Elsevier

Neurobiology of Disease

Volume 50, February 2013, Pages 59-68
Neurobiology of Disease

Critical role of neuronal pentraxin 1 in mitochondria-mediated hypoxic–ischemic neuronal injury

https://doi.org/10.1016/j.nbd.2012.10.003Get rights and content

Abstract

Developing brain is highly susceptible to hypoxic–ischemic (HI) injury leading to severe neurological disabilities in surviving infants and children. Previously, we have reported induction of neuronal pentraxin 1 (NP1), a novel neuronal protein of long-pentraxin family, following HI neuronal injury. Here, we investigated how this specific signal is propagated to cause the HI neuronal death. We used wild-type (WT) and NP1 knockout (NP1-KO) mouse hippocampal cultures, modeled in vitro following exposure to oxygen glucose deprivation (OGD), and in vivo neonatal (P9–10) mouse model of HI brain injury. Our results show induction of NP1 in primary hippocampal neurons following OGD exposure (4–8 h) and in the ipsilateral hippocampal CA1 and CA3 regions at 24–48 h post-HI compared to the contralateral side. We also found increased PTEN activity concurrent with OGD time-dependent (4–8 h) dephosphorylation of Akt (Ser473) and GSK-3β (Ser9). OGD also caused a time-dependent decrease in the phosphorylation of Bad (Ser136), and Bax protein levels. Immunofluorescence staining and subcellular fractionation analyses revealed increased mitochondrial translocation of Bad and Bax proteins from cytoplasm following OGD (4 h) and simultaneously increased release of Cyt C from mitochondria followed by activation of caspase-3. NP1 protein was immunoprecipitated with Bad and Bax proteins; OGD caused increased interactions of NP1 with Bad and Bax, thereby, facilitating their mitochondrial translocation and dissipation of mitochondrial membrane potentialΨm). This NP1 induction preceded the increased mitochondrial release of cytochrome C (Cyt C) into the cytosol, activation of caspase-3 and OGD time-dependent cell death in WT primary hippocampal neurons. In contrast, in NP1-KO neurons there was no translocation of Bad and Bax from cytosol to the mitochondria, and no evidence of ΔΨm loss, increased Cyt C release and caspase-3 activation following OGD; which resulted in significantly reduced neuronal death. Our results indicate a regulatory role of NP1 in Bad/Bax-dependent mitochondrial release of Cyt C and caspase-3 activation. Together our findings demonstrate a novel mechanism by which NP1 regulates mitochondria-driven hippocampal cell death; suggesting NP1 as a potential therapeutic target against HI brain injury in neonates.

Highlights

NP1 is induced in hippocampal neuronal death following OGD and HI. ► NP1 facilitates Bad and Bax translocation to mitochondria and regulates cell death. ► NP1-KO neurons showed no Bad and Bax translocation, ΔΨm and caspase-3 activation. ► Significant protection of the NP1-KO hippocampal neurons against the OGD insult. ► NP1 causes cell death by perturbing mitochondrial functions; uncover new mechanism.

Introduction

Brain injury due to fetal or neonatal asphyxia (Johnston, 1997, Lorenz et al., 1998, Northington et al., 2001a, Northington et al., 2001b) or neonatal stroke is a leading cause of morbidity and mortality in surviving infants and children worldwide with a high societal cost (Balduini et al., 2004, Gresham et al., 1997, Shiber et al., 2010, Sudlow and Warlow, 1997, Volpe, 2001), and has an incidence as high as 1 in 4000 (Ferriero, 2004, Nelson, 2007). Yet, there is no promising therapy for neonatal brain injury at present; very few trials have documented limited clinical benefit (Blomgren and Hagberg, 2006, Kalenderian et al., 2009, Pezzini and Padovani, 2009, Uchino et al., 2001), in part, owing to incomplete understanding of the pathological secondary injury mechanisms that proceed from the primary insult (Blomgren et al., 2003, Blomgren and Hagberg, 2006, Pezzini and Padovani, 2009, Smaha, 2004). The hippocampus is often injured in neonatal stroke (Osredkar et al., 2010), and that disruption of oxygen and glucose supply resulting from occlusion of a blood vessel ultimately leads to neuronal losses and necrosis (Banasiak et al., 2000). Previously, we demonstrated the induction of a novel neuronal protein neuronal pentraxin 1 (NP1) in in vivo rat model of hypoxic–ischemic (HI) brain injury (Hossain et al., 2004), and that NP1 induction in HI cortical neuronal cell death is regulated via a glycogen synthase kinase (GSK)-3α/β-dependent mechanism (Russell et al., 2011). However, how this specific signal of NP1 induction is propagated to cause the HI neuronal death remains to be elucidated.

The cellular decision as to whether cells will undergo death or survival process is determined by the integration of multiple survival and death signals. The PI3-K/Akt signaling pathway plays a critical role in the survival of neuronal cells (Brunet et al., 2001). Whereas, PTEN (phosphatase and tensin homolog deleted on chromosome 10), which negatively regulates PI3-K/Akt pathway, plays a central role in cell migration, survival and apoptosis (Salmena et al., 2008, Waite and Eng, 2002) For example, PTEN deletion has been reported to prevent ischemic brain injury (Shi et al., 2011), and enhances regenerative ability of adult corticospinal neurons (Liu et al., 2010). In addition, there are many downstream effectors of PI3-K/Akt signaling pathway such as glycogen synthase kinase 3 (GSK-3) and Bcl-2/Bcl-xL-associated death protein (Bad) that play crucial roles in the process of cellular survival or deaths (Cross et al., 1995, Datta et al., 1997, del Peso et al., 1997, Hetman et al., 2000). In particular, GSK-3 is a critical activator of cell death in numerous models of neuronal apoptosis (Hetman et al., 2000, Li et al., 2000, Phiel et al., 2003), including HI (Russell et al., 2011). GSK-3β exerts some of its pro-apoptotic effects in neurons by regulating the mitochondrial localization of Bax, a key component of the intrinsic apoptotic cascade (Linseman et al., 2004).

Neuronal cell death signaling pathways in mitochondria has been demonstrated in the ischemic brain (Chan, 2004, Pérez-Pinzón et al., 1999). This often occurs via an intrinsic cell death pathway triggered by the translocation of Bad and Bax, two proapoptotic Bcl-2 family members, to mitochondria (Armstrong and Jones, 2002, Yuan and Yankner, 2000, Zong et al., 2001), reduction in mitochondrial membrane potential (ΔΨm) (Deckworth and Johnson, 1993), opening of the mitochondrial permeability transition pore (PTP) (Zarotti and Szabo, 1995), release of mitochondrial cytochrome c (Cyt C) (Narita et al., 1998, Wang et al., 1999, Zong et al., 2001) and formation of apoptosome with Apaf-1 and pro-caspase-9 leading to activation of the downstream executioner caspases, including caspase-3 (Li et al., 1997a, Li et al., 1997b, Zou et al., 1999). These findings suggest potential mitochondrion-targeted strategies for preventing HI neuronal cell death. In this study, we have used primary hippocampal neuronal cultures, modeled in vitro, following oxygen glucose deprivation and in vivo neonatal mouse model of HI to investigate the role of NP1 in causing cell death in the hippocampus, and also the underlying mitochondrial mechanisms of the NP1-mediated death program. Furthermore, we used NP1-/- primary hippocampal neurons to specifically demonstrate the contribution of NP1 in mediating mitochondrial perturbation and cell death.

Section snippets

Mouse primary hippocampal culture and oxygen glucose deprivation

Primary hippocampal neuronal cultures were prepared from wild-type (WT), NP1 knockout (NP1-KO) mice (C57BL/6 background) and NP triple knockout (NP-TKO; all pentraxin family members-neuronal pentraxin 1, neuronal pentraxin 2 and neuronal pentraxin receptor are knocked down) mice (C57BL/6 and 129 mixed background) at postnatal day 1 or 2 (PND1 or 2) as described previously (Berbari et al., 2007) with modifications. NP1-KO and TKO mice were kindly provided by Dr. Paul Worley, Dept. of

NP 1 is induced in dissociated primary hippocampal cultures exposed to OGD and in the hippocampus of neonatal mice following HI brain injury

Hippocampal brain regions are highly susceptible to injury in the event of stroke (Osredkar et al., 2010). First, we asked whether NP1 is contributing to the neuronal death in the hippocampus. We used mature hippocampal cultures (12–14 DIV) to investigate OGD-induced neuronal death, because of the higher degree of sensitivity of mature hippocampal neurons (12–18 DIV) to hypoxic damage (Loreto and Balestrino, 1997). Increased induction of NP1 mRNA and protein levels were observed after exposure

Discussion

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

Acknowledgments

We gratefully acknowledge Dr. Paul Worley for providing the knockout mice. This work was supported by the National Institutes of Health grant RO1 NS046030 and the Cerebral Palsy International Research Foundation grant R-793-09.

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