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Val C Sheffield - One of the best experts on this subject based on the ideXlab platform.
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Ectopic expression of BBS1 rescues male infertility, but not retinal degeneration, in a BBS1 mouse model
Gene Therapy, 2021Co-Authors: Matthew R. Cring, Charles Searby, Kai Wang, Kacie J. Meyer, Adam Hedberg-buenz, Michael Cave, Michael G. Anderson, Val C SheffieldAbstract:Bardet–Biedl syndrome (BBS) is a rare ciliopathy for which there are no current effective treatments. BBS is a genetically heterogeneous disease, though the M390R mutation in BBS1 is involved in ~25% of all genetic diagnoses of BBS. The principle features of BBS include retinal degeneration, obesity, male infertility, polydactyly, intellectual disability, and renal abnormalities. Patients with mutations in BBS genes often present with night blindness within the first decade of life, which progresses to complete blindness. This is due to progressive loss of photoreceptor cells. Male infertility is caused by a lack of spermatozoa flagella, rendering them immobile. In this study, we have crossed the wild-type human BBS1 gene, driven by the CAG promoter, onto the Bbs1 ^ M390R/M390R mouse model to determine if ectopic expression of BBS1 rescues male infertility and retinal degeneration. qRT-PCR indicates that the BBS1 transgene is expressed in multiple tissues throughout the mouse, with the highest expression seen in the testes, and much lower expression in the eye and hypothalamus. Immunohistochemistry of the transgene in the eye showed little if any expression in the photoreceptor outer nuclear layer. When male Bbs1 ^ M30R/M390R ;BBS1 ^ TG+ mice are housed with WT females, they are able to sire offspring, indicating that the male infertility phenotype of BBS is rescued by the transgene. Using electroretinography (ERGs) to measure retinal function and optical coherence tomography to measure retinal thickness, we show that the transgene does not confer protection against retinal degeneration in Bbs1 ^ M300R/M390R ;BBS1 ^ TG+ mice. The results of this study indicate that the male infertility aspect of BBS is an attractive target for gene therapy.
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Absence of BBSome function leads to astrocyte reactivity in the brain
Molecular Brain, 2019Co-Authors: Minati Singh, Janelle E. Garrison, Kai Wang, Val C SheffieldAbstract:In humans, dysfunctional primary cilia result in Bardet-Biedl syndrome (BBS), which presents with clinical features including intellectual disabilities, obesity, and retinal degeneration, and, in mouse models, the added feature of hydrocephalus. We observed increased Glial Fibrillary Acidic Protein (GFAP) immunoreactivity in BBS mouse brains. Increased GFAP expression is a hallmark of astrocyte reactivity that is associated with microglia activation and neuro-inflammation. To gain a better understanding of reactive astrocytes observed in BBS mice, we used two mouse models of BBS8, a BBSome protein, to characterize the reactive astrocyte phenotype. The finding of reactive astrocytes in young BBS mouse brains led us to hypothesize that loss of BBSome function leads to reactive astrocytes prior to hydrocephalus and obesity. By using two mouse models of BBS8, a congenital BBS8 knockout with hydrocephalus, and a tamoxifen-inducible BBS8 knockout without hydrocephalus, we were able to molecularly phenotype the reactive astrocytes. Molecular phenotype of reactive astrocytes shows differential regulation of inducers of Pan, A1 neurotoxic, and A2 neuroprotective astrocytes that are significantly altered in brains of both congenital and induced knockouts of BBS8, but without microglia activation. We find evidence for neuroinflammation in the brains of congenital knockout mice, but not in induced knockout mice. Protein levels of GFAP, SERPINA3N and post-synaptic density 95 (PSD95) are significantly increased in congenital knockout mice, but remain unchanged in induced knockout mice. Thus, despite the reactive astrocyte phenotype being present in both models, the molecular signature of reactive astrocytes in BBS8 mice models are distinct. Together, these findings suggest that BBS8, and by extension the BBSome, plays a role in neuro-astrocyte functions independent of hydrocephalus, and its dysregulation is associated with astrocyte reactivity without microglia activation. (Total word count 278).
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BBS4 is required for intraflagellar transport coordination and basal body number in mammalian olfactory cilia.
Journal of cell science, 2019Co-Authors: Cedric R. Uytingco, Val C Sheffield, Darryl Y Nishimura, Corey L. Williams, Chao Xie, Dana T. Shively, Warren W. Green, Kirill Ukhanov, Lian Zhang, Jeffrey R. MartensAbstract:ABSTRACT Bardet–Beidl syndrome (BBS) manifests from genetic mutations encoding for one or more BBS proteins. BBS4 loss impacts olfactory ciliation and odor detection, yet the cellular mechanisms remain unclear. Here, we report that Bbs4 -/− mice exhibit shorter and fewer olfactory sensory neuron (OSN) cilia despite retaining odorant receptor localization. Within Bbs4 −/− OSN cilia, we observed asynchronous rates of IFT-A/B particle movements, indicating miscoordination in IFT complex trafficking. Within the OSN dendritic knob, the basal bodies are dynamic, with incorporation of ectopically expressed centrin-2 and γ-tubulin occurring after nascent ciliogenesis. Importantly, BBS4 loss results in the reduction of basal body numbers separate from cilia loss. Adenoviral expression of BBS4 restored OSN cilia lengths and was sufficient to re-establish odor detection, but failed to rescue ciliary and basal body numbers. Our results yield a model for the plurality of BBS4 functions in OSNs that includes intraciliary and periciliary roles that can explain the loss of cilia and penetrance of ciliopathy phenotypes in olfactory neurons.
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Nuclear/cytoplasmic transport defects in BBS6 underlie congenital heart disease through perturbation of a chromatin remodeling protein
2017Co-Authors: Charles Anthony Scott, Qihong Zhang, Charles Searby, Val C Sheffield, Xitiz Chamling, Autumn N. Marsden, Michael R. Rebagliati, Lisa M. Baye, Diane C SlusarskiAbstract:Mutations in BBS6 cause two clinically distinct syndromes, Bardet-Biedl syndrome (BBS), a syndrome caused by defects in cilia transport and function, as well as McKusick-Kaufman syndrome, a genetic disorder characterized by congenital heart defects. Congenital heart defects are rare in BBS, and McKusick-Kaufman syndrome patients do not develop retinitis pigmentosa. Therefore, the McKusick-Kaufman syndrome allele may highlight cellular functions of BBS6 distinct from the presently understood functions in the cilia. In support, we find that the McKusick-Kaufman syndrome disease-associated allele, BBS6H84Y; A242S, maintains cilia function. We demonstrate that BBS6 is actively transported between the cytoplasm and nucleus, and that BBS6H84Y; A242S, is defective in this transport. We developed a transgenic zebrafish with inducible bbs6 to identify novel binding partners of BBS6, and we find interaction with the SWI/SNF chromatin remodeling protein Smarcc1a (SMARCC1 in humans). We demonstrate that through this interaction, BBS6 modulates the sub-cellular localization of SMARCC1 and find, by transcriptional profiling, similar transcriptional changes following smarcc1a and bbs6 manipulation. Our work identifies a new function for BBS6 in nuclear-cytoplasmic transport, and provides insight into the disease mechanism underlying the congenital heart defects in McKusick-Kaufman syndrome patients.
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BBS1 protein interacts with the LRb and regulates its trafficking to the cells surface.
2016Co-Authors: Deng Fu Guo, Darryl Nishimura, Qihong Zhang, Val C Sheffield, Donald A Morgan, Daniel R. Thedens, Huxing Cui, Justin L. Grobe, Kamal RahmouniAbstract:(A) Interaction between the BBS1 protein and the endogenous LRb in brain lysate. Ability of Flag-tagged BBS1 and LRb to pull down each other by co-immunoprecipitation assays using brain lysates of transgenic (Tg) mice expressing a Flag-BBS1 protein. IP: immunoprecipitation, IB: immunoblot (representative of n = 3, 1 male and 2 females). (B–C) shRNA-mediated silencing of BBS1 reduces the surface levels of Flag-tagged LRb in a dose-dependent manner in HEK 293 cells transiently co-transfected with Flag-LRb and Bbs1-shRNA plasmids. This effect is specific as the surface expression of HA-tagged LRa (in separate experiments) and endogenous transferrin receptor (TrR, in same experiments as the Flag-LRb) were not altered (n = 6 per group). (D-E) Silencing of BBS2, another BBSome protein, lowers the surface levels of Flag-LRb, but not HA-LRa or endogenous TrR in HEK 293 cells (n = 6 per group). (F–G) Blockade of Rab8a using a dominant negative (DN) form decreases the surface expression of Flag-LRb, HA-LRa and endogenous TrR in HEK 293 cells (n = 6 per group). (H–I) siRNA-mediated silencing of Ift88 had no effect on the surface expression of Flag-LRb or endogenous TrR in HEK 293 cells (n = 8 per group). Data are means ± SEM, *P< 0.05 vs. control group.
Charles Searby - One of the best experts on this subject based on the ideXlab platform.
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Ectopic expression of BBS1 rescues male infertility, but not retinal degeneration, in a BBS1 mouse model
Gene Therapy, 2021Co-Authors: Matthew R. Cring, Charles Searby, Kai Wang, Kacie J. Meyer, Adam Hedberg-buenz, Michael Cave, Michael G. Anderson, Val C SheffieldAbstract:Bardet–Biedl syndrome (BBS) is a rare ciliopathy for which there are no current effective treatments. BBS is a genetically heterogeneous disease, though the M390R mutation in BBS1 is involved in ~25% of all genetic diagnoses of BBS. The principle features of BBS include retinal degeneration, obesity, male infertility, polydactyly, intellectual disability, and renal abnormalities. Patients with mutations in BBS genes often present with night blindness within the first decade of life, which progresses to complete blindness. This is due to progressive loss of photoreceptor cells. Male infertility is caused by a lack of spermatozoa flagella, rendering them immobile. In this study, we have crossed the wild-type human BBS1 gene, driven by the CAG promoter, onto the Bbs1 ^ M390R/M390R mouse model to determine if ectopic expression of BBS1 rescues male infertility and retinal degeneration. qRT-PCR indicates that the BBS1 transgene is expressed in multiple tissues throughout the mouse, with the highest expression seen in the testes, and much lower expression in the eye and hypothalamus. Immunohistochemistry of the transgene in the eye showed little if any expression in the photoreceptor outer nuclear layer. When male Bbs1 ^ M30R/M390R ;BBS1 ^ TG+ mice are housed with WT females, they are able to sire offspring, indicating that the male infertility phenotype of BBS is rescued by the transgene. Using electroretinography (ERGs) to measure retinal function and optical coherence tomography to measure retinal thickness, we show that the transgene does not confer protection against retinal degeneration in Bbs1 ^ M300R/M390R ;BBS1 ^ TG+ mice. The results of this study indicate that the male infertility aspect of BBS is an attractive target for gene therapy.
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Nuclear/cytoplasmic transport defects in BBS6 underlie congenital heart disease through perturbation of a chromatin remodeling protein
2017Co-Authors: Charles Anthony Scott, Qihong Zhang, Charles Searby, Val C Sheffield, Xitiz Chamling, Autumn N. Marsden, Michael R. Rebagliati, Lisa M. Baye, Diane C SlusarskiAbstract:Mutations in BBS6 cause two clinically distinct syndromes, Bardet-Biedl syndrome (BBS), a syndrome caused by defects in cilia transport and function, as well as McKusick-Kaufman syndrome, a genetic disorder characterized by congenital heart defects. Congenital heart defects are rare in BBS, and McKusick-Kaufman syndrome patients do not develop retinitis pigmentosa. Therefore, the McKusick-Kaufman syndrome allele may highlight cellular functions of BBS6 distinct from the presently understood functions in the cilia. In support, we find that the McKusick-Kaufman syndrome disease-associated allele, BBS6H84Y; A242S, maintains cilia function. We demonstrate that BBS6 is actively transported between the cytoplasm and nucleus, and that BBS6H84Y; A242S, is defective in this transport. We developed a transgenic zebrafish with inducible bbs6 to identify novel binding partners of BBS6, and we find interaction with the SWI/SNF chromatin remodeling protein Smarcc1a (SMARCC1 in humans). We demonstrate that through this interaction, BBS6 modulates the sub-cellular localization of SMARCC1 and find, by transcriptional profiling, similar transcriptional changes following smarcc1a and bbs6 manipulation. Our work identifies a new function for BBS6 in nuclear-cytoplasmic transport, and provides insight into the disease mechanism underlying the congenital heart defects in McKusick-Kaufman syndrome patients.
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The centriolar satellite protein AZI1 interacts with BBS4 and regulates ciliary trafficking of the BBSome.
PLoS genetics, 2014Co-Authors: Xitiz Chamling, Charles Searby, Diane C Slusarski, Seongjin Seo, Gunhee Kim, Val C SheffieldAbstract:Bardet-Biedl syndrome (BBS) is a well-known ciliopathy with mutations reported in 18 different genes. Most of the protein products of the BBS genes localize at or near the primary cilium and the centrosome. Near the centrosome, BBS proteins interact with centriolar satellite proteins, and the BBSome (a complex of seven BBS proteins) is believed to play a role in transporting ciliary membrane proteins. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. Here, we show that a centriolar satellite protein, AZI1 (also known as CEP131), interacts with the BBSome and regulates BBSome ciliary trafficking activity. Furthermore, we show that AZI1 interacts with the BBSome through BBS4. AZI1 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon AZI1 depletion. Under conditions in which the BBSome does not normally enter cilia, such as in BBS3 or BBS5 depleted cells, knock down of AZI1 with siRNA restores BBSome trafficking to cilia. Finally, we show that azi1 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer's vesicle abnormalities and melanosome transport delay. These findings associate AZI1 with the BBS pathway. Our findings provide further insight into the regulation of BBSome ciliary trafficking and identify AZI1 as a novel BBS candidate gene.
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AZI1 interacts with the BBSome through BBS4.
2014Co-Authors: Xitiz Chamling, Charles Searby, Diane C Slusarski, Seongjin Seo, Gunhee Kim, Val C SheffieldAbstract:A) A silver stained gel from GFP IP sample of testis lysate. Lane 1 shows the wild-type (WT) precipitate and lane 2 is the precipitate of testis lysate from transgenic (TG) animals. The band labeled with an asterisk is the 120 KDa band identified as Azi1 by mass spectrometry. B) Western blot showing interaction of BBS4 with AZI1 and PCM1. FLAG and S tagged BBSome subunits were transfected in 293T cells and Co-IP was performed using FLAG agarose beads. The top blot shows efficient transfection of the BBS subunits detected by anti-FLAG antibody. The lower two blots were immunoblotted with AZI1 and PCM1 antibody, respectively. C) FLAG-BBS4 stable cell (293T) was co-immunoprecipitated with FLAG agarose beads. Input, supernatant and eluate were immunoblotted with, antibody against GAPDH to show that equal amounts were loaded, antibody against FLAG to show efficient precipitation of BBS4 by FLAG beads, and antibody against AZI1 to show amounts of AZI1 precipitated by BBS4 in our cell line. D) 293T cells stably expressing the FLAG tagged BBSome subunits BBS4, 5, 8, and 9 were co-IPed with FLAG agarose beads and immunoblotted with AZI1 antibody. Although all the subunits were able to precipitate AZI1, BBS4 showed the most efficient pull-down. E) Co-IP using AZI1 antibody was performed in 293T cells followed by Western blotting with different BBS antibody to show interaction of endogenous BBS proteins and AZI1.
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AZI1 knockdown reduces ciliogenesis but increases ciliary localization of the BBSome.
2014Co-Authors: Xitiz Chamling, Charles Searby, Diane C Slusarski, Seongjin Seo, Gunhee Kim, Val C SheffieldAbstract:A) RPE-1 cells were transfected with siRNA against BBS4, PCM1, and AZI1. Approximately 500 cells per sample were counted. B) AZI1 depletion increases the ciliary localization of BBS9 compared to the control knockdown. Red staining represents BBS9, and cilia are stained with acetylated α-tubulin. Nuclei are stained blue with DAPI. C) Graph showing a significant increase in cells with ciliary BBS9 upon AZI1 knockdown by different siRNAs. D) BBS8 (red) is used as a BBSome marker to confirm increased ciliary localization of the BBSome upon AZI1 knockdown. Cilia (green) in the insets of figures B and D are slightly shifted to show ciliary localization of BBS proteins. E) The graph shows a significant increase in the number of ciliated cells with BBS8 or BBS9 upon AZI1 knockdown, and decrease in ciliary BBS8 or BBS9 localization upon AZI1 overexpression. Approximately 250 ciliated cells were counted in control as well as AZI1 knockdown culture of RPE-1 cells. All data are presented in mean +/− SEM. Significance is calculated using Student's t-test for C and E, and one way ANOVA for A. P
Darryl Y Nishimura - One of the best experts on this subject based on the ideXlab platform.
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BBS4 is required for intraflagellar transport coordination and basal body number in mammalian olfactory cilia.
Journal of cell science, 2019Co-Authors: Cedric R. Uytingco, Val C Sheffield, Darryl Y Nishimura, Corey L. Williams, Chao Xie, Dana T. Shively, Warren W. Green, Kirill Ukhanov, Lian Zhang, Jeffrey R. MartensAbstract:ABSTRACT Bardet–Beidl syndrome (BBS) manifests from genetic mutations encoding for one or more BBS proteins. BBS4 loss impacts olfactory ciliation and odor detection, yet the cellular mechanisms remain unclear. Here, we report that Bbs4 -/− mice exhibit shorter and fewer olfactory sensory neuron (OSN) cilia despite retaining odorant receptor localization. Within Bbs4 −/− OSN cilia, we observed asynchronous rates of IFT-A/B particle movements, indicating miscoordination in IFT complex trafficking. Within the OSN dendritic knob, the basal bodies are dynamic, with incorporation of ectopically expressed centrin-2 and γ-tubulin occurring after nascent ciliogenesis. Importantly, BBS4 loss results in the reduction of basal body numbers separate from cilia loss. Adenoviral expression of BBS4 restored OSN cilia lengths and was sufficient to re-establish odor detection, but failed to rescue ciliary and basal body numbers. Our results yield a model for the plurality of BBS4 functions in OSNs that includes intraciliary and periciliary roles that can explain the loss of cilia and penetrance of ciliopathy phenotypes in olfactory neurons.
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bardet biedl syndrome 3 bbs3 knockout mouse model reveals common bbs associated phenotypes and bbs3 unique phenotypes
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Qihong Zhang, Kevin Bugge, Charles Searby, Timothy W Vogel, Edwin M. Stone, Seongjin Seo, Donald A Morgan, Darryl Y Nishimura, Kamal Rahmouni, Val C SheffieldAbstract:Bardet-Biedl syndrome (BBS) is a heterogeneous disorder characterized by obesity, retinopathy, polydactyly, and congenital anomalies. The incidence of hypertension and diabetes are also increased in BBS patients. Mutation of 16 genes independently causes BBS, and seven BBS proteins form the BBSome that promotes ciliary membrane elongation. BBS3 (ARL6), an ADP ribosylation factor-like small GTPase, is not part of the BBSome complex. The in vivo function of BBS3 is largely unknown. Here we developed a Bbs3 knockout model and demonstrate that Bbs3−/− mice develop BBS-associated phenotypes, including retinal degeneration, male infertility, and increased body fat. Interestingly, Bbs3−/− mice develop some unique phenotypes not seen in other BBS knockout models: no overt obesity, severe hydrocephalus, and elevated blood pressure (shared by some but not all BBS gene knockout mice). We found that endogenous BBS3 and the BBSome physically interact and depend on each other for their ciliary localization. This finding explains the phenotypic similarity between Bbs3−/− mice and BBSome subunit knockout mice. Loss of Bbs3 does not affect BBSome formation but disrupts normal localization of melanin concentrating hormone receptor 1 to ciliary membranes and affects retrograde transport of Smoothened inside cilia. We also show that the endogenous BBSome and BBS3 associate with membranes and the membrane association of the BBSome and BBS3 are not interdependent. Differences between BBS mouse models suggest nonoverlapping functions to individual BBS protein.
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bardet biedl syndrome in denmark report of 13 novel sequence variations in six genes
Human Mutation, 2010Co-Authors: Tina Duelund Hjortshoj, Val C Sheffield, Darryl Y Nishimura, Karen Gronskov, Alisdair R Philp, Ruth Riise, Thomas Rosenberg, Karen BrondumnielsenAbstract:Bardet-Biedl syndrome (BBS) is an autosomal recessive disease characterized by retinal dystrophy, polydactyly, obesity, learning disabilities, renal involvement, and male hypogenitalism. BBS is genetically heterogeneous with mutations of 14 genes, accounting for approximately 70% of cases. Triallelic inheritance has been suggested in about 5% of cases. Forty-nine unrelated BBS patients were screened for mutations by DHPLC analysis in BBS1, BBS2, BBS4, BBS6/MKKS, BBS10, and BBS12. The selected genes either account for more than 5% of the mutational load or are commonly reported in triallelic inheritance. Eight patients with only one or no BBS mutation were further investigated by single nucleotide polymorphism (SNP) analysis. In total, mutations were detected in 44 patients. Twenty percent had two mutations in BBS1, 18% in BBS2, 4% in BBS9, 43% in BBS10, and 2% in BBS12. Five patients were heterozygous for a sequence variation in BBS6/MKKS. We found eight patients with three sequence variations in two genes, which could be explained by triallelic inheritance, by the prevalence of heterozygous carriers or the third sequence variations representing rare polymorphisms. All changes found in a second BBS gene were amino acid substitutions. Genotype–phenotype correlations suggest a milder phenotype for BBS1 compared to BBS2 and BBS10, which we ascribe to the hypomorphic p.Met390Arg-mutation. Hum Mutat 31:429–436, 2010. © 2010 Wiley-Liss, Inc.
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Leptin resistance contributes to obesity and hypertension in mouse models of Bardet-Biedl syndrome.
The Journal of clinical investigation, 2008Co-Authors: Kamal Rahmouni, Darryl Y Nishimura, Melissa A. Fath, Daniel R. Thedens, Christopher J. Berry, Robert M. Weiss, Val C SheffieldAbstract:Bardet-Biedl syndrome (BBS) is a heterogeneous genetic disorder characterized by many features, including obesity and cardiovascular disease. We previously developed knockout mouse models of 3 BBS genes: BBS2, BBS4, and BBS6. To dissect the mechanisms involved in the metabolic disorders associated with BBS, we assessed the development of obesity in these mouse models and found that BBS-null mice were hyperphagic, had low locomotor activity, and had elevated circulating levels of the hormone leptin. The effect of exogenous leptin on body weight and food intake was attenuated in BBS mice, which suggests that leptin resistance may contribute to hyperleptinemia. In other mouse models of obesity, leptin resistance may be selective rather than systemic; although mice became resistant to leptin's anorectic effects, the ability to increase renal sympathetic nerve activity (SNA) was preserved. Although all 3 of the BBS mouse models were similarly resistant to leptin, the sensitivity of renal SNA to leptin was maintained in Bbs4 -/- and Bbs6 -/- mice, but not in Bbs2 -/- mice. Consequently, Bbs4 -/- and Bbs6 -/- mice had higher baseline renal SNA and arterial pressure and a greater reduction in arterial pressure in response to ganglionic blockade. Furthermore, we found that BBS mice had a decreased hypothalamic expression of proopiomelanocortin, which suggests that BBS genes play an important role in maintaining leptin sensitivity in proopiomelanocortin neurons.
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loss of bardet biedl syndrome proteins alters the morphology and function of motile cilia in airway epithelia
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Alok S. Shah, Kevin Bugge, Charles Searby, Darryl Y Nishimura, Sara L. Farmen, Thomas O. Moninger, Thomas R. Businga, Michael P. Andrews, Kim A. Brogden, Joel N. KlineAbstract:Mutations in a group of genes that contribute to ciliary function cause Bardet–Biedl syndrome (BBS). Most studies of BBS have focused on primary, sensory cilia. Here, we asked whether loss of BBS proteins would also affect motile cilia lining the respiratory tract. We found that BBS genes were expressed in human airway epithelia, and BBS2 and BBS4 localized to cellular structures associated with motile cilia. Although BBS proteins were not required for ciliogenesis, their loss caused structural defects in a fraction of cilia covering mouse airway epithelia. The most common abnormality was bulges filled with vesicles near the tips of cilia. We discovered this same misshapen appearance in airway cilia from Bbs1, Bbs2, Bbs4, and Bbs6 mutant mice. The structural abnormalities were accompanied by functional defects; ciliary beat frequency was reduced in Bbs mutant mice. Previous reports suggested BBS might increase the incidence of asthma. However, compared with wild-type controls, neither airway hyperresponsiveness nor inflammation increased in Bbs2−/− or Bbs4−/− mice immunized with ovalbumin. Instead, these animals were partially protected from airway hyperresponsiveness. These results emphasize the role of BBS proteins in both the structure and function of motile cilia. They also invite additional scrutiny of motile cilia dysfunction in patients with this disease.
Philip L. Beales - One of the best experts on this subject based on the ideXlab platform.
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Bardet Biedl syndrome proteins 1 and 3 regulate the ciliary trafficking of polycystic kidney disease 1 protein
Human molecular genetics, 2014Co-Authors: Kaitlin Driscoll, Philip L. Beales, Gang Yao, Anas Raed, Jing ZhouAbstract:Bardet-Biedl syndrome (BBS) and autosomal dominant polycystic kidney disease (ADPKD) are two genetically distinct ciliopathies but share common phenotypes such as renal cysts. Seven BBS proteins form a complex called the BBSome which is localized at the basal body or ciliary axoneme and regulates the ciliary entry or flagellar exit of several signaling molecules. Here, we demonstrate that, unlike the seven-span somatostatin receptor 3 or the leptin receptor that interacts with all subunits of the BBSome, the ADPKD protein polycystin-1 (PC1) interacts with BBS1, BBS4, BBS5 and BBS8, four of the seven components of the BBSome. Only depletion or mutation of BBS1, but not depletion of BBS5 and BBS8, or knockout of BBS4, impairs ciliary trafficking of PC1 in kidney epithelial cells. Depletion of these BBS proteins affects neither the ciliary length nor the plasma membrane targeting of PC1. Expression of a pathogenic BBS3/Arl6 mutant (T31R) that locks Arl6 in the GDP form leads to stunted cilia and inhibition of PC1 on primary cilia. We propose that the 11-span membrane protein PC1 is a BBSome cargo and that the components of the BBSome may possess subunit-specific functions. Moreover, physical interactions between the BBS and ADPKD proteins may underline the overlapping renal phenotypes in these two diseases.
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bardet biedl syndrome proteins control the cilia length through regulation of actin polymerization
Human Molecular Genetics, 2013Co-Authors: Victor Hernandezhernandez, Helen Maysimera, Priyanka Pravincumar, Anna Diazfont, Dagan Jenkins, Martin M Knight, Philip L. BealesAbstract:Primary cilia are cellular appendages important for signal transduction and sensing the environment. Bardet-Biedl syndrome proteins form a complex that is important for several cytoskeleton-related processes such as ciliogenesis, cell migration and division. However, the mechanisms by which BBS proteins may regulate the cytoskeleton remain unclear. We discovered that Bbs4 and Bbs6 deficient renal medullary cells display a characteristic behaviour comprising poor migration, adhesion and division with an inability to form lamellipodial and filopodial extensions. Moreover, fewer mutant cells were ciliated (48% ± 6 for wild-type cells vs 23% ± 7 for Bbs4 null cells; P-value < 0.0001) and their cilia were shorter (2.55eμm ± 0.41 for wild-type cells vs 2.16eμm ± 0.23 for Bbs4 null cells; P-value < 0.0001). Whilst the microtubular cytoskeleton and cortical actin were intact, actin stress fibre formation was severely disrupted, forming abnormal apical stress fibre aggregates. Furthermore, we observed over-abundant focal adhesions in Bbs4, Bbs6 and Bbs8-deficient cells. In view of these findings and the role of RhoA in regulation of actin filament polymerisation, we showed that RhoA-GTP levels were highly upregulated in the absence of Bbs proteins. Upon treatment of Bbs4-deficient cells with chemical inhibitors of RhoA, we were able to restore cilia length and number as well as the integrity of the actin cytoskeleton. Together these findings indicate that Bbs proteins play a central role in the regulation of the actin cytoskeleton and control cilia length through alteration of RhoA levels.
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mkks bbs6 a divergent chaperonin like protein linked to the obesity disorder bardet biedl syndrome is a novel centrosomal component required for cytokinesis
Journal of Cell Science, 2005Co-Authors: Jun Chul Kim, Jose L. Badano, Carmen C Leitch, Nicholas Katsanis, Philip L. Beales, Muneer A Esmail, Elsa Fiedrich, John M Archibald, Jerome B Rattner, Michel R. LerouxAbstract:Chaperonins are multisubunit, cylinder-shaped molecular chaperones involved in folding newly synthesized polypeptides. Here we show that MKKS/BBS6, one of several proteins associated with Bardet-Biedl syndrome (BBS), is a Group II chaperonin-like protein that has evolved recently in animals from a subunit of the eukaryotic chaperonin CCT/TRiC, and diverged rapidly to acquire distinct functions. Unlike other chaperonins, cytosolic BBS6 does not oligomerize, and the majority of BBS6 resides within the pericentriolar material (PCM), a proteinaceous tube surrounding centrioles. During interphase, BBS6 is confined to the lateral surfaces of the PCM but during mitosis it relocalizes throughout the PCM and is found at the intercellular bridge. Its predicted substrate-binding apical domain is sufficient for centrosomal association, and several patient-derived mutations in this domain cause mislocalization of BBS6. Consistent with an important centrosomal function, silencing of the BBS6 transcript by RNA interference in different cell types leads to multinucleate and multicentrosomal cells with cytokinesis defects. The restricted tissue distribution of BBS6 further suggests that it may play important roles in ciliated epithelial tissues, which is consistent with the probable functions of BBS proteins in basal bodies (modified centrioles) and cilia. Our findings provide the first insight into the nature and cellular function of BBS6, and shed light on the potential causes of several ailments, including obesity, retinal degeneration, kidney dysfunction and congenital heart disease.
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heterozygous mutations in bbs1 bbs2 and bbs6 have a potential epistatic effect on bardet biedl patients with two mutations at a second bbs locus
Human Molecular Genetics, 2003Co-Authors: Jose L. Badano, Bethan E. Hoskins, Stephen J. Ansley, Philip L. Beales, Richard A. Lewis, Michel R. Leroux, Jun Chul Kim, David J Cutler, Claudio Castellan, Nicholas KatsanisAbstract:Bardet-Biedl syndrome (BBS) is a pleiotropic genetic disorder with substantial inter- and intrafamilial variability, that also exhibits remarkable genetic heterogeneity, with seven mapped BBS loci in the human genome. Recent data have demonstrated that BBS may be inherited either as a simple Mendelian recessive or as an oligogenic trait, since mutations at two loci are sometimes required for pathogenesis. This observation suggests that genetic interactions between the different BBS loci may modulate the phenotype, thus contributing to the clinical variability of BBS. We present three families with two mutations in either BBS1 or BBS2, in which some but not all patients carry a third mutation in BBS1, BBS2 or the putative chaperonin BBS6. In each example, the presence of three mutant alleles correlates with a more severe phenotype. For one of the missense alleles, we also demonstrate that the introduction of the mutation in mammalian cells causes a dramatic mislocalization of the protein compared with the wild-type. These data suggest that triallelic mutations are not always necessary for disease manifestation, but might potentiate a phenotype that is caused by two recessive mutations at an independent locus, thus introducing an additional layer of complexity on the genetic modeling of oligogenicity.
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Genetic Interaction of BBS1 Mutations with Alleles at Other BBS Loci Can Result in Non-Mendelian Bardet-Biedl Syndrome
American journal of human genetics, 2003Co-Authors: Philip L. Beales, Bethan E. Hoskins, Stephen J. Ansley, Jose L. Badano, Alison Ross, Peter J. Scambler, Brigitta Kirsten, Charles A. Mein, Philippe Froguel, Richard A. LewisAbstract:Bardet-Biedl syndrome is a genetically and clinically heterogeneous disorder caused by mutations in at least seven loci (BBS1-7), five of which are cloned (BBS1, BBS2, BBS4, BBS6, and BBS7). Genetic and mutational analyses have indicated that, in some families, a combination of three mutant alleles at two loci (triallelic inheritance) is necessary for pathogenesis. To date, four of the five known BBS loci have been implicated in this mode of oligogenic disease transmission. We present a comprehensive analysis of the spectrum, distribution, and involvement in non-Mendelian trait transmission of mutant alleles in BBS1, the most common BBS locus. Analyses of 259 independent families segregating a BBS phenotype indicate that BBS1 participates in complex inheritance and that, in different families, mutations in BBS1 can interact genetically with mutations at each of the other known BBS genes, as well as at unknown loci, to cause the phenotype. Consistent with this model, we identified homozygous M390R alleles, the most frequent BBS1 mutation, in asymptomatic individuals in two families. Moreover, our statistical analyses indicate that the prevalence of the M390R allele in the general population is consistent with an oligogenic rather than a recessive model of disease transmission. The distribution of BBS oligogenic alleles also indicates that all BBS loci might interact genetically with each other, but some genes, especially BBS2 and BBS6, are more likely to participate in triallelic inheritance, suggesting a variable ability of the BBS proteins to interact genetically with each other.
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bbs7 is required for bbsome formation and its absence in mice results in bardet biedl syndrome phenotypes and selective abnormalities in membrane protein trafficking
Journal of Cell Science, 2013Co-Authors: Qihong Zhang, Jianqiang Shao, Calvin S Carter, Kevin Bugge, Ruth E. Swiderski, Darryl Nishimura, Charles Searby, Timothy W Vogel, Edwin M. Stone, Val C SheffieldAbstract:Bardet-Biedl Syndrome (BBS) is a pleiotropic and genetically heterozygous disorder caused independently by numerous genes (BBS1–BBS17). Seven highly conserved BBS proteins (BBS1, 2, 4, 5, 7, 8 and 9) form a complex known as the BBSome, which functions in ciliary membrane biogenesis. BBS7 is both a unique subunit of the BBSome and displays direct physical interaction with a second BBS complex, the BBS chaperonin complex. To examine the in vivo function of BBS7, we generated Bbs7 knockout mice. Bbs7−/− mice show similar phenotypes to other BBS gene mutant mice including retinal degeneration, obesity, ventriculomegaly and male infertility characterized by abnormal spermatozoa flagellar axonemes. Using tissues from Bbs7−/− mice, we show that BBS7 is required for BBSome formation, and that BBS7 and BBS2 depend on each other for protein stability. Although the BBSome serves as a coat complex for ciliary membrane proteins, BBS7 is not required for the localization of ciliary membrane proteins polycystin-1, polycystin-2, or bitter taste receptors, but absence of BBS7 leads to abnormal accumulation of the dopamine D1 receptor to the ciliary membrane, indicating that BBS7 is involved in specific membrane protein localization to cilia.
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Ectopic Expression of Human BBS4 Can Rescue Bardet-Biedl Syndrome Phenotypes in Bbs4 Null Mice
PloS one, 2013Co-Authors: Xitiz Chamling, Kevin Bugge, Charles Searby, Kamal Rahmouni, Deng F. Guo, Arlene V. Drack, Val C SheffieldAbstract:Bardet-Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder characterized by obesity, retinal degeneration, polydactyly, hypogenitalism and renal defects. Recent findings have associated the etiology of the disease with cilia, and BBS proteins have been implicated in trafficking various ciliary cargo proteins. To date, 17 different genes have been reported for BBS among which BBS1 is the most common cause of the disease followed by BBS10, and BBS4. A murine model of Bbs4 is known to phenocopy most of the human BBS phenotypes, and it is being used as a BBS disease model. To better understand the in vivo localization, cellular function, and interaction of BBS4 with other proteins, we generated a transgenic BBS4 mouse expressing the human BBS4 gene under control of the beta actin promoter. The transgene is expressed in various tissues including brain, eye, testis, heart, kidney, and adipose tissue. These mice were further bred to express the transgene in Bbs4 null mice, and their phenotype was characterized. Here we report that despite tissue specific variable expression of the transgene, human BBS4 was able to complement the deficiency of Bbs4 and rescue all the BBS phenotypes in the Bbs4 null mice. These results provide an encouraging prospective for gene therapy for BBS related phenotypes and potentially for other ciliopathies.
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bardet biedl syndrome 3 bbs3 knockout mouse model reveals common bbs associated phenotypes and bbs3 unique phenotypes
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Qihong Zhang, Kevin Bugge, Charles Searby, Timothy W Vogel, Edwin M. Stone, Seongjin Seo, Donald A Morgan, Darryl Y Nishimura, Kamal Rahmouni, Val C SheffieldAbstract:Bardet-Biedl syndrome (BBS) is a heterogeneous disorder characterized by obesity, retinopathy, polydactyly, and congenital anomalies. The incidence of hypertension and diabetes are also increased in BBS patients. Mutation of 16 genes independently causes BBS, and seven BBS proteins form the BBSome that promotes ciliary membrane elongation. BBS3 (ARL6), an ADP ribosylation factor-like small GTPase, is not part of the BBSome complex. The in vivo function of BBS3 is largely unknown. Here we developed a Bbs3 knockout model and demonstrate that Bbs3−/− mice develop BBS-associated phenotypes, including retinal degeneration, male infertility, and increased body fat. Interestingly, Bbs3−/− mice develop some unique phenotypes not seen in other BBS knockout models: no overt obesity, severe hydrocephalus, and elevated blood pressure (shared by some but not all BBS gene knockout mice). We found that endogenous BBS3 and the BBSome physically interact and depend on each other for their ciliary localization. This finding explains the phenotypic similarity between Bbs3−/− mice and BBSome subunit knockout mice. Loss of Bbs3 does not affect BBSome formation but disrupts normal localization of melanin concentrating hormone receptor 1 to ciliary membranes and affects retrograde transport of Smoothened inside cilia. We also show that the endogenous BBSome and BBS3 associate with membranes and the membrane association of the BBSome and BBS3 are not interdependent. Differences between BBS mouse models suggest nonoverlapping functions to individual BBS protein.
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A Novel Protein LZTFL1 Regulates Ciliary Trafficking of the BBSome and Smoothened
PLoS genetics, 2011Co-Authors: Seongjin Seo, Kevin Bugge, Qihong Zhang, Charles Searby, Maxence V Nachury, David K Breslow, Val C SheffieldAbstract:Many signaling proteins including G protein-coupled receptors localize to primary cilia, regulating cellular processes including differentiation, proliferation, organogenesis, and tumorigenesis. Bardet-Biedl Syndrome (BBS) proteins are involved in maintaining ciliary function by mediating protein trafficking to the cilia. However, the mechanisms governing ciliary trafficking by BBS proteins are not well understood. Here, we show that a novel protein, Leucine-zipper transcription factor-like 1 (LZTFL1), interacts with a BBS protein complex known as the BBSome and regulates ciliary trafficking of this complex. We also show that all BBSome subunits and BBS3 (also known as ARL6) are required for BBSome ciliary entry and that reduction of LZTFL1 restores BBSome trafficking to cilia in BBS3 and BBS5 depleted cells. Finally, we found that BBS proteins and LZTFL1 regulate ciliary trafficking of hedgehog signal transducer, Smoothened. Our findings suggest that LZTFL1 is an important regulator of BBSome ciliary trafficking and hedgehog signaling.
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Requirement of Bardet-Biedl syndrome proteins for leptin receptor signaling
Human molecular genetics, 2009Co-Authors: Deng Fu Guo, Kevin Bugge, Donald A Morgan, Kamal Rahmouni, Val C SheffieldAbstract:Obesity is a major public health problem in most developed countries and a major risk factor for diabetes and cardiovascular disease. Emerging evidence indicates that ciliary dysfunction can contribute to human obesity but the underlying molecular and cellular mechanisms are unknown. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous human obesity syndrome associated with ciliary dysfunction. BBS proteins are thought to play a role in cilia function and intracellular protein/vesicle trafficking. Here, we show that BBS proteins are required for leptin receptor (LepR) signaling in the hypothalamus. We found that Bbs2 2/2 , Bbs4 2/2 and Bbs6 2/2 mice are resistant to the action of leptin to reduce body weight and food intake regardless of serum leptin levels and obesity. In addition, activation of hypothalamic STAT3 by leptin is significantly decreased in Bbs2 2/2 , Bbs4 2/2 and Bbs6 2/2 mice. In contrast, downstream melanocortin receptor signaling is unaffected, indicating that LepR signaling is specifically impaired in Bbs2 2/2 , Bbs4 2/2 and Bbs6 2/2 mice. Impaired LepR signaling in BBS mice was associated with decreased Pomc gene expression. Furthermore, we found that BBS1 protein physically interacts with the LepR and that loss of BBS proteins perturbs LepR trafficking. Our data indicate that BBS proteins mediate LepR trafficking and that impaired LepR signaling underlies energy imbalance in BBS. These findings represent a novel mechanism for leptin resistance and obesity.