The authors have declared that no competing interests exist.
Applied IPA analysis to microarray data: DRR. Planning the overall experimental design and execution of all experiments: HLH DRB JMC. Conceived and designed the experiments: HLH DSP BEM DSD. Performed the experiments: DRR DRB JMC. Analyzed the data: HLH DSP DRR MAM SLS DRB. Contributed reagents/materials/analysis tools: HLH DSP DSD DRR BEM. Wrote the paper: HLH DSP MAM SLS.
Developing new pharmacotherapies for traumatic brain injury (TBI) requires elucidation of the neuroprotective mechanisms of many structurally and functionally diverse compounds. To test our hypothesis that diverse neuroprotective drugs similarly affect common gene targets after TBI, we compared the effects of two drugs, metyrapone (MT) and carbenoxolone (CB), which, though used clinically for noncognitive conditions, improved learning and memory in rats and humans. Although structurally different, both MT and CB inhibit a common molecular target, 11β hydroxysteroid dehydrogenase type 1, which converts inactive cortisone to cortisol, thereby effectively reducing glucocorticoid levels. We examined injury-induced signaling pathways to determine how the effects of these two compounds correlate with pro-survival effects in surviving neurons of the injured rat hippocampus. We found that treatment of TBI rats with MT or CB acutely induced in hippocampal neurons transcriptional profiles that were remarkably similar (i.e., a coordinated attenuation of gene expression across multiple injury-induced cell signaling networks). We also found, to a lesser extent, a coordinated increase in cell survival signals. Analysis of injury-induced gene expression altered by MT and CB provided additional insight into the protective effects of each. Both drugs attenuated expression of genes in the apoptosis, death receptor and stress signaling pathways, as well as multiple genes in the oxidative phosphorylation pathway such as subunits of NADH dehydrogenase (Complex1), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V). This suggests an overall inhibition of mitochondrial function. Complex 1 is the primary source of reactive oxygen species in the mitochondrial oxidative phosphorylation pathway, thus linking the protective effects of these drugs to a reduction in oxidative stress. The net effect of the drug-induced transcriptional changes observed here indicates that suppressing expression of potentially harmful genes, and also, surprisingly, reduced expression of pro-survival genes may be a hallmark of neuroprotective therapeutic effects.
To date, all pharmacotherapeutic agents in clinical trials for treatment of traumatic brain injury (TBI) have failed to show efficacy, suggesting a need for more effective pre-clinical screening of novel therapeutic compounds
Ideally, in order to counteract hippocampal dysfunction, an effective therapeutic agent would have both neuroprotective and nootropic (memory and cognitive enhancing) properties. In order to focus on the drug-induced alterations in known cell signaling pathways, as opposed to the functional consequences of drug treatment, we investigated two compounds, metyrapone (MT) and carbenoxolone (CB), that possess neuroprotective and nootropic properties but have been used clinically for nonneurologic indications. Metyrapone is used to test for adrenal insufficiency
Additionally, glucocorticoid-mediated oxidative damage in the rat hippocampus has been associated with cognitive deficits
Although both MT and CB have undesirable side-effects that preclude their long-term clinical use
We found that the protective effects of MT and CB in the injured rat hippocampus were associated with similar patterns of changes in injury-induced gene expression, i.e., reduced oxidative stress response, suppressed expression of potentially harmful genes and, surprisingly, reduced expression of pro-survival genes.
Adult male Sprague-Dawley rats (400–500 g) from Charles River (Portland, Maine) were housed two per cage with food and water
Rats were randomly assigned to one of four treatment groups (n = 6 per group). All treatments were administered 30 min prior to moderate TBI: 1) sham injury plus saline (0.1 ml/kg sc; SHAM); 2) moderate TBI plus saline (0.1 ml/kg sc; TBI); 3) moderate TBI plus metyrapone (100 mg/kg; TBI+MT); 4) moderate TBI plus carbenoxolone (30 mg/kg; TBI+CB). Brains were collected at 4 and 24 h post surgery, immediately frozen on dry ice and stored at −80°C for later Fluoro-Jade (FJ) staining and laser capture microdissection (LCM) for gene expression analysis.
Moderate fluid percussion TBI was performed as previously described
Brains were embedded in OCT and 10 µm sections were stained with 0.001% FJ and counterstained with 1% cresyl violet. FJ-positive neurons in the CA1–CA3 hippocampal subfields were counted as previously described
Using a PixCell IIe laser capture microscope with an infrared diode laser (MDS Analytical Technologies, Sunnyvale, CA), we located surviving, FJ-negative neurons that were adjacent to dying, FJ-positive neurons as described in Rojo
Total RNA was prepared using the RNAqueous Micro kit (Ambion), and the RNA samples were sent to GenUs BioSystems (Northbrook, IL) for microarray analysis using 35,000-gene rat CodeLink oligonucleotide microarrays. Biotin-labeled cRNA was prepared by linear amplification of the poly(A)+ RNA population within the total RNA sample. Briefly, 100–500 pg of total RNA for each sample (quantified using Agilent Bioanalyzer) was amplified using the Arcturus RiboAmp HS kit (Molecular Devices). After a second round of cDNA synthesis and purification of double-stranded cDNA,
Each biological replicate sample was hybridized to duplicate arrays. Thus, a total of 32 arrays representing 4 and 24 h time points after TBI were processed with CodeLink Expression Analysis software (GE Healthcare), and data were normalized, filtered, and queried for gene ontology category enrichment with GeneSpring software (Agilent Technologies). To compare individual expression values across arrays, raw intensity data from each probe (generated from CodeLink Expression 4.0 software) was normalized to the median intensity of the array. Only genes with normalized expression values greater than background intensity in at least one condition were used for further analysis. The starting gene list was further limited by only including genes with technical and sample replicate values having less than 2-fold variation. The resulting qualified gene list was used in individual treatment comparisons to determine genes that were differentially expressed across treatments.
The numbers of FJ-positive neurons were quantified for each of the four treatment groups and reported as mean +/− SEM and analyzed using analysis of variance (ANOVA) followed by the Bonferroni-Dunn test with α = 0.05. Statistical computations were carried out using PROC GLM in SAS®, Release9.1
Gene expression levels in hippocampal neurons (considered both uninjured and surviving) of sham-injured rats were measured to establish baseline data for comparison of TBI-induced changes in known signaling pathways. All drug-induced gene expression changes in surviving hippocampal neurons were compared to mean gene expression levels in surviving neurons of TBI alone. Both TBI-induced changes >2-fold compared to the sham-injured baseline controls and the drug-treated samples were analyzed in GeneSpring for significant enrichment (hypergeometric p-values <0.05) of Gene Ontology categories as defined by the GO Consortium. All sham-injury and drug-induced gene expression changes >2-fold compared with TBI alone were uploaded into Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood City, CA) and mapped to the functional networks available in the Ingenuity Pathway Knowledge Base. Canonical pathway analysis identified the pathways from the Ingenuity Pathways Analysis library of canonical pathways that were most significant to the data set. The significance of the association between the data set and the canonical pathway was measured (in IPA) in two ways: 1) A ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules that map to that pathway. 2) The right-tailed Fisher's exact test was used to calculate a
Rats were pretreated with MT or CB 30 min before TBI, and neurodegeneration in the hippocampus was evaluated 24 h post-injury using FJ staining
(
To address the hypothesis that MT and CB act via a common set of genomic targets, we used LCM to collect 600 FJ-negative (surviving) CA3 hippocampal neurons from rats from each of the four treatment groups (Sham, TBI, TBI+MT, TBI+CB). Total RNA was pooled for each treatment group and microarray analysis was performed using rat CodeLink BioArrays (GE Healthcare). At 4 h after TBI, a total of 870 genes were found to have >2-fold differential expression in either Sham, TBI+MT, or TBI+CB relative to TBI alone. The drug-treated samples had broadly similar effects in gene expression at 4 h. Both MT and CB attenuated injury-induced increases in gene expression at 4 h after TBI (
Gene Ontology enrichment analysis revealed diverse biological processes and functions enriched with the 870 genes >2-fold at 4 h after TBI (
We used Ingenuity Pathway Analysis (Ingenuity Systems,
We found a common subset of injury-induced genes that were up-regulated in the TBI group but down-regulated in both the TBI+MT and TBI+CB groups (custom pathway shown in
(
We found that both MT and CB increased expression of genes in the NFAT signaling pathway after TBI (
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Gene expression is shown at 4 h (2 fold cut-off) and 24 h post-TBI (no cut-off). A key cell survival associated gene, NFκB1A, is upregulated by both drugs at 24 h compared with TBI alone. (See
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Both MT and CB altered metabolic pathways linked to oxidative stress. Although neurodegenerative diseases are associated with mitochondrial dysfunction, drugs that induce a mild uncoupling of oxidative phosphorylation (OxPhos), i.e., decrease the proton gradient and mitochondrial potential (ΔΨ)
(
This is the first molecular comparison of the potential mechanisms underlying the neuroprotective effects of MT and CB, and of the molecular targets and associated cell signaling pathways that mediate their protective effects on hippocampal neurons after TBI. Previous reports have shown that, in addition to their nootropic effects and common gene targets, both MT and CB reduce neuronal injury both
The complex pathogenesis of TBI is associated with injury-induced changes in multiple molecular pathways
The main finding of our study is that a distinctive common molecular signature characterizes the effects of MT and CB, thus suggesting an avenue for investigation of the pathophysiology of TBI, as well as evaluation of potential treatment strategies for TBI. Our data show that the cumulative genomic response to these drugs appears to be a homeostatic improvement or normalization in injury-induced signaling. Specifically, MT and CB attenuate injury-induced gene expression associated with neurodegeneration and cell death, and, contrary to our expectation that these drugs would increase mobilization of protective genes, both drugs attenuate injury-induced gene expression associated with cell survival.
Another important outcome of our study is that we show, for the first time, that MT and CB reduce the number of degenerating neurons after TBI in the hippocampus, a brain region critical for learning and memory. These data are concordant with previous studies showing that these drugs prevented ischemia-induced synaptic dysfunction in the hippocampus
Because the goal of this study was to understand drug-induced neuroprotection, it was essential to examine gene expression in only neurons that survived TBI, without the contaminating data deriving from dying neurons. In an earlier study, LCM made it possible for us to selectively capture only surviving, FJ-negative pyramidal neurons from the CA3 subfield of the rat hippocampus at 24 h post-TBI
While it is impossible to predict whether the surviving neurons in untreated TBI rats would have died or survived at a later time point, we know that, at 24 h post TBI, the neurons analyzed in our study were not undergoing neurodegeneration as determined by FJ staining. However, even surviving neurons are expected to show a transcriptional profile reflecting their exposure to TBI in the absence of drug treatment, and surviving neurons from treated animals would show gene expression profiles influenced by drug treatment.
Whether or not the final outcome is death or survival, hippocampal neurons mount a protective molecular response after TBI. In the case of the dying neurons, the response is inadequate. This suggests that neuronal survival or death is regulated by a cell survival rheostat that is determined based on a ratio of cell survival to cell death genes that, in turn, depends on the stochastic expression of these genes in hippocampal neurons before TBI
Pathway analysis revealed several biologically relevant events. Specifically, acute and long-term neurodegeneration is the result of TBI-induced dysregulation of genes in multiple cell signaling networks, and neurons regulate synaptic plasticity by regulating the expression of activity-induced gene expression
The attenuation or normalization of injury-induced gene expression is one of the defining molecular signatures in our study, suggesting that, paradoxically, neuroprotection is associated with an overall lack of mobilization of protective genes. Although we did observe upregulation of some survival-associated genes in the NFAT and PI3K pathways, the attenuating effects of both drugs on several pathways associated with cell death and stress signaling are also consistent with previous studies showing that improved levels of neuroprotection were obtained with therapeutic agents that targeted multiple pathways involved in cell death
Both CB and MT display inhibitory effects on 11βHSD1, and, hence, glucocorticoid production, resulting in inhibition of glucocorticoid-regulated targets such as neuropeptide Y and tyrosine hydroxylase
Extensive
Because TBI produces significant oxidative stress in the rat hippocampus
Interestingly, compounds that partially inhibit OxPhos can increase tolerance to subsequent hypoxia
Finally, one proposed neuroprotective strategy involves reduction of oxidative stress using therapeutic agents such as resveratrol
Although the undesirable side-effect profiles of these drugs preclude long-term use in TBI patients, the demonstration that, following a single treatment, MT or CB reduces neurodegeneration, indicates that acute, short-term treatment after TBI might safely mitigate some of the deleterious effects of the initial injury. These two drugs associated with neuroprotection and cognitive enhancement induce a distinctive molecular profile associated with a reduction in oxidative stress and cell death. Additionally, many drugs in clinical use for other purposes may exhibit similar effects. Thus, the screening of such drugs for efficacy in TBI patients is warranted. Drugs identified using these criteria may also be beneficial for other neurodegenerative disorders in which similar cell survival pathways are affected.
Microarray data is MIAMI compliant and has been deposited in the Gene Expression Omnibus under the accession number GSE31357.
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Supplementary References
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We thank Bridget Capra and Kristy Eidson for their assistance with the laser capture microdissection experiments. We also thank Andy Hall, Laurie Bolding and Christy Perry for their editorial support. We are very grateful to Christy Perry for her superb work on the pathway figures.