Caractérisation des mécanismes physiopathologiques associés aux mutations NEK8 / NPHP9 identifiées chez des patients atteints de ciliopathies rénales sévères

Les ciliopathies sont un groupe de maladies génétiques multi-systémiques liées à un dysfonctionnement du cil primaire, une structure sensorielle présente à la surface des cellules qui régule des voies de signalisation clés au cours du développement et de l'homéostasie tissulaire. Afin d'identifier de nouveaux gènes responsables de ciliopathies développementales sévères, ~ 500 patients / fétus ont été analysés par une approche de séquençage à haut débit de l'exome ciblant > 1 200 gènes ciliaires ("ciliome"). Nous avons identifié huit nouvelles mutations dans le gène NEK8/NPHP9 chez cinq familles dont les syndromes se chevauchent. NEK8/NPHP9 code une protéine kinase de la famille des NIMA qui se localise au niveau du compartiment Inversine du cil primaire et agit comme un régulateur de la signalisation Hippo, une voie essentielle contrôlant la taille des organes. Nous montrons pour la première fois que les mutations du gène NEK8 sont associées à une agénésie rénale et une hypodysplasie. De plus, notre travail met en évidence une corrélation génotype/phénotype: les mutations "perte de fonction" de NEK8 conduisant à reins élargies et kystiques, des kystes pancréatiques et hépatique, alors que les mutations faux-sens de NEK8 causent une hypodysplasie/agénésie rénale associée à une cardiopathie et une paucité des canaux biliaires. La première partie de mon projet de thèse porte sur l'étude de l'impact des mutations faux-sens de NEK8 sur divers processus cellulaires et des voies de signalisation dépendantes de NEK8. Nous avons démontré un effet "gain de fonction" des mutations faux-sens de NEK8 puisqu'elles affectent la ciliogenèse et la composition du compartiment Inversine (localisation ciliaire de ANKS6). De plus, ces mutations altèrent la localisation nucléaire de YAP, le principal acteur de la voie Hippo, ainsi que l'expression des gènes cibles de YAP dans les fibroblastes de patients et dans la lignée cellulaire rénale (mIMCD3) invalidée pour NEK8. De même, nous avons montré une accumulation anormale de YAP nucléaire dans les reins polykystiques de la souris Jck, porteuse d'une mutation faux-sens de Nek8. Un déséquilibre de la voie Hippo serait donc à l'origine des défauts de morphogenèses épithéliales. En effet, les cellules mIMCD3 invalidées pour NEK8 forment en culture 3D des structures anormales et/ou des sphères élargies qui s'accompagnent d'une persistance du marquage nucléaire de YAP et Ki-67 et forment de grandes sphères par rapport aux cellules contrôles. Des défauts plus sévères ont été observés pour les cellules ré-exprimant les différents mutants de NEK8, confirmant la pathogénicité de ces mutations et leur effet "gain de fonction". Enfin, le traitement par la Vertéporfine, un inhibiteur spécifique de l'activité transcriptionnelle de YAP, améliore non seulement le phénotype des fibroblastes de patients et des cellules rénales invalidées pour NEK8 en culture 3D, mais également in vivo les anomalies observées chez les embryons de poisson zèbre dues à la surexpression de la forme NEK8 humaine, confirmant ainsi l'implication d'une dérégulation de YAP dans les mécanismes physiopathologiques. Par ailleurs, nous avons observé que les mutants de NEK8 s'accumulent de manière anormale au niveau de l'appareil de Golgi dans les fibroblastes de patients, et que cet appareil de Golgi apparait dispersé. Nos résultats montrent que le recrutement de NEK8 au Golgi est sensible à la Brefeldine A et dépendrait donc de ARF1, une petite GTPase impliquée dans le trafic de protéines entre les compartiments du Golgi et du réticulum endoplasmique. Nous avons démontré que NEK8 interagit et co-localise préférentiellement avec la forme d'ARF1 liée au GDP, suggérant pour NEK8 une possible fonction de facteur d'échange d'ARF1 à des sites spécifiques (appareil de Golgi, membranes, cil) afin de promouvoir le trafic vésiculaire de protéines telles que les protéine ciliaires. (...)Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, ~500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of > 1200 ciliary genes. We have identified eight novel mutations in NEK8/NPHP9 in five independent families with severe overlapping syndromic disorders. NEK8/NPHP9 encodes a NIMA-related kinase that localizes at the inversin compartment of the primary cilium and acts as a regulator of Hippo signaling, a pathway that is essential for control of organ size during development. We show for the first time that NEK8 mutations are associated with renal agenesis and hypodysplasia, and our work highlights a genotype/phenotype correlation with NEK8 loss-of-function mutations leading to enlarged cystic kidney, pancreas and liver, whereas NEK8 gain-of-function (missense) mutations cause renal hypodysplasia, cardiopathy and paucity of bile ducts. The first part of my thesis project focuses on the study of the impact of these NEK8 missense mutations on various cellular processes and NEK8-dependent signaling pathways. We demonstrate that NEK8 missense mutations impair the Inversin (INVS) compartment composition and ciliogenesis, and also alter the nuclear localization of the main Hippo signalling effector, YAP, as well as expression of its target genes in patient fibroblasts and renal cell lines. We also demonstrated that this Hippo pathway imbalance causes epithelial morphogenesis defects in 3D matrigel culture. Indeed, mIMCD3 cells depleted for NEK8 showed persistent YAP and Ki-67 staining and formed bigger spheres compared to control cells. Abnormal sphere volume was also observed in cells re-expressing NEK8-GFP mutations, suggesting their pathogenicity. We confirm these data in vivo in Jck mice, a model of polycystic kidney disease bearing a Nek8 missense mutation. Finally, treatment with Verteporfin, a specific inhibitor of YAP transcriptional activity, improves the mutant phenotype of both cellular models and zebrafish embryos overexpressing human NEK8, further supporting the involvement of YAP dysregulation in the pathogenic cellular mechanisms. Surprisingly, in patient fibroblasts, we showed that mutated NEK8 accumulates at the Golgi that appeared dispersed. NEK8 recruitment at the Golgi apparatus is dependent on ARF1 (Brefeldin A sensitive), a small GTPase involved in protein trafficking between Golgi compartments and ER. We notably demonstrated that NEK8 mostly interacts and localizes with the dominant negative form of ARF1 (T31N), suggesting that NEK8 could act as an activator (GEF) of ARF1 to promote vesicular trafficking of ciliary proteins. The second part of my project focuses on a new candidate gene for which a missense homozygous mutation has been identified in 3 individuals presenting a late onset NPH with hepatic fibrosis. This gene encodes ANKS3, an evolutionarily conserved protein whose function is still poorly characterized. Interestingly, ANKS3 has been reported to be a partner of NEK8, even though we showed it does not localize at the INVS compartment with NEK8 but is rather present at the base of cilia in fibroblasts. We showed that the missense mutation does not affect ANKS3 localization but leads to longer cilia and abnormal accumulation of NEK8 at the cilium base in patient fibroblasts and kidney tubules. Altogether, my work focused on NEK8 and its partners, ANKS6 and ANKS3, each of whose related gene is mutated in patients presenting a broad clinical spectrum of phenotypes. (...

Rational CCL5 mutagenesis integration in a lactobacilli platform generates extremely potent HIV-1 blockers

Abstract Efforts to improve existing anti-HIV-1 therapies or develop preventatives have identified CCR5 as an important target and CCL5 as an ideal scaffold to sculpt potent HIV-1 entry inhibitors. We created novel human CCL5 variants that exhibit exceptional anti-HIV-1 features using recombinant lactobacilli (exploited for live microbicide development) as a screening platform. Protein design, expression and anti-HIV-1 activity flowed in iterative cycles, with a stepwise integration of successful mutations and refinement of an initial CCL5 mutant battery towards the generation of two ultimate CCL5 derivatives, a CCR5 agonist and a CCR5 antagonist with similar anti-HIV-1 potency. The CCR5 antagonist was tested in human macrophages and against primary R5 HIV-1 strains, exhibiting cross-clade low picomolar IC50 activity. Moreover, its successful combination with several HIV-1 inhibitors provided the ground for conceiving therapeutic and preventative anti-HIV-1 cocktails. Beyond HIV-1 infection, these CCL5 derivatives may now be tested against several inflammation-related pathologies where the CCL5:CCR5 axis plays a relevant role

Nephrocystins play a crucial role in renal epithelial morphogenesis via the regulation of Wnt/PCP components Dishevelled and Rho GTPases

International audienc

Dishevelled stabilization by the ciliopathy protein Rpgrip1l is essential for planar cell polarity.

International audienceCilia are at the core of planar polarity cellular events in many systems. However, the molecular mechanisms by which they influence the polarization process are unclear. Here, we identify the function of the ciliopathy protein Rpgrip1l in planar polarity. In the mouse cochlea and in the zebrafish floor plate, Rpgrip1l was required for positioning the basal body along the planar polarity axis. Rpgrip1l was also essential for stabilizing dishevelled at the cilium base in the zebrafish floor plate and in mammalian renal cells. In rescue experiments, we showed that in the zebrafish floor plate the function of Rpgrip1l in planar polarity was mediated by dishevelled stabilization. In cultured cells, Rpgrip1l participated in a complex with inversin and nephrocystin-4, two ciliopathy proteins known to target dishevelled to the proteasome, and, in this complex, Rpgrip1l prevented dishevelled degradation. We thus uncover a ciliopathy protein complex that finely tunes dishevelled levels, thereby modulating planar cell polarity processes

Novel <i>NEK8</i> Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation

<div><p>Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. <i>NEK8/NPHP9</i> mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, <i>situs inversus</i>, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of <i>NEK8</i> mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, proliferation/apoptosis/DNA damage response, as well as epithelial morphogenesis. Notably, missense mutations exacerbated some of the defects due to <i>NEK8</i> loss of function, highlighting their likely gain-of-function effect. We also showed that <i>NEK8</i> missense and loss-of-function mutations differentially affect the regulation of the main Hippo signaling effector, YAP, as well as the expression of its target genes in patient fibroblasts and renal cells. YAP imbalance was also observed in enlarged spheroids of <i>Nek8</i>-invalidated renal epithelial cells grown in 3D culture, as well as in cystic kidneys of <i>Jck</i> mice. Moreover, co-injection of <i>nek8</i> MO with WT or mutated <i>NEK8-GFP</i> RNA in zebrafish embryos led to shortened dorsally curved body axis, similar to embryos injected with human <i>YAP</i> RNA. Finally, treatment with Verteporfin, an inhibitor of YAP transcriptional activity, partially rescued the 3D spheroid defects of <i>Nek8</i>-invalidated cells and the abnormalities of NEK8-overexpressing zebrafish embryos. Altogether, our study demonstrates that <i>NEK8</i> human mutations cause major organ developmental defects due to altered ciliogenesis and cell differentiation/proliferation through deregulation of the Hippo pathway.</p></div

Mutant NEK8 proteins fail to localize at the ciliary axoneme in patient fibroblasts and in shNEK8 mIMCD3 cells re-expressing mutant constructs.

<p>(A) Fibroblasts from control (CTRL) and affected individuals II.1 of families 1 and 5 (PT1 and PT5) were grown for 6 days in standard medium followed by 2 days of serum starvation in order to induce cilia growth (high cell density). Cells were stained for endogenous NEK8 (red) and acetylated α-tubulin (green). Arrows (insets) indicate the presence and absence of NEK8 at the axoneme in control and patient fibroblasts respectively. Scale bar, 10 μm. (A’) Percentage of cells with NEK8 staining along the cilium in control and patient fibroblasts. (B-C) Graphs representing the percentage of ciliated cells (B) and cilia length (μm) (C) in control and patient fibroblasts. All graphs show the mean ± SEM of at least three independent experiments. ***p < 0.001, calculated via Kruskall-Wallis (B) or Bonferroni (C) post-hoc tests following ANOVA. (D) Ciliary localization of NEK8 in control pLKO and stable shNEK8 cells re-expressing GFP-tagged NEK8 wild type (WT) or patients’ variants. Cells were stained for acetylated α-tubulin (red, cilia), GFP (green) and nuclei (Hoechst, blue). Only NEK8-GFP WT and partially NEK8-GFP T87A are able to localize along the axoneme. Scale bar, 10 μm. (D’) Histogram showing the percentage of cells with positive NEK8-GFP staining at the primary cilium. (E) Graph showing the percentage of ciliated cells in shNEK8 and shNEK8 re-expressing the mutated proteins cells. Statistical analysis performed by Bonferroni post-hoc test following ANOVA, ns = non significant, *p < 0.05, **p < 0.01 when samples are compared to shNEK8+NEK-GFP WT and ^#p < 0.05, ^##p < 0.01to shNEK8.</p

YAP nuclear shuttling is impaired in patient fibroblasts and results in Hippo pathway dysregulation.

<p>(A) Fibroblasts from control (CTRL) and affected individual II.1 of family 1 and 5 (PT1 and PT5) were grown for 2 days in standard medium with serum (low cell density) or for 6 days then serum-starved for 2 days (high cell density). Cells were co-immunostained for YAP (red), acetylated α-tubulin (green) and nuclei (Hoechst, blue). Insets show higher magnification of YAP localization. Scale bar, 10 μm. (A’, A”) Graphs (mean ± SEM of three independent experiments) showing the nuclear YAP intensity in control and patient fibroblasts (A’) and the percentage of ciliated cells positive for YAP staining (A”). ns = not significant, *p < 0.05, ***p <0.001, calculated by Bonferroni or Kruskal-Wallis post-hoc test following ANOVA, respectively. (B, B’) Localization of phospho-YAP at the cilium in control and patient fibroblasts (B) and quantification (mean ± SEM of three independent experiments) of phospho-YAP-positive cilia (B’). *p < 0.05 calculated via Kruskal-Wallis test. (C, D) qPCR analyses (mean ± SEM of three independent experiments) of the expression of YAP (<i>TEAD4</i>, <i>CYR61</i>, <i>CTGF</i>, <i>JAG1</i>) and Notch (<i>HES1</i>) target genes, normalized to <i>GAPDH</i>, in control and patient fibroblasts at low and high cell density. Statistical analyses were performed using Kruskal- Wallis test, ns = non significant, *p < 0.05, **p < 0.01, ***p < 0.001.</p

NEK8 overexpression in zebrafish induces Hippo pathway deregulation.

<p>(A) Representative pictures of the different classes of body phenotype observed upon <i>nek8</i> morpholino (MO) or human <i>NEK8-GFP</i> RNA injections in zebrafish embryos. Arrowhead points out cyclopia observed in a subset of short embryos. (A’) Graph showing the distribution of the three classes of body axis curvature observed in rescue experiments with WT or mutated (T87A, R602W) <i>NEK8-GFP</i> RNA forms. 30% of embryos co-injected with <i>nek8</i> MO and WT <i>NEK8-GFP</i> RNA exhibited a stunted dorsally curved body axis similar to embryos injected with human <i>YAP</i> RNA. Data are from three independent experiments (n = 90 to 180 embryos). (A”) Graph showing the distribution of normal and stunted dorsally curved animals among <i>GFP</i> or WT <i>NEK8-GFP</i> RNA injected embryos upon Verteporfin (VP) treatment. A partial rescue of the phenotype can be observed in the presence of 20 μM Verteporfin. Data are from four independent experiments (n = 120 to 240 embryos).</p

Yap and Yap target gene expression are induced in <i>Jck</i> mice.

<p>(A) Immunohistochemistry of Yap in wild-type (Wt) and <i>Jck</i> mice at 5 weeks of age (n = 4 of each). (B-B’) Western blot analysis (B) and quantification of YAP and phospho-YAP (S¹²⁷) expression (B’) in Wt (n = 8) and <i>Jck</i> mice (n = 8) at 5 weeks of age. **p < 0.01, calculated by Mann-Whitney test. (C) Relative quantification of Yap targets <i>Ctgf</i>, <i>Cyr61</i>, <i>Ankrd1</i> and <i>Birc5</i> mRNA expression in wild-type (n = 10) and <i>Jck</i> mice (n = 8) at 5 weeks of age. **p < 0.01, ***p < 0.001, calculated by Mann-Whitney test.</p