it is widely accepted that the amyloid -protein ( a ) , which   exists in fibrillar forms as a major component of senile plaques , is central to the development of alzheimer 's disease ( ad ) [ 1 , 2 ] . the conversion of soluble , nontoxic a monomer to aggregated toxic a rich in -sheet structures ignites the neurotoxic cascade(s ) of a . the physiological concentration of a in biological fluids ( < 10  m )   is much lower than the concentration ( ~1  m ) above which a-(140 ) spontaneously forms fibrils . therefore , there should be mechanisms that facilitate the abnormal aggregation of a under pathological conditions . although   clusterin ( apo j )   and zn ions   were reported to facilitate the aggregation of a more than a decade ago , their aggregation - promoting mechanisms are yet to be elucidated . in addition to these soluble factors , jarrett and lansbury , jr . suggested that a fibrillizes via a nucleation - dependent polymerization mechanism and lipids could act as heterogeneous seeds for the polymerization .   in 1995 , yanagisawa and colleagues discovered a specific form of a bound to monosialoganglioside gm1 ( gm1 ) in brains exhibiting early pathological changes of ad and suggested that the gm1-bound form of a may serve as a seed for the formation of toxic amyloid aggregates / fibrils . we have been investigating the interaction of a with ganglioside - containing membranes for a dozen years and found that not the uniformly distributed but the clustered gangliosides mediate the formation of amyloid fibrils by a , the toxicity and physicochemical properties of which are different from those of a amyloids formed in solution . this review article summarizes a-ganglioside interaction in detail , including latest findings that were not covered in our previous reviews [ 10 , 11 ] . especially , differences between a-(140 ) and a-(142 ) in membrane interaction and amyloidogenesis are extensively discussed .   furthermore , a link between a aggregation and lipid metabolism is emphasized . early studies indicated that a-(140 ) associates with gm1 in egg yolk phosphatidylcholine ( pc ) vesicles only when the gm1 content exceeds 30% [ 12 , 13 ] . these findings suggest that gm1 molecules only in a specific state can recognize a.   to reveal the underlying mechanism , we systematically investigated the interaction of   dye - labeled - a-(140 ) [ 1416 ] and -a-(142 )   with membranes of various lipid compositions . the n - termini of as were labeled with the 7-diethylaminocoumarin-3-carbonyl group ( dac - a ) . dac - a is useful for fluorometrically monitoring protein - lipid interactions , because a significant blue shift and an enhancement in intensity are induced by a change in polarity upon membrane   binding . dac - as do not bind to major membrane lipids , including electrically neutral pc , sm , cholesterol , negatively charged phosphatidylserine , and phosphatidylglycerol under physiological conditions .   on the other hand , the proteins exhibit similar high affinity ( binding constant ~10  m ) for raft - like membranes composed of gm1 , cholesterol , and sm [ 14 , 15 ] . dac - a-(140 ) also binds to other gangliosides ( gd1a , gd1b , gt1b , and asialo gm1 ) and lactosyl ceramide in raft - like membranes with higher affinity for lipids having larger sugar chains [ 15 , 16 ] . we have proposed that as specifically bind to ganglioside clusters because a gm1 cluster is formed in gm1/sm / cholesterol membranes but not in gm1/pc membranes . a-(140 ) bound to ganglioside clusters assumes different conformations depending on the protein density on the membrane . circular dichroism measurements revealed that the protein forms an -helix - rich structure at lower protein - to - ganglioside ratios ( 0.025 )   whereas it changes its conformation to a -sheet - rich structure at higher ratios ( 0.05 ) [ 14 , 15 ] . only the -sheet form facilitates amyloidogenesis by a-(140 ) [ 15 , 1820 ] .        despite very similar initial protein - ganglioside interaction , that is , the binding behavior and the -helix - to--sheet conformational change , a large difference was observed in amyloidogenic activity ( amount of amyloids formed under certain conditions ) between a-(140 ) and a-(142 ) . as were incubated with gm1/cholesterol / sm liposomes at a a-to - gm1 ratio of 5 , and the aggregation of a was monitored as an increase in fluorescence of the amyloid - specific dye thioflavin - t ( th - t ) ( figure 1 ) . in contrast , a-(140 ) at 5  m did not form amyloids , at least not in 12  h.   at a 10-fold higher concentration , a-(140 ) started to aggregate after a lag time of ~2  h.   the effectiveness of a-(142 ) in fibrillogenesis is at least partly due to the fragility of fibrils , because the fragmentation greatly facilitates fibril growth   ( see also section 5 ) . other factors , such as the rapid formation of seeds and/or elongation , may also contribute to the difference . cell experiments also support the above mechanism of a-ganglioside interaction [ 22 , 23 ] . amyloids and gangliosides were detected by the amyloid - specific dye congo red and the fluorescent - labeled cholera toxin b subunit , respectively . amyloids were selectively formed on ganglioside - rich domains ( figure 2(a ) ) . depletion of cholesterol , either by methyl--cyclodextrin or the cholesterol synthesis inhibitor compactin , suppressed the accumulation of a.   the amyloidogenic activity of a-(142 ) was again more than 10-fold that of a-(140 ) on human sh - sy5y neuroblastoma cells expressing gangliosides ( figure 2(b ) ) . when cells were incubated with 5  m a-(142 ) , congo red - positive spots appeared later at 24  h and became prominent with time . in contrast , when cells were incubated with 5  m a-(140 ) , no fibrils were detected even after 72  h.   incubation with a 10-fold higher concentration of the protein , however , resulted in the appearance of congo red - positive spots at 48  h. the a fibrils formed on ganglioside clusters ( mem - fibrils ) are not identical to those formed in solution ( sol - fibrils ) in terms of physicochemical properties and cytotoxicity . transmission electron micrographs indicate that mem - fibrils are typical nonbranched fibrils ( 12.0  0.7  nm , width )   whereas sol - fibrils are thinner fibrils or protofilaments    ( 6.7  1.3  nm , width ) , and the protofilaments associate laterally and twist into rope - like fibrils ( 14.5  0.9  nm , width ) . the surface hydrophobicity of mem - fibrils as estimated by the binding of 1-anilinonaphthalene-8-sulfonate ( ans ) is larger than that of sol - fibrils ( figure 3(a ) ) , therefore mem - fibrils exhibit significantly stronger binding to cell membranes than sol - fibrils ( figure 3(b ) ) . consequently , mem - fibrils are cytotoxic   whereas sol - fibrils are much less toxic ( figure 3(c ) ) . recently , a correlation between ans - binding and cytotoxicity was reported for various amyloid species . the structure of mem - fibrils is suggested to be different from that of sol - fibrils , in which the cross- unit is a double - layered structure , with in - resister parallel -sheets formed by residues 1224 and 3040 . the amide i spectrum of the former shows , in addition to a major peak around 1630  cm characteristic of a -sheet , a weak peak at 1695  cm   whereas that of the latter shows a peak around 1660  cm . a fibrils were reported to trigger functional disorder in neuronal cells and cell death [ 2731 ]   whereas soluble a oligomers have been proposed to play a pivotal role in the onset of ad [ 6 , 28 , 3239 ] .   to obtain an insight into the cytotoxic mechanism of a , we established a multistaining visualization method using unlabeled as and antibodies   in contrast to conventional methods using fluorophore - labeled proteins [ 23 , 40 ] . the accumulation of a , the formation of amyloid fibrils , the formation of oligomers , and cell viability were visualized using the a monoclonal antibody 6e10 , the amyloid - staining dye congo red , the antioligomer antibody a11 , and calcein acetoxymethyl , respectively .   cell death was detected after the significant accumulation of fibrils ( figure 2(b ) ) and no a11-positive spot was detected , suggesting that fibril - induced physicochemical stress , such as the induction of a negative curvature   or membrane deformation upon fibril growth , leads to cytotoxicity . it should be noted , however , that at certain gm1 contents gm1-liposomes generate toxic soluble a-(140 ) oligomers .   for both as , similar levels of fibrils were required for cytotoxicity ( figure 2(b ) ) , indicating that the fibrils possess comparable intrinsic toxicity . the fibrillization process and cytotoxicity can be effectively blocked by small compounds , such as nordihydroguaiaretic acid and rifampicin . the morphology of amyloid fibrils formed on cell membranes was visualized by total internal reflection fluorescence microscopy ( tirfm ) . tirfm effectively reduces the background fluorescence and therefore is suitable for observing the cell surface . relatively long fibrillar structures were detected around the cell membrane for a-(140 )   whereas relatively short fibrils were coassembled in the case of a-(142 ) ( figure 4 ) . based on the above observations , we propose a novel model for a-membrane interaction as a mechanism for the abnormal aggregation of the protein ( figure 5 ) . a specifically binds to a ganglioside cluster , the formation of which is facilitated by cholesterol . the a undergoes a conformational transition from an -helix - rich structure to a -sheet - rich one with increasing protein density on the membrane . the -sheet form serves as a seed for the formation of amyloid fibrils , which are more toxic than and have different structures from those formed in solution .   depending on ganglioside contents in the membrane , toxic soluble oligomers the amyloidogenic activity of a-(142 ) is more than 10-fold that of a-(140 ) , although the initial interaction with gangliosides is similar between the two proteins . this model can explain roles of various risk factors in the pathogenesis of ad , especially from a viewpoint of lipid metabolism .   both the aging and the apolipoprotein e4 allele are strong risk factors for developing ad .   the amount of cholesterol in the exofacial leaflets of the synaptic plasma membrane increases in aged   as well as apolipoprotein e4-knock - in   mice . gm1 clustering occurs at presynaptic neuritic terminals in mouse brains in an age - dependent manner . diet - induced hypercholesterolemia accelerates the amyloid pathology in a transgenic mouse model .   a link between cholesterol , a , and human ad brains also show abnormality in lipid metabolism in accordance with our model [ 51 , 52 ] . that is , significant increase in gm1 was reported in a-positive nerve terminals from the ad cortex , and lipid rafts from the frontal cortex and the temporal cortex of ad brains were also found to contain a higher concentration of gm1 compared to an age - matched control . it   should be noted that , in addition to these modulations of a aggregation by lipids , a also in turn regulates lipid metabolism . the 10-fold higher amyloidogenic activity of a-(142 ) is in accordance with the facts ( 1 ) that genetic mutations in the presenilins causing early - onset ad increase the level of a-(142 )   and ( 2 ) that the protein is the major species in diffuse plaques , the earliest stage in the deposition of a   .        in conclusion , in addition to other biochemical cascades , a complex purely physicochemical cascade linked to lipid metabolism ( figure 5 ) appears to be also involved in the process of a aggregation .
it is widely accepted that the conversion of the soluble , nontoxic amyloid -protein ( a ) monomer to aggregated toxic a rich in -sheet structures is central to the development of alzheimer 's disease . 
 however , the mechanism of the abnormal aggregation of a in vivo is not well understood . 
 accumulating evidence suggests that lipid rafts ( microdomains ) in membranes mainly composed of sphingolipids ( gangliosides and sphingomyelin ) and cholesterol play a pivotal role in this process . 
 this paper summarizes the molecular mechanisms by which a aggregates on membranes containing ganglioside clusters , forming amyloid fibrils . 
 notably , the toxicity and physicochemical properties of the fibrils are different from those of a amyloids formed in solution . 
 furthermore , differences between a-(140 ) and a-(142 ) in membrane interaction and amyloidogenesis are also emphasized .