Fabry disease Schwann cells release p11 to induce sensory neuron hyperactivity.

Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a mechanism we believe to be novel in which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observed in a genetic rat model of Fabry disease. Using in vivo and in vitro electrophysiological recordings, we demonstrated that Fabry rat sensory neurons exhibited pronounced hyperexcitability. Schwann cells probably contributed to this finding because application of mediators released from cultured Fabry Schwann cells induced spontaneous activity and hyperexcitability in naive sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells released elevated levels of the protein p11 (S100A10), which induced sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media caused hyperpolarization of neuronal resting membrane potentials, indicating that p11 may contribute to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that sensory neurons from rats with Fabry disease exhibit hyperactivity caused in part by Schwann cell release of the protein p11.

Descending mechanism by which medial prefrontal cortex endocannabinoid signaling controls the development of neuropathic pain and neuronal activity of dorsal root ganglion.

ABSTRACT: Although regulation of nociceptive processes in the dorsal horn by deep brain structures has long been established, the role of cortical networks in pain regulation is minimally explored. The medial prefrontal cortex (mPFC) is a key brain area in pain processing that receives ascending nociceptive input and exerts top-down control of pain sensation. We have shown critical changes in mPFC synaptic function during neuropathic pain, controlled by endocannabinoid (eCB) signaling. This study tests whether mPFC eCB signaling modulates neuropathic pain through descending control. Intra-mPFC injection of cannabinoid receptor type 1 (CB1R) agonist WIN-55,212-2 (WIN) in the chronic phase transiently alleviates the pain-like behaviors in spared nerve injury (SNI) rats. By contrast, intra-mPFC injection of CB1R antagonist AM4113 in the early phase of neuropathic pain reduces the development of pain-like behaviors in the chronic phase. Spared nerve injury reduced the mechanical threshold to induce action potential firing of dorsal horn wide-dynamic-range neurons, but this was reversed in rats by WIN in the chronic phase of SNI and by mPFC injection of AM4113 in the early phase of SNI. Elevated dorsal root ganglion neuronal activity after injury was also diminished in rats by mPFC injection of AM4113, potentially by reducing antidromic activity and subsequent neuronal inflammation. These findings suggest that depending on the phase of the pain condition, both blocking and activating CB1 receptors in the mPFC can regulate descending control of pain and affect both dorsal horn neurons and peripheral sensory neurons, contributing to changes in pain sensitivity.

Schwann cell release of p11 induces sensory neuron hyperactivity in Fabry disease.

Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a novel mechanism by which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observe in a genetic rat model of Fabry disease. Using in vivo and in vitro electrophysiological recordings, we demonstrate that Fabry rat sensory neurons exhibit pronounced hyperexcitability. Schwann cells likely contribute to this finding as application of mediators released from cultured Fabry Schwann cells induces spontaneous activity and hyperexcitability in naïve sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells release elevated levels of the protein p11 (S100-A10) which induces sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media causes hyperpolarization of neuronal resting membrane potential, indicating that p11 contributes to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that rats with Fabry disease exhibit sensory neuron hyperexcitability caused in part by Schwann cell release of the protein p11.

Repetitive Mild Traumatic Brain Injury in Rats Impairs Cognition, Enhances Prefrontal Cortex Neuronal Activity, and Reduces Pre-synaptic Mitochondrial Function.

A major hurdle preventing effective interventions for patients with mild traumatic brain injury (mTBI) is the lack of known mechanisms for the long-term cognitive impairment that follows mTBI. The closed head impact model of repeated engineered rotational acceleration (rCHIMERA), a non-surgical animal model of repeated mTBI (rmTBI), mimics key features of rmTBI in humans. Using the rCHIMERA in rats, this study was designed to characterize rmTBI-induced behavioral disruption, underlying electrophysiological changes in the medial prefrontal cortex (mPFC), and associated mitochondrial dysfunction. Rats received 6 closed-head impacts over 2 days at 2 Joules of energy. Behavioral testing included automated analysis of behavior in open field and home-cage environments, rotarod test for motor skills, novel object recognition, and fear conditioning. Following rmTBI, rats spent less time grooming and less time in the center of the open field arena. Rats in their home cage had reduced inactivity time 1 week after mTBI and increased exploration time 1 month after injury. Impaired associative fear learning and memory in fear conditioning test, and reduced short-term memory in novel object recognition test were found 4 weeks after rmTBI. Single-unit in vivo recordings showed increased neuronal activity in the mPFC after rmTBI, partially attributable to neuronal disinhibition from reduced inhibitory synaptic transmission, possibly secondary to impaired mitochondrial function. These findings help validate this rat rmTBI model as replicating clinical features, and point to impaired mitochondrial functions after injury as causing imbalanced synaptic transmission and consequent impaired long-term cognitive dysfunction.

Dynamic Change of Endocannabinoid Signaling in the Medial Prefrontal Cortex Controls the Development of Depression After Neuropathic Pain.

Many patients with chronic pain conditions suffer from depression. The mechanisms underlying pain-induced depression are still unclear. There are critical links of medial prefrontal cortex (mPFC) synaptic function to depression, with signaling through the endocannabinoid (eCB) system as an important contributor. We hypothesized that afferent noxious inputs after injury compromise activity-dependent eCB signaling in the mPFC, resulting in depression. Depression-like behaviors were tested in male and female rats with traumatic neuropathy [spared nerve injury (SNI)], and neuronal activity in the mPFC was monitored using the immediate early gene c-fos and in vivo electrophysiological recordings. mPFC eCB Concentrations were determined using mass spectrometry, and behavioral and electrophysiological experiments were used to evaluate the role of alterations in eCB signaling in depression after pain. SNI-induced pain induced the development of depression phenotypes in both male and female rats. Pyramidal neurons in mPFC showed increased excitability followed by reduced excitability in the onset and prolonged phases of pain, respectively. Concentrations of the eCBs, 2-arachidonoylglycerol (2-AG) in the mPFC, were elevated initially after SNI, and our results indicate that this resulted in a loss of CB1R function on GABAergic interneurons in the mPFC. These data suggest that excessive release of 2-AG as a result of noxious stimuli triggers use-dependent loss of function of eCB signaling leading to excessive GABA release in the mPFC, with the final result being behavioral depression.SIGNIFICANCE STATEMENT Pain has both somatosensory and affective components, so the complexity of mechanisms underlying chronic pain is best represented by a biopsychosocial model that includes widespread CNS dysfunction. Many patients with chronic pain conditions develop depression. The mechanism by which pain causes depression is unclear. Although manipulation of the eCB signaling system as an avenue for providing analgesia per se has not shown much promise in previous studies. An important limitation of past research has been inadequate consideration of the dynamic nature of the connection between pain and depression as they develop. Here, we show that activity-dependent synthesis of eCBs during the initial onset of persistent pain is the critical link leading to depression when pain is persistent.

Dorsal root ganglion stimulation of injured sensory neurons in rats rapidly eliminates their spontaneous activity and relieves spontaneous pain.

ABSTRACT: Dorsal root ganglion field stimulation (GFS) relieves evoked and spontaneous neuropathic pain by use-dependent blockade of impulse trains through the sensory neuron T-junction, which becomes complete within less than 1 minute for C-type units, also with partial blockade of Aδ units. We used this tool in the spinal nerve ligation (SNL) rat model to selectively block sensory neuron spontaneous activity (SA) of axotomized neurons at the fifth lumbar (L5) level vs blockade of units at the L4 level that remain uninjured but exposed to inflammation. In vivo dorsal root single-unit recordings after SNL showed increased SA in L5 units but not L4 units. Ganglion field stimulation blocked this SA. Ganglion field stimulation delivered at the L5 dorsal root ganglion blocked mechanical hyperalgesia behavior, mechanical allodynia, and ongoing spontaneous pain indicated by conditioned place preference, whereas GFS at L4 blocked evoked pain behavior but not spontaneous pain. In vivo single-unit recordings of spinal cord dorsal horn (DH) wide-dynamic-range neurons showed elevated SA after SNL, which was reduced by GFS at the L5 level but not by GFS at the L4 level. In addition, L5 GFS, but not L4 GFS, increased mechanical threshold of DH units during cutaneous mechanical stimulation, while L5 GFS exceeded L4 GFS in reducing evoked firing rates. Our results indicate that SA in injured neurons supports increased firing of DH wide-dynamic-range neurons, contributing to hyperalgesia, allodynia, and ongoing pain. Ganglion field stimulation analgesic effects after nerve injury are at least partly attributable to blocking propagation of this SA.

Analgesic dorsal root ganglionic field stimulation blocks conduction of afferent impulse trains selectively in nociceptive sensory afferents.

Increased excitability of primary sensory neurons after peripheral nerve injury may cause hyperalgesia and allodynia. Dorsal root ganglion field stimulation (GFS) is effective in relieving clinical pain associated with nerve injury and neuropathic pain in animal models. However, its mechanism has not been determined. We examined effects of GFS on transmission of action potentials (APs) from the peripheral to central processes by in vivo single-unit recording from lumbar dorsal roots in sham injured rats and rats with tibial nerve injury (TNI) in fiber types defined by conduction velocity. Transmission of APs directly generated by GFS (20 Hz) in C-type units progressively abated over 20 seconds, whereas GFS-induced Aß activity persisted unabated, while Aδ showed an intermediate pattern. Activity generated peripherally by electrical stimulation of the sciatic nerve and punctate mechanical stimulation of the receptive field (glabrous skin) was likewise fully blocked by GFS within 20 seconds in C-type units, whereas Aß units were minimally affected and a subpopulation of Aδ units was blocked. After TNI, the threshold to induce AP firing by punctate mechanical stimulation (von Frey) was reduced, which was reversed to normal during GFS. These results also suggest that C-type fibers, not Aß, mainly contribute to mechanical and thermal hypersensitivity (von Frey, brush, acetone) after injury. Ganglion field stimulation produces use-dependent blocking of afferent AP trains, consistent with enhanced filtering of APs at the sensory neuron T-junction, particularly in nociceptive units.

AAV-encoded CaV2.2 peptide aptamer CBD3A6K for primary sensory neuron-targeted treatment of established neuropathic pain.

Transmission of pain signals from primary sensory neurons to secondary neurons of the central nervous system is critically dependent on presynaptic voltage-gated calcium channels. Calcium channel-binding domain 3 (CBD3), derived from the collapsin response mediator protein 2 (CRMP2), is a peptide aptamer that is effective in blocking N-type voltage-gated calcium channel (CaV2.2) activity. We previously reported that recombinant adeno-associated virus (AAV)-mediated restricted expression of CBD3 affixed to enhanced green fluorescent protein (EGFP) in primary sensory neurons prevents the development of cutaneous mechanical hypersensitivity in a rat neuropathic pain model. In this study, we tested whether this strategy is effective in treating established pain. We constructed AAV6-EGFP-CBD3A6K (AAV6-CBD3A6K) expressing a fluorescent CBD3A6K (replacing A to K at position 6 of CBD3 peptide), which is an optimized variant of the parental CBD3 peptide that is a more potent blocker of CaV2.2. Delivery of AAV6-CBD3A6K into lumbar (L) 4 and 5 dorsal root ganglia (DRG) of rats 2 weeks following tibial nerve injury (TNI) induced transgene expression in neurons of these DRG and their axonal projections, accompanied by attenuation of pain behavior. We additionally observed that the increased CaV2.2α1b immunoreactivity in the ipsilateral spinal cord dorsal horn and DRG following TNI was significantly normalized by AAV6-CBD3A6K treatment. Finally, the increased neuronal activity in the ipsilateral dorsal horn that developed after TNI was reduced by AAV6-CBD3A6K treatment. Collectively, these results indicate that DRG-restricted AAV6 delivery of CBD3A6K is an effective analgesic molecular strategy for the treatment of established neuropathic pain.

δ-Opioid Receptor Activation Attenuates the Oligomer Formation Induced by Hypoxia and/or α-Synuclein Overexpression/Mutation Through Dual Signaling Pathways.

We have recently demonstrated that δ-opioid receptor (DOR) activation attenuates α-synuclein expression/aggregation induced by MPP(+) and/or severe hypoxia. Since α-synuclein plays a critical role in the pathogenesis of Parkinson's disease, DOR activation may trigger an antiparkinson pathway(s) against α-synuclein-induced injury. However, the underlying mechanism is unknown yet. In HEK293T and PC12 cells, we investigated the effects of DOR activation on the oligomer formation induced by α-synuclein overexpression and mutation in normoxic and hypoxic conditions and explored the potential signaling pathways for DOR protection. We found that (1) increased expression of both wild-type and A53T-mutant α-synuclein led to the formation of α-synuclein oligomers and cytotoxic injury; (2) DOR activation largely attenuated the formation of toxic α-synuclein oligomers induced by α-synuclein overexpression/mutation and/or hypoxia; (3) DOR activation attenuated α-synuclein-induced cytotoxicity through TORC1/SIK1/CREB, but not the phospho-CREB pathway, while DOR activation reduced hypoxic cell injury through the phospho-CREB mechanism; and (4) the interaction of α-synuclein and the DJ-1 was involved in the mechanisms for DOR-mediated protection against α-synuclein oligomer formation. Our findings suggest that DOR attenuates the formation of toxic α-synuclein oligomers through the phos-CREB pathway under hypoxic conditions, and through TORC1/SIK1/CREB pathways in the conditions of α-synuclein overexpression and mutation. The DJ-1 gene was involved in the DOR protection against parkinsonian injury.

δ-Opioid Receptor-Nrf-2-Mediated Inhibition of Inflammatory Cytokines in Neonatal Hypoxic-Ischemic Encephalopathy.

Neonatal hypoxic-ischemic encephalopathy (HIE) causes serious neurological disability; there are, however, currently few promising therapies for it. We have recently shown that δ-opioid receptor (DOR) is neuroprotective by downregulating TNF-α. Since hypoxia-ischemia (HI) triggers a robust inflammatory response, which exacerbates HI brain damage, we investigated, in this study, whether DOR activation could regulate inflammatory cytokine expression, thereby playing a protective effect on the neonatal brain under HI. Twenty-five neonatal rats were randomly divided into five groups: (1) control (control); (2) HI; (3) HI with saline (HI + NS); (4) DOR activation with UFP-512 (a potent and specific DOR agonist) under HI conditions (HI + U); and (5) DOR inhibition using NT treatment under HI conditions (HI + NT). The rats were sacrificed by decapitation at 24 h after HI, and their brains were rapidly removed for measurements. The protein expression of TNF-α, IL-6, ICAM-1, IL-10, IL-18, NQO-1, Nrf-2, and HO-1 was measured using Western blot. In the hemispheres exposed to HI, DOR activation significantly decreased the expressions of TNF-α, IL-6, and ICAM-1 in the cortex, while it significantly increased IL-10 and had no effect on IL-18 in the same region. In contrast, DOR had no appreciable effect on inflammatory cytokine expression in non-cortical tissues including hippocampal, subcortical, and cerebellar tissues. Moreover, HI stress triggered an upregulation of Nrf-2 nuclear protein as well as some of its downstream anti-inflammatory genes such as HO-1 and NQO-1 in the cortex, while DOR activation further augmented such a protective reaction against HI injury. DOR plays an important role in protecting against HI by regulating the expression of inflammatory and anti-inflammatory cytokines in the cortex, which is likely mediated by the Nrf-2/HO-1/NQO-1 signaling.

Dorsal Root Ganglion Field Stimulation Prevents Inflammation and Joint Damage in a Rat Model of Rheumatoid Arthritis.

OBJECTIVES: Electrical stimulation of the dorsal root ganglion (DRG), referred to here as ganglionic field stimulation (GFS), is effective in reducing clinical pain, probably by interrupting transmission of afferent impulse trains on sensory neurons as they pass through the DRG. We therefore tested whether efferent impulse trains conveyed by sensory neurons, which contribute to neurogenic inflammation, may also be interrupted by GFS. MATERIALS AND METHODS: Collagen-induced arthritis, a model of clinical rheumatoid arthritis, was initiated in rats concurrently with the insertion of an electrode for GFS at the fourth lumbar DRG. Continuous GFS (20 Hz pulse rate, current at 80% of the motor threshold) was initiated 6 days later and continued for 14 days. Plantar pain sensitivity, ankle arthritis score, and dimensions of the foot and ankle were determined one hour after termination of GFS. RESULTS: The foot/ankle contralateral to GFS developed hypersensitivity to threshold and noxious mechanical stimulation, swelling, and high arthritis score, all of which were normalized in the foot/ankle ipsilateral with GFS. Histology showed GFS limited joint destruction. Electrophysiological recording showed GFS can block efferent impulse trains. CONCLUSIONS: Our findings show that GFS can reduce neurogenic inflammation and the resulting joint damage in a model of rheumatoid arthritis, probably by blocking the transit of impulse trains through the DRG. GFS may have clinical utility in limiting joint destruction in inflammatory arthritis such as rheumatoid arthritis.

δ-Opioid Receptor Activation and MicroRNA Expression in the Rat Heart Under Prolonged Hypoxia.

BACKGROUND: Hypoxic/ischemic injury to the heart is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs (miRNAs) are involved in hypoxic/ischemic events, and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering miRNA expression in the heart under hypoxic conditions. The present study aimed to test our hypothesis. METHODS: Male Sprague Dawley rats were exposed to hypoxia (9.5-10% O2) for 1, 5, or 10 days with or without DOR activation. The target miRNAs were selected from TaqMan low-density array (TLDA) data and were further analyzed by quantitative real-time PCR. RESULTS: We found that: 1) hypoxia alters the miRNA expression profiles depending on the hypoxic duration; 2) DOR activation shifts miRNA expression profiles in normoxic conditions and upregulates miR-128a-3p, miR-134-5p, miR-135a, miR-193a-3p, miR-196a, miR-324-3p, and miR-338; and 3) DOR activation modifies hypoxia-induced changes in miRNA expression and increases the levels of miR-128a-3p, miR-134-5p, miR-135a, miR-193a-3p, miR-196a, miR-324-3p, miR-141, miR-200b, and miR-324-3p. For example, miR-196c-5p decreased by 50% while miR-135a-5p increased 2.9 fold after 10 days under hypoxic conditions. Moreover, DOR activation further strengthened the hypoxia-induced increase of the levels of miR-7a-5p. When DOR was activated using UFP-512, the level of miR-107-3p significantly increased 1 day after the administration of UFP-512, but gradually decreased back to normal under normoxia. CONCLUSION: Hypoxia significantly modifies the miRNA profile in the heart, which can be mimicked or modified by DOR activation. Defining the targeted pathways that regulate the diverse cellular and molecular functions of miRNAs may provide new insights into potential therapies for hypoxic/ischemic injury of the heart.

Animal behavioral assessments in current research of Parkinson's disease.

Parkinson's disease (PD), a neurodegenerative disorder, is traditionally classified as a movement disorder. Patients typically suffer from many motor dysfunctions. Presently, clinicians and scientists recognize that many non-motor symptoms are associated with PD. There is an increasing interest in both motor and non-motor symptoms in clinical studies on PD patients and laboratory research on animal models that imitate the pathophysiologic features and symptoms of PD patients. Therefore, appropriate behavioral assessments are extremely crucial for correctly understanding the mechanisms of PD and accurately evaluating the efficacy and safety of novel therapies. This article systematically reviews the behavioral assessments, for both motor and non-motor symptoms, in various animal models involved in current PD research. We addressed the strengths and weaknesses of these behavioral tests and their appropriate applications. Moreover, we discussed potential mechanisms behind these behavioral tests and cautioned readers against potential experimental bias. Since most of the behavioral assessments currently used for non-motor symptoms are not particularly designed for animals with PD, it is of the utmost importance to greatly improve experimental design and evaluation in PD research with animal models. Indeed, it is essential to develop specific assessments for non-motor symptoms in PD animals based on their characteristics. We concluded with a prospective view for behavioral assessments with real-time assessment with mobile internet and wearable device in future PD research.

Attenuating Ischemic Disruption of K+ Homeostasis in the Cortex of Hypoxic-Ischemic Neonatal Rats: DOR Activation vs. Acupuncture Treatment.

Perinatal hypoxic-ischemic (HI) brain injury results in death or profound long-term neurologic disability in both children and adults. However, there is no effective pharmacological therapy due to a poor understanding of HI events, especially the initial triggers for hypoxic-ischemic injury such as disrupted ionic homeostasis and the lack of effective intervention strategy. In the present study, we showed that neonatal brains undergo a developmental increase in the disruption of K+ homeostasis during simulated ischemia, oxygen-glucose deprivation (OGD) and neonatal HI cortex has a triple phasic response (earlier attenuation, later enhancement, and then recovery) of disrupted K+ homeostasis to OGD. This response partially involves the activity of the δ-opioid receptor (DOR) since the earlier attenuation of ischemic disruption of K+ homeostasis could be blocked by DOR antagonism, while the later enhancement was reversed by DOR activation. Similar to DOR activation, acupuncture, a strategy to promote DOR activity, could partially reverse the later enhanced ischemic disruption of K+ homeostasis in the neonatal cortex. Since maintaining cellular K+ homeostasis and inhibiting excessive K+ fluxes in the early phase of hypoxic-ischemic insults may be of therapeutic benefit in the treatment of ischemic brain injury and related neurodegenerative conditions, and since many neurons and other cells can be rescued during the "window of opportunity" after HI insults, our first findings regarding the role of acupuncture and DOR in attenuating ischemic disruption of K+ homeostasis in the neonatal HI brain suggest a potential intervention therapy in the treatment of neonatal brain injury, especially hypoxic-ischemic encephalopathy.

Low Cerebral Glucose Metabolism: A Potential Predictor for the Severity of Vascular Parkinsonism and Parkinson's Disease.

This study explored the association between cerebral metabolic rates of glucose (CMRGlc) and the severity of Vascular Parkinsonism (VP) and Parkinson's disease (PD). A cross-sectional study was performed to compare CMRGlc in normal subjects vs. VP and PD patients. Twelve normal subjects, 22 VP, and 11 PD patients were evaluated with the H&Y and MMSE, and underwent 18F-FDG measurements. Pearson's correlations were used to identify potential associations between the severity of VP/PD and CMRGlc. A pronounced reduction of CMRGlc in the frontal lobe and caudate putamen was detected in patients with VP and PD when compared with normal subjects. The VP patients displayed a slight CMRGlc decrease in the caudate putamen and frontal lobe in comparison with PD patients. These decreases in CMRGlc in the frontal lobe and caudate putamen were significantly correlated with the VP patients' H&Y, UPDRS II, UPDRS III, MMSE, cardiovascular, and attention/memory scores. Similarly, significant correlations were observed in patients with PD. This is the first clinical study finding strong evidence for an association between low cerebral glucose metabolism and the severity of VP and PD. Our findings suggest that these changes in glucose metabolism in the frontal lobe and caudate putamen may underlie the pathophysiological mechanisms of VP and PD. As the scramble to find imaging biomarkers or predictors of the disease intensifies, a better understanding of the roles of cerebral glucose metabolism may give us insight into the pathogenesis of VP and PD.

A novel mechanism for cytoprotection against hypoxic injury: δ-opioid receptor-mediated increase in Nrf2 translocation.

BACKGROUND AND PURPOSE: Hypoxia/reoxygenation induces synthesis of reactive oxygen species (ROS) which can attack macromolecules and cause brain injury. The transcription factor, nuclear factor (erythroid-derived 2)-like 2, (Nrf2), ia potent activator of genes with an antioxidant responsive element and Nrf2 can counteract oxidative injury by increasing expression of several antioxidative genes in response to ROS stress. Here, we show that activation of the δ-opioid receptor (DOR) increasedNrf2 protein expression and translocation, thereby leading to cytoprotection. EXPERIMENTAL APPROACH: We used HEK293t cells exposed to 0.5% O2 for 16 h and then reoxygenated for 4 h as a model of hypoxia-reperfusion (H/R) injury. Real time PCR, Western blotting, siRNA and immunohistochemical techniques were used to follow Nrf2 expression and activity. Cell viability and damage (as LDH leakage) were also measured. KEY RESULTS: H/R injury triggered Nrf2 translocation into the nucleus and up-regulated expression of several downstream genes, relevant to antioxidation, such as NAD(P)H: quinone oxidoreductase (NQO1). Incubation with the DOR agonist UFP-512 enhanced Nrf2 protein expression and translocation and up-regulated its downstream genes in normoxia and further increased Nrf2 expression and translocation after H/R, protecting the cells against loss of viability and damage. The effect of UFP-512 on Nrf2 nuclear translocation was blocked by the DOR antagonist, naltrindole. Also, DOR-mediated cytoprotection was strongly inhibited after transfection of HEK293t cells with Nrf2 siRNA. CONCLUSIONS AND IMPLICATIONS: The DOR agonist UFP-512 was cytoprotective against H/R injury and this effect was partly dependent on DOR-mediated increase in Nrf2 function.

δ-Opioid receptors up-regulate excitatory amino acid transporters in mouse astrocytes.

BACKGROUND AND PURPOSE: Astrocytic excitatory amino acid transporters (EAATs) regulate extracellular glutamate concentrations and play a role in preventing neuroexcitotoxicity. As the δ-opioid receptor (DOP receptor) is neuroprotective against excitotoxic injury, we determined whether DOP receptor activation up-regulates EAAT expression and function. EXPERIMENTAL APPROACH: We measured mRNA and protein expression of EAAT1, EAAT2 and EAAT3 in cultured mouse astrocytes exposed to a specific DOP receptor agonist (UFP-512) with or without a DOP receptor antagonist, DOP receptor siRNA or inhibitors of PKC, PKA, PI3K, p38, MAPK, MEK and ERK, and evaluated the function of EAATs by measuring glutamate uptake. KEY RESULTS: Astrocytic DOP receptor mRNA and protein were suppressed by DOP receptor siRNA knockdown. DOP receptor activation increased mRNA and protein expression of EAAT1 and EAAT2, but not EAAT3, thereby enhancing glutamate uptake of astrocytes. DOP receptor-induced EAAT1 and EAAT2 expression was largely reversed by DOP receptor antagonist naltrindole or by DOP receptor siRNA knockdown, and suppressed by inhibitors of MEK, ERK and p38. DOP receptor-accelerated glutamate uptake was inhibited by EAAT blockers, DOP receptor siRNA knockdown or inhibitors of MEK, ERK or p38. In contrast, inhibitors of PKA, PKC or PI3K had no significant effect on DOP receptor-induced EAAT expression. CONCLUSIONS AND IMPLICATIONS: DOP receptor activation up-regulates astrocytic EAATs via MEK-ERK-p38 signalling, suggesting a critical role for DOP receptors in the regulation of astrocytic EAATs and protection against neuroexcitotoxicity. As decreased EAAT expression contributes to pathophysiology in many neurological diseases, including amyotrophic lateral sclerosis, our findings present a new platform for potential treatments of these diseases.

δ-Opioid receptors and inflammatory cytokines in hypoxia: differential regulation between glial and neuron-like cells.

Emerging evidence suggests that the δ-opioid receptor (DOR) is neuroprotective against hypoxic stress. Since inflammatory factors play an important role in hypoxic injury, we sought to determine if DOR modulates cellular inflammatory process in hypoxia. Since astrocytes produce inflammatory cytokines and play a major role in inflammatory response to hypoxia, we exposed cultured rat astrocytes to severe hypoxia (0.1% O2) for 3-6 h to examine if DOR activation altered hypoxia-induced expression of proinflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) along with cell viability. To examine a differential DOR-mediated modulation between glial and neuron-like cells, we also conducted the same experiments in differentiated PC-12 cells. We found that hypoxia reduced cell viability in both astrocytes and PC-12 cells, and DOR activation with UFP-512 (5 µM) effectively protected both the cells against hypoxic insult. Hypoxia markedly increased TNF-α levels in the astrocytes (p < 0.05); however, the same hypoxic stress reduced TNF-α levels in the PC-12 cells. Exogenous addition of TNF-α to the culture media further worsened the hypoxia-induced damage in PC-12 cells. Hypoxia increased the DOR-expression in astrocytes but not in PC-12 cells. DOR-activation reversed the hypoxia-induced changes in TNF-α levels in both the cells. In contrast, the IL-1ß levels did not significantly alter in astrocytes and PC-12 cells when exposed to the same hypoxic conditions or following DOR activation. Our novel data suggest that DOR activation inhibits TNF-α mediated inflammatory processes following exposure to severe hypoxia with a differential regulation between glial vs. neuron-like cells.

δ-Opioid receptor activation reduces α-synuclein overexpression and oligomer formation induced by MPP(+) and/or hypoxia.

Hypoxic/ischemic brain injury is a potential cause of Parkinson's disease (PD) with ɑ-synuclein playing a critical role in the pathophysiology. Since δ-opioid receptor (DOR) is neuroprotective against hypoxic/ischemic insults, we sought to determine if DOR regulates ɑ-synuclein under hypoxia and/or MPP(+) stress. We found that in HEK293 cells 1) MPP(+) in normoxia enhanced ɑ-synuclein expression and the formation of ɑ-synuclein oligomers thereby causing cytotoxic injury; 2) hypoxia at 1% O2 for 48h or at 0.5% O2 for 24h also induced ɑ-synuclein overexpression and its oligomer formation with cell injury; 3) however, hypoxia at 1% O2 for 24h, though increasing ɑ-synuclein expression, did not cause ɑ-synuclein oligomer formation and cell injury; 4) UFP-512 mediated DOR activation markedly attenuated the hypoxic cell injury and ɑ-synuclein overexpression, which was largely attenuated by DOR antagonism with naltrindole or siRNA "knock-down" of the DOR; and 5) DOR activation enhanced CREB phosphorylation and prevented the collapse of mitochondrial membrane potential (△ψm). These findings suggest that DOR activation attenuates MPP(+) or severe hypoxia induced ɑ-synuclein expression/aggregation via a CREB pathway.

Effect of δ-opioid receptor activation on BDNF-TrkB vs. TNF-α in the mouse cortex exposed to prolonged hypoxia.

We investigated whether δ-opioid receptor (DOR)-induced neuroprotection involves the brain-derived neurotrophic factor (BDNF) pathway. We studied the effect of DOR activation on the expression of BDNF and other proteins in the cortex of C57BL/6 mice exposed to hypoxia (10% of oxygen) for 1-10 days. The results showed that: (1) 1-day hypoxia had no appreciable effect on BDNF expression, while 3- and 10-day hypoxia progressively decreased BDNF expression, resulting in 37.3% reduction (p < 0.05) after 10-day exposure; (2) DOR activation with UFP-512 (1 mg/kg, i.p., daily) partially reversed the hypoxia-induced reduction of BDNF expression in the 3- or 10-day exposed cortex; (3) DOR activation partially reversed the hypoxia-induced reduction in functional TrkB (140-kDa) and attenuated hypoxia-induced increase in truncated TrkB (90-kDa) in the 3- or 10-day hypoxic cortex; and (4) prolonged hypoxia (10 days) significantly increased TNF-α level and decreased CD11b expression in the cortex, which was completely reversed following DOR activation; and (5) there was no significant change in pCREB and pATF-1 levels in the hypoxic cortex. We conclude that prolonged hypoxia down-regulates BDNF-TrkB signaling leading to an increase in TNF-α in the cortex, while DOR activation up-regulates BDNF-TrkB signaling thereby decreasing TNF-α levels in the hypoxic cortex.