Decreased extrasynaptic δ-GABAA receptors in PNN-associated parvalbumin interneurons correlates with anxiety in APP and tau mouse models of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is associated with gradual memory loss and anxiety which affects ~75% of AD patients. This study investigated whether AD-associated anxiety correlated with modulation of extrasynaptic δ-subunit-containing GABAA receptors (δ-GABAARs) in experimental mouse models of AD. EXPERIMENTAL APPROACH: We combined behavioural experimental paradigms to measure cognition performance, and anxiety with neuroanatomy and molecular biology, using familial knock-in (KI) mouse models of AD that harbour ß-amyloid (Aß) precursor protein App (AppNL-F) with or without humanized microtubule-associated protein tau (MAPT), age-matched to wild-type control mice at three different age windows. RESULTS: AppNL-F KI and AppNL-F/MAPT AD models showed a similar magnitude of cognitive decline and elevated magnitude of anxiety correlated with neuroinflammatory hallmarks, including triggering receptor expressed on myeloid cells 2 (TREM2), reactive astrocytes and activated microglia consistent with accumulation of Aß, tau and down-regulation of Wnt/ß-catenin signalling compared to aged-matched WT controls. In both the CA1 region of the hippocampus and dentate gyrus, there was an age-dependent decline in the expression of δ-GABAARs selectively expressed in parvalbumin (PV)-expressing interneurons, encapsulated by perineuronal nets (PNNs) in the AD mouse models compared to WT mice. In vivo positive allosteric modulation of the δ-GABAARs, using a δ-selective-compound DS2, decreased the level of anxiety in the AD mouse models, which was correlated with reduced hallmarks of neuroinflammation, and 'normalisation' of the expression of δ-GABAARs. CONCLUSIONS: Our data show that the δ-GABAARs could potentially be targeted for alleviating symptoms of anxiety, which would greatly improve the quality of life of AD individuals.

Dysregulated Wnt and NFAT signaling in a Parkinson's disease LRRK2 G2019S knock-in model.

Parkinson's disease (PD) is a progressive late-onset neurodegenerative disease leading to physical and cognitive decline. Mutations of leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. LRRK2 is a complex scaffolding protein with known regulatory roles in multiple molecular pathways. Two prominent examples of LRRK2-modulated pathways are Wingless/Int (Wnt) and nuclear factor of activated T-cells (NFAT) signaling. Both are well described key regulators of immune and nervous system development as well as maturation. The aim of this study was to establish the physiological and pathogenic role of LRRK2 in Wnt and NFAT signaling in the brain, as well as the potential contribution of the non-canonical Wnt/Calcium pathway. In vivo cerebral Wnt and NFATc1 signaling activity was quantified in LRRK2 G2019S mutant knock-in (KI) and LRRK2 knockout (KO) male and female mice with repeated measures over 28 weeks, employing lentiviral luciferase biosensors, and analyzed using a mixed-effect model. To establish spatial resolution, we investigated tissues, and primary neuronal cell cultures from different brain regions combining luciferase signaling activity, immunohistochemistry, qPCR and western blot assays. Results were analyzed by unpaired t-test with Welch's correction or 2-way ANOVA with post hoc corrections. In vivo Wnt signaling activity in LRRK2 KO and LRRK2 G2019S KI mice was increased significantly ~ threefold, with a more pronounced effect in males (~ fourfold) than females (~ twofold). NFATc1 signaling was reduced ~ 0.5-fold in LRRK2 G2019S KI mice. Brain tissue analysis showed region-specific expression changes in Wnt and NFAT signaling components. These effects were predominantly observed at the protein level in the striatum and cerebral cortex of LRRK2 KI mice. Primary neuronal cell culture analysis showed significant genotype-dependent alterations in Wnt and NFATc1 signaling under basal and stimulated conditions. Wnt and NFATc1 signaling was primarily dysregulated in cortical and hippocampal neurons respectively. Our study further built on knowledge of LRRK2 as a Wnt and NFAT signaling protein. We identified complex changes in neuronal models of LRRK2 PD, suggesting a role for mutant LRRK2 in the dysregulation of NFAT, and canonical and non-canonical Wnt signaling.

Astrocyte-neuron lactate transport in the ACC contributes to the occurrence of long-lasting inflammatory pain in male mice.

Lactate transport is an important means of communication between astrocytes and neurons and is implicated in a variety of neurobiological processes. However, the connection between astrocyte-neuron lactate transport and nociceptive modulation has not been well established. Here, we found that Complete Freund's adjuvant (CFA)-induced inflammation pain leads to a significant increase in extracellular lactate levels in the anterior cingulate cortex (ACC). Inhibition of glycogenolysis and lactate release in the ACC disrupted the persistent, but not acute, inflammation pain induced by CFA, and this effect was reversed by exogenous L-lactate administration. Knocking down the expression of lactate transporters (MCT1, MCT4, or MCT2) also disrupted the long lasting inflammation pain induced by CFA. Moreover, glycogenolysis in the ACC is critical for the induction of molecular changes related to neuronal plasticity, including the induction of phospho- (p-) ERK, p-CREB, and Fos. Taken together, our findings indicate that astrocyte-neuron lactate transport in the ACC is critical for the occurrence of persistent inflammation pain, suggesting a novel mechanism underlying chronic pain.

Transcribed ultraconserved noncoding RNA uc.153 is a new player in neuropathic pain.

Transcribed ultraconserved regions are a novel class of long noncoding RNAs and are completely conserved in humans, rats, and mice. Transcribed ultraconserved regions have been implicated in diverse biological processes; however, very little is currently known about their role in pain modulation. Here, we found that the level of the spinal transcribed ultraconserved region uc.153 was significantly increased in a mouse model of sciatic nerve chronic constriction injury (CCI)-induced chronic neuropathic pain. The knockdown of spinal uc.153 prevented and reversed chronic constriction injury-induced pain behaviours and spinal neuronal sensitization. By contrast, the overexpression of spinal uc.153 produced pain behaviours and neuronal sensitization in naive mice. Moreover, we found that uc.153 participates in the regulation of neuropathic pain by negatively modulating the processing of pre-miR-182-5p. Collectively, our findings reveal an important role for uc.153 in pain modulation and provide a novel drug target for neuropathic pain therapy.

METTL3 regulates inflammatory pain by modulating m6A-dependent pri-miR-365-3p processing.

N6-methyladenosine (m6A) modification in RNA has been implicated in diverse biological processes. However, very little is currently known about its role in nociceptive modulation. Here, we found that the level of spinal m6A modification was significantly increased in a mouse model of Complete Freund's Adjuvant (CFA)-induced chronic inflammatory pain, which was accompanied with the augmentation of methyltransferase-like 3 (METTL3) expression in the spinal cord. Knockdown of spinal METTL3 prevented and reversed CFA-induced pain behaviors and spinal neuronal sensitization. In contrast, overexpression of spinal METTL3 produced pain behaviors and neuronal sensitization in naive mice. Moreover, we found that METTL3 positively modulated the pri-miR-65-3p processing in a microprocessor protein DiGeorge critical region 8-dependent manner. Collectively, our findings reveal an important role of METTL3-mediated m6A modification in nociceptive sensitization and provide a novel perspective on m6A modification in the development of pathological pain.

PiRNA-DQ541777 Contributes to Neuropathic Pain via Targeting Cdk5rap1.

Piwi-Interacting RNA (piRNA) is the largest class of small noncoding RNA and is involved in various physiological and pathological processes. However, whether it has a role in pain modulation remains unknown. In the present study, we found that spinal piRNA-DQ541777 (piR-DQ541777) was significantly increased in the male mouse model of sciatic nerve chronic constriction injury (CCI)-induced neuropathic pain. Knockdown of spinal piR-DQ541777 alleviated CCI-induced thermal hyperalgesia and mechanical allodynia and spinal neuronal sensitization. However, the overexpression of spinal piR-DQ541777 in naive mice produced pain behaviors and increased spinal neuron sensitization. Furthermore, we found that piR-DQ541777 regulates pain behaviors by targeting CDK5 regulatory subunit-associated protein 1 (Cdk5rap1). CCI increased the methylation level of CpG islands in the cdk5rap1 promoter and consequently reduced the expression of Cdk5rap1, which was reversed by the knockdown of piR-DQ541777 and mimicked by the overexpression of piR-DQ541777 in naive mice. Finally, piR-DQ541777 increased the methylation level of CpG islands by recruiting DNA methyltransferase 3A (DNMT3a) to cdk5rap1 promoter. In conclusion, this study represents a novel role of piR-DQ541777 in the regulation of neuropathic pain through the methylation of cdk5rap1SIGNIFICANCE STATEMENT Chronic pain affects ∼20% of the population of the world and is a major global public health problem. Although we have studied the neurobiological mechanism of neuropathic pain for decades, there is still no ideal drug available to treat it. This work indicates that a novel role of Piwi-interacting RNA (piRNA) DQ541777 in the regulation of neuropathic pain through the methylation of cdk5rap1 Our findings provide the first evidence of the regulatory effect of piRNAs on neuropathic pain, which may improve our understanding of pain mechanisms and lead to the discovery of novel drug targets for the prevention and treatment of neuropathic pain.

COX-2 contributed to the remifentanil-induced hyperalgesia related to ephrinB/EphB signaling.

Background and Objectives： Studying the underlying mechanisms of opiate-induced hyperalgesia is fundamental to understanding and treating pain. Our previous study has proved that ephrinB/EphB signaling contributes to opiate-induced hyperagesia, but the manner in which ephrinB/EphB signaling acts on spinal nociceptive information networks to produce hyperalgesia remains unclear. Other studies have suggested that ephrinB/EphB signaling, NMDA receptor and COX-2 act together to participate in the modulation of nociceptive information processes at the spinal level. The objective of this research was to investigate the role of COX-2 in remifentanil-induced hyperalgesia and its relationship with ephrinB/EphB signaling. Methods： We characterized the remifentanil-induced pain behaviours by evaluating thermal hyperalgesia and mechanical allodynia in a mouse hind paw incisional model. Protein expression of COX-2 in spinal cord was assayed by western blotting and mRNA level of COX-2 was assayed by Real-time PCR (RT-PCR). Results： Continuing infusion of remifentanil produced thermal hyperalgesia and mechanical allodynia, which was accompanied by increased expression of spinal COX-2 protein and mRNA. This response was inhibited by pre-treatment with EphB2-Fc, an antagonist of ephrinB/EphB. SC58125 and NS398, inhibitors of COX-2, suppressed pain behaviours induced by remifentanil infusion and reversed the increased pain behaviours induced by intrathecal injection of ephrinB2-Fc, an agonist of ephrinB/EphB. Conclusions: Our findings confirmed that COX-2 is involved in remifentanil-induced hyperalgesia related to ephrinB/EphB signaling. EphrinB/EphB signaling might be the upstream of COX-2.

MicroRNA-182-5p Regulates Nerve Injury-induced Nociceptive Hypersensitivity by Targeting Ephrin Type-b Receptor 1.

BACKGROUND: The authors and others have previously shown that the up-regulation of spinal ephrin type-b receptor 1 plays an essential role in the pathologic process of nerve injury-induced nociceptive hypersensitivity, but the regulatory mechanism remains unclear. METHODS: Radiant heat and von Frey filaments were applied to assess nociceptive behaviors. Real-time quantitative polymerase chain reaction, Western blotting, fluorescence in situ hybridization, immunofluorescence, immunohistochemistry, dual-luciferase reporter gene assays, recombinant lentivirus, and small interfering RNA were used to characterize the likely mechanisms. RESULTS: Periphery nerve injury induced by chronic constriction injury of the sciatic nerve significantly reduced spinal microRNA-182-5p (miR-182-5p) expression levels, which were inversely correlated with spinal ephrin type-b receptor 1 expression (R = 0.90; P < 0.05; n = 8). The overexpression of miR-182-5p in the spinal cord prevented and reversed the nociceptive behaviors induced by sciatic nerve injury, accompanied by a decreased expression of spinal ephrin type-b receptor 1 (recombinant lentiviruses containing pre-microRNA-182: 1.91 ± 0.34 vs. 1.24 ± 0.31, n = 4; miR-182-5p mimic: 2.90 ± 0.48 vs. 1.51 ± 0.25, n = 4). In contrast, the down-regulation of spinal miR-182-5p facilitated the nociceptive behaviors induced by sciatic nerve injury and increased the expression of spinal ephrin type-b receptor 1 (1.0 ± 0.26 vs. 1.74 ± 0.31, n = 4). Moreover, the down-regulation of miR-182-5p and up-regulation of ephrin type-b receptor 1 caused by sciatic nerve injury were mediated by the N-methyl-D-aspartate receptor. CONCLUSIONS: Collectively, our findings reveal that the spinal ephrin type-b receptor 1 is regulated by miR-182-5p in nerve injury-induced nociceptive hypersensitivity.

Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain.

BACKGROUND: The analgesic potency of opioids is reduced in neuropathic pain. However, the molecular mechanism is not well understood. RESULTS: The present study demonstrated that increased methylation of the Mu opioid receptor (MOR) gene proximal promoter (PP) in dorsal root ganglion (DRG) plays a crucial role in the decreased morphine analgesia. Subcutaneous (s.c.), intrathecal (i.t.) and intraplantar (i.pl.), not intracerebroventricular (i.c.v.) injection of morphine, the potency of morphine analgesia was significantly reduced in nerve-injured mice compared with control sham-operated mice. After peripheral nerve injury, we observed a decreased expression of MOR protein and mRNA, accompanied by an increased methylation status of MOR gene PP, in DRG. However, peripheral nerve injury could not induce a decreased expression of MOR mRNA in the spinal cord. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC), inhibited the increased methylation of MOR gene PP and prevented the decreased expression of MOR in DRG, thereby improved systemic, spinal and periphery morphine analgesia. CONCLUSIONS: Altogether, our results demonstrate that increased methylation of the MOR gene PP in DRG is required for the decreased morphine analgesia in neuropathic pain.

[Pretreatment with cisplatin plus hyperthermia kills different tumor cells but preserves the erythrocyte function in the intra-operative blood salvage in vitro].

OBJECTIVE: To investigate the effects of cisplatin plus hyperthermia on erythrocytes and killing human hepatocarcinoma (HepG2), gastro carcinoma (SGC7901) and colonic carcinoma (SW620) cells in the intra-operative blood salvage from cancer surgery in vitro. METHODS: HepG2, SGC7901 or SW620 cells were mixed into the aliquot of erythrocyte concentrated from each intra-operative blood salvage of 30 patients subjected to gastrointestinal cancer surgery. The mixture cells were divided into the following groups (n = 30): A group (37 °C); B group (42 °C); C, D, E groups (50, 100, or 200 µg/ml DDP); F, G, H, I groups (42 °C, 25, 50, 100, or 200 µg/ml DDP). After treating for 60 min, tumor cells and erythrocytes were separated by density gradient centrifugation. The Na(+)-K(+)-ATPase activity, cell count, osmotic fragility, and blood gas variables were determined in erythrocytes. Cell viability and colony formation were determined in tumor cells. RESULTS: Compared with A [(0.30 ± 0.08) µmol Pi/10(7)/h], the Na(+)-K(+)-ATPase activity was significantly decreased in E, H and I groups [(0.24 ± 0.07), (0.25 ± 0.06) and (0.24 ± 0.07) µmol Pi/10(7)/h] (P < 0.05). Extra-erythrocytic K(+) in E, H and I groups [(2.16 ± 0.37), (2.16 ± 0.38) and (2.56 ± 0.50) mmol/L] were significantly increased compared with A group [(1.53 ± 0.43) mmol/L] (P < 0.05). Compared with A group, osmotic fragility in E, H and I groups was significantly increased (P < 0.05). Among B, C, D, E, F, G groups, only in G group colony formations of HepG2, SGC7901, and SW620 (0% ± 0%, 0% ± 0% and 0.01% ± 0.01%) at 14 d were completely inhibited (P < 0.01) compared with A group (78.54% ± 7.83%, 72.28% ± 6.58% and 66.69% ± 6.69%). CONCLUSION: Pretreatment with cisplatin (50 µg/ml) plus hyperthermia (42 °C) for 60 min in vitro might be an effective strategy to clear tumor cells contamination but preserve erythrocytes, which is worthy to be optimized and used in the intra-operative blood salvage in cancer surgery.

Hypercholesterolemia blocked sevoflurane-induced cardioprotection against ischemia-reperfusion injury by alteration of the MG53/RISK/GSK3ß signaling.

BACKGROUND: Recent studies have demonstrated that volatile anesthetic preconditioning confers myocardial protection against ischemia-reperfusion (IR) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired in hypercholesterolemia, we investigate whether anesthetic-induced cardiac protection was maintained in hypercholesterolemic rats. METHODS: Normocholesteolemic or hypercholesterolemic rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Animals received 2.4% sevoflurane during three 5 min periods with and without PI3K antagonist wortmannin (10 µg/kg, Wort) or the ERK inhibitor PD 98059 (1 mg/kg, PD). The infarct size, apoptosis, p-Akt, p-ERK1/2, p-GSK3ß were determined. RESULTS: Two hundred and six rats were analyzed in the study. In the healthy rats, sevoflurane significantly reduced infarct size by 42%, a phenomenon completely reversed by wortmannin and PD98059 and increased the phosphorylation of Akt, ERK1/2 and their downstream target of GSK3ß. In the hypercholesterolemic rats, sevoflurane failed to reduce infarct size and increase the phosphorylated Akt, ERK1/2 and GSK3ß. In contrast, GSK inhibitor SB216763 conferred cardioprotection against IR injury in healthy and hypercholesterolemic hearts. CONCLUSIONS: Hyperchoesterolemia abrogated sevoflurane-induced cardioprotection against IR injury by alteration of upstream signaling of GSK3ß and acute GSK inhibition may provide a novel therapeutic strategy to protect hypercholesterolemic hearts against IR injury.