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.
Highlights
Mitochondrial-mediated apoptosis has been shown to be involved in neuronal cell death after cerebral ischemia in studies of both patient samples and animal models of acute stroke,[5] with reduced expression of anti-apoptotic Bcl-2 and Bcl-w and induction of pro-apoptotic Bax observed within the ischemic core and surrounding penumbra.[6]
In order to identify which pro-apoptotic Bcl-2 homology domain 3 (BH3)-only proteins may have a role in ischemic neuronal injury, cultures of neocortical neurons were subjected to oxygen/glucose deprivation (OGD) in vitro
Mature cultures of neocortical neurons were subjected to 45 min of OGD and allowed to recover under normoxic conditions for various timepoints (4, 6, 24 h) at which points mRNA levels of BH3-only proteins were assessed by real-time quantitative PCR analysis. mRNA levels for noxa were found to be upregulated from 4 h onward, and levels were maintained significantly up to 24 h at 3.9-fold (Figure 1a)
Summary
Mitochondrial-mediated apoptosis has been shown to be involved in neuronal cell death after cerebral ischemia in studies of both patient samples and animal models of acute stroke,[5] with reduced expression of anti-apoptotic Bcl-2 and Bcl-w and induction of pro-apoptotic Bax observed within the ischemic core and surrounding penumbra.[6] Translocation of cytosolic Bax to the mitochondrial outer membrane is key for the activation of mitochondrial apoptosis in neurons.[7,8,9,10,11]. A role for several BH3-only proteins, in particular Bid .22,23 and Puma, .24 in ischemic neuronal injury has been previously suggested in studies using animals deficient in these genes. Both are known to act as indirect activators of apoptosis with roles as ‘de-repressors’, preventing sequestration of direct activators
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