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Abstract
Exposure to stress elicits excitoxicity and neuroinflammation in the brain, contributing to cell death and damage in stress-related neurological and neuropsychiatric diseases. The endocannabinoid system is present in stress-responsive neural circuits and has been proposed as an endogenous neuroprotective system activated in some neuropathological scenarios to restore homeostasis. To elucidate the possible regulatory role of cannabinoid receptor 1 (CB1) in stress-induced excitotoxicity and neuroinflammation, both genetic and pharmacological approaches were used alternatively: (1) wild-type (WT) and CB1 knockout mice (CB1-KO) were exposed to immobilization/acoustic stress (2 h/day for 4 days) and (2) to specifically activate CB1, the selective CB1 agonist Arachidonyl-2′-chloroethylamide (ACEA) (2.5 mg/kg) was intraperitoneally administered daily to some groups of animals. Stress exposure increased CB1 mRNA and protein expression in the prefrontal cortex of WT mice in a mechanism related to N-methyl-d-aspartate glutamate receptor activation. Daily ACEA pretreatment prevented stress-induced: (1) upregulation of CB1 mRNA and protein, (2) decrease in glutamate uptake and glutamate astroglial transporter excitatory amino acid transporter 2 expression, (3) increase in consecutive proinflammatory molecules, such as cytokines (tumor necrosis factor-α and MCP-1), nuclear factor kappa B, and enzymatic sources, such as inducible nitric oxide synthase (NOS-2) and cyclooxygenase-2 (COX-2), (4) increase in lipid peroxidation; although having no effect on plasma corticosterone. Interestingly, a possible related mechanism could be the positive ACEA modulation of the antiinflammatory pathway deoxyprostaglandin/peroxisome proliferator-activated receptor γ (15d-PGJ2/PPARγ). Conversely, KO animal experiments indicated that a lack of CB1 produces hypothalamic/pituitary/adrenal (HPA) axis dysregulation and exacerbates stress-induced excitotoxic/neuroinflammatory responses. These multifaceted neuroprotective effects suggest that CB1 activation could be a new therapeutic strategy against neurological/neuropsychiatric pathologies with HPA axis dysregulation and an excitotoxic/neuroinflammatory component in their pathophysiology.
INTRODUCTION
Uncontrolled excitotoxicity and neuroinflammation contribute to cell death and damage in neurological and neuropsychiatric diseases, including some that are related to stress exposure (neurodegenerative diseases, depression, post-traumatic stress disorder, and schizophrenia) (McLeod et al, 2001; Madrigal et al, 2006). Indeed, previous studies indicate that long-lasting stress affects synaptic plasticity, dendritic morphology, and neurogenesis in animals (reviewed in Kim and Yoon, 1998), and induces both clinical and anatomical features of neurotoxic damage in humans (Bremner et al, 1995). Moreover, the relationship between stressful events and the onset, evolution, and resolution of inflammatory-related diseases (cardiovascular, neurological, digestive, or immunological) is well documented (Baum and Posluszny, 1999; Slimmer et al, 2001). The precise molecular and cellular events induced by stress that are responsible for the brain damage found in these conditions are still a matter of debate. In this way, previous studies using stress models (physical, psychological, or mixed) reported a consistent brain response that is mainly characterized by excitotoxicity and a resulting activation of several consecutive inflammatory mediators (GarcÃa-Bueno et al, 2008a). Briefly, stress exposure elicits excitotoxicity due to the massive release of the excitatory amino acid, glutamate, in some brain areas (eg, the prefrontal cortex) (Moghaddam, 1993), inducing the release of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) or interleukin 1β (Madrigal et al, 2002). Stress also activates the nuclear transcription factor-kappa B (NF-κB) pathway in a TNF-α-dependent mechanism (Bierhaus et al, 2003; Madrigal et al, 2002). NF-κB activation elicits the expression and activity of proinflammatory enzymatic sources, such as inducible nitric oxide (NO) synthase (NOS-2), and cyclooxygenase-2 (COX-2), among others (Madrigal et al, 2001a, 2003). The result of this sequence of events is the accumulation of oxidative and nitrosative mediators, which can attack membrane phospholipids and cause cell damage in a process known as lipid peroxidation (McEwen, 1998; Madrigal et al, 2001b) (see Figure 9 for a schematic view of this stress-activated proinflammatory pathway). However, there remain unresolved issues concerning the mechanisms, regulation, and functional significance of the excitotoxic/inflammatory processes induced by stress in the brain.
In spite of stress-induced production and accumulation of potentially cytotoxic and/or proinflammatory mediators like glutamate, NO, peroxynitrite anion (ONOO−), or prostaglandin E2 (PGE2), various authors have discussed the possibility that some of the many changes caused by stress response effectors are not damaging to the neurons, but in fact predominantly beneficial to their structure and function (McEwen, 1998). One possible mechanism involved as a possible neuroprotective pathway that has received considerable experimental attention is led by some COX-2-derived lipid mediators, such as prostaglandin 15d-PGJ2 and its peroxisome proliferator-activated nuclear receptor γ (PPARγ). This COX-derived sub-pathway is activated in the brain in response to stress and other neuroinflammatory stimuli, constituting a possible endogenous antiinflammatory defense mechanism against excessive excitotoxicity and neuroinflammation (GarcÃa-Bueno et al, 2008a) (see Figure 8 for a schematic view of this stress-activated antiinflammatory pathway).
The study of alternative/related antiinflammatory pathways deserves further attention to understand pathophysiological inflammatory alterations as well as to develop possible new therapeutic approaches for the treatment of stress-related neuropathologies. Currently, the endocannabinoid system (ECS) is considered as an endogenous homeostatic system activated by different immune challenges, restoring brain balance at different levels (Mechoulam and Shohami, 2007) in different experimental settings (Galve-Roperh et al, 2008; Wolf et al, 2008; Bambico et al, 2009; Cabral and Griffin-Thomas, 2009). ECS refers to a group of endogenous arachidonate-based lipids (ie, anandamide and 2-arachidonoylglycerol), widely known as 'endocannabinoids'; their cannabinoid G protein-coupled receptors, namely cannabinoid receptor 1 (CB1) and CB2, and the enzymes fatty acid amide hydrolase and monoacylglycerol lipase that are responsible for their degradation and/or reuptake.
Interestingly, several epidemiological studies have identified that cannabinoids affect anxiety and stress responsivity (Ledent et al, 1999), and the presence of the ECS in stress-responsive neural circuits suggests that it may have a critical role in regulating hypothalamic/pituitary/adrenal (HPA) axis activation and the resulting excitotoxic/neuroinflammatory response. Although it is widely assumed that this response is mainly mediated by the CB2 receptor, which is expressed by a wide range of immune cells (Arévalo-MartÃn et al, 2003), recent evidence suggests that pharmacological enhancement of CB1 protects neurons from acute brain injury, demonstrating a potentially attractive alternative therapeutic strategy against CNS pathologies that deserves further investigation (Cabral and Griffin-Thomas, 2009).
Taking into account all this background, the aim of the present study was to explore the effect of sub-chronic stress exposure on CB1 expression in the prefrontal cortex in mice, and the possible regulatory role of CB1 in conditions of stress-induced excitotoxicity and neuroinflammation, using alternatively pharmacological (activation of CB1 with the selective agonist arachidonyl-2′-chloroethylamide (ACEA)) and genetic (CB1 receptor-deficient mice) strategies.
RESULTS
CB1 Expression in the Prefrontal Cortex of Control and Stressed WT Mice. Glutamate-Related Mechanism
Our first study was aimed to elucidate the possible effect of stress exposure on CB1 expression in the prefrontal cortex in mice. Western blot studies and subsequent densitometric analysis of the band of interest (Figure 1a) revealed a significant increase in CB1 protein expression (≈60 kDa band) elicited by stress. As expected, this band is absent in CB1 knockout (KO) mice in control and stress conditions, validating the specificity of the antibody used. This observed increase in protein expression is in agreement with CB1 mRNA upregulation, quantified by Q-PCR (Figure 1b).
Prestress daily ACEA administration blocked stress-induced CB1 mRNA upregulation (Figure 1b) and protein expression (Figure 1c) in the prefrontal cortex in mice. In control conditions, neither ACEA nor MK treatments were able to modify CB1 protein and mRNA expression (data not shown to simplify Figures 1b and c).
Stress exposure leads to the massive release of glutamate into the synaptic cleft, reaching excitotoxic levels in some brain areas (ie, hippocampus, prefrontal cortex) (Moghaddam, 1993). To elucidate whether stress-induced CB1 upregulation is related to this massive glutamate release, a group of animals was i.p. injected with the NMDA-selective blocker, MK-801, at 1 h before the onset of the stress exposure. Interestingly, daily administration of MK-801 partly inhibited stress-induced CB1 mRNA upregulation (Figure 1b) and protein overexpression (Figure 1c) in the prefrontal cortex of WT mice.
CB1-Dependent Effects on Stress-Induced Changes in Synaptosomal Glutamate Uptake and Expression of Glutamate Transporters
The ECS has been shown to confer neuroprotection inhibiting glutamatergic excitotoxic neurotransmission by mechanism/s still not completely understood. We decided to explore the possible effects of CB1 on glutamate transport in brain cortical synaptosomes from WT and CB1 KO mice submitted to stress.
As it has been previously described (GarcÃa-Bueno et al, 2007), stress exposure leads to a marked decrease in glutamate uptake compared with what was seen in the control group (Figure 2a). Pretreatment with ACEA fully prevented this stress-induced impairment in glutamate uptake function (Figure 2a). Conversely, CB1 KO mice showed dramatically reduced glutamate transport in cortical synaptosomes in both control and stress conditions (Figure 2a).
We also assessed the possibility that these effects might be due to expressional changes in the main brain glutamate transporter, EAAT-2 (astrocytic). Western blot analysis revealed that stress exposure reduced EAAT-2 expression (Figure 2b), and that treatment with ACEA prevented this stress-induced decrease. As in the case of synaptosomal glutamate transport, both stressed and nonstressed CB1 KO mice showed reduced EAAT-2 expression in brain cortical homogenates (Figure 2b).
Antiinflammatory Effects Elicited by CB1 Activation. Mechanisms Involved
In general, ECS has been proposed as an endogenous protective system against excessive inflammatory/immune responses in multiple CNS pathologies. Our following studies were aimed at clarifying the particular role of CB1 as a possible regulator of stress-induced inflammatory response.
Effects on HPA axis activity. Plasma corticosterone levels Our first study evaluated CB1 effects on glucocorticoid synthesis, the main stress hormones, widely known as classical regulators of the inflammatory/immune response in brain and periphery.
The quantification by RIA of plasma corticosterone levels at the time of blood extraction (approximately 1500 h) in all groups studied revealed an expected increase in corticosterone in all groups of stressed animals compared with their respective controls (Figure 3); however, this increase is statistically higher in KO mice than in WT animals (Figure 3). Interestingly, daily ACEA administration did not change plasma corticosterone levels after stress at the time of blood extraction (Figure 3), suggesting that the mechanism(s) of this compound in modulating stress response could be independent of peripheral corticosterone levels.
Brain proinflammatory cytokines and chemokines Proinflammatory cytokines, such as TNF-α or MCP-1, are the first proinflammatory mediators activated in the brain by the massive release of glutamate after stress exposure. Q-PCR studies showed a consistent increase in TNF-α mRNA in WT mouse prefrontal cortex after stress, an effect that is blocked by ACEA (Figure 4a). Conversely, stressed CB1 KO animals presented a reliable increase in the mRNA of the proinflammatory cytokine, suggesting a potentially deleterious chronic inflammatory status in brain. Stress also increased MCP-1 levels in the prefrontal cortex of WT mice and pharmacological stimulation of CB1 decreased them. CB1 KO mice showed no differences from WT in nonstressed animals. However, stress increased MCP-1 levels in brain in SKO group compared with SWT group (Figure 4b).
In this set of neuroinflammation-related experiments (Figures 4, 5, 6 and 7) there were no differences between CWT and CWT + ACEA groups and the data are not shown to simplify figures.
Nuclear factor-κB The release of TNF-α after stress also accounts for NF-κB activation, and we also tested the possible involvement of CB1 in the level of NF-κB expression.
Western blot studies of the inhibitory protein of NF-κB (IκBα) in cytosolic extracts (Figure 5a) and proinflammatory NFκB subunit (p65) in nuclear extracts (Figure 5b) revealed that CB1 regulated NF-κB activation after stress. As it can be observed, stress exposure induced a decrease in the expression of IκBα in brain extracts from WT mice, which was reversed by ACEA (Figure 5a). Levels of nuclear p65 in the different groups studied (Figure 5b) mirrored the expression of its inhibitory factor. CB1 KO animals presented lower levels of IκBα in cytosolic extracts than their respective controls (CWT) (Figure 5a) and a consequent increase in p65 protein in nuclear extracts (Figure 5b), suggesting a state of chronic NF-κB activation in these animals. Interestingly, stress exposure did not affect IκBα/NF-κB expression in the prefrontal cortex of CB1 KO mice.
Proinflammatory enzymes (NOS-2 and COX-2) NF-κB regulates the expression of genes involved in the accumulation of oxidative/nitrosative and inflammatory mediators after stress exposure. Among others, two main sources of these mediators dependent on NF-κB are inducible NOS-2 and COX-2. Their major products (NO and PGE2, respectively) are potent oxidant/proinflammatory molecules that have been widely connected with damage and even cellular death in multiple CNS pathologies. We decided to explore CB1 effects on the expression and/or activity of these enzymes.
NOS-2 expression increased in the cerebral cortex after stress exposure in WT mice (Figure 6a), although no changes in COX-2 expression were found after this stress protocol (Figure 6b). However, the presumed brain COX-2-derived product, PGE2, appeared in an increased quantity after the stress exposure Figure 6c).
CB1 receptor activation with ACEA was able to completely block NOS-2 upregulation produced by stress exposure (Figure 6a). As in the case of other proinflammatory mediators, CB1 KO mice, both in control and stress conditions, showed consistent NOS-2 upregulation compared with their WT control groups, and a higher increase after stress (Figure 6a).
The interaction between CB1 receptor and COX-2 is more complex, as suggested by our results. PGE2 cortical levels, a presumed COX-2 product in different tissues, including the brain, are affected by CB1 activation. As it can be observed in Figure 6c, ACEA reduced PGE2 tissue levels compared with those in the SWT animals, whereas CB1 SKO animals presented insignificant differences in PGE2 content compared with the SWT group.
Lipid peroxidation As a final index of stress-induced cellular damage that could be affected by CB1-selective modulation, we measured the accumulation of the lipid peroxidation marker MDA in the brain prefrontal cortex of the different groups of mice. Daily treatment with ACEA prevented MDA overaccumulation, observed after stress exposure in WT mice (Figure 6d). Conversely, CB1 KO mice presented higher levels of MDA after stress than the SWT group (Figure 6d).
CB1-Dependent Modulation of the Antiinflammatory Pathway L-PGDS/15d-PGJ2/PPARγ
So far, our results had shown that the daily administration of the selective CB1 receptor agonist ACEA prevented stress-induced excitotoxicity and proinflammatory mediator accumulation in the prefrontal cortex of WT mice, whereas CB1 KO mice presented an aggravated excitotoxic/neuroinflammatory process after stress exposure. Our final series of studies were aimed to elucidate possible alternative antiinflammatory mechanisms related to CB1 activation. One candidate of interest is the antiinflammatory pathway L-PGDS/15d-PGJ2/PPARγ, that is activated after inflammatory/immune stimuli (stress included) and whose activation (even by some cannabinoids) has been shown to confer neuroprotection after stress exposure, and in diverse CNS pathologies with an inflammatory component in their pathophysiology.
Western blot studies and subsequent densitometric analysis (Figure 7a) revealed a significant increase in the 15d-PGJ2 enzymatic source L-PGDS in the prefrontal cortex homogenates of mouse brains, daily treated with ACEA, compared with the SWT group. On the other hand, L-PGDS protein levels were reduced in the brain of CB1 KO mice in control conditions and after stress exposure (Figure 7a).
Consequently, 15d-PGJ2 levels, quantified by ELISA in brain prefrontal cortex samples, were increased by the administration of ACEA in WT mice compared with their controls (Figure 7b). Both in WT and CB1 KO mice, stress exposure produced a marked decrease in 15d-PGJ2 levels but there were no differences between WT and KO animals.
Western blot analysis of PPARγ protein revealed a significant increase of this 15d-PGJ2 nuclear receptor in prefrontal cortex homogenates of mouse brains, daily treated with ACEA, compared with their controls after stress exposure (SWT) (Figure 7c). CKO mice presented significant lower levels of PPARγ than the CWT group, but stress exposure did not affect PPARγ expression in the brains of CB1 KO animals (Figure 7c).
Validation of ACEA CB1-Dependent Antiexcitotoxic/Antiinflammatory Effects
Finally, to elucidate whether ACEA actions are CB1 mediated, we examined its effects in CB1 KO mice on some representative excitotoxic (EAAT-2 expression) and inflammatory parameters (NOS-2 expression and MDA levels) after sub-chronic stress exposure.
As has been previously shown (Figure 2), stress exposure downregulates EAAT-2 expression in mouse prefrontal cortex and this effect is completely prevented by ACEA pretreatment in WT mice, however this ACEA antiexcitotoxic effect is absent in CB1 KO mice (SKO + ACEA) (Figure 8a), suggesting the direct involvement of CB1 receptor. A similar pattern occurs with the proinflammatory parameter NOS-2 expression (Figure 8b) and MDA levels (Figure 8c), in which ACEA neuroprotective effects are lost in CB1 KO mice compared with WT. These results suggest that ACEA antiinflammatory effects are also dependent on CB1 in our model.
DISCUSSION
Our studies show for the first time to our knowledge, CB1 upregulation in mouse prefrontal cortex exposed to sub-chronic restraint/acoustic stress in a mechanism dependent on NMDA glutamate receptors. In addition, daily ACEA administration prevented stress-induced upregulation of CB1 mRNA and protein, and presented an antiexcitotoxic profile, regulating glutamate transport and uptake after stress exposure, at least in part by restoring protein expression of EAAT-2. CB1 activation also produces antiinflammatory effects, regulating stress-induced proinflammatory response at multiple levels. Notably, a possible related mechanism is the positive modulation of the antiinflammatory pathway L-PGDS/15d-PGJ2/PPARγ, although the precise mechanism/s responsible for this relationship needs to be further investigated (see Figure 9 for a schematic view of the regulatory role of CB1 in stress-induced excitotoxicity and neuroinflammatory balance).
Stress exposure is a major contributing factor to cell death and damage in neurological and neuropsychiatric diseases by three interrelated mechanisms: HPA axis dysregulation, excitotoxicity, and neuroinflammation. However, stress exposure also activates alternative mechanisms aimed at correctly resolving this response. In this way, the CB1 upregulation demonstrated here could be a mechanism related to this protective response of the brain. Similarly, endocannabinoids are released to negatively regulate HPA axis activation (Manzanares et al, 1999; Patel et al, 2004), and brain SEC activation has been considered as a protective mechanism aimed at reducing both neurodegenerative and inflammatory damage after an immune/inflammatory challenge (Centonze et al, 2008).
In particular, our data are in agreement with several studies that propose a role for CB1 as a main regulator of glutamate neurotransmission and metabolism in multiple pathological scenarios (Parmentier-Batteur et al, 2002; Marsicano et al, 2003; Veldhuis et al, 2003; Fernández-López et al, 2006; Kim et al, 2006; Gilbert et al, 2007; LorÃa et al, 2010). In relation to our findings, stress-induced CB1 upregulation could represent a compensatory response aimed at controlling the massive release of glutamate into the synaptic cleft that might produce cellular damage and even death by excitotoxicity after stress exposure (Lowy et al, 1993; Stein-Behrens et al, 1994; GarcÃa-Bueno et al, 2007). As reported here, CB1 receptor activation with ACEA enhances the main glutamate uptake mechanism, by restoring protein expression of the glutamate transporter EAAT-2. Astrocyte uptake of glutamate is a high-affinity process regulated by EAAT-2 (Hertz et al, 1983); the fact that stress decreases EAAT-2 expression in the cerebral cortex (GarcÃa-Bueno et al, 2007), indicates a specific damaging effect at this level, and makes the effect of ACEA more interesting. CB1 effects on EAAT-2 have also been demonstrated in other experimental settings, such as an in vitro model of multiple sclerosis (MS) (LorÃa et al, 2010), or after prenatal exposure to WIN 55,212-2 (Castaldo et al, 2007). It has been widely demonstrated that presynaptic CB1 inhibits glutamate release (Freund et al, 2003; Chevaleyre et al, 2006; Steiner and Wotjak, 2008), but our data suggests that glutamatergic neurotransmission is also regulated by CB1 at the level of postsynaptic reuptake clearance mechanisms.
How neuronal CB1 activation enhances glutamate glial uptake may be explained by neuron—actrocyte communication that regulates PPARγ activity in astrocytes. Notably, EAAT-2 glutamate transporter is a target gene of PPARγ in excitotoxic-related models, such as stress exposure (GarcÃa-Bueno et al, 2007) or brain ischemia (Romera et al, 2007). As well, a recent study identified endocannabinoids as PPARγ agonists affording neuroprotection targeting EAAT-2 in an in vitro model of MS (LorÃa et al, 2010).
Regarding stress-induced neuroinflammation, although cannabinoid-dependent antiinflammatory and antioxidant actions have been commonly related with CB2 receptor activation in brain immune cells (Cabral and Griffin-Thomas, 2009; Ashton and Glass, 2007; Arévalo-MartÃn et al, 2008), or even with receptor-independent mechanisms (Hampson et al, 2000; Marsicano et al, 2002)), our and other studies revealed a significant role for CB1 as well in inflammatory/immune responses in animal models of MS (Arévalo-MartÃn et al, 2003; Pryce et al, 2003; Maresz et al, 2007) and brain ischemia (Fernández-López et al, 2006).
Here, we demonstrated that CB1 activation interacted with the proinflammatory response elicited by stress at multiple levels. In the case of cytokines, CB1 activation has been shown to reduce TNFα and IL-1β synthesis in neurons, astrocytes, and microglia (Fernández-López et al, 2006; Maresz et al, 2007; Marchalant et al, 2009). Notably, the opposite relationship, in which cytokines presumably upregulate cannabinoid receptor expression, has also been suggested in some immune/inflammatory scenarios (Jean-Gilles et al, 2010), stress included (Lee et al, 2006).
Regarding other stress-induced proinflammatory mediators, like the key transcription factor NF-κB, our results coincide with others showing a role for CB1 in NF-κB regulation in injured mouse brains (Panikashvili et al, 2005); however regulation of NF-κB by cannabinoids has been commonly related with CB2 in some immune cells such as macrophages or monocytes (Jeon et al, 1996; Rajesh et al, 2008); and even with CB1/CB2 receptor-independent mechanisms, as is the case of cannabidiol in LPS-activated microglia (Kozela et al, 2010) or 2-arachidonyl glycerol through PPARγ (Rockwell et al, 2006).
Two main NF-κB-dependent enzymatic sources of oxidative/nitrosative mediators after stress in the brain are NOS-2 and COX-2. NOS-2 expression is downregulated by selective CB1 activation in neurons and astrocytes both in in vitro and in vivo inflammatory-related models (Fernández-López et al, 2006; Esposito et al, 2006; Sheng et al, 2005), as it is also reported in our study. However, cannabinoid—cyclooxygenase interactions may be more intricate. In our stress model, CB1 regulates the levels of the presumed COX-2 product PGE2 without altering enzyme expression. These results suggest possible effects of ACEA on the catalytic activity or protein stability of COX-2, or a possible effect on the activity of tissue-specific prostaglandin E2 synthases, which is yet to be explored. Some authors have reported opposite COX-2/CB1 interactions: protection afforded by 2-arachidonoylglycerol and THC by downregulation of COX-2 expression and activity in cultured neurons, and LPS stimulated J774 macrophages (Zhang and Chen, 2008; Chang et al, 2001), or THC neurotoxicity in hippocampal neurons in a mechanism attributable to COX-2 activity and the concomitant generation of free radicals (Chan et al, 1998).
Our results show expression changes in the antiinflammatory pathway L-PGDS/15d-PGJ2/PPARγ after CB1 pharmacological and genetic manipulations; although still speculative, the involvement of this antiinflammatory pathway in CB1 neuroprotective effects is an interesting idea that deserves further investigation. This potential relationship is especially relevant as some pharmacological and genetic studies using CB1/CB2 KO mice indicate possible CB1/CB2 alternative mechanisms of action. PPARs could be excellent candidates, taking into account that diverse cannabinoids are potent ligands of PPARs (O'Sullivan, 2007). Thus, the main endocannabinoids, anandamide, and 2-arachidonoylglycerol, and also ajulemic acid, a THC structural analog, have antiinflammatory properties mediated by PPARγ (Sun and Bennett, 2007). In addition, the endogenous acylethanolamines, oleoylethanolamide, and palmitoylethanolamide have neuroprotective effects mediated through PPARα. The possibility of these related acylethanolamines modulating the stress response through interaction with PPARα remains unexplored.
Our results show a significant upregulation of L-PGDS/15d-PGJ2 after ACEA administration. This upregulation has been also demonstrated after the administration of ajulemic acid to human synovial cells (Stebulis et al, 2008), inducing an antiinflammatory profile of eicosanoids. PPARγ regulation is not yet completely understood, but one of the main mechanisms may be ligand availability (Daynes and Jones, 2002). This kind of regulation could be important in our stress model because we also found increased 15d-PGJ2 levels after ACEA administration. Other authors have proposed alternative mechanisms by which cannabinoids can potentially lead to PPARs activation. For example, CB1 binding could stimulate MAPK pathways, as it occurs in other scenarios (Lépicier et al, 2006), which regulates PPARγ transcriptional activity by direct phosphorylation on serine residues (Burns and Vanden Heuvel, 2007).
In contrast to ACEA neuroprotective effects in stress conditions, we and others have showed a dysregulated HPA axis activity in basal and after stress exposure in CB1 KO mice (Barna et al, 2004; Cota, 2008; Aso et al, 2008). In addition, a status of excessive excitotoxicity and neuroinflammation is also presented, increasing their susceptibility to experience damage and even cellular death. Similarly, in an animal model of Parkinson's disease, other authors have also found an increased vulnerability to 6-hydroxydopamine lesion in CB1 KO mice, presenting a higher index of lipid peroxidation and NOS-2 expression in selected brain areas (Pérez-Rial et al, 2009).
In addition to HPA axis dysregulation, chronic excitotoxicity, and neuroinflammation, CB1 KO mice present defective adult neurogenesis (Jin et al, 2004), increased anxiety (Urigüen et al, 2004; Mikics et al, 2009), and deficiency in neuronal plasticity by decreasing BDNF levels in the hippocampus (Aso et al, 2008), suggesting a possible role of CB1 in the pathophysiology of depression and its pharmacological manipulation as a potential therapeutic strategy.
In summary, the multifaceted neuroprotective effects described here suggest that CB1 activation is an attractive therapeutic strategy against diverse neuropsychiatric pathologies with HPA axis dysregulation and an excitotoxic/neuroinflammatory component in their pathophysiology.
Source, Graphs and Figures: Neuropsychopharmacology - Regulatory Role of Cannabinoid Receptor 1 in Stress-Induced Excitotoxicity and Neuroinflammation
Exposure to stress elicits excitoxicity and neuroinflammation in the brain, contributing to cell death and damage in stress-related neurological and neuropsychiatric diseases. The endocannabinoid system is present in stress-responsive neural circuits and has been proposed as an endogenous neuroprotective system activated in some neuropathological scenarios to restore homeostasis. To elucidate the possible regulatory role of cannabinoid receptor 1 (CB1) in stress-induced excitotoxicity and neuroinflammation, both genetic and pharmacological approaches were used alternatively: (1) wild-type (WT) and CB1 knockout mice (CB1-KO) were exposed to immobilization/acoustic stress (2 h/day for 4 days) and (2) to specifically activate CB1, the selective CB1 agonist Arachidonyl-2′-chloroethylamide (ACEA) (2.5 mg/kg) was intraperitoneally administered daily to some groups of animals. Stress exposure increased CB1 mRNA and protein expression in the prefrontal cortex of WT mice in a mechanism related to N-methyl-d-aspartate glutamate receptor activation. Daily ACEA pretreatment prevented stress-induced: (1) upregulation of CB1 mRNA and protein, (2) decrease in glutamate uptake and glutamate astroglial transporter excitatory amino acid transporter 2 expression, (3) increase in consecutive proinflammatory molecules, such as cytokines (tumor necrosis factor-α and MCP-1), nuclear factor kappa B, and enzymatic sources, such as inducible nitric oxide synthase (NOS-2) and cyclooxygenase-2 (COX-2), (4) increase in lipid peroxidation; although having no effect on plasma corticosterone. Interestingly, a possible related mechanism could be the positive ACEA modulation of the antiinflammatory pathway deoxyprostaglandin/peroxisome proliferator-activated receptor γ (15d-PGJ2/PPARγ). Conversely, KO animal experiments indicated that a lack of CB1 produces hypothalamic/pituitary/adrenal (HPA) axis dysregulation and exacerbates stress-induced excitotoxic/neuroinflammatory responses. These multifaceted neuroprotective effects suggest that CB1 activation could be a new therapeutic strategy against neurological/neuropsychiatric pathologies with HPA axis dysregulation and an excitotoxic/neuroinflammatory component in their pathophysiology.
INTRODUCTION
Uncontrolled excitotoxicity and neuroinflammation contribute to cell death and damage in neurological and neuropsychiatric diseases, including some that are related to stress exposure (neurodegenerative diseases, depression, post-traumatic stress disorder, and schizophrenia) (McLeod et al, 2001; Madrigal et al, 2006). Indeed, previous studies indicate that long-lasting stress affects synaptic plasticity, dendritic morphology, and neurogenesis in animals (reviewed in Kim and Yoon, 1998), and induces both clinical and anatomical features of neurotoxic damage in humans (Bremner et al, 1995). Moreover, the relationship between stressful events and the onset, evolution, and resolution of inflammatory-related diseases (cardiovascular, neurological, digestive, or immunological) is well documented (Baum and Posluszny, 1999; Slimmer et al, 2001). The precise molecular and cellular events induced by stress that are responsible for the brain damage found in these conditions are still a matter of debate. In this way, previous studies using stress models (physical, psychological, or mixed) reported a consistent brain response that is mainly characterized by excitotoxicity and a resulting activation of several consecutive inflammatory mediators (GarcÃa-Bueno et al, 2008a). Briefly, stress exposure elicits excitotoxicity due to the massive release of the excitatory amino acid, glutamate, in some brain areas (eg, the prefrontal cortex) (Moghaddam, 1993), inducing the release of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) or interleukin 1β (Madrigal et al, 2002). Stress also activates the nuclear transcription factor-kappa B (NF-κB) pathway in a TNF-α-dependent mechanism (Bierhaus et al, 2003; Madrigal et al, 2002). NF-κB activation elicits the expression and activity of proinflammatory enzymatic sources, such as inducible nitric oxide (NO) synthase (NOS-2), and cyclooxygenase-2 (COX-2), among others (Madrigal et al, 2001a, 2003). The result of this sequence of events is the accumulation of oxidative and nitrosative mediators, which can attack membrane phospholipids and cause cell damage in a process known as lipid peroxidation (McEwen, 1998; Madrigal et al, 2001b) (see Figure 9 for a schematic view of this stress-activated proinflammatory pathway). However, there remain unresolved issues concerning the mechanisms, regulation, and functional significance of the excitotoxic/inflammatory processes induced by stress in the brain.
In spite of stress-induced production and accumulation of potentially cytotoxic and/or proinflammatory mediators like glutamate, NO, peroxynitrite anion (ONOO−), or prostaglandin E2 (PGE2), various authors have discussed the possibility that some of the many changes caused by stress response effectors are not damaging to the neurons, but in fact predominantly beneficial to their structure and function (McEwen, 1998). One possible mechanism involved as a possible neuroprotective pathway that has received considerable experimental attention is led by some COX-2-derived lipid mediators, such as prostaglandin 15d-PGJ2 and its peroxisome proliferator-activated nuclear receptor γ (PPARγ). This COX-derived sub-pathway is activated in the brain in response to stress and other neuroinflammatory stimuli, constituting a possible endogenous antiinflammatory defense mechanism against excessive excitotoxicity and neuroinflammation (GarcÃa-Bueno et al, 2008a) (see Figure 8 for a schematic view of this stress-activated antiinflammatory pathway).
The study of alternative/related antiinflammatory pathways deserves further attention to understand pathophysiological inflammatory alterations as well as to develop possible new therapeutic approaches for the treatment of stress-related neuropathologies. Currently, the endocannabinoid system (ECS) is considered as an endogenous homeostatic system activated by different immune challenges, restoring brain balance at different levels (Mechoulam and Shohami, 2007) in different experimental settings (Galve-Roperh et al, 2008; Wolf et al, 2008; Bambico et al, 2009; Cabral and Griffin-Thomas, 2009). ECS refers to a group of endogenous arachidonate-based lipids (ie, anandamide and 2-arachidonoylglycerol), widely known as 'endocannabinoids'; their cannabinoid G protein-coupled receptors, namely cannabinoid receptor 1 (CB1) and CB2, and the enzymes fatty acid amide hydrolase and monoacylglycerol lipase that are responsible for their degradation and/or reuptake.
Interestingly, several epidemiological studies have identified that cannabinoids affect anxiety and stress responsivity (Ledent et al, 1999), and the presence of the ECS in stress-responsive neural circuits suggests that it may have a critical role in regulating hypothalamic/pituitary/adrenal (HPA) axis activation and the resulting excitotoxic/neuroinflammatory response. Although it is widely assumed that this response is mainly mediated by the CB2 receptor, which is expressed by a wide range of immune cells (Arévalo-MartÃn et al, 2003), recent evidence suggests that pharmacological enhancement of CB1 protects neurons from acute brain injury, demonstrating a potentially attractive alternative therapeutic strategy against CNS pathologies that deserves further investigation (Cabral and Griffin-Thomas, 2009).
Taking into account all this background, the aim of the present study was to explore the effect of sub-chronic stress exposure on CB1 expression in the prefrontal cortex in mice, and the possible regulatory role of CB1 in conditions of stress-induced excitotoxicity and neuroinflammation, using alternatively pharmacological (activation of CB1 with the selective agonist arachidonyl-2′-chloroethylamide (ACEA)) and genetic (CB1 receptor-deficient mice) strategies.
RESULTS
CB1 Expression in the Prefrontal Cortex of Control and Stressed WT Mice. Glutamate-Related Mechanism
Our first study was aimed to elucidate the possible effect of stress exposure on CB1 expression in the prefrontal cortex in mice. Western blot studies and subsequent densitometric analysis of the band of interest (Figure 1a) revealed a significant increase in CB1 protein expression (≈60 kDa band) elicited by stress. As expected, this band is absent in CB1 knockout (KO) mice in control and stress conditions, validating the specificity of the antibody used. This observed increase in protein expression is in agreement with CB1 mRNA upregulation, quantified by Q-PCR (Figure 1b).
Prestress daily ACEA administration blocked stress-induced CB1 mRNA upregulation (Figure 1b) and protein expression (Figure 1c) in the prefrontal cortex in mice. In control conditions, neither ACEA nor MK treatments were able to modify CB1 protein and mRNA expression (data not shown to simplify Figures 1b and c).
Stress exposure leads to the massive release of glutamate into the synaptic cleft, reaching excitotoxic levels in some brain areas (ie, hippocampus, prefrontal cortex) (Moghaddam, 1993). To elucidate whether stress-induced CB1 upregulation is related to this massive glutamate release, a group of animals was i.p. injected with the NMDA-selective blocker, MK-801, at 1 h before the onset of the stress exposure. Interestingly, daily administration of MK-801 partly inhibited stress-induced CB1 mRNA upregulation (Figure 1b) and protein overexpression (Figure 1c) in the prefrontal cortex of WT mice.
CB1-Dependent Effects on Stress-Induced Changes in Synaptosomal Glutamate Uptake and Expression of Glutamate Transporters
The ECS has been shown to confer neuroprotection inhibiting glutamatergic excitotoxic neurotransmission by mechanism/s still not completely understood. We decided to explore the possible effects of CB1 on glutamate transport in brain cortical synaptosomes from WT and CB1 KO mice submitted to stress.
As it has been previously described (GarcÃa-Bueno et al, 2007), stress exposure leads to a marked decrease in glutamate uptake compared with what was seen in the control group (Figure 2a). Pretreatment with ACEA fully prevented this stress-induced impairment in glutamate uptake function (Figure 2a). Conversely, CB1 KO mice showed dramatically reduced glutamate transport in cortical synaptosomes in both control and stress conditions (Figure 2a).
We also assessed the possibility that these effects might be due to expressional changes in the main brain glutamate transporter, EAAT-2 (astrocytic). Western blot analysis revealed that stress exposure reduced EAAT-2 expression (Figure 2b), and that treatment with ACEA prevented this stress-induced decrease. As in the case of synaptosomal glutamate transport, both stressed and nonstressed CB1 KO mice showed reduced EAAT-2 expression in brain cortical homogenates (Figure 2b).
Antiinflammatory Effects Elicited by CB1 Activation. Mechanisms Involved
In general, ECS has been proposed as an endogenous protective system against excessive inflammatory/immune responses in multiple CNS pathologies. Our following studies were aimed at clarifying the particular role of CB1 as a possible regulator of stress-induced inflammatory response.
Effects on HPA axis activity. Plasma corticosterone levels Our first study evaluated CB1 effects on glucocorticoid synthesis, the main stress hormones, widely known as classical regulators of the inflammatory/immune response in brain and periphery.
The quantification by RIA of plasma corticosterone levels at the time of blood extraction (approximately 1500 h) in all groups studied revealed an expected increase in corticosterone in all groups of stressed animals compared with their respective controls (Figure 3); however, this increase is statistically higher in KO mice than in WT animals (Figure 3). Interestingly, daily ACEA administration did not change plasma corticosterone levels after stress at the time of blood extraction (Figure 3), suggesting that the mechanism(s) of this compound in modulating stress response could be independent of peripheral corticosterone levels.
Brain proinflammatory cytokines and chemokines Proinflammatory cytokines, such as TNF-α or MCP-1, are the first proinflammatory mediators activated in the brain by the massive release of glutamate after stress exposure. Q-PCR studies showed a consistent increase in TNF-α mRNA in WT mouse prefrontal cortex after stress, an effect that is blocked by ACEA (Figure 4a). Conversely, stressed CB1 KO animals presented a reliable increase in the mRNA of the proinflammatory cytokine, suggesting a potentially deleterious chronic inflammatory status in brain. Stress also increased MCP-1 levels in the prefrontal cortex of WT mice and pharmacological stimulation of CB1 decreased them. CB1 KO mice showed no differences from WT in nonstressed animals. However, stress increased MCP-1 levels in brain in SKO group compared with SWT group (Figure 4b).
In this set of neuroinflammation-related experiments (Figures 4, 5, 6 and 7) there were no differences between CWT and CWT + ACEA groups and the data are not shown to simplify figures.
Nuclear factor-κB The release of TNF-α after stress also accounts for NF-κB activation, and we also tested the possible involvement of CB1 in the level of NF-κB expression.
Western blot studies of the inhibitory protein of NF-κB (IκBα) in cytosolic extracts (Figure 5a) and proinflammatory NFκB subunit (p65) in nuclear extracts (Figure 5b) revealed that CB1 regulated NF-κB activation after stress. As it can be observed, stress exposure induced a decrease in the expression of IκBα in brain extracts from WT mice, which was reversed by ACEA (Figure 5a). Levels of nuclear p65 in the different groups studied (Figure 5b) mirrored the expression of its inhibitory factor. CB1 KO animals presented lower levels of IκBα in cytosolic extracts than their respective controls (CWT) (Figure 5a) and a consequent increase in p65 protein in nuclear extracts (Figure 5b), suggesting a state of chronic NF-κB activation in these animals. Interestingly, stress exposure did not affect IκBα/NF-κB expression in the prefrontal cortex of CB1 KO mice.
Proinflammatory enzymes (NOS-2 and COX-2) NF-κB regulates the expression of genes involved in the accumulation of oxidative/nitrosative and inflammatory mediators after stress exposure. Among others, two main sources of these mediators dependent on NF-κB are inducible NOS-2 and COX-2. Their major products (NO and PGE2, respectively) are potent oxidant/proinflammatory molecules that have been widely connected with damage and even cellular death in multiple CNS pathologies. We decided to explore CB1 effects on the expression and/or activity of these enzymes.
NOS-2 expression increased in the cerebral cortex after stress exposure in WT mice (Figure 6a), although no changes in COX-2 expression were found after this stress protocol (Figure 6b). However, the presumed brain COX-2-derived product, PGE2, appeared in an increased quantity after the stress exposure Figure 6c).
CB1 receptor activation with ACEA was able to completely block NOS-2 upregulation produced by stress exposure (Figure 6a). As in the case of other proinflammatory mediators, CB1 KO mice, both in control and stress conditions, showed consistent NOS-2 upregulation compared with their WT control groups, and a higher increase after stress (Figure 6a).
The interaction between CB1 receptor and COX-2 is more complex, as suggested by our results. PGE2 cortical levels, a presumed COX-2 product in different tissues, including the brain, are affected by CB1 activation. As it can be observed in Figure 6c, ACEA reduced PGE2 tissue levels compared with those in the SWT animals, whereas CB1 SKO animals presented insignificant differences in PGE2 content compared with the SWT group.
Lipid peroxidation As a final index of stress-induced cellular damage that could be affected by CB1-selective modulation, we measured the accumulation of the lipid peroxidation marker MDA in the brain prefrontal cortex of the different groups of mice. Daily treatment with ACEA prevented MDA overaccumulation, observed after stress exposure in WT mice (Figure 6d). Conversely, CB1 KO mice presented higher levels of MDA after stress than the SWT group (Figure 6d).
CB1-Dependent Modulation of the Antiinflammatory Pathway L-PGDS/15d-PGJ2/PPARγ
So far, our results had shown that the daily administration of the selective CB1 receptor agonist ACEA prevented stress-induced excitotoxicity and proinflammatory mediator accumulation in the prefrontal cortex of WT mice, whereas CB1 KO mice presented an aggravated excitotoxic/neuroinflammatory process after stress exposure. Our final series of studies were aimed to elucidate possible alternative antiinflammatory mechanisms related to CB1 activation. One candidate of interest is the antiinflammatory pathway L-PGDS/15d-PGJ2/PPARγ, that is activated after inflammatory/immune stimuli (stress included) and whose activation (even by some cannabinoids) has been shown to confer neuroprotection after stress exposure, and in diverse CNS pathologies with an inflammatory component in their pathophysiology.
Western blot studies and subsequent densitometric analysis (Figure 7a) revealed a significant increase in the 15d-PGJ2 enzymatic source L-PGDS in the prefrontal cortex homogenates of mouse brains, daily treated with ACEA, compared with the SWT group. On the other hand, L-PGDS protein levels were reduced in the brain of CB1 KO mice in control conditions and after stress exposure (Figure 7a).
Consequently, 15d-PGJ2 levels, quantified by ELISA in brain prefrontal cortex samples, were increased by the administration of ACEA in WT mice compared with their controls (Figure 7b). Both in WT and CB1 KO mice, stress exposure produced a marked decrease in 15d-PGJ2 levels but there were no differences between WT and KO animals.
Western blot analysis of PPARγ protein revealed a significant increase of this 15d-PGJ2 nuclear receptor in prefrontal cortex homogenates of mouse brains, daily treated with ACEA, compared with their controls after stress exposure (SWT) (Figure 7c). CKO mice presented significant lower levels of PPARγ than the CWT group, but stress exposure did not affect PPARγ expression in the brains of CB1 KO animals (Figure 7c).
Validation of ACEA CB1-Dependent Antiexcitotoxic/Antiinflammatory Effects
Finally, to elucidate whether ACEA actions are CB1 mediated, we examined its effects in CB1 KO mice on some representative excitotoxic (EAAT-2 expression) and inflammatory parameters (NOS-2 expression and MDA levels) after sub-chronic stress exposure.
As has been previously shown (Figure 2), stress exposure downregulates EAAT-2 expression in mouse prefrontal cortex and this effect is completely prevented by ACEA pretreatment in WT mice, however this ACEA antiexcitotoxic effect is absent in CB1 KO mice (SKO + ACEA) (Figure 8a), suggesting the direct involvement of CB1 receptor. A similar pattern occurs with the proinflammatory parameter NOS-2 expression (Figure 8b) and MDA levels (Figure 8c), in which ACEA neuroprotective effects are lost in CB1 KO mice compared with WT. These results suggest that ACEA antiinflammatory effects are also dependent on CB1 in our model.
DISCUSSION
Our studies show for the first time to our knowledge, CB1 upregulation in mouse prefrontal cortex exposed to sub-chronic restraint/acoustic stress in a mechanism dependent on NMDA glutamate receptors. In addition, daily ACEA administration prevented stress-induced upregulation of CB1 mRNA and protein, and presented an antiexcitotoxic profile, regulating glutamate transport and uptake after stress exposure, at least in part by restoring protein expression of EAAT-2. CB1 activation also produces antiinflammatory effects, regulating stress-induced proinflammatory response at multiple levels. Notably, a possible related mechanism is the positive modulation of the antiinflammatory pathway L-PGDS/15d-PGJ2/PPARγ, although the precise mechanism/s responsible for this relationship needs to be further investigated (see Figure 9 for a schematic view of the regulatory role of CB1 in stress-induced excitotoxicity and neuroinflammatory balance).
Stress exposure is a major contributing factor to cell death and damage in neurological and neuropsychiatric diseases by three interrelated mechanisms: HPA axis dysregulation, excitotoxicity, and neuroinflammation. However, stress exposure also activates alternative mechanisms aimed at correctly resolving this response. In this way, the CB1 upregulation demonstrated here could be a mechanism related to this protective response of the brain. Similarly, endocannabinoids are released to negatively regulate HPA axis activation (Manzanares et al, 1999; Patel et al, 2004), and brain SEC activation has been considered as a protective mechanism aimed at reducing both neurodegenerative and inflammatory damage after an immune/inflammatory challenge (Centonze et al, 2008).
In particular, our data are in agreement with several studies that propose a role for CB1 as a main regulator of glutamate neurotransmission and metabolism in multiple pathological scenarios (Parmentier-Batteur et al, 2002; Marsicano et al, 2003; Veldhuis et al, 2003; Fernández-López et al, 2006; Kim et al, 2006; Gilbert et al, 2007; LorÃa et al, 2010). In relation to our findings, stress-induced CB1 upregulation could represent a compensatory response aimed at controlling the massive release of glutamate into the synaptic cleft that might produce cellular damage and even death by excitotoxicity after stress exposure (Lowy et al, 1993; Stein-Behrens et al, 1994; GarcÃa-Bueno et al, 2007). As reported here, CB1 receptor activation with ACEA enhances the main glutamate uptake mechanism, by restoring protein expression of the glutamate transporter EAAT-2. Astrocyte uptake of glutamate is a high-affinity process regulated by EAAT-2 (Hertz et al, 1983); the fact that stress decreases EAAT-2 expression in the cerebral cortex (GarcÃa-Bueno et al, 2007), indicates a specific damaging effect at this level, and makes the effect of ACEA more interesting. CB1 effects on EAAT-2 have also been demonstrated in other experimental settings, such as an in vitro model of multiple sclerosis (MS) (LorÃa et al, 2010), or after prenatal exposure to WIN 55,212-2 (Castaldo et al, 2007). It has been widely demonstrated that presynaptic CB1 inhibits glutamate release (Freund et al, 2003; Chevaleyre et al, 2006; Steiner and Wotjak, 2008), but our data suggests that glutamatergic neurotransmission is also regulated by CB1 at the level of postsynaptic reuptake clearance mechanisms.
How neuronal CB1 activation enhances glutamate glial uptake may be explained by neuron—actrocyte communication that regulates PPARγ activity in astrocytes. Notably, EAAT-2 glutamate transporter is a target gene of PPARγ in excitotoxic-related models, such as stress exposure (GarcÃa-Bueno et al, 2007) or brain ischemia (Romera et al, 2007). As well, a recent study identified endocannabinoids as PPARγ agonists affording neuroprotection targeting EAAT-2 in an in vitro model of MS (LorÃa et al, 2010).
Regarding stress-induced neuroinflammation, although cannabinoid-dependent antiinflammatory and antioxidant actions have been commonly related with CB2 receptor activation in brain immune cells (Cabral and Griffin-Thomas, 2009; Ashton and Glass, 2007; Arévalo-MartÃn et al, 2008), or even with receptor-independent mechanisms (Hampson et al, 2000; Marsicano et al, 2002)), our and other studies revealed a significant role for CB1 as well in inflammatory/immune responses in animal models of MS (Arévalo-MartÃn et al, 2003; Pryce et al, 2003; Maresz et al, 2007) and brain ischemia (Fernández-López et al, 2006).
Here, we demonstrated that CB1 activation interacted with the proinflammatory response elicited by stress at multiple levels. In the case of cytokines, CB1 activation has been shown to reduce TNFα and IL-1β synthesis in neurons, astrocytes, and microglia (Fernández-López et al, 2006; Maresz et al, 2007; Marchalant et al, 2009). Notably, the opposite relationship, in which cytokines presumably upregulate cannabinoid receptor expression, has also been suggested in some immune/inflammatory scenarios (Jean-Gilles et al, 2010), stress included (Lee et al, 2006).
Regarding other stress-induced proinflammatory mediators, like the key transcription factor NF-κB, our results coincide with others showing a role for CB1 in NF-κB regulation in injured mouse brains (Panikashvili et al, 2005); however regulation of NF-κB by cannabinoids has been commonly related with CB2 in some immune cells such as macrophages or monocytes (Jeon et al, 1996; Rajesh et al, 2008); and even with CB1/CB2 receptor-independent mechanisms, as is the case of cannabidiol in LPS-activated microglia (Kozela et al, 2010) or 2-arachidonyl glycerol through PPARγ (Rockwell et al, 2006).
Two main NF-κB-dependent enzymatic sources of oxidative/nitrosative mediators after stress in the brain are NOS-2 and COX-2. NOS-2 expression is downregulated by selective CB1 activation in neurons and astrocytes both in in vitro and in vivo inflammatory-related models (Fernández-López et al, 2006; Esposito et al, 2006; Sheng et al, 2005), as it is also reported in our study. However, cannabinoid—cyclooxygenase interactions may be more intricate. In our stress model, CB1 regulates the levels of the presumed COX-2 product PGE2 without altering enzyme expression. These results suggest possible effects of ACEA on the catalytic activity or protein stability of COX-2, or a possible effect on the activity of tissue-specific prostaglandin E2 synthases, which is yet to be explored. Some authors have reported opposite COX-2/CB1 interactions: protection afforded by 2-arachidonoylglycerol and THC by downregulation of COX-2 expression and activity in cultured neurons, and LPS stimulated J774 macrophages (Zhang and Chen, 2008; Chang et al, 2001), or THC neurotoxicity in hippocampal neurons in a mechanism attributable to COX-2 activity and the concomitant generation of free radicals (Chan et al, 1998).
Our results show expression changes in the antiinflammatory pathway L-PGDS/15d-PGJ2/PPARγ after CB1 pharmacological and genetic manipulations; although still speculative, the involvement of this antiinflammatory pathway in CB1 neuroprotective effects is an interesting idea that deserves further investigation. This potential relationship is especially relevant as some pharmacological and genetic studies using CB1/CB2 KO mice indicate possible CB1/CB2 alternative mechanisms of action. PPARs could be excellent candidates, taking into account that diverse cannabinoids are potent ligands of PPARs (O'Sullivan, 2007). Thus, the main endocannabinoids, anandamide, and 2-arachidonoylglycerol, and also ajulemic acid, a THC structural analog, have antiinflammatory properties mediated by PPARγ (Sun and Bennett, 2007). In addition, the endogenous acylethanolamines, oleoylethanolamide, and palmitoylethanolamide have neuroprotective effects mediated through PPARα. The possibility of these related acylethanolamines modulating the stress response through interaction with PPARα remains unexplored.
Our results show a significant upregulation of L-PGDS/15d-PGJ2 after ACEA administration. This upregulation has been also demonstrated after the administration of ajulemic acid to human synovial cells (Stebulis et al, 2008), inducing an antiinflammatory profile of eicosanoids. PPARγ regulation is not yet completely understood, but one of the main mechanisms may be ligand availability (Daynes and Jones, 2002). This kind of regulation could be important in our stress model because we also found increased 15d-PGJ2 levels after ACEA administration. Other authors have proposed alternative mechanisms by which cannabinoids can potentially lead to PPARs activation. For example, CB1 binding could stimulate MAPK pathways, as it occurs in other scenarios (Lépicier et al, 2006), which regulates PPARγ transcriptional activity by direct phosphorylation on serine residues (Burns and Vanden Heuvel, 2007).
In contrast to ACEA neuroprotective effects in stress conditions, we and others have showed a dysregulated HPA axis activity in basal and after stress exposure in CB1 KO mice (Barna et al, 2004; Cota, 2008; Aso et al, 2008). In addition, a status of excessive excitotoxicity and neuroinflammation is also presented, increasing their susceptibility to experience damage and even cellular death. Similarly, in an animal model of Parkinson's disease, other authors have also found an increased vulnerability to 6-hydroxydopamine lesion in CB1 KO mice, presenting a higher index of lipid peroxidation and NOS-2 expression in selected brain areas (Pérez-Rial et al, 2009).
In addition to HPA axis dysregulation, chronic excitotoxicity, and neuroinflammation, CB1 KO mice present defective adult neurogenesis (Jin et al, 2004), increased anxiety (Urigüen et al, 2004; Mikics et al, 2009), and deficiency in neuronal plasticity by decreasing BDNF levels in the hippocampus (Aso et al, 2008), suggesting a possible role of CB1 in the pathophysiology of depression and its pharmacological manipulation as a potential therapeutic strategy.
In summary, the multifaceted neuroprotective effects described here suggest that CB1 activation is an attractive therapeutic strategy against diverse neuropsychiatric pathologies with HPA axis dysregulation and an excitotoxic/neuroinflammatory component in their pathophysiology.
Source, Graphs and Figures: Neuropsychopharmacology - Regulatory Role of Cannabinoid Receptor 1 in Stress-Induced Excitotoxicity and Neuroinflammation
