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Common molecular mechanism of amyloid pore formation by Alzheimer’s β-amyloid peptide and α-synuclein

Calcium-permeable pores formed by small oligomers of amyloid proteins are the primary pathologic species in Alzheimer’s and Parkinson’s diseases. However, the molecular mechanisms underlying the assembly of these toxic oligomers in the plasma membrane of brain cells remain unclear. Here we have analyzed and compared the pore-forming capability of a large panel of amyloid proteins including wild-type, variant and truncated forms, as well as synthetic peptides derived from specific domains of Aβ1-42 and α-synuclein. We show that amyloid pore formation involves two membrane lipids, ganglioside and cholesterol, that physically interact with amyloid proteins through specific structural motifs. Mutation or deletion of these motifs abolished pore formation. Moreover, α-synuclein (Parkinson) and Aβ peptide (Alzheimer) did no longer form Ca2+-permeable pores in presence of drugs that target either cholesterol or ganglioside or both membrane lipids. These results indicate that gangliosides and cholesterol cooperate to favor the formation of amyloid pores through a common molecular mechanism that can be jammed at two different steps, suggesting the possibility of a universal therapeutic approach for neurodegenerative diseases. Finally we present the first successful evaluation of such a new therapeutic approach (coined “membrane therapy”) targeting amyloid pores formed by Aβ1-42 and α-synuclein.


Fig. S1: Calcium flux studies.
Intracellular Ca 2+ levels were analyzed in SH-SY5Y cells preloaded with the Ca 2+ dye indicator Fluo-4AM. In a typical experiment, the amyloid protein (A1-42 in this case) was added at a concentration of 220 nM (blue curve). A control experiment with vehicle alone (either H 2 0 or 1% NH 4 OH) was conducted in parallel (red curve). Signals were expressed as fluorescence after treatment (F t ) divided by the fluorescence before treatment (F 0 ) and multiplied by 100. The results were then averaged and the fluorescence of control is subtracted of each value. Data are expressed as mean ± S.E.M. (n = 100).

Figure S2: Determination of residual methyl--cyclodextrin after two consecutive washes of cell cultures.
A. Calibration curve of methyl--cyclodextrin concentration as determined by the Coomassie Brilliant Blue G-250 method (Han Liu: A simple, rapid and sensitive method for the quantification of methyl--cyclodextrin, available at http://www.paper.edu.cn). The addition of methyl--cyclodextrin to a Coomassie blue solution (100 M) induces a dose-dependent increase of the optical density (O.D.) at 637 nm. The relationship between absorbance and methyl--cyclodextrin concentration is linear over the 0-50 M range (dotted red line). B. Determinations of methyl--cyclodextrin concentration in the incubation medium (1 mM) and in the washing medium after the 1 st (R1) and 2 nd (R2) washes. After the 1 st rinse the concentration of residual methyl--cyclodextrin is decreased by 83 times (12 vs 1000 M) to reach undetectable levels after the 2 nd rinse. From these data, it can be deduced that after the last (6 th ) wash preceding the addition of the amyloid proteins, residual methyl-cyclodextrin in the incubation medium is as low as 3.10 -15 M. In fact, only three washes are needed to reach a concentration of 1.74 nM, a value that is far below the concentration of amyloid proteins used in the amyloid pore-forming assay (220 nM). Data are expressed as mean ± S.E.M. (n = 2).

Figure S3: Effect of PPMP on cell surface expression of ganglioside GM1.
Cell surface detection of GM1 ganglioside was assessed by immunofluorescence staining of control or PPMP-treated SH-SY5Y cells (scale bar = 100 µm). These data showed that PPMP treatment (10 M, 48hr) induced a dramatic decrease of the cell surface expression of GM1. The anti-ganglioside GM1 antibody was revealed with goat anti-rabbit Alexa Fluor 488.

Figure S5: Additive binding of A1-42 and of the GBD-derived peptide A1-16 on model GM1 containing raft-like domains.
A ternary monolayer consisting of GM1/cholesterol/POPC (1/1/1, mol/mol/mol) was prepared at the air-water interface (initial surface pressure of 20 mN.m -1 ) as described in   1 . This reconstituted membrane forms a meniscus with a fixed area. After solvent evaporation, A1-42, A1-16 or an equimolar mixture of both (80 nmol each) was added in the aqueous subphase underneath the monolayer. The interaction with the GM1 raft-like membrane was followed kinetically (panel A) or after 30 minutes of incubation (panel B) by measuring the surface pressure increase induced by the proteins (). In both panels the arithmetic sum of the surface pressure increase induced by each individual protein is shown in red (theoretical value). Note that in every case the surface pressure increase induced by the A1-42/A1-16 mixture matches the theoretical values calculated by the addition of the induced by each single protein. These data show that A1-42 and the GBD peptide do not interact together in solution since both bind to GM1, which, in this experiment, is present in large excess in the monolayer compared to the protein concentration. On the surface of neural cells, the amounts of GM1 are much lower so that A1-42 and the GBD peptide would compete for GM1 binding. Data are expressed as mean ± S.E.M. (n = 3). GBD, ganglioside-binding domain; POPC, palmytoyl-oleoylphosphatidylcholine.

Figure S6: Preincubation of the cells with A1-16 followed by extensive washings prevents the formation of amyloid pores induced by full-length A1-42.
SH-SY5Y cells were preloaded with the Ca 2+ -sensitive dye Fluo-4AM, then with A1-16 (220 nM) for 30 minutes, washed three times and finally incubated with A1-42 (+). In parallel, control cells (-) were preincubated with vehicle alone instead of A1-16. Data are expressed as mean ± SEM (n = 71 for cells treated with A1-16 and 40 for cells treated with vehicle alone). These data showed that A1-16 binds to a cell component (GM1) and not to the fulllength A1-42 protein. By saturating the GM1 sites on the cell surface before the addition of A1-42, the GBD peptide prevents the specific attachment A1-42 on the cell surface and thus blocks amyloid pore formation.  The chimeric -syn/A peptide has been described previously 1 . It recognizes all human brain gangliosides including GM1, GM3, GM4, GD1a, GD1b, and GT1b and thus blocks the initial interaction of amyloid proteins with cell surface gangliosides. The activity of bexarotene against amyloid pores has been described 4 . Both cholesterol and bexarotene share a common amphipathic structure with a large polycyclic apolar domain and a small polar head. . Bexarotene has a higher affinity for amyloid proteins than cholesterol, which explains why it prevents amyloid pore formation when present in competition at equimolar concentrations 5 .
E, energy of interaction of the cholesterol/-synuclein and bexarotene/-synuclein complexes.