Assaying sensory ciliopathies using calcium biosensor expression in zebrafish ciliated olfactory neurons

Background: Primary cilia mediate signal transduction by acting as an organizing scaffold for receptors, signalling proteins and ion channels. Ciliated olfactory sensory neurons (OSNs) organize olfactory receptors and ion channels on cilia and generate a calcium influx as a primary signal in odourant detection. In the zebrafish olfactory placode, ciliated OSNs and microvillus OSNs constitute the major OSN cell types with distinct odourant sensitivity. Methods: Using transgenic expression of the calcium biosensor GCaMP5 in OSNs, we analysed sensory cilia-dependent odour responses in live zebrafish, at individual cell resolution. oval/ift88 mutant and ift172 knockdown zebrafish were compared with wild-type siblings to establish ciliated OSN sensitivity to different classes of odourants. Results: oval/ift88 mutant and ift172 knockdown zebrafish showed fewer and severely shortened OSN cilia without a reduction in OSN number. The fraction of responding OSNs and response amplitudes to bile acids and food odour, both sensed by ciliated OSNs, were significantly reduced in ift88 mutants and ift172-deficient embryos, while the amino acids responses were not significantly changed. Conclusions: Our approach presents a quantitative model for studying sensory cilia signalling using zebrafish OSNs. Our results also implicate ift172-deficiency as a novel cause of hyposmia, a reduced sense of smell, highlighting the value of directly assaying sensory cilia signalling in vivo and supporting the idea that hyposmia can be used as a diagnostic indicator of ciliopathies. Electronic supplementary material The online version of this article (10.1186/s13630-018-0056-1) contains supplementary material, which is available to authorized users

A Replication Study of the Association between Rheumatoid Arthritis and Deletion of the Late Cornified Envelope Genes LCE3B and LCE3C

OBJECTIVE: Two recent studies, in a Spanish and a Chinese population, point to an association between rheumatoid arthritis (RA) risk and the deletion of the Late Cornified Envelope (LCE) 3B and 3C genes (LCE3C_LCE3B-del), a known risk factor for psoriasis. We aimed to replicate these studies in a large Dutch cohort. METHODS: 1039 RA cases and 759 controls were genotyped for LCE3C_LCE3B-del. Association analysis was performed for the complete cohort and after stratification for the serologic markers anti-cyclic citrullinated peptide and rheumatoid factor. A meta-analysis was performed combining our data with the Spanish and Chinese datasets, resulting in an analysis including 2466 RA cases and 2438 controls. RESULTS: In the Dutch cohort we did not observe a significant association of LCE3C_LCE3B-del (p = 0.093) with RA risk. A stratified analysis for the serologic positive and negative group did not show an association between the genetic variant and disease risk, either. The meta-analysis, however, confirmed a significant association (p<0.0001, OR = 1.31, 95% confidence interval 1.16-1.47). CONCLUSION: Our meta-analysis confirms the association of the LCE3 deletion with RA, suggesting that LCE3C_LCE3B-del is a common risk factor for (auto)immune diseases

b-Defensin-2 Protein Is a Serum Biomarker for Disease Activity in Psoriasis and Reaches Biologically Relevant Concentrations in Lesional Skin

Abstract Background: Previous studies have extensively documented antimicrobial and chemotactic activities of beta-defensins. Human beta-defensin-2 (hBD-2) is strongly expressed in lesional psoriatic epidermis, and recently we have shown that high beta-defensin genomic copy number is associated with psoriasis susceptibility. It is not known, however, if biologically and pathophysiologically relevant concentrations of hBD-2 protein are present in vivo, which could support an antimicrobial and proinflammatory role of beta-defensins in lesional psoriatic epidermis

β-Defensin-2 Protein Is a Serum Biomarker for Disease Activity in Psoriasis and Reaches Biologically Relevant Concentrations in Lesional Skin

BACKGROUND: Previous studies have extensively documented antimicrobial and chemotactic activities of beta-defensins. Human beta-defensin-2 (hBD-2) is strongly expressed in lesional psoriatic epidermis, and recently we have shown that high beta-defensin genomic copy number is associated with psoriasis susceptibility. It is not known, however, if biologically and pathophysiologically relevant concentrations of hBD-2 protein are present in vivo, which could support an antimicrobial and proinflammatory role of beta-defensins in lesional psoriatic epidermis. METHODOLOGY/PRINCIPAL FINDINGS: We found that systemic levels of hBD-2 showed a weak but significant correlation with beta defensin copy number in healthy controls but not in psoriasis patients with active disease. In psoriasis patients but not in atopic dermatitis patients, we found high systemic hBD-2 levels that strongly correlated with disease activity as assessed by the PASI score. Our findings suggest that systemic levels in psoriasis are largely determined by secretion from involved skin and not by genomic copy number. Modelling of the in vivo epidermal hBD-2 concentration based on the secretion rate in a reconstructed skin model for psoriatic epidermis provides evidence that epidermal hBD-2 levels in vivo are probably well above the concentrations required for in vitro antimicrobial and chemokine-like effects. CONCLUSIONS/SIGNIFICANCE: Serum hBD-2 appears to be a useful surrogate marker for disease activity in psoriasis. The discrepancy between hBD-2 levels in psoriasis and atopic dermatitis could explain the well known differences in infection rate between these two diseases

Genetic compensation for cilia defects in cep290/NPHP6 mutants by upregulation of cilia-associated small GTPases

Mutations in CEP290, a large multidomain coiled coil protein, are associated with multiple cilia-associated syndromes. Over 130 CEP290 mutations have been linked to a wide spectrum of human ciliopathies, raising the question of how mutations in a single gene cause different disease syndromes. In zebrafish the expressivity of cep290 deficiencies were linked to the type of genetic ablation: acute cep290 morpholino knockdown caused severe cilia-related phenotypes while defects in a Crispr/Cas9 genetic mutant were restricted to photoreceptor defects. Here we show that milder phenotypes in genetic mutants were associated with upregulation of genes encoding the cilia-associated small GTPases arl3, arl13b, and unc119b. Upregulation of UNC119b was also observed in urine-derived renal epithelial cells from human JBTS CEP290 patients. Ectopic expression of arl3, arl13b and unc119b in cep290 morphant zebrafish embryos rescued Kupffer's vesicle cilia and partially rescued photoreceptor outer segment defects. The results suggest that genetic compensation by upregulation of genes involved in a common subcellular process, lipidated protein trafficking to cilia, may be a conserved mechanism contributing to genotype-phenotype variations observed in CEP290 deficiencies

Meta-analyses of association of the <i>LCE3C_LCE3B</i> deletion with RA in the Spanish, Chinese and Dutch cohort.

<p>Number of controls in the Spanish, Chinese and Dutch studies is 978, 681 and 779 respectively. The number of controls in the meta-analysis is 2438. <i>LCE3C_LCE3B</i> deletion: deletion of the <i>LCE3B</i> and <i>LCE3C</i> gene; del: deletion; CCP−: patients negative for anti cyclic citrullinated peptide; CCP+: patients positive for anti-cyclic citrullinated peptide; RF−: patients negative for rheumatoid factor; RF+ patients positive for rheumatoid factor. OR: odds ratio; L95: left border 95% confidence interval. R95: right border 95% confidence interval. I² (p): measure for heterogeneity and p-value of the chi-square test for heterogeneity.</p

No association of the <i>LCE3C_LCE3B</i> deletion with RA in the Dutch cohort.

<p><i>LCE3C_LCE3B</i> deletion: deletion of the <i>LCE3B</i> and <i>LCE3C</i> gene; del: deletion; CCP−: patients negative for anti cyclic citrullinated peptide; CCP+: patients positive for anti-cyclic citrullinated peptide; RF−: patients negative for rheumatoid factor; RF+ patients positive for rheumatoid factor. OR: odds ratio; 95% CI: 95% confidence interval.</p

Demographics of the rheumatoid arthritis cohort.

<p>Numbers are depicted as n(%) or mean ± standard deviation. Data available for ^#686, ^##717, ^###952, ^####451 patients. RA: rheumatoid arthritis; CCP: anti-cyclic citrullinated peptide; RF: rheumatoid factor.</p

Protein-protein interaction studies.

<p>(a) Schematic protein structure of DZANK1. Two different deletion constructs of DZANK1, one consisting of the ZNF_RBZ domains (244-317aa) and the other of the ANK domains (595-681aa), were used for co-transformation in yeast. (b) Results of the co-transformation in yeast. Yeast transformants were selected on low (SD/-Trp/-Leu/-His) and high-stringency (SD/-Trp/-Leu/-His-/-Ade) medium with observed growth, indicating interaction of the tested bait and prey proteins. In addition, the β-galactosidase filter lift assay was performed. The USH2A_intracellular region was used as a positive control. Empty prey vector was used as a negative control. Yeast-two-hybrid analysis revealed a specific interaction between DZANK1 ZNF_RBZ domains and both NINL^isoA/B. (c) GST pull-down assays, showing that Strep/FLAG-tagged DZANK1 was efficiently pulled down by GST-fused NINL^isoB, but not by GST alone. The first lane shows 5% input of the protein lysate. (d) Co-immunoprecipitation of DZANK1 Full Length (FL) with NINL^isoB, but not with LRRK2. The immunoblot (IB) in the top panel shows that HA-tagged NINL co-immunoprecipitated with Strep/FLAG-tagged DZANK1 (lane 2), whereas unrelated FLAG-tagged LRRK2 (lane 3) did not. The anti-HA immunoprecipitates are shown in the middle panel; protein input is shown in the bottom panel. (d’) Reciprocal IP experiments using anti-FLAG antibodies confirmed the co-immunoprecipitation of HA-tagged NINL^isoB with Strep/FLAG-tagged DZANK1 (lane 2) and not with LRRK2 (lane 3) shown in the top panel. The anti-FLAG immunoprecipitations are shown in the middle panel; protein input is shown in the bottom panel. ANK: ankyrin repeat domain; ZNF_RBZ: Zinc Finger domain like in Ran-binding proteins; aa: amino acids.</p