Synaptosomes secrete and uptake functionally active microRNAs via exocytosis and endocytosis pathways.
In this study, we first characterized synaptosome microRNA (miRNA) profiles using microarray and qRT-PCR. MicroRNAs were detected in isolated synaptic vesicles, and Ago2 immunoprecipitation studies revealed an association between miRNAs and Ago2. Second, we found that miR-29a, miR-99a, and miR-125a were significantly elevated in synaptosome supernatants after depolarization. MiRNA secretion by the synaptosome was Ca(2+)-dependent and was inhibited by the exocytosis inhibitor, okadaic acid. Furthermore, application of nerve growth factor increased miRNA secretion without altering the spontaneous release of miRNAs. Conversely, kainic acid decreased miRNA secretion and enhanced the spontaneous release of miRNAs. These results indicate that synaptosomes could secrete miRNAs. Finally, synthesized miRNAs were taken up by synaptosomes, and the endocytosis inhibitor Dynasore blocked this process. After incubation with miR-125a, additional miR-125a was bound to Ago2 in the synaptosome, and expression of the miR-125a target gene (PSD95 mRNA) was decreased; these findings suggest that the ingested miRNAs were assembled in the RNA-induced silencing complex, resulting in the degradation of target mRNAs. To our knowledge, this is the first study that demonstrates the secretion of miRNAs by synaptosomes under physiological stimulation and demonstrates that secreted miRNAs might be functionally active after being taken up by the synaptic fraction via the endocytic pathway.
Jiangsu Engineering Research Center for Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China.
Jie
J
Xu
Qun
Q
Chen
Ke
K
Zen
Chenyu
C
Zhang
Qipeng
Q
Zhang
D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism; D001921:Brain / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism; D000076122:Disks Large Homolog 4 Protein; D004705:Endocytosis / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D018698:Glutamic Acid / Q000494:pharmacology; D051528:Guanylate Kinases / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D053858:Metabolic Networks and Pathways / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D046529:Microscopy, Electron, Transmission; D020932:Nerve Growth Factor / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism* / Q000648:ultrastructure; D050863:Synaptotagmin I
C526735:Ago2 protein, mouse; D060565:Argonaute Proteins; D000076122:Disks Large Homolog 4 Protein; C501447:Dlg4 protein, mouse; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D035683:MicroRNAs; D012333:RNA, Messenger; D050863:Synaptotagmin I; C494311:Syt1 protein, mouse; D019319:Okadaic Acid; D018698:Glutamic Acid; D020932:Nerve Growth Factor; D007770:L-Lactate Dehydrogenase; D051528:Guanylate Kinases; D002118:Calcium