Bardet-Biedl Syndrome Proteins Research Articles

Adipocyte-specific disruption of the BBSome causes metabolic and autonomic dysfunction.

Obesity is a major public health issue due to its association with type 2 diabetes, hypertension, and other cardiovascular risks. The BBSome, a complex of eight conserved Bardet-Biedl syndrome (BBS) proteins, has emerged as a key regulator of energy and glucose homeostasis as well as cardiovascular function. However, the importance of adipocyte BBSome in controlling these physiological processes is not clear. Here, we show that adipocyte-specific constitutive disruption of the BBSome through selective deletion of the Bbs1 gene adiponectin (AdipoCre/Bbs1fl/fl mice) does not affect body weight under normal chow or high-fat and high-sucrose diet (HFHSD). However, constitutive BBSome deficiency caused impairment in glucose tolerance and insulin sensitivity. Similar phenotypes were observed after inducible adipocyte-specific disruption of the BBSome (AdipoCreERT2/Bbs1fl/fl mice). Interestingly, a significant increase in renal sympathetic nerve activity, measured using multifiber recording in the conscious state, was observed in AdipoCre/Bbs1fl/fl mice on both chow and HFHSD. A significant increase in tail-cuff arterial pressure was also observed in chow-fed AdipoCre/Bbs1fl/fl mice, but this was not reproduced when arterial pressure was measured by radiotelemetry. Moreover, AdipoCre/Bbs1fl/fl mice had no significant alterations in vascular reactivity. On the other hand, AdipoCre/Bbs1fl/fl mice displayed impaired baroreceptor reflex sensitivity when fed HFHSD, but not on normal chow. Taken together, these data highlight the relevance of the adipocyte BBSome for the regulation of glucose homeostasis and sympathetic traffic. The BBSome also contributes to baroreflex sensitivity under HFHSD, but not normal chow.NEW & NOTEWORTHY The current study show how genetic manipulation of fat cells impacts various functions of the body including sensitivity to the hormone insulin.

Mitochondrial involvement in the metabolic defects evoked by hepatocyte-specific BBSome deficiency

The BBSome, a protein complex of 8 Bardet-Biedl syndrome (BBS) proteins including BBS1, has emerged as an important regulator of metabolic function. BBSome deficient human and mice display several metabolic features including obesity and type 2 diabetes. Here, we show that mice lacking the BBSome specifically in liver which were generated by crossing the Bbs1fl/fl mice with liver-specific (albumin) AlbCre mice exhibit significantly higher lipid droplets in liver, by Oil Red-O staining, and in primary cultured hepatocytes (5.7%±0.4% vs 2.4%±0.6%, control), and by Transmission electron microscope (TEM). AlbCre/Bbs1flox also have elevated fasting glucose (169± 14 vs 136±8 mg/dL, p<0.05) and impaired insulin sensitivity. In addition, insulin-induced activation of AKT was significantly decreased in the liver (2.6±0.07 vs 6.6± 0.34 AU, p<0.05), but not in white adipose tissue and skeletal muscle, of AlbCre/Bbs1flox mice. Using immunohistochemistry, we found that insulin receptor level in plasma membrane was decreased by 24.2±6.8% in primary cultured hepatocytes derived from AlbCre/Bbs1flox mice which explains the insulin resistance. Our previous work implicating the BBSome in the regulation of mitochondria led us to hypothesize that mitochondria dysfunction contributes to the development of metabolic defects in AlbCre/Bbs1flox mice. TEM studies revealed that mitochondria were significantly larger and longer in hepatocytes of AlbCre/Bbs1flox mice than controls. This was associated with decreased levels of Dynamin-related protein 1 (DRP1), which critically regulates mitochondrial function through fission. Next, we asked whether the mitochondrial defects may explain the insulin receptor mistraffcking in BBSome deficient hepatocytes. To test this, hepatocytes were infected with feline immunodeficiency virus that express an active mutant form of DRP1 (FIV-DRP1S637A). Interestingly, insulin receptor localization in the plasma membrane was significantly higher in hepatocytes of Bbs1fl/fl/AlbCre­ mice infected with FIV-DRP1S637A pointing to mitochondrial defects as the underlying cause of insulin receptor mislocalization induced by hepatic BBSome deficiency. Taken together, these findings demonstrate that BBSome regulation of metabolic function involve mitochondria. NIH and VA. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Restoring retinal polyunsaturated fatty acid balance and retina function by targeting ceramide in AdipoR1-deficient mice

Mutations in the adiponectin receptor 1 gene (AdipoR1) lead to retinitis pigmentosa and are associated with age-related macular degeneration. This study explores the effects of AdipoR1 gene deficiency in mice, revealing a striking decline in ω3 polyunsaturated fatty acids (PUFA), an increase in ω6 fatty acids, and elevated ceramides in the retina. The AdipoR1 deficiency impairs peroxisome proliferator-activated receptor α signaling, which is crucial for FA metabolism, particularly affecting proteins associated with FA transport and oxidation in the retina and retinal pigmented epithelium. Our lipidomic and proteomic analyses indicate changes that could affect membrane composition and viscosity through altered ω3 PUFA transport and synthesis, suggesting a potential influence of AdipoR1 on these properties. Furthermore, we noted a reduction in the Bardet-Biedl syndrome proteins, which are crucial for forming and maintaining photoreceptor outer segments that are PUFA-enriched ciliary structures. Diminution in Bardet-Biedl syndrome-proteins content combined with our electron microscopic observations raises the possibility that AdipoR1 deficiency might impair ciliary function. Treatment with inhibitors of ceramide synthesis led to substantial elevation of ω3 LC-PUFAs, alleviating photoreceptor degeneration and improving retinal function. These results serve as the proof of concept for a ceramide-targeted strategy to treat retinopathies linked to PUFA deficiency, including age-related macular degeneration.

POMC Neuron BBSome Regulation of Body Weight is Independent of its Ciliary Function.

The BBSome, a complex of several Bardet-Biedl syndrome (BBS) proteins including BBS1, has emerged as a critical regulator of energy homeostasis. Although the BBSome is best known for its involvement in cilia trafficking, through a process that involve BBS3, it also regulates the localization of cell membrane receptors underlying metabolic regulation. Here, we show that inducible Bbs1 gene deletion selectively in proopiomelanocortin (POMC) neurons cause a gradual increase in body weight, which was associated with higher fat mass. In contrast, inducible deletion of Bbs3 gene in POMC neurons failed to affect body weight and adiposity. Interestingly, loss of BBS1 in POMC neurons led to glucose intolerance and insulin insensitivity, whereas BBS3 deficiency in these neurons is associated with slight impairment in glucose handling, but normal insulin sensitivity. BBS1 deficiency altered the plasma membrane localization of serotonin 5-HT2C receptor (5-HT2CR) and ciliary trafficking of neuropeptide Y2 receptor (NPY2R).In contrast, BBS3 deficiency, which disrupted the ciliary localization of the BBSome, did not interfere with plasma membrane expression of 5-HT2CR, but reduced the trafficking of NPY2R to cilia. We also show that deficiency in BBS1, but not BBS3, alters mitochondria dynamics and decreased total and phosphorylated levels of dynamin-like protein 1 (DRP1) protein. Importantly, rescuing DRP1 activity restored mitochondria dynamics and localization of 5-HT2CR and NPY2R in BBS1-deficient cells. The contrasting effects on energy and glucose homeostasis evoked by POMC neuron deletion of BBS1 versus BBS3 indicate that BBSome regulation of metabolism is not related to its ciliary function in these neurons.

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1.

Bardet-Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate.

Metabolic consequences of skeletal muscle- and liver-specific BBSome deficiency.

The BBSome is a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins including BBS1. Humans and mice lacking a functional BBSome display obesity and type 2 diabetes, highlighting the importance of this protein complex for metabolic regulation. However, the contribution of the BBSome in insulin-sensitive tissues such as skeletal muscle and liver to metabolic regulation is ill-defined. Here, we show that disruption of the BBSome through Bbs1 gene deletion in the skeletal muscle had no effect on body weight or glucose handling, but improved insulin sensitivity of female mice without changing insulin receptor signaling. Interestingly, when fed an obesogenic diet, male mice lacking the Bbs1 gene in skeletal muscle exhibited heightened insulin sensitivity despite the comparable weight gain and glucose tolerance relative to controls. On the other hand, normal chow-fed mice missing the Bbs1 gene in hepatocytes displayed increased body weight, as well as impaired glucose handling and insulin sensitivity. This was associated with attenuated insulin signaling in liver and hepatocytes, but not skeletal muscle and white adipose tissue. Moreover, hepatocytes lacking the Bbs1 gene displayed significant reduction in plasma membrane insulin receptor levels due to the mitochondrial dysfunction evoked by loss of the BBSome. Together, these findings demonstrate that myocyte BBSome is minimally involved in metabolic regulation, whereas the hepatic BBSome plays a critical role in the control of energy homeostasis and insulin sensitivity through its requirement for insulin receptor trafficking.NEW & NOTEWORTHY The ongoing epidemic of obesity and associated illnesses highlights the need to understand the biological processes that regulate energy balance. Here, we identified an important role for a protein complex called BBSome in the control of hepatic function. We show that the liver BBSome is necessary to maintain body weight and blood glucose levels due to its requirements to generate energy and detect insulin, a hormone that is essential for metabolic regulation.

Abstract 038: Adipocyte-specific BBSome Deficiency Impacts The Sympathetic Nervous System And Baroreceptor Reflex Sensitivity

The BBSome, a protein complex of 8 Bardet-Biedl syndrome (BBS) proteins including BBS1, has emerged as an important regulator of metabolic, cardiovascular, and autonomic functions. Patients with Bbs gene mutations display severe obesity, type II diabetes and hypertension. Previous studies demonstrated that neuronal BBSome contributes to metabolic homeostasis and blood pressure regulation, however the importance of adipocyte BBSome in controlling glucose homeostasis and blood pressure remains poorly understood. To address this, we generated mice lacking the BBSome specifically in adipocytes through selective deletion of the Bbs1 gene (Adipo Cre /Bbs1 fl/fl ) by crossing the adiponectin Cre (Adipo Cre ) male mice crossing with female Bbs1 floxed (Bbs1 fl/fl ) mice. Adipocyte-specific Cre recombinase was validated by further crossing the Adipo Cre mice with td-Tomato reporter mice. Bbs1 gene deletion in adipocytes was confirmed by measuring Bbs1 gene expression in adipose tissue using qRT-PCR. To induce obesity, mice were fed a high fat high sucrose diet (HFHSD) for 16 weeks starting at 4 weeks of age. Interestingly, the weight gain evoked by HFHSD was comparable between male and female Adipo Cre /Bbs1 fl/fl mice and littermate controls. Similarly, Adipo Cre /Bbs1 fl/fl mice displayed unaltered blood glucose as indicated by glucose and insulin tolerance tests. Using radiotelemetry, we found no significant difference in systolic, diastolic, and mean arterial pressure and heart rate between control and Adipo Cre /Bbs1 fl/fl mice fed HFHSD. Surprisingly, a significant increase in renal sympathetic nerve activity was observed in Adipo Cre /Bbs1 fl/fl mice (Control: 101.1 ± 16.23 spikes/sec, Adipo Cre /Bbs1 fl/fl : 133.2 ± 32.15 spikes/sec, p < 0.05). Next, we measured baroreceptor reflex sensitivity by analyzing the responses to sodium nitroprusside (SNP) and phenylephrine (PE). The slope analysis showed that Adipo Cre /Bbs1 fl/fl mice were less sensitive to SNP (Control: -2.56 ± 0.82, Adipo Cre /Bbs1 fl/fl : -1.746± 0.29, p < 0.05), demonstrating altered baroreflex. Taken together, our data implicate the adipocyte BBSome is a regulator of sympathetic nervous activation and baroreflex sensitivity.

352-OR: Regulation of Melanocortin Receptor 4 (MC4R) Ciliary Trafficking

The primary cilium, a hair-like projection at the surface of nearly every cell, functions as a signaling antenna in processes as diverse as vision and body weight homeostasis. Obesity is a cardinal symptom of many ciliopathies including Bardet-Biedl Syndrome (BBS). While we are beginning to understand how cilia participate in body weight homeostasis, the molecular pathway that leads to obesity in BBS patients remains unclear. We now know that the melanocortin receptor 4, a GPCR that regulates feeding behavior, localizes to cilia in hypothalamic neurons. Meanwhile, most BBS proteins assemble or regulate the BBSome, a protein complex that removes activated signaling receptors from cilia. To characterize the stimuli and mechanisms that regulate MC4R trafficking in and out of cilia, we recapitulated MC4R trafficking in ciliated cell lines amenable to rapid manipulation. Surprisingly, levels of MC4R in cilia remain under the detection limit in most cells unless treated with an inverse agonist of MC4R. In addition, interference with BBSome function also increases ciliary MC4R levels in all cells. Thus, the tonic activity of MC4R promotes the constitutive clearance of MC4R from cilia via BBSome-mediated exit. These results suggest that artificially increasing the ciliary levels of MC4R may augment its signaling activity. To selectively recognize activated GPCRs, the BBSome relies on specific ubiquitin chains linked at lysine 63 (K63Ub chains) that become attached onto activated GPCRs via β-arrestin-associated ubiquitin ligases. Depletion of β-arrestin blocked the constitutive exit of MC4R from cilia and mutation of ubiquitin acceptor lysines from MC4R and MRAP2 results in a measurable increase in the ciliary levels of MC4R. In addition, targeting a protease that specifically cleaves K63Ub chains to cilia completely blocks the exit of MC4R from cilia. We conclude that the tonically active MC4R is ubiquitinated in cilia and actively removed by the BBSome. Disclosure M.Nachury: None. Funding American Diabetes Association (1-20-VSN-03)

An evaluation of setmelanotide injection for chronic weight management in adult and pediatric patients with obesity due to Bardet–Biedl syndrome

ABSTRACT Introduction Bardet–Biedl Syndrome (BBS) is a rare, multisystemic ciliopathy with an incidence of obesity of 89%. Mutations in genes encoding BBS proteins are linked to reduced leptin sensitivity of hypothalamic POMC neurons and reduced activation of the melanocortin-4 receptor (MC4R) pathway due to deficient α-MSH production by hypothalamic POMC neurons. The MC4R pathway is involved in controlling body weight and energy metabolism, and its disruption is linked to hyperphagia and obesity. Setmelanotide is an MC4R agonist that counteracts deficiencies in the MC4R pathway of individuals with BBS. Areas covered Data from clinical trials were reviewed along with information available from setmelanotide’s approval for treatment of obesity in people ages ≥6y with a clinical diagnosis of BBS. Expert opinion Setmelanotide is available as a daily injectable that can be used for amelioration of obesity in people with Bardet–Biedl syndrome. Its cost is substantial, which may limit its use, but among those who respond, setmelanotide can reduce body mass dramatically and potentially improve comorbid conditions associated with obesity. Setmelanotide treatment has generally tolerable side effects, primarily injection site reactions and nausea/vomiting that generally improve with continued use; almost all people using setmelanotide experience marked skin darkening due to off-target activation of cutaneous MC1R.

Ciliary signaling proteins are mislocalized in the brains of Bardet-Biedl syndrome 1-null mice.

In the brain, primary cilia are found on most, if not all, central neurons. The importance of neuronal cilia is underscored by the fact that human diseases caused by primary cilia dysfunction, which are known as ciliopathies, are associated with neuropathologies, including neuropsychiatric disorders and learning and memory deficits. Neuronal cilia are enriched for certain G protein-coupled receptors and their downstream effectors, suggesting they sense and respond to neuromodulators in the extracellular milieu. GPCR ciliary localization is disrupted in neurons from mouse models of the ciliopathy Bardet-Biedl syndrome, with GPCRs failing to localize to cilia, indicating the Bardet-Biedl syndrome proteins are required for trafficking of G protein-coupled receptors into neuronal cilia. Yet, dopamine receptor 1 accumulates in cilia in the absence of Bardet-Biedl syndrome proteins, suggesting Bardet-Biedl syndrome proteins are required for normal ciliary import and export. To further explore the roles of the Bardet-Biedl syndrome proteins in neuronal cilia, we examined localization of ciliary signaling proteins in a new constitutive Bbs1 knockout mouse model. Interestingly, we find that two additional ciliary G protein-coupled receptors (Gpr161 and Gpr19) abnormally accumulate in cilia on Bardet-Biedl syndrome neurons. In addition, we find that the GPCR signaling protein β-arrestin accumulates in a subset of cilia in the brain, suggesting the presence of additional unidentified ciliary G protein-coupled receptors. These results confirm the importance of the Bardet-Biedl syndrome proteins in establishing ciliary GPCR pathways and indicate that loss of Bbs1 leads to complex changes in the localization of signaling proteins in the brain.

Retinal Degeneration Animal Models in Bardet-Biedl Syndrome and Related Ciliopathies.

Retinal degeneration due to photoreceptor ciliary-related proteins dysfunction accounts for more than 25% of all inherited retinal dystrophies. The cilium, being an evolutionarily conserved and ubiquitous organelle implied in many cellular functions, can be investigated by way of many models from invertebrate models to nonhuman primates, all these models have massively contributed to the pathogenesis understanding of human ciliopathies. Taking the Bardet-Biedl syndrome (BBS) as an emblematic example as well as other related syndromic ciliopathies, the contribution of a wide range of models has enabled to characterize the role of the BBS proteins in the archetypical cilium but also at the level of the connecting cilium of the photoreceptors. There are more than 24 BBS genes encoding for proteins that form different complexes such as the BBSome and the chaperone proteins complex. But how they lead to retinal degeneration remains a matter of debate with the possible accumulation of proteins in the inner segment and/or accumulation of unwanted proteins in the outer segment that cannot return in the inner segment machinery. Many BBS proteins (but not the chaperonins for instance) can be modeled in primitive organisms such as Paramecium, Chlamydomonas reinardtii, Trypanosoma brucei, and Caenorhabditis elegans These models have enabled clarifying the role of a subset of BBS proteins in the primary cilium as well as their relations with other modules such as the intraflagellar transport (IFT) module, the nephronophthisis (NPHP) module, or the Meckel-Gruber syndrome (MKS)/Joubert syndrome (JBTS) module mostly involved with the transition zone of the primary cilia. Assessing the role of the primary cilia structure of the connecting cilium of the photoreceptor cells has been very much studied by way of zebrafish modeling (Danio rerio) as well as by a plethora of mouse models. More recently, large animal models have been described for three BBS genes and one nonhuman primate model in rhesus macaque for BBS7 In completion to animal models, human cell models can now be used notably thanks to gene editing and the use of induced pluripotent stem cells (iPSCs). All these models are not only important for pathogenesis understanding but also very useful for studying therapeutic avenues, their pros and cons, especially for gene replacement therapy as well as pharmacological triggers.

The BBSome regulates mitochondria dynamics and function

ObjectiveThe essential role of mitochondria in regulation of metabolic function and other physiological processes has garnered enormous interest in understanding the mechanisms controlling the function of this organelle. We assessed the role of the BBSome, a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins, in the control of mitochondria dynamic and function. MethodsWe used a multidisciplinary approach that include CRISPR/Cas9 technology-mediated generation of a stable Bbs1 gene knockout hypothalamic N39 neuronal cell line. We also analyzed the phenotype of BBSome deficient mice in presence or absence of the gene encoding A-kinase anchoring protein 1 (AKAP1). ResultsOur data show that the BBSome play an important role in the regulation of mitochondria dynamics and function. Disruption of the BBSome cause mitochondria hyperfusion in cell lines, fibroblasts derived from patients as well as in hypothalamic neurons and brown adipocytes of mice. The morphological changes in mitochondria translate into functional abnormalities as indicated by the reduced oxygen consumption rate and altered mitochondrial distribution and calcium handling. Mechanistically, we demonstrate that the BBSome modulates the activity of dynamin-like protein 1 (DRP1), a key regulator of mitochondrial fission, by regulating its phosphorylation and translocation to the mitochondria. Notably, rescuing the decrease in DRP1 activity through deletion of one copy of the gene encoding AKAP1 was effective to normalize the defects in mitochondrial morphology and activity induced by BBSome deficiency. Importantly, this was associated with improvement in several of the phenotypes caused by loss of the BBSome such as the neuroanatomical abnormalities, metabolic alterations and obesity highlighting the importance of mitochondria defects in the pathophysiology of BBS. ConclusionsThese findings demonstrate a critical role of the BBSome in the modulation of mitochondria function and point to mitochondrial defects as a key disease mechanism in BBS.

Disruption of Dopamine Receptor 1 Localization to Primary Cilia Impairs Signaling in Striatal Neurons.

A rod-shaped appendage called a primary cilium projects from the soma of most central neurons in the mammalian brain. The importance of cilia within the nervous system is highlighted by the fact that human syndromes linked to primary cilia dysfunction, collectively termed ciliopathies, are associated with numerous neuropathologies, including hyperphagia-induced obesity, neuropsychiatric disorders, and learning and memory deficits. Neuronal cilia are enriched with signaling molecules, including specific G-protein-coupled receptors (GPCRs) and their downstream effectors, suggesting that they act as sensory organelles that respond to neuromodulators in the extracellular space. We previously showed that GPCR ciliary localization is disrupted in neurons from mouse models of the ciliopathy Bardet-Biedl syndrome (BBS). Based on this finding, we hypothesized that mislocalization of ciliary GPCRs may impact receptor signaling and contribute to the BBS phenotypes. Here, we show that disrupting localization of the ciliary GPCR dopamine receptor 1 (D1) in male and female mice, either by loss of a BBS protein or loss of the cilium itself, specifically in D1-expressing neurons, results in obesity. Interestingly, the weight gain is associated with reduced locomotor activity, rather than increased food intake. Moreover, the loss of a BBS protein or cilia on D1-expressing neurons leads to a reduction in D1-mediated signaling. Together, these results indicate that cilia impact D1 activity in the nervous system and underscore the importance of neuronal cilia for proper GPCR signaling.SIGNIFICANCE STATEMENT Most mammalian neurons possess solitary appendages called primary cilia. These rod-shaped structures are enriched with signaling proteins, such as G-protein-coupled receptors (GPCRs), suggesting that they respond to neuromodulators. This study examines the consequences of disrupting ciliary localization of the GPCR dopamine receptor 1 (D1) in D1-expressing neurons. Remarkably, mice that have either an abnormal accumulation of D1 in cilia or a loss of D1 ciliary localization become obese. In both cases, the obesity is associated with lower locomotor activity rather than overeating. As D1 activation increases locomotor activity, these results are consistent with a reduction in D1 signaling. Indeed, we found that D1-mediated signaling is reduced in brain slices from both mouse models. Thus, cilia impact D1 signaling in the brain.

LB1023 Deletion of bardet biedl syndrome 1 (BBS1) gene in T cells impairs wound healing in mice

Primary cilia play an important role in cell directional migration and tissue repair. Ciliary function depends on several proteins including the BBSome, a complex of eight conserved Bardet-Biedl syndrome (BBS) proteins, such as BBS1, which transport cargos to and from cilia. The immune synapse (IS), the nano-scale gap between T cells and antigen presenting cells, in non-ciliated T cells is functionally homologous to the primary cilium. During the formation of the IS, the BBSome appears to be critical for centrosome translocation toward the IS interface within T cells. Data from our lab demonstrate that BBS1 hypomorphic mice (containing a systemic homozygous knock-in missense mutation) characteristically exhibit impaired cilia function, altered fibroblast function, and reduced wound healing/tissue repair. The skin is residence for most lymphocytes in the body. To investigate the role of BBS1 in T cells in wound healing, we generated a novel transgenic mouse model in which the BBS1 gene was conditionally deleted in T cells. For in vivo wound healing, 2 mm full-thickness punch biopsy was performed on the shaved backs of 9 to 11 weeks old male and female T-BBS1-/- mice and their wild-type littermate controls and assessed for rate of wound closure by secondary intention between day 0 and day 7. The wounds formed fibrin clots at similar times in both T-BBS1-/- and littermate controls. However, male T-BBS1-/- mice demonstrated significantly delayed wound healing at day 7 compared to controls (4.061±1.79%, p=0.0342, n=10-12/group). The female mice show a trend toward reduced would healing at 7 days as well. These results demonstrate that BBS1 plays an important role in T cell mediated skin repair and that this effect may be sex specific.

A cilia-independent function of BBSome mediated by DLK-MAPK signaling in C.elegans photosensation.

Bardet-Biedl syndrome (BBS) is a genetic disorder that affects primary cilia. BBSome, a protein complex composed of eight BBS proteins, regulates the structure and function of cilia, and its malfunction causes BBS in humans. Here, we report a cilia-independent function of BBSome. To identify genes that regulate the C.elegans photoreceptor protein LITE-1 in ciliated ASH photosensory neurons, we performed a genetic screen and isolated bbs mutants. Functional analysis revealed that BBSome regulates LITE-1 protein stability independently of cilia. Through another round of genetic screening, we found that this cilia-independent function of BBSome is mediated by DLK-MAPK signaling, which acts downstream of BBSome to control LITE-1 stability via Rab5-mediated endocytosis. BBSome exerts its function by regulating the expression of DLK. BBSome also regulates the expression of LZK, a mammalian DLK in human cells. These studies identify a cilia-independent function of BBSome and uncover DLK as an evolutionarily conserved BBSome effector.

Investigation into the Role of the Skeletal Muscle BBSome in Insulin Sensitivity

The BBSome is a protein complex composed of eight Bardet‐Biedl syndrome (BBS) proteins including BBS1. Humans and mice lacking a functional BBSome display impaired insulin sensitivity, hyperglycemia, and type 2 diabetes, highlighting the importance of this protein complex in the control of insulin action and glucose homeostasis. However, the contribution of the BBSome in insulin sensitive tissues such as the skeletal muscle to the control of insulin sensitivity is not clear. We hypothesized that BBSome deficiency in the skeletal muscle will lead to insulin resistance and diabetes. To test this, we generated a novel conditional knockout mouse model lacking the Bbs1 gene specifically in the skeletal muscle. This was achieved by crossing mice expressing floxed alleles of the Bbs1 gene (Bbs1fl/fl) with mice expressing Cre‐recombinase in the skeletal muscle (MCKCre). These mice were further bred with tdTomato reporter mice to validate Cre‐recombinase using red tdTomato protein fluorescence. Body weight, glucose handling (measured using glucose tolerance test, GTT) and insulin sensitivity (measured using insulin tolerance test, ITT) of Bbs1fl/fl/MCKCre mice were compared to littermate controls. Furthermore, we used Western blot to test the ability of insulin to activate Akt, a key signaling pathway associated with the insulin receptor. We found that both male and female Bbs1fl/fl/MCKCre mice have no significant alteration in body weight relative to wild type controls (31.4±1.5 vs 31.1±1.0 g in males and 24.1±2.2 vs 23.0±1.7 g in females at 16 weeks of age). There was also no significant change in glucose handling (GTT AUC: 30608±2106 vs 30741±3480 in males and 28836±4466 vs 26530±3334 AUC in females) and insulin sensitivity (ITT AUC: 11017±1285 vs 9935±1080 in males and 8223±792 vs 5803±978 AUC in females) between Bbs1fl/fl/MCKCre mice and controls. This was further confirmed by the lack of difference in insulin‐induced Akt activation in skeletal muscle, liver, and adipose tissue. These findings suggest that BBS1 protein in skeletal muscle is not required for insulin sensitivity. Next, we assessed whether challenging the mice with high fat/high sucrose diet (HFHSD), starting at weaning, could reveal a role for the skeletal muscle BBSome in insulin sensitivity. The weight gain evoked by HFHSD was comparable between Bbs1fl/fl/MCKCre mice and controls (32.3±3.1 vs 33.3±1.0 g in males at 10 weeks of age). Once they reach 16 weeks of age, the HFHSD mice will be subject to glucose tolerance and insulin sensitivity tests to determine if obesity exaggerates hyperglycemia and insulin resistance in Bbs1fl/fl/MCKCre mice.

Conditional Disruption of the BBSome in POMC Neurons Alter Energy and Glucose Homeostasis

The BBSome, a protein complex composed of 8 Bardet‐Biedl syndrome (BBS) proteins including BBS1 has emerged as critical regulator of energy and glucose homeostasis. Humans and animals bearing disruption of the BBSome through loss of various BBS genes display numerous features including obesity and diabetes, but the underlying mechanisms are not well understood. We previously demonstrated that BBSome deficiency in the anorexigenic proopiomelanocortin (POMC) neurons through deletion of the Bbs1 gene lead to the development of obesity. To determine the effects BBSome deficiency in adulthood, we bred mice expressing an inducible POMCERCre with Bbs1fl/fl mice. tdTomato reporter mice were further breed with these mice to validate Cre recombinase via red tdTomato fluorescence protein expression. To induce Bbs1gene deletion we treated the POMCERCre/Bbs1fl/fl at age of 6 weeks with tamoxifen (75 mg/kg, IP, for 5 days). Importantly, both male and female POMCERCre/Bbs1fl/flmice displayed an obesity phenotype as indicated by the increased (P<0.05) body weight (41.0±1.3 vs 36.0±1.9 g in males and 29.0±1.1 vs 26.3±0.9 g in females at age of 20 weeks) and fat mass (10.8±1.2 vs. 7.8.±1.7g in males and 8.2±1.4 vs. 4.8±0.9 g in females). This was associated with increased (P<0.05) food intake (3.3.±0.3 vs. 2.9±0.2 g in males and 2.8±0.4g vs 2.5±0.2 g in females). These results indicate that hyperphagia contributes to the development of obesity in POMCERCre/Bbs1fl/fl mice. Moreover, using glucose tolerance and insulin tolerance tests we found that both male and female POMCERCre/Bbs1fl/flmice display glucose intolerance and insulin resistance. These findings demonstrate the importance of the BBSome in POMC neurons for the regulation of energy and glucose homeostasis.

Brainstem BBSome Regulates Glucose Metabolism and High Fat Diet Sensitivity

Bardet‐Biedl syndrome (BBS) proteins form a protein trafficking complex (BBSome) that regulates the composition of membrane proteins and contributes to neuron function. Patients harboring mutations in any BBS gene results in BBSome dysfunction and the development of metabolic complications including obesity and type 2 diabetes. Studies from our lab and others indicate that hypothalamic BBSome is a critical regulator of metabolic homeostasis, however the role of the BBSome in the brainstem is unknown. To determine whether hindbrain BBSome contributes to metabolic regulation, we crossed Phox2bCre mice with BBS1fl/fl mice (Phox2bCre/BBS1fl/fl) yielding BBS1 deletion specifically in brainstem nuclei. BBS1 deletion exclusively in hindbrain nuclei was validated by breeding these mice on a tdTomato Cre reporter background. Male Phox2bCre/BBS1fl/fl mice maintained on standard chow diet did not exhibit energy imbalance as evident by comparable body weight (37.2±2.8g vs. 35.8±4.4g), body composition (fat 20.8±2.8% vs 15.6±4.1%, lean 60.6±2.5% vs 64.2±3.0%), and food intake (3.5±0.4g/day vs 3.8±0.5g/day) relative to litter‐mate controls at 20 weeks of age. Interestingly, hindbrain BBSome disruption impairs glucose clearance and induces insulin resistance suggested by glucose (AUC 36,323±3027 vs 26,129±1972) and insulin (AUC 11,749±1678 vs 7,737±1559) tolerance tests. Strikingly, Phox2bCre/BBS1fl/fl male mice exhibit resistance to diet‐induced obesity indicated by decreased body weight (45.0±1.8g vs. 54.1±1.3g) when maintained on a high fat/high sugar diet independent of body composition (fat 42.5±1.6% vs 40.1±2.1%, lean 45.5±0.7 vs 48.4±1.5%) and food consumption (2.7±0.2g/day vs 3.0±0.1g/day). Both Phox2bCre/BBS1fl/fl and littermate controls developed diabetes when exposed to an obesogenic diet as indicated by glucose (AUC 63,645±4438 vs 62,477±2654) and insulin (AUC 14,303±3446 vs 16,406±2160) tolerance tests. These findings suggest that brainstem BBSome critically regulates glucose metabolism, but not energy balance, in mice maintained on a standard diet regimen. Furthermore, our studies indicate that hindbrain BBSome contributes to the development of diet‐induced obesity without effecting glucose handling and food intake.

Unraveling Molecular and Metabolic Gut‐Brain Signalling Disruptions and Potential Therapy in New Bardet‐Biedl Syndrome Mouse

Obesity is a cardinal feature of Bardet‐Biedl syndrome (BBS), a syndromic ciliopathy caused by mutations in genes for BBS 1‐21. BBS proteins control the function of primary cilia that act as a sensory antenna on virtually all mammalian cells. The mechanisms of BBS‐induced obesity remain poorly understood, resulting in a lack of therapeutic options for these patients. The overarching goal of the study was to define underlying mechanisms involved in the pathogenesis of BBS‐induced obesity and use this knowledge to identify novel therapeutic targets. Given the importance of cilia in interoceptive and homeostatic sensing, we hypothesized that disruption of cilia function by BBS mutation would impair negative feedback resulting in a proinflammatory microenvironment in adipose tissue and disruption of neuroendocrine control of feeding in hypothalamus.In this study, we first characterized the metabolic phenotype of a novel BBS5 knockout mouse (BBS5‐/‐; n=6‐12/strain/gender). Next, we performed immunophenotyping of adipocytes and unbiased RNA sequencing of the hypothalamus of BBS5‐/‐ and WT mice. To validate the predictions from transcriptomic data, we performed pharmacological‐based feeding experiments and tested a putative therapeutic for BBS‐induced obesity.We found that BBS5‐/‐ mice exhibits all the classic symptoms observed in human patients with BBS, including hyperphagia, cognitive impairments, glucose, and insulin intolerance, and 3 to 4‐fold increased fat mass on chow diet compared to wildtype mice (p<0.001). BBS5‐/‐ mice had increased proinflammatory M1 macrophages and decreased functional regulatory T‐cells in adipocytes suggesting that extrinsic factors promote fat mass gain. Pathway analysis of the transcriptomics data revealed altered endocrine receptor signaling in the hypothalamus of BBS5‐/‐ mice. Feeding experiments confirmed the development of both leptin and cholecystokinin resistance independent of body weight (p>0.05). The receptor for glucagon‐like‐peptide‐1 (GLP1) was found to be upregulated in the hypothalamus of BBS5‐/‐ mice (p<0.001). Chronic daily pharmacological administration of GLP1 receptor agonist (Semaglutide) for two weeks significantly reduced food intake, body weight, elevated glucose levels, and fat mass (p<0.001).In conclusion, BBS5‐/‐ mice are a valuable new translational tool to elucidate interoceptive and homeostatic gut‐brain axis alterations in BBS‐induced obesity. We identify adipose inflammation, and impaired hypothalamic leptin, insulin, and cholecystokinin signalling as underlying mechanisms for the metabolic pathogenesis of ciliopathy. Our results support using GLP1 receptor agonist as a novel therapeutic strategy for treating obesity in patients with BBS.

Bardet-Biedl syndrome: The pleiotropic role of the chaperonin-like BBS6, 10, and 12 proteins.

Bardet–Biedl syndrome (BBS) is a rare pleiotropic disorder known as a ciliopathy. Despite significant genetic heterogeneity, BBS1 and BBS10 are responsible for major diagnosis in western countries. It is well established that eight BBS proteins, namely BBS1, 2, 4, 5, 7, 8, 9, and 18, form the BBSome, a multiprotein complex serving as a regulator of ciliary membrane protein composition. Less information is available for BBS6, BBS10, and BBS12, three proteins showing sequence homology with the CCT/TRiC family of group II chaperonins. Even though their chaperonin function is debated, scientific evidence demonstrated that they are required for initial BBSome assembly in vitro. Recent studies suggest that genotype may partially predict clinical outcomes. Indeed, patients carrying truncating mutations in any gene show the most severe phenotype; moreover, mutations in chaperonin‐like BBS proteins correlated with severe kidney impairment. This study is a critical review of the literature on genetics, expression level, cellular localization and function of BBS proteins, focusing primarily on the chaperonin‐like BBS proteins, and aiming to provide some clues to understand the pathomechanisms of disease in this setting.