Focal cortical dysplasia (FCD) is a rare, genetic, non-syndromic developmental malformation of the cerebral cortex that accounts for 5-10% of patients with focal epilepsy. FCD represents the main cause of pharmacologically intractable epilepsy, the only treatment available consisting of invasive surgical resection of the epileptogenic zone, which results effective in only 62% of patients. Thus, a better understanding of this disorder is necessary to develop more effective and less invasive treatments. FCD type 2 (FCD2) is an mTORopathy caused by genetic mutations that cause hyperactivation of the mTOR pathway, leading to abnormal cell size (cytomegalic dysmorphic neurons and balloon cells) and disruption of the structure of the cortex. Recently, the supervisor S. Baulac and others have partially explained the focal nature of the disorder by identifying post-zygotic somatic (mosaic) mutations in a percentage of cells that correlates with the size of the lesion. Despite this, the underling genetics remains unknown for ~40% of FCD2 patients. My first objective is to perform deep whole-exome sequencing of a cohort of 60 unsolved FCD2 cases to explore alternative pathways and find possibly pathogenic variants, focusing on those with an established link with mTOR pathway and/or neurodevelopment. Abnormal cells found in FCD2 lesions have been hypothesized as the source of the epileptogenic activity. However, the developmental origins of these cells are still unclear, and there is a need to find clear biomarkers that can clarify the link between mTOR hyperactivation and neuronal hyperexcitability. This project aims to clarify these aspects by applying my expertise in single cell genomics and S. Baulac’s expertise in functional studies and disease modelling in human brain organoids. The MSCA IF will allow me to reintegrate in Europe after a 4-years+ postdoc in the US and complete my training with the objective of applying to set up my own lab at the end of the two years.

In France, about 5000 new people with a primary malignant brain tumor are diagnosed each year. The most common primary tumors are gliomas, originating from glial cells (astrocytomas and oligodendrogliomas). Low-grade gliomas are mildly aggressive, but they often evolve into a more malignant form. Mutations in the genes encoding isocitrate dehydrogenase (IDH) are found in about 80% of low-grade gliomas and secondary glioblastomas and are associated with a favorable prognosis. Remarkably, IDH-mutated gliomas are characterized by a specific cellular metabolism causing the accumulation of D-2-hydroxyglutarate (2HG) in tumor cells. 2HG can be detected in vivo using 1H magnetic resonance spectroscopy (MRS) and is recognized as a unique, noninvasive biomarker of IDH-mutated gliomas. Noninvasive detection of IDH mutations via 2HG MRS represents a crucial step for decision-making and patient care. Despite its clinical utility, however, 2HG detection is still not part of the clinical routine due to technical challenges and the fact that advanced MRS techniques for 2HG quantification are available only in a few research centres worldwide. A subset of IDH-mutated tumors also presents a complete deletion of 1p and 19q chromosome arms (1p/19q codeletion). The 1p/19q codeletion is specifically linked to the oligodendroglial histologic subtype and it has been associated with a better patient outcome. However, the biological effects of this genetic alteration are still unclear and in vivo markers are lacking. Recently, we reported the first in vivo detection of the cystathionine molecule in human brain gliomas using MRS and explored the association between cystathionine accumulation and 1p/19q codeletion in gliomas. We hypothesized that cystathionine overproduction is linked to a partial deletion of chromosome 1p, yielding specific effects on cancer cell metabolism, and that cystathionine could be a marker of the compensatory anti-oxidant activity of cancer cells. In this project, we will combine cutting edge MRI and MRS techniques for metabolic and microstructural characterization of brain tumors with the aim of providing novel reliable noninvasive biomarkers of tumor genetic subtypes. These methods will allow us to identify noninvasively IDH-mutated gliomas and, potentially, 1p/19q codeleted gliomas. In addition, we will investigate the utility of 2HG, cystathionine and MRI microstructural markers to monitor tumor response to anti-cancer treatments and tumor progression. The outputs of this project, altogether, may open new avenues to a better understanding of the pathophysiological mechanisms of oncogenesis and the design of new treatments for gliomas. Advanced MRI/MRS methods will be translated into a clinical setting and will be shortly exploitable for diagnosis, prognosis, and planning of personalized therapies in the clinic.

phase I/II. Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease, characterized by muscular paralysis due to the loss of motoneurons in the spinal cord, motor cortex and the brainstem. The majority of cases are sporadic (90%) and familial forms are associated with different genes C9ORF72, SOD1... Currently, no treatment stops the progression of the disease and the patients die within 3 to 5 years after symptom onset. Therefore it is crucial to develop new therapeutic approaches especially for symptomatic patients. Our laboratory has demonstrated therapeutic efficacy of CYP46A1 overexpression, an neuronal enzyme from the cholesterol pathway, in several neurodegenerative diseases. The aim of my project is to perform all the preclinical studies to validate a gene therapy strategy for Amyotrophic Lateral Sclerosis (ALS) to restore the neuronal cholesterol mechanism, which is affected in the disease, through the delivery in motoneurons of its key enzyme : CYP46A1 thanks to an AAV vector. I just established the proof of concept of this approach in the severe mouse model of the pathology (SOD1G93A). A unique intravenous delivery of AAV-CYP46A1 not only prevent but also rescue motor abnormalities and histopathological of ALS. My aims are, 1 ) Complete the proof of concept, that I already established in the SOD1 model, in C9ORF72 model this confirming that this approach could be efficiently applied in different forms (genetic and potentially sporadic) of the disease, and perform a dose response study in SOD1G93A mouse model 2) Decipher the molecular, cellular and metabolic mechanisms by which AAV-CYP46A1 targeted to motoneurons may rescue the pathophysiological hallmarks of ALS and 3) perform the translational steps towards clinical phase I/II application: optimize the delivery of the AAV-CYP46A1 vector in non-human primates to efficiently target cortical and spinal motoneurons, define the clinical protocol and demonstrate tolerance.

The study of spinal circuits underlying locomotion has a rich history, spanning over 100 years of research. Great efforts have been made to map out the connection patterns between motor neurons and different muscle targets. Yet, we know little about how premotor networks govern the recruitment of motor neurons and the control of speed. Recent advances in optogenetics, 3D light patterning and fluorescent-targeted patch clamp recordings of identified neurons now enable to map the functional connectome for speed control in spinal motor circuits. Here we will focus on the role of V2a excitatory premotor interneurons since these cells have the potential to act as a key nexus in the spinal network: i) spanning both in the hindbrain and spinal cord, ii) making direct connections with motor neurons and iii) modulating locomotor speed. Recent studies revealed piecemeal information related to the V2a to motor neurons connection pattern. By taking advantage of the optical clarity of the zebrafish larva, we will implement a comprehensive approach combining in vivo electrophysiology, state-of-the-art 3D optical stimulation of genetically targeted cells and functional calcium imaging to resolve the connectivity map of V2a neurons and answer 3 new key questions: 1. What are the synaptic connections among V2as and between V2a and motor neurons within both slow and fast locomotion? 2. What is the degree of convergence and divergence between V2a neurons and motor neurons? 3. Do supraspinal inputs to the spinal cord segregate based on speed? A talented young scientist, with a great track record and the right expertise to tackle these ambitious questions, will carry out the project in the prolific research environment offered by the Wyart lab at the Brain and Spine Institute in Paris. This effort will lead to high impact publications as well as to develop the necessary skills to launch an independent research career for the applicant.