The HMGB1-RAGE axis in nucleus accumbens facilitates cocaine-induced conditioned place preference via modulating microglial activation.

INTRODUCTION: Repeated exposure to cocaine induces microglial activation. Cocaine exposure also induces a release of high mobility group box-1 (HMGB1) from neurons into the extracellular space in the nucleus accumbens (NAc). HMGB1 is an important late inflammatory mediator of microglial activation. However, whether the secretion of HMGB1 acts on microglia or contributes to cocaine addiction is largely unknown. METHODS: Rats were trained by intraperitoneal cocaine administration and cocaine-induced conditioned place preference (CPP). Expression of HMGB1 was regulated by viral vectors. Activation of microglia was inhibited by minocycline. Interaction of HMGB1 and the receptor for advanced glycation end products (RAGE) was disrupted by peptide. RESULTS: Cocaine injection facilitated HMGB1 signaling, together with the delayed activation of microglia concurrently in the NAc. Furthermore, the inhibition of HMGB1 or microglia activation attenuated cocaine-induced CPP. Box A, a specific antagonist to interrupt the interaction of HMGB1 and RAGE, abolished the expression of cocaine reward memory. Meanwhile, the inhibition of HMGB1-RAGE interaction suppressed cocaine-induced microglial activation, as well as the consolidation of cocaine-induced memory. CONCLUSION: All above results suggest that the neural HMGB1 induces activation of microglia through RAGE, which contributes to the consolidation of cocaine reward memory. These findings offer HMGB1-RAGE axis as a new target for the treatment of drug addiction.

Correction: Orexin-A Promotes Cell Migration in Cultured Rat Astrocytes via Ca2+-Dependent PKCα and ERK1/2 Signals.

[This corrects the article DOI: 10.1371/journal.pone.0095259.].

Cell type-specific NRBF2 orchestrates autophagic flux and adult hippocampal neurogenesis in chronic stress-induced depression.

Dysfunctional autophagy and impairment of adult hippocampal neurogenesis (AHN) each contribute to the pathogenesis of major depressive disorder (MDD). However, whether dysfunctional autophagy is linked to aberrant AHN underlying MDD remains unclear. Here we demonstrate that the expression of nuclear receptor binding factor 2 (NRBF2), a component of autophagy-associated PIK3C3/VPS34-containing phosphatidylinositol 3-kinase complex, is attenuated in the dentate gyrus (DG) under chronic stress. NRBF2 deficiency inhibits the activity of the VPS34 complex and impairs autophagic flux in adult neural stem cells (aNSCs). Moreover, loss of NRBF2 disrupts the neurogenesis-related protein network and causes exhaustion of aNSC pool, leading to the depression-like phenotype. Strikingly, overexpressing NRBF2 in aNSCs of the DG is sufficient to rescue impaired AHN and depression-like phenotype of mice. Our findings reveal a significant role of NRBF2-dependent autophagy in preventing chronic stress-induced AHN impairment and suggest the therapeutic potential of targeting NRBF2 in MDD treatment.

CD200 in dentate gyrus improves depressive-like behaviors of mice through enhancing hippocampal neurogenesis via alleviation of microglia hyperactivation.

BACKGROUND: Neuroinflammation and microglia play critical roles in the development of depression. Cluster of differentiation 200 (CD200) is an anti-inflammatory glycoprotein that is mainly expressed in neurons, and its receptor CD200R1 is primarily in microglia. Although the CD200-CD200R1 pathway is necessary for microglial activation, its role in the pathophysiology of depression remains unknown. METHODS: The chronic social defeat stress (CSDS) with behavioral tests were performed to investigate the effect of CD200 on the depressive-like behaviors. Viral vectors were used to overexpress or knockdown of CD200. The levels of CD200 and inflammatory cytokines were tested with molecular biological techniques. The status of microglia, the expression of BDNF and neurogenesis were detected with immunofluorescence imaging. RESULTS: We found that the expression of CD200 was decreased in the dentate gyrus (DG) region of mice experienced CSDS. Overexpression of CD200 alleviated the depressive-like behaviors of stressed mice and inhibition of CD200 facilitated the susceptibility to stress. When CD200R1 receptors on microglia were knocked down, CD200 was unable to exert its role in alleviating depressive-like behavior. Microglia in the DG brain region were morphologically activated after exposure to CSDS. In contrast, exogenous administration of CD200 inhibited microglia hyperactivation, alleviated neuroinflammatory response in hippocampus, and increased the expression of BDNF, which in turn ameliorated adult hippocampal neurogenesis impairment in the DG induced by CSDS. CONCLUSIONS: Taken together, these results suggest that CD200-mediated alleviation of microglia hyperactivation contributes to the antidepressant effect of neurogenesis in dentate gyrus in mice.

BNIP3L/NIX-mediated mitophagy alleviates passive stress-coping behaviors induced by tumor necrosis factor-α.

Recent studies based on animal models of various neurological disorders have indicated that mitophagy, a selective autophagy that eliminates damaged and superfluous mitochondria through autophagic degradation, may be involved in various neurological diseases. As an important mechanism of cellular stress response, much less is known about the role of mitophagy in stress-related mood disorders. Here, we found that tumor necrosis factor-α (TNF-α), an inflammation cytokine that plays a particular role in stress responses, impaired the mitophagy in the medial prefrontal cortex (mPFC) via triggering degradation of an outer mitochondrial membrane protein, NIP3-like protein X (NIX). The deficits in the NIX-mediated mitophagy by TNF-α led to the accumulation of damaged mitochondria, which triggered synaptic defects and behavioral abnormalities. Genetic ablation of NIX in the excitatory neurons of mPFC caused passive coping behaviors to stress, and overexpression of NIX in the mPFC improved TNF-α-induced synaptic and behavioral abnormalities. Notably, ketamine, a rapid on-set and long-lasting antidepressant, reversed the TNF-α-induced behavioral abnormalities through activation of NIX-mediated mitophagy. Furthermore, the downregulation of NIX level was also observed in the blood of major depressive disorder patients and the mPFC tissue of animal models. Infliximab, a clinically used TNF-α antagonist, alleviated both chronic stress- and inflammation-induced behavioral abnormalities via restoring NIX level. Taken together, these results suggest that NIX-mediated mitophagy links inflammation signaling to passive coping behaviors to stress, which underlies the pathophysiology of stress-related emotional disorders.

Microglia-dependent excessive synaptic pruning leads to cortical underconnectivity and behavioral abnormality following chronic social defeat stress in mice.

Synapse loss in medial prefrontal cortex (mPFC) has been implicated in stress-related mood disorders, such as depression. However, the exact effect of synapse elimination in the depression and how it is triggered are largely unknown. Through repeated longitudinal imaging of mPFC in the living brain, we found both presynaptic and postsynaptic components were declined, together with the impairment of synapse remodeling and cross-synaptic signal transmission in the mPFC during chronic stress. Meanwhile, chronic stress also induced excessive microglia phagocytosis, leading to engulfment of excitatory synapses. Further investigation revealed that the elevated complement C3 during the stress acted as the tag of synapses to be eliminated by microglia. Besides, chronic stress induced a reduction of the connectivity between the mPFC and neighbor regions. C3 knockout mice displayed significant reduction of synaptic pruning and alleviation of disrupted functional connectivity in mPFC, resulting in more resilience to chronic stress. These results indicate that complement-mediated excessive microglia phagocytosis in adulthood induces synaptic dysfunction and cortical hypo-connectivity, leading to stress-related behavioral abnormality.

Deficiency of Glycosylated α-Dystroglycan in Ventral Hippocampus Bridges the Destabilization of Gamma-Aminobutyric Acid Type A Receptors With the Depressive-like Behaviors of Male Mice.

BACKGROUND: Depression is a common psychiatric disorder associated with defects in GABAergic (gamma-aminobutyric acidergic) neurotransmission. α-Dystroglycan (α-DG), a cell adhesion molecule known to be essential for skeletal muscle integrity, is also present at inhibitory synapses in the central nervous system and forms a structural element in certain synapses. However, the role of α-DG in the regulation of depressive-like behaviors remains largely unknown. METHODS: Depressive-like behaviors were induced by chronic social defeat stress in adult male mice. Surface protein was extracted by a biotin kit, and the expression of protein was detected by Western blotting. Intrahippocampal microinjection of the lentivirus or adeno-associated virus or agrin intervention was carried out using a stereotaxic instrument and followed by behavioral tests. Miniature inhibitory postsynaptic currents were recorded by whole-cell patch-clamp techniques. RESULTS: The expression of α-DG and glycosylated α-DG in the ventral hippocampus was significantly lower in chronic social defeat stress-susceptible male mice than in control mice, accompanied by a decreased surface expression of GABAA receptor γ2 subunit and reduced GABAergic neurotransmission. RNA interference-mediated knockdown of Dag1 increased the susceptibility of mice to subthreshold stress. Both in vivo administration of agrin and overexpression of like-acetylglucosaminyltransferase ameliorated depressive-like behaviors and restored the decrease in surface expression of GABAA receptor γ2 subunit and the amplitude of miniature inhibitory postsynaptic currents in chronic social defeat stress-exposed mice. CONCLUSIONS: Our findings demonstrate that glycosylated α-DG plays a role in the pathophysiological process of depressive-like behaviors by regulating the surface expression of GABAA receptor γ2 subunit and GABAergic neurotransmission in the ventral hippocampus.

The effects of Kctd12, an auxiliary subunit of GABAB receptor in dentate gyrus on behavioral response to chronic social defeat stress in mice.

Adaptive responses to stress are critical to enhance physical and mental well-being, but excessive or prolonged stress may cause inadaptability and increase the risks of psychiatric disorders, such as depression. GABABR signaling is fundamental to brain function and has been identified in neuropsychiatric disorders. KCTD12 is a critical auxiliary subunit in GABABR signaling, but its role in mental disorders, such as depression is unclear. In the present study, we used a well-validated mice model, chronic social defeat stress (CSDS) to investigate behavioral responses to stress and explore the role of Kctd12 in stress response, as well as the relevant mechanisms. We found that CSDS increased the expression of Kctd12 in the dentate gyrus (DG), a subregion of hippocampus. Overexpression of Kctd12 in DG induced higher responsiveness to acute stress and increased vulnerability to social stress in mice, whereas knock-down of Kctd12 in DG prevented the social avoidance. Furthermore, an increased expression of GABAB receptor 2 (GB2) in the DG of CSDS-treated mice was observed, and CGP35348, an antagonist of GABABR, improved the stress-induced behavior responses along with suppressing the excess expression of Kctd12. In addition, Kctd12 regulated the excitability of granule cell in DG, and the stimulation of neuronal activity by silencing Kctd12 contributed to the antidepressant-like effect of fluoxetine. These findings identify that the Kctd12 in DG works as a critical mediator of stress responses, providing a promising therapeutic target in stress-related psychiatric disorders, including depression.

Transcription Factor TWIST1 Integrates Dendritic Remodeling and Chronic Stress to Promote Depressive-like Behaviors.

BACKGROUND: Deficiency in neuronal structural plasticity is involved in the development of major depressive disorder. TWIST1, a helix-loop-helix transcription factor that is essential for morphogenesis and organogenesis, is normally expressed at low levels in mature neurons. However, it is poorly understood what role TWIST1 plays in the brain and whether it is involved in the pathophysiology of depression. METHODS: Depressive-like behaviors in C57BL/6J mice were developed by chronic social defeat stress. Genetic and pharmacological approaches were used to investigate the role of the TWIST1-miR-214-PPAR-δ signaling pathway in depressive-like behaviors. Molecular biological and morphological studies were performed to define the molecular mechanisms downstream of TWIST1. RESULTS: The expression of TWIST1 was positively correlated with depressive behaviors in humans and mice. Chronic stress elevated TWIST1 expression in the medial prefrontal cortex of mice, which was reversed by fluoxetine treatment. While the overexpression of TWIST1 increased susceptibility to stress, the knockdown of TWIST1 prevented the defective morphogenesis of dendrites of pyramidal neurons in layer II/III of the medial prefrontal cortex and alleviated depressive-like behaviors. Mechanistically, this prodepressant property of TWIST1 was mediated, at least in part, through the repression of miR-214-PPAR-δ signaling and mitochondrial function, which was also mimicked by genetic and pharmacological inhibition of PPAR-δ. CONCLUSIONS: These results suggest that TWIST1 in the medial prefrontal cortex mediates chronic stress-induced dendritic remodeling and facilitates the occurrence of depressive-like behavior, providing new information for developing drug targets for depression therapy.

SKF83959, an agonist of phosphatidylinositol-linked dopamine receptors, prevents renewal of extinguished conditioned fear and facilitates extinction.

Fear-related anxiety disorders, such as social phobia and post-traumatic stress disorder, are partly explained by an uncontrollable state of fear. An emerging literature suggests dopamine receptor-1 (D1 receptor) in the amygdala is involved in the regulation of fear memory. An early study has reported that amygdaloid D1 receptor (D1R) is not coupled to the classic cAMP-dependent signal transduction. Here, we investigated whether SKF83959, a typical D1R agonist that mainly activates a D1-like receptor-dependent phosphatidylinositol (PI) signal pathway, facilitates fear extinction and reduces the return of extinguished fear. Interestingly, long-term loss of fearful memories can be induced through a combination of SKF83959 (1 mg/kg/day, i.p., once daily for one week) pharmacotherapy and extinction training. Furthermore, sub-chronic administration of SKF83959 after fear conditioning reduced fear renewal and reinstatement in the mice. We found that the activation D1R and PI signaling in the amygdala was responsible for the effect of SKF83959 on fear extinction. Additionally, SKF83959 significantly promoted the elevation of brain-derived neurotrophic factor (BDNF) expression, possibly by the cAMP response element binding protein (CREB) -directed gene transcription. Given the beneficial effects on extinction, SKF83959 may emerge as a candidate pharmacological approach for improving cognitive-behavioral therapy on fear-related anxiety disorders.

Response by the authors.

Response to comments on Cui Q-Q et al: "Hippocampal CD 39/ENTPD 1 promotes mouse depression-like behavior through hydrolyzing extracellular ATP".

Angiotensin-Converting Enzyme Inhibitor Rapidly Ameliorates Depressive-Type Behaviors via Bradykinin-Dependent Activation of Mammalian Target of Rapamycin Complex 1.

BACKGROUND: Angiotensin-converting enzyme inhibitors (ACEIs) are widely prescribed antihypertensive agents. Intriguingly, case reports and clinical trials have indicated that ACEIs, including captopril and lisinopril, may have a rapid mood-elevating effect in certain patients, but few experimental studies have investigated their value as fast-onset antidepressants. METHODS: The present study consisted of a series of experiments using biochemical assays, immunohistochemistry, and behavioral techniques to examine the effect and mechanism of captopril on depressive-like behavior in 2 animal models, the chronic unpredictable stress model and the chronic social defeat stress model. RESULTS: Captopril (19.5 or 39 mg/kg, intraperitoneal injection) exerted rapid antidepressant activity in mice treated under the chronic unpredictable stress model and mice treated under the chronic social defeat stress model. Pharmacokinetic analysis revealed that captopril crossed the blood-brain barrier and that lisinopril, another ACEI with better blood-brain barrier permeability, exerted a faster and longer-lasting effect at a same molar equivalent dose. This antidepressant effect seemed to be independent of the renin-angiotensin system, but dependent on the bradykinin (BK) system, since the decreased BK detected in the stressed mice could be reversed by captopril. The hypofunction of the downstream effector of BK, Cdc42 (cell division control protein 42) homolog, contributed to the stress-induced loss of dendritic spines, which was rapidly reversed by captopril via activating the mTORC1 (mammalian target of rapamycin complex 1) pathway. CONCLUSIONS: Our findings indicate that the BK-dependent activation of mTORC1 may represent a promising mechanism underlying antidepressant pharmacology. Considering their affordability and availability, ACEIs may emerge as a novel fast-onset antidepressant, especially for patients with comorbid depression and hypertension.

Hippocampal CD39/ENTPD1 promotes mouse depression-like behavior through hydrolyzing extracellular ATP.

Emerging evidence implicates that low levels of ATP in the extracellular space may contribute to the pathophysiology of major depressive disorder (MDD). The concentration of extracellular ATP is regulated by its hydrolase ectonucleotide tri(di)phosphohydrolase (ENTPD). However, the role of ENTPD in depression remains poorly understood. Here we examine the role of CD39 (known as ENTPD1) in mouse depression-like behavior induced by chronic social defeat stress (CSDS). We demonstrate that CSDS enhances the expression and activity of CD39 in hippocampus. The CD39 functional analog apyrase also induces depression-like behavior, which can be ameliorated by ATP replenishment. Pharmacological inhibition and genetic silencing of CD39 has an antidepressant-like effect via increasing hippocampal extracellular ATP concentration, accompanied with an increase in hippocampal neurogenesis and dendritic spine numbers in defeated mice. These results suggest that hippocampal CD39 contributes to CSDS-induced depression-like behavior via hydrolyzing extracellular ATP, indicating that CD39 may be a promising new target for the treatment of depression.

Neuronal HMGB1 in nucleus accumbens regulates cocaine reward memory.

Cocaine is a common abused drug that can induce abnormal synaptic and immune responses in the central nervous system (CNS). High mobility group box 1 (HMGB1) is one kind of inflammatory molecules that is expressed both on neurons and immune cells. Previous studies of HMGB1 in the CNS have largely focused on immune function, and the role of HMGB1 in neurons and cocaine addiction remains unknown. Here, we show that cocaine exposure induced the translocation and release of HMGB1 in the nucleus accumbens (NAc) neurons. Gain and loss of HMGB1 in the NAc bidirectionally regulate cocaine-induced conditioned place preference. From the nucleus to the cytosol, HMGB1 binds to glutamate receptor subunits (GluA2/GluN2B) on the membrane, which regulates cocaine-induced synaptic adaptation and the formation of cocaine-related memory. These data unveil the role of HMGB1 in neurons and provide the evidence for the HMGB1 involvement in drug addiction.

A-Kinase Anchoring Protein 150 and Protein Kinase A Complex in the Basolateral Amygdala Contributes to Depressive-like Behaviors Induced by Chronic Restraint Stress.

BACKGROUND: The basolateral amygdala (BLA) has been widely implicated in the pathophysiology of major depressive disorder. A-kinase anchoring protein 150 (AKAP150) directs kinases and phosphatases to synaptic glutamate receptors, controlling synaptic transmission and plasticity. However, the role of the AKAP150 in the BLA in major depressive disorder remains poorly understood. METHODS: Depressive-like behaviors in C57BL/6J mice were developed by chronic restraint stress (CRS). Mice received either intra-BLA injection of lentivirus-expressing Akap5 short hairpin RNA or Ht-31, a peptide to disrupt the interaction of AKAP150 and protein kinase A (PKA), followed by depressive-like behavioral tests. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptor (AMPAR)-mediated miniature excitatory postsynaptic currents were recorded by whole-cell patch-clamp techniques. RESULTS: Chronic stress exposure induced depressive-like behaviors, which were accompanied by an increase in total and synaptic AKAP150 expression in the BLA. Accordingly, CRS facilitated the association of AKAP150 with PKA, but not of calcineurin in the BLA. Intra-BLA infusion of lentivirus-expressing Akap5 short hairpin RNA or Ht-31 prevented depressive-like behaviors and normalized phosphorylation of serine 845 and surface expression of AMPAR subunit 1 (GluA1) in the BLA of CRS mice. Finally, blockage of AKAP150-PKA complex signaling rescued the changes in AMPAR-mediated miniature excitatory postsynaptic currents in depressive-like mice. CONCLUSIONS: These results suggest that AKAP150-PKA directly modulates BLA neuronal synaptic strength, and that AKAP150-PKA-GluA1 streamline signaling complex is responsible for CRS-induced disruption of synaptic AMPAR-mediated transmission and depressive-like behaviors in mice.

Gephyrin Palmitoylation in Basolateral Amygdala Mediates the Anxiolytic Action of Benzodiazepine.

BACKGROUND: Benzodiazepines (BZDs) have been used to treat anxiety disorders for more than five decades as the allosteric modulator of the gamma-aminobutyric acid A receptor (GABAAR). Little is known about other mechanisms of BZDs. Here, we describe how the rapid stabilization of postsynaptic GABAAR is essential and sufficient for the anxiolytic effect of BZDs via a palmitoylation-dependent mechanism. METHODS: Palmitoylated proteins in the basolateral amygdala (BLA) of rats with different anxious states were assessed by a biotin exchange protocol. Both pharmacological and genetic approaches were used to investigate the role of palmitoylation in anxiety behavior. Electrophysiological recording, reverse transcription polymerase chain reaction, Western blotting, and coimmunoprecipitation were used to investigate the mechanisms. RESULTS: Highly anxious rats were accompanied by the deficiency of gephyrin palmitoylation and decreased the synaptic function of GABAAR in the BLA. We then identified that the dysfunction of DHHC12, a palmitoyl acyltransferase that specifically palmitoylates gephyrin, contributed to the high-anxious state. Furthermore, diazepam, as an anxiolytic drug targeting GABAARs, was found to increase gephyrin palmitoylation in the BLA via a GABAAR-dependent manner to activate DHHC12. The anxiolytic effect of diazepam was nearly abolished by the DHHC12 knockdown. Specifically, similar to the effect of BZD, the overexpression of DHHC12 in the BLA exerted a significant anxiolytic action, which was prevented by flumazenil. CONCLUSIONS: Our results support the view that the strength of inhibitory synapse was controlled by gephyrin palmitoylation in vivo and proposes a previously unknown palmitoylation-centered mode of BZD's action.

SAR405, a Highly Specific VPS34 Inhibitor, Disrupts Auditory Fear Memory Consolidation of Mice via Facilitation of Inhibitory Neurotransmission in Basolateral Amygdala.

BACKGROUND: Autophagy has been demonstrated to play an important role in memory deficits as well as the degradation of neurotransmitter receptors. SAR405 is a newly discovered inhibitor that can specifically inhibit vacuolar sorting protein 34 and prevent autophagosome biogenesis. However, the effects of SAR405 on memory processes remain largely unknown. METHODS: Western blotting, immunofluorescence, and transmission electron microscopy were used to assess the level of autophagy after fear conditioning and SAR405 treatment. Behavioral tests, biotinylation assay, electrophysiology, and co-immunoprecipitation were used to unravel the mechanisms of SAR405 in memory consolidation. RESULTS: SAR405 infusion into the basolateral amygdala impaired long-term memory through autophagy inhibition. Furthermore, the trafficking of gamma-aminobutyric acid type A receptors (GABAARs) following fear conditioning was disrupted by SAR405, and the decreased frequency and amplitude of miniature inhibitory postsynaptic currents induced by fear conditioning were also reversed by SAR405, suggesting that SAR405 disrupted memory consolidation through blockade of the downregulated inhibitory neurotransmission in basolateral amygdala. GABAAR-associated protein (GABARAP) and its interaction with GABAAR γ2 subunit were found to be upregulated after fear conditioning, and SAR405 could suppress this increased interaction. Moreover, disruption of the GABARAP-GABAAR binding by a trans-activating transcriptional activator-GABARAP inhibitory peptide blocked the decrease in surface expression of GABAARs and attenuated long-term memory. CONCLUSIONS: The present study suggests that SAR405 can prevent the memory consolidation via intervening autophagy and GABAAR trafficking and has a potential therapeutic value for disorders characterized by exaggerated fear memories, such as posttraumatic stress disorder.

Identification and Function of Acid-sensing Ion Channels in RAW 264.7 Macrophage Cells.

Activation of acid-sensing ion channels (ASICs) plays an important role in neuroinflammation. Macrophage recruitment to the sites of inflammation is an essential step in host defense. ASIC1 and ASIC3 have been reported to mediate the endocytosis and maturation of bone marrow derived macrophages. However, the expression and inflammation-related functions of ASICs in RAW 264.7 cells, another common macrophage, are still elusive. In the present study, we first demonstrated the presence of ASIC1, ASIC2a and ASIC3 in RAW 264.7 macrophage cell line by using reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence experiments. The non-specific ASICs inhibitor amiloride and specific homomeric ASICla blocker PcTxl reduced the production of iNOS and COX-2 by LPS-induced activating RAW 264.7 cells. Furthermore, not only amiloride but also PcTxl inhibited the migration and LPS-induced apoptosis of RAW 264.7 cells. Taken together, our findings suggest that ASICs promote the inflammatory response and apoptosis of RAW 264.7 cells, and ASICs may serve as a potential novel target for immunological disease therapy.

Pannexin-1 channel dysfunction in the medial prefrontal cortex mediates depressive-like behaviors induced by chronic social defeat stress and administration of mefloquine in mice.

Mefloquine (MFQ) is widely used for the treatment of malaria clinically. Apart from antimalarial effect, psychiatric side effects such as depression and anxiety of MFQ have been reported. Interestingly, MFQ is also known as a broad-spectrum pannexin-1 (Panx1) inhibitor. Panx1 is a new gap junction channel in the brain which mediates efflux of adenosine triphosphate (ATP). Although exogenous ATP has been known to produce a potential antidepressant-like effect, little is known about the role of Panx1 in pathophysiology of depression, especially the depression induced by administration of MFQ. Here, we used the chronic social defeat stress (CSDS) model and found a decrease in the expression and function of Panx1 in the medial prefrontal cortex (mPFC) of susceptible mice. Furthermore, pharmacological blockade of Panx1 in the mPFC with carbenoxolone (CBX) (100 mM) or 10Panx (100 µM) was sufficient to induce depressive-like behaviors and increase vulnerability to stress in mice, which were prevented by preconditioning with ATP (25 µM). Finally, systemic and intral-mPFC injection of MFQ both inhibited the activity of Panx1 and induced depressive-like and anxiety behaviors in mice with sub-threshold social defeat stress. Indeed, the behavioral abnormalities induced by MFQ were prevented by preconditioning with ATP in the mPFC. In conclusion, our study demonstrates a role of the Panx1 channel in chronic stress and MFQ-induced depressive-like and anxiety behaviors, which may provide a novel molecular mechanism for psychiatric side effects of MFQ.

ASIC1 and ASIC3 contribute to acidity-induced EMT of pancreatic cancer through activating Ca2+/RhoA pathway.

Extracellular acid can have important effects on cancer cells. Acid-sensing ion channels (ASICs), which emerged as key receptors for extracellular acidic pH, are differently expressed during various diseases and have been implicated in underlying pathogenesis. This study reports that ASIC1 and ASIC3 are mainly expressed on membrane of pancreatic cancer cells and upregulated in pancreatic cancer tissues. ASIC1 and ASIC3 are responsible for an acidity-induced inward current, which is required for elevation of intracellular Ca2+ concentration ([Ca2+]i). Inhibition of ASIC1 and ASIC3 with siRNA or pharmacological inhibitor significantly decreased [Ca2+]i and its downstream RhoA during acidity and, thus, suppressed acidity-induced epithelial-mesenchymal transition (EMT) of pancreatic cancer cells. Meanwhile, downregulating [Ca2+]i with calcium chelating agent BAPTA-AM or knockdown of RhoA with siRNA also significantly repressed acidity-induced EMT of pancreatic cancer cells. Significantly, although without obvious effect on proliferation, knockdown of ASIC1 and ASIC3 in pancreatic cancer cells significantly suppresses liver and lung metastasis in xenograft model. In addition, ASIC1 and ASIC3 are positively correlated with expression of mesenchymal marker vimentin, but inversely correlated with epithelial marker E-cadherin in pancreatic cancer cells. In conclusion, this study indicates that ASICs are master regulator of acidity-induced EMT. In addition, the data demonstrate a functional link between ASICs and [Ca2+]i/RhoA pathway, which contributes to the acidity-induced EMT.