高島 明彦

Tau-mediated neurodegeneration is a central event in Alzheimer's disease (AD) and other tauopathies. Consistent with suggestions that lifetime stress may be a clinically-relevant precipitant of AD pathology, we previously showed that stress triggers Tau hyperphosphorylation and accumulation; however, little is known about the etiopathogenic interaction of chronic stress with other AD risk factors, such as sex and aging. This study focused on how these various factors converge on the cellular mechanisms underlying Tau aggregation in the hippocampus of chronically stressed male and female (middle-aged and old) mice expressing the most commonly found disease-associated Tau mutation in humans, P301L-Tau. We report that environmental stress triggers memory impairments in female, but not male, P301L-Tau transgenic mice. Furthermore, stress elevates levels of caspase-3-truncated Tau and insoluble Tau aggregates exclusively in the female hippocampus while it also alters the expression of the molecular chaperones Hsp90, Hsp70, and Hsp105, thus favoring accumulation of Tau aggregates. Our findings provide new insights into the molecular mechanisms through which clinically-relevant precipitating factors contribute to the pathophysiology of AD. Our data point to the exquisite sensitivity of the female hippocampus to stress-triggered Tau pathology.

Senile plaques comprised of A beta aggregates and neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau filaments are the hallmarks of Alzheimer's disease (AD). A number of amyloid precursor protein (APP) transgenic (Tg) mice harboring APP mutations have been generated as animal models of AD. These mice successfully display amyloid plaque formation and subsequent tau hyperphosphorylation, but seldom induce NFT formations. We have demonstrated that the APP(OSK)-Tg mice, which possess the E693 Delta (Osaka) mutation in APP and thereby accumulate A beta oligomers without plaques, exhibit tau hyperphosphorylation at 8 months, but not NFT formation even at 24 months. We assumed that APP-Tg mice, including ours, failed to form NFTs because NFT formation requires human tau. To test this hypothesis, we crossbred APP(OSK)-Tg mice with tau-Tg mice (tau264), which express low levels of 3-repeat and 4-repeat wild-type human tau without any pathology. The resultant double Tg mice displayed tau hyperphosphorylation at 6 months and NFT formation at 18 months in the absence of tau mutations. Importantly, these NFTs contained both 3-repeat and 4-repeat human tau, similar to those in AD. Furthermore, the double Tg mice exhibited A beta oligomer accumulation, synapse loss, and memory impairment at 6 months and neuronal loss at 18 months, all of which appeared earlier than in the parent APP(OSK)-Tg mice. These results suggest that A beta and human tau synergistically interact to accelerate each other's pathology, that the presence of human tau is critical for NFT formation, and that A beta oligomers can induce NFTs in the absence of amyloid plaques.

In an aging society, the number of people with dementia has increased. Since Alzheimer's disease and a part of frontotemporal lober degeneration (FTLD-tau) have abnormal tau pathology in brain, these are called Tauopathy. Previously results showed that occurrence of abnormal tau has been involved in development of cognitive dysfunction and neuronal loss indicating that tau-focused drug (inhibitors of tau hyperphosphorylation, tau aggregation and so on) may be valuable in therapy for Tauopathy. This study collated data of clinical trials that evaluated tau-based drugs to help development of agent for dementia drugs in future. We discovered a novel tau aggregation inhibitor, and elucidated the inhibitory mechanism of the compound on tau aggregation. These results suggest that tau aggregation is an important target for therapy of dementia.

The microtubule-associated protein tau is a principal component of neurofibrillary tangles, and has been identified as a key molecule in Alzheimer's disease and other tauopathies. However, it is unknown how a protein that is primarily located in axons is involved in a disease that is believed to have a synaptic origin. To investigate a possible synaptic function of tau, we studied synaptic plasticity in the hippocampus and found a selective deficit in long-term depression (LTD) in tau knockout mice in vivo and in vitro, an effect that was replicated by RNAi knockdown of tau in vitro. We found that the induction of LTD is associated with the glycogen synthase kinase-3-mediated phosphorylation of tau. These observations demonstrate that tau has a critical physiological function in LTD.

Alzheimer's disease is a progressive dementia that is characterized by a loss of recent memory. Evidence has accumulated to support the hypothesis that synapses are critical storage sites for memory. However, it is still uncertain whether tau protein is involved in associative memory storage and whether tau is distributed in mature brain synapses. To address this question, we examined the synaptosomal distribution of tau protein in both JNPL3 transgenic mice expressing human P301L tau and non-transgenic littermates. The JNPL3 mouse line is known as one of the mouse models of human tauopathy that develop motor and behavioral deficits with intracellular tau aggregates in the spinal cord and brainstem. The phenotype of disease progression is highly dependent on strain background. In this study, we confirmed that male JNPL3 transgenic mice with C57BL/6J strain background showed neither any sign of motor deficits nor accumulation of hyper-phosphorylated tau in the sarkosyl-insoluble fraction until 18 months of age. Subcellular fractionation analysis showed that both mouse tau and human P301L tau were present in the synaptosomal fraction. Those tau proteins were less-phosphorylated than tau in the cytosolic fraction. Human P301L tau was preferentially distributed in the synaptosomal fraction while mouse endogenous tau was more distributed in the cytosolic fraction. Interestingly, a human-specific tau band with phosphorylation at Ser199 and Ser396 was observed in the synaptosomal fraction of JNPL3 mice. This tau was not identical to either tau species in cytosolic fraction or a prominent hyperphosphorylated 64 kDa tau species that was altered to tau pathology. These results suggest that exogenous human P301L tau induces synaptosomal distribution of tau protein with a certain phosphorylation. Regulating the synaptosomal tau level might be a potential target for a therapeutic intervention directed at preventing neurodegeneration.

Accumulation of intracellular tau fibrils has been the focus of research on the mechanisms of neurodegeneration in Alzheimer's disease (AD) and related tauopathies. Here, we have developed a class of tau ligands, phenyl/pyridinyl-butadienyl-benzothiazoles/benzothiazoliums (PBBs), for visualizing diverse tau inclusions in brains of living patients with AD or non-AD tauopathies and animal models of these disorders. In vivo optical and positron emission tomographic (PET) imaging of a transgenic mouse model demonstrated sensitive detection of tau inclusions by PBBs. A pyridinated PBB, [C-11]PBB3, was next applied in a clinical PET study, and its robust signal in the AD hippocampus wherein tau pathology is enriched contrasted strikingly with that of a senile plaque radioligand, [C-11]Pittsburgh Compound-B ([C-11]PIB. [C-11]PBB3-PET data were also consistent with the spreading of tau pathology with AD progression. Furthermore, increased [C-11]PBB3 signals were found in a corticobasal syndrome patient negative for [C-11]PIB-PET.

Neurofibrillarly tangles (NFTs) are seen most patients, who shows dementia, while b amyloid deposition observed specifically in AD patients. NFTs observe first from coeruleus and entorhinal cortex, and then spread to neocortex, which well explain clinical progression of AD, such as memory problem to dementia. Tau fibrils are the major component of NFT. Before forming tau fibrils, tau binds together, form soluble tau oligomer, and then granular tau oligomer. The soluble tau oligomer associates with synapse loss, and granular tau formation induces neuronal loss. Therefore, tau aggregation inhibitor is expected to halt the clinical progression of AD.

Oxidative stress is considered to promote aging and age-related disorders such as tauopathy. Although recent reports suggest that oxidative stress under certain conditions possesses anti-aging properties, no such conditions have been reported to ameliorate protein-misfolding diseases. Here, we used neuronal and murine models that overexpress human tau to demonstrate that mild oxidative stress generated by alloxan suppresses several phenotypes of tauopathy. Alloxan treatment reduced HSP90 levels and promoted proteasomal degradation of tau, c-Jun N-amino terminal kinase, and histone deacetylase (HDAC) 6. Moreover, reduced soluble tau (phosphorylated tau) levels suppressed the formation of insoluble tau in tau transgenic mice, while reduced HDAC6 levels contributed to microtubule stability by increasing tubulin acetylation. Age-dependent decreases in HDAC2 and phospho-tau levels correlated with spatial memory enhancement in alloxan-injected tau mice. These results suggest that mild oxidative stress, through adaptive stress responses, operates counteractively against some of the tauopathy phenotypes.

The binding of curcumin to senile plaques (SPs) and cerebral amyloid angiopathy (CAA) was examined in the aged brain of various animal species and a human patient with Alzheimer's disease (AD), together with its binding to neurotibrillary tangles (NFTs). Brain sections were immunostained with anti-amyloid beta protein 1-42 (A beta 42) and anti-amyloid beta protein 1-40 (A beta 40) antibodies. These sections were also stained with alkaline Congo red, periodic acid-methenamine silver (PAM), and curcumin (0.009% curcumin solution) with or without formic acid pretreatment. The sections from the AD brain were also immunostained for anti-paired helical filament-tau (PHF-tau), and were stained with Gallyas silver for NFTs. Some SPs in the AD, monkey, dog, bear, and amyloid precursor protein transgenic mouse (APP Tg-mouse) brains contained congophilic materials, and were intensely positive for curcumin. In addition, curcumin labeled some diffuse SPs negative for Congo red in the AD, monkey, bear, and APP Tg-mouse brains. In all animals. CAA was intensely positive for both Congo red and curcumin. The specific curcumin staining activity was lost by formic acid pretreatment. In the AD brain, NFTs positive for PHF-tau and Gallyas silver were moderately stained with curcumin. These findings indicate that curcumin specifically binds to the aggregated A beta molecules in various animals, and further to phosphorylatecl tau protein, probably according to its conformational nature.

Tauopathies differ in terms of the brain regions that are affected. In Alzheimer's disease, basal forebrain and hippocampus are mainly involved, while frontotemporal lobar degeneration affects the frontal and temporal lobes and subcortical nuclei including striatum. Over 90% of human cases of tauopathies are sporadic, although the majority of established tau-transgenic mice have had mutations. This prompted us to establish transgenic mice expressing wild-type human tau (Tg601). Old (> 14 months old) Tg601 mice displayed decreased anxiety in the elevated plus maze test and impaired place learning in the Morris water maze test. Immunoblotting of brain tissue identified that soluble tau multimer was increased with aging even though insoluble tau was not observed. In the striatum of old Tg601, the level of AT8- or AT180-positive tau was decreased compared with that of other regions, while PHF-1-positive tau levels remained equal. Phosphorylated tau-positive axonal dilations were present mainly in layers V and VI of the prefrontal cortex. Loss of synaptic dendritic spine and decreased immunohistochemical level of synaptic markers were observed in the nucleus accumbens. In vivo 2-[(18)F]fluoro-2-deoxy-D-glucose positron emission tomography analysis also showed decreased activity exclusively in the nucleus accumbens of living Tg601 mice. In Tg601 mice, the axonal transport defect in the prefrontal cortex-nucleus accumbens pathway may lead to decreased anxiety behavior. Differential distribution of hyperphosphorylated tau may cause region-specific neurodegeneration. (c) 2011 Elsevier Inc. All rights reserved.

Cap'n'Collar (CNC) proteins heterodimerize with small Maf proteins and regulate the transcription of various genes. Small Maf-deficient mice develop severe neurodegeneration, and it remains unclear whether CNC proteins are involved in this process. In this study, we examined the contribution of Nrf1, one of the CNC proteins, to neuronal homeostasis in vivo. As Nrf1 gene knockout mice are embryonic lethal, we developed a central nervous system (CNS)-specific Nrf1 knockout (CKO) mouse line using mice bearing an Nrf1flox allele and Nestin-Cre allele. At birth, the CKO mice appeared indistinguishable from control mice, but thereafter they showed progressive motor ataxia and severe weight loss. All Nrf1 CKO mice died within 3 weeks. These phenotypes are similar to those reported in small Maf-deficient mice, suggesting the presence of collaboration between Nrf1 and small Maf proteins. We also found aberrant accumulation of polyubiquitinated proteins in various CNS regions and apparent neuronal loss in the hippocampus of Nrf1 CKO mice. An oxidative stress marker was accumulated in the spinal cords of the mice, but the expression patterns of oxidative stress response genes regulated by Nrf2 did not change substantially. These results show that Nrf1 sustains the CNS homeostasis through regulating target genes distinct from those regulated by Nrf2.

Stressful life experiences are likely etiological factors in sporadic forms of Alzheimer's disease (AD). Many AD patients hypersecrete glucocorticoids (GCs), and their GC levels correlate with the rate of cognitive impairment and extent of neuronal atrophy. Severity of cognitive deficits in AD correlates strongly with levels of hyperphosphorylated forms of the cytoskeletal protein TAU, an essential mediator of the actions of amyloid beta (A beta), another molecule with a key pathogenic role in AD. Our objective was to investigate the sequential interrelationships between these various pathogenic elements, in particular with respect to the mechanisms through which stress might precipitate cognitive decline. We thus examined whether stress, through the mediation of GCs, influences TAU hyperphosphorylation, a critical and early event in the cascade of processes leading to AD pathology. Results from healthy, wild-type, middle-aged rats show that chronic stress and GC induce abnormal hyperphosphorylation of TAU in the hippocampus and prefrontal cortex (PFC), with contemporaneous impairments of hippocampus-and PFC-dependent behaviors. Exogenous GC potentiated the ability of centrally infused A beta to induce hyperphosphorylation of TAU epitopes associated with AD and cytoplasmic accumulation of TAU, while previous exposure to stress aggravated the biochemical and behavioral effects of GC in A beta-infused animals. Thus, lifetime stress/GC exposure may have a cumulative impact on the onset and progress of AD pathology, with TAU hyperphosphorylation serving to transduce the negative effects of stress and GC on cognition.

Neurofibrillary tangles (NFTs), which consist of highly phosphorylated tau, are hallmarks of neurodegenerative diseases including Alzheimer disease (AD). In neurodegenerative diseases, neuronal dysfunction due to neuronal loss and synaptic loss accompanies NFT formation, suggesting that a process associated with NFT formation may be involved in neuronal dysfunction. To clarify the relationship between the tau aggregation process and synapse and neuronal loss, we compared two lines of mice expressing human tau with or without an aggregation-prone P301L mutation. P301L tau transgenic (Tg) mice exhibited neuronal loss and produced sarcosyl-insoluble tau in old age but did not exhibit synaptic loss and memory impairment. By contrast, wild-type tau Tg mice neither exhibited neuronal loss nor produced sarcosyl-insoluble tau but did exhibit synaptic loss and memory impairment. Moreover, P301L tau was less phosphorylated than wild-type tau, suggesting that the tau phosphorylation state is involved in synaptic loss, whereas the tau aggregation state is involved in neuronal loss. Finally, increasing concentrations of insoluble tau aggregates leads to the formation of fibrillar tau, which causes NFTs to form. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Alzheimer's disease (AD) is a significant and growing health problem in the aging population. Although definitive mechanisms of pathogenesis remain elusive, genetic and histological clues have implicated the proteins presenilin (PS) and tau as key players in AD development. PS mutations lead to familial AD, and although tau is not mutated in AD, tau pathology is a hallmark of the disease. Axonal transport deficits are a common feature of several neurodegenerative disorders and may represent a point of intersection of PS and tau function. To investigate the contribution of wild-type, as opposed to mutant, tau to axonal transport defects in the context of presenilin loss, we used a mouse model postnatally deficient for PS (PS cDKO) and expressing wild-type human tau (WtTau). The resulting PS cDKO WtTau mice exhibited early tau pathology and axonal transport deficits that preceded development of these phenotypes in WtTau or PS cDKO mice. These deficits were associated with reduced neurotrophin signaling, defective learning and memory and impaired synaptic plasticity. The combination of these effects accelerated neurodegeneration in PS cDKO WtTau mice. Our results strongly support a convergent role for PS and tau in axonal transport and neuronal survival and function and implicate their misregulation as a contributor to AD pathogenesis. Copyright © 2010 the authors.

Neurofibrillary tangles (NFTs), which consist of a fibrillar aggregate of hyperphosphorylated tau, are commonly seen in aging brains and those with Alzheimer's disease. Based on Braak staging of NFTs, NFTs are first observed in the entorhinal cortex. Then, NFTs spread from the entorhinal cortex to the limbic and neocortex. NFT the formation in the entorhinal cortex may be correlated with memory loss in brain aging, because entorhinal cortex is involved in memory formation, and NFTs in the limbic and neocortex may cause dementia in AD, because the limbic and neocortex serve higher order brain functions. These suggest that regional development of NFTs is correlated with decline of brain functions in aging and AD. Recent reports suggested that the process of NFT formation, but not NFT itself, is involved in neuronal dysfunction. We found that there are three tau aggregation forms, soluble tau oligomer, granular tau, and fibrilar tau, before NFT formation. From the analysis of tau Tg mice, it was indicated that soluble oligomer tau may be involved in synapse loss, and insoluble granular tau aggregates may play a role in neuronal death. Therefore, inhibition of oligomer tau, and granular tau aggregation is expected to block the progression of AD symptoms by preventing synapse loss and neuronal loss.

Based on the amyloid hypothesis, studies on for Alzheimer disease (AD) therapy mostly focus on elimination of β-amyloid. However, results of recent studies on amyloid immunotherapy suggest that it may not be sufficient to target only β-amyloid for AD therapy. Neurofibrillary tangles (NFTs), which contain hyperphosphorylated tau are the other pathological hallmark of AD clinical progression of NFTs is from the entorhinal cortex to the limbic cortex, and neocortex. In a brain region showing NFTs, synapse loss and neuronal loss were observed this suggests the possibility that NFT formation is involved in brain dysfunction because of synapse loss and neuronal loss. In the process of NFT formation, tau formed different aggregation species - tau oligomers, granules, and fibrils. From the analysis of different human tau-expressing mouse lines, soluble hyperphosphorylated tau, including the tau oligomer, was found to be involved in synapse loss and granular tau formation was also found to be involved in neuronal loss. Therefore, inhibition of tau aggregation and tau phosphorylation is expected to prevent synapse loss and neuron loss, which may halt progressive dementia in AD.

Neurofibrillary tangle-bearing neurons, a pathological hallmark of Alzheimer's disease, are mostly devoid of normal microtubule (MT) structure and instead have paired helical filaments that are composed of abnormal hyperphosphorylated tau. However, a causal relationship between tau phosphorylation and MT disruption has not been clarified. To examine whether MT disruption induces tau phosphorylation, stathmin, an MT-disrupting protein, was co-expressed with tau in COS-7 cells. Stathmin expression induced apparent MT catastrophe and tau hyperphosphorylation at Thr-181, Ser-202, Thr-205, and Thr-231 sites. In contrast, c-Jun N-terminal kinase activation, or phosphatase inhibition, led to significant tau phosphorylation without affecting MT structure. These findings suggest that MT disruption induces subsequent tau phosphorylation. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Rationale: The number of patients with coronary heart disease, including myocardial infarction, is increasing and novel therapeutic strategy is awaited. Tumor suppressor protein p53 accumulates in the myocardium after myocardial infarction, causes apoptosis of cardiomyocytes, and plays an important role in the progression into heart failure. Objectives: We investigated the molecular mechanisms of p53 accumulation in the heart after myocardial infarction and tested whether anti-p53 approach would be effective against myocardial infarction. Methods and Results: Through expression screening, we found that CHIP (carboxyl terminus of Hsp70-interacting protein) is an endogenous p53 antagonist in the heart. CHIP suppressed p53 level by ubiquitinating and inducing proteasomal degradation. CHIP transcription was downregulated after hypoxic stress and restoration of CHIP protein level prevented p53 accumulation after hypoxic stress. CHIP overexpression in vivo prevented p53 accumulation and cardiomyocyte apoptosis after myocardial infarction. Promotion of CHIP function by heat shock protein (Hsp) 90 inhibitor, 17-allylamino-17-demethoxy geldanamycin (17-AAG), also prevented p53 accumulation and cardiomyocyte apoptosis both in vitro and in vivo. CHIP-mediated p53 degradation was at least one of the cardioprotective effects of 17-AAG. Conclusions: We found that downregulation of CHIP level by hypoxia was responsible for p53 accumulation in the heart after myocardial infarction. Decreasing the amount of p53 prevented myocardial apoptosis and ameliorated ventricular remodeling after myocardial infarction. We conclude that anti-p53 approach would be effective to treat myocardial infarction. (Circ Res. 2010; 106: 1692-1702.)

Methylmercury (MeHg) is a well-known neurotoxicant inducing neuronal degeneration in the central nervous system. This in vivo study investigated the involvement of tau hyperphosphorylation in MeHg-induced neuropathological changes in the mouse brain, because abnormal tau hyperphosphorylation causes significant pathological changes associated with some neurodegenerative diseases. Mice that were administrated to 30 ppm MeHg in drinking water for 8 weeks exhibited neuropathological changes, e.g. a decrease in the number of neuron an increase in the number of migratory astrocytes and microglia/macrophages necrosis and apoptosis in the cerebral cortex, particularly the deep layer of primary motor cortex and prelimbic cortex. Western blotting revealed that MeHg exposure increased tau phosphorylation at Thr-205, Ser-396 and Ser-422 in the cerebral cortex, consistent with the phosphorylation patterns noted in Alzheimer's disease and frontotemporal dementia. Immunohistochemical analyses revealed that the distribution of tau-phosphorylated (Thr-205) neurons corresponded with the areas showing considerable neuropathological changes. Among the kinases and phosphatases related to tau hyperphosphorylation, the activation of mitogen-activated protein kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK) was recognized. Neither neuropathological changes nor tau hyperphosphorylation was detected in the hippocampus in this study although the mercury concentration here was twice that in the cerebral cortex. These findings suggest that MeHg exposure induces tau hyperphosphorylation at specific sites of tau mainly through the activation of JNK pathways, leading to neuropathological changes in the cerebral cortex selectively, but not in the hippocampus of mouse brain. © 2009 Elsevier Inc. All rights reserved.

The deposition of amyloid β (Aβ) protein is a consistent pathological hallmark of Alzheimer's disease (AD) brains therefore, inhibition of Aβ fibril formation and destabilization of pre-formed Aβ fibrils is an attractive therapeutic and preventive strategy in the development of disease-modifying drugs for AD. This study demonstrated that Paeonia suffruticosa, a traditional medicinal herb, not only inhibited fibril formation of both Aβ1-40 and Aβ1-42 but it also destabilized pre-formed Aβ fibrils in a concentration-dependent manner. Memory function was examined using the passive-avoidance task followed by measurement of Aβ burden in the brains of Tg2576 transgenic mice. The herb improved long-term memory impairment in the transgenic mice and inhibited the accumulation of Aβ in the brain. Three-dimensional HPLC analysis revealed that a water extract of the herb contained several different chemical compounds including 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose (PGG). No obvious adverse/toxic were found following treatment with PGG. As was observed with Paeonia suffruticosa, PGG alone inhibited Aβ fibril formation and destabilized pre-formed Aβ fibrils in vitro and in vivo. Our results suggest that both Paeonia suffruticosa and its active constituent PGG have strong inhibitory effects on formation of Aβ fibrils in vitro and in vivo. PGG is likely to be a safe and promising lead compound in the development of disease-modifying drugs to prevent and/or cure AD. © 2009 International Society for Neurochemistry.

Background: The M5 muscarinic acetylcholine receptor is known to play a crucial role in mediating acetylcholine dependent dilation of cerebral blood vessels. Previously, we reported that male M5 muscarinic acetylcholine knockout mice (M5R-/- mice) suffer from a constitutive constriction of cerebral arteries, reduced cerebral blood flow, dendritic atrophy, and short-term memory loss, without necrosis and/or inflammation in the brain. Methodology/Principal Findings: We employed the Magnetic Resonance Angiography to study the area of the basilar artery in male and female M5R-/- mice. Here we show that female M5R-/- mice did not show the reduction in vascular area observed in male M5R-/- mice. However, ovariectomized female M5R-/- mice displayed phenotypic changes similar to male M5R-/- mice, strongly suggesting that estrogen plays a key role in the observed gender differences. We found that 17β-estradiol (E2) induced nitric oxide release and ERK activation in a conditional immortalized mouse brain cerebrovascular endothelial cell line. Agonists of ERα, ERβ, and GPR30 promoted ERK activation in this cell line. Moreover, in vivo magnetic resonance imaging studies showed that the cross section of the basilar artery was restored to normal in male M5R-/- mice treated with E2. Treatment with E2 also improved the performance of male M5R-/- mice in a cognitive test and reduced the atrophy of neural dendrites in the cerebral cortex and hippocampus. M5R-/- mice also showed astrocyte swelling in cortex and hippocampus using the three-dimensional reconstruction of electron microscope images. This phenotype was reversed by E2 treatment, similar to the observed deficits in dendrite morphology and the number of synapses. Conclusions/Significance: Our findings indicate that M5R-/- mice represent an excellent novel model system to study the beneficial effects of estrogen on cerebrovascular function and cognition. E2 may offer new therapeutic perspectives for the treatment of cerebrovascular insufficiency related memory dysfunction.

In an attempt to express human β-amyloid precursor protein (APP) in yeast, we fortuitously found that this protein is only O-glycosylated in yeast. APP was effectively expressed in yeast, processed by yeast α-secretases, members of the Yapsin family, to produce N-terminal (sAPPα) and C-terminal (CTFα) domains, when its signal sequence was replaced by that of the yeast α-mating factor. APP is known to acquire N- and O-glycosylation through the endoplasmic reticulum (ER) and the Golgi apparatus and is transported to the plasma membrane in mammalian cells. In spite of the presence of canonical N-glycosylation consensus sequences, APP was not N-glycosylated in the yeast system. Pulse-chase experiments demonstrated that APP received only O-mannosylation in yeast. Examination of yeast pmt mutants, which are defective in the initiation of O-mannosylation in the ER, revealed that Pmt4p is most responsible for the oligosaccharide modification of APP. Maturation of APP was slowed down and aggregated forms of APP were observed by sucrose density gradient fractionation of the Δpmt4 mutant lysate. This caused decreased production of CTFα. We conclude that O-mannosylation is required for the solubilization of exogenously expressed human APP. © 2009 The Authors Journal compilation © 2009 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Methylmercury (MeHg) has been recognized as a neurotoxicant targeted on the central nervous system including cerebellum and cerebral cortex. Some molecular targets of MeHg have been identified using cerebellar neuronal cells, but little is known in the cerebrocortical neuronal cells. In this study, the molecular mechanism underlying MeHg-induced cell death in cerebrocortical neurons was investigated using a primary culture of embryonic rat cortical neuronal cells. The cultured cells exhibited apoptosis 3 days after exposure to 100 nM MeHg, suggesting the involvement of caspase-dependent apoptotic pathways. We demonstrated for the first time that neuritic degeneration precedes MeHg-induced apoptotic death in neurons exposed to 100 nM MeHg. Immunocytochemical and ELISA analyses for neurite-specific proteins namely, tau and MAP2, showed that injury to tau-positive axons was first induced followed by damage to the dendrites and cellular bodies. To further investigate the factors responsible for neuronal death, we investigated the expression levels of Rho-family proteins (Rac1, Cdc42, and RhoA), which regulate neuritic functions and apoptosis in neurons. Western blot analysis demonstrated that MeHg downregulated the expression levels of Rac1 and Cdc42 but did not affect RhoA. The exposure concentration and time course studies confirmed that Rac1 is targeted during an early stage of MeHg-induced cytotoxicity. The results indicate that neuritic degeneration, in particular axonal degeneration triggered by the downregulation of Rac1 expression, contributes to MeHg-induced apoptotic cell death in cultured cerebrocortical neurons. © 2008 Elsevier Inc. All rights reserved.

Intracellular accumulation of filamentous tau proteins is a defining feature of neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, Pick's disease, and frontotemporal dementia with Parkinsonism linked to chromosome 17, all known collectively as tauopathies. Tau protein is a member of microtubule (MT)-associated proteins. Tau is a highly soluble and natively unfolded protein dominated by a random coil structure in solution. It is believed that aberrant modifications of tau, including phosphorylation, truncation, and conformational changes, induce filamentous aggregation. However, the mechanism underlying the conversion of tau protein from a soluble state to one of insoluble aggregates still remains elusive. The importance of tau aggregation intermediates (e.g. tau dimer, tau multimer, and granular tau oligomer) in disease pathogenesis was suggested by recent studies. Here, we review the latest developments in tracking the structural changes of tau protein and discuss the utility improving our understanding of tau aggregation pathway leading to human tauopathies. ©2008 Bentham Science Publishers Ltd.

In Alzheimer's disease, tau is hyperphosphorylated, which is thought to detach it from microtubules (MTs), induce MT destabilization, and promote aggregation. Using a previously described in vivo model, we investigated whether hyperphosphorylation impacts tau function in wild-type and transgenic mice. We found that after anesthesia-induced hypothermia, MT-free tau was hyperphosphorylated, which impaired its ability to bind MTs and promote MT assembly. MT-bound tau was more resistant to hyperphosphorylation compared with free tau and tau did not dissociate from MTs in wild-type mice. However, 3-repeat tau detached from MT in the transgenic mice. Surprisingly, dissociation of tau from MTs did not lead to overt depolymerization of tubulin, and there was no collapse, or disturbance of axonal MT networks. These results indicate that, in vivo, a subpopulation of tau bound to MTs does not easily dissociate under conditions that extensively phosphorylate tau. Tau remaining on the MTs under these conditions is sufficient to maintain MT network integrity. Copyright © 2008 Society for Neuroscience.

Activation of GSK-3β is presumed to be involved in various neurodegenerative diseases, including Alzheimer's disease (AD), which is characterized by memory disturbances during early stages of the disease. The normal function of GSK-3β in adult brain is not well understood. Here, we analyzed the ability of heterozygote GSK-3β knockout (GSK+/ 2) mice to form memories. In the Morris water maze (MWM), learning and memory performance of GSK+/2 mice was no different from that of wild-type (WT) mice for the first 3 days of training. With continued learning on subsequent days, however, retrograde amnesia was induced in GSK+/2 mice, suggesting that GSK+/2 mice might be impaired in their ability to form long-term memories. In contextual fear conditioning (CFC), context memory was normally consolidated in GSK+/2 mice, but once the original memory was reactivated, they showed reduced freezing, suggesting that GSK+/2 mice had impaired memory reconsolidation. Biochemical analysis showed that GSK-3β was activated after memory reactivation in WT mice. Intraperitoneal injection of a GSK-3 inhibitor before memory reactivation impaired memory reconsolidation in WT mice. These results suggest that memory reconsolidation requires activation of GSK-3β in the adult brain. © 2008 Kimura et al.

Recent in vitro and in vivo studies suggest that destabilized proteins with defective folding induce aggregation and toxicity in protein-misfolding diseases. One such unstable protein state is called amyloid oligomer, a precursor of fully aggregated forms of amyloid. Detection of various amyloid oligomers with A11, an anti-amyloid oligomer conformation-specific antibody, revealed that the amyloid oligomer represents a generic conformation and suggested that toxic β-aggregation processes possess a common mechanism. By using A11 antibody as a probe in combination with mass spectrometric analysis, we identified GroEL in bacterial lysates as a protein that may potentially have an amyloid oligomer conformation. Surprisingly, A11 reacted not only with purified GroEL but also with several purified heat shock proteins, including human Hsp27, 40, 70, 90 yeast Hsp104 and bovine Hsc70. The native folds of A11-reactive proteins in purified samples were characterized by their anti-β-aggregation activity in terms of both functionality and in contrast to the β-aggregation promoting activity of misfolded pathogenic amyloid oligomers. The conformation-dependent binding of A11 with natively folded Hsp27 was supported by the concurrent loss of A11 reactivity and anti-β-aggregation activity of heat-treated Hsp27 samples. Moreover, we observed consistent anti-β-aggregation activity not only by chaperones containing an amyloid oligomer conformation but also by several A11-immunoreactive non-chaperone proteins. From these results, we suggest that the amyloid oligomer conformation is present in a group of natively folded proteins. The inhibitory effects of A11 antibody on both GroEL/ES-assisted luciferase refolding and Hsp70-mediated decelerated nucleation of Aβ aggregation suggested that the A11-binding sites on these chaperones might be functionally important. Finally, we employed a computational approach to uncover possible A11-binding sites on these targets. Since the β-sheet edge was a common structural motif having the most similar physicochemical properties in the A11-reactive proteins we analyzed, we propose that the β-sheet edge in some natively folded amyloid oligomers is designed positively to prevent β aggregation. © 2008 Yoshiike et al.

Advanced age and mutations in the genes encoding amyloid precursor protein (APP) and presenilin (PS1) are two serious risk factors for Alzheimer's disease (AD). Finding common pathogenic changes originating from these risks may lead to a new therapeutic strategy. We observed a decline in memory performance and reduction in hippocampal long-term potentiation (LTP) in both mature adult (9-15 months) transgenic APP/PS1 mice and old (19-25 months) non-transgenic (nonTg) mice. By contrast, in the presence of bicuculline, a GABA(A) receptor antagonist, LTP in adult APP/PS1 mice and old nonTg mice was larger than that in adult nonTg mice. The increased LTP levels in bicuculline-treated slices suggested that GABA(A) receptor-mediated inhibition in adult APP/PS1 and old nonTg mice was upregulated. Assuming that enhanced inhibition of LTP mediates memory decline in APP/PS1 mice, we rescued memory deficits in adult APP/PS1 mice by treating them with another GABA(A) receptor antagonist, picrotoxin (PTX), at a non-epileptic dose for 10 days. Among the saline vehicle-treated groups, substantially higher levels of synaptic proteins such as GABA(A) receptor alpha 1 subunit, PSD95, and NR2B were observed in APP/PS1 mice than in nonTg control mice. This difference was insignificant among PTX-treated groups, suggesting that memory decline in APP/PS1 mice may result from changes in synaptic protein levels through homeostatic mechanisms. Several independent studies reported previously in aged rodents both an increased level of GABA(A) receptor alpha 1 subunit and improvement of cognitive functions by long term GABA(A) receptor antagonist treatment. Therefore, reduced LTP linked to enhanced GABA(A) receptor-mediated inhibition may be triggered by aging and may be accelerated by familial AD-linked gene products like A beta and mutant PS1, leading to cognitive decline that is pharmacologically treatable at least at this stage of disease progression in mice.

Neprilysin is an amyloid-β-degrading enzyme localized in the brain parenchyma. The involvement of neprilysin in the pathogenesis of Alzheimer's disease has recently received much attention. We examined the localization of neprilysin and amyloid-β, as well as the activity of neprilysin, in the brains of dogs and cats of various ages to clarify the relationship between neprilysin activity and amyloid-β deposition. The distribution of neprilysin was almost identical in dogs and cats, being high in the striatum, globus pallidus, and substantia nigra, but very low in the cerebral cortex. The white matter and hippocampus were negative. Neprilysin activity in the brain regions in dogs and cats was ranked from high to low as follows: thalamus/striatum &lt cerebral cortex lt hippocampus &lt white matter. Amyloid-p deposition was first detected at 7 and 10 years of age in dogs and cats, respectively, and both the quantity and frequency of deposition increased with age. In both species, amyloid-β deposition appeared in the cerebral cortex and the hippocampus. In summary, the localization of neprilysin and neprilysin activity, and that of amyloid-p, were complementary in the brains of dogs and cats.

Neurofibrillary tangles (NFTs), comprising human intracellular microtubule-associated protein tau, are one of the hallmarks of tauopathies, including Alzheimer's disease. Recently, a report that caspase-cleaved tau is present in NFTs has led to the hypothesis that the mechanisms underlying NFT formation may involve the apoptosis cascade. Here, we show that adenoviral infection of tau into COS-7 cells induces activation of c-jun N-terminal kinase (JNK), followed by excessive phosphorylation of tau and its cleavage by caspase. However, JNK activation alone was insufficient to induce sodium dodecyl sulfate (SDS)-insoluble tau aggregation and additional phosphorylation by GSK-3β was required. In SH-SY5Y neuroblastoma cells, overexpression of active JNK and GSK-3β increased caspase-3 activation and cytotoxicity more than overexpression of tau alone. Taken together, these results indicate that, although JNK activation may be a primary inducing factor, further phosphorylation of tau is required for neuronal death and NFT formation in neurodegenerative diseases, including those characterized by tauopathy. © The Authors (2008).

Pathological studies show that neurofibrillary tangles are found in regions where neuronal death occurs. This observation raises the question: Are neurofibrillary tangles toxic or protective? Findings from various mouse models expressing human tau with the FTDP17 mutation suggest that some component involved in the formation of neurofibrillary tangles, rather than the neurofibrillary tangles themselves, might be responsible for the toxicity leading to neuronal death. Here, I review our current understanding of a toxic species of tau and the mechanism by which it contributes to neuronal dysfunction and death. Recent studies suggest that, before forming fibrils but after becoming hyperphosphorylated, tau is involved in neurodegenerative disease.

To investigate how tau affects neuronal function during neurofibrillary tangle (NFT) formation, we examined the behavior, neural activity, and neuropathology of mice expressing wild-type human tau. Here, we demonstrate that aged (&gt 20 months old) mice display impaired place learning and memory, even though they do not form NFTs or display neuronal loss. However, soluble hyperphosphorylated tau and synapse loss were found in the same regions. Mn-enhanced MRI showed that the activity of the parahippocampal area is strongly correlated with the decline of memory as assessed by the Morris water maze. Taken together, the accumulation of hyperphosphorylated tau and synapse loss in aged mice, leading to inhibition of neural activity in parahippocampal areas, including the entorhinal cortex, may underlie place learning impairment. Thus, the accumulation of hyperphosphorylated tau that occurs before NFT formation in entorhinal cortex may contribute to the memory problems seen in Alzheimer's disease (AD). ©2007 European Molecular Biology Organization.

Hyperphosphorylated tau is the major component of paired helical filaments in neurofibrillary tangles found in Alzheimer's disease ( AD) brains, and tau hyperphosphorylation is thought to be a critical event in the pathogenesis of the disease. The large majority of AD cases is late onset and sporadic in origin, with aging as the most important risk factor. Insulin resistance, impaired glucose tolerance, and diabetes mellitus (DM) are other common syndromes in the elderly also strongly age dependent, and there is evidence supporting a link between insulin dysfunction and AD. To investigate the possibility that insulin dysfunction might promote tau pathology, we induced insulin deficiency and caused DM in mice with streptozotocin (STZ). A mild hyperphosphorylation of tau could be detected 10, 20, and 30 d after STZ injection, and a massive hyperphosphorylation of tau was observed after 40 d. The robust hyperphosphorylation of tau was localized in the axons and neuropil, and prevented tau binding to microtubules. Neither mild nor massive tau phosphorylation induced tau aggregation. Body temperature of the STZ-treated mice did not differ from control animals during 30 d, but dropped significantly thereafter. No change in beta-amyloid (A beta) precursor protein (APP), APP C-terminal fragments, or A beta levels were observed in STZ-treated mice; however, cellular protein phosphatase 2A activity was significantly decreased. Together, these data indicate that insulin dysfunction induced abnormal tau hyperphosphorylation through two distinct mechanisms: one was consequent to hypothermia; the other was temperature-independent, inherent to insulin depletion, and probably caused by inhibition of phosphatase activity.

Intracellular accumulation of filamentous tau proteins is a defining feature of neurodegenerative diseases termed tauopathies. The pathogenesis of tauopathies remains largely unknown. Molecular chaperones such as heat shock proteins (HSPs), however, have been implicated in tauopathies as well as in other neurodegenerative diseases characterized by the accumulation of insoluble protein aggregates. To search for in vivo evidence of chaperone-related tau protein metabolism, we analyzed human brains with varying degrees of neurofibrillary tangle (NFT) pathology, as defined by Braak NFT staging. Quantitative analysis of soluble protein levels revealed significant positive correlations between tau and Hsp90, Hsp40, Hsp27, α-crystallin, and CHIP. An inverse correlation was observed between the levels of HSPs in each specimen and the levels of granular tau oligomers, the latter of which were isolated from brain as intermediates of tau filaments. We speculate that HSPs function as regulators of soluble tau protein levels, and, once the capacity of this chaperone system is saturated, granular tau oligomers form virtually unabated. This is expressed pathologically as an early sign of NFT formation. The molecular basis of chaperone-mediated protection against neurodegeneration might lead to the development of therapeutics for tauopathies. © 2007 Wiley-Liss, Inc.

Degenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases are believed to be causally related to the accumulation of amyloid oligomers that exhibit a common structure and may be toxic by a common mechanism involving permeabilization of membranes. We discovered that amyloid oligomers and the pore-forming bacterial toxin, α-hemolysin (αHL), as well as human perforin from cytotoxic T lymphocytes, share a structural and functional homology at the level of their common reactivity with a conformation-dependent antibody that is specific for amyloid oligomers, A11. The αHL oligomeric pores and partially folded αHL protomer, but not the monomer αHL precursor reacts with A11 antibody. A11 antibody inhibits the hemolytic activity of αHL, indicating that the structural homology is functionally significant. Perforin oligomers were also recognized by A11. Amyloidogenic properties of αHL and perforin were confirmed spectroscopically and morphologically. These results indicate that pore forming proteins (PFP) and amyloid oligomers share structural homology and suggest that PFPs and amyloid oligomers share the same mechanism of membrane permeabilization. © Humana Press Inc. 2007.

Amyloid beta (A beta) toxicity has been hypothesized to initiate the pathogenesis of Alzheimer's disease (AD). The characteristic fibrillar morphology of A beta-aggregates, that constitute the main components of senile plaque, has long been considered to account for the neurotoxicity. But recent reports argue against a primary role for mature fibrils in AD pathogenesis because of the lack of a robust correlation between the severity of neurological impairment and the extent of amyloid deposition. Toxicity from the soluble prefibrillar intermediate entity of aggregates often called oligomer has recently proposed a plausible explanation for this inconsistency. An alternative explanation is based on the observation that certain amyloid fibril morphologies are more toxic than others, indicating that not all amyloid fibrils are equally toxic. Here, we report that it is not only the beta-sheeted fibrillar structure but also the surface physicochemical composition that affects the toxicity of A beta fibrils. For the first time, colloidal gold was used to visualize by electron microscopy positive-charge clusters on A beta fibrils. Chemical modifications as well as point-mutated A beta synthesis techniques were applied to change the surface structures of A beta and to show how local structure affects surface properties that are responsible for electrostatic and hydrophobic interactions with cells. We also report that covering the surface of A beta fibers with myelin basic protein, which has surface properties contrary to those of A beta, suppresses A beta toxicity. On the basis of these results, we propose that the surface structure of A beta fibrils plays an important role in A beta toxicity.

Two types of tau isoform, three- and four-repeat tau, are found in neurofibrillary tangles-a pathological hallmark of tauopathies. Which isoform is deposited in the affected tissues depends on the tauopathy. To study how and which tau isoforms contribute to neuronal degeneration, we have developed and characterized two novel conformation-sensitive antibodies, T3R and T4R. Two closely related synthetic peptides, PGGGKVQIVYK and PGGGSVQIVYK, respectively, were designed as antigens. The isoform-specific residues, 305K in three-repeat tau or 305S in four-repeat tau, and the PHF6 motif (VQIVYK) were identified as critical sequences. Despite the high similarity of the antigens, there was no cross-reactivity between T3R and T4R. Furthermore, T3R and T4R showed reduced binding to the thioflavin-positive β-structural form of their target. These features may enable these antibodies to act as novel indicators that allow us to observe and evaluate conformational changes in each distinct isoform of tau. © 2007 Elsevier Inc. All rights reserved.

Abnormal accumulation of tau as filamentous structures is a neuropathological hallmark of neurodegenerative diseases referred to as tauopathies. Little is known about the role of native cysteine residues in tau assembly because their substitution with other amino acids has no effect on tau filament morphology. To understand the process involved in tau oligomerization, we analysed both heparin-induced assembly of different forms of recombinant human tau and assembly of tau from COS-7 cells transiently expressing different human tau constructs. Here, we demonstrated that tau assembly involves two distinct dimers (cysteine-dependent and cysteine-independent) that differ in resistance to reduction. During assembly, an increase of cysteine-dependent tau oligomer was observed prior to detection of increased thioflavin T fluorescence signals. The latter event was accompanied by an increase of cysteine-independent dimer. Fewer higher-order oligomers and aggregates were assembled from four-repeat tau containing two amino-terminus inserts that have either the C291A/C322A mutation (cysless-4R2N) or a hexapeptide deletion at residues 306-311 (ΔPHF6-4R2N) compared with those assembled from wild-type tau. Assembly of distinct types of dimers was also observed in lysates from COS-7 cells expressing wild-type 4R2N and brain extracts from mice expressing P301L mutant tau. In contrast, COS-7 cells expressing cysless- or ΔPHF6-4R2N tau contained very little cysteine-dependent dimer. Together, the results indicate that intermolecular disulfide crosslinking along with PHF6 hexapeptide facilitates tau oligomerization and that this event is accompanied by cysteine-independent intermolecular bridging of microtubule-binding domain, leading to assembly of higher-order oligomers. The levels of these dimers may be used to gauge the potential for tau assembly. © The Authors (2007).

Neurofibrillary tangles (NFTs) are pathological hallmarks of several neurodegenerative disorders, including Alzheimer's disease (AD). NFTs are composed of microtubule-binding protein tau, which assembles to form paired helical filaments (PHFs) and straight filaments. Here we show by atomic force microscopy that AD brain tissue and in vitro tau form granular and fibrillar tau aggregates. CD spectral analysis and immunostaining with conformation-dependent antibodies indicated that tau may undergo conformational changes during fibril formation. Enriched granules generated filaments, suggesting that granular tau aggregates may be an intermediate form of tau fibrils. The amount of granular tau aggregates was elevated in prefrontal cortex of Braak stage I cases compared to that of Braak stage 0 cases, suggesting that granular tau aggregation precedes PHF formation. Thus, granular tau aggregates may be a relevant marker for the early diagnosis of tauopathy. Reducing the level of these aggregates may be a promising therapy for tauopathies and for promoting healthy brain aging. © 2007 American Chemical Society.

A clear understanding of cell fate regulation during differentiation is key in successfully using stem cells for therapeutic applications. Here, we report that mild electrical stimulation strongly influences embryonic stem cells to assume a neuronal fate. Although the resulting neuronal cells showed no sign of specific terminal differentiation in culture, they showed potential to differentiate into various types of neurons in vivo, and, in adult mice, contributed to the injured spinal cord as neuronal cells. Induction of calcium ion influx is significant in this differentiation system. This phenomenon opens up possibilities for understanding novel mechanisms underlying cellular differentiation and early development, and, perhaps more importantly, suggests possibilities for treatments in medical contexts.

Presenilin (PS) is a catalytic subunit of the γ-secretase complex that cleaves the intramembranous region of amyloid precursor protein (APP), producing amyloid β (Aβ) peptide. Familial Alzheimer's disease (FAD) results from PS mutations, which may alter γ-secretase activity to enhance the production of highly aggregable Aβ42. The precise pathogenic effects of mutant PS remain unclear. To exclude the effects of endogenous PS, we established cell lines from PS1/PS2-deficient (PS-/-) fibroblasts capable of stably expressing either wild-type (wt) PS1 or different mutant PS1s. Although both wt PS1 and mutant PS1 formed γ-secretase complexes of the same size and containing the same components, the amount of Aβ secreted by FAD mutant PS1-expressing cells was significantly reduced. The ratio of Aβ42 to Aβ40 (Aβ42/Aβ40) secreted by these cells, however, was significantly higher than that secreted by cells expressing wt PS1, which corroborated findings from a previous report. The elevated Aβ42/Aβ40 ratio observed with mutant PS1-expressing cells may be due to reduced Aβ40 production not increased Aβ42 production. © 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society.