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Keywords:

  • cation channels;
  • modeling;
  • molecular dynamics;
  • oligomers;
  • synuclein

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Accumulation of α-synuclein resulting in the formation of oligomers and protofibrils has been linked to Parkinson's disease and Lewy body dementia. In contrast, β-synuclein (β-syn), a close homologue, does not aggregate and reduces α-synuclein (α-syn)-related pathology. Although considerable information is available about the conformation of α-syn at the initial and end stages of fibrillation, less is known about the dynamic process of α-syn conversion to oligomers and how interactions with antiaggregation chaperones such as β-synuclein might occur. Molecular modeling and molecular dynamics simulations based on the micelle-derived structure of α-syn showed that α-syn homodimers can adopt nonpropagating (head-to-tail) and propagating (head-to-head) conformations. Propagating α-syn dimers on the membrane incorporate additional α-syn molecules, leading to the formation of pentamers and hexamers forming a ring-like structure. In contrast, β-syn dimers do not propagate and block the aggregation of α-syn into ring-like oligomers. Under in vitro cell-free conditions, α-syn aggregates formed ring-like structures that were disrupted by β-syn. Similarly, cells expressing α-syn displayed increased ion current activity consistent with the formation of Zn2+-sensitive nonselective cation channels. These results support the contention that in Parkinson's disease and Lewy body dementia, α-syn oligomers on the membrane might form pore-like structures, and that the beneficial effects of β-synuclein might be related to its ability to block the formation of pore-like structures.

Abbreviations
aa

amino acid

α-syn

α-synuclein

β-syn

β-synuclein

GFP

green fluorescent protein

LBD

Lewy body disease

PD

Parkinson's disease

POPC

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

tg

transgenic

In recent years, new hope for understanding the pathogenesis of Parkinson's disease (PD) and Lewy body dementia (LBD) has emerged with the discovery of mutations and duplications in the α-synuclein (α-syn) gene that are associated with rare familial forms of Parkinsonism [1–3]. Moreover, it has been shown that α-syn is centrally involved in the pathogenesis of both sporadic and inherited forms of PD and LBD because this molecule accumulates in Lewy bodies (LBs) [4–6], synapses, and axons, and its expression in transgenic (tg) mice [7–9] and Drosophila [10] mimics several aspects of PD.

The mechanisms through which α-syn leads to neurodegeneration and the characteristic symptoms of LBD are unclear. However, recent evidence indicates that abnormal accumulation of misfolded α-syn in the synaptic terminals and axons plays an important role [11–14]. These studies suggest that α-syn oligomers and protofibrils rather than fibrils might be the neurotoxic species [15].

α-syn is an abundant presynaptic molecule [16] that probably plays a role in modulating vesicular synaptic release [17]. Synucleins belong to a family of related proteins including α-, β-, and γ-synucleins. α-syn belongs to a class of so-called ‘naturally unfolded proteins’[13,18]. Such proteins do not have a stable tertiary structure and during their existence change their conformations. Human α-syn is a 140-amino acid (aa) protein, and β-syn is a 134-aa protein. Each of the synucleins is composed of an N-terminal lipid-binding domain containing 11 residue repeats and a C-terminal acidic domain that has been proposed to be involved in protein–protein interactions. It has been shown [19–22] that at the lipid–protein interface, α-syn has a conformation characterized by two helical domains interrupted by a short nonhelical turn. α-syn contains a highly amyloidogenic hydrophobic domain in the N-terminus region (aa 60–95), which is partially absent in β-syn and might explain why β-syn has a reduced ability to self-aggregate and form oligomers and fibrils [23,24]. Interestingly, although under physiological conditions β-syn is nonamyloidogenic, a recent study demonstrated that certain factors, namely, particular metals and pesticides, can cause rapid fibrillation of this molecule and of mixtures of α- and β-syn [25] under in vitro cell-free conditions. However, previous studies have shown that in the absence of metals, β-syn interacts with α-syn and is capable of preventing α-syn aggregation and related deficits both in vitro and in vivo[23,24].

Various lines of evidence support the contention that abnormal aggregates arise from a partially folded intermediate precursor that contains hydrophobic patches. It has been proposed that the intermediate α-syn oligomers form annular protofibrils and pore-like structures [26–29]. The mechanism through which monomeric α-syn converts into a toxic oligomer and later into fibrils is currently under intense investigation. Recent reviews indicate that the kinetics of α-syn fibrillation are consistent with a nucleation-dependent mechanism for which a partially folded intermediate is needed in the early stages of aggregation [30]. Factors leading to the formation of the folded intermediates include oxidation, phosphorylation, mutations, and lipids in the membrane [30–34]. α-syn oligomerization might occur on the membrane and involves interactions between hydrophobic residues of the amphipathic α-helices of α-syn [35]. These studies indicate that the hydrophobic lipid binding domains in the N-terminal region might be important in modulating α-syn aggregation [13,36–38]. There are several studies describing the effects of membranes and membrane-like structure on aggregation [21,39,40], however, less is known about the effects of membrane lipids on β-syn structure. In this context, a recent study has analyzed by NMR the micelle-bound structure and dynamics of β- and γ-syn [41].

Thus, better understanding of the steps involved in the process of α-syn aggregation is important in order to develop intervention strategies that might prevent or reverse α-syn oligomerization and toxic conversion. The conformational state of α-syn at the initial and end stages of fibrillation have been characterized in some detail and recent studies have shown that early stage oligomers are globular structures with variable height (2–6 nm) that after prolonged incubation results in the formation of elongated protofibrils which disappear upon fibril formation [42].

However, less is known about the dynamic process of conversion of α-syn at earlier stages and how interactions with antiaggregation chaperones such as β-syn and heat-shock proteins might occur. This is in part due to the transient nature of the oligomers and the difficulties in crystallizing such conformers. Therefore, the use of computer-based molecular modeling techniques in combination with biochemical and cell based assays might facilitate understanding the dynamic characteristics and structure of the synuclein aggregates. In this context, the main objective was to develop a dynamical model for the early steps of α-syn aggregation using computer simulations that includes the process of membrane docking and the potential mechanisms through which β-syn blocks α-syn aggregation.

Our studies suggest that at early stages, propagating α-syn dimers immersed in the membrane lead to the formation of pentamers and hexamers with a pore-like structure. These ring-like aggregates might correspond to Zn2+-sensitive nonselective cation channels whose formation is blocked by β-syn. The inhibitory effect of β-syn may result from its interaction with α-syn, which prevents formation of functional α-syn channels.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Conformational diversity of α-syn and β-syn molecules during molecular dynamics simulations

To better understand the conformational changes that α- and β-syn undergo over time and to model the homo and heterodimeric interactions preventing or leading to aggregation, molecular dynamics simulations in water were performed based on the micelle-bound structure of α-syn as resolved by NMR. This approach allows the investigation of the dynamic structural changes of the folded α-syn (micelle-derived) under simplified conditions. The curved N-terminal domain of this structure is divided into two regions (termed helix-N and helix-C) [21] connected by a short linker (Fig. 1A,B). In our baseline models, the two curved helical N-terminal domains of the micelle-derived α-syn molecular structure form an angle around 55 ± 3° that decreases to around 42–44° during the first 2.0 ns of the simulation, and then increases to 64–70° after 3.0–5.0 ns of simulation. During simulation (Fig. 1A,B), the initial two curved helical N-terminal domains (helices N and C) of α-syn transform into three uncurved N-terminal helical structures. The third helical region appears when the second curved helix (aa 46–84) converts into two uncurved helices, helix 2 (aa 46–63) and helix 3 (aa 74–84), linked by aa 64–73 (Fig. 1A,B). To confirm these results, we repeated the simulation in water, with different seed numbers, for 3.0 ns. These additional data corroborate the initial results, showing that over time α-syn acquires a more three-dimensional shape due to movement of the C-terminal domain relative to the N-terminus (Fig. 1A,B). It is worth noting that the micelle-derived helical structure of α-syn is highly stable and did not return to an unfolded state even though the molecular dynamics simulations were performed using the water box to simplify the procedure.

image

Figure 1.  Molecular dynamics simulations of α- and β-syn monomers in water. (A) Snapshots of molecular dynamics conformations of α-syn. (B) Superimposed α-syn conformers (area of superimposition: aa 1–15). (C) Snapshots of molecular dynamics conformations of β-syn. (D) Superimposed β-syn conformers (area of superimposition: aa 1–15).

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At time zero, β-syn has a structural organization close to the initial structure of α-syn (Fig. 1C,D). Unlike α-syn, the curved helix at residues 46–84 of β-syn does not undergo conversion into two distinct helices during the course of simulation of up to 5.0 ns. Instead, the curved helices adopt a relatively straight configuration after 2.0 ns (Fig. 1C,D) and this conformer increases in stability from 2.0 to 3.5 ns. Further simulation shows additional conformational changes, mostly in the C-terminal tail and the angle between the N-terminal helices (Fig, 1C,D). Compared with α-syn, the C-terminal tail of β-syn displayed greater motility.

Further analysis consisted of determining changes in secondary structure of α-syn and β-syn over time. After 500 ps of simulation for α-syn a coiled region appeared, interrupting the α-helix around residue 68 (Fig. S1A). Beginning at 750 ps, turns appeared in the α-helical structure around residue 47, then after 1.0 ns this region was transformed into a π-helix (Fig. S1A). The length of this π-helix increased with time, and from 3.0 ns covered the region from residues 45–55. In another part of the sequence, a second π-helix appeared from 2.0 ns that includes residues 74–83 (Fig. S1A).

Changes in β-syn secondary structure over time consisted of transformations from a bended α-helical structure to the structure with two straight helices with further conversion to π-helical structure around residue 30 and the N-terminus region (Fig. S1B). The C-terminal region beyond residue 70 showed limited changes in secondary structure (Fig. S1B). Overall, β-syn underwent significantly fewer changes in secondary structure than α-syn during molecular dynamics simulations.

Interactions of α-syn propagating dimers predict the formation of pore-like structures

The first studies of the interactions of α-syn were performed by docking the initial structures of two α-syn monomers on a flat surface without specific limitations. Under these conditions, some low energy complexes of two molecules formed a ‘head-to-tail’ position. This configuration is not favorable for further aggregation on the membrane. The appearance of such dimeric aggregates is caused mostly by electric charge profile complementarities between the N- and C-termini of α-syn monomers (Fig. 2A and 3A). These α-syn homodimers can interact with additional α-syn molecules, but further simulations indicate that the resulting higher order aggregates are not likely to produce continuously propagating multimers on the membrane. For the nonpropagating α-syn homodimers, usually only one α-syn has the membrane binding surface, such as for the 1.5 ns molecular dynamics conformers (Fig. 2A and 3A).

image

Figure 2.  Molecular modeling of nonpropagating and propagating α-syn aggregates on the membrane. (A) α-syn minimal energy nonpropagating dimers (head-to-tail). (B) α-syn conformer at 4.0 ns oriented to the membrane surface (membrane-contacting residues depicted in orange). (C–E) Propagating α-syn multimers on the membrane at 3.5 ns (C) Dimer (D) tetramer, and (E) hexamer. Multimers can be formed by docking of α-syn monomers to α-syn propagating dimers, or by addition of α-syn dimers to α-syn propagating dimers, with either scenario resulting in the same final hexamer structure. (F) Final configuration of the hexamer after 3.5 ns on the membrane (side view). (G) Modeling of multimers at various time points between 1.5 and 4.5 ns (top view). The table to the right margin indicates the inner diameters (ID) and outer diameters (OD) of the multimers created from the conformers obtained at the various molecular dynamics (MD) time points.

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image

Figure 3.  Modeling of docking of nonpropagating and propagating α-syn dimers and multimers on the membrane. Membrane-contacting N-terminal (n-term) regions are designated by boxes and C-terminal (c-term) regions by lines, as viewed perpendicular to the membrane surface. For docking, the second α-syn molecule (α-syn 2) docks to the first (α-syn 1), followed by docking of the third α-syn molecule (α-syn 3) to the second, etc., considering minimal docking energies from all possible docking positions. (A) Non-propagating conformation (head-to-tail) of two α-syn monomers that prevents low-energy docking of additional monomers. (B) Propagating conformation that allows low energy docking of additional monomers added sequentially (in the direction of the arrow). (C) Docking of two α-syn conformers at 4.5 ps on the POPC membrane. (D) Weakly propagating α-syn multimer, composed of four head-to-tail conformers at 4.5 ns (E) Propagating α-syn multimer, composed of five head-to-head conformers at 4.5 ns (F) Electrostatic energies of the complexes growing from one α-syn monomer to a five-monomer complex at 4.5 ns. The propagating multimer has more favorable electrostatic energy than the weakly propagating multimer.

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As previous studies have suggested that the assembly of α-syn into toxic oligomers might involve interactions with the membrane [26,43], we proceeded to simulate the docking of α-syn conformers on a flat surface representing the membrane. The α-syn conformations at 250 ps increments of molecular dynamics were docked with their surfaces facing the membrane (defined as membrane-contacting by the mapas program [44]). These membrane-contacting surfaces were distributed as expected along the N-terminal helices of the α-syn conformers (Fig. 2B). To further verify the conformational changes of α-syn dimers upon interactions with the membrane, we conducted docking of two α-syn 4 ps conformers onto a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane with a grid cell of 1 Å, including the membrane in calculations (Fig. 3C). The electrostatic energy of interaction is around 30–50 kcal·mol−1 for docking of two α-syn molecules. Only minimal differences (< 10% in docking energy values) were detected between molecules docked on the flat surface and molecules docked on the POPC membrane. In general, two possible initial docking configurations for α-syn molecules on the membrane were observed. In the first one, the dimer is arranged in a head-to-tail position and additional monomers cannot easily add to this complex to propagate toward higher order aggregates, as low-energy binding sites do not appear to exist for consecutive docking (Fig. 2A and 3A). It is possible for weakly propagating multimers to form over time up to 4.0 ns (Fig. 3D), however, the binding energies of the growing complexes (Fig. 3F) are significantly less favorable than for propagating configurations (Fig. 3E, Fig. S2), and these species would represent only a small fraction of the total multimers present. Therefore, for our purposes, this head-to-tail configuration of two α-syn monomers is designated a ‘nonpropagating dimer’. In all cases, the nonpropagating interactions involve regions of the N-terminus up to residue 75 of one molecule with residues located on the C-terminal region of the second molecule. In the second configuration, the pair of monomers is oriented ‘head-to-head’ (with tails oriented in similar directions) allowing consecutive docking with similar low energy for the successive molecules of α-syn. We designate this configuration a ‘propagating dimer’ (Figs 2C and 3B,E). Docking additional α-syn monomers (at specified time points) to a single initial propagating dimer resulted in the formation of energetically favorable trimers, tetramers, pentamers, and hexamers on the membrane (Figs 2C–G, 3E, and Fig. S2). We used the molecular dynamics conformations ranging from 1.5 to 5.0 ns for docking, and noted that with longer molecular dynamics simulation times (4.0 ns and later), more residues on the C-terminal tail (residue 110 and above) became involved in intermolecular interactions (Fig. S3). Because the tail of α-syn carries the majority of this protein's positive charge, this might help to explain why there was a significant enhancement of α-syn dimer docking energies (and accordingly the stability of the multimers) after 4.0 ns of simulation (Table 1). Moreover, Fig. S2 shows that the most stable conformation of α-syn occurs after 3.8 ns of molecular dynamics simulation time. For β-syn, the most stable conformations arise between 2.2 and 3.5 ns of simulation (Fig. S2). The most probable α-syn multimers were selected based on the conformers with the most favorable energies of intermolecular interaction between two monomers and the most stable conformers. Six distinct possible multimers were generated as the result of ‘propagating docking’ (Fig. 2G). These multimers formed low energy pentamers and hexamers with different configurations that generated ring-like structures with a central lumen (Fig. 2G). The most stable multimers of α-syn were generated with α-syn conformers from 4.0 ns simulation and later. The theoretical pentameric and hexameric conformations of the α-syn multimers on the membrane are reminiscent of the pore-like appearance of cell-free α-syn aggregates that have been reported by atomic force microscopy (AFM) [26].

Table 1.   Intermolecular interaction energies of propagating α-syn/α-syn dimers docked on the flat membrane. MD, molecular dynamics.
MD time (ns)Electrostatic energy (kcal·mol−1)
1.50−10.6
2.00−10.6
2.50−13.4
3.50−15.1
4.00−19.7
4.50−32.9

α-syn propagating dimers form pore-like structures that are embedded in the membrane

To further investigate how closely the simulation-derived model resembles α-syn aggregates generated in vitro, recombinant α-syn was incubated for various time periods at 65 °C and the preparations analyzed by western blot and electron microscopy. At 15 h of incubation, immunoblot analysis showed the appearance of multiple bands at molecular weight levels consistent with α-syn dimers, trimers, tetramers, and pentamers (Figs 4A,B). After 20 h, higher order aggregates consistent with hexamers were also detected (Fig. 4A,B). Ultrastructurally, after 10 h of incubation, ill-defined globular elements were noted, and around 15 h, ring-like structures ranging in diameter between 9 and 15 nm with a central channel of ∼2–5 nm were found (Fig. 4C–E), while after 20 h fibrils (9–12 nm in diameter) became more apparent (Fig. 4F). Remarkably, the ring-like structures that formed after 15 h of incubation were of similar dimension to the α-syn pentamers and hexamers generated after 4.0 ns of molecular dynamics simulation (Fig. 4K) and further simulations showed that they became embedded in the membrane after relatively short (350 ps) molecular dynamics simulation of the membrane–protein complex (Fig. 5A). During extended simulation times, the α-syn pentamer embeds progressively further into the membrane, reaching 16 Å in the membrane by 800 ps (Fig. 5B–E).

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Figure 4.  Biochemical and ultrastructural analysis of α-syn aggregation, interactions with β-syn, and modeling of ring-like structures. (A) In vitro cell-free aggregation of α-syn monomers into dimers, trimers, tetramers, pentamers, and hexamers over time without (left panel) and with (right panel) the addition of β-syn. (B) Semiquantitative analysis of levels of α-syn multimers over time. (C–F) Electron microscopy analysis of α-syn aggregation over time into ring-like structures and fibrils. (G–J) Electron microscopy analysis demonstrating reduction in α-syn aggregation over time in the presence of β-syn. Scale bar = 20 nm. (K) Superimposition of α-syn pentamer (4.5 ns) onto the ring-like structure detected by electron microscopy. Scale bar = 10 nm.

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image

Figure 5.  Modeling of the embedded α-syn complex in the membrane over time. (A) Top view (at the level of the uppermost membrane-associated atom) of the embedded portion of the α-syn pentamer (350 ps) on the POPC membrane (white, α-syn pentamer; green, membrane phospholipids). Note the penetration of the pentamer into the membrane and the exposed membrane in the center of the α-syn ring-like structure. (B–E) The steps of penetration of the α-syn pentamer into the POPC membrane during 0.8 ns molecular dynamics simulation (B, initial; C, 0.2 ns; D, 0.5 ns; E, 0.8 ns). The depth of protein insertion into the membrane was measured between the uppermost membrane-associated atom and the atom that is embedded deepest into the membrane.

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β-syn interrupts the formation of propagating α-syn dimers

We have previously shown that β-syn is capable of reducing α-syn accumulation and related deficits [23], however, the molecular characteristics for the interactions between these two molecules are unclear. For this purpose, we modeled β- and β-syn, and β- and α-syn heterodimeric interactions. Firstly, theoretical docking of various molecular dynamics conformers of α-syn to conformers of β-syn was performed. All of the docked α-syn–β-syn complexes displayed a significant level of negative electrostatic energy of formation (Table 2). In these simulations, β-syn was able to bind α-syn, creating stable nonpropagating heterodimers, similar to nonpropagating α-syn homodimers (Fig. 6A). Strong electrostatic interactions contributed to the formation of these α- and β-syn heterodimers. For example, complexes between α-syn (2.5 ns) and β-syn (2.2 ns) displayed a minimum intermolecular electrostatic energy of −31.6 kcal·mol−1, while the electrostatic energy of interaction between two α-syn (2.5 ns) conformers that can aggregate into hexamers on the membrane was −13.4 kcal·mol−1. Thus the binding energy between α- and β-syn was significantly lower, and more favorable, than the energy of interaction between two α-syn molecules located on the membrane-like surfaces. The net charge for β-syn (−16 e) at pH 7.0 is much lower than that of α-syn (– 9 e), which might help to explain why it is less likely for β-syn than for α-syn to form propagating dimers in the membrane [23,24].

Table 2.   Intermolecular interaction energies of the β-syn conformers with 1.5 ns molecular dynamics α-syn conformer docked on the flat membrane. MD, molecular dynamics.
β-syn conformer MD time (ns)Electrostatic energy (kcal·mol−1)
Initial−27.4
0.25−22.0
0.50−29.8
0.75−45.9
1.00−37.8
1.50−26.7
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Figure 6.  Molecular modeling of the interactions of β-syn with α-syn monomers and dimers. (A) α-syn and β-syn minimal energy nonpropagating heterodimers. (B) Primary electrostatic interactions in the minimal energy α-syn and β-syn dimer. (C) β-syn minimal energy complex with the α-syn dimer (4.5 ns simulation for α-syn and 2.2 ns simulation for β-syn). This complex does not support further propagation.

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In addition to binding to α-syn monomers, the simulations showed that β-syn interacts with α-syn monomers and propagating dimers (Fig. 6C), which can theoretically form annular-like structures on the membrane. In fact, β-syn binding to earlier α-syn conformers was stronger than that of the next α-syn molecule that participates in propagating membrane-facing pair-wise docking. One β-syn molecule (shown in green in Fig. 6C) docked to an α-syn dimer on the membrane has a position that conflicts with the neighboring α-syn molecules that can flank it from both sides in the possible multimeric complex. Further analysis of the electrostatic energies of interaction between heterodimers starting with the 1.5 ns molecular dynamics conformer showed that in most cases, the energy of interaction between β-syn and α-syn (Table 2) was significantly lower than for α-syn homodimers (−10.6 kcal·mol−1, Table 1). This supports the possibility that β-syn might be able to interrupt the assembly of propagating α-syn homodimers at various stages of the oligomerization process.

β-syn blocks the formation of α-syn ring-like structures and attenuates ion conductance alterations

Previous studies have shown that when β- and α-syn are incubated simultaneously, β-syn reduces α-syn aggregation over time [23,24,45]. However, it is unclear whether β-syn might decrease α-syn aggregation when added after the process of α-syn oligomerization has started. The theoretical model presented in the previous section predicts that under experimental in vitro conditions, addition of β-syn might prevent further aggregation of α-syn (Fig. 4). To investigate this possibility, α-syn was allowed to aggregate and then β-syn was added for various lengths of time. When β-syn was added 1 h after α-syn aggregation started, there was a significant decrease in the subsequent formation of α-syn multimers at the various time points analyzed (Fig. 4A,B). Consistent with the immunoblot studies, ultrastructural analysis showed that β-syn reduced the formation of globular, ring-like, and fibrillar structures (Fig. 4G–J).

As previous studies have suggested that the α-syn ring-like structures might form pores in the membrane that might be responsible for the neurotoxic effects of α-syn oligomers [26,29,46,47], we investigated whether abnormally high levels of ion currents are detected in cells overexpressing α-syn and if this process might be attenuated by β-syn. For this purpose, we recorded and compared whole-cell currents in HEK293T cells transiently transduced with lentiviral vectors expressing α-syn, β-syn, or α-syn and β-syn together (Fig. 7). Immunoblot analysis confirmed that cells expressed comparable levels of α-syn and β-syn (Fig. 7A). Double-labeling verified that in cotransduced cells, green fluorescent protein (GFP) was also expressed with either α-syn or β-syn (Fig. 7B). The target cells (displaying green fluorescence) for electrophysiological measurements were identified by cotransduction with a lenti-GFP vector (Fig. 7C). Cells expressing α-syn showed a significant increase in whole-cell currents elicited by depolarizing the cells from a holding potential of −50 mV to a series of test potentials ranging from −80 to +80 mV (Fig. 7D,E). The current density at +80 mV was 54.8 ± 4.3 in cells transduced with an empty vector, 181.1 ± 18.1 (P < 0.001 vs. vector control) picoamperes/picofarads in α-syn-expressing cells, 64.2 ± 5.3 (P = 0.21) picoamperes/picofarads in cells expressing β-syn, and 78.1 ± 10.4 (P = 0.07) pA/pF in cells transduced with α-syn + β-syn (Fig. 7D,E). Furthermore, the currents in α-syn transduced cells were sensitive to Zn2+ (Fig. 7F). Extracellular application of 5 µm Zn2+ reversibly decreased the currents; the maximal inhibition took place within 3 min (Fig. 7F). These data indicate that α-syn forms a Zn2+-sensitive nonselective cation channel and that coexpression of α-syn with β-syn significantly inhibited the amplitude of currents of the putative α-syn channels.

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Figure 7.  Studies of ion conductance in α-syn and β-syn transduced cells utilizing lentiviral vectors. (A) HEK293T cells transduced with lentiviral vectors encoding α-syn, β-syn, and GFP express comparable protein levels. (B) Double-labeling immunocytochemical analysis of 293T cells cotransduced with lenti-GFP and lenti-αsyn or lenti-βsyn. (C) 293T cells transduced with an empty GFP vector, lenti-αsyn, or lenti-βsyn and cells cotransfected with α-syn and β-syn. The transduced cells are indicated by green fluorescence. Scale bar = 20 µm. (D, E) Representative currents elicited by depolarizing the cells from a holding potential of −50mv to a series of test potentials ranging from −80 to +80mv, and corresponding current–voltage relationship (E; means ± SE) in tranduced cells. (F) Representative currents at +80mv (left panel) before (Cont), during (Zn2+) and after (Wash) application of 500 µm Zn2+. Time course (right panel) of the change in current density before, during, and after extracellular application of Zn2+. The arrows correspond to the currents shown in the left panel (Cont, a; Zn2+, b; and Washout, c).

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Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

The present study showed by utilizing molecular modeling and molecular dynamics simulations, in combination with biochemical and ultrastructural analysis, that α-syn can arrange into homodimers that can adopt nonpropagating and propagating conformations. The evidence predicts that propagating α-syn dimers dock on the membrane surface and can incorporate additional α-syn molecules, leading to the formation of pore-like structures. In contrast, β-syn dimers do not propagate, and when interacting with α-syn aggregates block the propagation of α-syn into multimeric structures. Recent studies have suggested that the transformation of α-syn into a neurotoxic molecule might involve the sequential conversion of α-syn monomers into globular oligomers and then protofibrils [46]. In contrast, α-syn fibrils, which are present in the LBs [6], might represent a mechanism for isolating toxic oligomers [15]. Previous studies have investigated the conformation of α-syn either at the very initial stages of aggregation [21] or during the process of fibril formation [42]. In micelles, α-syn monomers consist of two curved α-helices connected by a short linker in an antiparallel arrangement, followed by a short extended region and a predominantly unstructured mobile tail [21,48]. The molecular dynamics studies described here showed that this structure of α-syn displayed significant changes in the organization of the N-terminal helices from 2 to 3 helices over time, which might lead to more complex membrane interactions.

Computerized analysis predicted that these changes were accompanied over time by alterations in the secondary structure showing that a π-helical conformation appears in the N-terminus in addition to the α-helix. However, confirmation of this structural transformation awaits NMR analysis. Interestingly, molecular modeling of the misfolding of the Alzheimer's disease amyloid-β protein has shown a rapid transition of the N-terminal α-helix 1 into a π-helix [49]. Such conformational changes, in combination with β-hairpin structures, might be essential to the aggregation process [50] and the subsequent formation of pore-like structures.

Under basal conditions, both nonpropagating and propagating dimers might exist, with a higher proportion of dimers exhibiting a nonpropagating conformation. In disorders with α-syn aggregation such as PD it is possible that an increased proportion of propagating dimers might be present. The conditions that might favor an increased ratio of propagating α-syn complexes are unclear, but given the conformational instability of the proteins implied by both experimental and modeling results, it may be highly sensitive to local environmental influences. In support of this, closer association with the membrane has been suggested to induce α-syn oligomerization [35]. It has been reported that small oligomeric forms of α-syn preferentially associate with lipids and cell membranes [35], however, the conformations of α-syn multimers have been difficult to study due to the inability to crystallize the oligomeric form of this protein. Our molecular dynamics studies support the contention that oligomers of α-syn associate with membranes and suggest that propagating dimers might be thermodynamically stable on membranes in association with lipids. Moreover, the simulations and modeling suggest that anchoring of propagating α-syn dimers to the membrane facilitates the incorporation of additional α-syn monomers, leading to the formation of trimers, tetramers, pentamers, and hexamers, the latter oligomers forming ring-like structures.

Recent Raman and AFM studies showed that in vitro early stage oligomers have a globular structure that elongates over time to form protofibrils [42,51]. High-resolution ultrastructural and AFM have suggested that these aggregates might form pore-like structures that appear to be common to those produced by other molecules involved in neurodegeneration [52], including amyloid-β protein [53], British dementia peptide (ABri) [54], Danish dementia peptide (ADan), serum amyloid A, and amylin [55]. In such studies, the dimension of the α-syn disc-like structure was 8–10 nm (outer diameter) with a central pore of 1–2 nm [26]. Similarly, our theoretical studies show that the outer diameter of the α-syn multimers is 9–15 nm, with an inner diameter of 2–5 nm. Consistent with the possibility that these α-syn aggregates might represent active pores, cells transduced with α-syn displayed significant increases in Zn2+-sensitive ion channel activity that might correspond to Zn2+-sensitive nonselective cation channels. The Zn2+-sensitivity of the α-syn pore-like structures is likely to be a result of interactions between Zn2+ and cysteine, histidine or arginine residues, as has been previously shown in the case of Zn2+-sensitive Aβ-derived pore-like structures [56,57]. Specifically, the His residue located at position 50 in the α-syn monomer is a possible candidate for interaction with Zn2+ ions because it is situated near the putative pore region of the α-syn pentamer. The increased ion channel activity observed in the present study is in agreement with recent results showing that human neuronal cells expressing mutant α-syn have high plasma membrane ion permeability that was sensitive to calcium chelators [47]. Taken together, these results support the contention that α-syn aggregates might form functional ion-permeable channels that in turn might play a role in the mechanisms of neurodegeneration in LBD.

Therefore, developing strategies that might prevent α-syn aggregation and subsequent oligomerization into pore-like structures, or compounds that might block such potential ion channels could represent a viable approach to treating disorders characterized by α-syn aggregation. As in other neurodegenerative disorders, such as AD similar pore-like structures are formed [58,59], it is possible that generic antioligomer antibodies that can recognize these assemblies might be useful [60,61]. The presence of the oligomers in the membrane might also facilitate recognition by antibodies that promote clearance of aggregated α-syn [62]. Chaperone molecules such as heat sock proteins and β-syn might also be useful. Remarkably, in support of this possibility, we found that, via interactions with α-syn monomers and multimers, β-syn is capable of preventing further oligomerization. Moreover, we found that β-syn ameliorated the abnormal increase in plasma membrane ion permeability in cells expressing α-syn. These findings might help to explain previous in vitro and in vivo studies showing that β-syn is protective [63]. Therefore, developing molecules that might mimic the effects of β-syn based on the molecular structure observations described here may help in the development of therapeutic strategies to reduce α-syn aggregation in disorders with parkinsonism.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Molecular dynamics simulations and modeling of α-syn and β-syn

α-syn is a natively unfolded molecule [64] in the cytosol that, once it interacts with lipids in the membrane, adopts a helical structure [21,39,40]. Modeling and simulations were based on the previously reported NMR structure of the micelle-bound α-syn (PDB index 1xq8 [21]). The micelle-derived structure of α-syn was used as the starting point for the simulations because it was predicted that this conformation was most likely to lead to oligomerization. For β-syn, a homology model was generated using the Homology module of the insight ii program (Accelrys, San Diego, CA, USA). The molecule was then minimized for 10 000 iterations of steeped descent with the discover program (Accelrys). Molecular dynamics simulations of α-syn and β-syn molecules were conducted using periodic boundary conditions at constant pressure (1 atm) and temperature (310 K) with the water box in which the shortest distance between the protein molecule and the box walls was 30 Å. The simulation system contained 237826 atoms, 41 Na+and 32 Cl counter ions. The namd molecular dynamics program version 2.5 [65] was used with the CHARMM27 force-field parameters [66] to simulate the behavior of α-syn and β-syn molecules in water under normal conditions and the interaction of the POPC membrane with the α-syn aggregates. The temperature was maintained at 310 K by means of Langevin dynamics using a collision frequency of 1 ps−1. A fully flexible cell at constant pressure (1 atm) was employed using the Nosé–Hoover Langevin Piston algorithm [67,68], as used in the namd software package. Initial coordinates were taken from a previously equilibrated 500 ps system. The van der Waals’ interactions were switched smoothly to zero over the region 10 Å and electrostatic interactions were included via the smooth particle-mesh Ewald summation [69]. The impulse-based Verlet-I/r-RESPA method [70,71] was used for multiple time-stepping: 4 fs for the long-range electrostatic forces, 2 fs for short-range nonbonded forces, and 1 fs for bonded forces.

The simulation was done in four steps. Initially the system of protein and water molecules was minimized for 10 000 iterations. Then the system was heated in 0.1° increments and equilibrated for 10 ps; then the molecular dynamics simulation was conducted. Data for analysis were taken between 50 ps and 5.0 ns of the simulation.

Theoretical docking of synucleins to the membrane

Interactions between two α-syn and between α-syn and β-syn monomers were studied using programs hex 4.5 [72,73] and the Docking module of insight ii (Accelrys) with a 1 Å grid and cut-off distance of 20 Å. Taking into account previous studies [35] showing that α-syn aggregation occurs on the membrane, we also studied docking of synuclein molecules on a flat surface representing the plasma membrane. Because the docking configurations of the proteins were mostly open to solution, we used a dielectric constant of 16 to calculate the electrostatic energy for α-syn docking.

For predicting membrane-contacting surfaces of proteins we developed the program mapas, based on calculations of membrane association scores for proteins and protein aggregates (I. Tsigelny, personal communication). This program was applied to intermediate α-syn conformers and top scoring predicted membrane attractive surfaces were used to dock the protein molecules and to subsequently calculate possible α-syn–α-syn docking configurations on the membrane. Low energy α-syn/α-syn propagating complexes were used in simulations of consecutive docking of the next α-syn molecules. We also simulated the behavior of α-syn complexes on the POPC membrane. The explicit (all-atom) membrane models were utilized for simulation.

Immunoblot and electron microscopy studies

In previous studies, we have shown that α-syn can prevent β-syn aggregation in a cell-free system when both synucleins are incubated together at the same time [23]. In this study, we wanted to determine whether β-syn reduces α-syn aggregation after α-syn aggregation has already started. For this purpose, recombinant α-syn (1 µm; Calbiochem, San Diego, CA, USA) was incubated at 65 °C for time periods from 1 to 48 h [31]. Incubation at this temperature allows the study of α-syn aggregation over short periods of time [31]. After 1 h of incubation recombinant β-syn (16 µm, purified as previously described [23,63]) was added to the mix. Samples were subjected to immunoblot analysis with the mouse monoclonal antibody against α-syn (LB509, 1 : 1000; Zymed Laboratories, San Francisco, CA, USA) as previously described [31] and analyzed in the versadoc imaging system using the quantity one software (Bio-Rad, Hercules, CA, USA).

To investigate the ultrastructural characteristics of the synuclein aggregates, 1-µL aliquots of α-syn either alone or in combination with β-syn prepared under identical conditions as for immunoblotting were pipetted onto formvar coated grids, followed by 2% uranyl acetate staining. Grids were analyzed with a Zeiss OM 10 electron microscope as previously described [31].

Preparation and electrophysiological analysis of cells expressing α- and β-syn

HEK293T cells were grown on 25-mm coverslips at 50% confluence and were incubated with lentiviruses expressing α-syn, β-syn, or GFP (each at 1.0 × 107 TU) in 10% fetal calf serum for 24 h at 37 °C, 5% CO2. Lentiviruses were prepared as previously described [74]. The cells were then washed with NaCl/Pi and incubated in Dulbecco's modified Eagle's medium with 10% fetal calf serum for an additional 4 days. The efficiency of transduction of lenti-GFP was >90%. For electrophysiological measurements, whole-cell currents were recorded with an Axopatch-1D amplifier and a DigiData 1200 interface (Axon Instruments, Sunnyvale, CA, USA) using patch-clamp techniques. Patch pipettes (2–3 MΩ) were fabricated on a Sutter electrode puller using borosilicate glass tubes and fire polished on a Narishige microforge. Command voltage protocols and data acquisition were performed using pCLAMP-8 software (Axon Instruments). All experiments were performed at room temperature (22–24 °C). The ionic composition of the external solution was (in mm): NaCl 145, KCl 5, MgCl2 1, CaCl2 2, glucose 10, and Hepes 10 (pH = 7.4). During patch-clamp recording, tetrodotoxin (0.1 µm) and CdCl2 (0.1 mm) were added to the external solution to block voltage-dependent Na+ and Ca2+ channels. The ionic composition of the pipette solution was (in mm): CsCl 150 and Hepes 10 (pH = 7.2). The current–voltage (I–V) relationship was determined by a step voltage protocol of 50 ms duration. The membrane potential was held at −50 mV and stepped to levels between −80 mV and +80 mV in 20-mV increments.

For verification of synuclein expression after lentivirus infection, transduced cells were harvested in lysis buffer and analyzed by immunoblot with antibodies against α-syn (1 : 1000, Chemicon, Temecula, CA, USA), β-syn (prepared as previously described [75]) and GFP (1 : 1000, Chemicon). For immunocytochemistry, cells were cultured on coverslips until 50% confluence and treated as described above, fixed in 4% paraformaldehyde for 20 min, and blocked overnight at 4 °C in 10% fetal calf serum and 5% bovine serum albumin.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

This work was supported by NIH grants AG18440, HL066012, AG022074, AG5131 and DOE INCITE grant. The authors are also grateful to IBM for funding under its Institutes of Innovation program, and for computational support on its BlueGene computers at the San Diego Supercomputer Center and at the Argonne National Laboratory.

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  6. Acknowledgements
  7. References
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