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Primary Cilia in the Developing and Mature Brain

Alicia Guemez-Gamboa,1 Nicole G. Coufal,1 and Joseph G. Gleeson1,* 1Howard Hughes Medical Institute, Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2014.04.024

Primary cilia were the largely neglected nonmotile counterparts of their better-known cousin, the motile cilia. For years these nonmotile cilia were considered evolutionary remnants of little consequence to cellular func- tion. Fast forward 10 years and we now recognize primary cilia as key integrators of extracellular ligand- based signaling and cellular polarity, which regulate neuronal cell fate, migration, differentiation, as well as a host of adult behaviors. Important future questions will focus on structure-function relationships, their roles in signaling and disease and as areas of target for treatments.

Introduction son, 2011). However, cilia still form in cells with mutations in The role of primary cilia are well known in special sensory cells these cilia genes, although they may be altered morphologically. involving hearing, vision, taste, and olfaction as the location of Therefore, it is important to differentiate between the role of a stimulus transduction, but their identification in other neural specific gene and the role of the entire cilium in a cell. By study- types came as somewhat of a surprise. Evidence for primary cilia ing ciliopathies, we can begin to understand the key roles of cilia in the vertebrate nervous system was first documented during in development and homeostasis, but we must recognize that electron microscopic examination of brain sections. The obser- cilia may still maintain some function even in the setting of spe- vation was initially assumed to be simply evidence of an evolu- cific ciliopathy gene mutations. tionary remnant. The finding harkened back to our evolutionary More than a dozen disorders are now considered to be within roots as flagellates, when cells needed a motile flagella for move- the ciliopathy spectrum, with a plethora of names to distinguish ment. For decades the observation that most neural cells display the subtypes including named syndromes Joubert (JBTS), a primary cilia was ignored. Nephronophthisis (NPHP), Senior-Løken (SLS), Leber congenital All of that began to change with several key observations. (1) amaurosis (LCA), Meckel-Gruber (MKS), Alstro¨ m (AS), and The protein products of genes mutated in murine hydrocephalus Bardet-Biedl (BBS). Ciliopathies demonstrate a range of clinical are localized to the primary cilium. (2) Sonic hedgehog (Shh) features including early fetal lethality, polydactyly (extra fingers signaling in vertebrates is altered with mutations in flagellar or toes), cardiac defects, cystic kidneys, hepatic fibrosis, genes. (3) Human cerebellar disorders are due to mutations in obesity, and nervous system defects. Although most of the genes encoding ciliary proteins, thus defining the ciliopathies. specific subtypes can display involvement in any of these Recent studies have demonstrated diverse roles for primary cilia, organs, each has a classical presentation, and most patients which include regulation of signal transduction that goes well can be diagnosed specifically. Altogether, these conditions beyond their sensory roles (Goetz and Anderson, 2010). involve nearly every major body organ and emphasize the impor- The primary cilium is increasingly viewed as critical for tant role of the primary cilium in development and homeostasis the development of the vertebrate nervous system, playing (Goetz and Anderson, 2010). either essential or modulatory roles in specific neurodevelop- Although it is now clear that mutations in over 50 different mental signaling pathways such as Shh and Wnt (Han et al., genes lead to ciliopathies, much less is known about specific 2008). Cilia also play important roles in regulation of stem cells ciliary signaling pathways and the pathogenic principles that and regeneration in the adult nervous system, where cell-cycle ultimately result in disease phenotypes at the tissue and organ and specific morphogen pathways recapitulate developmental levels. The phenotypic characterization of the disorders, the functions to facilitate cell-fate decisions in ciliated tissues. More- identification of causative genes, and research focusing on over, primary cilia are implicated in neuronal signaling and gene function are providing new insights that link ciliary function central control of appetite (Berbari et al., 2013; Spassky et al., with key developmental signaling pathways. Nervous-system- 2008), but the specific mechanisms are still the subject of related defects include neural tube, migration and cerebellar debate. development, retinal degeneration, anosmia, and obesity. The plethora of nervous system defects suggests many potential Ciliopathies: Human Disease Class Related to Ciliary roles of cilia in development and function. Dysfunction JBTS is a genetically and phenotypically heterogeneous The ciliopathies are a group of genetic disorders caused by disorder with several subtypes united by the defining feature of defects in primary ciliary structure and/or function. Ciliopathies hindbrain defects observed on axial brain MRI sections, known are characterized by pleiotropic clinical features, with mutations as the ‘‘molar tooth’’ sign. JBTS is sometimes associated with disrupting ciliary function and leading to distinctive develop- hydrocephalus (i.e., enlarged cerebral ventricles), anatomical mental and/or degenerative phenotypes in several tissues, abnormalities of the cerebral cortex, retinal dystrophy, and including the retina, kidney, and nervous system (Lee and Glee- cognitive deficits including intellectual disability and autism

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spectrum disorders (Romani et al., 2013). BBS is also genetically et al., 2011). The consensus is that disruption of IFT-A may heterogeneous and is characterized by intellectual disability, disturb trafficking of Shh pathway components differentially, anosmia, obesity, polydactytly, hypogonadism, and retinal causing phenotypes distinct from those observed in mutants in degeneration (Zaghloul and Katsanis, 2009). MKS is an early which cilia are absent. lethal disorder, genetically overlapping with JBTS and BBS, The encoded ciliopathy proteins localize mostly to the ciliary with occipital encephalocele, cystic kidney, and polydactyly base or axoneme, now a standard assessment for confirmation (Barker et al., 2013). Alstro¨ m syndrome, caused by mutations of newly proposed ciliopathy factors. While initial observations in the ALMS1 gene, is associated with obesity and retinal degen- focused on simple reduction in the percent of ciliated cells or eration, but unlike BBS or JBTS, mental defect, polydactytly, and length of cilia in mutant cells, the field has come to appreciate hypogonadism are not featured (Collin et al., 2002). One might that this is too crude a measure of disrupted function. Recent expect that tissue expression of the responsible genes corre- observations point to important defects in specific signaling lates with disease organ specificity or time of disease onset, pathways in ciliary ultrastructure such as impaired axonemal but no such correlations have emerged to date. Thus, it is fasci- tubulin modifications or structure of the 9+0 arrangement (Lee nating to consider how these tiny subcellular organelles can and Gleeson, 2011). Making things more complicated is the mediate so many diverse cellular functions, and how they may finding that these same pathways can themselves regulate cilio- be potentially disrupted in so many different ways to produce genesis, spacing, and orientation of cilia, and whether a cell such specific syndromes. builds a motile or nonmotile cilium (Boskovski et al., 2013). For instance, most JBTS genes identified to date encode Structure-Function Relationships of the Cilium proteins localized predominantly to the transition zone or the The primary cilium is a slender extension of the cell membrane ciliary axoneme. One of these genes mutated in JBTS, protruding from the surface of most cells, most notable in epithe- ARL13B, encodes a small GTPase localized to the ciliary lial cells. The cilium is assembled within a ciliary membrane axoneme, which regulates the graded response to Shh signaling extended over the axoneme and is anchored to the cell by the (Caspary et al., 2007). In the absence of Arl13b in mice, Smo is basal body. Primary (i.e., nonmotile) ciliary axonemes classically constitutively enriched in cilia (Larkins et al., 2011). Mutant contain nine doublet microtubules (9+0 axoneme), whereas mammalian cells show reduced cilia length and defective secondary (i.e., motile) cilia axonemes contain nine doublet axonemal tubulin structure, whereas overexpression results in microtubules and an extra central pair of microtubules that are artificially lengthened cilia. In arl13 mutant worms, disrupted attached to a dynein motor to generate movement (9+2 IFT integrity is linked to dissociation between IFT-A and IFT-B axoneme). Therefore, the ultrastructure of the axoneme can and compromised ciliogenesis. Still many questions remain, predict whether a given cilium is likely to be motile or nonmotile. including how Arl13b controls tubulin structure, how the defect In the brain, motile cilia are restricted to ependymal cells lining in IFT contributes to defective Shh signaling, and the identity of the ventricle and some choroid plexus cells (Lee, 2013), whereas Arl13b guanine exchange factors (GEFs) and GTP-activating primary cilia are evident on virtually all brain cells including pro- proteins (GAPs). genitors, neurons, and astrocytes. A key feature of cilia is that they contain no vesicles and thus Cilia in Sonic Signaling utilize methods different from the rest of the cell to transport The importance of primary cilia in vertebrate development was lipids and transmembrane proteins. While the ciliary membrane first identified by genetic experiments demonstrating that ciliary is contiguous with the plasma membrane, it has a unique set proteins are required for murine embryonic patterning, linking of sensory and transduction proteins to respond to extracellular Shh signaling to cilia function in mammals (Huangfu et al., signals. The cytoplasm of cilia is mostly isolated from the rest of 2003). Shh signaling is essential for embryonic development of the cell by a transition zone (TZ) at the base, which acts as a numerous tissues, controlling the pattern of cellular differentia- selective pore, and by the GTPase Septin in the membrane, tion in a concentration-dependent fashion, and disruptions thereby establishing a barrier to protein diffusion as well as a lead to neural tube and dorsoventral patterning defects. loading-unloading zone for transport into and out of the cilium Although the Shh pathway can exhibit low-level activity in the (Reiter et al., 2012). absence of cilia, in vertebrates, cilia are nonetheless required Proteins selected for entry are carried along the axoneme by for signal amplification when Shh ligand is present. The result intraflagellar transport (IFT) (Kozminski et al., 1993), mediated is defects in spinal cord patterning and alterations in cerebellar by two protein complexes, IFT-B and IFT-A. IFT-B complex granule neuron pools expansion, both under Shh control. There moves cargo from the cilia base toward the tip under the control are also midline defects including optic colobomas, corpus of the anterograde kinesin-2 (Kif3 motor complex), whereas callosal defects, hydrocephalus, failed closure of the rostral neu- IFT-A moves cargo in the opposite direction utilizing the retro- ral tube (i.e., exencephaly), and open posterior neural tube (i.e., grade axonemal dynein motors. Mutations in IFT-B components occipital encephalocele). such as Ift172, Ift88, Ift52, and Ift57 lead to complete absence of Shh regulates the balance of Gli transcriptional activators and cilia and, surprisingly, severely blunted Shh signaling (Huangfu repressors, dependent upon the ability of Smo and Ptch et al., 2003). In contrast, some mutations in IFT-A proteins lead signaling components to travel through cilia (Humke et al., to the formation of abnormally bulbous or elongated ends, 2010). The ciliary-associated kinesin Kif7 is the top candidate consistent with a cargo backup, and, even more surprisingly, for ciliary transport of these factors, acting downstream of result in activation rather than repression of Shh signaling (Qin Smo and upstream of Gli2, and having both negative and

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pathway, vice versa, or both. The best evidence is from the Retrograde Vangl1/2 double knockout mouse, the two homologs of the transport Drosophila core PCP component, which shows intact ciliogene- sis, cilium motility, Shh signaling, and basal body docking but completely disrupted cellular polarity and, as a result, failed Anterograde left-right asymmetry at the node (Song et al., 2010). The results transport suggest that PCP controls cellular and ciliary positioning, but not signaling. Wild type IFTA mutant Are cilia involved in PCP or Wnt signal transduction? Inversin, a key cilia protein mutated in cystic kidney disease, acts in primary cilia to inactivate canonical Wnt signaling during kidney morphogenesis and promotes convergence extension, thus functioning as a switch between these two Wnt pathways (Simons et al., 2005). Ahi1, mutated in JBTS, is a Wnt potenti- ator, facilitating beta-catenin nuclear accumulation (Lancaster et al., 2009). On the other hand, some evidence points to a role for cilia in negative regulation of Wnt signaling, as embryos without cilia (i.e., kif3a nulls) show dramatically IFTB mutant enhanced Wnt reporter activation (Corbit et al., 2008). Kif7 mutant Dynein motor mutant The data suggest a role for cilia in repressing Wnt signaling, not by silencing the pathway, but by maintaining a discrete range of Wnt responsiveness (Lancaster et al., 2011). Yet not all studies are in agreement, most notably a comprehensive Floor plate Motor neurons V0 study of canonical Wnt signaling in mutant embryos that lack V3 V1 primary cilia due to loss of IFT components found no Wnt phenotype or signaling defects (Ocbina et al., 2009). Thus, re- Figure 1. Neural Patterning Phenotypes in Intraflagellar Transport sults vary, and we can at least conclude that if cilia regulate Mutants Wnt signaling, it may be in subtle, perhaps indirect ways or in Axonemal and membrane components are transported by intraflagellar specific settings. transport (IFT) particles (complexes B and A) along the axoneme toward the tip (anterograde transport) by kinesins and in the opposite direction (retrograde transport) by the motor dyneins, respectively. In WT embryos, ventral neural Primary Cilia, Autophagy, and Cell-Cycle Entry cell fates are specified by Shh gradient. Anterograde IFT-B mutants Ift88 or The assembly of primary cilia is a dynamic process initiated once Ift172 lack cilia, Shh signaling is reduced, and the neural tube is dorsalized. cells exit the cell cycle to enter quiescence. In cultured cells, Retrograde IFT-A mutants like Ift139 exhibit excess Shh signaling and expanded ventral cell pools. Mutations that disrupt the kinesin Kif7 cause a primary cilium assembly and autophagy are both triggered by partial activation of the Shh pathway, with an expansion of motor neurons that serum withdrawal, and thus it is not surprising that part of the require intermediate levels of Shh. V0, V1, motor neurons, V3, and floor plate molecular machinery involved in ciliogenesis also participates represent progressively more ventral cell types in the developing spinal cord (adapted from Goetz and Anderson, 2010). in the early steps of the autophagic process, and recent evidence links these processes together (Pampliega et al., 2013; Tang et al., 2013). In Parmpliega’s paper, a previous positive roles in Shh signal transduction (Goetz and Anderson, unknown reciprocal relationship between ciliogenesis and auto- 2010). The role of cilia in hedgehog signaling extends to phagy was identified, finding that autophagy inhibits ciliogenesis vertebrates other than mammals, and to hedgehogs other than by limiting ciliary trafficking of components required for ciliary sonic, suggesting ancient evolutionary connections. Several growth. In contrast, Tang’s paper demonstrates that autophagic other signaling proteins including protein kinase A, Gpr161, degradation of the ciliopathy protein OFD1 at centriolar satellites pituitary adenylate cyclase-activating polypeptide, and forkhead promotes primary ciliary biogenesis. These apparently con- transcription factor Foxj1 mediate cilia- and Gli-dependent tradictory findings remain unresolved but suggest crosstalk be- Shh signaling (Cruz et al., 2010; Mukhopadhyay et al., 2013; tween the pathways. Niewiadomski et al., 2013; Tuson et al., 2011) and a complete understanding of mechanisms is one of the holy grails of devel- Cilia Role in Neurogenesis, Migration, and Axon opmental biology (Figure 1). Guidance Shh signaling is the major proliferative driver for cerebellar Cilia Interplay with PCP and Wnt Signaling granule neuron precursors (CGPs), an effect directly mediated Disruptions of PCP protein function are also linked to neural tube by cilia and a likely explanation for the cerebellar defects defects (NTDs) but in more caudal areas, probably reflecting a observed in ciliopathies. CGPs display cilia and are essential requirement for cell polarity cues during complex convergent for Shh-dependent proliferation of CGPs during cerebellar devel- extension cell-cell interactions at the dorsal midline (Wallingford, opment (Spassky et al., 2008). Accordingly, the conditional 2012). But unlike cilia in regulation of Shh signaling, which is well removal of other Shh-targeted ciliary genes (e.g., Ift88 or established, it is less clear whether cilia regulate the PCP Stumpy) in CGPs gives rise to striking dysgenesis and abnormal

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Arl13b Figure 2. Primary Cilia Participate in Neural wildtype mutants Migration during Cortical Development via Arl13b Pial surface Pial surface Arl13b is critical for the formation of the polarized Progenitors radial progenitor scaffold (wild-type). Deletion of Reelin-producing VZ Arl13b leads to a reversal in radial glial apical-basal polarity, inversion of position of reelin-producing SVZ cells, and consequently to aberrant neuronal migration, i.e., from the pial surface to the ventric- ular surface (arrow indicates direction). wildtype

SVZ defect in JBTS patients. Moreover, func- tional imaging and diffusion tensor imaging support defects in axon guidance in JBTS VZ Reelin-producing patients (Poretti et al., 2007), but mouse Arl13b mutants Progenitors models have failed to show correlated Ventricular surface Ventricular surface defects (Lancaster et al., 2011). The obvious question is how cilia, located at the soma, influence direction or guid- foliation of the cerebellum due to decreased proliferation (Breu- ance of the growth cone. Thus, ciliary roles and mechanisms in nig et al., 2008; Chizhikov et al., 2007). In addition, mutations of axon guidance remain to be elucidated. ciliary genes including Fantom, Ofd1, and Smo, also show cerebellar defects (Ferrante et al., 2006; Spassky et al., 2008; Primary Cilia and Cortical Development Vierkotten et al., 2007). Moreover, the link between cilia and Convergent data point to a role for cilia-associated factors in CGP proliferation extends to medulloblastoma, as CGPs are mammalian brain patterning (i.e., the progressive subdivision the likely cell source of the tumor, and cilia regulate tumorogen- of the neural tube along the dorsal-ventral and rostral-caudal esis (Han et al., 2009). axis). Genetic screens have uncovered mutations in Ift88 (an Freely motile vertebrate cells like sperm use their modified anterograde IFT component essential for ciliogenesis), Ttc21b motile cilium (i.e., flagella) for motility, but can primary cilia also (encoding retrograde IFT139) and Slb (encoding anterograde regulate cellular motility? Cilia are critical for some forms of IFT172) in mice with profound Shh-related cortical pattern de- platelet-derived growth factor (PDGF) signaling, a stimulant for fects (Gorivodsky et al., 2009; Stottmann et al., 2009; Willaredt fibroblast migration (Schneider et al., 2010). Do neurons behave et al., 2008). similarly? Shh has potent chemotactic effects, but surprisingly It is important to differentiate between roles for primary cilia ciliary mutants show enhanced rather than diminished Shh and roles for the centrioles in cortical development, even though chemotactic responsiveness (Bijlsma et al., 2012), and thus the mother centriole forms the base of the cilium. Asymmetric any effects on migration are likely to be Shh independent. divisions of radial glia progenitors produce both self-renewing Although migration defects are not widely recognized in ciliopa- radial glia and differentiating neuroblasts simultaneously in the thies, evidence suggests that BBS patients have defective neural ventricular zone (VZ). Whereas differentiating neuroblasts leave crest cell migration as cause for facial dysmorphisms and gut the VZ to constitute the future neocortex, renewing radial glia motility defects, thus implicating primary cilia albeit indirectly progenitors stay in the VZ for subsequent divisions. In both fly (Tobin et al., 2008). and mouse, the cell that inherits the older centriole preferentially The best evidence for cilia in neuronal migration comes from remains proliferative, whereas the younger centriole marks the study of the ciliary protein Arl13b in the cerebral cortex, where differentiating neuroblast (Wang et al., 2009; Yamashita et al., interneuron mutants show a specific defect in directional naviga- 2007). In agreement, the vast majority of genes mutated in tion but not general motility. Ciliary receptors such as GPCRs human primary microcephaly encode centrosome-associated essential for guidance cue responsivity fail to localize in Arl13b proteins, suggesting nonciliary roles for the centrosome in prolif- mutant cells (Higginbotham et al., 2012), potentially as a result eration and stem cell maintenance, probably functioning at the of defective PKA-mediated phosphorylation of cilia-localized mother centriole. proteins (Niewiadomski et al., 2013). These defects in cilia- It is also obviously important to differentiate between the dependent interneuron migration may in part underlie the neuro- requirement for an organelle and the requirement for proteins logical defects in JBTS patients (Figure 2). localized to the organelle. While cilia are identified on nearly all In the JBTS patients studied at postmortem, a striking cortical projection and interneurons, many murine mutants with finding was failed decussation of the corticospinal tract aberrant ciliogenesis show remarkably normal neuronal migra- (CST) and superior cerebellar peduncles (SCP) (Yachnis and tion, laminar organization, and polarity (for example, the Stumpy Rorke, 1999). The CST is the main motor output of the cerebral mutant) (Arellano et al., 2012), whereas for other mutants there cortex, decussating at the cervicomedullary junction, and are notable developmental defects. One such example is the SCP is the main outflow tract of the cerebellum, decussating Arl13b, the deletion of which leads to a reversal in radial glial at the red nucleus, so this finding suggests an axon guidance apical-basal polarity and consequently aberrant neuronal

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migration (Higginbotham et al., 2013). The fascinating aspect of photoreceptor apoptose, a condition known as Leber congenital the phenotype, novel in mammalian cortical mutants to date, amaurosis (LCA) (if early onset) and retinitis pigmentosa (RP) was a complete inversion of the entire cortical organization, (if later onset) (Adams et al., 2007). LCA/RP as well anosmia i.e., the apical aspect was located basally and vice versa. are well described in ciliopathies including JBTS, NPHP, and Whether this phenotype is the result of exencephaly or hinting BBS, among others (Arts et al., 2007; Delous et al., 2007). Acous- a role for cilia in radial glial polarity is still controversial, but as ticovestibular cells show a primary cilium-like kinocilium, and a result, proliferation occurred adjacent to the pia and the thus it is not surprising that deafness is a frequent accompani- reelin-positive layer 1 was adjacent to the ventricle. The pheno- ment in ciliopathies such as in Usher syndrome. Although type was recapitulated with conditional removal using Foxg1 but GPCRs mediate gustatory responses, there is a paucity of liter- not Nestin or Gfap-driven Cre, suggesting a requirement during ature on gustatory cilia. polarization but not maintenance of radial glia. Interestingly, the targeted ablation of Arl13b in projection Primary Cilia in the Adult Nervous System: neurons did not alter their migration but did induce axonal Neurogenesis and Signaling? outgrowth and connectivity defects (Arellano et al., 2012). More- New neurons are continuously produced to control circuit over, disruption of ciliogenesis in layer 2/3 cortical neurons, as plasticity, learning, and memory in the hippocampal dentate well as in neurons destined for the deeper layers of the cortex, gyrus (DG) and anterior subventricular zone (aSVZ). Shh reduced dendritogenesis, supporting a link between cilia and signaling is not only crucial for neurodevelopment but also par- dendrite outgrowth (Guadiana et al., 2013). Together, these ticipates in the expansion of postnatal progenitors (Breunig results, pending validation, suggest that the impact of ciliogene- et al., 2008). Is adult neurogenesis under the control of cilia? sis on neuronal development and maturation is dependent on Treatment with Smo inhibitor decreases proliferation in vivo, both developmental age and neuronal subtype. and mice lacking Smo in GFAP-positive neural precursors Rotatin is a centrosome-associated protein that colocalizes exhibit hippocampal neurogenesis defects (Machold et al., with the basal bodies at the primary cilium (Kheradmand Kia 2003), and loss of ciliary genes (Kif3a, Ift88,orFtm) leads to et al., 2012). Humans with mutations show an abnormal cortical defective hippocampal neurogenesis due to premature cell- organization called polymicrogyria and structural abnormalities cycle exit (Amador-Arjona et al., 2011; Han et al., 2008). Interest- of the cilium. Some BBS patients also exhibit enlarged cerebral ingly, loss of either primary cilia or Shh signaling has no effect on ventricles, and a ciliopathy mouse model for BBS shows the generation of other cell types such as astrocytes, suggesting increased apoptosis and reduced neurogenesis progressing to that this mechanism is cell-type specific. neonatal hydrocephalus as a result of defective development Although hippocampal defects have not been extensively (Carter et al., 2012). These results hint at requirements for ciliary described in human ciliopathy patients, learning disabilities proteins in human cortical development, but specific mecha- are common, and reports suggest that up to half of BBS nisms will require more work. patients have neocortical and hippocampal volume loss. More- Defective neuronal migration and resulting aberrant con- over, there are connections between hippocampal dysgenesis nectivity may represent a key to understanding the various and variable neuropsychiatric phenotypes in patients with neurological symptoms exhibited by patients diagnosed with BBS, emphasizing the impact of primary cilia in cognition ciliopathies, including cognitive deficits, autism spectrum disor- (Baker et al., 2011; Bennouna-Greene et al., 2011). Murine ders, seizures, schizophrenia, and developmental dyslexia. For models also indicate a delay in spatial learning and alteration instance, the frequent cognitive impairments in JBST are likely of spatial novelty recognition with decreased adult neuro- to be accompanied by less obvious defects in the cerebral genesis by conditional ablation of cilia-required genes (Ama- cortex as nearly 40% of patients are diagnosed with autism dor-Arjona et al., 2011). Whether the cognitive deficits spectrum disorders (Alvarez Retuerto et al., 2008). The specific observed in ciliopathies are related to the alteration in Shh changes induced in the neural network or in signaling pathways signaling or other signaling pathways remains to be deter- by ciliary defects may depend on the nature of the defect and the mined. One interesting possibility is altered cilia-localized type of neuron affected. GPCR signaling. For instance, somatostatin receptor 3 (SSTR3) is localized to the neuronal cilium, and Sstr3 mouse Role of Cilia in Special Sensory Neurons mutants show impaired object recognition and hippocampal Sensory neurons in the olfactory, visual, and acousticovestibular LTP (Einstein et al., 2010; Stanic et al., 2009). systems display a highly modified and specialized primary Might GPCR or other receptors use cilia as signaling centers or cilium. Specialized sensory cilia are located between the outer hubs to integrate signals and regulate synapses? TULP1, and inner segments of the retinal rods and cones and on the mutated in humans with RP, is required for photoreceptor synap- dendrites of olfactory receptor neurons (Louvi and Grove, tic ribbon development in mice prior to the onset of retinal 2011). Primary cilia in olfactory neurons respond to odorants degeneration, showing disrupted spatial relationship between via olfactory G protein-coupled receptors (GPCRs), producing the ribbon-associated proteins, Bassoon and Piccolo (Gross- a cAMP-mediated depolarization, whereas photoreceptor outer man et al., 2010). Overexpression of dominant-negative Kif3a segments mediate phototransduction via the rhodopsin family of blocks ciliogenesis in adult hippocampal-derived neural progen- GPCR, producing a cGMP-mediated hyperpolarization (Hengl itors and as a result there is enhanced Wnt signaling and defects et al., 2010). The modified cilium of photoreceptors is especially in dendritic refinement and glutamatergic synapse formation sensitive to disruptions and without an intact connecting cilium (Kumamoto et al., 2012). Although these results point to potential

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Figure 3. Proposed Roles for Cilia in A -/- Δ/Δ -/- Δ/Δ -/- Δ/Δ wild-type Bbs or Ift88 Bbs or Ift88 Bbs or Ift88 Regulation of Weight and Predisposition to before weight gain after weight gain Obesity (A) Bbs knockout or Ift88 conditional knockout Weight: mice are obese and show reduced leptin respon- siveness. However, both before weight gain and after weight loss, the knockouts show intact leptin Leptin Response: + + + responsiveness, suggesting that the leptin insensi- tivity is secondary to obesity and is independent of ciliary function. NPY (B) Models for the roles of ciliopathy proteins in B obesity. Left: proposes leptin receptors at the cell NPYR2 membrane require BBS for trafficking and down- BBS stream Stat3 signaling. Right: proposes other satiety signals like NPY, signaling through its cilia- localized NPY2R GPCR receptor, require BBS components for trafficking and modulation of cAMP levels. Alternatively, cilia may modulate satiety through pathways such as mTOR or Mchr1.

ceptor trafficking and downstream Stat3 Leptin signaling leading to leptin resistance (Rahmouni et al., 2008), because obesity Leptin Receptor occurs despite elevations in leptin, a P satiety signal, and because loss of cilia cAMP Stat3 mTOR ? specifically in leptin-responsive proopio- Mchr1 ? BBS melanocortin (POMC) neurons results in obesity (Davenport et al., 2007). Bbs1 interacts with the leptin receptor and roles for neuronal cilia in synapse development, further confir- mediates its trafficking, and mice lacking Bbs2, Bbs4, and mation in other systems is missing. Bbs6 also present leptin resistance, failing to reduce food intake In the case of special sensory photoreceptor and olfactory in response to leptin (Seo et al., 2009)(Figure 3). neurons, signals must pass through the cilium to the synapse But more recent work suggests that Bbs4 and Ift88 mutant to relay information, but how much capacity do primary cilia mice present hyperphagia-associated obesity independent have for conveying signals to the cell body? Chlamydomonas from defective leptin signaling and ciliary function, as both uses compartmentalized calcium signaling in motile cilia to drive before weight gain and after weight loss the knockouts show intraflagellar transport (Collingridge et al., 2013), but visualizing intact leptin responsiveness. Furthermore, other phenotypes calcium fluctuations in cilia is only now possible. Cilium-localized associated with leptin signaling defects are not consistent be- calcium indicators now provide evidence of calcium signaling in tween cilia mutant mice and leptin-deficient ob/ob mice, sug- cilia upon chemical or mechanical stimulation (Su et al., 2013). gesting that leptin resistance is a secondary consequence of By patch clamping onto ciliary membrane (the difficulty of which the obesity in ciliopathy mutant mice (Berbari et al., 2013). There should not be underappreciated), the Polycystin proteins were are several posited mechanisms for the obesity observed in identified as ciliary TRP-like calcium channels, modulating Shh ciliopathies other than leptin-signaling defects. The melanin- signaling. However, the Pkd2l1 mutant mice present a mild concentrating hormone receptor 1 (Mchr1), present on hypotha- phenotype when compared to other Shh-deficient mutant lamic neurons, is mistargeted in Bbs mutant mice (Berbari et al., mice, suggesting that calcium is probably also adjusted by other 2008). Altered Mchr1 signaling in the absence of cilia could PKD members or even other as-yet-unidentified ion pumps or result in hyperphagia, inducing obesity. Another possibility is transporters in the cilium during development. The ciliary mem- alteration of mammalian target of rapamycin (mTOR) activity brane is richly populated with calcium-permeant, relatively due to cilia loss (Berbari et al., 2011; Boehlke et al., 2010). nonselective cation channels, but the ciliary compartment is Although treatment of cilia mutant mice with rapamycin can mostly isolated electrophysiologically and chemically from partially correct some of the ciliary phenotypes (Shillingford cellular compartments (DeCaen et al., 2013; Delling et al., et al., 2006), the connection between cilia, mTOR signaling, 2013). The degree to which these tools will enable new discov- and obesity requires further evaluation. Obesity in BBS mice eries in ciliary signaling is unknown. may alternatively result from defects of hypothalamus patterning, due to misregulated Shh signaling. Accordingly, Primary Cilia and Obesity Bbs mutant mice have a decreased number of POMC neurons Obesity is a common feature in syndromes such as BBS and (Seo et al., 2009). Thus, it will be interesting to evaluate whether Alstro¨ m, suggesting that cilia may modulate neural circuitry modulating cilia-dependent signaling activities can alter feeding that monitors food intake and appetite. Recent data indicate behavior and energy homeostasis. A recent paper proposes that obesity in BBS mutant mice is due to defects in leptin re- other satiety signals like NPY, signaling through its cilia-localized

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NPY2R receptor, requires BBS components for trafficking and restored cilia function in ex vivo and in vivo models of varying modulation of cAMP levels to control energy balance in mam- ciliopathies. Retina and olfactory epithelium is appealing for mals (Loktev and Jackson, 2013). gene delivery in ciliopathies, as these cells are accessible and Recently, homozygous mutations in CEP19, a highly show time-dependent degeneration. Adeno-associated virus conserved protein that localizes to the centrosome and primary (AAV) has successfully been utilized in several animal ciliopathy cilia, were found in a newly described morbid-obesity syndrome. models of retinal degeneration. AAV-mediated photoreceptor Moreover, Cep19 knockout mice are morbidly obese, hyper- transduction of RPE65 led to overtly improved vision in humans phagic, glucose intolerant, and insulin resistant (Shalata et al., (Maguire et al., 2008), and since then a plethora of RD animal 2013). The identification of CEP19 as the cause of morbid models and human trials have appeared. In Bbs4 mutants, obesity in both humans and mice provides an additional link AAV5-mediated restoration rescued rhodopsin mislocalization between cilia dysfunction and obesity. and downstream RD (Simons et al., 2011). Taken together, these studies show the promise of photoreceptor-directed gene Treatment of Ciliopathies therapy. Treatment of anosmia due to ciliary defects in olfactory Although the spectrum of mutations contributing to the ciliopa- sensory neurons is a potential target for gene delivery, via thies has advanced in recent years, current therapies are limited intranasal injection. AV-mediated IFT88 transduction in fully primarily to symptom management, and there are no curative differentiated olfactory sensory neurons in Ift88 mutant mice therapies currently available to patients. Challenges to the devel- saw restoration of ciliary structures and rescue of olfactory func- opment of treatments for the ciliopathies include the pleiotropy tion (McIntyre et al., 2012). of genes involved, including 89 described gene mutations (and Lentivirus has recently been proven beneficial for gene counting) and 23 known syndromes, variable penetrance result- therapy against childhood lysosomal disorders (Aiuti et al., ing in symptomatic heterogeneity within a single mutation, and 2013; Biffi et al., 2013); however, lentivirus has not yet been effects on multiple different organs and cell types such that widely utilized for gene delivery in ciliopathies except for clinical manifestations can be found in a multitude of different in vitro studies. One proof-of-concept study utilized cultured organ systems. Phenotypes may be due to neurodevelopmen- respiratory epithelial cells from a patient with primary ciliary tal-associated defects, sensory impairments in vision, hearing, dyskinesia with a mutation in DnaI1, showing restoration of and olfaction, or in progressive tissue degeneration as a result normal ciliary beating frequency in transduced cells (Chhin of liver or kidney disease. et al., 2009). Persistent difficulties of viral methods include A spectrum of clinical trials is underway aimed at improving limiting expression in unintentional cell types, potential detri- symptomatic treatment, specifically targeting polycystic kidney mental effects of gene overexpression/misregulation, and most disease and retinal degeneration (RD). Several treatments importantly the risk of malignancy due to viral integration into targeting the development of renal cysts in polycystic kidney deleterious gene loci. disease have shown promise in rodent models and are currently An alternative approach is manipulation of DNA splicing. This in clinical development (Bukanov et al., 2006; Shillingford et al., approach circumvents difficulties associated with full-length 2006). However, human clinical trials have shown only limited gene delivery. To this end, modified U1 small nuclear RNAs success to date (Chang and Ong, 2012; Torres et al., 2012). and antisense oligonucleotides have been engineered to correct These trials target pathways implicated in renal cyst devel- splicing defects due to donor site mutations, successfully opment, specifically inhibiting mTOR, cAMP, or cyclin-depen- utilized in two different ciliopathy models in vitro. The single dent kinases and therefore may not be efficacious in other organ most common cause of LCA is a founder intronic CEP290 splice systems affected in the ciliopathies. site mutation, which can be corrected by antisense oligonucleo- RD could be an ideal target for therapy, as the retina repre- tide therapy (Collin et al., 2012). The potential challenge is off- sents a convenient, immunologically privileged site for injection target splicing effects on other transcripts, yet to be addressed. of therapeutics. Ciliary neurotrophic factor (CNTF) slows RD in Thus RNA-based therapies, while potentially immunogenic due an animal model and has shown some promising results for a to activation of Toll-like receptors, represent an exciting avenue variety of RD syndromes by direct slow release injection of for treatment. encapsulated cell technology in clinical trials (Birch et al., 2013). Tauroursodeoxycholic acid, the active ingredient in bear Perspectives bile, has antiapoptotic effects in many RD models and in Bbs1 The link between the structure of primary cilia and signaling mutants rescues loss of photoreceptors (Drack et al., 2012). pathways has become a framework for understanding the Likewise, the Bbs12 mutant demonstrated slowed RD upon pathogenesis of ciliopathies. Until recently, studies utilized polytherapy to block the unfolded protein response (Mockel germline constitutive knockout animal models, which provided et al., 2012). Overall pharmacologic management of symptoms useful loss-of-function phenotypes but were limited in mecha- associated with ciliopathies offers the prospect of delayed dis- nistic interpretation. We are beginning to see more intricate anal- ease progression while lacking curative potential. ysis with cell-type-specific or temporal-specific gene loss. These will be especially important as we relate gene function to specific Gene Therapy ciliary defects such as the requirement of one gene on ciliary Gene therapy offers a promising avenue for long-term targeted localization of a different gene’s encoded protein, or the partic- rather than symptomatic treatment. Numerous gene delivery ular role of a gene on intricate 3D ciliary ultrastructure (Garcia- approaches have shown promise in early studies and have Gonzalo et al., 2011; Williams et al., 2011).

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Although the role of primary cilia during nervous system devel- Arellano, J.I., Guadiana, S.M., Breunig, J.J., Rakic, P., and Sarkisian, M.R. opment is now well established, many questions about their (2012). Development and distribution of neuronal cilia in mouse neocortex. J. Comp. Neurol. 520, 848–873. involvement in adult nervous system functions remain. Adult- specific ciliopathy gene mutations to date, with the exception Arts, H.H., Doherty, D., van Beersum, S.E., Parisi, M.A., Letteboer, S.J., Gorden, N.T., Peters, T.A., Ma¨ rker, T., Voesenek, K., Kartono, A., et al. of neurogenesis defects, have failed to show major neurological (2007). Mutations in the gene encoding the basal body protein RPGRIP1L, a abnormalities, yet the link with GPCR, Wnt, calcium, and other nephrocystin-4 interactor, cause Joubert syndrome. Nat. Genet. 39, 882–888. signaling pathways suggests discoveries remain, perhaps Baker, K., Northam, G.B., Chong, W.K., Banks, T., Beales, P., and Baldeweg, around complex behavior and brain plasticity. In this regard, T. (2011). Neocortical and hippocampal volume loss in a human ciliopathy: A neuropsychiatric risk genes were linked to ciliation in a provo- quantitative MRI study in Bardet-Biedl syndrome. Am. J. Med. Genet. A. cative study. Using RNA interference to broadly test expressed 155A, 1–8. candidate genes associated with schizophrenia, bipolar affec- Barker, A.R., Thomas, R., and Dawe, H.R. (2013). Meckel-Gruber syndrome tive disorder, autism spectrum disorder, and intellectual and the role of primary cilia in kidney, skeleton and central nervous system disability, approximately half of the candidates were found to development. Organogenesis 10, 10. produce a ciliary phenotype (Marley and von Zastrow, 2012). In Bennouna-Greene, V., Kremer, S., Stoetzel, C., Christmann, D., Schuster, C., this light, DISC1, one of the best genetic candidates for schizo- Durand, M., Verloes, A., Sigaudy, S., Holder-Espinasse, M., Godet, J., et al. phrenia, has been implicated in ciliogenesis (Marley and von (2011). Hippocampal dysgenesis and variable neuropsychiatric phenotypes in patients with Bardet-Biedl syndrome underline complex CNS impact of Zastrow, 2010). In addition, variable neuropsychiatric pheno- primary cilia. Clin. Genet. 80, 523–531. types in patients with BBS and JBTS suggest primary cilia may regulate complex CNS functions (Bennouna-Greene et al., Berbari, N.F., Lewis, J.S., Bishop, G.A., Askwith, C.C., and Mykytyn, K. (2008). Bardet-Biedl syndrome proteins are required for the localization of 2011). G protein-coupled receptors to primary cilia. Proc. Natl. Acad. Sci. USA Little attention has been focused on how primary cilia could 105, 4242–4246. influence other neurological diseases until recently. Mice lacking Berbari, N.F., Kin, N.W., Sharma, N., Michaud, E.J., Kesterson, R.A., and tau tubulin kinase 2 (TTBK2), a microtubule-associated kinase, Yoder, B.K. (2011). Mutations in Traf3ip1 reveal defects in ciliogenesis, embry- fail ciliogenesis and exhibit neural tube defects (Goetz et al., onic development, and altered cell size regulation. Dev. Biol. 360, 66–76. 2012). This same gene with truncating mutations around resi- Berbari, N.F., Pasek, R.C., Malarkey, E.B., Yazdi, S.M., McNair, A.D., Lewis, dues 122–137 causes dominant spinocerebellar ataxia type 11 W.R., Nagy, T.R., Kesterson, R.A., and Yoder, B.K. (2013). Leptin resistance (SCA11) (Houlden et al., 2007). Furthermore, when these trun- is a secondary consequence of the obesity in ciliopathy mutant mice. Proc. Natl. Acad. Sci. USA 110, 7796–7801. cated cDNAs are transfected into cells there is inhibition of cilio- genesis, suggesting SCA may relate to ciliary defects. Ataxin10, Biffi, A., Montini, E., Lorioli, L., Cesani, M., Fumagalli, F., Plati, T., Baldoli, C., displaying a trinucleotide expansion as the cause for spinocere- Martino, S., Calabria, A., Canale, S., et al. (2013). Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science bellar ataxia-10 (SCA10), showed a homozygous splice mutation 341, 1233158. in a patient with JBTS (Sang et al., 2011). These examples sug- gest a potential connection between cerebellar development Bijlsma, M.F., Damhofer, H., and Roelink, H. (2012). Hedgehog-stimulated chemotaxis is mediated by smoothened located outside the primary cilium. and degeneration linked by cilia but will require specific human Sci. Signal. 5, ra60. alleles introduced into in vivo models to clarify these links. Birch, D.G., Weleber, R.G., Duncan, J.L., Jaffe, G.J., and Tao, W.; Ciliary Neurotrophic Factor Retinitis Pigmentosa Study Groups (2013). Randomized trial of ciliary neurotrophic factor delivered by encapsulated cell intraocular ACKNOWLEDGMENTS implants for retinitis pigmentosa. Am. J. Ophthalmol. 156, 283–292, e1.

We wish to thank members of the J.G.G. lab for discussions and Eva Anton, Boehlke, C., Kotsis, F., Patel, V., Braeg, S., Voelker, H., Bredt, S., Beyer, T., Bradley Yoder, John Wallingford, Val Sheffield, and Peter Jackson for Janusch, H., Hamann, C., Go¨ del, M., et al. (2010). Primary cilia regulate comments. A.G.-G. is supported by a UC MEXUS-CONACYT postdoctoral mTORC1 activity and cell size through Lkb1. Nat. Cell Biol. 12, 1115–1122. fellowship. Boskovski, M.T., Yuan, S., Pedersen, N.B., Goth, C.K., Makova, S., Clausen, H., Brueckner, M., and Khokha, M.K. (2013). The heterotaxy gene GALNT11 glycosylates Notch to orchestrate cilia type and laterality. Nature 504, REFERENCES 456–459.

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