• 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • Although DAPK has been implicated in neuronal


    Although DAPK has been implicated in neuronal death in ischemic stroke and neurodegenerative diseases, such as Alzheimer’s disease (Shamloo et al., 2005, Duan et al., 2013), the mechanisms about how DAPK regulates cell death are still elusive. In the present study, we investigated the signal transduction pathways involved in DAPK-mediated neuronal death in response to oxygen glucose deprivation (OGD).
    Experimental procedures
    Discussion It has been suggested that DAPK is involved in neuronal death during ischemic Nimodipine injury (Schumacher et al., 2002, Bialik and Kimchi, 2004). However, molecular mechanisms about how DAPK mediates neuronal death are still unclear. Findings from the present study demonstrated that DAPK was activated during OGD and transfection of DAPK shRNA reduced neuronal death caused by OGD exposure. Findings also revealed that BimEL was a downstream effector of DAPK in its death-promoting process. Further analysis indicated that DAPK regulated BimEL through inactivation of ERK1/2 and activation of JNK1/2. Thus, these findings revealed novel molecular pathways mediating neuronal death in response to OGD. DAPK activation occurred rapidly after cells were exposed to OGD, with increased DAPK and decreased phospho-DAPK detected in cells received 0.5h OGD treatment. Although substantial cell death was observed in cells exposed to 4h or longer OGD exposure, no marked cell death was observed in cells treated with OGD for 2h or less (data not shown), suggesting DAPK activation occurred prior to the neuronal death. DAPK knockdown significantly increased cell survival in the cells treated with 8- or 16-h OGD, strongly implying that DAPK signaling pathway was indispensible in the cell death during OGD. A previous study has shown that DAPK-induced cell death involves ER stress activation (Gozuacik et al., 2008). During ER stress, DAPK is activated through dephosphorylation at Ser308. Primary fibroblasts from DAPK knockout mice are protected from ER stress-induced cell death. DAPK knockout mice are protected from kidney damage caused by ER stress (Gozuacik et al., 2008). In the experimental setting here, exposing the cells to OGD caused ER stress activation, as increased phospho-eIF2α and CHOP levels were detected. The increase of phospho-eIF2α and CHOP expressions were observed later than DAPK activation. Furthermore, DAPK shRNA transfection resulted in decreased phospho-eIF2α and CHOP expressions, suggesting ER stress activation was a downstream event of DAPK signaling upon OGD treatment. Bim is a pro-apoptotic member of Bcl-2 family and has been found essential for ER stress-induced cell death in different types of cells in in vitro or in vivo studies. For example, thymocytes and macrophages from Bim-deficient mice are more resistant to ER stress caused cell death (Puthalakath et al., 2007). In the present study, BimEL level was highly increased following OGD treatment. BimS also increased after OGD treatment, but the level of BimS was very low. The basal level of BimL was very low in these cells and it did not change following the OGD treatment (data not shown). Therefore, subsequent studies focused on the role of BimEL in DAPK-mediated neuronal death. Bax is a pro-apoptotic Bcl-2 family member. In some studies, Bax is up-regulated in its protein level during apoptosis, while in others only Bax mitochondrial translocation is found (Lang-Rollin et al., 2005). In the present study, Bax expression was not changed following the OGD exposure. Whether Bax mitochondrial translocation contributed to the OGD-induced cell death still needs to be investigated. Although Bcl-2 and Bcl-xl are the Bcl-2 members that protect cells from apoptosis, Bcl-2 and Bcl-xl levels did not change significantly following the OGD exposure. Similar results have also been observed during apoptosis in previous studies (Yang et al., 2000, Ribas and Boix, 2004), suggesting other mechanisms may be responsible for the cell death. A previous study also supports that Bim and Bcl-2 balance is critical in controlling memory T cell fate. Overexpression of Bim over Bcl-2 leads to more T cells being killed (Wojciechowski et al., 2007). In the present study, BimEL level was increased while Bcl-2 level was unchanged after OGD exposure, suggesting increased BimEL/Bcl-2 ratio may also played a role in the OGD-induced neuronal death.