Nouara Yahi

The Driving Force of Alpha-Synuclein Insertion and Amyloid Channel Formation in the Plasma Membrane of Neural Cells: Key Role of Ganglioside- and Cholesterol-Binding Domains

Alpha-synuclein is an amyloidogenic protein expressed in brain and involved in Parkinsons disease. It is an intrinsically disordered protein that folds into an alpha-helix rich structure upon binding to membrane lipids. Helical alpha-synuclein can penetrate the membrane and form oligomeric ion channels, thereby eliciting important perturbations of calcium fluxes. The study of alpha-synuclein/lipid interactions had shed some light on the molecular mechanisms controlling the targeting and functional insertion of alpha-synuclein in neural membranes. The protein first interacts with a cell surface glycosphingolipid (ganglioside GM3 in astrocytes or GM1 in neurons). This induces the folding of an alpha-helical domain containing a tilted peptide (67-78) that displays a high affinity for cholesterol. The driving force of the insertion process is the formation of a transient OH-Pi hydrogen bond between the ganglioside and the aromatic ring of the alpha-synuclein residue Tyr-39. The higher polarity of Tyr-39 vs. the lipid bilayer forces the protein to cross the membrane, allowing the tilted peptide to reach cholesterol. The tilted geometry of the cholesterol/alpha-synuclein complex facilitates the formation of an oligomeric channel. Interestingly, this functional cooperation between glycosphingolipids and cholesterol presents a striking analogy with virus fusion mechanisms.

Deciphering the glycolipid code of Alzheimer's and Parkinson's amyloid proteins allowed the creation of a universal ganglioside-binding peptide.

A broad range of microbial and amyloid proteins interact with cell surface glycolipids which behave as infectivity and/or toxicity cofactors in human pathologies. Here we have deciphered the biochemical code that determines the glycolipid-binding specificity of two major amyloid proteins, Alzheimers β-amyloid peptide (Aβ) and Parkinsons disease associated protein α-synuclein. We showed that both proteins interact with selected glycolipids through a common loop-shaped motif exhibiting little sequence homology. This 12-residue domain corresponded to fragments 34-45 of α-synuclein and 5-16 of Aβ. By modulating the amino acid sequence of α-synuclein at only two positions in which we introduced a pair of histidine residues found in Aβ, we created a chimeric α-synuclein/Aβ peptide with extended ganglioside-binding properties. This chimeric peptide retained the property of α-synuclein to recognize GM3, and acquired the capacity to recognize GM1 (an Aβ-inherited characteristic). Free histidine (but not tryptophan or asparagine) and Zn2+ (but not Na+) prevented this interaction, confirming the key role of His-13 and His-14 in ganglioside binding. Molecular dynamics studies suggested that the chimeric peptide recognized cholesterol-constrained conformers of GM1, including typical chalice-shaped dimers, that are representative of the condensed cholesterol-ganglioside complexes found in lipid raft domains of the plasma membrane of neural cells. Correspondingly, the peptide had a particular affinity for raft-like membranes containing both GM1 and cholesterol. The chimeric peptide also interacted with several other gangliosides, including major brain gangliosides (GM4, GD1a, GD1b, and GT1b) but not with neutral glycolipids such as GlcCer, LacCer or asialo-GM1. It could inhibit the binding of Aβ1-42 onto neural SH-SY5Y cells and did not induce toxicity in these cells. In conclusion, deciphering the glycolipid code of amyloid proteins allowed us to create a universal ganglioside-binding peptide of only 12-residues with potential therapeutic applications in infectious and neurodegenerative diseases that involve cell surface gangliosides as receptors.

Anandamide-ceramide interactions in a membrane environment: Molecular dynamic simulations data

Anandamide is a lipid neurotransmitter that interacts with various plasma membrane lipids. The data here consists of molecular dynamics simulations of anandamide, C18-ceramide and cholesterol performed in vacuo and within a hydrated palmitoyl-oleoyl-phosphatidylcholine (POPC)/cholesterol membrane. Several models of anandamide/cholesterol and anandamide/ceramide complexes are presented. The energy of interaction and the nature of the intermolecular forces involved in each of these complexes are detailed. The impact of water molecules hydrating the POPC/cholesterol membrane for the stability of the anandamide/cholesterol and anandamide/ceramide complexes is also analyzed. From a total number of 1920 water molecules stochatiscally merged with the lipid matrix, 48 were eventually redistributed around the polar head groups of the anandamide/ceramide complex, whereas only 15 reached with the anandamide/cholesterol complex. The interpretation of this dataset is presented in the accompanying article “Ceramide binding to anandamide increases its half-life and potentiates its cytotoxicity in human neuroblastoma cells” [1].

Broad neutralization of calcium-permeable amyloid pore channels with a chimeric Alzheimer/Parkinson peptide targeting brain gangliosides

Growing evidence supports a role for brain gangliosides in the pathogenesis of neurodegenerative diseases including Alzheimers and Parkinsons. Recently we deciphered the ganglioside-recognition code controlling specific ganglioside binding to Alzheimers β-amyloid (Aβ1-42) peptide and Parkinsons disease-associated protein α-synuclein. Cracking this code allowed us to engineer a short chimeric Aβ/α-synuclein peptide that recognizes all brain gangliosides. Here we show that ganglioside-deprived neural cells do no longer sustain the formation of zinc-sensitive amyloid pore channels induced by either Aβ1-42 or α-synuclein, as assessed by single-cell Ca(2+) fluorescence microscopy. Thus, amyloid channel formation, now considered a key step in neurodegeneration, is a ganglioside-dependent process. Nanomolar concentrations of chimeric peptide competitively inhibited amyloid pore formation induced by Aβ1-42 or α-synuclein in cultured neural cells. Moreover, this peptide abrogated the intracellular calcium increases induced by Parkinsons-associated mutant forms of α-synuclein (A30P, E46K and A53T). The chimeric peptide also prevented the deleterious effects of Aβ1-42 on synaptic vesicle trafficking and decreased the Aβ1-42-induced impairment of spontaneous activity in rat hippocampal slices. Taken together, these data show that the chimeric peptide has broad anti-amyloid pore activity, suggesting that a common therapeutic strategy based on the prevention of amyloid-ganglioside interactions is a reachable goal for both Alzheimers and Parkinsons diseases.

Ceramide binding to anandamide increases its half-life and potentiates its cytotoxicity in human neuroblastoma cells

Anandamide (AEA) is a ubiquitous lipid that exerts neurotransmitter functions but also controls important biological functions such as proliferation, survival, or programmed cell death. The latter effects are also regulated by ceramide, a lipid enzymatically generated from sphingomyelin hydrolysis by sphingomyelinase. Ceramide has been shown to increase the cellular toxicity of AEA, but the mechanisms controlling this potentiating effect remained unclear. Here we have used a panel of in silico, physicochemical, biochemical and cellular approaches to study the crosstalk between AEA and ceramide apoptotic pathways. Molecular dynamics simulations indicated that AEA and ceramide could form a stable complex in phosphatidylcholine membranes. Consistent with these data, we showed that AEA can specifically insert into ceramide monolayers whereas it did not penetrate into sphingomyelin membranes. Then we have studied the effects of ceramide on AEA-induced toxicity of human neuroblastoma cells. In these experiments, the cells have been either naturally enriched in ceramide by neutral sphingomyelinase pre-incubation or treated with C2-ceramide, a biologically active ceramide analog. Both treatments significantly increased the cytotoxicity of AEA as assessed by the MTS mitochondrial toxicity assay. This effect was correlated with the concomitant accumulation of natural ceramide (or its synthetic analog) and AEA in the cells. A kinetic study of AEA hydrolysis showed that ceramide inhibited the fatty acid amino hydrolase (FAAH) activity in cell extracts. Taken together, these data suggested that ceramide binds to AEA, increases its half-life and potentiates its cytotoxicity. Overall, these mechanisms account for a functional cross-talk between AEA and ceramide apoptotic pathways.

Comparison of the amyloid pore forming properties of rat and human Alzheimer’s beta-amyloid peptide 1-42: Calcium imaging data

The data here consists of calcium imaging of human neuroblastoma SH-SY5Y cells treated with the calcium-sensitive dye Fluo-4AM and then incubated with nanomolar concentrations of either human or rat Alzheimer’s β-amyloid peptide Aβ1-42. These data are both of a qualitative (fluorescence micrographs) and semi-quantitative nature (estimation of intracellular calcium concentrations of cells probed by Aβ1-42 peptides vs. control untreated cells). Since rat Aβ1-42 differs from its human counterpart at only three amino acid positions, this comparative study is a good assessment of the specificity of the amyloid pore forming assay. The interpretation of this dataset is presented in the accompanying study “Broad neutralization of calcium-permeable amyloid pore channels with a chimeric Alzheimer/Parkinson peptide targeting brain gangliosides” [1].