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Introduction Sarin is a highly toxic organophosphorus nerve agent that irreversibly inhibits neuronal enzyme acetylcholinesterase. In the prevailing scenario, it is of paramount importance to develop early diagnosis and medical countermeasures for sarin exposure. A deeper understanding of the molecular mechanism of sarin intoxication and perturbations in the associated cellular processes is likely to provide valuable clues for the elucidation of diagnostic markers and therapeutic targets for sarin exposure. Methods Present study, uncovered the changes in phosphorylation patterns of multiple proteins in different rat brain regions after sarin intoxication using 2-DE/MS approach. It provided a holistic view of the phosphorylation-mediated changes in the cellular proteome and highlighted various signaling and response pathways affected at an early time point of sarin intoxication. Results We found total 22 proteins in the cortex, 25 proteins in the corpus striatum, and 17 proteins in the hippocampus, showed ≥1.5 fold changes (hyper- or hypo- phosphorylated) with respect to control, either at 2.5 h or 1 d after sarin exposure. These results indicated the differential expression of phosphoproteins involved in protein folding in the endoplasmic reticulum, carbon metabolism, metabolic function, and energy metabolism. Conclusion Four candidates (protein disulfide-isomerase A3, heat shock cognate 71 kDa protein, alpha-enolase, and creatine kinase B-type), hyperphosphorylated in all three brain regions, can be further studied to understand the molecular mechanism behind neurodegenerative changes mediated by sarin exposure. The study sheds light on major pathogenic processes initiated during sarin intoxication and provides putative diagnostic markers/therapeutic targets for further validation.

Significance Chronic neurodegeneration, a major cause of the long-term disabilities that afflict survivors of traumatic brain injury (TBI), is linked to an increased risk for late-life neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, vascular dementia, and chronic traumatic encephalopathy. Here, we report on the restoration of blood–brain barrier (BBB) structure and function by P7C3-A20 when administered 12 mo after TBI. This pharmacotherapy was associated with cessation of chronic neurodegeneration and recovery of normal cognitive function, benefits that persisted long after treatment cessation. Pharmacologic renewal of BBB integrity may thus provide a new treatment option for patients who have suffered a remote TBI, or other neurological conditions associated with BBB deterioration., Chronic neurodegeneration in survivors of traumatic brain injury (TBI) is a major cause of morbidity, with no effective therapies to mitigate this progressive and debilitating form of nerve cell death. Here, we report that pharmacologic restoration of the blood–brain barrier (BBB), 12 mo after murine TBI, is associated with arrested axonal neurodegeneration and cognitive recovery, benefits that persisted for months after treatment cessation. Recovery was achieved by 30 d of once-daily administration of P7C3-A20, a compound that stabilizes cellular energy levels. Four months after P7C3-A20, electron microscopy revealed full repair of TBI-induced breaks in cortical and hippocampal BBB endothelium. Immunohistochemical staining identified additional benefits of P7C3-A20, including restoration of normal BBB endothelium length, increased brain capillary pericyte density, increased expression of BBB tight junction proteins, reduced brain infiltration of immunoglobulin, and attenuated neuroinflammation. These changes were accompanied by cessation of TBI-induced chronic axonal degeneration. Specificity for P7C3-A20 action on the endothelium was confirmed by protection of cultured human brain microvascular endothelial cells from hydrogen peroxide-induced cell death, as well as preservation of BBB integrity in mice after exposure to toxic levels of lipopolysaccharide. P7C3-A20 also protected mice from BBB degradation after acute TBI. Collectively, our results provide insights into the pathophysiologic mechanisms behind chronic neurodegeneration after TBI, along with a putative treatment strategy. Because TBI increases the risks of other forms of neurodegeneration involving BBB deterioration (e.g., Alzheimer’s disease, Parkinson’s disease, vascular dementia, chronic traumatic encephalopathy), P7C3-A20 may have widespread clinical utility in the setting of neurodegenerative conditions.

Diisopropyl fluorophosphate (DFP), a surrogate of nerve agent sarin, is an organophosphorus (OP) compound which inhibits neuronal enzyme acetylcholinesterase (AChE). Exposure of this compound leads to a wide range of toxic symptoms and survivors may exhibit long term neurotoxicity related to cognitive and memory defects. Due to ease of availability and similar mechanism of action to other highly toxic nerve agent, DFP is widely used as model compound to trace changes associated with nerve agent exposures. Proximal fluids are widely used for the elucidation of biomarkers for exposure to toxic substances and to study the mechanism of toxicity. Using a rat model of OP intoxication, the present study was carried out to elucidate proteomic changes in plasma associated with DFP intoxication. Rats were exposed to a single dose (0.5 LD50) of DFP and their plasma proteome was studied, one day post exposure by two dimensional gel electrophoresis - mass spectrometry (2DE-MS). Some of the milestone changes were validated by Western blot analysis. A total 15 proteins showed significant fold changes in expression with respect to control after 1 day of DFP intoxication. Most of the proteins showing changes in expression at initial stages were related to immunogenic function, acute phase response, blood coagulation, and stress response. Experiments reported here demonstrate that 0.5 LD50 DFP intoxication leads to AChE inhibition, modulation of immunogenic function, and generation of stress at an early stage. Although, some proteins and their putative functional ramifications indicated similarity with those observed in our previous plasma proteome study, neurodegenerative changes were not observed in plasma of 0.5 LD50 DFP treated animals.

Significance The unique aspects of brain mitochondria composition and function are incompletely understood, and new approaches are required to understand their relationship. We addressed this challenge through a dual-process isolation procedure that yields both semipure brain mitochondria suitable for functional studies but not protein localization and ultrapure brain mitochondria that are suitable for determining protein localization but functionally compromised. We observed that contrary to the published literature, N-methyl-D-aspartate receptor, ryanodine receptor 1, glyceraldehyde 3-phosphate dehydrogenase, monocarboxylate transporter 1, excitatory amino acid transporter 1, and the L-type calcium channel Cav1.2α1 subunit are absent from brain mitochondria. Utilization of this dual-process brain mitochondria isolation technique will foster clarity on mechanisms of brain health and disease., The brain requires continuously high energy production to maintain ion gradients and normal function. Mitochondria critically undergird brain energetics, and mitochondrial abnormalities feature prominently in neuropsychiatric disease. However, many unique aspects of brain mitochondria composition and function are poorly understood. Developing improved neuroprotective therapeutics thus requires more comprehensively understanding brain mitochondria, including accurately delineating protein composition and channel–transporter functional networks. However, obtaining pure mitochondria from the brain is especially challenging due to its distinctive lipid and cell structure properties. As a result, conflicting reports on protein localization to brain mitochondria abound. Here we illustrate this problem with the neuropsychiatric disease-associated L-type calcium channel Cav1.2α1 subunit previously observed in crude mitochondria. We applied a dual-process approach to obtain functionally intact versus compositionally pure brain mitochondria. One branch utilizes discontinuous density gradient centrifugation to isolate semipure mitochondria suitable for functional assays but unsuitable for protein localization because of endoplasmic reticulum (ER) contamination. The other branch utilizes self-forming density gradient ultracentrifugation to remove ER and yield ultrapure mitochondria that are suitable for investigating protein localization but functionally compromised. Through this process, we evaluated brain mitochondria protein content and observed the absence of Cav1.2α1 and other previously reported mitochondrial proteins, including the NMDA receptor, ryanodine receptor 1, monocarboxylate transporter 1, excitatory amino acid transporter 1, and glyceraldehyde 3-phosphate dehydrogenase. Conversely, we confirmed mitochondrial localization of several plasma membrane proteins previously reported to also localize to mitochondria. We expect this dual-process isolation procedure will enhance understanding of brain mitochondria in both health and disease.

Traumatic brain injury (TBI) is the largest non-genetic, non-aging related risk factor for Alzheimer's disease (AD). We report here that TBI induces tau acetylation (ac-tau) at sites acetylated also in human AD brain. This is mediated by S-nitrosylated-GAPDH, which simultaneously inactivates Sirtuin1 deacetylase and activates p300/CBP acetyltransferase, increasing neuronal ac-tau. Subsequent tau mislocalization causes neurodegeneration and neurobehavioral impairment, and ac-tau accumulates in the blood. Blocking GAPDH S-nitrosylation, inhibiting p300/CBP, or stimulating Sirtuin1 all protect mice from neurodegeneration, neurobehavioral impairment, and blood and brain accumulation of ac-tau after TBI. Ac-tau is thus a therapeutic target and potential blood biomarker of TBI that may represent pathologic convergence between TBI and AD. Increased ac-tau in human AD brain is further augmented in AD patients with history of TBI, and patients receiving the p300/CBP inhibitors salsalate or diflunisal exhibit decreased incidence of AD and clinically diagnosed TBI.

Sarin is a highly toxic organophosphonate and neural enzyme acetylcholinesterase (AChE) inhibitor. Inhibition of AChE causes large accumulation of acetylcholine at synaptic cleft leading to hyper activation of nicotinic and muscarinic acetylcholine receptors, causing excessive secretions, muscle fasciculation, nausea, vomiting, respiratory distress and neurological effects. There are cases in which long term psychomotor function deficiency, reduced learning and memory functions have been observed several years after exposure of sarin among survivors. This phenomenon is called Organophosphorus ester Induced Chronic Neurotoxicity (OPICN) and cannot be explained by AChE inhibition alone. Plasma proteomics at earlier stages was carried out to study changes reflected at blood level that can help predict possible neurological insults at an early time point to take proper therapeutic interventions against OPICN. In the present study, a 0.5 LD50 dose of sarin was administered to Wistar rats and possible changes in blood plasma proteomic profile were investigated after one and seven days of sarin exposure. Proteins were separated on 2-dimensional gel electrophoresis and identified by MALDI-TOF/MS. Expression profile of major proteins was validated by Western blot. Result showed that after exposure of sarin inhibition of AChE persisted after one week of exposure. There were 14 plasma proteins that showed significant changes in expression (>1.5-fold). It included proteins related to immune function, neurodegenerative condition and chaperone function. Interestingly sarin exposure caused decreased expression of plasma Apolipoprotein A-1 and Haptoglobin on day seven, which are the putative early molecular markers for cognitive impairment and neurodegenerative changes.

In addition to needing acute emergency management, blast-mediated traumatic brain injury (TBI) is also a chronic disorder with delayed-onset symptoms that manifest and progress over time. While the immediate consequences of acute blast injuries are readily apparent, chronic sequelae are harder to recognize. Indeed, the identification of individuals with mild-TBI or TBI-induced symptoms is greatly impaired in large part due to the lack of objective and robust biomarkers. The purpose of this study was to address these need by identifying candidates for serum-based biomarkers of blast TBI, and also to identify unique or differentially regulated protein expression in the thalamus in C57BL/6J mice exposed to blast using high throughput qualitative screens of protein expression. To identify thalamic proteins differentially or uniquely associated with blast exposure, we utilized an antibody-based affinity-capture strategy (referred to as “proteomics-based analysis of depletomes”; PAD) to deplete thalamic lysates from blast-treated mice of endogenous thalamic proteins also found in control mice. Analysis of this “depletome” detected 75 unique proteins, many with associations to the myelin sheath. To identify blast-associated proteins eliciting production of circulating autoantibodies, serum antibodies of blast-treated mice were immobilized, and their immunogens subsequently identified by proteomic analysis of proteins specifically captured following incubation with thalamic lysates (a variant of a strategy referred to as “proteomics-based expression library screening”; PELS). This analysis identified 46 blast-associated immunogenic proteins, including 6 shared in common with the PAD analysis (ALDOA, PHKB, HBA-A1, DPYSL2, SYN1, and CKB). These proteins and their autoantibodies are appropriate for further consideration as biomarkers of blast-mediated TBI., Biochemistry; Neuroscience; Proteins; Retinal ganglion cell; Biomarker; Blast injury; Traumatic brain injury.