A protective role for lipid raft cholesterol against amyloid-induced membrane damage in human neuroblastoma cells

Abstract

Increasing evidence supports the idea that the initial events of A beta oligomerization and cytotoxicity in Alzheimer's disease involve the interaction of amyloid A beta-derived diffusible ligands (ADDLs) with the cell membrane. This also indicates lipid rafts, ordered membrane microdomains enriched in cholesterol, sphingolipids and gangliosides, as likely primary interaction sites of ADDLs. To shed further light on the relation between ADDL-cell membrane interaction and oligomer cytotoxicity, we investigated the dependence of ADDLs binding to lipid rafts on membrane cholesterol content in human SH-SY5Y neuroblastoma cells. Confocal laser microscopy showed that A beta 1-42 oligomers markedly interact with membrane rafts and that a moderate enrichment of membrane cholesterol prevents their association with the monosialoganglioside GM1. Moreover, anisotropy fluorescence measurements of flotillin-1-positive rafts purified by sucrose density gradient suggested that the content of membrane cholesterol and membrane perturbation by ADDLs are inversely correlated. Finally, contact mode atomic force microscope images of lipid rafts in liquid showed that ADDLs induce changes in raft morphology with the appearance of large cavities whose size and depth were significantly reduced in similarly treated cholesterol-enriched rafts. Our data suggest that cholesterol reduces amyloid-induced membrane modifications at the lipid raft level by altering raft physicochemical features

Soluble Oligomers Require a Ganglioside to Trigger Neuronal Calcium Overload

“…These results suggest that the cholesterol content of the cell membrane is inversely correlated with the membrane perturbing effects of A␤ 42 oligomers. These data are also consistent with our previous reports showing that disruption of cholesterol homeostasis can be detrimental to neuronal cells, because toxic A␤ aggregates interact more easily with cholesterol-poor membranes [4,44]. In addition, these results indicate that A-oligomers can contribute to the pathogenic process.…”

“…To further study whether GM1 abundance could affect the binding capacity of A␤ 42 oligomers to the lipid bilayers, we used small unilamellar vesicles (SUVs) with a variable content of 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) (70-100%) and GM1 (0-30%) as a model of neuronal membranes [56]. We first analyzed the fluorescence anisotropy ratio (r) of 1,6-diphenyl-1,3,5-hexatriene (DPH), whose value correlates inversely to the degree of membrane fluidity [44]. A progressive reduction of membrane fluidity was observed in vesicles with increasing percentage of GM1 following treatment with either amyloid oligomer (Fig.…”

“…HypF-N protein was purified and converted into toxic (type A) or nontoxic (type B) aggregates as previously described [43]. The oligomeric status of A␤ 42 peptide and HypF-N protein was determined by tapping mode AFM, as previously reported [43,44]. Each type of oligomer was immediately diluted in the appropriate medium at a monomer equivalent concentration of 12 M and then added to the cell culture media or to calcein-loaded small unilamellar vesicles (SUV).…”

“…Fluorescence anisotropy (r) of 1,6-diphenyl-1,3,5-hexatriene (DPH, Sigma Aldrich) was used to measure the structural order of SUVs, using a Perkin-Elmer LS 55 luminescence spectrometer, as previously reported [44]. SUVs containing DOPC and different GM1 contents were incubated for 30 min with 12 M (monomer equivalents) A␤ 42 oligomers (A+ or A-) and then for 30 min with DPH in a 1:250 probe-to-SUV ratio.…”

Membrane lipid composition and its physicochemical properties define cell vulnerability to aberrant protein oligomers

“…Lyophilised Ab peptide was dissolved in HFIP to 1.0 mM and incubated for 1 hour at room temperature to allow complete peptide monomerisation. Aliquots of Ab 42 were dissolved in dimethylsulphoxide, incubated in F12 medium to 100 mM at 4˚C for 24 hours and centrifuged at 14,000 g for 10 minutes to obtain Ab 42 oligomers, according to the Lambert's protocol (Lambert et al, 2001), resuspended in cell culture medium to obtain a final peptide concentration of 12 mM and morphologically characterised by AFM analysis as previously reported (Cecchi et al, 2009). …”