Primary cilia are evolutionarily conserved organelles required in a number of signalling pathways influencing the development and behaviour of a diverse range of organisms. More recently, studies into a new class of human diseases known as ciliopathies have helped to shed light on the critical role of this once-ignored signalling centre. Bardet-Biedl syndrome (BBS) proteins localize to the primary cilium and participate in cilium biogenesis and function. BBS is a pleiotropic human disorder with variable severity that is suitable as a disease model for investigating the pathogenesis of a number of common ciliopathy features such as photoreceptor degeneration, renal cysts, and obesity. The C. elegans genome encodes a number of BBS proteins which undergo intraflagellar transport (IFT) at the primary cilium. Given the conservation between C. elegans and human BBS proteins, I hypothesize the existence of unidentified conserved genetic pathways related to the functions of these proteins. Using C. elegans, I characterize novel features of bbs mutants while identifying sources of genomic variation that may elucidate the variability of human BBS features. I show that C. elegans bbs mutants exhibit smaller body size, delayed development, and decreased exploration behaviour. Moreover, I identify a role for the soluble guanylate cyclases GCY-35/GCY-36 in modifying these bbs phenotypes. I conclude that BBS proteins non-cell autonomously influence a set of body cavity neurons in which GCY-35/GCY-36 function genetically upstream of a cGMP-dependent protein kinase (PKG), EGL-4, to control body size. Furthermore, the role of GCY-35/GCY-36 is unique amongst a large number of guanylate cyclases and BBS proteins may influence body size via an IFT-independent function. I explore the biological functions of EGL-4 and conclude that it may regulate body size through multiple cellular mechanisms. I also examine potential candidate genes related to cGMP production and turnover, confirming that additional cGMP-related factors can influence body size although not necessarily in body cavity neurons. In conclusion, I propose a model where BBS-expressing sensory neurons influence body size and development through cGMP-PKG signalling in body cavity neurons while functioning in parallel with additional sensory neurons (possibly BBS-independent) that use similar cGMP-PKG signalling dynamics.

Primary cilia are evolutionarily conserved organelles required in a number of signalling pathways influencing the development and behaviour of a diverse range of organisms. More recently, studies into a new class of human diseases known as ciliopathies have helped to shed light on the critical role of this once-ignored signalling centre. Bardet-Biedl syndrome (BBS) proteins localize to the primary cilium and participate in cilium biogenesis and function. BBS is a pleiotropic human disorder with variable severity that is suitable as a disease model for investigating the pathogenesis of a number of common ciliopathy features such as photoreceptor degeneration, renal cysts, and obesity. The C. elegans genome encodes a number of BBS proteins which undergo intraflagellar transport (IFT) at the primary cilium. Given the conservation between C. elegans and human BBS proteins, I hypothesize the existence of unidentified conserved genetic pathways related to the functions of these proteins. Using C. elegans, I characterize novel features of bbs mutants while identifying sources of genomic variation that may elucidate the variability of human BBS features. I show that C. elegans bbs mutants exhibit smaller body size, delayed development, and decreased exploration behaviour. Moreover, I identify a role for the soluble guanylate cyclases GCY-35/GCY-36 in modifying these bbs phenotypes. I conclude that BBS proteins non-cell autonomously influence a set of body cavity neurons in which GCY-35/GCY-36 function genetically upstream of a cGMP-dependent protein kinase (PKG), EGL-4, to control body size. Furthermore, the role of GCY-35/GCY-36 is unique amongst a large number of guanylate cyclases and BBS proteins may influence body size via an IFT-independent function. I explore the biological functions of EGL-4 and conclude that it may regulate body size through multiple cellular mechanisms. I also examine potential candidate genes related to cGMP production and turnover, confirming that additional cGMP-related factors can influence body size although not necessarily in body cavity neurons. In conclusion, I propose a model where BBS-expressing sensory neurons influence body size and development through cGMP-PKG signalling in body cavity neurons while functioning in parallel with additional sensory neurons (possibly BBS-independent) that use similar cGMP-PKG signalling dynamics.

BBSome is a protein complex crucial for trafficking of specific cargoes to the primary cilium. Although primary cilia are typically not present in cells of haematopoietic origin, such as T cells, recent research has revealed striking parallels between the primary cilium and the immunological synapse. Amongst other similarities, both structures are supposed to use the same transport machinery involving Rab8 and IFT20, the close interaction partners of BBSome. The first goal of this thesis was to investigate the role of BBSome in the biology of T cells. Using RT-qPCR, we have shown that BBSome subunits are expressed in lymphoid tissues and T cells. Studies of localization of BBSome subunits in Jurkat cell line and primary OT-I T cells revealed that the subunits have distinct localization patterns with BBS4 localizing to the centrosome and BBS1, BBS5, and BBip10 having dispersed localization. After the contact with an antigen presenting cell, BBS4 re-localizes to the immunological synapse. Mutations in BBSome encoding genes cause Bardet-Biedl syndrome (BBS), a rare ciliopathy presenting with multiorganic symptoms. The second goal of this thesis was to examine the associations between BBS and the immune system. Examination of medical records of more than 450 BBS patients revealed that autoimmune...

Indiana University-Purdue University Indianapolis (IUPUI) / Primary cilia are solitary cellular appendages that function as signaling centers for cells in adult energy homeostasis. Here in chapter 1, I introduce cilia and how dysfunction of these conserved organelles results in ciliopathies, such as Bardet-Biedl Syndrome (BBS), which present with childhood obesity. Furthermore, conditional loss of primary cilia from neurons in the hypothalamus leads to hyperphagia and obesity in mouse models of ciliopathies. Classically, cilia coordinate signaling often through specific G-protein coupled receptors (GPCRs) as is the case in both vision and olfaction. In addition, neurons throughout the brain including hypothalamic neurons possess primary cilia whose dysfunction contributes to ciliopathy-associated obesity. How neuronal cilia regulate the signaling of GPCRs remains unclear and many fundamental cell biology questions remain about cilia mediated signaling. For example, how cilia coordinate signaling to influence neuronal activity is unknown. To begin to address some of these cell biology questions around neuronal cilia, chapter 2, describes the development and use of a system for primary neuronal cultures from the hypothalamus. Using this system, we found that activation of the cilia regulated hedgehog pathway, which is critical in development, influenced the ability of neurons to respond to GPCR ligands. This result highlights the role of the developmentally critical hedgehog pathway on terminally differentiated hypothalamic neurons. One challenge facing the cilia field is our ability to assess cilia in large numbers without potential bias. This is especially true in tissues like the brain, where cilia appear to have region-specific characteristics. Work included in Chapter 3 describes the use of a computer-assisted artificial intelligence (Ai) approach to analyze cilia composition and morphology in a less biased and high throughput manner. Cilia length and intensities are important parameters for evaluation of cilia signaling. Evidence suggests that activation of some ciliary GPCRs results in shortening of cilia whereas deviations from normal cilia length in mutant phenotypes affects normal physiological processes such as decreased mucociliary clearance. Therefore, to analyze a large number of cilia, we describe the use of the Ai module from in vitro and in vivo samples in a reproducible manner that minimizes user bias. Using this approach, we identified that Mchr1 expression is significantly stronger in the cilia of paraventricular nucleus than that in the arcuate nucleus of adult mice. Work in Chapter 4 continues to explore the integration between hedgehog pathway and ciliary GPCR signaling in the central nervous system, and its relevance with energy homeostasis. We evaluated the hedgehog ligand in the plasma of mice in acute and long-term metabolic changes and identified that the activity of the ligand changed under altered metabolic conditions. We also developed a genetic mouse model where hedgehog signaling was constitutively active in neuronal cilia. These mice become hyperphagic and obese. These results further emphasize the potential role of the hedgehog signaling pathway in regulation of feeding behavior in adult vertebrates. Overall, results from this work will provide a better understanding of the defects not only underlying ciliopathy-associated obesity but may also reveal more common mechanisms of centrally mediated obesity. In addition, the tools I have developed will help in understanding how neuronal cilia are used for intercellular communications and ultimately how they regulate behaviors like feeding.

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Les ciliopathies rétiniennes sont un groupe de maladies rares causés par des mutations de gènes ciliaires. Les défauts des gènes ciliaires peuvent causer des défauts de trafic de protéines et induit l'apoptose des cellules photoréceptrices causés par le stress du réticulum endoplasmique (RE). On a étudié ciliopathies rétiniennes par modèle mourin, amaurose congénitale de Leber, rétinopathie pigmentaire liée à l’X, syndrome de Bardet-Biedl, syndrome d’Alström. Les souris Bbs1-/- , Bbs10-/- et CEP290-/- ont monté une diminution de la fonction rétinienne et sont causée par ER stress. Les souris Rd9/y et Alms1foz/foz présentent une apparition tardive et avec un faible taux de dégénérescence rétinienne et ils pourrait être causée par d'autres mécanismes. Le traitement GV-Ret basé sur le stress du RE pourrait sauver à la fois la fonction de et la morphologie de la rétine dans souris BBS. / Retinal ciliopathies are a group of rare diseases caused by mutations of ciliary genes. Defects in ciliary genes can cause defects in proteins traffics and induces apoptosis of photoreceptor cells caused by stress of the endoplasmic reticulum (ER) .We studied retinal ciliopathies by mice models, Leber congenital amaurosis, Xlinked retinitis pigmentosa, Bardet-Biedl syndrome and Alström Syndrome. The Bbs1-/-, Bbs10-/- and CEP290-/- mice exhibited a decrease in retinal function caused by ER stress. Rd9/y and Alms1foz/foz mice showed a late onset and a low rate of retinal degeneration and they could be caused by other mechanisms. The GV-Ret treatment based on ER stress could save both the function and morphology of the retina in BBS mice .

<p>  </p> <p>Cilia are cell appendages that sense our environment and are critical in cell-to-cell communication. Dysfunction of cilia can result in several disease states including obesity. While cilia in the brain are known to be important for feeding behavior, it is unclear how they regulate energy homeostasis. Classically, cilia coordinate signaling through surface receptors called G-protein coupled receptors (GPCRs). For example, cilia mediated GPCR signaling is critical for both our senses of vision and smell. How cilia regulate the signaling of GPCRs in other areas of the body including the brain is only now emerging. To answer cell biology questions around cilia mediated GPCR signaling in neurons, we developed a system for primary neuronal cultures. We discovered that the cilia mediated hedgehog pathway influences the ability of neurons to respond to GPCR ligands. For the first time, this result highlights the role of the hedgehog pathway in neurons. We continue to explore how cilia integrate the hedgehog pathway and GPCR signaling in the central nervous system, and the potential connections to energy homeostasis. We discovered that hedgehog pathway activity in feeding centers of the brain changes based upon feeding conditions like fasting. We also learned that activating the hedgehog pathway in these brain regions is sufficient to cause obesity in mice. These novel results highlight an unrecognized role for the hedgehog pathway in the regulation of feeding behavior. Overall, this work provides a better understanding of ciliopathy associated obesity and may reveal more common mechanisms of obesity in the general population. In addition, this work implicates the hedgehog pathway in regulating behaviors and new modes of cell-cell communication within the central nervous system.</p>