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.

Disturbance of the balance of long-range and short-range connections is thought to be associated with autism spectrum disorder (ASD). The core structure of the short-range connections is a set of U-shaped fiber bundles that circumvent the cortical folds. They have never been mapped at a large scale in the human brain, probably because of the large variability of the cortical folding patterns. We propose to leverage the emergence of a dictionary of the most frequent folding patterns to infer a specific U-bundle atlas for each such pattern. This dictionary of atlases inferred from the outstanding dataset of the Human Connectome Project will then be used to look for abnormal short-range connectivity in a large ASD dataset already collected in the context of a European project. We will finally validate the U-bundle organizations found abnormal using postmortem brains imaged with high resolution diffusion MRI performed at 11,7T and digitalized Klingler dissection.

Neurodevelopmental disorders (NDDs) represent a heterogeneous group of conditions that persist throughout life, and affect more than 3% of individuals worldwide. NDD has a major impact on the affected individuals, families and society as a whole. Due to high-throughput sequencing, up to 50% of NDD cases are diagnosed as a monogenic cause. Our consortium specifically focuses on pathogenic variants in genes encoding components of the ubiquitin-proteasome system (UPS) associated with NDDs. The UPS ensures the selective degradation of proteins through a complex ubiquitination process involving >1,000 distinct ubiquitin ligases, which prepare these proteins for degradation by the 26S proteasome. The UPS is essential for cellular homeostasis and a vast number of genes are involved, most of them abundantly expressed in brain. It is therefore not surprising that 10-15% of NDDs have been associated with UPS dysfunction. The partners of our UPS-NDDiag consortium have identified more than 250 pathogenic or likely pathogenic variants across >30 UPS genes associated with NDD. However, the complexity of the system causes major challenges in assessing the pathogenicity of genetic variants, and good biomarkers that indicate UPS dysfunction are largely lacking, hampering diagnosis. Our consortium is structured around six interconnected work packages. These will be addressed by six partners from five countries with complementary skills and outstanding expertise in advanced genetics, functional genomics; facial recognition for diagnosis of rare diseases; functional studies in hIPSCs, Drosophila and mice; bioinformatics; integrative analysis of multi-omics, and pharmaceutical nanotechnology. In addition, 26 international collaborators join the consortium to enrich its knowledge and skills. Our main delivery is to provide reliable biomarkers and functional assays to classify UPS-related variants. Besides, UPS-NDDiag will yield therapeutic targets that may support drug development for personalized medicine and shed light on our current understanding of the overall pathogenesis of disorders related to the UPS.

Targeted gene and drug delivery (delivery of a compound to a strictly localized site in the human body) is one of the most ambitious goals of modern therapy. Although a great amount of work is conducted worldwide on the research of various targeted gene and drug delivery systems, this goal still remains unachievable yet. In recent years, new promising possibilities for targeted delivery have been discovered based on the combination ultrasound and contrast agents (UCAs). UCAs are micron-sized encapsulated gas bubbles for an improved medical diagnosis. Besides, they can carry drugs and selectively adhere to specific sites in the human body. This capability, in combination with the effect known as sonoporation, provides great possibilities for localized gene and drug delivery. Sonoporation is a process in which ultrasonically activated UCAs, pulsating nearby biological barriers (cell membrane or endothelial layer), increase their permeability and thereby boost the penetration of external substances. In this way drugs and genes can be delivered inside individual cells without serious consequences for the cell viability. In addition, this delivery system promises to be a low-cost technology like other ultrasound technologies. However, the mechanisms of sonoporation are still today unknown. The aim of the proposed project is to determine the physical and cellular mechanisms responsible for cell membrane permeabilization caused by sonoporation, investigating theoretically and experimentally the interaction between ultrasound waves, contrast agent microbubbles and cell membranes. In the present project, it is first planned to unite all the problems related to sonoporation and to solve them jointly using complementary disciplines such as ultrasound physics, microbubble dynamics, cell biology and molecular dynamics simulations.

The objective of SATURN is the development of simulation tools dedicated to High Intensity Therapeutic Ultrasound. The project is based on the creation of a simulation platform from which will be developed software tools dedicated to targeted industrial applications: Simulation module used in the lab for design of probes and treatment and simulation module implemented in the FocalOne HIFU device (EDAP) for use by practitioners during the treatment. Basic research will be conducted on the propagation of ultrasounds and their interactions with biological tissues (non-linear effects for example) and technological innovations achieved (algorithmic optimization for implementation on parallel architecture). SATURN is a collaborative research project that brings together two INSERM labs, leaders in ultrasound therapy, the CEA LIST expert in numerical simulation and software technology and the company EDAP, French global leader in therapeutic ultrasound.