Inhibitor of Endocannabinoid Deactivation Protects Against In Vitro and In Vivo Neurotoxic Effects of Paraoxon

Abstract

The anticholinesterase paraoxon (Pxn) is related to military nerve agents that increase acetylcholine levels, trigger seizures, and cause excitotoxic damage in the brain. In rat hippocampal slice cultures, high-dose Pxn was applied resulting in a presynaptic vulnerability evidenced by a 64% reduction in synapsin IIb (syn IIb) levels, whereas the postsynaptic protein GluR1 was unchanged. Other signs of Pxn-induced cytotoxicity include the oxidative stress-related production of stable 4-hydroxynonenal (4-HNE)-protein adducts. Next, the Pxn toxicity was tested for protective effects by the fatty acid amide hydrolase (FAAH) inhibitor AM5206, a compound linked to enhanced repair signaling through the endocannabinoid pathway. The Pxn-mediated declines in syn IIb and synaptophysin were prevented by AM5206 in the slice cultures. To test if the protective results in the slice model translate to an in vivo model, AM5206 was injected i.p. into rats, followed immediately by subcutaneous Pxn administration. The toxin caused a pathogenic cascade initiated by seizure events, leading to presynaptic marker decline and oxidative changes in the hippocampus and frontal cortex. AM5206 exhibited protective effects including the reduction of seizure severity by 86%, and improving balance and coordination measured 24 h post-insult. As observed in hippocampal slices, the FAAH inhibitor also prevented the Pxn-induced loss of syn IIb in vivo. In addition, the AM5206 compound reduced the 4-HNE modifications of proteins and the β1 integrin activation events both in vitro and in vivo. These results indicate that Pxn exposure produces oxidative and synaptic toxicity that leads to the behavioral deficits manifested by the neurotoxin. In contrast, the presence of FAAH inhibitor AM5206 offsets the pathogenic cascade elicited by the Pxn anticholinesterase.

Early Synaptic Alterations and Selective Adhesion Signaling in Hippocampal Dendritic Zones Following Organophosphate Exposure

“…Transverse slices (400 μm thickness) were prepared and kept in cooled-buffer solution until 8–9 slices were placed on the Biopore PTFE membrane of each Millicell-CM culture insert (Millipore, Billerica, MA). Media was changed every 2–3 days, consisting of 50% basal medium Eagle (Sigma-Aldrich, St. Louis, MO), 25% Earle’s balanced salts (Sigma-Aldrich), 25% horse serum (Gemini Bio-products, Sacramento, CA), and defined supplements, as described previously 35–37 . The hippocampal slices were maintained in culture at 37 °C in 5% CO 2 -enriched atmosphere for a maturation period of 18–20 days before experiments were conducted.…”

“…Note that the Pxn concentration and conditions used were previously established 37 . Slice groups with Pxn exposure times of 3–48 h were staggered in order to ensure same-day harvesting, and vehicle control groups were incubated for 24–48 h. A subset of Pxn-treated hippocampal tissue was co-treated with the fatty acid amide hydrolase inhibitor AM5206, previously shown to have neuroprotectant activity against neurotoxin exposure 37 . The tissues were pre-treated for 1 h with 20 μM AM5206, and subsequently treated with Pxn with continued presence of AM5206.…”

Acetaminophen metabolites p-aminophenol and AM404 inhibit microglial activation

“…FAAH also breaks down anandamide to arachidonic acid and ethanolamine. The catabolic activity of FAAH on anandamide is counterbalanced by N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD), which re-synthesizes anandamide from arachidonic acid and ethanolamine a a cortex [34] . In summary, some of the pharmacological activity of acetaminophen is mediated by its bioactive metabolites p-aminophenol and AM404.…”

Paraoxon: An Anticholinesterase That Triggers an Excitotoxic Cascade of Oxidative Stress, Adhesion Responses, and Synaptic Compromise

“…In a recent study of Pxn exposure using stable explants of brain tissue, indications of oxidative stress and induced alterations to β1-class integrin adhesion molecules were found in correspondence with synaptic compromise (Farizatto et al, 2017). This synaptic compromise was characterized by selectivity of the presynaptic components it affected.…”

“…OP agents like Pxn may initiate synaptic deterioration in which the presynaptic markers appear to be more susceptible than postsynaptic markers (Kozhemyakin et al, 2010; Farizatto et al, 2017). The synaptic decline profile induced by Pxn can be compared to 1) synaptic disruption reported in excitotoxicity studies using hippocampal slice cultures (Bahr et al, 2002; Karanian et al, 2005; Piwońska et al, 2016) and 2) hippocampal slice synaptic declines from protein accumulation stress studies (Bendiske and Bahr, 2003; Butler et al, 2007; Wisniewski et al, 2011).…”