Báo cáo khoa học: Dynamics of a-synuclein aggregation and inhibition of pore-like oligomer development by b-synuclein doc

Although considerable information is available about the conformation of a-syn at the initial and end stages of fibrillation, less is known about the dynamic process of a-syn conversion t

Dynamics of a-synuclein aggregation and inhibition

of pore-like oligomer development by b-synuclein

Igor F Tsigelny1,2, Pazit Bar-On3, Yuriy Sharikov2, Leslie Crews4, Makoto Hashimoto3,

Mark A Miller2, Steve H Keller5, Oleksandr Platoshyn5, Jason X.-J Yuan5and Eliezer Masliah3,4

1 Departments of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, USA

2 San Diego Super Computer Center, University of California San Diego, La Jolla, CA, USA

3 Department of Neurosciences, University of California San Diego, La Jolla, CA, USA

4 Department of Pathology, University of California San Diego, La Jolla, CA, USA

5 Department of Medicine, University of California San Diego, La Jolla, CA, USA

In recent years, new hope for understanding the

patho-genesis of Parkinson’s disease (PD) and Lewy body

dementia (LBD) has emerged with the discovery of

mutations and duplications in the a-synuclein (a-syn)

gene that are associated with rare familial forms of

Parkinsonism [1–3] Moreover, it has been shown that

a-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 a-syn leads to neu-rodegeneration and the characteristic symptoms of LBD are unclear However, recent evidence indicates that abnormal accumulation of misfolded a-syn in the

Keywords

cation channels; modeling; molecular

dynamics; oligomers; synuclein

Correspondence

E Masliah, Department of Neurosciences,

University of California, San Diego, La Jolla,

CA 92093–0624, USA

Fax: +1 858 5346232

Tel: +1 858 5348992

E-mail: emasliah@UCSD.edu

(Received 1 December 2006, revised 26

January 2007, accepted 8 February 2007)

doi:10.1111/j.1742-4658.2007.05733.x

Accumulation of a-synuclein resulting in the formation of oligomers and protofibrils has been linked to Parkinson’s disease and Lewy body demen-tia In contrast, b-synuclein (b-syn), a close homologue, does not aggregate and reduces a-synuclein (a-syn)-related pathology Although considerable information is available about the conformation of a-syn at the initial and end stages of fibrillation, less is known about the dynamic process of a-syn conversion to oligomers and how interactions with antiaggregation chaper-ones such as b-synuclein might occur Molecular modeling and molecular dynamics simulations based on the micelle-derived structure of a-syn showed that a-syn homodimers can adopt nonpropagating (head-to-tail) and propagating (head-to-head) conformations Propagating a-syn dimers

on the membrane incorporate additional a-syn molecules, leading to the formation of pentamers and hexamers forming a ring-like structure In con-trast, b-syn dimers do not propagate and block the aggregation of a-syn into ring-like oligomers Under in vitro cell-free conditions, a-syn aggre-gates formed ring-like structures that were disrupted by b-syn Similarly, cells expressing a-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, a-syn oligomers on the membrane might form pore-like struc-tures, and that the beneficial effects of b-synuclein might be related to its ability to block the formation of pore-like structures

Abbreviations

aa, amino acid; a-syn, a-synuclein; b-syn, b-synuclein; GFP, green fluorescent protein; LBD, Lewy body disease; PD, Parkinson’s disease; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; tg, transgenic.

synaptic terminals and axons plays an important role

[11–14] These studies suggest that a-syn oligomers and

protofibrils rather than fibrils might be the neurotoxic

species [15]

a-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 a-, b-, and c-synucleins a-syn

belongs to a class of so-called ‘naturally unfolded

pro-teins’ [13,18] Such proteins do not have a stable

ter-tiary structure and during their existence change their

conformations Human a-syn is a 140-amino acid (aa)

protein, and b-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, a-syn has a

conformation characterized by two helical domains

interrupted by a short nonhelical turn a-syn contains

a highly amyloidogenic hydrophobic domain in the

N-terminus region (aa 60–95), which is partially absent

in b-syn and might explain why b-syn has a reduced

ability to self-aggregate and form oligomers and fibrils

[23,24] Interestingly, although under physiological

conditions b-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 a- and b-syn [25]

under in vitro cell-free conditions However, previous

studies have shown that in the absence of metals,

b-syn interacts with a-syn and is capable of preventing

a-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

pat-ches It has been proposed that the intermediate a-syn

oligomers form annular protofibrils and pore-like

structures [26–29] The mechanism through which

monomeric a-syn converts into a toxic oligomer and

later into fibrils is currently under intense

investiga-tion Recent reviews indicate that the kinetics of a-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] a-syn oligomerization

might occur on the membrane and involves

interac-tions between hydrophobic residues of the amphipathic

a-helices of a-syn [35] These studies indicate that the

hydrophobic lipid binding domains in the N-terminal

region might be important in modulating a-syn aggre-gation [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 b-syn struc-ture In this context, a recent study has analyzed

by NMR the micelle-bound structure and dynamics of b- and c-syn [41]

Thus, better understanding of the steps involved in the process of a-syn aggregation is important in order

to develop intervention strategies that might prevent

or reverse a-syn oligomerization and toxic conversion The conformational state of a-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 disap-pear upon fibril formation [42]

However, less is known about the dynamic process

of conversion of a-syn at earlier stages and how inter-actions with antiaggregation chaperones such as b-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 tech-niques in combination with biochemical and cell based assays might facilitate understanding the dynamic characteristics and structure of the synuclein aggre-gates In this context, the main objective was to develop a dynamical model for the early steps of a-syn aggregation using computer simulations that includes the process of membrane docking and the potential mechanisms through which b-syn blocks a-syn aggre-gation

Our studies suggest that at early stages, propaga-ting a-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 b-syn The inhibitory effect of b-syn may result from its interac-tion with a-syn, which prevents formainterac-tion of func-tional a-syn channels

Results

Conformational diversity of a-syn and b-syn molecules during molecular dynamics simulations

To better understand the conformational changes that a- and b-syn undergo over time and to model the homo and heterodimeric interactions preventing or

leading to aggregation, molecular dynamics

simula-tions in water were performed based on the

micelle-bound structure of a-syn as resolved by NMR This

approach allows the investigation of the dynamic

structural changes of the folded a-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 a-syn molecular structure form an angle

around 55 ± 3 that decreases to around 42–44

dur-ing the first 2.0 ns of the simulation, and then

increa-ses 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 a-syn

trans-form 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

dif-ferent seed numbers, for 3.0 ns These additional data corroborate the initial results, showing that over time a-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 a-syn is highly sta-ble and did not return to an unfolded state even though the molecular dynamics simulations were per-formed using the water box to simplify the procedure

At time zero, b-syn has a structural organization close to the initial structure of a-syn (Fig 1C,D) Unlike a-syn, the curved helix at residues 46–84 of b-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 conform-ational changes, mostly in the C-terminal tail and the angle between the N-terminal helices (Fig, 1C,D) Compared with a-syn, the C-terminal tail of b-syn displayed greater motility

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

Further analysis consisted of determining changes in

secondary structure of a-syn and b-syn over time

After 500 ps of simulation for a-syn a coiled region

appeared, interrupting the a-helix around residue 68

(Fig S1A) Beginning at 750 ps, turns appeared in the

a-helical structure around residue 47, then after 1.0 ns

this region was transformed into a p-helix (Fig S1A)

The length of this p-helix increased with time, and

from 3.0 ns covered the region from residues 45–55 In

another part of the sequence, a second p-helix

appeared from 2.0 ns that includes residues 74–83

(Fig S1A)

Changes in b-syn secondary structure over time

con-sisted of transformations from a bended a-helical

structure to the structure with two straight helices with

further conversion to p-helical structure around residue

30 and the N-terminus region (Fig S1B) The

C-ter-minal region beyond residue 70 showed limited

chan-ges in secondary structure (Fig S1B) Overall, b-syn

underwent significantly fewer changes in secondary

structure than a-syn during molecular dynamics

simu-lations

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

The first studies of the interactions of a-syn were per-formed by docking the initial structures of two a-syn monomers on a flat surface without specific limita-tions Under these conditions, some low energy com-plexes 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-ter-mini of a-syn monomers (Fig 2A and 3A) These a-syn homodimers can interact with additional a-syn molecules, but further simulations indicate that the resulting higher order aggregates are not likely to pro-duce continuously propagating multimers on the mem-brane For the nonpropagating a-syn homodimers, usually only one a-syn has the membrane binding sur-face, such as for the 1.5 ns molecular dynamics con-formers (Fig 2A and 3A)

Fig 2 Molecular modeling of nonpropagating and propagating a-syn aggregates on the membrane (A) a-syn minimal energy nonpropagating dimers (head-to-tail) (B) a-syn conformer at 4.0 ns oriented to the membrane surface (membrane-contacting residues depicted in orange) (C–E) Propagating a-syn multimers on the membrane at 3.5 ns (C) Dimer (D) tetramer, and (E) hexamer Multimers can be formed by dock-ing of a-syn monomers to a-syn propagatdock-ing dimers, or by addition of a-syn dimers to a-syn propagatdock-ing dimers, with either scenario result-ing in the same final hexamer structure (F) Final configuration of the hexamer after 3.5 ns on the membrane (side view) (G) Modelresult-ing 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.

As previous studies have suggested that the assembly

of a-syn into toxic oligomers might involve interactions

with the membrane [26,43], we proceeded to simulate

the docking of a-syn conformers on a flat surface

repre-senting the membrane The a-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 a-syn

conformers (Fig 2B) To further verify the

conforma-tional changes of a-syn dimers upon interactions with

the membrane, we conducted docking of two a-syn

4 ps conformers onto a

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane with a grid cell of

1 A˚, including the membrane in calculations (Fig 3C)

The electrostatic energy of interaction is around 30–50 kcalÆmol)1 for docking of two a-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 a-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 aggre-gates, 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 3 Modeling of docking of nonpropagating and propagating a-syn dimers and multimers on the membrane Membrane-contacting N-ter-minal (n-term) regions are designated by boxes and C-terN-ter-minal (c-term) regions by lines, as viewed perpendicular to the membrane surface For docking, the second a-syn molecule (a-syn 2) docks to the first (a-syn 1), followed by docking of the third a-syn molecule (a-syn 3) to the second, etc., considering minimal docking energies from all possible docking positions (A) Non-propagating conformation (head-to-tail) of two a-syn monomers that prevents low-energy docking of additional monomers (B) Propagating conformation that allows low energy dock-ing of additional monomers added sequentially (in the direction of the arrow) (C) Dockdock-ing of two a-syn conformers at 4.5 ps on the POPC membrane (D) Weakly propagating a-syn multimer, composed of four head-to-tail conformers at 4.5 ns (E) Propagating a-syn multimer, com-posed of five head-to-head conformers at 4.5 ns (F) Electrostatic energies of the complexes growing from one a-syn monomer to a five-monomer complex at 4.5 ns The propagating multimer has more favorable electrostatic energy than the weakly propagating multimer.

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

a-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

configur-ation, the pair of monomers is oriented ‘head-to-head’

(with tails oriented in similar directions) allowing

con-secutive docking with similar low energy for the

successive molecules of a-syn We designate this

confi-guration a ‘propagating dimer’ (Figs 2C and 3B,E)

Docking additional a-syn monomers (at specified time

points) to a single initial propagating dimer resulted in

the formation of energetically favorable trimers,

tetra-mers, pentatetra-mers, 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

dynam-ics 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 a-syn carries the majority of this

protein’s positive charge, this might help to explain

why there was a significant enhancement of a-syn dimer

docking energies (and accordingly the stability of the

multimers) after 4.0 ns of simulation (Table 1)

More-over, Fig S2 shows that the most stable conformation

of a-syn occurs after 3.8 ns of molecular dynamics

simulation time For b-syn, the most stable

conforma-tions arise between 2.2 and 3.5 ns of simulation

(Fig S2) The most probable a-syn multimers were

selected based on the conformers with the most

favora-ble energies of intermolecular interaction between two

monomers and the most stable conformers Six distinct

possible multimers were generated as the result of

‘pro-pagating docking’ (Fig 2G) These multimers formed

low energy pentamers and hexamers with different

con-figurations that generated ring-like structures with a

central lumen (Fig 2G) The most stable multimers of

a-syn were generated with a-syn conformers from 4.0 ns simulation and later The theoretical pentameric and hexameric conformations of the a-syn multimers

on the membrane are reminiscent of the pore-like appearance of cell-free a-syn aggregates that have been reported by atomic force microscopy (AFM) [26]

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

To further investigate how closely the simulation-derived model resembles a-syn aggregates generated

in vitro, recombinant a-syn was incubated for various time periods at 65C and the preparations analyzed by western blot and electron microscopy At 15 h of incu-bation, immunoblot analysis showed the appearance of multiple bands at molecular weight levels consistent with a-syn dimers, trimers, tetramers, and pentamers (Figs 4A,B) After 20 h, higher order aggregates consis-tent 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 diam-eter) became more apparent (Fig 4F) Remarkably, the ring-like structures that formed after 15 h of incubation were of similar dimension to the a-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 rel-atively short (350 ps) molecular dynamics simulation of the membrane–protein complex (Fig 5A) During extended simulation times, the a-syn pentamer embeds progressively further into the membrane, reaching 16 A˚

in the membrane by 800 ps (Fig 5B–E)

b-syn interrupts the formation of propagating a-syn dimers

We have previously shown that b-syn is capable of reducing a-syn accumulation and related deficits [23], however, the molecular characteristics for the interac-tions between these two molecules are unclear For this purpose, we modeled b- and b-syn, and b- and a-syn heterodimeric interactions Firstly, theoretical docking

of various molecular dynamics conformers of a-syn to conformers of b-syn was performed All of the docked a-syn–b-syn complexes displayed a significant level of negative electrostatic energy of formation (Table 2) In these simulations, b-syn was able to bind a-syn, cre-ating stable nonpropagcre-ating heterodimers, similar to nonpropagating a-syn homodimers (Fig 6A) Strong

Table 1 Intermolecular interaction energies of propagating

a-syn ⁄ a-syn dimers docked on the flat membrane MD, molecular

dynamics.

MD time (ns) Electrostatic energy (kcalÆmol)1)

electrostatic interactions contributed to the formation

of these a- and b-syn heterodimers For example,

com-plexes between a-syn (2.5 ns) and b-syn (2.2 ns)

dis-played a minimum intermolecular electrostatic energy

of )31.6 kcalÆmol)1, while the electrostatic energy of

interaction between two a-syn (2.5 ns) conformers that

can aggregate into hexamers on the membrane

was)13.4 kcalÆmol)1 Thus the binding energy between

a- and b-syn was significantly lower, and more

favora-ble, than the energy of interaction between two a-syn

molecules located on the membrane-like surfaces The

net charge for b-syn ()16 e) at pH 7.0 is much lower

than that of a-syn (– 9 e), which might help to explain

why it is less likely for b-syn than for a-syn to form

propagating dimers in the membrane [23,24]

In addition to binding to a-syn monomers, the

simu-lations showed that b-syn interacts with a-syn

mono-mers and propagating dimono-mers (Fig 6C), which can

theoretically form annular-like structures on the

mem-brane In fact, b-syn binding to earlier a-syn

conform-ers was stronger than that of the next a-syn molecule

that participates in propagating membrane-facing

pair-wise docking One b-syn molecule (shown in green in Fig 6C) docked to an a-syn dimer on the membrane has a position that conflicts with the neighboring a-syn molecules that can flank it from both sides in the poss-ible multimeric complex Further analysis of the elec-trostatic energies of interaction between heterodimers starting with the 1.5 ns molecular dynamics conformer showed that in most cases, the energy of interaction between b-syn and a-syn (Table 2) was significantly lower than for a-syn homodimers ()10.6 kcalÆmol)1, Table 1) This supports the possibility that b-syn might

be able to interrupt the assembly of propagating a-syn homodimers at various stages of the oligomerization process

b-syn blocks the formation of a-syn ring-like structures and attenuates ion conductance alterations

Previous studies have shown that when b- and a-syn are incubated simultaneously, b-syn reduces a-syn aggregation over time [23,24,45] However, it is unclear

Fig 4 Biochemical and ultrastructural analysis of a-syn aggregation, interactions with b-syn, and modeling of ring-like structures (A) In vitro cell-free aggregation of a-syn monomers into dimers, trimers, tetramers, pentamers, and hexamers over time without (left panel) and with (right panel) the addition of b-syn (B) Semiquantitative analysis of levels of a-syn multimers over time (C–F) Electron microscopy analysis of a-syn aggregation over time into ring-like structures and fibrils (G–J) Electron microscopy analysis demonstrating reduction in a-syn aggrega-tion over time in the presence of b-syn Scale bar ¼ 20 nm (K) Superimposition of a-syn pentamer (4.5 ns) onto the ring-like structure detec-ted by electron microscopy Scale bar ¼ 10 nm.

whether b-syn might decrease a-syn aggregation when

added after the process of a-syn oligomerization has

started The theoretical model presented in the

previ-ous section predicts that under experimental in vitro

conditions, addition of b-syn might prevent further

aggregation of a-syn (Fig 4) To investigate this

possi-bility, a-syn was allowed to aggregate and then b-syn

was added for various lengths of time When b-syn

was added 1 h after a-syn aggregation started, there

was a significant decrease in the subsequent formation

of a-syn multimers at the various time points analyzed (Fig 4A,B) Consistent with the immunoblot studies, ultrastructural analysis showed that b-syn reduced the formation of globular, ring-like, and fibrillar structures (Fig 4G–J)

As previous studies have suggested that the a-syn ring-like structures might form pores in the membrane that might be responsible for the neurotoxic effects of a-syn oligomers [26,29,46,47], we investigated whether abnormally high levels of ion currents are detected in cells overexpressing a-syn and if this process might be attenuated by b-syn For this purpose, we recorded and compared whole-cell currents in HEK293T cells transiently transduced with lentiviral vectors expressing a-syn, b-syn, or a-syn and b-syn together (Fig 7) Immunoblot analysis confirmed that cells expressed comparable levels of a-syn and b-syn (Fig 7A) Dou-ble-labeling verified that in cotransduced cells, green fluorescent protein (GFP) was also expressed with either a-syn or b-syn (Fig 7B) The target cells (dis-playing green fluorescence) for electrophysiological measurements were identified by cotransduction with a lenti-GFP vector (Fig 7C) Cells expressing a-syn

Fig 5 Modeling of the embedded a-syn

complex in the membrane over time (A)

Top view (at the level of the uppermost

membrane-associated atom) of the

embed-ded portion of the a-syn pentamer (350 ps)

on the POPC membrane (white, a-syn

pen-tamer; green, membrane phospholipids).

Note the penetration of the pentamer into

the membrane and the exposed membrane

in the center of the a-syn ring-like structure.

(B–E) The steps of penetration of the a-syn

pentamer into the POPC membrane during

0.8 ns molecular dynamics simulation (B,

ini-tial; C, 0.2 ns; D, 0.5 ns; E, 0.8 ns) The

depth of protein insertion into the

mem-brane was measured between the

upper-most membrane-associated atom and the

atom that is embedded deepest into the

membrane.

Table 2 Intermolecular interaction energies of the b-syn

conform-ers with 1.5 ns molecular dynamics a-syn conformer docked on the

flat membrane MD, molecular dynamics.

b-syn conformer MD time (ns)

Electrostatic energy (kcalÆmol)1)

showed a significant increase in whole-cell currents

eli-cited 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

con-trol) picoamperes⁄ picofarads in a-syn-expressing cells,

64.2 ± 5.3 (P¼ 0.21) picoamperes ⁄ picofarads in cells

expressing b-syn, and 78.1 ± 10.4 (P¼ 0.07) pA ⁄ pF

in cells transduced with a-syn + b-syn (Fig 7D,E)

Furthermore, the currents in a-syn transduced cells

were sensitive to Zn2+(Fig 7F) Extracellular

applica-tion of 5 lm Zn2+ reversibly decreased the currents;

the maximal inhibition took place within 3 min

(Fig 7F) These data indicate that a-syn forms a Zn2+

-sensitive nonselective cation channel and that

coexpres-sion of a-syn with b-syn significantly inhibited the

amplitude of currents of the putative a-syn channels

Discussion

The present study showed by utilizing molecular

mode-ling and molecular dynamics simulations, in

combina-tion with biochemical and ultrastructural analysis, that

a-syn can arrange into homodimers that can adopt nonpropagating and propagating conformations The evidence predicts that propagating a-syn dimers dock

on the membrane surface and can incorporate addi-tional a-syn molecules, leading to the formation of pore-like structures In contrast, b-syn dimers do not propagate, and when interacting with a-syn aggregates block the propagation of a-syn into multimeric struc-tures Recent studies have suggested that the transfor-mation of a-syn into a neurotoxic molecule might involve the sequential conversion of a-syn monomers into globular oligomers and then protofibrils [46] In contrast, a-syn fibrils, which are present in the LBs [6], might represent a mechanism for isolating toxic oligo-mers [15] Previous studies have investigated the con-formation of a-syn either at the very initial stages of aggregation [21] or during the process of fibril forma-tion [42] In micelles, a-syn monomers consist of two curved a-helices connected by a short linker in an anti-parallel 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 a-syn displayed significant changes in the organization of the

N-ter-Fig 6 Molecular modeling of the interactions of b-syn with a-syn monomers and dimers (A) a-syn and b-syn minimal energy nonpropagat-ing heterodimers (B) Primary electrostatic interactions in the minimal energy a-syn and b-syn dimer (C) b-syn minimal energy complex with the a-syn dimer (4.5 ns simulation for a-syn and 2.2 ns simulation for b-syn) This complex does not support further propagation.

minal 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

secon-dary structure showing that a p-helical conformation

appears in the N-terminus in addition to the a-helix

However, confirmation of this structural

transforma-tion awaits NMR analysis Interestingly, molecular

modeling of the misfolding of the Alzheimer’s disease

amyloid-b protein has shown a rapid transition of

the N-terminal a-helix 1 into a p-helix [49] Such

con-formational changes, in combination with b-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 propor-tion of dimers exhibiting a nonpropagating conforma-tion In disorders with a-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 a-syn com-plexes are unclear, but given the conformational insta-bility 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 a-syn oligomerization [35] It has been reported that small oligomeric forms of a-syn preferentially asso-ciate with lipids and cell membranes [35], however, the

Fig 7 Studies of ion conductance in a-syn and b-syn transduced cells utilizing lentiviral vectors (A) HEK293T cells transduced with lentiviral vectors encoding a-syn, b-syn, and GFP express comparable protein levels (B) Double-labeling immunocytochemical analysis of 293T cells cotransduced with lenti-GFP and lenti-asyn or lenti-bsyn (C) 293T cells transduced with an empty GFP vector, lenti-asyn, or lenti-bsyn and cells cotransfected with a-syn and b-syn The transduced cells are indicated by green fluorescence Scale bar ¼ 20 lm (D, E) Representa-tive 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 (Zn 2+ ) and after (Wash) application of 500 l M Zn 2+ Time course (right panel) of the change in current density before, during, and after extracellular application of Zn 2+ The arrows correspond to the currents shown in the left panel (Cont, a; Zn 2+ , b; and Washout, c).