The Endocannabinoid System and Oligodendrocytes in Health and Disease

Cannabinoid-based interventions are being explored for central nervous system (CNS) pathologies such as neurodegeneration, demyelination, epilepsy, stroke, and trauma. As these disease states involve dysregulation of myelin integrity and/or remyelination, it is important to consider effects of the endocannabinoid system on oligodendrocytes and their precursors. In this review, we examine research reports on the effects of the endocannabinoid system (ECS) components on oligodendrocytes and their precursors, with a focus on therapeutic implications. Cannabinoid ligands and modulators of the endocannabinoid system promote cell signaling in oligodendrocyte precursor survival, proliferation, migration and differentiation, and mature oligodendrocyte survival and myelination. Agonist stimulation of oligodendrocyte precursor cells (OPCs) at both CB1 and CB2 receptors counter apoptotic processes via Akt/PI3K, and promote proliferation via Akt/mTOR and ERK pathways. CB1 receptors in radial glia promote proliferation and conversion to progenitors fated to become oligodendroglia, whereas CB2 receptors promote OPC migration in neonatal development. OPCs produce 2-arachidonoylglycerol (2-AG), stimulating cannabinoid receptor-mediated ERK pathways responsible for differentiation to arborized, myelin basic protein (MBP)-producing oligodendrocytes. In cell culture models of excitotoxicity, increased reactive oxygen species, and depolarization-dependent calcium influx, CB1 agonists improved viability of oligodendrocytes. In transient and permanent middle cerebral artery occlusion models of anoxic stroke, WIN55212-2 increased OPC proliferation and maturation to oligodendroglia, thereby reducing cerebral tissue damage. In several models of rodent encephalomyelitis, chronic treatment with cannabinoid agonists ameliorated the damage by promoting OPC survival and oligodendrocyte function. Pharmacotherapeutic strategies based upon ECS and oligodendrocyte production and survival should be considered.


INTRODUCTION
Phytocannabinoid use in management of multiple sclerosis (MS) symptoms (Consroe et al., 1997) has led to clinical trial evidence for the efficacy of tetrahydrocannabinol (THC)/cannabidiol (CBD) oromucosal spray (Sativex) in controlling spasticity and pain (Wade et al., 2010;Giacoppo et al., 2017). MS, a demyelinating disease characterized by persistent neuroinflammation and progressive central nervous system (CNS) demyelination (Kutzelnigg and Lassmann, 2014), is only one of many demyelinating neurodegenerative diseases involving oligodendrocytes, the myelinating cells of the CNS. The endocannabinoid system (ECS) (Howlett et al., 2002;Pertwee et al., 2010) involvement in neuroprotection (Panikashvili et al., 2001;van der Stelt and Di Marzo, 2005;Martinez-Orgado et al., 2007;Sanchez and García-Merino, 2012) and the immune system in CNS diseases (Croxford and Yamamura, 2005;Rom and Persidsky, 2013;Chiurchiu et al., 2015;Olah et al., 2017) have been reviewed. Here, we address ECS effects on oligodendrocytes and their precursors, in order to evaluate the evolving research around cannabinoids in healthy development and in demyelinating neurodegenerative diseases.

CANNABINOIDS, OLIGODENDROCYTE PRECURSOR CELLS AND OLIGODENDROCYTES IN HEALTH
Oligodendrocytes, myelinating cells of the vertebrate CNS, enable neurons to signal more energy-efficiently and at higher speed due to saltatory conduction, and maintain axonal integrity through trophic and metabolic support (Michalski and Kothary, 2015;Simons and Nave, 2015). Generation of oligodendrocytes is an ongoing process, starting in embryonic development and continuing throughout life (Baumann and Pham-Dinh, 2001;Trotter et al., 2010;Dimou and Gallo, 2015;Michalski and Kothary, 2015). In brief, oligodendrocyte precursor cells (OPCs), also known as NG2-glia, O-2A progenitors, polydendrocytes, or synantocytes, arise from neural stem cells (NSCs), and preferentially populate distinct areas of the developing CNS in lineage-and time-specific waves (Richardson et al., 2006). Upon arrival, many undergo apoptosis, while many others either mature into myelinating oligodendrocytes or persist as progenitors and remain capable of self-renewal as well as production of mature oligodendrocytes well into adulthood (Dawson et al., 2003). These progenitors become distributed throughout gray and white matter and maintain their respective domains by continuously sampling their environment, able to expand to neighboring areas vacated by other OPCs (Kirby et al., 2006;Hughes et al., 2013).
Despite this interchangeability, it is becoming increasingly clear that oligodendrocyte precursors represent a heterogeneous group, distinct in their origin, signaling, and ability to revert differentiation to produce neurons and astrocytes (Trotter et al., 2010;Dimou and Gallo, 2015;Nishiyama et al., 2016;Vigano and Dimou, 2016). Regardless of the differences, all OPCs rely on the processes of proliferation, migration, and differentiation to become mature, functioning oligodendrocytes (Baumann and Pham-Dinh, 2001;Miron et al., 2011;Dubois et al., 2014;Sampaio-Baptista and Johansen-Berg, 2017). As these steps are differentially regulated (Marinelli et al., 2016), it is important to look at the effects of cannabinoid agonists on each (see Figure 1).

Survival
Reports of cannabinoid receptors in newborn rat white matter by immunostaining (Berrendero et al., 1999), led to subsequent studies exploring cells in vitro. OPCs were isolated from newborn Wistar rat forebrains and expanded by incubation in serumfree defined media with supplements including platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) (Molina-Holgado et al., 2002). OPCs could be differentiated into myelin basic protein (MBP)-producing mature oligodendrocytes by incubation in serum-free defined media in which PDGF and FGF were replaced by triiodothyronine (T3). As ascertained by RT-PCR, Western blot, and immunohistochemistry, both OPC and mature oligodendrocytes expressed both CB 1 and CB 2 cannabinoid receptors (Molina-Holgado et al., 2002). Because activation of cannabinoid receptors confers neuroprotection (van der Stelt and Di Marzo, 2005;Martinez-Orgado et al., 2007;Sanchez and García-Merino, 2012), the influence of cannabinoid agonists on viability of OPCs was investigated. Upon incubation in serum-free DMEM/F12 media for 12 h, nearly half of OPCs underwent apoptosis (Molina-Holgado et al., 2002). However, most were rescued by concurrent supplementation with CB 1 agonist arachidonyl-2 -chloroethylamide (ACEA, 25 nM) or CB 1 /CB 2 agonists WIN55212-2 (25 nM) or HU210 (500 nM). Co-treatment with CB 1 antagonist SR141716 (1 µM) abolished the anti-apoptotic effect of ACEA, but not of WIN55212-2 or HU210. Both SR141716 plus CB 2 antagonist SR144528 (1 µM) were required to nullify the pro-survival effect of HU210. These results show that the activation of either CB 1 or CB 2 receptors Frontiers in Neuroscience | www.frontiersin.org could be sufficient in promoting OPC survival under conditions of trophic factor deprivation. The mechanism includes activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, a known modulator of OPC survival (Vemuri and McMorris, 1996;Ebner et al., 2000). Applying each of the three agonists correlated with increased Akt phosphorylation, while co-treatment with PI3K inhibitors LY294002 (10 µM) or wortmannin (100 nM) nullified the effects of WIN55212-2 and HU210 on both Akt phosphorylation and cell survival (Molina-Holgado et al., 2002). Thus, stimulation of Akt/PI3K pathways via CB 1 and CB 2 receptors present in OPCs can curtail apoptotic processes and promote survival.
Studies have examined cannabinoid agonist impact on rodent SVZ proliferation beyond the newborn stage. In juvenile (PD35-PD48) Lewis rats, WIN55212-2 (2 mg/kg, IP bid, 2-weeks) increased SVZ BrdU staining, without changing the ratio of progenitors committed to neuronal or OPC fates, or the number of cells undergoing caspase-3 mediated apoptosis (Bortolato et al., 2014). These results are consistent with findings that genetic ablation of CB 1 receptors decreased progenitor proliferation in adult mouse SVZ (Jin et al., 2004;Kim et al., 2006). In adult mice, cannabidiol (CBD; 3 mg/kg IP daily for 14 days) increased SVZ proliferative markers Ki67 and BrdU staining (Schiavon et al., 2016). However, CBD at 30 mg/kg decreased proliferation markers (Schiavon et al., 2016), highlighting the importance of dose. Molina-Holgado et al. (2002) investigated OPC differentiation using isolated OPCs differentiated with T3 (30 ng/mL, 48 h) in the absence of PDGF/FGF (Gomez et al., 2011). Activating either cannabinoid receptor (ACEA for CB 1 and JWH133 for CB 2 , 0.5 µM) increased OPC branching and accumulation of MBP (Western blot). The CB 1 /CB 2 agonist HU210 (0.5 µM) evoked the same responses, which could be abolished by either AM281 (CB 1 ) or AM630 (CB 2 ; 1 µM). The mechanism for HU210-mediated OPC arborization and production of MBP involved PI3K/Akt and mTOR pathways, as these effects were blocked with LY290042 (2.5 µM) and rapamycin (0.75 nM), respectively. Gomez et al. (2010) found the Western blot level of DAGLs to be higher in OPCs, whereas the level of MAGL was higher in mature oligodendrocytes, culminating in the finding that OPCs accumulated a greater content of 2-AG than mature oligodendrocytes. Levels of anandamide (AEA) were low and did not differ between the cell stages . Differentiation, denoted by branching morphology and levels of MBP after 96 h, was increased by MAGL inhibitor JZL184 (1 µM), and decreased by DAGL inhibitor RHC80267 (5 µM), with exogenous 2-AG (2 µM) abolishing the effect of blocking its synthesis. Inhibition of the ERK pathway by the MEK blocker PD98059 (10 µM) abolished Western blot staining for MBP, implicating the ERK pathway in differentiation. In a CB 1 receptor mRNA-expressing human oligodendrocyte precursor line HOG16, WIN55212-2 (1 µM, 24 h) increased MBP mRNA expression, particularly in cells treated with T3-supplemented differentiating medium (Tomas-Roig et al., 2016). Collectively, these studies suggest that endogenous 2-AG in OPCs triggers the ERK pathway, leading to the maturation of arborized, MBPproducing oligodendrocytes.
The endocannabinoid 2-AG has been shown to be neuroprotective after traumatic brain injury (Panikashvili et al., 2001), and 2-AG is elevated after spinal trauma (Marsicano et al., 2003;Garcia-Ovejero et al., 2009). Thus, the impact of 2-AG on oligodendrocyte survival was explored in a model of contusive SCI generated by a dropped weight in male adult Wistar rats . 2-AG (5 mg/kg, IP 30 min after injury) preserved myelin integrity and reduced oligodendrocyte death at the epicenter (1 day post-injury), with the same effects seen as far as 10 mm rostral of epicenter (1 and 7 days post-injury). Co-administration of both CB 1 and CB 2 antagonists (AM281 and AM630, respectively; 3 mg/kg, IP), but not by either alone, could reverse the effects of 2-AG. These results support the idea that improved oligodendrocyte survival and preserved white matter integrity underlie the cannabinoidmediated improvement in SCI recovery (Arevalo-Martin et al., 2012).

ECS and Models of Demyelination
To investigate demyelination in a mouse model of experimental autoimmune encephalomyelitis (EAE), PD49 or PD56 C57BL/6 mice received a single injection of myelin oligodendrocyte glycoprotein peptide (MOG 35-55), followed by injections of pertussis toxin on the day of MOG inoculation and again 2 days afterward (Bernal-Chico et al., 2015). MAGL inhibitor JZL184 (8 mg/kg, IP daily for 3 weeks, starting on day 14 postinoculation) ameliorated the reduction in spinal cord white matter staining (Bernal-Chico et al., 2015). Similar results in the EAE model were achieved by THC (Moreno-Martet et al., 2015), CBD (Giacoppo et al., 2015), cannabigerol quinone (Carrillo-Salinas et al., 2014), and CB 2 agonist HU308 (Shao et al., 2014). The effects of CB52 on demyelination EAE (Ribeiro et al., 2013) showed that when initiated before symptom development (3 days) or after clinical disease onset (12 or 20 days), CB52 (2 mg/kg, IP daily) ameliorated the loss of staining for spinal cord myelin and mature oligodendrocytes at day-30. In contrast to CB52's action on cultured oligodendrocytes in vitro (Ribeiro et al., 2013), both of its effects in vivo were blocked by CB 1 antagonist AM281 (2 mg/kg), but not by CB 2 antagonist AM630 (2 mg/kg).
Microglia are an integral part of demyelinating diseases' neuroimmune complex (Gonzalez et al., 2014). In microglia, CB 1 receptors are expressed at low levels constitutively; however, CB 2 receptors become upregulated when microglia become activated (Cabral et al., 2008). Endocannabinoids 2-AG and AEA have been shown to drive microglia toward alternative, anti-inflammatory activation state, M2, and away from classic, pro-inflammatory polarization, M1, which in turn causes microglia to upregulate its own 2-AG synthesizing enzymes . Because microglial 2-AG has been shown to promote OPC differentiation (Miron et al., 2013), blocking its degradation could be of use in counteracting demyelination. This has been explored in a mouse model of EAE (Wen et al., 2015), by inhibiting the 2-AG hydrolyzing microglial enzyme ABHD6 (Li et al., 2007;Marrs et al., 2010;Murataeva et al., 2014) with WWL70 (10 mg/kg, IP daily starting at the onset of clinical symptoms on day-11 postinoculation). WWL70 increased cerebral 2-AG at day-21, and ameliorated the loss of staining of spinal cord myelin and mature oligodendrocytes in wildtype mice on day-28 (Wen et al., 2015). These results were not seen in CB 2 -KO mice, nor when WWL70 was co-administered with CB 2 antagonist AM630 (3 mg/kg), suggesting that microglial 2-AG accumulation is dependent upon CB 2 receptor signaling. Co-administration with CB 1 antagonist AM281 failed to interfere with WWL70's effects.
OPC gliogenesis in Borna Disease Virus (BDV) encephalomyelitis, generated in PD28 male Lewis rats (Solbrig et al., 2010), demonstrated that WIN55212-2 (1 mg/kg, IP daily for 7-days starting 1 week after virus inoculation) increased OPC proliferation in striatum, decreased apoptosis of proliferating cells, skewed precursor differentiation away from astrocytes and toward oligodendrocytes, and promoted OPC maturation. In uninfected controls, WIN55212-2 increased proliferation in both PFC and striatum.
In Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), PD28 female CJL/J mice received an intracerebral injection of the Daniel strain virus (Feliu et al., 2017). When started after symptom onset at day-75, a 10-day treatment with MAGL inhibitor UCM03025 (5 mg/kg, IP) increased the spinal cord populations of both mature oligodendrocytes and OPCs, and restored MBP level to that of sham controls (Feliu et al., 2017).

CBD and OPCs in Inflammation
CBD has been promoted for potential therapeutic applications (Devinsky et al., 2014;Blessing et al., 2015;Ibeas Bih et al., 2015) including anti-inflammation (Burstein, 2015). Inflammation underlies a range of pathologies including neurodegeneration (Glass et al., 2010;Cunningham, 2013), stroke (Turner and Vink, 2007;Ahmad et al., 2014), and demyelination (Popescu and Lucchinetti, 2012;Kutzelnigg and Lassmann, 2014). To examine CBD's anti-inflammatory impact on OPC survival, cultured OPCs isolated from the forebrain of newborn Wistar rats were exposed to inflammation-related stressors (Mecha et al., 2012). Treatment with CBD (1 µM) reduced: (1) caspase-3mediated apoptosis resulting from lipopolysaccharides (LPS) and interferon-γ (IFNγ)-mediated inflammation (48 h); (2) cell death induced by endoplasmic reticulum stress instigated by tunicamycin (1 µg/ml, 24 h); and (3) cell detachment and ROS production in response to hydrogen peroxide (2 h). CBD was unable to increase OPC proliferation in culture (Mecha et al., 2012), in contrast to its chronic administration in SVZ of adult Swiss mice (Schiavon et al., 2016). CBD did not promote apoptosis in culture, as observed in unstressed cultured oligodendrocytes (Mato et al., 2010). CBD's cellular mechanism(s) have yet to be established for OPC and oligodendrocyte function, but might counter the endocannabinoid responses at their receptors. Further, CBD may target other cell types in the neuro-immune complex, explaining differences between in vitro vs. in vivo models.

AUTHOR CONTRIBUTIONS
AI conceived the idea and approach to the review, wrote and edited the manuscript. CM and EP provided the feedback and edited the manuscript. AH developed approach to the review, structured, wrote and edited the manuscript.

FUNDING
This work was supported by NIDA grants R01-DA0042157 and P50-DA006634.