Impaired calcium signaling in astrocytes modulates autism spectrum disorder-like behaviors in mice.

Autism spectrum disorder (ASD) is a common neurodevelopmental disorder. The mechanisms underlying ASD are unclear. Astrocyte alterations are noted in ASD patients and animal models. However, whether astrocyte dysfunction is causal or consequential to ASD-like phenotypes in mice is unresolved. Type 2 inositol 1,4,5-trisphosphate 6 receptors (IP3R2)-mediated Ca2+ release from intracellular Ca2+ stores results in the activation of astrocytes. Mutations of the IP3R2 gene are associated with ASD. Here, we show that both IP3R2-null mutant mice and astrocyte-specific IP3R2 conditional knockout mice display ASD-like behaviors, such as atypical social interaction and repetitive behavior. Furthermore, we show that astrocyte-derived ATP modulates ASD-like behavior through the P2X2 receptors in the prefrontal cortex and possibly through GABAergic synaptic transmission. These findings identify astrocyte-derived ATP as a potential molecular player in the pathophysiology of ASD.

Impairments in remote memory caused by the lack of Type 2 IP3 receptors.

The second messenger inositol 1,4,5-trisphosphate (IP3 ) is paramount for signal transduction in biological cells, mediating Ca2+ release from the endoplasmic reticulum. Of the three isoforms of IP3 receptors identified in the nervous system, Type 2 (IP3 R2) is the main isoform expressed by astrocytes. The complete lack of IP3 R2 in transgenic mice was shown to significantly disrupt Ca2+ signaling in astrocytes, while leaving neuronal intracellular pathways virtually unperturbed. Whether and how this predominantly nonneuronal receptor might affect long-term memory function has been a matter of intense debate. In this work, we found that the absence of IP3 R2-mediated signaling did not disrupt normal learning or recent (24-48 h) memory. Contrary to expectations, however, mice lacking IP3 R2 exhibited remote (2-4 weeks) memory deficits. Not only did the lack of IP3 R2 impair remote recognition, fear, and spatial memories, but it also prevented naturally occurring post-encoding memory enhancements consequent to memory consolidation. Consistent with the key role played by the downscaling of synaptic transmission in memory consolidation, we found that NMDAR-dependent long-term depression was abnormal in ex vivo hippocampal slices acutely prepared from IP3 R2-deficient mice, a deficit that could be prevented upon supplementation with D-serine - an NMDA-receptor co-agonist whose synthesis depends upon astrocytes' activity. Our results reveal that IP3 R2 activation, which in the brain is paramount for Ca2+ signaling in astrocytes, but not in neurons, can help shape brain plasticity by enhancing the consolidation of newly acquired information into long-term memories that can guide remote cognitive behaviors.

Expression of the type 3 InsP3 receptor is a final common event in the development of hepatocellular carcinoma.

BACKGROUND & OBJECTIVES: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Several types of chronic liver disease predispose to HCC, and several different signalling pathways have been implicated in its pathogenesis, but no common molecular event has been identified. Ca2+ signalling regulates the proliferation of both normal hepatocytes and liver cancer cells, so we investigated the role of intracellular Ca2+ release channels in HCC. DESIGN: Expression analyses of the type 3 isoform of the inositol 1, 4, 5-trisphosphate receptor (ITPR3) in human liver samples, liver cancer cells and mouse liver were combined with an evaluation of DNA methylation profiles of ITPR3 promoter in HCC and characterisation of the effects of ITPR3 expression on cellular proliferation and apoptosis. The effects of de novo ITPR3 expression on hepatocyte calcium signalling and liver growth were evaluated in mice. RESULTS: ITPR3 was absent or expressed in low amounts in hepatocytes from normal liver, but was expressed in HCC specimens from three independent patient cohorts, regardless of the underlying cause of chronic liver disease, and its increased expression level was associated with poorer survival. The ITPR3 gene was heavily methylated in control liver specimens but was demethylated at multiple sites in specimens of patient with HCC. Administration of a demethylating agent in a mouse model resulted in ITPR3 expression in discrete areas of the liver, and Ca2+ signalling was enhanced in these regions. In addition, cell proliferation and liver regeneration were enhanced in the mouse model, and deletion of ITPR3 from human HCC cells enhanced apoptosis. CONCLUSIONS: These results provide evidence that de novo expression of ITPR3 typically occurs in HCC and may play a role in its pathogenesis.

Inositol 1,4,5-trisphosphate receptor type 2-independent Ca2+ release from the endoplasmic reticulum in astrocytes.

Accumulating evidence indicates that astrocytes are actively involved in the physiological and pathophysiological functions of the brain. Intracellular Ca2+ signaling, especially Ca2+ release from the endoplasmic reticulum (ER), is considered to be crucial for the regulation of astrocytic functions. Mice with genetic deletion of inositol 1,4,5-trisphosphate receptor type 2 (IP3 R2) are reportedly devoid of astrocytic Ca2+ signaling, and thus widely used to explore the roles of Ca2+ signaling in astrocytic functions. While functional deficits in IP3 R2-knockout (KO) mice have been found in some reports, no functional deficit was observed in others. Thus, there remains a controversy regarding the functional significance of astrocytic Ca2+ signaling. To address this controversy, we re-evaluated the assumption that Ca2+ release from the ER is abolished in IP3 R2-KO astrocytes using a highly sensitive imaging technique. We expressed the ER luminal Ca2+ indicator G-CEPIA1er in cortical and hippocampal astrocytes to directly visualize spontaneous and stimulus-induced Ca2+ release from the ER. We found attenuated but significant Ca2+ release in response to application of norepinephrine to IP3 R2-KO astrocytes. This IP3 R2-independent Ca2+ release induced only minimal cytosolic Ca2+ transients but induced robust Ca2+ increases in mitochondria that are frequently in close contact with the ER. These results indicate that ER Ca2+ release is retained and is sufficient to increase the Ca2+ concentration in close proximity to the ER in IP3 R2-KO astrocytes.

Long-term In Vivo Calcium Imaging of Astrocytes Reveals Distinct Cellular Compartment Responses to Sensory Stimulation.

Localized, heterogeneous calcium transients occur throughout astrocytes, but the characteristics and long-term stability of these signals, particularly in response to sensory stimulation, remain unknown. Here, we used a genetically encoded calcium indicator and an activity-based image analysis scheme to monitor astrocyte calcium activity in vivo. We found that different subcellular compartments (processes, somata, and endfeet) displayed distinct signaling characteristics. Closer examination of individual signals showed that sensory stimulation elevated the number of specific types of calcium peaks within astrocyte processes and somata, in a cortical layer-dependent manner, and that the signals became more synchronous upon sensory stimulation. Although mice genetically lacking astrocytic IP3R-dependent calcium signaling (Ip3r2-/-) had fewer signal peaks, the response to sensory stimulation was sustained, suggesting other calcium pathways are also involved. Long-term imaging of astrocyte populations revealed that all compartments reliably responded to stimulation over several months, but that the location of the response within processes may vary. These previously unknown characteristics of subcellular astrocyte calcium signals provide new insights into how astrocytes may encode local neuronal circuit activity.

Loss of IP3 Receptor-Mediated Ca2+ Release in Mouse B Cells Results in Abnormal B Cell Development and Function.

Intracellular calcium (Ca2+) mobilization after engagement of the BCR has been proposed to play an important role in B cell development and function. BCR activation causes an initial Ca2+ release from the endoplasmic reticulum that is mediated by inositol 1,4,5-trisphosphate receptor (IP3R) and then triggers store-operated Ca2+ entry once endoplasmic reticulum Ca2+ store is depleted. Store-operated Ca2+ entry has been shown to regulate B cell function but is dispensable for B cell development. By contrast, the function of IP3R-mediated Ca2+ release in B cells remains to be determined. In this study, we generated a B cell-specific IP3R triple-knockout (IP3R-TKO) mouse model and revealed that loss of IP3Rs increased transitional B cell numbers and reduced recirculating mature B cell numbers in bone marrow. In the peripheral tissues, the numbers of conventional B2 B cells and B1 B cells were both significantly decreased in IP3R-TKO mice. Ablation of IP3Rs also dramatically reduced BCR-mediated B cell proliferation and survival. Furthermore, T cell-dependent and T cell-independent Ab responses were altered in IP3R-TKO mice. In addition, deletion of IP3Rs reduced IL-10-producing regulatory B cell numbers and led to defects in NFAT activation, which together resulted in decreased IL-10 secretion. Taken together, our study demonstrated for the first time, to our knowledge, that IP3R-mediated Ca2+ release plays an essential role in regulating B cell development, proliferation, Ab production, and B cell regulatory function in vivo.

PTEN counteracts FBXL2 to promote IP3R3- and Ca2+-mediated apoptosis limiting tumour growth.

In response to environmental cues that promote IP3 (inositol 1,4,5-trisphosphate) generation, IP3 receptors (IP3Rs) located on the endoplasmic reticulum allow the 'quasisynaptical' feeding of calcium to the mitochondria to promote oxidative phosphorylation. However, persistent Ca2+ release results in mitochondrial Ca2+ overload and consequent apoptosis. Among the three mammalian IP3Rs, IP3R3 appears to be the major player in Ca2+-dependent apoptosis. Here we show that the F-box protein FBXL2 (the receptor subunit of one of 69 human SCF (SKP1, CUL1, F-box protein) ubiquitin ligase complexes) binds IP3R3 and targets it for ubiquitin-, p97- and proteasome-mediated degradation to limit Ca2+ influx into mitochondria. FBXL2-knockdown cells and FBXL2-insensitive IP3R3 mutant knock-in clones display increased cytosolic Ca2+ release from the endoplasmic reticulum and sensitization to Ca2+-dependent apoptotic stimuli. The phosphatase and tensin homologue (PTEN) gene is frequently mutated or lost in human tumours and syndromes that predispose individuals to cancer. We found that PTEN competes with FBXL2 for IP3R3 binding, and the FBXL2-dependent degradation of IP3R3 is accelerated in Pten-/- mouse embryonic fibroblasts and PTEN-null cancer cells. Reconstitution of PTEN-null cells with either wild-type PTEN or a catalytically dead mutant stabilizes IP3R3 and induces persistent Ca2+ mobilization and apoptosis. IP3R3 and PTEN protein levels directly correlate in human prostate cancer. Both in cell culture and xenograft models, a non-degradable IP3R3 mutant sensitizes tumour cells with low or no PTEN expression to photodynamic therapy, which is based on the ability of photosensitizer drugs to cause Ca2+-dependent cytotoxicity after irradiation with visible light. Similarly, disruption of FBXL2 localization with GGTi-2418, a geranylgeranyl transferase inhibitor, sensitizes xenotransplanted tumours to photodynamic therapy. In summary, we identify a novel molecular mechanism that limits mitochondrial Ca2+ overload to prevent cell death. Notably, we provide proof-of-principle that inhibiting IP3R3 degradation in PTEN-deregulated cancers represents a valid therapeutic strategy.

Signaling Pathway for Endothelin-1- and Phenylephrine-Induced cAMP Response Element Binding Protein Activation in Rat Ventricular Myocytes: Role of Inositol 1,4,5-Trisphosphate Receptors and CaMKII.

BACKGROUND/AIMS: Endothelin-1 (ET-1) and the α₁-adrenoceptor agonist phenylephrine (PE) activate cAMP response element binding protein (CREB), a transcription factor implicated in cardiac hypertrophy. The signaling pathway involved in CREB activation by these hypertrophic stimuli is poorly understood. We examined signaling pathways for ET-1- or PE-induced cardiac CREB activation. METHODS: Western blotting was performed with pharmacological and genetic interventions in rat ventricular myocytes. RESULTS: ET-1 and PE increased CREB phosphorylation, which was inhibited by blockade of phospholipase C, the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway, protein kinase C (PKC) or Ca2+-calmodulin-dependent protein kinase II (CaMKII). Intracellular Ca2+ buffering decreased ET-1- and PE-induced CREB phosphorylation by ≥80%. Sarcoplasmic reticulum Ca2+ pump inhibitor, inositol 1,4,5-trisphosphate receptor (IP₃R) blockers, or type 2 IP₃R (IP₃R2) knock-out abolished ET-1- or PE-induced CREB phosphorylation. ET-1 and PE increased phosphorylation of CaMKII and ERK1/2, which was eliminated by IP₃R blockade/knock-out or PKC inhibition. Activation of CaMKII, but not ERK1/2, by these agonists was sensitive to Ca2+ buffering or to Gö6976, the inhibitor of Ca2+-dependent PKC and protein kinase D (PKD). CONCLUSION: CREB phosphorylation by ET-1 and PE may be mainly mediated by IP₃R2/Ca2+-PKC-PKD-CaMKII signaling with a minor contribution by ERK1/2, linked to IP₃R2 and Ca2+-independent PKC, in ventricular myocytes.

Astrocytic calcium release mediates peri-infarct depolarizations in a rodent stroke model.

Stroke is one of the most common diseases and a leading cause of death and disability. Cessation of cerebral blood flow (CBF) leads to cell death in the infarct core, but tissue surrounding the core has the potential to recover if local reductions in CBF are restored. In these areas, detrimental peri-infarct depolarizations (PIDs) contribute to secondary infarct growth and negatively affect stroke outcome. However, the cellular pathways underlying PIDs have remained unclear. Here, we have used in vivo multiphoton microscopy, laser speckle imaging of CBF, and electrophysiological recordings in a mouse model of focal ischemia to demonstrate that PIDs are associated with a strong increase of intracellular calcium in astrocytes and neurons. We found that astroglial calcium elevations during PIDs are mediated by inositol triphosphate receptor type 2-dependent (IP3R2-dependent) release from internal stores. Importantly, Ip3r2-deficient mice displayed a reduction of PID frequency and overall PID burden and showed increased neuronal survival after stroke. These effects were not related to local CBF changes in response to PIDs. However, we showed that the release and extracellular accumulation of glutamate during PIDs is strongly curtailed in Ip3r2-deficient mice, resulting in ameliorated calcium overload in neurons and astrocytes. Together, these data implicate astroglial calcium pathways as potential targets for stroke therapy.

Disruption of IP3R2-mediated Ca²âº signaling pathway in astrocytes ameliorates neuronal death and brain damage while reducing behavioral deficits after focal ischemic stroke.

Inositol trisphosphate receptor (IP3R)-mediated intracellular Ca(2+) increase is the major Ca(2+) signaling pathway in astrocytes in the central nervous system (CNS). Ca(2+) increases in astrocytes have been found to modulate neuronal function through gliotransmitter release. We previously demonstrated that astrocytes exhibit enhanced Ca(2+) signaling in vivo after photothrombosis (PT)-induced ischemia, which is largely due to the activation of G-protein coupled receptors (GPCRs). The aim of this study is to investigate the role of astrocytic IP3R-mediated Ca(2+) signaling in neuronal death, brain damage and behavior outcomes after PT. For this purpose, we conducted experiments using homozygous type 2 IP3R (IP3R2) knockout (KO) mice. Histological and immunostaining studies showed that IP3R2 KO mice were indeed deficient in IP3R2 in astrocytes and exhibited normal brain cytoarchitecture. IP3R2 KO mice also had the same densities of S100ß+ astrocytes and NeuN+ neurons in the cortices, and exhibited the same glial fibrillary acidic protein (GFAP) and glial glutamate transporter (GLT-1) levels in the cortices and hippocampi as compared with wild type (WT) mice. Two-photon (2-P) imaging showed that IP3R2 KO mice did not exhibit ATP-induced Ca(2+) waves in vivo in the astrocytic network, which verified the disruption of IP3R-mediated Ca(2+) signaling in astrocytes of these mice. When subject to PT, IP3R2 KO mice had smaller infarction than WT mice in acute and chronic phases of ischemia. IP3R2 KO mice also exhibited less neuronal apoptosis, reactive astrogliosis, and tissue loss than WT mice. Behavioral tests, including cylinder, hanging wire, pole and adhesive tests, showed that IP3R2 KO mice exhibited reduced functional deficits after PT. Collectively, our study demonstrates that disruption of astrocytic Ca(2+) signaling by deleting IP3R2s has beneficial effects on neuronal and brain protection and functional deficits after stroke. These findings reveal a novel non-cell-autonomous neuronal and brain protective function of astrocytes in ischemic stroke, whereby suggest that the astrocytic IP3R2-mediated Ca(2+) signaling pathway might be a promising target for stroke therapy.

Ca(2+) signaling in astrocytes from Ip3r2(-/-) mice in brain slices and during startle responses in vivo.

Intracellular Ca(2+) signaling is considered to be important for multiple astrocyte functions in neural circuits. However, mice devoid of inositol triphosphate type 2 receptors (IP3R2) reportedly lack all astrocyte Ca(2+) signaling, but display no neuronal or neurovascular deficits, implying that astrocyte Ca(2+) fluctuations are not involved in these functions. An assumption has been that the loss of somatic Ca(2+) fluctuations also reflects a similar loss in astrocyte processes. We tested this assumption and found diverse types of Ca(2+) fluctuations in astrocytes, with most occurring in processes rather than in somata. These fluctuations were preserved in Ip3r2(-/-) (also known as Itpr2(-/-)) mice in brain slices and in vivo, occurred in end feet, and were increased by G protein-coupled receptor activation and by startle-induced neuromodulatory responses. Our data reveal previously unknown Ca(2+) fluctuations in astrocytes and highlight limitations of studies that used Ip3r2(-/-) mice to evaluate astrocyte contributions to neural circuit function and mouse behavior.

Mice lacking inositol 1,4,5-trisphosphate receptors exhibit dry eye.

Tear secretion is important as it supplies water to the ocular surface and keeps eyes moist. Both the parasympathetic and sympathetic pathways contribute to tear secretion. Although intracellular Ca2+ elevation in the acinar cells of lacrimal glands is a crucial event for tear secretion in both the pathways, the Ca2+ channel, which is responsible for the Ca2+ elevation in the sympathetic pathway, has not been sufficiently analyzed. In this study, we examined tear secretion in mice lacking the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3 (Itpr2-/-;Itpr3-/-double-knockout mice). We found that tear secretion in both the parasympathetic and sympathetic pathways was abolished in Itpr2-/-;Itpr3-/- mice. Intracellular Ca2+ elevation in lacrimal acinar cells after acetylcholine and epinephrine stimulation was abolished in Itpr2-/-;Itpr3-/- mice. Consequently, Itpr2-/-;Itpr3-/- mice exhibited keratoconjunctival alteration and corneal epithelial barrier disruption. Inflammatory cell infiltration into the lacrimal glands and elevation of serum autoantibodies, a representative marker for Sjögren's syndrome (SS) in humans, were also detected in older Itpr2-/-;Itpr3-/- mice. These results suggested that IP3Rs are essential for tear secretion in both parasympathetic and sympathetic pathways and that Itpr2-/-;Itpr3-/- mice could be a new dry eye mouse model with symptoms that mimic those of SS.

Disruption of endogenous purinergic signaling inhibits vascular endothelial growth factor- and glutamate-induced osmotic volume regulation of Müller glial cells in knockout mice.

BACKGROUND/AIMS: Osmotic swelling of Müller cells is a common phenomenon in animal models of ischemic and diabetic retinopathies. Müller cells possess a swelling-inhibitory purinergic signaling cascade which can be activated by various receptor ligands including vascular endothelial growth factor (VEGF) and glutamate. Here, we investigated whether deletion of P2Y1 (P2Y1R) and adenosine A1 receptors (A1AR), and of inositol-1,4,5-trisphosphate-receptor type 2 (IP3R2), in mice affects the inhibitory action of VEGF and glutamate on Müller cell swelling. METHODS: The cross-sectional area of Müller cell somata was recorded after a 4-min superfusion of retinal slices with a hypoosmotic solution. RESULTS: Hypoosmolarity induced a swelling of Müller cells from P2Y1R(-/-), A1AR(-/-) and IP3R2(-/-) mice, but not from wild-type mice. Swelling of wild-type Müller cells was induced by hypoosmotic solution containing barium chloride. Whereas VEGF inhibited the swelling of wild-type Müller cells, it had no swelling-inhibitory effect in cells from A1AR(-/-) and IP3R2(-/-) mice. Glutamate inhibited the swelling of wild-type Müller cells but not of cells from P2Y1R(-/-), A1AR(-/-) and IP3R2(-/-) animals. CONCLUSION: The swelling-inhibitory effects of VEGF and glutamate in murine Müller cells is mediated by transactivation of P2Y1R and A1AR, as well as by intracellular calcium signaling via activation of IP3R2.

TRPA1 channels are regulators of astrocyte basal calcium levels and long-term potentiation via constitutive D-serine release.

Astrocytes are found throughout the brain where they make extensive contacts with neurons and synapses. Astrocytes are known to display intracellular Ca(2+) signals and release signaling molecules such as D-serine into the extracellular space. However, the role(s) of astrocyte Ca(2+) signals in hippocampal long-term potentiation (LTP), a form of synaptic plasticity involved in learning and memory, remains unclear. Here, we explored a recently discovered novel TRPA1 channel-mediated transmembrane Ca(2+) flux pathway in astrocytes. Specifically, we determined whether block or genetic deletion of TRPA1 channels affected LTP of Schaffer collateral to CA1 pyramidal neuron synapses. Using pharmacology, TRPA1(-/-) mice, imaging, electrophysiology, and D-serine biosensors, our data indicate that astrocyte TRPA1 channels contribute to basal Ca(2+) levels and are required for constitutive D-serine release into the extracellular space, which contributes to NMDA receptor-dependent LTP. The findings have broad relevance for the study of astrocyte-neuron interactions by demonstrating how TRPA1 channel-mediated fluxes contribute to astrocyte basal Ca(2+) levels and neuronal function via constitutive D-serine release.

In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase.

Calcium-dependent release of vasoactive gliotransmitters is widely assumed to trigger vasodilation associated with rapid increases in neuronal activity. Inconsistent with this hypothesis, intact stimulus-induced vasodilation was observed in inositol 1,4,5-triphosphate (IP3) type-2 receptor (R2) knock-out (KO) mice, in which the primary mechanism of astrocytic calcium increase-the release of calcium from intracellular stores following activation of an IP3-dependent pathway-is lacking. Further, our results in wild-type (WT) mice indicate that in vivo onset of astrocytic calcium increase in response to sensory stimulus could be considerably delayed relative to the simultaneously measured onset of arteriolar dilation. Delayed calcium increases in WT mice were observed in both astrocytic cell bodies and perivascular endfeet. Thus, astrocytes may not play a role in the initiation of blood flow response, at least not via calcium-dependent mechanisms. Moreover, an increase in astrocytic intracellular calcium was not required for normal vasodilation in the IP3R2-KO animals.

Astrocytic adenosine 5'-triphosphate release regulates the proliferation of neural stem cells in the adult hippocampus.

Astrocytes are key components of the niche for neural stem cells (NSCs) in the adult hippocampus and play a vital role in regulating NSC proliferation and differentiation. However, the exact molecular mechanisms by which astrocytes modulate NSC proliferation have not been identified. Here, we identified adenosine 5'-triphosphate (ATP) as a proliferative factor required for astrocyte-mediated proliferation of NSCs in the adult hippocampus. Our results indicate that ATP is necessary and sufficient for astrocytes to promote NSC proliferation in vitro. The lack of inositol 1,4,5-trisphosphate receptor type 2 and transgenic blockage of vesicular gliotransmission induced deficient ATP release from astrocytes. This deficiency led to a dysfunction in NSC proliferation that could be rescued via the administration of exogenous ATP. Moreover, P2Y1-mediated purinergic signaling is involved in the astrocyte promotion of NSC proliferation. As adult hippocampal neurogenesis is potentially involved in major mood disorder, our results might offer mechanistic insights into this disease.

Astrocytic Ca(2+) waves mediate activation of extrasynaptic NMDA receptors in hippocampal neurons to aggravate brain damage during ischemia.

Excitotoxicity plays a central role in the neuronal damage during ischemic stroke. Although growing evidence suggests that activation of extrasynaptic NMDA receptors initiates neuronal death, no direct evidence demonstrated their activation during ischemia. Using rat hippocampal slices, we detected oxygen-glucose deprivation (OGD) induced slow inward currents (SICs) mediated by extrasynaptic NMDA receptors in CA1 pyramidal neurons. Moreover, Ca(2+) chelator BAPTA dialysis into astrocytic network decreased the frequency of OGD induced SICs, indicating that the activation of extrasynaptic NMDA receptors depended on astrocytic Ca(2+) activity. To further demonstrate the importance of astrocytic Ca(2+) activity, we tested hippocampal slices from inositol triphosphate receptor type 2 (IP3R2) knock-out mice which abolished the astrocytic Ca(2+) activity. As expected, the frequency of OGD induced SICs was reduced. Using two-photon Ca(2+) imaging, we characterized the astrocytic Ca(2+) dynamics. By controlling Ca(2+) level in the individual astrocytes using targeted photolysis, we found that OGD facilitated the propagation of intercellular Ca(2+) waves, which were inhibited by gap junction blocker carbenoxolone (CBX). CBX also inhibited the Ca(2+) activity of the astrocytic network and decreased the SIC frequency during OGD. Functionally, the infarct volumes from brain ischemia were reduced in IP3R2 knock-out mice and in rat intracerebrally delivered with CBX. Our results demonstrate that enhanced Ca(2+) activity of the astrocytic network plays a key role on the activation of extrasynaptic NMDA receptors in hippocampal neurons, which enhances brain damage during ischemia.

An IP3R3- and NPY-expressing microvillous cell mediates tissue homeostasis and regeneration in the mouse olfactory epithelium.

Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3) receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3) is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY). We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3(+/-), and IP3R3(-/-) mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3(-/-) mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3(-/-) mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3(-/-) mice. The reductions in both NPY release and number of progenitor cells in IP3R3(-/-) mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.

Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation.

During sepsis, acute lung injury (ALI) results from activation of innate immune cells and endothelial cells by endotoxins, leading to systemic inflammation through proinflammatory cytokine overproduction, oxidative stress, and intracellular Ca2+ overload. Despite considerable investigation, the underlying molecular mechanism(s) leading to LPS-induced ALI remain elusive. To determine whether stromal interaction molecule 1-dependent (STIM1-dependent) signaling drives endothelial dysfunction in response to LPS, we investigated oxidative and STIM1 signaling of EC-specific Stim1-knockout mice. Here we report that LPS-mediated Ca2+ oscillations are ablated in ECs deficient in Nox2, Stim1, and type II inositol triphosphate receptor (Itpr2). LPS-induced nuclear factor of activated T cells (NFAT) nuclear accumulation was abrogated by either antioxidant supplementation or Ca2+ chelation. Moreover, ECs lacking either Nox2 or Stim1 failed to trigger store-operated Ca2+ entry (SOCe) and NFAT nuclear accumulation. LPS-induced vascular permeability changes were reduced in EC-specific Stim1-/- mice, despite elevation of systemic cytokine levels. Additionally, inhibition of STIM1 signaling prevented receptor-interacting protein 3-dependent (RIP3-dependent) EC death. Remarkably, BTP2, a small-molecule calcium release-activated calcium (CRAC) channel blocker administered after insult, halted LPS-induced vascular leakage and pulmonary edema. These results indicate that ROS-driven Ca2+ signaling promotes vascular barrier dysfunction and that the SOCe machinery may provide crucial therapeutic targets to limit sepsis-induced ALI.

No contribution of IP3-R(2) to disease phenotype in models of dilated cardiomyopathy or pressure overload hypertrophy.

BACKGROUND: We investigated the contribution of inositol(1,4,5)-trisphosphate (Ins(1,4,5)P3 [IP3]) receptors (IP3-R) to disease progression in mouse models of dilated cardiomyopathy (DCM) and pressure overload hypertrophy. Mice expressing mammalian sterile 20-like kinase and dominant-negative phosphatidylinositol-3-kinase in heart (Mst1×dn-PI3K-2Tg; DCM-2Tg) develop severe DCM and conduction block, associated with increased expression of type 2 IP3-R (IP3-R(2)) and heightened generation of Ins(1,4,5)P3. Similar increases in Ins(1,4,5)P3 and IP3-R(2) are caused by transverse aortic constriction. METHODS AND RESULTS: To evaluate the contribution of IP3-R(2) to disease progression, the DCM-2Tg mice were further crossed with mice in which the type 2 IP3-R (IP3-R(2)-/-) had been deleted (DCM-2Tg×IP3-R(2)-/-) and transverse aortic constriction was performed on IP3-R(2)-/- mice. Hearts from DCM-2Tg mice and DCM-2Tg×IP3-R(2)-/- were similar in terms of chamber dilatation, atrial enlargement, and ventricular wall thinning. Electrophysiological changes were also similar in the DCM-2Tg mice, with and without IP3-R(2). Deletion of IP3-R(2) did not alter the progression of heart failure, because DCM-2Tg mice with and without IP3-R(2) had similarly reduced contractility, increased lung congestion, and atrial thrombus, and both strains died between 10 and 12 weeks of age. Loss of IP3-R(2) did not alter the progression of hypertrophy after transverse aortic constriction. CONCLUSIONS: We conclude that IP3-R(2) do not contribute to the progression of DCM or pressure overload hypertrophy, despite increased expression and heightened generation of the ligand, Ins(1,4,5)P3.