Jdp2 is a spatiotemporal transcriptional activator of the AhR via the Nrf2 gene battery.

BACKGROUND: Crosstalk between the aryl hydrocarbon receptor (AhR) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling is called the "AhR-Nrf2 gene battery", which works synergistically in detoxification to support cell survival. Nrf2-dependent phase II gene promoters are controlled by coordinated recruitment of the AhR to adjacent dioxin responsive element (DRE) and Nrf2 recruitment to the antioxidative response element (ARE). The molecular interaction between AhR and Nrf2 members, and the regulation of each target, including phase I and II gene complexes, and their mediators are poorly understood. METHODS: Knockdown and forced expression of AhR-Nrf2 battery members were used to examine the molecular interactions between the AhR-Nrf2 axis and AhR promoter activation. Sequential immunoprecipitation, chromatin immunoprecipitation, and histology were used to identify each protein complex recruited to their respective cis-elements in the AhR promoter. Actin fiber distribution, cell spreading, and invasion were examined to identify functional differences in the AhR-Jdp2 axis between wild-type and Jdp2 knockout cells. The possible tumorigenic role of Jdp2 in the AhR-Nrf2 axis was examined in mutant Kras-Trp53-driven pancreatic tumors. RESULTS: Crosstalk between AhR and Nrf2 was evident at the transcriptional level. The AhR promoter was activated by phase I ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the AhR-Jdp2-Nrf2 axis in a time- and spatial transcription-dependent manner. Jdp2 was a bifunctional activator of DRE- and ARE-mediated transcription in response to TCDD. After TCDD exposure, Jdp2 activated the AhR promoter at the DRE and then moved to the ARE where it activated the promoter to increase reactive oxygen species (ROS)-mediated functions such as cell spreading and invasion in normal cells, and cancer regression in mutant Kras-Trp53-driven pancreatic tumor cells. CONCLUSIONS: Jdp2 plays a critical role in AhR promoter activation through the AhR-Jdp2-Nrf2 axis in a spatiotemporal manner. The AhR functions to maintain ROS balance and cell spreading, invasion, and cancer regression in a mouse model of mutant Kras-Trp53 pancreatic cancer. These findings provide new insights into the roles of Jdp2 in the homeostatic regulation of oxidative stress and in the antioxidation response in detoxification, inflammation, and cancer progression.

Effect of 9,12-Octadecadiynoic Acid on Neurobehavioral Development in Caenorhabditis elegans.

Human breast milk lipids have major beneficial effects: they promote infant early brain development, growth and health. To identify the relationship between human breast milk lipids and infant neurodevelopment, multivariate analyses that combined lipidomics and psychological Bayley-III scales evaluation were utilized. We identified that 9,12-octadecadiynoic acid has a significantly positive correlation with infant adaptive behavioral development, which is a crucial neurodevelopment to manage risk from environmental stress. To further clarify the biological function of 9,12-octadecadiynoic acid in regulating neurodevelopment, Caenorhabditis elegans (C. elegans) was used as a model to investigate the effect of 9,12-octadecadiynoic acid on neurobehavioral development. Supplementation with 9,12-octadecadiynoic acid from the L1 to L4 stage in larvae affected locomotive behaviors and foraging ability that were not socially interactive, implying that 9,12-octadecadiynoic acid is involved in regulating the serotonergic neuronal ability. We found that supplementary 0.1 µM 9,12-octadecadiynoic acid accelerated the locomotive ability and foraging ability via increasing the expression of serotonin transporter mod-1. Antioxidant defense genes, sod-1, sod-3 and cyp-35A2 are involved in 9,12-octadecadiynoic acid-induced motor neuronal activity. Nevertheless, supplementary 9,12-octadecadiynoic acid at concentrations above 1 µM significantly attenuated locomotive behaviors, foraging ability, serotonin synthesis, serotonin-related gene expressions and stress-related gene expression, resulting in the decreased longevity of worms in the experiment. In conclusion, our study demonstrates the biological function of 9,12-octadecadiynoic acid in governing adaptive behavioral development.

2-Aminoethyl diphenylborinate inhibits bleomycin-induced skin and pulmonary fibrosis via interrupting intracellular Ca2+ regulation.

BACKGROUND: Systemic sclerosis (SSc) causes progressive fibrosis of multiple organs with the low efficacy of immunosuppressive therapies. Our previous study indicated the SSc pathological pathways are closely correlated with Ca2+ signals, and blockage of the intracellular Ca2+ elevation facilitates inhibition of SSc pathogenesis. OBJECTIVE: Transforming growth factor ß (TGF-ß)-modulated SMAD signaling is crucial in regulating SSc pathogenesis. Whether Ca2+ signals are involved in TGF-ß1/SMAD signaling-induced fibrotic process has been further investigated. METHODS: We utilized TGF-ß1-induced myofibroblasts as a model to detect how Ca2+ signals affected SSc pathogenesis, and investigated the combination of treatment with store-operated Ca2+ entry (SOCE) associated inhibitors, 2-aminoethyl diphenylborinate (2-APB) and SKF96365 to restrain the increased Ca2+ signaling in myofibroblasts. In addition, the SSc bleomycin mouse model was used to detect the effect of 2-APB on SSc pathogenesis in vivo. RESULTS: Our findings revealed increased levels of TGF-ß1 production in SSc was associated with intracellular Ca2+ activity, and inhibition of intracellular Ca2+ regulation by 2-APB resulted in the dedifferentiation of TGF-ß1-induced myofibroblasts. This was due to the fact that 2-APB restrained the expression fibrotic markers, α-SMA, fibronectin and vimentin through inhibiting TGF-ß1/SMAD3 signaling. Thus, subcutaneous injection of 2-APB improved bleomycin-induced skin and pulmonary fibrosis. CONCLUSION: 2-APB is a potential candidate for treating fibrosis, by disrupting intracellular Ca2+ regulation in SSc to induce the dedifferentiation of myofibroblasts and meliorates fibrosis pathogenesis via inhibiting TGF-ß1/SMAD3 signaling.

Peripheral-neuron-like properties of differentiated human dental pulp stem cells (hDPSCs).

Elucidating the mechanisms underlying human pain sensation requires the establishment of an in vitro model of pain reception comprising human cells expressing pain-sensing receptors and function properly as neurons. Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells and a promising candidate for producing human neuronal cells, however, the functional properties of differentiated hDPSCs have not yet been fully characterized. In this study, we demonstrated neuronal differentiation of hDPSCs via both their expression of neuronal marker proteins and their neuronal function examined using Ca2+ imaging. Moreover, to confirm the ability of nociception, Ca2+ responses in differentiated hDPSCs were compared to those of rat dorsal root ganglion (DRG) neurons. Those cells showed similar responses to glutamate, ATP and agonists of transient receptor potential (TRP) channels. Since TRP channels are implicated in nociception, differentiated hDPSCs provide a useful in vitro model of human peripheral neuron response to stimuli interpreted as pain.

A novel strategy for treating cancer: understanding the role of Ca2+ signaling from nociceptive TRP channels in regulating cancer progression.

Cancer is an aging-associated disease and caused by genomic instability that is driven by the accumulation of mutations and epimutations in the aging process. Although Ca2+ signaling, reactive oxygen species (ROS) accumulation, DNA damage response (DDR) and senescence inflammation response (SIR) are processed during genomic instability, the underlying mechanism for the cause of genomic instability and cancer development is still poorly understood and needs to be investigated. Nociceptive transient receptor potential (TRP) channels, which firstly respond to environmental stimuli, such as microbes, chemicals or physical injuries, potentiate regulation of the aging process by Ca2+ signaling. In this review, the authors provide an explanation of the dual role of nociceptive TRP channels in regulating cancer progression, initiating cancer progression by aging-induced genomic instability, and promoting malignancy by epigenetic regulation. Thus, therapeutically targeting nociceptive TRP channels seems to be a novel strategy for treating cancers.

Rapid differentiation of human dental pulp stem cells to neuron-like cells by high K+ stimulation.

As human-origin cells, human dental pulp stem cells (hDPSCs) are thought to be potentially useful for biological and medical experiments. They are easily obtained from lost primary teeth or extracted wisdom teeth, and they are mesenchymal stem cells that are known to differentiate into osteoblasts, chondrocytes, and adipocytes. Although hDPSCs originate from neural crest cells, it is difficult to induce hDPSCs to differentiate into neuron-like cells. To facilitate their differentiation into neuron-like cells, we evaluated various differentiation conditions. Activation of K+ channels is thought to regulate the intracellular Ca2+ concentration, allowing for manipulation of the cell cycle to induce the differentiation of hDPSCs. Therefore, in addition to a conventional neural cell differentiation protocol, we activated K+ channels in hDPSCs. Immunocyto-chemistry and real-time PCR revealed that applying a combination of 3 stimuli (high K+ solution, epigenetic reprogramming solution, and neural differentiation solution) to hDPSCs increased their expression of neuronal markers, such as ß3-tubulin, postsynaptic density protein 95, and nestin within 5 days, which led to their rapid differentiation into neuron-like cells. Our findings indicate that epigenetic reprogramming along with cell cycle regulation by stimulation with high K+ accelerated the differentiation of hDPSCs into neuron-like cells. Therefore, hDPSCs can be used in various ways as neuron-like cells by manipulating their cell cycle.

Impairment of cerebellar long-term depression and GABAergic transmission in prion protein deficient mice ectopically expressing PrPLP/Dpl.

Prion protein (PrPC) knockout mice, named as the "Ngsk" strain (Ngsk Prnp0/0 mice), show late-onset cerebellar Purkinje cell (PC) degeneration because of ectopic overexpression of PrPC-like protein (PrPLP/Dpl). Our previous study indicated that the mutant mice also exhibited alterations in cerebellum-dependent delay eyeblink conditioning, even at a young age (16 weeks of age) when neurological changes had not occurred. Thus, this electrophysiological study was designed to examine the synaptic function of the cerebellar cortex in juvenile Ngsk Prnp0/0 mice. We showed that Ngsk Prnp0/0 mice exhibited normal paired-pulse facilitation but impaired long-term depression of excitatory synaptic transmission at synapses between parallel fibres and PCs. GABAA-mediated inhibitory postsynaptic currents recorded from PCs were also weakened in Ngsk Prnp0/0 mice. Furthermore, we confirmed that Ngsk Prnp0/0 mice (7-8-week-old) exhibited abnormalities in delay eyeblink conditioning. Our findings suggest that these alterations in both excitatory and inhibitory synaptic transmission to PCs caused deficits in delay eyeblink conditioning of Ngsk Prnp0/0 mice. Therefore, the Ngsk Prnp0/0 mouse model can contribute to study underlying mechanisms for impairments of synaptic transmission and neural plasticity, and cognitive deficits in the central nervous system.

Nociceptive transient receptor potential canonical 7 (TRPC7) mediates aging-associated tumorigenesis induced by ultraviolet B.

Aging, cancer, and longevity have been linked to intracellular Ca2+ signaling and nociceptive transient receptor potential (TRP) channels. We found that TRP canonical 7 (TRPC7) is a nociceptive mechanoreceptor and that TRPC7 channels specifically mediate the initiation of ultraviolet B (UVB)-induced skin aging and tumor development due to p53 gene family mutations. Within 30 min after UVB irradiation, TRPC7 mediated UVB-induced Ca2+ influx and the subsequent production of reactive oxygen species in skin cells. Notably, this function was unique to TRPC7 and was not observed for other TRP channels. In TRPC7 knockout mice, we did not observe the significant UVB-associated pathology seen in wild-type mice, including epidermal thickening, abnormal keratinocyte differentiation, and DNA damage response activation. TRPC7 knockout mice also had significantly fewer UVB-induced cancerous tumors than did wild-type mice, and UVB-induced p53 gene family mutations were prevented in TRPC7 knockout mice. These results indicate that TRPC7 activity is pivotal in the initiation of UVB-induced skin aging and tumorigenesis and that the reduction in TRPC7 activity suppresses the UVB-induced aging process and tumor development. Our findings support that TRPC7 is a potential tumor initiator gene and that it causes cell aging and genomic instability, followed by a change in the activity of proto-oncogenes and tumor suppressor genes to promote tumorigenesis.

A novel role of oxytocin: Oxytocin-induced well-being in humans.

We review the involvement of a small molecule, oxytocin, in various effects of physical stimulation of somatosensory organs, mindfulness meditation, emotion and fragrance on humans, and then propose a hypothesis that complex human states and behaviors, such as well-being, social bonding, and emotional behavior, are explained by oxytocin. We previously reported that oxytocin can induce pain relief and described the possibility how oxytocin in the dorsal horn and/or the dorsal root ganglion relieves joint and muscle pain. In the present article, we expand our research target from the physical analgesic effects of oxytocin to its psychologic effects to upregulate well-being and downregulate stress and anxiety. For this purpose, we propose a "hypothalamic-pituitary-adrenal (HPA) axis-oxytocin model" to explain why mindfulness meditation, placebo, and fragrance can reduce stress and anxiety, resulting in contentment. This new proposed model of HPA axis-oxytocin in the brain also provides a target to address other questions regarding emotional behaviors, learning and memory, and excess food intake leading to obesity, aimed at promoting a healthy life.

A potential new approach for treating systemic sclerosis: Dedifferentiation of SSc fibroblasts and change in the microenvironment by blocking store-operated Ca2+ entry.

Transforming growth factor-ß (TGF-ß) is an important target for treating systemic sclerosis (SSc). However, our study revealed three levels of TGF-ß1 expression in SSc patients, indicating that inhibiting TGF-ß is not sufficient to treat SSc. A previous clinical trial also displayed disappointing results. Thus, our study attempted to search for a potential novel approach. Ingenuity Pathway Analysis (IPA) indicated that the SSc pathological pathways were closely associated with store-operated Ca2+ entry (SOCE)-regulated signals, and SOCE activity was found to be increased in SSc fibroblasts. Further treatment of SSc fibroblasts with SOCE inhibitors, 2APB, and associated calcium channel inhibitors SKF96365, and indomethacin, showed that the SOCE inhibitors selectively decreased fibrosis markers and altered the cell morphology. Consequently, SOCE inhibitors, especially 2APB and indomethacin, caused the dedifferentiation of SSc fibroblasts via cytoskeleton remodeling and altered collagen secretion and restored the cell mobility. We further explained SSc pathogenesis as fibroblast differentiation with SOCE. Treatment with exogenous factors, gelatin-1, FAM20A and human albumin, which were identified from the conditioned medium of SSc fibroblasts, was important for regulating the differentiation of fibroblasts with higher levels of SOCE and α-SMA. Conclusively, to treat SSc, blockage of the increased SOCE activity in SSc induces the dedifferentiation of SSc fibroblasts and simultaneously changes the extracellular matrix (ECM) structure to limit SSc pathogenesis.

Adhesive pyramidal thorn patches provide pain relief to athletes.

Pain in athletes is ideally treated without systemic medicine. Therefore, complementary and alternative medicine, including patch treatments, is often used. The physiologic mechanisms of pain relief produced by patch treatment, however, are not well elucidated. In the present study, we introduce a pyramidal thorn (PT) patch that we developed, demonstrate the effects of this PT patch for the treatment of various types of pain in 300 subjects, and suggest a physiologic mechanism for the pain relief effects. One treatment with the PT patch effectively relieved pain in almost half the subjects evaluated. Except for pain generated deeply under the skin, such as low-back pain, pain was eliminated within four treatments with the PT patch in almost all of the subjects. Interestingly, the pain-sensing region moved along the nerve fibers after each trial. Further, patches without PT also provided some pain relief. We considered that this effect was due to hair deflection on the skin; that is, adhesion of the PT patch activates Merkel cells directly as well as Merkel cell-neurite complexes around the hair follicles by deflecting the hair follicles, whereas adhesion of a patch without PT only activates the Merkel cell-neurite complexes. In any case, patch adhesion stimulates Aß fibers to alleviate pain. Finally, we found that the pain threshold is increased by electric stimulation, suggesting that the gentle adhesion of a PT patch would be more effective. To our knowledge, this is the first study to demonstrate physiologically the validity of an adherent patch for pain relief.

A Proposed Molecular Mechanism for Physical Analgesia in Chronic Pain.

Although pain is indispensable for survival, chronic pain places a heavy burden on humans. As the efficacy of opioid treatment is limited, the development of alternative methods of pain relief without medication is desirable. Recently, we have developed a novel method of physical analgesia using an adhesive "pyramidal thorn patch." When we apply about 3 trials of these patches on the skin of a pain region, the pain region moves toward the spinal cord like a "cutaneous rabbit," and finally, the pain vanishes. In the present review, we propose a molecular mechanism for this analgesic method or pain relief following application of the pyramidal thorn patch where firstly the mechanoreceptors and their related nerves under the skin are activated in response to touch. Transient receptor potential (TRP) channels serve as mechanosensitive channels within these mechanoreceptors. We further propose that activation of the nerves connected with the mechanoreceptors releases oxytocin, which has an antinociceptive function and activates TRP channels to hyperpolarize the pain signal nerves. We believe that our system will pave the way for alternative pain treatment.

Hydrogen gas protects IP3Rs by reducing disulfide bridges in human keratinocytes under oxidative stress.

Based on the oxidative stress theory, aging derives from the accumulation of oxidized proteins induced by reactive oxygen species (ROS) in the cytoplasm. Hydrogen peroxide (H2O2) elicits ROS that induces skin aging through oxidation of proteins, forming disulfide bridges with cysteine or methionine sulfhydryl groups. Decreased Ca2+ signaling is observed in aged cells, probably secondary to the formation of disulfide bonds among Ca2+ signaling-related proteins. Skin aging processes are modeled by treating keratinocytes with H2O2. In the present study, H2O2 dose-dependently impaired the adenosine triphosphate (ATP)-induced Ca2+ response, which was partially protected via co-treatment with ß-mercaptoethanol, resulting in reduced disulfide bond formation in inositol 1, 4, 5-trisphosphate receptors (IP3Rs). Molecular hydrogen (H2) was found to be more effectively protected H2O2-induced IP3R1 dysfunction by reducing disulfide bonds, rather than quenching ROS. In conclusion, skin aging processes may involve ROS-induced protein dysfunction due to disulfide bond formation, and H2 can protect oxidation of this process.

A Novel Strategy for TNF-Alpha Production by 2-APB Induced Downregulated SOCE and Upregulated HSP70 in O. tsutsugamushi-Infected Human Macrophages.

Orientia (O.) tsutsugamushi-induced scrub typhus is endemic across many regions of Asia and the Western Pacific, where an estimated 1 million cases occur each year; the majority of patients infected with O. tsutsugamushi end up with a cytokine storm from a severe inflammatory response. Previous reports have indicated that blocking tumor necrosis factor (TNF)-α reduced cell injury from a cytokine storm. Since TNF-α production is known to be associated with intracellular Ca2+ elevation, we examined the effect of store-operated Ca2+ entry (SOCE) inhibitors on TNF-α production in O. tsutsugamushi-infected macrophages. We found that 2-aminoethoxydiphenyl borate (2-APB), but not SKF96365, facilitates the suppression of Ca2+ mobilization via the interruption of Orai1 expression in O. tsutsugamushi-infected macrophages. Due to the decrease of Ca2+ elevation, the expression of TNF-α and its release from macrophages was repressed by 2-APB. In addition, a novel role of 2-APB was found in macrophages that causes the upregulation of heat shock protein 70 (HSP70) expression associated with ERK activation; upregulated TNF-α production in the case of knockdown HSP70 was inhibited with 2-APB treatment. Furthermore, elevated HSP70 formation unexpectedly did not help the cell survival of O. tsutsugamushi-infected macrophages. In conclusion, the parallelism between downregulated Ca2+ mobilization via SOCE and upregulated HSP70 after treatment with 2-APB against TNF-α production was found to efficiently attenuate an O. tsutsugamushi-induced severe inflammatory response.

Derinat Protects Skin against Ultraviolet-B (UVB)-Induced Cellular Damage.

Ultraviolet-B (UVB) is one of the most cytotoxic and mutagenic stresses that contribute to skin damage and aging through increasing intracellular Ca(2+) and reactive oxygen species (ROS). Derinat (sodium deoxyribonucleate) has been utilized as an immunomodulator for the treatment of ROS-associated diseases in clinics. However, the molecular mechanism by which Derinat protects skin cells from UVB-induced damage is poorly understood. Here, we show that Derinat significantly attenuated UVB-induced intracellular ROS production and decreased DNA damage in primary skin cells. Furthermore, Derinat reduced intracellular ROS, cyclooxygenase-2 (COX-2) expression and DNA damage in the skin of the BALB/c-nu mice exposed to UVB for seven days in vivo. Importantly, Derinat blocked the transient receptor potential canonical (TRPC) channels (TRPCs), as demonstrated by calcium imaging. Together, our results indicate that Derinat acts as a TRPCs blocker to reduce intracellular ROS production and DNA damage upon UVB irradiation. This mechanism provides a potential new application of Derinat for the protection against UVB-induced skin damage and aging.

Role of TRP channels in the induction of heat shock proteins (Hsps) by heating skin.

Transient receptor potential (TRP) channels in skin are crucial for achieving temperature sensitivity to maintain internal temperature balance and thermal homeostasis, as well as to protect skin cells from environmental stresses such as infrared (IR) or near-infrared (NIR) radiation via heat shock protein (Hsp) production. However, the mechanisms by which IR and NIR activate TRP channels and produce Hsps intracellularly have been independently reported. In this review, we discuss the relationship between TRP channel activation and Hsp production, and introduce the roles of several skin TRP channels in the regulation of HSP production by IR and NIR exposure.

Thermodynamic implications of high Q 10 of thermo-TRP channels in living cells.

The activity of thermo-transient receptor potential (TRP) channels is highly dependent on temperature, and thus thermo-TRP reactions have a high temperature coefficient Q 10. In thermodynamics, a high value of Q 10 indicates the existence of a large activation energy (i.e., a large enthalpy) over a short period during the transition process between the closed and open states of the channels. The Gibbs free energy equation shows that a large entropy is required to compensate for this large enthalpy and permit activation of the channels, suggesting a large conformational change of the channels. These large values of enthalpy and entropy seem to be a match for the values of the unfolding process of globular proteins. We outline these thermodynamic issues in thermo-TRPs.

Analysis for distinctive activation patterns of pain and itchy in the human brain cortex measured using near infrared spectroscopy (NIRS).

Pain and itch are closely related sensations, yet qualitatively quite distinct. Despite recent advances in brain imaging techniques, identifying the differences between pain and itch signals in the brain cortex is difficult due to continuous temporal and spatial changes in the signals. The high spatial resolution of positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) has substantially advanced research of pain and itch, but these are uncomfortable because of expensiveness, importability and the limited operation in the shielded room. Here, we used near infrared spectroscopy (NIRS), which has more conventional usability. NIRS can be used to visualize dynamic changes in oxygenated hemoglobin and deoxyhemoglobin concentrations in the capillary networks near activated neural circuits in real-time as well as fMRI. We observed distinct activation patterns in the frontal cortex for acute pain and histamine-induced itch. The prefrontal cortex exhibited a pain-related and itch-related activation pattern of blood flow in each subject. Although it looked as though that activation pattern for pain and itching was different in each subject, further cross correlation analysis of NIRS signals between each channels showed an overall agreement with regard to prefrontal area involvement. As a result, pain-related and itch-related blood flow responses (delayed responses in prefrontal area) were found to be clearly different between pain (τ = +18.7 sec) and itch (τ = +0.63 sec) stimulation. This is the first pilot study to demonstrate the temporal and spatial separation of a pain-induced blood flow and an itch-induced blood flow in human cortex during information processing.

Ca(2+) spiking activity caused by the activation of store-operated Ca(2+) channels mediates TNF-α release from microglial cells under chronic purinergic stimulation.

Cytokines released from microglia mediate defensive responses in the brain, but the underlying mechanisms are obscure. One proposed process is that nucleotide leakage or release from surrounding cells is sensed by metabotropic (P2Y) and ionotropic (P2X) purinergic receptors, which may trigger long-term intracellular Ca(2+) flux and tumor necrosis factor α (TNF-α) release. Indeed, 3h of exposure to ATP was required to evoke TNF-α release from a murine microglial cell line (MG5). A Ca(2+) chelator, ethylene glycol tetraacetic acid (EGTA), reduced ATP-induced TNF-α release, suggesting that intracellular Ca(2+) is important in this response. Therefore, Ca(2+) sensor genes (YC3.6) were transfected into MG5 cells to investigate the Ca(2+) dynamics underlying ATP-induced TNF-α release. The results demonstrated ATP-induced biphasic Ca(2+) mobilization mediated by P2Y (~5min) and P2X7 receptors (5-30min). Moreover, Ca(2+) spiking activity in cell processes progressively increased with a reduction in P2X7 receptor-mediated Ca(2+) elevation during 3-h ATP stimulation. Increased Ca(2+) spiking activity paralleled the reduction in thapsigargin-sensitive internal Ca(2+) stores, dendrite extension, and expression of macrophage scavenger receptors with collagenous structure. The Ca(2+) spiking activity was enhanced by a P2X7 receptor antagonist (A438079), but inhibited by a store-operated channel antagonist (SKF96365) or by co-transfection of small interference ribonucleic acid (siRNA) targeted on the channel component (Orai1). Furthermore, ATP-induced TNF-α release was enhanced by A438079 but was inhibited by SKF96365. Because store-operated channels (Stim1/Orai1) were expressed both in MG5 and primary microglial cultures, we suggest that P2X7 receptor signaling inhibits store-operated channels during ATP stimulation, and disinhibition of this process gates TNF-α release from microglial cells.

Age-dependent impairment of eyeblink conditioning in prion protein-deficient mice.

Mice lacking the prion protein (PrP(C)) gene (Prnp), Ngsk Prnp (0/0) mice, show late-onset cerebellar Purkinje cell (PC) degeneration because of ectopic overexpression of PrP(C)-like protein (PrPLP/Dpl). Because PrP(C) is highly expressed in cerebellar neurons (including PCs and granule cells), it may be involved in cerebellar synaptic function and cerebellar cognitive function. However, no studies have been conducted to investigate the possible involvement of PrP(C) and/or PrPLP/Dpl in cerebellum-dependent discrete motor learning. Therefore, the present cross-sectional study was designed to examine cerebellum-dependent delay eyeblink conditioning in Ngsk Prnp (0/0) mice in adulthood (16, 40, and 60 weeks of age). The aims of the present study were two-fold: (1) to examine the role of PrP(C) and/or PrPLP/Dpl in cerebellum-dependent motor learning and (2) to confirm the age-related deterioration of eyeblink conditioning in Ngsk Prnp (0/0) mice as an animal model of progressive cerebellar degeneration. Ngsk Prnp (0/0) mice aged 16 weeks exhibited intact acquisition of conditioned eyeblink responses (CRs), although the CR timing was altered. The same result was observed in another line of PrP(c)-deficient mice, ZrchI PrnP (0/0) mice. However, at 40 weeks of age, CR incidence impairment was observed in Ngsk Prnp (0/0) mice. Furthermore, Ngsk Prnp (0/0) mice aged 60 weeks showed more significantly impaired CR acquisition than Ngsk Prnp (0/0) mice aged 40 weeks, indicating the temporal correlation between cerebellar PC degeneration and motor learning deficits. Our findings indicate the importance of the cerebellar cortex in delay eyeblink conditioning and suggest an important physiological role of prion protein in cerebellar motor learning.