Kazuma Murakami

Modeling Alzheimer’s Disease with iPSCs Reveals Stress Phenotypes Associated with Intracellular Aβ and Differential Drug Responsiveness

Oligomeric forms of amyloid-β peptide (Aβ) are thought to play a pivotal role in the pathogenesis ofxa0Alzheimers disease (AD), but the mechanism involved is still unclear. Here, we generated induced pluripotent stem cells (iPSCs) from familial and sporadic AD patients and differentiated them into neural cells. Aβ oligomers accumulated in iPSC-derived neurons and astrocytes in cells from patients with a familial amyloid precursor protein (APP)-E693Δ mutation and sporadic AD, leading to endoplasmic reticulum (ER) and oxidative stress. The accumulated Aβ oligomers were not proteolytically resistant, and docosahexaenoic acid (DHA) treatment alleviated the stress responses in the AD neural cells. Differential manifestation of ER stress and DHA responsiveness may help explain variable clinical results obtained with the use of DHA treatment and suggests that DHA may in fact be effective forxa0a subset of patients. It also illustrates how patient-specific iPSCs can be useful for analyzing AD pathogenesis and evaluating drugs.

Site-specific Inhibitory Mechanism for Amyloid β42 Aggregation by Catechol-type Flavonoids Targeting the Lys Residues

Background: The inhibitory mechanism of Aβ42 aggregation by flavonoid is fully unknown. Results: The oxidant enhanced the inhibitory activity of (+)-taxifolin against Aβ42 aggregation by forming Aβ42-taxifolin adducts between the Lys residues and oxidized (+)-taxifolin. Conclusion: The inhibitory activity of catechol-type flavonoids requires autoxidation to form an o-quinone to react with Lys. Significance: These may help design promising inhibitors against Aβ42 aggregation for Alzheimer disease therapy. The aggregation of the 42-residue amyloid β-protein (Aβ42) is involved in the pathogenesis of Alzheimer disease (AD). Numerous flavonoids exhibit inhibitory activity against Aβ42 aggregation, but their mechanism remains unclear in the molecular level. Here we propose the site-specific inhibitory mechanism of (+)-taxifolin, a catechol-type flavonoid, whose 3′,4′-dihydroxyl groups of the B-ring plays a critical role. Addition of sodium periodate, an oxidant, strengthened suppression of Aβ42 aggregation by (+)-taxifolin, whereas no inhibition was observed under anaerobic conditions, suggesting the inhibition to be associated with the oxidation to form o-quinone. Because formation of the Aβ42-taxifolin adduct was suggested by mass spectrometry, Aβ42 mutants substituted at Arg5, Lys16, and/or Lys28 with norleucine (Nle) were prepared to identify the residues involved in the conjugate formation. (+)-Taxifolin did not suppress the aggregation of Aβ42 mutants at Lys16 and/or Lys28 except for the mutant at Arg5. In addition, the aggregation of Aβ42 was inhibited by other catechol-type flavonoids, whereas that of K16Nle-Aβ42 was not. In contrast, some non-catechol-type flavonoids suppressed the aggregation of K16Nle-Aβ42 as well as Aβ42. Furthermore, interaction of (+)-taxifolin with the β-sheet region in Aβ42 was not observed using solid-state NMR unlike curcumin of the non-catechol-type. These results demonstrate that catechol-type flavonoids could specifically suppress Aβ42 aggregation by targeting Lys residues. Although the anti-AD activity of flavonoids has been ascribed to their antioxidative activity, the mechanism that the o-quinone reacts with Lys residues of Aβ42 might be more intrinsic. The Lys residues could be targets for Alzheimer disease therapy.

Intracellular Accumulation of Toxic Turn Amyloid-β is Associated with Endoplasmic Reticulum Stress in Alzheimer’s Disease

Amyloid-β protein (Aβ) accumulates in the neurons of Alzheimers disease (AD) patients at an early stage of the disease. Recently, we found that Aβ with a toxic turn at positions 22 and 23 accumulates in neurons in AD brain. Here, we studied the accumulation of Aβ, toxic turn Aβ and high-molecular-weight Aβ oligomers in presenilin 1 (PS1) gene-transfected SH-SY5Y cells as well as in the brains of 3xTg-AD mice and AD patients. Immunostaining revealed that accumulation of toxic turn Aβ was promoted in G384A- and I143T-mutant PS1-transfected cells and further enhanced by co-transfection of cells with the Aβ-precursor protein (AβPP) gene. In contrast, accumulation of high-molecular-weight Aβ oligomers was promoted in mutant PS1 cells but attenuated by co-transfection of cells with the AβPP gene. Toxic turn Aβ was detected in the neurons of 3xTg-AD mice aged 2 months, when the mice were cognitively unimpaired. In contrast, high-molecular-weight Aβ oligomers were detected in the neurons of 7-month-old mice, when memory dysfunction is apparent. Furthermore, immunostaining and western blotting for Rab4, Rab6 and GRP78 revealed increased levels of these proteins in mutant PS1 cells and their accumulation in the neurons of 3xTg-AD mice. Remarkably, GRP78 immunoreactivity was increased at 2 months of age. Double-label immunostaining of AD brain revealed an apparent association between toxic turn Aβ and GRP78, an endoplasmic reticulum (ER) stress marker. Intraneuronal accumulation of toxic turn Aβ may be associated with ER stress in the brains of AD model mice and AD patients at an early stage.

[Monoclonal antibody against the turn of the 42-residue amyloid β-protein at positions 22 and 23].

Aggregation of the 42-mer amyloid β-protein (Aβ42) plays a critical role in the pathogenesis of Alzheimers disease (AD). We have proposed a toxic conformer with a turn at positions 22 and 23, as well as a nontoxic conformer with a turn at positions 25 and 26, in Aβ42 aggregates from systematic proline scanning and solid-state NMR studies. Although recent clinical trials of immunization targeting Aβ42 aggregates have proved useful, some adverse effects were reported. One of the reasons was hypothesized to be excessive immunoreactions derived from the unintended removal of nontoxic Aβ42, which plays an important role in the physiological function. To develop a monoclonal antibody for toxic Aβ42, E22P-Aβ10-35, a minimum moiety for neurotoxicity containing the turn at positions 22 and 23, was used for the generation of antibodies, following the selection of clones using Aβ42 mutants of E22P (turn-inducing) and E22V (turn-preventing). The obtained clone (11A1) showed a high binding affinity (K(D) = 10.3 nM) for Aβ42 using surface plasmon resonance. 11A1 also inhibited the neurotoxicity of Aβ42 in PC12 cells. Immunohistochemical studies showed that not only extracellular but intracellular amyloid was stained in human AD brains. In Western blotting analyses using human brains, low-molecular weight-oligomers rather than the monomer of Aβ were readily recognized by 11A1. These results imply that 11A1 could detect toxic Aβ42 oligomers with the turn at positions 22 and 23 and that 11A1 could be applicable for the therapeutic targeting of toxic Aβ42 in AD.

Silymarin Attenuated the Amyloid β Plaque Burden and Improved Behavioral Abnormalities in an Alzheimer’s Disease Mouse Model

Alzheimer’s disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid β-protein (Aβ) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aβ deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aβ oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.

Early accumulation of intracellular fibrillar oligomers and late congophilic amyloid angiopathy in mice expressing the Osaka intra-Aβ APP mutation

Pathogenic amyloid-β peptide precursor (APP) mutations clustered around position 693 of APP—position 22 of the Aβ sequence—are commonly associated with congophilic amyloid angiopathy (CAA) and intracerebral hemorrhages. In contrast, the Osaka (E693Δ) intra-Aβ APP mutation shows a recessive pattern of inheritance that leads to AD-like dementia despite low brain amyloid on in vivo positron emission tomography imaging. Here, we investigated the effects of the Osaka APP mutation on Aβ accumulation and deposition in vivo using a newly generated APP transgenic mouse model (E22ΔAβ) expressing the Osaka mutation together with the Swedish (K670N/M671L) double mutation. E22ΔAβ mice exhibited reduced α-processing of APP and early accumulation of intraneuronal fibrillar Aβ oligomers associated with cognitive deficits. In line with our in vitro findings that recombinant E22Δ-mutated Aβ peptides form amyloid fibrils, aged E22ΔAβ mice showed extracellular CAA deposits in leptomeningeal cerebellar and cortical vessels. In vitro results from thioflavin T aggregation assays with recombinant Aβ peptides revealed a yet unknown antiamyloidogenic property of the E693Δ mutation in the heterozygous state and an inhibitory effect of E22Δ Aβ42 on E22Δ Aβ40 fibrillogenesis. Moreover, E22Δ Aβ42 showed a unique aggregation kinetics lacking exponential fibril growth and poor seeding effects on wild-type Aβ aggregation. These results provide a possible explanation for the recessive trait of inheritance of the Osaka APP mutation and the apparent lack of amyloid deposition in E693Δ mutation carriers.

Conformation-specific antibodies to target amyloid β oligomers and their application to immunotherapy for Alzheimer's disease.

Amyloid β-protein (Aβ) oligomers, intermediates of Aβ aggregation, cause cognitive impairment and synaptotoxicity in the pathogenesis of Alzheimer’s disease (AD). Immunotherapy using anti-Aβ antibody is one of the most promising approaches for AD treatment. However, most clinical trials using conventional sequence-specific antibodies have proceeded with difficulty. This is probably due to the unintended removal of the non-pathological monomer and fibrils of Aβ as well as the pathological oligomers by these antibodies that recognize Aβ sequence, which is not involved in synaptotoxicity. Several efforts have been made recently to develop conformation-specific antibodies that target the tertiary structure of Aβ oligomers. Here, we review the recent findings of Aβ oligomers and anti-Aβ antibodies including our own, and discuss their potential as therapeutic and diagnostic tools. Graphical Abstract Toxic conformer of amyloid β42 protein with turn structure at Glu22 and Asp23 and C-terminal core, and its intraneuronal depositions in Alzheimer’s disease-affected brain.

The turn formation at positions 22 and 23 in the 42-mer amyloid β peptide: The emerging role in the pathogenesis of Alzheimer's disease

One hallmark of Alzheimers disease (AD) is the accumulation of amyloid β (Aβ) peptides in the brain; Aβ mainly consists of 42‐mer and 40‐mer peptides (Aβ42 and Aβ40). Aβ42 plays a more critical role in the pathogenesis of AD because Aβ42 aggregates much faster and is more toxic than Aβ40. Therefore, there is an urgent need to elucidate the mechanism of aggregation and neurotoxicity of Aβ42 to develop therapeutic agents. Here, we introduce the pathological role of Aβ42 in AD and review our recent findings of the structural analysis of Aβ42 using systematic proline replacement, electron spin resonance and solid‐state nuclear magnetic resonance, and the new mechanism of neurotoxicity of Aβ42 through the formation of radicals. Geriatr Gerontol Int 2010; 10 (Suppl. 1): S169–S179.

Stimulation of the Amyloidogenic Pathway by Cytoplasmic Superoxide Radicals in an Alzheimer's Disease Mouse Model

Oxidative stress is involved in the pathogenesis of neurodegeneration. Amyloid β (Aβ) oligomer as an intermediate of aggregates causes memory loss in Alzheimers disease (AD). We have suggested that oxidative stress plays an important role in Aβ oligomerization and cognitive impairment using a human amyloid precursor protein (hAPP) transgenic AD mice lacking cytoplasmic superoxide dismutase (hAPP/Sod1 −/−). Recently, clinical trials revealed inhibitors of Aβ production from hAPP as promising therapeutics, but the relationship between oxidative stress and Aβ metabolism remains unclear. Here we found that Sod1 deficiency enhanced β-cleavage of hAPP, suggesting that it increased Aβ production in hAPP/Sod1 −/− mice. In contrast, Aβ degradation did not decrease in hAPP/Sod1 −/− as compared with hAPP/Sod1 +/+ mice. Furthermore, we successfully detected in situ superoxide radicals associated with increased protein carbonylation in hAPP/Sod1 −/−. These results suggest that cytoplasmic oxidative stress is involved in Aβ production as well as aggregation during AD progression.

Insulin receptor mutation results in insulin resistance and hyperinsulinemia but does not exacerbate Alzheimer's-like phenotypes in mice.

Obesity is a risk factor for Alzheimers disease (AD), which is characterized by amyloid β depositions and cognitive dysfunction. Although insulin resistance is one of the phenotypes of obesity, its deleterious effects on AD progression remain to be fully elucidated. We previously reported that the suppression of insulin signaling in a mouse with a heterozygous mutation (P1195L) in the gene for the insulin receptor showed insulin resistance and hyperinsulinemia but did not develop diabetes mellitus [15]. Here, we generated a novel AD mouse model carrying the same insulin receptor mutation and showed that the combination of insulin resistance and hyperinsulinemia did not accelerate plaque formation or memory abnormalities in these mice. Interestingly, the insulin receptor mutation reduced oxidative damage in the brains of the AD mice. These findings suggest that insulin resistance is not always involved in the pathogenesis of AD.