Role of Aberrant α-Synuclein–Membrane Interactions in Parkinson’s Disease

Chapter 39

Role of Aberrant α-Synuclein–Membrane Interactions in Parkinson’s Disease Amy M. Griggs*, Daniel Ysselstein* and Jean-Christophe Rochet Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA *These authors contributed equally.

Chapter Outline Introduction443 Evidence for Membrane-Induced α-Syn Self-Assembly 444 Conformations of α-Syn at the Membrane Surface 445

INTRODUCTION Parkinson’s disease (PD) is a neurodegenerative disorder that involves a loss of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain. PD patients commonly display an array of motor symptoms, including resting tremors, bradykinesia, rigidity, and postural instability [1–3]. A hallmark neuropathologic feature in the PD brain is the presence of cytosolic inclusions enriched with fibrillar forms of the presynaptic protein α-synuclein (αsyn), called Lewy bodies [4,5]. Several autosomal-dominant mutations in the SNCA gene encoding α-syn have been linked to familial (and generally early-onset) forms of PD, including (i) point mutations resulting in A30P, E46K, H50Q, G51D, and A53T substitutions [6–12]; and (ii) duplication and triplication mutations that increase the expression level of the protein by 1.5- and 2-fold, respectively [13,14]. Studies carried out in cell-free systems revealed that the A30P, E46K, and A53T mutants have an increased intrinsic propensity to form high-molecular-weight oligomers [15–19]. The duplication and triplication mutations, in addition to polymorphisms in the SNCA gene associated with enhanced α-syn expression and increased risk of PD [20–22], are expected to stimulate α-syn aggregation via mass action [23]. Taken together, these neuropathologic and genetic data suggest that α-syn aggregation is a key event in PD pathogenesis. α-Syn accumulation is also a hallmark feature of other neurodegenerative diseases (e.g. dementia with Lewy bodies (DLB)), which, together with PD, are referred to as ‘synucleinopathy’ disorders [24]. Bio-nanoimaging. http://dx.doi.org/10.1016/B978-0-12-394431-3.00039-0 Copyright © 2014 Elsevier Inc. All rights reserved.

A Role for Aberrant α-Syn–Membrane Interactions in Neurotoxicity447 Concluding Remarks 447

α-Syn is commonly expressed as a 14 kDa, 140-residue protein, although smaller isoforms exist [25]. Within the 140 residues there are three major domains (Fig. 39.1). The N-terminal domain spanning residues 1–67 includes five of six highly conserved repeats composed of the sequence KTK(E/Q)GV. The central domain spanning residues 61–95, known as the ‘non-Aβ component of AD amyloid’ (NAC) region, has an abundance of hydrophobic residues that play a critical role in α-syn aggregation [26]. The C-terminal domain spanning residues 96–140 is enriched in proline, aspartate, and glutamate residues, and is thought to interfere with α-syn self-assembly through long-range intramolecular interactions that result in shielding of the central hydrophobic region [27–29]. Evidence from studies involving a range of biophysical techniques, including NMR, CD, EPR, and fluorescencebased methods, suggests that α-syn possesses no significant secondary structure in aqueous solution, and the protein is often referred to as being ‘natively unfolded’ [30–34]. α-Syn undergoes self-assembly upon extended incubation in physiologic buffers, yielding amyloid-like fibrils similar to those found in Lewy bodies [15,35–38]. α-Syn fibrillization involves the transient accumulation of intermediate, β-sheet-rich oligomers named ‘protofibrils’, and data from various studies suggest that these species may in fact be more toxic than mature fibrils [15,16,39,40]. Recent data from in-cell cross-linking studies suggest that α-syn can form an aggregation-resistant, α-helical tetramer in intact cells, although this tetrameric species is difficult to characterize using conventional biochemical techniques because of its

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EVIDENCE FOR MEMBRANE-INDUCED α-SYN SELF-ASSEMBLY

FIGURE 39.1  Amino-acid sequence of α-syn shown in one-letter code. The region that encompasses the imperfect six-residue repeats (KTK[E/Q]GV) spanning residues 1–85 is underlined in blue. The hydrophobic NAC segment spanning residues 61–95 is underlined in violet. The unfolded C-terminal region enriched with proline, aspartate, and glutamate residues spanning residues 96–140 is underlined in green. Red asterisks indicate sites of amino-acid substitutions (A30P, E46K, H50Q, G51D, and A53T) in mutant forms of α-synuclein involved in familial PD.

sensitivity to cell lysis conditions [41–43]. If an α-helical, tetrameric form of α-syn is indeed significantly populated in vivo, then the destabilization of this species may be a key initiating event that precedes self-assembly steps leading to the formation of pathologic α-syn aggregates. Although α-syn has been a major focus of research in PD for over 15 years, the protein’s biologic function remains poorly understood. The N-terminal lysine-rich repeats resemble lipid-binding motifs in amphipathic helical domains of exchangeable apolipoproteins, suggesting that α-syn interacts with phospholipid membranes as part of its normal function [44]. α-Syn knockout mice exhibit defects in dopamine release triggered by paired or repeated electrical stimuli [45–47], and knockdown of α-syn with antisense oligonucleotides induces a decrease in the size of the distal pool of synaptic vesicles in hippocampal neurons [48]. Modest over-expression of α-syn results in a disruption of neurotransmitter release in cultured neurons and chromaffin cells via a mechanism involving a depletion of the readily releasable and recycling pools of synaptic vesicles [49,50]. Collectively, these observations have led to the hypothesis that α-syn is involved in regulating the size of ‘reserve’ and/ or ‘resting’ pools of vesicles at the synapse [46]. In addition to its presumed role in modulating neurotransmission, membrane-associated α-syn can undergo self-assembly to form oligomers at the bilayer surface. Evidence suggests that the process of membrane-induced α-syn aggregation results in damage to bilayer lipids and thus may play a role in neurotoxicity. A number of recent studies have yielded insights into conformational properties of α-syn that favor the protein’s self-assembly at the membrane surface. Here we review these findings and describe how aberrant α-syn–membrane interactions may play a role in various reported mechanisms of α-syn neurotoxicity.

α-Syn is bound to vesicles isolated from neuronal cells or brain homogenates [51–53], and the protein has been shown to associate with membranes in intact neurons [54]. Biophysical analyses have shown that the N-terminal repeat region of α-syn has a high affinity for synthetic vesicles enriched with anionic phospholipids, and it adopts an amphipathic α-helical structure upon binding the membrane [44,55–58]. The fact that membrane-bound α-syn has an α-helical structure suggests that this form of the protein should have a low propensity to convert to β-sheet-rich protofibrils or fibrils. Indeed, phospholipids have been found to suppress α-syn fibrillization at low protein-to-lipid ratios [59,60]. In contrast, α-syn undergoes accelerated self-assembly in the presence of anionic phospholipid vesicles, synaptosomal membranes, or anionic detergent micelles at high protein-tolipid (or protein-to-detergent) ratios [53,60–62]. Moreover, data from our studies of α-syn interactions with supported phospholipid bilayers revealed that the segregation (‘demixing’) of anionic and zwitterionic lipids can provide a driving force for the early clustering of α-helical α-syn conformers at the membrane surface [63–65]. These observations can be interpreted to mean that encounters of α-syn molecules needed for oligomerization may occur with greater probability in a two-dimensional space on a phospholipid bilayer than in a three-dimensional space in solution [66,67]. Accordingly, membrane binding serves to increase the local concentration of α-syn, thereby increasing the probability of interactions favoring oligomerization. In a state with excess lipid, α-syn binding is sparser, and crowding-induced oligomerization is less likely to occur. Another interpretation is that a lipid-limited state favors a conformation of membranebound α-syn that is susceptible to aggregation at the membrane surface (this is discussed in greater detail below). A groundbreaking study by Lee and colleagues [68] revealed that α-syn formed SDS-resistant oligomers more rapidly in an isolated membrane fraction of a rat brain homogenate than in the corresponding cytosolic fraction. α-Syn self-assembly was inhibited in membrane fractions incubated in the presence of antioxidants, suggesting that oxidative stress promotes aggregation of the membranebound protein. The addition of cytosolic α-syn to the membrane fraction stimulated the formation of α-syn oligomers, suggesting that membrane-bound oligomers of α-syn recruited the unbound cytosolic protein to the membrane. α-Syn readily forms oligomers in the presence of lipids enriched with polyunsaturated fatty acids (PUFAs). An early study revealed that α-syn formed detergent-resistant oligomers at a faster rate when incubated at physiologic concentrations with lipid vesicles containing arachidonoyland docosahexaenoyl-phosphatidylcholine than in aqueous

Chapter | 39  α-Synuclein–Membrane Interactions in Parkinson’s Disease

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FIGURE 39.2  Model illustrating destabilization and aggregation of α-syn at the membrane. (Top) Schematic of the secondary structure of membranebound α-syn showing extension of the amphipathic α-helix through the aggregation-prone NAC domain. Secondary structural elements spanning the amphipathic N-domain (blue), the NAC domain (red), and the C-domain (green) are depicted above a map of the polypeptide chain with identical color coding. (Bottom) Destabilization of membrane-bound α-syn (e.g. by protein substitution or modification, a change in lipid content, or increased lipid oxidation) leads to a shift from the aggregation-resistant ‘hidden’ state to the aggregation-prone ‘exposed’ state. The hidden and exposed states are referred to by Bax and colleagues [89] as the SL2 and SL1 conformations, respectively. Adapted from reference 96.

solution [69]. α-Syn oligomers were also found to be preferentially bound to lipid droplets and cellular membranes in cells loaded with fatty acids [70]. Selkoe and colleagues [71] reported that α-syn formed SDS-resistant, soluble oligomers upon interacting with PUFAs, including docosahexaenoic acid (DHA). Similar lipid-associated oligomers were found to be elevated in PD and DLB brains and to accumulate in mesencephalic cells exposed to PUFAs and in the brains of α-syn transgenic mice. Another study revealed that DHA promotes the conversion of α-syn to amyloid-like fibrils and stable oligomers at relatively high and low protein-tolipid ratios, respectively [72]. α-Syn transgenic mice fed a diet rich in DHA showed an increase in the accumulation of insoluble α-syn [73]. Evidence that DHA promotes α-syn aggregation is particularly relevant given that DHA is abundant in the brain and found to be associated with α-syn aggregates in PD patients [74]. One reason why PUFAs stimulate α-syn self-assembly is that they are susceptible to oxidation, and evidence suggests that oxidized lipids promote α-syn oligomerization. Examples of lipid peroxidation products that trigger α-syn self-assembly include acrolein, 4-oxo-2-nonenal (ONE), and 4-hydroxy-2-nonenal (HNE) [75–77]. HNE was shown to react with α-syn at lysine and histidine residues, yielding protein–lipid adducts that readily converted to neurotoxic β-sheet-rich oligomers with a low propensity to form amyloid-like fibrils [76,78–80]. Basal lipid peroxidation is increased in the substantia nigra of PD patients [81], suggesting that the stimulatory effect of oxidized lipids on α-syn aggregation may play a role in PD pathogenesis.

CONFORMATIONS OF α-SYN AT THE MEMBRANE SURFACE The observed link between membrane binding and α-syn oligomerization has prompted biophysical studies aimed at understanding which conformations of membrane-associated

α-syn have a high propensity to undergo self-assembly. Several groups showed that α-syn binds SDS micelles by forming a broken α-helical structure, with residues ∼40–45 forming an unstructured loop separating the two helical segments [82–84]. SDS is commonly used as a lipid mimetic in vitro, yet these micelles have a much higher curvature than physiologic vesicles. The increased curvature could cause stress in the α-helical region spanning residues ∼1–100, forcing it to separate into two segments. Upon binding small unilamellar vesicles (SUVs) with a diameter of 30 nm, α-syn was found to adopt a predominant conformation of a single extended α-helix [85–87], although some ‘broken’ helical structure was also observed [88]. These results imply that membrane curvature affects the conformation adopted by α-syn upon interacting with different membranes in the cell. NMR data obtained by Bax and colleagues [89] suggest that α-syn binds more physiologically relevant SUVs by adopting two conformations, referred to here as the ‘exposed’ and ‘hidden’ states (Fig. 39.2). In the exposed state residues 3–25 are helical and in contact with the bilayer, whereas the hidden state consists of an extended, membrane-bound helix spanning residues 3–100. NMR is a valuable technique for examining the extent of binding at any given position in the α-syn polypeptide chain because the signal attenuation at each site corresponds to the fractional population of the residue in contact with the vesicle membrane. Further support for a two-state binding model was provided by Beyer and colleagues [90], who showed that the N-terminal segment spanning residues 1–25 formed a helix with a substantially greater membrane affinity than that of the central hydrophobic region. Removal of the first six N-terminal residues resulted in a marked decrease in αsyn binding, highlighting the importance of these residues for α-syn membrane affinity. The two-state model suggests a potential mechanism for α-syn oligomerization involving interactions between hydrophobic NAC regions of neighboring exposed conformers that may lead to the formation

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of β-sheet-rich oligomers (see below) [89]. In contrast, the NAC region is more closely associated with the lipid vesicle in the hidden state and is thus less likely to engage in interactions involved in α-syn oligomerization. The two-state model outlined above also accounts for the enhanced toxicity of familial α-syn mutants. Although A30P has a lower membrane affinity than wild-type α-syn, a greater proportion of membrane-bound A30P molecules adopt the exposed conformation [89]. A53T also exhibits a higher exposed to hidden ratio. E46K displays an increased affinity for the lipid membrane, and thus it populates a greater number of exposed conformers than wild-type α-syn, despite the fact that the two variants exhibit similar exposed to hidden ratios. This model also helps explain toxicity induced by the SNCA gene duplication and triplication because a higher concentration of α-syn is likely to produce an increase in the protein-to-lipid ratio (i.e. a lipid-limited situation) which should, in turn, favor the exposed conformation [91]. Although the interaction of α-syn with lipid vesicles is driven by the N-terminal region, post-translational modifications in the C-terminal region have been shown to modulate this interaction. Notably, nitration of tyrosine residues in the C-terminal domain is an abundant modification in Lewy bodies in the brains of PD and DLB patients [92] and has been shown to induce nigral dopaminergic cell death in a rat model [93]. Rhoades and colleagues [94] recently reported that nitration of the three C-terminal tyrosine residues reduced lipid binding though an allosteric mechanism, suggesting a potential link between nitration and altered lipid binding that could play a role in α-syn neurotoxicity in PD. The oligomers formed at the membrane have been difficult to characterize. Western blot analysis has revealed a laddering of SDS-resistant oligomers, but it is unlikely that the band sizes on a gel reflect the molecular weights of the intact membrane-bound oligomers. Polarized infrared spectroscopy experiments revealed that membrane-bound α-syn contains a significant β-sheet component [95], suggesting that the protein can undergo a conversion from α-helix to β-sheet at the membrane surface. This α-helix to β-sheet transition was recently monitored by magic-angle spinning solid state NMR in a study reported by Reinstra and colleagues [96]. The results revealed that α-helical α-syn conformers initially formed upon binding anionic phospholipids gradually convert to a β-sheet structure when the protein–lipid mixture is incubated at 37°C. After prolonged incubation, the α-syn secondary structure was found to consist predominantly of β-sheet, similar to mature α-syn fibrils. Interestingly, the intermediate states displayed a unique pattern of peak intensities compared to the mature fibril, suggesting the existence of distinct β-sheet-rich intermediate species in close association with lipid. The observation that membrane-bound α-syn oligomers are enriched with β-sheet structure [96] suggests that they are similar to soluble oligomeric species formed in the presence of lipid peroxidation products (e.g. ONE, 4-HNE, acrolein)

FIGURE 39.3  Schematic illustrating a mechanism by which α-syn aggregation may elicit membrane damage on a supported lipid bilayer. (A) α-Syn initially binds the membrane as an α-helical monomer. (B) α-Syn self-assembly at the membrane surface results in membrane disruption and lipid extraction near maturing α-syn aggregates. (C) After prolonged incubation, α-syn forms amyloid-like fibrils at the membrane surface. At this stage the membrane is extensively disrupted. Reproduced with permission from reference 98.

which have been better characterized and thus may provide insight into structural details of membrane bound α-syn. αSyn oligomers formed in the presence of lipid peroxidation products also display significant β-sheet structure, are predicted to have a mass of ∼2000 kDa, and are stable in SDSor guanidine-containing solutions [75,76]. Analysis of these oligomers by atomic force microscopy has revealed species with dimensions of 40–80 nm by 6–8 nm. These oligomers have been shown to inhibit proteasomal function linking αsyn oligomers to neurotoxicity [77]. A recent study showed that α-syn formed radiating amyloid fibrils (RAFs) on the surface of synthetic lipid membranes through a unit assembly process [97]. These RAFs developed and grew larger by the addition of oligomers, and this progression led to disruption of the membrane. Further evidence in support of this mechanism was reported by Seeger and colleagues [98], who used super critical angle fluorescence microscopy and Förster resonance energy transfer (FRET) to demonstrate that α-syn aggregates were formed on supported lipid bilayers and that these aggregates extracted lipids from

Chapter | 39  α-Synuclein–Membrane Interactions in Parkinson’s Disease

the bilayer as they increased in size (Fig. 39.3). Therefore, association of α-syn with the membrane may stimulate oligomer formation by increasing the effective concentration of the protein, and this process may trigger neurotoxicity via disruption of membrane integrity by the growing aggregates.

A ROLE FOR ABERRANT α-SYN–MEMBRANE INTERACTIONS IN NEUROTOXICITY Aberrant α-syn–membrane interactions may play a role in various mechanisms of α-syn neurotoxicity. As one example, α-syn has been shown to interfere with trafficking between the endoplasmic reticulum (ER) and Golgi, a defect that precedes ER stress [99]. The ER–Golgi vesicle trafficking gene Ypt1p/Rab1 rescued dopaminergic cell loss induced by α-syn in Drosophila, Caenorhabditis elegans, and rat primary midbrain cultures. A subsequent study revealed that (i) α-syn impaired the docking of vesicles to Golgi membranes, and (ii) two additional Rab proteins, Rab3a and Rab8a, alleviated α-syn neurotoxicity [100]. αSyn aggregates were also shown to induce clustering of ERGolgi transport vesicles in a yeast model system [101]. α-Syn has been demonstrated to interfere with mitochondrial function, a phenomenon that may involve disruption of the mitochondrial electron transport chain and a buildup of reactive oxygen species (ROS). α-Syn over-expression in SH-SY5Y neuroblastoma cells had damaging effects on mitochondria, causing defects in cellular respiration, impairment of mitochondrial transmembrane potential, a surge in protein tyrosine nitration, and an increase in ROS [102]. These disruptive effects may involve the association of αsyn with mitochondria. In a recent study, α-syn was found to bind isolated mitochondria in a concentration-dependent manner via a mechanism involving interactions between N-terminal α-syn residues and the mitochondrial membrane [103]. α-Syn was also shown to interact with mitochondria isolated from HeLa cells [104]. A FRET study revealed that α-syn undergoes a conformational change from a ‘closed’ to an ‘open’ state upon binding isolated mitochondria, thereby positioning the C-terminus away from the N-terminus [105]. Evidence that α-syn can fragment mitochondria further supports the idea that the protein may disrupt mitochondrial function through direct membrane interactions [106,107]. Alternatively, α-syn may disrupt mitochondrial function by affecting calcium transfer from the ER to mitochondria [108]. Under normal conditions, α-syn promotes ER–mitochondrial interactions necessary for calcium transfer. However, α-syn over-expression or aggregation can perturb this mechanism, thereby disrupting mitochondrial homeostasis. Aberrant α-syn–membrane interactions may also play a role in the prion-like transmission of α-syn aggregates. A number of recent studies have explored the idea that α-syn neuropathology spreads in the brains of PD patients via a prion-like mechanism [109–111]. α-Syn neuropathology

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first appears in the form of Lewy bodies in the lower brainstem and then spreads through the midbrain and mesocortex, finally extending to the neocortex [112]. Current data have led to a proposed mechanism for the spreading of αsyn aggregates throughout the brain. Detergent-insoluble αsyn aggregates were shown to be released from SH-SY5Y neuroblastoma cells exposed to various stresses via a nonclassic exocytosis mechanism [113]. Another study revealed that both monomeric and oligomeric α-syn were secreted from SH-SY5Y cells via a mechanism that depended on intracellular Ca2+ levels and the release of exosomes [114]. Additional data suggest that lysosomal impairment enhances exosome-mediated α-syn secretion [115]. Cellular treatment with HNE was found to increase the translocation of α-syn into vesicles, α-syn release from cells, and cell-to-cell transfer of α-syn oligomers, thus promoting the spread of neurotoxic α-syn aggregates [80]. Collectively, these observations imply that membrane-induced α-syn oligomerization could potentially play a role in prion-like transmission of the protein in PD and other synucleinopathy disorders.

CONCLUDING REMARKS The research findings reviewed in this chapter support a model in which the binding of α-syn to phospholipid membranes plays a role in the protein’s normal and pathologic functions (Fig. 39.4). Under normal physiologic conditions, α-syn adopts an amphipathic α-helical structure upon binding phospholipid vesicles as part of its role in modulating synaptic neurotransmission. However, under pathologic conditions, membrane-bound α-syn may alter its conformation from the highly α-helical hidden state to the less folded exposed state. Examples of perturbations that could promote the hidden to exposed conversion include familial α-syn substitutions, an increase in the α-syn to lipid ratio, α-syn oxidation (e.g. C-terminal tyrosine nitration), and changes in the membrane lipid composition (e.g. age-dependent lipid oxidation). An increase in the abundance of membrane-bound exposed conformers may lead to α-syn oligomerization or fibrillization at the membrane surface, a process that has been shown to trigger disruption of the phospholipid bilayer. Conceivably, aberrant α-syn–membrane interactions that promote membrane-induced α-syn aggregation coupled with membrane disruption could play a role in various mechanisms of α-syn neurotoxicity, including disruption of ER–Golgi trafficking, impairment of mitochondrial function, and the propagation of α-syn neuropathology via a prion-like mechanism. Alternatively, aberrant α-syn–membrane interactions may contribute to neurotoxicity via mechanisms that are independent of αsyn oligomerization at the membrane surface; for example, a shift from the hidden to exposed conformation could perturb interactions of α-syn with other membrane-bound proteins such as Rab3a [116]. A high priority for future studies will be to characterize membrane-bound conformations (e.g.

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FIGURE 39.4  Model depicting α-syn–membrane interactions and aggregation pathways leading to downstream neurotoxic effects. Natively unfolded monomeric α-syn (black line) can adopt an α-helical structure (blue cylinder) upon binding phospholipid membranes, whereas the C-terminal tail remains unstructured (purple wavy line). Once α-syn is bound to vesicles it can facilitate their docking to target membranes. Vesicles and other target membranes can serve as templates for α-syn aggregation. β-sheet rich assemblies (yellow) that form in the cytosol and/or at the membrane surface have detrimental downstream effects (represented by red dashed arrows) on mitochondria, lysosomes, the ubiquitin–proteasome system (UPS), and ER–Golgi trafficking, leading to cell death [117].

exposed or hidden conformers, membrane-bound oligomers) that are populated by α-syn under various conditions in neurons or in the brain. It will also be critical to identify α-syn– membrane or α-syn–protein interactions that account for the protein’s neurotoxic mechanisms. Ultimately, an improved understanding of α-syn conformations and interactions will stimulate the development of novel therapeutic strategies (e.g. strategies to stabilize the hidden conformation) to slow neurodegeneration in patients with PD and other synucleinopathy disorders.

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Chapter | 39  α-Synuclein–Membrane Interactions in Parkinson’s Disease

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