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Mitochondrial BNIP3 upregulation precedes endonuclease G translocation in hippocampal neuronal death following oxygen-glucose deprivation.

Zhao ST, Chen M, Li SJ, Zhang MH, Li BX, Das M, Bean JC, Kong JM, Zhu XH, Gao TM - BMC Neurosci (2009)

Bottom Line: Importantly, the mitochondrial upregulation of BNIP3 precedes the translocation of EndoG.Forced expression of BNIP3 increases the nuclear translocation of EndoG and neuronal death while knockdown of BNIP3 decreases the OGD-induced nuclear translocation of EndoG and neuronal death.These results suggest that BNIP3 and EndoG play important roles in hippocampal neuronal apoptosis following ischemia, and mitochondrial BNIP3 is a signal protein upstream of EndoG that can induce neuronal death.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology, Southern Medical University, Guangzhou, PR China. zhaoshenting2004@126.com

ABSTRACT

Background: Caspase-independent apoptotic pathways are suggested as a mechanism for the delayed neuronal death following ischemic insult. However, the underlying signalling mechanisms are largely unknown. Recent studies imply the involvement of several mitochondrial proteins, including endonuclease G (EndoG) and Bcl-2/adenovirus E1B 19 kDa-interacting protein (BNIP3), in the pathway of non-neuronal cells.

Results: In this report, using western blot analysis and immunocytochemistry, we found that EndoG upregulates and translocates from mitochondria to nucleus in a time-dependent manner in cultured hippocampal neurons following oxygen-glucose deprivation (OGD). Moreover, the translocation of EndoG occurs hours before the observable nuclear pyknosis. Importantly, the mitochondrial upregulation of BNIP3 precedes the translocation of EndoG. Forced expression of BNIP3 increases the nuclear translocation of EndoG and neuronal death while knockdown of BNIP3 decreases the OGD-induced nuclear translocation of EndoG and neuronal death.

Conclusion: These results suggest that BNIP3 and EndoG play important roles in hippocampal neuronal apoptosis following ischemia, and mitochondrial BNIP3 is a signal protein upstream of EndoG that can induce neuronal death.

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Related in: MedlinePlus

EndoG nuclear translocation preceded neuronal cell death following reoxygenation. (A-D) Confocal laser scanning microscope images of EndoG immunoreactivity (red) at control, EBSS, OGD/R6 h, OGD/R24 h, respectively. Co-staining with DAPI (blue) allowed the identification of nuclear translocation of EndoG. The white arrow indicates the nucleus with EndoG translocation but without pyknosis, and the white arrowhead indicates the pyknotic nucleus with EndoG translocation; (E) Percentage of damaged neurons and neurons displaying nuclear EndoG at different time points following reoxygenation. *p < 0.05 vs. control group, n = 9.
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Figure 3: EndoG nuclear translocation preceded neuronal cell death following reoxygenation. (A-D) Confocal laser scanning microscope images of EndoG immunoreactivity (red) at control, EBSS, OGD/R6 h, OGD/R24 h, respectively. Co-staining with DAPI (blue) allowed the identification of nuclear translocation of EndoG. The white arrow indicates the nucleus with EndoG translocation but without pyknosis, and the white arrowhead indicates the pyknotic nucleus with EndoG translocation; (E) Percentage of damaged neurons and neurons displaying nuclear EndoG at different time points following reoxygenation. *p < 0.05 vs. control group, n = 9.

Mentions: To evaluate the role of nuclear EndoG translocation in OGD, we investigated the relation between EndoG translocation and cell death. At different time points after reoxygenation, the intracellular localization of EndoG was detected by immunocytochemistry; the morphologically damaged neurons were identified by condensed and fragmented nuclei using DAPI nuclear staining. The result revealed that EndoG was mainly located in the intracellular space out of the nucleus shown as red fluorescence in normal control group or in EBSS-treated group under normoxic condition (Figure 3A and 3B). Consistent with the results from the previous western blot analysis, the nuclear translocation of EndoG started at 2 h after reoxygenation and increased progressively (Figure 3C-E). Significant numbers of neurons displaying EndoG nuclear staining were observed 6 h after reoxygenation, a time point when no obvious morphological neuronal damage could be observed (Figure 3C and 3E). Thereafter, although the number of morphologically damaged neurons also increased with time, a great quantity of pyknotic nuclei could not be observed until 12 h after reoxygenation. Furthermore, the proportions of morphologically damaged neurons were less than that of the EndoG translocated neurons at 12 h and 24 h, respectively (Figure 3D and 3E). These results demonstrate that EndoG translocation from mitochondria to nucleus precedes neuronal cell death.


Mitochondrial BNIP3 upregulation precedes endonuclease G translocation in hippocampal neuronal death following oxygen-glucose deprivation.

Zhao ST, Chen M, Li SJ, Zhang MH, Li BX, Das M, Bean JC, Kong JM, Zhu XH, Gao TM - BMC Neurosci (2009)

EndoG nuclear translocation preceded neuronal cell death following reoxygenation. (A-D) Confocal laser scanning microscope images of EndoG immunoreactivity (red) at control, EBSS, OGD/R6 h, OGD/R24 h, respectively. Co-staining with DAPI (blue) allowed the identification of nuclear translocation of EndoG. The white arrow indicates the nucleus with EndoG translocation but without pyknosis, and the white arrowhead indicates the pyknotic nucleus with EndoG translocation; (E) Percentage of damaged neurons and neurons displaying nuclear EndoG at different time points following reoxygenation. *p < 0.05 vs. control group, n = 9.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: EndoG nuclear translocation preceded neuronal cell death following reoxygenation. (A-D) Confocal laser scanning microscope images of EndoG immunoreactivity (red) at control, EBSS, OGD/R6 h, OGD/R24 h, respectively. Co-staining with DAPI (blue) allowed the identification of nuclear translocation of EndoG. The white arrow indicates the nucleus with EndoG translocation but without pyknosis, and the white arrowhead indicates the pyknotic nucleus with EndoG translocation; (E) Percentage of damaged neurons and neurons displaying nuclear EndoG at different time points following reoxygenation. *p < 0.05 vs. control group, n = 9.
Mentions: To evaluate the role of nuclear EndoG translocation in OGD, we investigated the relation between EndoG translocation and cell death. At different time points after reoxygenation, the intracellular localization of EndoG was detected by immunocytochemistry; the morphologically damaged neurons were identified by condensed and fragmented nuclei using DAPI nuclear staining. The result revealed that EndoG was mainly located in the intracellular space out of the nucleus shown as red fluorescence in normal control group or in EBSS-treated group under normoxic condition (Figure 3A and 3B). Consistent with the results from the previous western blot analysis, the nuclear translocation of EndoG started at 2 h after reoxygenation and increased progressively (Figure 3C-E). Significant numbers of neurons displaying EndoG nuclear staining were observed 6 h after reoxygenation, a time point when no obvious morphological neuronal damage could be observed (Figure 3C and 3E). Thereafter, although the number of morphologically damaged neurons also increased with time, a great quantity of pyknotic nuclei could not be observed until 12 h after reoxygenation. Furthermore, the proportions of morphologically damaged neurons were less than that of the EndoG translocated neurons at 12 h and 24 h, respectively (Figure 3D and 3E). These results demonstrate that EndoG translocation from mitochondria to nucleus precedes neuronal cell death.

Bottom Line: Importantly, the mitochondrial upregulation of BNIP3 precedes the translocation of EndoG.Forced expression of BNIP3 increases the nuclear translocation of EndoG and neuronal death while knockdown of BNIP3 decreases the OGD-induced nuclear translocation of EndoG and neuronal death.These results suggest that BNIP3 and EndoG play important roles in hippocampal neuronal apoptosis following ischemia, and mitochondrial BNIP3 is a signal protein upstream of EndoG that can induce neuronal death.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology, Southern Medical University, Guangzhou, PR China. zhaoshenting2004@126.com

ABSTRACT

Background: Caspase-independent apoptotic pathways are suggested as a mechanism for the delayed neuronal death following ischemic insult. However, the underlying signalling mechanisms are largely unknown. Recent studies imply the involvement of several mitochondrial proteins, including endonuclease G (EndoG) and Bcl-2/adenovirus E1B 19 kDa-interacting protein (BNIP3), in the pathway of non-neuronal cells.

Results: In this report, using western blot analysis and immunocytochemistry, we found that EndoG upregulates and translocates from mitochondria to nucleus in a time-dependent manner in cultured hippocampal neurons following oxygen-glucose deprivation (OGD). Moreover, the translocation of EndoG occurs hours before the observable nuclear pyknosis. Importantly, the mitochondrial upregulation of BNIP3 precedes the translocation of EndoG. Forced expression of BNIP3 increases the nuclear translocation of EndoG and neuronal death while knockdown of BNIP3 decreases the OGD-induced nuclear translocation of EndoG and neuronal death.

Conclusion: These results suggest that BNIP3 and EndoG play important roles in hippocampal neuronal apoptosis following ischemia, and mitochondrial BNIP3 is a signal protein upstream of EndoG that can induce neuronal death.

Show MeSH
Related in: MedlinePlus