Loss of the lysosomal lipid flippase ATP10B leads to progressive dopaminergic neurodegeneration and Parkinsonian motor deficits

Abstract

ABSTRACTBackgroundATP10B, a transmembrane lipid flippase located in late endosomes and lysosomes, facilitates the export of glucosylceramide and phosphatidylcholine by coupling this process to ATP hydrolysis. Recently, loss-of-function mutations in theATP10Bgene have been identified in Parkinson’s disease patients, pointing toATP10Bas a candidate genetic risk factor. Previous studies have shown compromised lysosomal functionality uponATP10Bknockdown in human cell lines and primary cortical neurons. However, its rolein vivoand specifically in the nigrostriatal dopaminergic system remains poorly understood.MethodsTo investigate the role ATP10B in PD neuropathology, we induced ATP10B knockdown specifically insubstantia nigra pars compactaneurons of rats using viral vector technology. Two different microRNA-based shRNA constructs targeting distinct regions of the ATP10B mRNA were used to cross-validate the findings. Behavioral evaluation, dopamine transporter18F-FE-PE2I positron emission tomography imaging and neuropathological examination of the nigrostriatal pathway at one year post-injection were conducted. Additionally, midbrain neuronal cultures derived from ATP10B knock-out human induced pluripotent stem cells clones were used to study the impact of ATP10B loss in dopaminergic neurons in a more translational model.ResultsATP10Bknockdown in rat brain induced Parkinsonian motor deficits, and longitudinal striatal dopamine transporter18F-FE-PE2I PET imaging revealed a progressive decrease in binding potential. Immunohistochemical analysis conducted one year post-injection confirmed the loss of dopaminergic terminals in thestriatum, alongside a loss of dopaminergic neurons in thesubstantia nigra pars compacta. The expression of LAMP1, LAMP2a, cathepsin B and glucocerebrosidase was studied by immunofluorescence in the surviving dopaminergic neurons. A decrease in lysosomal numbers and an increase in lysosomal volume were observed more consistently in one of the knockdown constructs. The vulnerability of dopaminergic neurons to ATP10B loss-of-function was also observed in midbrain neuronal cultures derived from ATP10B knock-out human induced pluripotent stem cells clones, which showed a significant reduction in TH-positive neurons.ConclusionTaken together, our findings demonstrate that ATP10B depletion detrimentally impacts the viability of dopaminergic neurons bothin vivoandin vitro. Moreover, a broader impact on the functionality of the nigrostriatal pathway was evidenced as rats withATP10Bknockdown exhibited motor impairments similar to those observed in PD patients.