Gene profiling identifies commonalities in neuronal pathways in excitotoxicity: Evidence favouring cell cycle re-activation in concert with oxidative stress

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Abstract

Excitotoxicity, induced by the aberrant rise in cytosolic Ca2+ level, is a major neuropathological process in numerous neurodegenerative disorders. It is triggered when extracellular glutamate (Glu) concentration reaches neuropathological levels resulting in dysregulation and hyper-activation of ionotropic glutamate receptor subtype (iGluRs). Even though all three members of the iGluRs, namely N-methyl-d-aspartate (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) and kainate (KAR) receptors are implicated in excitotoxicity, their individual contributions to downstream signaling transduction have not been explored. In this study, we report a comprehensive description of the recruitment of cellular processes in neurons upon iGluR activation during excitotoxicity through temporal (5 h, 15 h, and 24 h) global gene profiling of AMPA, KA, NMDA, and Glu excitotoxic models. DNA microarray analyses of mouse primary cortical neurons treated with these four pharmacological agonists are further validated via real-time PCR. Bi-model analyses against Glu model demonstrate that NMDARs and KARs play a more pivotal role in Glu-mediated excitotoxicity, with a higher degree of global gene profiling overlaps, as compared to that of AMPARs. Comparison of global transcriptomic profiles reveals aberrant calcium ion binding and homeostasis, organellar (lysosomal and endoplasmic reticulum) stress, oxidative stress, cell cycle re-entry and activation of cell death processes as the main pathways that are significantly modulated across all excitotoxicity models. Singular profile analyses demonstrate substantial transcriptional regulation of numerous cell cycle proteins. For the first time, we show that iGluR activation forms the basis of cell cycle re-activation, and together with oxidative stress fulfill the “two-hit” hypothesis that accelerates neurodegeneration.

Highlights

► Gene activation is profiled in neurons treated with ionotropic l-glutamate agonists. ► Common profiles in excitotoxicity show pivotal roles for NMDA and kainate receptors. ► Aberrant calcium ion homeostasis occurs with organelle stress and death signalling. ► Functional annotation revealed oxidative stress and major cell cycle re-activation. ► The latter downstream targets fulfill the “two-hit” hypothesis of neurodegeneration.

Introduction

l-Glutamate (Glu) is the most abundant excitatory neurotransmitter in the central nervous system (CNS) and is the only physiological agonist activating for glutamate receptors (GluRs) in the mammalian brain. GluRs play important roles in structuring neurocognitive processes underlying memory and learning (Mukherjee and Manahan-Vaughan, 2013). Therefore, when the regulation of GluR activation is impaired, not only its signaling properties are affected, but the consequences can also lead to cell death via excitotoxicity. Excitotoxicity is a general term that defines a damage-inflicting cellular process mediated via overstimulation of GluRs to effect a rise in cytosolic calcium ion level (Arundine and Tymianski, 2003). Because cellular indices reflecting excitotoxic damage are altered early in the pathogenesis of various neurodegenerative diseases such as Alzheimer’s disease (AD; (Hynd et al., 2004)), dementia associated with Down syndrome (DS; (Scheuer et al., 1996)) and acute neurological deficits due to traumatic brain injury (TBI) and stroke (Arundine and Tymianski, 2004), excitotoxicity, is believed to be one of the primary upstream events that induces neuronal injury at a cellular level.

Depending on their individual mode of activation, GluR are grouped into either metabotropic (mGluRs) or ionotropic (iGluRs) GluRs (Niciu et al., 2012). While mGluRs indirectly activate ion channel via signaling pathways that involve G proteins, iGluRs possess intrinsic ion channel activities. Therefore, during excitotoxicity, neuronal cell death is mediated by two concurrent yet distinct signaling processes determined by both iGluRs and mGluRs (Lea and Faden, 2003). In particular, iGluRs are made up of N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) receptor subtypes which are named after their specific pharmacological agonists active at different intrinsic ligand-gated ion channel activity that allows passage of Na+ and Ca2+ ions through a pore. All three subtypes of iGluRs (NMDAR, AMPAR and KAR) are actively involved during excitotoxic neuronal cell death. The NMDAR plays a major role due to its abundant expression and highest Ca2+ permeability (Hara and Snyder, 2007). Studies have shown that excessive NMDAR activation induces Ca2+ influx and release from intracellular stores, resulting in the activation of cytoplasmic proteases such as calcium-activated calpains (Simpkins et al., 2003) that hydrolyze cytoskeletal proteins. An example of such cytoskeletal protein is α-fodrin (Posner et al., 1995, Siman et al., 1989). Furthermore, NMDAR activation can result in the destabilization of lysosomes and the release of lysosomal cathepsins, causing cell death (Graber et al., 2004, Tenneti et al., 1998). Likewise, dysregulation of AMPAR and KAR also induces excitotoxicity in neurons (Jayakar and Dikshit, 2004, Sattler and Tymianski, 2001, Vincent and Mulle, 2009). Indeed, we were the first to demonstrate that AMPA alone could produce apoptotic-like injury (Larm et al., 1997), just as can KA, with injury likely to exert an apoptotic-necrotic continuum of programmed cell death (Cheung et al., 1998).

To date, the consequences of activation of individual iGluRs subtypes in downstream signal transduction during excitotoxicity has not been comprehensively and simultaneously explored in a comparative platform, impeding an understanding of the relative contributions of individual iGluRs, be that via convergent or divergent cellular pathways, towards excitotoxic damage. In our current study, global transcriptomic profiling was employed to elucidate downstream signaling pathways, in terms of amplification and diversification, subsequent to different iGluR activation to model the sequence of events subsequent to extracellular Glu in the brain reaching pathological concentrations. Here, the DNA microarray technique was applied to four excitotoxic models induced by (a) the general GluR agonist, Glu, (b) AMPAR agonist, AMPA, (c) NMDAR agonist, NMDA and (d) KAR agonist, KA. Subsequent comparative global gene profile analysis was performed to elucidate the major primary biological processes regulated by iGluRs in the trigger of excitotoxicity during Glu-mediated neuronal injury. Our study is the first to attempt global gene profiling of this type and scale to elucidate the pathobiology of excitotoxicity. Briefly, oxidative stress and cell cycle re-activation were identified as the primary cellular pathways that were significantly modulated. The specific association of cell cycle-reactivation with iGluR activation provided interesting evidence for the occurrence of a ‘two-hit’ hypothesis in excitotoxicity that has been previously postulated by others for the basis of neurodegeneration in AD pathogenesis (Zhu et al., 2004, Zhu et al., 2007).

Section snippets

Murine neocortical neuronal cultures

Neocortical neurons (gestational days 15 or 16) obtained from foetal cortices of Swiss albino mice were used to prepare the primary cultures as previous described with minor modifications (Cheung et al., 2000). Dissected cortices were subjected to trypsin digestion and mechanical trituration. Cells were collected by centrifugation and resuspended in Neurobasal™ (NB) medium containing 2.5% B27 supplement, 1% penicillin, 1% streptomycin, 0.25% GlutaMAX-1 supplement, and 10% dialyzed foetal calf

Results

Overall, cellular transcriptional regulation was assessed in excitotoxic models employing 250 μM Glu, 200 μM NMDA, 300 μM AMPA and 100 μM KA over a 24-h period using Illumina® Mouse Ref8 V1.1 genechips. The raw transcriptional signal data from individual arrays were then subjected to statistical filtering using one-way ANOVA, p < 0.05 and Benjamini–Hochberg FDR. Gene probes were considered to be significantly regulated when they demonstrated gene expression changes of at least ±1.5 folds in a minimum

Discussion

Excitotoxicity is well accepted to be a cell death process contributing to both acute and chronic neurodegenerative conditions (Doble, 1999). Although rises in intracellular Ca2+ subsequent to activation of NMDARs play a central role in most discussions of excitotoxicity, the recruitment of cell signaling cascades is likely to be more complex. Here, we sought to determine the individual contributions of over-stimulated iGluRs to the excitotoxic process by transcriptomic profiling. Simultaneous

Acknowledgement

This work is supported by: NSC and PMB, project grant funding from the National Health and Medical Research Council of Australia. PMB is a Research Fellow of the NHMRC (Australia).

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    These authors contributed equally to this study.

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