Mutations in genes belonging to the RHO GTPase pathway are responsible for intellectual disability (ID), psychiatric disorders and brain development anomalies. The great heterogeneity of phenotypes associated with these gene mutations renders the development of therapeutic strategies strenuous. Studying PAK3, a central gene of the RHO GTPase pathway, will help us establish a genotype/phenotype correlation, which is essential to 1- define the rules behind mutation pathogenicity, 2- understand the underlying mechanisms and 3- propose adapted therapeutic approaches. 1- Our project is to define the genotype/phenotype correlation using about 20 different PAK3 mutations in order to understand the origin of PAK3-linked ID degree of severity, as well as why ID may sometimes be associated with other neurodevelopmental defects. Thus, we will establish and characterise the broadest cohort of patients bearing PAK3 mutations ever built. In parallel, we will assess the functional defects of mutated PAK3 variants and their effects on cell biology (shape, adhesion, migration) as well as neuron differentiation (neurite growth, dendritic spine formation). Our hypothesis states that mutation pathogenicity is not simply a loss or gain of function but may involve more complex mechanisms of signalling interference. Indeed, the presence of a mutated protein is often more deleterious than the lack of a protein. 2- To go further in analysing the severe forms of PAK3-linked ID, we created a new knock-in model bearing a mutation clinically responsible for a severe ID associated with secondary microcephaly. This mouse model presents strong behavioural and cognitive anomalies, as well as secondary microcephaly, reminiscent of the clinical case. Our project consists in a more thorough analysis of the mouse model behavioural and cognitive defects, in order to compare our results with the patient’s clinical traits. Our ex-vivo and in-vitro preliminary analyses allowed us to propose a new molecular mechanism of mutation pathogenicity, which we will investigate thoroughly. 3- We will test two phenotypical rescue strategies with the aim of further developing therapeutic solutions. The first strategy concerns severe forms of the disease. The degradation of stable pathogenic PAK3 proteins should, at least partially, restore phenotypic anomalies usually associated with severe ID. This strategy of specifically degrading stable pathogenic variants was never explored in the context of neurodevelopmental disorders, even while it is being developed as potential cancer treatment. It would also be applicable to over-activating mutations in genes belonging to the RHO GTPase pathway. The second rescue approach targets Cofilin, a convergence point of the RHO GTPase pathway. Several strategies targeting this actin polymerisation regulator were already explored to rescue behavioural anomalies and synaptic plasticity defects. We aim to demonstrate that this approach would also correct neuronal differentiation anomalies appearing during post-natal development. Thus, the efficiency of a cofilin-blocking peptide to restore neuritic arborisation and dendritic spine formation in mutated mice will be evaluated. This project is based on strong preliminary results and an already operational consortium composed of 2 clinician teams and 3 research teams (1 team being knowledgeable in the two fields). This project will allow us to understand the genotype/phenotype relations regarding PAK3 gene mutations as well as mutations on other genes belonging to the RHO-GTPase pathway. Our results will greatly help advance genetic counselling and patient monitoring. The post genomic and preclinical aspects of this project will also enable us to pave the way for new therapeutic approaches in the optic of personalised medicine.