Preconditioning by voluntary wheel running attenuates later neuropathic pain via nuclear factor E2-related factor 2 antioxidant signaling in rats.

ABSTRACT: Animal and human studies have shown that exercise prior to nerve injury prevents later chronic pain, but the mechanisms of such preconditioning remain elusive. Given that exercise acutely increases the formation of free radicals, triggering antioxidant compensation, we hypothesized that voluntary running preconditioning would attenuate neuropathic pain by supporting redox homeostasis after sciatic nerve injury in male and female rats. We show that 6 weeks of voluntary wheel running suppresses neuropathic pain development induced by chronic constriction injury across both sexes. This attenuation was associated with reduced nitrotyrosine immunoreactivity-a marker for peroxynitrite-at the sciatic nerve injury site. Our data suggest that prior voluntary wheel running does not reduce the production of peroxynitrite precursors, as expression levels of inducible nitric oxide synthase and NADPH oxidase 2 were unchanged. Instead, voluntary wheel running increased superoxide scavenging by elevating expression of superoxide dismutases 1 and 2. Prevention of neuropathic pain was further associated with the activation of the master transcriptional regulator of the antioxidant response, nuclear factor E2-related factor 2 (Nrf2). Six weeks of prior voluntary wheel running increased Nrf2 nuclear translocation at the sciatic nerve injury site; in contrast, 3 weeks of prior wheel running, which failed to prevent neuropathic pain, had no effect on Nrf2 nuclear translocation. The protective effects of prior voluntary wheel running were mediated by Nrf2, as suppression was abolished across both sexes when Nrf2 activation was blocked during the 6-week running phase. This study provides insight into the mechanisms by which physical activity may prevent neuropathic pain. Preconditioning by voluntary wheel running, terminated prior to nerve injury, suppresses later neuropathic pain in both sexes, and it is modulated through the activation of Nrf2-antioxidant signaling.

Small molecule targeting NaV1.7 via inhibition of the CRMP2-Ubc9 interaction reduces pain in chronic constriction injury (CCI) rats.

The voltage-gated sodium channel isoform NaV1.7 is a critical player in the transmission of nociceptive information. This channel has been heavily implicated in human genetic pain disorders and is a validated pain target. However, targeting this channel directly has failed, and an indirect approach - disruption of interactions with accessory protein partners - has emerged as a viable alternative strategy. We recently reported that a small-molecule inhibitor of CRMP2 SUMOylation, compound 194, selectively reduces NaV1.7 currents in DRG neurons across species from mouse to human. This compound also reversed mechanical allodynia in a spared nerve injury and chemotherapy-induced model of neuropathic pain. Here, we show that oral administration of 194 reverses mechanical allodynia in a chronic constriction injury (CCI) model of neuropathic pain. Furthermore, we show that orally administered 194 reverses the increased latency to cross an aversive barrier in a mechanical conflict-avoidance task following CCI. These two findings, in the context of our previous report, support the conclusion that 194 is a robust inhibitor of NaV1.7 function with the ultimate effect of profoundly ameliorating mechanical allodynia associated with nerve injury. The fact that this was observed using both traditional, evoked measures of pain behavior as well as the more recently developed operator-independent mechanical conflict-avoidance assay increases confidence in the efficacy of 194-induced anti-nociception.

A role for neuroimmune signaling in a rat model of Gulf War Illness-related pain.

More than a quarter of veterans of the 1990-1991 Persian Gulf War suffer from Gulf War Illness (GWI), a chronic, multi-symptom illness that commonly includes musculoskeletal pain. Exposure to a range of toxic chemicals, including sarin nerve agent, are a suspected root cause of GWI. Moreover, such chemical exposures induce a neuroinflammatory response in rodents, which has been linked to several GWI symptoms in rodents and veterans with GWI. To date, a neuroinflammatory basis for pain associated with GWI has not been investigated. Here, we evaluated development of nociceptive hypersensitivity in a model of GWI. Male Sprague Dawley rats were treated with corticosterone in the drinking water for 7 days, to mimic high physiological stress, followed by a single injection of the sarin nerve agent surrogate, diisopropyl fluorophosphate. These exposures alone were insufficient to induce allodynia. However, an additional sub-threshold challenge (a single intramuscular injection of pH 4 saline) induced long-lasting, bilateral allodynia. Such allodynia was associated with elevation of markers for activated microglia/macrophages (CD11b) and astrocytes/satellite glia (GFAP) in the lumbar dorsal spinal cord and dorsal root ganglia (DRG). Additionally, Toll-like receptor 4 (TLR4) mRNA was elevated in the lumbar dorsal spinal cord, while IL-1ß and IL-6 were elevated in the lumbar dorsal spinal cord, DRG, and gastrocnemius muscle. Demonstrating a casual role for such neuroinflammatory signaling, allodynia was reversed by treatment with either minocycline, the TLR4 inhibitor (+)-naltrexone, or IL-10 plasmid DNA. Together, these results point to a role for neuroinflammation in male rats in the model of musculoskeletal pain related to GWI. Therapies that alleviate persistent immune dysregulation may be a strategy to treat pain and other symptoms of GWI.

Oral Dimethyl Fumarate Reduces Peripheral Neuropathic Pain in Rodents via NFE2L2 Antioxidant Signaling.

BACKGROUND: Available treatments for neuropathic pain have modest efficacy and significant adverse effects, including abuse potential. Because oxidative stress is a key mechanistic node for neuropathic pain, the authors focused on the master regulator of the antioxidant response-nuclear factor erythroid 2-related factor 2 (NFE2L2; Nrf2)-as an alternative target for neuropathic pain. The authors tested whether dimethyl fumarate (U.S. Food and Drug Administration-approved treatment for multiple sclerosis) would activate NFE2L2 and promote antioxidant activity to reverse neuropathic pain behaviors and oxidative stress-dependent mechanisms. METHODS: Male Sprague Dawley rats, and male and female wild type and Nfe2l2 mice were treated with oral dimethyl fumarate/vehicle for 5 days (300 mg/kg; daily) after spared nerve injury/sham surgery (n = 5 to 8 per group). Allodynia was measured in von Frey reflex tests and hyperalgesia in operant conflict-avoidance tests. Ipsilateral L4/5 dorsal root ganglia were assayed for antioxidant and cytokine/chemokine levels, and mitochondrial bioenergetic capacity. RESULTS: Dimethyl fumarate treatment reversed mechanical allodynia (injury-vehicle, 0.45 ± 0.06 g [mean ± SD]; injury-dimethyl fumarate, 8.2 ± 0.16 g; P < 0.001) and hyperalgesia induced by nerve injury (injury-vehicle, 2 of 6 crossed noxious probes; injury-dimethyl fumarate, 6 of 6 crossed; P = 0.013). The antiallodynic effect of dimethyl fumarate was lost in nerve-injured Nfe2l2 mice, but retained in nerve-injured male and female wild type mice (wild type, 0.94 ± 0.25 g; Nfe2l2, 0.02 ± 0.01 g; P < 0.001). Superoxide dismutase activity was increased by dimethyl fumarate after nerve injury (injury-vehicle, 3.96 ± 1.28 mU/mg; injury-dimethyl fumarate, 7.97 ± 0.47 mU/mg; P < 0.001). Treatment reduced the injury-dependent increases in cytokines and chemokines, including interleukin-1ß (injury-vehicle, 13.30 ± 2.95 pg/mg; injury-dimethyl fumarate, 6.33 ± 1.97 pg/mg; P = 0.022). Injury-impaired mitochondrial bioenergetics, including basal respiratory capacity, were restored by dimethyl fumarate treatment (P = 0.025). CONCLUSIONS: Dimethyl fumarate, a nonopioid and orally-bioavailable drug, alleviated nociceptive hypersensitivity induced by peripheral nerve injury via activation of NFE2L2 antioxidant signaling. Dimethyl fumarate also resolved neuroinflammation and mitochondrial dysfunction-oxidative stress-dependent mechanisms that drive nociceptive hypersensitivity after nerve injury.

Loss of Estrogen-Related Receptor Alpha Facilitates Angiogenesis in Endothelial Cells.

Estrogen-related receptors (ERRs) have emerged as major metabolic regulators in various tissues. However, their expression and function in the vasculature remains unknown. Here, we report the transcriptional program and cellular function of ERRα in endothelial cells (ECs), a cell type with a multifaceted role in vasculature. Of the three ERR subtypes, ECs exclusively express ERRα. Gene expression profiling of ECs lacking ERRα revealed that ERRα predominantly acts as a transcriptional repressor, targeting genes linked with angiogenesis, cell migration, and cell adhesion. ERRα-deficient ECs exhibit decreased proliferation but increased migration and tube formation. ERRα depletion increased basal as well as vascular endothelial growth factor A (VEGFA)- and ANG1/2-stimulated angiogenic sprouting in endothelial spheroids. Moreover, retinal angiogenesis is enhanced in ERRα knockout mice compared to that in wild-type mice. Surprisingly, ERRα is dispensable for the regulation of its classic targets, such as metabolism, mitochondrial biogenesis, and cellular respiration in the ECs. ERRα is enriched at the promoters of angiogenic, migratory, and cell adhesion genes. Further, VEGFA increased ERRα recruitment to angiogenesis-associated genes and simultaneously decreased their expression. Despite increasing its gene occupancy, proangiogenic stimuli decrease ERRα expression in ECs. Our work shows that endothelial ERRα plays a repressive role in angiogenesis and potentially fine-tunes growth factor-mediated angiogenesis.

Long-term PGC1ß overexpression leads to apoptosis, autophagy and muscle wasting.

Skeletal muscle wasting is prevalent in many chronic diseases, necessitating inquiries into molecular regulation of muscle mass. Nuclear receptor co-activator peroxisome proliferator-activated receptor co-activator 1 alpha (PGC1α) and its splice variant PGC1α4 increase skeletal muscle mass. However, the effect of the other PGC1 sub-type, PGC1ß, on muscle size is unclear. In transgenic mice selectively over-expressing PGC1ß in the skeletal muscle, we have found that PGC1ß progressively decreases skeletal muscle mass predominantly associated with loss of type 2b fast-twitch myofibers. Paradoxically, PGC1ß represses the ubiquitin-proteolysis degradation pathway genes resulting in ubiquitinated protein accumulation in muscle. However, PGC1ß overexpression triggers up-regulation of apoptosis and autophagy genes, resulting in robust activation of these cell degenerative processes, and a concomitant increase in muscle protein oxidation. Concurrently, PGC1ß up-regulates apoptosis and/or autophagy transcriptional factors such as E2f1, Atf3, Stat1, and Stat3, which may be facilitating myopathy. Therefore, PGC1ß activation negatively affects muscle mass over time, particularly fast-twitch muscles, which should be taken into consideration along with its known aerobic effects in the skeletal muscle.

Muscle Arnt/Hif1ß Is Dispensable in Myofiber Type Determination, Vascularization and Insulin Sensitivity.

Aryl Hydrocarbon Receptor Nuclear Translocator/ hypoxia-inducible factor 1 beta (ARNT/ HIF1ß), a member of bHLH-PAS family of transcriptional factors, plays a critical role in metabolic homeostasis, insulin resistance and glucose intolerance. The contributions of ARNT in pancreas, liver and adipose tissue to energy balance through gene regulation have been described. Surprisingly, the impact of ARNT signaling in the skeletal muscles, one of the major organs involved in glucose disposal, has not been investigated, especially in type II diabetes. Here we report that ARNT is expressed in the skeletal muscles, particularly in the energy-efficient oxidative slow-twitch myofibers, which are characterized by increased oxidative capacity, mitochondrial content, vascular supply and insulin sensitivity. However, muscle-specific deletion of ARNT did not change myofiber type distribution, oxidative capacity, mitochondrial content, capillarity, or the expression of genes associated with these features. Consequently, the lack of ARNT in the skeletal muscle did not affect weight gain, lean/fat mass, insulin sensitivity and glucose tolerance in lean mice, nor did it impact insulin resistance and glucose intolerance in high fat diet-induced obesity. Therefore, skeletal muscle ARNT is dispensable for controlling muscle fiber type and metabolic regulation, as well as diet-induced weight control, insulin sensitivity and glucose tolerance.

Exercise-like effects by Estrogen-related receptor-gamma in muscle do not prevent insulin resistance in db/db mice.

Dissecting exercise-mimicking pathways that can replicate the benefits of exercise in obesity and diabetes may lead to promising treatments for metabolic disorders. Muscle estrogen-related receptor gamma (ERRγ) is induced by exercise, and when over-expressed in the skeletal muscle mimics exercise by stimulating glycolytic-to-oxidative myofiber switch, mitochondrial biogenesis and angiogenesis in lean mice. The objective of this study was to test whether muscle ERRγ in obese mice mitigates weight gain and insulin resistance. To do so, ERRγ was selectively over-expressed in the skeletal muscle of obese and diabetic db/db mice. Muscle ERRγ over-expression successfully triggered glycolytic-to-oxidative myofiber switch, increased functional mitochondrial content and boosted vascular supply in the db/db mice. Despite aerobic remodeling, ERRγ surprisingly failed to improve whole-body energy expenditure, block muscle accumulation of triglycerides, toxic diacylglycerols (DAG) and ceramides or suppress muscle PKCε sarcolemmal translocation in db/db mice. Consequently, muscle ERRγ did not mitigate impaired muscle insulin signaling or insulin resistance in these mice. In conclusion, obesity and diabetes in db/db mice are not amenable to selective ERRγ-directed programming of classic exercise-like effects in the skeletal muscle. Other biochemical pathways or integrated whole-body effects of exercise may be critical for resisting diabetes and obesity.

Ligands for FKBP12 increase Ca2+ influx and protein synthesis to improve skeletal muscle function.

Rapamycin at high doses (2-10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 µg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca(2+) influx to enhance refilling of sarcoplasmic reticulum Ca(2+) stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function.

PGC1ß activates an antiangiogenic program to repress neoangiogenesis in muscle ischemia.

Revascularization of ischemic skeletal muscle is governed by a balance between pro- and antiangiogenic factors in multiple cell types but particularly in myocytes and endothelial cells. Whereas the regulators of proangiogenic factors are well defined (e.g., hypoxia-inducible factor [HIF]), the transcriptional pathways encoding antiangiogenic factors remain unknown. We report that the transcriptional cofactor PGC1ß drives an antiangiogenic gene program in muscle and endothelial cells. PGC1ß transcriptionally represses proangiogenic genes (e.g., Vegfc, Vegfd, Pdgfb, Angpt1, Angpt2, Fgf1, and Fgf2) and induces antiangiogenic genes (e.g., Thbs1, Thbs2, Angstat, Pedf, and Vash1). Consequently, muscle-specific PGC1ß overexpression impairs muscle revascularization in ischemia and PGC1ß deletion enhances it. PGC1ß overexpression or deletion in endothelial cells also blocks or stimulates angiogenesis, respectively. PGC1ß stimulates the antiangiogenic genes partly by coactivating COUP-TFI. Furthermore, proangiogenic stimuli such as hypoxia, hypoxia-mimetic agents, and ischemia decrease PGC1ß expression in a HIF-dependent manner. PGC1ß is an antiangiogenic transcriptional switch that could be targeted for therapeutic angiogenesis.

Muscle ERRγ mitigates Duchenne muscular dystrophy via metabolic and angiogenic reprogramming.

Treatment of Duchenne muscular dystrophy (DMD) by replacing mutant dystrophin or restoring dystrophin-associated glycoprotein complex (DAG) has been clinically challenging. Instead, identifying and targeting muscle pathways deregulated in DMD will provide new therapeutic avenues. We report that the expression of nuclear receptor estrogen-related receptor-γ (ERRγ), and its metabolic and angiogenic targets are down-regulated (50-85%) in skeletal muscles of mdx mice (DMD model) vs. wild-type mice. Corelatively, oxidative myofibers, muscle vasculature, and exercise tolerance (33%) are decreased in mdx vs. wild-type mice. Overexpressing ERRγ selectively in the dystrophic muscles of the mdx mice restored metabolic and angiogenic gene expression compared with control mdx mice. Further, ERRγ enhanced muscle oxidative myofibers, vasculature, and blood flow (by 33-66%) and improved exercise tolerance (by 75%) in the dystrophic mice. Restoring muscle ERRγ pathway ameliorated muscle damage and also prevented DMD hallmarks of postexercise muscle damage, hypoxia, and fatigue in mdx mice. Notably, ERRγ did not restore sarcolemmal DAG complex, which is thus dispensable for antidystrophic effects of ERRγ. In summary, ERRγ-dependent metabolic and angiogenic gene program is defective in DMD, and we demonstrate that its restoration is a potential strategy for treating muscular dystrophy.

Revascularization of ischemic skeletal muscle by estrogen-related receptor-γ.

RATIONALE: Oxidative myofibers in the skeletal muscles express high levels of angiogenic factors, have dense vasculature, and promptly revascularize during ischemia. Estrogen-related receptor-gamma (ERRγ) activates genes that govern metabolic and vascular features typical to oxidative myofibers. Therefore, ERRγ-dependent remodeling of the myofibers may promote neoangiogenesis and restoration of blood perfusion in skeletal muscle ischemia. OBJECTIVE: To investigate the muscle fiber type remodeling by ERRγ and its role in the vascular recovery of ischemic muscle. METHODS AND RESULTS: Using immunohistology, we show that skeletal muscle-specific transgenic overexpression of ERRγ increases the proportions of oxidative and densely vascularized type IIA and IIX myofibers and decreases glycolytic and less vascularized type IIB myofibers. This myofiber remodeling results in a higher basal blood flow in the transgenic skeletal muscle. By applying unilateral hind limb ischemia to transgenic and wild-type mice, we found accelerated revascularization (fluorescent microangiography), restoration of blood perfusion (laser Doppler flowmetry), and muscle repair (Evans blue dye exclusion) in transgenic compared to wild-type ischemic muscles. This ameliorative effect is linked to enhanced neoangiogenesis (CD31 staining and microfil perfusion) by ERRγ. Using cultured muscle cells in which ERRγ is inactivated, we show that the receptor is dispensable for the classical hypoxic response of transcriptional upregulation and secretion of vascular endothelial growth factor A. Rather, the ameliorative effect of ERRγ is linked to the receptor-mediated increase in oxidative myofibers that inherently express and secrete high levels of angiogenic factors. CONCLUSIONS: The ERRγ is a hypoxia-independent inducer of neoangiogenesis that can promote reparative revascularization.

Exercise and PGC-1α-independent synchronization of type I muscle metabolism and vasculature by ERRγ.

How type I skeletal muscle inherently maintains high oxidative and vascular capacity in the absence of exercise is unclear. We show that nuclear receptor ERRγ is highly expressed in type I muscle and, when transgenically expressed in anaerobic type II muscles (ERRGO mice), dually induces metabolic and vascular transformation in the absence of exercise. ERRGO mice show increased expression of genes promoting fat metabolism, mitochondrial respiration, and type I fiber specification. Muscles in ERRGO mice also display an activated angiogenic program marked by myofibrillar induction and secretion of proangiogenic factors, neovascularization, and a 100% increase in running endurance. Surprisingly, the induction of type I muscle properties by ERRγ does not involve PGC-1α. Instead, ERRγ genetically activates the energy sensor AMPK in mediating the metabovascular changes in ERRGO mice. Therefore, ERRγ represents a previously unrecognized determinant that specifies intrinsic vascular and oxidative metabolic features that distinguish type I from type II muscle.