This study aimed at illuminating that ASD children and ASD model rats exhibited disruption of eCB system and pharmacological modulators of eCB system may have a therapeutic potential in ASD. Our results of reduced eCBs content, elevated degrading enzymes together with a compensatory up-regulation of CBRs suggested lower eCB signaling in ASD. In the meantime, we observed that JZL184 treatment, through enhancing intrinsic 2-AG levels, ameliorated autistic behaviors in VPA-exposed offspring. This is characterized by a reduced repetitive and stereotypical behaviors in marble burying and self-grooming test, reduced hyperactivity in open field test, increased sociability and social preference in three-chamber test and improved cognitive functioning in Morris water maze test. This research is important in driving the identification of potential targets for improved therapeutic treatments in ASD.
To date, there were only two human evidence about the eCBs levels in ASD children’s blood sample[11, 12], and their findings that lower concentrations of AEA, PEA and OEA in autistic children were consistent with ours. Noteworthily, this is the first time that we found circulating 2-AG levels were reduced in autistic children. We also found the ASD children with lower PEA level represented more serious ASD traits. Interestingly, Kelly demonstrated that plasma eCBs levels were dysregulated at age one in children with poor communication scores at age three, which implicating eCBs may be potential biomarker for early diagnosis of ASD. Anecdotally, case reports from Italy firstly have corroborated that PEA, alone or combination with other natural supplements, can refine ASD core impairments . Subsequently, an Iranian randomized, double-blind placebo-controlled trial revealed that PEA (600 mg twice daily) may augment therapeutic effects of risperidone on ASD-related irritability and hyperactivity without serious side effects . The non-psychoactive, medical cannabis (e.g. cannabidiol, cannabidivarin) in ASD patients appears to be well tolerated, safe (a low side effects rate) and effective option to relieve autistic symptoms in some countries [30, 31], nevertheless, the cannabinoid treatment remains a controversial ethical issue in individuals with ASD, and any kind of cannabinoid consumption is illegal in China. Collectively, eCBs, either as diagnostic biomarker or as potential therapeutic target, their decreased levels were associated with ASD.
Confirming the human results, we found levels of two major eCBs, AEA and 2-AG, were reduced in the hippocampus of VPA-induced rats. However, Kerr and colleagues found levels of AEA, PEA, OEA and 2-AG in hippocampus did not differ between VPA and CON rats. Intriguingly, they demonstrated that eCBs levels were enhanced in the hippocampus of VPA-exposed rats immediately following sociability test, that is, the contents of eCBs were susceptible to the behavioral testing which is supported by many other studies[32, 33]. To this end, in the present study behavioral experiments were paralleled by biochemical measurement, whereas Kerr examined eCBs concentration 72 h after animals underwent behavioral experiments. This might account for the disparity in the two studies. In addition, several lines of work pointed out that eCB changes appeared to be region-specific, so we did not observe similar profile and magnitude of eCBs in the hippocampus and in the PFC. In line with that changes of eCB system are more pronounced in the hippocampus with respect to the PFC.
In light of PBMCs (lymphocytes, NK cells and monocytes) could serve as a tool to investigate eCB system changes in CNS in several neuropsychiatric disorders, we also examined eCB-associated receptors and enzymes in PBMCs from autistic children. CB1R is the most abundant G protein-coupled receptor located in the brain, while CB2R is sparsely expressed in the brain and instead principally in immunoregulatory cells, like microglia and peripheral immune cells. This could explain that the differences in CB1R mRNA expression in PBMCs and CB2R protein expression in the hippocampus were not observed between ASD cases and controls; even CB1R protein expression was not detected in PBMCs in the present study. Of note, we found that transcription and translation of CB2R in the PBMCs and CB1R in the hippocampus were all enhanced. Siniscalco showed similar results to ours, they found unchanged CB1 mRNA levels and upregulated CB2R mRNA and protein levels in ASD-PBMCs as compared to healthy subjects. That prenatal VPA exposure increased CB1R protein levels were also observed in Zamberletti study. Remarkably, CB1R and CB2R activation exert diverse consequences across cellular physiology. As the main molecular target of eCBs, CB1R is found high densities on presynaptic termini of glutamatergic and GABAergic neuron, thereby, the activation of CB1R is implicated in regulation of excitatory-inhibitory balance, synaptic strength and neurotransmitter release, ultimately mediating social functioning, learning and memory[35, 36]. In conjunction, genetic variants in CNR1 (encoding CB1R) gene were correlated to not only verbal but non-verbal social communication as well in ASD researches[37, 38]. Additionally, via activation of CB2R, eCB system exerts anti-inflammatory actions and decrease glial activity to prevent excessive inflammation and cell damage [34, 39]. We could not obtain brain tissue of patients, nonetheless, to some degree, the expression of CB2R on peripheral immune cells reflects changes in the CNS. Actually, there was also an increase in mRNA expression of CB2R in the hippocampus of VPA-exposed offspring in this study. Hence, our findings support the notion that eCB system played the protective role of inflammatory response in autistic children via elevating CB2R expression, however further experimental evidence towards neuroinflammation is needed. Since it is impossible that the changes in components of eCB system are independent from one another, researchers suggested there might be a negative feedback regulation between eCBs and CBRs densities [41, 42]. We deduced that upregulation of CBRs was in response to the lower levels of eCBs in autistic patients and model rats in the current study. Alterations in CBRs levels are transient adaptive reactions which attempt to re-establish normal homeostasis disrupted by the disease.
So far, only Siniscalco’s team previously investigated eCB system in PBMCs from individual with ASD, but they incorporated neither 2-AG metabolic enzymes nor protein expression of eCB-related enzymes into their study[13, 14]. To our knowledge, this is the first study to explore relatively entire components of eCB system in PBMCs from human. The biosynthesis of 2-AG can be catalyzed by two diacylglycerol lipase isoforms, namely DAGL-α and DAGL-β. DAGL-α is expressed throughout the brain and 2-AG levels dropped by up to 80–90% in the brain in the DAGL-α null mouse brain. Given that DAGL-α is the main 2-AG synthesizing enzyme in the brain, in this study DAGL-α expression was solely examined (DAGL for short). We surprisingly revealed DAGL mRNA expression was increased in the human and rat sample, but not protein. Meanwhile, we found concurrent increases in expression of FAAH and MAGL, responsible for AEA and 2-AG degradation, both in the PBMCs and in the hippocampus. The lower eCB levels in the present study may account for increased degraded enzymes. Interestingly, in Siniscalco’s study, FAAH mRNA expression did not significantly change and NAPE-PLD slightly decreased in 17 ASD cases and 22 healthy controls. Our findings from animals were in keeping with previous reports that upregulated expression of FAAH and MAGL in VPA-induced rats[18, 44]. As a whole, our results and other studies highlighted the presence of a reduced eCB signaling in ASD children and animal model that might be responsible for the deficits in the cognitive and social domain.
The animal model studies have shown that prenatal VPA exposure in rodents recapitulates ASD-like pathophysiology at a molecular, cellular and behavioral level. VPA-induced rats have been developed and became a widely used environmental preclinical model of ASD with strong face and construct validity, and also serve as a good platform for testing pharmacological reagents that might be use treating ASD. On behavioral level, our findings have confirmed that VPA-exposed rats exhibited the core symptoms of ASD, impaired social interaction and repetitive behavior, possibly co-occurred emotional and cognitive problems. Meanwhile, what we found that prenatal VPA exposure inducing similar disturbance of eCB system in offspring rats to ASD children was in accordance with prior researches[16, 18], which indicating a reduced eCB tone in ASD. Therefore, we evaluated efficacy of boosting 2-AG levels by hydrolysis inhibitor JZL184 toward repetitive and stereotypical behaviors, hyperactivity, deficits in social and cognitive functioning in VPA-induced rats. Results here presented showed either acute or chronic JZL184 administration was able to relieve ASD-like behaviors, which were in line with previous reports in Fmr1 knock-out mice and Shank3B−/− mice [15, 45, 46].
Rats treated with acute administration 40 mg/kg JZL184 exhibited reduced repetitive marble-burying and grooming behaviors and hyperactivity, and improvement in sociability and social preference induced by VPA exposure. We observed that single injection of JZL184 (40 mg/kg) did not change the levels of eCBs in the hippocampus and PFC, which was in consistence with the findings from Kerr  who did not detect the alternation in the levels of eCBs 2.5 h after injection JZL184 (10 mg/kg). Nonetheless, JZL184 at dose of 40 mg/kg could show loss of MAGL activity. Kruk-Slomka  showed the similar results that acute injection of JZL184 40 mg/kg significantly decreases locomotion and improved long-term acquisition of memory and learning process. Although the alternations of eCB system were not yet appeared, acute JZL184 treatment still exhibited its positive effect on behaviors. Repeated treatment with JZL184 at dose of 3 mg/kg displayed a restored effect on repetitive marble-burying and grooming behaviors, locomotor activity, social preference, learning and spatial memory. In contrast, high dose (10 mg/kg) only partially affected repetitive behaviors, social preference and learning, and low dose tested (1 mg/kg) was instead ineffective. In fact, the behavioral efficacious dose of 3 mg/kg robustly increased 2-AG in both hippocampus and PFC as well, whereas concomitantly with a little increase in AEA levels. In this regard, Dagla−/− animals showed an extensive reduction in 2-AG levels and concomitant decrease in AEA in the hippocampus and cortex, furthermore, administration of JZL184 to Dagla−/− mice increased not only 2-AG levels but also the level of AEA. Schlosburg also confirmed that chronic dosing also caused a modest elevation in AEA. These data, together with our similar results, suggests a crosstalk of 2-AG and AEA production in the brain, however, the underlying mechanism is not known. We should note that cumulative exposure to JZL184 likely generates a partial effect of blockade on FAAH, rather than MAGL contributes directly to the degradation of AEA. Convergent literature demonstrated that sustained elevations in brain 2-AG caused by either genetic deletion or chronic pharmacological blockade of MAGL led to CB1R desensitization and tolerance to CB1R agonists, significant decreases in CB1R number and function, this effect would limit therapeutic potential of JZL184. A chronic JZL184 dose of 16 mg/kg daily (typically 1 week) reliably produced tolerance. The current study observed the dose of 10 mg/kg daily (2 weeks) has induced reduced the CB1R and CB2R expression in the hippocampus. Thus, repeated administration of a low dose JZL184, 3 mg/kg in the current study, could produce elevated eCB brain levels without behavioral tolerance and CB1R desensitization. Additionally, acute treatment with JZL184 also has an important effect on CBRs. We hypothesized that JZL184 treatment could improve the ASD-like behaviors via CBRs-dependent and -independent way: (1) As 2-AG acting via CB1R generally suppresses synaptic transmission, neuronal excitability, and neurogenesis, the improvement of eCB-induced synaptic plasticity could ameliorate ASD-like behaviors. (2) 2-AG is an important metabolic intermediate in lipid synthesis and also serves as a major source of AA, which is required for pro-inflammatory prostaglandin synthesis. Pharmacological inactivation of MAGL induced not only elevations of 2-AG, but reductions in the product AA and downstream AA-derived eicosanoids as well. This impairment of eicosanoids production is a direct consequence of reduction in AA rather than the augmentation of eCB signaling, possibly relevant to cyclooxygenase enzymes. Furthermore, inactivation of MAGL could suppress the pro-inflammatory cytokines production and microglial activation induced by LPS[48, 51]. Eventually, independent of CBRs, increasing 2-AG generates protective function against neuroinflammation, and then ameliorates ASD-like behaviors.