Single-Molecule Kinetic Observation of Antibody Interactions with Growing Amyloid ß Fibrils.

Understanding the dynamic assembly process of amyloid ß (Aß) during fibril formation is essential for developing effective therapeutic strategies against Alzheimer's disease. Here, we employed high-speed atomic force microscopy to observe the growth of Aß fibrils at the single-molecule level, focusing specifically on their interaction with anti-Aß antibodies. Our findings show that fibril growth consists of intermittent periods of elongation and pausing, which are dictated by the alternating addition of Aß monomers to protofilaments. We highlight the distinctive interaction of antibody 4396C, which specifically binds to the fibril ends in the paused state, suggesting a unique mechanism to hinder fibril elongation. Through real-time visualization of fibril growth and antibody interactions combined with molecular simulation, this study provides a refined understanding of Aß assembly during fibril formation and suggests novel strategies for Alzheimer's therapy aimed at inhibiting the fibril elongation.

High performance plasma amyloid-ß biomarkers for Alzheimer's disease.

To facilitate clinical trials of disease-modifying therapies for Alzheimer's disease, which are expected to be most efficacious at the earliest and mildest stages of the disease, supportive biomarker information is necessary. The only validated methods for identifying amyloid-ß deposition in the brain-the earliest pathological signature of Alzheimer's disease-are amyloid-ß positron-emission tomography (PET) imaging or measurement of amyloid-ß in cerebrospinal fluid. Therefore, a minimally invasive, cost-effective blood-based biomarker is desirable. Despite much effort, to our knowledge, no study has validated the clinical utility of blood-based amyloid-ß markers. Here we demonstrate the measurement of high-performance plasma amyloid-ß biomarkers by immunoprecipitation coupled with mass spectrometry. The ability of amyloid-ß precursor protein (APP)669-711/amyloid-ß (Aß)1-42 and Aß1-40/Aß1-42 ratios, and their composites, to predict individual brain amyloid-ß-positive or -negative status was determined by amyloid-ß-PET imaging and tested using two independent data sets: a discovery data set (Japan, n = 121) and a validation data set (Australia, n = 252 including 111 individuals diagnosed using 11C-labelled Pittsburgh compound-B (PIB)-PET and 141 using other ligands). Both data sets included cognitively normal individuals, individuals with mild cognitive impairment and individuals with Alzheimer's disease. All test biomarkers showed high performance when predicting brain amyloid-ß burden. In particular, the composite biomarker showed very high areas under the receiver operating characteristic curves (AUCs) in both data sets (discovery, 96.7%, n = 121 and validation, 94.1%, n = 111) with an accuracy approximately equal to 90% when using PIB-PET as a standard of truth. Furthermore, test biomarkers were correlated with amyloid-ß-PET burden and levels of Aß1-42 in cerebrospinal fluid. These results demonstrate the potential clinical utility of plasma biomarkers in predicting brain amyloid-ß burden at an individual level. These plasma biomarkers also have cost-benefit and scalability advantages over current techniques, potentially enabling broader clinical access and efficient population screening.

Size and Shape of Amyloid Fibrils Induced by Ganglioside Nanoclusters: Role of Sialyl Oligosaccharide in Fibril Formation.

Ganglioside-enriched microdomains in the presynaptic neuronal membrane play a key role in the initiation of amyloid ß-protein (Aß) assembly related to Alzheimer's disease. We previously isolated lipids from a detergent-resistant membrane microdomain fraction of synaptosomes prepared from aged mouse brain and found that spherical Aß assemblies were formed on Aß-sensitive ganglioside nanoclusters (ASIGN) of reconstituted lipid bilayers in the synaptosomal fraction. In the present study, we investigated the role of oligosaccharides in Aß fibril formation induced by ganglioside-containing mixed lipid membranes that mimic the features of ASIGN. Ganglioside nanoclusters were constructed as ternary mixed lipid bilayers composed of ganglioside (GM1, GM2, GM3, GD1a, or GT1b), sphingomyelin, and cholesterol, and their surface topography was visualized by atomic force microscopy. Aß fibril formation on the nanocluster was strongly induced in the presence of 10 mol % ganglioside, and Aß-sensitive features were observed at cholesterol contents of 35-55 mol %. GM1-, GD1a-, and GT1b-containing membranes induced longer fibrils than those containing GD1b and GM2, indicating that the terminal galactose of GM1 along with N-acetylneuraminic acid accelerates protofibril elongation. These results demonstrate that Aß fibril formation is induced by ASIGN that are highly enriched ganglioside nanoclusters with a limited number of components and that the generation and elongation of Aß protofibrils are regulated by the oligosaccharide structure of gangliosides.

Retromer and Rab2-dependent trafficking mediate PS1 degradation by proteasomes in endocytic disturbance.

Accumulating evidence suggests that endocytic pathway deficits are involved in Alzheimer's disease pathogenesis. Several reports show that endocytic disturbance affects ß-amyloid peptide (Aß) cleavage from ß-amyloid precursor protein (APP). Presenilin-1 (PS1) is the catalytic core of the γ-secretase complex required for Aß generation. Previously, we showed that aging induces endocytic disturbance, resulting in the accumulation of Aß and APP in enlarged endosomes. It remains unclear, however, whether PS1 localization and function are affected with endocytic disturbance. Here, we report that in endocytic disturbance, PS1 is transported from endosomes to ER/Golgi compartments via retromer trafficking, and that PS1 interacts with vacuolar protein sorting-associated protein 35 both in vitro and in vivo. Moreover, PS1 is degraded by proteasomes via a Rab2-dependent trafficking pathway, only during endocytic disturbance. These findings suggest that PS1 levels and localization in endosomes are regulated by retromer trafficking and ER-associated degradation system, even if endocytic disturbance significantly induces the endosomal accumulation of APP and ß-site APP-cleaving enzyme 1. Results of this study also suggest that retromer deficiency can affect PS1 localization in endosomes, where Aß cleavage mainly occurs, possibly leading to enhanced Aß pathology. We proposed the following mechanism for intracellular transport of presenilin-1 (PS1). When endosome/lysosome trafficking is disturbed, PS1 is transported from endosome to endoplasmic reticulum (ER)/Golgi compartments via retromer and Rab2-mediated trafficking, and then degraded by endoplasmic reticulum-associated degradation (ERAD). Perturbations in this trafficking can cause abnormal endosomal accumulation of PS1, and then may lead to exacerbated Aß pathology. Cover Image for this issue: doi: 10.1111/jnc.13318.

GM1 ganglioside and Alzheimer's disease.

Assembly and deposition of amyloid ß-protein (Aß) is an invariable and fundamental event in the pathological process of Alzheimer's disease (AD). To decipher the AD pathogenesis and also to develop disease-modifying drugs for AD, clarification of the molecular mechanism underlying the Aß assembly into amyloid fibrils in the brain has been a crucial issue. GM1-ganglioside-bound Aß (GAß), with unique molecular characteristics such as having an altered conformation and the capability to accelerate Aß assembly, was discovered in an autopsied brain showing early pathological changes of AD in 1995. On the basis of these findings, it was hypothesized that GAß is an endogenous seed for amyloid fibril formation in the AD brain. A body of evidence that supports this GAß hypothesis has been growing over this past 20 years. In this article, seminal GAß studies that have been carried out to date, including recent ones using unique animal models, are reviewed.

Influence of APOE genotype and the presence of Alzheimer's pathology on synaptic membrane lipids of human brains.

The APOE genotype is the major risk factor for Alzheimer's disease (AD); however, it remains unclarified how the ε4 allele accelerates whereas the ε2 allele suppresses AD development, compared with the more common ε3 allele. On the basis of the previous finding that the assembly of the amyloid-ß protein (Aß) into fibrils in the brain, an early and invariable pathological feature of AD, depends on the lipid environment, we determined the levels of synaptic membrane lipids in aged individuals of different APOE genotypes. In the comparison between amyloid-free ε2/ε3 and ε3/ε3 brains, the presence of the ε2 allele significantly decreased the level of cholesterol. Alternatively, in the comparison among ε3/ε3 brains, the presence of AD pathology substantially decreased the levels of cholesterol. This study suggests that the ε2 allele suppresses the initiation of AD development by lowering the cholesterol levels in synaptic membranes.

Novel plasma biomarker surrogating cerebral amyloid deposition.

Alzheimer's disease (AD) is the most common and devastating dementia. Simple and practical biomarkers for AD are urgently required for accurate diagnosis and to facilitate the development of disease-modifying interventions. The subjects for the study were selected on the basis of PiB amyloid imaging by PET. Forty PiB-positive (PiB+) individuals, including cognitively healthy controls (HC), and mild cognitive impairment and AD individuals, and 22 PiB-negative (PiB-) HC participated. Employing our novel highly sensitive immunoprecipitation-mass spectrometry, we measured plasma amyloid ß-proteins (Aßs; Aß1-40 and Aß1-42) and Aß-approximate peptides (AßAPs), which were cleaved from amyloid precursor protein (APP). Among the AßAPs, APP669-711 appeared to be a good reference for deciphering pathological change of Aß1-42. We evaluated the performance of the ratio of APP669-711 to Aß1-42 (APP669-711/Aß1-42) as a biomarker. APP669-711/Aß1-42 significantly increased in the PiB+ groups. The sensitivity and specificity to discriminate PiB+ individuals from PiB- individuals were 0.925 and 0.955, respectively. Our plasma biomarker precisely surrogates cerebral amyloid deposition.

Prevention of amyloid ß fibril deposition on the synaptic membrane in the precuneus by ganglioside nanocluster-targeting inhibitors.

Alzheimer's disease (AD), a progressive neurodegenerative condition, is one of the most common causes of dementia. Senile plaques, a hallmark of AD, are formed by the accumulation of amyloid ß protein (Aß), which starts to aggregate before the onset of the disease. Gangliosides, sialic acid-containing glycosphingolipids, play a key role in the formation of toxic Aß aggregates. In membrane rafts, ganglioside-bound complexes (GAß) act as nuclei for Aß assembly, suggesting that GAß is a promising target for AD therapy. The formation of GAß-induced Aß assemblies has been evaluated using reconstituted planar lipid membranes composed of synaptosomal plasma membrane (SPM) lipids extracted from human and mouse brains. Although the effects of gangliosides on Aß accumulation in the precuneus have been established, effects on Aß fibrils have not been determined. In this study, Aß42 fibrils on reconstituted membranes composed of SPM lipids prepared from the precuneus cortex of human autopsied brains were evaluated by atomic force microscopy. In particular, Aß42 accumulation, as well as the fibril number and size were higher for membranes with precuneus lipids than for membranes with calcarine cortex lipids. In addition, artificial peptide inhibitors targeting Aß-sensitive ganglioside nanoclusters cleared Aß assemblies on synaptic membranes in the brain, providing a novel therapeutic strategy for AD.

Lysosomal dysfunction in a mouse model of Sandhoff disease leads to accumulation of ganglioside-bound amyloid-ß peptide.

Alterations in the lipid composition of endosomal-lysosomal membranes may constitute an early event in Alzheimer's disease (AD) pathogenesis. In this study, we investigated the possibility that GM2 ganglioside accumulation in a mouse model of Sandhoff disease might be associated with the accumulation of intraneuronal and extracellular proteins commonly observed in AD. Our results show intraneuronal accumulation of amyloid-ß peptide (Aß)-like, α-synuclein-like, and phospho-tau-like immunoreactivity in the brains of ß-hexosaminidase knock-out (HEXB KO) mice. Biochemical and immunohistochemical analyses confirmed that at least some of the intraneuronal Aß-like immunoreactivity (iAß-LIR) represents amyloid precursor protein C-terminal fragments (APP-CTFs) and/or Aß. In addition, we observed increased levels of Aß40 and Aß42 peptides in the lipid-associated fraction of HEXB KO mouse brains, and intraneuronal accumulation of ganglioside-bound Aß (GAß) immunoreactivity in a brain region-specific manner. Furthermore, α-synuclein and APP-CTFs and/or Aß were found to accumulate in different regions of the substantia nigra, indicating different mechanisms of accumulation or turnover pathways. Based on the localization of the accumulated iAß-LIR to endosomes, lysosomes, and autophagosomes, we conclude that a significant accumulation of iAß-LIR may be associated with the lysosomal-autophagic turnover of Aß and fragments of APP-containing Aß epitopes. Importantly, intraneuronal GAß immunoreactivity, a proposed prefibrillar aggregate found in AD, was found to accumulate throughout the frontal cortices of postmortem human GM1 gangliosidosis, Sandhoff disease, and Tay-Sachs disease brains. Together, these results establish an association between the accumulation of gangliosides, autophagic vacuoles, and the intraneuronal accumulation of proteins associated with AD.

Density of GM1 in nanoclusters is a critical factor in the formation of a spherical assembly of amyloid ß-protein on synaptic plasma membranes.

The deposition of amyloid ß-protein (Aß) is a pathological hallmark of Alzheimer's disease (AD). We previously found that the ganglioside-enriched microdomains (ganglioside clusters) in presynaptic neuronal membranes play a key role in the initiation of the Aß assembly process. However, not all ganglioside clusters accelerate Aß assembly. In the present study, we directly observed a spherical Aß in an atomic force microscopic study on the morphology of a reconstituted lipid bilayer composed of lipids that were extracted from a detergent-resistant membrane microdomain (DRM) fraction of synaptosomes prepared from aged mouse brain. The Aß assembly was generated on a distinctive GM1 domain, which was characterized as the Aß-sensitive ganglioside nanocluster (ASIGN). By using an artificial GM1 cluster-binding peptide, ASIGN was found to have a high density of GM1; therefore, there would be a critical density of GM1 in nanoclusters to induce Aß binding and assembly. These results suggest that ganglioside-bound Aß (GAß), which acts as an endogenous seed for Aß fibril formation in AD brains, is generated on ASIGN on synaptosomal membranes.

The Double-Layered Structure of Amyloid-ß Assemblage on GM1-Containing Membranes Catalytically Promotes Fibrillization.

Alzheimer's disease (AD) is associated with progressive accumulation of amyloid-ß (Aß) cross-ß fibrils in the brain. Aß species tightly associated with GM1 ganglioside, a glycosphingolipid abundant in neuronal membranes, promote amyloid fibril formation; therefore, they could be attractive clinical targets. However, the active conformational state of Aß in GM1-containing lipid membranes is still unknown. The present solid-state nuclear magnetic resonance study revealed a nonfibrillar Aß assemblage characterized by a double-layered antiparallel ß-structure specifically formed on GM1 ganglioside clusters. Our data show that this unique assemblage was not transformed into fibrils on GM1-containing membranes but could promote conversion of monomeric Aß into fibrils, suggesting that a solvent-exposed hydrophobic layer provides a catalytic surface evoking Aß fibril formation. Our findings offer structural clues for designing drugs targeting catalytically active Aß conformational species for the development of anti-AD therapeutics.

[Searching for biomarkers to diagnose dementia].

A decrease in the concentration of amyloid beta-protein-42 and an increase in that of tau or phosphorylated tau in addition to volumetry on MRI and amyloid imaging by PET are available biomarkers. However, we need better biomarkers to detect very early stage of Alzheimer disease to develop disease modifying drugs, which should be used 10 or 15 years prior to emergence of clinical symptoms. In terms of biomarkers for other dementing neurodegenerative diseases, including synucleinopathies such as dementia with Lewy bodies and multiple system atrophy, and tauopathies such as progressive supranuclear palsy, corticobasal degeneration, synuclein, TDP-43, progranulin and tau may be candidate proteins for possible biomarkers for these diseases.

[Strategy and perspectives for development of Alzheimer disease-modifying drugs].

Although there has been a much progress in the clarification of the pathophysiology of Alzheimer disease (AD) over the last two decades, bona fide drugs that suppress the emergence and progression of the disease are not available yet. Fortunately, in addition to donepezil, two other cholinesterase inhibitors, namely galantamine and rivastigmine, and an NMDA receptor antagonist, memantine, have recently been approved for the treatment of AD patients at mild to moderately severe and moderately severe to severe stages of the disease, respectively, in Japan. These drugs potentially improve symptoms of AD; however, their disease-modifying effects are limited. Thus, AD is still the largest unmet medical need in neurology. To develop disease-modifying drugs for AD, the molecular mechanisms underlying AD must be clarified. On the basis of the findings that amyloid pathology proceeds 10 to 15 years prior to the emergence of clinical symptoms, disease-modifying drugs, particularly anti-amyloid drugs should be administered much earlier than that in the case of current clinical trials. Attention should also be paid to AD-associated pathologic proteins other than amyloid beta-protein, e.g., tau, to control the progression of AD, because evidence is accumulating that tau is involved in the amyloid-dependent and-independent pathophysiology of AD.

Abeta polymerization through interaction with membrane gangliosides.

Clarification of the molecular and cellular mechanisms underlying the assembly of amyloid beta-protein (Abeta) into insoluble fibrils in the brain has been one of the biggest challenges in the research on Alzheimer disease (AD). We previously identified a novel Abeta species, which was characterized by its tight binding to GM1 ganglioside (GM1), in the brain showing early pathological changes of AD. The ganglioside-bound Abeta (GAbeta) possessed unique characteristics, including its altered immunoreactivity, which suggests its distinct conformation from native Abeta, and its strong potency to accelerate Abeta assembly into fibrils. On the basis of these characteristics, it was hypothesized that Abeta adopts an altered conformation following interaction with GM1, leading to the generation of GAbeta, and then GAbeta acts as an endogenous seed for Alzheimer amyloid in the brain. To date, various in vitro and in vivo studies on GAbeta have revealed how Abeta binds to gangliosides, i.e., what are the favorable physicochemical and neurobiological conditions for generating GAbeta, and what is the pathological significance of ganglioside-induced Abeta assembly in the development of AD. Interestingly, GAbeta is favorably generated in the unique ganglioside-enriched (clustered), raft-like microdomains; moreover, amyloid fibrils formed in the presence of gangliosides are neurotoxic. Furthermore, the conformational change of Abeta in the presence of ganglioside has been characterized by an NMR study. In this review, we focus on the recent progress of GAbeta studies and highlight the possibility that ganglioside binding is the initial and common step in the development of a part of human misfolding-type amyloidoses, including AD.

Surface-induced phase separation of a sphingomyelin/cholesterol/ganglioside GM1-planar bilayer on mica surfaces and microdomain molecular conformation that accelerates Abeta oligomerization.

Ganglioside GM1 mediates the amyloid beta (Abeta) aggregation that is the hallmark of Alzheimer's disease (AD). To investigate how ganglioside-containing lipid bilayers interact with Abeta, we examined the interaction between Abeta40 and supported planar lipid bilayers (SPBs) on mica and SiO(2) substrates by using atomic force microscopy, fluorescence microscopy, and molecular dynamics computer simulations. These SPBs contained several compositions of sphingomyelin, cholesterol, and GM1 and were treated at physiological salt concentrations. Surprisingly high-speed Abeta aggregation of fibril formations occurred at all GM1 concentrations examined on the mica surface, but on the SiO(2) surface, only globular agglomerates formed and they formed slowly. At a GM1 concentration of 20mol%, unique triangular regions formed on the mica surface and the rapidly formed Abeta aggregations were observed only outside these regions. We have found that some unique surface-induced phase separations are induced by the GM1 clustering effects and the strong interactions between the GM1 head group and the water layer adsorbed in the ditrigonal cavities on the mica surface. The speed of Abeta40 aggregation and the shape of the agglomerates depend on the molecular conformation of GM1, which varies depending on the substrate materials. We identified the conformation that significantly accelerates Abeta40 aggregation, and we think that the detailed knowledge about the GM1 molecular conformation obtained in this work will be useful to those investigating Abeta-GM1 interactions.

Pathological significance of ganglioside clusters in Alzheimer's disease.

One of the key questions regarding the pathogenesis of Alzheimer's disease (AD) is how amyloid ß-protein (Aß), a proteinaceous component of senile plaques, starts to assemble into amyloid fibrils in the brain. A body of evidence is growing to suggest that Aß binds to ganglioside on neuronal membranes, and then, is converted to an endogenous seed with an altered conformation (ganglioside-bound Aß, GAß) for amyloid fibril formation in the brain. Notably, the risk factors for the development of AD, including aging and apolipoprotein E4, likely facilitate the formation of ganglioside clusters in lipid raft-like membrane microdomains at pre-synaptic terminals, which provide a favorable milieu for the GAß generation. Furthermore, it has also been suggested that endocytic pathway abnormality of neurons is involved in the formation of the ganglioside clusters. In this review, the nature of the ganglioside clusters and how gangliosides behave in the clusters leading to the GAß generation are discussed.

Depletion of vitamin E increases amyloid beta accumulation by decreasing its clearances from brain and blood in a mouse model of Alzheimer disease.

Increased oxidative damage is a prominent and early feature in Alzheimer disease. We previously crossed Alzheimer disease transgenic (APPsw) model mice with alpha-tocopherol transfer protein knock-out (Ttpa(-/-)) mice in which lipid peroxidation in the brain was significantly increased. The resulting double-mutant (Ttpa(-/-)APPsw) mice showed increased amyloid beta (Abeta) deposits in the brain, which was ameliorated with alpha-tocopherol supplementation. To investigate the mechanism of the increased Abeta accumulation, we here studied generation, degradation, aggregation, and efflux of Abeta in the mice. The clearance of intracerebral-microinjected (125)I-Abeta(1-40) from brain was decreased in Ttpa(-/-) mice to be compared with wild-type mice, whereas the generation of Abeta was not increased in Ttpa(-/-)APPsw mice. The activity of an Abeta-degrading enzyme, neprilysin, did not decrease, but the expression level of insulin-degrading enzyme was markedly decreased in Ttpa(-/-) mouse brain. In contrast, Abeta aggregation was accelerated in Ttpa(-/-) mouse brains compared with wild-type brains, and well known molecules involved in Abeta transport from brain to blood, low density lipoprotein receptor-related protein-1 (LRP-1) and p-glycoprotein, were up-regulated in the small vascular fraction of Ttpa(-/-) mouse brains. Moreover, the disappearance of intravenously administered (125)I-Abeta(1-40) was decreased in Ttpa(-/-) mice with reduced translocation of LRP-1 in the hepatocytes. These results suggest that lipid peroxidation due to depletion of alpha-tocopherol impairs Abeta clearances from the brain and from the blood, possibly causing increased Abeta accumulation in Ttpa(-/-)APPsw mouse brain and plasma.

Plasma Amyloid-ß Biomarker Associated with Cognitive Decline in Preclinical Alzheimer's Disease.

BACKGROUND: Using immunoprecipitation-mass spectrometry, we recently developed and validated a plasma composite biomarker for the assessment of amyloid-ß (Aß) levels. However, as yet, its relationship with clinical outcomes remains unclear. OBJECTIVE: We aimed to examine the relationship between this plasma Aß composite biomarker and cognitive function in cognitively normal older adults in two independent cohorts. METHODS: Participants enrolled in the Australian Imaging, Biomarkers and Lifestyle (AIBL) study and the National Centre for Geriatrics and Gerontology (NCGG) study had undergone Aß neuroimaging using positron emission tomography (PET), cognitive assessments and provided blood samples. We derived a high-performance plasma Aß composite biomarker by immunoprecipitation with mass-spectrometry. RESULTS: Both continuous and categorical measures of the plasma Aß composite biomarker were significantly related to decline in episodic memory and executive function. The magnitude of effects of the plasma Aß composite on episodic memory and executive function were comparable to that observed for the effects of PET Aß levels on these same outcome measures. CONCLUSION: Several plasma Aß biomarkers have been developed, but none have yet been applied to investigate their relationship with cognitive outcomes. Our results have important implications for the use of this biomarker in the detection of at-risk individuals.

Age-dependent high-density clustering of GM1 ganglioside at presynaptic neuritic terminals promotes amyloid beta-protein fibrillogenesis.

The deposition of amyloid beta-protein (Abeta) is an invariable feature of Alzheimer's disease (AD); however, the biological mechanism underlying Abeta assembly into fibrils in the brain remains unclear. Here, we show that a high-density cluster of GM1 ganglioside (GM1), which was detected by the specific binding of a novel peptide (p3), appeared selectively on synaptosomes prepared from aged mouse brains. Notably, the synaptosomes bearing the high-density GM1 cluster showed extraordinary potency to induce Abeta assembly, which was suppressed by an antibody specific to GM1-bound Abeta, an endogenous seed for AD amyloid. Together with evidence that Abeta deposition starts at presynaptic terminals in the AD brain and that GM1 levels significantly increase in amyloid-positive synaptosomes prepared from the AD brain, our results suggest that the age-dependent high-density GM1 clustering at presynaptic neuritic terminals is a critical step for Abeta deposition in AD.

Late endocytic dysfunction as a putative cause of amyloid fibril formation in Alzheimer's disease.

The assembly of amyloid beta-protein to amyloid fibrils is a critical event in Alzheimer's disease. Evidence exists that endocytic pathway abnormalities, including the enlargement of early endosomes, precede the extraneuronal amyloid fibril deposition in the brain. We determined whether endocytic dysfunction potently promotes the assembly of amyloid beta-protein on the surface of cultured cells. Blocking the early endocytic pathway by clathrin suppression, inactivation of small GTPases, removal of membrane cholesterol, and Rab5 knockdown did not result in amyloid fibril formation on the cell surface from exogenously added soluble amyloid beta-protein. In contrast, blocking the late endocytic pathway by Rab7 suppression markedly induced the amyloid fibril formation in addition to the enlargement of early endosomes. Notably, a monoclonal antibody specific to GM1-ganglioside-bound amyloid beta-protein, an endogenous seed for Alzheimer amyloid, completely blocks the amyloid fibril formation. Our results suggest that late but not early endocytic dysfunction contributes to the amyloid fibril formation by facilitating the generation of amyloid seed in the Alzheimer's brain.