Neocortical development requires tightly regulated processes, and perturbations lead to malformations (MCDs). Our groups have demonstrated the importance of the cytoskeleton during these key developmental steps. Recently, Cytoplasmic dynein 1, heavy chain 1 (DYNC1H1) mutations were identified in MCDs, as well as in motor neuron degeneration, referred to as « Dyneinopathies ». The wide spectrum of these disorders, together with dynein’s pleiomorphic cellular functions, raise fundamental questions about the effect of mutations on different cellular partners and processes. In this transversal project, we unite molecular and cellular neurobiologists, with clinicians and geneticists. This project will address crucial elements concerning the dyneinopathies, i.e. to find out the various molecular and cellular mechanisms by which disease-related dynein mutations disrupt cellular functions. We will question how distinct mutations perturb dynein’s behavior in patient-derived cells, as well as in mouse progenitors and post-mitotic neurons. Individual genotype-phenotype correlations will advance comprehension of these severe and variable disorders. Our objectives are: · To refine the phenotypic spectrum and natural history of dyneinopathies ranging from fœtus to adulthood, and to understand how DYNC1H1 mutations generate a broad spectrum of phenotypes by correlating phenotype, genotype and protein modelling of mutations, in order to provide clues concerning key and distinct mechanisms perturbed in these disorders. Related to this, we will search for mutations in other genes involved in dynein-dependent pathways. · To assess the biochemical consequences of DYNC1H1 mutations on dynein complexes and to identify their cellular consequences and effects on major dynein-dependent processes in patient-derived fibroblasts, as well as in genome-edited cell lines with different versions of mutant DYNC1H1. · To characterize the effects of selected DYNC1H1 mutations in neuronal progenitors and post-mitotic neurons in the developing mouse cortex. To this end, we will begin by studying a new Dync1h1 knock-in mouse mutant carrying the p.Lys3334Asn mutation responsible for human MCD, identifying perturbed cellular mechanisms during cortical development. These data will be compared with an existing mouse mutant model (Legs at odd angles (Dync1h1 +/Loa) that shows subtle cortical defects and a slow motor neuron loss similar to « peripheral dyneinopathies ». We will thus shed light on perturbed mechanisms specifically affecting cortical development and leading to MCDs, versus other mutations affecting motoneuron survival and potentially disturbing different dynein functions. Importantly, we will determine whether individual mutations may lead to loss of function effects while others may lead to gain of function or dominant negative effects. These experiments will provide major information on the molecular mechanisms leading to the different pathologies as well as strongly increase our knowledge on the in vivo regulation of the dynein motor. We will characterize the critical roles of mutated DYNC1H1 during proliferation, migration and differentiation of neurons in the cortex. Such studies may distinguish MCD from peripheral (SMA-LED) phenotypes and help determine pharmacological interventions, necessary in each case. In addition to exploration of the proposed pathophysiological mechanisms, these studies could have diagnostic implications, especially to assess the pathogenic effect of some rare variants for which conclusions are difficult to draw. This information will be important to better classify patients and identify those requiring early specialized care. In addition, this work will pinpoint different facets of dynein function, involving its numerous partners and the microtubule cytoskeleton, in cortical development.