Parfitt, G. | bioRxiv Science

bioRxiv2024

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

BackgroundATP10B, 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 the ATP10B gene have been identified in Parkinsons disease patients, pointing to ATP10B as a candidate genetic risk factor. Previous studies have shown compromised lysosomal functionality upon ATP10B knockdown in human cell lines and primary cortical neurons. However, its role in vivo and specifically in the nigrostriatal dopaminergic system remains poorly understood. MethodsTo investigate the role ATP10B in PD neuropathology, we induced ATP10B knockdown specifically in substantia nigra pars compacta neurons 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 transporter 18F-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. ResultsATP10B knockdown in rat brain induced Parkinsonian motor deficits, and longitudinal striatal dopamine transporter 18F-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 the striatum, alongside a loss of dopaminergic neurons in the substantia 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 both in vivo and in vitro. Moreover, a broader impact on the functionality of the nigrostriatal pathway was evidenced as rats with ATP10B knockdown exhibited motor impairments similar to those observed in PD patients.

neuroscience↗

bioRxiv2020

KRASG12D mutant cells are outcompeted by wild type neighbours in adult pancreas in an EPHA2-dependent manner.

As we age, our tissues are repeatedly challenged by mutational insult, yet cancer occurrence is a relatively rare event. Cells carrying cancer-causing genetic mutations compete with normal neighbours for space and survival in tissues. However, the mechanisms underlying mutant-normal competition in adult tissues and the relevance of this process to cancer remain incompletely understood. Here, we investigate how the adult pancreas maintains tissue health in vivo following sporadic expression of oncogenic Kras (KrasG12D), the key driver mutation in human pancreatic cancer. We find that when present in tissues in low numbers, KrasG12D mutant cells are outcompeted and cleared from exocrine and endocrine compartments in vivo. Using quantitative 3D tissue imaging, we show that prior to being cleared, KrasG12D cells lose cell volume, segregate from normal cells and decrease E-cadherin-based cell-cell adhesions with normal neighbours. We identify EphA2 receptor is an essential signal in the clearance of KrasG12D cells from exocrine and endocrine tissues in vivo. In the absence of functional EphA2, KrasG12D cells no longer segregate, E-cadherin-based cell-cell adhesions increase and KrasG12D cells are retained in tissues. Retention of KRasG12D cells leads to an increased burden of premalignant pancreatic intraepithelial neoplasia (PanINs) in tissues. Our data show that adult pancreas tissues remodel to clear KrasG12D cells and maintain tissue health. This study provides evidence to support a conserved functional role of EphA2 in Ras-driven cell competition in epithelial tissues and suggests that EphA2 is a novel tumour suppressor in pancreatic cancer.

cell biology↗