Youichi Shinozaki

Microglia release ATP by exocytosis.

Microglia survey the brain environment by sensing several types of diffusible molecules, among which extracellular nucleotides released/leaked from damaged cells have central roles. Microglia sense ATP or other nucleotides by multiple P2 receptors, after which they change into several different phenotypes. However, so far, it is largely unknown whether microglia themselves release ATP and, if so, by what mechanism. Here we show that exocytosis is the mechanism by which microglia release ATP. When we stimulated microglia with ionomycin, they released ATP and the release was dependent on Ca2+, vesicular H+‐ATPase, or SNAREs but independent of connexin/pannexin hemichannels. VNUT was found to be expressed in microglia and exhibited no colocalization with lysosome. We also visualized the exocytosis of ATP by a quinacrine‐based fluorescent time‐lapse imaging. Moreover, we found that lipopolysaccharide increased the ionomycin‐induced release of ATP, which was dependent on the increase in VNUT. Taken together, our data suggested that exocytosis is the mechanism of ATP release from microglia. When activated, they would release ATP by increasing VNUT‐dependent exocytotic mechanisms. GLIA 2013;61:1320–1330

Cytoprotection against oxidative stress-induced damage of astrocytes by extracellular ATP via P2Y1 receptors

Oxidative stress is the main cause of neuronal damage in traumatic brain injury, hypoxia/reperfusion injury, and neurodegenerative disorders. Although extracellular nucleosides, especially adenosine, are well known to protect against neuronal damage in such pathological conditions, the effects of these nucleosides or nucleotides on glial cell damage remain largely unknown. We report that ATP but not adenosine protects against the cell death of cultured astrocytes induced by hydrogen peroxide (H2O2). ATP ameliorated the H2O2‐induced decrease in cell viability of astrocytes in an incubation time‐ and concentration‐dependent fashion. Protection by ATP was inhibited by P2 receptor antagonists and was mimicked by P2Y1 receptor agonists but not by adenosine. The expressions of P2Y1 mRNAs and functional P2Y1 receptors in astrocytes were confirmed. Thus, ATP, acting on P2Y1 receptors in astrocytes, showed a protective action against H2O2. The astrocytic protection by the P2Y1 receptor agonist 2‐methylthio‐ADP was inhibited by an intracellular Ca2+ chelator and a blocker of phospholipase C, indicating the involvement of intracellular signals mediated by Gq/11‐coupled P2Y1 receptors. The ATP‐induced protection was inhibited by cycloheximide, a protein synthesis inhibitor, and it took more than 12 h for the onset of the protective action. In the DNA microarray analysis, ATP induced a dramatic upregulation of various oxidoreductase genes. Taken together, ATP acts on P2Y1 receptors coupled to Gq/11, resulting in the upregulation of oxidoreductase genes, leading to the protection of astrocytes against H2O2.

Microglia trigger astrocyte-mediated neuroprotection via purinergic gliotransmission

Microglia are highly sensitive to even small changes in the brain environment, such as invasion of non-hazardous toxicants or the presymptomatic state of diseases. However, the physiological or pathophysiological consequences of their responses remain unknown. Here, we report that cultured microglia sense low concentrations of the neurotoxicant methylmercury (MeHglow) and provide neuroprotection against MeHg, for which astrocytes are also required. When exposed to MeHglow, microglia exocytosed ATP via p38 MAPK- and vesicular nucleotide transporter (VNUT)-dependent mechanisms. Astrocytes responded to the microglia-derived ATP via P2Y1 receptors and released interleukin-6 (IL-6), thereby protecting neurons against MeHglow. These neuroprotective actions were also observed in organotypic hippocampal slices from wild-type mice, but not in slices prepared from VNUT knockout or P2Y1 receptor knockout mice. These findings suggest that microglia sense and respond to even non-hazardous toxicants such as MeHglow and change their phenotype into a neuroprotective one, for which astrocytic support is required.

The Astrocyte-Targeted Therapy by Bushi for the Neuropathic Pain in Mice

Background There is accumulating evidence that the activation of spinal glial cells, especially microglia, is a key event in the pathogenesis of neuropathic pain. However, the inhibition of microglial activation is often ineffective, especially for long-lasting persistent neuropathic pain. So far, neuropathic pain remains largely intractable and a new therapeutic strategy for the pain is still required. Methods/Principal Findings Using Seltzer model mice, we investigated the temporal aspect of two types of neuropathic pain behaviors, i.e., thermal hyperalgesia and mechanical allodynia, as well as that of morphological changes in spinal microglia and astrocytes by immunohistochemical studies. Firstly, we analyzed the pattern of progression in the pain behaviors, and found that the pain consisted of an “early induction phase” and subsequent “late maintenance phase”. We next analyzed the temporal changes in spinal glial cells, and found that the induction and the maintenance phase of pain were associated with the activation of microglia and astrocytes, respectively. When Bushi, a Japanese herbal medicine often used for several types of persistent pain, was administered chronically, it inhibited the maintenance phase of pain without affecting the induction phase, which was in accordance with the inhibition of astrocytic activation in the spinal cord. These analgesic effects and the inhibition of astrocytic activation by Bushi were mimicked by the intrathecal injection of fluorocitrate, an inhibitor of astrocytic activation. Finally, we tested the direct effect of Bushi on astrocytic activation, and found that Bushi suppressed the IL-1β- or IL-18-evoked ERK1/2-phosphorylation in cultured astrocytes but not the ATP-evoked p38- and ERK1/2-phosphorylation in microglia in vitro. Conclusions Our results indicated that the activation of spinal astrocytes was responsible for the late maintenance phase of neuropathic pain in the Seltzer model mice and, therefore, the inhibition of astrocytic activation by Bushi could be a useful therapeutic strategy for treating neuropathic pain.

Cortical astrocytes rewire somatosensory cortical circuits for peripheral neuropathic pain

Long-term treatments to ameliorate peripheral neuropathic pain that includes mechanical allodynia are limited. While glial activation and altered nociceptive transmission within the spinal cord are associated with the pathogenesis of mechanical allodynia, changes in cortical circuits also accompany peripheral nerve injury and may represent additional therapeutic targets. Dendritic spine plasticity in the S1 cortex appears within days following nerve injury; however, the underlying cellular mechanisms of this plasticity and whether it has a causal relationship to allodynia remain unsolved. Furthermore, it is not known whether glial activation occurs within the S1 cortex following injury or whether it contributes to this S1 synaptic plasticity. Using in vivo 2-photon imaging with genetic and pharmacological manipulations of murine models, we have shown that sciatic nerve ligation induces a re-emergence of immature metabotropic glutamate receptor 5 (mGluR5) signaling in S1 astroglia, which elicits spontaneous somatic Ca2+ transients, synaptogenic thrombospondin 1 (TSP-1) release, and synapse formation. This S1 astrocyte reactivation was evident only during the first week after injury and correlated with the temporal changes in S1 extracellular glutamate levels and dendritic spine turnover. Blocking the astrocytic mGluR5-signaling pathway suppressed mechanical allodynia, while activating this pathway in the absence of any peripheral injury induced long-lasting (>1 month) allodynia. We conclude that reawakened astrocytes are a key trigger for S1 circuit rewiring and that this contributes to neuropathic mechanical allodynia.

Astrocytes Protect Neurons against Methylmercury via ATP/P2Y1 Receptor-Mediated Pathways in Astrocytes

Methylmercury (MeHg) is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y1 receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6)-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y1 receptor-mediated mechanisms were required because the IL-6 production was (i) inhibited by a P2Y1 receptor antagonist, MRS2179, (ii) abolished in astrocytes obtained from P2Y1 receptor-knockout mice, and (iii) mimicked by exogenously applied ATP. In addition, (iv) MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A1 receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y1 receptors to upregulate IL-6, thereby leading to A1 receptor-mediated neuro-protection against MeHg.

In Vitro Blood-Brain Barrier Models Using Brain Capillary Endothelial Cells Isolated from Neonatal and Adult Rats Retain Age-Related Barrier Properties

The blood–brain barrier (BBB) restricts the entry of circulating drugs and xenobiotics into the brain, and thus its permeability to substances is a critical factor that determines their central effects. The infant brain is vulnerable to neurotoxic substances partly due to the immature BBB. The employment of in vitro BBB models to evaluate permeability of compounds provides higher throughput than that of in vivo animal experiments. However, existing in vitro BBB models have not been able to simulate the intrinsic neonatal BBB. To establish a neonatal BBB model that mimics age-related BBB properties, the neonatal and adult in vitro BBB models were constructed with brain endothelial cells isolated from 2- and 8-week-old rats, respectively. To evaluate BBB functions, transendothelial electrical resistance, permeability of sodium fluorescein and Evans blue-albumin, and transport of rhodamine123 were measured. Radiolabelled drugs were used for BBB permeability studies in the neonatal and adult BBB models (in vitro) and in age-matched rats (in vivo). The neonatal BBB model showed lower barrier and p-glycoprotein (P-gp) functions than the adult BBB model; these were well associated with lower expressions of the barrier-related proteins and P-gp, and a different distribution pattern of immunostained barrier-related proteins. Verapamil (a P-gp inhibitor) significantly increased the influx of rhodamine 123, supporting functional P-gp expression in the neonatal BBB model. Valproic acid, but not nicotine, showed higher BBB permeability in the neonatal BBB model, which was well in accordance with the in vivo BBB property. We established a neonatal BBB model in vitro. This could allow us to assess the age-dependent BBB permeability of drugs.

Extracellular ATP counteracts the ERK1/2‐mediated death‐promoting signaling cascades in astrocytes

Oxidative stress is the main cause of neuronal death in pathological conditions. Hydrogen peroxide (H2O2), one of the reactive oxygen species, activates many intracellular signaling cascades including src family and mitogen‐activated protein kinases (MAPKs), some of which are critically involved in the induction of cellular damage. We previously showed that H2O2‐induced cell death in astrocytes and adenosine 5′‐triphosphate (ATP), acting on P2Y1 receptors, had a protective effect. Here, we examined the H2O2‐induced changes in intracellular signaling cascades that promote cell death in astrocytes, showing the molecular mechanisms by which the activation of P2Y1 receptors counteracts such signals. Although H2O2 activated three MAPKs including ERK1/2, p38, and JNK, only the activation of ERK1/2 participated in the H2O2‐evoked cell death. H2O2 induced a sustained activation of ERK1/2 mainly in the nucleus region, which was well in accordance with the H2O2‐induced cell death. H2O2 also activated the src tyrosine kinase family, which was an upstream signal for ERK1/2. Activation of P2Y1 receptors by 2methylthio‐ADP (2MeSADP) inhibited the H2O2‐evoked activation of src tyrosine kinase, resulting in the inhibition of the phosphorylated‐ERK1/2 accumulation in the nucleus. 2MeSADP enhanced the gene expression and activity of protein tyrosine phosphatase (PTP), which was responsible for the inhibition of src tyrosine kinase. Thioredoxin reductase, another cytoprotective gene we previously showed to be upregulated by 2MeSADP, also controlled the activity of PTP. Taken together, ATP, acting on P2Y1 receptors, upregulates the PTP expression and its activity, which counteracts the H2O2‐promoted death signaling cascades including ERK1/2 and its upstream signal src tyrosine kinase in astrocytes.

Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca(2+) ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca(2+) ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm(2), and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices.

Urothelial ATP exocytosis: Regulation of bladder compliance in the urine storage phase

The bladder urothelium is more than just a barrier. When the bladder is distended, the urothelium functions as a sensor to initiate the voiding reflex, during which it releases ATP via multiple mechanisms. However, the mechanisms underlying this ATP release in response to the various stretch stimuli caused by bladder filling remain largely unknown. Therefore, the aim of this study was to elucidate these mechanisms. By comparing vesicular nucleotide transporter (VNUT)-deficient and wild-type male mice, we showed that ATP has a crucial role in urine storage through exocytosis via a VNUT-dependent mechanism. VNUT was abundantly expressed in the bladder urothelium, and when the urothelium was weakly stimulated (i.e. in the early filling stages), it released ATP by exocytosis. VNUT-deficient mice showed reduced bladder compliance from the early storage phase and displayed frequent urination in inappropriate places without a change in voiding function. We conclude that urothelial, VNUT-dependent ATP exocytosis is involved in urine storage mechanisms that promote the relaxation of the bladder during the early stages of filling.