elucidating-tissue-plasma-specific-proteomic-alterations-health

Elucidating Tissue- and Plasma-Specific Proteomic Alterations in Health and Disease

Proteomics is the large-scale study of expressed proteins, focusing on their structure, function, interactions, abundance, and post-translational ...

Walter Reed Army Institute of Research

ORISE Fellow

Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980-1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using (a) surgically implanted radiotelemetry probes for electroencephalogram (EEG), (b) neurohistopathology of brain, (c) cholinesterase activities in the PNS and CNS, and (d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/status epilepticus (SE) in diisopropylfluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro-2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of brain AChE activity. Pro-2-PAM represents the first non-invasive means of administering a CNS therapeutic for the deleterious effects of OP poisoning by reactivating CNS AChE.

Pro-2-PAM therapy for central and peripheral cholinesterases.

Utilizing our previously reported in silico pharmacophore model for reactivation efficacy of oximes, we present here a discovery of twelve new non-oxime reactivators of diisopropylfluorophosphate (DFP)-inhibited acetylcholinesterase (AChE) obtained through virtual screening of an in-house compound database. Rate constant (kr) efficacy values of the non-oximes were found to be within ten-fold of pralidoxime (2-PAM) in an in vitro DFP inhibited eel AChE assay and one of them showed in vivo efficacy comparable to 2-PAM against brain symptoms for DFP induced neuropathology in guinea pigs. Short listing of the identified compounds were performed on the basis of in silico evaluations for favorable blood brain barrier penetrability, octanol-water partition (Clog P), toxicity (rat oral LD50) and binding affinity to the active site of the crystal structure of a OP- inhibited AChE.

Discovery of non-oxime reactivators using an in silico pharmacophore model of reactivators for DFP-inhibited acetylcholinesterase.

Exposure to blast overpressure waves is implicated as the major cause of ocular injuries and resultant visual dysfunction in veterans involved in recent combat operations. No effective therapeutic strategies have been developed so far for blast-induced ocular dysfunction. Lysophosphatidic acid (LPA) is a bioactive phospholipid generated by activated platelets, astrocytes, choroidal plexus cells, and microglia and is reported to play major roles in stimulating inflammatory processes. The levels of LPA in the cerebrospinal fluid have been reported to increase acutely in patients with traumatic brain injury (TBI) as well as in a controlled cortical impact (CCI) TBI model in mice. In the present study, we have evaluated the efficacy of a single intravenous administration of a monoclonal LPA antibody (25 mg/kg) given at 1 h post-blast for protection against injuries to the retina and associated ocular dysfunctions. Our results show that a single 19 psi blast exposure significantly increased the levels of several species of LPA in blood plasma at 1 and 4 h post-blast. The anti-LPA antibody treatment significantly decreased glial cell activation and preserved neuronal cell morphology in the retina on day 8 after blast exposure. Optokinetic measurements indicated that anti-LPA antibody treatment significantly improved visual acuity in both eyes on days 2 and 6 post-blast exposure. Anti-LPA antibody treatment significantly increased rod photoreceptor and bipolar neuronal cell signaling in both eyes on day 7 post-blast exposure. These results suggest that blast exposure triggers release of LPAs, which play a major role promoting blast-induced ocular injuries, and that a single early administration of anti-LPA antibodies provides significant protection.

Antibodies Against Lysophosphatidic Acid Protect Against Blast-Induced Ocular Injuries.

Traumatic brain injury (TBI) results in neuronal, axonal and glial damage. Interventions targeting neuroinflammation to enhance recovery from TBI are needed. Exercise is known to improve cognitive function in TBI patients. Omega-3 fatty acids and vitamin D reportedly reduce inflammation, and in combination, might improve TBI outcomes. This study examined how an anti-inflammatory diet affected plasma TBI biomarkers, voluntary exercise and behaviors following exposure to mild TBI (mTBI). Adult, male rats were individually housed in cages fitted with running wheels and daily running distance was recorded throughout the study. A modified weight drop method induced mTBI, and during 30 days post-injury, rats were fed diets supplemented with omega-3 fatty acids and vitamin D (AIDM diet), or non-supplemented AIN-76A diets (CON diet). Behavioral tests were periodically conducted to assess functional deficits. Plasma levels of Total tau (T-tau), glial fibrillary acidic protein (GFAP), ubiquitin c-terminal hydrolase L1 (UCH-L1) and neurofilament light chain (NF-L) were measured at 48 h, 14 days, and 30 days post-injury. Fatty acid composition of food, plasma, and brain tissues was determined. In rats exposed to mTBI, NF-L levels were significantly elevated at 48 h post-injury ( < 0.005), and decreased to levels seen in uninjured rats by 14 days post-injury. T-tau, GFAP, and UCH-L1 plasma levels did not change at 48 h or 14 days post-injury. However, at 30 days post-injury, T-tau, GFAP and UCH-L1 all significantly increased in rats exposed to mTBI and fed CON diets ( < 0.005), but not in rats fed AIDM diets. Behavioral tests conducted post-injury showed that exercise counteracted cognitive deficits associated with mTBI. The AIDM diets significantly increased docosahexaenoic acid levels in plasma and brain tissue ( < 0.05), and in serum levels of vitamin D ( < 0.05). The temporal response of the four injury biomarkers examined is consistent with studies by others demonstrating acute and chronic neural tissue damage following exposure to TBI. The anti-inflammatory diet significantly altered the temporal profiles of plasma T-tau, GFAP, and UCH-L1 following mTBI. Voluntary exercise protected against mTBI-induced cognitive deficits, but had no impact on plasma levels of neurotrauma biomarkers. Thus, the prophylactic effect of exercise, when combined with an anti-inflammatory diet, may facilitate recovery in patients with mTBI.

Omega-3 Fatty Acids and Vitamin D Decrease Plasma T-Tau, GFAP, and UCH-L1 in Experimental Traumatic Brain Injury.

Blast injury has been implicated as the major cause of traumatic brain injury (TBI) and ocular system injury, in military operations in Iraq and Afghanistan. Soldiers exposed to traumatic stress also have undiagnosed, chronic vision problems. Here we hypothesize that excessive intake of ω-6 fatty acid linoleic acid (LA) and insufficiency of dietary long chain ω-3 polyunsaturated fatty acids (PUFAs, e.g., docosahexaenoic acid; DHA) would dysregulate endocannabinoid-mediated neuronal plasticity and immune response. The study objective was to determine the effect of blast-TBI and traumatic stress on retinal gene expression and assess the role of dietary deficiency of long chain ω-3 PUFAs on the vulnerability to these injury models.

Impact of dietary changes on retinal neuronal plasticity in rodent models of physical and psychological trauma.

Animal models enable investigation of the impact of stress on emotional-behavioral performance to facilitate understanding of posttraumatic stress symptoms experienced in humans. Refinement of animal stress models could lead to a reduction in the number of subjects needed to detect statistically significant stress effects, in accordance with Russel and Burch's three Rs of research. We assessed whether performance of experimental procedures (that is, stress exposure and poststress behavioral testing) during the dark or light phases of the 12-h light/12 h-dark cycle is a refinement that could accomplish this reduction. At 3 h into either the light or dark phase, male and female adult Sprague-Dawley rats underwent a single-day traumatic stress exposure. Rats then underwent behavioral testing for exploratory behaviors, startle responses, and conditioned fear responses at 2 h, 1 d, and 9 d after stress exposure. Distance traveled in the elevated plus maze (EPM) by both male and female rats was significantly reduced in the dark phase compared with the light phase. Male rats of the dark phase group also spent less time in the open arms of the EPM, and traveled less, spent less time in the center, and spent more cumulative time freezing in the open field. Female rats of the dark phase group spent more cumulative time freezing in the EPM and exhibited significantly more tone-cued conditioned freezing responses. Our results suggest that performing experimental procedures during the dark phase of the light cycle may be a useful refinement mechanism, as procedures performed during this period had the greatest effect on behavioral outcomes in both males and females. Light cycle phase is an experimental variable that should be considered when designing experiments to maximize behavioral effects, including those in response to stress.

Article	Year
Pro-2-PAM therapy for central and peripheral cholinesterases. Chemico-biological interactions\| PubMed ID: 20156430	2010
Discovery of non-oxime reactivators using an in silico pharmacophore model of reactivators for DFP-inhibited acetylcholinesterase. European journal of medicinal chemistry\| PubMed ID: 25461321	2015
Antibodies Against Lysophosphatidic Acid Protect Against Blast-Induced Ocular Injuries. Frontiers in neurology\| PubMed ID: 33384658	2020
Omega-3 Fatty Acids and Vitamin D Decrease Plasma T-Tau, GFAP, and UCH-L1 in Experimental Traumatic Brain Injury. Frontiers in nutrition\| PubMed ID: 34150829	2021
Impact of dietary changes on retinal neuronal plasticity in rodent models of physical and psychological trauma. Frontiers in genetics\| PubMed ID: 39346777	2024
Light Cycle Phase Effects on Behavioral Responses to Stress Exposure in Rats. Journal of the American Association for Laboratory Animal Science : JAALAS\| PubMed ID: 40035271	2025

James C. DeMar

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