Metabotropic Glutamate Receptor 5 in the Dysgranular Zone of Primary Somatosensory Cortex Mediates Neuropathic Pain in Rats.

The primary somatosensory cortex (S1) plays a key role in the discrimination of somatic sensations. Among subdivisions in S1, the dysgranular zone of rodent S1 (S1DZ) is homologous to Brodmann's area 3a of primate S1, which is involved in the processing of noxious signals from the body. However, molecular changes in this region and their role in the pathological pain state have never been studied. In this study, we identified molecular alteration of the S1DZ in a rat model of neuropathic pain induced by right L5 spinal nerve ligation (SNL) surgery and investigated its functional role in pain symptoms. Brain images acquired from SNL group and control group in our previous study were analyzed, and behaviors were measured using the von Frey test, acetone test, and conditioned place preference test. We found that metabotropic glutamate receptor 5 (mGluR5) levels were significantly upregulated in the S1DZ contralateral to the nerve injury in the SNL group compared to the sham group. Pharmacological deactivation of mGluR5 in S1DZ ameliorated symptoms of neuropathic allodynia, which was shown by a significant increase in the mechanical paw withdrawal threshold and a decrease in the behavioral response to cold stimuli. We further confirmed that this treatment induced relief from the tonic-aversive state of chronic neuropathic pain, as a place preference memory associated with the treatment-paired chamber was formed in rats with neuropathic pain. Our data provide evidence that mGluR5 in the S1DZ is involved in the manifestation of abnormal pain sensations in the neuropathic pain state.

Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation

Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (ICALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as ICALHM1. Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect ICALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both ICALHM1 and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.

Multi-Layer Nanofibrous PCL Scaffold-Based Colon Cancer Cell Cultures to Mimic Hypoxic Tumor Microenvironment for Bioassay.

Three-dimensional (3D) cancer cell culture systems have been developed to aid the study of molecular mechanisms in cancer development, identify therapeutic targets, and test drug candidates. In this study, we developed a strategy for mimicking the hypoxic tumor microenvironment in a 3D cancer cell culture system using multi-layer, nanofibrous poly(ε-caprolactone) (PCL) scaffold (pNFS)-based cancer cell cultures. We found that human colon cancer cells infiltrated pNFS within 3 days and could be cultured three-dimensionally within the NFS. When incubated in four stacks of 30 µm-thick pNFS for 3 days, colon cancer cells in layer three showed partially reduced entry into the S phase, whereas those in layer four, located farthest from the media, showed a marked reduction in S-phase entry. As a consequence, cells in layer four exhibited hypoxia-induced disorganization of F-actin on day 3, and those in layers three and four showed an increase in the expression of the hypoxia-induced transcription factor HIF-1α and its target genes, Glut1, CA9, VEGF, and LDHA. Consistent with these results, doxorubicin- and ionizing radiation-induced cell death was reduced in colon cancer cells cultured in layers three and four. These results suggest that pNFS-based multi-layer colon cancer cell cultures mimic the hypoxic tumor microenvironment and are useful for bioassays.

Tumor Spheroids of an Aggressive Form of Central Neurocytoma Have Transit-Amplifying Progenitor Characteristics with Enhanced EGFR and Tumor Stem Cell Signaling.

Central neurocytoma (CN) has been known as a benign neuronal tumor. In rare cases, CN undergoes malignant transformation to glioblastomas (GBM). Here we examined its cellular origin by characterizing differentiation potential and gene expression of CN-spheroids. First, we demonstrate that both CN tissue and cultured primary cells recapitulate the hierarchal cellular composition of subventricular zone (SVZ), which is comprised of neural stem cells (NSCs), transit amplifying progenitors (TAPs), and neuroblasts. We then derived spheroids from CN which displayed EGFR+/ MASH+ TAP and BLBP+ radial glial cell (RGC) characteristic, and mitotic neurogenesis and gliogenesis by single spheroids were observed with cycling multipotential cells. CN-spheroids expressed increased levels of pluripotency and tumor stem cell genes such as KLF4 and TPD5L1, when compared to their differentiated cells and human NSCs. Importantly, Gene Set Enrichment Analysis showed that gene sets of GBM-Spheroids, EGFR Signaling, and Packaging of Telomere Ends are enriched in CN-spheroids in comparison with their differentiated cells. We speculate that CN tumor stem cells have TAP and RGC characteristics, and upregulation of EGFR signaling as well as downregulation of eph-ephrin signaling have critical roles in tumorigenesis of CN. And their ephemeral nature of TAPs destined to neuroblasts, might reflect benign nature of CN.

Neuron type-specific expression of a mutant KRAS impairs hippocampal-dependent learning and memory.

KRAS mutations are associated with rare cases of neurodevelopmental disorders that can cause intellectual disabilities. Previous studies showed that mice expressing a mutant KRAS have impaired the development and function of GABAergic inhibitory neurons, which may contribute to behavioural deficits in the mutant mice. However, the underlying cellular mechanisms and the role of excitatory neurons in these behavioural deficits in adults are not fully understood. Herein, we report that neuron type-specific expression of a constitutively active mutant KRASG12V in either excitatory or inhibitory neurons resulted in spatial memory deficits in adult mice. In inhibitory neurons, KRASG12V induced ERK activation and enhanced GABAergic synaptic transmission. Expressing KRASG12V in inhibitory neurons also impaired long-term potentiation in the hippocampal Shaffer-collateral pathway, which could be rescued by picrotoxin treatment. In contrast, KRASG12V induced ERK activation and neuronal cell death in excitatory neurons, which might have contributed to the severe behavioural deficits. Our results showed that both excitatory and inhibitory neurons are involved in mutant KRAS-associated learning deficits in adults via distinct cellular mechanisms.

Decoding neuropathic pain severity using distinct patterns of corticolimbic metabotropic glutamate receptor 5.

Susceptibility to neuropathic pain and the degree of pain amplification vary among individuals. However, methods for objective evaluation of pain status have not been well established. Using an animal model, we identified the brain signature of neuropathic pain, and developed a method for the objective evaluation of pain degree. We analyzed paw withdrawal thresholds from rats that were subjected to right L5 spinal nerve ligation (SNL) surgery, and regressed them to the metabotropic glutamate receptor 5 (mGluR5) availability levels in the brain using [11C] ABP688 PET image data from our previous research. We found clusters with a significant correlation to paw withdrawal threshold localized in brain areas involved in sensory, cognitive, and affective aspects of pain processing. Strikingly, mGluR5 availability levels in the identified brain regions showed distinct patterns in the neuropathic pain group but not in the control group. We successfully elucidated the degree of pain-sensing behavior using the neuropathic pain-specific pattern of the mGluR5 availability. Our study provides new insight into the signature of neuropathic pain in the brain, and offers a novel diagnostic method for objectively decoding the status of individual neuropathic pain.

TNF-α increases the intrinsic excitability of cerebellar Purkinje cells through elevating glutamate release in Bergmann Glia.

For decades, the glial function has been highlighted not only as the 'structural glue', but also as an 'active participant' in neural circuits. Here, we suggest that tumor necrosis factor α (TNF-α), a key inflammatory cytokine, alters the neural activity of the cerebellar Purkinje cells (PCs) by facilitating gliotransmission in the juvenile male rat cerebellum. A bath application of TNF-α (100 ng/ml) in acute cerebellar slices elevates spiking activity of PCs with no alterations in the regularity of PC firings. Interestingly, the effect of TNF-α on the intrinsic excitability of PCs was abolished under a condition in which the type1 TNF receptor (TNFR1) in Bergmann glia (BG) was genetically suppressed by viral delivery of an adeno-associated virus (AAV) containing TNFR1-shRNA. In addition, we measured the concentration of glutamate derived from dissociated cerebellar cortical astrocyte cultures treated with TNF-α and observed a progressive increase of glutamate in a time-dependent manner. We hypothesised that TNF-α-induced elevation of glutamate from BGs enveloping the synaptic cleft may directly activate metabotropic glutamate receptor1 (mGluR1). Pharmacological inhibition of mGluR1, indeed, prevented the TNF-α-mediated elevation of the intrinsic excitability in PCs. Taken together, our study reveals that TNF-α triggers glutamate release in BG, thereby increasing the intrinsic excitability of cerebellar PCs in a mGluR1-dependent manner.

Restoration of miR-29b exerts anti-cancer effects on glioblastoma.

BACKGROUND: Glioblastoma multiforme (GBM) is known as one of the most fatal forms of cancer. MicroRNAs have been widely implicated in the regulation of mammalian development and pathogenesis. The brain-enriched miR-29 subfamilies are known to be exclusively expressed in the developing brain, and they are aberrantly down-regulated in GBM. This study aims to elucidate the role of miR-29b in GBM development and the feasibility of therapeutic targeting using conjugated nanoparticles. METHODS: After confirmation of miR-29b expression levels in GBM tissues by analysis of open source data, the anticancer effect of miR-29b was tested by the introduction of syn-hsa-miR-29b-3p in the A172 GBM cell line. In vitro studies of cell viability and apoptosis and ex vivo study using GBM tissue slice cultures from 3 patients and nanoparticle delivery of miR-29b were performed. RESULTS: We discovered an increase in apoptotic cell populations with the introduction of miR-29b in the GBM cell line. An established human-derived GBM tissue slice culture system confirmed the anticancer effect of miR-29b-conjugated nanoparticles. Using PCR array, we found that exogenous miR-29b inhibits the expression of COL1A2, COL3A1, COL4A1, ELN, ITGA11, MMP24, and SPARC, which mediates an anticancer effect. CONCLUSIONS: miR-29b may serve as a putative therapeutic molecule when its expression is restored using a nanoparticle delivery system in GBM.

mGlu1 receptor mediates homeostatic control of intrinsic excitability through Ih in cerebellar Purkinje cells.

Homeostatic intrinsic plasticity is a cellular mechanism for maintaining a stable neuronal activity level in response to developmental or activity-dependent changes. Type 1 metabotropic glutamate receptor (mGlu1 receptor) has been widely known to monitor neuronal activity, which plays a role as a modulator of intrinsic and synaptic plasticity of neurons. Whether mGlu1 receptor contributes to the compensatory adjustment of Purkinje cells (PCs), the sole output of the cerebellar cortex, in response to chronic changes in excitability remains unclear. Here, we demonstrate that the mGlu1 receptor is involved in homeostatic intrinsic plasticity through the upregulation of the hyperpolarization-activated current (Ih) in cerebellar PCs. This plasticity was prevented by inhibiting the mGlu1 receptor with Bay 36-7620, an mGlu1 receptor inverse agonist, but not with CPCCOEt, a neutral antagonist. Chronic inactivation with tetrodotoxin (TTX) increased the components of Ih in the PCs, and ZD 7288, a hyperpolarization-activated cyclic nucleotide-gated channel selective inhibitor, fully restored reduction of firing rates in the deprived neurons. The homeostatic elevation of Ih was also prevented by BAY 36-7620, but not CPCCOEt. Furthermore, KT 5720, a blocker of protein kinase A (PKA), prevented the effect of TTX reducing the evoked firing rates, indicating the reduction in excitability of PCs due to PKA activation. Our study shows that both the mGlu1 receptor and the PKA pathway are involved in the homeostatic intrinsic plasticity of PCs after chronic blockade of the network activity, which provides a novel understanding on how cerebellar PCs can preserve the homeostatic state under activity-deprived conditions.

Impaired learning and memory in CD38 null mutant mice.

CD38 is an enzyme that catalyzes the formation of cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate, both of which are involved in the mobilization of Ca(2+) from intracellular stores. Recently, CD38 has been shown to regulate oxytocin release from hypothalamic neurons. Importantly, CD38 mutations are associated with autism spectrum disorders (ASD) and CD38 knockout (CD38(-/-)) mice display ASD-like behavioral phenotypes including deficient parental behavior and poor social recognition memory. Although ASD and learning deficits commonly co-occur, the role of CD38 in learning and memory has not been investigated. We report that CD38(-/-) mice show deficits in various learning and memory tasks such as the Morris water maze, contextual fear conditioning, and the object recognition test. However, either long-term potentiation or long-term depression is not impaired in the hippocampus of CD38(-/-) mice. Our results provide convincing evidence that CD38(-/-) mice show deficits in various learning and memory tasks including spatial and non-spatial memory tasks. Our data demonstrate that CD38 is critical for regulating hippocampus-dependent learning and memory without modulating synaptic plasticity.

FBXO11 represses cellular response to hypoxia by destabilizing hypoxia-inducible factor-1α mRNA.

The transcriptional factor hypoxia-inducible factor-1α (HIF-1α) is induced under hypoxia and plays crucial roles in cancer progression and angiogenesis. Protein arginine methyltransferases (PRMTs), 11 isoforms of which have been identified so far, modulates the functions of diverse proteins by catalyzing arginine methylation in post-translational level. PRMT9 (alternatively named FBXO11) and PRMT11 (FBXO10) are expected to have the E3 ubiquitin ligase activity through their F-box domains as well as the methyltrasferase activity. Given previous studies examining roles of 8 PRMT isoforms (PRMT1-8) in the HIF-1 signaling pathway, PRMT1 and PRMT5 were demonstrated to regulate HIF-1α expression in opposite ways. We herein examined if FBXO10 and FBXO11 participate in the HIF-1 signaling pathway. Consequently, the siRNA-mediated knockdown of FBXO11 facilitated HIF-1α expression in various cancer cells and HIF-1-driven gene expressions, but the FBXO10 knockdown did not. Mechanistically, FBXO11 was found to inhibit de novo synthesis of HIF-1α protein by destabilizing HIF-1α mRNA. Since a FBXO11 mutant lacking F-box failed to reverse the HIF-1α expression by FBXO11 knockdown, the FBXO11 regulation of HIF-1α may be attributed to the ubiquitination of some proteins controlling HIF-1α mRNA stability. Considering the oncogenic roles of HIF-1α, FBXO11 is suggested to act as a tumor suppressor and also to be a potential target for cancer therapy.

Toll-like receptor-2 deficiency induces schizophrenia-like behaviors in mice.

Dysregulation of the immune system contributes to the pathogenesis of neuropsychiatric disorders including schizophrenia. Here, we demonstrated that toll-like receptor (TLR)-2, a family of pattern-recognition receptors, is involved in the pathogenesis of schizophrenia-like symptoms. Psychotic symptoms such as hyperlocomotion, anxiolytic-like behaviors, prepulse inhibition deficits, social withdrawal, and cognitive impairments were observed in TLR-2 knock-out (KO) mice. Ventricle enlargement, a hallmark of schizophrenia, was also observed in TLR-2 KO mouse brains. Levels of p-Akt and p-GSK-3α/ß were markedly higher in the brain of TLR-2 KO than wild-type (WT) mice. Antipsychotic drugs such as haloperidol or clozapine reversed behavioral and biochemical alterations in TLR-2 KO mice. Furthermore, p-Akt and p-GSK-3α/ß were decreased by treatment with a TLR-2 ligand, lipoteichoic acid, in WT mice. Thus, our data suggest that the dysregulation of the innate immune system by a TLR-2 deficiency may contribute to the development and/or pathophysiology of schizophrenia-like behaviors via Akt-GSK-3α/ß signaling.

The molecular evidence of neural plasticity induced by cerebellar repetitive transcranial magnetic stimulation in the rat brain: a preliminary report.

Cerebellar repetitive transcranial magnetic stimulation (rTMS) has been applied to treat several pathological conditions with insufficient evidence of molecular mechanism. Neural plasticity is proposed as one of mechanism. This study aimed to (1) confirm the feasibility of focal stimulation over cerebellar cortex and (2) investigate cerebellar rTMS effects on molecular changes associated with neural plasticity in the rat. For feasibility, six male Sprague-Dawley rats underwent (18)F-FDG positron emission tomography (PET) to confirm focal stimulation on the cerebellar cortex after rTMS. For molecular evidence, thirty rats underwent a single (N=15) or 10 sessions (N=15) of rTMS with low-, high-frequency, or sham stimulation. In cerebellar cortex, reverse-transcriptase polymerase chain reaction and western blotting were performed on mRNA and proteins associated with neural plasticity: metabotrophic glutamate receptor 1 (GluR1), 2-amino-5-methyl-4-isoxazole-propionatic acid (AMPA) receptor (GluR2) and protein kinase C (PKC). As a result, (18)F-FDG-PET showed an increase of glucose metabolism in the cerebellar cortex. The transcription of mGluR1 decreased following a single session of high-frequency rTMS. Synthesis of mGluR, PKC and GluR2 was reduced after rTMS, especially high frequency stimulation. It is suggested that rTMS could focus on the cerebellar cortex in the rat and induce neural plasticity associated with long-term depression.

The ganglioside GQ1b regulates BDNF expression via the NMDA receptor signaling pathway.

Gangliosides are sialic acid-containing glycosphingolipids which play a role in neuronal functions. Among the gangliosides, tetrasialoganglioside GQ1b shows neurotrophic factor-like actions, such as increasing neurite outgrowth, cell proliferation, and long-term potentiation. In addition, we recently reported that GQ1b improves spatial learning and memory performance in naïve rats. However, it is still unknown how GQ1b exerts its diverse neuronal functions. Thus, we hypothesized that GQ1b might influence synaptic activity by regulating brain-derived neurotrophic factor (BDNF) expression, which is an important protein for synaptic plasticity and cognition. Interestingly, GQ1b treatment increased BDNF expression in GQ1b-null SH-SY5Y cell lines and rat primary cortical neurons. Additionally, we confirmed whether the observed effects were due to GQ1b or due to a ganglioside with fewer sialic acid molecules (GT1b and GD1b) created by the sialidases present on the plasma membranes, by directly applying GT1b and GD1b or GQ1b co-treated with a sialidase inhibitor. Treatment with GT1b or GD1b had no effect on BDNF expression, whereas co-treatment with a sialidase inhibitor and GQ1b significantly increased BDNF levels. Moreover, GQ1b restored the decreased BDNF expression induced by the ganglioside synthesis inhibitor, D-PDMP, in rat primary cortical neurons. GQ1b treatment significantly increased BDNF levels, whereas pretreatment with the N-methyl-d-aspartate (NMDA) receptor antagonist D-AP5 blocked the effects of GQ1b on BDNF expression, suggesting that GQ1b regulates BDNF expression via the NMDA receptor signaling. Finally, we performed an intracerebroventricular GQ1b injection, which resulted in increased prefrontal and hippocampal BDNF expression in vivo. These findings demonstrate, for the first time, that tetrasialoganglioside GQ1b regulates BDNF expression in vitro and in vivo.

Signal transducer and activator of transcription 3 (Stat3) contributes to T-cell homeostasis by regulating pro-survival Bcl-2 family genes.

The naive T-cell pool in peripheral lymphoid tissues is fairly stable in terms of number, diversity and functional capabilities in spite of the absence of prominent stimuli. This stability is attributed to continuous tuning of the composition of the T-cell pool by various homeostatic signals. Despite extensive research into the link between signal transducer and activator of transcription 3 (Stat3) and T-cell survival, little is known about how Stat3 regulates homeostasis by maintaining the required naive T-cell population in peripheral lymphoid organs. We assessed whether the elimination of Stat3 in T cells limits T-cell survival. We demonstrated that the proportion and number of single-positive thymocytes as well as T cells in the spleen and lymph nodes were significantly decreased in the Stat3-deficient group as a result of the enhanced susceptibility of Stat3-deleted T lymphocytes to apoptosis. Importantly, expression of the anti-apoptotic Bcl-2 and Bcl-xL was markedly decreased in Stat3-deleted single-positive thymocytes and T lymphocytes, suggesting that Stat3 helps to maintain the T-cell pool in the resting condition by promoting the expression of Bcl-2 family genes. These findings suggest the importance of Stat3 in the integration of homeostatic cues for the maintenance and functional tuning of the T-cell pool.

Pathogenic role of HIF-1α in prostate hyperplasia in the presence of chronic inflammation.

Benign prostatic hyperplasia (BPH) commonly occurs in older men with chronic prostatitis. Although BPH is frequently accompanied by inflammation, it is unclear whether inflammation underlies prostate enlargement. Recently, we reported that hypoxia-inducible factor 1α (HIF-1α), which is known to be induced by proinflammatory cytokines, is involved in testosterone-induced prostate hyperplasia. Therefore, we hypothesized that cytokines secreted from infiltrated macrophages under inflammatory conditions stimulate prostate enlargement by up-regulating HIF-1α. In the present study, we injected lipopolysaccharide (LPS) into rat prostates to mimic prostatitis and evaluated prostate hyperplasia 14days later. Epithelial cells of LPS-treated prostates were found to be highly proliferative and HIF-1α levels in prostate tissues to be elevated. When prostate epithelial cells were incubated in conditioned medium from macrophages activated with LPS, they robustly expressed HIF-1α, and under these conditions IL-1ß, IL-6, and TNF-α cytokines were found to mediate HIF-1α induction. In addition, HIF-1α was found to enhance the expression of Twist, which initiates epithelial-mesenchymal transition (EMT). Furthermore, profound EMT features were observed in LPS-treated rat prostates, and the natural HIF-1α inhibitors ascorbate and curcumin were found to attenuate EMT and prostate hyperplasia both in vivo and in vitro. Based on these results, we propose that HIF-1α mediates prostate enlargement under inflammatory conditions, and we suggest that HIF-1α be viewed as a promising target for blocking the transition from prostatitis to BPH.

Experimental and clinical factors influencing long-term stable in vitro expansion of multipotent neural cells from human adult temporal lobes.

Autologous adult human neural stem cells may be used for regenerative cell therapies bypass potential ethical problems. However, stable in vitro expansion protocols and experimental/clinical factors influencing primary cultures need to be further elucidated for clinically applicable techniques. To address these issues, we obtained biopsy specimens from 23 temporal lobe epilepsy patients and adult human multipotent neural cells (ahMNCs) were primarily cultured in a defined attachment culture condition. When the success of primary cultures was defined as stable expansion of cells (>ten in vitro passages) and expression of NSC markers, success rate of the primary culture was 39% (nine of 23 temporal lobes). During the long-term expansion, expressions of NSC markers and differentiation potentials into astrocytes and neurons were maintained. After the 18th sub-culture, spontaneous senescence and differentiation were observed, and the cultivated ahMNCs ceased their proliferation. The culture results were not affected by seizure characteristics; however, an older age (>40 years) and a smaller sample volume (<2 ml) were found to exert negative influences on the primary culture results. Furthermore therapeutic effects of ahMNCs against stroke were analyzed in an animal model. Transplantation of ahMNCs cells reduced infarction volumes and enhanced motor activity, significantly. The results here would provide promising experimental and clinical strategy of using patient-specific autologous ahMNCs in regenerative medicine in the future.

PI3Kγ is required for NMDA receptor-dependent long-term depression and behavioral flexibility.

Phosphatidylinositol 3-kinase (PI3K) has been implicated in synaptic plasticity and other neural functions in the brain. However, the role of individual PI3K isoforms in the brain is unclear. We investigated the role of PI3Kγ in hippocampal-dependent synaptic plasticity and cognitive functions. We found that PI3Kγ has a crucial and specific role in NMDA receptor (NMDAR)-mediated synaptic plasticity at mouse Schaffer collateral-commissural synapses. Both genetic deletion and pharmacological inhibition of PI3Kγ disrupted NMDAR long-term depression (LTD) while leaving other forms of synaptic plasticity intact. Accompanying this physiological deficit, the impairment of NMDAR LTD by PI3Kγ blockade was specifically correlated with deficits in behavioral flexibility. These findings suggest that a specific PI3K isoform, PI3Kγ, is critical for NMDAR LTD and some forms of cognitive function. Thus, individual isoforms of PI3Ks may have distinct roles in different types of synaptic plasticity and may therefore influence various kinds of behavior.

Mitochondrial Ca(2+) uptake is essential for synaptic plasticity in pain.

The increase of cytosolic free Ca(2+) ([Ca(2+)](c)) due to NMDA receptor activation is a key step for spinal cord synaptic plasticity by altering cellular signal transduction pathways. We focus on this plasticity as a cause of persistent pain. To provide a mechanism for these classic findings, we report that [Ca(2+)](c) does not trigger synaptic plasticity directly but must first enter into mitochondria. Interfering with mitochondrial Ca(2+) uptake during a [Ca(2+)](c) increase blocks induction of behavioral hyperalgesia and accompanying downstream cell signaling, with reduction of spinal long-term potentiation (LTP). Furthermore, reducing the accompanying mitochondrial superoxide levels lessens hyperalgesia and LTP induction. These results indicate that [Ca(2+)](c) requires downstream mitochondrial Ca(2+) uptake with consequent production of reactive oxygen species (ROS) for synaptic plasticity underlying chronic pain. These results suggest modifying mitochondrial Ca(2+) uptake and thus ROS as a type of chronic pain therapy that should also have broader biologic significance.

Anti-neuroinflammatory activity of nobiletin on suppression of microglial activation.

A growing body of evidence suggests that nobiletin (5,6,7,8,3',4'-hexamethoxy flavone) from the peel of citrus fruits, enhances the damaged cognitive function in disease animal models. However, the neuroprotective mechanism has not been clearly elucidated. Since nobiletin has shown anti-inflammatory effects in several tissues, we investigated whether nobiletin suppresses excessive microglial activation implicated in neurotoxicity in lipopolysaccharide (LPS)-stimulated BV-2 microglia cell culture models. Release of nitric oxide (NO), the major inflammatory mediator in microglia, was markedly suppressed in a dose-dependent manner following nobiletin treatment (1-50 µM) in LPS-stimulated BV-2 microglia cells. The inhibitory effect of nobiletin was similar to that of minocycline, a well-known microglial inactivator. Nobiletin significantly inhibited the release of the pro-inflammatory cytokine tumor necrosis factor (TNF-α) and interleukin-1ß (IL-1ß). LPS-induced phosphorylations of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) were also significantly inhibited by nobiletin treatment. In addition, nobiletin markedly inhibited the LPS-induced pro-inflammatory transcription factor nuclear factor κB (NF-κB) signaling pathway by suppressing nuclear NF-κB translocation from the cytoplasm and subsequent expression of NF-κB in the nucleus. Taken together, these results may contribute to further exploration of the therapeutic potential and molecular mechanism of nobiletin in relation to neuroinflammation and neurodegenerative diseases.