in 3T3-L1 inhibited adipogenesis [135]. Depletion of the ciliary proteins Pkd1 and BBS12 also resulted in enhanced adipogenesis while inhibition of the IFT anterograde molecular motor Kif3a impaired adipocyte differentiation [136,137]. Therefore, albeit the primary cilium plays a critical role mediating adipocyte differentiation, the identity of the affected protein and thus the specific way in which its absence affect ciliary and/or non-ciliary functions associated with adipogenesis is likely to explain important differences in the presentation of obesity. Obesity is commonly associated with the development of insulin resistance and type 2 diabetes (T2D). However, a study comparing BBS patients with BMI-matched controls showed that the former present better glucose tolerance, thus hinting at potentially important differences between BBS-related and common obesity [138]. Marion and colleagues showed that knockdown of BBS12 in hMSCs results in increased adipogenesis that is accompanied by increased expression of the insulin receptor (IR) and GLUT4, increased localization of GLUT4 to the plasma membrane, and overall increased glucose absorption, and Bbs12 / animals present increased insulin sensitivity, improved glucose metabolism and reduced inflammation [136]. Importantly, since adipocytes secrete leptin in response to insulin and glucose, the increased glucose absorption and the overall increased adipogenesis are likely to contribute to the hyperleptinemia that characterize BBS [21,138]. In contrast, Gerdes et al. have uncovered a critical role for cilia and BBS4 in pancreatic b-cell function whereby the insulin receptor is found on cilia upon stimulation, and Bbs4 / mice show impaired insulin secretion and signaling [139]. Altogether, these data is showing that obesity in BBS likely has multiple contributing factors where a CNS defect leading to hyperphagia, a peripheral tissue defect leading to altered adipogenesis and impaired b-cell function are likely to be contributing factors. Finally, the discrepancies between different BBS models again highlight the importance of understanding the specific functions of individual BBS proteins.

6. Concluding remarks Since the cloning of the first BBS genes an impressive amount of work has followed, both regarding the genetics and deciphering the function of the BBS proteins. The studies in BBS have been instrumental in shaping the concept of ciliopathies, which today encompasses a number of disorders and syndromes that were, not long ago, considered completely disconnected. In addition, we now recognize a set of phenotypes that are considered typical consequences of cilia dysfunction, findings that have facilitated the classification of additional human conditions as ciliopathies and have therefore catalyze the identification and initial functional characterization of causal genes. The identification of the BBSome was a milestone to understand the role of the BBS proteins in cilia biology and to start dissecting the cellular basis of the syndrome. In this context, the possibility of the BBSome presenting different intermediate complexes/isoforms is intriguing and raises questions both from a genetic and a cellular perspective [52]. Are there different BBSome complexes that could achieve different functions? Can mutations in patients affect the balance between BBSome complexes? In this scenario, the levels and balance of different BBSome ‘‘isoforms’’ could be affected in different ways by mutations found in patients and thus could contribute to disease phenotypic variability. Further work will be required to address these questions. Finally, an increasing body of data is demonstrating that at least some of the BBS proteins are likely not functionally restricted to the primary cilium, a factor that could explain why BBS is one of the most pleiotropic ciliopathies and also that could be relevant to fully understand the etiology of the syndrome. A more generalized role in intracellular transport is an example of this since the effect of disrupting the BBS proteins might affect trafficking of proteins destined to other regions of the plasma membrane, to a given organelle, or that depend on the endosomal and secretory pathways for their function as it has been shown for Notch [83]. The possible shuttling of BBS proteins in and out of the nucleus provides another means by which these proteins could modulate signaling for example. The regulation of proteasome-mediated degradation of important signaling moieties also provides critical insight to understand the etiology of BBS. Identifying target proteins and establishing whether this role depends or its linked to ciliary function are key aspects to understand this function. Dissecting cilia versus non-ciliary functions of the BBS proteins will be challenging but studying the function of BBS proteins in non-ciliated cells could provide important insight. An example could be studying the role of the BBS proteins on the proteasome-mediated degradation of NFkB in non-ciliated cells such as lymphocytes. Importantly, understanding the different functions of the BBS proteins will not only allow us to fully understand the cellular basis of this condition but will likely provide novel entry-points for improving patient diagnosis, management, and for the rational design of therapeutic interventions. Acknowledgments We apologize to all the researchers whose original contribution was not properly cited. This work was supported by ANII-Innova and FOCEM (MERCOSUR Structural Convergence Fund). R.N. and M.C.-R. were supported with scholarships from the ‘‘Agencia Nacional de Investigación e Innovación (ANII, Uruguay)’’ and ‘‘Comisión Académica de Posgrado, Universidad de la República, Uruguay’’. J.L.B., M.C.-R. and F.I. are supported by ANII and J.L.B. and F.I. are researcher from the Programa para el Desarrollo de las Ciencias Básicas (PEDECIBA, Uruguay).

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