This study identifies the cellular signaling pathways involved in the regulation of a novel neuronal protein NP1 expression, which we found are induced in hypoxic-ischemic neuronal death [9
]. The experiments presented here demonstrate that the GSK-3 α/β kinase signaling pathways, which is implicated in the apoptotic cell death mechanism, regulate NP1 induction. Our results provide direct evidence for both GSK-3α -and GSK-3β-specific activities as key regulators of NP1 induction participating in hypoxic-ischemic cell death program. Particularly interesting are those molecular manipulations to selectively silencing/inhibiting the GSK-3α and/or GSK-3β signaling significantly blocked NP1 induction, and thus, rescuing primary cortical neurons from hypoxic-ischemic cell death. Our findings provide a novel mechanism of regulating hypoxic-ischemic neuronal death in brain.
We observed a temporal pattern of NP1 induction in hypoxic-ischemic neuronal cells that preceded the time course of neuronal death observed in our experimental model. In demonstrating this we also found that NP1 gene silencing by NP1 target specific siRNA prevented this hypoxic-ischemic neuronal death, which is in agreement with our previous findings [9
]. The link between NP1 induction and neuronal death was further validated in NP1−/− cortical neurons. Furthermore, comparing the time period of NP1 induction relative to cell death, it appears that NP1 induction occurred before the actual appearance of morphological signs and biochemical evidences for neuronal death. This is consistent with a role for NP1 in the injury mechanisms. It is now widely accepted that the cell fate of mature neurons depends on the balance between the survival and death signals. In this report, we applied loss/gain-of-function strategies to examine the specific involvement of GSK-3α and GSK-3β cellular signaling pathways known to be associated with the apoptotic death mechanism [18
]. In addition, we examined the upstream survival promoting regulatory kinases Akt that suppresses neuronal death and negatively regulates the GSK-3 α/β function. We asked if Akt signaling influences NP1 expression. Previously, we observed activation of the Akt kinase involved in the neurotrophin FGF-1-elicited neuroprotection against NMDA-mediated excitotoxicity [34
]. On the other hand, we also found that dephosphorylation of Akt (e.g. deactivation) is a causal mediator of NMDA-elicited excitotoxicity [40
], a process that contributes to cell loss in brain following HI, trauma, stroke, epilepsy and in many neurodegenerative diseases [45
]. Interestingly, the present results show that the deactivation of Akt kinase signaling under hypoxic-ischemic conditions significantly enhanced NP1 induction. Blockade of Akt kinase function by the dominant negative inhibitor Akt-kd showed similar increase in NP1 induction. On the other hand, overexpression of the wtAkt or the constitutively active Akt-myr in cortical neurons significantly blocked NP1 induction in hypoxic-ischemic neurons. Our interpretation of these results is that inhibition of the survival promoting pathways potentially shifts the cell fate balance towards the prodeath signaling mechanism of GSK-3, which in turn triggers NP1 induction and ultimately lead to the cell death.
To investigate the intracellular signaling mechanism(s) regulating the NP1 expression, we next examined the role of the prodeath signaling kinase GSK-3. GSK-3 has been implicated in apoptotic neuronal death induced by ischemia [18
] and glutamate-induced excitotoxicity [20
]. Although, several studies have emphasized the role of GSK-3β isoforms in the mechanism of apoptotic cell death [18
], the efficacy of GSK-3α in the neuronal cell death program triggered by hypoxia-ischemia is unclear. GSK-3 is a dual specificity kinase that can be both activated or inhibited through phosphorylation and dephosphorylation of its α and the β isoforms [25
]. Enguita and co-workers (2005) have shown a role for GSK-3β in NP1 overexpression in cerebellar granule neurons undergoing apoptosis [30
], but the relative role of the GSK-3α isoform of this prodeath signaling kinase in their findings was not described. We found that Akt deactivation occurred in cortical neurons following exposure to hypoxic-ischemic condition as evidenced by decreased levels of phospho-Akt. Our findings suggest that Akt, under basal conditions, exerts a restraining effect on cytotoxic process. Hypoxic-ischemic exposure of cortical neurons deactivated Akt, terminating its protective effect. Moreover, this Akt deactivation is in agreement with Akt deactivation we previously observed in multiple cell death models including NMDA excitotoxicity [40
]. Since, GSK-3 is a downstream substrate for activated Akt, it is likely that deactivation of Akt leads to decrease in phosphorylation of GSK-3α and GSK-3β isoforms resulting in disinhibition of kinase activity of both GSK-3α and GSK-3β isoforms. The fact that this GSK-3 signaling pathway is activated in cortical neurons under our experimental conditions is further supported by our findings of increased kinase activity of both GSK-3α and GSK-3β isoforms with a time course that preceded that of NP1 induction. Activation of GSK-3 has been proposed to regulate transcription of many genes through directly phosphorylation or indirectly as a consequence of changes in GSK-3 activity, thus, shifting the cell fate towards the prodeath pathway [47
]. This is evident from our findings that silencing GSK-3α and/or inhibiting GSK-3β signaling significantly inhibited NP1 induction, resulting significant decreases in cell death.