In the past few years, the paradigm that associated directly amyloid fibril formation and the neurotoxicity of Alzheimer's β (Aβ) peptides has been seriously questioned (Lal et al. 2007; Fantini and Yahi 2010). The demonstration that various oligomers of Aβ peptides can form ion channels and perturb Ca2+ homeostasis in cultured neural cells has led to a re-examination of the dogma (Arispe et al. 1993, 2010; Jang et al. 2010; Fändrich 2012). Moreover, oligomeric Aβ species have been found in the brain of Alzheimer's disease patients (Shankar et al. 2008). Interestingly, Esparza et al. (2012) have recently reported that cognitively normal elderly patients can have comparable amounts of Aβ plaques than patients with clinical dementia of the Alzheimer type. In contrast, the concentration of Aβ oligomers in cortex samples was higher in Alzheimer's disease patients than in cognitively normal patients. These striking observations definitely suggest that Aβ oligomers, rather than Aβ fibrils or plaques, are the main neurotoxic species of Alzheimer's disease.
Even if a correlation between plasma cholesterol levels and the cholesterol content of neural membranes has not been formally established, it is important to assess the role of this lipid in the pathogenesis of Alzheimer's disease. Several studies suggest that plasma membrane cholesterol control the cytotoxicity of Aβ peptides (Arispe and Doh 2002; Abramov et al. 2003, 2011). Cholesterol is known to bind to the amyloid protein precursor (APP) (Beel et al. 2010), to regulate its membrane insertion (Lahdo and De La Fournière-Bessueille 2004) and to facilitate its processing into Aβ peptides in lipid raft microdomains (Ehehalt et al. 2003). Cholesterol also binds to monomeric Aβ peptides (Barrett et al. 2012), oligomers (Ashley et al. 2006), aggregates (Avdulov et al. 1997) and fibrils (Harris 2008). Recently, we identified the 20–35 fragment of Aβ1-42 as a linear cholesterol-binding domain and we showed that several peptides encompassing this domain bind cholesterol with high affinity (Di Scala et al. 2013). One of these peptides, i.e. Aβ22-35, has particularly retained our attention since it exerts neurotoxic effect on hippocampal neurons (Takadera et al. 1993), and it induces dementia in mice when injected intra-cerebroventricularly (Zhao et al. 2011). Although the Aβ22-35 peptide has been shown to self-aggregate into amyloid fibrils (Fraser et al. 1991), we wondered whether this peptide could also form oligomeric ion channels in the plasma membrane of neural cells and perturb cellular Ca2+ homeostasis. Using a combination of complementary techniques including intracellular Ca2+ levels measurements, lipid-protein interaction studies, and molecular dynamics simulations, we showed that cholesterol stimulates Aβ22-35 insertion and ion channel formation in the plasma membrane of neural cells. This effect was dependent upon the presence of membrane cholesterol. We used molecular dynamics simulation to examine the binding of cholesterol to Aβ22-35 in a realistic membrane environment. On this basis, we could construct a perfect annular channel resulting from the oligomerization of eight peptide/cholesterol subunits. This model was validated by the experimental data obtained with a series of synthetic Aβ22-35 peptides (wild-type, single, and double mutants). Overall, we propose a molecular mechanism accounting for the role of cholesterol as a key cofactor of amyloid ion channel formation.
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- Materials and methods
- Supporting Information
As recently pointed out by Lukiw (2013), Alzheimer's disease is primarily ‘a disorder of the plasma membrane’. The proteolytic cleavage of the amyloid protein precursor APP into neurotoxic Aβ peptides occurs in cholesterol-enriched lipid raft nanodomains (Ehehalt et al. 2003). Correspondingly, cholesterol depletion inhibits the generation of Aβ peptides (Simons et al. 1998). Cholesterol also stimulates Aβ deposition in neural tissues by modulating the activities of Aβ-producing enzymes (Wang et al. 2012). Both physico-chemical and microscopy techniques have shown that cholesterol physically interacts with APP (Beel et al. 2010), with Aβ peptides (Beel et al. 2010) and with fibrils (Harris 2008). Interestingly, APP and Aβ peptides have distinct, although partially overlapping cholesterol-binding domains, that is, Aβ17-40 for APP (Barrett et al. 2012) and Aβ20-35 for Aβ1-42 (Di Scala et al. 2013). Moreover, Gly residues play a key role in the interaction of APP with cholesterol (Barrett et al. 2012; Fantini and Barrantes 2013), whereas Val-24 and Lys-28 are critical for cholesterol binding to Aβ1-42 (Di Scala et al. 2013). Recently, we showed that the synthetic Aβ22-35 peptide encompassing most of the linear cholesterol-binding domain of Aβ1-42, binds cholesterol with high affinity (Di Scala et al. 2013). Because it displays both neurotoxicity properties (Takadera et al. 1993; Zhao et al. 2011) and cholesterol-binding potency, Aβ22-35 is a unique tool for studying the mechanistic relationship between cholesterol and Aβ toxicity (Hartmann 2001). Shorter neurotoxic peptides such as Aβ25-35 also have amyloid potential (Simmons and Schneider 1993), but do not contain key amino acid residues such as Val-24 that are critical for the binding of cholesterol to Aβ1-42. Thus, even if Aβ25-35 has retained some of the cholesterol-binding properties of full-length Aβ peptides, it could not be used as a surrogate for Aβ1-42 in mechanistic studies focused on cholesterol. Accordingly, only Aβ22-35 simultaneously offers the neurotoxicity potential, amyloidogenic properties and cholesterol-binding capability of full length Aβ peptides.
Our in silico data suggest that the driving force of the membrane insertion/oligomerization of Aβ22-35 is a combination of three parameters: (i) a cluster of apolar residues which limits the energetic cost of the membrane penetration process, (ii) the deprotonation of Lys-28 which finely tunes the helix tilt and facilitates the oligomerization process by its interaction with Asn27 through inter-peptide driven hydrogen-bond and (iii) cholesterol, which boosts the whole process, through direct binding to the peptide through three main anchoring sites including Val-24, Asn-27, and Lys-28, inducing a favorable tilted topology and the reorganization of acidic residues in the N-ter part of the peptide. An α-helical wheel representation of the Aβ22-35 peptide complexed to cholesterol (Figure S4) showed residues Glu-22, Asp-23, and Ser-26 in a relatively short arc. These polar residues are grouped in the hydrophilic 90° sector and constitute a pathway for Ca2+ permeation. The remaining 270° sector, which faces membrane lipids, is chiefly hydrophobic, except for the presence of Asn-27 and Lys-28 whose polar head groups are neutralized by the hydrogen bond network described above. For the sake of comparison, a similar hydrogen bond network retrieved from the structure of a ligand-bound dimerized Fibroblast growth factor receptor (Plotnikov et al. 1999) is shown in the Supplementary information (Figure S2). Finally, it is noteworthy that the helical topology of Aβ22-35 in the channel is strikingly similar to the α-helical transmembrane segments of a synthetic tetrameric calcium channel (Zhorov and Ananthanarayanan 1996).
Given the propensity of various truncated Aβ peptides to form oligomeric structures (Jang et al. 2010), we considered the possibility that the neurotoxic properties of Aβ22-35 could be due to the formation of oligomeric channels in the plasma membrane of neural cells. To evaluate the reliability of our docking study, we have analyzed calcium cellular fluxes induced by a collection of synthetic Aβ peptide. We showed that Aβ1-42 and Aβ22-35 rapidly induced a rapid, progressive, and time-dependent increase of intracellular Ca2+ levels. We believed that this effect is due to the cholesterol-dependent formation of an oligomeric Ca2+ channel in the plasma membrane of SH-SY5Y cells for the following reasons: the Ca2+ source was extracellular, as demonstrated by the lack of calcium fluxes changes in cells incubated in Ca2+-free medium; Aβ1-42 and Aβ22-35-induced Ca2+ flux were inhibited by zinc, a blocker of amyloid ion channel activity which also decreases the uptake of Ca2+ induced by Aβ11-42, Aβ17-42 (Jang et al. 2010); Aβ1-42 and Aβ22-35 had no effect in cholesterol-depleted cells treated with methyl-β-cyclodextrin prior to peptide addition; Aβ1-16, a truncated peptide which interacts with GM1 (Fantini and Yahi 2011) but not with cholesterol (Di Scala et al. 2013), had no effect on Ca2+ fluxes; mutations that affect cholesterol recognition (V24G/K28G and N27R/K28R) impaired channel formation. Overall, the Aβ22-35 channels were functionally similar to the zinc-sensitive Ca2+ channels formed by longer Aβ peptides (e.g. Aβ11-42, Aβ17-42) in the plasma membrane of neural cells. Thus, we have further delineated the minimal fragment of Aβ peptides able to form oligomeric ion channel to the 22–35 fragment, which also corresponds to the cholesterol-binding domain of Aβ1-42. Moreover, we demonstrated that Aβ22-35 channels are cholesterol-dependent, thereby suggesting a mechanistic link between cholesterol and amyloid ion channel formation.
Our molecular modeling data concerning cholesterol dependence of Aβ22-35 channel formation are strongly supported by the experiments performed with the mutant peptides Aβ22-35/V24G/K28G and Aβ22-35/N27R/K28R. These double mutants have lost the ability to form Ca2+ channels in neural cells (Fig. 4b) through both altered interaction with cholesterol and impaired oligomerization. Indeed, Val-24, Asn-27, and Lys-28 are involved in cholesterol recognition and Lys-28 plays a central role in the oligomerization process of Aβ22-35 (Fig. 1b). Interestingly, Aβ22-35/V24G/K28G and Aβ22-35/N27R/K28R have retained some capability of interaction with cholesterol (Fig. 5b) but have lost its aggregating properties. This might reflect a distinctive type of association with cholesterol, most likely a partial insertion, preventing channel formation. Conversely, the single mutants (V24G, N27R, K28G, and K28R), which have lost the cholesterol monolayer insertion potency in vitro, can still form Ca2+ channels in live cells. All together these data suggested that Aβ22-35 channel formation requires both a tight association with cholesterol and an oligomerization capacity in the membrane environment. This further strengthens the key role of cholesterol as a lipid cofactor for Aβ peptide membrane binding/insertion (Ji et al. 2002; Qiu et al. 2009; Yu and Zheng 2012) and as a universal regulator of ion channel function (Levitan et al. 2010).
In conclusion, our study provides a potential molecular mechanism accounting for the cholesterol-assisted amyloid ion channel formation. Apart from Aβ peptides, this mechanism could also account for the cholesterol-dependent membrane insertion/oligomerization of α-synuclein (Fantini and Yahi 2011) and amylin (Trikha and Jeremic 2011). Finally, the identification of a linear cholesterol-binding domain in Aβ suggests a new therapeutic strategy to reduce Aβ neurotoxicity. Indeed, any non-toxic molecule that specifically targets the 22–35 domain of Aβ has potential neuroprotective effects that should be evaluated in cellular and animal models of Alzheimer's disease. Molecular docking studies are currently conducted in our laboratory to identify such possible inhibitors of cholesterol/Aβ interactions that could prevent Aβ membrane insertion and amyloid channel formation.