Assembly of hereditary amyloid beta-protein variants in the presence of favorable gangliosides

The mechanisms underlying regional amyloid beta-protein (Abeta) deposition in brain remain unclear. Here we show that assembly of hereditary variant Dutch- and Italian-type Abetas, and Flemish-type Abeta was accelerated by GM3 ganglioside, and GD3 ganglioside, respectively. Notably, cerebrovascular smooth muscle cells, which compose the cerebral vessel wall at which the Dutch- and Italian-type Abetas deposit, exclusively express GM3 whereas GD3 is upregulated in the co-culture of endothelial cells and astrocytes, which forms the cerebrovascular basement membrane, the site of Flemish-type Abeta deposition. Our results suggest that regional Abeta deposition is induced by the local gangliosides in the brain.     

Environment- and mutation-dependent aggregation behavior of Alzheimer amyloid beta-protein

The deposition of amyloid beta-protein in the brain is a fundamental process in the development of Alzheimerís disease; however, the mechanism underlying aggregation of amyloid beta-protein remains to be determined. Here, we report that a membrane-mimicking environment, generated in the presence of detergents or a ganglioside, is sufficient per se for amyloid fibril formation from soluble amyloid beta-protein. Furthermore, hereditary variants of amyloid beta-protein, which are caused by amyloid precursor protein gene mutations, including the Dutch (E693Q), Flemish (A692G) and Arctic (E693G) types, show mutually different aggregation behavior in these environments. Notably, the Arctic-type amyloid beta-protein, in contrast to the wild-type and other variant forms, shows a markedly rapid and higher level of amyloid fibril formation in the presence of sodium dodecyl sulfate or GM1 ganglioside. These results suggest that there are favorable local environments for fibrillogenesis of amyloid beta-protein.     

GM1-ganglioside-induced Abeta assembly on synaptic membranes of cultured neurons

The cell-surface expression of GM1 ganglioside was studied using various cultured cells, including brain-derived endothelial cells, astrocytes, neuroblastoma cells (SH-SY5Y), and pheochromocytoma cells (PC12). GM1 ganglioside was detected only on the surface of native and nerve-growth-factor (NGF)-treated PC12 cells. We investigated whether GM1 ganglioside on the surface of these cells is sufficiently potent to induce the assembly of an exogenous soluble amyloid beta-protein (Abeta). A marked Abeta assembly was observed in the culture of NGF-treated PC12 cells. Notably, immunocytochemical study revealed that, despite the ubiquitous surface expression of GM1 ganglioside throughout cell bodies and neurites, Abeta assembly initially occurred at the terminals of SNAP25-immunopositive neurites. Abeta assembly in the culture was completely suppressed by the coincubation of Abeta with the subunit B of cholera toxin, a natural ligand for GM1 ganglioside, or 4396C, a monoclonal antibody specific to GM1-ganglioside-bound Abeta (GAbeta). In primary neuronal cultures, Abeta assembly initially occurred at synaptophysin-positive sites. These results suggest that the cell-surface expression of GM1 ganglioside is strictly cell-type-specific, and that expression of GM1 ganglioside on synaptic membranes is unique in terms of its high potency to induce Abeta assembly through the generation of GAbeta, which is an endogenous seed for Abeta assembly in Alzheimer brain.     

Role of gangliosides in Alzheimer's disease

One of the fundamental questions regarding the pathogenesis of Alzheimer's disease (AD) is how the monomeric, nontoxic amyloid beta-protein (Abeta) is converted to its toxic assemblies in the brain. A unique Abeta species was identified previously in an AD brain, which is characterized by its binding to the GM1 ganglioside (GM1). On the basis of the molecular characteristics of this GM1-bound Abeta (GAbeta), it was hypothesized that Abeta adopts an altered conformation through its binding to GM1, and GAbeta acts as a seed for Abeta fibrillogenesis in an AD brain. To date, various in vitro and in vivo studies of GAbeta have been performed, and their results support the hypothesis. Using a novel monoclonal antibody specific to GAbeta, it was confirmed that GAbeta is endogenously generated in the brain. Regarding the role of gangliosides in the facilitation of Abeta assembly, it has recently been reported that region-specific deposition of hereditary variant-type Abetas is determined by local gangliosides in the brain. Furthermore, it is likely that risk factors for AD, including aging and the expression of apolipoprotein E4, alter GM1 distribution on the neuronal surface, leading to GAbeta generation.     

GM1-ganglioside-induced Aβ assembly on synaptic membranes of cultured neurons

The cell-surface expression of GM1 ganglioside was studied using various cultured cells, including brain-derived endothelial cells, astrocytes, neuroblastoma cells (SH-SY5Y), and pheochromocytoma cells (PC12). GM1 ganglioside was detected only on the surface of native and nerve-growth-factor (NGF)-treated PC12 cells. We investigated whether GM1 ganglioside on the surface of these cells is sufficiently potent to induce the assembly of an exogenous soluble amyloid β-protein (Aβ). A marked Aβ assembly was observed in the culture of NGF-treated PC12 cells. Notably, immunocytochemical study revealed that, despite the ubiquitous surface expression of GM1 ganglioside throughout cell bodies and neurites, Aβ assembly initially occurred at the terminals of SNAP25-immunopositive neurites. Aβ assembly in the culture was completely suppressed by the coincubation of Aβ with the subunit B of cholera toxin, a natural ligand for GM1 ganglioside, or 4396C, a monoclonal antibody specific to GM1-ganglioside-bound Aβ (GAβ). In primary neuronal cultures, Aβ assembly initially occurred at synaptophysin-positive sites. These results suggest that the cell-surface expression of GM1 ganglioside is strictly cell-type-specific, and that expression of GM1 ganglioside on synaptic membranes is unique in terms of its high potency to induce Aβ assembly through the generation of GAβ, which is an endogenous seed for Aβ assembly in Alzheimer brain.     

Suppression of Abeta deposition in brain by peripheral administration of Fab fragments of anti-seed antibody

Assembly and deposition of amyloid beta-protein (Abeta) in the brain is a fundamental process of Alzheimer's disease (AD). We previously hypothesized that GM1 ganglioside-bound Abeta (GAbeta) is an endogenous seed for Abeta assembly in brain. Recently, we have succeeded in generation of a monoclonal antibody specific to GAbeta. Notably, this antibody, 4396C, per se substantially inhibits Abeta assembly in vitro. Here we report that the peripheral administration of Fab fragments of 4396C into transgenic mice expressing a mutant amyloid precursor protein gene, following the conjugation of the protein transduction domain of the Tat protein, markedly suppressed Abeta deposition in the brain. This result further supports our previous hypothesis and also provides a new insight into develop AD therapy through targeting seed Abeta in the brain, which selectively inhibits the initial step of the pathological process of AD.     

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.     

Synthesis,aggregation, neurotoxicity,and secondary structure of various A beta 1-42 mutants of familial Alzheimer's disease at positions 21-23

Cerebral amyloid angiopathy (CAA) due to amyloid beta (A beta) deposition is a key pathological feature of Alzheimer's disease (AD), especially in some form of familial Alzheimer's disease (FAD) including hereditary cerebral hemorrhage with amyloidosis-Dutch type. A beta mainly consists of 40- and 42-mer peptides (Abeta 1-40 and A beta 1-42), which accumulate in senile plaques of AD brains and show neurotoxicity for cultured nerve cells. We synthesized all variant forms of A beta 1-42 associated with reported FAD, such as A21G (Flemish), E22Q (Dutch), E22K (Italian), E22G (Arctic), and D23N (Iowa) along with three potential mutants by one point missense mutation (E22A, E22D, and E22V) in a highly pure form, and examined their ability to aggregate and their neurotoxicity in PC12 cells. The mutants at positions 22 and 23 showed potent aggregative ability and neurotoxicity whereas the potential mutants did not, indicating that A beta 1-42 mutants at positions 22 and 23 play a critical role in FAD of Dutch-, Italian-, Arctic-, and Iowa-types. However, Flemish-type FAD needs alternative explanation except the aggregation and neurotoxicity of the corresponding A beta 1-42 mutant.     

Accelerated accumulation of amyloid beta proteins on oxidatively damaged lipid membranes

The fully developed lesion of Alzheimer's Disease is a dense plaque composed of fibrillar amyloid beta-proteins with a characteristic and well-ordered beta-sheet secondary structure. Because the incipient lesion most likely develops when these proteins are first induced to form beta-sheet secondary structure, it is important to understand factors that induce amyloid beta-proteins to adopt this conformation. In this investigation we used a novel form of infrared spectroscopy that can characterize the conformation, orientation, and rate of accumulation of the protein on various lipid membranes to determine whether oxidatively damaged phospholipid membranes induce the formation of beta-sheet secondary structure in a 42-residue amyloid beta-protein. We found that membranes containing oxidatively damaged phospholipids accumulated amyloid beta-protein significantly faster than membranes containing only unoxidized or saturated phospholipids. Accelerated accumulation was also seen when 3 mol % G(M1) ganglioside was incorporated into a saturated phosphatidylcholine membrane. The accumulated protein more completely adopted a beta-sheet conformation on oxidized membranes, and the plane of the beta-sheet was oriented parallel to the plane of the membrane. These results indicate that oxidatively damaged phospholipid membranes promote beta-sheet formation by amyloid beta-proteins, and they suggest a possible role for lipid peroxidation in the pathogenesis of Alzheimer's Disease.     

Role of gangliosides in Alzheimer’s disease

One of the fundamental questions regarding the pathogenesis of Alzheimer’s disease (AD) is how the monomeric, nontoxic amyloid β-protein (Aβ) is converted to its toxic assemblies in the brain. A unique Aβ species was identified previously in an AD brain, which is characterized by its binding to the GM1 ganglioside (GM1). On the basis of the molecular characteristics of this GM1-bound Aβ (GAβ), it was hypothesized that Aβ adopts an altered conformation through its binding to GM1, and GAβ acts as a seed for Aβ fibrillogenesis in an AD brain. To date, various in vitro and in vivo studies of GAβ have been performed, and their results support the hypothesis. Using a novel monoclonal antibody specific to GAβ, it was confirmed that GAβ is endogenously generated in the brain. Regarding the role of gangliosides in the facilitation of Aβ assembly, it has recently been reported that region-specific deposition of hereditary variant-type Aβs is determined by local gangliosides in the brain. Furthermore, it is likely that risk factors for AD, including aging and the expression of apolipoprotein E4, alter GM1 distribution on the neuronal surface, leading to GAβ generation.     

Accelerated release of exosome-associated GM1 ganglioside (GM1) by endocytic pathway abnormality: another putative pathway for GM1-induced amyloid fibril formation

Exosomes are extracellularly released small vesicles that are derived from multivesicular bodies formed via the endocytic pathway. We treated pheochromocytoma PC12 cells with chloroquine, an acidotropic agent, which potently perturbs membrane trafficking from endosomes to lysosomes. Chloroquine treatment increased the level of GM1 ganglioside in cell media only when the cells were exposed to KCl for depolarization, which is known to enhance exosome release from neurons. In the sucrose-density-gradient fractionation of cell media, GM1 ganglioside was exclusively recovered with Alix, a specific marker of exosomes, in the fractions with the density corrresponding to that of exosomes. Notably, amyloid-β assembly was markedly accelerated when incubated with the exosome fraction prepared from the culture media of PC12 cells treated with chloroquine and KCl. Furthermore, amyloid-β assembly was significantly suppressed by the co-incubation with an antibody specific to GM1-bound amyloid-β, an endogenous seed for amyloid formation of Alzheimer's disease. Together with our previous finding that chloroquine treatment induces the accumulation of GM1 ganglioside in early endosomes, results of this study suggest that endocytic pathway abnormality accelerates the release of exosome-associated GM1 ganglioside following its accumulation in early endosomes. Furthermore, this study also suggests that extracellular amyloid fibril formation is induced by not only GM1 gangliosides accumulated on the surface of the cells but also those released in association with exosomes.     

Accelerated Abeta aggregation in the presence of GM1-ganglioside-accumulated synaptosomes of aged apoE4-knock-in mouse brain

Aging and apolipoprotein E4 (apoE4) expression are strong risk factors for the development of Alzheimer's disease (AD); however, their pathological roles remain to be clarified. In the process of AD development, the conversion of the nontoxic amyloid beta-protein (Abeta) monomer to its toxic aggregates is a fundamental process. We previously hypothesized that Abeta aggregation is accelerated through the generation of GM1 ganglioside (GM1)-bound Abeta which acts as a seed for Abeta fibril formation. Here we report that GM1 level in detergent-resistant membrane microdomains (DRMs) of synaptosomes increased with age and that this increase was significantly pronounced in the apoE4- than the apoE3-knock-in mouse brain. Furthermore, we show that Abeta aggregation is markedly accelerated in the presence of the synaptosomes of the aged apoE4-knock-in mouse brain. These observations suggest that aging and apoE4 expression cooperatively accelerate Abeta aggregation in the brain through an increase in the level of GM1 in neuronal membranes.     

Interactions of amyloid beta-protein with various gangliosides in raft-like membranes: importance of GM1 ganglioside-bound form as an endogenous seed for Alzheimer amyloid

GM1 ganglioside-bound amyloid beta-protein (GM1-Abeta), found in brains exhibiting early pathological changes of Alzheimer's disease (AD) plaques, has been suggested to accelerate amyloid fibril formation by acting as a seed. We have previously found using dye-labeled Abeta that Abeta recognizes a GM1 cluster, the formation of which is facilitated by cholesterol [Kakio, A., Nishimoto, S., Yanagisawa, K., Kozutsumi, Y., and Matsuzaki, K. (2001) J. Biol. Chem. 276, 24985-24990]. In this study, we investigated the ganglioside species-specificity in its potency to induce a conformational change of Abeta, by which ganglioside-bound Abeta acts as a seed for Abeta fibrillogenesis, using a major ganglioside occurring in brains (GM1, GD1a, GD1b, and GT1b) in raft-like membranes composed of cholesterol and sphingomyelin. Abeta recognized ganglioside clusters, the density of which increased with the number of sialic acid residues. Interestingly, however, mixing of gangliosides inhibited cluster formation. In contrast, the affinities of the protein for the clusters were similar irrespective of lipid composition and of the order of 10(6) M(-)(1) at 37 degrees C. Abeta underwent a conformational transition from an alpha-helix-rich structure to a beta-sheet-rich structure with the increase in protein density on the membrane. Ganglioside-bound Abeta proteins exhibited seeding abilities for amyloid formation. GM1-Abeta exhibited the strongest seeding potential, especially under beta-sheet-forming conditions. This study suggested that lipid composition including gangliosides and cholesterol strictly controls amyloid formation.     

Brain insulin resistance accelerates Aβ fibrillogenesis by inducing GM1 ganglioside clustering in the presynaptic membranes

Type 2 diabetes mellitus is thought to be a significant risk factor for Alzheimer's disease. Insulin resistance also affects the central nervous system by regulating key processes, such as neuronal survival and longevity, learning and memory. However, the mechanisms underlying these effects remain uncertain. To investigate whether insulin resistance is associated with the assembly of amyloid β-protein (Aβ) at the cell surface of neurons, we inhibited insulin-signalling pathways of primary neurons. The treatments of insulin receptor (IR)-knockdown and a phosphatidylinositol 3-kinase inhibitor (LY294002), but not an extracellular signal-regulated kinase inhibitor, induced an increase in GM1 ganglioside (GM1) levels in detergent-resistant membrane microdomains of the neurons. The aged db/db mouse brain exhibited reduction in IR expression and phosphorylation of Akt, which later induced an increase in the high-density GM1-clusters on synaptosomes. Neurons treated with IR knockdown or LY294002, and synaptosomes of the aged db/db mouse brains markedly accelerated an assembly of Aβs. These results suggest that ageing and peripheral insulin resistance induce brain insulin resistance, which accelerates the assembly of Aβs by increasing and clustering of GM1 in detergent-resistant membrane microdomains of neuronal membranes.     

The corneous layer of the claw in the lizard Anolis carolinensis mainly contains the glycine–cysteine-rich beta-protein HgGC3 in addition to hard keratins

The localization of specific claw beta-proteins among the 40 total corneous beta-proteins present in the lizard Anolis carolinensis is not known. The hardness of claws likely depends on glycine–cysteine-rich beta-proteins content, as suggested by previous immunoblot studies. Previous studies have indicated that glycine–cysteine-rich corneous beta-proteins in addition to cysteine-rich alpha-keratins are present in the claw. In order to detect at the ultrastructural level the presence of claw-specific corneous proteins immunofluorescence and electron microscopy immunogold have been utilized. More intense immunoreactivity is obtained for the HgGC3 beta-protein while less intense immunolabeling is seen for HgGC10 and HgG5 beta-proteins and no labeling for the cysteine-rich beta-protein HgC1. The HgGC3 beta-protein appears the prevalent type present in the claw and its numerous cysteines likely form intermolecular disulphide bonds while glycine contributes hydrophobic properties to the corneous material. Other antibodies tagging the core-box and pre-core box regions of beta-proteins label with less intensity the corneous layer. The presence of cysteine-rich alpha-keratins with high homology to some human hair keratins in the dorsal part of the claw suggests that HgGC3-like beta-proteins form numerous disulphide bonds with the larger alpha-keratins giving rise to the hard corneous material of the claw.     

Role of ganglioside metabolism in the pathogenesis of Alzheimer's disease--a review

Gangliosides are expressed in the outer leaflet of the plasma membrane of the cells of all vertebrates and are particularly abundant in the nervous system. Ganglioside metabolism is closely associated with the pathology of Alzheimer's disease (AD). AD, the most common form of dementia, is a progressive degenerative disease of the brain characterized clinically by progressive loss of memory and cognitive function and eventually death. Neuropathologically, AD is characterized by amyloid deposits or "senile plaques," which consist mainly of aggregated variants of amyloid beta-protein (Abeta). Abeta undergoes a conformational transition from random coil to ordered structure rich in beta-sheets, especially after addition of lipid vesicles containing GM1 ganglioside. In AD brain, a complex of GM1 and Abeta, termed "GAbeta," has been found to accumulate. In recent years, Abeta and GM1 have been identified in microdomains or lipid rafts. The functional roles of these microdomains in cellular processes are now beginning to unfold. Several articles also have documented the involvement of these microdomains in the pathogenesis of certain neurodegenerative diseases, such as AD. A pivotal neuroprotective role of gangliosides has been reported in in vivo and in vitro models of neuronal injury, Parkinsonism, and related diseases. Here we describe the possible involvement of gangliosides in the development of AD and the therapeutic potentials of gangliosides in this disorder.     

A ganglioside-induced toxic soluble Abeta assembly. Its enhanced formation from Abeta bearing the Arctic mutation

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.     

Formation of a membrane-active form of amyloid beta-protein in raft-like model membranes

The conversion of soluble, nontoxic amyloid beta-protein (A beta) to aggregated, toxic A beta rich in beta-sheet structures is considered to be the key step in the development of Alzheimer's disease. We have proposed that the aggregation proceeds in the lipid raft containing a ganglioside cluster, the formation of which is facilitated by cholesterol and for which A beta shows a specific affinity. In this study, using fluorescence resonance energy transfer, we found that after A beta binds to raft-like membranes composed of monosialoganglioside GM1/cholesterol/sphingomyelin (1/1/1), the protein can translocate to the phosphatidylcholine membranes to which soluble A beta does not bind. Furthermore, self-quenching experiments using fluorescein-labeled A beta revealed that the translocation process competes with the oligomerization of the protein in the raft-like membranes. These results suggest that the lipid raft containing a ganglioside cluster serves as a conformational catalyst or a chaperon generating a membrane-active form of A beta with seeding ability.     

Aβ polymerization through interaction with membrane gangliosides

Clarification of the molecular and cellular mechanisms underlying the assembly of amyloid β-protein (Aβ) 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 Aβ species, which was characterized by its tight binding to GM1 ganglioside (GM1), in the brain showing early pathological changes of AD. The ganglioside-bound Aβ (GAβ) possessed unique characteristics, including its altered immunoreactivity, which suggests its distinct conformation from native Aβ, and its strong potency to accelerate Aβ assembly into fibrils. On the basis of these characteristics, it was hypothesized that Aβ adopts an altered conformation following interaction with GM1, leading to the generation of GAβ, and then GAβ acts as an endogenous seed for Alzheimer amyloid in the brain. To date, various in vitro and in vivo studies on GAβ have revealed how Aβ binds to gangliosides, i.e., what are the favorable physicochemical and neurobiological conditions for generating GAβ, and what is the pathological significance of ganglioside-induced Aβ assembly in the development of AD. Interestingly, GAβ 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 Aβ in the presence of ganglioside has been characterized by an NMR study. In this review, we focus on the recent progress of GAβ 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.     

Chloroquine-induced endocytic pathway abnormalities: Cellular model of GM1 ganglioside-induced Abeta fibrillogenesis in Alzheimer's disease

Endocytic pathway abnormalities were previously observed in brains affected with Alzheimer’s disease (AD). To clarify the pathological relevance of these abnormalities to assembly of amyloid β-protein (Aβ), we treated PC12 cells with chloroquine, which potently perturbs membrane trafficking from endosomes to lysosomes. Chloroquine treatment induced accumulation of GM1 ganglioside (GM1) in Rab5-positive enlarged early endosomes and on the cell surface. Notably, an increase in GM1 level on the cell surface was sufficient to induce Aβ assembly. Our results suggest that endocytic pathway abnormalities in AD brain induce GM1 accumulation on the cell surface, leading to amyloid fibril formation in brain.