NeuroToxicology 48 (2015) 180–191

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NeuroToxicology

N-acetyl-aspartyl-glutamate and inhibition of glutamate carboxypeptidases protects against soman-induced neuropathology Huifu Guo a, Jiong Liu a, Kerry Van Shura c, HuaZhen Chen a, Michael N. Flora b, Todd M. Myers d, John H. McDonough c, Joseph T. McCabe a,* a

Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences (USUHS), 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA Biomedical Instrumentation Center, Uniformed Services University of the Health Sciences (USUHS), 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA c Pharmacology Branch, Research Division, United States Army Medical Research Institute of Chemical Defense (USAMRICD), 3100 Ricketts Point Road, Aberdeen Proving Ground, MD 21010-5400, USA d Neurobehavioral Toxicology Branch, Analytical Toxicology Division, United States Army Medical Research Institute of Chemical Defense (USAMRICD), 3100 Ricketts Point Road, Aberdeen Proving Ground, MD 21010-5400, USA b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 December 2014 Accepted 13 March 2015 Available online 28 March 2015

N-acetyl-aspartyl-glutamate (NAAG) is the most abundant neuropeptide in the mammalian brain. In a variety of animal models of brain injury, the administration of NAAG-related compounds, or inhibitors of glutamate carboxypeptidases (GCPs; the enzymes that hydrolyze NAAG), were shown to be neuroprotective. This study determined the impact of the administration of three NAAG-related compounds, NAAG, b-NAAG (a NAAG homologue resistant to degradation), and 2-phosphonomethyl pentanedioic acid (2-PMPA; an inhibitor of GCP enzymes), on the neuropathology that develops following exposure to the nerve agent, soman. When given 1 min after soman exposure, NAAG-related drug treatments did not alter the survival rate or body weight loss seen 24 h after rats were exposed to soman. Likewise, brain levels of both NAAG and its metabolite, N-acetyl-aspartate (NAA), were substantially decreased 24 h after soman, and in particularly vulnerable brain regions the drug treatments were unable to attenuate the reduction in NAA and NAAG levels. Histochemical study indicated there was a dramatic increase in Fluoro-Jade C (FJC) staining, indicative of neuron cell death, 24 h after soman exposure. However, in the amygdala and in the entorhinal and piriform limbic cortex, which sustained severe neuropathology following soman intoxication, single or combined injections of NAAG compounds and 2-PMPA significantly reduced the number of FJC-positive cells, and effect size estimates suggest that in some brain regions the treatments were effective. The findings suggest that NAAG neurotransmission in the central nervous system is significantly altered by soman exposure, and that the administration of NAAG-related compounds and 2-PMPA reduces neuron cell death in brain regions that sustain severe damage. Published by Elsevier Inc.

Keywords: 2-Phosphonomethyl pentanedioic acid (2PMPA) Brain injury High performance liquid chromatography Effect size

1. Introduction The threat of nerve agent casualty is a concern for military personnel and civilians. With no forewarning, a nerve agent could be released during an actual military engagement, an act of terrorism, or an accident, resulting in the death or serious neurological impairment of exposed warfighters and/or civilians (Dolgin, 2013; Yanagisawa et al., 2006). At present, antidotes for nerve agent exposure provide limited protection, and it is almost impossible to

* Corresponding author. Tel.: +1 301 295 3664; fax: +1 301 295 1786. E-mail address: [email protected] (J.T. McCabe). http://dx.doi.org/10.1016/j.neuro.2015.03.010 0161-813X/Published by Elsevier Inc.

reduce nerve-agent-induced seizures and subsequent brain injury. Therefore, new therapies to treat nerve agent-induced brain damage are warranted. The neuropharmacological processes underlying the initiation and maintenance of nerve-agent-induced seizures that ultimately result in neuropathology are believed to develop in three phases (McDonough and Shih, 1997). According to this model, seizures are initiated by a sudden increase in acetylcholine levels in susceptible brain structures following irreversible inhibition of brain cholinesterase by the nerve agent. Only cholinergic processes appear to be involved in the initiation and early maintenance phase. The acetylcholine-associated excitatory activity of the seizure, however, perturbs non-cholinergic neurotransmitter systems, most notably

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the excitatory amino acid, glutamatergic system, and enters into the second or transition phase. During this phase, both cholinergic and non-cholinergic influences modulate seizure activity. Gradually, a third phase emerges, where non-cholinergic excitatory activity mediates seizure activity independent of cholinergic activity. The activation of glutamatergic receptors, most importantly NMDA receptors, leads to the elevation of intracellular Ca2+ that eventually results in neuropathology. The majority of treatments for nerve agent neurotoxicity target the cholinergic, glutamatergic, and the inhibitory GABAergic neurotransmitter systems (Myhrer, 2010; Weissman and Raveh, 2008). With levels as much as 1000-fold higher than any other peptide in the CNS, N-acetyl-aspartyl-glutamate (NAAG) is the most abundant neuropeptide in the mammalian brain (Neale et al., 2000; Passani et al., 1997; Zhou et al., 2005). NAAG is synthesized primarily in neurons of the CNS and in peripheral nerves from the intracellular stores of N-acetyl-aspartate (NAA) and glutamate by the recently identified enzymes, NAAG synthetase I and II (Becker et al., 2010; Collard et al., 2010; Lodder-Gadaczek et al., 2011). NAAG meets the criteria for a neurotransmitter (Neale, 2011; Neale et al., 2005; Wroblewska, 2006). It is co-localized with a wide range of neurotransmitters in the CNS, including glutamate, GABA, serotonin, norepinephrine, dopamine and acetylcholine (Forloni et al., 1987; Neale et al., 2000), and is released from synaptic endings upon intense depolarizing activity (Neale et al., 2005). The release of NAAG is known to selectively activate presynaptic type 3 metabotropic glutamate receptors (mGluR3) (Neale et al., 2011; Wroblewska, 2006). mGluR3 action facilitates glutamate reuptake and suppresses release of glutamate, thereby decreasing synaptic excitability (Puttfarcken et al., 1993; Sanchez-Prieto et al., 1996; Sekiguchi et al., 1989). NAAG is also known to activate postsynaptic mGluR3 receptors to reduce cyclic AMP levels via an inhibitory G protein in neurons and astrocytes (Bruno et al., 1998). NAAG is catabolized to N-acetylaspartate (NAA) and glutamate by the glutamate carboxypeptidases II and III (GCPII and III); enzymes expressed on the plasma membrane of astrocytes in the extrasynaptic space (Robinson et al., 1987). NAA is also primarily localized in neurons. Measure of NAA levels in the brain by magnetic resonance spectroscopy has shown that NAA is reduced in a number of neurodegenerative diseases, such as Alzheimer’s disease, stroke, schizophrenia, multiple sclerosis, and Huntington’s disease (Madhavarao and Namboodiri, 2006; Schuff et al., 2006). Administration of NAAG or inhibitors of GCP enzymes was shown to be neuroprotective in a number of brain injury or traumatic conditions, and in cultured neuron models of cell stress and excitotoxicity (Zhong et al., 2014). Cai and colleagues showed that intraperitoneal (i.p.) administration of low doses of NAAG (2– 10 mg/kg), given before or even 1 h after hypoxic exposure, greatly reduced hypoxia-ischemia-induced brain injury (Cai et al., 2002). Lu and colleagues found that intracerebroventricular pre-infusion of 1–4 mM of NAAG significantly reduced the volume of rat brain injury from stroke by middle artery occlusion (Lu et al., 2000). NAAG also reduced the brain lesion or injury induced by quinolinic acid and endothelin-1 (Orlando et al., 1997; Van Hemelrijck et al., 2005). In addition, NAAG was shown to be highly protective in cultured cerebellar neurons and primary spinal cord cultures against hypoxia, hypoglycemia, and NMDA toxicity (Tortella et al., 2000; Yourick et al., 2003). Similarly, GCP inhibitors, such as 2-PMPA, ZJ-43, and GPI5232, administered either before or up to 90–120 min after stroke or other brain injury, dramatically increased NAAG levels, reduced glutamate levels in various brain substrates, and were strongly neuroprotective against various types of brain injury and neural trauma (Feng et al., 2011, 2012; Gurkoff et al., 2013; Neale et al., 2005; Slusher et al., 1999; Thomas et al., 2006; Zhong et al., 2005, 2006).

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NAAG and GCP-inhibitors reduce brain injury in several ways. As noted earlier, NAAG activates presynaptic mGluR3s, which decreases synaptic excitability by suppression of synaptic glutamate release and by facilitating the presynaptic reuptake of glutamate (Puttfarcken et al., 1993; Sekiguchi et al., 1989). Second, NAAG provides neuroprotection by interacting with NMDA receptors, and thereby interferes with glutamate binding (Bergeron et al., 2005; Carpenter and Dickenson, 2001). Third, GCP inhibition reduces extracellular glutamate levels by preventing NAAG hydrolysis. Fourth, NAAG stimulates neuroprotective pathways, such as the release of transforming growth factors (TGF-b) from glia (Thomas et al., 2001, 2006). Fifth, NAAG and NAA may function to transfer metabolic water from cells to the extracellular fluid (Baslow, 2010). NAAG’s activation of astrocyte mGluR3 initiates Ca2+ oscillations in astrocytes and releases second messengers to the capillary endothelial cells, resulting in a local hyperemic response and providing energy to neurons (Baslow, 2010). To our knowledge, the impact of nerve agents on the NAAG neuropeptide system has not been considered, either in terms of changes after exposure or regarding the possible effects of NAAGrelated compound administration. Given the critical role of glutamate toxicity in nerve agent-induced seizure and neuropathology, NAAG analogs and inhibition of GCP may provide effective therapy for nerve agent-induced neuronal injuries. In the current study, we examined the effects of the nerve agent, soman, on brain NAA and NAAG levels and evaluated the effects of NAAG, b-NAAG (a NAAG derivative that resists hydrolysis by GCP), and the GCP inhibitor, 2-PMPA, on soman-induced neuropathology. 2. Materials and methods 2.1. Animals Experiments were performed using male Sprague Dawley rats, weighing 250–300 g at the start of the experiments. Animals were individually housed in an environmentally controlled room (20– 23 8C, 12-h light/12-h dark cycle, lights on 06:00 h), with food and water available. All animal experiments were conducted following the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council), and were approved by the Institutional Animal Care and Use Committee. The animal care and use programs at the U.S. Army Medical Research Institute of Chemical Defense and the Uniformed Services University of the Health Sciences are fully accredited by AAALAC International. 2.2. Chemical agent administration Soman (pinacoyl methylphosphonofluoridate; obtained from the U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD, USA) was diluted in cold saline, and administered via a single subcutaneous injection (180 mg/kg, 1.6 LD50); a dose used as a standard in drug studies (Myhrer et al., 2004, 2011; Shih and McDonough, 1999). To increase survival rate, all rats were administered an i.p. injection of the oxime HI-6 (1-(2hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2oxapropane dichloride; 125 mg/kg), 30 min prior to soman. HI-6 is a bispyridinium oxime that can reactivate inhibited acetylcholinesterase, primarily in the periphery (Bajgar, 2005). Within 1 min after soman exposure, all rats also received an intramuscular (i.m.) injection of atropine methyl nitrate (2 mg/kg, Sigma, St. Louis, MO) to minimize peripheral toxic effects. All animals developed status epilepticus (SE), and were injected with diazepam (10 mg/kg, i.m.) 40 min after seizure onset to attenuate behavioral seizures. Animals in the sham or drug only (see below) control groups

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were not exposed to soman treatment but received all attendant injections (HI-6, atropine, diazepam). 2.3. Drug treatments of soman toxicity NAAG and b-NAAG were purchased from Bachem (G-1015 and G-4590; Torrance, CA). NAA was purchased from Sigma (A5625). The 2-PMPA compound was a gift of Dr. Barbara S. Slusher (Brain Science Institute NeuroTranslational Program, Johns Hopkins School of Medicine). The NAAG compounds, 2-PMPA or vehicle control saline were freshly prepared and were injected (i.p.) directly into control animals (sham or drug only groups) or at 1 min after soman exposure. Due to drug costs and attempts to use as few animals as feasible, a sequence of three studies were conducted to evaluate the effects of different drug treatments. In Experiment 1, rats received a single i.p. injection of either 2-PMPA (50 mg/kg) or a lower dose of NAAG (10 mg/kg) or b-NAAG (10 mg/kg). In Experiment 2, rats received higher doses of NAAG (50 mg/kg) or b-NAAG (50 mg/kg) or the combination of b-NAAG (50 mg/kg) and 2-PMPA (50 mg/kg). In Experiment 3, rats received 10 or 50 mg/kg of NAAG in combination with 50 mg/kg of 2-PMPA. As requested by the Institutional Animal Care and Use Committee, animals in Experiments 2 and 3 received an additional subcutaneous injection of 5 ml sterile normal saline late in the afternoon of the day of soman exposure to enhance overnight survival. To determine whether or not NAAG-related drugs have neuropathological effects, some animals in Experiment 2 were administered the combination of b-NAAG (50 mg/kg) and 2-PMPA (50 mg/kg) without soman exposure. In addition to the drug treatments after the soman exposure, other normal (naı¨ve) animals were administered i.p. injections of NAA, NAAG compounds, 2-PMPA or the combinations of these compounds and brain levels of NAA and NAAG were measured 3 h after the injections. 2.4. High performance liquid chromatography (HPLC) For HPLC assays, the animals were anesthetized with ketamine (100 mg/kg, i.p.) and euthanized by decapitation either 3 h after NAAG-related drug administration (normal animals with no soman exposure or HI-6, atropine or diazepam injection) or at 24 h after soman exposure and drug administration. The brains were quickly removed and submerged in ice, and then dissected on ice into six major brain regions: the dorsal lateral cortex (CTX), the amygdala, entorhinal cortex, and piriform cortex regions (AEP), hippocampus (HIP), diencephalon (DI), cerebellum (CB), and the brain stem (BS). Dissected tissues were frozen on dry ice and stored in liquid nitrogen. The tissue was homogenized in 10 volumes (tissue weight/ volume) of ice-cold 85% (volume/volume) methanol using a Kontes (Vineland, NJ) micro-ultrasonic cell disruptor and centrifuged at 17,968  g for 15 min to remove the insoluble fraction. The pellet was used to measure protein concentration using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL). The supernatant was evaporated to dryness using a speed-vac. The powder was dissolved in 0.4 ml of double-distilled water (ddH2O), and added to a Bio-Rad AG 50W-X8 column (Bio-Rad, Hercules, CA) in order to remove all primary amines, including aspartate and glutamate. The column was washed with 0.8 ml ddH2O, and the eluate (total of 1.2 ml) was collected, frozen, lyophilized, and stored at 20 8C until use. Prior to HPLC analysis, each of the samples was re-suspended in 65 ml ddH2O. A WhatmanTM (Clifton, NJ) PartisilTM 10 SAX anion-exchange column (250 mm  4.6 mm) was used for the HPLC assays (Agilent 1200 series HPLC system, Agilent Technologies, Santa Clara, CA). An isocratic condition was employed, with the mobile phase consisting of 0.1 M KH2PO4 and

0.025 M KCl at pH 4.5 with a flow rate of 1.5 ml/min. The flow from the column was monitored with an Agilent 1200 series diode array detector SL, scanning 190 nm to 230 nm, focusing on 210 nm. As shown below, the concentrations of NAA and NAAG external standards were linear to the peak area, which was measured automatically by the ChemStation B.04 software (Agilent Technologies). Concentrations of NAA and NAAG in brain tissue samples were calculated based on the external standards, and normalized to the internal protein concentration. 2.5. Mass spectrometry The specificity of the HPLC method was evaluated by obtaining pooled, fractionated brain samples from HPLC and determination of sample molecular weight by matrix-assisted laser desorption/ ionization (MALDI; Voyager-DETM STR BiospectrometryTM Workstation, PerSeptive Biosystems). In the first sample, HPLC fractions from seven brain samples were pooled. A second replication was performed by collecting HPLC fractions from eleven brain samples. The samples were each diluted in 50% methanol:50% water to a final volume of 60 ml, and two matrices from each pooled sample were prepared with a-cyano-4-hydroxy cinnamic acid (CHCA; 10 mg/ml) or sinapinic acid (SinA; 10 mg/ml). Bradykinin (MW 1060.2) was added as an internal standard and samples were purified with 5 ml of elution buffer using Millpore c18 Ziptips. Two microliters of each sample were diluted in 2 ml CHCA or SinA, and the sample/matrix mixtures were spotted on a stainless steel MALDI-TOF plate. Profiles were generated using the resident angiotensin reflector program (guide wire voltage 0.05, laser power 1600 in the positive mode, nitrogen laser, 337 nm) by collecting 50, 3-nanosecond-wide pulses (20 pulses/s) for each sample. The positive mode profile produced a high matrix response for both CHCA and SinA, but very low analyte response; not an unexpected finding since NAA and NAAG are negatively charged. The signal for the analytes was more definitive in the negative mode, where well-defined peaks were obtained with SinA, and no interfering peaks were observed within the immediate mass range for NAA and NAAG. The remainders of the samples were used for HPLC (WhatmanTM PartisilTM 10 SAX Column). Peaks with the same retention time were collected, their volume reduced by speed vacuum, and the samples were reanalyzed by MALDI-TOF using SinA in the negative mode. Profile masses confirmed the peaks collected were NAA and NAAG. 2.6. Fluoro-Jade C (FJC) histochemistry FJC staining (Histo-Chem, Jefferson, AR) was used to identify irreversibly degenerating neurons (Chidlow et al., 2009; Schmued et al., 2005). One day after soman exposure, rats were deeply anesthetized using ketamine (100 mg/kg, i.p.) and transcardially perfused with phosphate buffered saline (PBS, 100 ml) followed by 10% formalin (200 ml). The brains were removed and post-fixed overnight at 4 8C, then transferred to a solution of 30% sucrose in PBS for 72 h, and frozen with dry ice before storage at 80 8C until sectioning. Serial sections were prepared (40 mm thickness) on a sliding microtome and mounted on slides, air-dried overnight, and then immersed in a solution of 1% sodium hydroxide in 80% ethanol for 5 min. The slides were then rinsed for 2 min in 70% ethanol, 2 min in deionized H2O (dH2O) and incubated in 0.06% potassium permanganate solution for 10 min. After a 2-min rinse in dH2O, the slides were submerged for 10 min in a 0.0001% solution of FJC dissolved in 0.1% acetic acid. Following three 1-min rinses in dH2O, the slides were dried on a slide warmer, cleared in xylene for at least 1 min, and coverslipped with DPX (p-xylene-bis(Npyridinium bromide; Sigma).

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The extent of neurodegeneration was assessed in sections between the coordinates 2.56 mm to 3.80 mm (frontal plane through the piriform cortex region) and of 4.80 mm to 5.80 mm (level of the entorhinal cortex). Brain regions were chosen based on the Paxinos and Watson rat brain atlas (Paxinos and Watson, 2005). For each brain region (Fig. 3C), ImageJ software (Collins, 2007) was used to place a fixed-size (667 mm  528 mm) counting box over the brain region of interest. FJC-positive cells from three adjacent sections per animal were counted and averaged by an individual unaware of the experimental history of a given animal. A two-way, mixed-design ANOVA for the factors treatment groups  brain region, with brain region treated as a withinsubject factor, was used to compare FJC staining across all groups (SigmaStat, version 3.1). However, in almost all cases, even with data transformation, the mixed model did not meet the assumptions of normality or equal variances. A single-factor ANOVA was applied for data of each brain region, followed by multiple comparisons using Bonferroni’s t-test, as advised in these instances to perform consistent analyses across all experiments (Motulsky, 2010; Osborne, 2013). If the analysis did not meet the assumptions of normality or equal variances, a Kruskal–Wallis ANOVA on ranked data was performed, followed by multiple comparisons using Dunn’s method. Histogram bars in Figs. 2, 3 and 5 depict group means  standard error of the mean (SEM). Finally, to provide a summary of drug effects on neuropathology from the three experiments, effect size was computed using Hedge’s g effect size index (Borenstein et al., 2009) as follows. Cohen’s d index (Cohen, 1988) was first computed using the formula, d = (Msoman  Msoman+drug treatment)/spooled, where M refers to the mean number of FJC-positive cells in a brain region in soman and soman + drug treated animals, respectively, and spooled is an estimate of the standard deviation. For each experiment, spooled was computed using the square root of the mean square error from the ANOVA for each specific brain region (Grissom and Kim, 2012). Since d has an overestimation bias when computed for smaller sample sizes, the d index was multiplied by a correction factor for small sample sizes to compute an ‘‘unbiased’’ effect size statistic, Hedge’s g, using the formula g ffi J  d, where   J ¼ 1  4df31 . Finally, the standard error for g was computed by estimating the variance of d with the formula V d ¼

n1 þn2 n1 n2

2

þ 2ðn dþn Þ, 1

2

applying a correction for the variance of g, Vg = J2  Vd, to compute pffiffiffiffiffiffi the standard error of g as SEg ¼ V g and lower and upper 95% confidence intervals as LLg = g  1.96  SEg and ULg = g + 1.96  SEg, respectively (Borenstein et al., 2009).

3. Results 3.1. Rodent survival and body weight loss from soman exposure was not affected by the administration of NAAG-related drugs Table 1 summarizes survival rates in animals exposed to soman with or without a variety of treatments. The survival rates in Experiment 1 were sham group: 100% (15/15) and for the somanexposed groups ranged from 73 to 94%. In Experiment 2, the survival rates of different groups of animals were 100% for sham (14/14) and 75–85% for animals exposed to soman that received NAAG-related drugs. Finally, the treatments applied in Experiment 3 resulted in a 100% survival rate for the sham group (4/4), and survival rates of 58–90% in agent-exposed animals. Since in all cases there were instances where the expected values for observations were less than 5, application of the X2 test was not suitable. However, a Fisher exact test was performed to evaluate the largest observed difference in each experiment. Comparison of the survival rates for the soman-only and the NAAG-related drug

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Table 1 Survival rate and body weight loss after soman exposure. Experiment 1 Lower dose study

Survival

N*

Weight loss (g)#

Sham (no soman exposure) Soman Soman + NAAG (10 mg/kg) Soman + b-NAAG (10 mg/kg) Soman + 2-PMPA (50 mg/kg)

100% 73% 84% 83% 94%

15/15 16/22 16/19 15/18 15/16

10.8  2.1 41.7  2.6y 42.7  1.8y 43.1  2.2y 38.5  2.1y

Experiment 2 Higher dose study

Survival

N*

Weight loss (g)

Sham (no soman exposure) Soman Soman + NAAG (50 mg/kg) Soman + b-NAAG (50 mg/kg) Soman + b-NAAG (50 mg/kg) + 2-PMPA b-NAAG + 2-PMPA (No soman exposure)

100% 75% 85% 74% 85% 100%

14/14 18/24 17/20 15/20 19/23 16/16

8.7  1.8 41.7  1.3y 43.4  2.2y 41.5  1.6y 40.5  2.0y 12.3  1.4

Experiment 3 NAAG + 2-PMPA study

Survival

N*

Weight loss (g)

Sham (no soman exposure) Soman Soman + NAAG (10 mg/kg) + 2-PMPA Soman + NAAG (50 mg/kg) + 2-PMPA

100% 90% 58% 70%

4/4 9/10 7/12 7/10

15.0  3.4 39.4  1.7y 36.9  3.5y 37.6  2.7y

* Number of animals, out of total, in each treatment group that survived treatment. # Data summarizes reduction of body weight before and 24 h after experimental treatment  SEM in rats that survived. y Body weight loss was significantly different from the Sham group. Body weight loss measured in the drug-treated groups did not differ from the Soman (alone) group (statistics not shown).

treatment group that was exposed to soman and had the greatest survival rate indicated no significant difference in survival in Experiment 1 (73% vs. 94%, p = 0.20), Experiment 2 (75% vs. 85%, p = 0.242), or Experiment 3 (58% vs. 90%, p = 0.162), suggesting survival rates were not affected by drug treatment. Experiment 1 examined the effects of lower dosages of NAAG and b-NAAG (10 mg/kg). As shown in Table 1, drug treatment had no effect on body weight reduction as a result of soman exposure. Single-factor ANOVA indicated that all groups that received soman lost a significantly greater amount of weight than the sham group (F4,72 = 39.8, p < 0.001) while the soman-exposed groups lost approximately the same amount of body weight (Bonferroni t-test comparisons, p = 1.0). Results were similar for Experiment 2, where drug treatments had no significant effect on the body weight reduction from soman exposure. All groups that received soman lost a significantly greater amount of weight than the sham group and a second control group that received just treatments with bNAAG + 2-PMPA (F5,93 = 78.9, p < 0.001) while the sham-treated and second control group body weight measures did not differ (Bonferroni t-test, p = 1.0). In Experiment 3, all three groups of rats that were exposed to soman exhibited significant reductions in body weight 24 h after soman exposure, compared to the sham group (F3,41 = 7.3, p < 0.001, Bonferroni t-test comparisons to the sham group, p < 0.01), but were not different among themselves. Although not included in the main studies, it should be noted that the sham treatments with HI-6 and atropine methyl nitrate affect overnight body weight. A control experiment indicated shamtreated rats lose a significantly greater amount of weight in 24 h than naı¨ve animals that received no pharmacological treatments (16.0  4.02 g vs. +0.80  1.2 g, respectively, F1,6 = 13.984, p = 0.010). 3.2. Implementation of HPLC for measurement of NAA and NAAG levels The extraction and HPLC methods allowed for reliable quantitation of NAA and NAAG levels. Fig. 1A and B, respectively,

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Fig. 1. NAA and NAAG analysis by HPLC. Representative HPLC traces showing NAA and NAAG peaks in standard (A: left chromatogram) and brain extracts (B: right chromatogram). Standard curve profiles showing linear sensitivity for NAA (C) and NAAG (D). mAU: milli-absorption units.

are chromatograms from HPLC of standard NAA and NAAG (1A) and from a brain extract (1B). The run times for both analytes were similar in the standard and brain extract samples. Retention times were 5.5 min and 11.5 min for NAA and NAAG, respectively. Additional tests were performed to determine the detectable level of each analyte. As shown in Fig. 1C and 1D, a linear function was obtained for a 0.01–0.05 mg range of NAA and 0.45–22.5 mg range of NAAG (R2 = 0.9979 and 0.9996, respectively). Samples from the standard preparations and brain extracts were used for mass spectrometry. Negative mode MALDI-TOF analysis indicated NAA and NAAG control samples (Supplemental Figs. 1 and 2, respectively) produced identifiable peaks at 174.39 m/z for NAA (molecular mass 175.14 g/mol; 0.4% difference) and for NAAG at 303.80 m/z for NAAG (304.25 g/mol; 0.4% difference). Samples of extracted brain tissue samples identified almost identical peaks for NAA (175.66 m/z) and NAAG (303.80 m/z) (Supplemental Figs. 3 and 4, respectively), and pooling of brain samples resulted in identification of NAA and NAAG (Supplemental Fig. 5). 3.3. Soman administration decreased NAA and NAAG levels in the rat brain Following soman exposure, NAA and NAAG levels (Fig. 2A, B, respectively) were significantly decreased in the dorsal cortex (labeled CTX, F1,11 = 15.020, p = 0.003; F1,11 = 26.018, p < 0.001, NAA and NAAG levels, respectively), the AEP region (F1,11 = 43.702, p < 0.001; F1,11 = 33.722, p < 0.001), the hippocampus (labeled HIP, F1,11 = 9.613, p = 0.010; F1,11 = 83.912, p < 0.001), and the diencephalon (DI, F1,20 = 26.129, p < 0.001; F1,20 = 11.478, p = 0.003), but no changes were observed in the cerebellum (CB, F1,11 = 0.591, p = 0.458; F1,11 = 0.730, p = 0.411). In the brainstem (BS), soman exposure resulted in a significant increase in NAA levels (F1,11 = 11.307, p = 0.006), but no change in NAAG (F1,11 = 1.015, p = 0.335). The NAA/NAAG ratio (Fig. 2C) increased in the dorsal lateral cortex (CTX: Kruskal–Wallis ranks ANOVA H1 = 4.592, p = 0.035), the AEP region (AEP: F1,11 = 8.558, p = 0.014),

and the hippocampus (HIP: Kruskal–Wallis ranks ANOVA H1 = 9.000, p = 0.001), indicating that NAAG levels more dramatically decreased in these regions, relative to the decrease in NAA levels (Fig. 2C). In the cerebellum, diencephalon, and the brain stem, the NAA/NAAG levels were not altered after soman administration (all regions, p  0.118). 3.4. Impact of NAAG-related compounds and 2-PMPA (single injections or in combination) on NAA and NAAG brain content of normal (no soman-exposure) animals Before testing the treatment effects of NAAG, b-NAAG and 2PMPA in soman-exposed animals, a study was conducted to determine whether or not injections of NAAG, b-NAAG and 2PMPA, 3 h after administration, altered NAAG levels in the rat brain. Single injections of 10 mg/kg of NAAG or b-NAAG, a dose regularly used in previous rat studies, did not increase brain NAAG levels (data not shown). The GCP inhibitor, 2-PMPA, when given alone or in combination with NAAG or b-NAAG would inhibit the breakdown of NAAG to NAA and glutamate. Table 2 provides a summary of two studies where ANOVA indicated significant effects of drug treatment upon NAAG concentration levels in some brain areas that were tested 3 h after injection (first study summarized in upper portion of Table 2: AEP Region: F2,7 = 4.957, p = 0.046; HIP: F2,7 = 6.917, p = 0.022; CB: F2,7 = 6.086, p = 0.029; second study summarized in lower portion of table: AEP: F1,12 = 4.945, p = 0.046; HIP: Kruskal–Wallis ranks ANOVA H1 = 4.267, p = 0.043; CB: F1,12 = 12.301, p = 0.004). When 2-PMPA was given 3 h before animals were euthanized, it resulted in no significant change in measured levels of NAAG (Table 2). However, the administration of 10 mg/kg of NAAG in combination with 2-PMPA (50 mg/kg), resulted in increases in NAAG levels in the AEP samples, the hippocampus, and the cerebellum (upper portion of Table 2: Bonferroni t-tests, t = 3.076, p = 0.036, t = 3.676, p = 0.016, t = 3.067, p = 0.036, respectively). The combined administration of 2-PMPA with b-NAAG likewise increased NAAG levels in the AEP

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region, hippocampus, and cerebellum (Table 2, bottom row, t = 2.224, p = 0.046; Q = 2.066, p < 0.05; t = 3.507, p = 0.004, respectively). These treatments did not alter NAAG levels in the diencephalon or brain stem, and did not significantly change NAA levels in any brain region (data not shown). 3.5. Impact of NAAG-related compounds and 2-PMPA (single injections or in combination) on NAA and NAAG brain content following soman-exposure The effects of NAAG compounds and 2-PMPA on NAA and NAAG levels were measured in the cerebral cortex, AEP region, and the hippocampus 24 h after soman exposure (Table 3). In Experiment 1, compared to levels in sham-treated rats, soman exposure resulted in a significant reduction in brain levels of NAA in the cortex (F4,40 = 11.444, p < 0.001), and AEP region (F4,31 = 18.662, p < 0.001), but not in the hippocampus (F4,41 = 2.372, p = 0.068). However, NAAG levels were significantly decreased in all three sampled brain regions (cortex, F4,40 = 16.573, p < 0.001; AEP region, F4,31 = 33.92, p < 0.001, hippocampus, F4,41 = 8.145, p < 0.001). In Experiment 2, where higher dose levels were employed, soman exposure significantly reduced brain levels of NAA in all brain areas that were sampled (cortex, F5,38 = 16.376, p < 0.001; AEP region, F5,39 = 24.339, p < 0.001, hippocampus, F5,24 = 12.065, p < 0.001), as well as levels of NAAG (cortex F5,38 = 14.863, p < 0.001, AEP region, F5,39 = 18.388, p < 0.001, hippocampus (F5,24 = 6.080, p < 0.001; see minor exceptions indicated in Table 3). However, Bonferroni t-tests indicated that after soman exposure there were no significant differences in brain levels as a function of treatments with NAAG-related compounds compared to the animals exposed to soman alone. 3.6. Soman administration caused neuronal degeneration There were no observable FJC-positive cells in brain sections from naı¨ve animals or the sham group (that received HI-6 and atropine, but no soman exposure). In brain sections from animals that had been exposed to soman, however, there was extensive neuronal degeneration in various brain regions (Fig. 3). A high density of positive cells was seen in the amygdala, piriform cortex, entorhinal cortex, in the thalamic nucleus reuniens, and the lateral dorsal nucleus and medial dorsal nucleus in the thalamus. A large number of positive cells were also observed in the sampled dorsal and lateral cortex areas and the hippocampal CA1 region. In the hippocampal CA2/CA3 and dentate gyrus regions, there were a moderate number of positive cells. Scattered FJC-positive cells were also seen across other cortical and diencephalic structures (data not shown). 3.7. NAAG, b-NAAG and 2-PMPA reduced soman-induced neuropathology

Fig. 2. NAA and NAAG content in selected brain regions following soman exposure. (A, B) Compared to levels measured in sham animals, the concentration of NAA (A) and NAAG (B) in soman-treated animals was decreased in the cerebral cortex, amygdala/ entorhinal/piriform cortex (AEP), hippocampus, and diencephalon 24 h after exposure. The AEP region showed the greatest NAA and NAAG reductions, which decreased by 39% and 55%, respectively. The ordinate on the right relates to the DI and BS measures. (C) Comparison of the NAA/NAAG ratio between the sham group and soman treated animals. The ratio of NAA/NAAG was significantly greater in the cerebral cortex, amygdala/entorhinal/piriform cortex, and hippocampus following soman exposure. Data in each histogram were evaluated by a single-factor ANOVA, *p < 0.01 comparing sham-group to soman exposed animals. n = 6 and 7 rats/group for sham and all soman groups, respectively, except DI data from 10 and 12 samples. CTX: dorsal lateral cortex; AEP: amygdala, entorhinal cortex and piriform cortex; Hip: hippocampus; DI: diencephalon; CB: cerebellum; BS: brain stem.

Fig. 4 provides images from rats that received soman alone or soman followed by treatments with NAAG-related compounds. In all cases FJC-positive cells were observed. Fig. 5A–C summarizes FJC-positive cell counts from Experiments 1–3, respectively. The ANOVA from Experiment 1 (Fig. 5A) indicated there were overall changes in FJC staining in the amygdala, entorhinal cortex, and piriform cortex as a function of drug treatments (F3,20 = 7.994, p < 0.001; F3,20 = 5.475, p = 0.007; F3,20 = 4.597, p = 0.013, respectively). Bonferroni t-test comparisons of the number of FJC-positive cells in animals that received soman indicated that treatment with 10 mg/kg of NAAG resulted in fewer FJC-positive cells in the amygdala (45%: t = 4.040, p = 0.002) and piriform cortex (43%; t = 3.199, p = 0.014), but reduced levels were not significantly different in the entorhinal cortex (27%; t = 2.354, p = 0.087). After

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186 Table 2 NAAG levels (nmol/mg protein) 3 h after drug treatments. Drug treatment

NAAG concentration per brain region

Saline 2-PMPA (50 mg/kg) NAAG (10 mg/kg) + 2-PMPA (50 mg/mg) Saline b-NAAG (50 mg/kg) + 2-PMPA (50 mg/kg)

CTX

AEP

HIP

DI

CB

BS

2.8  0.029 3.1  0.029 2.9  0.16 3.5  0.11 3.8  0.074

2.6  0.062 2.7  0.12 3.0  0.11y 3.1  0.173 3.6  0.093*

2.6  0.054 2.8  0.095 2.9  0.051* 2.9  0.15 3.4  0.20*

6.1  0.27 6.5  0.42 7.1  0.041 10.7  0.96 11.0  0.74

5.0  0.15 5.7  0.075 5.8  0.29y 7.2  0.21 8.4  0.26*

14.5  1.1 16.1  1.7 15.8  0.81 24.4  0.87 25.0  1.1

CTX: dorsolateral cortex; AEP: amygdala, entorhinal cortex, and piriform cortex; HIP: hippocampus; DI: diencephalon; CB: cerebellum; BS: brainstem. * Significantly different than the saline group. y p = 0.054.

Table 3 NAA and NAAG levels (nmol/mg protein) after soman exposure and drug treatments. Experiment 1

NAA concentration

Lower dose study

CTX

AEP

HIP

NAAG concentration CTX

AEP

HIP

Sham (no soman exposure) Soman Soman + NAAG (10 mg/kg) Soman + b-NAAG (10 mg/kg) Soman + 2-PMPA (50 mg/kg)

145.5  5.1 113.2  5.5* 119.7  5.7* 98.0  4.8* 106.8  5.4*

134.2  6.2 78.8  6.2* 103.9  8.0* 82.8  4.1* 82.8  4.0*

114.4  7.0 86.0  7.0 93.3  6.5 95.3  7.8 87.4  7.0

3.5  0.15 2.3  0.11* 2.6  0.10* 2.2  0.14* 2.5  0.14*

3.6  0.17 1.8  0.10* 2.1  1.3* 2.2  0.089* 2.0  0.11*

3.1  0.26 1.8  0.14* 1.9  0.17* 1.9  0.20* 1.8  0.15*

Experiment 2

NAA concentration

NAAG concentration

Higher dose study

CTX

AEP

HIP

CTX

AEP

HIP

Sham (no soman exposure) Soman Soman + NAAG (50 mg/kg) Soman + b-NAAG (50 mg/kg) Soman + b-NAAG (50 mg/kg) + 2-PMPA b-NAAG + 2-PMPA (no soman exposure)

129.9  7.1 94.8  6.8* 105.0  6.6* 99.0  4.6* 100.2  3.2* 144.2  2.9

149.8  7.8 77.1  9.0* 85.0  4.2* 92.5  7.5* 88.8  6.6* 158.9  7.5

119.2  4.1 83.1  2.7* 87.8  4.8* 88.9  3.7* 90.1  4.2* 125.1  8.2

3.3  0.12 1.8  0.094* 2.4  0.22* 2.4  0.18* 2.4  0.20# 3.7  0.21

3.9  0.34 1.3  0.33* 2.3  0.30* 1.7  0.26* 2.1  0.20* 4.1  0.24

3.4  0.28 1.9  0.088* 1.9  0.22* 1.9  0.21y 2.2  0.22 3.2  0.42

CTX: dorsolateral cortex; AEP: amygdala, entorhinal cortex, and piriform cortex; HIP: hippocampus. * In Experiment 1, significantly different from the Sham (no soman exposure) or in Experiment 2, significantly different from the Sham group and the b-NAAG + 2-PMPA (no soman exposure) group. The groups indicated by * do not differ from each other. # Significantly different from the b-NAAG + 2-PMPA (No soman exposure) group. y Significantly different from the Sham group.

injection of b-NAAG (10 mg/kg), the number of FJC-positive cells in the amygdala (38%; t = 3.745, p = 0.004) and entorhinal cortex (34%; t = 3.291, p = 0.011) were significantly reduced, but not so in the piriform cortex (42%; t = 2.521, p = 0.061). The administration of 2-PMPA (50 mg/kg; Figs. 4A and 5A), reduced the number of FJC-positive cells by 42% (t = 4.165, p < 0.001), 43% (t = 3.694, p = 0.004) and 47% (t = 3.178, p = 0.014) in the amygdala, entorhinal cortex, and piriform cortex, respectively. These data indicate that overall lower doses of NAAG or b-NAAG (10 mg/kg), as well as administration of 2-PMPA (50 mg/kg), were associated with reduced soman-induced neuropathology in some of the most vulnerable brain regions. Similar results were found in Experiment 2. There were significant differences in the number of FJC-positive cells in certain brain regions, where higher doses (50 mg/kg) of NAAG or bNAAG reduced neuropathology (Figs. 4B and 5B). ANOVA indicated significant changes with drug treatment after soman in the amygdala (F3,29 = 9.887, p < 0.001), entorhinal cortex (F3,29 = 7.053, p < 0.001), piriform cortex (F3,29 = 5.974, p = 0.003), and the MD (F3,29 = 4.056, p = 0.016) and reuniens (Re; F3,29 = 2.987, p = 0.047) thalamic nuclei. Comparisons to FJC labeling in the Soman (alone) group indicated NAAG (50 mg/kg) administration resulted in fewer FJC-positive cells in the amygdala (39%; t = 4.280, p < 0.001) and piriform cortex (27%; t = 2.276, p = 0.030). Administration of b-NAAG (50 mg/kg), resulted in a reduction in the number of FJC-positive cells in the amygdala (45%; t = 4.732, p < 0.001), entorhinal cortex (50%; t = 4.564, p < 0.001), piriform cortex (48%; t = 3.985, p = 0.001), medial dorsal thalamic nucleus (38%; t = 3.472, p = 0.005), and the

reunien thalamic nucleus (33%; t = 2.991, p = 0.017). After injection of b-NAAG (50 mg/kg) plus 2-PMPA (50 mg/kg), the number of FJC-positive cells was reduced in the amygdala (35%; t = 3.975, p < 0.001), entorhinal cortex (27%; t = 2.602, p = 0.043), and piriform cortex (36%; t = 3.127, p = 0.008). Notably, in some brain regions, b-NAAG (50 mg/kg) appeared to reduce the number of FJC-positive cells to a greater extent than the combined treatment (entorhinal cortex 50% vs. 27%, the piriform cortex 48% vs. 36%, and amygdala 45% vs. 35%), but none of these comparisons were statistically significant. Experiment 3 was an evaluation of the combined effects of the administration of NAAG with 2-PMPA (Fig. 5C). Significant differences were seen in the number of FJC-positive cells as a function of drug treatment in the amygdala (F2,19 = 21.194, p < 0.001) and entorhinal cortex (F2,19 = 4.860, p = 0.020). In the amygdala, the combined administration of 10 or 50 mg/kg NAAG with 2-PMPA reduced FJC staining (30% t = 4.200, p < 0.001and 43%; t = 6.275, p < 0.001, respectively). When 50 mg/kg of NAAG was administered with 50 mg/kg 2-PMPA, there was a 25% reduction in FJC staining in the entorhinal cortex (t = 2.843, p = 0.021), but staining was not significantly reduced by the administration of 10 mg NAAG with 2-PMPA (t = 2.352, p = 0.059). 3.8. Effect size calculations suggest NAAG-related treatments result in a reduction of neuropathology from soman exposure Three independent experiments evaluated the effectiveness of NAAG-related compounds for reducing neuropathological changes that arise from soman. Table 4 provides a summary of effect sizes

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Fig. 3. FJC staining showing neuronal degeneration caused by soman administration. (A) Photomicrographs of FJC staining in sham group animals and soman-treated animals in various brain regions. Bar = 100 mm. (B) Summary of the mean number of the FJC-positive cells in different brain areas following soman administration (data are not shown for the sham group where no positive FJC cells were observed). Some of these data are also presented in Fig. 5B, but are shown here to illustrate the variation in average cell death per unit brain region after soman exposure. (C) A depiction of the brain regions that were sampled for assessment of FJC histochemical staining after soman exposure. Labels show the location of where cell counts were performed at Bregma 3.30 (top) and 5.20 (bottom), based upon the atlas of Paxinos and Watson (2005). RSG: retrosplenial granular cortex; LC1: lateral cortex region 1, superficial cortical area; LC2: lateral cortex region 2, deeper cortical regions; Ent: entorhinal cortex; Pir: piriform cortex; Amy: amygdala; LD: lateral dorsal thalamic nucleus; MD: medial dorsal thalamic nucleus; Re: reuniens thalamic nucleus; CA1: hippocampus CA1 region; CA2/CA3: hippocampus CA2/CA3 region; DG: hippocampus dentate gyrus.

Fig. 4. Representative photomicrographs showing the treatment effects of NAAG, b-NAAG and 2-PMPA on soman-induced neuropathology. (A) Effects of lower doses of NAAG (10 mg/kg), b-NAAG (10 mg/kg) and 2-PMPA (50 mg/kg). (B) Effects of higher doses of NAAG (50 mg/kg), b-NAAG (50 mg/kg), and the combination of b-NAAG (50 mg/kg) plus 2-PMPA (50 mg/kg). Amy: amygdala; Ent: entorhinal cortex; Pir: piriform cortex; MD: medial dorsal thalamus nucleus. Bar = 100 mm.

across experiments, where measurements of the difference between FJC staining in the soman-exposed animals (with no NAAG drug treatments) and drug-treated conditions are compared using Hedge’s g. Drug treatments were not identical in Experiments 1–3, but the results suggest (read across rows) that treatments with NAAG-related drugs had a consistent, large effect (g > 1) of reducing FJC levels in ‘‘AEP’’ limbic regions; areas that often exhibited the greatest degree of neurodegeneration after soman exposure (cf. Fig. 5).

4. Discussion 4.1. NAAG levels vary by brain region and administration of NAAGrelated compounds alters NAAG levels Consistent with previous reports, NAA levels in the rat brain of untreated animals were found to be similar across different brain areas, while NAAG levels exhibited greater variation (Fuhrman et al., 1994; Koller et al., 1984). The brain stem contains the highest

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The data suggest that in some brain regions peripheral administration of NAAG or b-NAAG, when given in combination with 2-PMPA, transiently altered brain NAAG levels 3 h later (Table 2). Specifically, at the dose levels tested 2-PMPA alone did not alter NAAG levels, but the administration of NAAG or b-NAAG with 2-PMPA resulted in significant elevations in measureable NAAG levels in some brain regions. It may be that the presence of 2-PMPA permits levels of NAAG to remain high as a result of the inhibition of GCPs. Although lower 10 mg/kg doses of NAAG or bNAAG (given without 2-PMPA) resulted in no change in NAAG or NAA levels, this dose level did significantly reduce the neuronal degeneration caused by soman in AEP brain areas (Fig. 5A). Previous studies have also shown that the administration of a 10 mg/kg dose NAAG or b-NAAG provided neuroprotection against traumatic brain injury (Cai et al., 2002; Fuhrman et al., 1994; Lu et al., 2000; Orlando et al., 1997; Slusher et al., 1999; Tortella et al., 2000; Van Hemelrijck et al., 2005; Yourick et al., 2003). 4.2. Soman reduced NAAG levels in vulnerable brain regions

Fig. 5. Summary of the effects of NAAG, b-NAAG, and 2-PMPA on soman-induced neuropathology in various brain regions. (A) Experiment 1: lower doses of NAAG, b-NAAG and 2-PMPA significantly reduced the number of FJC-positive cells in the entorhinal and piriform cortical regions and in the amygdala. Number of animals: 8, 9, 8, 7, 9, 7 per group for the sham, soman, soman + NAAG, soman + b-NAAG, soman + b-NAAG + PMPA, and b-NAAG + PMPA treatment groups, respectively. No FJC-positive cells were observed in tissue samples from sham-treated (no soman) animals (data not shown). *Horizontal bars above the three histogram bars for NAAG-related drug treatments were all p < 0.05 compared to the soman group. (B) Experiment 2: higher doses of NAAG, b-NAAG, and the combination of b-NAAG and 2-PMPA resulted in similar changes in the same brain regions (n = 9/group). *p < 0.05 compared to the soman group. There were no FJC-positive cells (data not shown) in brain tissue sections from the sham group or a control drug-only group (b-NAAG + PMPA but no soman exposure). (C) Experiment 3: administration of NAAG in combination with 2-PMPA significantly reduced the number of FJCpositive cells in the entorhinal cortex, piriform cortex, and the amygdala (*p < 0.05 compared to the soman group, number of animals per group: soman, n = 9, soman + 10 mg/kg NAAG + 2-PMPA, n = 6, soman + 50 mg/kg NAAG + PMPA, n = 7). Ent: entorhinal cortex; Pir: piriform cortex; Amy: amygdala; LD: lateral dorsal thalamic nucleus; MD: medial dorsal thalamic nucleus; Re: reuniens thalamic nucleus; RSG: retrosplenial granular cortex; LC1: lateral cortex region 1, superficial cortical area; LC2: lateral cortex region 2, deeper cortical regions; CA1: hippocampus CA1 region; CA2/CA3: hippocampus CA2/CA3 region; DG: hippocampus dentate gyrus.

level of NAAG, which was five to six times the levels in the dorsal lateral cortex, the AEP regions, and the hippocampus, and the diencephalon and the cerebellum contained two to three times higher NAAG concentrations than the cortex, hippocampus and the amygdala (Fig. 2B, saline histogram bars).

Here, for the first time, it was shown that nerve agent administration reduces NAA and NAAG content in several brain areas, including the diencephalon, the dorsal lateral cortex, the amygdala, entorhinal and piriform cortical regions and the hippocampus (Fig. 2A, B). The greatest reductions of NAA and NAAG levels were seen in temporal lobe structures (the ‘‘AEP’’ structures; amygdala, entorhinal cortex and piriform cortex). As NAA and NAAG reductions suggest the extent of neuronal injury, the observed changes appear to parallel the pathological findings examined by FJC in this study (Fig. 5), and other methods, that these regions typically display the most severe neuropathology (Apland et al., 2010; McDonough and Shih, 1997). Compared to levels measured in sham-treated animals, the ratio of NAA/NAAG was significantly elevated in the AEP samples, the cortex and hippocampus (Fig. 2C). Although not measured in this study, the change in this ratio could be associated with somaninduced seizures that result in added release and hydrolysis of NAAG in these regions. Considering reported neuroprotective properties of NAAG, such a state has the potential to reduce NAAGmediated inhibition of excitatory neurotransmission, but increase in glutamate content that furthers excitatory neurotoxicity and neuronal damage. It has been reported that these regions exhibit rapid increases in extracellular glutamate after nerve agent exposure (Lallement et al., 1991a, 1991b, 1992). In contrast, no significant reduction of NAA and NAAG content was seen in the brain stem and the cerebellum, and these regions have reportedly far less brain damage after agent exposure. High NAAG content in these brain areas may exert stronger inhibition on soman-induced epileptic activities, thereby providing stronger neuroprotection (see review in Section 1). Finally, the recently identified tripeptide, N-acetylaspartylglutamylglutamate (NAAG2), is preferentially expressed in the brainstem (Lodder-Gadaczek et al., 2011). Its functional significance requires further study, including whether or not it may also confer neuroprotection against neurotoxicity. 4.3. Administration of NAAG-related compounds reduced neuron cell death in some, but not all, vulnerable brain regions Single injections of 10 mg/kg of NAAG, b-NAAG or 50 mg/kg of 2-PMPA all conferred significant protection against neuronal degeneration in the entorhinal cortex, piriform cortex and the amygdala (Fig. 5A). Higher doses of NAAG, b-NAAG, or 2-PMPA given in combination with a higher dose of b-NAAG, likewise attenuated neuropathology (Fig. 5B). In Experiment 3, the impact of NAAG when given with 2-PMPA attenuated neuropathology (Fig. 5C). Table 4 provided a summary of the overall effects of

RSG: retrosplenial granular cortex; LC1: lateral cortex region 1, superficial cortical area; LC2: lateral cortex region 2, deeper cortical regions; Amy: amygdala; Ent: entorhinal cortex; Pir: piriform cortex; CA1: hippocampus CA1 region; CA2/CA3: hippocampus CA2/CA3 region; DG: hippocampus dentate gyrus; LD: lateral dorsal thalamic nucleus; MD: medial dorsal thalamic nucleus; Re: reuniens thalamic nucleus. * g provides a means for comparing effect sizes across experiments, where results of the difference between FJC staining in the soman-exposed animals (with no NAAG drug treatments) and drug-treated conditions are compared. In this case, drug treatments were not identical in Experiments 1–3, but results suggest (read across columns for each brain region) that treatments with NAAG-related drugs had a consistent, large effect (g > 1) of reducing FJC levels in AEP regions. The numbers in parentheses after each g value are LLg and ULg, the respective lower and upper limits for a 95% confidence interval.

0.30 (1.24, 0.64) 0.65 (0.31, 1.61) 0.30 (1.24, 0.64) 0.72 (0.29, 1.72) 0.50 (0.48, 1.49) 1.07 (0.03, 2.12) 0.32 (0.56, 1.21) 0.68 (0.23, 1.58) 0.66 (0.24, 1.57) 0.62 (0.30, 1.55) 0.83 (0.12, 1.77) 0.61 (0.32, 1.53) 0.45 (0.61, 1.51) 0.32 (0.73, 1.37) 0.17 (0.87, 1.22) Thalamic nuclei LD 0.61 (0.46, 1.69) MD 0.53 (0.54, 1.60) Re 0.00 (1.04, 1.04)

0.83 (0.27, 1.92) 0.91 (0.20, 2.01) 0.44 (0.62, 1.50)

0.65 (0.31, 1.61) 1.65 (0.56, 2.75) 1.42 (0.37, 2.48)

0.13 (1.04, 0.80) 0.43 (0.51, 1.38) 0.08 (1.02, 0.85) 0.54 (1.53, 0.45) 0.02 (0.99, 0.95) 0.77 (0.24, 1.78) 0.16 (0.72, 1.04) 0.59 (0.31, 1.49) 0.10 (0.98, 0.78) 0.38 (.053, 1.30) 0.74 (0.19, 1.68) 0.08 (0.83, 0.98) 0.21 (0.84, 1.26)  0.51 (0.55, 1.57) Hippocampal regions CA1 0.20 (1.25, 0.84) CA2/3  DG 0.18 (1.23, 0.86)

0.24 (1.29, 0.81)  0.11 (1.15, 0.94)

0.98 (0.01, 1.98) 0.61 (0.34, 1.57) 0.32 (1.26, 0.62)

2.99 (1.60, 4.38) 1.35 (0.31, 2.40) 0.78 (0.20, 1.75) 2.08 (0.86, 3.31) 1.17 (0.11, 2.22) 0.28 (0.70, 1.26) 1.78 (0.73, 2.84) 1.17 (0.21, 2.13) 1.40 (0.41, 2.40) 1.97 (0.85, 3.10) 0.95 (0.01, 1.91) 1.05 (0.08, 2.02) 2.00 (0.68, 3.31) 1.75 (0.50, 3.01) 1.34 (0.17, 2.52) AEP regions AMY 2.15 (0.80, 3.51) ENT 1.25 (0.10, 2.41) PIR 1.71 (0.46, 2.95)

2.22 (0.85, 3.59) 1.97 (0.66, 3.28) 1.69 (0.45, 2.94)

2.25 (1.04, 3.47) 2.17 (0.97, 3.37) 1.90 (0.76, 3.04)

0.01 (1.47, 0.50) 0.99 (0.01, 1.98) 0.39 (0.56, 1.32) 0.52 (0.47, 1.51) 0.83 (0.19, 1.85) 0.49 (1.47, 0.50) 0.68 (0.01, 1.94) 0.19 (0.69, 1.07) 0.88 (0.05, 1.80) 0.43 (0.48, 1.35) 0.30 (0.61, 1.21) 0.98 (0.01, 1.94) 0.56 (1.06, 1.02) 1.11 (0.03, 2.24) 0.50 (0.57, 1.56) Cortical regions RSG 0.02 (1.04, 1.04) LC1 0.08 (0.97, 1.12) LC2 0.33 (0.72, 1.39)

0.60 (0.47, 1.67) 0.52 (1.59, 0.54) 0.41 (1.47, 0.65)

0.14 (0.60, 1.21) 0.09 (0.84, 1.02) 0.77 (0.20, 1.74)

50 mg NAAG + 50 mg 2PMPA 10 mg NAAG + 50 mg 2PMPA

b-NAAG + 2PMPA b-NAAG NAAG

Experiment 2 (higher dose study, 50 mg each drug)

2PMPA

b-NAAG NAAG

Experiment 1 (lower dose study, 10 mg each drug)

Table 4 Summary of Hedge’s g effect size index after soman exposure and treatments with NAAG, b-NAAG and/or 2-PMPA.*

Experiment 3 (NAAG + 2PMPA study)

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NAAG-related drug treatments from the three experiments. Hedge’s g was used to allow comparison of drug effect size (treatment effect) across Experiments 1–3, where the g index is an assessment of group mean changes in FJC staining after soman compared to exposure followed by treatment. Mean change is standardized by dividing the mean difference by the standard deviation as well as other adjustments to compute g (see Section 2.6). As summarized in Table 4, conventional interpretation suggests that cortical regions (RSG, LC1, LC2) and hippocampal regions were not greatly affected by drug treatment. However, treatments invariably had a very large effect of reducing neuropathology in brain regions that exhibited higher levels of FJC staining; in this case measures of injury in the amygdala, the entorhinal and piriform cortical areas. To a lesser extent, thalamic regions (LD, MD, Re) were observed to have large (>0.80) to medium size effects (>0.50), but less reliably. From this summary, one can conclude that treatment with a variety of NAAG-related compounds, or combinatorial treatments, are effective in reducing FJC staining in regions that exhibit the greatest level of injury, but the effect is less robust in other areas that sustain damage. Based upon the idea that b-NAAG would be more resistant to degradation [kcat = 0.30/s vs. kcat = 1.1/s for NAAG by GCP II in vitro (Hlouchova´ et al., 2007)], it was predicted that the combination of 2-PMPA and the higher dose of b-NAAG would be most effective in reducing FJC staining. However, the combination was not any more effective than, for example, a higher dose of b-NAAG alone (Fig. 5B). There are at least two explanations for failing to observe further neuroprotection (evaluated by FJC staining). First, NAAGrelated drugs may ‘‘protect’’ just a portion of the neurons in a particularly vulnerable brain region, and the combination of bNAAG and 2-PMPA may not be more effective than when these compounds are given individually. Neurons that are not responsive to the protective effects of NAAG-related drugs would not further reduce observed neuropathology. Second, although it may not be harmful to the normal and sham control animals, it is possible that this combination, and the state of soman-induced seizure activity, causes extraordinarily high extracellular NAAG concentrations and that in this circumstance further neuroprotection is impeded. Additional experiments are needed to test the effect of drug combinations upon extracellular glutamate levels after nerve agent exposure. 4.4. Paradoxical effects of NAAG-related drug administration neuroprotection and reduced NAAG levels In contrast to the alleviated soman-induced neuropathology, soman exposure caused decreases in NAA and NAAG content that did not recover after the treatments with NAAG, b-NAAG, and 2PMPA (Section 3.5). An explanation for this finding is that the reduction of NAA and NAAG in the brain is not solely the result of neuronal death, but is also a parallel change caused by seizure activity. Besides neuronal loss, soman intoxication may reduce brain NAA and NAAG by several ways. First, it was shown that mitochondrial function was disrupted after soman exposure (Collombet et al., 2009), which may result in suppression of NAA synthesis in mitochondria. Second, as neuronal activity promotes the release of NAA and NAAG from axon terminals (Baslow et al., 2007; Tsai et al., 1990; Williamson et al., 1991), the abnormally high neuronal firing by soman-induced seizures may enhance the release of NAA from synapses, which is then catabolized by glial cells (Engel and Pedley, 2008; Schuff et al., 2006; Serles et al., 2001). Third, a marked increase in reactive astrocytes following soman exposure (Collombet et al., 2007; Zimmer et al., 1998) may accelerate the hydrolysis of NAA and NAAG. In epilepsy and seizure patients, for example, it was found that reductions in NAA are not merely attributable to neuronal loss

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(Petroff and Duncan, 2008), and decreased NAA levels in epilepsy patients is associated with the number of reactive astrocytes rather than neuronal loss (Hetherington et al., 2004). Furthermore, it was shown that epilepsy-induced NAA reduction returns to normal values after successful epilepsy surgery (Cendes et al., 1997). The upturn of NAA levels, however, occurs slowly after successful epilepsy surgery, taking 6 months to reach a 50% increase of the preoperative value (Serles et al., 2001). Thus, it may be necessary to conduct a long-term study to determine whether NAA and NAAG levels eventually recover after drug treatment for soman intoxication. A reduction in NAA—without neuronal loss—has been found in other circumstances besides epilepsy, including in cognitively impaired but non-demented elderly individuals and in brain samples from individuals with post-traumatic stress disorder (Schuff et al., 2006). Finally, axonal injury contributes to decreased NAA levels in the absence of neuronal loss (Fulham et al., 1994). Therefore, it is possible that seizure activity after soman exposure causes reduced NAA and NAAG levels in the majority of neurons in addition to those that succumbed to neuronal death. Alterations in soman-induced changes in enzymatic activities also must be considered. For example, enhanced NAA and NAAG hydrolysis could be the result of an increase in aspartoacylase (Moffett et al., 2011) or GCP enzyme activity or expression level changes in glial cells. Clinically, patients with seizure and epileptic status have been reported to show decreased brain NAA (and NAAG) content, measured by magnetic resonance spectroscopy, and seizure frequency correlates with the magnitude of the NAA decline at the seizure focus (for review, see Petroff and Duncan, 2008). Hence, the differences in NAAG levels between different brain regions, elevated seizure activity through certain neural pathways, and increased NAAG hydrolysis may all be contributing factors in determining the extent of nerve agent-induced neurotoxicity. The reduction of NAA and NAAG appears to be a parallel, initial change with the altered FJC profile, but secondary factors may also contribute to neuropathological changes in NAA and NAAG levels after soman intoxication. Further examination in animals depleted of NAAG (such as NAAG synthetase knockout mice) and NAAG2 would help elucidate these issues. 5. Conclusions As the most abundant neuropeptide in the central nervous system, NAAG performs a complex role in neurobiological functions. Findings indicated that soman-induced brain damage was most prominent in brain areas with the lowest NAAG content, while no damage was found in areas with the highest level of NAAG. Brain areas with the most dramatic neuropathological changes also exhibited the highest degree of NAAG degradation. In addition, results suggest that all three tested compounds, NAAG, b-NAAG, and 2-PMPA, significantly reduced neuron cell death in brain regions known to be particularly susceptible to agent exposure, suggesting that exogenously administered NAAG compounds and GCP enzyme inhibitors may be a partially effective therapy against neuropathology by nerve agents. These data suggest a critical role of physiological NAAG in neuroprotection against nerve agent toxicity, but further work is warranted to evaluate these compounds when they are administered at a delayed time after agent exposure and to determine the mechanisms that underlie their neuroprotective effectiveness. Conflict of interest The authors declare that there are no conflicts of interest.

Transparency document The Transparency document associated with this article can be found in the online version. Disclaimers The opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army, Department of Defense, the U.S. government, United States Army Medical Research Institute of Chemical Defense, or the Uniformed Services University of the Health Sciences. The use of trade names does not constitute an official endorsement or approval of the use of such reagents or commercial hardware or software. This document may not be cited for purposes of advertisement. Acknowledgements This work was supported by grant 1.E0044_08_US_C from the Defense Threat Reduction Agency to JTM. Experiments were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals, ILAR, National Research Council, DHEW Publ. # (NIH) 73-23. We thank Dr. Barbara S. Slusher, Brain Science Institute NeuroTranslational Program, Johns Hopkins School of Medicine, for the kind gift of 2-PMPA.

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