高島 明彦

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.

The mechanism underlying plaque-independent neuronal death in Alzheimer disease (AD), which is probably responsible for early cognitive decline in AD patients, remains unclarified. Here, we show that a toxic soluble Abeta assembly (TAbeta) is formed in the presence of liposomes containing GM1 ganglioside more rapidly and to a greater extent from a hereditary variant-type ("Arctic") Abeta than from wild-type Abeta. TAbeta is also formed from soluble Abeta through incubation with natural neuronal membranes prepared from aged mouse brains in a GM1 ganglioside-dependent manner. An oligomer-specific antibody (anti-Oligo) significantly suppresses TAbeta toxicity. Biophysical and structural analyses by atomic force microscopy and size exclusion chromatography revealed that TAbeta is spherical with diameters of 10-20 nm and molecular masses of 200-300 kDa. TAbeta induces neuronal death, which is abrogated by the small interfering RNA-mediated knockdown of nerve growth factor receptors, including TrkA and p75 neurotrophin receptor. Our results suggest that soluble Abeta assemblies, such as TAbeta, can cause plaque-independent neuronal death that favorably occurs in nerve growth factor-dependent neurons in the cholinergic basal forebrain in AD.

Etiological studies suggest that aluminum (Al) intake might increase an individual's risk of developing Alzheimer's disease (AD). Biochemical analysis data on the effects of Al, however, are inconsistent. Hence, the pathological involvement of Al in AD remains unclear. If Al is involved in AD, then it is reasonable to hypothesize that Al might be involved in the formation of either amyloid plaques or neurofibrillary tangles (NFTs). Here, we investigated whether Al might be involved in NFT formation by using an in vitro tau aggregation paradigm, a tau-overexpressing neuronal cell line (N2a), and a tau-overexpressing mouse model. Although Al induced tau aggregation in a heparin-induced tau assembly assay, these aggregates were neither thioflavin T positive nor did they resemble tau fibrils seen in human AD brains. With cell lysates from stable cell lines overexpressing tau, the accumulation of SDS-insoluble tau increased when the lysates were treated with at least 100 μM Al-maltolate. Yet Al-maltolate caused illness or death in transgenic mice overexpressing human tau and in non-transgenic littermates well before the Al concentration in the brain reached 100 μM. These results indicate that Al has no direct link to AD pathology. © 2007 - IOS Press and the authors. All rights reserved.

As Alzheimer's disease (AD) progresses, brain atrophy becomes conspicuous, and histologically there is neuronal loss, primarily with a deficit of cholinergic neurons observed. Hitherto, the view has been that cell death,apoptosis, plays a role in this neuronal loss. Apoptosis is characterized by the morphological changes of nuclear fragmentation, chromatin condensation and cell shrinkage, with activation of caspases, members of the cysteine protease family, resulting in considerable substrate cleavage. TUNEL positive neurons have in fact been detected in AD brain, indicating increased caspase activity and resulting substrate cleavage. In AD brain, amyloid beta peptides (Aβ), the main constituent of senile plaque, are a specific pathological hallmark observed in extracellular spaces. In contrast, the main constituent of intracellularly observed neurofibrillary tangles (NFT) is hyperphosphorylated tau, which is observed in various neurodegenerative disorders other than AD. The viewpoint of many studies is that the Aβ and NFT that cause these neuropathological changes probably participate in neuronal death. However, up until now it has been thought that there was no hypothesis offering a comprehensive explanation of how the accumulation of extracellular Aβ and intracellular NFT leads to neuronal death. This report first covers the mechanism of apoptosis as clarified by molecular biological methods, and provides an explanation of how apoptosis could be involved in AD pathology. The subject of autophagic cell death, a type of cell death morphology that has recently been the focus of attention, is also addressed. © 2006 The Authors Journal compilation © 2006 The Japanese Psychogeriatric Society.

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. © 2006 Elsevier Inc. All rights reserved.

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT) gene family comprises three human homologs that phosphorylate and inactivate glycogen synthase kinase 3β (GSK3β). Studies have reported the genetic association of AKT1 with schizophrenia. Additionally, decreased AKT1 protein expression and the reduced phosphorylation of GSK3β were reported in this disease, leading to a new theory of attenuated AKT1-GSK3β signaling in schizophrenia pathogenesis. We have evaluated this theory by performing both genetic and protein expression analyses. A family based association test of AKT1 did not show association with schizophrenia in Japanese subjects. The expression levels of total AKT, AKT1 and phosphorylated GSK3β detected in the schizophrenic brains from two different brain banks also failed to support the theory. In addition, no attenuated AKT-GSK3β signaling was observed in the lymphocytes from Japanese schizophrenics, contrasting with previous findings. Importantly, we found that the level of phosphorylated GSK3β at Ser9 tended to be inversely correlated with postmortem intervals, and that the phosphorylation levels of AKT were inversely correlated with brain pH, issues not assessed in the previous study. These data introduce a note of caution when estimating the phosphorylation levels of GSK3β and AKT in postmortem brains. Collectively, this study failed to support reduced signaling of the AKT-GSK3β molecular cascade in schizophrenia. © 2006 The Authors.

Trisomy 21 or Down syndrome (DS) is the most common genetic birth defect associated with mental retardation. The over-expression of genes on chromosome 21, including SOD1 (Cu/Zn superoxide dismutase) and APP (amyloid-beta precursor protein) is believed to underlie the increased oxidative stress and neurodegeneration commonly described in DS. However, a segmental trisomy 16 mouse model for DS, Ts1Cje, has a subset of triplicated human chromosome 21 gene orthologs that exclude APP and SOD1. Here, we report that Ts1Cje brain shows decreases of mitochondrial membrane potential and ATP production, increases of reactive oxygen species, hyperphosphorylation of tau without NFT formation, increase of GSK3 beta and JNK/SAPK activities and unaltered A beta PP metabolism. Our findings suggest that genes on the trisomic Ts1Cje segment other than APP and SOD1 can cause oxidative stress, mitochondrial dysfunction and hyperphosphorylation of tau, all of which may play critical roles in the pathogenesis of mental retardation in DS.

Because the deposition of β-amyloid protein (Aβ) is a consistent pathological hallmark of Alzheimer's disease (AD) brains, inhibition of Aβ generation, prevention of Aβ fibril formation, or destabilization of preformed Aβ fibrils would be attractive therapeutic strategies for the treatment of AD. We examined the effects of several medicinal herbs used in traditional Chinese medical formulae on the formation and destabilization of Aβ fibrils by using the thioflavin T binding assay, atomic force microscopic imaging, and electrophoresis. Our study demonstrates that several of these herbs have potent inhibitory effects on fibril formation of both Aβ1-40 and Aβ1-42 in concentration-dependent manners in particular, Uncaria rhynchophylla inhibited Aβ aggregation most intensively. Significant destabilization of preformed Aβ1-40 and Aβ1-42 fibrils was also induced by Uncaria rhynchophylla as well as some other herb extracts. Three-dimensional HPLC analysis indicated that the water extract of this herb contains several different chemical compounds, including oxindole and indol alkaloids, which have been regarded as neuroprotective. Our results suggest that Uncaria rhynchophylla has remarkably inhibitory effects on the regulation of Aβ fibrils, and we conclude that this medicinal herb could have the potency to be a novel therapeutic agent to prevent and/or cure AD. © 2006 Wiley-Liss, Inc.

Development of neurofibrillary tangles (NFTs) is a pathological hallmark in various neurodegenerative disorders including Alzheimer's disease (AD). Recently, we identified a granular tau oligomer having a pre-filamentous structure. To determine the role of this oligomer in NFT formation, we quantified the amount of granular tau oligomer in 21 frontal cortex samples, each displaying varying degrees of Braak-staged NFT pathology. Here we report that granular tau oligomer levels in frontal cortex were significantly increased, even in brains displaying Braak-stage I neuropathology, a stage at which clinical symptoms of AD and NFTs in frontal cortex are believed to be absent. This suggests that increases in granular tau oligomer levels occur before NFTs form and before individuals manifest clinical symptoms of AD. Increased granular tau oligomer levels, therefore, may lead to NFT formation in frontal cortex, eventually leading to the development of AD. Thus, increases in granular tau oligomer levels may represent a very early sign of NFT formation and AD. © 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Mutations in the presenilin 1 (PS1) gene are responsible for the early onset of familial Alzheimer disease (FAD). Accumulating evidence shows that PS1 is involved in γ-secretase activity and that FAD-associated mutations of PS1 commonly accelerate Aβ1-42 production, which causes Alzheimer disease (AD). Recent studies suggest, however, that PS1 is involved not only in Aβ production but also in other processes that lead to neurodegeneration. To better understand the causes of neurodegeneration linked to the PS1 mutation, we analyzed the development of tau pathology, another key feature of AD, in PS1 knock-in mice. Hippocampal samples taken from FAD mutant (I213T) PS1 knock-in mice contained hyperphosphorylated tau that reacted with various phosphodependent tau antibodies and with Alz50, which recognizes the conformational change of PHF tau. Some neurons exhibited Congo red birefringence and Thioflavin T reactivity, both of which are histological criteria for neurofibrillary tangles (NFTs). Biochemical analysis of the samples revealed SDS-insoluble tau, which under electron microscopy examination, resembled tau fibrils. These results indicate that our mutant PS1 knock-in mice exhibited NFT-like tau pathology in the absence of Aβ deposition, suggesting that PS1 mutations contribute to the onset of AD not only by enhancing Aβ1-42 production but by also accelerating the formation and accumulation of filamentous tau. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

The presenilin (PS) complex, including PS, nicastrin (NCT), APH-1 and PEN-2, is essential for γ-secretase activity. Previously, the PS C-terminal tail was shown to be essential for γ-secretase activity. Here, to further understand the precise mechanism underlying the activation of γ-secretase regulated by PS cofactors, we focused on the role of the PS1 C-terminal region including transmembrane domain (TM) 8 in γ-secretase activity. For this purpose, we co-expressed C-terminally truncated PS1 (PS1ΔC) completely lacking γ-secretase activity and the PS1 C-terminal short fragment in PS-null cells, because the successful reconstitution of γ-secretase activity in PS-null cells by the co-expression of PS1ΔC and the PS1 C-terminal short fragment would allow us to investigate the role of the PS1 C-terminal region in γ-secretase activity. We found that the exogenous expression of the PS1 C-terminal short fragment with NCT and APH-1 completely rescued a defect of the γ-secretase activity of PS1ΔC in PS-null cells. With this reconstitution system, we demonstrate that both TM8 and the PS1 C-terminal seven-amino-acid-residue tail are involved in the formation of the active γ-secretase complex via the assembly of PS1 with NCT and APH-1. © 2005 The Author(s) Journal compilation © 2005 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Glycogen synthase kinase-3 (GSK-3) is a pivotal molecule in the development of Alzheimer's disease (AD). GSK-3 beta is involved in the formation of paired helical filament (PHF)-tau, which is an integral component of the neurofibrillary tangle (NFT) deposits that disrupt neuronal function, and a marker of neurodegeneration in AD. GSK-3 beta has exactly the same oligonucleotide sequence as tau-protein kinase I (TPKI), which was first purified from the microtubule fraction of bovine brain. Initially, we discovered that GSK-3 beta was involved in amyloid-beta (A beta)-induced neuronal death in rat hippocampal cultures. In the present review, we discuss our initial in vitro results and additional investigations showing that A beta activates GSK-3 beta through impairment of phosphatidylinositol-3 (PI3)/Akt signaling; that A beta-activated GSK-3 beta induces hyperphosphorylation of tau, NFT formation, neuronal death, and synaptic loss (all found in the AD brain); that GSK-3 beta can induce memory deficits in vivo; and that inhibition of GSK-3 alpha (an isoform of GSK-3 beta) reduces A beta production. These combined results strongly suggest that GSK-3 activation is a critical step in brain aging and the cascade of detrimental events in AD, preceding both the NFT and neuronal death pathways. Therefore, therapeutics targeted to inhibiting GSK-3 may be beneficial in the treatment of this devastating disease.

Mutant R406W human tau was originally identified in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and causes a hereditary tauopathy that clinically resembles Alzheimer's disease (AD). In the current study, we examined the performance of R406W transgenic (Tg) mice in the forced swimming test, a test with high predictivity of antidepressant efficacy in human depression, and found an enhancement of the immobility time. In contrast, the motor function and anxiety-related emotional response of R406W Tg mice were normal. Furthermore, a selective serotonin reuptake inhibitor (SSRI), fluvoxamine (100 mg/kg, p.o.), significantly reduced this enhancement of the immobility time, whereas a noradrenaline reuptake inhibitor, desipramine, had no effect. In an in vivo microdialysis study, R406W Tg mice exhibited a significantly decreased extracellular 5-hydroxyindoleacetic acid (5-HIAA) level in the frontal cortex and also exhibited a tendency toward a decreased extracellular 5-hydroxytryptamine (5-HT) level. Moreover, fluvoxamine, which reduced the enhancement of the immobility time, significantly increased the extracellular 5-HT level in R406W Tg mice. These results suggest that R406W Tg mice exhibit changes in depression-related behavior involving serotonergic neurons and provide an animal model for investigating AD with depression. © 2005 Elsevier B.V. All rights reserved.

It is well known that tau is a good in vitro substrate for Ca2+/ calmodulin-dependent protein kinase II (CaM kinase II). However, it is not clear at present whether CaM kinase II phosphorylates tau in vivo or not. Serine 416, numbered according to the longest h uman tau isoform, has been reported to be one of the major phosphorylation sites by CaM kinase II in vitro. In this study, we produced a specific antibody against tau phosphorylated at serine 416 (PS416-tau). Immunoblot analysis revealed that the antibody reacted with tau in the rat brain extract which was prepared in the presence of protein phosphatase inhibitors. Developmental study indicated that serine 416 was strongly phosphorylated at early developmental stages in rat brain. We examined the localization of PS416-tau in primary cultured hippocampal neurons and the immortalized GnRH neurons (GT1-7 cells), which were stably transfected with CaM kinase IIα cDNA. Immunostaining of these cells indicated that tau was phosphorylated mainly in neuronal soma. Interestingly, tau in neuronal soma in Alzheimer's disease (AD) brain was strongly immunostained by the antibody. These results suggest that CaM kinase II is involved in the accumulation of tau in neuronal soma in AD brain. © 2005 International Society for Neurochemistry.

The carboxyl terminus of heat-shock cognate (Hsc)70-interacting protein (CHIP) is a ubiquitin E3 ligase that can collaborate with molecular chaperones to facilitate protein folding and prevent protein aggregation. Previous studies showed that, together with heat-shock protein (Hsp)70, CHIP can regulate tau ubiquitination and degradation in a cell culture system. Ubiquitinated tau is one component in neurofibrillary tangles (NFTs), which are a major histopathological feature of Alzheimer's disease (AD). However, the precise sequence of events leading to NFT formation and the mechanisms involved remain unclear. To confirm CHIP'S role in suppressing NFT formation in vivo, we performed a quantitative analysis of CHIP in human and mouse brains. We found increased levels of CHIP and Hsp70 in AD compared with normal controls. CHIP levels in both AD and controls corresponded directly to Hsp90 levels, but not to Hsp70 or Hsc70 levels. In AD samples, CHIP was inversely proportional to sarkosyl-insoluble tau accumulation. In a JNPL3 mouse brain tauopathy model, CHIP was widely distributed but weakly expressed in spinal cord, which was the most prominent region for tau inclusions and neuronal loss. Protein levels of CHIP in cerebellar regions of JNPL3 mice were significantly higher than in non-transgenic littermates. Human tau was more highly expressed in this region of mouse brains, but only moderate levels of sarkosyl-insoluble tau were detected. This was confirmed when increased insoluble tau accumulation was found in mice lacking CHIP. These findings suggest that increases in CHIP may protect against NFT formation in the early stages of AD. If confirmed, this would indicate that the quality-control machinery in a neuron might play an important role in retarding the pathogenesis of tauopathies. © 2005 International Society for Neurochemistry.

Neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated and ubiquitylated tau, are exhibited at regions where neuronal loss occurs in neurodegenerative diseases however, the mechanisms of NFT formation remain unknown. Molecular studies of frontotemporal dementia with parkinsonism-17 demonstrated that increasing the ratio of tau with exon 10 insertion induced fibrillar tau accumulation. Here, we show that carboxyl terminus of Hsc70-interacting protein (CHIP), a U-box protein, recognizes the microtubule-binding repeat region of tau and preferentially ubiquitylates four-repeat tau compared with three-repeat tau. Overexpression of CHIP induced the prompt degradation of tau, reduced the formation of detergent-insoluble tau and inhibited proteasome inhibitor-induced cell death. NFT bearing neurons in progressive supranuclear palsy, in which four-repeat tau is a component, showed the accumulation of CHIP. Thus, CHIP is a ubiquitin ligase for four-repeat tau and maintains neuronal survival by regulating the quality control of tau in neurons.

Cerebral deposition of amyloid β-peptide (Aβ) in the brain is an invariant feature of Alzheimer disease (AD). Plasma or cerebrospinal fluid concentrations of antioxidant vitamins and carotenoids, such as vitamins A, C, E, and β-carotene, have been reported to be lower in AD patients, and these vitamins clinically have been demonstrated to slow the progression of dementia. In this study, we used fluorescence spectroscopy with thioflavin T (ThT) and electron microscopy to examine the effects of vitamin A (retinol, retinal, and retinoic acid), β-carotene, and vitamins B2, B6, C, and E on the formation, extension, and destabilization of β-amyloid fibrils (fAβ) in vitro. Among them, vitamin A and β-carotene dose-dependently inhibited formation of fAβ from fresh Aβ, as well as their extension. Moreover, they dose-dependently destabilized preformed fAβs. The overall activity of the molecules examined was in the order of retinol = retinal &gt β-carotene &gt retinoic acid. Although the exact mechanisms are still unclear, vitamins A and β-carotene could be key molecules for the prevention and therapy of AD. © 2004 Elsevier Inc. All rights reserved.

Alzheimer's disease (AD) brains contain neurofibrillary tangles (NFTs) composed of abnormally hyperphosphorylated tau protein. Regional reductions in cerebral glucose metabolism correlating to NFT densities have been reported in AD brains. Assuming that reduced glucose metabolism might cause abnormal tau hyperphosphorylation, we induced in vivo alterations of glucose metabolism in mice by starvation or intraperitoneal injections of either insulin or deoxyglucose. We found that the treatments led to abnormal tau hyperphosphorylation with patterns resembling those in early AD brains and also resulted in hypothermia. Surprisingly, tau hyperphosphorylation could be traced down to a differential effect of low temperatures on kinase and phosphatase activities. These data indicate that abnormal tau hyperphosphorylation is associated with altered glucose metabolism through hypothermia. Our results imply that serine-threonine protein phosphatase 2A plays a major role in regulating tau phosphorylation in the adult brain and provide in vivo evidence for its crucial role in abnormal tau hyperphosphorylation in AD.

Presenilin (PS)1 and its mutants, which consist of the N-terminal and C-terminal fragments, cause certain familial forms of Alzheimer's disease (FAD). Our earlier studies found that FAD-linked M146L-PS1 causes neuronal cell death through nitrogen oxide synthase (NOS) and that FAD-linked N141I-PS2, another member of the PS family, causes neuronal cell death through NADPH oxidase. In this study, we examined 27 different FAD-linked mutants of PS1, and found that PS1 mutants with mutations in the N-terminal fragment caused NOS inhibitor (NOSI)-sensitive neuronal cell death in contrast, the PS1 mutants with mutations in the C-terminal fragment caused NOSI-resistant neuronal cell death. The former toxicity was resistant to the specific NADPH oxidase inhibitor apocynin and was inhibited by Humanin (HN), a newly identified neuroprotective factor against Alzheimer's disease (AD)-relevant insults, but not by insulin-like growth factor-I (IGF-I). In contrast, the latter toxicity was sensitive to apocynin and inhibited by both IGF-I and HN. This study indicates for the first time that N- and C-terminal fragment PS1 mutants can generate distinct neurotoxic signals, which will provide an important clue to the understanding of the entire array of neurotoxic signals generated by FAD-causative mutations of PS1. © 2003 Wiley-Liss, Inc.

We characterized senile plaques (SPs) immunohistochemically in cynomolgus monkey brains and also examined age-related biochemical changes of Alzheimer's disease (AD)-associated proteins in these brains from monkeys of various ages. In the neocortex of aged monkeys (&gt 20 years old), we found SPs but no neurofibrillary tangles (NFTs). Antibodies against β-amyloid precursor protein (APP) or apolipoprotein E (ApoE) stained SPs however, the pattern of immunostaining was different for the two antigens. APP was present only in swollen neurites, but ApoE was present throughout all parts of SPs. Western blot analysis revealed that the pattern of APP expression changed with age. Although full-length APP695 protein was mainly expressed in brains from young monkeys (4 years old), the expression of full-length APP751 protein was increased in brains from older monkeys (&gt 20 years old). Biochemical analyses also showed that levels of various AD-associated proteins increased significantly with age in nerve ending fractions. Both SP-associated (APP) and NFT-associated proteins (tau, activated glycogen synthase kinase 3β, cyclin dependent kinase 5, p35, and p25) accumulated in the nerve ending fraction with increasing age however, we found no NFTs or paired helical filaments of tau in aged cynomolgus monkey brains. This age-related accumulation of these proteins in the nerve ending fraction was similar to that observed in our laboratory previously for presenilin-1 (PS-1). The accumulation of these SP-associated proteins in this fraction may be a causal event in the spontaneous formation of SPs thus, SPs may be formed initially in nerve endings. Taken together, these results suggest that intensive investigation of age-related changes in the nerve ending and in axonal transport will contribute to a better understanding of the pathogenesis of neurodegenerative disorders such as AD. © 2003 Elsevier Inc. All rights reserved.

Cerebral deposition of amyloid β-peptide (Aβ) in the brain is an invariant feature of Alzheimer's disease (AD). A consistent protective effect of wine consumption on AD has been documented by epidemiological studies. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of wine-related polyphenols (myricetin, morin, quercetin, kaempferol (+)-catechin and (-)-epicatechin) on the formation, extension, and destabilization of β-amyloid fibrils (fAβ) at pH 7.5 at 37°C in vitro. All examined polyphenols dose-dependently inhibited formation of fAβ from fresh Aβ(1-40) and Aβ(1-42), as well as their extension. Moreover, these polyphenols dose-dependently destabilized preformed fAβs. The overall activity of the molecules examined was in the order of: myricetin = morin = quercetin &gt kaempferol &gt (+)-catechin = (-)-epicatechin. The effective concentrations (EC50) of myricetin, morin and quercetin for the formation, extension and destabilization of fAβs were in the order of 0.1-1 μM. In cell culture experiments, myricetin-treated fAβ were suggested to be less toxic than intact fAβ, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Although the mechanisms by which these polyphenols inhibit fAβ formation from Aβ, and destabilize pre-formed fAβ in vitro are still unclear, polyphenols could be a key molecule for the development of preventives and therapeutics for AD.

Presenilin1 (PS1), a protein implicated in Alzheimer's disease (AD), forms complexes with N-cadherin, a transmembrane protein with important neuronal and synaptic functions. Here, we show that a PS1-dependent γ-secretase protease activity promotes an ε-like cleavage of N-cadherin to produce its intracellular domain peptide, N-Cad/CTF2. NMDA receptor agonists stimulate N-Cad/CTF2 production suggesting that this receptor regulates the ε-cleavage of N-cadherin. N-Cad/CTF2 binds the transcription factor CBP and promotes its proteasomal degradation, inhibiting CRE-dependent transactivation. Thus, the PS1-dependent ε-cleavage product N-Cad/CTF2 functions as a potent repressor of CBP/CREB-mediated transcription. Importantly, PS1 mutations associated with familial AD (FAD) and a γ-secretase dominant-negative mutation inhibit N-Cad/CTF2 production and upregulate CREB-mediated transcription indicating that FAD mutations cause a gain of transcriptional function by inhibiting production of transcriptional repressor N-Cad/CTF2. These data raise the possibility that FAD mutation-induced transcriptional abnormalities maybe causally related to the dementia associated with FAD.

Alzheimer's disease (AD) may be caused by toxic aggregates formed from amyloid-β (Aβ) peptides. By using Thioflavin T, a dye that specifically binds to β-sheet structures, we found that highly toxic forms of Aβ-aggregates were formed at the initial stage of fibrillogenesis, which is consistent with recent reports on Aβ oligomers. Formation of such aggregates depends on factors that affect both nucleation and elongation. As reported previously, addition of Aβ42 systematically accelerated the nucleation of Aβ40, most likely because of the extra hydrophobic residues at the C terminus of Aβ42. At Aβ42-increased specific ratio (Aβ40: Aβ42 = 10: 1), on the other hand, not only accelerated nucleation but also induced elongation were observed, suggesting pathogenesis of early-onset AD. Because a larger proportion of Aβ40 than Aβ42 was still required for this phenomenon, we assumed that elongation does not depend only on hydrophobic interactions. Without any change in the C-terminal hydrophobic nature, elongation was effectively induced by mixing wild type Aβ40 with Italian variant Aβ40 (E22K) or Dutch variant (E22Q). We suggest that Aβ peptides in specific compositions that balance hydrophilic and hydrophobic interactions promote the formation of toxic β-aggregates. These results may introduce a new therapeutic approach through the disruption of this balance.

Protein folding defects have been demonstrated in a variety of neurodegenerative diseases, including Parkinson's disease and Huntington's disease, and molecular chaperones appear to play role in mediating these pathologies. It remains unclear whether chaperones also play a role in Alzheimer's disease (AD), which is characterized by both extracellular aggregates, consisting of β-amyloid, and intracellular aggregates known as neurofibrillary tangles, consisting mainly of tau proteins. We report an inverse relationship between accumulated tau and levels of the molecular chaperone Hsp90 in transgenic tau mouse brain. In cell culture, we demonstrate direct association of Hsp70 and Hsp90 with tau proteins and show that tau solubility and binding to microtubules is promoted by increased levels of these chaperones. Furthermore, increasing chaperone levels reduces tau aggregation and phosphorylation. The opposite results from depletion of the molecular chaperones. In addition, we show that chaperones facilitate degradation of tau, especially mutant tau. Our results suggest that molecular chaperones may suppress formation of neurofibrillary tangles by preventing tau aggregation and partitioning tau into a productive folding pathway and by accelerating degradation of aberrantly folded tau. © 2003, Elsevier Science B.V. All rights reserved.

A commercially available atomic force microscope (AFM) equipped with a hand made simple self-oscillation circuit was used in imaging biomolecular samples in liquid environments, i.e. under physiological conditions. Assembled tau proteins, which are the major component of the neurofibrillary deposits in Alzheimer's disease, was taken as a trial sample. In order to image its native structure, the protein was physically absorbed on a cleaved mica surface without fixation. Using the frequency feedback imaging with a self-oscillation technique, the structure of protein fibers was clearly imaged even in a wide scanning range (3.75 μm) with a contact force less than 100 pN. Furthermore, no damage of the proteins was observed in successive imagings. This indicates that the deformation of proteins was negligible in our method. In contrast, the proteins were destroyed when the vertical applied force of above 300 pN was applied using the amplitude feedback imaging with the self-oscillation technique. © 2002 Elsevier Science B.V. All rights reserved.

The mutation L271V in exon 8 of the presenilin-1 (PS-i) gene was detected in an Alzheimer's disease pedigree. Neuropathological examination of affected individuals identified variant, large, non-cored plaques without neuritic dystrophy, reminiscent of cotton wool plaques. Biochemical analysis of L271V mutation showed that it increased secretion of the 42-amino acid amyloid-β peptide, suggesting a pathogenic mutation. Analysis of PS-1 transcripts from the brains of two mutation carriers revealed a 17-50% increase in PS-1 transcripts with deletion of exon 8 (PS-1Δexon8) compared with unrelated Alzheimer's disease brains. Exon trapping analysis confirmed that L271V mutation enhanced the deletion of exon 8. Western blots of brain lysates indicated that PS-1Δexon8 was overexpressed in an affected individual. Biochemical analysis of PS-1Δexon8 in COS and BD8 cells indicate the splice isoform is not intrinsically active but interacts with wild-type PS-1 to generate amyloid-β. Western blots of cell lysates immunoprecipitated with anti-Tau or anti-GSK-3β antibodies indicated that PS-1Δexon8, unlike wild-type PS-1, does not interact directly with Tau or GSK-3β, potential modifiers of neuritic dystrophy. We postulate that variant plaques observed in this family are due in part to the effects of PS-1Δexon8 and that interaction between PS-1 and various protein complexes are necessary for neuritic plaque formation.

Molecular chaperones and their functions in protein folding have been implicated in several neurodegenerative diseases, including Parkinson's disease and Huntington's disease, which are characterized by accumulation of protein aggregates (e.g., α-synuclein and huntingtin, respectively). These aggregates have been shown in various experimental systems to respond to changes in levels of molecular chaperones suggesting the possibility of therapeutic intervention and a role for chaperones in disease pathogenesis. It remains unclear whether chaperones also play a role in Alzheimer's disease, a neurodegenerative disorder characterized by β-amyloid and tau protein aggregates. Here, we report an inverse relationship between aggregated tau and the levels of heat shock protein (Hsp)70/90 in tau transgenic mouse and Alzheimer's disease brains. In various cellular models, increased levels of Hsp70 and Hsp90 promote tau solubility and tau binding to microtubules, reduce insoluble tau and cause reduced tau phosphorylation. Conversely, lowered levels of Hsp70 and Hsp90 result in the opposite effects. We have also demonstrated a direct association of the chaperones with tau proteins. Our results suggest that up-regulation of molecular chaperones may suppress formation of neurofibrillary tangles by partitioning tau into a productive folding pathway and thereby preventing tau aggregation.

Neurofibrillary tangles (NFTs) are found in a wide range of neurodegenerative disorders, including Alzheimer's disease. The major component of NFTs is aberrantly hyperphosphorylated microtubule-associated protein tau. Because appropriate in vivo models have been lacking, the role of tau phosphorylation in NFTs formation has remained elusive. Here, we describe a new model in which adenovirus-mediated gene expression of tau, ΔMEKK, JNK3, and GSK-3β in COS-7 cells produces most of the pathological phosphorylation epitopes of tau including AT100. Furthermore, this co-expression resulted in the formation of tau aggregates having short fibrils that were detergent-insoluble and Thioflavin-S-reactive. These results suggest that aberrant tau phosphorylation by the combination of these kinases may be involved in "pretangle," oligomeric tau fibril formation in vivo.

The R406W tau mutation found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) causes a hereditary tauopathy clinically resembling Alzheimer's disease. Expression of modest levels of the longest human tau isoform with this mutation under the control of the α-calcium-calmodulin-dependent kinase-II promoter in transgenic (Tg) mice resulted in the development of congophilic hyperphosphorylated tau inclusions in forebrain neurons. These inclusions appeared as early as 18 months of age. As with human cases, tau inclusions were composed of both mutant and endogenous wild-type tau, and were associated with microtubule disruption and flame-shaped transformations of the affected neurons. Straight tau filaments were recovered from Sarkosyl-insoluble fractions from only the aged Tg brains. Behaviorally, aged Tg mice had associative memory impairment without obvious sensorimotor deficits. Therefore, these mice that exhibit a phenotype mimicking R406W FTDP-17 provide an animal model for investigating the adverse properties associated with this mutation, which might potentially recapitulate some etiological events in Alzheimer's disease.

To differentiate multiple activities of presenilin 1 (PS1), we generated transgenic mice expressing two human PS1 alleles: one with the aspartate to alanine mutation at residue 257 (hPS1D257A) that impairs the proteolytic activity of PS1, and the other deleting amino acids 340-371 of the hydrophilic loop sequence (hPS1Δcat) essential for β-catenin interaction. We show here that although hPS1Δcat is fully competent in rescuing the PS1-null lethal phenotype, hPS1D257A does not exhibit developmental activity. hPS1D257A also leads to the concurrent loss of the proteolytic processing of Notch and β-amyloid precursor protein (APP) and the generation of β-amyloid peptides (Aβ). Further, by measuring the levels of endogenous AβX-40 and AβX-42 in primary neuronal cultures, we confirmed the concept that PS1 is indispensable for the production of secreted Aβ.

To examine the regulation of amyloid secretion in more detail, Aβ sandwich ELISAs with high sensitivity and specificity were developed. Using this technique, we measured Aβ secreted from COS7 cells transiently transfected with APP C100 in the presence of LiCl, a potent glycogen synthase kinase (GSK)-3β inhibitor. We found that both Aβx-40 and Aβx-42 secretion were reduced by LiCl treatment in a dose-dependent manner. Diminished amyloid secretion was associated with GSK-3β activity. These results suggest that GSK-3β might function as a possible mediator for regulating both amyloid deposition and tau pathology in Alzheimer's disease (AD), and that lithium should be re-evaluated as a candidate reagent for preventing AD pathology. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Formation of neurofibrillary tangles (NFTs) is a common neuropathological feature found in several neurodegenerative diseases, including Alzheimer's disease. We have developed a transgenic (Tg) mouse expressing mutant human tau (V337M), derived from frontotemporal dementia parkinsonism-17. V337M Tg mice revealed tau aggregations in the hippocampus, which fulfills the histological criteria for NFTs in human neuro-degenerative diseases. Concurrent with the accumulation of RNA and phosphorylated tau, neurons exhibited morphological characteristics of degenerating neurons, which include a loss of microtubules, accumulation of ribosomes, plasma and nuclear membrane ruffling, and swelling of the Golgi network. Thus, mutant tau induces neuronal degeneration associated with the accumulation of RNA and phosphorylated tau. The functional consequences of this neuronal degeneration was evidenced by the reduction of hippocampal neural activity and behavioral abnormality in Tg mice.

Intracellular Aβ was examined in both a neuronal cell line (B103) expressing human APP with Swedish mutation and a non-neuronal cell line (Chinese hamster ovary, CHO) expressing wild human APP. Exposure of the APP695sw-transfected B103 cells to okadaic acid for 3 h, Aβ immunostaining was enhanced, as demonstrated by two independent anti-Aβ antibodies. The confocal microscopic study revealed that the immunoreactivity of Aβ was mainly colocalized with a Golgi marker and partially with an ER marker. Quantitative analyses, using Aβ sandwich ELISA, showed significantly increased intracellular Aβ. False positive detection of Aβ by antibody cross-reaction with APP was ruled out by extracting the fraction with formic acid and making it alkaline before subjecting it to ELISA. This procedure resulted in a fraction that contained little APP. Using CHO cells, OA treatment was also shown to be effective in increasing Aβ, as demonstrated by Western blot. The increased full-length APP and decreased APPC99 were also observed. This is the first study to demonstrate that OA treatment significantly increases intracellular Aβ. © 2002 Elsevier Science Inc. All rights reserved.

Inhibition of the accumulation of amyloid β-peptide (Aβ) and the formation of β-amyloid fibrils (fAβ) from Aβ, as well as the degradation of pre-formed fAβ in the CNS would be attractive therapeutic objectives for the treatment of Alzheimer's disease (AD). We previously reported that nordihydroguaiaretic acid (NDGA) inhibited fAβ formation from Aβ(1-42) and Aβ(1-42) dose-dependently in the range of 10-30 μM in vitro. Utilizing fluorescence spectroscopic analysis with thioflavin T and electron microscopic study, we show here that NDGA dose dependently breaks down fAβ(1-40) and fAβ(1-42) within a few hours at pH 7.5 at 37°C. At 4 h, the fluorescence of fAβ(1-40) and fAβ(1-42) incubated with 50 μM NDGA was 5% and 10% of the initial fluorescence, respectively. The activity of NDGA to break down these fAβs was observed even at a low concentration of 0.1 μM. At 1 h, many short, sheared fibrils were observed in the mixture incubated with 50 μM NDGA, and at 4 h, the number of fibrils reduced markedly, and small amorphous aggregates were observed. We next compared the activity of NDGA to break down fAβ(1-40) and fAβ(1-42), with other molecules reported to inhibit fAβ formation from Aβ and/or to degrade pre-formed fAβ both in vivo and vivoand in vitro. At a concentration of 50 μM, the overall activity of the molecules examined in this study was in the order of: NDGA &gt &gt rifampicin = tetracycline &gt poly(vinylsulfonic acid, sodium salt) = 1,3-propanedisulfonic acid, disodium salt &gt β-sheet breaker peptide (iAβ5). In cell culture experiments, fAβ disrupted by NDGA were less toxic than intact fAβ, as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Although the mechanisms by which NDGA inhibits fAβ formation from Aβ, as well as breaking down preformed fAβ in vitro, are still unclear, NDGA could be a key molecule for the development of therapeutics for AD.

Age-related changes in PS-1 localization were examined in the brains of 22 cynomolgus monkeys ranging in age from embryonic day 87 to 35 years. In embryonic monkey brains, anti-PS-1 antibody N12, which recognizes the PS-1 N-terminal fragment (Ntf) and holo protein, stained immature neuronal cells. In juvenile monkeys, N12 stained large pyramidal neurons, cerebral neocortical neurons, and cerebellar Purkinje's cells. Cytoplasmic staining of these cells was granular in appearance. In aged monkeys, N12 stained neurons in all layers of the neocortex. In contrast, regardless of the age of the animals examined, M5, an anti-PS-1 antibody that specifically recognizes only the PS-1 C-terminal fragment (Ctf), stained neurons in all layers of the neocortex and neurons in the cerebellum. M5 also stained neuropil and white matter, and in aged monkeys, M5 stained swollen neurites of mature senile plaques. Age-related changes in PS-1 expression were further examined using Western blot analysis of mitochondrial, myelin, microsomal, nuclear, synaptosomal, and cytosol fractions isolated from 10 monkey brains ranging in age from embryonic day 87 to 32 years. In all brains, Ntf and Ctf were expressed most abundantly in the microsome fraction. The amount of PS-1 in the nuclear fraction dramatically increased with age. We conclude that the transport of PS-1 diminished with age and that PS-1 fragments accumulated in endoplasmic reticulum (ER) associated with the nuclear membrane. © 2001 Elsevier Science B.V. All rights reserved.

Dysfunction and filamentous microtubule-binding tau protein are key markers of neurodegenerative pathologies, including the pathology and neural degeneration associated with Alzheimer's disease (AD). Immunocytochemical studies of NFT-bearing neurons showed that NFTs are composed of ubiquitin and phosphorylation- dependent tau. Congo-red birefringency and thioflavin-S reactivity in NFT-bearing neurons also demonstrated that the tau aggregation forms a β-sheet structure. Discovery of the molecular mechanisms of NFT formation may lead to more insight about events occurring during neurodegeneration. In frontotemporal dementia parkinsonism 17 (FTDP17), genetic studies indicated that tau is a causative gene, and mutation is found in exons and introns of tau gene. A patient who possesses this mutation exhibits pathologically NFT and clinically personality change and cognitive dysfunction. Then, we produced the Tg mice expressing human longest tau with misssense mutation V337M. In the present study, neurons of hippocampus and cerebral cortex in our Tg mice showed phosphorylated and ubiquitinated tau aggregations with a β-sheet structure. This was demonstrated by Congo-red and thioflavin-S positive staining, a histological criterion used to identify NFTs observed in neurodegenerative disorders. The mice also displayed altered behaviors that were associated with NFT formation. Thus, V337M mice provide a first animal model exhibiting altered behavior due to NFTs.

Presenilin 1 (PS1) is linked with Alzheimer's disease but exhibits functional roles regulating growth and development. For instance, PS1 binds to β-catenin and modulates β-catenin signaling. In the current study, we observed that knockout of PS1 inhibited β-catenin-mediated transcription by 35%, as shown by a luciferase reporter driven by the hTcf-4 promoter. Overexpressing wild-type PS1 increased β-catenin-mediated transcription by 37.5%, and overexpressing PS1 with mutations associated with Alzheimer's disease decreased β-catenin-mediated transcription by 66%. To examine whether regulation of β-catenin by PS1 requires phosphorylation by glycogen synthase kinase 3β (GSK 3β), we examined whether inhibiting GSK 3β activity overcomes the inhibition of β-catenin transcription induced by mutant PS1 constructs. Cells expressing wild-type or mutant PS1 were treated with LiC1, which inhibits GSK 3β, or transfected with β-catenin constructs that lack the GSK 3β phosphorylation sites. Neither treatment overcame PS1-mediated inhibition of β-catenin signaling, suggesting that regulation of β-catenin by PS1 was not affected by the activity of GSK 3β. To investigate how PS1 might regulate β-catenin signaling, we determined whether PS1 interacts with other elements of the β-catenin signaling cascade, such as the Tcf-4 transcription factor. Coimmunoprecipitation studies showed binding of PS1 and hTcf-4, and examining nuclear isolates indicated that nuclear hTcf-4 was decreased in cells expressing mutant PS1. These data show that PS1 interacts with multiple components of the β-catenin signaling cascade and suggest that PS1 regulates β-catenin in a manner independent of GSK 3β activity.

Amyloid-β protein (Aβ) aggregates in the brain to form senile plaques. By using thioflavin T, a dye that specifically binds to fibrillar structures, we found that metals such as Zn(II) and Cu(II) normally inhibit amyloid β-aggregation. Another method for detecting Aβ, which does not distinguish the types of aggregates, showed that these metals induce a non-β-sheeted aggregation, as reported previously. Secondary structural analysis and microscopic studies revealed that metals induced Aβ to make non-fibrillar aggregates by disrupting β-sheet formation. These non-fibrillar Aβ aggregates displayed much weaker Congo Red birefringence, and in separate cell culture experiments, were less toxic than self β-aggregates, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The toxicity of soluble Aβ was enhanced in the presence of Cu(II), which suggests the previously hypothesized role of Aβ in generating oxidative stress. Finally, under an acidic condition, similar to that in the inflammation associated with senile plaques, β-aggregation was robustly facilitated at one specific concentration of Zn(II) in the presence of heparin. However, because a higher concentration of Zn(II) virtually abolished this abnormal phenomenon, and at normal pH any concentrations strongly inhibit β-aggregation and its associated cytotoxicity, including its anti-oxidative nature we suggest that Zn(II) has an overall protective effect against β-amyloid toxicity.

P35 or its truncated fragment p25 is required for cyclin dependent kinase (Cdk)5 activation. It has been reported that p25 is accumulated in the brain of Alzheimer's disease (AD) patients and that p25/Cdk5 induces high phosphorylation of tau and apoptosis in cultured neurons (Nature 402 (1999) 615). Our investigation of AD brain did not show specific accumulation of p25. Exposure to Ca ionophore (A23187) at 10-6 M induced p25 accumulation in rat primary hippocampal neurons, causing neuronal death without showing hyperphosphorylation of tau. Transgenic mice expressing p25 showed the accumulation of p25 but neither hyperphosphorylation of tau nor neuronal death was shown in these mice. The feature of these mice was the progression of cell growth in pituitary gland. These results suggest that overexpression of p25 lead to the activation of cell cycle but not to the direct phosphorylation of tau. © 2001 Elsevier Science Ireland Ltd.

Formation of neurofibrillary tangles (NFTs) is the most common feature in several neurodegenerative diseases, including Alzheimer's disease (AD). Here we report the formation of filamentous tau aggregations having a β-sheet structure in transgenic mice expressing mutant human tau. These mice contain a tau gene with a mutation of the frontotemporal dementia parkinsonism (FTDP-17) type, in which valine is substituted with methionine residue 337. The aggregation of tau in these transgenic mice satisfies all histological criteria used to identify NFTs common to human neurodegenerative diseases. These mice, therefore, provide a preclinical model for the testing of therapeutic drugs for the treatment of neurodegenerative disorders that exhibit NFTs. © 2001 Elsevier Science.