Long-term adenosine A1 receptor activation-induced sortilin expression promotes α-synuclein upregulation in dopaminergic neurons

Prolonged activation of adenosine A1 receptor likely leads to damage of dopaminergic neurons and subsequent development of neurodegenerative diseases.However,the pathogenesis underlying long-term adenosine A1 receptor activation-induced neurodegeneration remains unclear.In this study,rats were intraperitoneally injected with 5 mg/kg of the adenosine A1 receptor agonist N6-cyclopentyladenosine(CPA)for five weeks.The mobility of rats was evaluated by forced swimming test,while their cognitive capabilities were evaluated by Y-maze test.Expression of sortilin,α-synuclein,p-JUN,and c-JUN proteins in the substantia nigra were detected by western blot analysis.In addition,immunofluorescence staining of sortilin andα-synuclein was performed to detect expression in the substantia nigra.The results showed that,compared with adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine(5 mg/kg)+CPA co-treated rats,motor and memory abilities were reduced,surface expression of sortin andα-synuclein in dopaminergic neurons was reduced,and total sortilin and totalα-synuclein were increased in CPA-treated rats.MN9D cells were incubated with 500 nM CPA alone or in combination with 10μM SP600125(JNK inhibitor)for 48 hours.Quantitative real-time polymerase chain reaction analysis of sortilin andα-synuclein mRNA levels in MN9D cells revealed upregulated sortilin expression in MN9D cells cultured with CPA alone,but the combination of CPA and SP600125 could inhibit this expression.Predictions made using Jasper,PROMO,and Alibaba online databases identified a highly conserved sequence in the sortilin promoter that was predicted to bind JUN in both humans and rodents.A luciferase reporter assay of sortilin promoter plasmid-transfected HEK293T cells confirmed this prediction.After sortilin expression was inhibited by sh-SORT1,expression of p-JUN and c-JUN was detected by western blot analysis.Long-term adenosine A1 receptor activation levels upregulatedα-synuclein expression at the post-transcriptional level by affecting sortilin expression.The online tool Raptor-X-Binding and Discovery Studio 4.5 prediction software predicted that sortilin can bind toα-synuclein.Co-immunoprecipitation revealed an interaction between sortilin andα-synuclein in MN9D cells.Our findings indicate that suppression of prolonged adenosine A1 receptor activation potently inhibited sortilin expression andα-synuclein accumulation,and dramatically improved host cognition and kineticism.This study was approved by the University Committee of Animal Care and Supply at the University of Saskatchewan(approval No.AUP#20070090)in March 2007 and the Animals Ethics Committee of University of South China(approval No.LL0387-USC)in June 2017.

Bioinformatics analysis of epitope-based vaccine design against the novel SARS-CoV-2

Background An outbreak of infection caused by SARS-CoV-2 recently has brought a great challenge to public health.Rapid identification of immune epitopes would be an efficient way to screen the candidates for vaccine development at the time of pandemic.This study aimed to predict the protective epitopes with bioinformatics methods and resources for vaccine development.Methods The genome sequence and protein sequences of SARS-CoV-2 were retrieved from the National Center for Biotechnology Information(NCBI)database.ABCpred and BepiPred servers were utilized for sequential B-cell epitope analysis.Discontinuous B-cell epitopes were predicted via DiscoTope 2.0 program.IEDB server was utilized for HLA-1 and HLA-2 binding peptides computation.Surface accessibility,antigenicity,and other important features of forecasted epitopes were characterized for immunogen potential evaluation.Results A total of 63 sequential B-cell epitopes on spike protein were predicted and 4 peptides(Spike315–324,Spike333–338,Spike648–663,Spike1064–1079)exhibited high antigenicity score and good surface accessibility.Ten residues within spike protein(Gly496,Glu498,Pro499,Thr500,Leu1141,Gln1142,Pro1143,Glu1144,Leu1145,Asp1146)are forecasted as components of discontinuous B-cell epitopes.The bioinformatics analysis of HLA binding peptides within nucleocapsid protein produced 81 and 64 peptides being able to bind MHC class I and MHC class II molecules respectively.The peptides(Nucleocapsid66–75,Nucleocapsid104–112)were predicted to bind a wide spectrum of both HLA-1 and HLA-2 molecules.Conclusions B-cell epitopes on spike protein and T-cell epitopes within nucleocapsid protein were identified and recommended for developing a protective vaccine against SARS-CoV-2.