Study of LIMK2 and RNF135, involved in neurofibromatosis type 1, in autism and mental deficiency

L'autisme et la déficience mentale (DM) sont des pathologies neurodéveloppementales fréquentes qui partagent des facteurs génétiques communs. Afin de mieux comprendre leur étiologie, nous avons étudié les mécanismes moléculaires de la neurofibromatose de type 1 (NF1), qui est souvent associée à l'autisme et à la DM. La neurofibromine, dont le gène est muté dans la NF1 interagit avec LIMK2. Cette protéine fait partie de la voie des Rho-GTPases dont des mutations de plusieurs membres ont été trouvés mutés dans des cas d'autisme et de DM. Chez le rat, nous avons montré que l’expression de Limk2d, une isoforme sans domaine kinase, augmente la croissance des neurites des cellules neuronales NSC-34. Chez l'homme, LIMK2-1 est la seule isoforme qui comporte un domaine inhibiteur de la phosphatase 1 (PP1i). Nous avons montré que l’expression de cette protéine diminue la longueur des neurites des cellules NSC-34 in vitro. Nous avons observé l'association de la variation située dans le domaine PP1i à la DM (p.S668P, rs151191437) (p=0,04, test de Fisher, OR = 3,29). Elle abolit l’effet inhibiteur de croissance des neurites de l'isoforme LIMK2-1 diminue l'interaction de LIMK2-1 avec la neurofibromine. La fréquence de l'autisme est plus élevée chez les patients atteints ayant des délétions de 14 gènes du locus NF1. Nous avons observé une association entre la variation R115K (rs111902263) du gène RNF135 de ce locus et l'autisme (p=0,00014, test de Fisher) ainsi qu’une anomalie du nombre de copies située dans l'intron 2 de ce gène chez un d’entre eux. Ce travail souligne la spécificité de deux isoformes de LIMK2 sur la croissance des neurites. Il renforce l’intérêt d’étudier l’implication du gène RNF135 dans l’autisme. Des études fonctionnelles seront entreprises afin de confirmer le rôle de LIMK2 et de RNF135 dans l'étiologie de l'autisme et de la DM.Autism and mental deficiency (MD) are two neurodevelopemental diseases which share genetic factors in common. To better understand their etiologies, we studied the molecular mechanisms of neurofibromatosis type 1, a pathology frequently associated with autism and MD. Neurofibromatosis type 1 is due to deletions or mutations of the NF1 gene which encodes neurofibromin. This protein interacts with several proteins such as LIMK2. This protein belongs to the Rho-GTPases pathway in wich mutations of numerous members have been associated with autism and MD. In our study, we showed that LIMK2 isoforms do not only have important structural differencies but have also functional specificities. Limk2d, which lacks the kinase domain, promotes neurite outgrowth of NSC-34 cells. On the contrary, LIMK2-1, which is primate specific and has a C-terminal PP1i domain, inhibits neurite outgrowth. Analysis of the LIMK2-1 coding sequence, revealed the association between MD and a variation located in the PP1i domain, S668P (rs151191437) (p=0.04, Fisher test, OR = 3.29). This variation abrogated the LIMK2-1 effect on neurite outgrowth and inhibited LIMK2-1 interaction with neurofibromin. Deletions occuring in neurofibromatosis type 1 which include the NF1 gene and 13 others are associated with a higher frequency of autism. Mutations of one of them, RNF135, have been identified in patients with MD and overgrowth syndrome. Two of these patients also presented autistic features. By analysing RNF135 gene in autistic patients, we showed the association of the variation R115K (rs111902263) with autism. We also identified a duplication of a region located in RNF135 gene intron 2 in one patient presenting autism and MD. Our results highlight the importance and specificity of LIMK2 isoforms on neurite outgrowth and strengthen the importance to analyze both the sequence and copy-number of RNF135 gene. Further functional experiments will be undertaken to confirm the implication of LIMK2 and RNF135 in autism and MD etiology

Anatomy and Cell Biology of Autism Spectrum Disorder: Lessons from Human Genetics.

International audienceUntil recently autism spectrum disorder (ASD) was regarded as a neurodevelopmental condition with unknown causes and pathogenesis. In the footsteps of the revolution of genome technologies and genetics, and with its high degree of heritability, ASD became the first neuropsychiatric disorder for which clues towards molecular and cellular pathogenesis were uncovered by genetic identification of susceptibility genes. Currently several hundreds of risk genes have been assigned, with a recurrence below 1% in the ASD population. The multitude and diversity of known ASD genes has extended the clinical notion that ASD comprises very heterogeneous conditions ranging from severe intellectual disabilities to mild high-functioning forms. The results of genetics have allowed to pinpoint a limited number of cellular and molecular processes likely involved in ASD including protein synthesis, signal transduction, transcription/chromatin remodelling and synaptic function all playing an essential role in the regulation of synaptic homeostasis during brain development. In this context, we highlight the role of protein synthesis as a key process in ASD pathogenesis as it might be central in synaptic deregulation and a potential target for intervention. These current insights should lead to a rational design of interventions in molecular and cellular pathways of ASD pathogenesis that may be applied to affected individuals in the future

LIMK2-1, a new primate-specific isoform of LIMK2, is associated with intellectual disability

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LIMK2d, a truncated isoform of Lim kinase 2 regulates neurite growth in absence of the LIM kinase domain

Lim kinase 2 isoforms, LIMK2a and LIMK2b, phosphorylate cofilin leading to remodeling of actin cytoskeleton during neuronal differentiation. The expression and function of the LIMK2d isoform, missing the kinase domain, remain unknown. We analyzed the expression of LIMK2 splice variants in adult rat brain and in cultures of rat neural stem cells by RT-QPCR. All three splice variants were expressed in adult cortex, hippocampus and cerebellum. Limk2a and Limk2d expression, but not Limk2b, increased during neuronal differentiation. We studied the localization and function of LIMK2d isoform by transfecting Hela, NSC-34, and hippocampal rat neuron cultures. Similarly to LIMK2b, LIMK2d was expressed in the cytoplasm, neurites and dendritic spines, but not in the nucleus. Similarly to LIMK2a, LIMK2d over-expression in NSC-34 cells increased neurite length, but independently of cofilin phosphorylation or of direct interaction with actin. Overall, these results indicate that LIMK2d is a third LIMK2 isoform which regulates neurite extension and highlights the possible existence of a kinase independent function of LIMK2

LIMK2-1 is a Hominidae-Specific Isoform of LIMK2 Expressed in Central Nervous System and Associated with Intellectual Disability

International audienceLIMK2 is involved in neuronal functions by regulating actin dynamics. Different isoforms of LIMK2 are described in databanks. LIMK2a and LIMK2b are the most characterized. A few pieces of evidence suggest that LIMK2 isoforms might not have overlapping functions. In this study, we focused our attention on a less studied human LIMK2 isoform, LIMK2-1. Compared to the other LIMK2 isoforms, LIMK2-1 contains a supplementary C-terminal phosphatase 1 inhibitory domain (PP1i). We found out that this isoform was hominidae-specific and showed that it was expressed in human fetal brain and faintly in adult brain. Its coding sequence was sequenced in 173 patients with sporadic non-syndromic intellectual disability (ID), and we observed an association of a rare missense variant in the PP1i domain (rs151191437, p.S668P) with ID. Our results also suggest an implication of LIMK2-1 in neurite outgrowth and neurons arborization which appears to be affected by the p.S668P variation. Therefore our results suggest that LIMK2-1 plays a role in the developing brain, and that a rare variation of this isoform is a susceptibility factor in ID

Mutation screening of the ubiquitin ligase gene RNF135 in French patients with autism

International audienceMany genes are now thought to confer susceptibility to autism. Despite the fact that this neuropsychiatric disease appears to be related to several different causes, common cellular and molecular pathways have emerged and point to synaptic dysfunction or cellular growth. Several studies have indicated the importance of the ubiquitin pathway in synaptic function and the aetiology of autism. Here, we focused on the ring finger protein 135 (RNF135) gene, encoding an E3 ubiquitin ligase expressed in the cortex and cerebellum, and located in the NF1 gene locus in 17q11.2, a region linked to autism. We carried out a genetic analysis of the coding sequence of RFN135 in a French cohort of patients with autism and observed a significantly increased frequency of genotypes carrying the rare allele of the rs111902263 (p.R115K) missense variant in patients (P=0.0019, odds ratio : 4.23, 95% confidence interval : 1.87-9.57). Particularly, three unrelated patients showed a homozygous genotype for K115, a situation not observed in the 1812 control individuals. Further cellular and molecular studies are required to elucidate the role of this gene and the variant K115 in brain development and neuronal function