The present study demonstrates induction of NP1 in primary hippocampal neuronal cultures following OGD and in the ipsilateral hippocampal CA1 and CA3 areas following HI in neonatal mice, and further delineates how induction of NP1 is involved in mediating neuronal death. Firstly, OGD caused activation of PTEN with subsequent dephosphorylation of Akt (i.e. inactivation), and prodeath kinase GSK-3β (i.e. activation) in WT hippocampal cultures. Secondly, OGD induced down-regulation of Bax and decreased phosphorylation of Bad that resulted in enhanced mitochondrial translocation of the Bax and Bad proteins. This was also evident by the dissipation of ΔΨm, increased mitochondrial release of Cyt C with subsequent activation of caspase -3 ultimately leading to neuronal death. Particularly interesting is our identification of the association of NP1 with the prodeath proteins Bad and Bax, which further facilitated Bax and Bad mitochondrial localization and OGD-induced neuronal death. Thirdly, in contrast to the WT cultures, the NP1−/− null hippocampal cultures showed no significant change in mitochondrial translocation of Bad and Bax, ΔΨm, and no apparent increase in mitochondrial release of Cyt C and caspase-3; resulting in significant protection of the NP−/− neurons against the OGD insult. This is the first demonstration of significant neuroprotection in NP1-KO hippocampal neuronal cultures under ischemic stroke condition, further supporting a crucial role of NP1 in neuronal death.
The brain is mostly susceptible to oxidative stress because it consumes a large quantity of oxygen, and hippocampus, in particular, is often vulnerable in hypoxia-ischemia (Osredkar et al., 2010
). Previous report from our laboratory showed NP1 induction in hypoxic-ischemic brain injury (Hossain et al., 2004
), and GSK-3α/β regulated the expression of the NP1 in cultured cortical neurons (Russell et al., 2011
). In this study, we show the specificity of NP1 induction in OGD-induced neuronal death using a homogenous population of mature hippocampal neuronal cultures. We also found HI-time dependent induction of NP1 in the hippocampal (pyramidal layers of CA3 and CA1) regions; the same brain areas that developed infarction (i.e. injury) following HI. In fact, NP1 induction occurred prior (24 h and prior) to the development of the infarction (data not shown), indicating NP1 as an upstream component of the death cascade. This was evidenced by OGD time-dependent expression of NP1 mRNA and protein in cultured hippocampal neurons and the observed OGD-induced neuronal death. Live immunostaining of hippocampal neurons with NP1 antibody further consolidated our findings that NP1 induction occurs before the actual cell death. In addition, the intact morphological characteristics and significantly less cell death observed in NP1-KO and NP-TKO neurons following OGD specifically establish a role of NP1 inhibition in neuroprotection. Taken together our results provide strong evidence that NP1 induction plays a key role in OGD-induced hippocampal neuronal death.
Ample evidence suggests that apoptotic and anti-apoptotic signaling pathways are activated after cerebral HI, and that a shift in the balance between apoptotic and anti-apoptotic cellular factors determines cells fate. The PI3-K/Akt pathway regulates the survival response against pro-apoptotic stimuli (Cross et al., 1995
; Hetman et al., 2000
), whereas, PTEN, which acts as an antagonist of PI3-K (Oudit et al., 2004
) has been reported to play a role in mediating mitochondria-dependent apoptosis (Zhu et al., 2006
) and ischemic brain damage (Ning et al., 2004
). We found OGD time-dependent decrease in the phosphorylation of PTEN and Akt, abolishing their protective effects. Whereas, decrease in the phosphorylation of the downstream target GSK-3β results in activation of the death kinase, which is consistent with previous reports (Enguita et al., 2005
; Russell et al., 2011
). Similarly, blockade of GSK-3 activity by pharmacological inhibitor decreased the OGD-induced expression of NP1 and its interaction with Bad and Bax, further supporting the GSK-3-dependent induction of NP1. Interestingly, the activation of GSK-3β appears to be suppressed in NP-KO cultures, at least during the early time-points of OGD. This is possibly because of the fact that knock down of NP1 gene is neuroprotective- a condition where prosurvival PI3-K/Akt pathways are active that may have contributed to the observed phosphorylation of GSK-3β (inactivation), shifting the balance toward the survival path.
The Bcl-2 family of proteins Bad and Bax play a crucial role in intracellular apoptotic signal transduction by regulating the permeability of mitochondrial membrane (Yuan and Yankner, 2000
; Zong et al., 2001
). Akt phosphorylates Bad (Datta et al., 1997
) and phosphorylation of Bad promotes binding to 14-3-3 proteins to be sequestered in the cytosol (Datta et al., 2000
; Yang et al., 1995
; Zha et al., 1996
). Thus, Akt deactivation and decreased phosphorylation of Bad following OGD resulted in enhanced mitochondrial localization of Bad. Similarly, Bax translocation to mitochondria triggers neuronal apoptosis (Zong et al., 2001
). Bax is a direct target of GSK-3β that modulates Bax expression and function at the transcriptional and posttranslational levels to promote mitochondrial apoptosis (Linseman et al., 2004
; Watcharasit et al., 2003
). Our findings suggest that activated GSK-3β following OGD resulted in decreased Bax protein levels in the cytoplasm with concurrent increase in the mitochondrial fraction. Importantly, OGD exposure of NP1−/− null hippocampal neurons did not display any change in Bax protein and phospho-Bad levels. Also, knockdown of NP1 prevented the translocation of Bad and Bax from cytosol to mitochondria; indicating a specific involvement of NP1 in mitochondrial localization of Bad/Bax proteins following OGD.
The cell death signaling pathway in mitochondria has been demonstrated in the ischemic brain and that mitochondrial membrane depolarization and Cyt C release from inner membrane determine the final steps of apoptosis (Merry and Korsmeyer, 1997
; Narita et al., 1998
). Mitochondrial membrane potential is a very important marker for the function of mitochondria and a decrease in ΔΨm
predicts cell injury (Green and Reed, 1998
). To specifically demonstrate the role of NP1 in reduction of ΔΨm
, we found that NP1-KO cultures resisted the OGD-induced dissipation of ΔΨm
and there was significant inhibition of mitochondrial release of Cyt C. While we cannot rule out the contribution of other stressors on OGD-induced mitochondrial outer membrane permeabilization, our results clearly showed stabilization and neuroprotection from mitochondrial cell death pathway as a specific characteristic of NP1-KO hippocampal neurons. Most interestingly, our co-immunoprecipitation experiments revealed that NP1 formed protein complexes with Bad and Bax, and that OGD caused an increase in the interactions of both NP1-Bad and NP1-Bax. However, the increased association of NP1/Bad and NP1/Bax following OGD was reversed by pretreatment with SB216763, which further confirmed the GSK-3 regulation on NP1 expression and ensuing interactions with Bad and Bax proteins. Our current results are also in agreement with the report that shows NP1 facilitates the accumulation of Bax in mitochondria during apoptosis in cerebellar granule neurons in culture (Clayton et al., 2012
). Therefore, we suggest that NP1 influences the translocation of these proapoptotic proteins to the mitochondria, thereby contributing to mitochondrial outer membrane permeabilization. We also observed cell death after OGD was associated with robust increase in activated caspase-3 in WT neurons but not in the NP1-KO cultures under identical condition, supporting a role for NP1 in mitochondria-mediated caspase-3-dependent cell death pathway as evident by LDH release in WT vs. NP1-KO neurons. It appears that absence of NP1 desensitizes neurons to the ischemic injury by preventing mitochondria-mediated activation of cell death pathway.
In summary, our results clearly demonstrate a mechanism () by which NP1 influences mitochondria-mediated cell death by facilitating the translocation of proapoptotic Bad and Bax proteins to the mitochondria, decreasing ΔΨm and releasing Cyt C into the cytosol with subsequent activation of caspase-3. Our results present a novel mechanism of neuronal death involving PTEN/PI3K/Akt/GSK-3-dependent NP1 induction and its contribution to mitochondria-mediated hippocampal neuronal death following HI insult. Taken together, our findings suggest a novel neuroprotective strategy in neonates suffering from HI brain injury.
Fig. 7 Schematic diagram proposing a potential NP1-mediated mitochondrial cell death mechanism. Ischemia causes inactivation of Akt by activating PTEN followed by GSK-3 stimulation, which induces NP1 expression. NP1 promotes apoptotic pathways by facilitating (more ...)