Les neurodégénérescences avec accumulation de fer dans le cerveau (Neurodegeneration with Brain Iron Accumulation, NBIA) sont des maladies neurodégénératives progressives, génétiquement hétérogènes. On connait actuellement 11 gènes de ces maladies mais pour la plupart d’entre eux leur lien avec l’accumulation en fer est encore incompris. Ce travail de thèse présente deux nouveaux gènes de NBIA identifiés par séquençage d’exome dans deux familles indépendantes. Le premier gène, REPS1, est impliqué dans le recyclage de l’endosome. Les fibroblastes de patients sont caractérisés par une accumulation de fer qui est corrigée par l’expression de l’ADNc de REPS1 dans ces cellules. Le deuxième gène, CRAT, code une carnitine acétyltransferase et le déficit de β-oxydation détecté dans les fibroblastes du patient a été corrigé par l’expression de l’ADNc CRAT normal. Le rôle de REPS1 dans le recyclage de l’endosome a mis sur la voie du mécanisme physiopathologique des NBIA. En effet, les fibroblastes des patients REPS1 et CRAT mais aussi d’autres patients avec des mutations d’autres gènes connus de NBIA (PANK2, PLA2G6, FA2H, C19ORF12) ont une accumulation massive en fer et une anomalie de recyclage du récepteur à la transferrine (TfR1). TfR1 permet l’entrée du fer par endocytose et son expression est régulée par le contenu en fer des cellules. La seule régulation connue de l’homéostasie du fer se fait au niveau post-transcriptionnel par le système IRP/IRE qui est fonctionnel dans les fibroblastes NBIA alors que la protéine TfR1 s’accumule. Cette accumulation de fer montre ainsi qu’il existe une régulation post-traductionnelle, jusqu’ici inconnue, et qui n’est pas fonctionnelle dans les NBIA. Nous avons pu montrer que cette régulation se faisait par une palmitoylation du TfR1, déficitaire dans les NBIA, mais restaurée par l’artesunate. Ainsi quel que soit le gène muté, tous les NBIA résultent d’une anomalie de recyclage du TfR1 permettant de les définir comme des maladies du trafic intracellulaire. La deuxième partie de la thèse s’intéresse au mécanisme physiopathologique de l’ataxie de Friedreich (FRDA) caractérisée elle aussi par une accumulation de fer dans le cerveau. FRDA est due à des expansions de triplets dans le premier intron du gène FXN conduisant à l’extinction de FXN et de PIP5K1B situé en amont. L’étude de modèles cellulaires dans lesquels le gène FXN et/ou PIP5K1B ont été éteints par siRNA et de fibroblastes de patients a permis de mettre en évidence une anomalie de l’homéostasie du fer qui rappelle celle observée dans les NBIA. L’ensemble de ces résultats a permis de comprendre le mécanisme physiopathologique des NBIA, de mettre à jour une régulation encore inconnue de l’homéostasie du fer mais aussi d’envisager une voie de traitement des NBIA. / Neurodegeneration with brain iron accumulation (NBIA) encompasses a group of rare neurogenerative disorders with different clinical and molecular features, underlined by progressive extrapyramidal dysfunction and iron accumulation in the brain. To date, mutations in 11 genes are currently known. Nevertheless for most of them their link with iron accumulation is still misunderstood. This work presents two novel NBIA genes identified by exome sequencing in two independent families. The first gene, REPS1, is involved in endosome recycling. Patient’s fibroblasts are characterized by iron overload corrected by wild-type REPS1 cDNA overexpression. The second gene, CRAT, encodes a carnitine acetyltransferase and a β-oxidation deficit in patient’s fibroblasts has been fixed by overexpression of wild-type CRAT cDNA. The function of REPS1 in endosome recycling put on the path of the NBIA pathophysiological mechanism. Indeed, fibroblasts of REPS1 patients but also from other patients mutated in various NBIA genes (CRAT, PANK2, PLA2G6, FA2H, C19ORF12) present massive iron accumulation and abnormal transferrin receptor (TfR1) recycling. TfR1 allows iron uptake by endocytosis and its expression is regulated by the iron cellular status. The only known regulation of iron homeostasis occurs at the posttranscriptional level by the IRE/IRP system which is functional in NBIA fibroblasts whereas TfR1 protein accumulates. This iron accumulation highlights a yet unknown posttranslational regulation which is not functional in NBIA. We have been able to demonstrate that this regulation occurs via TfR1 palmitoylation, which is defective in NBIA, but restored by artesunate. Hence, whatever the disease gene, all NBIA gave rise to abnormal TfR1 recycling which allows defining NBIA as intracellular trafficking disease. The second part of the thesis focused on the pathophysiological mechanism of the Friedreich ataxia (FRDA) also characterized by brain iron overload . FRDA is related to triplets expansions in the first intron of FXN gene leading to the extinction of FXN and PIP5K1B upstream gene. Studying cellular models knocked down for FXN and/or PIP5K1B by siRNA and patients’ fibroblasts of patients allowed to detect abnormal iron homeostasis reminiscent of NBIA. All these results allowed to decipher the NBIA pathophysiological mechanism, to highlight a yet unknown iron homeostasis regulation and to open possible ways towards therapeutic drugs for NBIA.

Neurodegeneration with Brain Iron Accumulation (NBIA) disorders, such as Phospholipase A2G6-Associated Neurodegeneration (PLAN) and Pantothenate Kinase-Associated Neurodegeneration (PKAN), are a group of rare early-onset, genetic disorders characterized by neurodegeneration and iron accumulation inside of the basal ganglia (BG), which is accompanied by progressive motor symptoms. In order to address the limitations in available models of NBIA, a B6.C3-Pla2g6m1J/CxRwb mouse model of PLAN was characterized. This model demonstrated key hallmarks of the disease presentation in NBIA, including a severe and early-onset motor deficit, neurodegeneration inside of the substantia nigra (SN) including a loss of dopaminergic function and the formation of abnormal spheroid inclusions as well as iron accumulation. The capture of these hallmarks of NBIA makes this an ideal animal research model for NBIA. Additionally, exploration of candidate systemic biomarkers of NBIA was performed in a case study of a patient with PLAN and in a cohort of 30 patients with PKAN. These investigations demonstrated reductions in transfer and slight, but not significant elevations in soluble transferrin receptor. No significant difference was seen in serum iron parameters. A systemic disease burden including chronic oxidative stress; elevated malondialdehyde, and inflammation; elevated C-reactive protein (CRP), IL-6 and TNFα was noted in both investigations. A number of candidate protein biomarkers including: fibrinogen, transthyretin, zinc alpha-2 glycoprotein and retinol binding protein were also identified. These markers correlated with measures of the severity of iron loading in the globus pallidus (GP); based on R2* magnetic resonance imaging (MRI) and the severity of motor symptoms (Barry-Albright Dystonia Rating Scale) making them potential candidates markers of dysfunction in NBIA. In the patient with PLAN, 37 weeks of therapy with the iron chelator deferiprone (DFP) as well as 20 months of therapy with the antioxidants alpha lipoic acid (ALA) and n-acetylcysteine (NAC) were efficacious in reducing the systemic oxidative and inflammatory disease burden, but it did not significantly alter the progression of the disease. In the antioxidant therapy, this efficacy was primarily due to ALA. When the cohort of patients with PKAN were treated with DFP for 18 months it was highly efficacious in lowering brain iron accumulation in the GP. No significant reduction in the speed of disease progression was seen in DFP treated patients compared to placebo based on initial analysis. Similar to the PLAN patient, DFP also mitigated the systemic disease burden in PKAN patients. In both cases DFP was well tolerated and had minimal impact on serum iron levels, TIBC and transferrin saturation. Collectively these investigations provide valuable insights into disease progression in NBIA. They also provide tools to aid further investigations in NBIA. These are provided in the form of a well-characterized B6.C3-Pla2g6m1J/CxRwb model of PLAN, which robustly captures the disease presentation seen in patients, as well as a panel of systemic blood-based markers of disease burden in NBIA and candidate markers of dysfunction in NBIA. These markers were used to assess two novel therapies in NBIA chelation with DFP and antioxidant therapy with ALA and NAC. / Graduate / 2019-04-19