The gene PRICKLE1 is important for human brain function, as mutations in PRCKLE1 are associated with progressive myoclonus epilepsy (PME). Mutations in prickle orthologs could cause seizures in flies, zebrafish and mice, suggesting a conserved role of Prickle protein in seizure from fruit flies to humans. The underlying molecular mechanism how PRICKLE1 mutation causes PME is still unknown. Prickle1 is part of the planar cell polarity (PCP) pathway, which regulates cell polarity within plane of cell sheets. In Drosophila, prickle is recruited to one side of the cell by another PCP protein, Van Gogh. This asymmetric protein localization of Van Gogh/prickle establishes cell polarity. In zebrafish and Xenopus, loss of Prickle or Van Gogh like (Vangl) genes causes PCP phenotypes, which seemingly supports the Prickle/Vangl protein interaction and the role of Prickle in PCP pathway. The function of Prickle in mammals has not been analyzed. It is possible that mammalian Prickle also interacts with Vangl to mediate PCP signaling based on the conserved role of prickle from Drosophila to Xenopus. If Prickle1 interacts with Vangl and regulate PCP pathway, the PME we observed in humans might be associated with loss of neuronal polarity and impaired neuron activity. Therefore, to understand whether Prickle1 mediates Vangl signaling in mammals could be a step toward revealing the etiology of PME in human patients. Therefore, I analyzed the function of Prickle1 in three developmental processes, the limb development, the palate development, and the caudal migration of facial branchimotor neurons (FBMs), in which the function of PCP pathway, especially Vangl2, has been described. Supporting the interaction between Prickle1 and Vangl, mutations in either Prickle1 or Vangl2 leads to shorter limbs. However, Prickle1 and Vangl2 only have limited overlap in mRNA expression in the digit tips. This raises the question as to how impaired Prickle1/Vangl2 protein interaction in these cells in the digit tips cause defective growth of the whole limb. It also suggests alternate function of Prickle1 other than mediating Vangl2 function. This interaction between Prickle1 and Vangl2 is further challenged by the limited function of Vangl but the essential role of Prickle1 in palate development, which suggests that the function of Prickle1 is independent of Vangl2. In the caudal migration of FBMs, Prickle1 mutation impairs this migration process dose-dependently. This is different from Vangl2 mutation, which completely blocks the caudal migration and partially impairs the lateral migration of FBMs. More importantly, Prickle1 is expressed by the neurons, while Vangl2 functions in the surrounding cells, which again raises the question as to whether and how the two proteins could interact if they are not expressed in the same cell. These results together question the model that Prickle1 is the intracellular partner of Vangl2, but support Prickle1 function might be independent of Vangl. Actually, it is possible that Prickle1 is part of gene expression regulation machinery: Prickle1 mutation affects Wnt5a expression in the limb and Shh expression in the palate. Although this regulation mechanism is still unknown, it suggests that defective gene expression might be related to PME caused by PRICKLE1 mutation.

Epilepsy and Autism Spectrum disorders (ASD) are both complex neurodevelopmental disorders which share approximately 30% comorbidity. Epilepsy is characterized by unprovoked recurrent seizures and affects ~1% of the population while ASDs are characterized by deficits in language, social, and behavior and found 1 in 68 people. Variants in synaptic genes suggest disruptions in synaptic regulation underlie both conditions. PRICKLE1 and PRICKLE2 are known core WNT/ PCP genes implicated in Progressive myoclonic epilepsy in families and in the general population. Humans, mice, zebrafish, and Drosophila with disrupted Prickle exhibit epileptic behavior and other neurological deficits. Prickle is implicated in several aspects of neuronal development and mutated proteins display aberrant activity in vivo and in vitro. Recently, variations in PRICKLE1 were associated with ASDs in humans. The mechanisms by which PRICKLE could contribute to ASDs are unknown. Results presented here show Prickle1+/- mice exhibit ASD-like behavior. Prickle1 associates with Synapsin I; a phosphoprotein important for synaptogenesis, axonogenesis, and neurotransmitter release. Mutant R104QPRICKLE1 protein causes a reduction in sizes of dense-core vesicles in neuronal-like PC12 cells. Results indicate PRICKLE1 may be associated with ASDs and possibly involved in synaptic homeostasis. Prickle deregulation has also been associated with neural tube defects and cancers. The mechanism(s) by which Prickle is regulated is incompletely understood. To further elucidate the role of PRICKLE in disease, immunoprecipitates from PRICKLE-expressing stable tetracycline-regulated neuronal-like PC12 cells were identified by mass spectrometry. The deubiquitinating enzyme USP9X was identified as a novel interacting PRICKLE protein. USP9X is a substrate-specific deubiquitinating enzyme implicated in several aspects of neuronal development, associated with X-linked intellectual disability and a candidate gene for epilepsy. Results show that USP9X robustly deubiquitinates and protects PRICKLE from proteasomal degradation. USP9X variants found in the ARRA ASD cohort directly associates USP9X with ASDs. The identified USP9X mutations delete the PRICKLE-interacting domain and provide a possible mechanism for PRICKLE deregulation. Already a target for treating cancer, USP9X can serve as a therapeutic target to regulate PRICKLE levels.

La voie de la polarité planaire cellulaire (PCP), aussi connue sous le nom de la voie non-canonique du Frizzled/Dishevelled, contrôle le processus morphogénétique de l'extension convergente (CE) qui est essentiel pour la gastrulation et la formation du tube neural pendant l'embryogenèse. La signalisation du PCP a été récemment associée avec des anomalies du tube neural (ATN) dans des modèles animaux et chez l'humain. Prickle1 est une protéine centrale de la voie PCP, exprimée dans la ligne primitive et le mésoderme pendant l'embryogenèse de la souris. La perte ou le gain de fonction de Prickle1 mène à des mouvements de CE fautifs chez le poisson zèbre et la grenouille. PRICKLE1 interagit directement avec deux autres membres de la voie PCP, Dishevelled et Strabismus/Vang. Dans notre étude, nous avons investigué le rôle de PRICKLE1 dans l'étiologie des ATN dans une cohorte de 810 patients par le re-séquençage de son cadre de lecture et des jonctions exon-intron. Le potentiel pathogénique des mutations ainsi identifiées a été évalué par des méthodes bioinformatiques, suivi par une validation fonctionnelle in vivo dans un système poisson zèbre. Nous avons identifié dans notre cohorte un total de 9 nouvelles mutations dont sept: p.Ile69Thr, p.Asn81His, p.Thr275Met, p.Arg682Cys et p.Ser739Phe, p.Val550Met et p.Asp771Asn qui affectent des acides aminés conservés. Ces mutations ont été prédites in silico d’affecter la fonction de la protéine et sont absentes dans une large cohorte de contrôles de même origine ethnique. La co-injection de ces variantes avec le gène prickle1a de type sauvage chez l’embryon de poisson zèbre a démontré qu’une mutation, p.Arg682Cys, modifie dans un sens négatif le phénotype du défaut de la CE produit par pk1 de type sauvage. Notre étude démontre que PK1 peut agir comme facteur prédisposant pour les ATN chez l’humain et élargit encore plus nos connaissances sur le rôle des gènes de la PCP dans la pathogenèse de ces malformations. / The planar cell polarity pathway (PCP) or the non-canonical Frizzled/Dishevelled pathway controls the morphogenetic process of convergent extension (CE) that is essential during embryogenesis for gastrulation and neural tube formation. Recently, PCP signalling was associated with neural tube defects (NTD) in humans and animal models. The core PCP protein, Prickle1, is expressed in the primitive streak and mesoderm during mouse embryogenesis. Both gain and loss of function of Prickle1 cause faulty CE movements in zebrafish and the frog. PRICKLE1 physically interacts with two other core PCP members, Dishevelled and Strabismus/Vang. In the present study we investigated the role of PRICKLE1 in the aetiology of NTDs in a large cohort of 810 patients through resequencing of its open reading frame and exon-intron junctions. The pathogenicity of the identified mutations was assessed through bioinformatics methods followed by a functional validation in a zebrafish system, in vivo. We identified in our cohort a total of nine novel mutations, of which seven affected conserved amino acids: p.Ile69Thr, p.Asn81His, p.Thr275Met, p.Arg682Cys, p.Ser739Phe, p.Val550Met and p.Asp771As. These mutations were predicted to affect the function of the protein in silico and were absent in a large cohort of ethnically-matched controls. Co-injection of these variants with the wild type pk1 in zebrafish oocytes revealed that one mutation, p.Arg682Cys, antagonized the CE phenotype induced by the wild-type zebrafish prickle1a in a dominant fashion. Our study demonstrates that PRICKLE1 can represent a predisposing factor for human NTDs and further expands our knowledge on the role that PCP genes in the pathogenesis of these malformations.

La voie de la polarité planaire cellulaire (PCP), aussi connue sous le nom de la voie non-canonique du Frizzled/Dishevelled, contrôle le processus morphogénétique de l'extension convergente (CE) qui est essentiel pour la gastrulation et la formation du tube neural pendant l'embryogenèse. La signalisation du PCP a été récemment associée avec des anomalies du tube neural (ATN) dans des modèles animaux et chez l'humain. Prickle1 est une protéine centrale de la voie PCP, exprimée dans la ligne primitive et le mésoderme pendant l'embryogenèse de la souris. La perte ou le gain de fonction de Prickle1 mène à des mouvements de CE fautifs chez le poisson zèbre et la grenouille. PRICKLE1 interagit directement avec deux autres membres de la voie PCP, Dishevelled et Strabismus/Vang. Dans notre étude, nous avons investigué le rôle de PRICKLE1 dans l'étiologie des ATN dans une cohorte de 810 patients par le re-séquençage de son cadre de lecture et des jonctions exon-intron. Le potentiel pathogénique des mutations ainsi identifiées a été évalué par des méthodes bioinformatiques, suivi par une validation fonctionnelle in vivo dans un système poisson zèbre. Nous avons identifié dans notre cohorte un total de 9 nouvelles mutations dont sept: p.Ile69Thr, p.Asn81His, p.Thr275Met, p.Arg682Cys et p.Ser739Phe, p.Val550Met et p.Asp771Asn qui affectent des acides aminés conservés. Ces mutations ont été prédites in silico d’affecter la fonction de la protéine et sont absentes dans une large cohorte de contrôles de même origine ethnique. La co-injection de ces variantes avec le gène prickle1a de type sauvage chez l’embryon de poisson zèbre a démontré qu’une mutation, p.Arg682Cys, modifie dans un sens négatif le phénotype du défaut de la CE produit par pk1 de type sauvage. Notre étude démontre que PK1 peut agir comme facteur prédisposant pour les ATN chez l’humain et élargit encore plus nos connaissances sur le rôle des gènes de la PCP dans la pathogenèse de ces malformations. / The planar cell polarity pathway (PCP) or the non-canonical Frizzled/Dishevelled pathway controls the morphogenetic process of convergent extension (CE) that is essential during embryogenesis for gastrulation and neural tube formation. Recently, PCP signalling was associated with neural tube defects (NTD) in humans and animal models. The core PCP protein, Prickle1, is expressed in the primitive streak and mesoderm during mouse embryogenesis. Both gain and loss of function of Prickle1 cause faulty CE movements in zebrafish and the frog. PRICKLE1 physically interacts with two other core PCP members, Dishevelled and Strabismus/Vang. In the present study we investigated the role of PRICKLE1 in the aetiology of NTDs in a large cohort of 810 patients through resequencing of its open reading frame and exon-intron junctions. The pathogenicity of the identified mutations was assessed through bioinformatics methods followed by a functional validation in a zebrafish system, in vivo. We identified in our cohort a total of nine novel mutations, of which seven affected conserved amino acids: p.Ile69Thr, p.Asn81His, p.Thr275Met, p.Arg682Cys, p.Ser739Phe, p.Val550Met and p.Asp771As. These mutations were predicted to affect the function of the protein in silico and were absent in a large cohort of ethnically-matched controls. Co-injection of these variants with the wild type pk1 in zebrafish oocytes revealed that one mutation, p.Arg682Cys, antagonized the CE phenotype induced by the wild-type zebrafish prickle1a in a dominant fashion. Our study demonstrates that PRICKLE1 can represent a predisposing factor for human NTDs and further expands our knowledge on the role that PCP genes in the pathogenesis of these malformations.