Translational readthrough of ciliopathy genes BBS2 and ALMS1 restores protein, ciliogenesis and function in patient fibroblasts

Background: Ciliary dysfunction underlies a range of genetic disorders collectively termed ciliopathies, for which there are no treatments available. Bardet-Biedl syndrome (BBS) is characterised by multisystemic involvement, including rod-cone dystrophy and renal abnormalities. Together with Alström syndrome (AS), they are known as the ‘obesity ciliopathies’ due to their common phenotype. Nonsense mutations are responsible for approximately 11% and 40% of BBS and AS cases, respectively. Translational readthrough inducing drugs (TRIDs) can restore full-length protein bypassing in-frame premature termination codons, and are a potential therapeutic approach for nonsense-mediated ciliopathies. Methods: Patient fibroblasts harbouring nonsense mutations from two different ciliopathies (Bardet-Biedl Syndrome and Alström Syndrome) were treated with PTC124 (ataluren) or amlexanox. Following treatment, gene expression, protein levels and ciliogenesis were evaluated. The expression of intraflagellar transport protein IFT88 and G-protein coupled receptor SSTR3 was investigated as a readout of ciliary function. Findings: mRNA expression was significantly increased in amlexanox-treated patient fibroblasts, and full-length BBS2 or ALMS1 protein expression was restored in PTC124- and amlexanox-treated fibroblasts. Treatment with TRIDs significantly improved ciliogenesis defects in BBS2Y24*/R275* fibroblasts. Treatment recovered IFT88 expression and corrected SSTR3 mislocalisation in BBS2Y24*/R275* and ALMS1S1645*/S1645* fibroblasts, suggesting rescue of ciliary function. Interpretation: The recovery of full-length BBS2 and ALMS1 expression and correction of anatomical and functional ciliary defects in BBS2Y24*/R275* and ALMS1S1645*/S1645* fibroblasts suggest TRIDs are a potential therapeutic option for the treatment of nonsense-mediated ciliopathies

Improving planning, design, reporting and scientific quality of animal experiments by using the Gold Standard Publication Checklist, in addition to the ARRIVE guidelines

Several studies have demonstrated serious omissions in the way research that use animals is reported. In order to improve the quality of reporting of animal experiments, the Animals in research: reporting in vivo experiments (ARRIVE) Guidelines were published in the British Journal of Pharmacology in August 2010

Neofunctionalization of ciliary BBS proteins to nuclear roles is likely a frequent innovation across eukaryotes

The eukaryotic BBSome is a transport complex within cilia and assembled by chaperonin-like BBS proteins. Recent work indicates nuclear functions for BBS proteins in mammals, but it is unclear how common these are in extant proteins or when they evolved. We screened for BBS orthologues across a diverse set of eukaryotes, consolidated nuclear association via signal sequence predictions and permutation analysis, and validated nuclear localization in mammalian cells via fractionation and immunocytochemistry. BBS proteins are—with exceptions— conserved as a set in ciliated species. Predictions highlight five most likely nuclear proteins and suggest that nuclear roles evolved independently of nuclear access during mitosis. Nuclear localization was confirmed in human cells. These findings suggest that nuclear BBS functions are potentially not restricted tomammals, but may be a common frequently co-opted eukaryotic feature. Understanding the functional spectrum of BBS proteins will help elucidating their role in gene regulation, development, and disease

Mutational and functional analyses of Bardet-Biedl syndrome

This thesis investigates some of the underlying causes of Bardet-Biedl syndrome, a leading example of emerging ciliopathies. Bardet-Biedl syndrome (BBS) is a heterogeneous disorder with primary features that include age-related retinal dystrophy, obesity, Polydactyly, renal dysplasia, reproductive tract anomalies and cognitive impairment. To date 12 loci have been found to be causative for the disease (BBS1-12) and evidence suggests that aspects of the BBS phenotype may result from defective ciliogenesis/basal body function. The aims of this project were to identify new genes involved with the disease, analyse the phenotype of the Bbs6"7" mouse and further elucidate the mechanism of the disease in a zebrafish model. Pathogenic sequence mutations identified in candidate genes aided the identifictaion of BBS3 and BBS5. Further candidate genes were identified through the mapping of translocation breakpoints in a BBS patient. A yeast two-hybrid screen, using Bbs6 as bait, revealed several potential protein interactions. Phenotypical analysis of the Bbs&A mouse showed features comparable to BBS patients and other Bbs null mice, confirming its value as a model for further study. Further investigations of BBS proteins in the cochlea suggested a role beyond that of cilia and basal body function, namely that BBS proteins play distinct roles in the processes of microtubule nucleation and growth. Observed disruption of cochlear stereociliary bundles, along with other phenotypes associated with planar cell polarity (PCP) mutants, implied cilia might be involved in PCP signalling. This observation was supported by further analysis in the zebrafish. Disruption of bbs8 resulted in similar phenotypes to other PCP mutants, and laterality defects, thought to arise from defective cilia function at the Kupffer's Vesicle, were enhanced on a PCP mutant background. The results presented in this thesis pave the way for further investigation with a view to broadening the knowledge of the developmental processes behind BBS and also the processes behind the development of other signaling pathways, tissues and organ systems

VEGF is indirectly associated with NO production

Background? Increased levels of vascular endothelial growth factor (VEGF) have been observed in patients with metabolic syndrome (MetS). Nitric oxide (NO) formation is reduced in MetS, but its relationship to VEGF production remains poorly defined. We evaluated the association between VEGF/NO synthesis and insulin sensitivity in obese subjects and investigated the secretory response of VEGF to an acute elevation of glucose.Materials and methods? Seven healthy normal-weight subjects, seven obese subjects without MetS and seven obese subjects with MetS were recruited. Anthropometry, body composition and cardiometabolic functions (blood pressure, glucose, insulin, triglycerides, total cholesterol, HDL-C and VEGF) were measured, and a novel stable isotope method was used to assess in vivo rates of NO production. A frequent sampling intravenous glucose tolerance test was performed to study the dynamics of VEGF release.Results? Fasting VEGF levels were significantly higher in the two obese groups compared to the control group (P for trend = 0·02), but the difference was not significant after adjustment for age. Vascular endothelial growth factor levels were associated with systolic blood pressure (? = 0·54; P = 0·01) and NO production (? = ?0·44; P = 0·04). Vascular endothelial growth factor levels increased in response to acute hyperglycaemia in normal-weight and obese subjects (P < 0·001).Conclusions? Vascular endothelial growth factor levels rapidly increase during hyperglycaemia and are inversely related to NO production at steady state. The potential link between the acute secretion of VEGF and atherosclerotic risk in subjects with poorly controlled glycaemia as well as the potential of lowering elevated VEGF levels by increasing NO production and/or availability warrants further investigation

The actin-bundling protein Fascin-1 modulates ciliary signalling

Primary cilia are microtubule-based cell organelles important for cellular communication. Since they are involved in the regulation of numerous signalling pathways, defects in cilia development or function are associated with genetic disorders, collectively called ciliopathies. Besides their ciliary functions, recent research has shown that several ciliary proteins are involved in the coordination of the actin cytoskeleton. Although ciliary and actin phenotypes are related, the exact nature of their interconnection remains incompletely understood. Here, we show that the protein BBS6, associated with the ciliopathy Bardet–Biedl syndrome, cooperates with the actin-bundling protein Fascin-1 in regulating filopodia and ciliary signalling. We found that loss of Bbs6 affects filopodia length potentially via attenuated interaction with Fascin-1. Conversely, loss of Fascin-1 leads to a ciliary phenotype, subsequently affecting ciliary Wnt signalling, possibly in collaboration with BBS6. Our data shed light on how ciliary proteins are involved in actin regulations and provide new insight into the involvement of the actin regulator Fascin-1 in ciliogenesis and cilia-associated signalling. Advancing our knowledge of the complex regulations between primary cilia and actin dynamics is important to understand the pathogenic consequences of ciliopathies

Bardet-Biedl syndrome proteins control cilia length through regulation of actin polymerisation.

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 (2.55&emsp14;μm ± 0.41 for wild-type cells vs 2.16&emsp14;μm ± 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

A proposed framework for developing quality assessment tools

Background Assessment of the quality of included studies is an essential component of any systematic review. A formal quality assessment is facilitated by using a structured tool. There are currently no guidelines available for researchers wanting to develop a new quality assessment tool. Methods This paper provides a framework for developing quality assessment tools based on our experiences of developing a variety of quality assessment tools for studies of differing designs over the last 14 years. We have also drawn on experience from the work of the EQUATOR Network in producing guidance for developing reporting guidelines. Results We do not recommend a single ‘best’ approach. Instead, we provide a general framework with suggestions as to how the different stages can be approached. Our proposed framework is based around three key stages: initial steps, tool development and dissemination. Conclusions We recommend that anyone who would like to develop a new quality assessment tool follow the stages outlined in this paper. We hope that our proposed framework will increase the number of tools developed using robust methods.</p