Ayumi Takamura

Extracellular and intraneuronal HMW-AbetaOs represent a molecular basis of memory loss in Alzheimer's disease model mouse.

BackgroundSeveral lines of evidence indicate that memory loss represents a synaptic failure caused by soluble amyloid β (Aβ) oligomers. However, the pathological relevance of Aβ oligomers (AβOs) as the trigger of synaptic or neuronal degeneration, and the possible mechanism underlying the neurotoxic action of endogenous AβOs remain to be determined.ResultsTo specifically target toxic AβOs in vivo, monoclonal antibodies (1A9 and 2C3) specific to them were generated using a novel design method. 1A9 and 2C3 specifically recognize soluble AβOs larger than 35-mers and pentamers on Blue native polyacrylamide gel electrophoresis, respectively. Biophysical and structural analysis by atomic force microscopy (AFM) revealed that neurotoxic 1A9 and 2C3 oligomeric conformers displayed non-fibrilar, relatively spherical structure. Of note, such AβOs were taken up by neuroblastoma (SH-SY5Y) cell, resulted in neuronal death. In humans, immunohistochemical analysis employing 1A9 or 2C3 revealed that 1A9 and 2C3 stain intraneuronal granules accumulated in the perikaryon of pyramidal neurons and some diffuse plaques. Fluoro Jade-B binding assay also revealed 1A9- or 2C3-stained neurons, indicating their impending degeneration. In a long-term low-dose prophylactic trial using active 1A9 or 2C3 antibody, we found that passive immunization protected a mouse model of Alzheimers disease (AD) from memory deficits, synaptic degeneration, promotion of intraneuronal AβOs, and neuronal degeneration. Because the primary antitoxic action of 1A9 and 2C3 occurs outside neurons, our results suggest that extracellular AβOs initiate the AD toxic process and intraneuronal AβOs may worsen neuronal degeneration and memory loss.ConclusionNow, we have evidence that HMW-AβOs are among the earliest manifestation of the AD toxic process in mice and humans. We are certain that our studies move us closer to our goal of finding a therapeutic target and/or confirming the relevance of our therapeutic strategy.

Amyloid β accelerates phosphorylation of tau and neurofibrillary tangle formation in an amyloid precursor protein and tau double-transgenic mouse model

In Alzheimers disease, Aβ deposits are considered the initial cardinal events that induce tauopathy secondarily. However, the relationship between Aβ amyloidosis and tauopathy has not been determined in detail. We produced double transgenic mice, 2×TgTau+/–APP+/–, by mating Tg2576 mice that exhibit Aβ amyloidosis and TgTauP301L mice that show tauopathy, and statistically analyzed the effect of Aβ accumulation on tauopathy. There was no significant difference in theprogression of Aβ accumulation among 2×TgTau+/–APP+/– and 1×TgTau−/–APP+/–, and tau accumulation among 2×TgTau+/−APP+/− and 1×Tg Tau+/–APP−/–. The appearance rates of phosphorylated tau developing in neurons and processes were significantly accelerated in 2×TgTau+/–APP+/– mice compared with those in 1×TgTau+/–APP−/– mice at 23 months of age. Accumulation of phosphorylated and confomationally altered tau and GSK3β in neuronal processes was accelerated in the white matter in 2×TgTau+/–APP+/–. The level of phosphorylated tau in the sarkosyl‐insoluble fraction was increased in 2×TgTau+/–APP+/– brains compared with that in 1×TgTau+/–APP−/– brains. Thus, Aβ amyloid partially enhances tauopathy through accumulation of insoluble, phosphorylated, and conformationally changed tau in neuronal cytoplasm and processes in the late stage.

Factors responsible for neurofibrillary tangles and neuronal cell losses in tauopathy

TgTauP301L mice that overexpress the mutant human tauP301L present in FTDP‐17 reproduce neurofibrillary tangles (NFTs), neuronal cell losses, memory disturbance, and substantial phenotypic variation. To demonstrate factors responsible for NFT formation and neuronal cell losses, sets of TgTauP301L for comparison with or without NFTs and neuronal cell losses were studied with oligonucleotide microarrays. Gene expressions were altered in biological pathways, including oxidative stress, apoptosis, mitochondrial fatty acid betaoxidation, inflammatory response pathway, and complement and coagulation cascade pathways. Among 24 altered genes, increased levels of apolipoprotein D (ApoD) and neuronal PAS domain protein 4 (Npas4) and decreased levels of doublecortin (DCX) and potassium channel, voltage‐gated, shaker‐related subfamily, β member 1 (Kcnab1) were found in the TgTauP301L with NFTs and neuronal cell losses, Alzheimers brains, and tauopathy brains. Thus, many biological pathways and novel molecules are associated with NFT formation and neuronal cell losses in tauopathy brains.

Disease Modifying Therapies for Alzheimer's Disease Targeting Aβ Oligomers: Implications for Therapeutic Mechanisms

Several lines of evidence indicate that amyloid β (Aβ), particularly Aβ oligomers (AβOs), plays a causative role in Alzheimers disease. However, the mechanisms underlying the action of an anti-AβO antibody to clarify the toxic action of AβOs remain elusive. Here, we showed that the anti-AβO antibody (monoclonal 72D9) can modify the Aβ aggregation pathway. We also found that 72D9 directly sequesters both extracellular and intraneuronal AβOs in a nontoxic state. Thus, therapeutic intervention targeting AβOs is a promising strategy for neuronal protection in Alzheimers disease.

Dissociation of β-amyloid from lipoprotein in cerebrospinal fluid from Alzheimer's disease accelerates β-amyloid-42 assembly

Monoclonal 2C3 specific to β‐amyloid (Aβ) oligomers (AβOs) enabled us to test our hypothesis that the alteration of lipoprotein–Aβ interaction in the central nervous system (CNS) initiates and/or accelerates the cascade favoring Aβ assembly. Immunoprecipitation of frontal cortex employing 2C3 unequivocally detected soluble 4‐, 8‐, and 12‐mers in Alzheimers disease (AD) brains. Immunoblot analysis of the entorhinal cortex employing 2C3 revealed that the accumulation of soluble 12‐mers precedes the appearance of neuronal loss or cognitive impairment and is enhanced as the Braak neurofibrially tangle (NFT) stages progress. The dissociation of soluble Aβ from lipoprotein particles occurs in cerebrospinal fluid (CSF), and the presence of lipoprotein‐free oligomeric 2C3 conformers (4‐ to 35‐mers) was evident, which mimic CNS environments. Such CNS environments may strongly affect conformation of soluble Aβ peptides, resulting in the conversion of soluble Aβ42 monomers into soluble Aβ42 assembly. The findings suggest that functionally declined lipoproteins may accelerate the generation of metabolic conditions leading to higher levels of soluble Aβ42 assembly in the CNS.

An Unusual Case of Elderly-Onset Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy (CADASIL) With Multiple Cerebrovascular Risk Factors

Here we report a female patient with elderly-onset cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). At age 71, she developed gait disturbance, followed by memory disturbance 1 year later. She had been treated for hypertension and diabetes mellitus for 19 years. There apparently was low penetrance of disease. Magnetic resonance imaging (MRI) findings showed typical features of CADASIL, and the R607C mutation was detected in exon 11 in NOTCH3. This case strongly indicates that CADASIL should be considered when typical findings are observed on MRI even in cases of elderly onset with multiple cerebrovascular risk factors.

Molecular Mechanism Underlying Aβ Immunotherapy: Implications for the Toxic Action of Aβ Oligomers

Aβ immunotherapy brought us not only hope for but also led to greater attention to the mechanism underlying the clearance of amyloid β (Aβ), which provided fascinating insights into disease-relevant molecules such as toxic Aβ oligomers (AβOs). Accumulated lines of evidence indicate that AβOs play a causative role in the pathogenesis of Alzheimer’s disease (AD), leading to a synaptic failure, which is considered a major cellular mechanism underlying the cognitive deficits in patients with mild cognitive impairment and AD. In this mini review, we focus on recent knowledge of the possible mechanisms underlying the action of the anti-Aβ antibody to clarify the toxic action of AβOs.

Alzheimer neuronal degeneration: Extracellular versus intracellular Aß oligomers

age-matched control (n 1⁄4 8) and Alzheimer’s (n1⁄4 18) patients and in transgenic APP mutant mice (J9a) and age-matched wild-type littermate controls (4mo and approx 1yr). Amyloid was detected using immunohistochemical approaches with the antibodies 4G8 and 6F3D. The extent of immunostaining, both intracellular and extracellular, was semi-quantified in white matter using a grading system and differences compared using Mann Whitney. Results: In human post-mortem brain, amyloid was detected in the white matter in both controls and AD cases. However there was significantly more intracellular and extracellular amyloid detected in the white matter in AD brains as compared to controls. There was no evidence of amyloid accumulation in white matter in young wild-type mice and at an older age only weak expression of amyloid was observed in oligodendrocytes. In contrast in young mutant APP mice cellular expression of amyloid was detetected in white matter and this was most marked in older mice in which increased expression of amyloid within oligodendrocytes was observed and marked amyloid deposition. Old APP mutant mice also exhibited white matter pathology, disruption of axonal and myelin integrity. Conclusions: There is marked amyloid accumulation in white matter in AD and mutant mice brains that could contribute to a loss of white matter integrity and functional impairment.

Factors responsible for neurofibrillary tangle formation and neuronal cell death in tauopathy brains

Background: TgTauP301L that overexpresses mutant human tauP301L present in FTDP-17 reproduces neurofibrillary tangles (NFT), neuronal death, memory disturbance and substantial phenotypic variation. Methods: To reveal factors responsible for NFT formation and neuronal death, comparison sets of TgTauP301L with or without NFT and neuronal cell losses were studied using oligonucleotide microarrays. Results: Results indicate that alteration of gene expressions in biological pathways including oxidative stress, apoptosis, mitochondrial fatty acid betaoxidation, inflammatory response pathway, complement and coagulation cascades pathway and EGFR1pathway. Reactive increased Apolipoprotein D for NFT neurotoxicity and decreased doublecortin according to suppressed neurogenesis were observed. Conclusions: These findings indicate that NFT formations and neuronal cell losses occur under alterations of many biological pathways, especially oxidative stress, apoptosis, mitochondrial dysfunction, inflammation, cell cycle and signal transductions in tauopathy brains.