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MicroRNA-1（miR-1）是一种在肌肉中高表达的miRNA，且其在脑中的水平比在心脏中低100倍。近日，有研究人员发现心脏中miR-1的过表达通过外泌体运输参与SNAP-25转录调节并减弱海马中突触小泡的胞吐作用，这将有助于人们理解心血管疾病与脑功能障碍之间的关系。 该研究使用了两种动物模型，即microRNA-1-2转基因（Tg）小鼠的心脏特异性过表达模型和左冠状动脉结扎（leftcoronary artery ligation，LCA）诱导心肌梗塞小鼠模型。首先，研究人员使用原位杂交和透射电子显微镜（transmission electron microscopy，TEM）观察海马中的miR-1水平和突触小泡（synaptic vesicles，SV）分布，并通过蛋白质印迹评估囊泡胞吐相关蛋白的表达，再使用双荧光素酶报告基因测定来鉴定miR-1对Snap25基因的转录后调节作用，FM1-43染色以研究miR-1对突触小泡胞吐作用的影响，最后使用GW4869抑制外泌体的生物发生和分泌，以确定外泌体对miR-1从心脏到大脑的转运效果的影响。结果发现，与年龄匹配的WT小鼠中的水平相比，Tg小鼠的心脏和海马中的miR-1水平增加，伴随着SV的重新分布和胞吐作用相关蛋白SNAP-25表达减少。体外研究显示，SNAP-25蛋白表达分别通过miR-1过表达或抑制而下调或上调，然而，通过miRNA结合Snap25基因的3'UTR却没有改变。通过miR-1过表达抑制SV胞吐作用，这可以通过与抗miR-1寡核苷酸片段（AMO-1）的共转染而拮抗。通过携带AMO-1的慢病毒载体（lenti-pre-AMO-1）进行海马立体定向注射敲除miR-1，会使SNAP-25表达上调并抑制Tg小鼠海马突触中的SV浓度。 这项研究为揭示心脏与大脑连接的分子机制提出新见解，并表明心脏通过外泌体转运miR-1调节海马突触小泡的胞吐作用，两者之间存在潜在沟通。
推荐阅读原文：
Overexpression of miR-1 in the heart attenuates hippocampal synaptic vesicle exocytosis by the posttranscriptional regulation of SNAP-25 through the transportation of exosomes.
BACKGROUND:
The link between cardiac diseases and cognitive deterioration has been accepted from the concept of 'cardiogenic dementia', which was proposed in the late 1970s. However, the molecular mechanism is unclarified.
METHODS:
The two animal models used in this study were cardiac-specific overexpression of microRNA-1-2 transgenic (Tg) mice and a myocardial infarction mouse model generated by left coronary artery ligation (LCA). First, we observed the microRNA-1 (miR-1) level and synaptic vesicles (SV) distribution in the hippocampus using in situ hybridization and transmission electron microscopy (TEM) and evaluated the expression of vesicle exocytosis related proteins by western blotting. Second, we used dual luciferase reporter assay as well as antagonist and miRNA-masking techniques to identify the posttranscriptional regulatory effect of miR-1 on the Snap25 gene. Third, FM1-43 staining was performed to investigate the effect of miR-1 on synaptic vesicle exocytosis. Lastly, we used GW4869 to inhibit the biogenesis and secretion of exosomes to determine the transportation effect of exosomes for miR-1 from the heart to the brain.
RESULTS:
Compared with the levels in age-matched WT mice, miR-1 levels were increased in both the hearts and hippocampi of Tg mice, accompanied by the redistribution of SVs and the reduction in SV exocytosis-related protein SNAP-25 expression. In vitro studies showed that SNAP-25 protein expression was down- or upregulated by miR-1 overexpression or inhibition, respectively, however, unchanged by miRNA-masking the 3'UTR of the Snap25 gene. SV exocytosis was inhibited by miR-1 overexpression, which could be prevented by co-transfection with an anti-miR-1 oligonucleotide fragment (AMO-1). The knockdown of miR-1 by hippocampal stereotaxic injection of AMO-1 carried by a lentivirus vector (lenti-pre-AMO-1) led to the upregulation of SNAP-25 expression and prevented SV concentration in the synapses in the hippocampi of Tg mice. The application of GW4869 significantly reversed the increased miR-1 level in the blood and hippocampi as well as reduced the SNAP-25 protein levels in the hippocampi of both Tg and LCA mice.
CONCLUSION:
The overexpression of miR-1 in the heart attenuated SV exocytosis in the hippocampus by posttranscriptionally regulating SNAP-25 through the transportation of exosomes. This study contributes to the understanding of the relationship between cardiovascular disease and brain dysfunction.
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