Language：English   Publishing type：Research paper (scientific journal)  

Non-coding cis-regulatory elements are essential determinants of development, but their exact impacts on behavior and physiology in adults remain elusive. Cis-element-based transcriptional regulation is believed to be crucial for generating circadian rhythms in behavior and physiology. However, genetic evidence supporting this model is based on mutations in the protein-coding sequences of clock genes. Here, we report generation of mutant mice carrying a mutation only at the E'-box cis-element in the promoter region of the core clock gene Per2. The Per2 E'-box mutation abolishes sustainable molecular clock oscillations and renders circadian locomotor activity and body temperature rhythms unstable. Without the E'-box, Per2 messenger RNA and protein expression remain at mid-to-high levels. Our work delineates the Per2 E'-box as a critical nodal element for keeping sustainable cell-autonomous circadian oscillation and reveals the extent of the impact of the non-coding cis-element in daily maintenance of animal locomotor activity and body temperature rhythmicity.

DOI： 10.1038/s41467-019-10532-2

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To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.

DOI： 10.1016/j.cell.2019.04.007

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Circuit refinement during postnatal development is finely regulated by neuron-neuron interactions. Recent studies suggest participation of microglia in this process but it is unclear how microglia cooperatively act with neuronal mechanisms. To examine roles of microglia, we ablate microglia by microglia-selective deletion of colony-stimulating factor 1 receptor (Csf1r) by crossing floxed-Csf1r and Iba1-iCre mice (Csf1r-cKO). In Csf1r-cKO mice, refinement of climbing fiber (CF) to Purkinje cell (PC) innervation after postnatal day 10 (P10)-P12 is severely impaired. However, there is no clear morphological evidence suggesting massive engulfment of CFs by microglia. In Csf1r-cKO mice, inhibitory synaptic transmission is impaired and CF elimination is restored by diazepam, which suggests that impairment of CF elimination is caused by a defect of GABAergic inhibition on PCs, a prerequisite for CF elimination. These results indicate that microglia primarily promote GABAergic inhibition and secondarily facilitate the mechanism for CF elimination inherent in PCs.

DOI： 10.1038/s41467-018-05100-z

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Language：English   Publishing type：Research paper (scientific journal)  

The idea of utilizing unused oocytes present in the ovaries has been tested in various ways to produce offspring. However, only a limited number of studies succeeded in offspring generation. They include transplantation of ovaries into autologous or allogeneic animals, and acquisition of pups from oocytes obtained by transplanting mouse ovaries into immunodeficient rats. Here we report successful production of rat oocytes by transplanting rat ovaries under the kidney capsule of immunodeficient mice with addition of hormone administration to the mice. In addition, these oocytes were developed by in vitro fertilization, and transplanted into the oviducts of pseudopregnant rats, resulting in successful delivery of pups. The modified gene of the donor rat was confirmed to be correctly inherited to the pups. These results show that xenotransplantation of ovarian tissue makes it possible to leave offspring, beginning a new phase in developmental engineering.

DOI： 10.1038/s41598-024-71030-0

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Language：English   Publishing type：Research paper (scientific journal)  

Metabolic dysfunction-associated steatohepatitis (MASH, previously termed non-alcoholic steatohepatitis (NASH)), is a major complication of obesity that promotes fatty liver disease. MASH is characterized by progressive tissue fibrosis and sterile liver inflammation that can lead to liver cirrhosis, cancer, and death. The molecular mechanisms of fibrosis in MASH and its systemic control remain poorly understood. Here, we identified the secreted-type pro-fibrotic protein, procollagen C-endopeptidase enhancer-1 (PCPE-1), as a brown adipose tissue (BAT)-derived adipokine that promotes liver fibrosis in a murine obesity-induced MASH model. BAT-specific or systemic PCPE-1 depletion in mice ameliorated liver fibrosis, whereas, PCPE-1 gain of function in BAT enhanced hepatic fibrosis. High-calorie diet-induced ER stress increased PCPE-1 production in BAT through the activation of IRE-1/JNK/c-Fos/c-Jun signaling. Circulating PCPE-1 levels are increased in the plasma of MASH patients, suggesting a therapeutic possibility. In sum, our results uncover PCPE-1 as a novel systemic control factor of liver fibrosis.

DOI： 10.1038/s44318-024-00196-0

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Developmental synapse elimination is crucial for shaping mature neural circuits. In the neonatal mouse cerebellum, Purkinje cells (PCs) receive excitatory synaptic inputs from multiple climbing fibers (CFs) and synapses from all but one CF are eliminated by around postnatal day 20. Heterosynaptic interaction between CFs and parallel fibers (PFs), the axons of cerebellar granule cells (GCs) forming excitatory synapses onto PCs and molecular layer interneurons (MLIs), is crucial for CF synapse elimination. However, mechanisms for this heterosynaptic interaction are largely unknown. Here we show that deletion of AMPA-type glutamate receptor functions in GCs impairs CF synapse elimination mediated by metabotropic glutamate receptor 1 (mGlu1) signaling in PCs. Furthermore, CF synapse elimination is impaired by deleting NMDA-type glutamate receptors from MLIs. We propose that PF activity is crucial for CF synapse elimination by directly activating mGlu1 in PCs and indirectly enhancing the inhibition of PCs through activating NMDA receptors in MLIs.

DOI： 10.1038/s42003-024-06447-4

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Proper regulation of N-methyl-D-aspartate-type glutamate receptor (NMDA receptor) expression is responsible for excitatory synaptic functions in the mammalian brain. NMDA receptor dysfunction can cause various neuropsychiatric disorders and neurodegenerative diseases. Posttranslational protein S-palmitoylation, the covalent attachment of palmitic acid to intracellular cysteine residues via thioester bonds, occurs in the carboxyl terminus of GluN2B, which is the major regulatory NMDA receptor subunit. Mutations of three palmitoylatable cysteine residues in the membrane-proximal cluster of GluN2B to non-palmitoylatable serine (3CS) lead to the dephosphorylation of GluN2B Tyr1472 in the hippocampus and cerebral cortex, inducing a reduction in the surface expression of GluN2B-containig NMDA receptors. Furthermore, adult GluN2B 3CS homozygous mice demonstrated a definite clasping response without abnormalities in the gross brain structure, other neurological reflexes, or expression levels of synaptic proteins in the cerebrum. This behavioral disorder, observed in the GluN2B 3CS knock-in mice, indicated that complex higher brain functions are coordinated through the palmitoylation-dependent regulation of NMDA receptors in excitatory synapses.

DOI： 10.1007/s12035-024-04166-9

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Kainate receptors (KARs) are one of the ionotropic glutamate receptors in the central nervous system (CNS) comprised of five subunits, GluK1-GluK5. There is a growing interest in the association between KARs and psychiatric disorders, and there have been several studies investigating the behavioral phenotypes of KAR deficient mice, however, the difference in the genetic background has been found to affect phenotype in multiple mouse models of human diseases. Here, we examined GluK1-5 single KO mice in a pure C57BL/6N background and identified that GluK3 KO mice specifically express anxiolytic-like behavior with an alteration in dopamine D2 receptor (D2R)-induced anxiety, and reduced D2R expression in the striatum. Biochemical studies in the mouse cortex confirmed that GluK3 subunits do not assemble with GluK4 and GluK5 subunits, that can be activated by lower concentration of agonists. Overall, we found that GluK3-containing KARs function to express anxiety, which may represent promising anti-anxiety medication targets.

DOI： 10.1038/s41598-024-55063-z

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Brain-enriched guanylate kinase-associated protein (BEGAIN) is highly enriched in the post-synaptic density (PSD) fraction and was identified in our previous study as a protein associated with neuropathic pain in the spinal dorsal horn. PSD protein complexes containing N-methyl-D-aspartate receptors are known to be involved in neuropathic pain. Since these PSD proteins also participate in learning and memory, BEGAIN is also expected to play a crucial role in this behavior. To verify this, we first examined the distribution of BEGAIN in the brain. We found that BEGAIN was widely distributed in the brain and highly expressed in the dendritic regions of the hippocampus. Moreover, we found that BEGAIN was concentrated in the PSD fraction of the hippocampus. Furthermore, immunoelectron microscopy confirmed that BEGAIN was localized at the asymmetric synapses. Behavioral tests were performed using BEGAIN-knockout (KO) mice to determine the contribution of BEGAIN toward learning and memory. Spatial reference memory and reversal learning in the Barns circular maze test along with contextual fear and cued fear memory in the contextual and cued fear conditioning test were significantly impaired in BEGAIN-KO mice compared to with those in wild-type mice. Thus, this study reveals that BEGAIN is a component of the post-synaptic compartment of excitatory synapses involved in learning and memory.

DOI： 10.1038/s41598-023-49537-9

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

The common marmoset (Callithrix jacchus) has attracted attention as a valuable primate model for the analysis of human diseases. Despite the potential for primate genetic modification, however, its widespread lab usage has been limited due to the requirement for a large number of eggs. To make up for traditional oocyte retrieval methods such as hormone administration and surgical techniques, we carried out an alternative approach by utilizing ovarian tissue from deceased marmosets that had been disposed of. This ovarian tissue contains oocytes and can be used as a valuable source of follicles and oocytes. In this approach, the ovarian tissue sections were transplanted under the renal capsules of immunodeficient mice first. Subsequent steps consist of development of follicles by hormone administrations, induction of oocyte maturation and fertilization, and culture of the embryo. This method was first established with rat ovaries, then applied to marmoset ovaries, ultimately resulting in the successful acquisition of the late-stage marmoset embryos. This approach has the potential to contribute to advancements in genetic modification research and disease modeling through the use of primate models, promoting biotechnology with non-human primates and the 3Rs principle in animal experimentation.

DOI： 10.1038/s41598-023-45224-x

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Other Link： https://www.nature.com/articles/s41598-023-45224-x

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The circadian clock is a biological timekeeping system that oscillates with a circa-24-h period, reset by environmental timing cues, especially light, to the 24-h day-night cycle. In mammals, a "central" clock in the hypothalamic suprachiasmatic nucleus (SCN) synchronizes "peripheral" clocks throughout the body to regulate behavior, metabolism, and physiology. A key feature of the clock's oscillation is resistance to abrupt perturbations, but the mechanisms underlying such robustness are not well understood. Here, we probe clock robustness to unexpected photic perturbation by measuring the speed of reentrainment of the murine locomotor rhythm after an abrupt advance of the light-dark cycle. Using an intersectional genetic approach, we implicate a critical role for arginine vasopressin pathways, both central within the SCN and peripheral from the anterior pituitary.

DOI： 10.1073/pnas.2308489120

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Fatty acids have long been considered essential to brain development; however, the involvement of their synthesis in nervous system formation is unclear. We generate mice with knockout of GPSN2, an enzyme for synthesis of very-long-chain fatty acids (VLCFAs) and investigate the effects. Both GPSN2-/- and GPSN2+/- mice show abnormal neuronal networks as a result of impaired neuronal polarity determination. Lipidomics of GPSN2-/- embryos reveal that ceramide synthesis is specifically inhibited depending on FA length; namely, VLCFA-containing ceramide is reduced. We demonstrate that lipid rafts are highly enriched in growth cones and that GPSN2+/- neurons lose gangliosides in their membranes. Application of C24:0 ceramide, but not C16:0 ceramide or C24:0 phosphatidylcholine, to GPSN2+/- neurons rescues both neuronal polarity determination and lipid-raft density in the growth cone. Taken together, our results indicate that VLCFA synthesis contributes to physiological neuronal development in brain network formation, in particular neuronal polarity determination through the formation of lipid rafts.

DOI： 10.1016/j.celrep.2023.113195

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Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

The spinal dorsal horn comprises heterogeneous neuronal populations, that interconnect with one another to form neural circuits modulating various types of sensory information. Decades of evidence has revealed that transcription factors expressed in each neuronal progenitor subclass play pivotal roles in the cell fate specification of spinal dorsal horn neurons. However, the development of subtypes of these neurons is not fully understood in more detail as yet and warrants the investigation of additional transcription factors. In the present study, we examined the involvement of the POU domain-containing transcription factor Brn3a in the development of spinal dorsal horn neurons. Analyses of Brn3a expression in the developing spinal dorsal horn neurons in mice demonstrated that the majority of the Brn3a-lineage neurons ceased Brn3a expression during embryonic stages (Brn3a-transient neurons), whereas a limited population of them continued to express Brn3a at high levels after E18.5 (Brn3a-persistent neurons). Loss of Brn3a disrupted the localization pattern of Brn3a-persistent neurons, indicating a critical role of this transcription factor in the development of these neurons. In contrast, Brn3a overexpression in Brn3a-transient neurons directed their localization in a manner similar to that in Brn3a-persistent neurons. Moreover, Brn3a-overexpressing neurons exhibited increased axonal extension to the ventral and ventrolateral funiculi, where the axonal tracts of Brn3a-persistent neurons reside. These results suggest that Brn3a controls the soma localization and axonal extension patterns of Brn3a-persistent spinal dorsal horn neurons.

DOI： 10.1371/journal.pone.0285295

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Although there is a substantial amount of data on the clinical characteristics, diagnostic criteria, and pathogenesis of myelin oligodendrocyte glycoprotein (MOG) autoantibody-associated disease (MOGAD), there is still uncertainty regarding the MOG protein function and the pathogenicity of anti-MOG autoantibodies in this disease. It is important to note that the disease characteristics, immunopathology, and treatment response of MOGAD patients differ from those of anti-aquaporin 4 antibody-positive neuromyelitis optica spectrum disorders (NMOSDs) and multiple sclerosis (MS). The clinical phenotypes of MOGAD are varied and can include acute disseminated encephalomyelitis, transverse myelitis, cerebral cortical encephalitis, brainstem or cerebellar symptoms, and optic neuritis. The frequency of optic neuritis suggests that the optic nerve is the most vulnerable lesion in MOGAD. During the acute stage, the optic nerve shows significant swelling with severe visual symptoms, and an MRI of the optic nerve and brain lesion tends to show an edematous appearance. These features can be alleviated with early extensive immune therapy, which may suggest that the initial attack of anti-MOG autoantibodies could target the structures on the blood–brain barrier or vessel membrane before reaching MOG protein on myelin or oligodendrocytes. To understand the pathogenesis of MOGAD, proper animal models are crucial. However, anti-MOG autoantibodies isolated from patients with MOGAD do not recognize mouse MOG efficiently. Several studies have identified two MOG epitopes that exhibit strong affinity with human anti-MOG autoantibodies, particularly those isolated from patients with the optic neuritis phenotype. Nonetheless, the relations between epitopes on MOG protein remain unclear and need to be identified in the future.

DOI： 10.3390/ijms241713368

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Publishing type：Research paper (scientific journal)   Publisher：The Endocrine Society  

Abstract

Patients with secondary adrenal insufficiency can present with impaired free water excretion and hyponatremia, which is due to the enhanced secretion of vasopressin (AVP) despite increased total body water. AVP is produced in magnocellular neurons in the paraventricular nucleus of the hypothalamus (PVH) and supraoptic nucleus and in parvocellular corticotropin-releasing factor (CRF) neurons in the PVH. This study aimed to elucidate whether magnocellular AVP neurons or parvocellular CRF neurons coexpressing AVP are responsible for the pathogenesis of hyponatremia in secondary adrenal insufficiency. The number of CRF neurons expressing copeptin, an AVP gene product, was significantly higher in adrenalectomized AVP-floxed mice (AVPfl/fl) than in sham-operated controls. Adrenalectomized AVPfl/fl mice supplemented with aldosterone showed impaired water diuresis under ad libitum access to water or after acute water loading. They became hyponatremic after acute water loading, and it was revealed under such conditions that aquaporin-2 (AQP2) protein levels were increased in the kidney. Furthermore, translocation of AQP2 to the apical membrane was markedly enhanced in renal collecting duct epithelial cells. Remarkably, all these abnormalities observed in the mouse model for secondary adrenal insufficiency were ameliorated in CRF-AVP−/− mice that lacked AVP in CRF neurons. Our study demonstrates that CRF neurons in the PVH are responsible for the pathogenesis of impaired water excretion in secondary adrenal insufficiency.

DOI： 10.1210/endocr/bqad109

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Other Link： https://academic.oup.com/endo/article-pdf/164/8/bqad109/50976215/bqad109.pdf

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NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.

DOI： 10.15252/embj.2022113349

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Language：Japanese   Publisher：(公社)日本精神神経学会  

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The cytomatrix at the active zone-associated structural protein (CAST) and its homologue, named ELKS, being rich in glutamate (E), leucine (L), lysine (K), and serine (S), belong to a family of proteins that organize presynaptic active zones at nerve terminals. These proteins interact with other active zone proteins, including RIMs, Munc13s, Bassoon, and the β subunit of Ca2+ channels, and have various roles in neurotransmitter release. A previous study showed that depletion of CAST/ELKS in the retina causes morphological changes and functional impairment of this structure. In this study, we investigated the roles of CAST and ELKS in ectopic synapse localization. We found that the involvement of these proteins in ribbon synapse distribution is complex. Unexpectedly, CAST and ELKS, in photoreceptors or in horizontal cells, did not play a major role in ribbon synapse ectopic localization. However, depletion of CAST and ELKS in the mature retina resulted in degeneration of the photoreceptors. These findings suggest that CAST and ELKS play critical roles in maintaining neural signal transduction in the retina, but the regulation of photoreceptor triad synapse distribution is not solely dependent on their actions within photoreceptors and horizontal cells.

DOI： 10.3390/ijms24087251

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NMDA-type glutamate receptors (NMDARs) are tetrameric channel complex composed of two subunits of GluN1, which is encoded by a single gene and diversified by alternative splicing, and two subunits from four subtypes of GluN2, leading to various combinations of subunits and channel specificities. However, there is no comprehensive quantitative analysis of GluN subunit proteins for relative comparison, and their compositional ratios at various regions and developmental stages have not been clarified. Here we prepared six chimeric subunits, by fusing an N-terminal side of the GluA1 subunit with a C-terminal side of each of two splicing isoforms of GluN1 subunit and four GluN2 subunits, with which titers of respective NMDAR subunit antibodies could be standardized using common GluA1 antibody, thus enabling quantification of relative protein levels of each NMDAR subunit by western blotting. We determined relative protein amounts of NMDAR subunits in crude, membrane (P2) and microsomal fractions prepared from the cerebral cortex, hippocampus and cerebellum in adult mice. We also examined amount changes in the three brain regions during developmental stages. Their relative amounts in the cortical crude fraction were almost parallel to those of mRNA expression, except for some subunits. Interestingly, a considerable amount of GluN2D protein existed in adult brains, although its transcription level declines after early postnatal stages. GluN1 was larger in quantity than GluN2 in the crude fraction, whereas GluN2 increased in the membrane component-enriched P2 fraction, except in the cerebellum. These data will provide the basic spatio-temporal information on the amount and composition of NMDARs.

DOI： 10.1016/j.neuint.2023.105517

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

Optogenetics is a powerful tool for manipulating neuronal activity by light illumination with high temporal and spatial resolution. Anion-channelrhodopsins (ACRs) are light-gated anion channels that allow researchers to efficiently inhibit neuronal activity. A blue light-sensitive ACR2 has recently been used in several in vivo studies; however, the reporter mouse strain expressing ACR2 has not yet been reported. Here, we generated a new reporter mouse strain, LSL-ACR2, in which ACR2 is expressed under the control of Cre recombinase. We crossed this strain with a noradrenergic neuron-specific driver mouse (NAT-Cre) to generate NAT-ACR2 mice. We confirmed Cre-dependent expression and function of ACR2 in the targeted neurons by immunohistochemistry and electrophysiological recordings in vitro, and confirmed physiological function using an in vivo behavioral experiment. Our results show that the LSL-ACR2 mouse strain can be applied for optogenetic inhibition of targeted neurons, particularly for long-lasting continuous inhibition, upon crossing with Cre-driver mouse strains. The LSL-ACR2 strain can be used to prepare transgenic mice with homogenous expression of ACR2 in targeted neurons with a high penetration ratio, good reproducibility, and no tissue invasion.

DOI： 10.1038/s41598-023-30907-2

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Other Link： https://www.nature.com/articles/s41598-023-30907-2

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Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.

DOI： 10.1038/s41467-022-35501-0

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Nna1/CCP1 is generally known as a causative gene for a spontaneous autosomal recessive mouse mutation, Purkinje cell degeneration (pcd). There is enough evidence that the cytosolic function of the zinc carboxypeptidase (CP) domain at the C-terminus of the Nna1 protein is associated with cell death. On the other hand, this molecule’s two nuclear localization signals (NLSs) suggest some other functions exist. We generated exon 3-deficient mice (Nna1N KO), which encode a portion of the N-terminal NLS. Despite the frameshift occurring in these mice, there was an expression of the Nna1 protein lacking the N-terminal side. Surprisingly, the pcd phenotype did not occur in the Nna1N KO mouse. Behavioral analysis revealed that they were less anxious when assessed by the elevated plus maze and the light/dark box tests compared to the control. Furthermore, they showed impairments in context-dependent and sound stimulus-dependent learning. Biochemical analysis of Nna1N KO mice revealed a reduced level of the AMPA-type glutamine receptor GluA2 in the hippocampal synaptosomal fraction. In addition, the motor protein kinesin-1, which transports GluA2 to dendrites, was also decreased. These results indicate that Nna1 is also involved in emotion and memory learning, presumably through the trafficking and expression of synaptic signaling molecules, besides a known role in cell survival.

DOI： 10.3390/ijms232112961

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Receptor transporter protein 4 (RTP4), one of the receptor chaperone proteins, contributes to the maturation and membrane trafficking of opioid receptor heteromers consisting of mu (MOPr) and delta (DOPr) opioid receptors (MOPr-DOPr). Although MOPr-DOPr is known to mediate the development of morphine tolerance, the extent to which RTP4 plays a role in this process has not been elucidated. Given that RTP4 can be upregulated by repeated administration of morphine, especially in the hypothalamus, here we investigated the effect of hypothalamus-selective ablation of RTP4 on the development of antinociceptive tolerance to morphine. In this study, we generated RTP4flox mice and selectively knocked-out RTP4 using local injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the hypothalamus. The AAV-Cre injection partially, but significantly, decreased the level of RTP4 expression, and suppressed the development of antinociceptive tolerance to morphine. Next, we examined the mechanism of regulation of RTP4 and found that, in neuronal cells, Rtp4 induction is via Gi and MAPK activation, while, in microglial cells, the induction is via Toll-like receptor 4. Together, these studies highlight the role of MOR activity in regulating RTP4, which, in turn, plays an important role in modulating morphine effects in vivo.

DOI： 10.3390/biom12101471

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00125-022-05800-8

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Other Link： https://link.springer.com/article/10.1007/s00125-022-05800-8/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/ijms232112961

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Language：English   Publishing type：Research paper (scientific journal)  

Failure of the right ventricle plays a critical role in any type of heart failure. However, the mechanism remains unclear, and there is no specific therapy. Here, we show that the right ventricle predominantly expresses alternative complement pathway-related genes, including Cfd and C3aR1. Complement 3 (C3)-knockout attenuates right ventricular dysfunction and fibrosis in a mouse model of right ventricular failure. C3a is produced from C3 by the C3 convertase complex, which includes the essential component complement factor D (Cfd). Cfd-knockout mice also show attenuation of right ventricular failure. Moreover, the plasma concentration of CFD correlates with the severity of right ventricular failure in patients with chronic right ventricular failure. A C3a receptor (C3aR) antagonist dramatically improves right ventricular dysfunction in mice. In summary, we demonstrate the crucial role of the C3-Cfd-C3aR axis in right ventricular failure and highlight potential therapeutic targets for right ventricular failure.

DOI： 10.1038/s41467-022-33152-9

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Low body temperature predicts a poor outcome in patients with heart failure, but the underlying pathological mechanisms and implications are largely unknown. Brown adipose tissue (BAT) was initially characterised as a thermogenic organ, and recent studies have suggested it plays a crucial role in maintaining systemic metabolic health. While these reports suggest a potential link between BAT and heart failure, the potential role of BAT dysfunction in heart failure has not been investigated. Here, we demonstrate that alteration of BAT function contributes to development of heart failure through disorientation in choline metabolism. Thoracic aortic constriction (TAC) or myocardial infarction (MI) reduced the thermogenic capacity of BAT in mice, leading to significant reduction of body temperature with cold exposure. BAT became hypoxic with TAC or MI, and hypoxic stress induced apoptosis of brown adipocytes. Enhancement of BAT function improved thermogenesis and cardiac function in TAC mice. Conversely, systolic function was impaired in a mouse model of genetic BAT dysfunction, in association with a low survival rate after TAC. Metabolomic analysis showed that reduced BAT thermogenesis was associated with elevation of plasma trimethylamine N-oxide (TMAO) levels. Administration of TMAO to mice led to significant reduction of phosphocreatine and ATP levels in cardiac tissue via suppression of mitochondrial complex IV activity. Genetic or pharmacological inhibition of flavin-containing monooxygenase reduced the plasma TMAO level in mice, and improved cardiac dysfunction in animals with left ventricular pressure overload. In patients with dilated cardiomyopathy, body temperature was low along with elevation of plasma choline and TMAO levels. These results suggest that maintenance of BAT homeostasis and reducing TMAO production could be potential next-generation therapies for heart failure.

DOI： 10.1038/s41598-022-19245-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Brown adipose tissue (BAT) has a role in maintaining systemic metabolic health in rodents and humans. Here, we show that metabolic stress induces BAT to produce coagulation factors, which then-together with molecules derived from the circulation-promote BAT dysfunction and systemic glucose intolerance. When mice were fed a high-fat diet (HFD), the levels of tissue factor, coagulation Factor VII (FVII), activated coagulation Factor X (FXa), and protease-activated receptor 1 (PAR1) expression increased significantly in BAT. Genetic or pharmacological suppression of coagulation factor-PAR1 signaling in BAT ameliorated its whitening and improved thermogenic response and systemic glucose intolerance in mice with dietary obesity. Conversely, the activation of coagulation factor-PAR1 signaling in BAT caused mitochondrial dysfunction in brown adipocytes and systemic glucose intolerance in mice fed normal chow. These results indicate that BAT produces endogenous coagulation factors that mediate pleiotropic effects via PAR1 signaling under metabolic stress.

DOI： 10.1016/j.isci.2022.104547

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Long-lasting fear-related disorders depend on the excessive retention of traumatic fear memory. We previously showed that the palmitoylation-dependent removal of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors prevents hyperexcitation-based epileptic seizures and that AMPA receptor palmitoylation maintains neural network stability. In this study, AMPA receptor subunit GluA1 C-terminal palmitoylation-deficient (GluA1C811S) mice were subjected to comprehensive behavioral battery tests to further examine whether the mutation causes other neuropsychiatric disease-like symptoms. The behavioral analyses revealed that palmitoylation-deficiency in GluA1 is responsible for characteristic prolonged contextual fear memory formation, whereas GluA1C811S mice showed no impairment of anxiety-like behaviors at the basal state. In addition, fear generalization gradually increased in these mutant mice without affecting their cued fear. Furthermore, fear extinction training by repeated exposure of mice to conditioned stimuli had little effect on GluA1C811S mice, which is in line with augmentation of synaptic transmission in pyramidal neurons in the basolateral amygdala. In contrast, locomotion, sociability, depression-related behaviors, and spatial learning and memory were unaffected by the GluA1 non-palmitoylation mutation. These results indicate that impairment of AMPA receptor palmitoylation specifically causes posttraumatic stress disorder (PTSD)-like symptoms.

DOI： 10.1038/s41386-022-01347-9

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Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.

DOI： 10.1042/BCJ20220033

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Language：Japanese   Publisher：(公社)日本精神神経学会  

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.crmeth.2022.100168

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A germ line copy number duplication of chromosome 14q32, which contains ATG2B and GSKIP, was identified in families with myeloproliferative neoplasm (MPN). Here, we show that mice lacking both Atg2b and Gskip, but not either alone, exhibited decreased hematopoiesis, resulting in death in utero accompanied by anemia. In marked contrast to MPN patients with duplication of ATG2B and GSKIP, the number of hematopoietic stem cells (HSCs), in particular long-term HSCs, in double-knockout fetal livers was significantly decreased due to increased cell death. Although the remaining HSCs still had the ability to differentiate into hematopoietic progenitor cells, the differentiation efficiency was quite low. Remarkably, mice with knockout of Atg2b or Gskip alone did not show any hematopoietic abnormality. Mechanistically, while loss of both genes had no effect on autophagy, it increased the expression of genes encoding enzymes involved in oxidative phosphorylation. Taken together, our results indicate that Atg2b and Gskip play a synergistic effect in maintaining the pool size of HSCs.

DOI： 10.1128/MCB.00024-21

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>Olig2 is indispensable for motoneuron and oligodendrocyte fate-specification in the pMN domain of embryonic spinal cords, and also involved in the proliferation and differentiation of several cell types in the nervous system, including neural progenitor cells (NPCs) and oligodendrocytes. However, how Olig2 controls these diverse biological processes remains unclear. Here, we demonstrated that a novel Olig2-binding protein, DEAD-box helicase 20 (Ddx20), is indispensable for the survival of NPCs and oligodendrocyte progenitor cells (OPCs). A central nervous system (CNS)-specific <italic>Ddx20</italic> conditional knockout (cKO) demonstrated apoptosis and cell cycle arrest in NPCs and OPCs, through the potentiation of the p53 pathway in DNA damage-dependent and independent manners, including SMN complex disruption and the abnormal splicing of <italic>Mdm2</italic> mRNA. Analyzes of <italic>Olig2</italic> null NPCs showed that Olig2 contributed to NPC proliferation through Ddx20 protein stabilization. Our findings provide novel mechanisms underlying the Olig2-mediated proliferation of NPCs, via the Ddx20-p53 axis, in the embryonic CNS.

DOI： 10.1038/s41418-021-00915-8

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Other Link： https://www.nature.com/articles/s41418-021-00915-8

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We earlier reported female-biased, sex-specific involvement of the dorsolateral bed nucleus of the stria terminalis (dl BST) in the formalin-induced pain response in rats. The present study investigated pain effects on mice behaviors. Because the dl BST is densely populated with corticotropin-releasing hormone (CRH) neurons, we examined sex differences in these parameters for the dl BST CRH neurons in male and female mice of a mouse line for which the CRH gene promoter (corticotropin-releasing factor [CRF]-Venus ΔNeo) controls the expression of the modified yellow fluorescent protein (Venus). Approximately 92% of Venus-positive cells in the dl BST were also CRH mRNA-positive, irrespective of sex. Therefore, the cells identified using Venus fluorescence were regarded as CRH neurons. A female-biased sex difference was observed in pain-induced behaviors during the interphase (5-15 min after formalin injection) but not during the later phase (phase 2, 15-60 min) in wild-type mice. In CRF-Venus ΔNeo mice, a female-biased difference was observed in either the earlier phase (phase 1, 0-5 min) or the interphase, but not in phase 2. Patch-clamp recordings taken using an acute BST slice obtained from a CRF-Venus ΔNeo mouse after formalin injection showed miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs). Remarkably, the mEPSCs frequency was higher in the Venus-expressing cells of formalin-injected female mice than in vehicle-treated female mice. Male mice showed no increase in mEPSC frequency by formalin injection. Formalin injection had no effect on mEPSC or mIPSC amplitudes in either sex. Pain-induced changes in mEPSC frequency in putative CRH neurons were phase-dependent. Results show that excitatory synaptic inputs to BST CRH neurons are temporally enhanced along with behavioral sex differences in pain response, suggesting that pain signals alter the BST CRH neurons excitability in a sex-dependent manner.

DOI： 10.1016/j.brainres.2021.147688

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.celrep.2021.110107

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Experience-dependent plasticity is a key feature of brain synapses for which neuronal N-Methyl-D-Aspartate receptors (NMDARs) play a major role, from developmental circuit refinement to learning and memory. Astrocytes also express NMDARs, although their exact function has remained controversial. Here, we identify in mouse hippocampus, a circuit function for GluN2C NMDAR, a subtype highly expressed in astrocytes, in layer-specific tuning of synaptic strengths in CA1 pyramidal neurons. Interfering with astrocyte NMDAR or GluN2C NMDAR activity reduces the range of presynaptic strength distribution specifically in the stratum radiatum inputs without an appreciable change in the mean presynaptic strength. Mathematical modeling shows that narrowing of the width of presynaptic release probability distribution compromises the expression of long-term synaptic plasticity. Our findings suggest a novel feedback signaling system that uses astrocyte GluN2C NMDARs to adjust basal synaptic weight distribution of Schaffer collateral inputs, which in turn impacts computations performed by the CA1 pyramidal neuron.

DOI： 10.7554/eLife.70818

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Language：Japanese   Publisher：(公社)日本精神神経学会  

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Male germline development involves choreographed changes to mitochondrial number, morphology and organization. Mitochondrial reorganization during spermatogenesis was recently shown to require mitochondrial fusion and fission. Mitophagy, the autophagic degradation of mitochondria, is another mechanism for controlling mitochondrial number and physiology, but its role during spermatogenesis is largely unknown. During post-meiotic spermatid development, restructuring of the mitochondrial network results in packing of mitochondria into a tight array in the sperm midpiece to fuel motility. Here, we show that disruption of mouse Fis1 in the male germline results in early spermatid arrest that is associated with increased mitochondrial content. Mutant spermatids coalesce into multinucleated giant cells that accumulate mitochondria of aberrant ultrastructure and numerous mitophagic and autophagic intermediates, suggesting a defect in mitophagy. We conclude that Fis1 regulates mitochondrial morphology and turnover to promote spermatid maturation.

DOI： 10.1242/dev.199686

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/glia.24058

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/glia.24058

Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.celrep.2021.109427

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Kainate receptors (KARs) are members of the glutamate receptor family that regulate synaptic function in the brain. Although they are known to be associated with psychiatric disorders, how they are involved in these disorders remains unclear. KARs are tetrameric channels assembled from a combination of GluK1-5 subunits. Among these, GluK2 and GluK5 subunits are the major heteromeric subunits in the brain. To determine the functional similarities and differences between GluK2 and GluK5 subunits, we generated GluK2 KO and GluK5 KO mice on a C57BL/6N background, a well-characterized inbred strain, and compared their behavioral phenotypes. We found that GluK2 KO and GluK5 KO mice exhibited the same phenotypes in many tests, such as reduced locomotor activity, impaired motor function, and enhanced depressive-like behavior. No change was observed in motor learning, anxiety-like behavior, or sociability. Additionally, we identified subunit-specific phenotypes, such as reduced motivation toward their environment in GluK2 KO mice and an enhancement in the contextual memory in GluK5 KO mice. These results revealed that GluK2 and GluK5 subunits not only function in a coordinated manner but also have a subunit-specific role in regulating behavior. To summarize, we demonstrated subunit-specific and common behavioral effects of GluK2 and GluK5 subunits for the first time. Moreover, to the best of our knowledge, this is the first evidence of the involvement of the GluK5 subunit in the expression of depressive-like behavior and contextual memory, which strongly indicates its role in psychiatric disorders.

DOI： 10.1016/j.bbr.2021.113194

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Ca2+-dependent activator protein for secretion 2 (CAPS2) regulates dense-core vesicle (DCV) exocytosis to facilitate peptidergic and catecholaminergic transmitter release. CAPS2 deficiency in mice has mild neuronal effects but markedly impairs social behavior. Rare de novo Caps2 alterations also occur in autism spectrum disorder, although whether CAPS2-mediated release influences social behavior remains unclear. Here, we demonstrate that CAPS2 is associated with DCV exocytosis-mediated release of the social interaction modulatory peptide oxytocin (OXT). CAPS2 is expressed in hypothalamic OXT neurons and localizes to OXT nerve projection and OXT release sites, such as the pituitary. Caps2 KO mice exhibited reduced plasma albeit increased hypothalamic and pituitary OXT levels, indicating insufficient release. OXT neuron-specific Caps2 conditional KO supported CAPS2 function in pituitary OXT release, also affording impaired social interaction and recognition behavior that could be ameliorated by exogenous OXT administered intranasally. Thus, CAPS2 appears critical for OXT release, thereby being associated with social behavior.Significance StatementThe role of the neuropeptide, oxytocin, in enhancing social interaction and social bonding behavior has attracted considerable public and neuroscientific attention. A central issue in oxytocin biology concerns how oxytocin release is regulated. Our study provides an important insight into the understanding of oxytocin-dependent social behavior from the perspective of the CAPS2-regulated release mechanism.

DOI： 10.1523/JNEUROSCI.3240-20.2021

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The semaphorin family is a well-characterized family of secreted or membrane-bound proteins that are involved in activity-independent neurodevelopmental processes, such as axon guidance, cell migration, and immune functions. Although semaphorins have recently been demonstrated to regulate activity-dependent synaptic scaling, their roles in Hebbian synaptic plasticity as well as learning and memory remain poorly understood. Here, using a rodent model, we found that an inhibitory avoidance task, a hippocampus-dependent contextual learning paradigm, increased secretion of semaphorin 3A in the hippocampus. Furthermore, the secreted semaphorin 3A in the hippocampus mediated contextual memory formation likely by driving AMPA receptors into hippocampal synapses via the neuropilin1-plexin A4-semaphorin receptor complex. This signaling process involves alteration of the phosphorylation status of collapsin response mediator protein 2, which has been characterized as a downstream molecule in semaphorin signaling. These findings implicate semaphorin family as a regulator of Hebbian synaptic plasticity and learning.

DOI： 10.1111/ejn.15210

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Sleep is regulated in a homeostatic manner. Sleep deprivation increases sleep need, which is compensated mainly by increased EEG δ power during non-rapid eye movement sleep (NREMS) and, to a lesser extent, by increased sleep amount. Although genetic factors determine the constitutive level of sleep need and sleep amount in mice and humans, the molecular entity behind sleep need remains unknown. Recently, we found that a gain-of-function Sleepy (Slp) mutation in the salt-inducible kinase 3 (Sik3) gene, which produces the mutant SIK3(SLP) protein, leads to an increase in NREMS EEG δ power and sleep amount. Since Sik3Slp mice express SIK3(SLP) in various types of cells in the brain as well as multiple peripheral tissues from the embryonic stage, the cell type and developmental stage responsible for the sleep phenotype in Sik3Slp mice remain to be elucidated. Here, we generated two mouse lines, synapsin1CreERT2 and Sik3ex13flox mice, which enable inducible Cre-mediated, conditional expression of SIK3(SLP) in neurons on tamoxifen administration. Administration of tamoxifen to synapsin1CreERT2 mice during late infancy resulted in higher recombination efficiency than administration during adolescence. SIK3(SLP) expression after late infancy increased NREMS and NREMS δ power in male synapsin1CreERT2; Sik3ex13flox/+ mice. The expression of SIK3(SLP) after adolescence led to a higher NREMS δ power without a significant change in NREMS amounts. Thus, neuron-specific expression of SIK3(SLP) after late infancy is sufficient to increase sleep.SIGNIFICANCE STATEMENT The propensity to accumulate sleep need during wakefulness and to dissipate it during sleep underlies the homeostatic regulation of sleep. However, little is known about the developmental stage and cell types involved in determining the homeostatic regulation of sleep. Here, we show that Sik3Slp allele induction in mature neurons in late infancy is sufficient to increase non-rapid eye movement sleep amount and non-rapid eye movement sleep δ power. SIK3 signaling in neurons constitutes an intracellular mechanism to increase sleep.

DOI： 10.1523/JNEUROSCI.1004-20.2020

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Immature neurons undergo morphological and physiological maturation in order to establish neuronal networks. During neuronal maturation, a large number of genes change their transcriptional levels, and these changes may be mediated by chromatin modifiers. In this study, we found that the level of Ezh1, a component of Polycomb repressive complex 2 (PRC2), increases during neuronal maturation in mouse neocortical culture. In addition, conditional knockout of Ezh1 in post-mitotic excitatory neurons leads to downregulation of a set of genes related to neuronal maturation. Moreover, the locus encoding Cpg15/Neuritin (Nrn1), which is regulated by neuronal activity and implicated in stabilization and maturation of excitatory synapses, is a direct target of Ezh1 in cortical neurons. Together, these results suggest that elevated expression of Ezh1 contributes to maturation of cortical neurons.

DOI： 10.5582/ddt.2021.01017

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During rapid eye movement (REM) sleep, anti-gravity muscle tone and bodily movements are mostly absent, because somatic motoneurons are inhibited by descending inhibitory pathways. Recent studies showed that glycine/GABA neurons in the ventromedial medulla (VMM; GlyVMM neurons) play an important role in generating muscle atonia during REM sleep (REM-atonia). However, how these REM-atonia-inducing neurons interconnect with other neuronal populations has been unknown. In the present study, we first identified a specific subpopulation of GlyVMM neurons that play an important role in induction of REM-atonia by virus vector-mediated tracing in male mice in which glycinergic neurons expressed Cre recombinase. We found these neurons receive direct synaptic input from neurons in several brain stem regions, including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD; GluSLD neurons). Silencing this circuit by specifically expressing tetanus toxin light chain (TeTNLC) resulted in REM sleep without atonia. This manipulation also caused a marked decrease in time spent in cataplexy-like episodes (CLEs) when applied to narcoleptic orexin-ataxin-3 mice. We also showed that GlyVMM neurons play an important role in maintenance of sleep. This present study identified a population of glycinergic neurons in the VMM that are commonly involved in REM-atonia and cataplexy.SIGNIFICANCE STATEMENT We identified a population of glycinergic neurons in the ventral medulla that plays an important role in inducing muscle atonia during rapid eye movement (REM) sleep. It sends axonal projections almost exclusively to motoneurons in the spinal cord and brain stem except to those that innervate extraocular muscles, while other glycinergic neurons in the same region also send projections to other regions including monoaminergic nuclei. Furthermore, these neurons receive direct inputs from several brainstem regions including glutamatergic neurons in the sublaterodorsal tegmental nucleus (SLD). Genetic silencing of this pathway resulted in REM sleep without atonia and a decrease of cataplexy when applied to narcoleptic mice. This work identified a neural population involved in generating muscle atonia during REM sleep and cataplexy.

DOI： 10.1523/JNEUROSCI.0688-20.2020

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AIM: Sphingosine-1-phosphate (S1P) and ceramide are bioactive sphingolipids known to be important in regulating numerous processes involved in cancer progression. The aim of this study was to determine the absolute levels of sphingolipids in hepatocellular carcinoma (HCC) utilizing data obtained from surgical specimens. In addition, we explored the clinical significance of S1P in patients with HCC and the biological role of S1P in HCC cells. METHODS: Tumors and normal liver tissues were collected from 20 patients with HCC, and sphingolipids were measured by mass spectrometry. The Cancer Genome Atlas (TCGA) cohort was utilized to evaluate gene expression of enzymes related to sphingolipid metabolism. Immunohistochemistry of phospho-sphingosine kinase 1 (SphK1), an S1P-producing enzyme, was performed for 61 surgical specimens. CRISPR/Cas9-mediated SphK1 knockout cells were used to examine HCC cell biology. RESULTS: S1P levels were substantially higher in HCC tissue compared with normal liver tissue. Levels of other sphingolipids upstream of S1P in the metabolic cascade, such as sphingomyelin, monohexosylceramide and ceramide, were also considerably higher in HCC tissue. Enzymes involved in generating S1P and its precursor, ceramide, were found in higher levels in HCC compared with normal liver tissue. Immunohistochemical analysis found that phospho-SphK1 expression was associated with tumor size. Finally, in vitro assays indicated that S1P is involved in the aggressiveness of HCC cells. CONCLUSIONS: Sphingolipid levels, including S1P and ceramide, were elevated in HCC compared with surrounding normal liver tissue. Our findings suggest S1P plays an important role in HCC tumor progression, and further examination is warranted.

DOI： 10.1111/hepr.13625

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BACKGROUND: Activin A receptor type 2A (ACVR2A) is one of the most frequently mutated genes in microsatellite instability-high (MSI-H) gastric cancer. However, the clinical relevance of the ACVR2A mutation in MSI-H gastric cancer patients remains unclear. The aims of this study were to explore the effect of ACVR2A mutation on the tumor behavior and to identify the clinicopathological characteristics of gastric cancer patients with ACVR2A mutations. METHODS: An in vitro study was performed to investigate the biological role of ACVR2A via CRISPR/Cas9-mediated ACVR2A knockout MKN74 human gastric cancer cells. One hundred twenty-four patients with gastric cancer were retrospectively analyzed, and relations between MSI status, ACVR2A mutations, and clinicopathological factors were evaluated. RESULTS: ACVR2A knockout cells showed less aggressive tumor biology than mock-transfected cells, displaying reduced proliferation, migration, and invasion (P < 0.05). MSI mutations were found in 10% (13/124) of gastric cancer patients, and ACVR2A mutations were found in 8.1% (10/124) of patients. All ACVR2A mutations were accompanied by MSI. The 5-year overall survival rates of ACVR2A wild-type patients and ACVR2A-mutated patients were 57% and 90%, respectively (P = 0.048). Multivariate analysis revealed that older age (P = 0.015), distant metastasis (P < 0.001), and ACVR2A wild-type status (P = 0.040) were independent prognostic factors for overall survival. CONCLUSIONS: Our study demonstrated that gastric cancer patients with ACVR2A mutation have a significantly better prognosis than those without. Dysfunction of ACVR2A in MKN74 human gastric cancer cells caused less aggressive tumor biology, indicating the importance of ACVR2A in the progression of MSI-H tumors.

DOI： 10.1007/s11605-020-04889-9

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Reduced expression of glutamate decarboxylase 67 (GAD67), encoded by the Gad1 gene, is a consistent finding in postmortem brains of patients with several psychiatric disorders, including schizophrenia, bipolar disorder and major depressive disorder. The dysfunction of GAD67 in the brain is implicated in the pathophysiology of these psychiatric disorders; however, the neurobiological consequences of GAD67 dysfunction in mature brains are not fully understood because the homozygous Gad1 knockout is lethal in newborn mice. We hypothesized that the tetracycline-controlled gene expression/suppression system could be applied to develop global GAD67 knockdown mice that would survive into adulthood. In addition, GAD67 knockdown mice would provide new insights into the neurobiological impact of GAD67 dysfunction. Here, we developed Gad1tTA/STOP-tetO biallelic knock-in mice using Gad1STOP-tetO and Gad1tTA knock-in mice, and compared them with Gad1+/+ mice. The expression level of GAD67 protein in brains of Gad1tTA/STOP-tetO mice treated with doxycycline (Dox) was decreased by approximately 90%. The GABA content was also decreased in the brains of Dox-treated Gad1tTA/STOP-tetO mice. In the open-field test, Dox-treated Gad1tTA/STOP-tetO mice exhibited hyper-locomotor activity and decreased duration spent in the center region. In addition, acoustic startle responses were impaired in Dox-treated Gad1tTA/STOP-tetO mice. These results suggest that global reduction in GAD67 elicits emotional abnormalities in mice. These GAD67 knockdown mice will be useful for elucidating the neurobiological mechanisms of emotional abnormalities, such as anxiety symptoms associated with psychiatric disorders.

DOI： 10.1186/s13041-020-00713-2

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The properties of microglia largely differ depending on aging as well as on brain regions. However, there are few studies that investigated the functional importance of such heterogeneous properties of microglia at the molecular level. Voltage-gated proton channel, Hv1/VSOP, could be one of the candidates which confers functional heterogeneity among microglia since it regulates brain oxidative stress in age-dependent manner. In this study, we found that Hv1/VSOP shows brain region-dependent heterogeneity of gene expression with the highest level in the striatum. We studied the importance of Hv1/VSOP in two different brain regions, the cerebral cortex and striatum, and examined their relationship with aging (using mice of different ages). In the cortex, we observed the age-dependent impact of Hv1/VSOP on oxidative stress, microglial morphology, and gene expression profile. On the other hand, we found that the age-dependent significance of Hv1/VSOP was less obvious in the striatum than the cortex. Finally, we performed a battery of behavioral experiments on Hv1/VSOP-deficient mice both at young and aged stages to examine the effect of aging on Hv1/VSOP function. Hv1/VSOP-deficient mice specifically showed a marked difference in behavior in light/dark transition test only at aged stages, indicating that anxiety state is altered in aged Hv1/VSOP mice. This study suggests that a combination of brain region heterogeneity and animal aging underscores the functional importance of Hv1/VSOP in microglia.

DOI： 10.1111/jnc.15292

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Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.

DOI： 10.1038/s41467-020-20185-1

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KEY POINTS: Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so-called 'resonant properties'). Here, we report that heterologous expression of mouse Kv11 voltage-dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties. Kv11.3-expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor. The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice. This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons. ABSTRACT: The plasma membranes of some neurons preferentially respond to current inputs with a specific frequency, and output as large voltage changes. This property is called resonance, and is thought to be mediated by ion channels that show inductor-like behaviour. However, details of the candidate ion channels remain unclear. In this study, we mainly focused on the functional roles of Kv11 potassium (K+ ) channels, encoded by ether-á-go-go-related genes, in resonance in mouse inferior olivary (IO) neurons. We transfected HEK293 cells with long or short splice variants of Kv11.1 (Merg1a and Merg1b) or Kv11.3, and examined membrane properties using whole-cell recording. Transfection with Kv11 channels reproduced resonance at membrane potentials depolarized from the resting state. Frequency ranges of Kv11.3-, Kv11.1(Merg1b)- and Kv11.1(Merg1a)-expressing cells were 2-6 Hz, 2-4 Hz, and 0.6-0.8 Hz, respectively. Responses of Kv11.3 currents to step voltage changes were essentially similar to those of inductor currents in the resistor-inductor-capacitor circuit. Furthermore, Kv11 transfections generated membrane potential oscillations. We also confirmed the contribution of HCN1 channels as a major mediator of resonance at more hyperpolarized potentials by transfection into HEK293 cells. The Kv11 current kinetics and properties of Kv11-dependent resonance suggested that Kv11.3 mediated resonance in IO neurons. This finding was confirmed by the impairment of resonance and oscillation at -30 to -60 mV in Kcnh7 (Kv11.3) knockout mice. These results suggest that Kv11 channels have important roles in inducing frequency-dependent responses in a subtype-dependent manner from resting to depolarized membrane potentials.

DOI： 10.1113/JP280342

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The lateral habenula (LHb) attracts a growing interest as a regulator of monoaminergic activity which were frequently reported to be defective in depression. Here we found that chronic social defeat stress (CSDS) increased production of pro-inflammatory cytokines in LHb associated with mobilization of monocytes and remodeling of extracellular matrix by increased matrix metalloproteinase (MMP) activity. RNA-seq analysis identified proprotein convertase Pcsk5 as an upstream regulator of MMP activation, with upregulation in LHb neurons of mice with susceptibility to CSDS. PCSK5 facilitated motility of microglia in vitro by converting inactive pro-MMP14 and pro-MMP2 to their active forms, highlighting its role in mobilization of microglia and monocytes in neuroinflammation. Suppression of Pcsk5 expression via small interfering RNA (siRNA) ameliorated depressive-like behaviors and pathological mobilization of monocytes in mice with susceptibility to CSDS. PCSK5-MMPs signaling pathway could be a target for development of the antidepressants targeting the inflammatory response in specific brain regions implicated in depression.

DOI： 10.1038/s41386-020-00843-0

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Synapse formation and regulation require signaling interactions between pre- and postsynaptic proteins, notably cell adhesion molecules (CAMs). It has been proposed that the functions of neuroligins (Nlgns), postsynaptic CAMs, rely on the formation of trans-synaptic complexes with neurexins (Nrxns), presynaptic CAMs. Nlgn3 is a unique Nlgn isoform that localizes at both excitatory and inhibitory synapses. However, Nlgn3 function mediated via Nrxn interactions is unknown. Here we demonstrate that Nlgn3 localizes at postsynaptic sites apposing vesicular glutamate transporter 3-expressing (VGT3+) inhibitory terminals and regulates VGT3+ inhibitory interneuron-mediated synaptic transmission in mouse organotypic slice cultures. Gene expression analysis of interneurons revealed that the αNrxn1+AS4 splice isoform is highly expressed in VGT3+ interneurons as compared with other interneurons. Most importantly, postsynaptic Nlgn3 requires presynaptic αNrxn1+AS4 expressed in VGT3+ interneurons to regulate inhibitory synaptic transmission. Our results indicate that specific Nlgn-Nrxn signaling generates distinct functional properties at synapses.

DOI： 10.7554/eLife.59545

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Recent evidence suggests that the central nervous system (CNS) regulates plasma glucose levels, but the underlying mechanism is unclear. The present study investigated the role of dopaminergic function in the CNS in regulation of plasma glucose levels in mice. I.c.v. injection of neither the dopamine D1 receptor agonist SKF 38393 nor the antagonist SCH 23390 influenced plasma glucose levels. In contrast, i.c.v. injection of both the dopamine D2 receptor agonist quinpirole and the antagonist l-sulpiride increased plasma glucose levels. Hyperglycemia induced by quinpirole and l-sulpiride was absent in dopamine D2 receptor knockout mice. I.c.v. injection of quinpirole and l-sulpiride each increased mRNA levels of hepatic glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, which are the key enzymes for hepatic gluconeogenesis. Systemic injection of the β2 adrenoceptor antagonist ICI 118,551 inhibited hyperglycemia induced by l-sulpiride, but not by quinpirole. In contrast, hyperglycemia induced by quinpirole, but not by l-sulpiride, was inhibited by hepatic vagotomy. These results suggest that stimulation of central dopamine D2 receptors increases plasma glucose level by increasing hepatic glucose production through parasympathetic nerves, whereas inhibition of central dopamine D2 receptors increases plasma glucose level by increasing hepatic glucose production through sympathetic nerves.

DOI： 10.1038/s41598-020-79292-0

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Autism susceptibility candidate 2 (AUTS2), a risk gene for autism spectrum disorders (ASDs), is implicated in telencephalon development. Because AUTS2 is also expressed in the cerebellum where defects have been linked to ASDs, we investigated AUTS2 functions in the cerebellum. AUTS2 is specifically localized in Purkinje cells (PCs) and Golgi cells during postnatal development. Auts2 conditional knockout (cKO) mice exhibited smaller and deformed cerebella containing immature-shaped PCs with reduced expression of Cacna1a. Auts2 cKO and knock-down experiments implicated AUTS2 participation in elimination and translocation of climbing fiber synapses and restriction of parallel fiber synapse numbers. Auts2 cKO mice exhibited behavioral impairments in motor learning and vocal communications. Because Cacna1a is known to regulate synapse development in PCs, it suggests that AUTS2 is required for PC maturation to elicit normal development of PC synapses and thus the impairment of AUTS2 may cause cerebellar dysfunction related to psychiatric illnesses such as ASDs.

DOI： 10.1016/j.isci.2020.101820

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Voltage-gated proton channels (Hv1/VSOP), encoded by Hvcn1, are important regulator of reactive oxygen species (ROS) production in many types of immune cells. While in vitro studies indicate that Hv1/VSOP regulates ROS production by maintaining pH homeostasis, there are few studies investigating the functional importance of Hv1/VSOP in vivo. In the present study, we first show that Hv1/VSOP is functionally expressed in liver resident macrophage, Kupffer cells, regulating the hepatic oxidative stress in vivo. Our immunocytochemistry and electrophysiology data showed that Hvcn1 is specifically expressed in Kupffer cells, but not in hepatocytes. Furthermore, Hvcn1-deficiency drastically altered the hepatic oxidative stress. The Hvcn1-deficient mice showed high blood glucose and serum insulin but normal insulin sensitivity, indicating that these phenotypes were not linked to insulin resistance. Transcriptome analysis indicated that the gene expression of glycogen phosphorylase (Pygl) and Glucose-6-phosphatase, catalytic subunit (G6pc) were upregulated in Hvcn1-deficient liver tissues, and quantitative PCR confirmed the result for Pygl. Furthermore, we observed higher amount of glucose-6-phosphate, a key sugar intermediate for glucose in Hvcn1-deficient liver than WT, suggesting that glucose production in liver is accelerated in Hvcn1-deficient mice. The present study sheds light on the functional importance of Kupffer cells in hepatic oxidative stress and its potential relationship with glucose metabolism.

DOI： 10.1096/fj.202001056RRR

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Dystonin (Dst) is a causative gene for Dystonia musculorum (dt) mice, which is an inherited disorder exhibiting dystonia-like movement and ataxia with sensory degeneration. Dst is expressed in a variety of tissues, including the central nervous system and the peripheral nervous system (PNS), muscles, and skin. However, the Dst-expressing cell type(s) for dt phenotypes have not been well characterized. To address the questions whether the disruption of Dst in Schwann cells induces movement disorders and how much impact does it have on dt phenotypes, we generated Dst conditional knockout (cKO) mice using P0-Cre transgenic mice and Dst gene trap mice. First, we assessed the P0-Cre transgene-dependent Cre recombination using tdTomato reporter mice and then confirmed the preferential tdTomato expression in Schwann cells. In the Dst cKO mice, Dst mRNA expression was significantly decreased in Schwann cells, but it was intact in most of the sensory neurons in the dorsal root ganglion. Next, we analyzed the phenotype of Dst cKO mice. They exhibited a normal motor phenotype during juvenile periods, and thereafter, started exhibiting an ataxia. Behavioral tests and electrophysiological analyses demonstrated impaired motor abilities and slowed motor nerve conduction velocity in Dst cKO mice, but these mice did not manifest dystonic movements. Electron microscopic observation of the PNS of Dst cKO mice revealed significant numbers of hypomyelinated axons and numerous infiltrating macrophages engulfing myelin debris. These results indicate that Dst is important for normal PNS myelin organization and Dst disruption in Schwann cells induces late-onset neuropathy and sensory ataxia. MAIN POINTS: Dystonin (Dst) disruption in Schwann cells results in late-onset neuropathy and sensory ataxia. Dst in Schwann cells is important for normal myelin organization in the peripheral nervous system.

DOI： 10.1002/glia.23843

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Thalidomide exerts its teratogenic and immunomodulatory effects by binding to cereblon (CRBN) and thereby inhibiting/modifying the CRBN-mediated ubiquitination pathway consisting of the Cullin4-DDB1-ROC1 E3 ligase complex. The mechanism of thalidomide's classical hypnotic effect remains largely unexplored, however. Here we examined whether CRBN is involved in the hypnotic effect of thalidomide by generating mice harboring a thalidomide-resistant mutant allele of Crbn (Crbn YW/AA knock-in mice). Thalidomide increased non-REM sleep time in Crbn YW/AA knock-in homozygotes and heterozygotes to a similar degree as seen in wild-type littermates. Thalidomide similarly depressed excitatory synaptic transmission in the cortical slices obtained from wild-type and Crbn YW/AA homozygous knock-in mice without affecting GABAergic inhibition. Thalidomide induced Fos expression in vasopressin-containing neurons of the supraoptic nucleus and reduced Fos expression in the tuberomammillary nuclei. Thus, thalidomide's hypnotic effect seems to share some downstream mechanisms with general anesthetics and GABAA-activating sedatives but does not involve the teratogenic CRBN-mediated ubiquitin/proteasome pathway.

DOI： 10.1073/pnas.1917701117

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Impairments in synapse development are thought to cause numerous psychiatric disorders. Autism susceptibility candidate 2 (AUTS2) gene has been associated with various psychiatric disorders, such as autism and intellectual disabilities. Although roles for AUTS2 in neuronal migration and neuritogenesis have been reported, its involvement in synapse regulation remains unclear. In this study, we found that excitatory synapses were specifically increased in the Auts2-deficient primary cultured neurons as well as Auts2 mutant forebrains. Electrophysiological recordings and immunostaining showed increases in excitatory synaptic inputs as well as c-fos expression in Auts2 mutant brains, suggesting that an altered balance of excitatory and inhibitory inputs enhances brain excitability. Auts2 mutant mice exhibited autistic-like behaviors including impairments in social interaction and altered vocal communication. Together, these findings suggest that AUTS2 regulates excitatory synapse number to coordinate E/I balance in the brain, whose impairment may underlie the pathology of psychiatric disorders in individuals with AUTS2 mutations.

DOI： 10.1016/j.isci.2020.101183

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Gastrin-releasing peptide (GRP) receptor-expressing (GRPR)+ neurons have a central role in the spinal transmission of itch. Because their fundamental regulatory mechanisms are not yet understood, it is important to determine how such neurons are excited and integrate itch sensation. In this study, we investigated the mechanisms for the activation of itch-responsive GRPR+ neurons in the spinal dorsal horn (SDH). GRPR+ neurons expressed the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) containing the GluR2 subunit. In mice, peripherally elicited histaminergic and non-histaminergic itch was prevented by intrathecal (i.t.) administration of the AMPAR antagonist NBQX, which was consistent with the fact that firing of GRPR+ neurons in SDH under histaminergic and non-histaminergic itch was completely blocked by NBQX, but not by the GRPR antagonist RC-3095. Because GRP+ neurons in SDH contain glutamate, we investigated the role of GRP+ (GRP+/Glu+) neurons in regulating itch. Chemogenetic inhibition of GRP+ neurons suppressed both histaminergic and non-histaminergic itch without affecting the mechanical pain threshold. In nonhuman primates, i.t. administration of NBQX also attenuated peripherally elicited itch without affecting the thermal pain threshold. In a mouse model of diphenylcyclopropenone (DCP)-induced contact dermatitis, GRP, GRPR, and AMPAR subunits were upregulated in SDH. DCP-induced itch was prevented by either silencing GRP+ neurons or ablation of GRPR+ neurons. Altogether, these findings demonstrate that GRP and glutamate cooperatively regulate GRPR+ AMPAR+ neurons in SDH, mediating itch sensation. GRP-GRPR and the glutamate-AMPAR system may play pivotal roles in the spinal transmission of itch in rodents and nonhuman primates.

DOI： 10.1016/j.neuropharm.2020.108025

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The shortage of donor lungs hinders lung transplantation, the only definitive option for patients with end-stage lung disease. Blastocyst complementation enables the generation of transplantable organs from pluripotent stem cells (PSCs) in animal models. Pancreases and kidneys have been generated from PSCs by blastocyst complementation in rodent models. Here, we report the generation of lungs using mouse embryonic stem cells (ESCs) in apneumic Fgf10 Ex1mut/Ex3mutmice by blastocyst complementation. Complementation with ESCs enables Fgf10-deficient mice to survive to adulthood without abnormalities. Both the generated lung alveolar parenchyma and the interstitial portions, including vascular endothelial cells, vascular and parabronchial smooth muscle cells, and connective tissue, largely originate from the injected ESCs. These data suggest that Fgf10 Ex1mut/Ex3mutblastocysts provide an organ niche for lung generation and that blastocyst complementation could be a viable approach for generating whole lungs.

DOI： 10.1016/j.celrep.2020.107626

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In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD-95-expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS-digested freeze-fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular-anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions.

DOI： 10.1002/cne.24792

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In the adult mouse brain, neurogenesis occurs mainly in the ventricular-subventricular zone (V-SVZ) and the subgranular zone of the hippocampal dentate gyrus. Neuroblasts generated in the V-SVZ migrate to the olfactory bulb via the rostral migratory stream (RMS) in response to guidance molecules, such as netrin-1. We previously showed that the related netrin-5 (NTN5) is expressed in Mash1-positive transit-amplifying cells and doublecortin-positive neuroblasts in the granule cell layer of the olfactory bulb, the RMS, and the subgranular zone of the adult mouse brain. However, the precise role of NTN5 in adult neurogenesis has not been investigated. In this study, we show that proliferation in the neurogenic niche is impaired in NTN5 knockout mice. The number of proliferating (EdU-labeled) cells in NTN5 KO mice was significantly lower in the V-SVZ, whereas the number of Ki67-positive proliferating cells was unchanged, suggesting a longer cell cycle and decreased cell division in NTN5 KO mice. The number of EdU-labeled cells in the RMS and olfactory bulb was unchanged. By contrast, the numbers of EdU-labeled cells in the cortex, basal ganglia/lateral septal nucleus, and corpus callosum/anterior commissure were increased, which largely represented oligodendrocyte lineage cells. Lastly, we found that chain migration in the RMS of NTN5 KO mice was disorganized. These findings suggest that NTN5 may play important roles in promoting proliferation in the V-SVZ niche, organizing proper chain migration in the RMS, and suppressing oligodendrogenesis in the brain.

DOI： 10.3389/fnins.2020.570974

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The generation of mature, functional, thyroid follicular cells from pluripotent stem cells would potentially provide a therapeutic benefit for patients with hypothyroidism, but in vitro differentiation remains difficult. We earlier reported the in vivo generation of lung organs via blastocyst complementation in fibroblast growth factor 10 (Fgf10), compound, heterozygous mutant (Fgf10 Ex1mut/Ex3mut) mice. Fgf10 also plays an essential role in thyroid development and branching morphogenesis, but any role thereof in thyroid organogenesis remains unclear. Here, we report that the thyroids of Fgf10 Ex1mut/Ex3mut mice exhibit severe hypoplasia, and we generate thyroid tissues from mouse embryonic stem cells (ESCs) in Fgf10 Ex1mut/Ex3mut mice via blastocyst complementation. The tissues were morphologically normal and physiologically functional. The thyroid follicular cells of Fgf10 Ex1mut/Ex3mut chimeric mice were derived largely from GFP-positive mouse ESCs although the recipient cells were mixed. Thyroid generation in vivo via blastocyst complementation will aid functional thyroid regeneration.

DOI： 10.3389/fendo.2020.609697

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Language：Japanese   Publisher：THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT  

DOI： 10.14882/jrds.113.0_P-11

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Monoamine neurotransmitters are released by specialized neurons regulating behavioral, motor, and cognitive functions. Although the localization of monoaminergic neurons in the brain is well known, the distribution and kinetics of monoamines remain unclear. Here, we generated a murine brain atlas of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) levels using mass spectrometry imaging (MSI). We found several nuclei rich in both 5-HT and a catecholamine (DA or NE) and identified the paraventricular nucleus of the thalamus (PVT), where 5-HT and NE are co-localized. The analysis of 5-HT fluctuations in response to acute tryptophan depletion and infusion of isotope-labeled tryptophan in vivo revealed a close kinetic association between the raphe nuclei, PVT, and amygdala but not the other nuclei. Our findings imply the existence of a highly dynamic 5-HT-mediated raphe to PVT pathway that likely plays a role in the brain monoamine system.

DOI： 10.1016/j.isci.2019.09.036

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Orexins are hypothalamic neuropeptides that regulate feeding, energy expenditure, and sleep. Although orexin-deficient mice are susceptible to obesity, little is known about the roles of the orexin receptors in long-term energy metabolism. Here, we performed the metabolic characterization of orexin receptor-deficient mice. Ox1r-deficient mice were resistant to diet-induced obesity, and their food intake was similar between chow and high-fat food. Ox2r-deficient mice exhibited less energy expenditure than wild-type mice when fed a high-fat diet. Neither Ox1r-deficient nor Ox2r-deficient mice showed body weight gain similar to orexin-deficient mice. Although the presence of a running wheel suppressed diet-induced obesity in wild-type mice, the effect was weaker in orexin neuron-ablated mice. Finally, we did not detect abnormalities in brown adipose tissues of orexin-deficient mice. Thus, each orexin receptor signaling has a unique role in energy metabolism, and orexin neurons are involved in the interactive effect of diet and exercise on body weight gain.

DOI： 10.1016/j.isci.2019.09.003

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Intracellular mislocalization of TAR DNA-binding protein 43 (TDP-43), a nuclear DNA/RNA-binding protein involved in RNA metabolism, is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Although the aggregation-prone, TDP-43 C-terminal domain is widely considered as a key component of TDP-43 pathology in ALS, recent studies including ours suggest that TDP-43 N-terminal fragments (TDP-∆C) may also contribute to the motor dysfunction in ALS. However, the specific pathological functions of TDP-43 N-terminal fragments in mice have not been elucidated. Here, we established TDP-∆C knock-in mice missing a part of exon 6 of murine Tardbp gene, which encodes the C-terminal region of TDP-43. Homozygous TDP-∆C mice showed embryonic lethality, indicating that the N-terminal domain of TDP-43 alone is not sufficient for normal development. In contrast, heterozygous TDP-∆C mice developed normally but exhibited age-dependent mild motor dysfunction with a loss of C-boutons, large cholinergic synaptic terminals on spinal α-motor neurons. TDP-∆C protein broadly perturbed gene expression in the spinal cords of aged heterozygous TDP-∆C mice, including downregulation of Notch1 mRNA. Moreover, the level of Notch1 mRNA was suppressed both by TDP-43 depletion and TDP-∆C expression in Neuro2a cells. Decreased Notch1 mRNA expression in aged TDP-∆C mice was associated with the age-dependent motor dysfunction and loss of Akt surviving signal. Our findings indicate that the N-terminal region of TDP-43 derived from TDP-∆C induces the age-dependent motor dysfunction associated with impaired Notch1-Akt axis in mice.

DOI： 10.1186/s40478-019-0776-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Neurochemistry  

Oligodendrocytes (OLs) are myelinating cells of the central nervous system. Recent studies have shown that mechanical factors influence various cell properties. Mechanical stimulation can be transduced into intracellular biochemical signals through mechanosensors, such as integrin, p130Cas, talin and vinculin. However, the molecular mechanisms underlying the mechanical regulation of OLs by mechanosensors remain largely unknown. We found that morphology of OL was affected by knockdown of the mechanosensors p130Cas or talin1. Stretching of OL precursor cells induced the phosphorylation of p130Cas and talin-associated assembly of vinculin. Shear stress decreased the number of OL processes, whereas these effects were mechanically suppressed by dominant-negative (DN) p130Cas, but not by DN-talin1. To investigate the roles of p130Cas in post-natal OLs in vivo, we constructed a novel p130Cas knock-in mouse and found overexpression of p130Cas in vivo affected the number of mature OLs in the cortex. These results indicate that the mechanosensor p130Cas controls both OL morphogenesis and maturation.

DOI： 10.1111/jnc.14657

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Uninterrupted arousal is important for survival during threatening situations. Activation of orexin/hypocretin neurons is implicated in sustained arousal. However, orexin neurons produce and release orexin as well as several co-transmitters including dynorphin and glutamate. To disambiguate orexin-dependent and -independent physiological functions of orexin neurons, we generated a novel Orexin-flippase (Flp) knock-in mouse line. Crossing with Flp-reporter or Cre-expressing mice showed gene expression exclusively in orexin neurons. Histological studies confirmed that orexin was knock-out in homozygous mice. Orexin neurons without orexin showed altered electrophysiological properties, as well as received decreased glutamatergic inputs. Selective chemogenetic activation revealed that both orexin and co-transmitters functioned to increase wakefulness, however, orexin was indispensable to promote sustained arousal. Surprisingly, such activation increased the total time spent in cataplexy. Taken together, orexin is essential to maintain basic membrane properties and input-output computation of orexin neurons, as well as to exert awake-sustaining aptitude of orexin neurons.

DOI： 10.7554/eLife.44927

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Sleep/wakefulness cycle is regulated by coordinated interactions between sleep- and wakefulness-regulating neural circuitry. However, the detailed mechanism is far from understood. Here, we found that glutamic acid decarboxylase 67-positive GABAergic neurons in the ventral tegmental area (VTAGad67+) are a key regulator of non-rapid eye movement (NREM) sleep in mice. VTAGad67+ project to multiple brain areas implicated in sleep/wakefulness regulation such as the lateral hypothalamus (LH). Chemogenetic activation of VTAGad67+ promoted NREM sleep with higher delta power whereas optogenetic inhibition of these induced prompt arousal from NREM sleep, even under highly somnolescent conditions, but not from REM sleep. VTAGad67+ showed the highest activity in NREM sleep and the lowest activity in REM sleep. Moreover, VTAGad67+ directly innervated and inhibited wake-promoting orexin/hypocretin neurons by releasing GABA. As such, optogenetic activation of VTAGad67+ terminals in the LH promoted NREM sleep. Taken together, we revealed that VTAGad67+ play an important role in the regulation of NREM sleep.

DOI： 10.7554/eLife.44928

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BACKGROUND: AMPA receptors predominantly mediate fast excitatory synaptic transmission in the mammalian brain. Post-translational protein S-palmitoylation of AMPA receptor GluA subunits at their C-termini reversibly controls the receptors trafficking to and from excitatory glutamatergic synapses. Excitatory inputs to neurons induce the expression of immediate early genes (IEGs), including Arc, with particular spatial patterns. In the hippocampal dentate gyrus, Arc is mainly expressed in the upper (dorsal) blade at the basal state. GluA1 C-terminal palmitoylation-deficient (GluA1C811S) mice showed enhanced seizure susceptibility and disturbed synaptic plasticity without impaired gross anatomy or basal synaptic transmission. These mutant mice also exhibited an increased expression of IEG products, c-Fos and Arc proteins, in the hippocampus and cerebral cortex. In this report, we further analyzed excitability and Arc expression pattern in the dentate gyrus of GluA1C811S mice. METHODS AND RESULTS: Electrophysiological analysis of granule neurons to measure the evoked excitatory postsynaptic current/evoked inhibitory postsynaptic current ratio revealed that excitatory/inhibitory (E/I) balance was normal in GluA1C811S mice. In contrast, immunohistochemical staining showed an abnormal distribution of Arc-positive cells between upper and lower (ventral) blades of the dentate gyrus in these mutant mice. These data suggest that deficiency of GluA1 palmitoylation causes perturbed neuronal inputs from the entorhinal cortex to the dentate gyrus, which potentially underlies the excessive excitability in response to seizure-inducing stimulation. CONCLUSION: Our findings conclude that an appropriate regulation of Arc expression in the dentate gyrus, ensured by AMPA receptor palmitoylation, may be critical for stabilizing hippocampal neural circuits and may suppress excess excitation.

DOI： 10.1002/npr2.12044

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Multiple sclerosis (MS) is an autoimmune disease characterized by immune-mediated inflammation, which attacks the myelin sheath. MS pursues a relapsing and remitting course with varying intervals between symptoms. The main clinical pathological features include inflammation, myelin sheath destruction and plaque formation in the central nervous system (CNS). We previously reported that cystatin F (CysF) expression is induced in demyelinating lesions that are accompanied by active remyelination (referred to as shadow plaques) but is down-regulated in chronic demyelinated lesions (plaques) in the spinal cord of MS patients and in several murine models of demyelinating disease. CysF is a cathepsin protease inhibitor whose major target is cathepsin C (CatC), which is co-expressed in demyelinating regions in Plp4e/- mice, a model of chronic demyelination. Here, we report the time course of CatC and CysF expression and describe the symptoms in a mouse experimental autoimmune encephalomyelitis (EAE) model using CatC knockdown (KD) and CatC over-expression (OE) mice. In myelin oligodendrocyte glycoprotein (MOG)-EAE, CatC positive cells were found to infiltrate the CNS at an early stage prior to any clinical signs, in comparison to WT mice. CysF expression was not observed at this early stage, but appeared later within shadow plaques. CatC expression was found in chronic demyelinated lesions but was not associated with CysF expression, and CatCKD EAE mouse showed delayed demyelination. Whereas, CatCOE in microglia significantly increased severity of demyelination in the MOG-EAE model. Thus, these results demonstrate that CatC plays a major role in MOG-EAE.

DOI： 10.1111/jnc.14581

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Pancreatic β cells secrete insulin by Ca2+-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, is used in β cells to regulate Ca2+ influx for insulin secretion. β cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca2+ channel (VDCC) current density. In situ Ca2+ imaging of β cells within islets expressing a membrane-bound G-CaMP8b Ca2+ sensor demonstrated initial local Ca2+ signals at the ELKS-localized vascular side of the β cell plasma membrane, which were markedly decreased in ELKS-KO β cells. Mechanistically, ELKS directly interacted with the VDCC-β subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the β cell plasma membrane for polarized Ca2+ influx and first-phase insulin secretion from pancreatic islets.

DOI： 10.1016/j.celrep.2018.12.106

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CaMKII is a pivotal kinase that plays essential roles in synaptic plasticity. Apart from its signaling function, the structural function of CaMKII is becoming clear. CaMKII - F-actin interaction stabilizes actin cytoskeleton in a dendritic spine. A transient autophosphorylation at the F-actin binding region during LTP releases CaMKII from F-actin and opens a brief time-window of actin reorganization. However, the physiological relevance of this finding in learning and memory was not presented. Using a knock-in (KI) mouse carrying phosphoblock mutations in the actin-binding domain of CaMKIIβ, we demonstrate that proper regulation of CaMKII - F-actin interaction is important for fear conditioning memory tasks. The KI mice show poor performance in contextual and cued versions of fear conditioning test. These results suggest the importance of CaMKII - F-actin interactions in learning and memory.

DOI： 10.1016/j.nlm.2018.12.003

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Many inflammatory mediators are involved in the process of carcinogenesis and cancer progression. In addition to cytokines and chemokines, lipid mediators have recently attracted attention as signaling molecules associated with inflammatory diseases. Sphingosine-1-phosphate (S1P) is a pleiotropic lipid mediator that regulates cell survival and migration, immune cell recruitment, angiogenesis and lymphangiogenesis. S1P also plays a significant role in inflammation and cancer. The gradation of S1P concentration in the blood, lymph and tissue regulates lymphocyte trafficking, an important component of inflammation. Furthermore, cancer cells produce elevated levels of S1P, contributing to the tumor microenvironment and linking cancer and inflammation. Future technological advances may reveal greater detail about the mechanisms of S1P regulation in the tumor microenvironment and the contribution of S1P to cancer progression. Considering the critical role of S1P in linking inflammation and cancer, it is possible that the S1P signaling pathway could be a novel therapeutic target for cancers with chronic inflammation.

DOI： 10.1111/cas.13802

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BACKGROUND: Pancreatic cancer is a disease with poor prognosis, and development of new treatments is necessary. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays a critical role in progression of many types of cancer. However, little is known about the role of sphingosine kinases in pancreatic cancer. This study investigated the roles of sphingosine kinases in pancreatic cancer progression. MATERIALS AND METHODS: S1P levels in pancreatic cancer and noncancerous pancreatic tissue were measured in 10 patients. We generated PAN02 murine pancreatic cancer cell lines with a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system genes 9 (Cas9)-mediated deletion of SphK1 or SphK2 and assessed cell growth and migration. In an animal model, we assessed the survival of mice injected with PAN02 cells intraperitoneally. RESULTS: S1P levels in the pancreatic cancer tissue were significantly higher than those in noncancerous tissue. SphK1 knockout (KO) cells showed greater proliferation and migration than wild type (WT) cells, and SphK2 KO cells showed less proliferation and migration than WT cells. Animal experiments showed that the survival of mice injected with SphK1 KO cells was significantly shorter than those injected with WT cells, and the survival of mice injected with SphK2 KO cells was longer than those injected with WT cells. Surprisingly, cytotoxic assay using gemcitabine showed that SphK1 KO cells survived less than WT cells, and SphK2 KO cells survived more than WT cells. CONCLUSIONS: S1P produced by SphK1 and SphK2 may have different functions in pancreatic cancer cells. Targeting both SphK1 and SphK2 may be a potential strategy for pancreatic cancer treatment.

DOI： 10.1016/j.jss.2018.06.019

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Synaptic AMPAR expression controls the strength of excitatory synaptic transmission and plasticity. An excess of synaptic AMPARs leads to epilepsy in response to seizure-inducible stimulation. The appropriate regulation of AMPARs plays a crucial role in the maintenance of the excitatory/inhibitory synaptic balance; however, the detailed mechanisms underlying epilepsy remain unclear. Our previous studies have revealed that a key modification of AMPAR trafficking to and from postsynaptic membranes is the reversible, posttranslational S-palmitoylation at the C-termini of receptors. To clarify the role of palmitoylation-dependent regulation of AMPARs in vivo, we generated GluA1 palmitoylation-deficient (Cys811 to Ser substitution) knock-in mice. These mutant male mice showed elevated seizure susceptibility and seizure-induced neuronal activity without impairments in synaptic transmission, gross brain structure, or behavior at the basal level. Disruption of the palmitoylation site was accompanied by upregulated GluA1 phosphorylation at Ser831, but not at Ser845, in the hippocampus and increased GluA1 protein expression in the cortex. Furthermore, GluA1 palmitoylation suppressed excessive spine enlargement above a certain size after LTP. Our findings indicate that an abnormality in GluA1 palmitoylation can lead to hyperexcitability in the cerebrum, which negatively affects the maintenance of network stability, resulting in epileptic seizures.SIGNIFICANCE STATEMENT AMPARs predominantly mediate excitatory synaptic transmission. AMPARs are regulated in a posttranslational, palmitoylation-dependent manner in excitatory synapses of the mammalian brain. Reversible palmitoylation dynamically controls synaptic expression and intracellular trafficking of the receptors. Here, we generated GluA1 palmitoylation-deficient knock-in mice to clarify the role of AMPAR palmitoylation in vivo We showed that an abnormality in GluA1 palmitoylation led to hyperexcitability, resulting in epileptic seizure. This is the first identification of a specific palmitoylated protein critical for the seizure-suppressing process. Our data also provide insight into how predicted receptors such as AMPARs can effectively preserve network stability in the brain. Furthermore, these findings help to define novel key targets for developing anti-epileptic drugs.

DOI： 10.1523/JNEUROSCI.1590-18.2018

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At the presynaptic active zone (AZ), the related cytomatrix proteins CAST and ELKS organize the presynaptic release machinery. While CAST is known to regulate AZ size and neurotransmitter release, the role of ELKS and the integral system of CAST/ELKS together is poorly understood. Here, we show that CAST and ELKS have both redundant and unique roles in coordinating synaptic development, function, and maintenance of retinal photoreceptor ribbon synapses. A CAST/ELKS double knockout (dKO) mouse showed high levels of ectopic synapses and reduced responses to visual stimulation. Ectopic formation was not observed in ELKS conditional KO but progressively increased with age in CAST KO mice with higher rates in the dKO. Presynaptic calcium influx was strongly reduced in rod photoreceptors of CAST KO and dKO mice. Three-dimensional scanning EM reconstructions showed structural abnormalities in rod triads of CAST KO and dKO. Remarkably, AAV-mediated acute ELKS deletion after synapse maturation induced neurodegeneration and loss of ribbon synapses. These results suggest that CAST and ELKS work in concert to promote retinal synapse formation, transmission, and maintenance.

DOI： 10.1083/jcb.201704076

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Purkinje cell degeneration (pcd) was first identified in a spontaneous mouse mutant showing cerebellar ataxia. In addition to cerebellar Purkinje cells (PCs), retinal photoreceptors, mitral cells in the olfactory bulb, and a discrete subpopulation of thalamic neurons also degenerate in the mutant brains. The gene responsible for the pcd mutant is Nna1, also known as ATP/GTP binding protein 1 or cytosolic carboxypeptidase-like 1, which encodes a zinc carboxypeptidase protein. To investigate pathogenesis of the pcd mutation in detail, we generated a conditional Nna1 allele targeting the carboxypeptidase domain at C-terminus. After Cre recombination and heterozygous crossing, we generated Nna1 knockout (KO) mice and found that the Nna1 KO mice began to show cerebellar ataxia at postnatal day 20 (P20). Most PCs degenerated until 4-week-old, except lobule X. Activated microglia and astrocytes were also observed in the Nna1 KO cerebellum. In the mutant brain, the Nna1 mRNA level was dramatically reduced, suggesting that nonsense-mediated mRNA decay occurs in it. Since the Nna1 protein acts as a de-glutamatase on the C-terminus of α-tubulin and β-tubulin, increased polyglutamylated tubulin was detected in the Nna1 KO cerebellum. In addition, the endoplasmic reticulum stress marker, C/EBP homologous protein (CHOP), was up-regulated in the mutant PCs. We report the generation of a functional Nna1 conditional allele and possible mechanisms of PC death in the Nna1 KO in the cerebellum. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

DOI： 10.1111/jnc.14591

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Calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1 (Caskin1) is a direct binding partner of the synaptic adaptor protein CASK. Because Caskin1 forms homo-multimers and binds not only CASK but also other neuronal proteins in vitro, it is anticipated to have neural functions; but its exact role in mammals remains unclear. Previously, we showed that the concentration of Caskin1 in the spinal dorsal horn increases under chronic pain. To characterize this protein, we generated Caskin1-knockout (Caskin1-KO) mice and specific anti-Caskin1 antibodies. Biochemical and immunohistochemical analyses demonstrated that Caskin1 was broadly distributed in the whole brain and spinal cord, and that it primarily localized at synapses. To elucidate the neural function of Caskin1 in vivo, we subjected Caskin1-KO mice to comprehensive behavioral analysis. The mutant mice exhibited differences in gait, enhanced nociception, and anxiety-like behavior relative to their wild-type littermates. In addition, the knockouts exhibited strong freezing responses, with or without a cue tone, in contextual and cued-fear conditioning tests as well as low memory retention in the Barnes Maze test. Taken together, these results suggest that Caskin1 contributes to a wide spectrum of behavioral phenotypes, including gait, nociception, memory, and stress response, in broad regions of the central nervous system.

DOI： 10.1186/s13041-018-0407-2

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Configuration of tripartite synapses, comprising the pre-, post-, and peri-synaptic components (axon terminal or bouton, dendritic spine, and astroglial terminal process), is a critical determinant of neurotransmitter kinetics and hence synaptic transmission. However, little is known about molecular basis for the regulation of tripartite synapse morphology. Previous studies showed that CDC42EP4, an effector protein of a cell morphogenesis regulator CDC42, is expressed exclusively in Bergmann glia in the cerebellar cortex, that it forms tight complex with the septin heterooligomer, and that it interacts indirectly with the glutamate transporter GLAST and MYH10/nonmuscle myosin ΙΙB. Scrutiny of Cdc42ep4-/- mice had revealed that the CDC42EP4-septins-GLAST interaction facilitates glutamate clearance, while the role for CDC42EP4-septins-MYH10 interaction has remained unsolved. Here, we find anomalous configuration of the tripartite synapses comprising the parallel fiber boutons, dendritic spines of Purkinje cells, and Bergmann glial processes in Cdc42ep4-/- mice. The complex anomalies include 1) recession of Bergmann glial membranes from the nearest active zones, and 2) extension of nonactive synaptic contact around active zone. In line with the recession of Bergmann glial membranes by the loss of CDC42EP4, overexpression of CDC42EP4 in heterologous cells promotes cell spreading and partitioning of MYH10 to insoluble (i.e., active) fraction. Paradoxically, however, Cdc42ep4-/- cerebellum contained significantly more MYH10 and N-cadherin, which is attributed to secondary neuronal response mainly in Purkinje cells. Given cooperative actions of N-cadherin and MYH10 for adhesion between neurons, we speculate that their augmentation may reflect the extension of nonactive synaptic contacts in Cdc42ep4-/- cerebellum. Transcellular mechanism that links the absence of CDC42EP4 in Bergmann glia to the augmentation of N-cadherin and MYH10 in neurons is currently unknown, but the phenotypic similarity to GLAST-null mice indicates involvement of the glutamate intolerance. Together, the unique phenotype of Cdc42ep4-/- mice provides a clue to novel molecular network underlying tripartite synapse configuration.

DOI： 10.1016/j.neuint.2018.01.003

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Language：English   Publisher：(一社)日本癌学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

DOI： 10.3389/fnins.2018.00535

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The visual cortex of mice is a useful model for investigating the mammalian visual system. In primates, higher visual areas are classified into two parts, the dorsal stream ("where" pathway) and ventral stream ("what" pathway). The ventral stream is known to include a part of the temporal cortex. In mice, however, some cortical areas adjacent to the primary visual area (V1) in the occipital cortex are thought to be comparable to the ventral stream in primates, although the whole picture of the mouse ventral stream has never been elucidated. We performed wide-field Ca2+ imaging in awake mice to investigate visual responses in the mouse temporal cortex, and found that the postrhinal cortex (POR), posterior to the auditory cortex (AC), and the ectorhinal and temporal association cortices (ECT), ventral to the AC, showed clear visual responses to moving visual objects. The retinotopic maps in the POR and ECT were not clearly observed, and the amplitudes of the visual responses in the POR and ECT were less sensitive to the size of the objects, compared to visual responses in the V1. In the ECT, objects of different sizes activated different subareas. These findings strongly suggest that the mouse ventral stream extends to the ECT ventral to the AC, and that it has characteristic response properties that are markedly different from the response properties in the V1.

DOI： 10.1038/s41598-018-29530-3

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In the presynaptic terminal, the magnitude and location of Ca2+ entry through voltage-gated Ca2+ channels (VGCCs) regulate the efficacy of neurotransmitter release. However, how presynaptic active zone proteins control mammalian VGCC levels and organization is unclear. To address this, we deleted the CAST/ELKS protein family at the calyx of Held, a CaV2.1 channel-exclusive presynaptic terminal. We found that loss of CAST/ELKS reduces the CaV2.1 current density with concomitant reductions in CaV2.1 channel numbers and clusters. Surprisingly, deletion of CAST/ELKS increases release probability while decreasing the readily releasable pool, with no change in active zone ultrastructure. In addition, Ca2+ channel coupling is unchanged, but spontaneous release rates are elevated. Thus, our data identify distinct roles for CAST/ELKS as positive regulators of CaV2.1 channel density and suggest that they regulate release probability through a post-priming step that controls synaptic vesicle fusogenicity.

DOI： 10.1016/j.celrep.2018.06.024

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Although obesity with associated inflammation is now recognized as a risk factor for breast cancer and distant metastases, the functional basis for these connections remain poorly understood. Here, we show that in breast cancer patients and in animal breast cancer models, obesity is a sufficient cause for increased expression of the bioactive sphingolipid mediator sphingosine-1-phosphate (S1P), which mediates cancer pathogenesis. A high-fat diet was sufficient to upregulate expression of sphingosine kinase 1 (SphK1), the enzyme that produces S1P, along with its receptor S1PR1 in syngeneic and spontaneous breast tumors. Targeting the SphK1/S1P/S1PR1 axis with FTY720/fingolimod attenuated key proinflammatory cytokines, macrophage infiltration, and tumor progression induced by obesity. S1P produced in the lung premetastatic niche by tumor-induced SphK1 increased macrophage recruitment into the lung and induced IL6 and signaling pathways important for lung metastatic colonization. Conversely, FTY720 suppressed IL6, macrophage infiltration, and S1P-mediated signaling pathways in the lung induced by a high-fat diet, and it dramatically reduced formation of metastatic foci. In tumor-bearing mice, FTY720 similarly reduced obesity-related inflammation, S1P signaling, and pulmonary metastasis, thereby prolonging survival. Taken together, our results establish a critical role for circulating S1P produced by tumors and the SphK1/S1P/S1PR1 axis in obesity-related inflammation, formation of lung metastatic niches, and breast cancer metastasis, with potential implications for prevention and treatment.Significance: These findings offer a preclinical proof of concept that signaling by a sphingolipid may be an effective target to prevent obesity-related breast cancer metastasis. Cancer Res; 78(7); 1713-25. ©2018 AACR.

DOI： 10.1158/0008-5472.CAN-17-1423

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Language：Japanese   Publisher：(一社)日本外科学会  

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Elimination of redundant synapses formed early in development and strengthening of necessary connections are crucial for shaping functional neural circuits. Purkinje cells (PCs) in the neonatal cerebellum are innervated by multiple climbing fibers (CFs) with similar strengths. A single CF is strengthened whereas the other CFs are eliminated in each PC during postnatal development. The underlying mechanisms, particularly for the strengthening of single CFs, are poorly understood. Here we report that progranulin, a multi-functional growth factor implicated in the pathogenesis of frontotemporal dementia, strengthens developing CF synaptic inputs and counteracts their elimination from postnatal day 11 to 16. Progranulin derived from PCs acts retrogradely onto its putative receptor Sort1 on CFs. This effect is independent of semaphorin 3A, another retrograde signaling molecule that counteracts CF synapse elimination. We propose that progranulin-Sort1 signaling strengthens and maintains developing CF inputs, and may contribute to selection of single "winner" CFs that survive synapse elimination.

DOI： 10.1016/j.neuron.2018.01.018

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Sleep/wake behavior is regulated by distinct groups of neurons, such as dopaminergic, noradrenergic, and orexinergic neurons. Although monoaminergic neurons are usually considered to be wake-promoting, the role of serotonergic neurons in sleep/wake behavior remains inconclusive because of the effect of serotonin (5-HT)-deficiency on brain development and the compensation for inborn 5-HT deficiency by other sleep/wake-regulating neurons. Here, we performed selective ablation of central 5-HT neurons in the newly developed Rosa-diphtheria toxin receptor (DTR)-tdTomato mouse line that was crossed with Pet1Cre/+ mice to examine the role of 5-HT neurons in the sleep/wake behavior of adult mice. Intracerebroventricular administration of diphtheria toxin completely ablated tdTomato-positive cells in Pet1Cre/+; Rosa-DTR-tdTomato mice. Electroencephalogram/electromyogram-based sleep/wake analysis demonstrated that central 5-HT neuron ablation in adult mice decreased the time spent in rapid eye movement (REM) sleep, which was associated with fewer transitions from non-REM (NREM) sleep to REM sleep than in control mice. Central 5-HT neuron-ablated mice showed attenuated wake response to a novel environment and increased theta power during wakefulness compared to control mice. The current findings indicated that adult 5-HT neurons work to support wakefulness and regulate REM sleep time through a biased transition from NREM sleep to REM sleep.

DOI： 10.3389/fnins.2018.00535

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

Neurons expressing neuropeptide orexins (hypocretins) in the lateral hypothalamus (LH) and serotonergic neurons in the dorsal raphe nucleus (DR) both play important roles in the regulation of sleep/wakefulness states, and show similar firing patterns across sleep/wakefulness states. Orexin neurons send excitatory projections to serotonergic neurons in the DR, which express both subtypes of orexin receptors (Mieda et al., 2011), while serotonin (5-HT) potently inhibits orexin neurons through activation of 5HT1A receptors (5HT1ARs). In this study, we examined the physiological importance of serotonergic inhibitory regulation of orexin neurons by studying the phenotypes of mice lacking the 5HT1A receptor gene (Htr1a) specifically in orexin neurons (ox5HT1ARKO mice). ox5HT1ARKO mice exhibited longer NREM sleep time along with decreased wakefulness time in the later phase of the dark period. We also found that restraint stress induced a larger impact on REM sleep architecture in ox5HT1ARKO mice than in controls, with a larger delayed increase in REM sleep amount as compared with that in controls, indicating abnormality of REM sleep homeostasis in the mutants. These results suggest that 5HT1ARs in orexin neurons are essential in the regulation of sleep/wakefulness states, and that serotonergic regulation of orexin neurons plays a crucial role in the appropriate control of orexinergic tone to maintain normal sleep/wake architecture.

DOI： 10.3389/fnins.2018.00892

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Genetic studies point to a major role of de novo mutations in neurodevelopmental disorders of intellectual disability, autism spectrum disorders, and epileptic encephalopathy. The STXBP1 gene encodes the syntaxin-binding protein 1 (Munc18-1) that critically controls synaptic vesicle exocytosis and synaptic transmission. This gene harbors a high frequency of de novo mutations, which may play roles in these neurodevelopmental disorders. However, the system and behavioral-level pathophysiological changes caused by these genetic defects remain poorly understood. Constitutional (Stxbp1+/-), dorsal-telencephalic excitatory (Stxbp1fl/+/Emx), or global inhibitory neuron-specific (Stxbp1fl/+/Vgat) mice were subjected to a behavioral test battery examining locomotor activity, anxiety, fear learning, and social interactions including aggression. Furthermore, measurements of local field potentials in multiple regions of the brain were performed. Stxbp1+/- male mice exhibited enhanced aggressiveness and impaired fear learning associated with elevated gamma activity in several regions of the brain including the prefrontal cortex. Stxbp1fl/+/Emx mice showed fear-learning deficits, but neither Stxbp1fl/+/Emx nor Stxbp1fl/+/Vgat mice showed increased aggressiveness. Pharmacological potentiation of the excitatory transmission at active synapses via the systemic administration of ampakine CX516, which enhances the excitatory postsynaptic function, ameliorated the aggressive phenotype of Stxbp1+/- mice. These findings suggest that synaptic impairments of the dorsal telencephalic and subcortical excitatory neurons cause learning deficits and enhanced aggression in Stxbp1+/- mice, respectively. Additionally, normalizing the excitatory synaptic transmission is a potential therapeutic option for managing aggressiveness in patients with STXBP1 mutations.

DOI： 10.1093/hmg/ddx379

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Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

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Language：English   Publisher：生命科学系学会合同年次大会運営事務局  

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Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

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Local regulation of synaptic efficacy is thought to be important for proper networking of neurons and memory formation. Dysregulation of global translation influences long-term memory in mice, but the relevance of the regulation specific for local translation by RNA granules remains elusive. Here, we demonstrate roles of RNG105/caprin1 in long-term memory formation. RNG105 deletion in mice impaired synaptic strength and structural plasticity in hippocampal neurons. Furthermore, RNG105-deficient mice displayed unprecedentedly severe defects in long-term memory formation in spatial and contextual learning tasks. Genome-wide profiling of mRNA distribution in the hippocampus revealed an underlying mechanism: RNG105 deficiency impaired the asymmetric somato-dendritic localization of mRNAs. Particularly, RNG105 deficiency reduced the dendritic localization of mRNAs encoding regulators of AMPAR surface expression, which was consistent with attenuated homeostatic AMPAR scaling in dendrites and reduced synaptic strength. Thus, RNG105 has an essential role, as a key regulator of dendritic mRNA localization, in long-term memory formation.

DOI： 10.7554/eLife.29677

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Emotionally salient information activates orexin neurons in the lateral hypothalamus, leading to increase in sympathetic outflow and vigilance level. How this circuit alters animals' behavior remains unknown. Here we report that noradrenergic neurons in the locus coeruleus (NALC neurons) projecting to the lateral amygdala (LA) receive synaptic input from orexin neurons. Pharmacogenetic/optogenetic silencing of this circuit as well as acute blockade of the orexin receptor-1 (OX1R) decreases conditioned fear responses. In contrast, optogenetic stimulation of this circuit potentiates freezing behavior against a similar but distinct context or cue. Increase of orexinergic tone by fasting also potentiates freezing behavior and LA activity, which are blocked by pharmacological blockade of OX1R in the LC. These findings demonstrate the circuit involving orexin, NALC and LA neurons mediates fear-related behavior and suggests inappropriate excitation of this pathway may cause fear generalization sometimes seen in psychiatric disorders, such as PTSD.

DOI： 10.1038/s41467-017-01782-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner.

DOI： 10.1007/s00429-017-1408-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Netrin-1 (Ntn1) emanating from the ventral midline has been thought to act as a long-range diffusible chemoattractant for commissural axons (CAs). However, CAs still grow towards the midline in the absence of the floor plate (FP), a glial structure occupying the midline. Here, using genetically loss-of-function approaches in mice, we show that Ntn1 derived from the ventricular zone (VZ), but not the FP, is crucial for CA guidance in the mouse hindbrain. During the period of CA growth, Ntn1 is expressed in the ventral two-thirds of the VZ, in addition to the FP. Remarkably, deletion of Ntn1 from the VZ and even from the dorsal VZ highly disrupts CA guidance to the midline, whereas the deletion from the FP has little impact on it. We also show that the severities of CA guidance defects found in the Ntn1 conditional mutants were irrelevant to their FP long-range chemoattractive activities. Our results are incompatible with the prevailing view that Ntn1 is an FP-derived long-range diffusible chemoattractant for CAs, but suggest a novel mechanism that VZ-derived Ntn1 directs CAs to the ventral midline by its local actions.

DOI： 10.1038/s41598-017-12269-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states.

DOI： 10.1101/gad.300822.117

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

It has been established that voltage-gated proton channels (VSOP/Hv1), encoded by Hvcn1, support reactive oxygen species (ROS) production in phagocytic activities of neutrophils (El Chemaly et al. ) and antibody production in B lymphocytes (Capasso et al. ). VSOP/Hv1 is a potential therapeutic target for brain ischemia, since Hvcn1 deficiency reduces microglial ROS production and protects brain from neuronal damage (Wu et al. ). In the present study, we report that VSOP/Hv1 has paradoxical suppressive role in ROS production in microglia. Extracellular ROS production was lower in neutrophils of Hvcn1-/- mice than WT mice as reported. In contrast, it was drastically enhanced in isolated Hvcn1-/- microglia as compared with cells from WT mice. Actin dynamics was altered in Hvcn1-/- microglia and intracellular distribution of cytosolic NADPH oxidase subunit, p67, was changed. When expression levels of oxidative stress responsive antioxidant genes were compared between WT and Hvcn1-/- in cerebral cortex at different ages of animals, they were slightly decreased in Hvcn1-/- mice at younger stage (1 day, 5 days, 3 weeks old), but drastically increased at aged stage (6 months old), suggesting that the regulation of microglial ROS production by VSOP/Hv1 is age-dependent. We also performed brain ischemic stroke experiments and found that the neuroprotective effect of VSOP/Hv1deficiency on infarct volume depended on the age of animals. Taken together, regulation of ROS production by VSOP/Hv1 is more complex than previously thought and significance of VSOP/Hv1 in microglial ROS production depends on age.

DOI： 10.1111/jnc.14106

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Language：Japanese   Publisher：日本生物学的精神医学会・日本神経精神薬理学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Knock-in mice lacking PKN1 kinase activity were generated by introducing a T778A point mutation in the catalytic domain. PKN1[T778A] mutant mice developed to adulthood without apparent external abnormalities, but exhibited lower T and B lymphocyte counts in the peripheral blood than those of wild-type (WT) mice. T and B cell development proceeded in an apparently normal fashion in bone marrow and thymus of PKN1[T778A] mice, however, the number of T and B cell counts were significantly higher in the lymph nodes and spleen of mutant mice in those of WT mice. After transfusion into WT recipients, EGFP-labelled PKN1[T778A] donor lymphocytes were significantly less abundant in the peripheral circulation and more abundant in the spleen and lymph nodes of recipient mice compared with EGFP-labelled WT donor lymphocytes, likely reflecting lymphocyte sequestration in the spleen and lymph nodes in a cell-autonomous fashion. PKN1[T778A] lymphocytes showed significantly lower chemotaxis towards chemokines and sphingosine 1-phosphate (S1P) than WT cells in vitro. The biggest migration defect was observed in response to S1P, which is essential for lymphocyte egress from secondary lymphoid organs. These results reveal a novel role of PKN1 in lymphocyte migration and localization.

DOI： 10.1038/s41598-017-07936-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

In demyelinating diseases such as multiple sclerosis (MS), an imbalance between the demyelination and remyelination rates underlies the degenerative processes. Microglial activation is observed in demyelinating lesions; however, the molecular mechanism responsible for the homeostatic/environmental change remains elusive. We previously found that cystatin F (CysF), a cysteine protease inhibitor, is selectively expressed in microglia only in actively demyelinating/remyelinating lesions but ceases expression in chronic lesions, suggesting its role in remyelination. Here, we report the effects of manipulating the expression of CysF and cathepsin C (CatC), a key target of CysF, in a murine model of transgenic demyelinating disease, Plp4e/- . During the active remyelinating phase, both CysF knockdown (CysFKD) and microglial-selective CatC overexpression (CatCOE) showed a worsening of the demyelination in Plp4e/- transgenic mice. Conversely, during the chronic demyelinating phase, CatC knockdown (CatCKD) ameliorated the demyelination. Our results suggest that the balance between CatC and CysF expression controls the demyelination and remyelination process.

DOI： 10.1002/glia.23134

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

We examined the morphological features of corticotropin-releasing factor (CRF) neurons in a mouse line in which modified yellow fluorescent protein (Venus) was expressed under the CRF promoter. We previously generated the CRF-Venus knock-in mouse, in which Venus is inserted into the CRF gene locus by homologous recombination. In the present study, the neomycin phosphotransferase gene (Neo), driven by the pgk-1 promoter, was deleted from the CRF-Venus mouse genome, and a CRF-Venus∆Neo mouse was generated. Venus expression is much more prominent in the CRF-Venus∆Neo mouse when compared to the CRF-Venus mouse. In addition, most Venus-expressing neurons co-express CRF mRNA. Venus-expressing neurons constitute a discrete population of neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) that project to the median eminence. Venus-expressing neurons were also found in brain regions outside the neuroendocrine PVH, including the olfactory bulb, the piriform cortex (Pir), the extended amygdala, the hippocampus, the neocortices, Barrington's nucleus, the midbrain/pontine dorsal tegmentum, the periaqueductal gray, and the inferior olivary nucleus (IO). Venus-expressing perikarya co-expressing CRF mRNA could be observed clearly even in regions where CRF-immunoreactive perikarya could hardly be identified. We demonstrated that the CRF neurons contain glutamate in the Pir and IO, while they contain gamma-aminobutyric acid in the neocortex, the bed nucleus of the stria terminalis, the hippocampus, and the amygdala. A population of CRF neurons was demonstrated to be cholinergic in the midbrain tegmentum. The CRF-Venus∆Neo mouse may be useful for studying the structural and functional properties of CRF neurons in the mouse brain.

DOI： 10.1007/s00429-016-1303-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Oligodendrocytes (OLs) are myelinating cells of the central nervous system. Recent studies have shown that mechanical factors influence various cell properties. Mechanical stimuli can be transduced into intracellular biochemical signals through mechanosensors and intracellular mechanotransducers, such as YAP. However, the molecular mechanisms underlying mechanical regulation of OLs by YAP remain unknown. We found that OL morphology and interactions between OLs and neuronal axons were affected by knocking down YAP. Mechanical stretching of OL precursor cells induced nuclear YAP accumulation and assembly of focal adhesion, key platforms for mechanotransduction. Shear stress decreased the number of OL processes, whereas a dominant-negative form of YAP suppressed these effects. To investigate the roles of YAP in postnatal OLs in vivo, we constructed a novel YAP knock-in mouse and found that in vivo overexpression of YAP widely affected OL maturation. These results indicate that YAP regulates OL morphology and maturation in response to mechanical factors. GLIA 2017;65:360-374.

DOI： 10.1002/glia.23096

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PKN2, a member of the protein kinase N (PKN) family, has been suggested by in vitro culture cell experiments to bind to Rho/Rac GTPases and contributes to cell-cell contact and cell migration. To unravel the in vivo physiological function of PKN2, we targeted the PKN2 gene. Constitutive disruption of the mouse PKN2 gene resulted in growth retardation and lethality before embryonic day (E) 10.5. PKN2-/- embryo did not undergo axial turning and showed insufficient closure of the neural tube. Mouse embryonic fibroblasts (MEFs) derived from PKN2-/- embryos at E9.5 failed to grow. Cre-mediated ablation of PKN2 in PKN2flox/flox MEFs obtained from E14.5 embryos showed impaired cell proliferation, and cell cycle analysis of these MEFs showed a decrease in S-phase population. Our results show that PKN2 is essential for mouse embryonic development and cell-autonomous proliferation of primary MEFs in culture. Comparison of the PKN2-/- phenotype with the phenotypes of PKN1 and PKN3 knockout strains suggests that PKN2 has distinct nonredundant functions in vivo, despite the structural similarity and evolutionary relationship among the three isoforms.

DOI： 10.1111/gtc.12470

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

AMPA glutamate receptor complexes with fast kinetics conferred by subunits like GluA3 and GluA4 are essential for temporal precision of synaptic transmission. The specific role of GluA3 in auditory processing and experience related changes in the auditory brainstem remain unknown. We investigated the role of the GluA3 in auditory processing by using wild type (WT) and GluA3 knockout (GluA3-KO) mice. We recorded auditory brainstem responses (ABR) to assess auditory function and used electron microscopy to evaluate the ultrastructure of the auditory nerve synapse on bushy cells (AN-BC synapse). Since labeling for GluA3 subunit increases on auditory nerve synapses within the cochlear nucleus in response to transient sound reduction, we investigated the role of GluA3 in experience-dependent changes in auditory processing. We induced transient sound reduction by plugging one ear and evaluated ABR threshold and peak amplitude recovery for up to 60 days after ear plug removal in WT and GluA3-KO mice. We found that the deletion of GluA3 leads to impaired auditory signaling that is reflected in decreased ABR peak amplitudes, an increased latency of peak 2, early onset hearing loss and reduced numbers and sizes of postsynaptic densities (PSDs) of AN-BC synapses. Additionally, the lack of GluA3 hampers ABR threshold recovery after transient ear plugging. We conclude that GluA3 is required for normal auditory signaling, normal ultrastructure of AN-BC synapses in the cochlear nucleus and normal experience-dependent changes in auditory processing after transient sound reduction.

DOI： 10.1016/j.heares.2016.12.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Sleep is conserved from invertebrates to vertebrates, and is tightly regulated in a homeostatic manner. The molecular and cellular mechanisms that determine the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remain unknown. Here we identify two dominant mutations that affect sleep and wakefulness by using an electroencephalogram/electromyogram-based screen of randomly mutagenized mice. A splicing mutation in the Sik3 protein kinase gene causes a profound decrease in total wake time, owing to an increase in inherent sleep need. Sleep deprivation affects phosphorylation of regulatory sites on the kinase, suggesting a role for SIK3 in the homeostatic regulation of sleep amount. Sik3 orthologues also regulate sleep in fruitflies and roundworms. A missense, gain-of-function mutation in the sodium leak channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the use of a forward-genetics approach for studying sleep behaviours in mice, and demonstrate the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.

DOI： 10.1038/nature20142

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

The cerebellum is thought to be involved in cognitive functions in addition to its well established role in motor coordination and motor learning in humans. Cerebellin 1 (Cbln1) is predominantly expressed in cerebellar granule cells and plays a crucial role in the formation and function of parallel fiber-Purkinje cell synapses. Although genes encoding Cbln1 and its postsynaptic receptor, the delta2 glutamate receptor (GluD2), are suggested to be associated with autistic-like traits and many psychiatric disorders, whether such cognitive impairments are caused by cerebellar dysfunction remains unclear. In the present study, we investigated whether and how Cbln1 signaling is involved in non-motor functions in adult mice. We show that acquisition and retention/retrieval of cued and contextual fear memory were impaired in Cbln1-null mice. In situ hybridization and immunohistochemical analyses revealed that Cbln1 is expressed in various extracerebellar regions, including the retrosplenial granular cortex and the hippocampus. In the hippocampus, Cbln1 immunoreactivity was present at the molecular layer of the dentate gyrus and the stratum lacunosum-moleculare without overt mRNA expression, suggesting that Cbln1 is provided by perforant path fibers. Retention/retrieval, but not acquisition, of cued and contextual fear memory was impaired in forebrain-predominant Cbln1-null mice. Spatial learning in the radial arm water maze was also abrogated. In contrast, acquisition of fear memory was affected in cerebellum-predominant Cbln1-null mice. These results indicate that Cbln1 in the forebrain and cerebellum mediates specific aspects of fear conditioning and spatial memory differentially and that Cbln1 signaling likely regulates motor and non-motor functions in multiple brain regions. SIGNIFICANCE STATEMENT: Despites its well known role in motor coordination and motor learning, whether and how the cerebellum is involved in cognitive functions remains less clear. Cerebellin 1 (Cbln1) is highly expressed in the cerebellum and serves as an essential synaptic organizer. Although genes encoding Cbln1 and its receptor are associated with many psychiatric disorders, it remains unknown whether such cognitive impairments are caused by cerebellar dysfunction. Here, we show that Cbln1 is also expressed in the forebrain, including the hippocampus and retrosplenial granular cortex. Using forebrain- and cerebellum-predominant conditional Cbln1-null mice, we show that Cbln1 in the forebrain and cerebellum mediates specific aspects of fear conditioning and spatial memory differentially, indicating that Cbln1 signaling regulates both motor and non-motor functions in multiple brain regions.

DOI： 10.1523/JNEUROSCI.0322-16.2016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Short-term synaptic depression (STD) is a common form of activity-dependent plasticity observed widely in the nervous system. Few molecular pathways that control STD have been described, but the active zone (AZ) release apparatus provides a possible link between neuronal activity and plasticity. Here, we show that an AZ cytomatrix protein CAST and an AZ-associated protein kinase SAD-B coordinately regulate STD by controlling reloading of the AZ with release-ready synaptic vesicles. SAD-B phosphorylates the N-terminal serine (S45) of CAST, and S45 phosphorylation increases with higher firing rate. A phosphomimetic CAST (S45D) mimics CAST deletion, which enhances STD by delaying reloading of the readily releasable pool (RRP), resulting in a pool size decrease. A phosphonegative CAST (S45A) inhibits STD and accelerates RRP reloading. Our results suggest that the CAST/SAD-B reaction serves as a brake on synaptic transmission by temporal calibration of activity and synaptic depression via RRP size regulation.

DOI： 10.1016/j.celrep.2016.08.020

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Society for Neuroscience  

Maintenance of neuropathic pain caused by peripheral nerve injury crucially depends on the phosphorylation of GluN2B, a subunit of the N-methyl-d-aspartate (NMDA) receptor, at Tyr1472 (Y1472) and subsequent formation of a postsynaptic density (PSD) complex of superficial spinal dorsal horn neurons. Here we took advantage of comparative proteomic analysis based on isobaric stable isotope tags (iTRAQ) between wild-type and knock-in mice with a mutation of Y1472 to Phe of GluN2B (Y1472F-KI) to search for PSD proteins in the spinal dorsal horn that mediate the signaling downstream of phosphorylated Y1472 GluN2B. Among several candidate proteins, we focused on brain-enriched guanylate kinase-associated protein (BEGAIN), which was specifically up-regulated in wild-type mice after spared nerve injury (SNI). Immunohistochemical analysis using the generated antibody demonstrated that BEGAIN was highly localized at the synapse of inner lamina II in the spinal dorsal horn and that its expression was up-regulated after SNI in wild-type, but not in Y1472F-KI, mice. In addition, alteration of the kinetics of evoked excitatory postsynaptic currents for NMDA but not those for _-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in spinal lamina II was demonstrated by BEGAIN deletion. We demonstrated that mechanical allodynia, a condition of abnormal pain induced by innocuous stimuli, in the SNI model was significantly attenuated in BEGAIN-deficient mice. However, there was no significant difference between naive wild-type and BEGAIN-knockout mice in terms of physiological threshold for mechanical stimuli. These results suggest that BEGAIN was involved in pathological pain transmission through NMDA receptor activation by the phosphorylation of GluN2B at Y1472 in spinal inner lamina II.

DOI： 10.1523/ENEURO.0110-16.2016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

Nine outer doublet microtubules in axonemes of flagella and cilia are heterogeneous in structure and biochemical properties. In mammalian sperm flagella, one of the factors to generate the heterogeneity is tubulin polyglutamylation, although the importance of the heterogeneous modification is unclear. Here, we show that a tubulin polyglutamylase Ttll9 deficiency (Ttll9(-/-)) causes a unique set of phenotypes related to doublet heterogeneity. Ttll9(-/-) sperm axonemes had frequent loss of a doublet and reduced polyglutamylation. Intriguingly, the doublet loss selectively occurred at the distal region of doublet 7, and reduced polyglutamylation was observed preferentially on doublet 5. Ttll9(-/-) spermatozoa showed aberrant flagellar beating, characterized by frequent stalls after anti-hook bending. This abnormal motility could be attributed to the reduction of polyglutamylation on doublet 5, which probably occurred at a position involved in the switching of bending. These results indicate that mammalian Ttll9 plays essential roles in maintaining the normal structure and beating pattern of sperm flagella by establishing normal heterogeneous polyglutamylation patterns.

DOI： 10.1242/jcs.185983

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Language：Japanese   Publisher：(一社)日本神経精神薬理学会  

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Postsynaptic kainate-type glutamate receptors (KARs) regulate synaptic network activity through their slow channel kinetics, most prominently at mossy fiber (MF)-CA3 synapses in the hippocampus. Nevertheless, how KARs cluster and function at these synapses has been unclear. Here, we show that C1q-like proteins C1ql2 and C1ql3, produced by MFs, serve as extracellular organizers to recruit functional postsynaptic KAR complexes to the CA3 pyramidal neurons. C1ql2 and C1ql3 specifically bound the amino-terminal domains of postsynaptic GluK2 and GluK4 KAR subunits and the presynaptic neurexin 3 containing a specific sequence in vitro. In C1ql2/3 double-null mice, CA3 synaptic responses lost the slow, KAR-mediated components. Furthermore, despite induction of MF sprouting in a temporal lobe epilepsy model, KARs were not recruited to postsynaptic sites in C1ql2/3 double-null mice, leading to reduced recurrent circuit activities. C1q family proteins, broadly expressed, are likely to modulate KAR function throughout the brain and represent promising antiepileptic targets.

DOI： 10.1016/j.neuron.2016.04.001

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UNLABELLED: The number of AMPA-type glutamate receptors (AMPARs) at synapses is the major determinant of synaptic strength and varies from synapse to synapse. To clarify the underlying molecular mechanisms, the density of AMPARs, PSD-95, and transmembrane AMPAR regulatory proteins (TARPs) were compared at Schaffer collateral/commissural (SCC) synapses in the adult mouse hippocampal CA1 by quantitative immunogold electron microscopy using serial sections. We examined four types of SCC synapses: perforated and nonperforated synapses on pyramidal cells and axodendritic synapses on parvalbumin-positive (PV synapse) and pravalbumin-negative interneurons (non-PV synapse). SCC synapses were categorized into those expressing high-density (perforated and PV synapses) or low-density (nonperforated and non-PV synapses) AMPARs. Although the density of PSD-95 labeling was fairly constant, the density and composition of TARP isoforms was highly variable depending on the synapse type. Of the three TARPs expressed in hippocampal neurons, the disparity in TARP γ-2 labeling was closely related to that of AMPAR labeling. Importantly, AMPAR density was significantly reduced at perforated and PV synapses in TARP γ-2-knock-out (KO) mice, resulting in a virtual loss of AMPAR disparity among SCC synapses. In comparison, TARP γ-8 was the only TARP expressed at nonperforated synapses, where AMPAR labeling further decreased to a background level in TARP γ-8-KO mice. These results show that synaptic inclusion of TARP γ-2 potently increases AMPAR expression and transforms low-density synapses into high-density ones, whereas TARP γ-8 is essential for low-density or basal expression of AMPARs at nonperforated synapses. Therefore, these TARPs are critically involved in AMPAR density control at SCC synapses. SIGNIFICANCE STATEMENT: Although converging evidence implicates the importance of transmembrane AMPA-type glutamate receptor (AMPAR) regulatory proteins (TARPs) in AMPAR stabilization during basal transmission and synaptic plasticity, how they control large disparities in AMPAR numbers or densities across central synapses remains largely unknown. We compared the density of AMPARs with that of TARPs among four types of Schaffer collateral/commissural (SCC) hippocampal synapses in wild-type and TARP-knock-out mice. We show that the density of AMPARs correlates with that of TARP γ-2 across SCC synapses and its high expression is linked to high-density AMPAR expression at perforated type of pyramidal cell synapses and synapses on parvalbumin-positive interneurons. In comparison, TARP γ-8 is the only TARP expressed at nonperforated type of pyramidal cell synapses, playing an essential role in low-density or basal AMPAR expression.

DOI： 10.1523/JNEUROSCI.4178-15.2016

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Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KAR subunits are predominant in the brain, with distinct subunit combinations between the hippocampus and cerebellum. Kainate receptors, an unconventional member of the iGluR receptor family, have a tetrameric structure assembled from low-affinity (GluK1-3) and high-affinity (GluK4 and GluK5) subunits. We used a simple but novel procedure to measure the relative abundance of both low- and high-affinity subunits. This method revealed that the relative amount of GluK2 plus GluK3 subunits was several times higher than that of GluK4 plus GluK5 subunits, in both the hippocampus and cerebellum.

DOI： 10.1111/jnc.13384

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The small GTPase-effector proteins CDC42EP1-5/BORG1-5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4(-/-) mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (γDGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1-5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance.

DOI： 10.1038/ncomms10090

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

DOI： 10.1016/j.neuroscience.2015.06.064

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The transcription factor STAT5, which is activated by IL-7R, controls chromatin accessibility and rearrangements of the TCRγ locus. Although STAT-binding motifs are conserved in Jγ promoters and Eγ enhancers, little is known about their precise roles in rearrangements of the TCRγ locus in vivo. To address this question, we established two lines of Jγ1 promoter mutant mice: one harboring a deletion in the Jγ1 promoter, including three STAT motifs (Jγ1P(Δ/Δ)), and the other carrying point mutations in the three STAT motifs in that promoter (Jγ1P(mS/mS)). Both Jγ1P(Δ/Δ) and Jγ1P(mS/mS) mice showed impaired recruitment of STAT5 and chromatin remodeling factor BRG1 at the Jγ1 gene segment. This resulted in severe and specific reduction in germline transcription, histone H3 acetylation, and histone H4 lysine 4 methylation of the Jγ1 gene segment in adult thymus. Rearrangement and DNA cleavage of the segment were severely diminished, and Jγ1 promoter mutant mice showed profoundly decreased numbers of γδ T cells of γ1 cluster origin. Finally, compared with controls, both mutant mice showed a severe reduction in rearrangements of the Jγ1 gene segment, perturbed development of γδ T cells of γ1 cluster origin in fetal thymus, and fewer Vγ3(+) dendritic epidermal T cells. Furthermore, interaction with the Jγ1 promoter and Eγ1, a TCRγ enhancer, was dependent on STAT motifs in the Jγ1 promoter. Overall, this study strongly suggests that direct binding of STAT5 to STAT motifs in the Jγ promoter is essential for local chromatin accessibility and Jγ/Eγ chromatin interaction, triggering rearrangements of the TCRγ locus.

DOI： 10.4049/jimmunol.1302456

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In the mammalian olfactory epithelium (OE), olfactory receptor neurons (ORNs) are continuously regenerated throughout the animal's lifetime. Horizontal basal cells (HBCs) in the OE express the epithelial marker keratin 5 (K5) and the stem cell marker Pax6 and are considered relatively quiescent tissue stem cells in the OE. Pax6 is a key regulator of several developmental processes in the central nervous system and in sensory organs. Although Pax6 is expressed in the OE, its precise role remains unknown, particularly with respect to stem cell-like HBCs. To investigate the function of Pax6 in the developmental and regenerative processes in the OE, we generated conditional Pax6-knockout mice carrying a loxP-floxed Pax6 gene. Homozygous Pax6-floxed mice were crossed with K5-Cre transgenic mice to generate HBC-specific Pax6-knockout (Pax6-cKO) mice. We confirmed that the deletion of Pax6 expression in HBCs was sufficiently achieved in zone 1 of the OE in Pax6-cKO mice 3 days after methimazole-induced severe damage. In this condition, regeneration of the OE was dramatically impaired; both OE thickness and the number of ORNs were significantly decreased in the regenerated OE of Pax6-cKO mice. These results suggest that Pax6 expression is essential for HBCs to differentiate into neuronal cells during the regeneration process following severe injury.

DOI： 10.1089/scd.2015.0011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Neuropeptide B and W (NPB and NPW) are cognate peptide ligands for NPBWR1 (GPR7), a G protein-coupled receptor. In rodents, they have been implicated in the regulation of energy homeostasis, neuroendocrine/autonomic responses, and social interactions. Although localization of these peptides and their receptors in adult rodent brain has been well documented, their expression in mouse brain during development is unknown. Here we demonstrate the transient expression of NPW mRNA in the dorsomedial hypothalamus (DMH) of postnatal mouse brain and its co-localization with neuropeptide Y (NPY) mRNA. Neurons expressing both NPW and NPY mRNAs begin to emerge in the DMH at about postnatal day 0 (P-0) through P-3. Their expression is highest around P-14, declines after P-21, and by P-28 only a faint expression of NPW and NPY mRNA remains. In P-18 brains, we detected NPW neurons in the region spanning the subincertal nucleus (SubI), the lateral hypothalamic (LH) perifornical (PF) areas, and the DMH, where the highest expression of NPW mRNA was observed. The majority of these postnatal hypothalamic NPW neurons co-express NPY mRNA. A cross of NPW-iCre knock-in mice with a Cre-dependent tdTomato reporter line revealed that more than half of the reporter-positive neurons in the adult DMH, which mature from the transiently NPW-expressing neurons, are sensitive to peripherally administrated leptin. These data suggest that the DMH neurons that transiently co-express NPW and NPY in the peri-weaning period might play a role in regulating energy homeostasis during postnatal development. (C) 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.neuroscience.2015.06.014

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SWI/SNF chromatin remodeling complexes constitute a highly related family of multi-subunit complexes to modulate transcription, and SWI/SNF subunit genes are collectively mutated in 20% of all human cancers. Bcl11b is a SWI/SNF subunit and acts as a haploinsufficient tumor suppressor in leukemia/lymphomas. Here, we show expression of Bcl11b in intestinal crypt cells and promotion of intestinal tumorigenesis by Bcl11b attenuation in Apc (min/+) mice. Of importance, mutations or allelic loss of BCL11B was detected in one-third of human colon cancers. We also show that attenuated Bcl11b activity in the crypt base columnar (CBC) cells expressing the Lgr5 stem cell marker enhanced regeneration of intestinal epithelial cells after the radiation-induced injury. Interestingly, BCL11B introduction in human cell lines downregulated transcription of β-catenin target genes, whereas Bcl11b attenuation in Lgr5(+) CBCs increased expression of β-catenin targets including c-Myc and cyclin D1. Together, our results argue that Bcl11b impairment promotes tumor development in mouse and human intestine at least in part through deregulation of β-catenin pathway.

DOI： 10.1093/carcin/bgv044

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Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases.

DOI： 10.1002/eji.201444977

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If Bcl11b activity is compromised, CD4(+)CD8(+) double-positive (DP) thymocytes produce a greatly increased fraction of innate CD8(+) single-positive (SP) cells highly producing IFN-γ, which are also increased in mice deficient of genes such as Itk, Id3 and NF-κB1 that affect TCR signaling. Of interest, the increase in the former two is due to the bystander effect of IL-4 that is secreted by promyelocytic leukemia zinc finger-expressing NKT and γδT cells whereas the increase in the latter is cell intrinsic. Bcl11b zinc-finger proteins play key roles in T cell development and T cell-mediated immune response likely through TCR signaling. We examined thymocytes at and after the DP stage in Bcl11b (F/S826G) CD4cre, Bcl11b (F/+) CD4cre and Bcl11b (+/S826G) mice, carrying the allele that substituted serine for glycine at the position of 826. Here we show that Bcl11b impairment leads to an increase in the population of TCRαβ(high)CD44(high)CD122(high) innate CD8SP thymocytes, together with two different developmental abnormalities: impaired positive and negative selection accompanying a reduction in the number of CD8SP cells, and developmental arrest of NKT cells at multiple steps. The innate CD8SP thymocytes express Eomes and secrete IFN-γ after stimulation with PMA and ionomycin, and in this case their increase is not due to a bystander effect of IL-4 but cell intrinsic. Those results indicate that Bcl11b regulates development of different thymocyte subsets at multiple stages and prevents an excess of innate CD8SP thymocytes.

DOI： 10.1093/intimm/dxu104

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Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as a crucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesion G-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions.

DOI： 10.1016/j.neuron.2014.12.020

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Mutations in the Autism susceptibility candidate 2 gene (AUTS2) have been associated with a broad range of psychiatric illnesses including autism spectrum disorders, intellectual disability and schizophrenia. We previously demonstrated that the cytoplasmic AUTS2 acts as an upstream factor for the Rho family small GTPase Rac1 and Cdc42 that regulate the cytoskeletal rearrangements in neural cells. Moreover, genetic ablation of the Auts2 gene in mice has resulted in defects in neuronal migration and neuritogenesis in the developing cerebral cortex caused by inactivation of Rac1-signaling pathway, suggesting that AUTS2 is required for neural development. In this study, we conducted a battery of behavioral analyses on Auts2 heterozygous mutant mice to examine the involvement of Auts2 in adult cognitive brain functions. Auts2-deficient mice displayed a decrease in exploratory behavior as well as lower anxiety-like behaviors in the absence of any motor dysfunction. Furthermore, the capability for novel object recognition and cued associative memory were impaired in Auts2 mutant mice. Social behavior and sensory motor gating functions were, however, normal in the mutant mice as assessed by the three-chamber test and prepulse inhibition test, respectively. Together, our findings indicate that AUTS2 is critical for the acquisition of neurocognitive function.

DOI： 10.1371/journal.pone.0145979

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Mutations in the Autism susceptibility candidate 2 gene (AUTS2), whose protein is believed to act in neuronal cell nuclei, have been associated with multiple psychiatric illnesses, including autism spectrum disorders, intellectual disability, and schizophrenia. Here we show that cytoplasmic AUTS2 is involved in the regulation of the cytoskeleton and neural development. Immunohistochemistry and fractionation studies show that AUTS2 localizes not only in nuclei, but also in the cytoplasm, including in the growth cones in the developing brain. AUTS2 activates Rac1 to induce lamellipodia but downregulates Cdc42 to suppress filopodia. Our loss-of-function and rescue experiments show that a cytoplasmic AUTS2-Rac1 pathway is involved in cortical neuronal migration and neuritogenesis in the developing brain. These findings suggest that cytoplasmic AUTS2 acts as a regulator of Rho family GTPases to contribute to brain development and give insight into the pathology of human psychiatric disorders with AUTS2 mutations.

DOI： 10.1016/j.celrep.2014.11.045

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The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes of protein and organelle clearance. The role of the proteasome pathway in mammalian muscle has not been examined in vivo. In this study, we report that the muscle-specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4), resulted in profound muscle growth defects and a decrease in force production in mice. Specifically, developing muscles in conditional Rpt3-knockout animals showed dysregulated proteasomal activity. The autophagy pathway was upregulated, but the process of autophagosome formation was impaired. A microscopic analysis revealed the accumulation of basophilic inclusions and disorganization of the sarcomeres in young adult mice. Our results suggest that appropriate proteasomal activity is important for muscle growth and for maintaining myofiber integrity in collaboration with autophagy pathways. The deletion of a component of the proteasome complex contributed to myofiber degeneration and weakness in muscle disorders that are characterized by the accumulation of abnormal inclusions.

DOI： 10.1242/jcs.150961

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

IL-1 receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic intellectual disability and is associated with autism. IL1RAPL1 mediates excitatory synapse formation through trans-synaptic interaction with PTPδ. Here, we showed that the spine density of cortical neurons was significantly reduced in IL1RAPL1 knockout mice. The spatial reference and working memories and remote fear memory were mildly impaired in IL1RAPL1 knockout mice. Furthermore, the behavioural flexibility was slightly reduced in the T-maze test. Interestingly, the performance of IL1RAPL1 knockout mice in the rotarod test was significantly better than that of wild-type mice. Moreover, IL1RAPL1 knockout mice consistently exhibited high locomotor activity in all the tasks examined. In addition, open-space and height anxiety-like behaviours were decreased in IL1RAPL1 knockout mice. These results suggest that IL1RAPL1 ablation resulted in spine density decrease and affected not only learning but also behavioural flexibility, locomotor activity and anxiety.

DOI： 10.1038/srep06613

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ENDOCRINE SOC  

Corticotropin-releasing factor (CRF) is the key regulator of the hypothalamic-pituitary-adrenal axis. CRF neurons cannot be distinguished morphologically from other neuroendocrine neurons in the paraventricular nucleus of the hypothalamus (PVH) without immunostaining. Thus, we generated a knock-in mouse that expresses modified yellow fluorescent protein (Venus) in CRF neurons (CRF-Venus), and yet its expression is driven by the CRF promoter and responds to changes in the interior milieu. In CRF-Venus, Venus-expressing neurons were distributed in brain regions harboring CRF neurons, including the PVH. The majority of Venus-expressing neurons overlapped with CRF-expressing neurons in the PVH, but many neurons expressed only Venus or CRF in a physiological glucocorticoid condition. After glucocorticoid deprivation, however, Venus expression intensified, and most Venus neurons coexpressed CRF. Conversely, Venus expression was suppressed by excess glucocorticoids. Expression of copeptin, a peptide encoded within the vasopressin gene, was induced in PVH-Venus neurons by glucocorticoid deprivation and suppressed by glucocorticoid administration. Thus, Venus neurons recapitulated glucocorticoid-dependent vasopressin expression in PVH-CRF neurons. Noradrenaline increased the frequency of glutamate-dependent excitatory postsynaptic currents recorded from Venus-expressing neurons in the voltage clamp mode. In addition, the CRF-iCre knock-in mouse was crossed with a CAG-CAT-EGFP reporter mouse to yield the Tg(CAG-CAT-EGFP/wt);CRF(iCre/wt) (EGFP/CRF-iCre) mouse, in which enhanced green fluorescent protein (EGFP) is driven by the CAG promoter. EGFP was expressed more constitutively in the PVH of EGFP/CRF-iCre mice. Thus, CRF-Venus may have an advantage for monitoring dynamic changes in CRF neurons and CRF networks in different glucocorticoid states.

DOI： 10.1210/en.2014-1182

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Previous studies have implicated the role of Purkinje cells in motor learning and the underlying mechanisms have also been identified in great detail during the last decades. Here we report that cyclin-dependent kinase 5 (Cdk5)/p35 in Purkinje cell also contributes to synaptic plasticity. We previously showed that p35(-/-) (p35 KO) mice exhibited a subtle abnormality in brain structure and impaired spatial learning and memory. Further behavioral analysis showed that p35 KO mice had a motor coordination defect, suggesting that p35, one of the activators of Cdk5, together with Cdk5 may play an important role in cerebellar motor learning. Therefore, we created Purkinje cell-specific conditional Cdk5/p35 knockout (L7-p35 cKO) mice, analyzed the cerebellar histology and Purkinje cell morphology of these mice, evaluated their performance with balance beam and rota-rod test, and performed electrophysiological recordings to assess long-term synaptic plasticity. Our analyses showed that Purkinje cell-specific deletion of Cdk5/p35 resulted in no changes in Purkinje cell morphology but severely impaired motor coordination. Furthermore, disrupted cerebellar long-term synaptic plasticity was observed at the parallel fiber-Purkinje cell synapse in L7-p35 cKO mice. These results indicate that Cdk5/p35 is required for motor learning and involved in long-term synaptic plasticity.

DOI： 10.1111/jnc.12756

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Tandem duplications are concentrated within the Pcdh cluster throughout vertebrate evolution and as copy number variations (CNVs) in human populations, but the effects of tandem duplication in the Pcdh cluster remain elusive. To investigate the effects of tandem duplication in the Pcdh cluster, here we generated and analyzed a new line of the Pcdh cluster mutant mice. In the mutant allele, a 218-kb region containing the Pcdh-α2 to Pcdh-αc2 variable exons with their promoters was duplicated and the individual duplicated Pcdh isoforms can be disctinguished. The individual duplicated Pcdh-α isoforms showed diverse expression level with stochastic expression manner, even though those have an identical promoter sequence. Interestingly, the 5'-located duplicated Pcdh-αc2, which is constitutively expressed in the wild-type brain, shifted to stochastic expression accompanied by increased DNA methylation. These results demonstrate that tandem duplication in the Pcdh cluster expands the stochastic expression repertoire irrespective of sequence divergence.

DOI： 10.1038/srep06263

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Aldolase C (Aldoc, also known as "zebrin II"), a brain type isozyme of a glycolysis enzyme, is expressed heterogeneously in subpopulations of cerebellar Purkinje cells (PCs) that are arranged longitudinally in a complex striped pattern in the cerebellar cortex, a pattern which is closely related to the topography of input and output axonal projections. Here, we generated knock-in Aldoc-Venus mice in which Aldoc expression is visualized by expression of a fluorescent protein, Venus. Since there was no obvious phenotypes in general brain morphology and in the striped pattern of the cerebellum in mutants, we made detailed observation of Aldoc expression pattern in the nervous system by using Venus expression in Aldoc-Venus heterozygotes. High levels of Venus expression were observed in cerebellar PCs, cartwheel cells in the dorsal cochlear nucleus, sensory epithelium of the inner ear and in all major types of retinal cells, while moderate levels of Venus expression were observed in astrocytes and satellite cells in the dorsal root ganglion. The striped arrangement of PCs that express Venus to different degrees was carefully traced with serial section alignment analysis and mapped on the unfolded scheme of the entire cerebellar cortex to re-identify all individual Aldoc stripes. A longitudinally striped boundary of Aldoc expression was first identified in the mouse flocculus, and was correlated with the climbing fiber projection pattern and expression of another compartmental marker molecule, heat shock protein 25 (HSP25). As in the rat, the cerebellar nuclei were divided into the rostrodorsal negative and the caudoventral positive portions by distinct projections of Aldoc-positive and negative PC axons in the mouse. Identification of the cerebellar Aldoc stripes in this study, as indicated in sample coronal and horizontal sections as well as in sample surface photos of whole-mount preparations, can be referred to in future experiments.

DOI： 10.1371/journal.pone.0086679

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Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.

DOI： 10.1074/jbc.M113.504050

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Bcl11b is a haploinsufficient tumor suppressor, mutations or deletion of which has been found in 10-16% of T-cell acute lymphoblastic leukemias. Bcl11bKO/+ heterozygous mice are susceptible to thymic lymphomas, a model of T-cell acute lymphoblastic leukemia, when γ-irradiated, and irradiated Bcl11bKO/+ mice generate clonally expanding or premalignant thymocytes before thymic lymphoma development. Cells with radiation-induced DNA damages are assumed to be the cells of origin in tumors
however, which thymocyte is the tumor cell origin remains obscure. In this study we generated Bcl11bflox/+
Lck-Cre and Bcl11bflox/+
CD4-Cre mice
in the former, loss of one Bcl11b allele occurs in thymocytes at the immature CD4-CD8- stage, whereas in the latter the loss occurs in the more differentiated CD4+CD8+ double-positive stage. We examined clonal expansion and differentiation of thymocytes in mice 60 days after 3 Gy γ-irradiation. Half (9/18) of the thymuses in the Bcl11bflox/+
Lck-Cre group showed limited rearrangement sites at the T-cell receptor-β (TCRβ) locus, indicating clonal cell expansion, but none in the Bcl11bflox/+
CD4-Cre group did. This indicates that the origin of the premalignant thymocytes is not in double-positive cells but immature thymocytes. Interestingly, those premalignant thymocytes underwent rearrangement at various different sites of the TCRα locus and the majority showed a higher expression of TCRβ and CD8, and more differentiated phenotypes. This suggests the existence of a subpopulation of immature cells within the premalignant cells that is capable of proliferating and continuously producing differentiated thymocytes. © 2013 Japanese Cancer Association.

DOI： 10.1111/cas.12193

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

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Populations of neurons in the hypothalamic preoptic area (POA) fire rapidly during sleep, exhibiting sleep/waking state-dependent firing patterns that are the reciprocal of those observed in the arousal system. The majority of these preoptic "sleep-active" neurons contain the inhibitory neurotransmitter GABA. On the other hand, a population of neurons in the lateral hypothalamic area (LHA) contains orexins, which play an important role in the maintenance of wakefulness, and exhibit an excitatory influence on arousal-related neurons. It is important to know the anatomical and functional interactions between the POA sleep-active neurons and orexin neurons, both of which play important, but opposite roles in regulation of sleep/wakefulness states. In this study, we confirmed that specific pharmacogenetic stimulation of GABAergic neurons in the POA leads to an increase in the amount of non-rapid eye movement (NREM) sleep. We next examined direct connectivity between POA GABAergic neurons and orexin neurons using channelrhodopsin 2 (ChR2) as an anterograde tracer as well as an optogenetic tool. We expressed ChR2-eYFP selectively in GABAergic neurons in the POA by AAV-mediated gene transfer, and examined the projection sites of ChR2-eYFP-expressing axons, and the effect of optogenetic stimulation of ChR2-eYFP on the activity of orexin neurons. We found that these neurons send widespread projections to wakefulness-related areas in the hypothalamus and brain stem, including the LHA where these fibers make close appositions to orexin neurons. Optogenetic stimulation of these fibers resulted in rapid inhibition of orexin neurons. These observations suggest direct connectivity between POA GABAergic neurons and orexin neurons.

DOI： 10.3389/fncir.2013.00192

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Evidence suggests that protein misfolding is crucially involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, controversy still exists regarding the involvement of proteasomes or autophagy in ALS due to previous conflicting results. Here, we show that impairment of the ubiquitin-proteasome system, but not the autophagy-lysosome system in motor neurons replicates ALS in mice. Conditional knock-out mice of the proteasome subunit Rpt3 in a motor neuron-specific manner (Rpt3-CKO) showed locomotor dysfunction accompanied by progressive motor neuron loss and gliosis. Moreover, diverse ALS-linked proteins, including TAR DNA-binding protein 43 kDa (TDP-43), fused in sarcoma (FUS), ubiquilin 2, and optineurin were mislocalized or accumulated in motor neurons, together with other typical ALS hallmarks such as basophilic inclusion bodies. On the other hand, motor neuron-specific knock-out of Atg7, a crucial component for the induction of autophagy (Atg7-CKO), only resulted in cytosolic accumulation of ubiquitin and p62, and no TDP-43 or FUS pathologies or motor dysfunction was observed. These results strongly suggest that proteasomes, but not autophagy, fundamentally govern the development of ALS in which TDP-43 and FUS proteinopathy may play a crucial role. Enhancement of proteasome activity may be a promising strategy for the treatment of ALS.

DOI： 10.1074/jbc.M112.417600

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The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic suppression. Although the mechanisms of 2-AG production are well characterized, how 2-AG is degraded is less clearly understood. Here we found that expression of the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MGL) was highly heterogeneous in the cerebellum, being rich within parallel fiber (PF) terminals, weak in Bergman glia (BG), and absent in other synaptic terminals. Despite this highly selective MGL expression pattern, 2-AG-mediated retrograde suppression was significantly prolonged at not only PF-Purkinje cell (PC) synapses but also climbing fiber-PC synapses in granule cell-specific MGL knockout (MGL-KO) mice whose cerebellar MGL expression was confined to the BG. Virus-mediated expression of MGL into the BG of global MGL-KO mice significantly shortened 2-AG-mediated retrograde suppression at PF-PC synapses. Furthermore, contribution of MGL to termination of 2-AG signaling depended on the distance from MGL-rich PFs to inhibitory synaptic terminals. Thus, 2-AG is degraded in a synapse-type independent manner by MGL present in PFs and the BG. The results of the present study strongly suggest that MGL regulates 2-AG signaling rather broadly within a certain range of neural tissue, although MGL expression is heterogeneous and limited to a subset of nerve terminals and astrocytes.

DOI： 10.1073/pnas.1204404109

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The Arc/Arg3.1 gene product is rapidly upregulated by strong synaptic activity and critically contributes to weakening synapses by promoting AMPA-R endocytosis. However, how activity-induced Arc is redistributed and determines the synapses to be weakened remains unclear. Here, we show targeting of Arc to inactive synapses via a high-affinity interaction with CaMKIIβ that is not bound to calmodulin. Synaptic Arc accumulates in inactive synapses that previously experienced strong activation and correlates with removal of surface GluA1 from individual synapses. A lack of CaMKIIβ either in vitro or in vivo resulted in loss of Arc upregulation in the silenced synapses. The discovery of Arc's role in "inverse" synaptic tagging that is specific for weaker synapses and prevents undesired enhancement of weak synapses in potentiated neurons reconciles essential roles of Arc both for the late phase of long-term plasticity and for reduction of surface AMPA-Rs in stimulated neurons.

DOI： 10.1016/j.cell.2012.02.062

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In the adult cerebellum, each Purkinje cell (PC) is innervated by a single climbing fiber (CF) in proximal dendrites and 10(5)-10(6) parallel fibers (PFs) in distal dendrites. This organized wiring is established postnatally through heterosynaptic competition between PFs and CFs and homosynaptic competition among multiple CFs. Using PC-specific Cav2.1 knock-out mice (PC-Cav2.1 KO mice), we have demonstrated recently that postsynaptic Cav2.1 plays a key role in the homosynaptic competition by promoting functional strengthening and dendritic translocation of single "winner" CFs. Here, we report that Cav2.1 in PCs, but not in granule cells, is also essential for the heterosynaptic competition. In PC-Cav2.1 KO mice, the extent of CF territory was limited to the soma and basal dendrites, whereas PF territory was expanded reciprocally. Consequently, the proximal somatodendritic domain of PCs displayed hyperspiny transformation and fell into chaotic innervation by multiple CFs and numerous PFs. PC-Cav2.1 KO mice also displayed patterned degeneration of PCs, which occurred preferentially in aldolase C/zebrin II-negative cerebellar compartments. Furthermore, the mutually complementary expression of phospholipase Cβ3 (PLCβ3) and PLCβ4 was altered such that their normally sharp boundary was blurred in the PCs of PC-Cav2.1 KO mice. This blurring was caused by an impaired posttranscriptional downregulation of PLCβ3 in PLCβ4-dominant PCs during the early postnatal period. A similar alteration was noted in the banded expression of the glutamate transporter EAAT4 in PC-Cav2.1 KO mice. Therefore, Cav2.1 in PCs is essential for competitive synaptic wiring, cell survival, and the establishment of precise boundaries and reciprocity of biochemical compartments in PCs.

DOI： 10.1523/JNEUROSCI.2755-11.2012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

CD8 T cells play a critical role in protection against viral infections. During effector differentiation, CD8 T cells dramatically change chromatin structure and cellular metabolism, but how energy production increases in response to these epigenetic changes is unknown. We found that loss of basic leucine zipper transcription factor, ATF-like (BATF) inhibited effector CD8 T-cell differentiation. At the late effector stage, BATF was induced by IL-12 and required for IL-12-mediated histone acetylation and survival of effector T cells. BATF, together with c-Jun, transcriptionally inhibited expression of the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase Sirt1, resulting in increased histone acetylation of the T-bet locus and increased cellular NAD(+), which increased ATP production. In turn, high levels of T-bet expression and ATP production promoted effector differentiation and cell survival. These results suggest that BATF promotes effector CD8 T-cell differentiation by regulating both epigenetic remodeling and energy metabolism through Sirt1 expression.

DOI： 10.1073/pnas.1105133108

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately five times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor δ2 (GluRδ2 or GluD2), which is known to be efficiently trafficked to and selectively expressed at PF synapses in PCs. We applied postembedding immunogold electron microscopy to GluRδ2 knock-out (KO) and control mice, and measured labeling density for GluA1-4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluRδ2-KO mice, GluA1-3 were localized at PF and CF synapses in PCs, while GluA2-4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1-3 was four to six times lower at PF-PC synapses than at CF-PC synapses. In GluRδ2-KO mice, however, their labeling density displayed a three- to fivefold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathway-dependent disparity between the two PC synapses. Furthermore, we found an unexpected twofold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluRδ2. These results suggest that GluRδ2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons.

DOI： 10.1523/JNEUROSCI.5601-10.2011

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Lysophosphatidic acid (LPA) is a potent lipid mediator with a wide variety of biological actions mediated through G protein-coupled receptors (LPA(1-6)). LPA(4) has been identified as a G(13) protein-coupled receptor, but its physiological role is unknown. Here we show that a subset of LPA(4)-deficient embryos did not survive gestation and displayed hemorrhages and/or edema in many organs at multiple embryonic stages. The blood vessels of bleeding LPA(4)-deficient embryos were often dilated. The recruitment of mural cells, namely smooth muscle cells and pericytes, was impaired. Consistently, Matrigel plug assays showed decreased mural cell coverage of endothelial cells in the neovessels of LPA(4)-deficient adult mice. In situ hybridization detected Lpa4 mRNA in the endothelium of some vasculatures. Similarly, the lymphatic vessels of edematous embryos were dilated. These results suggest that LPA(4) regulates establishment of the structure and function of blood and lymphatic vessels during mouse embryogenesis. Considering the critical role of autotaxin (an enzyme involved in LPA production) and Gα(13) in vascular development, we suggest that LPA(4) provides a link between these 2 molecules.

DOI： 10.1182/blood-2010-03-272443

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CS (chondroitin sulfate) is a glycosaminoglycan species that is widely distributed in the extracellular matrix. To understand the physiological roles of enzymes involved in CS synthesis, we produced CSGalNAcT1 (CS N-acetylgalactosaminyltransferase 1)-null mice. CS production was reduced by approximately half in CSGalNAcT1-null mice, and the amount of short-chain CS was also reduced. Moreover, the cartilage of the null mice was significantly smaller than that of wild-type mice. Additionally, type-II collagen fibres in developing cartilage were abnormally aggregated and disarranged in the homozygous mutant mice. These results suggest that CSGalNAcT1 is required for normal CS production in developing cartilage.

DOI： 10.1042/BJ20100847

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Calpains constitute a superfamily of Ca2+-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8(-/-) and Capn9(-/-) mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8(-/-) stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9(-/-). Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed "G-calpain," in which both were essential for activity. This is the first example of a "hybrid" calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S "knock-in" (Capn8(CS/CS)) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8(-/-) stomach, that of the Capn8(CS/CS) mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.

DOI： 10.1371/journal.pgen.1001040

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The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, gamma-2 and gamma-7 are the two major TARPs expressed in the cerebellum. In the present study, we pursued their roles in synaptic expression of cerebellar AMPA receptors. In the cerebellar cortex, gamma-2 and gamma-7 were preferentially localized at various asymmetrical synapses. Using quantitative Western blot and immunofluorescence, we found severe reductions in GluA2 and GluA3 and mild reduction in GluA4 in gamma-2-knockout (KO) cerebellum, whereas GluA1 and GluA4 were moderately reduced in gamma-7-KO cerebellum. GluA2, GluA3 and GluA4 were further reduced in gamma-2/gamma-7 double-KO (DKO) cerebellum. The large losses of GluA2 and GluA3 in gamma-2-KO mice and further reductions in DKO mice were confirmed at all asymmetrical synapses examined with postembedding immunogold. Most notably, the GluA2 level in the postsynaptic density fraction, GluA2 labeling density at parallel fiber-Purkinje cell synapses, and AMPA receptor-mediated currents at climbing fiber-Purkinje cell synapses were all reduced to approximately 10% of the wild-type levels in DKO mice. On the other hand, the reduction in GluA4 in gamma-7-KO granular layer reflected its loss at mossy fiber-granule cell synapses, whereas that of GluA1 and GluA4 in gamma-7-KO molecular layer was caused, at least partly, by their loss in Bergmann glia. Therefore, gamma-2 and gamma-7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and the latter also promotes glial expression.

DOI： 10.1111/j.1460-9568.2010.07254.x

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Endocannabinoids are released from postsynaptic neurons and cause retrograde suppression of synaptic transmission. Anandamide and 2-arachidonoylglycerol (2-AG) are regarded as two major endocannabinoids. To determine to what extent 2-AG contributes to retrograde signaling, we generated and analyzed mutant mice lacking either of the two 2-AG synthesizing enzymes diacylglycerol lipase alpha (DGLalpha) and beta (DGLbeta). We found that endocannabinoid-mediated retrograde synaptic suppression was totally absent in the cerebellum, hippocampus, and striatum of DGLalpha knockout mice, whereas the retrograde suppression was intact in DGLbeta knockout brains. The basal 2-AG content was markedly reduced and stimulus-induced elevation of 2-AG was absent in DGLalpha knockout brains, whereas the 2-AG content was normal in DGLbeta knockout brains. Morphology of the brain and expression of molecules required for 2-AG production other than DGLs were normal in the two knockout mice. We conclude that 2-AG produced by DGLalpha, but not by DGLbeta, mediates retrograde suppression at central synapses.

DOI： 10.1016/j.neuron.2010.01.021

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GluN2B (GluRepsilon2/NR2B) subunit is involved in synapse development, synaptic plasticity, and cognitive function. However, its roles in synaptic expression and function of NMDA receptors (NMDARs) in the brain remain mostly unknown because of the neonatal lethality of global knock-out mice. To address this, we generated conditional knock-out mice, in which GluN2B was ablated exclusively in hippocampal CA3 pyramidal cells. By immunohistochemistry, GluN2B disappeared and GluN1 (GluRzeta1/NR1) was moderately reduced, whereas GluN2A (GluRepsilon1/NR2A) and postsynaptic density-95 (PSD-95) were unaltered in the mutant CA3. This was consistent with protein contents in the CA3 crude fraction: 9.6% of control level for GluN2B, 47.7% for GluN1, 90.6% for GluN2A, and 98.0% for PSD-95. Despite the remaining NMDARs, NMDAR-mediated currents and long-term potentiation were virtually lost at various CA3 synapses. Then, we compared synaptic NMDARs by postembedding immunogold electron microscopy and immunoblot using the PSD fraction. In the mutant CA3, GluN1 was severely reduced in both immunogold (20.6-23.6%) and immunoblot (24.6%), whereas GluN2A and PSD-95 were unchanged in immunogold but markedly reduced in the PSD fraction (51.4 and 36.5%, respectively), indicating increased detergent solubility of PSD molecules. No such increased solubility was observed for GluN2B in the CA3 of GluN2A-knock-out mice. Furthermore, significant decreases were found in the ratio of filamentous to globular actin (49.5%) and in the density of dendritic spines (76.2%). These findings suggest that GluN2B is critically involved in NMDAR channel function, organization of postsynaptic macromolecular complexes, formation or maintenance of dendritic spines, and regulation of the actin cytoskeleton.

DOI： 10.1523/JNEUROSCI.5531-08.2009

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Cysteinyl leukotrienes (CysLTs) are potent mediators of inflammation synthesized by the concerted actions of 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), leukotriene C(4) synthase, and additional downstream enzymes, starting with arachidonic acid substrate. CysLTs produced by macrophages, eosinophils, mast cells, and other inflammatory cells activate 3 different high-affinity CysLT receptors: CysLT(1)R, CysLT(2)R, and GPR 17. We sought to investigate vascular sites of CysLT(2)R expression and the role and mechanism of this receptor in mediating vascular permeability events. Vascular expression of CysLT(2)R was investigated by reporter gene expression in a novel CysLT(2)R deficient-LacZ mouse model. CysLT(2)R was expressed in small, but not large, vessels in mouse brain, bladder, skin, and cremaster muscle. Intravital, in addition to confocal and electron, microscopy investigations using FITC-labeled albumin in cremaster postcapillary venule preparations indicated rapid CysLT-mediated permeability, which was blocked by application of BAY-u9773, a dual CysLT(1)R/CysLT(2)R antagonist or by CysLT(2)R deficiency. Endothelial human CysLT(2)R overexpression in mice exacerbated vascular leakage even in the absence of exogenous ligand. The enhanced vascular permeability mediated by CysLT(2)R takes place via a transendothelial vesicle transport mechanism as opposed to a paracellular route and is controlled via Ca(2+) signaling. Our results reveal that CysLT(2)R can mediate inflammatory reactions in a vascular bed-specific manner by altering transendothelial vesicle transport-based vascular permeability.

DOI： 10.1096/fj.08-113274

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D-Serine is the endogenous ligand for the glycine binding site of the N-methyl-D-aspartate (NMDA)-type glutamate receptor (GluR) channel and is involved in the regulation of synaptic plasticity, neural network formation, and neurodegenerative disorders. D-Serine is synthesized from L-serine by serine racemase (SR), which was first reported to be localized in astrocytes. However, recently, SR mRNA and its protein have been detected in neurons. In this study, we examined the SR distribution in the brain during postnatal development and in cultured cells by using novel SR knockout mice as negative controls. We found that SR is predominantly localized in pyramidal neurons in the cerebral cortex and hippocampal CA1 region. Double immunofluorescence staining revealed that SR signals colocalized with those of the neuron-specific nuclear protein, but not with the astrocytic markers glial fibrillary acid protein and 3-phosphoglycerate dehydrogenase. In the striatum, we observed SR expression in gamma-aminobutyric acid (GABA)ergic medium-spiny neurons. Furthermore, in the adult cerebellum, we detected weak but significant SR signals in GABAergic Purkinje cells. From these findings, we conclude that SR is expressed predominantly in many types of neuron in the brain and plays a key role in the regulation of brain functions under physiological and pathological conditions via the production of the neuromodulator D-serine.

DOI： 10.1002/cne.21822

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Transmembrane alpha-amino-3-hydroxyl-5-isoxazolepropionate (AMPA) receptor regulatory proteins (TARPs) play pivotal roles in AMPA receptor trafficking and gating. Here we examined cellular and subcellular distribution of TARP gamma-8 in the mouse brain. Immunoblot and immunofluorescence revealed the highest concentration of gamma-8 in the hippocampus. Immunogold electron microscopy demonstrated dense distribution of gamma-8 on the synaptic and extrasynaptic surface of hippocampal neurons with very low intracellular labeling. Of the neuronal surface, gamma-8 was distributed at the highest level on asymmetrical synapses of pyramidal cells and interneurons, whereas their symmetrical synapses selectively lacked immunogold labeling. Then, the role of gamma-8 in AMPA receptor expression was pursued in the hippocampus using mutant mice defective in the gamma-8 gene. In the mutant cornu ammonis (CA)1 region, synaptic and extrasynaptic AMPA receptors on dendrites and spines were severely reduced to 35-37% of control levels, whereas reduction was mild for extrasynaptic receptors on somata (74%) and no significant decrease was seen for intracellular receptors within spines. In the mutant CA3 region, synaptic AMPA receptors were reduced mildly at asymmetrical synapses in the stratum radiatum (67% of control level), and showed no significant decrease at mossy fiber-CA3 synapses. Therefore, gamma-8 is abundantly distributed on hippocampal excitatory synapses and extrasynaptic membranes, and plays an important role in increasing the number of synaptic and extrasynaptic AMPA receptors on dendrites and spines, particularly, in the CA1 region. Variable degrees of reduction further suggest that other TARPs may also mediate this function at different potencies depending on hippocampal subregions, input sources and neuronal compartments.

DOI： 10.1111/j.1460-9568.2006.05081.x

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Language：English   Publisher：ELSEVIER IRELAND LTD  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

In NMDA receptors, GluRepsilon/NR2 subunits strictly require the GluRzeta1/NR1 subunit to exit from endoplasmic reticulum (ER) to the cell surface in vitro and to the postsynapse in vivo, whereas C terminus-dependent self-surface delivery has been demonstrated for the GluRzeta1 subunit in vitro. To test whether this leads to C terminus-dependent self-postsynaptic expression in neurons in vivo, we investigated the GluRzeta1 subunit in cerebellar granule cells lacking two major GluRepsilon subunits, GluRepsilon1/NR2A and GluRepsilon3/NR2C. In the mutant cerebellum, synaptic labeling for the GluRzeta1 subunit containing the C2 (GluRzeta1-C2) or C2' (GluRzeta1-C2') cassette was reduced at mossy fiber-granule cell synapses to the extrasynaptic level. The loss was not accompanied by decreased transcription and translation levels, increased extrasynaptic labeling, or ER accumulation. Quantitative immunoblot revealed substantial reductions in the mutant cerebellum of GluRzeta1-C2 and GluRzeta1-C2'. The most severe deficit was observed in the postsynaptic density (PSD) fraction: mutant levels relative to the wild-type level were 12.3 +/- 3.3% for GluRzeta1-C2 and 17.0 +/- 4.6% for GluRzeta1-C2'. The GluRzeta1 subunit carrying the C1 cassette (GluRzeta1-C1) was, although low in cerebellar content, also reduced to 12.7 +/- 3.5% in the mutant PSD fraction. Considering a trace amount of other GluRepsilon subunits in the mutant cerebellum, the severe reductions thus represent that the GluRzeta1 subunit, by itself, is virtually unable to accumulate at postsynaptic sites, regardless of C-terminal forms. By protein turnover analysis, the degradation of the GluRzeta1 subunit was accelerated in the mutant cerebellum, being particularly rapid for that carrying the C2 cassette. Therefore, accompanying expression of GluRepsilon subunits is essential for postsynaptic localization and protein stability of the GluRzeta1 subunit.

DOI： 10.1523/JNEUROSCI.1261-04.2004

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Glutamate receptor interacting protein (GRIP) is a member of the PDZ domain-containing protein family that is localized in the postsynaptic density area. This protein has been reported to interact specifically with the C-termini of AMPA-selective glutamate receptor channel subunits, GluR alpha2 and GluR alpha3 through its PDZ domains. To clarify the physiological functions of GRIP, we cloned mouse GRIP1, and found that there are three sites for alternative splicing and two putative translational start codons by characterizing GRIP1 cDNA clones and reverse transcription-polymerase chain reaction products. Metabolic labeling of COS-7 cells expressing two N-terminal GRIP1 proteins demonstrated that these proteins differed in their pattern of palmitoylation. These findings suggested that the molecular diversity of GRIP1 underlies the localization and functional heterogeneity of this protein. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-3940(01)01766-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

Spatiotemporally restricted gene targeting is needed for analyzing the functions of various molecules in a variety of biological phenomena. We have generated an inducible cerebellar Purkinje cell-specific gene targeting system. This was achieved by establishing a mutant mouse line (D2CPR) from a C57BL/6 mouse ES cell line, which expressed a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR). The Purkinje cell-specific expression of CrePR was attained by inserting CrePR into the glutamate receptor delta2 subunit (GluR delta2) gene, which was expressed specifically in the Purkinje cells. Using the transgenic mice carrying the Cre-mediated reporter gene, we showed that the antiprogesterone RU486 could induce recombinase activity of the CrePR protein specifically in the mature cerebellar Purkinje cells of the D2CPR line. Thus this mutant line will be a useful tool for studying the molecular function of mature Purkinje cells by manipulating gene expression in a temporally restricted manner. (C) 2001 Academic Press.

DOI： 10.1006/bbrc.2001.4492

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：STOCKTON PRESS  

1 We have previously found that intrathecal administration of prostaglandins E-2 (PGE(2)) and D-2 (PGD(2)) into conscious mice induced hyperalgesia by the hut plate test, The present study investigated the involvement of N-methyl-D-aspartate (NMDA) receptor in the prostaglandin-induced hyperalgesia by use of mice lacking NMDA receptor epsilon 1, epsilon 4, Or epsilon 1/epsilon 4 subunits,
2 PGE(2) induced hyperalgesia over a wide rang of doses from 50 pg to 500 ng kg (1)in wild-type mice, But PGE(2) could not induce hyperalgesia. in epsilon 1, epsilon 4, Or epsilon 1/epsilon 4 subunit knockout mice;
3 The NMDA receptor antagonist D-(-)-2-amino-5-phosphonovaleric acid (D-AP5), the nan-NP IDA receptor antagonist gamma-D-glutamylaminomethyl sulphonic acid (GAMS), and the nitric oxide synthase inhibitor N-omega-nitro-L-arginine methyl ester (L-NAME) inhibited the PGE(2)-induced hyperalgesia in wild-type mice.
4 PGD(2) induced hyperalgesia al doses of 25 ng to 250 ng kg(-1) in both wild-type and epsilon 1/epsilon 4 subunit knockout mice. The substance P receptor antagonist CP 96,345 blocked the PGD(2)-induced hyperalgesia in wild-type and epsilon 1/epsilon 4 subunit knockout mice.
5 These results demonstrate that the pathways leading to hyperalgesia are different; between PGD(2) and PGE(2).and that both epsilon 1 and epsilon 4 subunits of the NMDA receptor are involved in the PGE(2)-induced hyperalgesia.

DOI： 10.1038/sj.bjp.0701067

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Language：Japanese   Publisher：(一社)日本外科学会  

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Language：English   Publisher：生命科学系学会合同年次大会運営事務局  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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DOI： 10.7600/jspfsm.63.142

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S197_4

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DOI： 10.7600/jspfsm.63.142

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DOI： 10.1016/j.neures.2011.07.195

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DOI： 10.1016/j.neures.2011.07.1719

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DOI： 10.1016/j.neures.2011.07.399

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DOI： 10.1016/j.neures.2009.09.694

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DOI： 10.1016/j.neures.2009.09.693

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Publisher：日本生化学会  

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Language：Japanese   Publisher：Niigata University  

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Other Link： http://hdl.handle.net/10191/48383