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.

Leveraging publicly available coronavirus data to identify new therapeutic targets for COVID-19.

Many important questions remain regarding severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the viral pathogen responsible for COVID-19. These questions include the mechanisms explaining the high percentage of asymptomatic but highly infectious individuals, the wide variability in disease susceptibility, and the mechanisms of long-lasting debilitating effects. Bioinformatic analysis of four coronavirus datasets representing previous outbreaks (SARS-CoV-1 and MERS-CoV), as well as SARS-CoV-2, revealed evidence of diverse host factors that appear to be coopted to facilitate virus-induced suppression of interferon-induced innate immunity, promotion of viral replication and subversion and/or evasion of antiviral immune surveillance. These host factors merit further study given their postulated roles in COVID-19-induced loss of smell and brain, heart, vascular, lung, liver, and gut dysfunction.

Principal component analysis of blood microRNA datasets facilitates diagnosis of diverse diseases.

Early, ideally pre-symptomatic, recognition of common diseases (e.g., heart disease, cancer, diabetes, Alzheimer's disease) facilitates early treatment or lifestyle modifications, such as diet and exercise. Sensitive, specific identification of diseases using blood samples would facilitate early recognition. We explored the potential of disease identification in high dimensional blood microRNA (miRNA) datasets using a powerful data reduction method: principal component analysis (PCA). Using Qlucore Omics Explorer (QOE), a dynamic, interactive visualization-guided bioinformatics program with a built-in statistical platform, we analyzed publicly available blood miRNA datasets from the Gene Expression Omnibus (GEO) maintained at the National Center for Biotechnology Information at the National Institutes of Health (NIH). The miRNA expression profiles were generated from real time PCR arrays, microarrays or next generation sequencing of biologic materials (e.g., blood, serum or blood components such as platelets). PCA identified the top three principal components that distinguished cohorts of patients with specific diseases (e.g., heart disease, stroke, hypertension, sepsis, diabetes, specific types of cancer, HIV, hemophilia, subtypes of meningitis, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, mild cognitive impairment, aging, and autism), from healthy subjects. Literature searches verified the functional relevance of the discriminating miRNAs. Our goal is to assemble PCA and heatmap analyses of existing and future blood miRNA datasets into a clinical reference database to facilitate the diagnosis of diseases using routine blood draws.

MicroRNA sequencing of rat hippocampus and human biofluids identifies acute, chronic, focal and diffuse traumatic brain injuries.

High-throughput sequencing technologies could improve diagnosis and classification of TBI subgroups. Because recent studies showed that circulating microRNAs (miRNAs) may serve as noninvasive markers of TBI, we performed miRNA-seq to study TBI-induced changes in rat hippocampal miRNAs up to one year post-injury. We used miRNA PCR arrays to interrogate differences in serum miRNAs using two rat models of TBI (controlled cortical impact [CCI] and fluid percussion injury [FPI]). The translational potential of our results was evaluated by miRNA-seq analysis of human control and TBI (acute and chronic) serum samples. Bioinformatic analyses were performed using Ingenuity Pathway Analysis, miRDB, and Qlucore Omics Explorer. Rat miRNA profiles identified TBI across all acute and chronic intervals. Rat CCI and FPI displayed distinct serum miRNA profiles. Human miRNA profiles identified TBI across all acute and chronic time points and, at 24 hours, discriminated between focal and diffuse injuries. In both species, predicted gene targets of differentially expressed miRNAs are involved in neuroplasticity, immune function and neurorestoration. Chronically dysregulated miRNAs (miR-451a, miR-30d-5p, miR-145-5p, miR-204-5p) are linked to psychiatric and neurodegenerative disorders. These data suggest that circulating miRNAs in biofluids can be used as "molecular fingerprints" to identify acute, chronic, focal or diffuse TBI and potentially, presence of neurodegenerative sequelae.

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.

Development of a novel imaging system for cell therapy in the brain.

INTRODUCTION: Stem cells have been evaluated as a potential therapeutic approach for several neurological disorders of the central and peripheral nervous system as well as for traumatic brain and spinal cord injury. Currently, the lack of a reliable and safe method to accurately and non-invasively locate the site of implantation and track the migration of stem cells in vivo hampers the development of stem cell therapy and its clinical application. In this report, we present data that demonstrate the feasibility of using the human sodium iodide symporter (hNIS) as a reporter gene for tracking neural stem cells (NSCs) after transplantation in the brain by using single-photon emission tomography/computed tomography (SPECT/CT) imaging. METHODS: NSCs were isolated from the hippocampus of adult rats (Hipp-NSCs) and transduced with a lentiviral vector containing the hNIS gene. Hipp-NSCs expressing the hNIS (NIS-Hipp-NSCs) were characterized in vitro and in vivo after transplantation in the rat brain and imaged by using technetium-99m ((99m)Tc) and a small rodent SPECT/CT apparatus. Comparisons were made between Hipp-NSCs and NIS-Hipp-NSCs, and statistical analysis was performed by using two-tailed Student's t test. RESULTS: Our results show that the expression of the hNIS allows the repeated visualization of NSCs in vivo in the brain by using SPECT/CT imaging and does not affect the ability of Hipp-NSCs to generate neuronal and glial cells in vitro and in vivo. CONCLUSIONS: These data support the use of the hNIS as a reporter gene for non-invasive imaging of NSCs in the brain. The repeated, non-invasive tracking of implanted cells will accelerate the development of effective stem cell therapies for traumatic brain injury and other types of central nervous system injury.

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.

Traumatic brain injury in vivo and in vitro contributes to cerebral vascular dysfunction through impaired gap junction communication between vascular smooth muscle cells.

Gap junctions (GJs) contribute to cerebral vasodilation, vasoconstriction, and, perhaps, to vascular compensatory mechanisms, such as autoregulation. To explore the effects of traumatic brain injury (TBI) on vascular GJ communication, we assessed GJ coupling in A7r5 vascular smooth muscle (VSM) cells subjected to rapid stretch injury (RSI) in vitro and VSM in middle cerebral arteries (MCAs) harvested from rats subjected to fluid percussion TBI in vivo. Intercellular communication was evaluated by measuring fluorescence recovery after photobleaching (FRAP). In VSM cells in vitro, FRAP increased significantly (p<0.05 vs. sham RSI) after mild RSI, but decreased significantly (p<0.05 vs. sham RSI) after moderate or severe RSI. FRAP decreased significantly (p<0.05 vs. sham RSI) 30 min and 2 h, but increased significantly (p<0.05 vs. sham RSI) 24 h after RSI. In MCAs harvested from rats 30 min after moderate TBI in vivo, FRAP was reduced significantly (p<0.05), compared to MCAs from rats after sham TBI. In VSM cells in vitro, pretreatment with the peroxynitrite (ONOO(-)) scavenger, 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron[III], prevented RSI-induced reductions in FRAP. In isolated MCAs from rats treated with the ONOO(-) scavenger, penicillamine, GJ coupling was not impaired by fluid percussion TBI. In addition, penicillamine treatment improved vasodilatory responses to reduced intravascular pressure in MCAs harvested from rats subjected to moderate fluid percussion TBI. These results indicate that TBI reduced GJ coupling in VSM cells in vitro and in vivo through mechanisms related to generation of the potent oxidant, ONOO(-).

Effects of trauma, hemorrhage and resuscitation in aged rats.

Traumatic brain injury (TBI) is a leading cause of death in the elderly and the incidence of mortality and morbidity increases with age. This study tested the hypothesis that, after TBI followed by hemorrhagic hypotension (HH) and resuscitation, cerebral blood flow (CBF) would decrease more in aged compared with young rats. Young adult (4-6 months) and aged (20-24 months) male Sprague-Dawley rats were anesthetized with isoflurane, prepared for parasagittal fluid percussion injury (FPI) and randomly assigned to receive either moderate FPI (2.0 atm) only, moderate FPI+severe HH (40 mm Hg for 45 min) followed by return of shed blood, or sham FPI. Intracranial pressure (ICP), CBF, and mean arterial pressure (MAP) were measured and, after twenty-four hours survival, the rats were euthanized and their brains were sectioned and stained with Fluoro-Jade (FJ), a dye that stains injured neurons. After moderate FPI, severe HH and reinfusion of shed blood, MAP and CBF were significantly reduced in the aged group, compared to the young group. Both FPI and FPI+HH groups significantly increased the numbers of FJ-positive neurons in hippocampal cell layers CA1, CA2 and CA3 (p<0.05 vs Sham) in young and aged rats. Despite differences in post-resuscitation MAP and CBF, there were no differences in the numbers of FJ-positive neurons in aged compared to young rats after FPI, HH and blood resuscitation. Although cerebral hypoperfusion in the aged rats was not associated with increased hippocampal cell injury, the trauma-induced reductions in CBF and post-resuscitation blood pressure may have resulted in damage to brain regions that were not examined or neurological or behavioral impairments that were not assessed in this study. Therefore, the maintenance of normal blood pressure and cerebral perfusion would be advisable in the treatment of elderly patients 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.

Cerebrovascular connexin expression: effects of traumatic brain injury.

Traumatic brain injury (TBI) results in dysfunction of the cerebrovasculature. Gap junctions coordinate vasomotor responses and evidence suggests that they are involved in cerebrovascular dysfunction after TBI. Gap junctions are comprised of connexin proteins (Cxs), of which Cx37, Cx40, Cx43, and Cx45 are expressed in vascular tissue. This study tests the hypothesis that TBI alters Cx mRNA and protein expression in cerebral vascular smooth muscle and endothelial cells. Anesthetized (1.5% isoflurane) male Sprague-Dawley rats received sham or fluid-percussion TBI. Two, 6, and 24 h after, cerebral arteries were harvested, fresh-frozen for RNA isolation, or homogenized for Western blot analysis. Cerebral vascular endothelial and smooth muscle cells were selected from frozen sections using laser capture microdissection. RNA was quantified by ribonuclease protection assay. The mRNA for all four Cx genes showed greater expression in the smooth muscle layer compared to the endothelial layer. Smooth muscle Cx43 mRNA expression was reduced 2 h and endothelial Cx45 mRNA expression was reduced 24 h after injury. Western blot analysis revealed that Cx40 protein expression increased, while Cx45 protein expression decreased 24 h after injury. These studies revealed significant changes in the mRNA and protein expression of specific vascular Cxs after TBI. This is the first demonstration of cell type-related differential expression of Cx mRNA in cerebral arteries, and is a first step in evaluating the effects of TBI on gap junction communication in the cerebrovasculature.

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.

Gastrin-releasing peptide receptor in breast cancer mediates cellular migration and interleukin-8 expression.

BACKGROUND: Breast cancers aberrantly express gastrin-releasing peptide (GRP) hormone and its cognate receptor, gastrin-releasing peptide receptor (GRP-R). Experimental evidence suggests that bombesin (BBS), the pharmacological homologue of GRP, promotes breast cancer growth and progression. The contribution of GRP-R to other poor prognostic indicators in breast cancer, such as the expression of the EGF-R family of growth factors and hormone insensitivity, is unknown. MATERIALS AND METHODS: Two estrogen receptor (ER)-negative breast cancer cell lines were used. MDA-MB-231 overexpress both EGFR and GRPR, whereas SK-BR-3 cells express EGF-R but lack GRP-R. Cellular proliferation was assessed by Coulter counter. Chemotactic migration was performed using Transwell chambers, and the migrated cells were quantified. Northern blot and real-time PCR were used to evaluate proangiogenic factor interleukin-8 (IL-8) mRNA expression. RESULTS: In MDA-MB-231 cells, GRP-R and EGF-R synergize to regulate cell migration, IL-8 expression, but not cell proliferation. In SK-BR-3 cells, ectopic expression of GRP-R was sufficient to increase migration and IL-8 mRNA. CONCLUSIONS: These data suggest relevant roles for GRP-R in ER-negative breast cancer progression. Future mechanistic studies to define the molecular role of GRP-R in breast cancer metastasis provide novel targets for the treatment of ER-negative breast cancers.