Kimberly J. Newsom

Human Traumatic Brain Injury Induces Autoantibody Response against Glial Fibrillary Acidic Protein and Its Breakdown Products

The role of systemic autoimmunity in human traumatic brain injury (TBI) and other forms of brain injuries is recognized but not well understood. In this study, a systematic investigation was performed to identify serum autoantibody responses to brain-specific proteins after TBI in humans. TBI autoantibodies showed predominant immunoreactivity against a cluster of bands from 38–50 kDa on human brain immunoblots, which were identified as GFAP and GFAP breakdown products. GFAP autoantibody levels increased by 7 days after injury, and were of the IgG subtype predominantly. Results from in vitro tests and rat TBI experiments also indicated that calpain was responsible for removing the amino and carboxyl termini of GFAP to yield a 38 kDa fragment. Additionally, TBI autoantibody staining co-localized with GFAP in injured rat brain and in primary rat astrocytes. These results suggest that GFAP breakdown products persist within degenerating astrocytes in the brain. Anti-GFAP autoantibody also can enter living astroglia cells in culture and its presence appears to compromise glial cell health. TBI patients showed an average 3.77 fold increase in anti-GFAP autoantibody levels from early (0–1 days) to late (7–10 days) times post injury. Changes in autoantibody levels were negatively correlated with outcome as measured by GOS-E score at 6 months, suggesting that TBI patients with greater anti-GFAP immune-responses had worse outcomes. Due to the long lasting nature of IgG, a test to detect anti-GFAP autoantibodies is likely to prolong the temporal window for assessment of brain damage in human patients.

characterization of Antibodies that Detect Human GFAp after Traumatic Brain Injury

After traumatic brain injury (TBI), glial fibrillary acidic protein (GFAP) and other brain-derived proteins and their breakdown products are released into biofluids such as CSF and blood. Recently, a sandwich ELISA was constructed that measured GFAP concentrations in CSF or serum from human mild-moderate TBI patients. The goals of the present study were to characterize the same two antibodies used in this ELISA, and to determine which GFAP bands are detected by this antibody combination. Here, both antibodies recognized GFAP specifically in human brain and post-TBI CSF in a cluster of bands ranging from 50–38 kDa, that resembled bands from calpain-cleaved GFAP. By immunoprecipitation, the anti-GFAP Capture antibody recovered full length GFAP and its breakdown products from human brain lysate and post-TBI CSF. These findings demonstrate that the anti-GFAP ELISA antibodies non-preferentially detect intact GFAP and GFAP breakdown products, underscoring their utility for detecting brain injury in human patients.

Biomarkers Track Damage after Graded Injury Severity in a Rat Model of Penetrating Brain Injury

The goal of this project was to determine whether biochemical markers of brain damage can be used to diagnose and assess the severity of injury in a rat model of penetrating ballistic-like brain injury (PBBI). To determine the relationship between injury magnitude and biomarker levels, rats underwent three discrete PBBI severity levels defined by the magnitude of the ballistic component of the injury, calibrated to equal 5%, 10%, or 12.5% of total rat brain volume. Cortex, cerebrospinal fluid (CSF), and blood were collected at multiple time points. Levels of three biomarkers (αII-spectrin breakdown product [SBDP150], glial fibrillary acidic protein [GFAP], and ubiquitin C-terminal hydrolase-L1 [UCH-L1]), were measured using quantitative immunoblotting and/or enzyme-linked immunosorbent assays. In injured cortex, SBDP150 and GFAP levels were increased significantly over controls. Cortical SBDP150 was elevated at 1 day but not 7 days, and GFAP at 7 days but not 1 day. At their respective time points, mean levels of SBDP150 and GFAP biomarkers in the cortex rose stepwise as injury magnitude increased. In the CSF, increasing severity of PBBI was associated with increasing concentrations of both neuronal and glial biomarkers acutely at 1 day after injury, but no trends were observed at 7 days. In plasma, SBDP150 was elevated at 5 min after 10% PBBI and at 6 h after 12.5% PBBI. UCH-L1 levels in plasma were elevated acutely at 5 min post-injury reflecting injury severity and rapidly decreased within 2 h. Overall, our results support the conclusion that biomarkers are effective indicators of brain damage after PBBI and may also aid in the assessment of injury magnitude.

In vitro MS-based proteomic analysis and absolute quantification of neuronal-glial injury biomarkers in cell culture system.

MS‐based proteomics has been the method of choice for biomarker discovery in the field of traumatic brain injury (TBI). Due to its high sensitivity and specificity, MS is now being explored for biomarker quantitative validation in tissue and biofluids. In this study, we demonstrate the use of MS in both qualitative protein identification and targeted detection of acute TBI biomarkers released from degenerating cultured rat cortical mixed neuronal cells, mimicking intracellular fluid in the central nervous system after TBI. Calpain activation was induced by cell treatment with maitotoxin (MTX), a known calcium channel opener. Separate plates of mixed neuronal‐glial culture were subjected to excitotoxin N‐methyl‐D‐aspartate (NMDA) and apoptotic inducer staurosporine. Acute TBI biomarkers, GFAP and UCH‐L1, were first detected and assessed in the culture media by Western blot. The cell‐conditioned media were then trypsinized and subjected to bottom up proteomic analysis. GFAP was readily detected by data‐dependent scanning but not UCH‐L1. As a proof‐of‐principle study, rat glia‐enriched cell cultures treated with MTX were used to investigate the time‐dependent release of GFAP breakdown product by Western blot and for isotope dilution MS absolute quantitation method development. Absolute quantitation of the GFAP release was conducted using the three cortical mixed neuronal cell cultures treated with different agents. Other differentially expressed proteins identified in the glial‐enriched and cortical mixed neuronal cell culture models were further analyzed by bioinformatic tools. In summary, this study demonstrates the use of MS in both protein identification and targeted quantitation of acute TBI biomarkers and is the preliminary step toward development of TBI biomarker validation by targeted MS.

Induction of Group IVC Phospholipase A2 in Allergic Asthma: Transcriptional Regulation by TNF-α in Bronchoepithelial Cells

Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus Aspergillus fumigatus in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway. We observed the most significant induction in the group IVC PLA2 (phospholipase A2) [also known as cPLA2γ (cytosolic PLA2γ) or PLA2G4C]. Our results infer that A. fumigatus extract can induce cPLA2γ levels directly in eosinophils, whereas induction in lung epithelial cells is most likely to be a consequence of TNFα (tumour necrosis factor α) secretion by A. fumigatus-activated macrophages. The mechanism of TNFα-dependent induction of cPLA2γ gene expression was elucidated through a combination of promoter deletions, ChIP (chromatin immunoprecipitation) and overexpression studies in human bronchoepithelial cells, leading to the identification of functionally relevant CRE (cAMP-response element), NF-κB (nuclear factor κB) and E-box promoter elements. ChIP analysis demonstrated that RNA polymerase II, ATF-2 (activating transcription factor 2)-c-Jun, p65-p65 and USF (upstream stimulating factor) 1-USF2 complexes are recruited to the cPLA2γ enhancer/promoter in response to TNFα, with overexpression and dominant-negative studies implying a strong level of co-operation and interplay between these factors. Overall, our results link cytokine-mediated alterations in cPLA2γ gene expression with allergic asthma and outline a complex regulatory mechanism.

Regulation of human microsomal prostaglandin E synthase-1 by IL-1β requires a distal enhancer element with a unique role for C/EBPβ.

The studies of PGE2 (prostaglandin E2) biosynthesis have focused primarily on the role of cyclo-oxygenases. Efforts have shifted towards the specific PGE2 terminal synthases, particularly mPGES-1 (microsomal PGE synthase 1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by pro-inflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1 (early growth-response factor 1). Numerous studies demonstrate that the mPGES-1 promoter (PTGES) alone cannot account for the level of IL-1β (interleukin 1β) induction. We identified two DNase I-hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near -8.6 kb. Functional analysis of the distal site revealed two elements that co-operate with basal promoter expression and a stimulus-dependent enhancer. A specific binding site for C/EBPβ (CCAAT/enhancer-binding protein β) in the enhancer was directly responsible for inducible enhancer activity. ChIP (chromatin immunoprecipitation) analysis demonstrated constitutive Egr-1 binding to the promoter and induced RNA polymerase II and C/EBPβ binding to the promoter and enhancer respectively. Knockout/knockdown studies established a functional role for C/EBPβ in mPGES-1 gene regulation and the documented interaction between Egr-1 and C/EBPβ highlights the proximal promoter co-operation with a novel distal enhancer element in regulating inducible mPGES-1 expression.

cPLA2α Gene Activation by IL-1β is Dependent on an Upstream Kinase pathway, Enzymatic Activation and Downstream 15-lipoxygenase Activity: A Positive Feedback Loop

Cytosolic phospholipase A(2)α (cPLA(2)α) is the most widely studied member of the Group IV PLA(2) family. The enzyme is Ca(2+)-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA(2)α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA(2)α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca(2+) levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1β stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA(2)α is phosphorylated within 10min of IL-1β treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA(2)α and a downstream lipoxygenase (15-LOX2) are required for IL-1β-dependent induction of cPLA(2)α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA(2)α→15-LOX2-dependent, positive feedback loop where a proteins enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus.

A distal enhancer controls cytokine-dependent human cPLA2α gene expression

Specific control of group IVA cytosolic phospholipase A2 (cPLA2α or PLA2G4A) expression modulates arachidonic acid production, thus tightly regulating the downstream effects of pro- and anti-inflammatory eicosanoids. The significance of this pathway in human disease is apparent in a range of pathologies from inflammation to tumorigenesis. While much of the regulation of cPLA2α has focused on posttranslational phosphorylation of the protein, studies on transcriptional regulation of this gene have focused only on proximal promoter regions. We have identified a DNase I hypersensitive site encompassing a 5′ distal enhancer element containing a highly conserved consensus AP-1 site involved in transcriptional activation of cPLA2α by interleukin (IL)-1β. Chromatin immunoprecipitation (ChIP), knockdown, knockout, and overexpression analyses have shown that c-Jun acts both in a negative and positive regulatory role. Transcriptional activation of cPLA2α occurs through the phosphorylation of c-Jun in conjunction with increased association of C/EBPβ with the distal novel enhancer. The association of C/EBPβ with the transcriptional activation complex does not require an obvious DNA binding site. These data provide new and important contributions to the understanding of cPLA2α regulation at the transcriptional level, with implications for eicosanoid metabolism, cellular signaling, and disease pathogenesis.

A TEAD1/p65 complex regulates the eutherian-conserved MnSOD intronic enhancer, eRNA transcription and the innate immune response.

Manganese superoxide dismutase (MnSOD), a critical anti-oxidant enzyme, detoxifies the mitochondrial-derived reactive oxygen species, superoxide, elicited through normal respiration or the inflammatory response. Proinflammatory stimuli induce MnSOD gene expression through a eutherian-conserved, intronic enhancer element. We identified two prototypic enhancer binding proteins, TEAD1 and p65, that when co-expressed induce MnSOD expression comparable to pro-inflammatory stimuli. TEAD1 causes the nuclear sequestration of p65 leading to a novel TEAD1/p65 complex that associates with the intronic enhancer and is necessary for cytokine induction of MnSOD. Unlike typical NF-κB-responsive genes, the induction of MnSOD does not involve p50. Beyond MnSOD, the TEAD1/p65 complex regulates a subset of genes controlling the innate immune response that were previously viewed as solely NF-κB-dependent. We also identified an enhancer-derived RNA (eRNA) that is induced by either proinflammatory stimuli or the TEAD1/p65 complex, potentially linking the intronic enhancer to intra- and interchromosomal gene regulation through the inducible eRNA.

Characterization of a mechanism to inhibit ovarian follicle activation

OBJECTIVE To demonstrate that a small molecule can induce the transcription factor Foxo3 in the ovary and lead to inhibition of follicle activation. DESIGN Cell culture, organ culture, and animal studies. SETTING University-based laboratory. ANIMAL(S) 23 female C57BL/6 mice. INTERVENTION(S) Human ovary cells and mouse ovaries in culture treated with 2-deoxyglucose (2-DG) to mimic glucose deprivation, and mice intraperitoneally injected with 100 mg/kg, 300 mg/kg, or 600 mg/kg 2-DG daily for 2 weeks. MAIN OUTCOME MEASURE(S) In cell and organ culture, Foxo3 expression analyzed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR); in treated animals, expression of genes regulated by nutrient deprivation (Foxo3, ATF4, GRP78, CHOP, ASNS, c-Myc) measured in brain, kidney, and ovary by qRT-PCR; and ovarian follicles histologically classified and counted. RESULT(S) Foxo3 expression is induced by 2-DG at both the mRNA and protein level in human ovarian cell culture, possibly through ATF4-dependent gene regulation. Foxo3 expression is also induced by 2-DG in ovarian organ culture. Treatment of mice with 100 mg/kg 2-DG resulted in a 2.6 fold induction of Foxo3 in the ovary and a 58% decrease in type 3a primary follicles. CONCLUSION(S) Expression of Foxo3 is induced by nutrient deprivation in cell culture, organ culture, and in vivo. In mice, 2-DG treatment results in an inhibition of primordial follicle activation. These data indicate that Foxo3 induction by 2-DG may be useful for fertility preservation.