1 4306 123 MICRORNA-29A MITIGATES LAMINECTOMY-INDUCED SPINAL EPIDURAL FIBROSIS AND GAIT DYSREGULATION BY REPRESSING TGF-BETA1 AND IL-6. SPINAL EPIDURAL FIBROSIS IS ONE OF THE TYPICAL FEATURES ATTRIBUTABLE TO FAILED BACK SURGERY SYNDROME, WITH EXCESSIVE SCAR DEVELOPMENT IN THE DURA AND NERVE ROOTS. THE MICRORNA-29 FAMILY (MIR-29S) HAS BEEN FOUND TO ACT AS A FIBROGENESIS-INHIBITORY FACTOR THAT REDUCES FIBROTIC MATRIX OVERPRODUCTION IN VARIOUS TISSUES. HOWEVER, THE MECHANISTIC BASIS OF MIRNA-29A UNDERLYING THE OVERABUNDANT FIBROTIC MATRIX SYNTHESIS IN SPINAL EPIDURAL SCARS POST-LAMINECTOMY REMAINED ELUSIVE. THIS STUDY REVEALED THAT MIR-29A ATTENUATED LUMBAR LAMINECTOMY-INDUCED FIBROGENIC ACTIVITY, AND EPIDURAL FIBROTIC MATRIX FORMATION WAS SIGNIFICANTLY LESSENED IN THE TRANSGENIC MICE (MIR-29ATG) AS COMPARED WITH WILD-TYPE MICE (WT). MOREOVER, MIR-29ATG LIMITS LAMINECTOMY-INDUCED DAMAGE AND HAS ALSO BEEN DEMONSTRATED TO DETECT WALKING PATTERNS, FOOTPRINT DISTRIBUTION, AND MOVING ACTIVITY. IMMUNOHISTOCHEMISTRY STAINING OF EPIDURAL TISSUE SHOWED THAT MIR-29ATG WAS A REMARKABLY WEAK SIGNAL OF IL-6, TGF-BETA1, AND DNA METHYLTRANSFERASE MARKER, DNMT3B, COMPARED TO THE WILD-TYPE MICE. TAKEN TOGETHER, THESE RESULTS HAVE FURTHER STRENGTHENED THE EVIDENCE THAT MIR-29A EPIGENETIC REGULATION REDUCES FIBROTIC MATRIX FORMATION AND SPINAL EPIDURAL FIBROTIC ACTIVITY IN SURGERY SCARS TO PRESERVE THE INTEGRITY OF THE SPINAL CORD CORE. THIS STUDY ELUCIDATES AND HIGHLIGHTS THE MOLECULAR MECHANISMS THAT REDUCE THE INCIDENCE OF SPINAL EPIDURAL FIBROSIS, ELIMINATING THE RISK OF GAIT ABNORMALITIES AND PAIN ASSOCIATED WITH LAMINECTOMY. 2023 2 1692 31 DURATION OF EXPOSURE TO EPIDURAL ANESTHESIA AT DELIVERY, DNA METHYLATION IN UMBILICAL CORD BLOOD AND THEIR ASSOCIATION WITH OFFSPRING ASTHMA IN NON-HISPANIC BLACK WOMEN. EPIDURAL ANESTHESIA IS AN EFFECTIVE PAIN RELIEF MODALITY, WIDELY USED FOR LABOR ANALGESIA. CHILDHOOD ASTHMA IS ONE OF THE COMMONEST CHRONIC MEDICAL ILLNESSES IN THE USA WHICH PLACES A SIGNIFICANT BURDEN ON THE HEALTH-CARE SYSTEM. WE RECENTLY DEMONSTRATED A NEGATIVE ASSOCIATION BETWEEN THE DURATION OF EPIDURAL ANESTHESIA AND THE DEVELOPMENT OF CHILDHOOD ASTHMA; HOWEVER, THE UNDERLYING MOLECULAR MECHANISMS STILL REMAIN UNCLEAR. IN THIS STUDY OF 127 MOTHER-CHILD PAIRS COMPRISED OF 75 NON-HISPANIC BLACK (NHB) AND 52 NON-HISPANIC WHITE (NHW) FROM THE NEWBORN EPIGENETIC STUDY, WE TESTED THE HYPOTHESIS THAT UMBILICAL CORD BLOOD DNA METHYLATION MEDIATES THE ASSOCIATION BETWEEN THE DURATION OF EXPOSURE TO EPIDURAL ANESTHESIA AT DELIVERY AND THE DEVELOPMENT OF CHILDHOOD ASTHMA AND WHETHER THIS DIFFERED BY RACE/ETHNICITY. IN THE MOTHER-CHILD PAIRS OF NHB ANCESTRY, THE DURATION OF EXPOSURE TO EPIDURAL ANESTHESIA WAS ASSOCIATED WITH A MARGINALLY LOWER RISK OF ASTHMA (ODDS RATIO = 0.88, 95% CONFIDENCE INTERVAL = 0.76-1.01) FOR EACH 1-H INCREASE IN EXPOSURE TO EPIDURAL ANESTHESIA. OF THE 20 CPGS IN THE NHB POPULATION SHOWING THE STRONGEST MEDIATION EFFECT, 50% DEMONSTRATED AN AVERAGE MEDIATION PROPORTION OF 52%, WITH DIRECTIONAL CONSISTENCY OF DIRECT AND INDIRECT EFFECTS. THESE TOP 20 CPGS MAPPED TO 21 GENES ENRICHED FOR PATHWAYS ENGAGED IN ANTIGEN PROCESSING, ANTIGEN PRESENTATION, PROTEIN UBIQUITINATION AND REGULATORY NETWORKS RELATED TO THE MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) CLASS I COMPLEX AND NUCLEAR FACTOR KAPPA-B (NFKB) COMPLEX. OUR FINDINGS SUGGEST THAT DNA METHYLATION IN IMMUNE-RELATED PATHWAYS CONTRIBUTES TO THE EFFECTS OF THE DURATION OF EXPOSURE TO EPIDURAL ANESTHESIA ON CHILDHOOD ASTHMA RISK IN NHB OFFSPRING. 2023 3 2272 32 EPIGENETIC REDUCTION OF MIR-214-3P UPREGULATES ASTROCYTIC COLONY-STIMULATING FACTOR-1 AND CONTRIBUTES TO NEUROPATHIC PAIN INDUCED BY NERVE INJURY. EMERGING EVIDENCE HAS INDICATED THAT COLONY-STIMULATING FACTOR-1 (CSF1) MODULATES NEUROINFLAMMATION IN THE CENTRAL NERVOUS SYSTEM AND THE DEVELOPMENT OF NEUROPATHIC PAIN, WHILE THE UNDERLYING MECHANISM REMAINS UNKNOWN. HERE, WE IDENTIFIED THE INCREASED EXPRESSION OF CSF1 DERIVED FROM ACTIVATED ASTROCYTES IN THE IPSILATERAL DORSAL HORN IN RATS WITH SPINAL NERVE LIGATION (SNL). SUPPRESSION OF CSF1 EXPRESSION ALLEVIATED NEUROINFLAMMATION, NEURONAL HYPEREXCITABILITY, AND GLUTAMATERGIC RECEPTOR SUBUNIT UPREGULATION IN THE DORSAL HORN AND IMPROVED SNL-INDUCED PAIN BEHAVIOR. WE ALSO FOUND REDUCED MIR-214-3P EXPRESSION IN THE IPSILATERAL DORSAL HORN FOLLOWING AN SNL PROCEDURE; MIR-214-3P DIRECTLY BOUND TO THE 3'-UTR OF CSF1 MRNA AND NEGATIVELY REGULATED CSF1 EXPRESSION. INTRATHECAL DELIVERY OF MIR-214-3P MIMIC REVERSED THE ENHANCED EXPRESSION OF CSF1 AND ASTROCYTE OVERACTIVITY AND ALLEVIATED THE IL-6 UPREGULATION AND PAIN BEHAVIOR INDUCED BY SNL. MOREOVER, SUPPRESSION OF SPINAL MIR-214-3P INCREASED ASTROCYTE REACTIVITY, PROMOTED CSF1 AND IL-6 PRODUCTION, AND INDUCED PAIN HYPERSENSITIVITY IN NAIVE ANIMALS. FURTHERMORE, SNL INDUCED THE EXPRESSION OF DNA METHYLTRANSFERASE 3A (DNMT3A) THAT WAS ASSOCIATED WITH THE HYPERMETHYLATION OF THE MIR-214-3P PROMOTER, LEADING TO REDUCED MIR-214-3P EXPRESSION IN THE MODEL RODENTS. TREATMENT WITH THE DNMT INHIBITOR ZEBULARINE SIGNIFICANTLY REDUCED CYTOSINE METHYLATION IN THE MIR-214-3P PROMOTER; THIS REDUCED METHYLATION CONSEQUENTLY INCREASED THE EXPRESSION OF MIR-214-3P AND DECREASED THE CONTENT OF CSF1 IN THE IPSILATERAL DORSAL HORN AND, FURTHER, ATTENUATED IL-6 PRODUCTION AND PAIN BEHAVIOR IN RATS WITH SNL. TOGETHER, OUR DATA INDICATE THAT THE DNMT3A-MEDIATED EPIGENETIC SUPPRESSION OF MIR-214-3P ENHANCED CSF1 PRODUCTION IN ASTROCYTES, WHICH SUBSEQUENTLY INDUCED NEUROINFLAMMATION AND PAIN BEHAVIOR IN SNL MODEL RATS. 2020 4 3810 27 INTRATHECAL 5-AZACYTIDINE INHIBITS GLOBAL DNA METHYLATION AND METHYL- CPG-BINDING PROTEIN 2 EXPRESSION AND ALLEVIATES NEUROPATHIC PAIN IN RATS FOLLOWING CHRONIC CONSTRICTION INJURY. THE PATHOGENESIS OF NEUROPATHIC PAIN REMAINS LARGELY UNKNOWN. EPIGENETIC MECHANISMS MAY PLAY A MAJOR ROLE IN REGULATING EXPRESSION OF PRO- OR ANTINOCICEPTIVE GENES. DNA METHYLATION IS A MAJOR EPIGENETIC MECHANISM IN VERTEBRATES, AND METHYL- CPG-BINDING PROTEIN 2 (MECP2) IS DIRECTLY INVOLVED IN METHYLATION-MEDIATED GENE SILENCING. TO DETERMINE HOW CHANGES IN GLOBAL DNA METHYLATION AND MECP2 EXPRESSION OCCUR FOLLOWING CHRONIC CONSTRICTION INJURY (CCI) AND HOW REPRESSION OF DNA METHYLATION AFFECTS THESE CHANGES AND ATTENUATES NEUROPATHIC PAIN, WE USED INTRATHECAL 5-AZACYTIDINE, A DNA METHYLTRANSFERASE INHIBITOR, IN CCI RATS. RATS RECEIVED 0.9% SALINE OR 5-AZACYTIDINE (10MUMOL.D(-1)) VIA SPINAL INJECTION ONCE DAILY FROM DAY 3 TO DAY 14 AFTER CCI SURGERY. GLOBAL DNA METHYLATION AND MECP2 EXPRESSION INCREASED IN THE SPINAL CORD IN CCI RATS ON DAY 14 AFTER CCI SURGERY. MECHANICAL ALLODYNIA AND THERMAL HYPERALGESIA INDUCED BY CCI WERE ATTENUATED BY INTRATHECAL 5-AZACYTIDINE FROM DAY 5 TO DAY 14 AFTER CCI SURGERY. THE INCREASES IN GLOBAL DNA METHYLATION AND MECP2 EXPRESSION IN THE SPINAL CORD IN CCI RATS WERE ALSO SIGNIFICANTLY INHIBITED BY INTRATHECAL 5-AZACYTIDINE. THESE RESULTS DEMONSTRATE THAT INCREASED GLOBAL DNA METHYLATION AND MECP2 EXPRESSION IN THE SPINAL CORD AFTER NERVE DAMAGE MAY PLAY AN IMPORTANT ROLE IN NEUROPATHIC PAIN. 5-AZACYTIDINE SHOWS POTENTIAL FOR TREATING NEUROPATHIC PAIN. 2011 5 4617 27 NERVE INJURY-INDUCED CHRONIC PAIN IS ASSOCIATED WITH PERSISTENT DNA METHYLATION REPROGRAMMING IN DORSAL ROOT GANGLION. NERVE INJURY-INDUCED HYPERACTIVITY OF PRIMARY SENSORY NEURONS IN THE DORSAL ROOT GANGLION (DRG) CONTRIBUTES TO CHRONIC PAIN DEVELOPMENT, BUT THE UNDERLYING EPIGENETIC MECHANISMS REMAIN POORLY UNDERSTOOD. HERE WE DETERMINED GENOME-WIDE CHANGES IN DNA METHYLATION IN THE NERVOUS SYSTEM IN NEUROPATHIC PAIN. SPINAL NERVE LIGATION (SNL), BUT NOT PACLITAXEL TREATMENT, IN MALE SPRAGUE DAWLEY RATS INDUCED A CONSISTENT LOW-LEVEL HYPOMETHYLATION IN THE CPG SITES IN THE DRG DURING THE ACUTE AND CHRONIC PHASES OF NEUROPATHIC PAIN. DNA METHYLATION REMODELING IN THE DRG OCCURRED EARLY AFTER SNL AND PERSISTED FOR AT LEAST 3 WEEKS. SNL CAUSED DNA METHYLATION CHANGES AT 8% OF CPG SITES WITH PREVAILING HYPOMETHYLATION OUTSIDE OF CPG ISLANDS, IN INTRONS, INTERGENIC REGIONS, AND REPETITIVE SEQUENCES. IN CONTRAST, SNL CAUSED MORE GAINS OF METHYLATION IN THE SPINAL CORD AND PREFRONTAL CORTEX. THE DNA METHYLATION CHANGES IN THE INJURED DRGS RECAPITULATED DEVELOPMENTAL REPROGRAMMING AT THE NEONATAL STAGE. METHYLATION REPROGRAMMING WAS CORRELATED WITH INCREASED GENE EXPRESSION VARIABILITY. A DIET DEFICIENT IN METHYL DONORS INDUCED HYPOMETHYLATION AND PAIN HYPERSENSITIVITY. INTRATHECAL ADMINISTRATION OF THE DNA METHYLTRANSFERASE INHIBITOR RG108 CAUSED LONG-LASTING PAIN HYPERSENSITIVITY. DNA METHYLATION REPROGRAMMING IN THE DRG THUS CONTRIBUTES TO NERVE INJURY-INDUCED CHRONIC PAIN. RESTORING DNA METHYLATION MAY REPRESENT A NEW THERAPEUTIC APPROACH TO TREAT NEUROPATHIC PAIN.SIGNIFICANCE STATEMENT EPIGENETIC MECHANISMS ARE CRITICALLY INVOLVED IN THE TRANSITION FROM ACUTE TO CHRONIC PAIN AFTER NERVE INJURY. HOWEVER, GENOME-WIDE CHANGES IN DNA METHYLATION IN THE NERVOUS SYSTEM AND THEIR ROLES IN NEUROPATHIC PAIN DEVELOPMENT REMAIN UNCLEAR. HERE WE USED DIGITAL RESTRICTION ENZYME ANALYSIS OF METHYLATION TO QUANTITATIVELY DETERMINE GENOME-WIDE DNA METHYLATION CHANGES CAUSED BY NERVE INJURY. WE SHOWED THAT NERVE INJURY CAUSED DNA METHYLATION CHANGES AT 8% OF CPG SITES WITH PREVAILING HYPOMETHYLATION OUTSIDE OF CPG ISLANDS IN THE DORSAL ROOT GANGLION. REDUCING DNA METHYLATION INDUCED PAIN HYPERSENSITIVITY, WHEREAS INCREASING DNA METHYLATION ATTENUATED NEUROPATHIC PAIN. THESE FINDINGS EXTEND OUR UNDERSTANDING OF THE EPIGENETIC MECHANISM OF CHRONIC NEUROPATHIC PAIN AND SUGGEST NEW STRATEGIES TO TREAT NERVE INJURY-INDUCED CHRONIC PAIN. 2018 6 1631 35 DNMT3A METHYLATION IN NEUROPATHIC PAIN. BACKGROUND: MU OPIOID RECEPTOR (MOR) PLAYS A CRUCIAL ROLE IN MEDIATING ANALGESIC EFFECTS OF OPIOIDS AND IS CLOSELY ASSOCIATED WITH THE PATHOLOGIES OF NEUROPATHIC PAIN. PREVIOUS STUDIES HAVE REPORTED THAT PERIPHERAL NERVE INJURY DOWNREGULATES MOR EXPRESSION, BUT THE EPIGENETIC MECHANISMS REMAIN UNKNOWN. OBJECTIVE: THEREFORE, WE INVESTIGATED DNA METHYLTRANSFERASE3A (DNMT3A) EXPRESSION OR METHYLATION CHANGES WITHIN MOR PROMOTER IN THE SPINAL CORD IN A NEUROPATHIC PAIN INDUCED BY A CHRONIC CONSTRICTION INJURY (CCI) MOUSE MODEL AND FURTHER DETERMINED WHETHER THESE INJURY-ASSOCIATED CHANGES ARE REVERSIBLE BY PHARMACOLOGICAL INTERVENTIONS. METHODS: A CCI MOUSE MODEL WAS ESTABLISHED AND TISSUE SPECIMENS OF LUMBAR SPINAL CORDS WERE COLLECTED. THE NOCICEPTION THRESHOLD WAS EVALUATED BY A MODEL HEATED 400 BASE. DNMT3A AND MOR MRNA AND PROTEIN LEVEL WERE DETECTED BY REAL-TIME-POLYMERASE CHAIN REACTION AND WESTERN BLOT, RESPECTIVELY. METHYLATION OF DNMT3A GENE WAS MEASURED BY METHYLATION-SPECIFIC PCR. RESULTS: OUR DATA SHOWED THAT CHRONIC NERVE INJURY LED TO A SIGNIFICANT UPREGULATION OF DNMT3A EXPRESSION THAT WAS ASSOCIATED WITH INCREASED METHYLATION OF MOR GENE PROMOTER AND DECREASED MOR PROTEIN EXPRESSION IN THE SPINAL CORD. INHIBITION OF DNMT3A CATALYTIC ACTIVITY WITH DNMT INHIBITOR RG108 SIGNIFICANTLY BLOCKED THE INCREASE IN METHYLATION OF THE MOR PROMOTER, AND THEN UPREGULATED MOR EXPRESSION AND ATTENUATED THERMAL HYPERALGESIA IN NEUROPATHIC PAIN MICE. CONCLUSION: THIS STUDY DEMONSTRATES THAT AN INCREASE OF DNMT3A EXPRESSION AND MOR METHYLATION EPIGENETICALLY PLAY AN IMPORTANT ROLE IN NEUROPATHIC PAIN. TARGETING DNMT3A TO THE PROMOTER OF MOR GENE BY DNMT INHIBITOR MAY BE A PROMISING APPROACH TO THE DEVELOPMENT OF NEW NEUROPATHIC PAIN THERAPY. 2017 7 1559 31 DNA METHYLATION MODULATES NOCICEPTIVE SENSITIZATION AFTER INCISION. DNA METHYLATION IS A KEY EPIGENETIC MECHANISM CONTROLLING DNA ACCESSIBILITY AND GENE EXPRESSION. BLOCKADE OF DNA METHYLATION CAN SIGNIFICANTLY AFFECT PAIN BEHAVIORS IMPLICATED IN NEUROPATHIC AND INFLAMMATORY PAIN. HOWEVER, THE ROLE OF DNA METHYLATION WITH REGARD TO POSTOPERATIVE PAIN HAS NOT YET BEEN EXPLORED. IN THIS STUDY WE SOUGHT TO INVESTIGATE THE ROLE OF DNA METHYLATION IN MODULATING INCISIONAL PAIN AND IDENTIFY POSSIBLE TARGETS UNDER DNA METHYLATION AND CONTRIBUTING TO INCISIONAL PAIN. DNA METHYLTRANFERASE (DNMT) INHIBITOR 5-AZA-2'-DEOXYCYTIDINE SIGNIFICANTLY REDUCED INCISION-INDUCED MECHANICAL ALLODYNIA AND THERMAL SENSITIVITY. AZA-2'-DEOXYCYTIDINE ALSO REDUCED HINDPAW SWELLING AFTER INCISION, SUGGESTING AN ANTI-INFLAMMATORY EFFECT. GLOBAL DNA METHYLATION AND DNMT3B EXPRESSION WERE INCREASED IN SKIN AFTER INCISION, BUT NONE OF DNMT1, DNMT3A OR DNMT3B WAS ALTERED IN SPINAL CORD OR DRG. THE EXPRESSION OF PROOPIOMELANOCORTIN POMC ENCODING BETA-ENDORPHIN AND OPRM1 ENCODING THE MU-OPIOID RECEPTOR WERE UPREGULATED PERIPHERALLY AFTER INCISION; MOREOVER, OPRM1 EXPRESSION WAS FURTHER INCREASED UNDER DNMT INHIBITOR TREATMENT. FINALLY, LOCAL PERIPHERAL INJECTION OF THE OPIOID RECEPTOR ANTAGONIST NALOXONE SIGNIFICANTLY EXACERBATED INCISION-INDUCED MECHANICAL HYPERSENSITIVITY. THESE RESULTS SUGGEST THAT DNA METHYLATION IS FUNCTIONALLY RELEVANT TO INCISIONAL NOCICEPTIVE SENSITIZATION, AND THAT MU-OPIOID RECEPTOR SIGNALING MIGHT BE ONE METHYLATION REGULATED PATHWAY CONTROLLING SENSITIZATION AFTER INCISION. 2015 8 4618 24 NERVE INJURY-INDUCED EPIGENETIC SILENCING OF OPIOID RECEPTORS CONTROLLED BY DNMT3A IN PRIMARY AFFERENT NEURONS. OPIOIDS ARE THE GOLD STANDARD FOR PHARMACOLOGICAL TREATMENT OF NEUROPATHIC PAIN, BUT THEIR ANALGESIC EFFECTS ARE UNSATISFACTORY IN PART DUE TO NERVE INJURY-INDUCED DOWNREGULATION OF OPIOID RECEPTORS IN DORSAL ROOT GANGLIA (DRG) NEURONS. HOW NERVE INJURY DRIVES SUCH DOWNREGULATION REMAINS ELUSIVE. DNA METHYLTRANSFERASE (DNMT)-TRIGGERED DNA METHYLATION REPRESSES GENE EXPRESSION. WE SHOW HERE THAT BLOCKING THE NERVE INJURY-INDUCED INCREASE IN DRG DNMT3A (A DE NOVO DNMT) RESCUED THE EXPRESSION OF OPRM1 AND OPRK1 MRNAS AND THEIR RESPECTIVE ENCODING MU-OPIOID RECEPTOR (MOR) AND KAPPA-OPIOID RECEPTOR (KOR) PROTEINS IN THE INJURED DRG. BLOCKING THIS INCREASE ALSO PREVENTED THE NERVE INJURY-INDUCED INCREASE IN DNA METHYLATION IN THE PROMOTER AND 5'-UNTRANSLATED REGION OF THE OPRM1 GENE IN THE INJURED DRG, RESTORED MORPHINE OR LOPERAMIDE (A PERIPHERAL ACTING MOR PREFERRING AGONIST) ANALGESIC EFFECTS, AND ATTENUATED THE DEVELOPMENT OF THEIR ANALGESIC TOLERANCE UNDER NEUROPATHIC PAIN CONDITIONS. MIMICKING THIS INCREASE REDUCED THE EXPRESSION OF OPRM1 AND OPRK1 MRNAS AND THEIR CODING MOR AND KOR IN DRG AND AUGMENTED MOR-GATED NEUROTRANSMITTER RELEASE FROM THE PRIMARY AFFERENTS. MECHANISTICALLY, DNMT3A REGULATION OF OPRM1 GENE EXPRESSION REQUIRED THE METHYL-CPG-BINDING PROTEIN 1, MBD1, AS MBD1 KNOCKOUT RESULTED IN THE DECREASED BINDING OF DNMT3A TO THE OPRM1 GENE PROMOTER AND BLOCKED THE DNMT3A-TRIGGERED REPRESSION OF OPRM1 GENE EXPRESSION IN DRG NEURONS. THESE DATA SUGGEST THAT DNMT3A IS REQUIRED FOR NERVE INJURY-INDUCED AND MBD1-MEDIATED EPIGENETIC SILENCING OF THE MOR AND KOR IN THE INJURED DRG. DNMT3A INHIBITION MAY SERVE AS A PROMISING ADJUVANT THERAPY FOR OPIOID USE IN NEUROPATHIC PAIN MANAGEMENT. 2017 9 4615 26 NERVE INJURY DIMINISHES OPIOID ANALGESIA THROUGH LYSINE METHYLTRANSFERASE-MEDIATED TRANSCRIPTIONAL REPRESSION OF MU-OPIOID RECEPTORS IN PRIMARY SENSORY NEURONS. THE MU-OPIOID RECEPTOR (MOR, ENCODED BY OPRM1) AGONISTS ARE THE MAINSTAY ANALGESICS FOR TREATING MODERATE TO SEVERE PAIN. NERVE INJURY CAUSES DOWN-REGULATION OF MORS IN THE DORSAL ROOT GANGLION (DRG) AND DIMINISHES THE OPIOID EFFECT ON NEUROPATHIC PAIN. HOWEVER, THE EPIGENETIC MECHANISMS UNDERLYING THE DIMINISHED MOR EXPRESSION CAUSED BY NERVE INJURY ARE NOT CLEAR. G9A (ENCODED BY EHMT2), A HISTONE 3 AT LYSINE 9 METHYLTRANSFERASE, IS A KEY CHROMATIN REGULATOR RESPONSIBLE FOR GENE SILENCING. IN THIS STUDY, WE DETERMINED THE ROLE OF G9A IN DIMINISHED MOR EXPRESSION AND OPIOID ANALGESIC EFFECTS IN ANIMAL MODELS OF NEUROPATHIC PAIN. WE FOUND THAT NERVE INJURY IN RATS INDUCED A LONG-LASTING REDUCTION IN THE EXPRESSION LEVEL OF MORS IN THE DRG BUT NOT IN THE SPINAL CORD. NERVE INJURY CONSISTENTLY INCREASED THE ENRICHMENT OF THE G9A PRODUCT HISTONE 3 AT LYSINE 9 DIMETHYLATION IN THE PROMOTER OF OPRM1 IN THE DRG. G9A INHIBITION OR SIRNA KNOCKDOWN FULLY REVERSED MOR EXPRESSION IN THE INJURED DRG AND POTENTIATED THE MORPHINE EFFECT ON PAIN HYPERSENSITIVITY INDUCED BY NERVE INJURY. IN MICE LACKING EHMT2 IN DRG NEURONS, NERVE INJURY FAILED TO REDUCE THE EXPRESSION LEVEL OF MORS AND THE MORPHINE EFFECT. IN ADDITION, G9A INHIBITION OR EHMT2 KNOCKOUT IN DRG NEURONS NORMALIZED NERVE INJURY-INDUCED REDUCTION IN THE INHIBITORY EFFECT OF THE OPIOID ON SYNAPTIC GLUTAMATE RELEASE FROM PRIMARY AFFERENT NERVES. OUR FINDINGS INDICATE THAT G9A CONTRIBUTES CRITICALLY TO TRANSCRIPTIONAL REPRESSION OF MORS IN PRIMARY SENSORY NEURONS IN NEUROPATHIC PAIN. G9A INHIBITORS MAY BE USED TO ENHANCE THE OPIOID ANALGESIC EFFECT IN THE TREATMENT OF CHRONIC NEUROPATHIC PAIN. 2016 10 5574 23 ROLE OF MICRORNA-143 IN NERVE INJURY-INDUCED UPREGULATION OF DNMT3A EXPRESSION IN PRIMARY SENSORY NEURONS. PERIPHERAL NERVE INJURY INCREASED THE EXPRESSION OF THE DNA METHYLTRANSFERASE 3A (DNMT3A) MRNA AND ITS ENCODING DNMT3A PROTEIN IN INJURED DORSAL ROOT GANGLIA (DRG). THIS INCREASE IS CONSIDERED AS AN ENDOGENOUS INSTIGATOR IN NEUROPATHIC PAIN GENESIS THROUGH EPIGENETIC SILENCING OF PAIN-ASSOCIATED GENES (SUCH AS OPRM1) IN INJURED DRG. HOWEVER, HOW DRG DNMT3A IS INCREASED FOLLOWING PERIPHERAL NERVE INJURY IS STILL ELUSIVE. WE REPORTED HERE THAT PERIPHERAL NERVE INJURY CAUSED BY THE FIFTH SPINAL NERVE LIGATION (SNL) DOWNREGULATED MICRORNA (MIR)-143 EXPRESSION IN INJURED DRG. THIS DOWNREGULATION WAS REQUIRED FOR SNL-INDUCED DRG DNMT3A INCREASE AS RESCUING MIR-143 DOWNREGULATION THROUGH MICROINJECTION OF MIR-143 MIMICS INTO INJURED DRG BLOCKED THE SNL-INDUCED INCREASE IN DNMT3A AND RESTORED THE SNL-INDUCED DECREASES IN OPRM1 MRNA AND ITS ENCODING MU OPIOID RECEPTOR (MOR) IN INJURED DRG, IMPAIRED SPINAL CORD CENTRAL SENSITIZATION AND NEUROPATHIC PAIN, AND IMPROVED MORPHINE ANALGESIC EFFECTS FOLLOWING SNL. MIMICKING SNL-INDUCED DRG MIR-143 DOWNREGULATION THROUGH DRG MICROINJECTION OF MIR143 INHIBITORS IN NAIVE RATS INCREASED THE EXPRESSION OF DNMT3A AND REDUCED THE EXPRESSION OF OPRM1 MRNA AND MOR IN INJECTED DRG AND PRODUCED NEUROPATHIC PAIN-LIKE SYMPTOMS. THESE FINDINGS SUGGEST THAT MIR-143 IS A NEGATIVE REGULATOR IN DNMT3A EXPRESSION IN THE DRG UNDER NEUROPATHIC PAIN CONDITIONS AND MAY BE A POTENTIAL TARGET FOR THERAPEUTIC MANAGEMENT OF NEUROPATHIC PAIN. 2017 11 3489 33 IDENTIFICATION OF EPIGENETIC INTERACTIONS BETWEEN MICRORNA-30C-5P AND DNA METHYLTRANSFERASES IN NEUROPATHIC PAIN. NEUROPATHIC PAIN IS A PREVALENT AND SEVERE CHRONIC SYNDROME, OFTEN REFRACTORY TO TREATMENT, WHOSE DEVELOPMENT AND MAINTENANCE MAY INVOLVE EPIGENETIC MECHANISMS. WE PREVIOUSLY DEMONSTRATED A CAUSAL RELATIONSHIP BETWEEN MIR-30C-5P UPREGULATION IN NOCICEPTION-RELATED NEURAL STRUCTURES AND NEUROPATHIC PAIN IN RATS SUBJECTED TO SCIATIC NERVE INJURY. FURTHERMORE, A SHORT COURSE OF AN MIR-30C-5P INHIBITOR ADMINISTERED INTO THE CISTERNA MAGNA EXERTS LONG-LASTING ANTIALLODYNIC EFFECTS VIA A TGF-BETA1-MEDIATED MECHANISM. HEREIN, WE SHOW THAT MIR-30C-5P INHIBITION LEADS TO GLOBAL DNA HYPER-METHYLATION OF NEURONS IN THE LUMBAR DORSAL ROOT GANGLIA AND SPINAL DORSAL HORN IN RATS SUBJECTED TO SCIATIC NERVE INJURY. SPECIFICALLY, THE INHIBITION OF MIR-30-5P SIGNIFICANTLY INCREASED THE EXPRESSION OF THE NOVO DNA METHYLTRANSFERASES DNMT3A AND DNMT3B IN THOSE STRUCTURES. FURTHERMORE, WE IDENTIFIED THE MECHANISM AND FOUND THAT MIR-30C-5P TARGETS THE MRNAS OF DNMT3A AND DNMT3B. QUANTITATIVE METHYLATION ANALYSIS REVEALED THAT THE PROMOTER REGION OF THE ANTIALLODYNIC CYTOKINE TGF-BETA1 WAS HYPOMETHYLATED IN THE SPINAL DORSAL HORN OF NERVE-INJURED RATS TREATED WITH THE MIR-30C-5P INHIBITOR, WHILE THE PROMOTER OF NFYC, THE HOST GENE OF MIR-30C-5P, WAS HYPERMETHYLATED. THESE RESULTS ARE CONSISTENT WITH LONG-TERM PROTECTION AGAINST NEUROPATHIC PAIN DEVELOPMENT AFTER NERVE INJURY. ALTOGETHER, OUR RESULTS HIGHLIGHT THE KEY ROLE OF MIR-30C-5P IN THE EPIGENETIC MECHANISMS' UNDERLYING NEUROPATHIC PAIN AND PROVIDE THE BASIS FOR MIR-30C-5P AS A THERAPEUTIC TARGET. 2022 12 4579 18 N(6)-METHYLADENOSINE METHYLASE METTL3 CONTRIBUTES TO NEUROPATHIC PAIN BY EPIGENETIC SILENCING OF MU OPIOID RECEPTOR. WE AIMED AT EXPLORING THE ROLE AND MECHANISM OF METTL3-MEDIATED M(6)A MODIFICATION IN NEUROPATHIC PAIN. MALE SPRAGUE-DAWLEY RATS WERE RANDOMLY DIVIDED INTO FOUR GROUPS: SHAM OPERATION GROUP (SHAM GROUP), CHRONIC CONSTRICTION INJURY (CCI) OF THE SCIATIC NERVE MODEL GROUP (NPP GROUP), INTRATHECAL INJECTION OF VIRUS DOWN-REGULATED METTL3 + CCI MODEL GROUP (M3 + NPP GROUP) AND INTRATHECAL INJECTION OF NEGATIVE CONTROL VIRUS + CCI MODEL GROUP (SCR + NPP GROUP). THE M3 + NPP GROUP AND THE SCR + NPP GROUP WERE INTRATHECALLY INJECTED WITH VIRUS NINETEEN DAYS BEFORE OPERATION. THE PAW WITHDRAWAL MECHANICAL THRESHOLDS AND PAW WITHDRAWAL LATENCY WERE RESPECTIVELY RECORDED ONE DAY BEFORE OPERATION, THREE DAYS, FIVE DAYS AND SEVEN DAYS AFTER OPERATION. THE RATS WERE SACRIFICED ON THE SEVENTH DAY AFTER OPERATION, AND THEIR SPINAL CORD TISSUES WERE TAKEN. THE FROZEN SECTIONS OF RATS WERE PERFORMED TO OBSERVE THE EXPRESSION OF GREEN FLUORESCENT PROTEIN OF THE VIRUS. THE METHYLATION LEVEL OF RNA, THE PROTEIN EXPRESSION OF M(6)A-RELATED ENZYME (METTL3) AND MU OPIOID RECEPTOR (MOR) IN SPINAL CORD TISSUES OF THE FOUR GROUPS WERE MEASURED. DOWNREGULATION OF METTL3 HAD NO EFFECT ON THE OVERALL METHYLATION LEVEL OF THE SPINAL CORD, BUT IT COULD REGULATE THE METHYLATION LEVEL OF THE OPRM1 GENE RNA ENCODING MOR, PARTIALLY RESTORE THE EXPRESSION OF MOR, AND RELIEVE PAIN IN RATS. IN THE PROCESS OF NPP, METTL3 MAY INHIBIT THE EXPRESSION OF MOR BY REGULATING THE METHYLATION LEVEL OF OPRM1 GENE RNA ENCODING MOR, AND ULTIMATELY PROMOTE THE OCCURRENCE AND DEVELOPMENT OF NPP. 2023 13 1654 23 DORSAL ROOT GANGLIA COACTIVATOR-ASSOCIATED ARGININE METHYLTRANSFERASE 1 CONTRIBUTES TO PERIPHERAL NERVE INJURY-INDUCED PAIN HYPERSENSITIVITIES. NEUROPATHIC PAIN IS ASSOCIATED WITH GENE EXPRESSION CHANGES WITHIN THE DORSAL ROOT GANGLION (DRG) AFTER PERIPHERAL NERVE INJURY, WHICH INVOLVES EPIGENETIC MECHANISMS. COACTIVATOR-ASSOCIATED ARGININE METHYLTRANSFERASE 1 (CARM1), AN EPIGENETIC ACTIVATOR, REGULATES GENE TRANSCRIPTIONAL ACTIVITY BY PROTEIN POSTTRANSLATIONAL MODIFICATIONS. HOWEVER, WHETHER CARM1 PLAYS AN ESSENTIAL ROLE IN THE DEVELOPMENT AND MAINTENANCE OF NEUROPATHIC PAIN IS UNKNOWN. WE REPORT HERE THAT PERIPHERAL NERVE INJURY INDUCED THE UPREGULATION OF THE MRNA AND PROTEIN EXPRESSION OF CARM1 IN THE INJURED DRG, AND BLOCKING ITS EXPRESSION THROUGH SMALL INTERFERING RNA (SIRNA) IN THE INJURED DRG ATTENUATED THE DEVELOPMENT AND MAINTENANCE OF NEUROPATHIC PAIN. FURTHERMORE, PHARMACOLOGICAL INHIBITION OF CARM1 MITIGATED PERIPHERAL NERVE INJURY-INDUCED MECHANICAL ALLODYNIA AND THERMAL HYPERALGESIA. GIVEN THAT CARM1 INHIBITION OR KNOCKDOWN ATTENUATED THE INDUCTION AND MAINTENANCE OF NEUROPATHIC PAIN AFTER PERIPHERAL NERVE INJURY, OUR FINDINGS SUGGEST THAT CARM1 MAY SERVE AS A PROMISING THERAPEUTIC TARGET FOR NEUROPATHIC PAIN TREATMENT IN CLINICAL APPLICATIONS. 2018 14 5657 27 SEX-DEPENDENT PRONOCICEPTIVE ROLE OF SPINAL ALPHA(5) -GABA(A) RECEPTOR AND ITS EPIGENETIC REGULATION IN NEUROPATHIC RODENTS. EXTRASYNAPTIC ALPHA(5) -SUBUNIT CONTAINING GABA(A) (ALPHA(5) -GABA(A) ) RECEPTORS PARTICIPATE IN CHRONIC PAIN. PREVIOUSLY, WE REPORTED A SEX DIFFERENCE IN THE ACTION OF ALPHA(5) -GABA(A) RECEPTORS IN DYSFUNCTIONAL PAIN. HOWEVER, THE UNDERLYING MECHANISMS REMAIN UNKNOWN. THE AIM OF THIS STUDY WAS TO EXAMINE THIS SEXUAL DIMORPHISM IN NEUROPATHIC RODENTS AND THE MECHANISMS INVOLVED. FEMALE AND MALE WISTAR RATS OR ICR MICE WERE SUBJECTED TO NERVE INJURY FOLLOWED BY ALPHA(5) -GABA(A) RECEPTOR INVERSE AGONIST INTRATHECAL ADMINISTRATION, L-655,708. THE DRUG PRODUCED AN ANTIALLODYNIC EFFECT IN NERVE-INJURED FEMALE RATS AND MICE, AND A LOWER EFFECT IN MALES. WE HYPOTHESIZED THAT CHANGES IN ALPHA(5) -GABA(A) RECEPTOR, PROBABLY INFLUENCED BY HORMONAL AND EPIGENETIC STATUS, MIGHT UNDERLIE THIS SEX DIFFERENCE. THUS, WE PERFORMED QPCR AND WESTERN BLOT. NERVE INJURY INCREASED ALPHA(5) -GABA(A) MRNA AND PROTEIN IN FEMALE DORSAL ROOT GANGLIA (DRG) AND DECREASED THEM IN DRG AND SPINAL CORD OF MALES. TO INVESTIGATE THE HORMONAL INFLUENCE OVER ALPHA(5) -GABA(A) RECEPTOR ACTIONS, WE PERFORMED NERVE INJURY TO OVARIECTOMIZED RATS AND RECONSTITUTED THEM WITH 17BETA-ESTRADIOL (E2). OVARIECTOMY ABROGATED L-655,708 ANTIALLODYNIC EFFECT AND E2 RESTORED IT. OVARIECTOMY DECREASED ALPHA(5) -GABA(A) RECEPTOR AND ESTROGEN RECEPTOR ALPHA PROTEIN IN DRG OF NEUROPATHIC FEMALE RATS, WHILE E2 ENHANCED THEM. SINCE DNA METHYLATION MIGHT CONTRIBUTE TO ALPHA(5) -GABA(A) RECEPTOR DOWN-REGULATION IN MALES, WE EXAMINED CPG ISLAND DNA METHYLATION OF ALPHA(5) -GABA(A) RECEPTOR CODING GENE THROUGH PYROSEQUENCING. NERVE INJURY INCREASED METHYLATION IN MALE, BUT NOT FEMALE RATS. PHARMACOLOGICAL INHIBITION OF DNA METHYLTRANSFERASES INCREASED ALPHA(5) -GABA(A) RECEPTOR AND ENABLED L-655,708 ANTINOCICEPTIVE EFFECT IN MALE RATS. THESE RESULTS SUGGEST THAT ALPHA(5) -GABA(A) RECEPTOR IS A SUITABLE TARGET TO TREAT CHRONIC PAIN IN FEMALES. 2021 15 3809 28 INTRAPERITONEAL 5-AZACYTIDINE ALLEVIATES NERVE INJURY-INDUCED PAIN IN RATS BY MODULATING DNA METHYLATION. TO INVESTIGATE THE ROLE OF DNA METHYLATION IN MODULATING CHRONIC NEUROPATHIC PAIN (NPP), IDENTIFY POSSIBLE TARGET GENES OF DNA METHYLATION INVOLVED IN THIS PROCESS, AND PRELIMINARILY CONFIRM THE MEDICINAL VALUE OF THE DNA METHYLTRANSFERASES (DNMTS) INHIBITOR 5-AZACYTIDINE (5-AZA) IN NPP BY TARGETING GENE METHYLATION. TWO RAT NPP MODELS, CHRONIC CONSTRICTION INJURY (CCI) AND SPINAL NERVE LIGATION (SNL), WERE USED. THE DNA METHYLATION PROFILES IN THE LUMBAR SPINAL CORD WERE ASSAYED USING AN ARRAYSTAR RAT REFSEQ PROMOTER ARRAY. THE UNDERLYING GENES WITH DIFFERENTIAL METHYLATION WERE THEN IDENTIFIED AND SUBMITTED TO GENE ONTOLOGY AND PATHWAY ANALYSIS. METHYL-DNA IMMUNOPRECIPITATION QUANTITATIVE PCR (MEDIP-QPCR) AND QUANTITATIVE REVERSE TRANSCRIPTION-PCR (RT-QPCR) WERE USED TO CONFIRM GENE METHYLATION AND EXPRESSION. THE PROTECTIVE FUNCTION OF 5-AZA IN NPP AND GENE EXPRESSION WERE EVALUATED VIA BEHAVIORAL ASSAYS AND RT-QPCR, RESPECTIVELY. ANALYSIS OF THE DNA METHYLATION PATTERNS IN THE LUMBAR SPINAL CORD INDICATED THAT 1205 DIFFERENTIALLY METHYLATED FRAGMENTS IN CCI RATS WERE LOCATED WITHIN DNA PROMOTER REGIONS, INCLUDING 638 HYPERMETHYLATED FRAGMENTS AND 567 HYPOMETHYLATED FRAGMENTS. THE METHYLATION LEVELS OF GRM4, HTR4, ADRB2, KCNF1, GAD2, AND PPARG, WHICH ARE ASSOCIATED WITH LONG-TERM POTENTIATION (LTP) AND GLUTAMATERGIC SYNAPSE PATHWAYS, WERE INCREASED WITH A CORRESPONDING DECREASE IN THEIR MRNA EXPRESSION, IN THE SPINAL CORDS OF CCI RATS. MOREOVER, WE FOUND THAT THE INTRAPERITONEAL INJECTION OF 5-AZA (4 MG/KG) ATTENUATED CCI- OR SNL-INDUCED MECHANICAL ALLODYNIA AND THERMAL HYPERALGESIA. FINALLY, THE MRNA EXPRESSION OF HYPERMETHYLATED GENES SUCH AS GRM4, HTR4, ADRB2, KCNF1, AND GAD2 WAS REVERSED AFTER 5-AZA TREATMENT. CCI INDUCED WIDESPREAD METHYLATION CHANGES IN THE DNA PROMOTER REGIONS IN THE LUMBAR SPINAL CORD. INTRAPERITONEAL 5-AZA ALLEVIATED HYPERALGESIA IN CCI AND SNL RATS, AN EFFECT ACCOMPANIED BY THE REVERSED EXPRESSION OF HYPERMETHYLATED GENES. THUS, DNA METHYLATION INHIBITION REPRESENTS A PROMISING EPIGENETIC STRATEGY FOR PROTECTION AGAINST CHRONIC NPP FOLLOWING NERVE INJURY. OUR STUDY LAYS A THEORETICAL FOUNDATION FOR 5-AZA TO BECOME A CLINICAL TARGETED DRUG. 2023 16 2407 26 EPIGENETIC RESTORATION OF VOLTAGE-GATED POTASSIUM CHANNEL KV1.2 ALLEVIATES NERVE INJURY-INDUCED NEUROPATHIC PAIN. VOLTAGE-GATED POTASSIUM CHANNELS (KV) ARE IMPORTANT REGULATORS OF NEURONAL EXCITABILITY FOR ITS ROLE OF REGULATING RESTING MEMBRANE POTENTIAL AND REPOLARIZATION. RECENT STUDIES SHOW THAT KV CHANNELS PARTICIPATE IN NEUROPATHIC PAIN, BUT THE DETAILED UNDERLYING MECHANISMS ARE FAR FROM BEING CLEAR. IN THIS STUDY, WE USED SIRNA, MIR-137 AGOMIR, AND ANTAGOMIR TO REGULATE THE EXPRESSION OF KV1.2 IN SPINAL CORD AND DORSAL ROOT GANGLIA (DRG) OF NAIVE AND CHRONIC CONSTRICTION INJURY (CCI) RATS. KV CURRENTS AND NEURON EXCITABILITY IN DRG NEURONS WERE EXAMINED BY PATCH-CLAMP WHOLE-CELL RECORDING TO VERIFY THE CHANGE IN KV1.2 FUNCTION. THE RESULTS SHOWED THAT KV1.2 WAS DOWN-REGULATED IN DRG AND SPINAL DORSAL HORN (SDH) BY CCI. KNOCKDOWN OF KV1.2 BY INTRATHECALLY INJECTING KCNA2 SIRNA INDUCED SIGNIFICANT MECHANICAL AND THERMAL HYPERSENSITIVITY IN NAIVE RATS. CONCOMITANT WITH THE DOWN-REGULATION OF KV1.2 WAS AN INCREASE IN THE EXPRESSION OF THE MIR-137. THE TARGETING AND REGULATING OF MIR-137 ON KCNA2 WAS VERIFIED BY DUAL-LUCIFERASE REPORTER SYSTEM AND INTRATHECAL INJECTING MIR-137 AGOMIR. FURTHERMORE, RESCUING THE EXPRESSION OF KV1.2 IN CCI RATS, ACHIEVED THROUGH INHIBITING MIR-137, RESTORED THE ABNORMAL KV CURRENTS AND EXCITABILITY IN DRG NEURONS, AND ALLEVIATED MECHANICAL ALLODYNIA AND THERMAL HYPERALGESIA. THESE RESULTS INDICATE THAT THE MIR-137-MEDIATED KV1.2 IMPAIRMENT IS A CRUCIAL ETIOPATHOGENESIS FOR THE NERVE INJURY-INDUCED NEUROPATHIC PAIN AND CAN BE A NOVEL POTENTIAL THERAPEUTIC TARGET FOR NEUROPATHIC PAIN MANAGEMENT. 2021 17 5976 25 TET1-DEPENDENT EPIGENETIC MODIFICATION OF BDNF EXPRESSION IN DORSAL HORN NEURONS MEDIATES NEUROPATHIC PAIN IN RATS. TEN-ELEVEN TRANSLOCATION METHYLCYTOSINE DIOXYGENASE 1 (TET1) MEDIATES THE CONVERSION OF 5-METHYLCYTOSINE (5 MC) TO 5-HYDROXYMETHYLCYTOSINE (5 HMC), HENCE PROMOTING DNA DEMETHYLATION. ALTHOUGH RECENT STUDIES HAVE LINKED THE DNA DEMETHYLATION OF SPECIFIC GENES TO PAIN HYPERSENSITIVITY, THE ROLE OF SPINAL TET1-DEPENDENT DNA DEMETHYLATION IN NOCICEPTION HYPERSENSITIVITY DEVELOPMENT REMAINS ELUSIVE. HERE, WE REPORT CORRELATED WITH BEHAVIORAL ALLODYNIA, SPINAL NERVE LIGATION (SNL) UPREGULATED TET1 EXPRESSION IN DORSAL HORN NEURONS THAT HYDROXYLATE 5 MC TO 5 HMC AT CPG DINUCLEOTIDES IN THE BDNF PROMOTER TO PROMOTE SPINAL BDNF EXPRESSION AT DAY 7 AFTER OPERATION. FOCAL KNOCKDOWN OF SPINAL TET1 EXPRESSION DECREASED TET1 BINDING AND 5 HMC ENRICHMENT, FURTHER INCREASED 5 MC ENRICHMENT AT CPG SITES IN THE BDNF PROMOTER AND DECREASED SPINAL BDNF EXPRESSION ACCOMPANIED BY THE ALLEVIATION OF THE DEVELOPED ALLODYNIA. MOREOVER, AT DAY 7 AFTER OPERATION, SNL-ENHANCED TET1 EXPRESSION ALSO INHIBITED THE BINDING OF DNA METHYLTRANSFERASES (DNMTS, I.E., DNMT1, DNMT3A, AND DNMT3B) TO THE BDNF PROMOTER, A REQUIREMENT FOR TRANSCRIPTIONAL SILENCING BY CATALYSING 5-CYTOSINE (5C) TO 5 MC. TOGETHER, THESE DATA SUGGEST AT CPG SITES OF THE BDNF PROMOTER, SNL-ENHANCED TET1 EXPRESSION PROMOTES DNA DEMETHYLATION BOTH BY CONVERTING 5 MC TO 5 HMC AND INHIBITING DNMT BINDING TO REGULATE SPINAL BDNF EXPRESSION, HENCE CONTRIBUTING TO BEHAVIORAL ALLODYNIA DEVELOPMENT. 2016 18 2351 25 EPIGENETIC REGULATION OF NEWBORNS' IMPRINTED GENES RELATED TO GESTATIONAL GROWTH: PATTERNING BY PARENTAL RACE/ETHNICITY AND MATERNAL SOCIOECONOMIC STATUS. BACKGROUND: CHILDREN BORN TO PARENTS WITH LOWER INCOME AND EDUCATION ARE AT RISK FOR OBESITY AND LATER-LIFE RISK OF COMMON CHRONIC DISEASES, AND EPIGENETICS HAS BEEN HYPOTHESISED TO LINK THESE ASSOCIATIONS. HOWEVER, EPIGENETIC TARGETS ARE UNKNOWN. WE FOCUS ON A CLUSTER OF WELL-CHARACTERISED GENOMICALLY IMPRINTED GENES BECAUSE THEIR MONOALLELIC EXPRESSION IS REGULATED BY DNA METHYLATION AT DIFFERENTIALLY METHYLATED REGIONS (DMRS), ARE CRITICAL IN FETAL GROWTH, AND DNA METHYLATION PATTERNS AT BIRTH HAVE BEEN ASSOCIATED WITH INCREASED RISK OF BIRTH WEIGHT EXTREMES AND OVERWEIGHT STATUS OR OBESITY IN EARLY CHILDHOOD. METHODS: WE MEASURED DNA METHYLATION AT DMRS REGULATING GENOMICALLY IMPRINTED DOMAINS (IGF2/H19, DLK1/MEG3, NNAT AND PLAGL1) USING UMBILICAL CORD BLOOD LEUCOCYTES FROM 619 INFANTS RECRUITED IN DURHAM, NORTH CAROLINA IN 2010-2011. WE EXAMINED DIFFERENCES IN DNA METHYLATION LEVELS BY RACE/ETHNICITY OF BOTH PARENTS, AND THE ROLE THAT MATERNAL SOCIOECONOMIC STATUS (SES) MAY PLAY IN THE ASSOCIATION BETWEEN RACE/ETHNIC EPIGENETIC DIFFERENCES. RESULTS: UNADJUSTED RACE/ETHNIC DIFFERENCES ONLY WERE EVIDENT FOR DMRS REGULATING MEG3 AND IGF2; RACE/ETHNIC DIFFERENCES PERSISTED IN IGF2/H19 AND NNAT AFTER ACCOUNTING FOR INCOME AND EDUCATION. CONCLUSIONS: RESULTS SUGGEST THAT PARENTAL FACTORS MAY NOT ONLY INFLUENCE DNA METHYLATION, BUT ALSO DO SO IN WAYS THAT VARY BY DMR. FINDINGS SUPPORT THE HYPOTHESIS THAT EPIGENETICS MAY LINK THE OBSERVED LOWER SES DURING THE PRENATAL PERIOD AND POOR OUTCOMES SUCH AS LOW BIRTH WEIGHT; LOWER BIRTH WEIGHT HAS PREVIOUSLY BEEN ASSOCIATED WITH ADULT-ONSET CHRONIC DISEASES AND CONDITIONS THAT INCLUDE CARDIOVASCULAR DISEASES, DIABETES, OBESITY AND SOME CANCERS. 2015 19 2756 31 EXPRESSION OF DNA METHYLTRANSFERASES IN ADULT DORSAL ROOT GANGLIA IS CELL-TYPE SPECIFIC AND UP REGULATED IN A RODENT MODEL OF NEUROPATHIC PAIN. NEUROPATHIC PAIN IS ASSOCIATED WITH HYPEREXCITABILITY AND INTRINSIC FIRING OF DORSAL ROOT GANGLIA (DRG) NEURONS. THESE PHENOTYPICAL CHANGES CAN BE LONG LASTING, POTENTIALLY SPANNING THE ENTIRE LIFE OF ANIMAL MODELS, AND DEPEND ON ALTERED EXPRESSION OF NUMEROUS PROTEINS, INCLUDING MANY ION CHANNELS. YET, HOW DRGS MAINTAIN LONG-TERM CHANGES IN PROTEIN EXPRESSION IN NEUROPATHIC CONDITIONS REMAINS UNCLEAR. DNA METHYLATION IS A WELL-KNOWN MECHANISM OF EPIGENETIC CONTROL OF GENE EXPRESSION AND IS ACHIEVED BY THE ACTION OF THREE ENZYMES: DNA METHYLTRANSFERASE (DNMT) 1, 3A, AND 3B, WHICH HAVE BEEN STUDIED PRIMARILY DURING DEVELOPMENT. WE FIRST PERFORMED IMMUNOHISTOCHEMICAL ANALYSIS TO ASSESS WHETHER THESE ENZYMES ARE EXPRESSED IN ADULT RAT DRGS (L4-5) AND FOUND THAT DNMT1 IS EXPRESSED IN BOTH GLIA AND NEURONS, DNMT3A IS PREFERENTIALLY EXPRESSED IN GLIA AND DNMT3B IS PREFERENTIALLY EXPRESSED IN NEURONS. A RAT MODEL OF NEUROPATHIC PAIN WAS THEN USED TO DETERMINE WHETHER NERVE INJURY MAY INDUCE EPIGENETIC CHANGES IN DRGS AT MULTIPLE TIME POINTS AFTER PAIN ONSET. REAL-TIME RT PCR ANALYSIS REVEALED ROBUST AND TIME-DEPENDENT CHANGES IN DNMT TRANSCRIPT EXPRESSION IN IPSILATERAL DRGS FROM SPARED NERVE INJURY (SNI) BUT NOT SHAM RATS. INTERESTINGLY, DNMT3B TRANSCRIPT SHOWED A ROBUST UPREGULATION THAT APPEARED ALREADY 1 WEEK AFTER SURGERY AND PERSISTED AT 4 WEEKS (OUR ENDPOINT); IN CONTRAST, DNMT1 AND DNMT3A TRANSCRIPTS SHOWED ONLY MODERATE UPREGULATION THAT WAS TRANSIENT AND DID NOT APPEAR UNTIL THE SECOND WEEK. WE SUGGEST THAT DNMT REGULATION IN ADULT DRGS MAY BE A CONTRIBUTOR TO THE PAIN PHENOTYPE AND MERITS FURTHER STUDY. 2014 20 5266 34 PROMOTED INTERACTION OF C/EBPALPHA WITH DEMETHYLATED CXCR3 GENE PROMOTER CONTRIBUTES TO NEUROPATHIC PAIN IN MICE. DNA METHYLATION HAS BEEN IMPLICATED IN THE PATHOGENESIS OF CHRONIC PAIN. HOWEVER, THE SPECIFIC GENES REGULATED BY DNA METHYLATION UNDER NEUROPATHIC PAIN CONDITION REMAIN LARGELY UNKNOWN. HERE WE INVESTIGATED HOW CHEMOKINE RECEPTOR CXCR3 IS REGULATED BY DNA METHYLATION AND HOW IT CONTRIBUTES TO NEUROPATHIC PAIN INDUCED BY SPINAL NERVE LIGATION (SNL) IN MICE. SNL INCREASED CXCR3 MRNA AND PROTEIN EXPRESSION IN THE NEURONS OF THE SPINAL CORD. MEANWHILE, THE CPG (5'-CYTOSINE-PHOSPHATE-GUANINE-3') ISLAND IN THE CXCR3 GENE PROMOTER REGION WAS DEMETHYLATED, AND THE EXPRESSION OF DNA METHYLTRANSFERASE 3B (DNMT3B) WAS DECREASED. SNL ALSO INCREASED THE BINDING OF CCAAT (CYTIDINE-CYTIDINE-ADENOSINE-ADENOSINE-THYMIDINE)/ENHANCER BINDING PROTEIN ALPHA (C/EBPALPHA) WITH CXCR3 PROMOTER AND DECREASED THE BINDING OF DNMT3B WITH CXCR3 PROMOTER IN THE SPINAL CORD. C/EBPALPHA EXPRESSION WAS INCREASED IN SPINAL NEURONS AFTER SNL, AND INHIBITION OF C/EBPALPHA BY INTRATHECAL SMALL INTERFERING RNA ATTENUATED SNL-INDUCED PAIN HYPERSENSITIVITY AND REDUCED CXCR3 EXPRESSION. FURTHERMORE, SNL-INDUCED MECHANICAL ALLODYNIA AND HEAT HYPERALGESIA WERE MARKEDLY REDUCED IN CXCR3(-/-) MICE. SPINAL INHIBITION OF CXCR3 BY SHRNA OR CXCR3 ANTAGONIST ALSO ATTENUATED ESTABLISHED NEUROPATHIC PAIN. MOREOVER, CXCL10, THE LIGAND OF CXCR3, WAS INCREASED IN SPINAL NEURONS AND ASTROCYTES AFTER SNL. SUPERFUSING SPINAL CORD SLICES WITH CXCL10 ENHANCED SPONTANEOUS EPSCS AND POTENTIATED NMDA-INDUCED AND AMPA-INDUCED CURRENTS OF LAMINA II NEURONS. FINALLY, INTRATHECAL INJECTION OF CXCL10 INDUCED CXCR3-DEPENDENT PAIN HYPERSENSITIVITY IN NAIVE MICE. COLLECTIVELY, OUR RESULTS DEMONSTRATED THAT CXCR3, INCREASED BY DNA DEMETHYLATION AND THE ENHANCED INTERACTION WITH C/EBPALPHA, CAN BE ACTIVATED BY CXCL10 TO FACILITATE EXCITATORY SYNAPTIC TRANSMISSION AND CONTRIBUTE TO THE MAINTENANCE OF NEUROPATHIC PAIN. SIGNIFICANCE STATEMENT: PERIPHERAL NERVE INJURY INDUCES CHANGES OF GENE EXPRESSION IN THE SPINAL CORD THAT MAY CONTRIBUTE TO THE PATHOGENESIS OF NEUROPATHIC PAIN. CXCR3 IS A CHEMOKINE RECEPTOR. WHETHER IT IS INVOLVED IN NEUROPATHIC PAIN AND HOW IT IS REGULATED AFTER NERVE INJURY REMAIN LARGELY UNKNOWN. OUR STUDY DEMONSTRATES THAT SPINAL NERVE LIGATION DOWNREGULATES THE EXPRESSION OF DNMT3B, WHICH MAY CAUSE DEMETHYLATION OF CXCR3 GENE PROMOTER AND FACILITATE THE BINDING OF CCAAT/ENHANCER BINDING PROTEIN ALPHA WITH CXCR3 PROMOTER AND FURTHER INCREASE CXCR3 EXPRESSION IN SPINAL NEURONS. THE UPREGULATED CXCR3 MAY CONTRIBUTE TO NEUROPATHIC PAIN BY FACILITATING CENTRAL SENSITIZATION. OUR STUDY REVEALS AN EPIGENETIC MECHANISM UNDERLYING CXCR3 EXPRESSION AND ALSO SUGGESTS THAT TARGETING THE EXPRESSION OR ACTIVATION OF CXCR3 SIGNALING MAY OFFER NEW THERAPEUTICS FOR NEUROPATHIC PAIN. 2017