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.

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.

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.

Induction of ganglioside synthesis in Drosophila brain accelerates assembly of amyloid ß protein.

The assembly and deposition of amyloid ß protein (Aß) is a fundamental event during the early stages of Alzheimer's disease (AD) and cerebral amyloid angiopathy. A growing body of evidence indicates that gangliosides form a pathological platform for the generation of ganglioside-bound Aß, which facilitates the assembly of soluble Aßs; however, the molecular mechanisms underlying the binding of Aß to gangliosides in the brain remain unclear due to the lack of an in vivo system that may address this issue. In insects, including the fruit fly Drosophila melanogaster, gangliosides are not intrinsically present at a detectable level. We herein demonstrate that ganglioside expression is inducible in Drosophila via the expression of transgenes of ganglioside synthesis enzymes and the feeding of exogenous sialic acid, and also that the induction of ganglioside synthesis significantly accelerates Aß assembly in vivo. Our results support the hypothesis that gangliosides are responsible for Aß assembly in vivo and also provide an opportunity to develop a valuable model for basic research as well as a therapeutic strategy for AD.

Ganglioside-Mediated Assembly of Amyloid ß-Protein: Roles in Alzheimer's Disease.

Assembly and deposition of amyloid ß-protein (Aß) is an early and invariable pathological event of Alzheimer's disease (AD), a chronic neurodegenerative disease affecting the neurons in the brain of aging population. Thus, clarification of the molecular mechanism underlying Aß assembly is crucial not only for understanding the pathogenesis of AD, but also for developing disease-modifying remedies. In 1995, ganglioside-bound Aß (GAß), with unique molecular characteristics, including its altered immunoreactivity and its conspicuous ability to accelerate Aß assembly, was discovered in an autopsied brain showing early pathological changes of AD. Based on 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 the GAß hypothesis has been growing for over 20years as follows. First, the conformational changes of Aß from a random coil to an α-helix, and then to a ß-sheet in the presence of ganglioside were validated by several techniques. Second, the seed activity of GAß to accelerate the assembly of soluble Aß into amyloid fibrils was confirmed by various in vitro and in vivo experiments. Third, it was found that the Aß binding to ganglioside to form GAß occurs under limited conditions, which were provided by the lipid environment surrounding ganglioside. Fourth, the region-specific Aß deposition in the brain appeared to be dependent on the presence of the lipid environment that was in favor of GAß generation. In this chapter, further progress of the study of ganglioside-mediated Aß assembly, especially from the aspects of physicochemistry, structural biology, and neuropathology, is reviewed.

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.

Traffic jam hypothesis: Relationship between endocytic dysfunction and Alzheimer's disease.

Membrane trafficking pathways, like the endocytic pathway, carry out fundamental cellular processes that are essential for normal functioning. One such process is regulation of cell surface receptor signaling. A growing body of evidence suggests that ß-amyloid protein (Aß) plays a key role in Alzheimer's disease (AD) pathogenesis. Cleavage of Aß from its precursor, ß-amyloid precursor protein (APP), occurs through the endocytic pathway in neuronal cells. In early-stage AD, intraneuronal accumulation of abnormally enlarged endosomes is common, indicating that endosome trafficking is disrupted. Strikingly, genome-wide association studies reveal that several endocytosis-related genes are associated with AD onset. Also, recent studies demonstrate that alteration in endocytosis induces not only Aß pathology but also the propagation of tau protein pathology, another key pathological feature of AD. Endocytic dysfunction can disrupt neuronal physiological functions, such as synaptic vesicle transport and neurotransmitter release. Thus, "traffic jams" in the endocytic pathway may be involved in AD pathogenesis and may serve as a novel target for the development of new therapeutics.

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.

Imbalance in fatty-acid-chain length of gangliosides triggers Alzheimer amyloid deposition in the precuneus.

Amyloid deposition, a crucial event of Alzheimer's disease (AD), emerges in distinct brain regions. A key question is what triggers the assembly of the monomeric amyloid ß-protein (Aß) into fibrils in the regions. On the basis of our previous findings that gangliosides facilitate the initiation of Aß assembly at presynaptic neuritic terminals, we investigated how lipids, including gangliosides, cholesterol and sphingomyelin, extracted from synaptic plasma membranes (SPMs) isolated from autopsy brains were involved in the Aß assembly. We focused on two regions of the cerebral cortex; precuneus and calcarine cortex, one of the most vulnerable and one of the most resistant regions to amyloid deposition, respectively. Here, we show that lipids extracted from SPMs isolated from the amyloid-bearing precuneus, but neither the amyloid-free precuneus nor the calcarine cortex, markedly accelerate the Aß assembly in vitro. Through liquid chromatography-mass spectrometry of the lipids, we identified an increase in the ratio of the level of GD1b-ganglioside containing C20:0 fatty acid to that containing C18:0 as a cause of the enhanced Aß assembly in the precuneus. Our results suggest that the local glycolipid environment play a critical role in the initiation of Alzheimer amyloid deposition.

Diabetes mellitus accelerates Aß pathology in brain accompanied by enhanced GAß generation in nonhuman primates.

Growing evidence suggests that diabetes mellitus (DM) is one of the strongest risk factors for developing Alzheimer's disease (AD). However, it remains unclear why DM accelerates AD pathology. In cynomolgus monkeys older than 25 years, senile plaques (SPs) are spontaneously and consistently observed in their brains, and neurofibrillary tangles are present at 32 years of age and older. In laboratory-housed monkeys, obesity is occasionally observed and frequently leads to development of type 2 DM. In the present study, we performed histopathological and biochemical analyses of brain tissue in cynomolgus monkeys with type 2 DM to clarify the relationship between DM and AD pathology. Here, we provide the evidence that DM accelerates Aß pathology in vivo in nonhuman primates who had not undergone any genetic manipulation. In DM-affected monkey brains, SPs were observed in frontal and temporal lobe cortices, even in monkeys younger than 20 years. Biochemical analyses of brain revealed that the amount of GM1-ganglioside-bound Aß (GAß)--the endogenous seed for Aß fibril formation in the brain--was clearly elevated in DM-affected monkeys. Furthermore, the level of Rab GTPases was also significantly increased in the brains of adult monkeys with DM, almost to the same levels as in aged monkeys. Intraneuronal accumulation of enlarged endosomes was also observed in DM-affected monkeys, suggesting that exacerbated endocytic disturbance may underlie the acceleration of Aß pathology due to DM.

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.

Soluble Aß oligomers are rapidly sequestered from brain ISF in vivo and bind GM1 ganglioside on cellular membranes.

Soluble Aß oligomers contribute importantly to synaptotoxicity in Alzheimer's disease, but their dynamics in vivo remain unclear. Here, we found that soluble Aß oligomers were sequestered from brain interstitial fluid onto brain membranes much more rapidly than nontoxic monomers and were recovered in part as bound to GM1 ganglioside on membranes. Aß oligomers bound strongly to GM1 ganglioside, and blocking the sialic acid residue on GM1 decreased oligomer-mediated LTP impairment in mouse hippocampal slices. In a hAPP transgenic mouse model, substantial levels of GM1-bound Aß42 were recovered from brain membrane fractions. We also detected GM1-bound Aß in human CSF, and its levels correlated with Aß42, suggesting its potential as a biomarker of Aß-related membrane dysfunction. Together, these findings highlight a mechanism whereby hydrophobic Aß oligomers become sequestered onto GM1 ganglioside and presumably other lipids on neuronal membranes, where they may induce progressive functional and structural changes.

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.

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 γ-secretase inhibitor DAPT increases the levels of gangliosides at neuritic terminals of differentiating PC12 cells.

Mutations in presenilins are the major cause of early onset familial Alzheimer disease. It has recently been argued that clinical presenilin mutations work as loss-of-function but not toxic gain-of-function. To investigate whether presenilins are involved in the regulation of the distribution of neuronal membrane lipids, we treated neuronally differentiated PC12 cells with DAPT, an inhibitor of presenilin-dependent γ-secretase, and performed lipid analyses of neuritic terminals, which is an initial site of Aß deposition in brains, using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). With DAPT treatment, levels of sphingomyelin, phosphatidylcholine, and cholesterol remained unchanged. However, DAPT treatment increased the ganglioside levels in PC12 neuritic terminals. Together with a previous finding that accumulation of gangliosides at neuritic terminals facilitates Aß assembly and deposition, the present data suggest that the loss-of-function of presenilins, i.e., a decrease in γ-secretase activity, has an impact on neuronal membrane architecture in a way that eventually exacerbates Alzheimer pathology.

[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.

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.