Activation of G protein-coupled receptor 30 protects neurons by regulating autophagy in astrocytes.

Ischemic stroke leads to neuronal damage induced by excitotoxicity, inflammation, and oxidative stress. Astrocytes play diverse roles in stroke and ischemia-induced inflammation, and autophagy is critical for maintaining astrocytic functions. Our previous studies showed that the activation of G protein-coupled receptor 30 (GPR30), an estrogen membrane receptor, protected neurons from excitotoxicity. However, the role of astrocytic GPR30 in maintaining autophagy and neuroprotection remained unclear. In this study, we found that the neuroprotection induced by G1 (GPR30 agonist) in wild-type mice after a middle cerebral artery occlusion was completely blocked in GPR30 conventional knockout (KO) mice but partially attenuated in astrocytic or neuronal GPR30 KO mice. In cultured primary astrocytes, glutamate exposure induced astrocyte proliferation and decreased astrocyte autophagy by activating mammalian target of rapamycin (mTOR) and c-Jun N-terminal kinase (JNK) and inhibiting p38 mitogen-activated protein kinase (MAPK) pathway. G1 treatment restored autophagy to its basal level by regulating the p38 pathway but not the mTOR and JNK signaling pathways. Our findings revealed a key role of GPR30 in neuroprotection via the regulation of astrocyte autophagy and support astrocytic GPR30 as a potential drug target against ischemic brain damage.

Cyclic AMP-dependent positive feedback signaling pathways in the cortex contributes to visceral pain.

Cortical areas including the anterior cingulate cortex (ACC) play critical roles in different types of chronic pain. Most of previous studies focus on the sensory inputs from somatic areas, and less information about plastic changes in the cortex for visceral pain. In this study, chronic visceral pain animal model was established by injection with zymosan into the colon of adult male C57/BL6 mice. Whole cell patch-clamp recording, behavioral tests, western blot, and Cannulation and ACC microinjection were employed to explore the role of adenylyl cyclase 1 (AC1) in the ACC of C57/BL6 and AC1 knock out mice. Integrative approaches were used to investigate possible changes of neuronal AC1 in the ACC after the injury. We found that AC1, a key enzyme for pain-related cortical plasticity, was significantly increased in the ACC in an animal model of irritable bowel syndrome. Inhibiting AC1 activity by a selective AC1 inhibitor NB001 significantly reduced the up-regulation of AC1 protein in the ACC. Furthermore, we found that AC1 is required for NMDA GluN2B receptor up-regulation and increases of NMDA receptor-mediated currents. These results suggest that AC1 may form a positive regulation in the cortex during chronic visceral pain. Our findings demonstrate that the up-regulation of AC1 protein in the cortex may underlie the pathology of chronic visceral pain; and inhibiting AC1 activity may be beneficial for the treatment of visceral pain.

Effects of CPEB1 in the anterior cingulate cortex on visceral pain in mice.

Patients with irritable bowel syndrome suffer from chronic visceral pain, and in some of them, this is accompanied by anxiety comorbidity. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) mediates the cytoplasmic polyadenylation of mRNAs and facilitates their translation. Our previous studies have shown that CPEB1 knockdown in the amygdala exerts anxiolytic but not analgesic effects in a mouse model of inflammatory pain. However, the roles of CPEB1 in the anterior cingulate cortex (ACC) in visceral pain modulation remain unclear. In this study, a visceral pain mouse model was established by injecting zymosan into the colon of mice. Zymosan injection significantly induced visceral pain- and anxiety-like behaviors in mice and increased the levels of GluA1, phosphorylated GluA1 at S845 and S831, and CPEB1 in the ACC. CPEB1 knockdown in the ACC by AAV-CPEB1-shRNA reduced zymosan-induced pain- and anxiety-like behaviors in mice. This observation was closely correlated with reduced AMPA receptor, synaptophysin, and PSD95 levels. These data suggest that CPEB1 in the ACC is a potential therapeutic target for visceral pain and anxiety comorbidity.

Anxiolytic effects of Formononetin in an inflammatory pain mouse model.

Chronic pain is commonly accompanied with anxiety disorder, which complicates treatment. In this study, we investigated the analgesic and anxiolytic effects of Formononetin (FMNT), an active component of traditional Chinese medicine red clover (Trifolium pratense L.) that is capable of protecting neurons from N-methyl-D-aspartate (NMDA)-evoked excitotoxic injury, on mice suffering from complete Freund's adjuvant (CFA)-induced chronic inflammatory pain. The results show that FMNT administration significantly reduces anxiety-like behavior but does not affect the nociceptive threshold in CFA-injected mice. The treatment reverses the upregulation of NMDA, GluA1, and GABAA receptors, as well as PSD95 and CREB in the basolateral amygdala (BLA). The effects of FMNT on NMDA receptors and CREB binding protein (CBP) were further confirmed by the potential structure combination between these compounds, which was analyzed by in silico docking technology. FMNT also inhibits the activation of the NF-κB signaling pathway and microglia in the BLA of mice suffering from chronic inflammatory pain. Therefore, the anxiolytic effects of FMNT are partially due to the attenuation of inflammation and neuronal hyperexcitability through the inhibition of NMDA receptor and CBP in the BLA.

Activation of G-Protein-Coupled Receptor 30 Protects Neurons against Excitotoxicity through Inhibiting Excessive Autophagy Induced by Glutamate.

Autophagy is a protecting intracellular pathway to transmit unnecessary or dysfunctional components to the lysosome for degeneration. Autophagic imbalance is connected with neurodegeneration. Neurodegenerative diseases including Parkinson's disease, Alzheimer's disease, and Huntington's disease are closely related to excitotoxicity and neuronal loss. Activation of G-protein-coupled receptor 30 (GPR30), an estrogen membrane receptor, protects neurons from excitotoxicity-induced cell death. However, whether autophagy is involved in the neuroprotective effect of GPR30 activation is not well-known. In this study, methyl thiazolyl tetrazolium (MTT), Western blot, monodansylcadaverine (MDC) staining, and immunofluorescent staining were employed to detect the role of autophagy in cultured primary cortical neurons after glutamate exposure and G1 treatment. Pretreatment of G1 (GPR30 specific agonist) reduced neuronal loss through inhibiting excessive autophagy induced by glutamate exposure, which was blocked by GPR30 antagonist G15, phosphatidylinositol-3-kinase (PI3K), and the mammalian target of rapamycin (mTOR) inhibitors. These data suggest that GPR30 protects neurons from cell loss primarily by modulating PI3K-AKT-mTOR signaling pathway. In addition, G1 alone did not affect the basal autophagy and cell viability. We conclude that GPR30 activation reduces glutamate-induced excessive autophagy in neurons and protects neurons against excitotoxicity.

Chronic minocycline treatment reduces the anxiety-like behaviors induced by repeated restraint stress through modulating neuroinflammation.

Anxiety disorders are chronic, disabling conditions across the world, and bring a great burden to individuals and society. Although advances have been made in understanding of the pathophysiology of these diseases, no mechanistically new drugs for anxiety disorders have reached the market in the past two decades. Some evidence indicates that stress increases neuroinflammatory signaling, which is related to the development of anxiety and depression. Minocycline, a broad-spectrum tetracycline-antibiotic, has been reported to suppress microglia activation-mediated brain endogenous inflammation. However, it is still unknown whether minocycline can be developed to treat stress-induced anxiety disorders and what is the underlying mechanisms. We chose the anxiety model induced by repeated stress consisting of 2 h of restraint on each of 7 consecutive days. The behavioral test results showed that chronic minocycline treatment, not acute minocycline treatment, increased the time spent in the center area in the open field test and the number of open arm entries and time spent in open arms in the elevated plus maze test, which were comparable with the effect of buspirone. Further mechanism studies demonstrated that chronic minocycline treatment inhibited the microglia activation and decreased the levels of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α). In addition, peroxisome proliferator-activated receptor gamma/ nuclear factor kappa B (PPAR-γ/NF-κB) signaling pathway was also modulated by chronic minocycline treatment. In conclusion, our findings support the hypothesis that immune dysregulation plays an important role in stress-induced anxiety disorders, and minocycline can be developed to be used in these diseases.

Anxiolytic effect of CPEB1 knockdown on the amygdala of a mouse model of inflammatory pain.

Anxiety disorders are a category of mental disorders characterized by feelings of anxiety, stress, and fear attached to various sources. However, their pathogenesis is complicated and has not been fully elucidated. The amygdala is a vital brain region that regulates anxiety and mental disorders. Cytoplasmic polyadenylation element binding protein 1 (CPEB1) mediates the extension of the mRNA polyadenylation tail and facilitates the translation of target RNA. CPEB1 is closely related to neuronal diseases, such as Fragile X Syndrome, learning and memory disorders, and chronic pain. In this study, the role of CPEB1 in anxiety development was determined in a pain-mediated anxiety mouse model. The anxiety model was established in mice by injecting with Complete Freund's Adjuvant (CFA) into the hindpaw. CFA injection then led to anxiety-like behaviors and increased the CPEB1 levels in the mouse basolateral amygdala (BLA). CPEB1 enhancement facilitated the translation of GluA1, GluN2A, GluN2B, PSD95, and GABA receptors, which disturbed the E/I balance in the BLA as shown by enhanced excitatory presynaptic release and reduced inhibitory presynaptic release. CPEB1 knockdown with AAV-CPEB1-shRNA alleviated the anxiety-like behaviors but not the pain-like behaviors by enhancing inhibitory transmission in the BLA of model mice. The data suggest that CPEB1 participates in anxiety development by regulating excitatory/inhibitory synaptic transmission in the BLA.