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Yasuo Uchiyama

Publications and source records attributed to Yasuo Uchiyama.

At least 19 recordsLinked to original sources
PubMed2007

Regulation of autophagy by extracellular signal-regulated protein kinases during 1-methyl-4-phenylpyridinium-induced cell death.

Increased autophagic vacuoles (AVs) occur in injured or degenerating neurons, under both developmental and pathological situations. Although regulation of starvation-induced autophagy has been extensively studied, less is known about autophagic responses to pathological damage. The neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) produces mitochondria-targeted injury, which contributes to parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine in mammals. Here, we demonstrate that MPP(+) elicited increased autophagy in SH-SY5Y cells, as assessed by electron microscopy, immunofluorescence for the autophagy protein LC3/Atg8, LC3 electrophoretic mobility shift, mitochondrial degradation, and monodansylcadaverine staining for late AVs/autolysosomes. During nutrient deprivation, class III phosphatidylinositol-3 kinase (PI3K) stimulates autophagy in concert with the autophagy-regulatory protein beclin 1/Atg6. Although PI3K inhibitors and RNA interference knockdown of beclin 1 effectively inhibited autophagy elicited by amino acid deprivation, neither reduced MPP+-induced autophagic stress. In contrast, inhibition of mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase reduced AV content, mitochondrial degradation, and cell death in MPP+-treated cells. RNA interference studies targeting core Atg proteins also reduced AV content and cell death. Likewise, in primary midbrain dopaminergic neurons, MPP+ elicited increased AV content, which was reversed by inhibition of mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase but not PI3K. These results implicate a role for extracellular signal-regulated protein kinase (ERK) signaling upstream of MPP+-elicited autophagic stress. Moreover, pathological stimulation of beclin 1-independent autophagy is associated with neuronal cell death.

1-Methyl-4-phenylpyridinium↗
PubMed2006

Kainic acid induces early and transient autophagic stress in mouse hippocampus.

Kainic acid (KA) treatment is a well-established model of hippocampal neuron death mediated in large part by KA receptor-induced excitotoxicity. KA-induced, delayed neuron death has been shown previously to follow the induction of seizures and exhibit characteristics of both apoptosis and necrosis. Growing evidence supports a role of autophagic stress-induced death of neurons in several in vitro and in vivo models of neuron death and neurodegeneration. However, whether autophagic stress also plays a role in KA-induced excitotoxicity has not been previously investigated. To examine whether KA alters the levels of proteins associated with or known to regulate the formation of autophagic vacuoles, we isolated hippocampal extracts from control mice and in mice following 2-16 h KA injection. KA induced a significant increase in the amount of LC3-II, a specific marker of autophagic vacuoles, at 4-6h following KA, which indicates a transient induction of autophagic stress. Levels of autophagy-associated proteins including ATG5 (conjugated to ATG12), ATG6 and ATG7 did not change significantly after treatment with KA. However, ratios of phospho-mTOR/mTOR were elevated from 6 to 16 h, and ratios of phospho-Akt/Akt were elevated at 16 h following KA treatment, suggesting a potential negative feedback loop to inhibit further stimulation of autophagic stress. Together these data indicate the transient induction of autophagic stress by KA which may serve to regulate excitotoxic death in mouse hippocampus.

Animals↗
PubMed2006

Uncoupling protein 2 negatively regulates neurite extensions in PC12h cells.

Uncoupling protein 2 (UCP2) distributes in many organs including the brain. Though recent reports suggest that UCP2 is involved in the neuroprotection and the regulation of neurosecretion, the roles of UCP2 in the central nervous systems remain largely unclear. In order to clarify the significance of UCP2 in the brain especially at developmental stage, subcellular localizations of rat UCP2 (rUCP2) in the developing cerebellar Purkinje cells were immunochemically examined. The rUCP2-like immunoreactivities observed axon or its terminal during axonal maturation. This result implies that rUCP2 contributes to the neurite development. In the PC12h cells overexpressing rUCP2 or active mutant of rUCP2, the neurite outgrowth was significantly inhibited along with a reduction of cellular ATP level. These findings suggest a possibility that UCP2 is involved in negative regulation of neurite extensions through repression of the energy supply.

Animals↗
PubMed2006

The luminal domain participates in the endosomal trafficking of the cation-independent mannose 6-phosphate receptor.

Although the role of the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor (CIMPR) has been well established in the receptor trafficking, that of the luminal domain is still controversial. We noticed that the peripheral distribution of GFP, fused to the transmembrane and cytoplasmic domains of CIMPR (G-CIMPR-tail), was distinct from that of endogenous CIMPR or of GFP fused to the full-length CIMPR (G-CIMPR-full). By live-cell imaging, trans-Golgi-network (TGN)-derived transport carriers containing G-CIMPR-full more frequently stopped and overlapped with transferrin-containing endosomes in the peripheral region than those containing G-CIMPR-tail. G-CIMPR-full was recycled back to the perinuclear TGN more slowly than that for G-CIMPR-tail, evidenced by fluorescence recovery after photobleaching analysis. Moreover, endogenous CIMPR and G-CIMPR-full, but not GFP-CIMPR-tail, drastically altered the characteristic distribution after treatment with chloroquine. A mutant receptor, G-CIMPR-full R/A, that cannot recognize the mannose 6-phosphate (M6P)-signal, behaved similarly to G-CIMPR-full, indicating that these differences are not attributable to the M6P-ligands binding situation. Interestingly, we also found that U18666A treatment was able to discriminate the M6P-ligand binding-dependent trafficking of CIMPR. Based on these findings, we propose that the CIMPR luminal domain is required for tight interaction with endocytic compartments, and retention by them, and that there are additional transport steps, in which the binding to M6P-ligands is involved.

Androstenes↗
PubMed2006

Effects of RNA interference of Atg4B on the limited proteolysis of LC3 in PC12 cells and expression of Atg4B in various rat tissues.

Atg4B, a mammalian homologue of yeast Atg4, has been shown to play an important role in the processing of LC3, a mammalian homologue of yeast Atg8, but the tissue distribution of Atg4B remains unknown. To better understand the role of Atg4B in rat tissue cells, we prepared antibodies against Atg4B, and PC12 cells in which the expression of Atg4B was knocked down by RNA interference. In the RNA interference-treated PC12 cells, for which the expression of Atg4B was 10% of wild-type PC12 cells, the expression of cytosolic LC3-I was similar to that in wild-type cells. Knockdown cell lysates, however, suppressed the cleavage of recombinant proLC3 to LC3-I. Moreover, the expression of Atg4B protein and mRNA was ubiquitous in rat tissues; however, the expression levels were not identical, but were dependent on the tissue, with the expression high in brain and testicular tissue, and low in muscular and heart tissue. In brain tissue, the expression of Atg4B protein and mRNA was higher in neurons, especially in the cerebellum and olfactory bulb, as evidenced by immunohistochemistry and in situ hybridization. These lines of evidence suggest that Atg4B plays a major role in the processing of LC3 and is widely distributed in rat tissues. In particular, in brain tissues, autophagy may be deeply associated with the metabolism of neurons, especially in the cerebellum.

Animals↗
PubMed2006

Loss of autophagy in the central nervous system causes neurodegeneration in mice.

Protein quality-control, especially the removal of proteins with aberrant structures, has an important role in maintaining the homeostasis of non-dividing neural cells. In addition to the ubiquitin-proteasome system, emerging evidence points to the importance of autophagy--the bulk protein degradation pathway involved in starvation-induced and constitutive protein turnover--in the protein quality-control process. However, little is known about the precise roles of autophagy in neurons. Here we report that loss of Atg7 (autophagy-related 7), a gene essential for autophagy, leads to neurodegeneration. We found that mice lacking Atg7 specifically in the central nervous system showed behavioural defects, including abnormal limb-clasping reflexes and a reduction in coordinated movement, and died within 28 weeks of birth. Atg7 deficiency caused massive neuronal loss in the cerebral and cerebellar cortices. Notably, polyubiquitinated proteins accumulated in autophagy-deficient neurons as inclusion bodies, which increased in size and number with ageing. There was, however, no obvious alteration in proteasome function. Our results indicate that autophagy is essential for the survival of neural cells, and that impairment of autophagy is implicated in the pathogenesis of neurodegenerative disorders involving ubiquitin-containing inclusion bodies.

Animals↗
PubMed2006

Gene transfer of hepatocyte growth factor gene improves learning and memory in the chronic stage of cerebral infarction.

There is no specific treatment to improve the functional recovery in the chronic stage of ischemic stroke. To provide the new therapeutic options, we examined the effect of overexpression of hepatocyte growth factor (HGF) in the chronic stage of cerebral infarction by transferring the HGF gene into the brain using hemagglutinating virus of Japan envelope vector. Sixty rats were exposed to permanent middle cerebral artery occlusion (day 1). Based on the sensorimotor deficits at day 7, the rats were divided equally into control vector or HGF-treated rats. At day 56, rats transfected with the HGF gene showed a significant recovery of learning and memory in Morris water maze tests (control vector 50+/-4 s; HGF 33+/-5 s; P<0.05) and passive avoidance task (control vector 132.4+/-37.5 s; HGF 214.8+/-26.5 s; P<0.05). Although the total volume of cerebral infarction was not related to the outcome, immunohistochemical analysis for Cdc42 and synaptophysin in the peri-infarct region revealed that HGF enhanced the neurite extension and increased synapses. Immunohistochemistry for glial fibriary acidic protein revealed that the formation of glial scar was also prevented by HGF gene treatment. Additionally, the number of the arteries was increased in the HGF group at day 56. These data demonstrated that HGF has a pivotal role for the functional recovery after cerebral infarction through neuritogenesis, improved microcirculation, and the prevention of gliosis. Our results also provide evidence for the feasibility of gene therapy in the chronic stage of cerebral infarction.

Animals↗
PubMed2006

Aggregate formation and phosphorylation of neurofilament-L Pro22 Charcot-Marie-Tooth disease mutants.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral nerve disorder. The causative gene for axonal type CMT2E has been identified as neurofilament light (NF-L) chain. Using cultured cells and in vitro assays, we analyzed the filament formation ability of Pro22 CMT mutant proteins of NF-L, P22S and P22T. NF-L Pro22 mutant proteins formed large aggregates in SW13- cells and cortical neurons and assembled into short twisty threads thinner than 10 nm filaments in vitro. Those threads associated with each other at their ends and entangled into large aggregates, also abnormalities, were detected at steps in oligomer formation. Pro22 mutations abolished Thr21 phosphorylation by cyclin-dependent kinase 5 and external signal regulated kinase, which suppressed filament assembly, but phosphorylation by protein kinase A (PKA) inhibited aggregate formation in vitro and alleviated aggregates in cortical neurons. These results indicate that the Pro22 CMT mutation induces abnormal filament aggregates by disrupting proper oligomer formation and the aggregates are mitigated by phosphorylation with PKA, which makes it a viable target for the development for therapeutics.

Amino Acid Substitution↗
PubMed2006

Different apoptotic mechanisms are activated in male and female brains after neonatal hypoxia-ischaemia.

Sex-related brain injury was evaluated after unilateral hypoxia-ischaemia (HI) in C57/BL6 mice on postnatal day (P) 5, 9, 21 or 60, corresponding developmentally to premature, term, juvenile and adult human brains. There was no sex difference in brain injury when the insult was severe, as evaluated by pathological scoring or tissue loss, but when the insult was moderate, adult (P60) females displayed less injury. In the immature (P9) male brains, neurones displayed a more pronounced translocation of apoptosis-inducing factor (AIF) (loss of AIF from the mitochondrial fraction and increase in nuclear AIF) after HI, whereas the female brain neurones displayed a stronger activation of caspase 3 (more pronounced loss of pro-caspase 3, increase in cleaved caspase 3 and increase in caspase 3 enzymatic activity). Two other mechanisms of injury, peroxynitrite-induced formation of nitrotyrosine and autophagy, were no different between males and females at P9. These data show that the CNS is more resistant to HI in adult females compared with males, whereas no sex differences were found in the extent of injury in neonatal mice. However, critical sex-dependent differences were demonstrated in vivo with regard to cellular, apoptosis-related mechanisms.

Animals↗
PubMed2006

Bafilomycin A1 inhibits chloroquine-induced death of cerebellar granule neurons.

Treatment of cells with the macrolide antibiotic bafilomycin A1, an inhibitor of vacuolar (V)-ATPase, or with the lysosomotropic agent chloroquine, has been shown to pharmacologically inhibit autophagy as evidenced by an accumulation of autophagosomes, which in turn causes Bax-dependent apoptosis. However, bafilomycin A1 has also been reported to inhibit chloroquine-induced apoptosis, suggesting a complex interrelationship between these two inhibitors of autophagy. To determine whether the cytoprotective effect of bafilomycin A1 on chloroquine-treated cells was dependent on inhibition of V-ATPase, we examined the single and combined effects of bafilomycin and chloroquine on cultured cerebellar granule neurons. When added separately, chloroquine or high concentrations of bafilomycin A1 (> or =10 nM) induced a dose-dependent inhibition of autophagy (as measured by an increase in LC3-II, a marker specific for autophagosomes), followed by caspase-3 activation and cell death. When added in combination, bafilomycin A1 potently inhibited chloroquine-induced caspase-3 activity and cell death at concentrations (< or =1 nM) that neither altered vacuolar acidification nor inhibited autophagy. The neuroprotective effects of bafilomycin A1 against chloroquine were substantially greater than those produced by Bax deficiency. Bafilomycin A1-induced neuroprotection seemed to be stimulus-specific, in that staurosporine-induced death was not attenuated by coaddition of bafilomycin A1. Together, these data suggest that in addition to promoting death via inhibition of V-ATPase and autophagy, bafilomycin A1 possesses novel, neuroprotective properties that inhibit Bax-dependent activation of the intrinsic apoptotic pathway resulting from the pharmacological inhibition of autophagy.

Animals↗
PubMed2006

Sleeping beauty transposon-based phenotypic analysis of mice: lack of Arpc3 results in defective trophoblast outgrowth.

The Sleeping Beauty (SB) transposon system has generated many transposon-insertional mutant mouse lines, some of which have resulted in embryonic lethality when bred to homozygosity. Here we report one such insertion mapped to the mouse actin-related protein complex subunit 3 gene (Arpc3). Arpc3 is a component of the Arp2/3 complex, which plays a major role in actin nucleation with Y-shaped branching from the mother actin filament in response to migration signaling. Arpc3 transposon-inserted mutants developed only to the blastocyst stage. In vitro blastocyst culture of Arpc3 mutants exhibited severe spreading impairment of trophoblasts. This phenotype was also observed in compound heterozygotes generated using conventional gene-targeted and transposon-inserted alleles. Arpc3-deficient mutants were shown to lack actin-rich structures in the spreading trophoblast. Electron microscopic analysis demonstrated the lack of mesh-like structures at the cell periphery, suggesting a role of Arpc3 in Y-shaped branching formation. These data indicate the importance of Arpc3 in the Arp2/3 complex for trophoblast outgrowth and suggest that Arpc3 may be indispensable for implantation.

Actin-Related Protein 2-3 Complex↗
PubMed2006

RANKL-induced expression of tetraspanin CD9 in lipid raft membrane microdomain is essential for cell fusion during osteoclastogenesis.

UNLABELLED: We showed that CD9, a member of tetraspanin superfamily proteins, is expressed in a specific membrane microdomain, called "lipid raft," and is crucial for cell fusion during osteoclastogenesis after activation of the RANK/RANKL system. INTRODUCTION: Osteoclasts are bone-resorbing multinuclear polykaryons that are essential for bone remodeling and are formed through cell fusion of mononuclear macrophage/monocyte lineage precursors. Although osteoclastogenesis has been shown to be critically regulated by the RANK/RANKL system, the mechanism how precursor cells fuse with each other remains unclear. We examined the function of CD9, a member of tetraspanin superfamily, which has previously been shown to form macromolecular membrane microdomains and to regulate cell-cell fusion in various cell types. MATERIALS AND METHODS: We used RAW264.7, a macrophage/monocyte lineage cell line, which can differentiate into osteoclast-like polykaryons on the application of RANKL. Expression and distribution of CD9 was assessed by Western blotting, fluorescence-assorted cell sorting (FACS) and immunohistochemistry with light and electron microscopy. A specific neutralizing antibody and RNA interference were used to inhibit the function of CD9, and green fluorescent protein (GFP)-CD9 was exogenously expressed to enhance the effect of CD9. The distribution of CD9 in lipid microdomain was examined by biochemical (sucrose density gradient) isolation and imaging technique. RESULTS: CD9 is expressed on cell surfaces of RAW264.7, which is enhanced by RANKL. Targeted inhibition of CD9 decreases the number of osteoclast-like cells. On the other hand, overexpression of CD9 promotes spontaneous cell fusion even in the absence of RANKL. CD9 is localized in detergent-insoluble "lipid raft" microdomain in RANKL stimulation, and disruption of lipid rafts markedly reduces the formation of osteoclast-like polykaryons. Immunohistochemical studies of bone tissues revealed the expression of CD9 in osteoclasts in vivo. CONCLUSIONS: These data suggest that function of tetraspanin CD9 and its expression in lipid rafts are crucial for cell fusion during osteoclastogenesis.

Animals↗
PubMed2005

Macroautophagy--a novel Beta-amyloid peptide-generating pathway activated in Alzheimer's disease.

Macroautophagy, which is a lysosomal pathway for the turnover of organelles and long-lived proteins, is a key determinant of cell survival and longevity. In this study, we show that neuronal macroautophagy is induced early in Alzheimer's disease (AD) and before beta-amyloid (Abeta) deposits extracellularly in the presenilin (PS) 1/Abeta precursor protein (APP) mouse model of beta-amyloidosis. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of intracellular Abeta. Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity. Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production. Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.

Adult↗
PubMed2005

Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells.

Definitive erythropoiesis usually occurs in the bone marrow or fetal liver, where erythroblasts are associated with a central macrophage in anatomical units called 'blood islands'. Late in erythropoiesis, nuclei are expelled from the erythroid precursor cells and engulfed by the macrophages in the blood island. Here we show that the nuclei are engulfed by macrophages only after they are disconnected from reticulocytes, and that phosphatidylserine, which is often used as an 'eat me' signal for apoptotic cells, is also used for the engulfment of nuclei expelled from erythroblasts. We investigated the mechanism behind the enucleation and engulfment processes by isolating late-stage erythroblasts from the spleens of phlebotomized mice. When these erythroblasts were cultured, the nuclei protruded spontaneously from the erythroblasts. A weak physical force could disconnect the nuclei from the reticulocytes. The released nuclei contained an undetectable level of ATP, and quickly exposed phosphatidylserine on their surface. Fetal liver macrophages efficiently engulfed the nuclei; masking the phosphatidylserine on the nuclei with the dominant-negative form of milk-fat-globule EGF8 (MFG-E8) prevented this engulfment.

Adenosine Triphosphate↗
PubMed2005

Roles of ARFRP1 (ADP-ribosylation factor-related protein 1) in post-Golgi membrane trafficking.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a small GTPase with significant similarity to the ARF family. However, little is known about the function of ARFRP1 in mammalian cells, although knockout mice of its gene are embryonic lethal. In the present study, we demonstrate that ARFRP1 is associated mainly with the trans-Golgi compartment and the trans-Golgi network (TGN) and is an essential regulatory factor for targeting of Arl1 and GRIP domain-containing proteins, golgin-97 and golgin-245, onto Golgi membranes. Furthermore, we show that, in concert with Arl1 and GRIP proteins, ARFRP1 is implicated in the Golgi-to-plasma membrane transport of the vesicular stomatitis virus G protein as well as in the retrograde transport of TGN38 and Shiga toxin from endosomes to the TGN.

ADP-Ribosylation Factors↗
PubMed2005

Application of fluorophore-assisted carbohydrate electrophoresis to analysis of disaccharides and oligosaccharides derived from glycosaminoglycans.

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.

Aminoacridines↗
PubMed2005

Proteolytic degradation of glutamate decarboxylase mediates disinhibition of hippocampal CA3 pyramidal cells in cathepsin D-deficient mice.

Although of clinical importance, little is known about the mechanism of seizure in neuronal ceroid lipofuscinosis (NCL). In the present study, we have attempted to elucidate the mechanism underlying the seizure of cathepsin D-deficient (CD-/-) mice that show a novel type of lysosomal storage disease with a phenotype resembling late infantile NCL. In hippocampal slices prepared from CD-/- mice at post-natal day (P)24, spontaneous burst discharges were recorded from CA3 pyramidal cells. At P24, the mean amplitude of IPSPs after stimulation of the mossy fibres was significantly smaller than that of wild-type mice, which was substantiated by the decreased level of gamma-aminobutyric acid (GABA) contents in the hippocampus measured by high-performance liquid chromatography (HPLC). At this stage, activated microglia were found to accumulate in the pyramidal cell layer of the hippocampal CA3 subfield of CD-/- mice. However, there was no significant change in the numerical density of GABAergic interneurons in the CA3 subfield of CD-/- mice at P24, estimated by counting the number of glutamate decarboxylase (GAD) 67-immunoreactive somata. In the hippocampus and the cortex of CD-/- mice at P24, some GABAergic interneurons displayed extremely high somatic granular immunoreactivites for GAD67, suggesting the lysosomal accumulation of GAD67. GAD67 levels in axon terminals abutting on to perisomatic regions of hippocampal CA3 pyramidal cells was not significantly changed in CD-/- mice even at P24, whereas the total protein levels of GAD67 in both the hippocampus and the cortex of CD-/- mice after P24 were significantly decreased as a result of degradation. Furthermore, the recombinant human GAD65/67 was rapidly digested by the lysosomal fraction prepared from the whole brain of wild-type and CD-/- mice. These observations strongly suggest that the reduction of GABA contents, presumably because of lysosomal degradation of GAD67 and lysosomal accumulation of its degraded forms, are responsible for the dysfunction of GABAergic interneurons in the hippocampal CA3 subfield of CD-/- mice.

Animals↗