Dual Modulation of Endocannabinoid Transport and Fatty Acid Amide Hydrolase Protects against Excitotoxicity

Abstract

The endocannabinoid system has been suggested to elicit signals that defend against several disease states including excitotoxic brain damage. Besides direct activation with CB 1 receptor agonists, cannabinergic signaling can be modulated through inhibition of endocannabinoid transport and fatty acid amide hydrolase (FAAH), two mechanisms of endocannabinoid inactivation. To test whether the transporter and FAAH can be targeted pharmacologically to modulate survival/repair responses, the transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404) and the FAAH inhibitor palmitylsulfonyl fluoride (AM374) were assessed for protection against excitotoxicity in vitro and in vivo. AM374 and AM404 both enhanced mitogen-activated protein kinase (MAPK) activation in cultured hippocampal slices. Interestingly, combining the distinct inhibitors produced additive effects on CB 1 signaling and associated neuroprotection. After an excitotoxic insult in the slices, infusing the AM374/AM404 combination protected against cytoskeletal damage and synaptic decline, and the protection was similar to that produced by the stable CB 1 agonist AM356 (R-methanandamide). AM374/ AM404 and the agonist also elicited cytoskeletal and synaptic protection in vivo when coinjected with excitotoxin into the dorsal hippocampus. Correspondingly, potentiating endocannabinoid responses with the AM374/AM404 combination prevented behavioral alterations and memory impairment that are characteristic of excitotoxic damage. The protective effects mediated by AM374/AM404 were (1) evident 7 d after insult, (2) correlated with the preservation of CB 1 -linked MAPK signaling, and (3) were blocked by a selective CB 1 antagonist. These results indicate that dual modulation of the endocannabinoid system with AM374/AM404 elicits neuroprotection through the CB 1 receptor. The transporter and FAAH are modulatory sites that may be exploited to enhance cannabinergic signaling for therapeutic purposes.

Equipotent Inhibition of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase – Dual Targets of the Endocannabinoid System to Protect against Seizure Pathology

“…KA was purchased from Tocris (Ellisville, MO). Affinity-purified antibodies to the amino-terminal of calpain-mediated spectrin breakdown product (BDP N ), and to the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subunit glutamate receptor 1 ([GluA1], also known as GluR1) were used as previously described [13,33,38,39]. A phosphorylation state-specific antibody against the AMPA receptor GluA1 subunit at serine-845 (pGluA1-Ser-845) was obtained from Millipore (Bedford, MA).…”

“…Hippocampus sections were rapidly removed from Sprague-Dawley rat pups 11 to 12 days postnatal and transversely sectioned into 400-μm thick slices, as previously described [13,33,43]. Nine hippocampal slices were then carefully transferred to each of 6 Millicell-CM membrane inserts (Millipore Corporation, Bedford, MA) containing 50% basal medium Eagle, 25% Earle's balanced salts, 25% horse serum, and defined supplements [39].…”

“…In the excitotoxicity field, much attention has been applied to potential strategies to preserve learning and memory and other brain functions, including the neuroprotective abilities of specific signaling lipids that make up the family of endocannabinoids (for more detail see Hwang J, et al [1] Zanettini C, et al [2], Pacher and Hasko [3], and Janero DR, et al [4]). Multiple studies have linked the principal endocannabinoids in the central nervous system, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), with protection against numerous diseases, including Alzheimer's, Parkinson's, and Huntington's disease, epileptic seizures, and stroke/ischemic brain damage [5][6][7][8][9][10][11][12][13][14]. In the nervous system, endocannabinoids AEA and 2-AG are synthesized and released on demand from membrane-bound phospholipids, and act at presynaptic Gi-coupled CB1 and CB2 receptors [15][16][17][18].…”

“…In the nervous system, endocannabinoids AEA and 2-AG are synthesized and released on demand from membrane-bound phospholipids, and act at presynaptic Gi-coupled CB1 and CB2 receptors [15][16][17][18]. CB1 receptors are localized predominantly to nerve terminals and function to suppress neurotransmitter release and excitotoxic signaling [13,14,[19][20][21][22]. Consequently, promoting cannabinergic signaling by inhibiting endocannabinoid inactivation is a neuroprotective strategy that is being intensively pursued.…”

“…Related to this response to injury, FAAH inhibition enhanced the salutary effects of cannabinergic signaling in the rat hippocampus, mediated predominantly via CB1 receptors [1,14,34,35]. It has been suggested that CB1 receptor activation reduces excessive glutamatergic signaling and excitotoxic progression thereby protecting hippocampal cells from cytoskeletal and synaptic damage [13,14,32,33,36]. These findings suggest a key role of endocannabinoids in attenuating excitotoxicity in the brain.…”