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Metabotropic glutamate (mGlu) receptors are involved in the experience-dependent neuroplasticity in the mesolimbic reward circuit. A Gα

Activation of protein kinases after cocaine administration controls psychomotor behaviours by interacting with metabotropic receptors in the brain. This study identified how c-Jun N-terminal kinase (JNK) interacts with metabotropic glutamate receptor 5 (mGluR5) in vitro and in the caudate and putamen (CPu). The potential role of this interaction in the regulation of psychomotor behaviour was also evaluated after administration of cocaine. Active JNK phosphorylates a threonine residue at position 1055 in the carboxyl terminus (CT) of mGluR5 in vitro. The binding of active JNK to the D-motif within CT2 is necessary for that phosphorylation. Interaction of phosphorylated JNK and mGluR5 occurs in the CPu. Unilateral interference of the interaction decreases the repeated cocaine-induced increases in locomotor activity and conditioned place preference. These findings suggest that activation of JNK has the capability to interact with mGluR5 in the CPu. Phosphorylation of mGluR5 following the JNK-mGluR5 interaction may be responsible for the potentiation of behavioural sensitisation and cocaine-wanting behaviour in response to cocaine administration.

Adenosine A(2A) receptors are G(olf)-coupled receptors and are predominantly expressed in the striatum of mammalian brains. As a mostly postsynaptic receptor, A(2A) receptors are implicated in the regulation of a variety of intracellular signaling pathways in striatopallidal output neurons and are linked to the pathogenesis of various neuropsychiatric and neurological disorders. This study investigated the possible role of A(2A) receptors in the modulation of the Src family kinase (SFK) in the adult rat striatum. In acutely prepared striatal slices, adding the A(2A) receptor agonist PSB-0777 induced a significant increase in phosphorylation of SFKs at a conserved autophosphorylation site (Y416) in the caudate putamen (CPu). This increase was also seen in the nucleus accumbens (NAc). Another A(2A) agonist CGS-21680 showed the similar ability to elevate SFK Y416 phosphorylation in the striatum. Treatment with the A(2A) receptor antagonist KW-6002 blocked the effect of PSB-0777 on SFK Y416 phosphorylation. In addition, PSB-0777 enhanced kinase activity of two key SFK members (Src and Fyn) immunoprecipitated from the striatum. These data demonstrate a positive linkage from A(2A) receptors to the SFK signaling pathway in striatal neurons. Activation of A(2A) receptors leads to the upregulation of phosphorylation of SFKs (Src and Fyn) at an activation-associated autophosphorylation site and kinase activity of these SFK members.

Major depressive disorder is a chronic psychiatric disease with a high prevalence. Brain mechanisms for depression at cellular and molecular levels are far from clear. Increasing evidence from clinical and preclinical studies reveals critical roles of the non-receptor tyrosine kinase (nRTK) superfamily in the pathophysiology, symptomatology, and therapy of depression. To date, several nRTK members from three nRTK subfamilies, i.e., the Src family kinase (SFK), the Janus tyrosine kinase (JAK) and the focal adhesion kinase (FAK) subfamilies, may connect to the intracellular, intranuclear, and synaptic signaling network linking chronic stress to depression- and anxiety-like behavior. These SFK/JAK/FAK nRTKs are abundantly expressed in the prefrontal cortex and hippocampus, two core limbic regions implicated in depression, and are enriched at synaptic sites. In various acute or chronic animal models of depression, the nRTKs were significantly altered (up- or downregulated) in their phosphorylation, expression, subcellular/subsynaptic distribution, and/or function. Stress that precipitates depressive behavior also influenced the interaction of nRTKs with other signaling molecules and downstream substrates, including ionotropic and metabotropic glutamate receptors. The commonly-used antidepressants showed the ability to alter nRTK activity. In sum, the limbic SFK/JAK/FAK nRTKs are sensitive to stress and undergo drastic adaptations in response to chronic depression. These long-lasting adaptations contribute to the remodeling of signaling network or synaptic plasticity critical for the vulnerability to depression and the therapeutic efficacy of antidepressants.

Doxorubicin is a widely used anthracycline-based anti-tumor agent for both solid and liquid tumors. Mounting evidence has demonstrated that microRNAs (miRNAs) are involved in chemoresistance and tumorigenesis. However, the roles of microRNA-501-5p (miR-501) in doxorubicin resistance and gastric cancer cell proliferation and invasion are still not fully understood. In this study, we identified that BLID (BH3-like motif-containing protein, cell death inducer) was directly regulated by miR-501 at the post-transcriptional level in multiple gastric cancer cell lines. Endogenous miR-501 was higher, whereas BLID was lower, in doxorubicin-resistant gastric cancer SGC7901/ADR cells compared with their parental SGC7901 cells. miR-501 suppressed gastric cancer cell apoptosis, induced resistance to doxorubicin, and enhanced cell proliferation, migration, and invasion. Subcutaneous injection of miR-501 lentivirus-infected SGC7901 cells resulted in rapid growth of xenograft tumors and resistance to doxorubicin treatment, unlike injection of negative miRNA lentivirus-infected SGC7901 cells. This is achieved at least partially by directly targeting BLID and subsequent inactivation of caspase-9 and caspase-3 and phosphorylation of Akt. Taken together, miR-501 induces doxorubicin resistance and enhances the tumorigenesis of gastric cancer cells by suppressing BLID. miR-501 might be a potential target for doxorubicin resistance and gastric cancer therapy. Keywords: microRNA-501-5p, chemoresistance, doxorubicin, BLID, gastric cancer

The extracellular signal-regulated kinase (ERK) is enriched in the central nervous system, including the dopamine responsive regions such as the striatum and medial prefrontal cortex (mPFC). The kinase is sensitive to changing cellular and synaptic input and is implicated in the regulation of synaptic transmission and plasticity. In this study, the role of a G(αi/o) protein-coupled adenosine A(1) receptor in the regulation of ERK1/2 was investigated in the rat brain in vivo. We found that an A(1) agonist CPA after an intraperitoneal injection reduced ERK1/2 phosphorylation in the nucleus accumbens (NAc) and mPFC. In contrast, a single dose of an A(1) antagonist DPCPX induced a rapid and transient increase in ERK1/2 phosphorylation in the caudate putamen (CPu), NAc, and mPFC. Pretreatment with a dopamine D(1) receptor antagonist SCH23390 abolished the DPCPX-induced ERK1/2 phosphorylation in the striatum and mPFC. Coadministration of DPCPX and a D(1) agonist SKF81297 at a low dose induced a greater elevation of ERK1/2 phosphorylation. Activation or blockade of A(1) receptors had no effect on total ERK1/2 expression in the striatum and mPFC. These results reveal an existence of an inhibitory linkage from adenosine A(1) receptors to ERK1/2 in striatal and mPFC neurons. This inhibitory linkage seems to form a dynamic balance with positive dopamine D(1) receptor signaling to control the ERK1/2 pathway.

Background Cervical cancer is the common gynecological deadly malignancy worldwide. Here we attempted to evaluate the effects and mechanisms of microRNA-501-5p (miR-501) on the cell proliferation, migration, invasion and the clinical significance in the cervical cancer. Methods Cervical cancer HeLa cells were transfected with miR-501 mimic or inhibitor or siRNA against Cylindromatosis (CYLD) using Lipofectamine 2000. miR-501 expression was assessed in HeLa cells and cervical cancer specimens by real-time qRT-PCR. The functional roles of miR-501 were determined by CCK-8, colony formation, scratch wound healing and transwell assays. The apoptosis rate was detected by flow cytometry assay. CYLD, BCL-2, BAX, NF- κ B p65 and phosphorylated p65 (p-p65) proteins were examined by Western blotting. CYLD expression was evaluated by immunohistochemistry in cervical cancer tissues. Results miR-501 was upregulated, whereas CYLD protein was downregulated in cervical cancer tissues compared to normal cervical tissues. miR-501 overexpression and CYLD protein downregulation were positively correlated with poor differentiation, tumor size, International Federation of Gynecology and Obstetrics (FIGO) stage and lymph node metastasis. CYLD was downregulated by miR-501 at both mRNA and protein levels in HeLa cells. miR-501 promoted cell proliferation, migration and invasion in cervical cancer, while inhibited the apoptosis. This is possibly due to the downregulation of CYLD and subsequent activation of NF-κB p65. Conclusions miR-501 upregulation and CYLD downregulation are associated with the development and progression of cervical cancer. miR-501 promotes cervical cancer cell proliferation, migration and invasion possibly via downregulating CYLD and subsequently activating NF-κB p65. miR-501 might be a potential therapeutic target for cervical cancer.

Several non-receptor tyrosine kinase (nRTK) members are expressed in neurons of mammalian brains. Among these neuron-enriched nRTKs, two Src family kinase members (Src and Fyn) are particularly abundant at synaptic sites and have been most extensively studied for their roles in the regulation of synaptic activity and plasticity. Increasing evidence shows that the synaptic subpool of nRTKs interacts with a number of local substrates, including glutamate receptors (both ionotropic and metabotropic glutamate receptors), postsynaptic scaffold proteins, presynaptic proteins, and synapse-enriched enzymes. By phosphorylating specific tyrosine residues in the intracellular domains of these synaptic proteins either constitutively or in an activity-dependent manner, nRTKs regulate these substrates in trafficking, surface expression, and function. Given the high sensitivity of nRTKs to changing synaptic input, nRTKs are considered to act as a critical regulator in the determination of the strength and efficacy of synaptic transmission.

Several key members of the non-receptor tyrosine kinase (nRTK) family are abundantly present within excitatory synapses in the mammalian brain. These neuron-enriched nRTKs interact with glutamate receptors and phosphorylate the receptors at tyrosine sites. The N-methyl-D-aspartate receptor is a direct substrate of nRTKs and has been extensively investigated in its phosphorylation responses to nRTKs. The α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor is the other glutamate receptor subtype that is subject to nRTK-mediated tyrosine phosphorylation. Recently, group I metabotropic glutamate receptors (mGluR1/5) were found to be sensitive to nRTKs. Robust tyrosine phosphorylation may occur in C-terminal tails of mGluR5. Tyrosine phosphorylation of glutamate receptors is either constitutive or induced activity-dependently by changing cellular and/or synaptic input. Through inducing tyrosine phosphorylation, nRTKs regulate trafficking, subcellular distribution, and function of modified receptors. Available data show that nRTK-glutamate receptor interactions and tyrosine phosphorylation of the receptors undergo drastic adaptations in mood disorders such as major depressive disorder. The remodeling of the nRTK-glutamate receptor interplay contributes to the long-lasting pathophysiology and symptomology of depression. This review summarizes the recent progress in tyrosine phosphorylation of glutamate receptors and analyzes the role of nRTKs in regulating glutamate receptors and depression-like behavior.

Dopamine and acetylcholine are two principal transmitters in the striatum and are usually balanced to modulate local neural activity and maintain striatal homeostasis. In this study, we investigated the role of dopamine and muscarinic acetylcholine receptors in the regulation of a central signaling protein, i.e., the mitogen-activated protein kinase (MAPK). We focused on the synaptic pool of MAPKs based on the fact that these kinases reside in peripheral synaptic structures in addition to their somatic locations. We found that a systemic injection of a dopamine D1 receptor (D1R) agonist SKF81297 enhanced phosphorylation of extracellular signal-regulated kinases (ERKs), a prototypic subclass of MAPKs, in the adult rat striatum. Similar results were observed in another dopamine responsive region, the medial prefrontal cortex (mPFC). The dopamine D2 receptor agonist quinpirole had no such effects. Pretreatment with a positive allosteric modulator (PAM) of muscarinic acetylcholine M4 receptors (M4Rs), VU0152100, attenuated the D1R agonist-stimulated ERK phosphorylation in the two regions, while the PAM itself did not alter basal ERK phosphorylation. All drug treatments had no effect on phosphorylation of c-Jun N-terminal kinases (JNKs), another MAPK subclass, in the striatum and mPFC. These results demonstrate that dopamine and acetylcholine are integrated to control synaptic ERK, although not JNK, activation in striatal and mPFC neurons in vivo. Activation of M4Rs exerts an inhibitory effect on the D1R-mediated upregulation of synaptic ERK phosphorylation.

Eighteen novel 2-(1-aryl-5-methyl-1,2,3-triazol-4-yl)-1,3,4-oxadiazole derivatives and two acylhydrazone intermediate compounds were synthesized by various pathways starting from I -aryl-5-methyl-1,2,3-triazol-4-formhydrazide (1). All products were identified by spectroscopic analysis, and 2-(l -aryl-5-methyl- 1,2,3-triazol-4-yl)-5-benzalthio-1,3,4-oxadiazole was further validated by X-ray crystallography. Results from primary antibacterial activity tests indicated that most of the compounds were effective against E coli, P. aeruginosa, B, subtilis and S. aureus.

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is the most abundant kinase within excitatory synapses in the mammalian brain. It interacts with and phosphorylates a large number of synaptic proteins, including major ionotropic glutamate receptors (iGluRs) and group I metabotropic glutamate receptors (mGluRs), to constitutively and/or activity-dependently regulate trafficking, subsynaptic localization, and function of the receptors. Among iGluRs, the N-methyl-D-aspartate receptor (NMDAR) is a direct target of CaMKII. By directly binding to an intracellular C-terminal (CT) region of NMDAR GluN2B subunits, CaMKII phosphorylates a serine residue (S1303) in the GluN2B CT. CaMKII also phosphorylates a serine site (S831) in the CT of α-amino-3-hydroxy-5- methylisoxazole-4-propionic acid receptors. This phosphorylation enhances channel conductance and is critical for synaptic plasticity. In addition to iGluRs, CaMKII binds to the proximal CT region of mGluR1a, which enables the kinase to phosphorylate threonine 871. Agonist stimulation of mGluR1a triggers a CaMKII-mediated negative feedback to facilitate endocytosis and desensitization of the receptor. CaMKII also binds to the mGluR5 CT. This binding seems to anchor and accumulate inactive CaMKII at synaptic sites. Active CaMKII dissociates from mGluR5 and may then bind to adjacent GluN2B to mediate the mGluR5-NMDAR coupling. Together, glutamate receptors serve as direct substrates of CaMKII. By phosphorylating these receptors, CaMKII plays a central role in controlling the number and activity of the modified receptors and determining the strength of excitatory synaptic transmission.

Background N-methyl-D-aspartate (NMDA) receptors play a central role in glutamatergic synaptic transmission in the mammalian brain and are linked to the pathophysiology and symptomatology of Parkinson’s disease (PD). However, changes in NMDA receptor expression in distinct subcellular compartments in PD have not been elucidated. In this study, we investigated changes in subcellular expression of NMDA receptors in striatal neurons in a rodent PD model. Methods Intracranial injection of the neurotoxin 6-hydroxydopamine (6-OHDA) was selectively lesioned into the nigrostriatal dopaminergic pathway in adult Sprague Dawley rats, which is a common rat model of PD. A surface receptor crosslinking assay was conducted to examine the response of individual NMDA receptor subunits to dopamine depletion in isolated and confined surface and intracellular compartments of striatal neurons. Results In PD rats where 6-OHDA was selectively lesioned, surface expression of NMDA receptor GluN1 subunits as detected by surface protein crosslinking assays was increased in the striatum. In contrast, intracellular levels of GluN1 were decreased in the lesioned region. The NMDA receptor GluN2B subunit was elevated in its abundance in the surface pool of the lesioned striatum, while intracellular GluN2B levels were not altered. GluN2A subunits in both surface and intracellular fractions remained stable. In addition, total cellular levels of striatal GluN1 and GluN2A were not changed in lesioned tissue, while total GluN2B proteins showed an increase. Conclusion These results demonstrate the differential sensitivity of principal NMDA receptor subunits to dopamine depletion. GluN1 and GluN2B expression in the distinct surface compartment underwent upregulation in striatal neurons after selective lesions of the dopaminergic pathway by 6-OHDA.

There is little ideal predictor available on evaluating the lymph node metastatic potential of breast carcinoma. This study was designed to determine the expression of gene products of E-cadherin (epithelial), N-cadherin (nerve), and matrix metalloproteinase-9 (MMP-9) in breast carcinoma tissue and investigate their association with the invasion and metastasis of breast carcinoma.The authors examined the expressions of E-cadherin, N-cadherin, and MMP-9 in 72 cases of breast carcinoma(39 cases with lymph node metastasis and 33 cases without lymph node metastasis) by immunohistochemistry. Multivariable Cox proportional hazards model was used to analyze the patients' prognosis.The average ranks of E-cadherin in lymph node metastasis group and no lymph node metastasis group were 29.19 and 45.14, respectively, with significant difference (P0.001). The expression of E-cadherin was correlated inversely with the metastasis of breast carcinoma. The average ranks of N-cadherin and MMP-9 were 40.04 and 42.97 in lymph node metastasis group, and 32.32 and 28.85 in no lymph node metastasis group, both with significant difference (P0.05), and these expressions were positively correlated with the lymph node metastasis of breast carcinoma. The patients who had high expression of E-cadherin had a longer survival time.Expression of E-cadherin, N-cadherin, and MMP-9 are associated strongly with lymph node metastasis of breast carcinoma. These proteins are indicators of metastasis potential and prognosis of breast carcinoma.

Jing Gan,1 Chen Qi,1 Li-Min Mao,2 Zhenguo Liu11Department of Neurology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China; 2Department of Basic Medical Science, University of Missouri-Kansas City School of Medicine, Kansas City, MO, USABackground: N-methyl-D-aspartate (NMDA) receptors play a central role in glutamatergic synaptic transmission in the mammalian brain and are linked to the pathophysiology and symptomatology of Parkinson's disease (PD). However, changes in NMDA receptor expression in distinct subcellular compartments in PD have not been elucidated. In this study, we investigated changes in subcellular expression of NMDA receptors in striatal neurons in a rodent PD model.Methods: Intracranial injection of the neurotoxin 6-hydroxydopamine (6-OHDA) was selectively lesioned into the nigrostriatal dopaminergic pathway in adult Sprague Dawley rats, which is a common rat model of PD. A surface receptor crosslinking assay was conducted to examine the response of individual NMDA receptor subunits to dopamine depletion in isolated and confined surface and intracellular compartments of striatal neurons.Results: In PD rats where 6-OHDA was selectively lesioned, surface expression of NMDA receptor GluN1 subunits as detected by surface protein crosslinking assays was increased in the striatum. In contrast, intracellular levels of GluN1 were decreased in the lesioned region. The NMDA receptor GluN2B subunit was elevated in its abundance in the surface pool of the lesioned striatum, while intracellular GluN2B levels were not altered. GluN2A subunits in both surface and intracellular fractions remained stable. In addition, total cellular levels of striatal GluN1 and GluN2A were not changed in lesioned tissue, while total GluN2B proteins showed an increase.Conclusion: These results demonstrate the differential sensitivity of principal NMDA receptor subunits to dopamine depletion. GluN1 and GluN2B expression in the distinct surface compartment underwent upregulation in striatal neurons after selective lesions of the dopaminergic pathway by 6-OHDA.Keywords: glutamate, excitatory amino acid, NMDA, GluN, dopamine, 6-hydroxydopamine, caudate putamen, nucleus accumbens

The analgesic effects induced by two different kinds of peripheral conditioning stimulations, electroacupuncture (EA) and transcutaneous electrical nerve stimulation (TENS), were compared in the rat using the latency of radiant heat-evoked tail flick reflex as nociceptive index. The parallel elevations of withdrawal latency of tail flick were produced by EA and TENS administrations at the acupoints of S36 and Sp6 with low intensity (1-2-3 mA) and one of three different frequencies (2, 15 and 100 Hz). Analgesic effects of EA or TENS were characterized by slow-on and slow-off nature, and a significant linear correlation was found between both at any one of three frequencies. Systemic naloxone hydrochloride (2 mg/kg) almost completely and partially antagonized 2 and 15 Hz EA- or TENS-induced analgesia, respectively, but failed to affect those induced by 100 Hz EA or TENS. Tolerance to EA stimulation with one of three frequencies reduced the corresponding frequency TENS-induced analgesia and vice versa. These data indicate that: (a) there is no significant difference in producing antinociception for two different peripheral conditioning stimulations when applied at the same sites and (b) the common neural mechanisms most likely process the analgesic effects of EA and TENS. The involvement of (an) endogenous opiate mechanism in the management of different frequency EA and TENS analgesia is discussed in detail.

AIM: In order to obtain lymphogenous metastasis-associated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential. METHODS: Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e., biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip® MOE430A (containing 22690 transcripts, including 14500 known mouse genes and 4371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by bioinformatics. RESULTS: Out of the 14500 known genes investigated, 110 (0.8%) were up regulated at least 23 fold. Among the total 4371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 23 fold. According to the Gene Ontology and TreeView analysis, the 110 genes were further classified into two groups: differential biological process profile and molecular function profile. CONCLUSION: Using high-throughput gene chip method, a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription, chaperone activity, motor activity, protein kinase activity, receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation. Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments. ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as the diagnostic markers and the therapeutic targets for lymphatic metastasis.