Biomedical subjects

Shigeyoshi Itohara

Publications and source records attributed to Shigeyoshi Itohara.

At least 19 recordsLinked to original sources

PubMed2006

Physiological mouse brain Abeta levels are not related to the phosphorylation state of threonine-668 of Alzheimer's APP.

BACKGROUND: Amyloid-beta peptide species ending at positions 40 and 42 (Abeta40, Abeta42) are generated by the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Abeta peptides accumulate in the brain early in the course of Alzheimer's disease (AD), especially Abeta42. The cytoplasmic domain of APP regulates intracellular trafficking and metabolism of APP and its carboxyl-terminal fragments (CTFalpha, CTFbeta). The role of protein phosphorylation in general, and that of the phosphorylation state of APP at threonine-668 (Thr668) in particular, has been investigated in detail by several laboratories (including our own). Some investigators have recently proposed that the phosphorylation state of Thr668 plays a pivotal role in governing brain Abeta levels, prompting the current study. METHODOLOGY: In order to evaluate whether the phosphorylation state of Thr668 controlled brain Abeta levels, we studied the levels and subcellular distributions of holoAPP, sAPPalpha, sAPPbeta, CTFalpha, CTFbeta, Abeta40 and Abeta42 in brains from "knock-in" mice in which a non-phosphorylatable alanyl residue had been substituted at position 668, replacing the threonyl residue present in the wild-type protein. CONCLUSIONS: The levels and subcellular distributions of holoAPP, sAPPalpha, sAPPbeta, CTFalpha, CTFbeta, Abeta40 and Abeta42 in the brains of Thr668Ala mutant mice were identical to those observed in wild-type mice. These results indicate that, despite speculation to the contrary, the phosphorylation state of APP at Thr668 does not play an obvious role in governing the physiological levels of brain Abeta40 or Abeta42 in vivo.

Alzheimer Disease↗

PubMed2006

Pael receptor induces death of dopaminergic neurons in the substantia nigra via endoplasmic reticulum stress and dopamine toxicity, which is enhanced under condition of parkin inactivation.

Selective loss of dopaminergic neurons is the final common pathway in Parkinson's disease. Expression of Parkin associated endothelin-receptor like receptor (Pael-R) in mouse brain was achieved by injecting adenoviral vectors carrying a modified neuron-specific promoter and Cre recombinase into the striatum. Upregulation of Pael-R in the substantia nigra pars compacta of mice by retrograde infection induced endoplasmic reticulum (ER) stress leads to death of dopaminergic neurons. The role of ER stress in dopaminergic neuronal vulnerability was highlighted by their decreased survival in mice deficient in the ubiquitin-protein ligase Parkin and the ER chaperone ORP150 (150 kDa oxygen-regulated protein). Dopamine-related toxicity was also a key factor, as a dopamine synthesis inhibitor blocked neuronal death in parkin null mice. These data suggest a model in which ER- and dopamine-related stress are major contributors to decreased viability of dopaminergic neurons in a setting relevant to Parkinson's disease.

Adenoviridae↗

PubMed2006

Enhanced amyloidogenic metabolism of the amyloid beta-protein precursor in the X11L-deficient mouse brain.

X11L, a neuronal adaptor protein, associates with the cytoplasmic domain of APP and suppresses APP cellular metabolism. APP is the precursor of Abeta, whose metabolism is strongly implicated in Alzheimer disease pathogenesis. To examine the roles of X11L function in APP metabolism, including the generation of Abeta in the brain, we produced X11L-deficient mutant mice on the C57BL/6 background. The mutant mice did not exhibit histopathological alterations or compensatory changes in the expression of other X11 family proteins, X11 and X11L2. The expression level and distribution of APP in the brain of mutant mice were also identical to those in wild-type mice. However, in the hippocampus, where substantial levels of X11L and APP are expressed, the mutant mice exhibited a significant increase in the level of the C-terminal fragments of APP produced by cleavage with beta-secretase but not alpha-secretase. The levels of Abeta were increased in the hippocampus of aged mutant mice as compared with age-matched controls. These observations clearly indicate that X11L suppresses the amyloidogenic but not amyloidolytic processing of APP in regions of the brain such as the hippocampus, which express significant levels of X11L.

Aging↗

PubMed2006

Matrix metalloproteinase-2 plays a critical role in the pathogenesis of white matter lesions after chronic cerebral hypoperfusion in rodents.

BACKGROUND AND PURPOSE: Cerebrovascular white matter (WM) lesions contribute to cognitive impairment and motor dysfunction in the elderly. A disruption of the blood-brain barrier (BBB) is believed to be a critical early event leading to these WM lesions. Previous studies have suggested the involvement of matrix metalloproteinase-2 (MMP-2) in BBB disruptions and the upregulation of MMP-2 after chronic cerebral hypoperfusion in a rat model. In the present study, we asked whether MMP-2 is involved in the BBB disruption and the subsequent WM lesions after chronic cerebral hypoperfusion. METHODS: We compared the severity of white matter lesions in rats after chronic cerebral hypoperfusion with or without an MMP inhibitor. Then, we also induced the chronic cerebral hypoperfusion in wild-type and MMP-2-null mice. RESULTS: In the rats treated with a relatively selective MMP-2 inhibitor, AG3340, the WM lesions after chronic cerebral hypoperfusion were significantly less severe, and the number of activated astroglia and microglia were also significantly lower as compared with the vehicle-treated rats. Gene knockout of MMP-2 also reduced the severity of the WM lesions and the number of activated astroglia and microglia in a mice system. In both rodents, the disruption of BBB function, as assessed by IgM staining and the Evans blue extravasation test, was less severe when MMP-2 activity was attenuated. CONCLUSIONS: These findings indicate that MMP-2 plays a critical role in the BBB disruption, glial cell activation, and WM lesions after chronic cerebral hypoperfusion and suggest the potential value of MMP-2 inhibitors as a therapeutic tool in cerebrovascular WM lesions.

Animals↗

PubMed2006

Induction of matrix metalloproteinase-2 enhances systemic arterial contraction after hypoxia.

This study was carried out to determine the role of increased vascular matrix metalloproteinase-2 (MMP-2) expression in the changes in systemic arterial contraction after prolonged hypoxia. Rats and mice were exposed to hypoxia (10% and 8% O(2), respectively) or normoxia (21% O(2)) for 16 h, 48 h, or 7 days. Aortae and mesenteric arteries were either mounted in organ bath myographs or frozen in liquid nitrogen. MMP-2 inhibition with cyclic CTTHWGFTLC (CTT) reduced contraction to phenylephrine (PE) in aortae and mesenteric arteries from rats exposed to hypoxia for 7 days but not in vessels from normoxic rats. Similarly, CTT reduced contraction to Big endothelin-1 (Big ET-1) in aortae from rats exposed to hypoxia for 7 days. Responses to PE were reduced in hypoxic MMP-2(-/-) mice compared with MMP-2(+/+) mice. Increased contraction to Big ET-1 after hypoxia was observed in MMP-2(+/+) mice but not in MMP-2(-/-) mice. Rat aortic MMP-2 and membrane type 1 (MT1)-MMP protein levels and MMP activity were increased after 7 days of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. We conclude that hypoxic induction of MMP-2 expression potentiates contraction in systemic conduit and resistance arteries. This may preserve the capacity to regulate the systemic circulation in the transition between the alterations in vascular tone and structural remodeling that occurs during prolonged hypoxic epochs.

Animals↗

PubMed2006

Loss of M5 muscarinic acetylcholine receptors leads to cerebrovascular and neuronal abnormalities and cognitive deficits in mice.

The M5 muscarinic acetylcholine receptor (M5R) has been shown to play a crucial role in mediating acetylcholine-dependent dilation of cerebral blood vessels. We show that male M5R-/- mice displayed constitutive constriction of cerebral arteries using magnetic resonance angiography in vivo. Male M5R-/- mice exhibited a significantly reduced cerebral blood flow (CBF) in the cerebral cortex, hippocampus, basal ganglia, and thalamus. Cortical and hippocampal pyramidal neurons from M5R-/- mice showed neuronal atrophy. Hippocampus-dependent spatial and nonspatial memory was also impaired in M5R-/- mice. In M5R-/- mice, CA3 pyramidal cells displayed a significantly attenuated frequency of the spontaneous postsynaptic current and long-term potentiation was significantly impaired at the mossy fiber-CA3 synapse. Our findings suggest that impaired M5R signaling may play a role in the pathophysiology of cerebrovascular deficits. The M5 receptor may represent an attractive novel therapeutic target to ameliorate memory deficits caused by impaired cerebrovascular function.

Acetylcholine↗

PubMed2006

A crucial role for matrix metalloproteinase 2 in osteocytic canalicular formation and bone metabolism.

Extracellular matrix production and degradation by bone cells are critical steps in bone metabolism. Mutations of the gene encoding MMP-2, an extracellular matrix-degrading enzyme, are associated with a human genetic disorder characterized by subcutaneous nodules, arthropathy, and focal osteolysis. It is not known how the loss of MMP-2 function results in the pathology. Here, we show that Mmp2(-/-) mice exhibited opposing bone phenotypes caused by an impaired osteocytic canalicular network. Mmp2(-/-) mice showed decreased bone mineral density in the limb and trunk bones but increased bone volume in the calvariae. In the long bones, there was moderate disruption of the osteocytic networks and reduced bone density throughout life, whereas osteoblast and osteoclast function was normal. In contrast, aged but not young Mmp2(-/-) mice had calvarial sclerosis with osteocyte death. Severe disruption of the osteocytic networks preceded osteocyte loss in Mmp2(-/-) calvariae. Successful transplantation of wild-type periosteum restored the osteocytic canalicular networks in the Mmp2(-/-) calvariae, suggesting local roles of MMP-2 in determining bone phenotypes. Our results indicate that MMP-2 plays a crucial role in forming and maintaining the osteocytic canalicular network, and we propose that osteocytic network formation is a determinant of bone remodeling and mineralization.

Animals↗

PubMed2006

The Rho-GTPase cdc42 regulates neural progenitor fate at the apical surface.

Stem cell persistence into adulthood requires self-renewal from early developmental stages. In the developing mouse brain, only apical progenitors located at the ventricle are self-renewing, whereas basal progenitors gradually deplete. However, nothing is known about the mechanisms regulating the fundamental difference between these progenitors. Here we show that the conditional deletion of the small Rho-GTPase cdc42 at different stages of neurogenesis in mouse telencephalon results in an immediate increase in basal mitoses. Whereas cdc42-deficient progenitors have normal cell cycle length, orientation of cell division and basement membrane contact, the apical location of the Par complex and adherens junctions are gradually lost, leading to an increasing failure of apically directed interkinetic nuclear migration. These cells then undergo mitoses at basal positions and acquire the fate of basal progenitors. Thus, cdc42 has a crucial role at the apical pole of progenitors, thereby regulating the position of mitoses and cell fate.

Adherens Junctions↗

PubMed2006

Cortical 5-HT2A receptor signaling modulates anxiety-like behaviors in mice.

Serotonin [5-hydroxytryptamine (5-HT)] neurotransmission in the central nervous system modulates depression and anxiety-related behaviors in humans and rodents, but the responsible downstream receptors remain poorly understood. We demonstrate that global disruption of 5-HT2A receptor (5HT2AR) signaling in mice reduces inhibition in conflict anxiety paradigms without affecting fear-conditioned and depression-related behaviors. Selective restoration of 5HT2AR signaling to the cortex normalized conflict anxiety behaviors. These findings indicate a specific role for cortical 5HT2AR function in the modulation of conflict anxiety, consistent with models of cortical, "top-down" influences on risk assessment.

Animals↗

PubMed2006

Molecular evidence for two-stage learning and partial laterality in eyeblink conditioning of mice.

The anterior interpositus nucleus (AIN) is the proposed site of memory formation of eyeblink conditioning. A large part of the underlying molecular events, however, remain unknown. To elucidate the molecular mechanisms, we examined transcriptional changes in the AIN of mice trained with delay eyeblink conditioning using microarray, quantitative real-time RT-PCR, and in situ hybridization techniques. Microarray analyses suggested that transcriptionally up-regulated gene sets were largely different between early (3-d training) and late (7-d) stages. Quantitative real-time RT-PCR aided by laser microdissection indicated that the expression of representative EARLY genes (Sgk, IkBa, and Plekhf1) peaked at 1-d training in both the paired and unpaired conditioning groups, and was maintained at a higher level in the paired group than in the unpaired group after 3-d training. In situ hybridization revealed increased expression of these genes in broad cerebellar areas, including the AIN, with no hemispheric preferences. In contrast, the expression of representative LATE genes (Vamp1, Camk2d, and Prkcd) was selectively increased in the AIN of the 7-d paired group, with dominance in the ipsilateral AIN. Increased Vamp1 mRNA expression was restricted to the ipsilateral dorsolateral hump, a subregion of the AIN. These expression patterns of two distinct subsets of genes fit well with the two-stage learning theory, which proposes emotional and motor learning phases, and support the notion that AIN has a crucial role in memory formation of eyeblink conditioning.

Animals↗

PubMed2006

Effect of MMP-2 deficiency on atherosclerotic lesion formation in apoE-deficient mice.

OBJECTIVE: Although it has been reported that matrix metalloproteinase (MMP)-2 is a major proteinase in atherosclerotic plaque lesions, there is no direct evidence of the role of MMP-2 in atherosclerotic lesion formation. In the present study we determined the role of MMP-2 in atherosclerosis plaque development using apolipoprotein E-deficient (apoE(-/-)) mice. METHODS AND RESULTS: To generate MMP-2-deficient, apoE-deficient mice (MMP-2(-/-):apoE(-/-)), MMP-2(-/-) mice were crossed with apoE(-/-) mice. After 8 weeks of feeding with a lipid-rich diet, morphological and biochemical studies of the aortic sinus and arch were conducted. A significant reduction of the atherosclerotic plaque in the aortic sinus and arch with the decrease in smooth muscle cell-positive area was observed in MMP-2(-/-):apoE(-/-) mice compared with that of MMP-2(+/+):apoE(-/-) mice. Macrophage- and collagen-positive areas were less in aortic sinus but not in aortic arch in MMP-2(-/-):apoE(-/-) mice. There was no difference of MMP-9 mRNA expression in the plaque lesion between the 2 genotypes. A much lower level of mRNA expression of TIMP-1 and TIMP-2 was detected in the atherosclerotic plaque lesions of MMP-2(-/-):apoE(-/-) mice than in those of MMP-2(+/+):apoE(-/-) mice. CONCLUSIONS: MMP-2 contributes to the development of atherosclerosis in apoE(-/-) mice.

Animals↗

PubMed2006

Fusion of Doppel to octapeptide repeat and N-terminal half of hydrophobic region of prion protein confers resistance to serum deprivation.

Our previous studies have shown an essential role played by the octapeptide repeat region (OR) and the N-terminal half of hydrophobic region (HR) in the anti-apoptotic activity of prion protein (PrP). As PrP-like protein Doppel (Dpl), which structurally resembles an N-terminally truncated PrP, did not show any anti-apoptotic activity, we examined apoptosis of HpL3-4 cells expressing Dpl fused to various lengths of the N-terminal region of PrP to investigate whether the PrP/Dpl fusion proteins retain anti-apoptotic function. HpL3-4 cells expressing Dpl fused to PrP(1-124) with the OR and N-terminal half of HR of PrP showed anti-apoptotic function, whereas Dpl fused to PrP(1-95) with OR did not rescue cells from apoptotic cell death induced by serum deprivation. These results indicate that the OR and N-terminal half of HR of PrP retains anti-apoptotic activity similar to full-length PrP.

Animals↗

PubMed2006

Decreased basal mucus secretion by Slp2-a-deficient gastric surface mucous cells.

Synaptotagmin-like protein (Slp) 2-a is a putative Rab27A/B-effector protein and is implicated in intracellular membrane transport. However, the precise tissue distribution of Slp2-a protein and its functions remain largely unknown. In this study we used a specific anti-Slp2-a antibody to investigate the tissue distribution of Slp2-a in mice and found that Slp2-a is most abundantly expressed in mouse stomach. Co-immunoprecipitation experiments indicated that Slp2-a interacts with Rab27A/B in vivo. We also discovered that Slp2-a and Rab27A/B are predominantly localized at the apical region of gastric-surface mucous cells, where mucus granules are accumulated. Analysis of Slp2-a mutant mice generated by homologous recombination showed a reduced number of mucus granules, a deficiency of granule docking with the apical plasma membrane in the gastric-surface mucous cells and reduction of mucus secretion by Slp2-a-deficient gastric primary cells. Based on these results, we propose that Slp2-a is part of the mucin secretory machinery in surface mucous cells of mouse stomach.

Amino Acid Sequence↗

PubMed2006

High-efficiency CAG-FLPe deleter mice in C57BL/6J background.

The increasing popularity of conditional knockout (KO) technology has resulted in the demand for efficient FLP deleter mice. In addition, FLP deleters are needed in genetic backgrounds that are suited to behavioral studies. We generated CAG-FLPe transgenic (Tg) mice with the C57BL/6J genetic background, which is one of the most commonly-used strains in behavioral studies. We assessed the recombination efficiency of the CAG-FLPe-Tg lines by crossing them with a mouse line carrying a FRT-PGK-neo-FRT cassette. Four of five independent CAG-FLPe lines induced recombination in most (91%-100%) of their progenies, although a small fraction (0%-30%, depending on the line) showed mosaic recombination patterns. These animals are highly potent as deleters of FRT cassettes and are useful for behavioral studies involving conditional KO mice.

Animals↗

PubMed2005

Granuphilin molecularly docks insulin granules to the fusion machinery.

The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin-null mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin-null beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.

Adenoviridae↗

PubMed2005

Cell-autonomous PrP-Doppel interaction regulates apoptosis in PrP gene-deficient neuronal cells.

The Prnd-encoded prion protein (PrP)-like protein, Doppel (Dpl), is a homologue of Prnp-encoded PrP, and is N-glycosylated protein with glycosylphosphatidylinositol anchor like PrP. Recently, ectopic expressions of Prnp/Prnd chimeric mRNAs have been identified as the cause of late-onset ataxia observed in several lines of Prnp-knockout mice such as ZrchII, Ngsk, Rcm0, and Rikn mice. However, it remains unclear whether the toxic effect of Dpl expression is a cell-autonomous mechanism but rather reflect a systemic process of heterogeneous cell population in the brain. In this study, the cell-autonomous role of Dpl was estimated by investigating PrP-deficient cells (HpL3-4)-the SV40 large T-antigen immortalized and Rikn Prnp(-/-) mice-derived neuronal cell line expressing Prnp/Prnd chimeric mRNAs. The reverse transcription polymerase chain reaction revealed that serum deprivation did not increase Prnp/Prnd chimeric mRNAs, which in fact was translated into a small amount of Dpl in HpL3-4 cells, whereas serum deprivation induced apoptotic cell death of HpL3-4 cells. Dpl overexpression enhanced apoptotic cell death, whereas the toxic effect of Dpl on apoptotic cell death was neutralized by PrP expression. These results indicate that Dpl elicited dose-dependently toxic effects on PrP-deficient cells without affecting on PrP-expressing cells, suggesting that the PrP-Dpl interaction can regulate cell death in a cell-autonomous manner.

Amyloid↗

PubMed2005

Exuberant thalamocortical axon arborization in cortex-specific NMDAR1 knockout mice.

Development of whisker-specific neural patterns in the rodent somatosensory system requires NMDA receptor (NMDAR)-mediated activity. In cortex-specific NR1 knockout (CxNR1KO) mice, while thalamocortical afferents (TCAs) develop rudimentary whisker-specific patterns in the primary somatosensory (barrel) cortex, layer IV cells do not develop barrels or orient their dendrites towards TCAs. To determine the role of postsynaptic NMDARs in presynaptic afferent development and patterning in the barrel cortex, we examined the single TCA arbors in CxNR1KO mice between postnatal days (P) 1-7. Sparsely branched TCAs invade the cortical plate on P1 in CxNR1KO mice as in control mice. In control animals, TCAs progressively elaborate patchy terminals, mostly restricted to layer IV. In CxNR1KO mice, TCAs develop far more extensive arbors between P3-7. Their lateral extent is twice that of controls from P3 onwards. By P7, CxNR1KO TCAs have significantly fewer branch points and terminal endings in layers IV and VI but more in layers II/III and V than control mouse TCAs. Within expansive terminal arbors, CxNR1KO TCAs develop focal terminal densities in layer IV, corresponding to the rudimentary whisker-specific patches. Given that thalamic NMDARs are spared in CxNR1KO mice, the present results show that postsynaptic NMDARs play an important role in refinement of presynaptic afferent arbors and whisker-specific patterning in the developing barrel cortex.

Afferent Pathways↗

PubMed2005

Pathological cell-cell interactions elicited by a neuropathogenic form of mutant Huntingtin contribute to cortical pathogenesis in HD mice.

Expanded polyglutamine (polyQ) proteins in Huntington's disease (HD) as well as other polyQ disorders are known to elicit a variety of intracellular toxicities, but it remains unclear whether polyQ proteins can elicit pathological cell-cell interactions which are critical to disease pathogenesis. To test this possibility, we have created conditional HD mice expressing a neuropathogenic form of mutant huntingtin (mhtt-exon1) in discrete neuronal populations. We show that mhtt aggregation is a cell-autonomous process. However, progressive motor deficits and cortical neuropathology are only observed when mhtt expression is in multiple neuronal types, including cortical interneurons, but not when mhtt expression is restricted to cortical pyramidal neurons. We further demonstrate an early deficit in cortical inhibition, suggesting that pathological interactions between interneurons and pyramidal neurons may contribute to the cortical manifestation of HD. Our study provides genetic evidence that pathological cell-cell interactions elicited by neuropathogenic forms of mhtt can critically contribute to cortical pathogenesis in a HD mouse model.

Animals↗