担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Neuroinflammation  

Background: Neuropathic pain is caused by sensory nerve injury, but effective treatments are currently lacking. Microglia are activated in the spinal dorsal horn after sensory nerve injury and contribute to neuropathic pain. Accordingly, molecules expressed by these cells are considered potential targets for therapeutic strategies. Our previous gene screening study using a mouse model of motor nerve injury showed that the G-protein-coupled receptor 34 gene (GPR34) is induced by nerve injury. Because GPR34 is now considered a microglia-enriched gene, we explored the possibility that it might be involved in microglial activation in the dorsal horn in a mouse model of neuropathic pain. Methods: mRNA expression of GPR34 and pro-inflammatory molecules was determined by quantitative real-time PCR in wild-type and GPR34-deficient mice with L4 spinal nerve injury. In situ hybridization was used to identify GPR34 expression in microglia, and immunohistochemistry with the microglial marker Iba1 was performed to examine microglial numbers and morphology. Mechanical sensitivity was evaluated by the von Frey hair test. Liquid chromatography-tandem mass spectrometry quantified expression of the ligand for GPR34, lysophosphatidylserine (LysoPS), in the dorsal horn, and a GPR34 antagonist was intrathecally administrated to examine the effect of inhibiting LysoPS-GPR34 signaling on mechanical sensitivity. Results: GPR34 was predominantly expressed by microglia in the dorsal horn after L4 nerve injury. There were no histological differences in microglial numbers or morphology between WT and GPR34-deficient mice. However, nerve injury-induced pro-inflammatory cytokine expression levels in microglia and pain behaviors were significantly attenuated in GPR34-deficient mice. Furthermore, the intrathecal administration of the GPR34 antagonist reduced neuropathic pain. Conclusions: Inhibition of GPR34-mediated signal by GPR34 gene deletion reduced nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia without affecting their morphology. Therefore, the suppression of GPR34 activity may have therapeutic potential for alleviating neuropathic pain.

DOI： 10.1186/s12974-019-1458-8

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：GLIA  

Several types of myeloid cell are resident in the CNS. In the steady state, microglia are present in the CNS parenchyma, whereas macrophages reside in boundary regions of the CNS, such as perivascular spaces, the meninges and choroid plexus. In addition, monocytes infiltrate into the CNS parenchyma from circulation upon blood–brain barrier breakdown after CNS injury and inflammation. Although several markers, such as CD11b and ionized calcium-binding adapter molecule 1 (Iba1), are frequently used as microglial markers, they are also expressed by other types of myeloid cell and microglia-specific markers were not defined until recently. Previous transcriptome analyses of isolated microglia identified a transmembrane lectin, sialic acid-binding immunoglobulin-like lectin H (Siglec-H), as a molecular signature for microglia; however, this was not confirmed by histological studies in the nervous system and the reliability of Siglec-H as a microglial marker remained unclear. Here, we demonstrate that Siglec-H is an authentic marker for microglia in mice by immunohistochemistry using a Siglec-H-specific antibody. Siglec-H was expressed by parenchymal microglia from developmental stages to adulthood, and the expression was maintained in activated microglia under injury or inflammatory condition. However, Siglec-H expression was absent from CNS-associated macrophages and CNS-infiltrating monocytes, except for a minor subset of cells. We also show that the Siglech gene locus is a feasible site for specific targeting of microglia in the nervous system. In conclusion, Siglec-H is a reliable marker for microglia that will allow histological identification of microglia and microglia-specific gene manipulation in the nervous system.

DOI： 10.1002/glia.23204

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Neuroinflammation  

Background: Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood. Methods: We used endogenous, natural, and surrogate agonists for GPR84 (capric acid, embelin, and 6-OAU, respectively) and examined their effect on mouse primary cultured microglia in vitro. Results: 6-n-Octylaminouracil (6-OAU), embelin, and capric acid rapidly induced membrane ruffling and motility in cultured microglia obtained from C57BL/6 mice, although these agonists failed to promote microglial pro-inflammatory cytokine expression. Concomitantly, 6-OAU suppressed forskolin-induced increase of cAMP in cultured microglia. Pertussis toxin, an inhibitor of Gi-coupled signaling, completely suppressed 6-OAU-induced microglial membrane ruffling and motility. In contrast, no 6-OAU-induced microglial membrane ruffling and motility was observed in microglia from DBA/2 mice, a mouse strain that does not express functional GPR84 protein due to endogenous nonsense mutation of the GPR84 gene. Conclusions: GPR84 mediated signaling causes microglial motility and membrane ruffling but does not promote pro-inflammatory responses. As GPR84 is a known receptor for medium-chain fatty acids, those released from damaged brain cells may be involved in the enhancement of microglial motility through GPR84 after neuronal injury.

DOI： 10.1186/s12974-017-0970-y

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/ccdis.2017.212

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1523/JNEUROSCI.3811-15.2016

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1523/JNEUROSCI.1238-16.2016

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/srep28331

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.15252/emmm.201606403

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbrc.2016.09.077

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.neuroscience.2015.11.028

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/srep25317

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jchemneu.2015.11.003

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：GLIA  

The unfolded protein response (UPR) is a signal transduction network that responds to endoplasmic reticulum (ER) stress by coordinating protein homeostasis to maintain cell viability. The UPR can also trigger cell death when adaptive responses fail to improve protein homeostasis. Despite accumulating evidence suggesting that the UPR plays a role in neurodegenerative diseases and brain insults, our understanding of how ER stress is induced under neuropathological conditions is limited. Here, we investigated the cell- and time-specific patterns of the ER stress response after brain injury using ER stress-activated indicator (ERAI) mice, which enable monitoring of the UPR in vivo via increased fluorescence of a spliced XBP-1 protein fused with the green fluorescent protein (GFP) variant Venus. Following cortical stab injury of ERAI mice, the GFP signal and number of GFP+ cells increased in the ipsilateral cortex throughout the observation period (6 h to 7 days post-injury), confirming the induction of the UPR. GFP signals were observed in injured neurons early (from 6 h) after brain injury. However, non-neuronal cells, mainly endothelial cells followed by astrocytes, accounted for the majority of GFP+ cells after brain injury. Similar results were obtained in a mouse model of focal cerebral ischemia. These findings suggest that activation of the UPR in both neuronal and non-neuronal cells, especially endothelial cells and astrocytes, may play an important role in and could be a potential therapeutic target for acute brain injuries.

DOI： 10.1002/glia.24303

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Chemical Neuroanatomy  

Axonal regeneration requires changes in the lipid dynamics of the axon membrane for growth and extension. Here, we examined the expression of genes associated with lipid transport after nerve injury. The expression of ATP-binding cassette transporter-A1 (ABCA1), which participates in the transport of cholesterol from the plasma membrane, was markedly upregulated in motor and sensory neurons after nerve injury. Stimulation of PC12 cells with the nerve growth factor induced neurite extension and ABCA1 expression predominantly in regions proximal to the neurite tip. To clarify the functional role of ABCA1 in neurite elongation, we examined the morphology of neurons cultured from conditionally-injured dorsal root ganglia from ABCA1-deficient mice. We found a significant increase in neurite branch formation in these neurons. In addition, the neurite tips of ABCA1-deficient neurons appeared excessively ruffled, and the direction of neurite elongation was unsteady. In contrast, the neurite tips of wild-type neurons were not excessively ruffled, and the neurites elongated rapidly in a stable directionally-oriented manner. Together, these findings suggest that ABCA1 plays an important role in regulating the membrane lipid composition of injured neurons and in axonal regeneration following nerve injury.

DOI： 10.1016/j.jchemneu.2022.102164

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Diabetes  

Leptin, a hormone secreted by adipocytes, exhibits therapeutic potential for the treatment of type 1 diabetes (T1D). Protein tyrosine phosphatase 1B (PTP1B) is a key enzyme that negatively regulates leptin receptor signaling. Here, the role of PTP1B in the treatment of T1D was investigated using PTP1B-deficient (knockout [KO]) mice and a PTP1B inhibitor. T1D wild-type (WT) mice induced by streptozotocin showed marked hyperglycemia compared with nonT1D WT mice. KO mice displayed significantly improved glucose metabolism equivalent to non-T1D WT mice, whereas peripheral or central administration of leptin partially improved glucose metabolism in T1D WT mice. Peripheral combination therapy of leptin and a PTP1B inhibitor in T1D WT mice improved glucose metabolism to the same level as non-T1D WT mice. Leptin was shown to act on the arcuate nucleus in the hypothalamus to suppress gluconeogenesis in liver and enhance glucose uptake in both brown adipose tissue and soleus muscle through the sympathetic nervous system. These effects were enhanced by PTP1B deficiency. Thus, treatment of T1D with leptin, PTP1B deficiency, or a PTP1B inhibitor was shown to enhance leptin activity in the hypothalamus to improve glucose metabolism. These findings suggest a potential alternative therapy for T1D.

DOI： 10.2337/db21-0953

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Neuroscience Research  

Microglia, which migrate into the central nervous system (CNS) during the early embryonic stages, are considered to play various roles in CNS development. However, their embryonic roles are largely unknown, partly due to the lack of an effective microglial ablation system in the embryo. Here, we show a microglial ablation model by injecting diphtheria toxin (DT) into the amniotic fluid of Siglechdtr mice, in which the gene encoding DT receptor is knocked into the microglia-specific gene locus Siglech. We revealed that embryonic microglia were depleted for several days throughout the CNS, including some regions where microglia transiently accumulated, at any embryonic time point from embryonic day 10.5, when microglia colonize the CNS. This ablation system was specific for microglia because CNS-associated macrophages, which are a distinct population from microglia that reside in the CNS interfaces such as meninges, were unaffected. Therefore, this microglial ablation system is highly effective for studying the embryonic functions of microglia.

DOI： 10.1016/j.neures.2021.06.002

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Comparative Neurology  

The axon initial segment (AIS) is structurally and functionally distinct from other regions of the axon, yet alterations in the milieu of the AIS after brain injury have not been well characterized. In this study, we have examined extracellular and intracellular changes in the AIS after hypoglossal nerve injury. Microglial adhesions to the AIS were rarely observed in healthy controls, whereas microglial adhesions to the AIS became apparent in the axonal injury model. Regarding intra-AIS morphology, we focused on mitochondria because mitochondrial flow into the injured axon appears critical for axonal regeneration. To visualize mitochondria specifically in injured axons, we used Atf3:BAC transgenic mice whose mitochondria were labeled with GFP in response to nerve injury. These mice clearly showed mitochondrial localization in the AIS after nerve injury. To precisely confirm the light microscopic observations, we performed three-dimensional ultrastructural analysis using focused ion beam/scanning electron microscopy (FIB/SEM). Although the healthy AIS was not surrounded by microglia, tight microglial adhesions with thick processes adhering to the AIS were observed after injury. FIB/SEM simultaneously allowed the observation of mitochondrial localization in the AIS. In the AIS of non-injured neurons, few mitochondria were observed, whereas mitochondria were abundantly localized in the cell body, axon hillock, and axon. Intriguingly, in the injured AIS, numerous mitochondria were observed throughout the AIS. Taken together, axonal injury changes the extracellular glial environment surrounding the AIS and intracellular mitochondrial localization in the AIS. These changes would be crucial responses, perhaps for injured neurons to regenerate after axonal injury.

DOI： 10.1002/cne.25212

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Neurochemistry  

Intracellular signaling pathways that promote axon regeneration are closely linked to the mechanism of neurite outgrowth. TC10, a signaling molecule that acts on neurite outgrowth through membrane transport, is a member of the Rho family G proteins. Axon injury increases the TC10 levels in motor neurons, suggesting that TC10 may be involved in axon regeneration. In this study, we tried to understand the roles of TC10 in the nervous system using TC10 knock-out mice. In cultured hippocampal neurons, TC10 ablation significantly reduced axon elongation without affecting ordinary polarization. We determined a role of TC10 in microtubule stabilization at the growth cone neck; therefore, we assume that TC10 limits axon retraction and promotes in vitro axon outgrowth. In addition, there were no notable differences in the size and structure of brains during prenatal and postnatal development between wild-type and TC10 knock-out mice. In motor neurons, axon regeneration after injury was strongly suppressed in mice lacking TC10 (both in conventional and injured nerve specific deletion). In retinal ganglion cells, TC10 ablation suppressed the axon regeneration stimulated by intraocular inflammation and cAMP after optic nerve crush. These results show that TC10 plays an important role in axon regeneration in both the peripheral and central nervous systems, and the role of TC10 in peripheral axon regeneration is neuron-intrinsic. (Figure presented.).

DOI： 10.1111/jnc.15235

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cell and Tissue Research  

The dura mater contains abundant macrophages whose functions remain largely elusive. Recent studies have demonstrated the origin, as well as the gene expression pattern, of dural macrophages (dMΦs). However, their histological features have not been explored yet. In this study, we performed immunohistochemistry and electron microscopy to elucidate their precise morphology, localization, and postnatal development in mice. We found that the morphology, as well as the localization, of dMΦs changed during postnatal development. In neonatal mice, dMΦ exhibited an amoeboid morphology. During postnatal development, their cell bodies elongated longitudinally and became aligned along dural blood vessels. In adulthood, nearly half of the dMΦs aligned along blood vessel networks. However, most of these cells were not directly attached to vessels; pericytes and fibroblasts interposed between dMΦs and vessels. This morphological information may provide further indications for the functional significance of dMΦs.

DOI： 10.1007/s00441-020-03346-y

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Life Sciences  

Neuropathic pain is caused by a lesion or a functional impairment of the sensory nervous system and allodynia is one of the frequently observed symptoms in neuropathic pain. Allodynia represents abnormal pain due to a non-noxious stimulus that does not normally provoke pain. Cellular mechanisms underlying neuropathic pain remain mostly elusive, and partial pain relief can be achieved in a limited number of patients by antidepressants, anticonvulsants topical anesthetics, and others. Zonisamide (ZNS) is widely used as an anti-epileptic and anti-Parkinson's disease drug. A recent report shows that ZNS suppresses neuropathic pain associated with diabetes mellitus in a mouse model. We made a mouse model of neuropathic pain in the hindlimb by cutting the nerve at the intervertebral canal at lumbar level 4 (L4). At 28 days after nerve injury, ZNS ameliorated allodynic pain, and reduced the expression of inflammatory cytokines and the nerve injury-induced increase of Iba1-positive microglia in the spinal dorsal horn at L4. In BV2 microglial cells, ZNS reduced the number of lipopolysaccharide-induced amoeboid-shaped cells, representing activated microglia. These results suggest that ZNS is a potential therapeutic agent for neuropathic pain partly by suppressing microglia-mediated neuroinflammation.

DOI： 10.1016/j.lfs.2020.118577

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Neurochemistry International  

Triggering receptor expressed on myeloid cells 2 (TREM2) forms a receptor complex with DNAX-activating protein of 12 kDa (DAP12) on the microglial plasma membrane. A wide variety of protein and non-protein ligands, including lipids and DNA, can bind to TREM2, inducing the activation of microglia via DAP12. Both Trem2 and Dap12 have been identified as causative genes for Nasu-Hakola disease, which causes presenile dementia in association with bone cysts. Furthermore, TREM2/DAP12 signaling represents an essential inducer of the activated microglial phenotype in neuronal diseases, including Alzheimer's disease. Therefore, most previous studies examining TREM2/DAP12 have focused on their roles in microglia under pathological conditions. However, a growing body of evidence has demonstrated the involvement of TREM2/DAP12 signaling in the regulation of physiological functions in microglia. Accordingly, by examining the importance of TREM2/DAP12 in the regulation of microglial activity during development, homeostasis, and aging in the brain, this review elucidates the roles played by this complex in the healthy brain.

DOI： 10.1016/j.neuint.2020.104878

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：EMBO Journal  

Microglia are the principal phagocytes that clear cell debris in the central nervous system (CNS). This raises the question, which cells remove cell debris when microglial phagocytic activity is impaired. We addressed this question using Siglechdtr mice, which enable highly specific ablation of microglia. Non-microglial mononuclear phagocytes, such as CNS-associated macrophages and circulating inflammatory monocytes, did not clear microglial debris. Instead, astrocytes were activated, exhibited a pro-inflammatory gene expression profile, and extended their processes to engulf microglial debris. This astrocytic phagocytosis was also observed in Irf8-deficient mice, in which microglia were present but dysfunctional. RNA-seq demonstrated that even in a healthy CNS, astrocytes express TAM phagocytic receptors, which were the main astrocytic phagocytic receptors for cell debris in the above experiments, indicating that astrocytes stand by in case of microglial impairment. This compensatory mechanism may be important for the maintenance or prolongation of a healthy CNS.

DOI： 10.15252/embj.2020104464

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Communications  

Microglia survey brain parenchyma, responding to injury and infections. Microglia also respond to systemic disease, but the role of blood–brain barrier (BBB) integrity in this process remains unclear. Using simultaneous in vivo imaging, we demonstrated that systemic inflammation induces CCR5-dependent migration of brain resident microglia to the cerebral vasculature. Vessel-associated microglia initially maintain BBB integrity via expression of the tight-junction protein Claudin-5 and make physical contact with endothelial cells. During sustained inflammation, microglia phagocytose astrocytic end-feet and impair BBB function. Our results show microglia play a dual role in maintaining BBB integrity with implications for elucidating how systemic immune-activation impacts neural functions.

DOI： 10.1038/s41467-019-13812-z

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Mediators of Inflammation  

Reg (regenerating gene) family proteins are known to be overexpressed in gastrointestinal (GI) tissues under conditions of inflammation. However, the pathophysiological significance of Reg family protein overexpression and its regulation is still unclear. In the present study, we investigated the profile of Reg family gene expression in a colitis model and focused on the regulation of Reg IIIβ and IIIγ, which are overexpressed in inflamed colonic mucosa. C57BL/6 mice were administered 2% dextran sulfate sodium (DSS) in drinking water for five days, and their colonic tissues were investigated histopathologically at interval for up to 12 weeks. Gene expression of the Reg family and cytokines (IL-6, IL-17, and IL-22) was evaluated by real-time RT-PCR, and Reg IIIβ/γ expression was examined by immunohistochemistry. The effects of cytokines on STAT3 phosphorylation and HIP/PAP (type III REG) expression in Caco2 and HCT116 cells were examined by Western blot analysis. Among Reg family genes, Reg IIIβ and IIIγ were alternatively overexpressed in the colonic tissues of mice with DSS-induced colitis. The expression of STAT3-associated cytokines (IL-6, IL-17, and IL-22) was also significantly increased in those tissues, being significantly correlated with that of Reg IIIβ/γ. STAT3 phosphorylation and HIP/PAP expression were significantly enhanced in Caco2 cells upon stimulation with IL-6, IL-17, and IL-22. In HCT116 cells, those enhancements were also observed by IL-6 and IL-22 stimulations but not IL-17. The link between type III Reg and STAT3-associated cytokines appears to play a pivotal role in the pathophysiology of DSS-induced colitis.

DOI： 10.1155/2019/7859460

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：International Journal of Developmental Neuroscience  

Microglia colonize the central nervous system (CNS) parenchyma during embryogenesis and contribute to various developmental processes leading to the formation of refined neural circuits. In this review, we focus on the bidirectional function of microglia during normal CNS development and discuss recent perspectives on the functions of microglia in neural circuit formation. Microglia participate in neurogenesis, migration, axonal growth, and synapse formation and remodeling, all of which are fundamental for the establishment of neural networks, by secreting a variety of molecules toward neurons and phagocytosing both live and dying neurons or their debris. Intriguingly, microglia play dual roles in each of the neurodevelopmental processes that they affect. For instance, microglia modulate synapse numbers by both promoting the formation of new synapses and eliminating unnecessary synapses. The study of the developmental roles of microglia is essential not only for understanding normal CNS development but also for preventing developmental brain disorders caused by microglial dysfunction.

DOI： 10.1016/j.ijdevneu.2018.09.009

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Neurochemical Research  

Our understanding of the physiological relevance of unique Damage-induced neuronal endopeptidase (DINE) [also termed Endothelin-converting enzyme-like 1 (ECEL1)] has recently expanded. DINE/ECEL1 is a type II membrane-bound metalloprotease, belonging to a family including the neprilysin (NEP) and endothelin-converting enzyme (ECE). The family members degrade and/or process peptides such as amyloid β and big-endothelins, which are closely associated with pathological conditions. Similar to NEP and ECE, DINE has been expected to play an important role in injured neurons as well as in developing neurons, because of its remarkable transcriptional response to neuronal insults and predominant neuronal expression from the embryonic stage. However, the physiological significance of DINE has long remained elusive. In the last decade, a series of genetically manipulated mice have driven research progress to elucidate the physiological aspects of DINE. The mice ablating Dine fail to arborize the embryonic motor axons in some subsets of muscles, including the respiratory muscles, and die immediately after birth. The abnormal phenotype of motor axons is also caused by one amino acid exchanges of DINE/ECEL1, which are responsible for distal arthrogryposis type 5 in a group of human congenital movement disorders. Furthermore, the mature Dine-deficient mice in which the lethality is rescued by genetic manipulation have shown the involvement of DINE in central nervous system regeneration. Here we describe recent research advances that DINE-mediated proteolytic processes are critical for nerve development, regeneration and pathogenesis, and discuss the future potential for DINE as a therapeutic target for axonal degeneration/disorder.

DOI： 10.1007/s11064-018-2665-x

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：iScience  

Immunology; Immune Response; Cell Biology; Functional Aspects of Cell Biology

DOI： 10.1016/j.isci.2019.05.011

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Neuroinflammation  

Background: Patients diagnosed with chronic fatigue syndrome (CFS) or fibromyalgia experience chronic pain. Concomitantly, the rat model of CFS exhibits microglial activation in the lumbar spinal cord and pain behavior without peripheral tissue damage and/or inflammation. The present study addressed the mechanism underlying the association between pain and chronic stress using this rat model. Methods: Chronic or continuous stress-loading (CS) model rats, housed in a cage with a thin level of water (1.5 cm in depth), were used. The von Frey test and pressure pain test were employed to measure pain behavior. The neuronal and microglial activations were immunohistochemically demonstrated with antibodies against ATF3 and Iba1. Electromyography was used to evaluate muscle activity. Results: The expression of ATF3, a marker of neuronal hyperactivity or injury, was first observed in the lumbar dorsal root ganglion (DRG) neurons 2 days after CS initiation. More than 50% of ATF3-positive neurons simultaneously expressed the proprioceptor markers TrkC or VGluT1, whereas the co-expression rates for TrkA, TrkB, IB4, and CGRP were lower than 20%. Retrograde labeling using fluorogold showed that ATF3-positive proprioceptive DRG neurons mainly projected to the soleus. Substantial microglial accumulation was observed in the medial part of the dorsal horn on the fifth CS day. Microglial accumulation was observed around a subset of motor neurons in the dorsal part of the ventral horn on the sixth CS day. The motor neurons surrounded by microglia were ATF3-positive and mainly projected to the soleus. Electromyographic activity in the soleus was two to three times higher in the CS group than in the control group. These results suggest that chronic proprioceptor activation induces the sequential activation of neurons along the spinal reflex arc, and the neuronal activation further activates microglia along the arc. Proprioceptor suppression by ankle joint immobilization significantly suppressed the accumulation of microglia in the spinal cord, as well as the pain behavior. Conclusion: Our results indicate that proprioceptor-induced microglial activation may be a key player in the initiation and maintenance of abnormal pain in patients with CFS.

DOI： 10.1186/s12974-019-1456-x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Neuroscience Research  

Over the last decade, mitochondrial dynamics beyond function during axon regeneration/degeneration have received attention. Axons have an effective delivery system of mitochondria shuttling between soma and axonal terminals, due to their polarized structure. The proper axonal transport of mitochondria, coordinated with mitochondrial fission/fusion and clearance, is vital for supplying high power energy in injured axons. Many researchers have studied mitochondrial dynamics using in vitro cultured cells with significant progress reported. However, the in vitro culture system is missing a physiological environment including glial cells, immune cells, and endothelial cells, whose communications are indispensable to nerve regeneration/degeneration. In line with this, the understanding of mitochondrial behavior in injured axon in vivo is necessary for promoting the physiological understanding of damaged axons and the development of a therapeutic strategy. In this review, we focus on recent insights into in vivo mitochondrial dynamics during axonal regeneration/degeneration, and introduce the advances of mouse strains to visualize mitochondria in a neuron-specific or an injury-specific manner, which are extremely useful for nerve regeneration/degeneration studies.

DOI： 10.1016/j.neures.2018.08.014

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cell Structure and Function  

Proper N-glycosylation of proteins is important for normal brain development and nervous system function. Identification of the localization, carrier proteins and interacting partners of N-glycans is essential for understanding the roles of glycoproteins. The present study examined the N-glycan A2G'2F (Galβ1-3GlcNAcβ1-2Manα1-6[Galβ1-3GlcNAcβ1-2Manα1-3]Manβ1-4GlcNAcβ1-4[Fucα1-6]GlcNAc-). A2G'2F has a branched sialic acid structural feature, and branched sialylated A2G'2F is a major N-glycan in the mouse brain. Its expression in the mouse brain increases during development, suggesting that branched sialylated Nglycans play essential roles during brain development. However, the carrier proteins, interacting partners and localization of branched sialylated N-glycans remain unknown. We previously improved our method for analyzing N-glycans from trace samples, and here we succeeded in detecting A2G'2F in small fragments excised from the two-dimensional electrophoresis gels of subcellular fractionated mouse brain proteins. A2G'2F was accumulated in mouse brain synaptosomes. We identified calreticulin as one of the candidate A2G'2F carriers and found calreticulin expression in both the endoplasmic reticulum and synaptosomal fractions. Calreticulin was observed in dendritic spines of cultured cortical neurons. Synthesized branched sialylated glycan clusters interacted with sialic acid-binding immunoglobulin-like lectin H (Siglec-H), which is known to be a microgliaspecific molecule. Taken together, these results suggest that branched sialylated A2G'2F in synaptosomes plays a role in the interaction of dendritic spines with microglia.

DOI： 10.1247/csf.18009

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PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Frontiers in Cellular Neuroscience  

Microglia are activated after neuronal injury and in neurodegenerative diseases, and trigger neuroinflammation in the central nervous system (CNS). Microglia-derived neuroinflammation has both beneficial and detrimental effects on neurons. Because the timing and magnitude of microglial activation is thought to be a critical determinant of neuronal fate, understanding the molecular mechanisms underlying microglial activation is required to enable establishment of microglia-targeted therapies for neural diseases. Plasma membrane receptors play primary roles as activators of microglia and in this review, we focus on a receptor complex involving triggering receptor expressed on myeloid cells 2 (TREM2) and DNAX-activating protein of 12 kDa (DAP12), both of which are causative genes for Nasu-Hakola disease, a dementia with bone cysts. Recent transcriptome approaches demonstrated TREM2/DAP12 signaling as the principal regulator that transforms microglia from a homeostatic to a neural disease-associated state. Furthermore, animal model studies revealed critical roles for TREM2/DAP12 in the regulation of microglial activity, including survival, phagocytosis, and cytokine production, not only in Alzheimer's disease but also in other neural diseases, such as Parkinson's disease, demyelinating disease, ischemia, and peripheral nerve injury. Intriguingly, while TREM2/DAP12-mediated microglial activation is detrimental for some diseases, including peripheral nerve injury, it is beneficial for other diseases. As the role of activated microglia differs among disease models, TREM2/DAP12 signaling may result in different outcomes in different diseases. In this review we discuss recent perspectives on the role of TREM2/DAP12 in microglia and their contribution to neural diseases.

DOI： 10.3389/fncel.2018.00206

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PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Tissue Engineering and Regenerative Medicine  

Neurogenesis in the adult peripheral nervous system remains to be demonstrated. We transplanted embryonic neural stem cells into a Wallerian degenerating nerve graft and observed development of a nodular structure consisting of neurons, glia, and Schwann cells. Histological analysis revealed a structure loosely resembling the spinal cord, including a synaptic network that formed along the neuron. Furthermore, the new axons reinnervated the paralysed muscle, forming both de novo and revived neuromuscular junctions. Reinnervation of the paralysed muscle resulted in significantly greater mean wet muscle weight and muscle fibre cross-sectional area on the cell transplantation side than on the surgical control side (body weight 0.071 ± 0.011% vs. 0.051 ± 0.007%, p =.006; area 355.6 ± 345.2 vs. 114.0 ± 132.0 μm2, p <.001). Electrophysiological experiments demonstrated a functional connection between the neurons and muscle; hence, we identified this nodule as an ectopic ganglion. Surprisingly, in green rat experiments, most of these glial cells, but none of the neurons, expressed enhanced green fluorescent protein, suggesting that the cells constituting the ectopic ganglion were derived from both transplanted stem cells and endogenous stem cells. Such adult neurogenesis in a peripheral nerve related to neural stem cell transplantation has not been reported previously, and these results form the basis for a novel regenerative medicine approach in paralysed muscle.

DOI： 10.1002/term.2679

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PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Scientific Reports  

FGF receptor 2 is involved in the formation of the neuromuscular junction (NMJ), but its in vivo ligand remains to be determined. Laser capture microdissection of the mouse spinal motor neurons (SMNs) revealed that Fgf18 mRNA is highly expressed in SMNs in adults. Expression of Fgf18 mRNA was the highest in the spinal cord at embryonic day (E) 15.5, which gradually decreased to postnatal day 7. FGF18 protein was localized at the NMJs of the tibialis anterior muscle at E18.5 and in adults. Fgf18-/- mice at E18.5 showed decreased expressions of the NMJ-specific Chrne and Colq genes in the diaphragm. In Fgf18-/- diaphragms, the synaptophysin-positive areas at the nerve terminals and the acetylcholine receptor (AChR)-positive areas at the motor endplates were both approximately one-third of those in wild-type embryos. Fgf18-/- diaphragms ultrastructurally showed abnormal aggregation of multiple nerve terminals making a gigantic presynapse with sparse synaptic vesicles, and simplified motor endplates. In Fgf18-/- diaphragms, miniature endplate potentials were low in amplitude with markedly reduced frequency. In C2C12 myotubes, FGF18 enhanced AChR clustering, which was blocked by inhibiting FGFRs or MEK1. We propose that FGF18 plays a pivotal role in AChR clustering and NMJ formation in mouse embryogenesis.

DOI： 10.1038/s41598-017-18753-5

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jns.2017.08.334

Web of Science

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Comparative Neurology  

Mitochondria undergo morphological changes through fusion and fission for their quality control, which are vital for neuronal function. In this study, we examined three-dimensional morphologies of mitochondria in motor neurons under normal, nerve injured, and nerve injured plus fission-impaired conditions using the focused ion beam/scanning electron microscopy (FIB/SEM), because the FIB/SEM technology is a powerful tool to demonstrate both 3D images of whole organelle and the intra-organellar structure simultaneously. Crossing of dynamin-related protein 1 (Drp1) gene-floxed mice with neuronal injury-specific Cre driver mice, Atf3:BAC Tg mice, allowed for Drp1 ablation specifically in injured neurons. FIB/SEM analysis demonstrated that somatic mitochondrial morphologies in motor neurons were not altered before or after nerve injury. However, the fission impairment resulted in prominent somatic mitochondrial enlargement, which initially induced complex morphologies with round regions and long tubular processes, subsequently causing a decrease in the number of processes and further enlargement of the round regions, which eventually resulted in big spheroidal mitochondria without processes. The abnormal mitochondria exhibited several degradative morphologies: local or total cristae collapse, vacuolization, and mitophagy. These suggest that mitochondrial fission is crucial for maintaining mitochondrial integrity in injured motor neurons, and multiple forms of mitochondria degradation may accelerate neuronal degradation.

DOI： 10.1002/cne.24213

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cell Death and Disease  

Damage-induced neuronal endopeptidase (DINE)/endothelin-converting enzyme-like 1 (ECEL1) is a membrane-bound metalloprotease that we identified as a nerve regeneration-associated molecule. The expression of DINE is upregulated in response to nerve injury in both the peripheral and central nervous systems, while its transcription is regulated by the activating transcription factor 3 (ATF3), a potent hub-transcription factor for nerve regeneration. Despite its unique hallmark of injury-induced upregulation, the physiological relevance of DINE in injured neurons has been unclear. In this study, we have demonstrated that the expression of DINE is upregulated in injured retinal ganglion cells (RGCs) in a coordinated manner with that of ATF3 after optic nerve injury, whereas DINE and ATF3 are not observed in any normal retinal cells. Recently, we have generated a mature DINE-deficient (KOTg ) mouse, in which exogenous DINE is overexpressed specifically in embryonic motor neurons to avoid aberrant arborization of motor nerves and lethality after birth that occurs in the conventional DINE KO mouse. The DINE KOTg mice did not show any difference in retinal structure and the projection to brain from that of wild–type (wild type) mice under normal conditions. However, injured RGCs of DINE KOTg mice failed to regenerate even after the zymosan treatment, which is a well-known regeneration-promoting reagent. Furthermore, a DINE KOTg mouse crossed with a Atf3:BAC Tg mouse, in which green fluorescent protein (GFP) is visualized specifically in injured RGCs and optic nerves, has verified that DINE deficiency leads to regeneration failure. These findings suggest that injury-induced DINE is a crucial endopeptidase for injured RGCs to promote axonal regeneration after optic nerve injury. Thus, a DINE-mediated proteolytic mechanism would provide us with a new therapeutic strategy for nerve regeneration.

DOI： 10.1038/cddis.2017.212

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PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

Web of Science

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Neuroscience  

Diphtheria toxin (DT) administration into transgenic mice that express the DT receptor (DTR) under control of specific promoters is often used for cell ablation studies in vivo. Because DTR is not expressed in mice, DT injection has been assumed to be nontoxic to cells in vivo. In this study, we demonstrated that DT application during the juvenile stage leads to hearing loss in wild-type mice. Auditory brainstem response measurement showed severe hearing loss in C57BL/6 mice administered DT during the juvenile period, and the hearing loss persisted into adulthood. However, ototoxicity did not occur when DT was applied on postnatal day 28 or later. Histological studies demonstrated that hearing loss was accompanied by significant degeneration of inner and outer hair cells (HCs), as well as spiral ganglion neurons. Scanning electron microscopy showed quick degeneration of inner HCs within 3 days and gradual degeneration of outer HCs within 1 week. These results demonstrated that DT has ototoxic action on C57BL/6 mice during the juvenile period, but not thereafter, and the hearing loss was due to degeneration of inner and outer HCs by unknown DT-related mechanisms.

DOI： 10.1016/j.neuroscience.2017.03.028

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：GLIA  

Under a quiescent state, microglia exhibit a ramified shape, rather than the amoeboid-like morphology following injury or inflammation. The manipulation of microglial morphology in vitro has not been very successful, which has impeded the progress of microglial studies. We demonstrate that lysophosphatidylserine (LysoPS), a kind of lysophospholipids, rapidly and substantially alters the morphology of primary cultured microglia to an in vivo-like ramified shape in a receptor independent manner. This mechanism is mediated by Cdc42 activity. LysoPS is incorporated into the plasma membrane and converted to phosphatidylserine (PS) via the Lands' cycle. The accumulated PS on the membrane recruits Cdc42. Both Cdc42 and PS colocalize predominantly in primary and secondary processes, but not in peripheral branches or tips of microglia. Along with the morphological changes LysoPS suppresses inflammatory cytokine production and NF-kB activity. The present study provides a tool to manipulate a microglial phenotype from an amoeboid to a fully ramified in vitro, which certainly contributes to studies exploring microglial physiology and pathology.

DOI： 10.1002/glia.23123

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PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

ゼミ形式

医学研究法の指導、論文作成

講義と実習

ゼミ形式

富山市立山室中学で生徒父兄対象に講演した。