WWL70 protects against chronic constriction injury-induced neuropathic pain in mice by cannabinoid receptor-independent mechanisms.

BACKGROUND: Targeting the endocannabinoid system has emerged as an effective strategy for the treatment of inflammatory and neurological diseases. Unlike the inhibition of the principal 2-arachidonyl glycerol (2-AG) hydrolytic enzyme monoacylglycerol lipase (MAGL), which leads to 2-AG overload and cannabinoid receptor desensitization, selective inhibition of the minor 2-AG hydrolytic enzyme alpha, beta-hydrolase domain 6 (ABHD6) can provide therapeutic benefits without producing cannabimimetic side effects. We have shown that inhibition of ABHD6 significantly reduces neuroinflammation and exerts neuroprotection in animal models of traumatic brain injury and multiple sclerosis. However, the role of ABHD6 inhibition on neuropathic pain has not been explored. METHODS: Neuropathic pain was induced by chronic constriction injury (CCI) of the mouse sciatic nerve and examined by Hargreaves and Von Frey tests. Activation of inflammatory cells and the production of cytokines and chemokines in the spinal cord dorsal horn, dorsal root ganglion (DRG), and sciatic nerve were assessed by qRT-PCR, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. The levels of 2-AG and arachidonic acid (AA) in sciatic nerve were quantified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). RESULTS: Treatment with the selective ABHD6 inhibitor WWL70 significantly alleviated CCI-induced thermal hyperalgesia and mechanical allodynia. Microglia activation, macrophage infiltration, and the production of nociceptive mediators were reduced in the ipsilateral lumbar spinal cord dorsal horn, DRG, and sciatic nerve of WWL70-treated animals. The diminished cytokine and chemokine production is likely due to the inhibitory effect of WWL70 on NF-κB phosphorylation. Surprisingly, the anti-nociceptive and anti-inflammatory effects of WWL70 were not reversed by addition of the cannabinoid receptor antagonists. Treatment with WWL70 did not alter the levels of 2-AG, AA, and the phosphorylation of cytosolic phospholipase A2 (cPLA2), but significantly reduced the production of prostaglandin E2 (PGE2) and the expression of cyclooxygenase-2 (COX-2) and prostaglandin E synthase-2 (PGES2) in the injured sciatic nerve. CONCLUSIONS: This study reveals a novel mechanism for the antinociceptive effect of the 2-AG catabolic enzyme ABHD6 inhibitor WWL70. Understanding the interaction between endocannabinoid and eicosanoid pathways might provide a new avenue for the treatment of inflammatory and neuropathic pain.

WWL70 attenuates PGE2 production derived from 2-arachidonoylglycerol in microglia by ABHD6-independent mechanism.

BACKGROUND: α/ß-Hydrolase domain 6 (ABHD6) is one of the major enzymes for endocannabinoid 2-arachidonoylglycerol (2-AG) hydrolysis in microglia cells. Our recent studies have shown that a selective ABHD6 inhibitor WWL70 has anti-inflammatory and neuroprotective effects in animal models of traumatic brain injury and multiple sclerosis. However, the role of ABHD6 in the neuroinflammatory response and the mechanisms by which WWL70 suppresses inflammation has not yet been elucidated in reactive microglia. METHODS: The hydrolytic activity and the levels of 2-AG in BV2 cells were measured by radioactivity assay and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) synthases in microglia treated with lipopolysaccharide (LPS) with/without WWL70 was determined by western blot and quantitative RT-PCR. The conversion of 2-AG to PGE2 or PGE2-glyceryl ester (PGE2-G) was assessed by enzyme-linked immunoassay (EIA) or LC-MS/MS. The involvement of ABHD6 in PGE2 production was assessed using pharmacological inhibitors and small interfering RNA (siRNA). The effect of WWL70 on PGE2 biosynthesis activity in the microsome fraction from BV2 cells and experimental autoimmune encephalopathy (EAE) mouse brain was also examined. RESULTS: We found that WWL70 suppressed PGE2 production in LPS-activated microglia via cannabinoid receptor-independent mechanisms, although intracellular levels of 2-AG were elevated by WWL70 treatment. This reduction was not attributable to WWL70 inhibition of ABHD6, given the fact that downregulation of ABHD6 by siRNA or use of KT182, an alternative ABHD6 inhibitor failed to suppress PGE2 production. WWL70 attenuated the expression of COX-2 and PGES-1/2 leading to the downregulation of the biosynthetic pathways of PGE2 and PGE2-G. Moreover, PGE2 production from arachidonic acid was reduced in the microsome fraction, indicating that WWL70 also targets PGE2 biosynthetic enzymes, which are likely to contribute to the therapeutic mechanisms of WWL70 in the EAE mouse model. CONCLUSIONS: WWL70 is an anti-inflammatory therapeutic agent capable of inhibiting PGE2 and PGE2-G production, primarily due to its reduction of COX-2 and microsomal PGES-1/2 expression and their PGE2 biosynthesis activity in microglia cells, as well as in the EAE mouse brain.

Therapeutic Effects of Combined Treatment with the AEA Hydrolysis Inhibitor PF04457845 and the Substrate Selective COX-2 Inhibitor LM4131 in the Mouse Model of Neuropathic Pain.

Chronic neuropathic pain resulting from peripheral nerve damage is a significant clinical problem, which makes it imperative to develop the mechanism-based therapeutic approaches. Enhancement of endogenous cannabinoids by blocking their hydrolysis has been shown to reduce inflammation and neuronal damage in a number of neurological disorders and neurodegenerative diseases. However, recent studies suggest that inhibition of their hydrolysis can shift endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide (AEA) toward the oxygenation pathway mediated by cyclooxygenase-2 (COX-2) to produce proinflammatory prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs). Thus, blocking both endocannabinoid hydrolysis and oxygenation is likely to be more clinically beneficial. In this study, we used the chronic constriction injury (CCI) mouse model to explore the therapeutic effects of simultaneous inhibition of AEA hydrolysis and oxygenation in the treatment of neuropathic pain. We found that the fatty acid amide hydrolase (FAAH) inhibitor PF04457845 and the substrate-selective COX-2 inhibitor LM4131 dose-dependently reduced thermal hyperalgesia and mechanical allodynia in the CCI mice. In addition to ameliorating the pain behaviors, combined treatment with subeffective doses of these inhibitors greatly attenuated the accumulation of inflammatory cells in both sciatic nerve and spinal cord. Consistently, the increased proinflammatory cytokines IL-1ß, IL-6, and chemokine MCP-1 in the CCI mouse spinal cord and sciatic nerve were also significantly reduced by combination of low doses of PF04457845 and LM4131 treatment. Therefore, our study suggests that simultaneous blockage of endocannabinoid hydrolysis and oxygenation by using the substrate-selective COX-2 inhibitor, which avoids the cardiovascular and gastrointestinal side effects associated with the use of general COX-2 inhibitors, might be a suitable strategy for the treatment of inflammatory and neuropathic pain.

Preclinical Studies of Posttraumatic Headache and the Potential Therapeutics.

Posttraumatic headache (PTH) attributed to traumatic brain injury (TBI) is a secondary headache developed within 7 days after head injury, and in a substantial number of patients PTH becomes chronic and lasts for more than 3 months. Current medications are almost entirely relied on the treatment of primary headache such as migraine, due to its migraine-like phenotype and the limited understanding on the PTH pathogenic mechanisms. To this end, increasing preclinical studies have been conducted in the last decade. We focus in this review on the trigeminovascular system from the animal studies since it provides the primary nociceptive sensory afferents innervating the head and face region, and the pathological changes in the trigeminal pathway are thought to play a key role in the development of PTH. In addition to the pathologies, PTH-like behaviors induced by TBI and further exacerbated by nitroglycerin, a general headache inducer through vasodilation are reviewed. We will overview the current pharmacotherapies including calcitonin gene-related peptide (CGRP) monoclonal antibody and sumatriptan in the PTH animal models. Given that modulation of the endocannabinoid (eCB) system has been well-documented in the treatment of migraine and TBI, the therapeutic potential of eCB in PTH will also be discussed.

Quantification of oxidized levels of specific RNA species using an aldehyde reactive probe.

Emerging evidence has shown that oxidation of RNA, including messenger RNA (mRNA), is elevated in several age-related diseases, although investigation of oxidized levels of individual RNA species has been limited. Recently we reported that an aldehyde reactive probe (ARP) quantitatively reacts with oxidatively modified depurinated/depyrimidinated (abasic) RNA. Here we report a novel method to isolate oxidized RNA using ARP and streptavidin beads. An oligo RNA containing abasic sites that were derivatized with ARP was pulled down by streptavidin beads, whereas a control oligo RNA was not. In vitro oxidized RNA, as well as total cellular RNA, isolated from oxidatively stressed cells was also pulled down, dependent on oxidation level, and concentrated in the pull-down fraction. Quantitative reverse transcription polymerase chain reaction (RT-PCR) using RNA in the pull-down fraction demonstrated that several gene transcripts were uniquely increased in the fraction by oxidative stress. Thus, our method selectively concentrates oxidized RNA by pull-down and enables the assessment of oxidation levels of individual RNA species.

Oxidative Modifications of RNA and Its Potential Roles in Biosystem.

Elevated level of oxidized RNA was detected in vulnerable neurons in Alzheimer patients. Subsequently, several diseases and pathological conditions were reported to be associated with RNA oxidation. In addition to several oxidized derivatives, cross-linking and unique strand breaks are generated by RNA oxidation. With a premise that dysfunctional RNA mediated by oxidation is the pathogenetic molecular mechanism, intensive investigations have revealed the mechanism for translation errors, including premature termination, which gives rise to aberrant polypeptides. To this end, we and others revealed that mRNA oxidation could compromise its translational activity and fidelity. Under certain conditions, oxidized RNA can also induce several signaling pathways, to mediate inflammatory response and induce apoptosis. In this review, we focus on the oxidative modification of RNA and its resulting effect on protein synthesis as well as cell signaling. In addition, we will also discuss the potential roles of enzymatic oxidative modification of RNA in mediating cellular effects.

The Novel Monoacylglycerol Lipase Inhibitor MJN110 Suppresses Neuroinflammation, Normalizes Synaptic Composition and Improves Behavioral Performance in the Repetitive Traumatic Brain Injury Mouse Model.

Modulation of the endocannabinoid system has emerged as an effective approach for the treatment of many neurodegenerative and neuropsychological diseases. However, the underlying mechanisms are still uncertain. Using a repetitive mild traumatic brain injury (mTBI) mouse model, we found that there was an impairment in locomotor function and working memory within two weeks post-injury, and that treatment with MJN110, a novel inhibitor of the principal 2-arachidononyl glycerol (2-AG) hydrolytic enzyme monoacylglycerol lipase dose-dependently ameliorated those behavioral changes. Spatial learning and memory deficits examined by Morris water maze between three and four weeks post-TBI were also reversed in the drug treated animals. Administration of MJN110 selectively elevated the levels of 2-AG and reduced the production of arachidonic acid (AA) and prostaglandin E2 (PGE2) in the TBI mouse brain. The increased production of proinflammatory cytokines, accumulation of astrocytes and microglia in the TBI mouse ipsilateral cerebral cortex and hippocampus were significantly reduced by MJN110 treatment. Neuronal cell death was also attenuated in the drug treated animals. MJN110 treatment normalized the expression of the NMDA receptor subunits NR2A and NR2B, the AMPA receptor subunits GluR1 and GluR2, and the GABAA receptor subunits α1, ß2,3 and γ2, which were all reduced at 1, 2 and 4 weeks post-injury. The reduced inflammatory response and restored glutamate and GABA receptor expression likely contribute to the improved motor function, learning and memory in the MJN110 treated animals. The therapeutic effects of MJN110 were partially mediated by activation of CB1 and CB2 cannabinoid receptors and were eliminated when it was co-administered with DO34, a novel inhibitor of the 2-AG biosynthetic enzymes. Our results suggest that augmentation of the endogenous levels of 2-AG can be therapeutically useful in the treatment of TBI by suppressing neuroinflammation and maintaining the balance between excitatory and inhibitory neurotransmission.

Endocannabinoid Modulation of Microglial Phenotypes in Neuropathology.

Microglia, the resident immune cells of the central nervous system, mediate brain homeostasis by controlling neuronal proliferation/differentiation and synaptic activity. In response to external signals from neuropathological conditions, homeostatic (M0) microglia can adopt one of two activation states: the classical (M1) activation state, which secretes mediators of the proinflammatory response, and the alternative (M2) activation state, which presumably mediates the resolution of neuroinflammation and tissue repair/remodeling. Since chronic inflammatory activation of microglia is correlated with several neurodegenerative diseases, functional modulation of microglial phenotypes has been considered as a potential therapeutic strategy. The endocannabinoid (eCB) system, composed of cannabinoid receptors and ligands and their metabolic/biosynthetic enzymes, has been shown to activate anti-inflammatory signaling pathways that modulate immune cell functions. Growing evidence has demonstrated that endogenous, synthetic, and plant-derived eCB agonists possess therapeutic effects on several neuropathologies; however, the molecular mechanisms that mediate the anti-inflammatory effects have not yet been identified. Over the last decade, it has been revealed that the eCB system modulates microglial activation and population. In this review, we thoroughly examine recent studies on microglial phenotype modulation by eCB in neuroinflammatory and neurodegenerative disease conditions. We hypothesize that cannabinoid 2 receptor (CB2R) signaling shifts the balance of expression between neuroinflammatory (M1-type) genes, neuroprotective (M2-type) genes, and homeostatic (M0-type) genes toward the latter two gene expressions, by which microglia acquire therapeutic functionality.

Anti-Inflammatory Effects by Pharmacological Inhibition or Knockdown of Fatty Acid Amide Hydrolase in BV2 Microglial Cells.

Fatty acid amide hydrolase (FAAH) has been recognized as a therapeutic target for several neurological diseases because its inhibition can exert neuroprotective and anti-inflammatory effects by boosting the endogenous levels of N-acylethanolamines. However, previous studies have shown inconsistent results by pharmacological inhibition and genetic deletion of FAAH in response to inflammation. In this study we used two inhibitors, PF3845 and URB597, together with siRNA knockdown to characterize further the effects of FAAH inhibition in BV2 microglial cells. Treatment with PF3845 suppressed lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) production, and down-regulated cyclooxygenase-2 and microsomal PGE synthase. PF3845 reduced the expression of pro-inflammatory cytokines but had no effect on the expression of anti-inflammatory cytokines. The anti-inflammatory effects of URB597 were not as potent as those of PF3845. Knockdown of FAAH also suppressed PGE2 production and pro-inflammatory gene expression. Interestingly, FAAH knockdown enhanced expression of anti-inflammatory molecules in both the absence and presence of LPS treatment. The anti-inflammatory effects of FAAH inhibition and knockdown were not affected by the cannabinoid receptor antagonists or the peroxisome proliferator-activated receptor (PPAR) antagonists. Although inhibition and knockdown of FAAH have potent anti-inflammatory effects and possibly lead to the dynamic change of microglial gene regulation, the underlying mechanisms remain to be elucidated.

Therapeutic Effect of a Novel Fatty Acid Amide Hydrolase Inhibitor PF04457845 in the Repetitive Closed Head Injury Mouse Model.

Concussive traumatic brain injury (TBI) is the predominant type of brain injury in young adults and is a risk factor for the development of chronic traumatic encephalopathy and other neurodegenerative diseases late in life. Using a repetitive closed head injury mouse model, we found that treatment with PF04457845, a novel fatty acid amide hydrolase (FAAH) inhibitor that selectively elevated the brain levels of anandamide, improved locomotor function, learning, and memory in TBI mice examined by beam walk, Y-maze, and Morris water maze tests. The accumulation of microglia and astrocytes and the expression of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor alpha (TNF-α), in the ipsilateral TBI mouse cortex and hippocampus were significantly reduced by drug treatment. The increased expression of amyloid precursor protein (APP), phosphorylated Tau (p-Tau), phosphorylated glycogen synthase kinase 3 beta (pGSK3ß) and p35/p25 subunits and the decreased expression of the pre-synaptic proteins, synaptophysin, synaptosome-associated protein of 25 kDa (SNAP25), and cysteine string protein alpha (α-CSP), in TBI mouse brain were also normalized by PF04458745 treatment. The improved locomotor function and working memory were partially mediated by activation of both cannabinoid (CB)1 and CB2 receptors, whereas the improvement on spatial learning and memory seemed to be CB1 receptor dependent. Interestingly, the blockage of PF04457845 on the reduced expression of synaptophysin, but not SNAP25 and α-CSP, was reversed by coadministration of the CB1 receptor antagonist. These results suggest that the therapeutic effect of PF04457845 is mediated by both cannabinoid receptor dependent and independent mechanisms, and selective inhibition of FAAH possesses a great potential for the treatment of TBI.

Region- and time-dependent gene regulation in the amygdala and anterior cingulate cortex of a PTSD-like mouse model.

Posttraumatic stress disorder is developed by exposure to a threatening and/or a horrifying event and characterized by the presence of anxiety, hyperarousal, avoidance, and sleep abnormality for a prolonged period of time. To elucidate the potential molecular mechanisms, we constructed a mouse model by electric foot shock followed by situational reminders and performed transcriptome analysis in brain tissues. The stressed mice acquired anxiety-like behavior after 2 weeks and exaggerated startle response after 4 weeks. Avoidance latency and freezing behavior were sustained up to 5 weeks post stress and abnormal static behavior was observed during the sleep period. RNA sequencing was performed in two of the emotional regulatory regions, anterior cingulate cortex and amygdala, at 2 and 5 weeks post stress. More than 1000 differentially expressed genes were identified at 2 weeks in both regions. The number of the regulated genes remained constant in amygdala at 5 weeks post stress, whereas those in anterior cingulate cortex were plummeted. Although synaptic remodeling and endocrine system were the most enriched signaling pathways in both anterior cingulate cortex and amygdala, the individual gene expression profile was regulated in a region- and time-dependent manner. In addition, several genes associated with PTSD involved in Hypothalamic-Pituitary-Adrenal axis were differentially regulated. These findings suggested that global gene expression profile was dynamically regulated in accordance with the disease development stage, and therefore targeting the distinct signaling molecules in different region and development stage might be critical for effective treatment to PTSD.

Correction to: Region- and time-dependent gene regulation in the amygdala and anterior cingulate cortex of a PTSD-like mouse model.

Following publication of the original article [1], the authors reported that one of the authors' names was spelled incorrectly.

Therapeutic Effect of the Substrate-Selective COX-2 Inhibitor IMMA in the Animal Model of Chronic Constriction Injury.

Enhancement of endocannabinoid signaling has emerged as an attractive strategy for the treatment of pain. In addition to the well-characterized hydrolytic pathways, cyclooxygenase-2 (COX-2) mediated oxygenation is thought to be an alternative route for endocannabinoid metabolism and therefore provides a new avenue for drug intervention. In this study, we examined the therapeutic effect of indomethacin morpholinamide (IMMA), a novel substrate-selective COX-2 inhibitor, in the chronic constriction injury (CCI) mouse model. Treatment with IMMA significantly alleviated hyperalgesia and mechanical allodynia demonstrated by increased thermal withdrawal latency in Hargreaves test and tactile thresholds in Von Frey test. Accumulation of astrocytes and microglia in spinal cord dorsal horn and infiltration of macrophages into the dorsal root ganglion and sciatic nerve were reduced by drug treatment. Co-administration of the CB2 receptor antagonist, but not the CB1 receptor antagonist partially reversed the inhibitory effect of IMMA on pain sensitivity and inflammatory infiltrates. IMMA downregulated the mRNA expression of TNF-α and IL-1ß and the production of IL-6 and MCP-1 proteins in the ipsilateral sciatic nerve. The enhanced NF-κB DNA binding activity in the CCI mouse dorsal spinal cord was also significantly reduced, suggesting that inactivation of NF-κB contributes to the anti-inflammatory property of IMMA. However, different from the previous reports showing that IMMA can increase endocannabinoids without interfering with arachidonic acid metabolism, treatment with IMMA failed to elevate the endogenous levels of AEA and 2-AG, but significantly reduced the production of prostaglandin E2 (PGE2). Furthermore, the mRNA expression of enzymes involved in PGE2 production, COX-2 and prostaglandin E synthase 2 in the ipsilateral sciatic nerve was also suppressed by IMMA treatment. Taken together, these results suggested that IMMA might exert anti-nociceptive effects through multiple mechanisms which include, but are not limited to, CB2 receptor activation and reduced PGE2 production.

Mammalian APE1 controls miRNA processing and its interactome is linked to cancer RNA metabolism.

Mammalian apurinic/apyrimidinic endonuclease 1 is a DNA repair enzyme involved in genome stability and expression of genes involved in oxidative stress responses, tumor progression and chemoresistance. However, the molecular mechanisms underlying the role of apurinic/apyrimidinic endonuclease 1 in these processes are still unclear. Recent findings point to a novel role of apurinic/apyrimidinic endonuclease 1 in RNA metabolism. Through the characterization of the interactomes of apurinic/apyrimidinic endonuclease 1 with RNA and other proteins, we demonstrate here a role for apurinic/apyrimidinic endonuclease 1 in pri-miRNA processing and stability via association with the DROSHA-processing complex during genotoxic stress. We also show that endonuclease activity of apurinic/apyrimidinic endonuclease 1 is required for the processing of miR-221/222 in regulating expression of the tumor suppressor PTEN. Analysis of a cohort of different cancers supports the relevance of our findings for tumor biology. We also show that apurinic/apyrimidinic endonuclease 1 participates in RNA-interactomes and protein-interactomes involved in cancer development, thus indicating an unsuspected post-transcriptional effect on cancer genes.APE1 plays an important role in the cellular response to oxidative stress, and mutations are linked to tumor progression and chemoresistance. Here, the authors characterize the interactions of APE1 with RNA and demonstrate a role in microRNA processing.

Efficacy of N-Acetylserotonin and Melatonin in the EAE Model of Multiple Sclerosis.

Melatonin and N-acetylserotonin (NAS) are tryptophan metabolites that have potent anti-oxidant, anti-inflammatory and neuroprotective properties in several animal models of neurological injury and disease including multiple sclerosis (MS). The therapeutic effect of NAS has not been reported previously in experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of MS. Using a MOG-peptide induced EAE mouse model we examined the effects of melatonin and NAS on clinical score, inflammatory markers, free radical generation, and sparing of axons, oligodendrocytes and myelin. We found that NAS and melatonin reduced clinical scores when administered prior to or after symptom onset. This effect was more pronounced when melatonin and NAS were administrated prior to symptom onset whereby the appearance of motor symptoms was significantly delayed. Activated microglia and CD4+ T-cells were increased in the white matter of untreated EAE mice, with a return to near control levels after melatonin or NAS treatment. The expression of the NADPH oxidase component p67phox and inducible nitric oxide synthase (iNOS) was increased in the EAE mice as compared with controls, and both drug treated groups had significant reductions in their expression. Melatonin and NAS treatment significantly reduced the loss of mature oligodendrocytes, demyelination and axonal injury. Both compounds also significantly attenuated iNOS induction and reactive oxygen species (ROS) generation in lipopolysaccharide-activated microglia in culture. Our results show for the first time the therapeutic effects of NAS and confirm previous reports on the effectiveness of melatonin in the EAE model of MS.

Activation of CB2 receptor is required for the therapeutic effect of ABHD6 inhibition in experimental autoimmune encephalomyelitis.

Alpha/beta-hydrolase domain 6 (ABHD6) is a novel 2-arachidonoylglycerol (2-AG) hydrolytic enzyme, that can fine-tune the endocannabinoid signaling in the central nervous system. Recently we and others have demonstrated the protective effect of ABHD6 inhibition in the animal models of traumatic brain injury and epileptic seizures. In this study, we investigated the role of targeting ABHD6 in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Post-symptom treatment with an ABHD6 inhibitor WWL70 increased the brain levels of 2-AG and ameliorated the clinical signs of EAE, T cells infiltration, microglia activation and the expression of activated leukocyte cell adhesion molecules. The production of iNOS, COX-2, TNF-α and IL-1ß and the phosphorylation of NF-κB were also significantly reduced by WWL70 treatment. The neuroprotective effect of WWL70 was demonstrated by increased survival of mature oligodendrocytes, reduced demyelination and axonal loss in WWL70 treated EAE mouse spinal cord. The therapeutic effect of WWL70 on EAE was absent by co-administration of CB2 receptor antagonist, but not CB1 receptor antagonist. Consistently, WWL70 did not afford any protection in CB2 receptor knockout mice after EAE induction. Given the increased expression of ABHD6 in microglia/macrophages, but not in T cells, we speculated that inhibition of ABHD6 might enhance 2-AG signaling particularly in microglia/macrophages to exert anti-inflammatory effects via activation of CB2 receptors. These results suggest that inhibition of ABHD6 might be used as an ideal strategy for the treatment of MS and other neurodegenerative diseases.

Heparin facilitates glial cell line-derived neurotrophic factor signal transduction.

Glial cell-line neurotrophic factor (GDNF), a neurotrophic factor with heparin binding affinity, promotes the survival and differentiation of a variety of neuronal cells including dopaminergic neuron. The effect of heparin on GDNF signaling was investigated based on the expression of the tyrosine hydroxyrase (TH) gene in neurobalstoma cells. Up-regulation of TH gene mRNA by GDNF was enhanced by co-administration of heparin. This facilitation by heparin was particularly evident at suboptimal levels of GDNF, which was consistent with the luciferase assay using TH gene promoter. Pretreatment with heparitinase decreased TH promoter activity in the absence of heparin. Phosphorylation of extracellular regulated kinase was increased in the presence of heparin, although tyrosine phosphorylation of Ret receptor tyrosine kinase was not affected by heparin. Expression of early response genes such as c-fos or Egr1 increased and sustained in the presence of heparin more than that without heparin. These results indicate that interaction with glycosaminoglycans such as heparin affects GDNF signal transduction positively.

A rapid assay for glial cell line-derived neurotrophic factor and neurturin based on transfection of cells with tyrosine hydroxylase promoter-luciferase construct.

Glial cell line-derived neurotrophic factor (GDNF), a potent survival and trophic factor for various neuronal cells, has been measured by assaying its bioactivity based on neurite outgrowth or cell proliferation. We describe herein a sensitive and simple non-radioactive quantitative bioassay for GDNF family proteins based on their ability to induce tyrosine hydroxylase (TH) gene expression. Human neuroblastoma SK-N-MC cells were stably transfected with expression constructs of c-ret and with a luciferase reporter gene driven by 2 kb of the rat TH gene promoter region. In the presence of GDNF, luciferase activity increased with 20 h of incubation. A dose-dependent increase in luciferase activity was observed in the presence of GDNF between 1 and 300 ng/ml. This assay was also applicable for the quantification of the bioactivity of neurturin, another member of the GDNF family showing an even more sensitive profile of dose dependency than GDNF. Typical culture media were applicable in this assay. This method can be easily applied to numerous samples of conditioned medium in a short time.

Distinct patterns of expression of traumatic brain injury biomarkers after blast exposure: role of compromised cell membrane integrity.

Glial fibrillary acidic protein (GFAP), a protein enriched in astrocytes, and Tau, a protein abundant in neuronal microtubules, are being widely studied as biomarkers of brain injury, and persistent severity-dependent increases in brain and blood have been reported. Studies on the acute changes of these proteins after blast exposure are limited. Using a mouse model of closely-coupled repeated blast exposures, we have evaluated acute changes in the levels of GFAP and total Tau by Western blotting. Brain levels of GFAP and Tau proteins decreased significantly at 6 h and increased considerably at 24 h after repeated blast exposures. Plasma samples showed a similar initial decrease and later increase over this timeframe. This biphasic pattern points to possible absorption or sequestration of these proteins from plasma immediately after repeated blast exposures. Liver and spleen tissue showed significant increases in the levels of GFAP and Tau protein at 6 and 24 h post-blast exposures whereas semi-quantitative RT-PCR analysis of liver showed no significant changes in the levels of GFAP or Tau mRNAs. These results suggest that blast exposure causes transient changes in cell membrane integrity in multiple organs leading to abnormal migration of proteins from the tissues to the plasma and vice versa. This transient changes in cell membrane permeability and subsequent bidirectional movement of molecules may contribute to the pathophysiology of TBI and polytrauma after blast exposure.

Posttrauma cotreatment with lithium and valproate: reduction of lesion volume, attenuation of blood-brain barrier disruption, and improvement in motor coordination in mice with traumatic brain injury.

OBJECT: Although traumatic brain injury (TBI) is the leading cause of death and morbidity in young adults, no effective pharmaceutical treatment is available. By inhibiting glycogen synthase kinase-3 (GSK-3) and histone deacetylases (HDACs), respectively, lithium and valproate (VPA) have beneficial effects in diverse neurodegenerative diseases. Furthermore, in an excitotoxic neuronal model and in animal models of amyotrophic lateral sclerosis, Huntington disease, and stroke, combined treatment with lithium and VPA produces more robust neuroprotective effects than treatment with either agent alone. Building on previous work that establishes that therapeutic doses of either lithium or VPA have beneficial effects in mouse models of TBI, this study evaluated the effects of combined treatment with subeffective doses of lithium and VPA in a mouse model of TBI. METHODS: Male C57BL/6 mice underwent TBI and were subsequently treated with lithium, VPA, or a combination of lithium and VPA 15 minutes post-TBI and once daily thereafter for up to 3 weeks; all doses were subeffective (1 mEq/kg of lithium and 200 mg/kg of VPA). Assessed parameters included lesion volume via H & E staining; blood-brain barrier (BBB) integrity via immunoglobulin G extravasation; neurodegeneration via Fluoro-Jade B staining; motor coordination via a beam-walk test; and protein levels of acetylhistone H3, phospho-GSK-3ß, and ß-catenin via Western blotting. RESULTS: Posttrauma treatment with combined subeffective doses of lithium and VPA significantly reduced lesion volume, attenuated BBB disruption, and mitigated hippocampal neurodegeneration 3 days after TBI. As expected, subeffective doses of lithium or VPA alone did not have these beneficial effects. Combined treatment also improved motor coordination starting from Day 7 and persisting at least 21 days after TBI. Acetylation of histone H3, an index of HDAC inhibition, was robustly increased by the combined treatment 3 days after TBI. CONCLUSIONS: Cotreatment with subeffective doses of lithium and VPA significantly attenuated TBI-induced brain lesion, BBB disruption, and neurodegeneration, and robustly improved long-term functional recovery. These findings suggest that potentiating histone acetylation by HDAC inhibition is probably part of the mechanism underlying the beneficial effects associated with this combined treatment for TBI. Because both lithium and VPA have a long history of safe clinical use, the results suggest that using a combination of these 2 agents at subtherapeutic doses to treat patients with TBI may also reduce side effects and enhance tolerability.