Excitotoxic stimulation activates distinct pathogenic and protective expression signatures in the hippocampus.

Excitotoxic events underlying ischaemic and traumatic brain injuries activate degenerative and protective pathways, particularly in the hippocampus. To understand opposing pathways that determine the brain's response to excitotoxicity, we used hippocampal explants, thereby eliminating systemic variables during a precise protocol of excitatory stimulation. N-methyl-d-aspartate (NMDA) was applied for 20 min and total RNA isolated one and 24 h later for neurobiology-specific microarrays. Distinct groups of genes exhibited early vs. delayed induction, with 63 genes exclusively reduced 24-h post-insult. Egr-1 and NOR-1 displayed biphasic transcriptional modulation: early induction followed by delayed suppression. Opposing events of NMDA-induced genes linked to pathogenesis and cell survival constituted the early expression signature. Delayed degenerative indicators (up-regulated pathogenic genes, down-regulated pro-survival genes) and opposing compensatory responses (down-regulated pathogenic genes, up-regulated pro-survival genes) generated networks with temporal gene profiles mirroring coexpression network clustering. We then used the expression profiles to test whether NF-κB, a potent transcription factor implicated in both degenerative and protective pathways, is involved in the opposing responses. The NF-κB inhibitor MG-132 indeed altered NMDA-mediated transcriptional changes, revealing components of opposing expression signatures that converge on the single response element. Overall, this study identified counteracting avenues among the distinct responses to excitotoxicity, thereby suggesting multi-target treatment strategies and implications for predictive medicine.

Genomic approaches for investigating mechanisms of genotoxicity.

The current genetic toxicity testing battery enables an accurate and effective detection of genotoxicity associated with exposure to chemicals. However, the interpretation of the data in light of the relevant risk to humans is often difficult due to limited insight into underlying genotoxic mechanisms. Thus, the development of experimental approaches capable of differentiating genotoxic mechanisms is expected to facilitate risk assessment. Recent progress in science and technology has enabled the investigation of the stress response associated with chemical exposure at the genomic level. For instance, gene expression profile analysis, transcriptomics, was proposed as a tool for evaluating toxic mechanisms. In addition, the advancement in genetic tools has allowed the study of stress response on a functional level, functional genomics. This review will outline a number of recent developments in genomic analyses of genotoxic stress response and provide a perspective on their application in genetic toxicology.

Gene expression profile analysis: an emerging approach to investigate mechanisms of genotoxicity.

The response to stress triggers transcriptional activation of genes involved in cell survival and/or cell death. Thus, the monitoring of gene expression levels in large gene sets or whole genomes in response to various agents (toxicogenomics) has been proposed as a tool for investigating mechanisms of toxicity. Although standard in vitro genetic toxicity testing provides relatively simple and accurate hazard detection, interpretation of positive findings, i.e., in vitro chromosome aberrations, in terms of relevant risk to humans is difficult, due to the limited insight into the underlying mechanisms. Therefore, the development of experimental approaches capable of differentiating a wide range of genotoxic mechanisms is expected to significantly improve risk assessment. The goal of this review is to summarize current developments in toxicogenomic analysis of genotoxic stress, and to provide a perspective on the application of gene expression profile analysis in genetic toxicology.

Differentiating mechanisms of toxicity using global gene expression analysis in Saccharomyces cerevisiae.

Genotoxic stress triggers a variety of biological responses including the transcriptional activation of genes regulating DNA repair, cell survival and cell death. Genomic approaches, which monitor gene expressions across large numbers of genes, can serve as a powerful tool for exploring mechanisms of toxicity. Here, using five different agents, we investigated whether the analysis of genome-wide expression profiles in Saccharomyces cerevisiae could provide insights into mechanisms of genotoxicity versus cytotoxicity. To differentiate the genotoxic stress-associated expression signatures from that of a general cytotoxic stress, we compared gene expression profiles following the treatment with DNA-reactive (cisplatin, MMS, bleomycin) and DNA non-reactive (ethanol and sodium chloride) compounds. Although each of the tested chemicals produced a distinct gene expression profile, we were able to identify a gene expression signature consisting of a relatively small number of biologically relevant genes capable of differentiating genotoxic and cytotoxic stress. The gene set includes such upregulated genes as HUG1, ECM4 and previously uncharacterized gene, YLR297W in the genotoxic and GAP1, CGR1 in the cytotoxic group. Our results indicate the potential of gene expression profile analysis for elucidating mechanism of action of genotoxic agents.

Biphasic NF-kappaB activation in the excitotoxic hippocampus.

Excitotoxic stimulation of NMDA receptors results in the activation of a variety of cellular responses. The inducible transcription factor NF-kappaB is known to be involved in excitotoxic responses by neurons. Here, we show that NF-kappaB activation occurs in a biphasic manner in hippocampal slices following a 20-min N-methyl- d-aspartate (NMDA) exposure. The biphasic activation profile consists of an early, rapid phase at 0.5-1 h post-insult, and a delayed phase evident 10-24 h post-insult. Endogenous inhibitors of NF-kappaB, IkappaBs, were examined for their involvement in the biphasic activation. IkappaBbeta exhibited marked degradation in response to the excitotoxity, while changes in the levels of IkappaBalpha and p105 isoforms were not detected. The initial decline in IkappaBbeta occurred in as little as 30 min post-NMDA exposure, coinciding with early NF-kappaB activity. A second, more gradual phase of IkappaBbeta degradation was also evident, possibly giving rise to the delayed activation of the transcription factor. While both phases of NF-kappaB activation were disrupted by the NMDA receptor antagonist AP5, they were distinct with regard to the composition of activated complexes and their responsiveness to altered culture conditions. The two phases of NF-kappaB activity also were associated with distinct gene regulation events. Up-regulation of bcl-2 message occurred early after the excitotoxic insult and remained up-regulated for an extended period. In contrast, bax message initially remained unchanged after the insult, but then exhibited an increase 24 h later, corresponding with the second phase of the NF-kappaB response. These results indicate that distinct phases of NF-kappaB activation are generated in the excitotoxic hippocampus, and that the phases may be involved in opposing cellular responses.

Intracellular deposition, microtubule destabilization, and transport failure: an "early" pathogenic cascade leading to synaptic decline.

Protein deposition is a common event in age-related neurological diseases that are characterized by neuronal dysfunction and eventual cell death. Here, cultured hippocampal slices were infused with the lysosomal disrupter chloroquine to examine the link between abnormal protein processing/deposition and early synaptopathogenesis. Tau species of 55 to 69 kDa increased over several days of treatment with chloroquine, while the protein and message levels of synaptic markers were selectively reduced. Neurons of subfields CA1, CA3, and dentate gyrus accumulated protein deposits recognized by antibodies against paired helical filaments and ubiquitin, and this was accompanied by tubulin fragmentation and deacetylation. The deposition filled the basal pole of pyramidal neurons, encompassing the area of the axon hillock and initial dendritic branching but without causing overt neuronal atrophy. Neurons containing the polar aggregates exhibited severely impaired transport along basal dendrites. Transport capability was also lost along apical dendrites, the opposite direction of deposited material in the basal pole; thus, perpetuating the problem beyond physical blockage must be the associated loss of microtubule integrity. These data indicate that transport failure forms a link between tau deposition and synaptic decline, thus shedding light on how protein aggregation events disrupt synaptic and cognitive functions before the ensuing cellular destruction.

Peptidyl alpha-keto amide inhibitor of calpain blocks excitotoxic damage without affecting signal transduction events.

The cysteine protease calpain is activated by calcium and has a wide range of substrates. Calpain-mediated cellular damage is associated with many neuropathologies, and calpain also plays a role in signal transduction events that are essential for cell maintenance, including the activation of important kinases and transcription factors. In the present study, the hippocampal slice culture was used as a model of excitotoxicity to test whether the neuroprotection elicited by selective calpain inhibition is associated with changes in cell signaling. Peptidyl alpha-keto amide and alpha-keto acid inhibitors reduced both calpain-mediated cytoskeletal damage and the concomitant synaptic deterioration resulting from an N-methyl-D-aspartate exposure. The alpha-keto amide CX295 was protective when infused into slice cultures before or after the excitotoxic episode. The slices protected with CX295 exhibited normal activation levels of mitogen-activated protein kinase and the transcription factor nuclear factor-kappaB. Thus, selective inhibition of calpain provides neuroprotection without influencing critical signaling pathways.

Survival signaling and selective neuroprotection through glutamatergic transmission.

In the brain, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate glutamatergic neurotransmission and, when intensely activated, can induce excitotoxic cell death. In addition to their ionotropic properties, however, AMPA receptors have been functionally coupled to a variety of signal transduction events involving Src-family kinases, G-proteins, and the mitogen-activated protein kinase (MAPK). In the present study, we tested whether AMPA receptors are linked to appropriate signaling events in order to prevent neuronal injury and/or enhance recovery. AMPA stimulation in hippocampal slice cultures caused the selective activation of MAPK through the upstream activator MAPK kinase (MEK). Inhibition of either component of the AMPA receptor--MAPK pathway potentiated cellular damage due to serum deprivation, suggesting that this pathway facilitates compensatory signals in response to injury. Correspondingly, positive modulation of AMPA receptors with the Ampakine 1-(quinoxalin-6-ylcarbonyl)piperidine (CX516) enhanced MAPK activation and reduced the extent of synaptic and neuronal degeneration resulting from excitotoxic episodes. CX516 was neuroprotective when infused into slices either before or after the insult. The Ampakine derivative also elicited neuroprotection in an in vivo model of excitotoxicity as evidenced by reduction in lesion size and preservation of two different types of neurons. Interestingly, the AMPA receptor--MAPK pathway selectively protects against excitotoxicity since enhancing the pathway did not protect against the nonexcitotoxic, slow pathology initiated by lysosomal dysfunction. The results indicate that glutamatergic communication is important for cellular maintenance and that AMPA receptors activate survival signals to counterpoise their own excitotoxic potential.