竹村 晶子

Sorting nexin 25 (SNX25) belongs to the sorting nexin superfamily, whose members are responsible for membrane attachment to organelles of the endocytic system. Recent reports point to critical roles for SNX25 as a negative regulator of transforming growth factor β signaling, but the expression patterns of SNX25 in the central nervous system (CNS) remain almost uncharacterized. Here, we show widespread neuronal expression of SNX25 protein and Snx25 mRNA using immunohistochemistry and in situ hybridization. As an exception, SNX25 was present in the Bergmann glia of the cerebellum. SNX25 immunoreactivity was found in cholinergic and catecholaminergic neurons. Moreover, SNX25 colocalized with tropomyosin receptor kinase B (TrkB) in the neurons of the cortex and hippocampus. In vitro, SNX25 can interact with full-length TrkB, but not with its C-terminal-truncated isoform. Overexpression of SNX25 accelerated degradation of full-lengh TrkB, indicating that SNX25 promotes the trafficking of TrkB for lysosomal degradation. These findings suggest that SNX25 is a new actor in endocytic signaling, perhaps contributing to the regulation of BDNF-TrkB signaling in the CNS.

Entrainment of mammalian circadian rhythms requires receptor-mediated signaling in the hypothalamic suprachiasmatic nucleus (SCN), the site of the master circadian pacemaker. Receptor-mediated signaling is regulated by endocytosis, indicating that endocytosis-related proteins contribute to SCN pacemaking. Sorting nexin 25 (SNX25) belongs to the sorting nexin superfamily, whose members are responsible for membrane attachment to organelles of the endocytic system. In this study, we showed that Snx25 mRNA and SNX25 protein are highly expressed and exhibit remarkable circadian rhythms in the SCN of adult mice. Expression was maximal at about zeitgeber time (ZT) 16 in the subjective night and minimal at ZT8 in the subjective day. Prominent SNX25 immunoreactivity was found in the arginine vasopressin-positive neurons of the SCN. These findings suggest that SNX25 is a new actor in endocytic signaling, perhaps contributing to the circadian pacemaking system.

The arcuate nucleus (Arc) integrates circulating hormonal and metabolic signals to control energy expenditure and intake. One of the most important routes that enables the Arc to sense circulating molecules is through the median eminence (ME), which lacks a typical blood-brain barrier. However, the mechanism by which circulating molecules reach the Arc neurons remains unclear. This review focuses on what is known to date regarding the special structure and permeability of the ME vasculature and active transport of circulating molecules from the ME to the Arc. Recent studies have demonstrated that the ME displays angiogenic behavior that is expected to provide high vascular permeability. Parenchymal diffusion of circulating molecules from the ME vasculature is size-dependent, and tanycytes actively transport circulating molecules from the ME to the Arc. Finally, we highlight structural plasticity of the Arc and ME as playing an important role in maintaining energy balance homeostasis.

The subfornical organ (SFO) has highly permeable fenestrated vasculature and is a key site for immune-to-brain communications. Recently, we showed the occurrence of continuous angiogenesis in the SFO. In the present study, we found that systemic administration of bacterial lipopolysaccharide (LPS) reduced the vascular permeability and endothelial cell proliferation. In LPS-administered mice, the SFO vasculature showed a significant decrease in the immunoreactivity of plasmalemma vesicle associated protein-1, a marker of endothelial fenestral diaphragms. These data suggest that vasculature undergoes structural change to decrease vascular permeability in response to systemic LPS administration. (C) 2016 Published by Elsevier B.V.

Fenestrated capillaries of the sensory circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis, the subfornical organ and the area postrema, lack completeness of the blood-brain barrier (BBB) to sense a variety of blood-derived molecules and to convey the information into other brain regions. We examine the vascular permeability of blood-derived molecules and the expression of tight-junction proteins in sensory CVOs. The present tracer assays revealed that blood-derived dextran 10 k (Dex10k) having a molecular weight (MW) of 10,000 remained in the perivascular space between the inner and outer basement membranes, but fluorescein isothiocyanate (FITC; MW: 389) and Dex3k (MW: 3000) diffused into the parenchyma. The vascular permeability of FITC was higher at central subdivisions than at distal subdivisions. Neither FITC nor Dex3k diffused beyond the dense network of glial fibrillar acidic protein (GFAP)-positive astrocytes/tanycytes. The expression of tight-junction proteins such as occludin, claudin-5 and zonula occludens-1 (ZO-1) was undetectable at the central subdivisions of the sensory CVOs but some was expressed at the distal subdivisions. Electron microscopic observation showed that capillaries were surrounded with numerous layers of astrocyte processes and dendrites. The expression of occludin and ZO-1 was also observed as puncta on GFAP-positive astrocytes/tanycytes of the sensory CVOs. Our study thus demonstrates the heterogeneity of vascular permeability and expression of tight-junction proteins and indicates that the outer basement membrane and dense astrocyte/tanycyte connection are possible alternative mechanisms for a diffusion barrier of blood-derived molecules, instead of the BBB.

The sensory circumventricular organs (CVOs), which comprise the organum vasculosum of the lamina terminalis (OVLT), the subfornical organ (SFO) and the area postrema (AP), lack a typical blood-brain barrier (BBB) and monitor directly blood-derived information to regulate body fluid homeostasis, inflammation, feeding and vomiting. Until now, almost nothing has been documented about vascular features of the sensory CVOs except fenestration of vascular endothelial cells. We therefore examine whether continuous angiogenesis occurs in the sensory CVOs of adult mouse. The angiogenesis-inducing factor vascular endothelial growth factor-A (VEGF-A) and the VEGF-A-regulating transcription factor hypoxia-inducible factor-1 alpha were highly expressed in neurons of the OVLT and SFO and in both neurons and astrocytes of the AP. Expression of the pericyte-regulating factor platelet-derived growth factor B was high in astrocytes of the sensory CVOs. Immunohistochemistry of bromodeoxyuridine and Ki-67, a nuclear protein that is associated with cellular proliferation, revealed active proliferation of endothelial cells. Moreover, immunohistochemistry of caspase-3 and the basement membrane marker laminin showed the presence of apoptosis and sprouting of endothelial cells, respectively. Treatment with the VEGF receptor-associated tyrosine kinase inhibitor AZD2171 significantly reduced proliferation and filopodia sprouting of endothelial cells, as well as the area and diameter of microvessels. The mitotic inhibitor cytosine-b-D-arabinofuranoside reduced proliferation of endothelial cells and the vascular permeability of blood-derived low-molecular-weight molecules without changing vascular area and microvessel diameter. Thus, our data indicate that continuous angiogenesis is dependent on VEGF signaling and responsible for the dynamic plasticity of vascular structure and permeability.

Accumulating evidence indicates that the medial prefrontal cortex (mPFC) is a site of myelin and oligodendrocyte abnormalities that contribute to psychotic symptoms of schizophrenia. The development of therapeutic approaches to enhance remyelination, a regenerative process in which new myelin sheaths are formed on demyelinated axons, may be an attractive remedial strategy. Geissoschizine methyl ether (GM) in the Uncaria hook, a galenical constituent of the traditional Japanese medicine yokukansan (Yi-gan san), is one of the active components responsible for the psychotropic effects of yokukansan, though little is known about the mechanisms underlying the effects of either that medicine or GM itself. In the present study, we employed a cuprizone (CPZ)-induced demyelination model and examined the cellular changes in response to GM administration during the remyelination phase in the mPFC of adult mice. Using the mitotic marker 5-bromo-2'-deoxyuridine (BrdU), we demonstrated that CPZ treatment significantly increased the number of BrdU-positive NG2 cells, as well as microglia and mature oligodendrocytes in the mPFC. Newly formed oligodendrocytes were increased by GM administration after CPZ exposure. In addition, GM attenuated a decrease in myelin basic protein immunoreactivity caused by CPZ administration. Taken together, our findings suggest that GM administration ameliorated the myelin deficit by mature oligodendrocyte formation and remyelination in the mPFC of CPZ-fed mice. The present findings provide experimental evidence supporting the role for GM and its possible use as a remedy for schizophrenia symptoms by promoting the differentiation of progenitor cells to and myelination by oligodendrocytes.

The blood-brain barrier (BBB) is a barrier that prevents free access of blood-derived substances to the brain through the tight junctions and maintains a specialized brain environment. Circumventricular organs (CVOs) lack the typical BBB. The fenestrated vasculature of the sensory CVOs, including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows parenchyma cells to sense a variety of blood-derived information, including osmotic ones. In the present study, we utilized immunohistochemistry to examine changes in the expression of NG2 and platelet-derived growth factor receptor beta (PDGFRB) in the OVLT, SFO and AP of adult mice during chronic osmotic stimulation. The expression of NG2 and PDGFRB was remarkably prominent in pericytes, although these angiogenesis-associated proteins are highly expressed at pericytes of developing immature vasculature. The chronic salt loading prominently increased the expression of NG2 in the OVLT and SFO and that of PDGFRB in the OVLT, SFO and AP. The vascular permeability of low-molecular-mass tracer fluorescein isothiocyanate was increased significantly by chronic salt loading in the OVLT and SFO but not AP. In conclusion, the present study demonstrates changes in pericyte expression of NG2 and PDGFRB and vascular permeability in the sensory CVOs by chronic osmotic stimulation, indicating active participation of the vascular system in osmotic homeostasis. Copyright (c) 2013 John Wiley & Sons, Ltd.

Blood-derived molecules are able to access to the median eminence (ME) and arcuate hypothalamic nucleus (Arc) due to the lack of the blood-brain barrier. In the present study, we examined the accessibility of low-molecular-mass (LMM) molecules into parenchyma in the ME and Arc of adult mice by administration of Dextran 3000 (Dex3k), Dex10k, Evans blue (EB) and fluorescein isothiocyanate (FITC). In the external zone of the ME, the fluorescence of Dex3k, EB and FITC tracers generated an intensity gradient from fenestrated capillary, but that of Dex10k was detected only between the inner and outer basement membrane of pericapillary space. The fluorescence of FITC in the external zone of the ME was closely associated with axonal terminals and surrounded by cellular processes of tanycytes-like cells and astrocytes. In the ependymal/internal zone of the ME and Arc, the fluorescence of all LMM tracers was seen at tanycytes-like cells and neurons. The fluorescence of EB and FITC in these regions was not detected when brains were fixed during or before the administration of tracers. The inhomogeneity of accessibility for fluorescent tracers depended on routes for tracer administration. Thus, the present study indicates that the accessibility of LMM blood-derived molecules to parenchyma depends on fenestration of the capillary in the external zone of the ME and active transport of ependymal cells in the ependymal/internal zone of the ME and Arc. Copyright (c) 2013 John Wiley & Sons, Ltd.

Growth-associated protein 43 (GAP-43), a novel axonal phosphoprotein, is originally identified as a growth-cone-specific protein of developing neurons in vitro. The expression of GAP-43 is also shown to be up-regulated concomitant with increased synaptic plasticity in the brains in vivo, but how GAP-43 is concerned with synaptic plasticity is not well understood. In the present study, therefore, we aimed to elucidate subcellular localization of GAP-43 as culture development of rat hippocampal neurons. Western blotting showed that the expression of GAP-43 in the cerebral and hippocampal tissues was prominently high at postnatal days 14 and 21 or the active period of synaptogenesis. Double-labelling immunohistochemistry with an axonal marker Tau revealed that the immunoreactivity of GAP-43 was seen throughout axons of cultured hippocampal neurons but stronger at axonal puncta of developing neurons than axonal processes. Double-labelling immunohistochemistry with presynaptic terminal markers of synapsin and synaptotagmin revealed that the immunoreactivity of GAP-43 was observed mostly at weak synapsin- and synaptotagmin-positive puncta rather than strong ones. The quantitative analysis of immunofluorescent intensity showed a clear inverse correlation between GAP-43 and either synapsin or synaptotagmin expression. These data indicate that GAP-43 is highly expressed at immature growing axonal terminals and its expression is decreased along with the maturation of synaptogenesis. Copyright (c) 2012 John Wiley & Sons, Ltd.

The circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP) sense a variety of blood-borne molecules because they lack typical blood-brain barrier. Though a few signaling pathways are known, it is not known how endogenous ligands for transient receptor potential vanilloid receptor 1 ion channel (TRPV1) are sensed in the CVOs. In this study, we aimed to examine whether or not astrocytic TRPV1 senses directly blood-borne molecules in the OVLT, SFO, and AP of adult mice. The reverse transcription-polymerase chain reaction and Western analysis revealed the expression of TRPV1 in the CVOs. Confocal microscopic immunohistochemistry further showed that TRPV1 was localized prominently at thick cellular processes of astrocytes rather than fine cellular processes and cell bodies. TRPV1-expressing cellular processes of astrocytes surrounded the vasculature to constitute dense networks. The expression of TRPV1 was also found at neuronal dendrites but not somata in the CVOs. The intravenous administration of a TRPV1 agonist resiniferatoxin (RTX) prominently induced Fos expression at astrocytes in the OVLT, SFO, and AP and neurons in adjacent related nuclei of the median preoptic nuclei (MnPO) and nucleus of the solitary tract (Sol) of wild-type but not TRPV1-knockout mice. The intracerebroventricular infusion of RTX induced Fos expression at both astrocytes and neurons in the CVOs, MnPO, and Sol. Thus, this study demonstrates that blood-borne molecules are sensed directly by astrocytic TRPV1 of the CVOs in adult mammalians.

Brain vasculature forms the bloodbrain barrier (BBB) that restricts the movement of molecules between the brain and blood, but the capillary of the median eminence (ME) lacks the BBB for secretion of adenohypophysial hormone-releasing peptides. In the present study, we aimed to elucidate whether continuous angiogenesis occurs in the ME of adult mice. By using a mitotic marker, bromodeoxyuridine (BrdU), we demonstrated that new endothelial cells were born continuously in the ME of adults. Prominent expression of NG2, platelet-derived growth factor receptor B (PDGFRB), and delta-like ligand 4 was observed at pericytes of adults, although the expression of these angiogenesis-associated proteins has been shown to be at low or trace levels in adult mature capillary. In addition, vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, was expressed highly in the nervous parenchyma of the ME. Expression of VEGF receptor 2 (VEGFR2) was observed at endothelial cells in the external zone and at somatodendrites in the internal zone. Finally, a VEGFR- and PDGFR-associated tyrosine kinase inhibitor, SU11248, significantly decreased the number of BrdU-positive proliferating endothelial cells and parenchyma cells. In conclusion, the present study demonstrates VEGF-dependent continuous angiogenesis in the ME of adult mouse brains under normal conditions, which provides new insight into our understanding of neurosecretion in the ME.

The blood-brain barrier (BBB) prevents free access of circulating molecules to the brain and maintains a specialized brain environment to protect the brain from blood-derived bioactive and toxic molecules; however, the circumventricular organs (CVOs) have fenestrated vasculature. The fenestrated vasculature in the sensory CVOs, including the organum vasculosum of lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows neurons and astrocytes to sense a variety of plasma molecules and convey their information into other brain regions and the vasculature in the secretory CVOs, including median eminence (ME) and neurohypophysis (NH), permits neuronal terminals to secrete many peptides into the blood stream. The present study showed that vascular permeability of low-molecular-mass tracers such as fluorescein isothiocyanate (FITC) and Evans Blue was higher in the secretory CVOs and kidney as compared with that in the sensory CVOs. On the other hand, vascular permeability of high-molecular-mass tracers such as FITC-labeled bovine serum albumin and Dextran 70,000 was lower in the CVOs as compared with that in the kidney. Prominent vascular permeability of low- and high-molecular-mass tracers was also observed in the arcuate nucleus. These data demonstrate that vascular permeability for low-molecular-mass molecules is higher in the secretory CVOs as compared with that in the sensory CVOs, possibly for large secretion of peptides to the blood stream. Moreover, vascular permeability for high-molecular-mass tracers in the CVOs is smaller than that of the kidney, indicating that the CVOs are not totally without a BBB.

We described a new method for the visualization of vasculature and endothelial cells and the assessment of extravascular leakage in adult mouse brain by using fluorescein isothiocyanate (FITC), or a reactive fluorescent dye. FITC is the fluorescein derivative that reacts covalently with amine groups at alkaline pH. In this method, strong fluorescence of FITC was seen at vasculature throughout the brain and spinal cord, when mice received intracardiac perfusion with FITC-containing saline at pH 7.0 followed by paraformaldehyde (PFA) fixative at pH 8.0. The fluorescence of FITC was faint when animals were fixed with PFA fixative at pH 7.0 after the perfusion of FITC-containing saline at pH 7.0. The fluorescence of FITC was not detected when mice was fixed with PFA fixative before the perfusion of FITC-containing saline. Double labeling immunohistochemistry using an endothelial cell marker CD31 or a pericyte marker desmin revealed that FITC was accumulated at nuclei of endothelial cells but riot at those of pericytes. Extravascular leakage of FITC was prominent in the area postrema or a brain region of the circumventricular organs that lacks the blood-brain barrier. Moreover, strong extravascular leakage of FITC was detected at damaged sites of the cerebral cortex with cryoinjury. Thus. FITC method is useful technique for examining the architecture of brain vasculature and endothelial cells and the assessment of extravascular leakage in adult rodents. Moreover. FITC binds covalently to cellular components, so that makes it possible to perform double labeling immunohistochemistry and long-term storage of the preparation. (C) 2011 Elsevier B.V. All rights reserved.

The hypothalamo neurohypophysial system (HNS) consisting of arginine vasopressin (AVP) and oxytocin (OXT) magnocellular neurons shows the structural plasticity including the rearrangement of synapses, dendrites, and neurovascular contacts during chronic physiological stimulation. In this study, we examined the remodeling of chondroitin sulfate proteoglycans (CSPGs), main extracellular matrix (ECM), in the HNS after salt loading known as a chronic stimulation to cause the structural plasticity. In the supraoptic nucleus (SON), confocal microscopic observation revealed that the immunoreactivity of 6B4 proteoglycans (PG) was observed mainly at AVP-positive magnocellular neurons but that of neurocan was seen chiefly at OXT-positive magnocellular neurons. The immunoreactivity of phosphacan and aggrecan was seen at both AVP- and OXT-positive magnocellular neurons. Electron microscopic observation further showed that the immunoreactivity of phosphacan and neurocan was observed at astrocytic processes to surround somata, dendrites, and terminals, but not synaptic junctions. In the neurohypophysis (NH), the immunoreactivity of phosphacan, 6B4 PGs, and neurocan was observed at AVP-positive magnocellular terminals, but the reactivity of Wisteria floribunda agglutinin lectin was seen at OXT-positive ones. The immunoreactivity of versican was found at microvessel and that of aggrecan was not detected in the NH. Quantitative morphometrical analysis showed that the chronic physiological stimulation by 7-day salt loading decreased the level of 6B4 PGs in the SON and the level of phosphacan, 6B4 PGs, and neurocan in the NH. These results suggest that the extracellular microenvironment of CSPGs is different between AVP and OXT magnocellular neurons and activity-dependent remodeling of CSPGs could be involved in the structural plasticity of the HNS. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.