Extensive uptake of α-synuclein oligomers in astrocytes results in sustained intracellular deposits and mitochondrial damage

Accepted Manuscript Extensive uptake of α-synuclein oligomers in astrocytes results in sustained intracellular deposits and mitochondrial damage

Veronica Lindström, Gabriel Gustafsson, Laurie H. Sanders, Evan H. Howlett, Jessica Sigvardson, Alex Kasrayan, Martin Ingelsson, Joakim Bergström, Anna Erlandsson PII: DOI: Reference:

S1044-7431(16)30184-1 doi: 10.1016/j.mcn.2017.04.009 YMCNE 3186

To appear in:

Molecular and Cellular Neuroscience

Received date: Revised date: Accepted date:

10 October 2016 12 April 2017 20 April 2017

Please cite this article as: Veronica Lindström, Gabriel Gustafsson, Laurie H. Sanders, Evan H. Howlett, Jessica Sigvardson, Alex Kasrayan, Martin Ingelsson, Joakim Bergström, Anna Erlandsson , Extensive uptake of α-synuclein oligomers in astrocytes results in sustained intracellular deposits and mitochondrial damage. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ymcne(2017), doi: 10.1016/j.mcn.2017.04.009

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ACCEPTED MANUSCRIPT Extensive uptake of α-synuclein oligomers in astrocytes results in sustained intracellular deposits and mitochondrial damage

Veronica Lindström1*, Gabriel Gustafsson1, Laurie H. Sanders2, Evan H. Howlett2, Jessica

Department of Public Health and Caring Sciences/Geriatrics, Rudbeck Laboratory, Uppsala

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Sigvardson3, Alex Kasrayan3, Martin Ingelsson1, Joakim Bergström1, Anna Erlandsson1

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University, Dag Hammarskjölds väg 20, S-751 85 Uppsala, Sweden

Pittsburgh Institute for Neurodegenerative Diseases and Department of Neurology,

BioArctic AB, Warfvinges väg 35, S-112 51 Stockholm, Sweden

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University of Pittsburgh, Pittsburgh, PA 15260, USA

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Corresponding author:

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Department of Public Health and Caring Sciences/Geriatrics, Uppsala University, Rudbeck Laboratory, Dag Hammarskjölds väg 20, S-751 85, Uppsala, Sweden Telephone: +46 18 4715030

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E-mail: [email protected]

Running title:

Accumulation of α-synuclein oligomers in astrocytes

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ACCEPTED MANUSCRIPT Abbreviations: PD, Parkinson’s disease; DLB, dementia with Lewy bodies; MSA, multiple system atrophy; HNE, 4-hydroxy-2-nonenal; GFAP, glial fibrillary acidic protein; CNPase, 2’,3’-cyclic-nucleotide

3’-phosphodiesterase;

mAb,

monoclonal

antibody;

IgG,

immunoglobulin G; LAMP-1, lysosomal-associated membrane protein 1; DAPI, 4’,6diamidino-2-phenylindole; PFA, paraformaldehyde; FGF2, Fibroblast Growth Factor 2; EGF,

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Epidermal Growth Factor; Drp1, dynamin-related protein 1; GFP, green fluorescent protein;

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TEM, transmission electron microscopy; Thio S, thioflavin S; QPCR, quantitative polymerase

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chain reaction; mtDNA, mitochondrial DNA

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ACCEPTED MANUSCRIPT Abstract The presence of Lewy bodies, mainly consisting of aggregated α-synuclein, is a pathological hallmark of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). The αsynuclein inclusions are predominantly found in neurons, but also appear frequently in astrocytes. However, the pathological significance of α-synuclein inclusions in astrocytes and

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the capacity of glial cells to clear toxic α-synuclein species remain unknown. In the present

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study we investigated uptake, degradation and toxic effects of oligomeric α-synuclein in a co-

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culture system of primary neurons, astrocytes and oligodendrocytes. Alpha-synuclein oligomers were found to co-localize with the glial cells and the astrocytes were found to

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internalize particularly large amounts of the protein. Following ingestion, the astrocytes started to degrade the oligomers via the lysosomal pathway but, due to incomplete digestion,

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large intracellular deposits remained. Moreover, the astrocytes displayed mitochondrial abnormalities. Taken together, our data indicate that astrocytes play an important role in the

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clearance of toxic α-synuclein species from the extracellular space. However, when their

Keywords:

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cellular processes.

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degrading capacity is overburdened, α-synuclein deposits can persist and result in detrimental

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α-synuclein oligomers, astrocytes, oligodendrocytes, Parkinson’s disease, glia, mitochondria

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Introduction Brains from patients with Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are characterized by Lewy bodies and Lewy neurites, intracellular inclusions predominantly consisting of insoluble α-synuclein fibrils (Spillantini et al., 1997). In healthy neurons, α-

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synuclein is highly abundant in the cytosol and presynaptic terminals, but its exact function

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remains unclear. During fibril formation, α-synuclein generates soluble intermediate

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aggregates, i.e. oligomers, which are particularly neurotoxic. For example, oligomeric αsynuclein has been shown to disrupt cellular membranes (Danzer et al., 2007; Winner et al.,

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2011) and induce mitochondrial dysfunction (Chinta et al., 2010; Luth et al., 2014).

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Although α-synuclein deposits are primarily found in neurons, they also appear frequently in astrocytes at advanced disease stages (Braak et al., 2007; Croisier and Graeber, 2006; Terada

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et al., 2003; Tu et al., 1998; Wakabayashi et al., 2000). In contrast to neurons, astrocytes

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express very low levels of α-synuclein (Mori et al., 2002) and the glial inclusions therefore likely stem from adjacent neurons. Possibly, α-synuclein can spread from neurons to glial

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cells via the extracellular space or via direct cell-to-cell transfer (Angot et al., 2012; Hansen et al., 2011; Reyes et al., 2015). In line with this hypothesis, astrocytes have been demonstrated

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to readily take up extracellular α-synuclein in vitro (Fellner et al., 2013; Lee et al., 2010b; Rannikko et al., 2015), a process that also activates pro-inflammatory responses (Fellner et al., 2013; Lee et al., 2010b). Moreover, astroglial inclusions have been found in transgenic αsynuclein mice after intracerebral injections of fibrillar or soluble forms of α-synuclein (Sacino et al., 2014).

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ACCEPTED MANUSCRIPT Astrocytes respond to pathological conditions through a process referred to as reactive astrogliosis. Thereby they upregulate their intermediate filaments, become hypertrophic, secrete various inflammatory mediators and transform to a phagocytic state (Buffo et al., 2010; Lööv et al., 2012; Lööv et al., 2015). We and others have shown that reactive astrogliosis is closely connected to α-synuclein pathology in mouse models of PD and DLB

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(Lindström et al., 2014; Neumann et al., 2002; Rockenstein et al., 2002). Moreover, reactive

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astrocytes have been demonstrated to be intimately associated with α-synuclein pathology in

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the human PD/DLB brain (Miklossy et al., 2006; Thannickal et al., 2007). Although there is growing evidence that astrocytes are highly involved in the pathology of PD/DLB, the

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functional consequence of α-synuclein deposition in astrocytes and their role in the disease

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progression remain unknown.

In the present study, we investigated the role of astrocytes in uptake, degradation and toxicity

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of α-synuclein oligomers. We found that these cells rapidly ingested large amounts of

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oligomers that were not efficiently degraded, resulting in the formation of ubiqutinylated, intracellular inclusions. The α-synuclein containing astrocytes remained viable, but displayed

Animals

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Methods

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mitochondrial impairment.

All experiments were approved by the Uppsala County Animal Ethics Board, following the rules and regulations of the Swedish Animal Welfare Agency, and in compliance with the European Communities Council Directive (2010/63/EU). The mice were housed in a 12:12 dark:light cycle, kept in an enriched environment and given water and food ad libitum. Embryonic C57Bl6 mice were used for cell culture experiments and brains from adult (Thy1)-h[A30P]α-synuclein mice (Kahle et al., 2000) were used for immunohistochemistry. 5

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Neural cell culture Embryonic cortical stem cells were isolated and expanded as neurospheres as previously described (Loov et al., 2012; Sollvander et al., 2016). The cells were passaged every third day, followed by seeding of dissociated cells (1.5x105 cells/ml) on cell culture dishes or cover

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slips coated with poly-L-ornithine (Sigma) and laminin (Invitrogen). During the first 24 h

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cells were maintained in EGF and FGF2 supplemented media before being replaced with

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mitogen-free medium to initiate cell differentiation. The media was replaced in full every second to third day during the differentiation period of seven days. After one week of

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differentiation the culture consisted of a mixed population of astrocytes (75%), neurons (20%) and oligodendrocytes (5%), without any microglia. This cell model has previously been well

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characterized (Davis and Temple, 1994; Johe et al., 1996; Lööv et al., 2012; Ravin et al.,

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2008). Neurospheres from passage 1–3 were used for the experiments.

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Generation of α-synuclein oligomers, fibrils and labeling Recombinant α-synuclein was produced as previously described (Nasstrom et al., 2011).

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Monomeric α-synuclein was incubated with 4-hydroxy-2-nonenal (HNE, Cayman Chemicals) in a HNE:α-synuclein ratio of 30:1 at 37 °C for 72 h. Size exclusion chromatography

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revealed a near complete conversion from monomers to oligomers (Figure 1A). The characteristics of the oligomers have been thoroughly described previously (Nässtrom et al., 2011). In short, the size of the oligomers is about 2000 kDa and their width is 100-200 nm. Moreover, they are stable and β-sheet rich but do not form fibrils. For the present experiments the α-synuclein oligomers were labeled with Cy3, using Lightning-Link Cy3 Antibody Labeling Kit (Novus Biologicals, 78-0015). Unbound excess Cy3 was removed by filtration

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ACCEPTED MANUSCRIPT in a Zeba spin desalting column (Thermo Scientific). Alpha-synuclein fibrils were generated as previously described (Nasstrom et al., 2011).

Alpha-synuclein exposure The mixed cell cultures containing neurons, astrocytes and oligodendrocytes were exposed to

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0.5 µM Cy3-labeled α-synuclein oligomers for 24 h. This concentration was chosen based on

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preliminary dose studies, using 0, 0.05 and 0.5 µM of oligomers (data not shown), and the exposure time was determined based on performed time-lapse experiments. Parallel control

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cultures were left untreated or exposed to 0.5 µM Cy3-labeled α-synuclein fibrils. Following

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exposure, cells were thoroughly washed two times in culture medium and continuously cultured in α-synuclein-free medium. At 0, 2, 6 and 12 days after exposure the cells were

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either fixed or lysed for further analyses. For visualization of mitochondria, cultures were transfected with CellLight Mitochondria-GFP (BacMam 2.0, Life Technologies) for 24 h,

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simultaneously with oligomer exposure.

Cells were recorded using time-lapse microscopy (Nikon Biostation IM Cell Recorder).

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Images were taken at 40X magnification every 10 min for 48 h of α-synuclein oligomer exposure (0.5 µM) or for 72 h after 24 h α-synuclein oligomer exposure and wash. Unexposed

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control cultures were recorded for 72 h.

Immunocytochemistry For the studies of uptake, intracellular location and toxic effects of α-synuclein, cells were cultured on coverslips and fixed with 4% PFA in PBS for 15 min. The cells were stained with antibodies specific for neurons (mouse monoclonal anti-βIII-tubulin, MMS-435P Covance, 1:200), astrocytes (polyclonal rabbit anti-GFAP (Z0334, DAKO) or monoclonal mouse anti-

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ACCEPTED MANUSCRIPT GFAP (G3893, Sigma), 1:400), oligodendrocytes (monoclonal mouse anti-CNPase, C5922, Sigma, 1:500), the lysosomal marker Lamp-1 (rabbit polyclonal anti-Lamp-1, ab24170, Abcam, 1:200), the mitochondrial markers mitofusin 1 (rabbit polyclonal anti-mitofusin 1, NBP1-51841, Novus Biologicals, 1:100) and the fission related protein, Drp1 (mouse monoclonal anti-Drp1, ab56788, Abcam, 1:200). The fixed cells were permeabilized and

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blocked in 0.1% Triton X-100 + 5% normal goat serum (NGS) in PBS for 30 min at room

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temperature (RT). The cells were then incubated for 1 h, at RT, with primary antibodies

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(diluted in 0.1% Triton X-100 + 0.5% NGS in PBS), followed by washes in PBS. After 1 h incubation with the secondary antibodies (goat anti-rabbit 488, goat anti-mouse 488 or goat

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anti-mouse 647 (Alexa, Life Technologies, 1:200)) in 0.1% Triton X-100 + 0.5% NGS in PBS at RT, the specimens were washed and mounted using Vectashield Hard Set with DAPI

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(Vector Laboratories). A wide-field microscope (Zeiss AxioImager Z1) was used for quantification of α-synuclein degradation (40X magnification) and confocal micrographs

ELISA measurements

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were taken with a Zeiss 510 confocal microscope (63X magnification).

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Cells were exposed to α-synuclein oligomers as described above. At 0, 2, 6 and 12 days after exposure the media was removed and 300 µl lysis buffer (20 mM Tris pH 7.5, 0.5% Triton X

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100, 0.5% deoxycholic acid, 150 mM NaCl, 10 mM EDTA, 30 mM NaPyroP, 500 µM Sodium orthovanadate and protease inhibitor (Roche)) per 10 cm2 well was added. The lysed cells were transferred to eppendorf tubes, incubated on ice for 30 min and centrifuged (30 min, 4 °C, 12 000 g). The supernatant was collected and kept in -70 °C until analysis.

The amount of α-synuclein oligomers was analyzed in cell lysates from three different experiments (n=3), using an oligomer-selective ELISA (Fagerqvist et al., 2013). High binding

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ACCEPTED MANUSCRIPT 96-well polystyrene plates (Costar 3590) were coated with the oligomer-selective antibody mAb38F (100 ng/well) diluted in PBS. After incubation at 4 °C overnight the plate was blocked with 1% bovine serum albumin for 2 h. Alpha-synuclein oligomer standards (0-250 pM) or cell lysate samples were added to the wells and incubated for 2 h, followed by incubation with biotinylated mAb38F for 1 h and Streptavidin-HRP (1:5000, Mabtech) for 45

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min. Finally, TMB (Neogen) was used as a substrate and the plates were analyzed at 450 nm

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(Infinite M1000, Tecan). The same protocol was performed to detect the total amount of α-

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(sc-10717, Santa Cruz Biotechnology) for detection.

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synuclein, using the mAb1338 (MAB1338, R&D Systems) as capture antibody and FL-140

Western blot

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Cell lysates were prepared as described above. Two sets of experiments were performed; in the first set (n=3) the cells were lysed at day 0, 2 and 6 after 24 h exposure and in the second

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set (n=3) the cells were lysed at day 0, 1, 2, 4 and 6 after 24 h exposure. The protein

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concentration was measured using the BCA Protein Assay Kit (Thermo Scientific). For analysis of total lysates 9.5 µg was loaded to each well of a 4-12% Bis-Tris Gel (NuPage, Life

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Technologies). Novex® Sharp Standard (Life technologies) was used as a standard. The gel was run for 1 h at 175 V in MES buffer (NuPage, Life Technologies), followed by transfer for

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1 h at 30 V onto a transfer membrane (Invitrogen). The membrane was blocked with 5% BSA in TBS with 0.2% Tween (TBS-T) for 1 h (RT), followed by washes in TBS-T and incubation with primary antibody in 0.5% BSA in TBS-T overnight, at 4 °C. After extensive washes in TBS-T the membrane was incubated with a peroxidase-conjugated secondary antibody in 0.5% BSA in TBS-T for 1 h (RT) and then washed again in TBS-T. The enhanced chemiluminescence (ECL) system (GE Healthcare) was used for development. The primary antibodies used were FL-140 (sc-10717, Santa Cruz Biotechnology, 1:500), 211 (sc-12767,

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ACCEPTED MANUSCRIPT Santa Cruz Technology, 1:500) anti-Lamp-1 (ab24170, Abcam, 1:1000) and anti-β-actin (8H10D10, Cell Signaling Technology). HRP-conjugated goat anti-rabbit IgG (Pierce, 1:20 000) or HRP-conjugated goat anti-mouse IgG, IgM (Pierce, 1:20 000) were used as secondary antibodies.

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For immunoprecipitation of Drp1, the Immunoprecipitation Dynabeads protein G kit (Novex,

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Life Technologies) was used. First, 200 µg of protein was incubated with 10 µg anti-Drp1

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antibody (mouse monoclonal anti-Drp1, ab 56788, Abcam) on rotation, at 4 °C, overnight. Then, 3 mg magnetic beads were added and the mixture was incubated on rotation for 2 h at

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RT. The beads were washed three times and the protein was eluted for 10 min at 70 °C, in the presence of LDS sample buffer and sample reducing agent (Bolt, Life technologies). Gel

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electrophoresis was run on a 4-12% Bis-Tris Plus gel (Bolt, Life Technologies) for 28 min at 200 V in SDS MES running buffer (Bolt, Life Technologies). A pre-stained protein ladder

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(Chameleon, Li-Cor) was used as a standard. Transfer was made for 1 h at 20 V onto a PVDF

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membrane (Novex, Life Technologies). The membrane was blocked with 5% BSA in TBS-T for 1 h (RT). After brief washing in TBS-T, the primary anti-Drp1 antibody (1:500) was

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added and the membrane was incubated in 0.5% BSA in TBS-T at 4 °C overnight. This was followed by washes and incubation with HRP-conjugated protein G (ab7460, Abcam, 1:10

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000) in 0.5% BSA in TBS-T for 1 h (RT). The ECL system was used for development.

Transmission electron microscopy Prior to fixation in 2.5% glutaraldehyde (1 h), cells were exposed to 0 or 0.5 µM α-synuclein oligomers for 24 h and briefly washed in PBS. The dishes were incubated in 1% osmium tetroxide in cacodylate buffer (SCB) at RT for 1 h followed by 10 min in SCB. Dehydration was performed with 70% ethanol for 30 min, 95% ethanol for 30 min and 99.7% ethanol for 1

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ACCEPTED MANUSCRIPT h. The dishes were rinsed with plastic (Agar 100 resin kit, Agar Scientific Ltd) and a new, thin layer of plastic was added to the cells for 2–4 h to permit evaporation of the alcohol. A second plastic layer was poured on and left overnight before a thicker, newly made plastic layer was added. The dishes were incubated in RT for 1 h before polymerization in oven (60

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°C) for 48 h. The cells were studied in a Hitachi H-7100 transmission electron microscope.

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Luminescent ATP detection assay

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Cells were grown in 24-well plates and exposed to 0.5 µM α-synuclein oligomers for 24 h. The total ATP levels were analyzed with the Luciferase based Luminescent ATP Detection

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Assay kit (ab113849, Abcam). The manufacturer’s instructions were followed. As a control, 1µM of the mitochondrial electron transport inhibitor Antimycin A (A8674, Sigma) was used

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(1-2 h incubation prior to measurement). Luminescence was measured with an Infinite M1000 plate reader (Tecan). The assay was performed in a biological replicate of n=6, i.e. from 6

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different batches of cells. Luminescence values are presented as the relative change as

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compared to untreated control cultures.

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Quantification of mitochondrial DNA damage using a QPCR-based assay Following α-synuclein oligomer exposure (0.5 µM), cell cultures were washed (three times)

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in cell media and harvested in lysis buffer (1 ml/10 cm2 dish). The cell suspension was incubated on ice for 10 min, followed by the addition of another 1 ml buffer solution after which the samples were mixed and centrifuged at 10 000 g for 20 min at 4 °C. The supernatant was removed and the remaining pellet was frozen and stored at -20 °C until analysis. Levels of mtDNA damage were measured using a quantitative polymerase chain reaction (qPCR)-based assay as previously described (Sanders et al., 2014a; Sanders et al., 2014b) with KAPA Long Range Hot Start DNA Polymerase Kit (KAPA Biosystems), instead

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ACCEPTED MANUSCRIPT of the GeneAmp XL PCR kit (Rooney et al., 2015). A total of 20 ng of DNA was used for amplification of a 10.0 kb mouse mitochondrial fragment with the following protocol: 10 min at 75 °C, 1 min at 94 °C, 26 x 15 s at 94 °C, 10 min at 67 °C and 10 min at 72 °C. Primers 13337 and 3278 (with previously described sequences (Ayala-Torres et al., 2000)) were used

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performed in technical triplicate for each biological replicate.

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for the amplification of the mouse mitochondrial fragment. All qPCR-based experiments were

Immunofluorescence of mouse brain tissue

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(Thy-1)-h [A30P] α-synuclein transgenic mice were anaesthetized at 19 months of age (n=2)

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and perfused with 0.9% NaCl. Brains were dissected, fixed in 4% phosphate-buffered formaldehyde and stored in 30% sucrose until sectioning. The left hemisphere was sectioned

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sagitally (20 µm) and specimens were stored in anti-freeze buffer at -20 °C. The sections were stained as previously described (Fagerqvist et al., 2013, Lindström et al., 2014) using the

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primary antibodies C20 (sc7011Santa Cruz), mAb1338 (MAB1338, R&D), GFAP (Z0334,

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DAKO or G3893, Sigma) and anti-βIII-tubulin (MRB-435P, Nordic Biosite). Secondary antibody (Alexa 488/594 anti-rabbit/anti-mouse (Life Technologies) diluted in M.O.M.

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diluents 1:250 was used. The specimens were mounted with Vectashield Hard set with DAPI (Vector Laboratories). As controls, adjacent sections were stained in parallel with either the

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primary or secondary antibody omitted from the protocol. Confocal images were captured using a Zeiss confocal laser scanning microscope (LSM700) and the software Zen 2012 was used for image processing.

Immunofluorescence of human brain tissue Paraffin embedded brain sections (7 µm) from the temporal cortex and substantia nigra of four patients with PD/DLB were used (kindly provided from Uppsala Biobank by Professor

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ACCEPTED MANUSCRIPT Irina Alafuzoff). Sections were deparaffinized and stained as previously described (Fagerqvist et al., 2013) with a few modifications using the primary antibodies Syn-1 (610787, BD Biosciences, 1:500) and rabbit anti-GFAP (Z0334, DAKO, 1:500) diluted in 0.1% Tween-20 PBS, (overnight, shaking, 4 °C). To quench autofluorescence sections were incubated for 3 min in 0.3% Sudan Black in 70% ethanol. Secondary antibodies (594 anti-mouse and 488

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anti-rabbit Alexa, Life Technologies, 1:250, diluted in 0.1% Tween-20 in PBS) was used and

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sections were mounted using Hard Set Vectashield with DAPI (Vector Laboratories). As

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controls, adjacent sections were stained in parallel with either the primary or secondary antibody omitted from the protocol. Confocal images were captured using a Zeiss confocal

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laser scanning microscope (LSM700) and the software Zen 2012 was used for image

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processing.

Analyses and statistics

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When applicable, the results are presented as mean +/- standard deviation. For statistical

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analyses Mann-Whitney U-test (GraphPad Prism) was used and the levels of significance

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were set to *P<0.05, **<0.01 and ***<0.001.

Images for quantifications were taken with 40X objective on a Zeiss AxioImager Z1. The area

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and intensity of the fluorescence of the Cy3-labeled α-synuclein were analyzed using ZEN 2012 software. The experiment was performed in independent cell cultures, derived from embryos of three different mice. In total of 36 images (12 images/per cell culture) were included in the analysis.

The number of astrocytes, neurons and oligodendrocytes containing α-synuclein deposits were counted manually. Depending on the amount of intracellular α-synuclein the cells were

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ACCEPTED MANUSCRIPT divided into four categories; cells with no detectable levels of α-synuclein (-), cells with one intracellular α-synuclein inclusion ≤ 2 µm (+), cells containing several α-synuclein inclusions ≤ 10 µm (++) and cells containing at least one large α-synuclein inclusion ≥ 10 µm (+++). Also, live astrocytes and neurons were counted in the culture at two different time points (24h and 24h+12d) to estimate the cell viability. The experiment was performed in independent

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cell cultures, derived from embryos of three different mice. In total of 36 images (12

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images/per cell culture) were included in the analysis.

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The number of live astrocytes and neurons in α-synuclein exposed and unexposed cell

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cultures were counted manually. Cell nuclei were identified with DAPI incorporation. Dead cells could easily be distinguished from live cells, based on their smaller and condensed

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nuclei that stain intensively with DAPI (Loov et al., 2015; Sollvander et al., 2016). The experiment was performed in independent cell cultures, derived from embryos of three

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different mice. In total of 30 images (10 images/per cell culture) were included in the

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analysis.

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Images for quantification of mitofusin 1 staining were captured with a 63X objective. The area or intensity of fluorescence was measured and normalized against the number of live

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cells. Mitochondrial morphology in CellLight Mitochondria-GFP transfected cells was quantified manually. The experiment was performed in independent cell cultures, derived from embryos of three different mice. In total of 30 images (10 images/per cell culture) were included in the analysis. The number of cells with fragmented mitochondria was counted and normalized against the total number of transfected cells. The experiment was performed in independent cell cultures, derived from embryos of three different mice. In total of 45 images (15 images/per cell culture) were included in the analysis.

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Quantification of the Western blot gel bands was performed on triplicate experiments using the ImageJ software or Image LabTM Software (Bio-Rad).

Results

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Alpha-synuclein oligomers accumulate in astrocytes and oligodendrocytes

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Co-cultures of astrocytes, neurons and oligodendrocytes were treated with 0.5 µM Cy3labeled α-synuclein oligomers (Figure 1A) for 24 h prior to fixation. Immunocytochemistry

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could not detect any uptake of α-synuclein oligomers in neurons (Figure 1B). In contrast,

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immunocytochemical staining to GFAP and CNPase showed that α-synuclein oligomers colocalized with both astrocytes and oligodendrocytes (Figure 1C-D). Confocal 3D images

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(Figure 1E-F and Supplementary Figure 1A-B) and Imaris 3D projection (Figure 1G-H) confirmed that the inclusions found in astrocytes and oligodendrocytes had an intracellular

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location. Immunostainings of cell cultures exposed to Cy3-labeled α-synuclein oligomers,

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using the oligomer-selective antibody mAb38F (Fagerqvist et al., 2013), displayed a total overlap with Cy3, verifying that no dissociation of Cy3 dye from the oligomers had taken

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place (Figure 1I).

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Quantification of the number of cells with intracellular α-synuclein deposits demonstrated that a majority of astrocytes (79.2 ± 14.9%) and oligodendrocytes (88.3 ± 18.2%) contained αsynuclein 24 h after oligomer exposure. The cells were divided into four categories; cells with no detectable levels of α-synuclein (-), cells with one intracellular α-synuclein inclusion ≤ 2 µm (+), cells containing several α-synuclein inclusions ≤ 10 µm (++) and cells containing at least one large α-synuclein inclusion ≥ 10 µm (+++). Representative images of the three αsynuclein positive categories in combination with GFAP staining are shown in Figure 2A. It is

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ACCEPTED MANUSCRIPT important to note that GFAP is expressed in the cytoskeleton of the astrocyte, which only constitutes 15-20% of the cell (Middeldorp and Hol, 2011). Therefore, the α-synuclein found very close to the GFAP staining is likely situated inside the cell. A majority of the glial cells were found to contain one small inclusion ≤ 2 µm (+) (44.1% of the astrocytes and 42.0% of the oligodendrocytes) or medium sized inclusions ≤ 10 µm (++) of α-synuclein (25.0% of the

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astrocytes and 40.6% of the oligodendrocytes), whereas large inclusions ≥ 10 µm (+++) were

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found in 11.4% of the astrocytes and in 5.7% of the oligodendrocytes (Figure 2 B-C). The α-

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synuclein inclusions in neurons were below the detection level and were therefore not

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included in the analyses.

To verify previous observations of astrocytic α-synuclein inclusions in vivo, we performed

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double immunostainings, using antibodies specific to GFAP, βIII-tubulin and α-synuclein, of tissue sections from (Thy-1)-h [A30P] α-synuclein transgenic mice and human PD/DLB

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brains, followed by confocal imaging. In α-synuclein transgenic mice, accumulated α-

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synuclein could be demonstrated inside both astrocytes and neurons in the midbrain and brainstem (Supplementary Figure 2A-B). The α-synuclein deposits in the astrocytes were a

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result of astrocytic engulfment, since the overexpression of human α-synuclein in the mice was under a neuronal specific promotor. In line with these findings, astrocytes with

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intracellular α-synuclein deposits were found to be present in substantia nigra of PD/DLB patients (Supplementary Figure 2C).

Alpha-synuclein inclusions are ubiquitinylated To further characterize the intracellular α-synuclein inclusions, cells treated with Cy3-labeled α-synuclein were stained with Thioflavin S (Thio S) and specific antibodies against ubiquitin (Figure 3A-E). As a positive control for the Thio S analyses, cell cultures were exposed to 0.5

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ACCEPTED MANUSCRIPT µM of previously characterized α-synuclein fibrils (Nasstrom et al., 2011). Immunostainings with antibodies specific to GFAP showed that while oligomeric α-synuclein accumulated intracellularly around the astrocytic nuclei, α-synuclein fibrils were attached to the astrocytic branches (Figure 3A-B and Supplementary Figure 3). In contrast to cells treated with αsynuclein fibrils (Figure 3C), the inclusions in α-synuclein oligomer exposed cultures did not

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stain positively for Thio S (Figure 3D). However, the intracellular deposits of aggregated α-

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synuclein co-localized with the ubiquitin staining, demonstrating that the ingested oligomers

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were partly ubiquitinylated (Figure 3E).

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Rapid uptake of α-synuclein oligomers

Time-lapse microscopy recordings, starting instantly after exposure, revealed a rapid uptake

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of Cy3-labeled α-synuclein oligomers in astrocytes (identified by their phenotype of “an egg sunny side up”; large nuclei and multi-vesicular cytoplasm) (Figure 4A). Already within 15

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min, large amounts of oligomers could be observed in astrocytes (Figure 4B) and over the

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next 20 h the amount increased even further (Figure 4C-D). In contrast to the astrocytes, oligomer co-localization with neurons (identified by their oval cell bodies, distinct axons and

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active migration) was very sparse (Figure 4E-H). In additional time-lapse experiments cultures were exposed to α-synuclein oligomers for 24 h, followed by rinsing in α-synuclein-

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free medium. The α-synuclein oligomers could then still be detected in astrocytes (Figure 4I), in contrast to neurons (Figure 4J). Analyses of oligodendrocytes were not possible using timelapse microscopy, due to the low percentage of oligodendrocytes in the culture.

Partial degradation of ingested α-synuclein To investigate the degradation of α-synuclein oligomer inclusions in the cell culture, cells were exposed to α-synuclein oligomers for 24 h, washed and fixed after 0, 2, 6 and 12 days in

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ACCEPTED MANUSCRIPT α-synuclein-free medium. Immediately after the 24 h exposure, Cy3-labeled oligomers formed large inclusions (Figure 5A). Although no cell marker staining was performed, it can be presumed that the inclusions were located to astrocytes and oligodendrocytes, due to the previous observation of relatively low uptake in neurons of this co-culture. Measurements using the Zen 2012 software showed a higher number of Cy3-labeled inclusions at 12 d

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following α-synuclein withdrawal (24 h+12 d). However, many of the inclusions were

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smaller, indicating that some degradation had occurred in the glial cells (Figure 5B). Further measurements of the Cy3 intensity confirmed the increase in number (Figure 5C) and

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decrease in size (Figure 5D) of α-synuclein inclusions over time. The total Cy3 intensity

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increased during the first days following α-synuclein removal, possibly due to ingestion of αsynuclein attached to the cell membrane. After 12 d in α-synuclein-free medium, the Cy3

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intensity had started to decrease (Figure 5E), although a majority of α-synuclein aggregates

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still remained in the cells.

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Western blot analysis of cell lysates, using an α-synuclein antibody (FL-140), showed a clear decline in total α-synuclein levels at six days following oligomer exposure (Figure 6 A-B).

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This degradation pattern was confirmed using an antibody specific to human α-synuclein (211) (Supplementary Figure 4). Moreover, the oligomer-selective ELISA confirmed that the

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oligomer levels decreased during the first six days after exposure (Figure 6C). However, using an antibody against the C-terminal of α-synuclein, measurable levels of α-synuclein could be detected even at twelve days (Figure 6D).

Lysosomal degradation of α-synuclein oligomers in astrocytes is not completed To study the involvement of the lysosomal pathway in the truncation or degradation of αsynuclein oligomers, the cells were stained for the endosomal/lysosomal protein Lamp-1.

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ACCEPTED MANUSCRIPT Cy3-labeled α-synuclein oligomers, ingested by the astrocytes were found to be situated close to Lamp-1 positive vesicles directly after the 24 h exposure (Figure 7A). Notably, at day 12 after exposure the co-localization of α-synuclein and Lamp-1 positive vesicles was reduced, although the α-synuclein inclusions had not been degraded (Figure 7B). Western blot analysis of total cell lysates from three different batches (Supplementary Figure 5A-D) showed that

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the total amount of Lamp-1 protein was not significantly changed over time, indicating that

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the reduced co-localisation was due to re-distribution of the protein within the cells. Double staining with antibodies to Lamp-1 and GFAP confirmed the co-localization of Lamp-1 and

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α-synuclein in astrocytes directly after exposure (Figure 7C). Thus, α-synuclein oligomers

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internalized by the astrocytes entered the lysosomal pathway, but the degradation was not

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completed.

Alpha-synuclein oligomers induce mitochondrial impairment

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Exposure to α-synuclein oligomers for 24 h was found to induce visual mitochondrial damage

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in the astrocytes, as demonstrated by transmission electron microscopy (TEM) (Figure 8A-C). In particular, the mitochondria had lost the structure of their outer membrane as well as of the

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inner cristae and appeared condensed or fragmented. Counting the number of live astrocytes and neurons showed that there was no significant increase in astrocytic (p=0.189) or neuronal

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(p=0.956) cell death at this time point. However, a significant decrease (p=0.026) in live neurons was observed at day twelve after α-synuclein oligomer exposure.

To further assess the mitochondrial effects of α-synuclein oligomer exposure on cell energetics, total ATP levels were measured. Cell cultures exposed to 0.5 µM α-synuclein oligomers for 24 h displayed an 11.2% decrease in total ATP level (p=0.0405), as compared

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ACCEPTED MANUSCRIPT to untreated cultures (Figure 8D). The positive control substance Antimycin A gave a 44.3% decrease in ATP levels (p=0.0003) (Figure 8D).

The extent of mitochondrial DNA (mtDNA) lesions was measured in α-synuclein oligomer exposed cells and in control cultures. Interestingly, cells treated with α-synuclein oligomers

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displayed fewer mtDNA lesions, compared to untreated cells (-0.11 ± 0.02 lesions/10kb; p =

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0.03) (Figure 8E). To control for a potential loss of mtDNA in response to α-synuclein

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oligomer treatment, a short mtDNA fragment (117 bp) was amplified that due to its small size was less likely to contain a lesion. We found that the mitochondrial DNA copy number was

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comparable between cells that had been subjected to α-synuclein oligomers and control cells.

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Next, the mitochondrial morphology was studied by transfection with CellLight Mitochondria-GFP (Figure 9A). Cells in control cultures displayed a normal mitochondrial

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network, whereas α-synuclein oligomer exposed cultures contained significantly more cells

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with fragmented mitochondria (Figure 9B) (n=3, p<0.0001). Interestingly, we noted that the

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cells with pathological mitochondria were primarily astrocytes.

Immunocytochemical stainings using specific antibodies to the mitochondrial fusion marker

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mitofusin 1 (Figure 9C) revealed a significant increase in staining area and intensity following α-synuclein oligomer exposure (Figure 9D-E). Moreover, Drp1, a protein involved in the regulation of mitochondrial fission, was differently expressed in α-synuclein oligomer exposed cultures compared to untreated cultures. In the control cultures, Drp1 displayed a highly organized and dotted staining pattern, that was partly localized to the elongated GFPlabelled mitochondria (Figure 9F). In α-synuclein oligomer exposed cells, the Drp1 staining was less organized and appeared in close proximity with the fragmented mitochondria (Figure

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ACCEPTED MANUSCRIPT 9F). Western blot analyses showed that the total Drp1 expression was similar in untreated cultures and in cultures exposed to α-synuclein oligomers for 24 h (Figure 9G-H).

Discussion Astrocytes, the most abundant glial cell type in the brain, have multiple functions that are

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tightly linked to pathological processes. Yet, their role in neurodegenerative diseases has been

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sparsely studied and the consequences of astrocytic α-synuclein inclusions remain unknown. The aim of the present study was to investigate the capacity of astrocytes to clear and degrade

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oligomers of the PD and DLB related protein α-synuclein. Moreover, we sought to clarify if

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astrocytic α-synuclein inclusions result in toxic effects in a co-culture system, containing mainly astrocytes but also neurons and oligodendrocytes. For this investigation it was crucial

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that the culture was completely devoid of professional phagocytes. We solved this problem by using a cell culture system based on embryonic, cortical stem cells that exclusively

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differentiate to cells within the neural lineage and, hence, do not form microglia or

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macrophages (Davis and Temple, 1994; Johe et al., 1996; Ravin et al., 2008).

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In the co-culture system, we found that 80% of astrocytes had ingested and accumulated αsynuclein oligomers within the 24 h exposure time. Moreover, 90% of the oligodendrocytes in

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the culture contained α-synuclein oligomers at this time point. However, since the number of oligodendrocytes in the co-culture was very low we focused on the astrocytic α-synuclein accumulation. In contrast to the glial cells, the uptake of oligomers in neurons was very limited and could not be analyzed in the co-culture setup.

The internalization of α-synuclein oligomers by astrocytes occurred within minutes, emphasizing a rapid phagocytic capacity of these cells. Directly after the exposure, the

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ACCEPTED MANUSCRIPT ingested oligomers were located around pyknotic cell nuclei and co-localized with vesicles expressing the endosomal-lysosomal marker Lamp-1. Lysosomal degradation is considered as the main pathway for eliminating α-synuclein in neurons, both under normal and pathological conditions (Lee et al., 2004; Mak et al., 2010). Interestingly, we found that the lysosomal digestion in the astrocytes was not completed. Increasing evidence indicates that insufficient

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lysosomal degradation is involved in the pathogenesis of different neurodegenerative

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diseases, including PD (Appelqvist et al., 2013; Nixon et al., 2008). The cellular dysfunction

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could be caused directly by remaining α-synuclein inclusions or indirectly by secondary

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events.

The co-localization with Lamp-1 positive vesicles declined drastically over time, indicating

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that the persisting astrocytic inclusions could be situated in aggresomes. Such cytoplasmic aggregates of misfolded proteins can be found in conditions where the degradation system has

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become overwhelmed (Hyttinen et al., 2014) and have been presumed to be cytoprotective, by

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isolating the misfolded protein from the rest of the cell. For example, aggresomes have been suggested to be involved in the formation of inclusion bodies in both PD and MSA (Chiba et

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al., 2012; Wakabayashi et al., 2013). During aggresome formation, the misfolded proteins aggregate step-wise and become ubiquitin-labeled (Hyttinen et al., 2014). In accordance, we

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found that the astrocytic α-synuclein deposits were at least partly ubiquitinylated.

Although an initial degradation of the α-synuclein deposits occurred, as indicated by the reduced signal in the oligomer-selective ELISA, considerable amounts of Cy3-labeled αsynuclein remained in the cell culture system even after twelve days. At this time point the material was no longer detected by the oligomer-selective ELISA, indicating that the oligomers after ingestion and initial degradation either had changed conformation or had

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ACCEPTED MANUSCRIPT become truncated. However, low levels of total α-synuclein were still detectable even after twelve days. Although the nature of these remaining species is not clear, we can conclude that they had not formed fibrillary structures since they were Thio S negative.

Toxic α-synuclein has previously been shown to cause mitochondrial morphology, increased

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mitochondrial fragmentation and affected mitophagy in neuronal cells (Devi et al., 2008;

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Martin et al., 2006; Nakamura, 2013). However, mitochondrial effects of pathological α-

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synuclein in astrocytes have barely been investigated. In a previous study, α-synuclein was suggested to locate to mitochondria and cause reduced oxygen consumption in human

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primary astrocytes (Braidy et al., 2013).

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In the present investigation, α-synuclein oligomers were shown to induce mitochondrial damage, as demonstrated by TEM, immunocytochemistry and ATP measurements. In line

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with previous reports of α-synuclein uptake in neurons, the mitochondrial morphology was

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changed, showing a fragmented pattern. Also, the expression patterns of the fusion protein Mitofusin 1 and the fission protein Drp1, were clearly affected in oligomer exposed cells,

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indicating a disturbance of the mitochondria fission-fusion dynamics. Overall, the total levels of Drp1 did not differ between exposed and unexposed cells. However, in α-synuclein

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exposed cells the Drp1 pattern was less organized and localized to the fragmented mitochondria, indicating that the abnormal mitochondrial phenotype may, at least partly, be due to increased mitochondrial fragmentation. In addition, the ATP analysis demonstrated that α-synuclein exposure significantly reduced the functionality of the mitochondria. Interestingly, in spite of that the astrocytes were observed to survive more than twelve days after exposure, implying that they are capable of compensating for the impaired energetics.

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ACCEPTED MANUSCRIPT Supporting this theory, we found that oligomer-exposed cells contained higher amounts of mitochondria and exhibited reduced mtDNA lesions, compared to control cells.

Interestingly, we observed an increase in neuronal cell death twelve days after α-synuclein exposure, but not immediately after the treatment, indicating a secondary rather than direct

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mechanism of neurotoxicity. A possible explanation for the delayed neurotoxicity could be

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that harmful α-synuclein species are secreted from the phagocytic astrocytes or spread to

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neighbouring cells by membrane contact. Alternatively, the toxicity could be mediated by cytokine release from the glial cells. Previous studies have shown that astrocytes react to

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exposure of extracellular α-synuclein released from neurons by an increased expression of pro-inflammatory cytokines, such as IL-1α and IL-1β (Lee et al., 2010b). This up-regulation

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causes an increased production of reactive oxygen species and induces various inflammatory processes (Fellner et al., 2013; Rannikko et al., 2015), thus affecting neighbouring neurons

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and microglia (Lee et al., 2010a).

In conclusion, the rapid and extensive uptake of α-synuclein oligomers in astrocytes indicates

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that these cells play an important role in sequestering toxic α-synuclein species from the extracellular space. At an initial stage the α-synuclein uptake may be neuroprotective, by

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preventing α-synuclein toxicity and disease progression. However, a more prolonged storage of α-synuclein in the astrocytes affects their mitochondrial integrity and lead to neurotoxicity. Our findings highlight the importance of additional studies to further address the role of astrocytes in the pathogenesis behind brain disorders with α-synuclein pathology.

Acknowledgements / Conflict of interest disclosure

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ACCEPTED MANUSCRIPT This study was supported by grants from the Swedish Research Council, the Swedish Parkinson Foundation, the Swedish Alzheimer Foundation, the U4 Ageing Brain network, the Åhlén Foundation, the Dementia Association Foundation, Hedlunds Foundation, Lennart and Christina Kalén, William N. & Bernice E. Bumpus Foundation Innovation Award, Marianne and Marcus Wallenberg Foundation, Swedish Brain Foundation, Parkinson Research

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Foundation, King Gustaf V and Queen Victoria's Foundation of Freemasons and the Uppsala

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Berzelii Center. We would like to thank Professor Irina Alafuzoff for kindly providing us

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with human brain sections. None of the authors have any conflicts of interest with the

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contents of this article.

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ACCEPTED MANUSCRIPT Figure 1. Uptake of α-synuclein oligomers. A) SEC-HPLC chromatogram of α-synuclein oligomers and monomers. SEC-HPLC analysis showed an almost complete conversion of monomers into oligomers (97%). The peaks of oligomers and monomers are indicated in the figure. B) No uptake of α-synuclein oligomers was visible in neurons (III-tubulin, green). Immunostainings against C) GFAP (green) and D) CNPase (green) revealed an extensive

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uptake of α-synuclein oligomers (red) in astrocytes and oligodendrocytes after a 24 h

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exposure to 0.5 µM oligomers. 3D confocal microscopy imaging confirmed that the α-

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synuclein oligomer inclusions (red) in E) astrocytes and F) oligodendrocytes were situated intracellularly. Imaris 3D projections (G-H) displayed intracellular inclusions (red) in an

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astrocyte (green). The stars in G and H indicate the same position in the cell. I) Immunocytochemistry, using the mAb38F antibody, demonstrated that the antibody signal co-

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F, I = 10 µm. Scale bar in G-H = 5µm.

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localized with the Cy3-labeling. Blue: DAPI. Arrowheads indicate inclusions. Scale bar in B-

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Figure 2. Quantifications of internalized α-synuclein oligomers in astrocytes and oligodendrocytes. A) Representative images of the four categories; cells with no detectable

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levels of α-synuclein (-), cells with one intracellular α-synuclein inclusion ≤ 2 µm (+), cells

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containing several α-synuclein inclusions ≤ 10 µm (++) and cells containing at least one large α-synuclein inclusion ≥ 10 µm (+++). (Green: GFAP, red: α-synuclein and blue: DAPI). The

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number of B) astrocytes and C) oligodendrocytes containing α-synuclein inclusions of various sizes were counted and plotted as the percentage of the total number of each cell type. After

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24 h exposure, 44.1% of the astrocytes and 42.0% of the oligodendrocytes contained one small inclusion ≤ 2 µm (+), 25.0% of the astrocytes and 40.6% of the oligodendrocytes contained medium sized inclusions ≤ 10 µm (++), and 11.4% of the astrocytes and 5.7% of the oligodendrocytes contained large inclusions ≥ 10 µm (+++). The graphs show mean± SD. Scale bar = 10 µm.

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Figure 3. Characterization of intracellular α-synuclein inclusions. Cortical co-cultures were exposed to Cy3 labelled α-synuclein fibrils or oligomers for 24 h. A) Fibrils were internalized by the astrocytes to a lower degree than B) oligomers. Instead, the fibrils were found to be attached to the cell membrane of the astrocytic processes (red: α-synuclein, green:

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GFAP, blue: DAPI). Immunocytochemistry revealed that the C) fibrils (red) were Thio S positive (green), while the D) oligomer inclusions (red) did not display any Thio S

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fluorescence (green). E) The ingested oligomers (red) were at least partly ubiquitinylated

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(green). Blue: DAPI. Scale bars = 10 µm.

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Figure 4. Time-lapse microscopy of cell cultures exposed to α-synuclein oligomers. A)

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Astrocytes were identified by their phenotype of an “egg sunny side up”; large nuclei and multi-vesicular cytoplasm. B-H) Time-lapse microscopy, started instantly after exposure to

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0.5 µM Cy3-labelled α-synuclein oligomers. A rapid uptake was observed in astrocytes already after B) 15 min, and further increasing at C) 4 h and D) 20 h. E-H). The oligomers

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were attached to the surface of the neurons. In cultures rinsed after the 24 h exposure the astrocytes still contained visible α-synuclein oligomers (I) in contrast to the neurons (J). A = astrocyte, N = neuron. Scale bars = 10 µm.

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Figure 5. Degradation of α-synuclein oligomers. Fluorescence images of the cell culture A)

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24 h or B) 24h+12d after exposure to Cy3-labeled oligomers. A) Large aggregates of α-

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synuclein oligomers (red) were located close to dead cell nuclei (blue: DAPI) after 24 h. B) Twelve days after α-synuclein removal, smaller Cy3-labeled inclusions were observed.

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Arrowheads indicate pyknotic dead cell nuclei (small arrowheads) and live cell nuclei (large arrowheads). Measurements of the Cy3-fluorescence showed an increase in the C) number of

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α-synuclein inclusions, and a decrease in D) inclusion size over time. E) The intensity of the inclusions increased with time until 24h+6d and then declined. p<0.05=*, p<0.0005=***. Scale bars = 10 µm.

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Figure 6. Degradation or modification of α-synuclein oligomers. A) Western blot analysis

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(α-synuclein antibody FL-140) of cells exposed to 0 (lane C) or 0.5 µM α-synuclein

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oligomers for 24 h + 0, 2, 6 or 12 days in oligomer-free medium. B). Quantification of relevant bands (2, 4, 5, 6) showed a decrease in α-synuclein signal between 2 and 6 days after

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exposure. Band 1 and 3 are artifacts that also appeared in the untreated cells. These bands were therefore not quantified. C) Sandwich-ELISA of cells exposed to α-synuclein oligomers

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for 24 h + 0, 1, 2, 4 or 6 d, using the oligomer-selective antibody mAb38F. The oligomer signal was rapidly reduced during the first 6 d after exposure. D) ELISA using mAb1338/FL140 of cells exposed to α-synuclein oligomers for 24 h + 0, 2, 6 or 12 d.

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Figure 7. Lysosomal degradation of α-synuclein oligomers. Cells were exposed to Cy3labeled α-synuclein oligomers (red) for 24 h and stained against Lamp-1 (green). A) Directly after exposure (24 h) there was a clear co-localization between α-synuclein and Lamp-1 38

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24 h of exposure. Blue: DAPI. Scale bars = 10 µm.

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Figure 8. Mitochondrial effects caused by α-synuclein oligomers. Transmission electron microscopy of A) an untreated astrocyte or B-C) an astrocyte exposed to α-synuclein

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oligomers for 24 h (the two separate pictures B-C are captured from the same cell, and B

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shows the mitochondria relative to the nucleus). The mitochondria in the α-synuclein exposed astrocyte were clearly affected, showing loss of structure of the outer membrane as well as the

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inner cristae. Magnification: 50 000X. D) Cells exposed to α-synuclein oligomers for 24 h displayed an 11.2% decrease in total ATP levels (p=0.0405), compared to untreated cells

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(Figure 8D). Antimycin A reduced the ATP levels with 44.3% (p=0.0003). E) qPCR-based analysis showed that α-synuclein oligomer exposed cells had fewer mtDNA lesions, compared to control cells (-0.11 ± 0.02 lesions/10kb; p=0.03).

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ACCEPTED MANUSCRIPT Figure 9. Mitochondrial effects caused by α-synuclein oligomers. CellLight mitochondriaGFP transfection (A) revealed increased mitochondrial (green) fragmentation in astrocytes (GFAP, red) after 24 h of oligomer exposure, as compared to control cells (B). C) Immunostainings displayed an increase in the expression of the mitochondrial marker mitofusin 1 (green) following α-synuclein oligomer exposure (Red = Cy3-labeled α-synuclein

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oligomers, blue = DAPI). D-E) Quantifications of the immunostainings demonstrated that the

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mitofusin 1 expression in exposed cultures was significantly increased compared to control cultures (p=0.0001). F) In control cells, Drp1 (red) displayed a highly organized, dotted

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staining pattern, partly localized to mitochondrial network (green). In α-synuclein oligomer

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exposed cells, the Drp1 staining was less organized (blue: DAPI, right: close ups). G-H) Western blot analysis of total cell lysates using Drp1 antibodies, demonstrated that the protein

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expression was unchanged following α-synuclein exposure. Scale bars = 10 µm.

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Supplementary Figure 1. Cells were exposed to α-synuclein for 24 h and analyzed with confocal microscopy. Z-stacks of cells stained with specific antibodies to GFAP (A-B) or CNPase (C-D) confirmed that the Cy-3 labelled α-synuclein oligomers were accumulated intracellularly in the astrocytes and oligodendrocytes. Two different intersections per z-stack are monitored. Red: Cy3-oligomers, green: GFAP or CNPase, blue: DAPI scale bar = 10 µm. 43

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Supplementary Figure 2. Immunohistochemical detection of accumulated α-synuclein in vivo. A) Confocal microscopy showed that α-synuclein (red, mAb1338) co-localized with GFAP (green) in 19 months old (Thy-1)-h [A30P] α-synuclein transgenic mice. Blue: DAPI, scale bar = 5 µm. B) Co-localization was also seen for α-synuclein (red, Syn-1) and the neuronal marker βIII-tubulin (blue) in transgenic mice. Neurons were intimately surrounded by astrocytes (green, GFAP). Scale bar = 10 µm. C) Astrocytes (green, GFAP) were also found to have internalized α-synuclein (red, Syn-1) in substantia nigra of a human PD/LBD brain. Blue: DAPI, scale bar = 5 µm. 44

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ACCEPTED MANUSCRIPT Supplementary Figure 3. Cells were exposed to α-synuclein fibrils for 24 h and analyzed with confocal microscopy. Z-stacks of cells stained with specific antibodies to GFAP confirmed that the Cy-3 labelled fibrils were attached to the astrocytic processes (arrowheads). Two different intersections are shown (A-B). Red: Cy3-fibrils, green: GFAP,

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blue: DAPI, scale bar = 10 µm.

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Supplementary Figure 4. Western blot analysis of total cell lysates from cultures exposed to α-synuclein oligomers for 24 h, + 0, 2, 6 and 12 d, using the α-synuclein antibody 211.

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Samples were run in triplicates (with an exception; +2 d were in duplicates). The two gels/membranes were run in parallel.

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Supplementary Figure 5. Western blot analysis of Lamp-1 in total cell lysates. A) Representative blot of Lamp-1 (120 kDa) in cultures exposed to α-synuclein oligomers for 24 h, +2, 6 and 12 d or vehicle only (C). Lane 1-5 shows batch A and lane 6-9 shows batch B.

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No significant difference in Lamp-1 expression could be detected over time (B). The bars

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represent the mean and standard deviation from the quantification of three independent cell batches. C) Representative blot of Lamp-1 (120 kDa) and the loading control β-actin (45 kDa) in cultures exposed to α-synuclein oligomers for 24 h, +1, 2, 4, 6 d or vehicle only (C). D) The ratio of the intensity of the bands of Lamp-1 and β-actin revealed no significant difference between different time points and the control (t-test). The bars represent mean and standard deviation from the quantification of three independent cell batches.

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Highlights  Astrocytes rapidly ingest large amounts of oligomeric α-synuclein.  Due to incomplete lysosomal degradation, the α-synuclein is intracellularly stored.  The accumulation of α-synuclein induces astrocytic mitochondrial impairments.  Our results emphasize an important role of astrocytes in α-synucleinopathies.

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