[Show abstract][Hide abstract] ABSTRACT: Glutaredoxin1 (GRX1) is a glutathione (GSH)-dependent thiol oxidoreductase. The GRX1/GSH system is important for the protection of proteins from oxidative damage and in the regulation of protein function. Previously we demonstrated that GRX1/GSH regulates the activity of the essential copper-transporting P1B-Type ATPases (ATP7A, ATP7B) in a copper-responsive manner. It has also been established that GRX1 binds copper with high affinity and regulates the redox chemistry of the metallochaperone ATOX1, which delivers copper to the copper-ATPases. In this study, to further define the role of GRX1 in copper homeostasis, we examined the effects of manipulating GRX1 expression on copper homeostasis and cell survival in mouse embryonic fibroblasts and in human neuroblastoma cells (SH-SY5Y). GRX1 knockout led to cellular copper retention (especially when cultured with elevated copper) and reduced copper tolerance, while in GRX1-overexpressing cells challenged with elevated copper, there was a reduction in both intracellular copper levels and copper-induced reactive oxygen species, coupled with enhanced cell proliferation. These effects are consistent with a role for GRX1 in regulating ATP7A-mediated copper export, and further support a new function for GRX1 in neuronal copper homeostasis and in protection from copper-mediated oxidative injury.
[Show abstract][Hide abstract] ABSTRACT: Excitotoxicity, induced by the aberrant rise in cytosolic Ca(2+) 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 (5h, 15h and 24h) 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.
Neurochemistry International 01/2013; · 2.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial dysfunction and oxidative stress are currently considered two key mechanisms contributing to pathobiology in neurodegenerative conditions. The current study investigated the temporal molecular events contributing to programmed cell death after treatment with the mitochondrial complex I inhibitor rotenone. Microarray analysis was performed using cultured neocortical neurons treated with 10 nM rotenone for 8, 15, and 24h. Genes showing at least ± 1.2-fold change in expression at one time point were considered significant. Transcriptomic analysis of the 4178 genes probes revealed major changes to nine biological processes, including those eliciting mitochondrial dysfunction, activation of calcium signaling, increased expression of apoptotic genes, and downplay of chaperones/co-chaperones, ubiquitin-proteasome system and autophagy. These data define targets for intervention where mitochondrial complex I dysfunction plays a substantial role, most notably Parkinson's disease.
Neurochemistry International 11/2012; · 2.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Studies have shown similarities between the histopathological characteristics of NPC and Alzheimer's disease (AD) including amyloid and tau pathologies. While dysfunction in insulin signaling was widely detected in AD brain, the function of insulin signaling proteins has not been examined in NPC disease. In this study, we have examined the expression and phosphorylation of proteins linked to the insulin signaling pathway in the brain of 9 weeks old NPC(nih) mice. Our results showed lower expression of insulin receptor substrate 2 (IRS2) in the NPC(nih) mice, and insulin receptor substrate 1 (IRS1) expression was almost non-detectable in this NPC mouse model. This reduction was associated with the loss of expression for the regulatory p85 subunit of phosphatidylinositol 3-kinase (p85/PI3K). Interestingly, the impairment was observed to link to a greater reduction of Akt phosphorylation at residue T308 than S473. This aberrant Akt phosphorylation could be contributing to lower GSK3β phosphorylation detected in the NPC(nih) mouse brain. To our knowledge, this is the first report documenting impaired insulin signaling in the brain of a NPC mouse model.
Biochemical and Biophysical Research Communications 07/2012; 424(3):482-7. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reduced glucose utilization is likely to precede the onset of cognitive deficits in Alzheimer's disease (AD). Similar aberrant glucose metabolism can also be detected in the brain of several AD mouse models. Although the cause of this metabolic defect is not well understood, it could be related to impaired insulin signaling that is increasingly being reported in AD brain. However, the temporal relationship between insulin impairment and amyloid-β (Aβ) biogenesis is unclear. In this study using female AβPPsw/PS1ΔE9 mice, we found that the level of Aβ40 was fairly constant in 6- to 15-month-old brains, whereas Aβ42 was only significantly increased in the 15-month-old brain. In contrast, increased levels of IRβ, IGF-1R, IRS1, and IRS-2, along with reduced glucose and insulin content, were detected earlier in the 12-month-old brains of AβPPsw/PS1ΔE9 mice. The reduction in brain glucose content was accompanied by increased GLUT3 and GLUT4 levels. Importantly, these changes precede the significant upregulation of Aβ42 level in the 15-month-old brain. Interestingly, reduction in the p85 subunit of PI3K was only apparent in the 15-month-old AβPPsw/PS1ΔE9 mouse brain. Furthermore, the expression profile of IRβ, IRS-2, and p85/PI3K in AβPPsw/PS1ΔE9 was distinct in wild-type mice of a similar age. Although the exact mechanisms underlining this connection remain unclear, our results suggest a possible early role for insulin signaling impairment leading to amyloid accumulation in AβPPsw/PS1ΔE9 mice.
[Show abstract][Hide abstract] ABSTRACT: Axotomized neurons have the innate ability to undergo regenerative sprouting but this is often impeded by the inhibitory central nervous system environment. To gain mechanistic insights into the key molecular determinates that specifically underlie neuronal regeneration at a transcriptomic level, we have undertaken a DNA microarray study on mature cortical neuronal clusters maintained in vitro at 8, 15, 24 and 48 hrs following complete axonal severance. A total of 305 genes, each with a minimum fold change of ± 1.5 for at least one out of the four time points and which achieved statistical significance (one-way ANOVA, P < 0.05), were identified by DAVID and classified into 14 different functional clusters according to Gene Ontology. From our data, we conclude that post-injury regenerative sprouting is an intricate process that requires two distinct pathways. Firstly, it involves restructuring of the neurite cytoskeleton, determined by compound actin and microtubule dynamics, protein trafficking and concomitant modulation of both guidance cues and neurotrophic factors. Secondly, it elicits a cell survival response whereby genes are regulated to protect against oxidative stress, inflammation and cellular ion imbalance. Our data reveal that neurons have the capability to fight insults by elevating biological antioxidants, regulating secondary messengers, suppressing apoptotic genes, controlling ion-associated processes and by expressing cell cycle proteins that, in the context of neuronal injury, could potentially have functions outside their normal role in cell division. Overall, vigilant control of cell survival responses against pernicious secondary processes is vital to avoid cell death and ensure successful neurite regeneration.
Journal of Cellular and Molecular Medicine 06/2011; 16(4):789-811. · 4.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inhibitors of Rho kinase (ROCK) have potential for management of neurological disorders by inhibition of glial scarring. Since astrocytes play key roles in brain physiology and pathology, we determined changes in the astrocytic transcriptome produced by the ROCK inhibitor Fasudil to obtain mechanistic insights into its beneficial action during brain injury. Cultured murine astrocytes were treated with Fasudil (100 µM) and morphological analyses revealed rapid stellation by 1 h and time-dependent (2-24 h) dissipation of F-actin-labelled stress fibres. Microarray analyses were performed on RNA and the time-course of global gene profiling (2, 6, 12 and 24 h) provided a comprehensive description of transcriptomic changes. Hierarchical clustering of differentially expressed genes and analysis for over-represented gene ontology groups using the DAVID database focused attention on Fasudil-induced changes to major biological processes regulating cellular shape and motility (actin cytoskeleton, axon guidance, transforming growth factor-β (TGFβ) signalling and tight junctions). Bioinformatic analyses of transcriptomic changes revealed how these biological processes contributed to changes in astrocytic motility and cytoskeletal reorganisation. Here genes associated with extracellular matrix were also involved, but unexpected was a subset of alterations (EAAT2, BDNF, anti-oxidant species, metabolic and signalling genes) indicative of adoption by astrocytes of a pro-survival phenotype. Expression profiles of key changes with Fasudil and another ROCK inhibitor Y27632 were validated by real-time PCR. Although effects of ROCK inhibition have been considered to be primarily cytoskeletal via reduction of glial scarring, we demonstrate additional advantageous actions likely to contribute to their ameliorative actions in brain injury.
Journal of Cellular Physiology 05/2011; 227(3):1199-211. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transient cerebral ischemia often results in secondary ischemic/reperfusion injury, the pathogenesis of which remains unclear. This study provides a comprehensive, temporal description of the molecular events contributing to neuronal injury after transient cerebral ischemia. Intraluminal middle cerebral artery occlusion (MCAO) was performed to induce a 2-h ischemia with reperfusion. Microarray analysis was then performed on the infarct cortex of wild-type (WT) and glutathione peroxidase-1 (a major antioxidant enzyme) knockout (Gpx1(-/-)) mice at 8 and 24h postreperfusion to identify differential gene expression profile patterns and potential alternative injury cascades in the absence of Gpx1, a crucial antioxidant enzyme, in cerebral ischemia. Genes with at least ±1.5-fold change in expression at either time point were considered significant. Global transcriptomic analyses demonstrated that 70% of the WT-MCAO profile overlapped with that of Gpx1(-/-)-MCAO, and 28% vice versa. Critical analysis of the 1034 gene probes specific to the Gpx1(-/-)-MCAO profile revealed regulation of additional novel pathways, including the p53-mediated proapoptotic pathway and Fas ligand (CD95/Apo1)-mediated pathways; downplay of the Nrf2 antioxidative cascade; and ubiquitin-proteasome system dysfunction. Therefore, this comparative study forms the foundation for the establishment of screening platforms for target definition in acute cerebral ischemia intervention.
Free Radical Biology and Medicine 03/2011; 50(6):736-48. · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide is implicated in the pathogenesis of various neuropathologies characterized by oxidative stress. Although nitric oxide has been reported to be involved in the exacerbation of oxidative stress observed in several neuropathologies, existent data fail to provide a holistic description of how nitrergic pathobiology elicits neuronal injury. Here we provide a comprehensive description of mechanisms contributing to nitric oxide induced neuronal injury by global transcriptomic profiling. Microarray analyses were undertaken on RNA from murine primary cortical neurons treated with the nitric oxide generator DETA-NONOate (NOC-18, 0.5 mM) for 8-24 hrs. Biological pathway analysis focused upon 3672 gene probes which demonstrated at least a ±1.5-fold expression in a minimum of one out of three time-points and passed statistical analysis (one-way anova, P < 0.05). Numerous enriched processes potentially determining nitric oxide mediated neuronal injury were identified from the transcriptomic profile: cell death, developmental growth and survival, cell cycle, calcium ion homeostasis, endoplasmic reticulum stress, oxidative stress, mitochondrial homeostasis, ubiquitin-mediated proteolysis, and GSH and nitric oxide metabolism. Our detailed time-course study of nitric oxide induced neuronal injury allowed us to provide the first time a holistic description of the temporal sequence of cellular events contributing to nitrergic injury. These data form a foundation for the development of screening platforms and define targets for intervention in nitric oxide neuropathologies where nitric oxide mediated injury is causative.
Journal of Cellular and Molecular Medicine 02/2011; 16(1):41-58. · 4.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inhibition of proteasome degradation pathway has been implicated in neuronal cell death leading to neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. We and others demonstrated that treatment of cortical neurons with the proteasomal inhibitor lactacystin leads to apoptosis. We discovered by microarray analysis that lactacystin treatment modulates the expression of both potentially neuroprotective as well as pro-apoptotic genes in neurons. However, the significance of the genes which upon transcriptional modulation contributed to proteasomal inhibition-induced apoptosis, remained unidentified. By employing microarray analysis to decipher the time-dependent changes in transcription of these genes in cultured cortical neurons, we discovered different groups of genes were transcriptionally regulated in the early and late phase of lactacystin-induced cell death. In the early phase, several neuroprotective genes such as those encoding the proteasome subunits and ubiquitin-associated enzymes, as well as the heat-shock proteins (HSP) were up-regulated. However, the pro-apoptotic endoplasmic reticulum (ER) stress-associated genes were also up-regulated at the early phase of lactacystin-induced neuronal cell death. In the late phase, genes encoding antioxidants and calcium-binding proteins were up-regulated while those associated with cholesterol biosynthesis were down-regulated. The data suggest that ER stress may participate in mediating the apoptotic responses induced by proteasomal inhibition. The up-regulation of the neuroprotective antioxidant genes and calcium-binding protein genes and down-regulation of the cholesterol biosynthesis genes in the later phase are likely consequences of stimulation of the pro-apoptotic signaling pathways in the early phase of lactacystin treatment.
Journal of Cellular Physiology 02/2011; 226(2):494-510. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The involvement of cyclin-dependent kinase-5 (Cdk5) and p25, the proteolytic fragment of activator p35, has long been implicated in the development of neuron-fibrillary tangles (NFTs), a hallmark of Alzheimer's disease (AD). Findings in this area over the past decade have been highly controversial and inconclusive. Here we report unprecedented detection of endogenous p10, the smaller proteolytic fragment of the Cdk5 activator p35 in treated primary cortical neurons that underwent significant apoptosis, triggered by proteasome inhibitors MG132 and lactacystin, and protein kinase inhibitor staurosporine (STS). p10 appeared exclusively in the detergent-resistant fraction made up of nuclear matrix, membrane-bound organelles, insoluble membrane proteins, and cytoskeletal components. Intriguingly, transient overexpression of p10 in neural cells induced apoptotic morphologies, suggesting that p10 may play an important role in mediating neuronal cell death in neurodegenerative diseases. We demonstrated for the first time that p10-mediated apoptosis occurred via a caspases-independent pathway. Furthermore, as p10 may contain the myristoylation signal for p35 which is responsible for binding p35 to several intracellular components and the membrane, all in all these novel results present that the accumulation of p10 to the detergent-insoluble fraction may be a crucial pathological event to triggering neuronal cell death.
Journal of Cellular Biochemistry 12/2010; 111(5):1359-66. · 3.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Massive neuronal apoptosis and accumulation of protein aggregates in the cortex and hippocampus of the brain are hallmarks of several neurodegenerative disorders, indicating ubiquitin proteasome system (UPS) dysfunction. Lactacystin, a classical proteasome inhibitor, is used to simulate ubiquitin proteasome system dysfunction in neurons to mimic pathological features of neurodegenerative disorders. Based on Western blot analyses, we reported for the first time that annexin A3 (AnxA3) is not only endogenously expressed in mouse cortical neurons but also more importantly, by gene expression microarray and real-time RT-PCR that it is greatly transcriptional up-regulated to approximately 11- and 15-fold, respectively in murine primary cortical neurons with 1μM lactacystin for 24h. Up-regulation of AnxA3 expression occurred after 12-15h post-lactacystin treatment, which corresponded with the onset of neuronal injury, with approximately 25% of the neurons being non-viable by that time interval. Western blot analysis with anti-AnxA3 antibodies further validated that up-regulation of AnxA3 only occurs with onset of neuronal death, and not with the onset of proteasome inhibition, which occurs at 4.5h post-lactacystin treatment. Over-expression studies suggested AnxA3 might be involved in death promotion during lactacystin-mediated neuronal death, since caspase-3 activation was significantly stronger upon neuronal AnxA3 over-expression. We propose AnxA3 up-regulation may have significant relevance in the elucidation of neurodegenerative pathophysiology.
[Show abstract][Hide abstract] ABSTRACT: With the identification of hypochlorous acid (HOCl) as a biomarker in diseased brains and endogenous detection of its modified proteins, HOCl might be implicated in the development of neurodegenerative disorders. However, its effect on neuronal cell death has not yet been investigated at gene expression level.
Therefore, DNA microarray was performed for screening of HOCl-responsive genes in primary mouse cortical neurons. Neurotoxicity caused by physiological relevant HOCl (250μM) exhibited several biochemical markers of apoptosis.
The biological processes affected during HOCl-mediated apoptosis included cell death, response to stress, cellular metabolism, and cell cycle. Among them, mRNAs level of cell death and stress response genes were up-regulated while expression of metabolism and cell cycle genes were down-regulated.
Our results showed, for the first time, that HOCl induces apoptosis in cortical neurons by upregulating apoptotic genes and gene expression of stress response such as heat shock proteins and antioxidant proteins were enhanced to provide protection. These data form a foundation for the development of screening platforms and define targets for intervention in HOCl neuropathologies where HOCl-mediated injury is causative.
Life sciences 10/2010; 87(15-16):457-67. · 2.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently the role of hydrogen sulphide (H(2) S) as a gasotransmitter stimulated wide interest owing to its involvement in Alzheimer's disease and ischemic stroke. Previously we demonstrated the importance of functional ionotropic glutamate receptors (GluRs) by neurons is critical for H(2) S-mediated dose- and time-dependent injury. Moreover N-methyl-D-aspartate receptor (NMDAR) antagonists abolished the consequences of H(2) S-induced neuronal death. This study focuses on deciphering the downstream effects activation of NMDAR on H(2) S-mediated neuronal injury by analyzing the time-course of global gene profiling (5, 15, and 24 h) to provide a comprehensive description of the recruitment of NMDAR-mediated signaling. Microarray analyses were performed on RNA from cultured mouse primary cortical neurons treated with 200 µM sodium hydrosulphide (NaHS) or NMDA over a time-course of 5-24 h. Data were validated via real-time PCR, western blotting, and global proteomic analysis. A substantial overlap of 1649 genes, accounting for over 80% of NMDA global gene profile present in that of H(2) S and over 50% vice versa, was observed. Within these commonly occurring genes, the percentage of transcriptional consistency at each time-point ranged from 81 to 97%. Gene families involved included those related to cell death, endoplasmic reticulum stress, calcium homeostasis, cell cycle, heat shock proteins, and chaperones. Examination of genes exclusive to H(2) S-mediated injury (43%) revealed extensive dysfunction of the ubiquitin-proteasome system. These data form a foundation for the development of screening platforms and define targets for intervention in H(2) S neuropathologies where NMDAR-activated signaling cascades played a substantial role.
Journal of Cellular Physiology 10/2010; 226(5):1308-22. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The neuronal Cdk5 activator p35 is involved in a multitude of neuronal activities, including cytoskeletal organization. We show here that p35 directly interacts with filamentous actin (F-actin) but not with monomeric actin (G-actin). Through binding, p35 induces the formation of actin bundles and stabilizes F-actin against dilution-induced depolymerization. p35 forms intermolecular self-associations, suggesting that p35 cross-links actin filaments into bundles via its intermolecular self-association. p35 dimerization and association with F-actin occur at the N-terminal region that is absent in the calpain-cleaved product p25, indicating that such p35 properties are lost by its truncation induced under neurotoxic conditions. Using p35 phosphorylated by Cdk5 and a mutational approach, we demonstrate that the phosphorylation of p35 promotes its homodimerization and p35-induced formation of F-actin bundles. In addition, the phosphorylation regulates p35 distribution to microtubule and actin cytoskeletons. Together, these observations define a novel function for p35 in cytoskeletal regulation.
Cellular and Molecular Life Sciences CMLS 10/2010; 68(9):1633-43. · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress plays a central role in neuronal injury and cell death in acute and chronic pathological conditions. The cellular responses to oxidative stress embrace changes in mitochondria and other organelles, notably endoplasmic reticulum, and can lead to a number of cell death paradigms, which cover a spectrum from apoptosis to necrosis and include autophagy. In Alzheimer's disease, and other pathologies including Parkinson's disease, protein aggregation provides further cellular stresses that can initiate or feed into the pathways to cell death engendered by oxidative stress. Specific attention is paid here to mitochondrial dysfunction and programmed cell death, and the diverse modes of cell death mediated by mitochondria under oxidative stress. Novel insights into cellular responses to neuronal oxidative stress from a range of different stressors can be gained by detailed transcriptomics analyses. Such studies at the cellular level provide the key for understanding the molecular and cellular pathways whereby neurons respond to oxidative stress and undergo injury and death. These considerations underpin the development of detailed knowledge in more complex integrated systems, up to the intact human bearing the neuropathology, facilitating therapeutic advances.
[Show abstract][Hide abstract] ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neurotrophic peptide. Here, we show that PACAP recruits Rap1 into caveolin-enriched membrane subdomains in PC12 cells and activates Rap1, nuclear ERK1/2, Elk-1 and CREB in a caveolae-dependent manner. We reveal that GSK3beta is a novel modulator in PACAP signalling. PACAP induces phosphorylation of serine 9 in GSK3beta, which is inhibited by silencing Rap1. Lithium and valproate promote but wortmannin and LY294002 attenuate PACAP-induced phosphorylation of both GSK3beta and ERK1/2, whereas MEK inhibitor PD98059 inhibits nerve growth factor- but not PACAP-induced phosphorylation of GSK3beta, suggesting that GSK3beta operates downstream of Rapt 1 but upstream of ERK1/2 in PACAP signalling. Inhibition or stimulation of GSK3beta results in a 2-fold increase and 6-fold decrease in PACAP-induced neurite outgrowth, respectively. These results reveal an important role of caveolae in the signal transduction of PACAP and that GSK3beta is a critical regulator in PACAP-induced neuronal differentiation.
[Show abstract][Hide abstract] ABSTRACT: Programmed cell death (PCD), is a highly regulated and sophisticated cellular mechanism that commits cell to isolated death fate. PCD has been implicated in the pathogenesis of numerous neurodegenerative disorders. Countless molecular events underlie this phenomenon, with each playing a crucial role in death commitment. A precedent event, apoptotic volume decrease (AVD), is ubiquitously observed in various forms of PCD induced by different cellular insults. Under physiological conditions, cells when subjected to osmotic fluctuations will undergo regulatory volume increase/decrease (RVI/RVD) to achieve homeostatic balance with neurons in the brain being additionally protected by the blood-brain-barrier. However, during AVD following apoptotic trigger, cell undergoes anistonic shrinkage that involves the loss of water and ions, particularly monovalent ions e.g. K(+), Na(+) and Cl(-). It is worthwhile to concentrate on the molecular implications underlying the loss of these cellular components which posed to be significant and crucial in the successful propagation of the apoptotic signals. Microarray and real-time PCR analyses demonstrated several ion and water channel genes are regulated upon the onset of lactacystin (a proteosomal inhibitor)-mediated apoptosis. A time course study revealed that gene expressions of water and ion channels are being modulated just prior to apoptosis, some of which are aquaporin 4 and 9, potassium channels and chloride channels. In this review, we shall looked into the molecular protein machineries involved in the execution of AVD in the central nervous system (CNS), and focus on the significance of movements of each cellular component in affecting PCD commitment, thus provide some pharmacological advantages in the global apoptotic cell death.
Current Neuropharmacology 07/2008; 6(2):102-16. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiotoxin-4b (CTX-4b), isolated from Naja naja sputatrix venom, shows lethality in several cell types. Employing murine primary cortical neurons, this study was undertaken to investigate the molecular mechanisms of CTX-4b in the induction of neuronal death. CTX-4b induced a dose- and time-dependent neuronal death. Strong induction of calpains as early as 4h post-CTX-4b 75 nM treatment was detected in neurons with negligible caspase 3 activation. For the first time in cultured murine primary cortical neurons, it was noted that CTX-4b-mediated cell death triggered oxidative stress with an increase in reactive oxygen species (ROS) levels, and that application of antioxidants showed effective attenuation of cell death. Taken together, these results indicate that CTX-4b-mediated neuronal death is associated with (i) early calpain activation and (ii) oxidative stress. Most importantly, antioxidants have proved to be a promising therapeutic avenue against CTX-4b-induced neuronal death.
[Show abstract][Hide abstract] ABSTRACT: Nitric oxide (NO), ubiquitously expressed in the central nervous system, has been perceived to be a potential neuromodulator. Employing cultured murine primary cortical neurons, NO resulted in an inhibition of the ubiquitin-proteasome system (UPS) with a dose- and time-dependent decrease in cell viability. This is consistent with a previous study that reported a dysfunction of UPS with consequential apoptotic death in macrophage cell with NO treatment. However, it cannot be unclear if the drop in UPS efficiency is directly imposed on by NO. Therefore by using microarray analysis, our study revealed an early down-regulation or non-significant differential expression of genes encoding UPS proteins in NOC-18 (NO donor)-treated neurons as compared to an observed elevation of corresponding gene expression genes in lactacystin (classical proteasome inhibitor)-treated neurons (conducted earlier). Furthermore, time-course analysis of proteasome activity in NOC-18-treated neurons demonstrated a late onset of reduction. This is intriguing as it is well established that in an exclusive proteasome dysfunction-induced cell death, a compensatory feedback mechanism will be activated with an initial and concerted up-regulation of genes encoding proteins involved in UPS as seen when neurons were treated with lactacystin. Thus, it is highly suggestive that NO-triggered neuronal death takes on a different signaling cascade from that of a classical proteasome inhibitor, and that the late reduction of proteasome activity is a downstream event following the activation of apoptotic cellular signaling cascade. In intracellular condition, the proteasome is not NO preferred primary target responsible for the trigger of the cell death machinery. In conclusion, we presented novel findings that shed light into NO-induced cell death signaling cascade, which would be important in understanding the pathogenesis of neurodegenerative disorders such as Parkinson's disease.