Publications by authors named "Katsuhiko Mikoshiba"

347 Publications

A non-canonical role for pyruvate kinase M2 as a functional modulator of Ca signalling through IP receptors.

Biochim Biophys Acta Mol Cell Res 2022 Jan 11;1869(4):119206. Epub 2022 Jan 11.

Division of Hematology/Oncology, Dept. Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA; Dept of Medicine, University Hospitals Cleveland Medical Center, Cleveland, OH 44106, USA; Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. Electronic address:

Pyruvate kinase isoform M2 (PKM2) is a rate-limiting glycolytic enzyme that is widely expressed in embryonic tissues. The expression of PKM2 declines in some tissues following embryogenesis, while other pyruvate kinase isozymes are upregulated. However, PKM2 is highly expressed in cancer cells and is believed to play a role in supporting anabolic processes during tumour formation. In this study, PKM2 was identified as an inositol 1,4,5-trisphosphate receptor (IPR)-interacting protein by mass spectrometry. The PKM2:IPR interaction was further characterized by pull-down and co-immunoprecipitation assays, which showed that PKM2 interacted with all three IPR isoforms. Moreover, fluorescence microscopy indicated that both IPR and PKM2 localized at the endoplasmic reticulum. PKM2 binds to IPR at a highly conserved 21-amino acid site (corresponding to amino acids 2078-2098 in mouse type 1 IPR isoform). Synthetic peptides (denoted 'TAT-D5SD' and 'D5SD'), based on the amino acid sequence at this site, disrupted the PKM2:IPR interaction and potentiated IPR-mediated Ca release both in intact cells (TAT-D5SD peptide) and in a unidirectional Ca flux assay on permeabilized cells (D5SD peptide). The TAT-D5SD peptide did not affect the enzymatic activity of PKM2. Reducing PKM2 protein expression using siRNA increased IPR-mediated Ca signalling in intact cells without altering the ER Ca content. These data identify PKM2 as an IPR-interacting protein that inhibits intracellular Ca signalling. The elevated expression of PKM2 in cancer cells is therefore not solely connected to its canonical role in glycolytic metabolism, rather PKM2 also has a novel non-canonical role in regulating intracellular signalling.
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http://dx.doi.org/10.1016/j.bbamcr.2021.119206DOI Listing
January 2022

Metabolic adaptation to the chronic loss of Ca signaling induced by KO of IP receptors or the mitochondrial Ca uniporter.

J Biol Chem 2021 Nov 19;298(1):101436. Epub 2021 Nov 19.

Department of Pathology, MitoCare Center, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. Electronic address:

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IPRs) and the mitochondrial Ca uniporter (MCU) are key proteins that regulate intracellular Ca concentration. Mitochondrial Ca accumulation activates Ca-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IPR isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca signaling. Our results show that TKO cells (exhibiting total loss of Ca signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca signaling is dispensable for the bioenergetic needs of both IPR TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.
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http://dx.doi.org/10.1016/j.jbc.2021.101436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8672050PMC
November 2021

Bcl-xL acts as an inhibitor of IPR channels, thereby antagonizing Ca-driven apoptosis.

Cell Death Differ 2021 Nov 8. Epub 2021 Nov 8.

KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, and Leuven Kanker Instituut, Campus Gasthuisberg O/N-1 Box 802, Herestraat 49, 3000, Leuven, Belgium.

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca dynamics by controlling IP receptor (IPR) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IPRs and preventing pro-apoptotic Ca release and Bcl-xL sensitizing IPRs to low [IP] and promoting pro-survival Ca oscillations. We here demonstrate that Bcl-xL too inhibits IPR-mediated Ca release by interacting with the same IPR regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2's IPR-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IPR and abrogated Bcl-xL's inhibitory effect on IPRs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xL, suppressed IPR single-channel openings stimulated by sub-maximal and threshold [IP]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IPRs contributes to its anti-apoptotic properties against Ca-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca elevations in wild-type but not in IPR-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca signals and cell death, while Bcl-xL was much less effective in doing so. In the absence of IPRs, Bcl-xL and Bcl-xL were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IPR activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IPR-mediated Ca release and increased the sensitivity towards STS, without altering the ER Ca content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IPR-mediated Ca release and IPR-driven cell death. Our work further underpins that IPR inhibition is an integral part of Bcl-xL's anti-apoptotic function.
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http://dx.doi.org/10.1038/s41418-021-00894-wDOI Listing
November 2021

Ten-eleven translocation 1 mediated-DNA hydroxymethylation is required for myelination and remyelination in the mouse brain.

Nat Commun 2021 08 24;12(1):5091. Epub 2021 Aug 24.

Department of Neurobiology, School of Basic Medicine, Fourth Military Medical University, Xi'an, Shaanxi, China.

Ten-eleven translocation (TET) proteins, the dioxygenase for DNA hydroxymethylation, are important players in nervous system development and diseases. However, their role in myelination and remyelination after injury remains elusive. Here, we identify a genome-wide and locus-specific DNA hydroxymethylation landscape shift during differentiation of oligodendrocyte-progenitor cells (OPC). Ablation of Tet1 results in stage-dependent defects in oligodendrocyte (OL) development and myelination in the mouse brain. The mice lacking Tet1 in the oligodendrocyte lineage develop behavioral deficiency. We also show that TET1 is required for remyelination in adulthood. Transcriptomic, genomic occupancy, and 5-hydroxymethylcytosine (5hmC) profiling reveal a critical TET1-regulated epigenetic program for oligodendrocyte differentiation that includes genes associated with myelination, cell division, and calcium transport. Tet1-deficient OPCs exhibit reduced calcium activity, increasing calcium activity rescues the differentiation defects in vitro. Deletion of a TET1-5hmC target gene, Itpr2, impairs the onset of OPC differentiation. Together, our results suggest that stage-specific TET1-mediated epigenetic programming and intracellular signaling are important for proper myelination and remyelination in mice.
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http://dx.doi.org/10.1038/s41467-021-25353-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8385008PMC
August 2021

Astrocytic IPRs: Beyond IPR2.

Front Cell Neurosci 2021 30;15:695817. Epub 2021 Jul 30.

University of Bordeaux, INSERM, Neurocentre Magendie, U1215, Bordeaux, France.

Astrocytes are sensitive to ongoing neuronal/network activities and, accordingly, regulate neuronal functions (synaptic transmission, synaptic plasticity, behavior, etc.) by the context-dependent release of several gliotransmitters (e.g., glutamate, glycine, D-serine, ATP). To sense diverse input, astrocytes express a plethora of G-protein coupled receptors, which couple, via G and G, to the intracellular Ca release channel IP-receptor (IPR). Indeed, manipulating astrocytic IPR-Ca signaling is highly consequential at the network and behavioral level: Depleting IPR subtype 2 (IPR2) results in reduced GPCR-Ca signaling and impaired synaptic plasticity; enhancing IPR-Ca signaling affects cognitive functions such as learning and memory, sleep, and mood. However, as a result of discrepancies in the literature, the role of GPCR-IPR-Ca signaling, especially under physiological conditions, remains inconclusive. One primary reason for this could be that IPR2 has been used to represent all astrocytic IPRs, including IPR1 and IPR3. Indeed, IPR1 and IPR3 are unique Ca channels in their own right; they have unique biophysical properties, often display distinct distribution, and are differentially regulated. As a result, they mediate different physiological roles to IPR2. Thus, these additional channels promise to enrich the diversity of spatiotemporal Ca dynamics and provide unique opportunities for integrating neuronal input and modulating astrocyte-neuron communication. The current review weighs evidence supporting the existence of multiple astrocytic-IPR isoforms, summarizes distinct sub-type specific properties that shape spatiotemporal Ca dynamics. We also discuss existing experimental tools and future refinements to better recapitulate the endogenous activities of each IPR isoform.
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http://dx.doi.org/10.3389/fncel.2021.695817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363081PMC
July 2021

ERAD components Derlin-1 and Derlin-2 are essential for postnatal brain development and motor function.

iScience 2021 Jul 19;24(7):102758. Epub 2021 Jun 19.

Laboratory of Biochemistry and Molecular Biology, Department of Medical Sciences, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.

Derlin family members (Derlins) are primarily known as components of the endoplasmic reticulum-associated degradation pathway that eliminates misfolded proteins. Here we report a function of Derlins in the brain development. Deletion of or in the central nervous system of mice impaired postnatal brain development, particularly of the cerebellum and striatum, and induced motor control deficits. Derlin-1 or Derlin-2 deficiency reduced neurite outgrowth and and surprisingly also inhibited sterol regulatory element binding protein 2 (SREBP-2)-mediated brain cholesterol biosynthesis. In addition, reduced neurite outgrowth due to Derlin-1 deficiency was rescued by SREBP-2 pathway activation. Overall, our findings demonstrate that Derlins sustain brain cholesterol biosynthesis, which is essential for appropriate postnatal brain development and function.
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http://dx.doi.org/10.1016/j.isci.2021.102758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324814PMC
July 2021

Reactive astrocyte-driven epileptogenesis is induced by microglia initially activated following status epilepticus.

JCI Insight 2021 05 10;6(9). Epub 2021 May 10.

Department of Neuropharmacology, Interdisciplinary Graduate School of Medicine.

Extensive activation of glial cells during a latent period has been well documented in various animal models of epilepsy. However, it remains unclear whether activated glial cells contribute to epileptogenesis, i.e., the chronically persistent process leading to epilepsy. Particularly, it is not clear whether interglial communication between different types of glial cells contributes to epileptogenesis, because past literature has mainly focused on one type of glial cell. Here, we show that temporally distinct activation profiles of microglia and astrocytes collaboratively contributed to epileptogenesis in a drug-induced status epilepticus model. We found that reactive microglia appeared first, followed by reactive astrocytes and increased susceptibility to seizures. Reactive astrocytes exhibited larger Ca2+ signals mediated by IP3R2, whereas deletion of this type of Ca2+ signaling reduced seizure susceptibility after status epilepticus. Immediate, but not late, pharmacological inhibition of microglial activation prevented subsequent reactive astrocytes, aberrant astrocyte Ca2+ signaling, and the enhanced seizure susceptibility. These findings indicate that the sequential activation of glial cells constituted a cause of epileptogenesis after status epilepticus. Thus, our findings suggest that the therapeutic target to prevent epilepsy after status epilepticus should be shifted from microglia (early phase) to astrocytes (late phase).
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http://dx.doi.org/10.1172/jci.insight.135391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262323PMC
May 2021

Both IRBIT and long-IRBIT bind to and coordinately regulate Cl/HCO exchanger AE2 activity through modulating the lysosomal degradation of AE2.

Sci Rep 2021 03 16;11(1):5990. Epub 2021 Mar 16.

Department of Pharmacotherapeutics, Showa Pharmaceutical University, Machida, Tokyo, 194-8543, Japan.

Anion exchanger 2 (AE2) plays crucial roles in regulating cell volume homeostasis and cell migration. We found that both IRBIT and Long-IRBIT (L-IRBIT) interact with anion exchanger 2 (AE2). The interaction occurred between the conserved AHCY-homologous domain of IRBIT/L-IRBIT and the N-terminal cytoplasmic region of AE2. Interestingly, AE2 activity was reduced in L-IRBIT KO cells, but not in IRBIT KO cells. Moreover, AE2 activity was slightly increased in IRBIT/L-IRBIT double KO cells. These changes in AE2 activity resulted from changes in the AE2 expression level of each mutant cell, and affected the regulatory volume increase and cell migration. The activity and expression level of AE2 in IRBIT/L-IRBIT double KO cells were downregulated if IRBIT, but not L-IRBIT, was expressed again in the cells, and the downregulation was cancelled by the co-expression of L-IRBIT. The mRNA levels of AE2 in each KO cell did not change, and the downregulation of AE2 in L-IRBIT KO cells was inhibited by bafilomycin A1. These results indicate that IRBIT binding facilitates the lysosomal degradation of AE2, which is inhibited by coexisting L-IRBIT, suggesting a novel regulatory mode of AE2 activity through the binding of two homologous proteins with opposing functions.
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http://dx.doi.org/10.1038/s41598-021-85499-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966362PMC
March 2021

Spinal astrocytes in superficial laminae gate brainstem descending control of mechanosensory hypersensitivity.

Nat Neurosci 2020 11 5;23(11):1376-1387. Epub 2020 Oct 5.

Department of Life Innovation, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.

Astrocytes are critical regulators of CNS function and are proposed to be heterogeneous in the developing brain and spinal cord. Here we identify a population of astrocytes located in the superficial laminae of the spinal dorsal horn (SDH) in adults that is genetically defined by Hes5. In vivo imaging revealed that noxious stimulation by intraplantar capsaicin injection activated Hes5 SDH astrocytes via α-adrenoceptors (α-ARs) through descending noradrenergic signaling from the locus coeruleus. Intrathecal norepinephrine induced mechanical pain hypersensitivity via α-ARs in Hes5 astrocytes, and chemogenetic stimulation of Hes5 SDH astrocytes was sufficient to produce the hypersensitivity. Furthermore, capsaicin-induced mechanical hypersensitivity was prevented by the inhibition of descending locus coeruleus-noradrenergic signaling onto Hes5 astrocytes. Moreover, in a model of chronic pain, α-ARs in Hes5 astrocytes were critical regulators for determining an analgesic effect of duloxetine. Our findings identify a superficial SDH-selective astrocyte population that gates descending noradrenergic control of mechanosensory behavior.
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http://dx.doi.org/10.1038/s41593-020-00713-4DOI Listing
November 2020

Astrocytic STAT3 activation and chronic itch require IPR1/TRPC-dependent Ca signals in mice.

J Allergy Clin Immunol 2021 04 8;147(4):1341-1353. Epub 2020 Aug 8.

Department of Life Innovation, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan. Electronic address:

Background: Chronic itch is a debilitating symptom of inflammatory skin diseases, but the underlying mechanism is poorly understood. We have recently demonstrated that astrocytes in the spinal dorsal horn become reactive in models of atopic and contact dermatitis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) and critically contribute to chronic itch. In general, STAT3 is transiently activated; however, STAT3 activation in reactive astrocytes of chronic itch model mice persistently occurs via an unknown mechanism.

Objective: We aimed to determine the mechanisms of persistent activation of astrocytic STAT3 in chronic itch conditions.

Methods: To determine the factors that are required for persistent activation of astrocytic STAT3, Western blotting and calcium imaging with cultured astrocytes or spinal cord slices were performed. Thereafter, chronic itch model mice were used for genetic and behavioral experiments to confirm the role of the factors determined to mediate persistent STAT3 activation from in vitro and ex vivo experiments in chronic itch.

Results: IP receptor type 1 (IPR1) knockdown in astrocytes suppressed IL-6-induced persistent STAT3 activation and expression of lipocalin-2 (LCN2), an astrocytic STAT3-dependent inflammatory factor that is required for chronic itch. IPR1-dependent astrocytic Ca responses involved Ca influx through the cation channel transient receptor potential canonical (TRPC), which was required for persistent STAT3 activation evoked by IL-6. IL-6 expression was upregulated in dorsal root ganglion neurons in a mouse model of chronic itch. Dorsal root ganglion neuron-specific IL-6 knockdown, spinal astrocyte-specific IPR1 knockdown, and pharmacologic spinal TRPC inhibition attenuated LCN2 expression and chronic itch.

Conclusion: Our findings suggest that IPR1/TRPC channel-mediated Ca signals elicited by IL-6 in astrocytes are necessary for persistent STAT3 activation, LCN2 expression, and chronic itch, and they may also provide new targets for therapeutic intervention.
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http://dx.doi.org/10.1016/j.jaci.2020.06.039DOI Listing
April 2021

The molecular mechanism of synaptic activity-induced astrocytic volume transient.

J Physiol 2020 10 8;598(20):4555-4572. Epub 2020 Aug 8.

Center for Glia-Neuron Interaction, Korea Institute of Science and Technology (KIST), Seoul, 02792, Republic of Korea.

Key Points: Neuronal activity causes astrocytic volume change via K uptake through TREK-1 containing two-pore domain potassium channels. The volume transient is terminated by Cl efflux through the Ca -activated anion channel BEST1. The source of the Ca required to open BEST1 appears to be the stretch-activated TRPA1 channel. Intense neuronal activity is synaptically coupled with a physical change in astrocytes via volume transients.

Abstract: The brain volume changes dynamically and transiently upon intense neuronal activity through a tight regulation of ion concentrations and water movement across the plasma membrane of astrocytes. We have recently demonstrated that an intense neuronal activity and subsequent astrocytic AQP4-dependent volume transient are critical for synaptic plasticity and memory. We have also pharmacologically demonstrated a functional coupling between synaptic activity and the astrocytic volume transient. However, the precise molecular mechanisms of how intense neuronal activity and the astrocytic volume transient are coupled remain unclear. Here we utilized an intrinsic optical signal imaging technique combined with fluorescence imaging using ion sensitive dyes and molecular probes and electrophysiology to investigate the detailed molecular mechanisms in genetically modified mice. We report that a brief synaptic activity induced by a train stimulation (20 Hz, 1 s) causes a prolonged astrocytic volume transient (80 s) via K uptake through TREK-1 containing two-pore domain potassium (K2P) channels, but not Kir4.1 or NKCC1. This volume change is terminated by Cl efflux through the Ca -activated anion channel BEST1, but not the volume-regulated anion channel TTYH. The source of the Ca required to open BEST1 appears to be the stretch-activated TRPA1 channel in astrocytes, but not IP R2. In summary, our study identifies several important astrocytic ion channels (AQP4, TREK-1, BEST1, TRPA1) as the key molecules leading to the neuronal activity-dependent volume transient in astrocytes. Our findings reveal new molecular and cellular mechanisms for the synaptic coupling of intense neuronal activity with a physical change in astrocytes via volume transients.
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http://dx.doi.org/10.1113/JP279741DOI Listing
October 2020

Author Correction: Structural basis of astrocytic Ca signals at tripartite synapses.

Nat Commun 2020 05 18;11(1):2541. Epub 2020 May 18.

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-16453-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7235234PMC
May 2020

Inhibitory synaptic transmission tuned by Ca and glutamate through the control of GABA R lateral diffusion dynamics.

Dev Growth Differ 2020 Aug 20;62(6):398-406. Epub 2020 May 20.

Laboratory for Developmental Neurobiology, RIKEN Center for Brain Science, Wako, Japan.

The GABAergic synapses, a primary inhibitory synapse in the mammalian brain, is important for the normal development of brain circuits, and for the regulation of the excitation-inhibition balance critical for brain function from the developmental stage throughout life. However, the molecular mechanism underlying the formation, maintenance, and modulation of GABAergic synapses is less understood compared to that of excitatory synapses. Quantum dot-single particle tracking (QD-SPT), a super-resolution imaging technique that enables the analysis of membrane molecule dynamics at single-molecule resolution, is a powerful tool to analyze the behavior of proteins and lipids on the plasma membrane. In this review, we summarize the recent application of QD-SPT in understanding of GABAergic synaptic transmission. Here we introduce QD-SPT experiments that provide further insights into the molecular mechanism supporting GABAergic synapses. QD-SPT studies revealed that glutamate and Ca signaling is involved in (a) the maintenance of GABAergic synapses, (b) GABAergic long-term depression, and GABAergic long-term potentiation, by specifically activating signaling pathways unique to each phenomenon. We also introduce a novel Ca imaging technique to describe the diversity of Ca signals that may activate the downstream signaling pathways that induce specific biological output.
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http://dx.doi.org/10.1111/dgd.12667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496684PMC
August 2020

Structural basis of astrocytic Ca signals at tripartite synapses.

Nat Commun 2020 04 20;11(1):1906. Epub 2020 Apr 20.

University of Bordeaux, Bordeaux, France.

Astrocytic Ca signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the spongiform domain of astrocytes where most tripartite synapses are located. Using 3D-STED microscopy in living organotypic brain slices, we imaged the spongiform domain of astrocytes and observed a reticular meshwork of nodes and shafts that often formed loop-like structures. These anatomical features were also observed in acute hippocampal slices and in barrel cortex in vivo. The majority of dendritic spines were contacted by nodes and their sizes were correlated. FRAP experiments and Ca imaging showed that nodes were biochemical compartments and Ca microdomains. Mapping astrocytic Ca signals onto STED images of nodes and dendritic spines showed they were associated with individual synapses. Here, we report on the nanoscale organization of astrocytes, identifying nodes as a functional astrocytic component of tripartite synapses that may enable synapse-specific communication between neurons and astrocytes.
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http://dx.doi.org/10.1038/s41467-020-15648-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170846PMC
April 2020

Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides.

Immunity 2020 04;52(4):683-699.e11

Institute for Anatomy and Cell Biology, German Center for Lung Research, Justus Liebig University, 35385 Giessen, Germany. Electronic address:

Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.
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http://dx.doi.org/10.1016/j.immuni.2020.03.005DOI Listing
April 2020

Inositol 1,4,5-Trisphosphate Receptor Type 3 Regulates Neuronal Growth Cone Sensitivity to Guidance Signals.

iScience 2020 Mar 5;23(3):100963. Epub 2020 Mar 5.

RIKEN Center for Brain Science, 2-1 Hirosawa, Wako City, Saitama 351-0198, Japan. Electronic address:

During neurodevelopment, the growth cone deciphers directional information from extracellular guidance cues presented as shallow concentration gradients via signal amplification. However, it remains unclear how the growth cone controls this amplification process during its navigation through an environment in which basal cue concentrations vary widely. Here, we identified inositol 1,4,5-trisphosphate (IP) receptor type 3 as a regulator of axonal sensitivity to guidance cues in vitro and in vivo. Growth cones lacking the type 3 subunit are hypersensitive to nerve growth factor (NGF), an IP-dependent attractive cue, and incapable of turning toward normal concentration ranges of NGF to which wild-type growth cones respond. This is due to globally, but not asymmetrically, activated Ca signaling in the hypersensitive growth cones. Remarkably, lower NGF concentrations can polarize growth cones for turning if IP receptor type 3 is deficient. These data suggest a subtype-specific IP receptor function in sensitivity adjustment during axon navigation.
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http://dx.doi.org/10.1016/j.isci.2020.100963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7082556PMC
March 2020

Type 3 Inositol 1,4,5-Trisphosphate Receptor is a Crucial Regulator of Calcium Dynamics Mediated by Endoplasmic Reticulum in HEK Cells.

Cells 2020 01 22;9(2). Epub 2020 Jan 22.

Beijing Key Laboratory of Gene Resource and Molecular Development, College of Life Sciences, Beijing Normal University, Beijing 100875, China.

Being the largest the Ca store in mammalian cells, endoplasmic reticulum (ER)-mediated Ca signalling often involves both Ca release via inositol 1, 4, 5-trisphosphate receptors (IPR) and store operated Ca entries (SOCE) through Ca release activated Ca (CRAC) channels on plasma membrane (PM). IPRs are functionally coupled with CRAC channels and other Ca handling proteins. However, it still remains less well defined as to whether IPRs could regulate ER-mediated Ca signals independent of their Ca releasing ability. To address this, we generated IPRs triple and double knockout human embryonic kidney (HEK) cell lines (IPRs-TKO, IPRs-DKO), and systemically examined ER Ca dynamics and CRAC channel activity in these cells. The results showed that the rate of ER Ca leakage and refilling, as well as SOCE were all significantly reduced in IPRs-TKO cells. And these TKO effects could be rescued by over-expression of IPR3. Further, results showed that the diminished SOCE was caused by NEDD4L-mediated ubiquitination of Orai1 protein. Together, our findings indicate that IPR3 is one crucial player in coordinating ER-mediated Ca signalling.
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http://dx.doi.org/10.3390/cells9020275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072192PMC
January 2020

An ultra-stable cytoplasmic antibody engineered for in vivo applications.

Nat Commun 2020 01 17;11(1):336. Epub 2020 Jan 17.

Laboratory for Developmental Neurobiology, Center for Brain Science, Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.

Targeting cytoplasmic protein-protein interactions with antibodies remains technically challenging, since antibodies expressed in the cytosol frequently form insoluble aggregates. Existing engineering methods are based on the notion that the estimated net charge at pH 7.4 affects stability; as such, they are unable to overcome this problem. Herein, we report a versatile method for engineering an ultra-stable cytoplasmic antibody (STAND), with a strong estimated net negative charge at pH 6.6, by fusing peptide tags with a highly negative charge and a low isoelectric point. Without the need for complicated amino acid substitutions, we convert aggregation-prone antibodies to STANDs that are useful for inhibiting in vivo transmitter release, modulating animal behaviour, and inhibiting in vivo cancer proliferation driven by mutated Kras-long recognised as an "undruggable" oncogenic protein. The STAND method shows promise for targeting endogenous cytoplasmic proteins in basic biology and for developing future disease treatments.
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http://dx.doi.org/10.1038/s41467-019-13654-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969036PMC
January 2020

Depotentiation depends on IP receptor activation sustained by synaptic inputs after LTP induction.

Learn Mem 2020 02 16;27(2):52-66. Epub 2020 Jan 16.

Laboratory for Developmental Neurobiology, Riken Brain Science Institute, Wako, Saitama 351-0198, Japan.

In CA1 neurons of guinea pig hippocampal slices, long-term potentiation (LTP) was induced in field excitatory postsynaptic potentials (EPSPs) or population spikes (PSs) by the delivery of high-frequency stimulation (HFS, 100 pulses at 100 Hz) to CA1 synapses, and was reversed by the delivery of a train of low-frequency stimulation (LFS, 1000 pulses at 2 Hz) at 30 min after HFS (depotentiation), and this effect was inhibited when test synaptic stimulation was halted for a 19-min period after HFS or for a 20-min period after LFS or applied over the same time period in the presence of an antagonist of N-methyl-D-aspartate receptors (NMDARs), group I metabotropic glutamate receptors (mGluRs), or inositol 1, 4, 5-trisphosphate receptors (IPRs). Depotentiation was also blocked by the application of a Ca/calmodulin-dependent protein kinase II (CaMKII) inhibitor or a calcineurin inhibitor applied in the presence of test synaptic input for a 10-min period after HFS or for a 20-min period after LFS. These results suggest that, in postsynaptic neurons, the coactivation of NMDARs and group I mGluRs due to sustained synaptic activity following LTP induction results in the activation of IPRs and CaMKII, which leads to the activation of calcineurin after LFS and depotentiation of CA1 synaptic responses.
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http://dx.doi.org/10.1101/lm.050344.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970427PMC
February 2020

Inositol 1,4,5-trisphosphate receptor 2 as a novel marker of vasculature to delineate processes of cardiopulmonary development.

Dev Biol 2020 02 20;458(2):237-245. Epub 2019 Nov 20.

Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan. Electronic address:

Congenital heart diseases (CHDs) involving the outflow tract (OFT), such as persistent truncus arteriosus (PTA), lead to mortality and morbidity with implications not only in the heart, but also in the pulmonary vasculature. The mechanisms of pulmonary artery (PA) development and the etiologies underlying PA disorders associated with CHD remain poorly understood partly because of a specific marker for PA development is nonexistent. The three subtypes of inositol 1,4,5-trisphosphate receptors (IPR1, 2, and 3) are intracellular Ca channels that are essential for many tissues and organs. We discovered that IPR2 was expressed in the vasculature and heart during development using transgenic mice, in which a LacZ marker gene was knocked into the IPR2 locus. Whole-mount and section LacZ staining showed that IPR2-LacZ-positive cells were detectable exclusively in the smooth muscle cells, or tunica media, of PA, merging into αSMA-positive cells during development. Furthermore, our analyses suggested that IPR2-LacZ positive PA smooth muscle layers gradually elongate from the central PA to the peripheral PAs from E13.5 to E18.5, supporting the distal angiogenesis theory for the development of PA, whereas IPR2-LacZ was rarely expressed in smooth muscle cells in the pulmonary trunk. Crossing IPR-LacZ mice with mice hypomorphic for Tbx1 alleles revealed that PTA of Tbx1 mutants may result from agenesis or hypoplasia of the pulmonary trunk; thus, the left and right central to peripheral PAs connect directly to the dorsal side of the truncus arteriosus in these mutants. Additionally, we found hypercellular interstitial mesenchyme and delayed maturation of the lung endoderm in the Tbx1 mutant lungs. Our study identifies IPR2 as a novel marker for clear visualization of PA during development and can be utilized for studying cardiopulmonary development and disease.
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http://dx.doi.org/10.1016/j.ydbio.2019.11.011DOI Listing
February 2020

IP Receptor Plasticity Underlying Diverse Functions.

Annu Rev Physiol 2020 02 15;82:151-176. Epub 2019 Nov 15.

Laboratory of Cell Calcium Signaling, Shanghai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, Shanghai, 201210, China; email:

In the body, extracellular stimuli produce inositol 1,4,5-trisphosphate (IP), an intracellular chemical signal that binds to the IP receptor (IPR) to release calcium ions (Ca) from the endoplasmic reticulum. In the past 40 years, the wide-ranging functions mediated by IPR and its genetic defects causing a variety of disorders have been unveiled. Recent cryo-electron microscopy and X-ray crystallography have resolved IPR structures and begun to integrate with concurrent functional studies, which can explicate IP-dependent opening of Ca-conducting gates placed ∼90 Å away from IP-binding sites and its regulation by Ca. This review highlights recent research progress on the IPR structure and function. We also propose how protein plasticity within IPR, which involves allosteric gating and assembly transformations accompanied by rapid and chronic structural changes, would enable it to regulate diverse functions at cellular microdomains in pathophysiological states.
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http://dx.doi.org/10.1146/annurev-physiol-021119-034433DOI Listing
February 2020

Histamine H receptor on astrocytes and neurons controls distinct aspects of mouse behaviour.

Sci Rep 2019 11 11;9(1):16451. Epub 2019 Nov 11.

Department of Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan.

Histamine is an important neurotransmitter that contributes to various processes, including the sleep-wake cycle, learning, memory, and stress responses. Its actions are mediated through histamine H-H receptors. Gene knockout and pharmacological studies have revealed the importance of H receptors in learning and memory, regulation of aggression, and wakefulness. H receptors are abundantly expressed on neurons and astrocytes. However, to date, studies selectively investigating the roles of neuronal and astrocytic H receptors in behaviour are lacking. We generated novel astrocyte- and neuron-specific conditional knockout (cKO) mice to address this gap in knowledge. cKO mice showed cell-specific reduction of H receptor gene expression. Behavioural assessment revealed significant changes and highlighted the importance of H receptors on both astrocytes and neurons. H receptors on both cell types played a significant role in anxiety. Astrocytic H receptors were involved in regulating aggressive behaviour, circadian rhythms, and quality of wakefulness, but not sleep behaviour. Our results emphasise the roles of neuronal H receptors in recognition memory. In conclusion, this study highlights the novel roles of H receptors on astrocytes and neurons in various brain functions.
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http://dx.doi.org/10.1038/s41598-019-52623-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6848115PMC
November 2019

IP receptor isoforms differently regulate ER-mitochondrial contacts and local calcium transfer.

Nat Commun 2019 08 19;10(1):3726. Epub 2019 Aug 19.

MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA.

Contact sites of endoplasmic reticulum (ER) and mitochondria locally convey calcium signals between the IP receptors (IP3R) and the mitochondrial calcium uniporter, and are central to cell survival. It remains unclear whether IP3Rs also have a structural role in contact formation and whether the different IP3R isoforms have redundant functions. Using an IP3R-deficient cell model rescued with each of the three IP3R isoforms and an array of super-resolution and ultrastructural approaches we demonstrate that IP3Rs are required for maintaining ER-mitochondrial contacts. This role is independent of calcium fluxes. We also show that, while each isoform can support contacts, type 2 IP3R is the most effective in delivering calcium to the mitochondria. Thus, these studies reveal a non-canonical, structural role for the IP3Rs and direct attention towards the type 2 IP3R that was previously neglected in the context of ER-mitochondrial calcium signaling.
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http://dx.doi.org/10.1038/s41467-019-11646-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700175PMC
August 2019

Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na, K-ATPase control of cell adhesion, proliferation, and survival.

FASEB J 2019 09 10;33(9):10193-10206. Epub 2019 Jul 10.

Department of Women's and Children's Health, Karolinska Institutet, Solna, Sweden.

The ion pump Na, K-ATPase (NKA) is a receptor for the cardiotonic steroid ouabain. Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. However, it is controversial whether ouabain-bound NKA is considered a signal transducer. To address this question, we performed a global analysis of protein phosphorylation in COS-7 cells, identifying 2580 regulated phosphorylation events on 1242 proteins upon 10- and 20-min treatment with ouabain. Regulated phosphorylated proteins include the inositol triphosphate receptor and stromal interaction molecule, which are essential for initiating calcium oscillations. Hierarchical clustering revealed that ouabain triggers a structured phosphorylation response that occurs in a well-defined, time-dependent manner and affects specific cellular processes, including cell proliferation and cell-cell junctions. We additionally identify regulation of the phosphorylation of several calcium and calmodulin-dependent protein kinases (CAMKs), including 2 sites of CAMK type II-γ (CAMK2G), a protein known to regulate apoptosis. To verify the significance of this result, CAMK2G was knocked down in primary kidney cells. CAMK2G knockdown impaired ouabain-dependent protection from apoptosis upon treatment with high glucose or serum deprivation. In conclusion, we establish NKA as the coordinator of a broad, tightly regulated phosphorylation response in cells and define CAMK2G as a downstream effector of NKA.-Panizza, E., Zhang, L., Fontana, J. M., Hamada, K., Svensson, D., Akkuratov, E. E., Scott, L., Mikoshiba, K., Brismar, H., Lehtiö, J., Aperia, A. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na, K-ATPase control of cell adhesion, proliferation, and survival.
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http://dx.doi.org/10.1096/fj.201900445RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704450PMC
September 2019

Bcl-2 and IP compete for the ligand-binding domain of IPRs modulating Ca signaling output.

Cell Mol Life Sci 2019 Oct 16;76(19):3843-3859. Epub 2019 Apr 16.

Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Leuven Cancer Institute (LKI), KU Leuven, Campus Gasthuisberg O/N-1 Bus 802, Herestraat 49, 3000, Leuven, Belgium.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca dynamics by directly targeting and inhibiting the IP receptor (IPR), a major intracellular Ca-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IPR activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IPR function provoked by low concentration of agonist or IP, while fails to attenuate against high concentration of agonist or IP. Surprisingly, Bcl-2 remained capable of inhibiting IPR1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380-1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IPR1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP and adenophostin A, ligands of the IPR. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IPR activity at the level of the LBD. This allows for exquisite modulation of Bcl-2's inhibitory properties on IPRs that is tunable to the level of IP signaling in cells.
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http://dx.doi.org/10.1007/s00018-019-03091-8DOI Listing
October 2019

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators.

J Vis Exp 2019 03 22(145). Epub 2019 Mar 22.

Laboratory for Developmental Neurobiology, RIKEN Center for Brain Science.

Calcium ion (Ca) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca signal sources-"Ca influx" from outside the cell and "Ca release" from the intracellular Ca2+ store endoplasmic reticulum (ER)-is considered to underlie the diverse spatio-temporal patterns of Ca signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca imaging method that enables monitoring of the very moment of "Ca influx" and "Ca release". OER-GCaMP6f is a genetically encoded Ca indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.
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http://dx.doi.org/10.3791/59246DOI Listing
March 2019

Dual-FRET imaging of IP and Ca revealed Ca-induced IP production maintains long lasting Ca oscillations in fertilized mouse eggs.

Sci Rep 2019 03 18;9(1):4829. Epub 2019 Mar 18.

Laboratory for Developmental Neurobiology, Center for Brain Sciences, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.

In most species, fertilization induces Ca transients in the egg. In mammals, the Ca rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP generated by PLCζ induces Ca release from the intracellular Ca store through IP receptor, termed IP-induced Ca release. Here, we developed new fluorescent IP sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.047-1.7 μM) than that we developed previously. IRIS-2s employed green fluorescent protein and Halo-protein conjugated with the tetramethylrhodamine ligand as fluorescence resonance energy transfer (FRET) donor and acceptor, respectively. For simultaneous imaging of Ca and IP, using IRIS-2s as the IP sensor, we developed a new single fluorophore Ca sensor protein, DYC3.60. With IRIS-2s and DYC3.60, we found that, right after fertilization, IP concentration ([IP]) starts to increase before the onset of the first Ca wave. [IP] stayed at the elevated level with small peaks followed after Ca spikes through Ca oscillations. We detected delays in the peak of [IP] compared to the peak of each Ca spike, suggesting that Ca-induced regenerative IP production through PLC produces small [IP] rises to maintain [IP] over the basal level, which results in long lasting Ca oscillations in fertilized eggs.
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http://dx.doi.org/10.1038/s41598-019-40931-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423007PMC
March 2019

A novel multi lines analysis tool of Ca dynamics reveals the nonuniformity of Ca propagation.

Cell Calcium 2019 03 4;78:76-80. Epub 2019 Jan 4.

Laboratory for Developmental Neurobiology, Center for Brain Science, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama, Japan. Electronic address:

Extracellular stimuli evoke a robust increase in the concentration of intracellular Ca ([Ca]) throughout the cell to trigger various cellular responses, such as gene expression and apoptosis. This robust expansion of [Ca] is called Ca propagation. To date, it is thought that intracellular second messengers, such as inositol 1,4,5-trisphosphate (IP) and intracellular Ca, and clusters of IP receptors (IPRs) regulate Ca propagation. However, little is known about how the elevation in the [Ca] spreads throughout the cell, especially in non-polar cell, including HeLa cell. In this study, we developed a novel multi lines analysis tool. This tool revealed that the velocity of Ca propagation was inconstant throughout cell and local concentration of intracellular Ca did not contribute to the velocity of Ca propagation. Our results suggest that intracellular Ca propagation is not merely the result of diffusion of intracellular Ca, and that, on the contrary, intracellular Ca propagation seems to be regulated by more complicated processes.
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http://dx.doi.org/10.1016/j.ceca.2019.01.001DOI Listing
March 2019

Type 2 inositol 1,4,5-trisphosphate receptor inhibits the progression of pulmonary arterial hypertension via calcium signaling and apoptosis.

Heart Vessels 2019 Apr 20;34(4):724-734. Epub 2018 Nov 20.

Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.

Pulmonary arterial hypertension (PAH) is a progressive disease associated with vasoconstriction and remodeling. Intracellular Ca signaling regulates the contraction of pulmonary arteries and the proliferation of pulmonary arterial smooth muscle cells (PASMCs); however, it is not clear which molecules related to Ca signaling contribute to the progression of PAH. In this study, we found the specific expression of type 2 inositol 1,4,5-trisphosphate receptor (IPR2), which is an intracellular Ca release channel, on the sarco/endoplasmic reticulum in mouse PASMCs, and demonstrated its inhibitory role in the progression of PAH using a chronic hypoxia-induced PAH mouse model. After chronic hypoxia exposure, IPR2 mice exhibited the significant aggravation of PAH, as determined by echocardiography and right ventricular hypertrophy, with significantly greater medial wall thickness by immunohistochemistry than that of wild-type mice. In IPR2 murine PASMCs with chronic hypoxia, a TUNEL assay revealed the significant suppression of apoptosis, whereas there was no significant change in proliferation. Thapsigargin-induced store-operated Ca entry (SOCE) was significantly enhanced in IPR2 PASMCs in both normoxia and hypoxia based on in vitro fluorescent Ca imaging. Furthermore, the enhancement of SOCE in IPR2 PASMCs was remarkably suppressed by the addition of DPB162-AE, an inhibitor of the stromal-interacting molecule (STIM)-Orai complex which is about 100 times more potent than 2-APB. Our results indicate that IPR2 may inhibit the progression of PAH by promoting apoptosis and inhibiting SOCE via the STIM-Orai pathway in PASMCs. These findings suggest a previously undetermined role of IPR in the development of PAH and may contribute to the development of targeted therapies.
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http://dx.doi.org/10.1007/s00380-018-1304-4DOI Listing
April 2019

Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington's Disease.

Front Cell Neurosci 2018 26;12:381. Epub 2018 Oct 26.

Laboratory of Neurodegeneration, International Institute of Molecular and Cell Biology in Warsaw (IIMCB), Warsaw, Poland.

Huntington's disease (HD) is a hereditary neurodegenerative disease that is caused by polyglutamine expansion within the huntingtin (HTT) gene. One of the cellular activities that is dysregulated in HD is store-operated calcium entry (SOCE), a process by which Ca release from the endoplasmic reticulum (ER) induces Ca influx from the extracellular space. HTT-associated protein-1 (HAP1) is a binding partner of HTT. The aim of the present study was to examine the role of HAP1A protein in regulating SOCE in YAC128 mice, a transgenic model of HD. After Ca depletion from the ER by the activation of inositol-(1,4,5)triphosphate receptor type 1 (IPR1), we detected an increase in the activity of SOC channels when HAP1 protein isoform HAP1A was overexpressed in medium spiny neurons (MSNs) from YAC128 mice. A decrease in the activity of SOC channels in YAC128 MSNs was observed when HAP1 protein was silenced. In YAC128 MSNs that overexpressed HAP1A, an increase in activity of IPR1 was detected while the ionomycin-sensitive ER Ca pool decreased. 6-Bromo--(2-phenylethyl)-2,3,4,9-tetrahydro-1-carbazol-1-amine hydrochloride (CHBrClN), identified in our previous studies as a SOCE inhibitor, restored the elevation of SOCE in YAC128 MSN cultures that overexpressed HAP1A. The IP sponge also restored the elevation of SOCE and increased the release of Ca from the ER in YAC128 MSN cultures that overexpressed HAP1A. The overexpression of HAP1A in the human neuroblastoma cell line SK-N-SH (i.e., a cellular model of HD (SK-N-SH HTT138Q)) led to the appearance of a pool of constitutively active SOC channels and an increase in the expression of STIM2 protein. Our results showed that HAP1A causes the activation of SOC channels in HD models by affecting IPR1 activity.
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http://dx.doi.org/10.3389/fncel.2018.00381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6231533PMC
October 2018
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