Studies On The Function Of NUS1,a Risk Factor For Parkinson’s Disease,in Drosophila

Background:NgBR is the Nogo-B receptor,encoded by NUS1 gene.Ng BR was speculated to stabilize nascent Niemann-Pick type C 2(NPC2)to facilitate cholesterol transport out of lysosomes.Mutations in the NUS1 were known as risk factors for Parkinson’s disease(PD).PD is a common neurodegenerative disorder characterized by a constellation of motor symptoms including bradykinesia,rest tremor and rigidity.The pathological hallmarks are loss of dopaminergic(DA)neurons in the substantia nigra pars compacta(SNc),which is accompanied by the formation of α-synuclein(α-syn)fibrils in neurons.Genetic,aging and environmental factors are known risk factors for PD.However,the underlying mechanisms of PD remain to be fully understood.Our previous study showed that knockdown of Drosophila NUS1 orthologous gene tango14 causes typical PD phenotype,including decreased climbing ability and loss of dopaminergic neurons.It suggests that tango14 plays an important role in the pathogenesis of PD.In this study,we will establish the Drosophila model to clarify the function of NUS1 in the regulatory mechanism of NUS1 in PD.Objective: To illustrate the function of NUS1 in neurodegeneration and explore the role of NUS1 in the pathogenesis of PD.Methods: Tango14 mutant flies were generated by CRISPR/Cas9.The UAS/Gal4 system was used to knockdown of tango14 RNAi lines.These two models were used in following experiments.First,life span and locomotion were recorded.To clarify the effect of tango14 on neurodegeneration,immunostaining and ultrastructure of electron microscope were used to measure pathological changes of vacuolation,apoptosis and cholesterol accumulation,Second,the changes of tango14 mutant dopaminergic neurons and cholesterol accumulation in dopaminergic neurons were analyzed by high performance liquid chromatography(HPLC)and immunostaining.In the PD model Drosophila with α-synuclein(α-syn)overexpression,we used tango14 knockdown to study α-synuclein locomotion,lifespan,dopaminergic neurons,and α-synuclein aggregation,to analyze the function of tango14 in PD.Results: In this study,tango14 mutant flies were generated with a mutation in the C-terminal enzyme activity region using CRISPR/ Cas9.Tango14 mutant showed reduced lifespan with locomotive defects.Neurodegenerative-related brain vacuolization and apoptosis were also detected in tango14 knockdown flies.Multilamellar bodies(MLBs)and cholesterol accumulation were found in Malpighian tubules and brains in tango14 mutants and knockdown flies.Cholesterol accumulation phenotype in the brain was rescued by feeding 2H-β-CD.These results indicate that tango14 is important for regulating cholesterol trafficking in neurons and could induce neurodegeneration.In addition,DA content decreases and cholesterol accumulation in DA neurons in tango14 mutants brain were rescued by feeding 2H-β-CD.These results suggest that DA neuron injury was caused by abnormal cholesterol transport in tango14 mutants.Tango14 knockdown increased α-synuclein neurotoxicity in α-syn-overexpressing flies,with decreased lifespan and locomotive activities,reduced dopaminergic neurons,and the aggregate of Proteinase K(PK)resistant α-synuclein.These results indicate that tango14 regulated cholesterol transport in neurons.Tango14 mutation or knockdown can lead to the neurotoxicity of dopaminergic neurons,and exacerbate the aggregation of α-synuclein.Conclusion: We successfully made a tango14 mutant in Drosophila,presenting typical neurodegeneration features and hallmark pathological of PD.Tango14 mutants induced cholesterol accumulation and led to loss of dopaminergic neurons,revealing the mechanism of neurodegeneration caused by tango14 mutants.Tango14 is involved in the regulation ofα-synuclein neurotoxicity and protein aggregation,leading to pathogenesis.