Abstract
Ubiquitinating enzymes catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme (DUB) action. Ubiquitin-specific protease 4 (USP4) is a member of the ubiquitin-specific protease (USP) family of DUBs that has a role in spliceosome regulation. In the present study, we demonstrated that USP4 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). We obtained a significant up-regulation of USP4 in neurons adjacent to the hematoma following ICH by the results of Western blot, immunohistochemistry, and immunofluorescence. Increasing USP4 level was found to be accompanied by the up-regulation of active caspase-3, γH2AX, Bax, and decreased expression of Bcl-2. In addition, USP4 co-localized well with γH2AX in the nucleus in the ICH model and hemin-induced apoptosis model. Moreover, in vitro study, knocking down USP4 by USP4-specific siRNA in PC12 cells reduced active caspase-3 expression. All these results above suggested that USP4 may be involved in neuronal apoptosis after ICH.
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Acknowledgments
This work was supported by the National Basic Research Program of China (973 Program, No.2012CB822104); the National Natural Science Foundation of China (No.31270802); Technology Innovation Programme of Nantong University (YKC15068), Technology Innovation Programme of Jiangsu Province (KYZZ15_0351), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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Chao Liu and Chun Liu contributed equally to this work.
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10571_2016_375_MOESM1_ESM.tif
Primary neurons were incubated with hemin at 100 μmol/L for different times. USP4 and active caspase-3 were up-regulated (a). The bar graph indicated the relative density of USP4 and active caspase-3 versus GAPDH at each time point (b). Data are presented as mean ± SEM (n = 3, *#P < 0.05)(TIFF 25519 kb)
10571_2016_375_MOESM2_ESM.tif
PC12 cells were incubated with hemin at 100 μmol/L for different times. USP4, active caspase-3 and γH2AX were up-regulated. (a). The bar graph indicated the relative density of USP4, active caspase-3, and γH2AX versus GAPDH at each time point (b). Immunofluorescent analysis of the co-localization between USP4 and γH2AX in PC12 cells after hemin treatment (c). The knockdown of USP4 induced decreasing levels of γH2AX expression (d). The bar graph indicated the relative density of γH2AX versus GAPDH (e). Data are presented as mean ± SEM (n = 3, *,^,#P < 0.05)(TIFF 25526 kb)
10571_2016_375_MOESM3_ESM.tif
The expression of USP4, active caspase-3 and γH2AX were relatively low in the sham group, however, up-regulated gradually following ICH, peaked at day 2 and declined thereafter (A). The bar graph indicated the density of USP4, active caspase-3, and γH2AX versus GAPDH at each time point (B). Data are presented as mean ± SEM (n = 3, *,^,#P < 0.05) Immunofluorescent analysis of the co-localization between USP4 and γH2AX around the hematoma at 2d after ICH (C)(TIFF 25522 kb)
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Liu, C., Liu, C., Liu, H. et al. Increased Expression of Ubiquitin-Specific Protease 4 Participates in Neuronal Apoptosis After Intracerebral Hemorrhage in Adult Rats. Cell Mol Neurobiol 37, 427–435 (2017). https://doi.org/10.1007/s10571-016-0375-y
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DOI: https://doi.org/10.1007/s10571-016-0375-y