Mechanistic insights gained from cell and molecular analysis of the neuroprotective potential of bioactive natural compounds in an immortalized hippocampal cell line.

Evaluating novel compounds for neuroprotective effects in animal models of traumatic brain injury (TBI) is a protracted, labor-intensive and costly effort. However, the present lack of effective treatment options for TBI, despite decades of research, shows the critical need for alternative methods for screening new drug candidates with neuroprotective properties. Because natural products have been a leading source of new therapeutic agents for human diseases, we used an in vitro model of stretch injury to rapidly assess pro-survival effects of three bioactive compounds, two isolated from natural products (clovanemagnolol [CM], vinaxanthone [VX]) and the third, a dietary compound (pterostilbene [PT]) found in blueberries. The stretch injury experiments were not used to validate drug efficacy in a comprehensive manner but used primarily, as proof-of-principle, to demonstrate that the neuroprotective potential of each bioactive agent can be quickly assessed in an immortalized hippocampal cell line in lieu of comprehensive testing in animal models of TBI. To gain mechanistic insights into potential molecular mechanisms of neuroprotective effects, we performed a pathway-specific PCR array analysis of the effects of CM on the rat hippocampus and microRNA sequencing analysis of the effects of VX and PT on cultured hippocampal progenitor neurons. We show that the neuroprotective properties of these natural compounds are associated with altered expression of several genes or microRNAs that have functional roles in neurodegeneration or cell survival. Our approach could help in quickly assessing multiple natural products for neuroprotective properties and expedite the process of new drug discovery for TBI therapeutics.

MicroRNA profiling identifies a novel compound with antidepressant properties.

Patients with traumatic brain injury (TBI) are frequently diagnosed with depression. Together, these two leading causes of death and disability significantly contribute to the global burden of healthcare costs. However, there are no drug treatments for TBI and antidepressants are considered off-label for depression in patients with TBI. In molecular profiling studies of rat hippocampus after experimental TBI, we found that TBI altered the expression of a subset of small, non-coding, microRNAs (miRNAs). One known neuroprotective compound (17ß-estradiol, E2), and two experimental neuroprotective compounds (JM6 and PMI-006), reversed the effects of TBI on miRNAs. Subsequent in silico analyses revealed that the injury-altered miRNAs were predicted to regulate genes involved in depression. Thus, we hypothesized that drug-induced miRNA profiles can be used to identify compounds with antidepressant properties. To confirm this hypothesis, we examined miRNA expression in hippocampi of injured rats treated with one of three known antidepressants (imipramine, fluoxetine and sertraline). Bioinformatic analyses revealed that TBI, potentially via its effects on multiple regulatory miRNAs, dysregulated transcriptional networks involved in neuroplasticity, neurogenesis, and circadian rhythms- networks known to adversely affect mood, cognition and memory. As did E2, JM6, and PMI-006, all three antidepressants reversed the effects of TBI on multiple injury-altered miRNAs. Furthermore, JM6 reduced TBI-induced inflammation in the hippocampus and depression-like behavior in the forced swim test; these are both properties of classic antidepressant drugs. Our results support the hypothesis that miRNA expression signatures can identify neuroprotective and antidepressant properties of novel compounds and that there is substantial overlap between neuroprotection and antidepressant properties.

Traumatic brain injury induces long-lasting changes in immune and regenerative signaling.

There are no existing treatments for the long-term degenerative effects of traumatic brain injury (TBI). This is due, in part, to our limited understanding of chronic TBI and uncertainty about which proposed mechanisms for long-term neurodegeneration are amenable to treatment with existing or novel drugs. Here, we used microarray and pathway analyses to interrogate TBI-induced gene expression in the rat hippocampus and cortex at several acute, subchronic and chronic intervals (24 hours, 2 weeks, 1, 2, 3, 6 and 12 months) after parasagittal fluid percussion injury. We used Ingenuity pathway analysis (IPA) and Gene Ontology enrichment analysis to identify significantly expressed genes and prominent cell signaling pathways that are dysregulated weeks to months after TBI and potentially amenable to therapeutic modulation. We noted long-term, coordinated changes in expression of genes belonging to canonical pathways associated with the innate immune response (i.e., NF-κB signaling, NFAT signaling, Complement System, Acute Phase Response, Toll-like receptor signaling, and Neuroinflammatory signaling). Bioinformatic analysis suggested that dysregulation of these immune mediators-many are key hub genes-would compromise multiple cell signaling pathways essential for homeostatic brain function, particularly those involved in cell survival and neuroplasticity. Importantly, the temporal profile of beneficial and maladaptive immunoregulatory genes in the weeks to months after the initial TBI suggests wider therapeutic windows than previously indicated.

Publisher Correction: Evidence linking microRNA suppression of essential prosurvival genes with hippocampal cell death after traumatic brain injury.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Laser Capture Microdissection in Traumatic Brain Injury Research: Obtaining Hippocampal Subregions and Pools of Injured Neurons for Genomic Analyses.

The methods presented here are based on our laboratory's 15 years of experience using laser capture microdissection to obtain samples for the study of gene expression after traumatic brain injury (TBI) using a well-established rat model of experimental TBI. Here, we describe how to use the ArcturusXT laser capture microdissection system to capture swaths of specific regions of the rat hippocampus as well as specific neuronal populations defined by Fluoro-Jade C staining. Staining with Fluoro-Jade C identifies a neuron that is in the process of degeneration. We have optimized our protocols for Fluoro-Jade C tissue staining and laser capture microdissection to maintain RNA integrity which is essential for a variety of downstream applications, such as microarray, PCR array, and quantitative real-time PCR analyses.

Effects of AAV-mediated knockdown of nNOS and GPx-1 gene expression in rat hippocampus after traumatic brain injury.

Virally mediated RNA interference (RNAi) to knock down injury-induced genes could improve functional outcome after traumatic brain injury (TBI); however, little is known about the consequences of gene knockdown on downstream cell signaling pathways and how RNAi influences neurodegeneration and behavior. Here, we assessed the effects of adeno-associated virus (AAV) siRNA vectors that target two genes with opposing roles in TBI pathogenesis: the allegedly detrimental neuronal nitric oxide synthase (nNOS) and the potentially protective glutathione peroxidase 1 (GPx-1). In rat hippocampal progenitor cells, three siRNAs that target different regions of each gene (nNOS, GPx-1) effectively knocked down gene expression. However, in vivo, in our rat model of fluid percussion brain injury, the consequences of AAV-siRNA were variable. One nNOS siRNA vector significantly reduced the number of degenerating hippocampal neurons and showed a tendency to improve working memory. GPx-1 siRNA treatment did not alter TBI-induced neurodegeneration or working memory deficits. Nevertheless, microarray analysis of laser captured, virus-infected neurons showed that knockdown of nNOS or GPx-1 was specific and had broad effects on downstream genes. Since nNOS knockdown only modestly ameliorated TBI-induced working memory deficits, despite widespread genomic changes, manipulating expression levels of single genes may not be sufficient to alter functional outcome after TBI.

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

Evidence linking microRNA suppression of essential prosurvival genes with hippocampal cell death after traumatic brain injury.

The underlying molecular mechanisms of how dysregulated microRNAs (miRNAs) cause neurodegeneration after traumatic brain injury (TBI) remain elusive. Here we analyzed the biological roles of approximately 600 genes - we previously found these dysregulated in dying and surviving rat hippocampal neurons - that are targeted by ten TBI-altered miRNAs. Bioinformatic analysis suggests that neurodegeneration results from a global miRNA-mediated suppression of genes essential for maintaining proteostasis; many are hub genes - involved in RNA processing, cytoskeletal metabolism, intracellular trafficking, cell cycle progression, repair/maintenance, bioenergetics and cell-cell signaling - whose disrupted expression is linked to human disease. Notably, dysregulation of these essential genes would significantly impair synaptic function and functional brain connectivity. In surviving neurons, upregulated miRNA target genes are co-regulated members of prosurvival pathways associated with cellular regeneration, neural plasticity, and development. This study captures the diversity of miRNA-regulated genes that may be essential for cell repair and survival responses after TBI.

Stochastic fluctuations in gene expression in aging hippocampal neurons could be exacerbated by traumatic brain injury.

Traumatic brain injury (TBI) is a risk factor for age-related dementia and development of neurodegenerative disorders such as Alzheimer's disease that are associated with cognitive decline. The exact mechanism for this risk is unknown but we hypothesized that TBI is exacerbating age-related changes in gene expression. Here, we present evidence in an animal model that experimental TBI increases age-related stochastic gene expression. We compared the variability in expression of several genes associated with cell survival or death, among three groups of laser capture microdissected hippocampal neurons from aging rat brains. TBI increased stochastic fluctuations in gene expression in both dying and surviving neurons compared to the naïve neurons. Increases in random, stochastic fluctuations in prosurvival or prodeath gene expression could potentially alter cell survival or cell death pathways in aging neurons after TBI which may lead to age-related cognitive decline.

Pathway-focused PCR array profiling of enriched populations of laser capture microdissected hippocampal cells after traumatic brain injury.

Cognitive deficits in survivors of traumatic brain injury (TBI) are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays) to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive) or surviving (Fluoro-Jade-negative) pyramidal neurons obtained by laser capture microdissection (LCM). In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER) stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.

Laser capture microdissection of enriched populations of neurons or single neurons for gene expression analysis after traumatic brain injury.

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function. Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al., has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types. We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 °C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

Pathway analysis reveals common pro-survival mechanisms of metyrapone and carbenoxolone after traumatic brain injury.

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, 11ß 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.

Traumatic brain injury-induced dysregulation of the circadian clock.

Circadian rhythm disturbances are frequently reported in patients recovering from traumatic brain injury (TBI). Since circadian clock output is mediated by some of the same molecular signaling cascades that regulate memory formation (cAMP/MAPK/CREB), cognitive problems reported by TBI survivors may be related to injury-induced dysregulation of the circadian clock. In laboratory animals, aberrant circadian rhythms in the hippocampus have been linked to cognitive and memory dysfunction. Here, we addressed the hypothesis that circadian rhythm disruption after TBI is mediated by changes in expression of clock genes in the suprachiasmatic nuclei (SCN) and hippocampus. After fluid-percussion TBI or sham surgery, male Sprague-Dawley rats were euthanized at 4 h intervals, over a 48 h period for tissue collection. Expression of circadian clock genes was measured using quantitative real-time PCR in the SCN and hippocampus obtained by laser capture and manual microdissection respectively. Immunofluorescence and Western blot analysis were used to correlate TBI-induced changes in circadian gene expression with changes in protein expression. In separate groups of rats, locomotor activity was monitored for 48 h. TBI altered circadian gene expression patterns in both the SCN and the hippocampus. Dysregulated expression of key circadian clock genes, such as Bmal1 and Cry1, was detected, suggesting perturbation of transcriptional-translational feedback loops that are central to circadian timing. In fact, disruption of circadian locomotor activity rhythms in injured animals occurred concurrently. These results provide an explanation for how TBI causes disruption of circadian rhythms as well as a rationale for the consideration of drugs with chronobiotic properties as part of a treatment strategy for TBI.

Influence of stochastic gene expression on the cell survival rheostat after traumatic brain injury.

Experimental evidence suggests that random, spontaneous (stochastic) fluctuations in gene expression have important biological consequences, including determination of cell fate and phenotypic variation within isogenic populations. We propose that fluctuations in gene expression represent a valuable tool to explore therapeutic strategies for patients who have suffered traumatic brain injury (TBI), for which there is no effective drug therapy. We have studied the effects of TBI on the hippocampus because TBI survivors commonly suffer cognitive problems that are associated with hippocampal damage. In our previous studies we separated dying and surviving hippocampal neurons by laser capture microdissection and observed unexplainable variations in post-TBI gene expression, even though dying and surviving neurons were adjacent and morphologically identical. We hypothesized that, in hippocampal neurons that subsequently are subjected to TBI, randomly increased pre-TBI expression of genes that are associated with neuroprotection predisposes neurons to survival; conversely, randomly decreased expression of these genes predisposes neurons to death. Thus, to identify genes that are associated with endogenous neuroprotection, we performed a comparative, high-resolution transcriptome analysis of dying and surviving hippocampal neurons in rats subjected to TBI. We found that surviving hippocampal neurons express a distinct molecular signature--increased expression of networks of genes that are associated with regeneration, cellular reprogramming, development, and synaptic plasticity. In dying neurons we found decreased expression of genes in those networks. Based on these data, we propose a hypothetical model in which hippocampal neuronal survival is determined by a rheostat that adds injury-induced genomic signals to expression of pro-survival genes, which pre-TBI varies randomly and spontaneously from neuron to neuron. We suggest that pharmacotherapeutic strategies that co-activate multiple survival signals and enhance self-repair mechanisms have the potential to shift the cell survival rheostat to favor survival and therefore improve functional outcome after TBI.

Chelation of neurotoxic zinc levels does not improve neurobehavioral outcome after traumatic brain injury.

Increases of synaptically released zinc and intracellular accumulation of zinc in hippocampal neurons after traumatic or ischemic brain injury is neurotoxic and chelation of zinc has been shown to reduce neurodegeneration. Although our previous studies showed that zinc chelation in traumatically brain-injured rats correlated with an increase in whole-brain expression of several neuroprotective genes and reduced numbers of apoptotic neurons, the effect on functional outcome has not been determined, and the question of whether this treatment may actually be clinically relevant has not been answered. In the present study, we show that treatment of TBI rats with the zinc chelator calcium EDTA reduces the numbers of injured, Fluoro-Jade-positive neurons in the rat hippocampus 24 h after injury but does not improve neurobehavioral outcome (spatial memory deficits) 2 weeks post-injury. Our data suggest that zinc chelation, despite providing short-term histological neuroprotection, fails to improve long-term functional outcome, perhaps because long-term disruptions in homeostatic levels of zinc adversely influence hippocampus-dependent spatial memory.

Injured Fluoro-Jade-positive hippocampal neurons contain high levels of zinc after traumatic brain injury.

Hippocampal damage contributes to cognitive dysfunction after traumatic brain injury (TBI). We previously showed that Fluoro-Jade, a fluorescent stain that labels injured, degenerating brain neurons, quantifies the extent of hippocampal injury after experimental fluid percussion TBI in rats. Coincidentally, we observed that injured neurons in the rat hippocampus also stained with Newport Green, a fluorescent dye specific for free ionic zinc. Here, we show that, regardless of injury severity or therapeutic intervention, the post-TBI population of injured neurons in rat hippocampal subfields CA1, CA3 and dentate gyrus is indistinguishable, both in numbers and anatomical distribution, from the population of neurons containing high levels of zinc. Treatment with lamotrigine, which inhibits presynaptic release of glutamate and presumably zinc that is co-localized with glutamate, reduced numbers of Fluoro-Jade-positive and Newport Green-positive neurons equally as did treatment with nicardipine, which blocks voltage-gated calcium channels through which zinc enters neurons. To confirm using molecular techniques that Fluoro-Jade and Newport Green-positive neurons are equivalent populations, we isolated total RNA from 25 Fluoro-Jade-positive and 25 Newport Green-positive pyramidal neurons obtained by laser capture microdissection (LCM) from the CA3 subfield, linearly amplified the mRNA and used quantitative ribonuclease protection analysis to demonstrate similar expression of mRNA for selected TBI-induced genes. Our data suggest that therapeutic interventions aimed at reducing neurotoxic zinc levels after TBI may reduce hippocampal neuronal injury.