A new generation fatty acid amide hydrolase inhibitor protects against kainate-induced excitotoxicity

Abstract

Endocannabinoids, including anandamide (AEA), have been implicated in neuroprotective on-demand responses. Related to such a response to injury, an excitotoxic kainic acid (KA) injection (i.p.) was found to increase AEA levels in the brain. To modulate the endocannabinoid response during events of excitotoxicity in vitro and in vivo, we utilized a new generation compound (AM5206) that selectively inhibits the AEA deactivating enzyme fatty acid amide hydrolase (FAAH). KA caused calpain-mediated spectrin breakdown, declines in synaptic markers, and disruption of neuronal integrity in cultured hippocampal slices. FAAH inhibition with AM5206 protected against the neurodegenerative cascade assessed in the slice model 24 h postinsult. In vivo, KA administration induced seizures and the same neurodegenerative events exhibited in vitro. When AM5206 was injected immediately after KA in rats, the seizure scores were markedly reduced as were levels of cytoskeletal damage and synaptic protein decline. The pre- and postsynaptic proteins were protected by the FAAH inhibitor to levels comparable to those found in healthy control brains. These data support the idea that endocannabinoids are released and converge on pro-survival pathways that prevent excitotoxic progression.

Fig. 1

Exposure to the excitotoxin KA increases AEA levels in the brain. Vehicle (a) or 9.5 mg/kg KA (b) was systemically administered to rats. At the 2 h postinjection time, cortical tissue was rapidly dissected, flash-frozen in liquid nitrogen, and assayed for AEA levels by mass spectrometry as described in the “Materials and Methods” section. Time of elution (minutes) from the reverse phase column is represented by the X-axes and is noted for reference peaks and AEA (shaded). AEA levels (nanograms per gram of wet tissue weight) were determined using an AEA standard curve and means ± SEM are shown (c). Unpaired t test; triple asterisk, P<0.001

Fig. 2

AM5206 selectively inhibits FAAH activity. Recombinant FAAH and MAGL were treated with varying concentrations of AM5206 in triplicate, and the two hydrolytic activities were determined using specific fluorogenic substrates. Data were normalized to 100% fluorescence activity in the absence of drug and are presented as means ± SEM

Fig. 3

AM5206 affords neuronal protection in the hippocampus after KA-induced excitotoxicity in vitro. Organotypic hippocampal slice cultures were used, and a low-power photomicrograph shows their characteristic maintenance of native cellular organization (a). Nissl staining was also used to assess cellular integrity across treatment groups. CA3 pyramidal zones are shown from slices treated consistently with vehicle (b), those pretreated with vehicle before a 2-h exposure to 60 μM KA (c), and slices pretreated with 10 μM AM5206 be fore the KA insult (d). After a washout step, the cultured slices were then incubated with vehicle or AM5206 for 24 h before the tissue was fixed, sectioned, and stained. The KA insult resulted in neuronal loss and obvious pyknotic changes that were reduced by the FAAH inhibitor. DG, dentate gyrus; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bar: a, 400 μm; b–d, 45 μm

Fig. 4

Treatment with AM5206 elicits several indicators of neuroprotection in the hippocampal slice model. The slices were treated consistently with vehicle (veh), pretreated with vehicle before a 2-h KA exposure, or pretreated with AM5206 before the KA insult. After the KA exposure and a washout step, slices were returned to their prior condition of the absence or presence of AM5206. Slices were harvested 24 h postinsult in groups of six to eight and assessed by immunoblotting for calpain-mediated spectrin breakdown product BDP_N, synaptic markers GluR1 and synapsin II (syn II), and protein load control actin (see blots in a). Integrated optical densities for BDP_N (b) and GluR1 (c) across the treatment groups are compared as means ± SEM. Unpaired t test compared to KA+vehicle data, single asterisk P<0.05; double asterisk P<0.01. Treated slices (25–27 per condition) divided into three groups were also assessed for LDH activity released into the culture medium (d). LDH release is expressed as the colorimetric assay absorbance (mean ± SEM) corrected for background activity present in fresh medium. AM5206 effect: single asterisk, P=0.026

Fig. 5

AM5206 affords seizure and neuronal protection after KA-induced excitotoxicity in vivo. Seizures were induced by i.p. injection of 9.5 mg/kg KA (n=12 rats), and following the KA administration animals were immediately injected with either vehicle or 8 mg/kg AM5206. Vehicle-treated control rats (veh, n=11) did not receive KA or AM5206. Seizures were scored by blinded raters over a 4-h period (a) and mean scores ± SEM are shown (ANOVA, P<0.0001). At 48 h postinjection, hippocampal tissue was rapidly dissected, homogenized, and equal protein aliquots assessed by immunoblot for BDP_N, GluR1, synapsin II (syn II), and actin (b). Mean integrated optical densities for GluR1 (c) are shown (±SEM; ANOVA, P<0.001). Post hoc tests compared to KA+ vehicle data, single asterisk P<0.05; triple asterisk, P<0.001

Fig. 6

Repeated administrations of AM5206 were assessed for signs of toxicity in rats. Animals in groups of four were injected i.p. with vehicle (0 mg/kg) or 5–15 mg/kg AM5206 daily for 14 days. They were evaluated by seizure scorers during the 2-h postinjection periods on the last 3 days of treatment (a); overall mean scores ± SD are shown. Blood was subsequently collected for measures of ALT activity (b) and BUN levels (c) in plasma, which are plotted as means ± SEM