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Cytotoxicity is attributed to a soluble aSN oligomeric species that permeabilizes membranes significantly more than monomers and fibrils. In humans, t...
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#-synuclein from animal species show low fibrillation propensity and low oligomer membrane disruption Cagla Sahin, Lars Kjær, Mette Solvang Christensen, Jannik Nedergaard Pedersen, Gunna Christiansen, Adriana-Michelle Wolf Pérez, Ian Max Moller, Jan Enghild, Jan Skov Pedersen, Knud Larsen, and Daniel Erik Otzen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00627 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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Biochemistry

α-synuclein from animal species show low fibrillation propensity and low oligomer membrane disruption Cagla Sahina,b§, Lars Kjær1§, Mette Solvang Christensena, Jannik N. Pedersena, Gunna Christiansenc, Adriana-Michelle Wolf Péreza, Ian Max Møllerb, Jan J. Enghildb, Jan S. Pedersena,d, Knud Larsene and Daniel E. Otzena,b* a

iNANO, Aarhus University, Gustav Wieds Vej 14, DK – 8000 Aarhus C

b

Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK –

8000 Aarhus C c

Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4, DK – 8000 Aarhus C

d

Department of Chemistry, Aarhus University, Langelandsgade 140, DK – 8000 Aarhus C

e

Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, DK –

8000 Aarhus C §

Equal contributors.

* To whom correspondence should be addressed. E-mail [email protected]

Running title: Reduced aggregation of animal α-synuclein

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ABSTRACT The intrinsically disordered protein α-synuclein (aSN) forms insoluble aggregates in the brains of Parkinson’s Disease patients. Cytotoxicity is attributed to a soluble aSN oligomeric species which permeabilizes membranes significantly more than monomers and fibrils. In humans, the mutation A53T induces early-onset PD and increases aSN oligomerization and fibrillation propensity, but Thr53 occurs naturally in aSN of most animals. We compared aSN from elephant, bowhead whale and pig with human aSN. While all three animal aSN showed significantly reduced fibrillation behavior, elephant aSN formed much more oligomer, and pig aSN much less, than human aSN. However, all animal aSN oligomers showed decreased permeabilization towards anionic lipid vesicles, indicative of lowered cytotoxicity. These animal aSN share three substitutions compared to human aSN: A53T, G68E and V95G. We analyzed aggregation and membrane binding of all 8 mutants combining these three mutations. While G68E is particularly important in reducing the fibrillation and possible toxicity, the greatest effect is seen when all three mutations are present. Thus a small number of mutations can significantly reduce aSN toxicity.

Key words: phylogenetic substitutions; animals; aggregation; membrane permeabilization; mutagenesis

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Biochemistry

INTRODUCTION Alpha-synuclein (aSN) is a 140-residue intrinsically disordered protein with a potential role in neurotransmitter vesicle release 1, 2. Binding of aSN to anionic lipid vesicles 3 or anionic micelles 4 in vitro also induces α-helical structure in the N-terminal region. aSN is also associated with neuropathological conditions in both Alzheimer’s disease and Parkinson’s disease (PD) duplication and triplication of the SNCA gene lead to PD H50Q, G51D and A53T induce early-onset PD

7, 8

:

while mutations such as A30P, E46K,

6, 9-13

. All these mutations except A30P are localized

in α-helix 2 of aSN, formed when aSN binds to lipid vesicles stabilizes and protects its target membrane

5, 6

14-16

. Lipid-mediated folding of aSN

17

. In turn, membrane binding prevents aSN from

18

aggregating . Hence the mutations E46K, H50Q, G51D and A53T could also affect lipid-binding, contributing to aSN pathology. Under pathological conditions, aSN adopts a β-sheet rich conformation in misfolded oligomers, fibrils and Lewy bodies 19. Both wildtype (WT) and mutated (A30P and A53T) aSN form insoluble fibrillary aggregates with anti-parallel β-sheet structure upon incubation in vitro

20, 21

. Aggregate

formation is accelerated in both of these PD-linked mutations 20; A53T aggregates the fastest while A30P has a higher propensity than WT to form dense bundled fibril networks 22. For several neurodegenerative diseases including PD, oligomers rather than fibrils have been proposed to be the toxic species, since they can perturb and permeabilize membranes oligomers are highly stable and consist of around 30 aSN monomers compact core surrounded by a more unstructured corona

23, 25

23, 24

. aSN

forming a relatively

26

. Hydrogen-deuterium exchange MS

identifies the protected (i.e. hydrogen-bonded) region to span residues 18-90, while residues 1-17 and 90-140 are not significantly protected, indicating lack of persistent structure. Yet only residues 100-140 are visible in the oligomeric structure by solution NMR

27

, indicating that this region is

highly mobile. The N-terminal region of aSN is involved in membrane interactions in both the monomeric

28, 29

and oligomeric state29, and this may affect the mobility of this region of the

oligomer. In contrast to the extensive studies of PD-accelerating aSN mutations, there has been little focus on mutations which delay it. We reasoned that such mutations could be identified in organisms with a comparable or longer life expectancy than humans. Thr is found naturally in position 53 in all nonhuman species 30, ranging from fish to mammals, many of which are known for their longevity. In silico studies have identified twelve phylogenetic substitutions in aSN; apart from the PD-related 3

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T53A substitution they are all in the NAC region or C-terminal tail

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31

. Nevertheless, they are

predicted to have significant effects on the structure (backbone torsion angle) in the N-terminal region of aSN (res. 32-58). Yet the connection between mutations found in humans, short-lived and long-lived organisms, and the aggregative properties of aSN remains unclear. Here we examine the aggregative properties of human aSN versus aSN from three different animals, bowhead whale (Balaena mysticetus), a species known to live for up to 200 years 32, African elephant (Loxodonta africana) which has a maximum life-span of 60 years under natural conditions (which compares favorably with the estimated 30-year life expectancy of Paleolithic man), and as a control for a shorter lived organism (~20 years), domestic pig (Sus scrofa). The three substitutions which all three animals share relative to human aSN (A53T, G68E, V95G) completely abolish both fibrillation and oligomerization in pig and severely reduce elephant and whale oligomers’ membrane permeabilization (despite an increase in oligomerization in elephant aSN). We also dissect the contributions of the three mutations and identify G68E as the main contributor. Taken together, the present data support the proposal that residues at position 53, 68 and 95 protect from neurodegenerative diseases compared to the human variety, by decreased aggregation propensities and lowered oligomeric vesicle permeabilization.

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Biochemistry

MATERIALS AND METHODS Materials: Human aSN was purified by recombinant expression in E. coli with a codon-optimized expression vector as described 26. All chemicals were of the highest possible purity and were from Sigma Aldrich (St. Louis, MO) unless otherwise stated. Lipids were from Avanti Polar Lipids (Alabaster, AL). Extraction of nucleic acids and cDNA synthesis: RNA used for molecular cloning of SNCA encoding aSN was isolated from pig frontal cortex, elephant lymph node and bowhead kidney. Tissues were pulverized in liquid nitrogen after dissection. Total RNA was isolated by the RNeasy method (Qiagen, Hilden, Germany). The integrity of the RNA samples was verified by ethidium bromide staining of the ribosomal RNA on 1% agarose gels. Synthesis of cDNA used for cloning was conducted with 5 µg of total RNA using SuperScript II RNase H- reverse transcriptase (Invitrogen, Carlsbad, CA). cDNA synthesis was initiated by heating of total RNA, oligo(dT)12-18 primer, dNTP at 65 oC for 5 min, followed by the addition of 200 U reverse transcriptase and then incubation at 42 oC for 50 min, followed by 70 oC for 15 min. Cloning of pig SNCA cDNA: Pig, elephant and bowhead SNCA cDNA were isolated using a RTPCR cloning approach. PCR primers for molecular cloning of bowhead SNCA were derived from genomic and transcript sequences 32. The RT-PCR reaction mix contained 2.5 µl cDNA synthesized from RNA isolated from frontal cortex and pituitary gland, 1.5 mM MgCl2, 0.2 mM dNTP, 0.5 µM of each SNCA sense and antisense species-specific primers (Table S1) and 1U Phusion DNA polymerase (Finnzymes, Thermo Fisher Scientific, Waltham, MA), in a total volume of 25 µl. PCR amplification was accomplished by employing the following program: denaturation at 95 oC for 2 min., 10 cycles of touchdown (-0.5 oC per cycle) 95 oC for 20 s, 60 oC for 30 s, 72 oC for 45 s, followed by 25 cycles of 95 oC for 20 s, 55 oC for 30 s, 72 oC for 45 s. The PCR program was finished with an extension step at 72 oC for 5 min. 25 µl of the amplification product was applied to a 1% agarose gel and visualized after electrophoresis by ethidium bromide staining. Fluorescent bands of approx. 420 bp for elephant and bowhead SNCA and an 800 bp band for pig SNCA were isolated and eluted using the Qiaquick Gel Extraction kit from Qiagen. The eluted PCR products were cloned directly into the pCR TOPO 2.1 vector (Invitrogen) and sequenced in both directions. The cloned pig SNCA sequence was used as template in a subsequent PCR reaction containing linkers for subcloning into the expression vector pET11d using restriction sites BamHI and NcoI which are compatible with BclI and PciI, respectively. Primers for subcloning SNCAPCI-F and SNCA-BCL-R are shown in Table 1. Elephant and whale cDNA were cloned into a pET28a vector 5

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from the previously purified SNCA by Mutagenex (Suwanee, GA) and mutated to replace any TAC Tyr codons with TAT (TAC codons can cause misincorporation of Cys into recombinant aSN 33). The vectors were transformed into E. coli BL21 DE3 T1 cells by electroporation and selected with kanamycin, and aSN was expressed and purified as described

26

. Identity was confirmed by MS

-1

analysis. The theoretical extinction coefficient of 0.412 (mg/mL) known for human aSN, was also applicable to all variants for concentration determination. Analysis of fibrils: Fibrillation of 1mg/mL aSN was carried out as described

34

. Transmission

electron microscopy (TEM) pictures were recorded using phosphotungstic acid staining as described 35. FTIR and far-UV CD spectra were recorded as described 26 with the modification that samples for CD were sonicated using a Qsonica sonicator with 1/16” tip at 20% intensity on 5 s on / 5 s off for 1 min to reduce light scattering. CD spectra were measured from 250 nm to 195 nm at a bandwidth of 2 nm, a scanning speed of 50 nm/min and a response time of 2 s using a 0.1 cm quartz cuvette. To determine the amount of soluble material left after fibrillation, samples were spun down for 10 min at 21 000 g at room temperature and the pellet and supernatant run on a non-reducing 415 % SDS-PAGE gel (Bio-Rad, Hercules, CA). Gel bands were quantified using imageJ. Binding of monomeric aSN to vesicles: Vesicles were made by suspending 1,2-dimyristoyl-sn-3phosphatidylglycerol (DMPG) in PBS buffer to 2 mg/mL and freeze thawing the solution 10 times, followed by extrusion through a 200 nm filter 10 times

27

. Solutions of 0.2 mg/mL aSN and 1

mg/mL DMPG vesicles were incubated at the start temperature for >1 min prior to measurement. CD spectra and thermal scans were recorded on a Jasco J-810 spectrometer

36

. Spectral data are

excluded where the PMT voltage was < 600 volts. A combined temperature and wavelength scan was carried out with a scan rate of 30 ˚C/h at 220 nm for the temperature scan, and wavelength scans were measured from 250 nm to 195 nm at bandwidth of 2 nm, a scanning speed of 50 nm/min and a response of 2 seconds using a 0.1 cm quartz cuvette. Oligomer preparation: aSN oligomers were purified on an ÄKTA Basic system (GE Healthcare, Little Chalfont, UK). 3 mg/mL aSN was incubated on a shaking incubator for 0, 5, 10, and 26 h at 37 °C. The solution was then filtered and separated on a Superose 6 10/300 column with PBS buffer at a flow rate of 0.5 mL/min, while monitoring absorbance at 215 nm. Integration was carried out on half peaks (right hand half for oligomer, left hand half for monomer) to avoid overlap with void volume and monomer degradation species. For consistency, integration was carried out even in the absence of overlapping peaks. Oligomer peaks were collected in 0.5 mL fractions and concentrated in 15 mL Amicon spin filters with 10 kDa cut-off (aSN oligomer mass ≈ 420 kDa). 6

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Biochemistry

Calcein Release: Calcein release was either done at a concentration series from 10-6 – 10-1 mg/mL oligomer (animal variants), or measured at a final concentration of 4.14 µg/ml oligomer (mutants). All measurements were in triplicate. Calcein-filled vesicles were prepared with 10 mg/mL of DOPG and 40 mM calcein in milliQ water which was subjected to 10 freeze-thawing cycles, followed by 20 rounds of extrusion through a 200 nm filter. The solution was passed over a PD 10 column (GE Healthcare) and fractions containing calcein were identified by fluorescence (excitation/emission at 495/515 nm). Typically the first 0.5 mL was discarded and the second 0.5 mL were collected for the assay. 2 µL of calcein vesicles were added to 138 µL of PBS and fluorescence was monitored on a Varioskan Flash fluorimeter (Thermo Fisher Scientific) with excitation/emission at 495/515 nm, 5 nm bandwith, 100 ms measurement using top optics with autorange. Subsequently 10 µL oligomer was added and fluorescence was measured over 40 min. Finally 1 µL of 1% Triton X-100 was added to each well to lyse all vesicles, and the fluorescence was allowed to stabilize for 10 min. Calcein release was calculated using the equation: Percentage release= 100*(F40-Fstart)/(Ffinal-Fstart)

(1)

where F40 is fluorescence after 40 min of exposure to the oligomers, Fstart is fluorescence prior to adding oligomers and Ffinal is fluorescence 10 min after addition of Triton X-100. Experiments were carried out on 3 independent occasions with different batches of oligomer. Small-angle X-ray scattering (SAXS): aSN oligomer samples for SAXS were purified by gel filtration and concentrated as above. SAXS experiments were performed at Aarhus University 37 on a NanoSTAR SAXS camera from Bruker AXS with a rotating Cu anode x-ray source with a wavelength of λ = 1.54 Å and home-built scatterless slits 38. Samples were measured for 1 h at 20 °C. PBS buffer was used for background subtraction and water used as a calibration standard using the SUPERSAXS program package (Oliveira, C.L.P. and Pedersen J.S, unpublished). Data is plotted as a function of the scattering vector q = 4sin(θ)/λ. Using the Indirect Fourier Transformation procedure (IFT)

39

with the home written program WIFT

40, 41

, we determined the

pair-distance distribution function (p(r)) to obtain model-independent information on our system. The p(r) function gives information on real-space distances as a histogram of distances between points, weighted by the excess electron density and the distance. The SAXS data from the oligomers were fitted to a core-shell model similar to the one described in 23

with a prolate ellipsoidal core surrounded by a shell of flexible protein.

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Let M be the total mass of a monomeric protein and f, the fraction of the protein in the shell. Denoting the number of monomers in the oligomer by N, the mass in the shell is Mshell = fMN and the mass in the core is Mcore = (1 − f)MN. By multiplying these masses by the scattering length per unit mass ρm, the scattering mass (SM) of shell and core are, respectively, SMshell = Mshell ρm and SMcore = Mcore ρm. For the core-shell ellipsoidal model, the scattering form factor of the average structure is: " !

 =   #

  

    !  − 

−    

. = and



!   $%%  ! ( sin, -,

30sin. − . cos.3 .4

(3)

 = 5 sin! , + 6 ! cos! ,½

(4)

 = 5 sin! , + 6 ! cos ! ,½

where  =

9" 4

4 5 ,  =

9" 4

(2)

 $%%  ! &

+     where



(5)

4 5 , ε is the eccentricity of the ellipsoidal core, Rtot is the outer

radius of the particle, Rcore is the core radius and Rtot – Rcore=εtotRtot – εRcore = D, where D is the shell thickness. The parameter εtot is the eccentricity of the shell-water interface. In the model, the interfaces are graded and the terms  

   /! $

are the interface smearing functions that make the

interface between core and shell, and between shell and solvent, respectively, graded. The parameter σi is the width of the smearing function. The scattering from the internal polymer-like structure of the shell also has to be included, so that the total intensity of the model is 42: ; =



> + < =

 ?=@ABB

10oC does not show two transitions between 10 and 30 oC, but only one transition coincident with the melting point of DMPG (23 °C). The trough for these mutants is much shallower and narrower than for human aSN. Relative to human aSN, the change in ellipticity (pre-transition level minus trough level) is only around 13, 24, 32 and 34% for pig, whale and elephant aSN. Also, all mutants 14

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Biochemistry

undergo a sharp melting out transition over the 25-40 oC range, with midpoint temperatures of approximately 26, 28, 30 and 34 oC for pig, whale and elephant aSN, followed by a more diffuse melting out at higher temperatures. These data highlight the mutants’ reduced ability to bind to vesicles (leading to helical structure) and consequent lower thermal resistance. Based on these data, we can rank the vesicle binding of the aSN variants as human >> elephant > whale > pig. This ranking corresponds nicely to fibrillation propensities in ThT and EM experiments and β-sheet levels detected by CD. Oligomers of elephant aSN are larger, more stable and show decreased membrane permeabilization compared to human aSN We next turned to an analysis of the different species’ ability to form aSN oligomers by size exclusion chromatography (SEC) (Table 1 and Fig. 3A). Elephant aSN forms the highest amount of oligomer while pig aSN forms the lowest amount. Human aSN monomer elutes at 17.5 mL, whereas the oligomer elutes at ~12 mL. Remarkably, elephant aSN increases both the amount and the size of the oligomer species, shifting elution volume to ~11 mL as well as a small peak at 8-9 mL. Whale aSN forms less oligomers than human and elephant, and produces larger amounts of degraded monomer eluting around 20 mL. A peak eluting around 20 mL contained a 103 amino acid long protein, corresponding to degraded aSN according to MS studies of human aSN (C. Sahin et al., unpublished data). Porcine aSN does not form oligomers (data not shown). The SEC purified oligomers showed no change in secondary structure by CD spectroscopy (Fig. S2), but the elephant oligomers were slightly larger and more prone to associate into bundles according to TEM (Fig. S3), while human aSN and whale aSN largely form spherical structures. More detailed structural information was provided by SAXS, which has previously shown human aSN oligomers to consist of ~29 monomers forming a compact ellipsoidal core surrounded by a shell of less structured protein23. Based on the forward scattering, the human aSN oligomer was estimated to have a molecular weight of 407 kDa corresponding to 28 monomers which agreed well with our earlier estimates. While the elephant aSN oligomer also fitted the ellipsoidal model (Fig. 3B and Table 2), its molecular weight was 689±14 kDa, corresponding to 47 monomers (Table 2). Consistent with this, the p(r) function (Fig. 3C) showed the elephant variant to have a slightly larger maximum diameter (24 nm) than human aSN oligomer (20 nm) as well as a larger Rg of 8.1±0.2 nm and 7.0±0.5 nm correspondingly. The fraction of protein in the flexible region of the structure is ~0.5 for both elephant and human oligomer, showing that the core forming part of the protein has likely not changed but rather structural rearrangements in the core caused the size difference. While both 15

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the elephant and human oligomer remained intact at elevated temperatures (Fig. S2), they showed different urea sensitivity. Elephant oligomer remained stable up to 8 M urea, but human aSN oligomers started to dissociate above 5 M urea (Fig. S4) as observed previously25. We examined the membrane permeabilizing effects of the oligomer variants, measured as the release of calcein from DOPG vesicles. It was not possible to purify sufficient amounts of pig aSN oligomers to carry out the assays for this variant due to its low yield. Compared to human aSN oligomers at 0.1 mg/ml, elephant oligomers only gave rise to 51 % release of calcein and only 27 % release for the whale variant of aSN (Fig. 3D).

Table 2 Comparison of parameters obtained from IFT analysis and from model fitting of SAXS data. Human

Elephant

χ2

0.96

2.3

Rg (nm)

7.0 ± 0.5

8.1 ± 0.2

Rg chain (nm)a

2.4

2.3

Nmonomer (IFT)

28 ± 1

47 ± 1

Nmonomer (model)

29 ± 1

51 ± 1

εb

1.9 ± 0.1

2.0 ± 0.1

fshellc

0.52

0.49

Rcore (nm)

3.2 ± 0.1

4.0 ± 0.1

Rshell (nm)

5.0 ± 0.2

6.0 ± 0.2

Notes: a

Radius of gyration of aSN coils in solution.

b

width:hight ratio of ellipsoid.

c

Fraction of protein in flexible region.

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Biochemistry

Figure 3: Properties of aSN oligomers from different species. (A) SEC chromatograms of oligomer purification of human, elephant and whale aSN. (B) SAXS measurements of human (black) and elephant (blue) aSN oligomers together with model fits. (C) p(r) functions obtained by SAXS measurements of human (black) and elephant (blue) aSN oligomers. Data has been normalized. (D) Calcein release assay of human (black) and elephant (blue) aSN oligomers. Data is normalized so human aSN at 0.1 mg/mL gives a 100 % release.

Dissection of the properties of individual mutations separating human and animal aSN Having shown a consistent change in fibrillation propensity and oligomer’s membrane perturbation across the three aSN variants, we examined the three mutations which the animals shared relative to human aSN, namely A53T, G68E and V95G (summarized in Table 4). All of the eight available mutant combinations (including human and pig aSN as the two end point species) were prepared and their aggregation and vesicle permeabilization properties were determined. At 0.25 mg/mL (Fig. 4A), fibrillation took place at the same rate for V95G, A53T and A53/V95G as for WT, with a lag time of ≈ 11 hrs. The double mutants A53T/G68E and G68E/V95G exhibited lag times of 17 17

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and 25 hrs, respectively, even slower fibrillation was observed for G68E (lag time 50 hrs), and no detectable fibrillation was obtained for the pig-WT aSN. Similar behavior was seen at 0.5-1 mg/ml (data not shown). After 150 hrs of fibrillation, A53T gives rise to the most pronounced far-UV CD minimum at 218 nm, characteristic of β-sheet structure, while pig aSN retains a random coil structure (cfr. Fig. 2C). Deconvolution of the spectra based on linear combinations of A53T and pig A53 (Fig. S5) gives the ranking (in terms of extent of β-sheet structure) A53T > V95G > human > G68E/V95G ≈ A53T/V95G > G68E > A53T/G68E > pig aSN. This ranking corresponds satisfactorily to the various fibrillation lag times (Fig. 4A): the four fastest fibrillating mutants have the highest levels of

β-sheet structure along with G68E/V95E, while pig aSN and G68E have the lowest levels and the longest lag times. A53T/G68E is a slight anomaly as it has a relatively short lag time but also rather low amounts of β-sheet structure. As depicted in Fig. S6, this mutant shows the same classical unbranched fibrils as all other mutants except pig aSN (which forms no fibrils, just small amounts of amorphous aggregates) and G68E (low levels of relatively short fibrils).

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Biochemistry

Figure 4: Fibrillation and seeding of different aSN variants. (A) Fibrillation observed for the 8 variants of aSN when shaken at a protein concentration of 0.25 mg/mL. Fibrillation is monitored with ThT fluorescence. (B) Degree of βsheet structure after 150 h of fibrillation determined with Circular dichroism. (C) Cross-seeding with 5% sonicated fibrils from the mutants in human WT monomer at a concentration of 0.25 mg/mL. The fibrillation is monitored with ThT fluorescence at unshaken conditions. The error bars are standard deviations over 3 replicates. (D) Self-seeding with 5% sonicated fibrils from the mutants in its own monomer at a concentration of 0.25 mg/mL. The fibrillation is monitored with ThT fluorescence at unshaken conditions.

Cross-seeding between the eight aSN mutants was examined by seeding human WT monomer with sonicated fibrils from the mutants (Fig. 4C) and by self-seeding monomers of aSN mutants with sonicated fibrils of the same mutants (Fig. 4D, Table 3). Data show a general reduction in seeding compared to human aSN. A53T seeds human WT monomers to a significantly reduced extent. V95G seeds induce fibrillation in both itself and human WT, but in both cases at a reduced rate 19

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compared to the WT control which seeds itself very well. G68E seeds both human WT and G68E monomers poorly. A53T/G68E does not seed well when added to human WT monomer, since limited aggregation is obtained. However, A53T/G68E seeds itself well, though the rate of fibrillation is slower than for human WT. Human WT monomer seeded with A53T/V95G aggregates essentially as well as human WT seeded with itself though A53T/V95G seeds itself poorly. G68E/V95G does not significantly seed either WT monomer or itself. We investigated the binding of the 8 aSN variants to DMPG vesicles by far-UV CD at 37oC (Fig. 5A) which showed a slight reduction in the amount of induced structure in the ranking order WT > G68E/V95G > G68E > V95G > A53T/G68E > A53T/V95G > A53T >>Pig. Pig aSN undergoes no significant change in structure in the presence of DMPG vesicles, suggesting a very low level of binding. The ranking is confirmed by temperature scans (Fig. 5B), which also show a significant correlation between the depth of the trough and the midpoint temperature of melting in the 40-70oC temperature range (insert to Fig. 5B), indicating that the stronger the aSN binds to the lipid, the more it is stabilized against denaturation 47. The mutants generally follow the trend provided by the individual animal aSN investigated in Fig. 2D. Oligomers were purified from each of the 8 mutants, and the amount of oligomer/monomer was quantified (Fig. 5C). The formation of oligomers varies between experimental runs but there are some consistent trends: 1) Human WT, A53T, G68E and V95G broadly form the same levels of oligomers. 2) G68E/V95G shows slightly higher oligomer formation. 3) A53T/G68E and A53T/V95G show 2-6 fold higher oligomer formation. The membrane permeabilization ability of the oligomers also varies between the different mutants, with A53T, G68E, A53T/G68E, and G68E/V95G showing approximately half the permeabilization ability of human aSN in the calcein release assays, whereas V95G shows the same permeabilization as human WT, and A53T/V95G shows approximately twice as high permeabilization (Fig. 5D). When both oligomer concentration and membrane permeabilization are taken into consideration, A53T/G68E gives rise to more aggregate toxicity as the increased amount of oligomer formed outweighs the reduced permeabilization. The reduced membrane interaction could be explained by a change in secondary structure that affects the oligomer’s membrane binding properties. A53T/V95G is formed in higher amounts and leads to higher permeabilization, suggesting that it would be much more toxic than human WT. 20

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Biochemistry

Table 3. Ability of aSN mutants to cross-seed using wt aSN monomers together with 8 different fibril seeds as well as self-seeding. Monomer WT

WT

+++

A53/V95G

++

V95G

++

Seed G68E

+

A53T

+

A53/G68E

-

A53/V95G V95G G68E A53T A53/G68E G68E/V95G

+ ++ +++ ++

G68E/V95G Pig aSN

-

-

21

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Figure 5: Lipid interactions of aSN variants as monomers and oligomers. (A) Structual analysis of aSN structure formation by wavelength scans on DMPG vesicles surfaces at 37 °C using Circular dichroism. (B) Circular dichroism analysis of the development of structure over temperature on DMPG vesicles. Insert: The correlation between the depth of the trough and the midpoint temperature of melting is shown for the 40-70 oC temperature range. (C) Oligomer formation in aSN mutants at 5h incubations of 3 mg/mL solutions over two seperate experimental analyses. (D) Calcein release by oligomers of the various mutants at 0.2 µM.

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Biochemistry

Table 4. Different properties for the aSN variants. The symbol +++ indicates highest signal/most signal, ++ is a medium signal, + is a smaller signal and – is equal to no signal. WT

V95G

A53/V95G

A53/G68E

G68E/V95G

G68E

A53T

(Human)

ThT lag time (hrs)

a

A53T/G68E/V95G (pig)

11

11

11

17

25

50

11

-

+++

+++

+++

+++

++

+

+++

-

+++

+++

++

+

++

+

+++

-

as 0.71

0.84

0.55

0.16

0.62

0.23

1.00

0.00

++

++

-

-

+

+

-

b

ThT end signal (1 mg/mL)

Beta sheet content CD

spectrum

fraction of A53Tc Cross –seeding with +++ wt monomer (Lag phase) Self-seeding ability

+++

++

+

++

-

-

+++

-

Lipid binding

+++

+++

++

+++

+++

+++

++

-

Oligomer formation

++

++

+++

+++

+++

++

++

-

Vesicle

++

++

+++

+

+

+

+

N/A

permeabilization

Notes: a

The time it takes for aSN to show a significant increase in ThT fluorescence intensity compared to the baseline level.

b c

The level of ThT intensity in its saturated phase (end-point).

β-sheet content normalized to the β-sheet content of A53T based CD spectra. 23

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DISCUSSION Reduced fibrillation in animal species centers around position 68 Since several animal species live significantly longer than humans prior to the emergence of modern civilization, they may be expected to have evolved to withstand old age better than humans, e.g. in protein fibrillation diseases where the sequence of the offending protein is a straightforward target for evolution. We do not claim that lack of fibrillation by itself leads to extended life spans, but suggest that animals which in various ways have evolved to have long life spans have also had to deal with the challenge of protein aggregation. (The remarkably low fibrillation and oligomerization properties of porcine aSN, derived from a relatively short-lived species, illustrates that short-lived species do not necessarily possess highly fibrillogenic aSN; in contrast, highly fibrillogenic aSN would a priori not be compatible with long life). Humans are not on a particularly long-lived branch of the mammalian family tree. Their closest relatives are apes (chimpanzee 50-60 years maximum in the wild, orangutan 59 years), monkeys (bolivian spider monkey 18 years, white cheek gibbon 28 years, American spider monkey 47 years), shrews (Chinese tree shrews 3 years, tree shrew 10 years), bats (6-30 years) lemurs (27 years), and mice (4 years). Most longer lived animals on this branch share residues A53 and V95 with humans 30. A53T in human aSN leads to increased oligomerization toxicity and familial early-onset PD but occurs in all other examined species ranging from fish to mammals, several of which are known for their longevity, e.g. the bowhead whale (200 years) and large land mammals (camels 50 years and elephants 60 years). The protective mutations present in our specific branch of the tree of life center around position 68 which is Gly on the human branch but Glu in most animals. Indeed, our fibrillation experiments, confirmed by CD, SDS-PAGE and EM, showed substantially reduced fibrillation by all of four animal aSN. In a similar manner, Nyström and Hammarström compared Prion proteins from several mammalian species, including human, in relation to prion-resistant species such as pig48. However, prions from all species seemed to fibrillate as monitored by ThT and Congo-red stain assays as well as by TEM. Minor sequence variations were identified in the C-terminal region that makes up the fibril core of the prion proteins, but these do not have major impact on prion aggregation properties. This contrasts to our studies where sequence alignment identified one residue at position 68 to be crucial for the fibrillation and oligomerization properties of aSN. Residue 68 in aSN is located in the NAC region, which is involved both in membrane interactions and in the formation of the hydrophobic fibril core. 24

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Biochemistry

A correlation between α-helical structure, membrane binding and aggregation propensity What consequence does this have in vivo? While aSN’s natural function is not yet firmly established, it is probably related to the mobilization and formation of signaling reserve vesicles 49, consistent with the formation of α-helical structures on the surface of lipid vesicles. Interestingly, for the different animal species, the extent of fibrillation correlates with the level of α-helix formation, with the most fibrillation prone version of aSN (human, followed by elephant) also showing the most α-helical structure, in turn most stabilized against thermal unfolding. Whale and pig aSN show very little lipid binding propensity, despite the lack of substitutions in the N-terminal 50 residues involved in lipid contacts. Lowering the percentage of anionic lipid to mimic the composition of biological membranes reduces both helicity and melting transition temperature even further

47

. Thus helicity can be considered a measure of the degree of binding of aSN to anionic

phospholipid vesicles, which in turn could suggest a positive link between lipid binding and fibrillation. This is also confirmed by the human A30P mutant which binds more poorly to lipids than wt human aSN

50, 51

and fibrillates more slowly

52

; its ability to induce early onset PD is

attributed to its enhanced oligomerization propensity53. Note that the non-human aSN structures all lose their helical structure in the presence of DMPG vesicles well below the body temperature of the animals involved, i.e. 35.6 °C for elephant 54, 33.8 °C for Bowhead whale 55 and 37.1 °C for pig 56. This suggests that most of the helical structure will not be present under physiological conditions even under optimal lipid binding conditions. Remarkably, the degree of oligomerization varied across the different aSN variants, with elephant aSN forming significantly more oligomer than human WT aSN, and pig aSN forming significantly less. Yet these animal oligomers show strongly reduced calcein release, indicating low cell toxicity consistent with the low monomeric aSN binding to vesicles. This makes the high (> 10%) fraction of elephant aSN oligomer intriguing. It is not likely to be toxic to membranes, though it conforms to our previously reported overall core-shell oligomer model for human aSN with slightly larger dimensions. The reduced membrane binding is likely linked to the monomer’s poor lipid binding, assuming that the same regions of aSN are involved in lipid contacts in monomer and oligomer. Non-toxic oligomers could constitute a sink for aSN that is just as innocuous as fibrils but could be mobilized more readily for the release of monomeric aSN. The importance of A53, G68 and V95 in fibrillation, oligomerization and toxicity The point mutations in positions 53, 68 and 95 shared by all 3 animals are predicted to increase the solubility of monomeric aSN compared to human aSN and thus decrease the relative stability of the 25

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insoluble state. The stability of the fibrillary species can also be affected more directly. The solidstate NMR structure of human aSN fibrils shows Gly68 and Val95 to be in close contact (bridging the two ends of the Greek-key motif, though the Cα of Gly68 is pointing away from Val95), with Ala53 in a completely separate part of the fibril57. Modifications at the 68-95 positions likely perturb fibril structure, in particular introduction of a polar moiety in position 68 which is placed in a place where the fibrils undergo a sharp 90o turn and thus require the flexibility provided by Gly in this position; in contrast the substitution Gly95 introduces a strand-destabilizing side chain in the middle of an existing β-sheet. Rather than a regular reduction in the level of fibrillation and oligomer toxicity from the highly fibrillogenic human aSN to the non-fibrillogenic pig aSN, we find that all mutations which increase the lag time of aSN fibrillation include G68E. Half-lives are ranked A53T/G68E < G68E/V95G