Quantitative Profiling of S-Nitrosylated Proteins in Parkinson's Disease Paradigms for the Effects of Botanical Phenolics [abstract]

Neuroscience - Vision & Functional Brain Imaging Poster SessionA convergent feature for most aging-related neurological diseases, such as Parkinson's Disease (PD), is excessive generation of free radicals - reactive nitrogen and oxygen species, which can contribute to neuronal cell death and link to the disease pathogenesis. Free radical nitric oxide (NO) is a signaling molecule involving in the regulation of a wide range of cellular functions from development to disease. Emerging evidence suggests that nitrosative stress due to NO over-production induces post-translational modifications of protein cysteine and modulates protein enzymatic activity in cells. S-Nitrosylation, the covalent adduction of NO to specific protein cysteine thiol, is considered as a predominant, redox-based prototypical mechanism for cell signaling. Previously, endogenous protein S-nitrosylation was detected by the biotin switch assay. Taking the advantages of both biotin switch assay and differential in-gel electrophoresis (DIGE), we developed a gel-based proteomics method, named as NitroDIGE, to globally and quantitatively investigate protein S-nitrosylation. Using this method, we identified a subset of S-nitrosylated proteins from both in vitro and in vivo models of Parkinsonism including pesticide rotenone-induced PD-relevant insults in SH-SY5Y cells. Moreover, we determined whether protein S-nitrosylation in cellular PD models could be modulated by different botanical phenolic compounds, including epigallocatechin gallate (EGCG) from green tea, and apocynin from Picrorhiza kurrooa, a herbal plant grown in the Himalayan. The NitroDIGE results demonstrated that the treatment of botanical compounds could reduce excessive S-nitrosylated proteins in SH-SY5Y cells exposed to rotenone, indicating that these botanical phenolics could serve as effective NO scavengers to attenuate nitrosative stress and PD-relevant insults

Cytosolic phospholipase A 2 plays a crucial role in ROS/NO signaling during microglial activation through the lipoxygenase pathway

BACKGROUND: Oxidative stress and inflammation are important factors contributing to the pathophysiology of numerous neurological disorders, including Alzheimer’s disease, Parkinson’s disease, acute stroke, and infections of the brain. There is well-established evidence that proinflammatory cytokines and glutamate, as well as reactive oxygen species (ROS) and nitric oxide (NO), are produced upon microglia activation, and these are important factors contributing to inflammatory responses and cytotoxic damage to surrounding neurons and neighboring cells. Microglial cells express relatively high levels of cytosolic phospholipase A(2) (cPLA(2)), an enzyme known to regulate membrane phospholipid homeostasis and release of arachidonic acid (AA) for synthesis of eicosanoids. The goal for this study is to elucidate the role of cPLA(2)IV in mediating the oxidative and inflammatory responses in microglial cells. METHODS: Experiments involved primary microglia cells isolated from transgenic mice deficient in cPLA(2)α or iPLA(2)β, as well as murine immortalized BV-2 microglial cells. Inhibitors of cPLA(2)/iPLA(2)/cyclooxygenase (COX)/lipoxygenase (LOX) were used in BV-2 microglial cell line. siRNA transfection was employed to knockdown cPLA(2) expression in BV-2 cells. Griess reaction protocol was used to determine NO concentration, and CM-H2DCF-DA was used to detect ROS production in primary microglia and BV-2 cells. WST-1 assay was used to assess cell viability. Western blotting was used to assess protein expression levels. Immunocytochemical staining for phalloidin against F-actin was used to demonstrate cell morphology. RESULTS: In both primary and BV-2 microglial cells, stimulation with lipopolysaccharide (LPS) or interferon gamma (IFNγ) resulted in a time-dependent increase in phosphorylation of cPLA(2) together with ERK1/2. In BV-2 cells, LPS- and IFNγ-induced ROS and NO production was inhibited by arachidonyl trifluoromethyl ketone (AACOCF3) and pyrrophenone as well as RNA interference, but not BEL, suggesting a link between cPLA(2), and not iPLA(2), on LPS/IFNγ-induced nitrosative and oxidative stress in microglial cells. Primary microglial cells isolated from cPLA(2)α-deficient mice generated significantly less NO and ROS as compared with the wild-type mice. Microglia isolated from iPLA(2)β-deficient mice did not show a decrease in LPS-induced NO and ROS production. LPS/IFNγ induced morphological changes in primary microglia, and these changes were mitigated by AACOCF3. Interestingly, despite that LPS and IFNγ induced an increase in phospho-cPLA(2) and prostaglandin E2 (PGE2) release, LPS- and IFNγ-induced NO and ROS production were not altered by the COX-1/2 inhibitor but were suppressed by the LOX-12 and LOX-15 inhibitors instead. CONCLUSIONS: In summary, the results in this study demonstrated the role of cPLA(2) in microglial activation with metabolic links to oxidative and inflammatory responses, and this was in part regulated by the AA metabolic pathways, namely the LOXs. Further studies with targeted inhibition of cPLA(2)/LOX in microglia during neuroinflammatory conditions can be valuable to investigate the therapeutic potential in ameliorating neurological disease pathology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12974-015-0419-0) contains supplementary material, which is available to authorized users

Perspectives on Primary Blast Injury of the Brain: Translational Insights Into Non-inertial Low-Intensity Blast Injury

Most traumatic brain injuries (TBIs) during military deployment or training are clinically “mild” and frequently caused by non-impact blast exposures. Experimental models were developed to reproduce the biological consequences of high-intensity blasts causing moderate to severe brain injuries. However, the pathophysiological mechanisms of low-intensity blast (LIB)-induced neurological deficits have been understudied. This review provides perspectives on primary blast-induced mild TBI models and discusses translational aspects of LIB exposures as defined by standardized physical parameters including overpressure, impulse, and shock wave velocity. Our mouse LIB-exposure model, which reproduces deployment-related scenarios of open-field blast (OFB), caused neurobehavioral changes, including reduced exploratory activities, elevated anxiety-like levels, impaired nesting behavior, and compromised spatial reference learning and memory. These functional impairments associate with subcellular and ultrastructural neuropathological changes, such as myelinated axonal damage, synaptic alterations, and mitochondrial abnormalities occurring in the absence of gross- or cellular damage. Biochemically, we observed dysfunctional mitochondrial pathways that led to elevated oxidative stress, impaired fission-fusion dynamics, diminished mitophagy, decreased oxidative phosphorylation, and compensated cell respiration-relevant enzyme activity. LIB also induced increased levels of total tau, phosphorylated tau, and amyloid β peptide, suggesting initiation of signaling cascades leading to neurodegeneration. We also compare translational aspects of OFB findings to alternative blast injury models. By scoping relevant recent research findings, we provide recommendations for future preclinical studies to better reflect military-operational and clinical realities. Overall, better alignment of preclinical models with clinical observations and experience related to military injuries will facilitate development of more precise diagnosis, clinical evaluation, treatment, and rehabilitation

Oxidation of the cysteine-rich regions of parkin perturbs its E3 ligase activity and contributes to protein aggregation

<p>Abstract</p> <p>Background</p> <p>Accumulation of aberrant proteins to form Lewy bodies (LBs) is a hallmark of Parkinson's disease (PD). Ubiquitination-mediated degradation of aberrant, misfolded proteins is critical for maintaining normal cell function. Emerging evidence suggests that oxidative/nitrosative stress compromises the precisely-regulated network of ubiquitination in PD, particularly affecting parkin E3 ligase activity, and contributes to the accumulation of toxic proteins and neuronal cell death.</p> <p>Results</p> <p>To gain insight into the mechanism whereby cell stress alters parkin-mediated ubiquitination and LB formation, we investigated the effect of oxidative stress. We found significant increases in oxidation (sulfonation) and subsequent aggregation of parkin in SH-SY5Y cells exposed to the mitochondrial complex I inhibitor 1-methyl-4-phenlypyridinium (MPP^<b>+</b>), representing an <it>in vitro </it>cell-based PD model. Exposure of these cells to direct oxidation via pathological doses of H₂O₂induced a vicious cycle of increased followed by decreased parkin E3 ligase activity, similar to that previously reported following S-nitrosylation of parkin. Pre-incubation with catalase attenuated H₂O₂accumulation, parkin sulfonation, and parkin aggregation. Mass spectrometry (MS) analysis revealed that H₂O₂reacted with specific cysteine residues of parkin, resulting in sulfination/sulfonation in regions of the protein similar to those affected by parkin mutations in hereditary forms of PD. Immunohistochemistry or gel electrophoresis revealed an increase in aggregated parkin in rats and primates exposed to mitochondrial complex I inhibitors, as well as in postmortem human brain from patients with PD with LBs.</p> <p>Conclusion</p> <p>These findings show that oxidative stress alters parkin E3 ligase activity, leading to dysfunction of the ubiquitin-proteasome system and potentially contributing to LB formation.</p

Mechanism-based Inhibitor of Matrix Metalloproteinase-9 Rescues Brain from Focal Cerebral Ischemia-induced Damage and Improve Neurological Outcomes in Mice [abstract]

Neuroscience - Vision & Functional Brain Imaging Poster SessionStroke is the third leading cause of death in the US and the primary cause of long-term disability. Acute ischemic stroke, the most common form of stroke, is caused by clotting in the cerebral arteries leading to brain oxygen deprivation and cerebral infarction. The events involved in stroke include brain cell injury or death, breakdown of the blood-brain barrier (BBB), edema, and hemorrhage, which are associated with the expression and activation of matrix metalloproteinases (MMPs), particularly MMP-9. In two focal cerebral ischemia paradigms - the filament-induced transient middle cerebral artery occlusion (MCAo) and the embolus-induced permanent MCAo in mice, we examined MMP-9 proteolysis of extracellular matrix (ECM) components and the neuroprotective effects of the highly selective mechanism-based inhibitor of MMP-9, SB-3CT, which is activated by MMP-9 under pathological conditions. We demonstrated that MMP-9 degrades the ECM protein laminin and that this degradation induces neuronal apoptosis in a transient focal cerebral ischemia model in mice. SB-3CT dramatically blocks MMP-9 activity and decreases MMP-9-mediated laminin cleavage, thus rescuing neurons from apoptosis and ameliorating neurobehavioral outcomes. Significant therapeutic activity of SB-3CT is seen up to 6 h after initial brain damage. Moreover, treatment with SB-3CT attenuates brain MMP-9 activity and protects against delayed neuronal cell death in the embolus-induced permanent MCAo in mice. We conclude that MMP-9 is a highly promising drug target and that SB-3CT has significant therapeutic potential in stroke patients

Prolonged exposure of cortical neurons to oligomeric amyloid-β impairs NMDA receptor function via NADPH oxidase-mediated ROS production: protective effect of green tea (–)-epigallocatechin-3-gallate

Excessive production of Aβ (amyloid β-peptide) has been shown to play an important role in the pathogenesis of AD (Alzheimer's disease). Although not yet well understood, aggregation of Aβ is known to cause toxicity to neurons. Our recent study demonstrated the ability for oligomeric Aβ to stimulate the production of ROS (reactive oxygen species) in neurons through an NMDA (N-methyl-d-aspartate)-dependent pathway. However, whether prolonged exposure of neurons to aggregated Aβ is associated with impairment of NMDA receptor function has not been extensively investigated. In the present study, we show that prolonged exposure of primary cortical neurons to Aβ oligomers caused mitochondrial dysfunction, an attenuation of NMDA receptor-mediated Ca2+ influx and inhibition of NMDA-induced AA (arachidonic acid) release. Mitochondrial dysfunction and the decrease in NMDA receptor activity due to oligomeric Aβ are associated with an increase in ROS production. Gp91ds-tat, a specific peptide inhibitor of NADPH oxidase, and Mn(III)-tetrakis(4-benzoic acid)-porphyrin chloride, an ROS scavenger, effectively abrogated Aβ-induced ROS production. Furthermore, Aβ-induced mitochondrial dysfunction, impairment of NMDA Ca2+ influx and ROS production were prevented by pre-treatment of neurons with EGCG [(−)-epigallocatechin-3-gallate], a major polyphenolic component of green tea. Taken together, these results support a role for NADPH oxidase-mediated ROS production in the cytotoxic effects of Aβ, and demonstrate the therapeutic potential of EGCG and other dietary polyphenols in delaying onset or retarding the progression of AD

Low-Intensity Blast Induces Acute Glutamatergic Hyperexcitability in Mouse Hippocampus Leading to Long-Term Learning Deficits and Altered Expression of Proteins Involved in Synaptic Plasticity and Serine Protease Inhibitors

Neurocognitive consequences of blast-induced traumatic brain injury (bTBI) pose significant concerns for military service members and veterans with the majority of invisible injury. However, the underlying mechanism of such mild bTBI by low-intensity blast (LIB) exposure for long-term cognitive and mental deficits remains elusive. Our previous studies have shown that mice exposed to LIB result in nanoscale ultrastructural abnormalities in the absence of gross or apparent cellular damage in the brain. Here we tested the hypothesis that glutamatergic hyperexcitability may contribute to long-term learning deficits. Using brain slice electrophysiological recordings, we found an increase in averaged frequencies with a burst pattern of miniature excitatory postsynaptic currents (mEPSCs) in hippocampal CA3 neurons in LIB-exposed mice at 1- and 7-days post injury, which was blocked by a specific NMDA receptor antagonist AP5. In addition, cognitive function assessed at 3-months post LIB exposure by automated home-cage monitoring showed deficits in dynamic patterns of discrimination learning and cognitive flexibility in LIB-exposed mice. Collected hippocampal tissue was further processed for quantitative global-proteomic analysis. Advanced data-independent acquisition for quantitative tandem mass spectrometry analysis identified altered expression of proteins involved in synaptic plasticity and serine protease inhibitors in LIB-exposed mice. Some were correlated with the ability of discrimination learning and cognitive flexibility. These findings show that acute glutamatergic hyperexcitability in the hippocampus induced by LIB may contribute to long-term cognitive dysfunction and protein alterations. Studies using this military-relevant mouse model of mild bTBI provide valuable insights into developing a potential therapeutic strategy to ameliorate hyperexcitability-modulated LIB injuries

Proteomic analysis and biochemical correlates of mitochondrial dysfunction following low-intensity primary blast exposure

Service members during military actions or combat training are frequently exposed to primary blasts by weaponry. Most studies have investigated moderate or severe brain injuries from blasts generating overpressures over 100-kPa, while understanding the pathophysiology of low-intensity blast (LIB)-induced mild traumatic brain injury (mTBI) leading to neurological deficits remains elusive. Our recent studies, using an open-field LIB-induced mTBI mouse model with an peak overpressure at 46.6-kPa, demonstrated behavioral impairments and brain nanoscale damages, notably mitochondrial and axonal ultrastructural changes. In this study, we used tandem mass tagged (TMT) quantitative proteomics and bioinformatics analysis to seek insights into the molecular mechanisms underlying ultrastructural pathology. Changes in global- and phospho-proteomes were determined at 3 and 24 hours, 7 and 30 days post injury (DPI), and to investigate the biochemical and molecular correlates of mitochondrial dysfunction. Results showed striking dynamic changes in a total of 2216 global and 459 phosphorylated proteins at vary time points after blast. Disruption of key canonical pathways included evidence of mitochondrial dysfunction, oxidative stress, axonal/cytoskeletal/synaptic dysregulation, and neurodegeneration. Bioinformatic analysis identified blast induced trends in networks related to cellular growth/development/movement/assembly and cell-to-cell signaling interactions. With observations of proteomic changes, we found LIB-induced oxidative stress associated with mitochondrial dysfunction mainly at 7 and 30 DPI. These dysfunctions included impaired fission-fusion dynamics, diminished mitophagy, decreased oxidative phosphorylation, and compensated respiration-relevant enzyme activities. Insights on the early pahtogenesis of primary LIB-induced brain damage provide a template for further characterization of its chronic effects, identification of potential biomarkers and targets for intervention.Hailong song (1), Mei Chen (6), Chen Chen (2), Jiankun Cui (1,7), Catherine Johnson (3), Jianlin Cheng (2), Xiaowan Wang (4), Russell H. Swerdlow (4), Ralph DePalma (5), Weiming Xia (6), Zezong Gu (1,7) ; 1. Department of Pathology & Anatomical Sciences, University of Missouri School of Medicine; 2. Department of Computer Sciences, University of Missouri; 3. Department of Mining and Nuclear Engineering, Missouri University of Science and Technology; 4. Department of Neurology, University of Kansas Medical Center; 5. Office of Research and Development, Department of Veterans Affairs; 6. Bedford VA Medical Center; 7. Truman VA Hospital Research Servic