Abstract
Nonmuscle myosin ⅡA, a kind of ATP-dependent molecular motor, binds actin to form the molecular motors of the cell. We found that interfering with nonmuscle myosin heavy chain (NMMHC) ⅡA could affect the exosome release from microglial cells stimulated by LPS. LPS could enhance exosome release from microglial cells by increasing exosome concentration, elevating the rate of positively labeled CD9 and CD81 proteins and protein expression. The myosin inhibitor, blebbistatin, could decrease the concentration of released exosome and reduce CD9 and CD81 protein expression on the exosome surface compared with that in the LPS group. To further determine the exact subtype of myosin Ⅱ responsible for these effects, we transfected microglial cells with siRNA for MYH9, MYH10, and MYH14. The data showed that only the transfection of siRNA-MYH9, but not MYH10 or MYH14 could decrease the released exosome concentration and particle size compared with those in the LPS group. siRNA-MYH9 would also weaken the CD9 and CD81 protein positive rate and protein expression compared with that in the LPS group by the quantification of CD9 and CD81 fluorescence intensities and by western blotting. Western blots and immunofluorescence assays indicated that NMMHC ⅡA might trigger the ROCK1/MLC/actin signaling pathway of microglial cells upon stimulation by LPS, which might be the potential mechanism of exosome release. These observations demonstrated that NMMHC ⅡA might be the potential target required for exosome release.
Highlights
In the early stage of acute cerebral ischemia injury, the immune system is activated in response to ischemia damage
A large number of inflammatory mediators are released from microglial cells, which encapsulate proteins, mRNA, nonmuscle myosin heavy chain (NMMHC) IIA Mediated Exosome Release miRNAs, cytokines, and other substances that are released into the circulatory system through immune defense cells (Jeppesen et al, 2019)
The data showed that the NMMHC IIA could interfere with exosome release from microglia stimulated by LPS
Summary
In the early stage of acute cerebral ischemia injury, the immune system is activated in response to ischemia damage. Our previous research indicated that compared with neurons, the microglial cells demonstrated earlier apoptosis in response to LPS stimulation (Lv et al, 2016). Extensive research has been done on the function of exosomes, such as the confinement of miRNA, mRNA, and proteins in cells or tissues that secreted them. Our previous study indicated that microglia could release microvesicles containing abnormally elevated expression levels of microRNAs, such as miR-146a (Lv et al, 2016) and miR-27a (Lv et al, 2018). There have been limited reports on the mechanisms of intervention in the process of exosome release by microglia cells under the stimulation of LPS. This study mainly focused on the explanation of release mechanisms of exosomes in the context of ischemia stroke diseases
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