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Analysis of BH3-only proteins upregulated in response to oxygen/glucose deprivation in cortical neurons identifies Bmf but not Noxa as potential mediator of neuronal injury.

Pfeiffer S, Anilkumar U, Chen G, Ramírez-Peinado S, Galindo-Moreno J, Muñoz-Pinedo C, Prehn JH - Cell Death Dis (2014)

Bottom Line: We observed a potent and early upregulation of noxa at mRNA and protein level, and a significant increase in Bmf protein levels during OGD in neocortical neurons and in the ipsilateral cortex of mice subjected to transient middle cerebral artery occlusion (tMCAO).Surprisingly, gene deficiency in noxa reduced neither OGD- nor glutamate-induced neuronal injury in cortical neurons and failed to influence infarct size or neurological deficits after tMCAO.In contrast, bmf deficiency induced significant protection against OGD- or glutamate-induced injury in cultured neurons, and bmf-deficient mice showed reduced neurological deficits after tMCAO in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 123 St. Stephen's Green, Dublin 2, Ireland.

ABSTRACT
Stress signaling in response to oxygen/glucose deprivation (OGD) and ischemic injury activates a group of pro-apoptotic genes, the Bcl-2 homology domain 3 (BH3)-only proteins, which are capable of activating the mitochondrial apoptosis pathway. Targeted studies previously identified the BH3-only proteins Puma, Bim and Bid to have a role in ischemic/hypoxic neuronal injury. We here investigated the transcriptional activation of pro-apoptotic BH3-only proteins after OGD-induced injury in murine neocortical neurons. We observed a potent and early upregulation of noxa at mRNA and protein level, and a significant increase in Bmf protein levels during OGD in neocortical neurons and in the ipsilateral cortex of mice subjected to transient middle cerebral artery occlusion (tMCAO). Surprisingly, gene deficiency in noxa reduced neither OGD- nor glutamate-induced neuronal injury in cortical neurons and failed to influence infarct size or neurological deficits after tMCAO. In contrast, bmf deficiency induced significant protection against OGD- or glutamate-induced injury in cultured neurons, and bmf-deficient mice showed reduced neurological deficits after tMCAO in vivo. Collectively, our data not only point to a role of Bmf as a BH3-only protein contributing to excitotoxic and ischemic neuronal injury but also demonstrate that the early and potent induction of noxa does not influence ischemic neuronal injury.

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Increased Bmf protein expression following OGD in cortical neurons and transient focal cerebral ischemia in WT mice. (a and b) Western blotting and densitometric analysis comparing the levels of Bmf induction and Spectrin cleavage in cortical neurons, (a) confirming Bmf protein induction at 24 h and (b) demonstrating an increased accumulation of calpain/caspase-3-generated αII spectrin breakdown products (145/150 kDa) in OGD-treated cultures. Significant increases in Bmf protein induction and Spectrin cleavage were observed at 24 h compared with sham-treated cultures. Data presented as mean±S.E.M. from n=5 independent experiments from n=5 independent cultures carried out in triplicate. *P<0.05 compared with sham-treated controls. (c and d) Western blotting and densitometric analysis comparing levels of Bmf induction and Spectrin cleavage in the ipsilateral cortex 3 and 24 h following ischemia in WT mice, (c) confirming Bmf protein induction and (d) demonstrating an increased accumulation of αII spectrin breakdown products at 24 h reperfusion. Significant increases in Bmf protein induction and Spectrin cleavage were observed at 24 h compared with matched controls. Densitometry data are expressed as Bmf or as a ratio of the 145 kDa spectrin breakdown product (BP) and the 280 kDa full length (FL) protein normalized to β-actin. Data presented as mean±S.E.M. from n=4 per group. *P<0.05 compared with matched controls
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fig5: Increased Bmf protein expression following OGD in cortical neurons and transient focal cerebral ischemia in WT mice. (a and b) Western blotting and densitometric analysis comparing the levels of Bmf induction and Spectrin cleavage in cortical neurons, (a) confirming Bmf protein induction at 24 h and (b) demonstrating an increased accumulation of calpain/caspase-3-generated αII spectrin breakdown products (145/150 kDa) in OGD-treated cultures. Significant increases in Bmf protein induction and Spectrin cleavage were observed at 24 h compared with sham-treated cultures. Data presented as mean±S.E.M. from n=5 independent experiments from n=5 independent cultures carried out in triplicate. *P<0.05 compared with sham-treated controls. (c and d) Western blotting and densitometric analysis comparing levels of Bmf induction and Spectrin cleavage in the ipsilateral cortex 3 and 24 h following ischemia in WT mice, (c) confirming Bmf protein induction and (d) demonstrating an increased accumulation of αII spectrin breakdown products at 24 h reperfusion. Significant increases in Bmf protein induction and Spectrin cleavage were observed at 24 h compared with matched controls. Densitometry data are expressed as Bmf or as a ratio of the 145 kDa spectrin breakdown product (BP) and the 280 kDa full length (FL) protein normalized to β-actin. Data presented as mean±S.E.M. from n=4 per group. *P<0.05 compared with matched controls

Mentions: Early and significant induction of noxa mRNA observed both in vitro after OGD and in vivo in response to ischemia prompted investigation of Noxa protein induction and the effects of deletion of noxa in ischemic neuronal injury. Levels of Noxa were examined in WT primary cortical neurons after OGD treatment followed by recovery for 0, 1, 4, 6 and 24 h by western blotting. Controls were sham-exposed and maintained under normoxic conditions. Forty-five minutes of OGD in healthy primary cortical neurons was sufficient to induce a significant increased expression of Noxa protein from 0 h after treatment and maintained significantly up to 24 h (Figure 3a) compared with normoxic controls, consistent with the observed upregulation of noxa mRNA. As a positive control for OGD-induced neuronal injury, we observed a significant increase in the accumulation of caspase/calpain-mediated II-spectrin breakdown cleavage products37, 38, 39 in cortical neurons after OGD (see below; Figure 5b).


Analysis of BH3-only proteins upregulated in response to oxygen/glucose deprivation in cortical neurons identifies Bmf but not Noxa as potential mediator of neuronal injury.

Pfeiffer S, Anilkumar U, Chen G, Ramírez-Peinado S, Galindo-Moreno J, Muñoz-Pinedo C, Prehn JH - Cell Death Dis (2014)

Increased Bmf protein expression following OGD in cortical neurons and transient focal cerebral ischemia in WT mice. (a and b) Western blotting and densitometric analysis comparing the levels of Bmf induction and Spectrin cleavage in cortical neurons, (a) confirming Bmf protein induction at 24 h and (b) demonstrating an increased accumulation of calpain/caspase-3-generated αII spectrin breakdown products (145/150 kDa) in OGD-treated cultures. Significant increases in Bmf protein induction and Spectrin cleavage were observed at 24 h compared with sham-treated cultures. Data presented as mean±S.E.M. from n=5 independent experiments from n=5 independent cultures carried out in triplicate. *P<0.05 compared with sham-treated controls. (c and d) Western blotting and densitometric analysis comparing levels of Bmf induction and Spectrin cleavage in the ipsilateral cortex 3 and 24 h following ischemia in WT mice, (c) confirming Bmf protein induction and (d) demonstrating an increased accumulation of αII spectrin breakdown products at 24 h reperfusion. Significant increases in Bmf protein induction and Spectrin cleavage were observed at 24 h compared with matched controls. Densitometry data are expressed as Bmf or as a ratio of the 145 kDa spectrin breakdown product (BP) and the 280 kDa full length (FL) protein normalized to β-actin. Data presented as mean±S.E.M. from n=4 per group. *P<0.05 compared with matched controls
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Related In: Results  -  Collection

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fig5: Increased Bmf protein expression following OGD in cortical neurons and transient focal cerebral ischemia in WT mice. (a and b) Western blotting and densitometric analysis comparing the levels of Bmf induction and Spectrin cleavage in cortical neurons, (a) confirming Bmf protein induction at 24 h and (b) demonstrating an increased accumulation of calpain/caspase-3-generated αII spectrin breakdown products (145/150 kDa) in OGD-treated cultures. Significant increases in Bmf protein induction and Spectrin cleavage were observed at 24 h compared with sham-treated cultures. Data presented as mean±S.E.M. from n=5 independent experiments from n=5 independent cultures carried out in triplicate. *P<0.05 compared with sham-treated controls. (c and d) Western blotting and densitometric analysis comparing levels of Bmf induction and Spectrin cleavage in the ipsilateral cortex 3 and 24 h following ischemia in WT mice, (c) confirming Bmf protein induction and (d) demonstrating an increased accumulation of αII spectrin breakdown products at 24 h reperfusion. Significant increases in Bmf protein induction and Spectrin cleavage were observed at 24 h compared with matched controls. Densitometry data are expressed as Bmf or as a ratio of the 145 kDa spectrin breakdown product (BP) and the 280 kDa full length (FL) protein normalized to β-actin. Data presented as mean±S.E.M. from n=4 per group. *P<0.05 compared with matched controls
Mentions: Early and significant induction of noxa mRNA observed both in vitro after OGD and in vivo in response to ischemia prompted investigation of Noxa protein induction and the effects of deletion of noxa in ischemic neuronal injury. Levels of Noxa were examined in WT primary cortical neurons after OGD treatment followed by recovery for 0, 1, 4, 6 and 24 h by western blotting. Controls were sham-exposed and maintained under normoxic conditions. Forty-five minutes of OGD in healthy primary cortical neurons was sufficient to induce a significant increased expression of Noxa protein from 0 h after treatment and maintained significantly up to 24 h (Figure 3a) compared with normoxic controls, consistent with the observed upregulation of noxa mRNA. As a positive control for OGD-induced neuronal injury, we observed a significant increase in the accumulation of caspase/calpain-mediated II-spectrin breakdown cleavage products37, 38, 39 in cortical neurons after OGD (see below; Figure 5b).

Bottom Line: We observed a potent and early upregulation of noxa at mRNA and protein level, and a significant increase in Bmf protein levels during OGD in neocortical neurons and in the ipsilateral cortex of mice subjected to transient middle cerebral artery occlusion (tMCAO).Surprisingly, gene deficiency in noxa reduced neither OGD- nor glutamate-induced neuronal injury in cortical neurons and failed to influence infarct size or neurological deficits after tMCAO.In contrast, bmf deficiency induced significant protection against OGD- or glutamate-induced injury in cultured neurons, and bmf-deficient mice showed reduced neurological deficits after tMCAO in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 123 St. Stephen's Green, Dublin 2, Ireland.

ABSTRACT
Stress signaling in response to oxygen/glucose deprivation (OGD) and ischemic injury activates a group of pro-apoptotic genes, the Bcl-2 homology domain 3 (BH3)-only proteins, which are capable of activating the mitochondrial apoptosis pathway. Targeted studies previously identified the BH3-only proteins Puma, Bim and Bid to have a role in ischemic/hypoxic neuronal injury. We here investigated the transcriptional activation of pro-apoptotic BH3-only proteins after OGD-induced injury in murine neocortical neurons. We observed a potent and early upregulation of noxa at mRNA and protein level, and a significant increase in Bmf protein levels during OGD in neocortical neurons and in the ipsilateral cortex of mice subjected to transient middle cerebral artery occlusion (tMCAO). Surprisingly, gene deficiency in noxa reduced neither OGD- nor glutamate-induced neuronal injury in cortical neurons and failed to influence infarct size or neurological deficits after tMCAO. In contrast, bmf deficiency induced significant protection against OGD- or glutamate-induced injury in cultured neurons, and bmf-deficient mice showed reduced neurological deficits after tMCAO in vivo. Collectively, our data not only point to a role of Bmf as a BH3-only protein contributing to excitotoxic and ischemic neuronal injury but also demonstrate that the early and potent induction of noxa does not influence ischemic neuronal injury.

Show MeSH
Related in: MedlinePlus