Prasad 2018

THE
JOURNAL • RESEARCH • www.fasebj.org
Monitoring a-synuclein multimerization in vivo

Vibha Prasad,* Yasmine Wasser,* Friederike Hans,† Anand Goswami,‡ Istvan Katona,‡ Tiago F. Outeiro,§,{,k
Philipp J. Kahle,† Jörg B. Schulz,*,# and Aaron Voigt*,#,1
*Department of Neurology and ‡Institute of Neuropathology, University Medical Center, Rheinisch-Westfälische Technische Hochschule
(RWTH) Aachen University, Aachen, Germany; †Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute
for Clinical Brain Research, German Center for Neurodegenerative Diseases, Tübingen, Germany; §Department of Experimental
Neurodegeneration, Center of Molecular Physiology of the Brain, Center for Biostructural Imaging of Neurodegeneration, University
Medical Center Göttingen, Göttingen, Germany; {Max Planck Institute for Experimental Medicine, Göttingen, Germany; kInstitute of
Neuroscience, The Medical School, Newcastle University, Newcastle Upon Tyne, United Kingdom; and #Jülich-Aachen Research Alliance
(JARA)–Brain Institute Molecular Neuroscience and Neuroimaging, Forschungszentrum Jülich GmbH and RWTH Aachen University,
Aachen, Germany
ABSTRACT: The pathophysiology of Parkinson’s disease is characterized by the abnormal accumulation of a-synuclein
(a-Syn), eventually resulting in the formation of Lewy bodies and neurites in surviving neurons in the brain.
Although a-Syn aggregation has been extensively studied in vitro, there is limited in vivo knowledge on a-Syn
aggregation. Here, we used the powerful genetics of Drosophila melanogaster and developed an in vivo assay to
monitor a-Syn accumulation by using a bimolecular fluorescence complementation assay. We found that both
genetic and pharmacologic manipulations affected a-Syn accumulation. Interestingly, we also found that alterations
in the cellular protein degradation mechanisms strongly influenced a-Syn accumulation. Administration of com-
pounds identified as risk factors for Parkinson’s disease, such as rotenone or heavy metal ions, had only mild or even
no impact on a-Syn accumulation in vivo. Finally, we show that increasing phosphorylation of a-Syn at serine 129
enhances the accumulation and toxicity of a-Syn. Altogether, our study establishes a novel model to study a-Syn
accumulation and illustrates the complexity of manipulating proteostasis in vivo.—Prasad, V., Wasser, Y., Hans, F.,
Goswami, A., Katona, I., Outeiro, T. F., Kahle, P. J., Schulz, J. B., Voigt, A. Monitoring a-synuclein multimerization in
vivo. FASEB J. 33, 000–000 (2019). www.fasebj.org
KEY WORDS: Parkinson’s disease • synucleinopathy • protein aggregation • BiFC
Neuronal accumulations of the presynaptic protein mutations in a-Syn were linked to PD, including amino acid
a-synuclein (a-Syn) represent a hallmark of Parkinson’s substitutions A30P, E46K, and A53T (6–8). It is still being
disease (PD). In addition to PD, there are at least 2 other debated whether these mutant a-Syn variants have differ-
neurodegenerative diseases in which a-Syn aggregates are ent aggregation propensity than wild-type (WT) a-Syn
the main pathologic hallmark: multiple system atrophy and (9–11). We rely only on the a-Syn variant A53T here, as this
dementia with Lewy bodies. This classification is why these variant was regularly used in our previous research (12).
diseases are summarized as synucleinopathies (1). PD rep- Depending on the model system and a-Syn variant
resents the most common synucleinopathy, and research used, detailed analysis revealed that a-Syn forms a variety
efforts are predominantly focused on PD etiology. Dupli- of different aggregate species. The highly heterogeneous
cations and triplications of the gene SCNA locus coding for and dynamic nature of these a-Syn aggregates does not
a-Syn are known to cause PD (2–5). In addition, several allow for a clear distinction of different aggregation states
in vivo in various model systems (13). However, compared
with the natively unfolded monomeric a-Syn, the follow-
ABBREVIATIONS: a-Syn, a-synuclein; 3MA, 3-methyladenine; BiFC, bimo-
lecular fluorescent complementation; CK1, casein kinase 1; Dco, discs over-
ing aggregation states have been established: oligomers,
grown; FRA, filter retardation assay; H2O2, hydrogen peroxide; Hsp70, 70 fibrils, and Lewy bodies. Oligomers (from dimer to x-mer)
kDa heat shock protein; PD, Parkinson’s disease; pS129, a- synuclein phos- are considered to be soluble, whereas insoluble amyloi-
phorylated at serine 129; pS6K, phosphorylated S6 kinase; ROS, reactive dogenic fibrils are of higher MW. These fibrils ultimately
oxygen species; S6K, S6 kinase; WT, wild type
1
accumulate in large cytoplasmic visible conglomerates, the
Correspondence: Department of Neurology, University Medical Center, Lewy bodies (14). In several disease model systems,
RWTH Aachen, Pauwelsstr. 30, 52074 Aachen, Germany. E-mail:
avoigt@ukaachen.de a-Syn–induced toxicity is observed without the formation
of Lewy body–like structures (15–17). This outcome sup-
doi: 10.1096/fj.201800148RRR
This article includes supplemental data. Please visit http://www.fasebj.org to ports the idea that either a-Syn oligomers or fibrils trigger
obtain this information. cytotoxic events in neurons (18). In Lewy bodies, a large
0892-6638/19/0033-0001 © FASEB 1
asebj.org by Iowa State University Serials Acquisitions Dept (129.186.138.35) on January 10, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNu
portion of a-Syn is phosphorylated at serine 129 (pS129). In In flies, 6 nearly identical copies of Hsp70 genes exist per haploid
contrast, ,10% of a-Syn is phosphorylated in healthy in- genotype. We used chromosomal deficiencies to remove 1 set of
dividuals (19). Thus far, it is still unclear whether pS129 these 6 known 70 kDa heat shock proteins (Hsp70s) in the diploid
fly. The exact human orthologs cannot be easily determined be-
influences the endogenous function of a-Syn, affecting its cause all the 6 fly genes share the highest sequence homology to
neurotoxicity or changing its aggregation propensities. human HSPA1A and HSPA1B. w[1118]; w[*];P{w[+mC]=UAS-
The effect of pS129 on a-Syn has not been studied in vivo RFP.W}2 (BL 30556) and w[*]; P{w[+mC]=UAS-Hsap\MJD.tr-
in great detail, and in most cases, phospho-mimics Q27}N18.3d (BL 8149) served as overexpression controls.
(S129D/E) and non-phosphorylatable (S129A) variants P{w[+mW.hs]=GawB}elav[C155] (BL 458) was used to drive pan
of a-Syn were used (16, 20). Thus, our strategy overcomes neural expression of UAS-constructs. w[1118];;P{w[+mC]=Ddc-
the problem of phosphomimetic mutants, which possibly GAL4.L}Lmpt[4.36], P(acman){w[+]=UAS-[A53T]SCNA} (ddc .
A53T in text) was used to drive untagged a-Syn in aminergic
does not perfectly reflect authentic serine phosphorylation neurons (12).
in all accounts. Although the presence of aggregated a-Syn The fly stocks w[*];; P{w[+mC]=UAS-dco[K38R]} and w[*];;
is doubtlessly linked to PD and the other synucleino- P{w[+mC]=UAS-dco[WT]} were a kind gift from J. L. Price
pathies, it remains unclear whether and which of these (University of Missouri-Kansas City, School of Biological
a-Syn species is causative for neuronal decline. More- Sciences, Division of Molecular Biology and Biochemistry,
over, epidemiologic studies suggest that certain envi- Kansas City, Missouri, USA) and have been used to over-
ronmental factors increase the risk for PD. Chronic express either WT or inactive [K38R] variants of Dco (25).
exposure to pesticides (e.g., rotenone) and metal ions The site-directed and random-inserted UAS-Hsap\
[A53T]SNCA transgenic lines were generated by germline
(e.g., zinc, mercury) are established risk factors for PD
transmission (BestGene, Chino Hills, CA, USA). Strain
(21, 22). Although the effect of chemicals mentioned PBac{yellow[+]-attP-3B}VK00002 (cytologic region 28E7 on
earlier on PD etiology has been discussed for some time, second chromosome; strain identifier at BestGene: 9723) was used
it is still unknown whether these chemicals have an to generate the site-directed insertions. We generated the follow-
effect on a-Syn aggregation in vivo. ing stock: P{w[+mW.hs]=GawB}elav[C155]; P{w[+]=UAS-Hsap\
The present article describes a fast, inexpensive, and [A53T]SNCA:VC}, PBac{attB[+mC]=UAS-Hsap\[A53T]SNCA:VN}/
powerful method to test the effect of chemicals and/or CyO. This stock with stable expression of BiFC-SNCA was used
in all genetic analysis. In experiments in which the food was
genetic manipulations on a-Syn aggregation in Drosophila supplemented with compounds, we crossed P{w[+mW.hs]=
melanogaster neurons. We used the well-established bi- GawB}elav[C155]; PBac{attB[+mC]=UAS-Hsap\[A53T]SNCA:
molecular fluorescent complementation (BiFC) assay (23, VN} with w[*]/Y; P{w[+]=UAS-Hsap\[A53T]SNCA:VC}ICyO and
24) to assess the multimerization of a-Syn in the RIPA- used males from the F1 P{w[+mW.hs]=GawB}elav[C155]/Y;
soluble fraction and relative quantification of RIPA- P{w[+]=UAS-Hsap\[A53T]SNCA:VC}/ P{w[+]=UAS-Hsap\
insoluble a-Syn by using a filter retardation assay (FRA). [A53T]SNCA:VN} for analysis (elav . a-Syn in text). For genetic
As proof of principle, we were able to show that alterations analysis, we recombined PBac{attB[+mC]=UAS-Hsap\[A53T]SNCA:
VN} with P{w[+]=UAS-Hsap\[A53T]SNCA:VC} and generated a
of cellular degradation pathways had an impact on a-Syn stable stock P{w[+mW.hs]=GawB}elav[C155]; P{w[+]=UAS-Hsap\
aggregate abundance in flies. We subsequently analyzed [A53T]SNCA:VC}, PBac{attB[+mC]=UAS-Hsap\[A53T]SNCA:
the impact of agents implicated as increasing or decreasing VN}/CyO (elav . a-Syn in text).
the risk of developing PD with regard to a-Syn aggrega-
tion. As risk factors for PD, we tested several metal ions,
the complex I inhibitor rotenone, and general oxidative Negative geotaxis assay
stress–inducing hydrogen peroxide (H2O2). Among all the
tested agents, most had little to no effect on the abundance Negative geotaxis was performed with minor changes as pre-
of soluble and/or insoluble a-Syn aggregate formation. viously described (26, 27). In brief, 10 male flies (n = 30) were
allowed to settle in a polypropylene plastic tube that was marked
Finally, we addressed the effect of enhanced phosphory- at a height of 8 cm distance from the bottom. A gentle tap at the
lation of a-Syn at Ser129 on a-Syn aggregate formation. tube bottom invokes a climbing response, and the number of flies
The Drosophila genome codes an ortholog of the mam- that crossed the 8 cm mark in 10 s was counted. The procedure
malian casein kinase 1 (CK1) family of serine/threonine was repeated 10 times with a resting interval of 50 s. The climbing
kinases, named discs overgrown (Dco). Expression of Dco ability of flies was determined at indicated time points, referring
increased Ser129 phosphorylation and coincided with to d posteclosion.
enhanced aggregation of a-Syn. In addition, elevated
a-Syn phosphorylation at Ser129 increased the detrimen-
Dose finding and administration of compounds
tal effects of a-Syn in our fly model. and metal ions
Compounds and metal ions were administered in artificial fly

MATERIALS AND METHODS food (2% agar, 4% sucrose) to single male flies in polycarbonate
tubes (5 mm in diameter and 65 mm in length). Final concen-
Fly stocks trations of compounds or metal ions were as follows: 3MA,
0.5 mM; rapamycin, 4 mM; MG132, 5 mM; rotenone, 0.25 mM;
In general, fly stocks were raised and maintained on standard MnCl2×4H2O, 0.5 mM; ZnCl2, 1 mM; CuCl2×2H2O, 0.75 mM;
cornmeal food. The following fly stocks were obtained from the HgCl2, 0.17 mM; and Fe3+, Fe2+, and Al3+, 2 mM. Solvent-only
Bloomington Drosophila Stock Centre (Bloomington, IN, USA): treated flies served as control. All metal ions and most com-
w[1118];;P{w[+mC]=UAS-HsapHSPA1L.W}41.1 (BL 7454) was pounds were dissolved in H2O. Exceptions were rotenone
used for over-expression of human Hsp70. w[1118]; Df(3R) and rapamycin, which were dissolved in DMSO. To detect the
Hsp70A, Df(3R)Hsp70B (BL 8841) is a 6 copy deletion of fly Hsp70. optimal concentration of administered compounds, titration of
2 Vol. 33 February 2019 The FASEB Journal x www.fasebj.org PRASAD ET AL.

reported bioactive concentrations was performed. Groups of 10 synapsin (3C11, 1:2000; Developmental Studies Hybridoma
male elav . a-Syn flies were exposed to a given compound Bank, University of Iowa, Department of Biology, Iowa City,
concentration, and survival was monitored over a time period of Iowa, USA).
5 d using a DAM2 Drosophila Activity Monitor (TriKinetics, After several washing steps in TBS-T, the membrane was in-
Waltham, MA, USA) with a 1 h reading interval. The first hour of cubated with an appropriate secondary anti-mouse or anti-rabbit
continuous absence of locomotion was considered as the time horseradish peroxidase–linked antibody (1:10,000; GE Health-
point of death. Concentrations were reduced by 50% as long as all care). Membranes were incubated with the secondary antibody
flies survived the 5-d period (Supplemental Figs. S2 and S3). for 1 h, followed by signal detection using ECL SuperSignal
Administration of H2O2 was achieved by placing groups of 10 (Thermo Fisher Scientific, Waltham, MA, USA) in combina-
male flies into an empty vial (dimensions: ∅ = 28.5 mm, h = tion with a chemiluminescence detection apparatus (Alliance
95 mm) containing a liquid-soaked Whatman paper (2 3 4 cm LD4.77WL.Auto; Biometra, Göttingen, Germany).
pieces; MilliporeSigma, Burlington, MA, USA). The liquid (4% We tried to roughly quantify the amount of a-Syn present in
sucrose, 0.75% H2O2 in water) served as the nutrition and water the fly head lysates. In brief, we loaded different concentrations of
source. Fresh liquid was added every other day until maximal commercially available a-Syn (rPeptide S-1001-2; 1–5 mg) and fly
absorbency of the Whatman paper was reached. head lysates (9 mg total protein) on a gel, separated proteins by
SDS-PAGE, and performed Western blotting for protein detection
(rabbit anti–a-Syn antibody; 2642S; 1:500; Cell Signaling Tech-
Preparation of fly head lysates
nology). Based on signal intensities of loaded pure a-Syn, a straight
calibration line was generated. From this process, we estimated
Flies were collected in 1.5 ml Eppendorf tubes and snap-frozen in the amount of a-Syn in our fly head lysates. According to the
liquid nitrogen. Fly heads (usually 10) were separated from the procedure described earlier, we determined a concentration of
body by vortexing the frozen flies. Heads were collected and ho- ;0.02 mg a-Syn per micrograms total protein in our fly head
mogenized in 10 ml/head RIPA [50 mM Tris, pH 8.0, 0.15 M NaCl, lysates.
0.1% (v/v) SDS, 1% NP-40, 0.5% sodium deoxycholate, protease
inhibitor; Roche, Basel, Switzerland] using a speed mill (Analytic
Jena, Jena, Germany). The lysates were centrifuged at 13,000 g for Relative quantification of the insoluble a-synuclein
120 s at 4°C. The supernatant was collected into a new Eppendorf fraction in fly head lysates: FRA
tube, and the volume was determined and labeled as the soluble
protein fraction. The remaining pellet was washed thrice with The insoluble aggregates were relatively quantified via the FRA.
RIPA (10 ml/head) for 20 min on a rotating wheel and centrifuged RIPA-insoluble pellet was dissolved in urea buffer (30 mM Tris
at 13,000 g at 4°C. The supernatant of the washing steps was Buffer, 7 M urea, 2 M thiourea, 4% CHAPS, pH 8.5; 10 ml/fly
discarded. The remaining pellet was finally dissolved in an equal head) by sonication (incubation in Ultrasonic Cleaner Bath; VWR
volume as was recovered as soluble fraction, in urea buffer International, Lutterworth, United Kingdom) for 30 min fol-
(30 mM Tris Buffer, 7 M urea, 2 M thiourea, 4% CHAPS, pH 8.5) lowed by centrifugation .15,000 g for 30 min at 4°C. The su-
and considered as the RIPA-insoluble fraction (discussed later). pernatant was transferred to an Eppendorf tube and considered
the (RIPA-) insoluble fraction. Relative to the total protein con-
centration in the soluble fraction, we loaded equal amounts of
Protein concentration measurements total protein (between 10 and 100 mg). The volume was adjusted
to 25 ml by addition of RIPA buffer supplemented with 7 ml dot
The protein concentration in RIPA-soluble fly head lysates was blot buffer (0.5 M Tris, pH 6.8, 0.4% SDS, 20% glycerol, 0.2 M
determined by using the DC Protein Assay Kit (Bio-Rad, Her- DTT). The samples were transferred to a 0.2 mM nitrocellulose
cules, CA, USA) according to the manufacturer’s instructions. In membrane (ProtranBA 83; GE Healthcare) by sucking the probes
brief, 5 ml of the lysate was treated with 24.5 ml of reagent A and through the membrane using a vacuum pump attached to a dot
0.5 ml of reagent S. After adding 200 ml of reagent B, samples were blot chamber (Camlab, Cambridge, United Kingdom). The
incubated for 15 min at room temperature, and absorbance was membrane was washed several times with TBS-T and blocked for
determined at 750 nm by using a plate reader (Infinite 200 Pro 1 h by using 5% skim milk in TBS-T. The membrane was then
Reader; Tecan, Männedorf, Switzerland). treated as described for Western blotting.
Determination of Venus signal intensities Determination of reactive oxygen species levels

in fly head lysates
The RIPA-soluble fractions of fly head lysates were adjusted to a
final concentration of 3 mg/ml total protein. Venus signal in- The oxidative stress levels were determined in freshly prepared
tensities were quantified (in triplicate, 5 measures per sample) at a fly head lysates (derived from flies after a 5 d exposure to 0.75%
wavelength of 530 nm by using a NanoQuant plate in combi- H2O2) using the reactive oxygen species (ROS) indicator, carboxy
nation with an Infinite 200 Pro Reader. H2DCFDA (Thermo Fisher Scientific). Twenty micrograms total
protein was supplemented with a ROS indicator solution (5 mM
final concentration) in a final volume of 20 ml. The fluorescence
Western blot analysis was measured over time (in intervals of 5 min over a time period
of 30 min total) using a NanoQuant plate inserted into an Infinite
Protein samples were separated via SDS-PAGE and then trans- 200 Pro Reader using excitation at 494 nm and emission at 520
ferred onto 0.2 mM nitrocellulose membrane (ProtranBA 83; nm.
GE Healthcare, Little Chalfont, United Kingdom). After blocking
with 5% skim milk in TBS-T (25 mM Tris, 140 mM NaCl, 0.05%
Tween, pH 7.5), membranes were incubated at 4°C overnight with Quantification of the sulfhydryl groups in the fly
the primary antibodies in indicated concentrations. The primary head lysates using cysteine standard method
antibodies (also in FRA analysis) used were as follows: rabbit
anti–a-Syn (2642S, 1:200, Cell Signaling Technology, Danvers, The freshly prepared fly head lysates (after a 5-d treatment of
MA, USA), rabbit anti-pSer129 a-Syn (ab51253, 1:2500; Abcam, H2O2) were used to determine the redox state of the sample via
Cambridge, United Kingdom), and mouse anti-Drosophila the thiol group quantification method, using an absorbance
MONITORING a-SYNUCLEIN MULTIMERIZATION 3

measure at 412 nm. Cysteine standards were prepared by using comparisons of individual bars in graphs are noted in the figure
cysteine (Thermo Fisher Scientific) dissolved in reaction buffer legends. Unless otherwise stated, the number of independent
(0.1 M sodium phosphate, pH 8.0 with 1 mM EDTA). The redox experiments summarized is n $ 3, and at least a number of n $ 5
state of the fly head lysate was determined by using Ellman’s technical replicates are summarized. Values of P , 0.05 were
reagent (Thermo Fisher Scientific) according to the manufac- considered significant.
turer’s instructions. FRA analysis was performed as follows: samples for com-
parison were analyzed on a single nitrocellulose membrane.
According to the great variability between different membranes,
Confirmation of amyloidogenic structure of a-Syn signal intensities were normalized to control. The vast majority
by the Thioflavin-T assay of data was parametric (D’Agostino and Pearson omnibus nor-
mality test) with the exception of rapamycin, rotenone, and
Whole PD fly brains were prepped in 1x TBS with 2% Triton-X. aluminum. Accordingly, we performed paired Student’s t tests
These fly brains were then fixed in 4% paraformaldehyde for for all datasets as presented. Significant differences are indicated
20 min on a spin wheel. This process was followed by 2 washes in line plots. Normalized data are plotted as scatter plots for
lasting 10 min each, with 1x TBS containing 2% Triton-X. After the better visualization of absolute effects between different
last wash, the brains were subject to tissue permeabilization with treatments.
1x TBS (with 2% Triton-X) and 1.6% normal goat serum (S1000;
Vector Laboratories, Burlingame, CA, USA) for 2 h on a spin wheel.
After permeabilization, the fly brains were incubated overnight at
4°C, with 0.1% Thioflavin-T dye (T3516-5G; MilliporeSigma) (28) RESULTS
and 1x TBS (2% Triton-X) in the ratio 1:2. The following day, the fly
brains were destained twice using 50% ethanol, for a 10-min Quantification of soluble and insoluble a-Syn
time interval. After destaining, the fly brains were incubated aggregates from in vivo samples
2 times for 10 min each with 1x TBS (2% Triton-X). The fly brain
whole mounts were then mounted on a glass side by using The BiFC system has been established as a useful tool for
Mowiol R 4-88 (0713.1; Carl Roth GmbH, Karlsruhe, Germany). visualizing a-Syn aggregates in cultured cells (23, 29). This
The images were captured by using a Zeiss Axio Observer Z1
technique is based on the reconstitution of a fluorescent
LSM 700 with EC-Plan-Neofluor 340/1.30 oil DIC M27 ob-
jective (Carl Zeiss AG, Oberkochen, Germany). The excitation Venus protein only as a consequence of the interaction of at
laser used was at 488 nm, and the images were taken by using least 2 a-Syn proteins (Fig. 1A). We have generated trans-
Zen software (Carl Zeiss AG). genic Drosophila models expressing PD-linked a-Syn[A53T]
tagged with BiFC-Venus under the control of the UAS/
Gal4-system. Although Drosophila models do not express
Visualization of a-Syn fibrils
endogenous a-Syn, the fly has been frequently used to model
Fly head lysates were prepared with soluble and insoluble fraction
aspects of a-Syn toxicity and aggregation (16, 30–41). a-
dissolved in RIPA lysis buffer and 8 M urea buffer, respectively, Syn[A53T] was chosen because it is causing PD and has been
and immediately fixed in 2.5% glutaraldehyde in 0.1 M phosphate shown in human cells to form visible Venus-positive punctae
buffer for 24 h and embedded in 2% agarose (at 60 1C; 05073; representing a-Syn aggregates (42). Pan neural expression
Fluka, St. Louis, MO, USA). Small blocks of embedded cells were (elavC155-Gal4) of these a-Syn[A53T] variants resulted in a
sliced and postfixed in 2.5% glutaraldehyde for 24 h followed by detectable fluorescent Venus signal in whole mount prepa-
washing in 0.1 M phosphate buffer for 24 h. Agarose blocks were rations of fly brains. As expected, signal intensities increased
then incubated in 1% OsO4 (in 0.2 M phosphate buffer) for a 3 h
time period, washed twice in distilled water, and dehydrated by over time, indicative of a continuous aggregation of a-Syn.
using ascending alcohol concentrations (25, 35, 50, 70, 85, 95, and Staining fly brains with Thioflavin-T revealed the amyloido-
100%; 5 min each). Dehydrated blocks were incubated in propy- genic nature of the a-Syn aggregates (Supplemental Fig. S1).
lenoxide followed by subsequent 20-min incubation in a 1:1 The flies with pan-neural expression of a-Syn[A53T] tagged
mixture of Epon [47.5% glycidether, 26.5% dodenylsuccinic acid with BiFC-Venus were used for all subsequent analyses. For
anhydride, 24.5% methylnadic anhydride, and 1.5% Tris (dime- simplicity, we refer to these as elav . a-Syn flies henceforth. In
thylaminomethyl) phenol] and propylenoxide. The samples were
head lysates from our elav . a-Syn flies, ;2% of total protein
then incubated in epoxy resin for 1 h at room temperature fol-
lowed by polymerization (28 1C for 8 h, 80 1C for 2.5 h, and finally corresponds to a-Syn (0.02 mg a-Syn per 1 mg total protein).
at room temperature for 4 h). Epon blocks were further processed The high abundance of a-Syn might explain the short time
for transmission electron microscopy: ultra-thin sections (70 nm) interval of aggregate formation in our in vivo model.
were mounted on grids, and the contrast was enhanced with a-Syn is known to form RIPA soluble aggregates (most
uranyl acetate and lead citrate, followed by examination with a likely low MW oligomers) and RIPA insoluble aggregates
Philips EM 400 transmission electron microscope (Philips GmbH, (most likely high MW oligomers and fibrils). To separate and
Hamburg, Germany) as previously described. The images were
captured by using a Morada digital camera (Olympus, Tokyo,
visualize these 2 a-Syn fractions, a simple approach (sum-
Japan). In cases of light microscopy, ultra-thin (1 mM) sections marized in Fig. 1) was used. We lysed heads of elav . a-Syn
stained with toluidine blue were examined by using a Zeiss Axi- flies and separated the RIPA-soluble and RIPA-insoluble
oplan microscope with a Zeiss Axiocam 506 color camera (Carl a-Syn fractions by centrifugation. The supernatants con-
Zeiss AG) with 3100 lens. taining RIPA-soluble a-Syn aggregates were used for optical
assessment of Venus fluorescent signal intensities. The pellets
Statistical analysis containing RIPA-insoluble a-Syn aggregates were dissolved
in urea/thiourea buffer, sonicated, and subjected to FRA, a
GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, method commonly used to visualize and quantify insoluble
USA) was used for statistical analysis and data presentation. protein aggregates (43–46). This approach allowed us to
Means 6 SD are presented in all graphs. Tests used for detect changes in abundance of soluble and insoluble a-Syn

Figure 1. Schematic representation of the BiFC principle and sample generation for subsequent analysis. A) Diagram illustrating the used
a-Syn[A53T] coupled BiFC system. A reconstitution of a fluorescent Venus protein is driven by the accumulation of a-Syn[A53T]. B)
Summary of the standardized readout system used to determine the effect of a given manipulation (genetic manipulation and/or
supplementing bioactive agents to the fly food) on a-Syn[A53T] aggregation properties comparing untreated control (C) vs. treated (T)
samples. Individual 0–2-d-old male flies with pan-neural (elav-Gal4C155) expression of BiFC a-Syn[A53T] were transferred to compound-
containing food vials. Subsequently, fly heads were lysed in RIPA, and the soluble and insoluble fractions were separated by centrifugation at
13,000 g. Abundance of a-Syn aggregates in the soluble fraction was assessed by determination of Venus signal intensities. The RIPA-
insoluble pellet was dissolved in urea buffer by sonication, and the amount of a-Syn was visualized by FRA utilizing a-Syn–specific antibodies.
C) Diagram depicting the detailed experimental design analyzing the soluble and insoluble fractions. The Venus signal intensities in the
soluble fraction are shown in bar graphs throughout the article. For every single treatment, we assessed whether the treatment had an
impact on general transcript/translation by using a simple Ponceau stain. The abundance of soluble a-Syn was controlled by Western blot
analysis. When no obvious changes (total protein and a-Syn abundance) were observed, we proceeded with the analysis of the insoluble
fraction. Protein was extracted from the pellet by urea solubilization and submitted to FRA. With regard to the insoluble pellet, the band
intensities derived from lysates of untreated control flies were used for normalization. Signal intensities determined in lysates of treated flies
were quantified in relation to untreated control, and the results are presented as a scatter plot. The raw data (not normalized) from each
dataset is also presented as a line plot, and significant differences (paired Student’s t test) are indicated. D) Toluidine blue–stained ultra-thin
sections (before ultra-structural analysis) of fly lysates derived from a-Syn–expressing flies. In the soluble fraction, a few fibrillar structures
(arrows) were visible, whereas the insoluble fractions showed abundant fibrillar species (arrows), probably denoting insoluble a-Syn fibrils.
In negative controls (fly head lysates from Canton-S), we were unable to detect similar structures either in the soluble or in the insoluble
fraction (data not shown). Scale bars, 2 mM. E) Ultra-thin sections were processed for EM; soluble fraction from the negative control (top
panel) shows no fibrillar structure consistent with the results seen in D, whereas the insoluble fraction from the a-Syn–expressing flies
consistently showed several fibrillar structures denoting insoluble a-Syn fibrils. Scale bars, 1 mM.
aggregates in flies. In addition, we confirmed the presence of We used this experimental outline to analyze the effect
fibrillar structures in the insoluble a-Syn fractions both in of different substances on a-Syn aggregate formation. To
ultra-thin sections (Fig. 1D) and via electron microscopy detect the optimal concentration of administered com-
(Fig. 1E), which were absent in the soluble a-Syn fraction pounds, titration of reported bioactive concentrations has
(Fig. 1D, E). Consistent with the electron microscopic find- been performed. Ten male elav . a-Syn flies were exposed to
ings, Thioflavin-T staining in a-Syn expressing fly brains a given compound concentration, and survival was moni-
(Supplemental Fig. S1) also showed the presence of fibrillar, tored in an automated fashion over a time period of
amyloidogenic a-Syn, in insoluble protein fractions. 5 d (using a DAM2 Drosophila Activity Monitor; TriKinetics).

Recording of movement was performed by using a 1-h significant increase in the abundance of soluble as well as
reading interval. The first hour of continuous absence of insoluble a-Syn aggregates (Fig. 3B). In contrast, we
locomotion was considered as the time point of death. treated elav . a-Syn flies with rapamycin, an inducer of
Concentrations were reduced by 50% as long as all flies autophagy (58–60). As expected, rapamycin treatment had
survived the 5-d period (Supplemental Figs. S2 and S3). In no impact on the abundance of soluble a-Syn. However,
addition, Western blot analysis of treated vs. untreated insoluble a-Syn species were significantly reduced by
elav . a-Syn flies was performed to determine whether ;50% upon treatment with rapamycin (Fig. 3C). The ef-
administration of the tested compounds had an effect on ficacy of treatment with 3MA or rapamycin was verified
a-Syn expression and abundance of a-Syn in the soluble by determining the phosphorylation levels of S6 kinase
fraction. After normalization of a-Syn levels (to synapsin), (S6K and pS6K, respectively). Activation of mechanistic
no obvious changes were observed on soluble a-Syn levels target of rapamycin causes phosphorylation/activation of
upon administration of compounds (Supplemental Fig. S4). S6K. Thus, pS6K levels should be reduced after rapamycin
treatment and enhanced after 3MA treatment (61, 62). We
confirmed these changes in pS6K abundance after re-
Soluble a-Syn aggregates are removed by the spective treatments (Supplemental Fig. S5).
proteasome, insoluble a-Syn aggregates In summary, our in vivo data strongly suggest that
by autophagy soluble a-Syn aggregates are removed via the ubiquitin/
proteasome system. Insoluble a-Syn aggregates are in turn
There are 2 major cellular pathways suggested to remove removed by the autophagic/lysosomal pathway.
aggregated a-Syn, the ubiquitin/proteasomal degrada-
tion pathway and autophagy (we are not discriminating
here between chaperone-mediated autophagy and mac- a-Syn aggregation is largely unaffected by
agents implicated as causing PD
roautophagy) (47). We decided to interfere with these
degradation pathways by using well-established chem-
Based on several epidemiologic studies, exposure to cer-
icals. Proteasomal inhibition using MG132 is established in
tain agents such as rotenone or oxidative stress–inducing
flies (48–51). As expected, treatment of elav . a-Syn flies
agents is believed to cause or at least represent risk factors
with the proteasomal inhibitor MG132 resulted in an in-
for the development of PD (22). To test whether these
crease of soluble a-Syn multimers and insoluble a-Syn
agents interfere with a-Syn aggregation, we exposed elav .
(Fig. 2A). In case soluble a-Syn aggregates are not effi-
a-Syn flies to an almost lethal dose of the complex I in-
ciently removed, these aggregates are believed to continue
hibitor rotenone. Doubling the dose of rotenone caused
the aggregation process and eventually become insoluble,
lethality within 5 d of treatment (Supplemental Fig. S2D).
which would explain the ;7-fold increase of insoluble
After administration of rotenone, no change in the soluble
a-Syn aggregates observed after inhibition of the
fraction of a-Syn aggregates was observed compared with
proteasome. To further validate our system and support
controls (Fig. 4A). However, rotenone-treated flies dis-
our previous findings, we used the powerful genetics in
played a ;1.5-fold increase in the abundance of insoluble
flies. Hsp70 is a chaperone that assists in protein folding
a-Syn aggregates. Rotenone treatment in flies causes an
and refolding of misfolded proteins in an ATP-dependent
increase in ROS as a secondary effect of complex I in-
manner. In addition, Hsp70 in conjunction with the E3
hibition (63–67). Thus, we assessed the effect of the general
ubiquitin ligase CHIP targets misfolded proteins to pro-
ROS-inducer H2O2 on a-Syn aggregation. In this case as
teasomal degradation by supporting ubiquitinylation of
well, we used an almost lethal dose of H2O2. Doubling the
the substrate (reviewed elsewhere [52, 53]). According to
dose used caused lethality within 5 d of treatment (Sup-
these functions, overexpression of human Hsp70 re-
plemental Fig. S6A). As was observed after rotenone
portedly protects flies in the context of a-Syn–induced
treatment, H2O2 administration did not change the abun-
detrimental effects (31, 54). The Drosophila model possesses
dance of soluble a-Syn aggregates. In contrast, there was an
12 copies of Hsp70, and reduction in Hsp70 copy number
;2.0-fold increase in the abundance of insoluble a-Syn
impaired the fly’s ability to compensate for certain stresses
aggregates after H2O2 treatment (Fig. 4B). However, when
(55). Accordingly, we reduced the gene copy number of
we tested redox state in the fly head lysates of H2O2-treated
Hsp70 in elav . a-Syn flies. Removing 50% of Hsp70 copy
flies, only slight increases in ROS were detected (Supple-
numbers (6 copies) caused an increase in the abundance of
mental Fig. S6B, C).
soluble and insoluble a-Syn aggregates (Fig. 2B). In con-
trast, overexpression of human Hsp70 (hHsp70) reduced
both soluble and insoluble a-Syn aggregates (Fig. 2C). Metal ions and a-Syn aggregation in vivo
In the next step, we inhibited autophagy by using 3-
methyladenine (3MA), also a well-known inhibitor of Exposure to heavy metal ions is discussed as enhancing
autophagy in flies (56–58). 3MA treatment of elav . a-Syn the risk of developing PD, and metal ions reportedly en-
flies had no effect on soluble a-Syn and no significant in- hance a-Syn aggregation in vitro (68). Thus, we treated elav .
crease in accumulation of insoluble a-Syn species in our fly a-Syn flies with several divalent metal ions. We first ti-
model system. However, a slight trend toward an increase trated the dose of all tested metal ion salts to permit
in insoluble a-Syn was observed (Fig. 3A). Thus, we ge- survival for 5 d. Effective uptake of these ions was as-
netically blocked autophagosome formation by silencing sumed as administration thereof strongly reduced fly
Atg9 using RNA interference. Here, we observed a locomotion and resulted in early lethality (Supplemental

A B ** C
1.2 1.2
1.2
**
1.0
0.9 0.9
at 530nm (AU)
Signal intensity
at 530nm (AU)
Signal intensity
0.8
at 530nm (AU)
Signal intensity
0.6 0.6
0.6
***
0.4
0.3 0.3
0.2
0.0 0.0 0.0
FP
27
70
l
n
2
l
tro
tro
tio
13
-Q
sp
R
on
on
le
G
hH
.tr
de
M
C
C
JD
py
M
co
6
25 2.5 1.5
20 2.0
Band intensity relative

to control (AU)
to control (AU)
1.0
to control (AU)
15
1.5
10 1.0
0.5
5 0.5
0 0.0 0.0
l
n
l
FP
27
tro
tro
70
tio
13
-Q
R
on
on
sp
le
G
.tr
de
C
M
hH
C
JD
py
M
co
6
ns ns
2.5 *** 1.5 2.5
2.0
2.0
1.0
Band intensity
Band intensity
Band intensity
1.5
(AU)
(AU)
(AU)
1.5
1.0
0.5
1.0
0.5
0.0 0.0
0.0
l
n
l
27
tro
tro
FP
70
tio
13
-Q
on
on
sp
R
le
G
.tr
m
de
hH
C
C
JD
py
M
co
6
Figure 2. a-Syn aggregation and modulation of cellular protein degradation pathways. elav . a-Syn flies were either fed with
the proteasome inhibitor MG132 or genetically modified with Hsp70 (deletion or overexpression). The effects of these alterations on the
abundance of soluble a-Syn aggregates was determined by the assessment of Venus signal intensities in fly head lysates (upper row). The
abundance of a-Syn aggregates in the insoluble fraction was determined by FRA and subsequent quantification of band intensities. Band
intensities relative to control (lower row) are displayed. A) Administration of MG132 increased the abundance of both soluble as well as
insoluble a-Syn aggregates. Significance determined by Student’s t test. B) The Drosophila model has 12 copies of Hsp70. On removing 6
copies of Drosophila Heat shock protein 70 (dHsp70) (reducing the dHsp70 levels to half), a strong increase in the abundance of soluble
protein fraction and a trend in the increase in the insoluble a-Syn aggregates were observed. elav . a-Syn flies served as an unaltered
control (black bar). Significance determined by Student’s t test. C) Overexpression of human Hsp70 (hHsp70) reduced soluble and
insoluble a-Syn aggregate abundance. Expression of the monomeric red fluorescent protein (mRFP) or a nontoxic ataxin-3–derived
peptide with 27 glutamines (controls, black bars) had no impact on a-Syn aggregation. One-way ANOVA followed by Dunnett’s multiple
comparison test was used to determine significance compared with control (C). Ns, not significant. Only significant differences are
indicated. **P , 0.01, ***P , 0.001.

Soluble Fraction
A 1.2
B 2.4
C 1.2
***
1.0 2.0
0.9
1.6
at 530nm (AU)
at 530nm (AU)
Signal intensity
Signal intensity
at 530nm (AU) 0.8
Signal intensity
0.6 1.2 0.6
0.4 0.8
0.3
0.2 0.4
0.0 0.0 0.0
Ai
Ai
l
l
tro
tro
in
3M
yc
on
on
lR
-R
am
C
C
g9
tro
ap
At
on
R
C
Insoluble Fraction
8 10 1.5
8

6
1.0
to control (AU)
to control (AU)
to control (AU)
6
4
4
0.5
2
2
0 0 0.0
Ai
Ai
l
l
A
tro
tro
in
3M
yc
on
on
lR
-R
am
C
C
g9
tro
ap
At
on
R
C
2.0 15 2.5
** ***
2.0
1.5
10
Band intensity
Band intensity
Band intensity
1.5
(AU)
(AU)
(AU)
1.0
1.0
5
0.5
0.5
0.0 0 0.0
Ai
Ai
l
l
tro
tro
in
3M
yc
on
on
lR
-R
am
C
C
g9
tro
ap
At
on
R
C
Figure 3. Effect of alterations in autophagy on a-Syn aggregation. Decreasing or increasing autophagy in elav . a-Syn flies had opposing
effects on the abundance of insoluble a-Syn aggregates. The effect of authophagy alterations on abundance of soluble a-Syn aggregates was
determined by the assessment of Venus signal intensities in fly head lysates (upper row). The abundance of a-Syn aggregates in the
insoluble fraction was determined by FRA followed by quantification of band intensities. Displayed are band intensities relative to control
(lower row). A) Administration of the autophagy inhibitor 3MA did not affect the abundance of soluble a-Syn aggregates in fly head lysates.
FRA revealed that 3MA treatment caused a slight increase in insoluble a-Syn aggregates. B) Genetic ablation of autophagy by silencing of
Atg9 via RNA interference (RNAi). Atg9 serves as an important initiator that is essential in autophagosome formation. Down-regulation of
Atg9 revealed a marked increase in soluble as well as insoluble a-Syn aggregates. C) Induction of autophagy by administration of rapamycin
had no effect on soluble a-Syn aggregates. However, the amount of insoluble a-Syn aggregates was decreased by ;50% upon
administration of rapamycin. Significance determined by Student’s t test. The Student’s t test revealed no significant differences in Venus
signal intensities of treated flies compared with controls. Solvent-only treated flies served as control. **P , 0.01, ***P , 0.001

A Soluble Fraction Insoluble Fraction
3 2.5
1.2
2.0
1.0

2
to control (AU)
Band intensity
0.8 1.5
at 530nm (AU)
Signal intensity
(AU)
0.6
1.0
1
0.4
0.5
0.2
0.0 0 0.0
l
e
tro
tro
tro
on
on
on
on
on
on
en
en
en
C
C
ot
ot
ot
R
R
B ***
6 1.5
1.2
1.0
4 1.0
to control (AU)
Band intensity
0.8
at 530nm (AU)
Signal intensity
(AU)
0.6
0.4 2 0.5
0.2
0.0 0 0.0
l
l
tro
tro
tro
O
O
2
2
on
on
on
on
H
H
2
2
en
C
C
ot
R
Figure 4. Oxidative stress induces accumulation of insoluble a-Syn aggregates. A) Administration of rotenone did not cause an increase
in soluble (upper row) and only mildly enhanced the abundance of insoluble (lower row) a-Syn aggregates. B) Induction of oxidative
stress by administration of H2O2 had no impact on the abundance of soluble a-Syn aggregates in fly neurons; however, it did result in a
robust increase in insoluble a-Syn aggregates. Significance determined by Student’s t test. ***P , 0.001.
Fig. S3). Following our standardized protocol, we ana- consistent mild increase in soluble and insoluble a-Syn
lyzed whether supplementation of heavy metal ions had aggregates (Fig. 6A). We next questioned whether treat-
an impact on the amount of soluble and insoluble a-Syn ment with another trivalent ion, aluminum (Al3+), had a
aggregates after 5 d of exposure. Administration of man- similar effect on a-Syn aggregation. In contrast to Fe3+,
ganese and mercury to elav . a-Syn flies caused a subtle elav . a-Syn flies fed with Al3+ displayed no changes in
but significant increase in soluble a-Syn aggregates. the abundance of soluble a-Syn aggregates. However, the
Treatment with zinc and divalent iron had no effect on the abundance of insoluble a-Syn aggregates was strongly
abundance of soluble a-Syn aggregates, whereas admin- reduced (;70%) compared with controls in the presence of
istration of copper caused a slight but significant decrease Al3+ (Fig. 6B).
in soluble a-Syn aggregates. The observed changes of
treatment with divalent metal ions on the abundance of Phosphorylation of a-Syn promotes
insoluble a-Syn aggregates were minor. Compared with aggregation and enhances toxicity
control, we detected a maximum 30% increase or decrease
in abundance of insoluble a-Syn aggregates (Fig. 5). CK1 d has been reported to phosphorylate a-Syn at serine
a-Syn possesses an iron-binding capacity, and it is 129 (70–72). Thus, we overexpressed Dco (dco), the fly
established that trivalent ferric ion (Fe3+) promotes a-Syn ortholog of CK1 d /e in our elav . a-Syn flies (73, 74). Sub-
aggregation in vitro (68, 69). We therefore administered sequent Western blot analysis using a phospho-serine 129
trivalent Fe3+ to elav . a-Syn flies. This treatment caused a (pSer129)-specific antibody revealed a significant increase in

A B C D E
1.2 1.2 1.2 1.2 1.2
** ***
Signal intensity *
at 530nm (AU) 0.9 0.9 0.9 0.9 0.9
0.6 0.6 0.6 0.6 0.6
0.3 0.3 0.3 0.3 0.3
0.0 0.0 0.0 0.0 0.0

l
Zn
2+
tro
tro
tro
tro
tro
C
Fe
on
on
on
on
on
C
C
5 2.0 3 4 2.0
to control (AU)
4
1.5 3 1.5
2
3
1.0 2 1.0
2
1
0.5 1 0.5
1
0 0.0 0 0 0.0
l
Zn
2+
tro
tro
tro
tro
tro
C
Fe
on
on
on
on
on
C
C
* **
1.5 2.0 2.0 32 10
16 8
Band intensity
1.5 1.5
1.0
8 6
(AU)
1.0 1.0
4 4
0.5
0.5 0.5
2 2
0.0 0.0 0.0 0 0

l
Zn
2+
tro
tro
tro
tro
tro
C
Fe
on
on
on
on
on
C
C
Figure 5. Effect of divalent metal ions on a-Syn aggregation. Administration of divalent metal ions Cu2+ (A), Mn2+ (B), Hg2+ (C), Zn2+
(D), and Fe2+ (E) did not result in an robust increase in soluble (upper row) and insoluble (lower row) abundance of aggregated a-Syn.
Although administration of Mn2+ and Hg2+ did significantly increase the abundance of soluble a-Syn aggregates in flies, the effects are
relatively subtle compared with treatment with MG132 or alterations of Hsp70 levels (compare with Fig. 2). In addition, the mild increase
in the abundance of soluble aggregates did not result in a robust increase in insoluble aggregates. Only significant differences are
indicated. Significance determined by Student’s t test. *P , 0.05, **P , 0.01, ***P , 0.001.
a-Syn phosphorylation at serine 129 with Dco coexpression. revealed variation with day 10 Dco[WT] flies exhibiting
This outcome was not observed when an inactive variant of poor climbing ability (Supplemental Fig. S7). This finding
Dco (Dco[K38R]) was expressed (Fig. 7A–C). Similarly, implies that phosphorylation of a-Syn by Dco not only en-
coexpression of Dco and a-Syn in flies resulted in enhanced hances formation of soluble and insoluble a-Syn aggregates
oligomerization of a-Syn and increased its abundance in the but also increases a-Syn–dependent neuronal dysfunction.
soluble fraction followed by a trend in the increase of the
insoluble fraction (Fig. 7D–F). The next step was to de-
termine if an increase in phosphorylation has an impact on DISCUSSION
the detrimental effects of a-Syn expression. We therefore
performed negative geotaxis analysis of flies. We found that The presence of a-Syn aggregates (namely, Lewy bodies
pan neural expression of neither Dco[WT] nor inactive Dco and Lewy neurites) is one of the pathologic hallmarks of
[K38R] altered locomotion. In contrast, elav . a-Syn flies PD (75). Assessing the formation and the removal of these
coexpressing either Dco[WT] or the inactive Dco[K38R] structures in vivo is challenging. Lately, BiFC has been used
exhibited marked differences in locomotion. Ten-d post- to efficiently monitor a-Syn aggregation in cultured cells
eclosion, elav . a-Syn flies coexpressing Dco[K38R] per- and even rodent models (11, 23, 29). Using BiFC to address
formed significantly better compared with elav . a-Syn flies aggregation properties of a-Syn, it has the advantage that
coexpressing Dco[WT] (Fig. 7G). A comparison of motor only a-Syn aggregates (referring to dimers as minimal
performance on day 1 vs. day 10 posteclosion among the fluorescent species up to higher order multimers) are de-
elav . a-Syn flies expressing GFP, Dco[K38R], or Dco[WT] tected by their fluorescent signals, whereas monomeric

A Soluble Fraction
1.2 4 2.0
***

0.9 3 1.5
Signal Intensity
at 530nm (AU)
to control (AU)
Band intensity
(AU)
0.6 2 1.0
0.3 1 0.5
0.0 0 0.0
l
3+
3+
3+
tro
tro
tro
Fe
Fe
Fe
on
on
on
C
C
B **
Insoluble Fraction
1.2 1.5 2.7
0.9 6.7
1.0
Signal Intensity
at 530nm (AU)
to control (AU)
Band intensity
(AU)
0.6 1.7
0.5
0.3 0.4
0.0 0.0 0.1

l
AI
AI
AI
tro
tro
tro
on
on
on
C
C
Figure 6. Effect of trivalent metal ions on a-Syn aggregation. A) Administration of trivalent ferric ions caused a slight increase in the
abundance of soluble (upper row) fraction and a trend in the increase in insoluble (lower row) a-Syn aggregates. B) Presence of trivalent
aluminum ions in the fly food did not affect the amount of soluble a-Syn aggregates. Insoluble aggregates were strongly reduced in
elav . a-Syn flies. Only significant differences are indicated. Significance determined by Student’s t test. **P , 0.01, ***P , 0.001.
a-Syn does not provide any fluorescent signal. However, agreement with multiple studies, we expect that a-Syn
based on BiFC detection, discrimination of dimers or oligomers (from dimer to x-mer) are mainly present in the
higher order oligomers is not possible. BiFC-Venus frag- soluble fraction. The insoluble fraction should be enriched
ments attached to a-Syn might influence the speed of ag- with fibrillar a-Syn aggregate species (76–78). This as-
gregate formation and might also affect the kinetics of sumption is further substantiated by our results obtained
monomer exchanges within formed a-Syn oligomers. Be- after inhibition of cellular degradation pathways. Inhibit-
cause we always perform an analysis of flies (siblings) ing the proteasome by administration of MG132 to flies, an
comparing treated vs. untreated situations, we can only increase was noted in the abundance of soluble as well
expect that the effect by Venus itself on a-Syn aggregation as insoluble a-Syn aggregates. Compared with the mild
should be similar/identical in treated as well as in un- but significant increase in the soluble aggregates, a strong
treated situations. Relying on previous knowledge, we (;7-fold) increase was observed in the insoluble a-Syn
applied the BiFC system to monitor neuronal aggregation aggregates. This finding suggests that soluble a-Syn ag-
of a-Syn in flies. By the generation of a stable fly stock with gregates seem to quickly accumulate to form higher order
pan neural expression of BiFC-competent a-Syn, we were insoluble aggregates in vivo. An alternative explanation
able to analyze whether different treatments have an effect would also be that insoluble aggregates could be degraded
on a-Syn aggregation in vivo. by the proteasome. This theory, however, is unlikely
In addition, we further divided aggregated a-Syn into 2 according to the sterical size exclusion. The proteasome
fractions, RIPA soluble and RIPA insoluble (Fig. 1B, C). diameter is very small (entrance: ;1–2 nm; inner core di-
Although we cannot directly prove which aggregates are ameter: 5–6 nm) and requires partial unfolding of peptides
present in the soluble and insoluble fractions in detail, in so as to be degraded (79). a-Syn oligomers are usually

A B 1.5
C 1.5 *

K38R
to Synapsin (AU)
WT
to Synapsin (AU)
Dco 1.0 1.0
Synapsin
G ***
VN-Syn
pSer129 0.5 0.5 100
Syn-VC 80
Motor Performance (%)

60
40
20
0.0 0.0 0
(10 dpe)
]
T]
T]
R
R
[W
[W
38
38
[K
co
[K
co
co
co
D
D
D
D
D E F
*** 4 20
1.4
1.2
FP
T]
R
at 530nm (AU)
Signal intensity
[W
3 15
38
to Control (AU)
G
Band intensity
[K
co
1.0
co
D
D
(AU)
0.8
2 10
0.6
0.4 1 5
0.2
0.0 0 0
]
T]
T]
T]
R
R
[W
[W
[W
38
38
38
[K
co
[K
co
[K
co
co
co
co
D
D
D
Figure 7. Phosphorylation of a-Syn at serine129, aggregation and toxicity in vivo. Assessment of the effect of Dco expression on
a-Syn phosphorylation and aggregation. A) Representative Western blot using an antibody specifically recognizing pSer129 on
the soluble a-Syn fraction revealed that expression of wild-type Dco enhanced serine 129 phosphorylation of a-Syn. B)
Quantification of pSer129 a-Syn abundance relative to loading control (synapsin), represented by scatter plot. C ) Quantification
of pSer129 a-Syn abundance relative to loading control (synapsin), represented by line plot. Significance in Student’s t test. D)
The effect of pSer129 on the abundance of soluble a-Syn aggregates was determined by the assessment of Venus signal intensities
in fly head lysates. Significance determined by Student’s t test. E ) The abundance of a-Syn aggregates in the insoluble fraction
determined by quantification of FRA band intensities, represented by scatter plot. F ) The abundance of a-Syn aggregates in the
insoluble fraction determined by quantification of FRA band intensities, represented by line plot. G) Assessment of climbing
ability of elav . a-Syn flies, coexpressing either Dco[WT] or Dco[K53R].The Dco[WT] coexpressing flies displayed a reduced
climbing ability at 10-d posteclosion. Significance determined by 1 way ANOVA followed by Bonferroni’s multiple comparison
test. Graph including time point 1- vs. 10-d posteclosion and subsequent statistical analysis (2-way ANOVA followed by
Bonferroni’s multiple comparison test is provided in Supplemental Fig. S6). *P , 0.05, ***P , 0.001
larger structures (.10 nm), and insoluble oligomers and Interestingly, both treatments (3MA and rapamycin) had
fibrils have even more elevated size ranges reaching a no impact on the abundance of soluble a-Syn aggregates
micrometers range (80). Thus, such structures do not fit the (Fig. 3A, C). These findings support the theory that soluble
proteasome. In the next step, we used the powerful ge- a-Syn aggregates are mainly degraded by the ubiquitin/
netics of flies. By ubiquitinylation, Hsp70/CHIP target proteasome pathway, and insoluble a-Syn aggregates are
misfolded proteins for proteasomal degradation and predominantly removed by autophagy.
overexpression of human Hsp70 has been shown to protect Our data also indicate that soluble a-Syn aggregates
from a-Syn–induced toxicity in flies (31, 53, 81). In our continue the aggregation process, resulting in the forma-
model, reduction in Hsp70 levels (by 50%) caused an in- tion of insoluble aggregates. This assumption would ex-
crease in soluble aggregated a-Syn and a mild enhance- plain the increase of soluble as well as insoluble aggregates
ment in the formation of insoluble a-Syn aggregates. In upon inhibition of the proteasome observed after MG132
contrast, overexpression of Hsp70 had the opposite effect treatment (Fig. 2A). A transition of insoluble a-Syn ag-
(Fig. 2B, C). We observed a decrease in soluble aggregates gregates toward soluble aggregates does not seem to be a
of ;50%. In contrast, there was only a mild decrease in the frequent event. Otherwise, we would expect that an in-
insoluble fraction of a-Syn aggregates. hibition of autophagy by 3MA would not only affect the
Conversely, inhibition of autophagy by 3MA caused a amount of insoluble but also soluble a-Syn aggregates.
very slight increase in insoluble aggregates, whereas acti- This outcome has not been observed (Fig. 3A).
vation of autophagy by administration of rapamycin de- In humans, an acute exposure to toxins (e.g., heavy
creased the abundance of insoluble a-Syn aggregates. metal ions or pesticides such as rotenone) is not implicated

Soluble fraction Insoluble fractionFigure 8. Schematic representation of a-Syn
aggregation in cells and cellular degradation
Monomer Oligomer Fibril pathways involved in the removal of aggregated
a-Syn. Monomeric a-Syn will form soluble,
LB
oligomeric aggregates. Upon further aggrega-
LN
tion and/or structural changes, oligomeric
a-Syn additionally accumulates and forms in-
Chaperones
soluble amyloidogenic fibrils. Sequestration of
these structures will eventually result in the
Autophagy/ formation of Lewy bodies (LB) and/or Lewy
Proteasome
Lysosome neuritis (LN). According to our data, soluble
oligomers are mainly degraded by the
Effector Abundance of Abundance of
soluble aggregates insoluble aggregates proteasome or might to a certain extent be
dissolved to monomers by chaperones (blue
Hsp70 oe down down
arrows). Once soluble oligomers are formed,
Hsp70 reduction up up these seem to continue aggregation fast and
become insoluble. Insoluble a-Syn aggregates
MG132 up strong upregulation
seem to be removed mainly via autophagy. The
3MA - up table summarizes the effects of alterations in
cellular protein degradation pathways on the
Rapamycin - down
abundance of soluble and insoluble a-Syn
aggregates. Overexpression (oe) of the human chaperone Hsp70 reduced the abundance of both soluble and insoluble a-Syn
aggregates. Reduction of endogenous Hsp70 (50%) had the opposite effect. Inhibition of the proteasome by MG132
administration increased soluble a-Syn aggregates and had a strong impact on insoluble a-Syn aggregates, increasing these
;7-fold. Alteration of autophagy had no impact on soluble a-Syn aggregates. Abundance of insoluble a-Syn aggregates was
slightly increased upon inhibition of autophagy (3MA) and decreased upon autophagy enhancement (rapamycin).
in the cause of PD. However, chronic exposure in low similar insoluble fraction differences were observed for Cu2+
doses has been discussed as a risk factor of PD (in- and Fe2+, with respect to divalent metal ion salts. Observed
discernible from sporadic PD) and a-Syn aggregation. In changes were ,30% increase or decrease, and no congruent
our acute fly model, formation of soluble and insoluble tendency was observed (Fig. 5). However, administration of
a-Syn aggregates was already observed after 5 d; conse- Fe3+ caused a slight but significant increase in the abundance
quently, the impact of toxins on aggregation of a-Syn of soluble (;10%) fraction and a trend in the insoluble (;1.4-
could be analyzed. Rotenone-treated flies displayed a very fold) a-Syn aggregate increase. The mild increase in soluble
slight increase (not significant) in the abundance of in- a-Syn aggregates suggests that Fe3+ either stabilizes a a-Syn
soluble a-Syn aggregates and in soluble aggregates. Ro- conformation that supports the formation of aggregates or
tenone is a rather specific inhibitor of complex I within the accelerates aggregation of soluble a-Syn. The soluble ag-
electron transport chain of mitochondria. Here, rotenone gregates continue the aggregation process and eventually
blocks the transfer of electrons from complex I to ubiqui- accumulate into insoluble a-Syn aggregates. This scenario
none. As a secondary effect of this action, ROS are pro- would explain the mild increase in the abundance of both
duced, and these ROS exert oxidative stress (82). Thus, we soluble and insoluble a-Syn aggregates. Surprisingly, we
directly induced oxidative stress by administration of found that administration of Al3+ strongly reduced the
H2O2. Similar to rotenone-treated flies, H2O2-treated flies abundance of insoluble a-Syn aggregates (by ;70%) with-
did not present significant changes in the abundance of out affecting the abundance of soluble a-Syn aggregates. We
soluble a-Syn aggregates. However, the abundance of can only speculate about the nature of this effect. Possible
insoluble a-Syn was increased in rotenone-treated flies explanations would be that high concentrations of Al3+ ions
(;1.3-fold), and even more insoluble a-Syn (;2.0-fold) enhance autophagy in an unknown manner. Alternatively,
was detected in H2O2-treated flies. This finding implies Al3+ ions could block the formation of insoluble a-Syn ag-
that ROS-induced oxidative stress enhances the accumu- gregates. A more detailed analysis of how Al3+ ions reduce
lation of insoluble a-Syn aggregates. The ability to handle the abundance of insoluble a-Syn aggregates is required,
ROS is decreasing with age (reviewed elsewhere [83]). especially as in vitro analysis revealed that Al3+ effectively
Age, in turn, is the main risk factor for developing sporadic enhanced a-Syn fibril formation (68).
PD. Our results are in agreement with the theory that the Finally, we addressed the impact of serine 129-phos-
accumulation of ROS during aging might contribute to the phorylation on a-Syn aggregation. In flies, phosphorylation
increased risk of developing PD. An imbalance in redox of serine 129 is implicated to increase a-Syn–induced tox-
state of cells might, either directly or indirectly, support icity and impact aggregation (16). In most analyses, phos-
a-Syn aggregation. It is noteworthy that ROS levels are phomimetic mutants (S129D/E) and not phosphorylatable
usually kept more or less constant by ROS scavengers, mutants (S129A) of a-Syn have been used. It is possible that
allowing cells to compensate for increased ROS to a certain these artificial mutant variants might not perfectly reflect
degree. authentic serine phosphorylation in all accounts.
Administration of divalent metal ions (Mn2+, Zn2+, Hg2+, CK1 d has been reported to phosphorylate a-Syn at serine
or Cu2+) to the fly food had some impact on the abundance 129 (71, 72). Drosophila models possess an ortholog of serine/
of soluble and insoluble aggregates. Significant differences threonine kinase family named Dco. Coexpression of ac-
in soluble fraction were observed for Mn2+ and Hg2+, and tive Dco enhanced a-Syn phosphorylation at serine

129. This enhanced phosphorylation coincided with in- microscopy images; and V. Prasad, J. B. Schulz, and A. Voigt
creased abundance of a-Syn aggregates (a trend in the wrote the paper, and all authors read and approved the
increase in the insoluble fraction) and increased a-Syn– final manuscript.
dependent toxicity shown by the reduced locomotor ac-
tivity in flies with coexpression of a-Syn and Dco (Fig. 7).
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JOURNAL • RESEARCH • www.fasebj.org

Monitoring a-synuclein multimerization in vivo

Compounds and metal ions were administered in artificial fly

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Determination of Venus signal intensities Determination of reactive oxygen species levels

MONITORING a-SYNUCLEIN MULTIMERIZATION 3

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0.0 0.0 0.0

Band intensity relative

Band intensity relative

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0.6 1.2 0.6

0.0 0.0 0.0

Band intensity relative

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Band intensity relative

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0.6 0.6 0.6 0.6 0.6

0.3 0.3 0.3 0.3 0.3

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0.0 0.0 0.0 0 0

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Band intensity relative

0.0 0.0 0.1

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Band intensity relative

Motor Performance (%)

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