N-arachidonoyl-L-serine is neuroprotective after traumatic brain injury by reducing apoptosis

Abstract

N-arachidonoyl-L-serine (AraS) is a brain component structurally related to the endocannabinoid family. We investigated the neuroprotective effects of AraS following closed head injury induced by weight drop onto the exposed fronto-parietal skull and the mechanisms involved. A single injection of AraS following injury led to a significant improvement in functional outcome, and to reduced edema and lesion volume compared with vehicle. Specific antagonists to CB2 receptors, transient receptor potential vanilloid 1 (TRPV1) or large conductance calcium-activated potassium (BK) channels reversed these effects. Specific binding assays did not indicate binding of AraS to the GPR55 cannabinoid receptor. N-arachidonoyl-L-serine blocked the attenuation in phosphorylated extracellular-signal-regulated kinase 1/2 (ERK) levels and led to an increase in pAkt in both the ipsilateral and contralateral cortices. Increased levels of the prosurvival factor Bcl-xL were evident 24 hours after injury in AraS-treated mice, followed by a 30% reduction in caspase-3 activity, measured 3 days after injury. Treatment with a CB2 antagonist, but not with a CB1 antagonist, reversed this effect. Our results suggest that administration of AraS leads to neuroprotection via ERK and Akt phosphorylation and induction of their downstream antiapoptotic pathways. These protective effects are related mostly to indirect signaling via the CB2R and TRPV1 channels but not through CB1 or GPR55 receptors.

Figure 1

N-arachidonoyl--serine (AraS) reduces neurologic disability after closed head injury (CHI). Neurologic severity score (NSS) of mice treated with vehicle, AraS, AraS+rimonabant (SR141716A), AraS+SR144528, AraS+capsazepine, or AraS+paxilline (^**P<0.01 versus vehicle, ^***P<0.001 versus vehicle, ^#P<0.05 versus AraS, ^##P<0.01 versus AraS, ^###P<0.001 versus AraS). AraS induced better neurobehavioral function after CHI was abolished by CB2 and transient receptor potential vanilloid 1 (TRPV1) antagonists SR144528 and capsazepine, respectively. The CB1 receptor antagonist rimonabant (SR141716A) did not alter the neuroprotective effect of AraS.

Figure 2

N-arachidonoyl--serine (AraS) reduces lesion volume after closed head injury (CHI). Lesion volumes measured 24 hours after CHI from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brains after treatment with vehicle, AraS, AraS+rimonabant (SR141716A), AraS+SR144528, AraS+capsazepine, or AraS+paxilline (^**P<0.01 versus vehicle, ^#P<0.05 versus AraS, ^##P<0.01 versus AraS, ^###P<0.001 versus AraS). AraS reduced hemispheric lesion volume after CHI, which was completely abolished by CB2 and transient receptor potential vanilloid 1 (TRPV1) antagonists SR144528 and capsazepine, respectively, and to some extent by BK channels blocker (paxilline). The reduction was not affected by CB1 antagonist (SR141716A).

Figure 3

N-arachidonoyl--serine (AraS) prevents reduction in phosphorylated extracellular-signal-regulated kinase 1/2 (pERK) levels 2 hours after closed head injury (CHI). (A) Western blots for phosphorylated extracellular-signal-regulated kinase (pERK) at different time points after CHI from brains treated with AraS or vehicle. (B) Quantitative measurements of optical density of the bands shown in (A) (^*P<0.05 versus sham, ^**P<0.01 versus sham, ^***P<0.001 versus sham, ^##P<0.01 versus AraS 2 hours, ^###P<0.001 versus AraS 2 hours). GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

Figure 4

N-arachidonoyl--serine (AraS) increases pAkt levels 4 hours after closed head injury (CHI). (A) Western blots for phosphorylated protein kinase B/Akt (pAkt) at the ipsilateral (ipsi) and contralateral (contra) cortex to the lesion, 4 and 24 hours after CHI. (B) Quantitative measurements of the optical density of the bands shown in (A) (^*P<0.05 versus vehicle, ^***P<0.01 versus vehicle).

Figure 5

N-arachidonoyl--serine (AraS) increases Bcl-xL levels after closed head injury (CHI). (A) Western blots for Bcl-xL levels, 4 and 24 hours after CHI. (B) Quantitative measurements of the optical density of the bands shown in (A) (^***P<0.001 versus vehicle, ipsi—cortex ipsilateral to the lesion, contra—cortex contralateral to the lesion).

Figure 6

N-arachidonoyl--serine (AraS) reduces active caspase-3 levels after closed head injury (CHI). (A) The effect of AraS treatment on caspase-3 activity (expressed as fluorescence units of DEVD-AFC cleaved product) in the brains of sham-operated controls and CHI animals treated with vehicle, AraS, AraS+SR141716A, AraS+SR144528, or AraS+capsazepine 72 hours after injury (&P<0.05 versus AraS, ^##P<0.01 versus vehicle, ^***P<0.001 versus sham). (B) Scheme describing the suggested mechanism triggered by AraS (Adapted from Cudaback et al, 2010; Zhuang et al, 2004; Datta et al, 1997; Yang et al, 1995; Chen et al, 2005).