Cannabinoid CB2 receptors are upregulated via bivalent histone modifications and control primary afferent input to the spinal cord in neuropathic pain

Abstract

Type-2 cannabinoid receptors (CB2, encoded by the Cnr2 gene) are mainly expressed in immune cells, and CB2 agonists normally have no analgesic effect. However, nerve injury upregulates CB2 in the dorsal root ganglion (DRG), following which CB2 stimulation reduces neuropathic pain. It is unclear how nerve injury increases CB2 expression or how CB2 activity is transformed in neuropathic pain. In this study, immunoblotting showed that spinal nerve ligation (SNL) induced a delayed and sustained increase in CB2 expression in the DRG and dorsal spinal cord synaptosomes. RNAscope in situ hybridization also showed that SNL substantially increased CB2 mRNA levels, mostly in medium and large DRG neurons. Furthermore, we found that the specific CB2 agonist JWH-133 significantly inhibits the amplitude of dorsal root–evoked glutamatergic excitatory postsynaptic currents in spinal dorsal horn neurons in SNL rats, but not in sham control rats; intrathecal injection of JWH-133 reversed pain hypersensitivity in SNL rats, but had no effect in sham control rats. In addition, chromatin immunoprecipitation–qPCR analysis showed that SNL increased enrichment of two activating histone marks (H3K4me3 and H3K9ac) and diminished occupancy of two repressive histone marks (H3K9me2 and H3K27me3) at the Cnr2 promoter in the DRG. In contrast, SNL had no effect on DNA methylation levels around the Cnr2 promoter. Our findings suggest that peripheral nerve injury promotes CB2 expression in primary sensory neurons via epigenetic bivalent histone modifications and that CB2 activation reduces neuropathic pain by attenuating nociceptive transmission from primary afferent nerves to the spinal cord. Type-2 cannabinoid receptors (CB2, encoded by the Cnr2 gene) are mainly expressed in immune cells, and CB2 agonists normally have no analgesic effect. However, nerve injury upregulates CB2 in the dorsal root ganglion (DRG), following which CB2 stimulation reduces neuropathic pain. It is unclear how nerve injury increases CB2 expression or how CB2 activity is transformed in neuropathic pain. In this study, immunoblotting showed that spinal nerve ligation (SNL) induced a delayed and sustained increase in CB2 expression in the DRG and dorsal spinal cord synaptosomes. RNAscope in situ hybridization also showed that SNL substantially increased CB2 mRNA levels, mostly in medium and large DRG neurons. Furthermore, we found that the specific CB2 agonist JWH-133 significantly inhibits the amplitude of dorsal root–evoked glutamatergic excitatory postsynaptic currents in spinal dorsal horn neurons in SNL rats, but not in sham control rats; intrathecal injection of JWH-133 reversed pain hypersensitivity in SNL rats, but had no effect in sham control rats. In addition, chromatin immunoprecipitation–qPCR analysis showed that SNL increased enrichment of two activating histone marks (H3K4me3 and H3K9ac) and diminished occupancy of two repressive histone marks (H3K9me2 and H3K27me3) at the Cnr2 promoter in the DRG. In contrast, SNL had no effect on DNA methylation levels around the Cnr2 promoter. Our findings suggest that peripheral nerve injury promotes CB2 expression in primary sensory neurons via epigenetic bivalent histone modifications and that CB2 activation reduces neuropathic pain by attenuating nociceptive transmission from primary afferent nerves to the spinal cord. Neuropathic pain is a chronic debilitating condition, and current treatments are unsatisfactory. Endogenous endocannabinoids, including N-arachidonoylethanolamine and 2-arachidonoylglycerol, produce biological effects mainly through activation of two cannabinoid receptors, type-1 cannabinoid receptors (CB1, encoded by the Cnr1 gene) and type-2 cannabinoid receptors (CB2, encoded by Cnr2) (1Donvito G. Nass S.R. Wilkerson J.L. Curry Z.A. Schurman L.D. Kinsey S.G. et al.The endogenous cannabinoid system: a budding source of targets for treating inflammatory and neuropathic pain.Neuropsychopharmacology. 2018; 43: 52-79Crossref PubMed Scopus (144) Google Scholar). CB1 are widely distributed in the peripheral and central nervous system (2Tsou K. Brown S. Sañudo-Peña M.C. Mackie K. Walker J.M. Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system.Neuroscience. 1998; 83: 393-411Crossref PubMed Scopus (1323) Google Scholar, 3Veress G. Meszar Z. Muszil D. Avelino A. Matesz K. Mackie K. et al.Characterisation of cannabinoid 1 receptor expression in the perikarya, and peripheral and spinal processes of primary sensory neurons.Brain Struct. Funct. PubMed Scopus Google Scholar). CB1 activation in et via peripheral 1 cannabinoid receptors in PubMed Scopus Google Scholar, S.R. G. et expression of cannabinoid receptors in primary sensory neurons in neuropathic PubMed Scopus Google CB1 agonists are not for of CB2 are in immune cells, including and but are expressed in the nervous system for PubMed Scopus Google Scholar, S. S. D. D. et of central and peripheral cannabinoid receptors in immune and PubMed Scopus Google Scholar). peripheral nerve injury increases CB2 expression in the dorsal root ganglion S.R. G. et expression of cannabinoid receptors in primary sensory neurons in neuropathic PubMed Scopus Google Scholar, G. A. S. S. nerve injury cannabinoid receptor expression in rat sensory PubMed Scopus Google Scholar). Furthermore, CB2 agonists have no analgesic effect but produce analgesic effects in of neuropathic pain S.R. et for cannabinoid PubMed Scopus Google Scholar, A. et of CB2 cannabinoid receptors by inhibits neuropathic pain by receptors not in the S. A. PubMed Scopus Google Scholar, et and peripheral of for receptor analgesic activity in chronic inflammatory and neuropathic pain in PubMed Scopus Google Scholar, et CB2 agonist pain and and PubMed Scopus Google Scholar). However, is unclear how the CB2 is transformed in neuropathic pain and how nerve injury upregulates CB2 in the DRG. injury and of a large of which in of primary afferent nerves and DRG neurons and nociceptive to the spinal cord S.R. nerve and in neuropathic Google Scholar, G. S.R. et is for epigenetic of in pain PubMed Scopus Google Scholar). histone modifications methylation and and DNA a in the of of DRG neurons and chronic pain nerve nerve injury increases the expression and activity of a histone which of including receptors, and by enrichment of a repressive histone at in DRG neurons S.R. G. et expression of cannabinoid receptors in primary sensory neurons in neuropathic PubMed Scopus Google Scholar, G. S.R. et is for epigenetic of in pain PubMed Scopus Google Scholar, S.R. G. injury through of receptors in primary sensory PubMed Scopus Google Scholar). from DRG neurons not chronic pain nerve injury G. S.R. et is for epigenetic of in pain PubMed Scopus Google Scholar, S.R. G. injury through of receptors in primary sensory PubMed Scopus Google Scholar). Furthermore, nerve injury reduces enrichment of activating histone at the of and receptors but increases at the promoter in the DRG in neuropathic pain G. S.R. et is for epigenetic of in pain PubMed Scopus Google Scholar, S.R. G. injury through of receptors in primary sensory PubMed Scopus Google Scholar, G. S.R. in the dorsal root ganglion to neuropathic pain PubMed Scopus Google Scholar). In addition, nerve injury of DNA methylation in the DRG G. S.R. et chronic pain is DNA methylation in dorsal root 2018; PubMed Scopus Google Scholar). However, is histone modifications DNA methylation a in the of CB2 expression in the DRG in chronic neuropathic pain. in the study, we CB2 expression in DRG neurons and in nociceptive transmission at the spinal cord in neuropathic pain. also epigenetic of Cnr2 in the DRG. S.R. G. et expression of cannabinoid receptors in primary sensory neurons in neuropathic PubMed Scopus Google Scholar, G. A. S. S. nerve injury cannabinoid receptor expression in rat sensory PubMed Scopus Google that nerve injury a increase in CB2 expression in DRG which inhibits glutamatergic from primary to spinal dorsal horn neurons in neuropathic pain. Furthermore, nerve CB2 is histone but not DNA at the Cnr2 promoter in the DRG. of CB2 expression in primary sensory neurons and analgesic in neuropathic pain. immunoblotting to the of in the expression levels of CB2 in the DRG nerve showed that that in sham rats, the of CB2 in the DRG significantly increased in spinal nerve ligation (SNL) at and but not However, SNL had no effect on the of CB2 in the dorsal spinal cord CB2 is in immune S. S. D. D. et of central and peripheral cannabinoid receptors in immune and PubMed Scopus Google CB2 expressed in DRG neurons nerve injury G. A. S. S. nerve injury cannabinoid receptor expression in rat sensory PubMed Scopus Google Scholar). the CB2 for immunoblotting in we RNAscope to the expression of CB2 mRNA in DRG neurons nerve CB2 in neurons and of of CB2 mRNA in the DRG significantly in SNL in sham and the of CB2 mRNA in DRG neurons significantly increased in SNL that in sham and Furthermore, the of CB2 mRNA significantly in and but not in DRG neurons in SNL in sham that peripheral nerve injury sustained of CB2 in primary sensory neurons. CB2 activation at the spinal cord neuropathic pain G. A. S. S. nerve injury cannabinoid receptor expression in rat sensory PubMed Scopus Google Scholar, S. S. effects of cannabinoid receptor agonists on neuropathic PubMed Scopus Google Scholar). in spinal cord to the of CB2 at primary afferent central in the control of glutamatergic to the spinal cord in SNL and sham to excitatory postsynaptic currents of neurons by stimulation of the dorsal to we S.R. et al.The receptor is in neuropathic pain and 2018; PubMed Scopus Google Scholar, S.R. receptor activity in spinal and pain hypersensitivity induced by nerve PubMed Scopus Google the amplitude of significantly in SNL in sham and In sham rats, of a specific CB2 agonist S. J.L. et and of for the CB2 PubMed Scopus Google Scholar, A. et CB2 cannabinoid receptors and PubMed Scopus Google at of to had no effect on the amplitude of of spinal neurons and In contrast, in SNL rats, of to of JWH-133 significantly the amplitude of of neurons in a and of a of S.R. receptor activity is increased through in neuropathic PubMed Scopus Google Scholar, S.R. receptor glutamatergic to spinal dorsal horn neurons in neuropathic PubMed Scopus Google significantly in SNL in sham and In SNL rats, of to JWH-133 a in the amplitude in the the in a increase in the of in neurons and However, JWH-133 at to had no effect on the of in spinal neurons in sham and In addition, we from the dorsal spinal cord to nerve injury increases CB2 showed that CB2 in spinal cord significantly in SNL in sham control and suggest that activation of CB2 at primary afferent central by nerve injury inhibits glutamatergic to spinal dorsal horn neurons. the CB2 agonist JWH-133 to the of CB2 in the spinal cord in the control of nerve neuropathic pain. via intrathecal to the DRG and spinal cord S.R. G. of and receptor in the spinal control of in PubMed Scopus Google Scholar, S.R. and of in nociceptive control in neuropathic PubMed Scopus Google Scholar). In sham and SNL to we the effect of intrathecal injection of and of JWH-133 on the of in to and In SNL rats, intrathecal of and but not or of JWH-133 significantly and However, in sham control rats, intrathecal injection of to of JWH-133 had no effect on or Furthermore, intrathecal injection of of JWH-133 had no effect on pain hypersensitivity SNL no increased CB2 expression at nerve injury suggest that CB2 activation at the spinal cord peripheral nerve neuropathic pain at the CB2 expression is increased in DRG neurons and central expression in the DRG is histone modifications at the promoter S.R. G. et expression of cannabinoid receptors in primary sensory neurons in neuropathic PubMed Scopus Google Scholar, G. S.R. et is for epigenetic of in pain PubMed Scopus Google Scholar, S.R. G. injury through of receptors in primary sensory PubMed Scopus Google Scholar, G. S.R. in the dorsal root ganglion to neuropathic pain PubMed Scopus Google Scholar). nerve CB2 in the DRG histone rat Cnr2 in the the the of we that at two of Cnr2 in the 1 and have encoded from two DNA on However, have 1 and the CB2 to the a CB2 a Cnr2 in and is via at two to a expression of two Cnr2 and A. A. et in cannabinoid receptor of and CB2 expression and by cannabinoid receptor PubMed Scopus Google Scholar). of and CB2 a and Our immunoblotting a expression of a but in the spinal CB2 and are Furthermore, analysis showed that the is expressed in the DRG and that the of promoter significantly increased by nerve injury we this Cnr2 promoter for chromatin of histone marks at is which mainly in et bivalent chromatin marks in PubMed Scopus Google Scholar). and are in a and are to expression G. S.R. et is for epigenetic of in pain PubMed Scopus Google Scholar, of histone PubMed Scopus Google Scholar). to the enrichment of two activating histone and and two histone and at the Cnr2 promoter in and SNL or sham from to the at the rat Cnr2 promoter for enrichment of histone enrichment of in at the Cnr2 promoter in the and significantly in SNL in sham the at the to and at the to of the Cnr2 promoter in SNL to sham rats. the enrichment of at the Cnr2 promoter significantly increased in in the DRG of SNL that in sham control in the to and in the to In contrast, the occupancy of the repressive histone at the Cnr2 promoter in the DRG significantly by nerve injury in the to in the to and in the to Furthermore, nerve injury significantly the enrichment of at the Cnr2 promoter findings suggest that nerve CB2 in the DRG from a increase in activating histone marks and in repressive histone marks at the Cnr2 promoter. nerve injury also DNA methylation in the DRG G. S.R. et chronic pain is DNA methylation in dorsal root 2018; PubMed Scopus Google and DNA methylation expression A. et DNA methylation and in the PubMed Scopus Google we to nerve injury DNA methylation of Cnr2 in the DRG. to the methylation of at the rat Cnr2 promoter from to and to in and SNL or sham promoter of Cnr2 is of in the Our showed that SNL not the methylation of at the Cnr2 promoter in the DRG the to and to the of in the DRG in SNL and sham not and we also DNA a specific J.L. et of in the of PubMed Scopus Google by no in the enrichment of the to or the to at the rat Cnr2 promoter in the DRG sham and SNL suggest that the of around the of nerve CB2 in the DRG is by the DNA methylation at the Cnr2 promoter. In the study, we found that nerve injury a delayed and sustained increase in CB2 expression in the DRG. CB2 is expressed in the DRG but is nerve injury S.R. G. et expression of cannabinoid receptors in primary sensory neurons in neuropathic PubMed Scopus Google Scholar, G. A. S. S. nerve injury cannabinoid receptor expression in rat sensory PubMed Scopus Google Scholar, S. D. of CB2 receptor expression in the rat spinal cord of neuropathic but not inflammatory chronic pain PubMed Scopus Google Scholar). nerve CB2 expression in primary sensory we RNAscope in situ hybridization to the CB2 mRNA levels in DRG neurons. showed that nerve injury increased the CB2 mRNA in and DRG neurons. Furthermore, we found that intrathecal injection of the CB2 agonist JWH-133 reversed the in the and in SNL CB2 is in the DRG but had no effects in sham control and SNL nerve suggest that CB2 activation at the spinal cord not but reduces neuropathic pain by nerve JWH-133 is a CB2 agonist a of and a for CB2 CB1 S. J.L. et and of for the CB2 PubMed Scopus Google Scholar, A. et CB2 cannabinoid receptors and PubMed Scopus Google Scholar). effect of JWH-133 is by the CB2 and is in CB2 of central cannabinoid CB2 receptor in in a of neuropathic PubMed Scopus Google Scholar, D. CB2 receptor activation inhibits of dorsal horn neurons in and in rat of inflammatory and neuropathic PubMed Scopus Google Scholar). Our findings are that CB2 agonists neuropathic pain S.R. et for cannabinoid PubMed Scopus Google Scholar, et and peripheral of for receptor analgesic activity in chronic inflammatory and neuropathic pain in PubMed Scopus Google Scholar, et CB2 agonist pain and and PubMed Scopus Google Scholar). to DRG neurons and primary afferent in the spinal cord S.R. G. of and receptor in the spinal control of in PubMed Scopus Google Scholar, S.R. receptors in primary sensory neurons are for analgesic effect on and inflammatory pain and PubMed Scopus Google the effect of JWH-133 on neuropathic pain is to increased CB2 expression in DRG neurons and central at the spinal cord of is that activation of CB2 at the spinal cord inhibits nociceptive glutamatergic to dorsal horn neurons in neuropathic which the of nerve CB2 in DRG neurons. glutamatergic to spinal dorsal horn neurons is in the of chronic neuropathic pain S.R. et al.The receptor is in neuropathic pain and 2018; PubMed Scopus Google Scholar, S.R. receptor activity in spinal and pain hypersensitivity induced by nerve PubMed Scopus Google Scholar, S.R. receptor activity is increased through in neuropathic PubMed Scopus Google Scholar). CB2 agonists the of spinal dorsal horn neurons in S. effects of CB1 and CB2 receptor agonists on of DRG neurons and dorsal horn neurons in neuropathic PubMed Scopus Google Scholar, S. receptor activity in neuropathic of spinal and peripheral PubMed Scopus Google Scholar). Our in spinal cord showed that the CB2 agonist JWH-133 the amplitude of from the dorsal root central of DRG and increased the of in spinal dorsal horn neurons of SNL rats. However, JWH-133 had no effect in control rats. suggest that activation of CB2 expressed at primary afferent central inhibits glutamatergic to spinal dorsal horn neurons by nerve injury from DRG neurons to central in the spinal dorsal horn S.R. et al.The receptor is in neuropathic pain and 2018; PubMed Scopus Google Scholar, S.R. 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