Synucleinopathies are a group of neurodegenerative disorders characterized by the presence of aggregated and fibrillar forms of alpha-synuclein (α-syn). Here, we analyze the effect of different species of α-syn, including monomeric, oligomeric and fibrillar forms of the protein, on rat astrocytes. Astrocytes treated with these distinct forms of α-syn showed an increase in long and thin processes and glial fibrillary acidic protein expression, indicating cell activation, high levels of intracellular oxidants and increased expression of cytokines. Moreover, astrocytes incubated with the different species induced hippocampal neuronal death in co-culture, and cytotoxicity was particularly enhanced by exposure to fibrillar α-syn. Further exploration of the mechanisms behind astrocyte activation and cytotoxicity revealed differences between the assessed α-syn species. Only oligomers induced mitochondrial dysfunction in astrocytes and significantly increased extracellular hydrogen peroxide production by these cells. Besides, TNF-α and IL-1β (interleukin 1β) expression presented different kinetics and levels depending on which species induced the response. Our data suggest that α-syn species (monomeric, oligomeric and fibrillar) induce astrocyte activation that can lead to neuronal death. Nevertheless, the tested α-syn species act through different preferential mechanisms and potency. All together these results help to understand the effect of α-syn species on astrocyte function and their potential impact on the pathogenesis of Parkinson's disease and related α-synucleinopathies.

Introduction

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by a massive loss of dopaminergic neurons in the substantia nigra and the presence of alpha-synuclein (α-syn) protein inclusions in neuronal cell bodies known as Lewy bodies [1,2]. α-Syn is also involved in the development of a group of diseases collectively known as synucleinopathies that include multiple system atrophy, pure autonomic failure and dementia with Lewy bodies [35].

α-Syn is a small and intrinsically disordered protein, composed by 140 amino acids, which is abundant in many regions of the brain [6,7]. It is a cytosolic protein, and although it has no known signaling sequence, it can be released from neuronal cells in small amounts, via unconventional exocytosis, under normal physiological conditions [8]. However, in pathological situations, α-syn monomers and aggregates may be released in larger quantities and endocyted by neighboring neurons, as well as by the surrounding astrocytes and microglia [810].

Astrocytes outnumber neurons in the central nervous system (CNS) and are responsible for a wide variety of important functions, including regulation of blood flow, maintenance of the blood–brain barrier, synaptic function and maintenance of the composition of the extracellular environment of ions, fluids and neurotransmitters [11]. Damage to the CNS due to injury or disease may result in functional changes in astrocytes, leading to ‘reactive astrogliosis’, a phenotype characterized by morphological changes, hypertrophy, proliferation and alterations in gene expression, including increased expression of the cytoskeleton glial fibrillary acidic protein (GFAP) and pro-inflammatory cytokines. Evidence indicates that reactive astrocytes contribute to neuronal death in amyotrophic lateral sclerosis animal models [12,13] as well as in patients [14,15], suggesting that these cells may promote the progression of neuronal death in other neurodegenerative diseases. Previous work from our group indicates that astrocyte-mediated toxicity is associated with mitochondrial dysfunction and oxidant formation [16,17]. A relationship between mitochondrial dysfunction and α-syn has been previously reported in PD. PD patients present accumulation of α-syn in mitochondria and decreased complex I activity [18], while mice overexpressing the mutated A53T α-syn have reduced complex IV activity [19]. However, most mitochondrial studies in PD were performed in neuronal cells [18,20,21], and there is scarce information concerning mitochondrial function in astrocytes and its relevance for neuronal survival, in this disease.

In the case of genetic forms of PD, many of the mutated genes, such as DJ-1 protein (PARK7 gene), have been identified in astrocytes [22,23]. Mutated α-syn overexpressed in mouse astrocytes lead to the development of a neurodegenerative disorder and astrogliosis, similar to human PD [24]. Since the expression of α-syn gene in astrocytes is very low, most studies consider α-syn protein as an exogenous stimulator of astrocytes [25].

Increasing evidence suggests that internalization and accumulation of α-syn in astrocytes may activate the cells and induce an inflammatory response [26,27]. Neuroinflammation, which is mainly mediated by activated astrocytes and microglia, has been suggested to play a critical role in PD progression [2830]. In this scenario, reactive astrocytes could secrete pro-inflammatory cytokines and produce reactive oxygen species (ROS), leading to an inflammatory state that would affect neuronal survival affecting on synaptic transmission and plasticity [26].

α-Syn can be found in different conformational species: monomers, oligomers and fibrils, since fibril formation from monomers involves the formation of oligomeric intermediates of variable size and morphology [3133]. Several reports propose that the oligomeric species are responsible for α-syn cytotoxicity [3436]. Nevertheless, α-syn fibrils have also been shown to induce toxicity [3739], raising doubts on the nature of the main pathogenic species involved in PD.

Here, we generated and carefully characterized monomeric, oligomeric and fibrillar α-syn species, and assessed their ability to induce a reactive and neurotoxic astrocyte phenotype. Our results help to understand the effect of α-syn species on astrocyte function and their potential impact on the pathogenesis of PD and related α-synucleinopathies.

Experimental

Animals

Procedures using laboratory animals were in accordance with international guidelines and were approved by the Institutional Animal Committee (Comisión Honoraria de Experimentación Animal de la Universidad de la República, CHEA; https://chea.edu.uy/).

Cell cultures

Astrocytes

Primary rat cortical astrocytes were prepared from 1- to 2-day-old pups following the protocol described by Saneto and De Vellis [40], with some modifications [41]. Briefly, brain cortexes were dissociated in 0.25% trypsin (Gibco) for 30 min at 37°C. Cells were collected by centrifugation, filtered through a 80 µm mesh cell dissociation sieve and plated at a density of 1.5 × 106 cells per 25 cm2 Nunc flask in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), HEPES (3.6 g/l), penicillin (100 IU/ml) and streptomycin (100 g/ml). When confluent, cultures were shaken for 48 h at 250 rpm at 37°C, incubated for another 48 h with 10 µM cytosine arabinoside and then plated at a 3.7 × 104 cells/well in 24-multiwell plates and maintained in DMEM supplemented with 10% FBS, HEPES (3.6 g/l), penicillin (100 IU/ml) and streptomycin (100 µg/ml), until confluence. The astrocyte monolayers were >98% pure as determined by GFAP immunoreactivity. The cultures were devoid of OX42-positive microglial cells.

Neurons

The hippocampal neuron preparations were obtained from E18 rat embryos. Hippocampi were dissected and mechanically dissociated with a glass Pasteur pipette in Neurobasal medium, 2% B27, and 2 mM glutamine until obtain a suspension of 400 000 cells/ml. Neurons were then plated on a confluent monolayer of astrocytes and maintained for 48 h in DMEM, 5% FBS and 5% horse serum. Hippocampal neurons were selected because the isolation procedure is well characterized and yields a fairly uniform culture consisting of a single type of neuron.

Co-cultures

A suspension of hippocampal neurons (10 000 cells/well) was seeded on top of astrocyte confluent monolayers pretreated for 16 h with α-syn species or left untreated (control). Astrocytes were washed before adding the neurons to remove the remaining α-syn and other components of the media. Hippocampal neurons on the astrocyte monolayer were identified by immunocytochemistry with an antibody against βIII tubulin (1 : 4000 monoclonal mouse antibody, Promega, Madison, WI, U.S.A.). After 3 days in vitro co-culture, the cells were fixed with 4% paraformaldehyde in PBS at 4°C, blocked with 0.1% Triton X-100, 10% FBS in PBS for 30 min and incubated with primary anti-βIII tubulin overnight at 4°C. After washing with PBS, the co-cultures were incubated 1 h at room temperature with a secondary anti-mouse antibody conjugated to biotin (1 : 500, Jackson Immune Research Inc., West Grove, PA, U.S.A.). Finally, the co-cultures were washed with PBS and incubated with streptavidin-peroxidase (1 : 250, Jackson ImmunoResearch Inc., West Grove, PA, U.S.A.) for 1 h at room temperature and revealed with diaminobenzidine. Hippocampal neuron survival was assessed by counting all βIII tubulin-positive cells displaying neurites longer than four cell diameters in an area of 0.90 cm2 along a diagonal in 24-well plates. The mean density of hippocampal neurons in control co-cultures was 88 ± 5 cell/cm2.

Protein expression and purification

Escherichia coli BL21 (DE3) were transformed with a pT7 plasmid containing the α-syn gene and recombinant α-syn was produced as described in ref. [42]. Briefly, bacterial cells were incubated at 37°C in LB (Luria-Bertani) broth containing kanamycin (30 µg/ml) until OD at 600 nm reached 0.6–0.8. Gene expression was induced using 0.5 mM IPTG (isopropyl β-d-thiogalactopyranoside). Cells were cultured at 37°C for 4 h and centrifuged at 4000 rpm during 20 min at 4°C. Pellets were resuspended in 20 ml of lysis buffer (750 mM NaCl, 10 mM Tris, 1 mM PMSF and 1 mM EDTA pH 7.5) and sonicated. The cell suspension was boiled during 10 min and placed immediately on ice. Cells were centrifuged at 8000 rpm for 15 min at 4°C and the supernatant was filtered using 4M whatman filter paper. The sample was loaded into a column resource Q ionic exchange, and α-syn was purified by HPLC using a NaCl gradient 0–1 M to elute the protein with a retention time of ∼25–28 min. The fraction obtained was dialyzed against a 5 mM Tris, 1 mM EDTA, pH 7.5, overnight at 4°C. The purification was confirmed using 12% SDS–PAGE, and the pure protein was diluted in 10 mM HEPES (pH 7.4) and stored at −80°C. α-Syn concentration was resolved by absorbance using an ε275 of 5600 M−1 cm−1 [43].

Oligomer preparation

Different methods are available in the literature to prepare α-syn oligomers [44], we selected that proposed by Apetri et al. [45]. Briefly, freeze-dried monomeric α-syn (300 µM) was incubated in 10 mM HEPES buffer (pH 7.4) at 37°C without stirring. After 20 h of incubation, the oligomers were separated and purified from the monomeric forms of the protein through filtration, using an Amicon 100 KDa cut-off filter.

Fibril preparation and Thioflavin T fluorescence assay

Fibrillar α-syn was generated stirring the protein in glass vials (140 µM monomeric α-syn) using Teflon magnetic microbars in 10 mM HEPES buffer (pH 7.4) at 37°C during 3 days, similar as Celej et al. [43]. Fibril formation was followed by an increase in thioflavin T (Thio-T) fluorescence. Aliquots were taken at different time points and mixed with 10 µM Thio-T in 90 mM Gly-NaOH (pH 8.5). Fluorescence emission spectra were recorded from 465 to 600 nm with excitation at 450 nm and an emission wavelength of 490 nm. Thio-T fluorescence measurements were performed on an Aminco Bowman Series 2 spectrofluorimeter using a 10 mm light path quartz cuvette (Supplementary Figure S1).

Transmission electron microscopy

Samples were stained with 1% uranyl acetate during 2 min and images were obtained in a JEOL JEM-1010 transmission electron microscope using Formvar-coated carbon grids (200 mesh).

Dynamic light scattering

Size distribution of monomeric and oligomeric α-syn species was determined by dynamic light scattering (DLS; Zetasizer NanoS, Malvern Instruments Ltd). For each sample (25 μM in 25 mM HEPES, 100 mM NaCl, pH 8.0), the total light scattered at an angle of 90° was collected using a 10 s acquisition time. Particle translational diffusion coefficients were calculated from the autocorrelated light intensity data and converted to hydrodynamic radius (RH) using the Stokes–Einstein equation.

Treatment with α-syn species

Astrocyte confluent monolayers were incubated with 0.02 mg/ml solutions of monomeric, oligomeric or fibrillar α-syn or left untreated (control condition for 16 h). We tested two different α-syn concentrations (0.02 and 0.04 mg/ml) according to previous reports [46], and selected the lowest concentration that induced changes in the astrocyte morphology. Conditioned astrocyte media were obtained from astrocytes treated with the different α-syn species during 16 h, the culture media were changed and the cells were incubated during 24 h, without α-syn. The conditioned media were diluted with fresh media and added to hippocampal neurons, and the neuronal survival was assessed as described above.

Griess assay

Nitrite () formation was measured in supernatants from treated astrocytes using the Griess assay [47]. This method is based on the reaction between sulfanilamide and N-1-napthylethylenediamine dihydrochloride under acidic conditions to form an azo dye that can be quantified spectrophotometrically at 540 nm.

Amplex red assay

Astrocyte confluent monolayers were seeded in 24-well plates and treated with different α-syn species as indicated above, then washed with PBS and 300 µl of a mixture of 20 µM Amplex Red and 0.01 mg/ml horseradish peroxidase in PBS was added to each well. Fluorescence was measured at different time points in a plate reader (Fluostar) at 37°C with a 530 nm excitation wavelength and a 590 nm emission wavelength.

DCFH2DA oxidation

Astrocyte confluent monolayers seeded in 24-well plates were treated with different α-syn species as indicated above. After treatment astrocytes were incubated with 30 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2DA) for 15 min, rinsed and cells were collected using a scraper. The oxidation product of DCFH2, 2′,7′-dichlorofluorescein (DCF), was analyzed by flow cytometry (FACS Calibur, Benton Dickinson).

Immunofluorescence and immunohistochemistry

Astrocytes were fixed with 4% paraformaldehyde in PBS at 4°C, permeabilized with 0.2% Triton X-100 in PBS, blocked with 10% goat serum, 2% BSA, and 0.2% Triton X-100 in PBS. The primary monoclonal antibody anti-GFAP (Sigma–Aldrich, St. Louis, MO, U.S.A.) was diluted (1 : 1000) in blocking solution and incubated overnight at 4°C. After washing with PBS, astrocytes were incubated with secondary goat anti-mouse Alexa Fluor 488 (Invitrogen, Carlsbad, CA, U.S.A.) 1 : 1000 in blocking solution for 1 h and then with DAPI 2 µg/ml for 15 min. The images were obtained using a confocal Leica DMI6000 TCS-SP5 microscope.

Quantification of the number of flat and process-bearing astrocytes

The relative numbers of flat and process-bearing astrocytes in each culture were quantified as described in Shobha et al. [48] on a four-well Lab-Tek™ chambered coverglass (ThermoFischer Scientific™, Nunc™, Waltham, MA, U.S.A.). Quantification was performed on 16 images for each Lab-Tek™ coverglass using a Leica DMI6000 TCS-SP5 confocal microscope under 40× magnification.

Cytokine gene expression

Total RNA was extracted from astrocyte confluent monolayers seeded in 24-well plates after treatment with different α-syn species as indicated above, using the Trizol reagent followed by chloroform extraction and isopropanol precipitation. Total RNA (1 µg) was converted into cDNA using the SuperScript III Reverse Transcriptase (Invitrogen); 100 ng of the cDNA were used for quantitative PCR (qPCR) performed with Quantimix Easy Master Mix (Biotools, B&M Labs, S.A.) and detected using a Rotor Gene 6000 PCR system (Qiagen). The sequences of quantitative primers used are as follows: for GAPDH F: 5′-CAC TGA GCA TCT CCC TCA CAA-3′ and R: 5′-TGG TAT TCG AGA GAA GGG AGG-3′, for β-actin (ACTB) F: 5′-CAG CCT TCC TTC CTG GGT AT-3′ and R: 5′-CTG TGT TGG CAT AGA GGT CTT-3′ for TNF-α F: 5′-TGA TCG GTC CCA ACA AGG A-3′ and R: 5′-TGC TTG GTG GTT TGC TAC GA-3′ and for IL-1 β F: 5′-TGC TGA ACC AGT TGG GG-3′ and R: 5′-CTC CAT GAG CTT TGT ACA AG-3′. Data analysis was performed using the 1.7 Rotor Gene 6000 Software.

Mitochondrial dynamics

Following incubation with different α-syn species, astrocyte confluent monolayers were incubated for 30 min with 100 nM MitoTracker® Green FM (Invitrogen, ThermoFischer Scientific™, Waltham, MA, U.S.A.) in DMEM 2% FBS at 37°C in a 5% CO2 atmosphere. After washing with PBS, astrocytes were maintained in DMEM 2% FBS. In vivo images were obtained using a Leica DMI6000 TCS-SP5 confocal microscope. To automate the measurements, we used the analysis and processing software Fiji (ImageJ). We developed a plugin for Fiji that could automatically segment the mitochondria in an image, allow validation by the user and extract form descriptors and morphological parameters in validated mitochondria. The segmentation stage is as follows: (1) application of contrast enhancement through the Fiji/ImageJ Process > Enhance Contrast command; (2) binarization of the result by thresholding using the Otsu method for two classes; (3) filtering according to size and shape with the Analyze Particles plugin from Fiji/ImageJ and (4) saving the mask of each mitochondria. Then, the length of the mitochondria was obtained for each mask as follows: (1) generation of the skeleton of the mask of each mitochondrion using the Fiji/ImageJ Skeletonize and Analyze Skeleton plugin; (2) extrapolation that allows extending the obtained skeleton until the corresponding mask is cut; (3) measurement of the skeleton lengths and parameters of form.

Oxygen consumption rate and extracellular acidification rate

Astrocytes (1 × 104 cells/well) were seeded into 20 wells of an XFe24 cell culture microplate (Seahorse Bioscience, Agilent). Culture medium was replaced with 600 µl of Seahorse medium composed of DMEM (Sigma D5030) supplemented with 32 mM NaCl, 5 mM d-glucose, 2 mM pyruvate, 2 mM glutamine and 15 mg/l phenol red (pH 7.4). Plates were kept at 37°C for 1 h and loaded into the Seahorse XFe24 extracellular flux analyzer (Seahorse Bioscience, Agilent); all experiments were performed at 37°C. Baseline measurements of oxygen consumption rate and extracellular acidification rate were obtained at the beginning of the assay; these were followed by the sequential addition of an ATP synthase inhibitor (0.5 μM oligomycin), an uncoupler of oxidative phosphorylation (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, FCCP) and an inhibitor of Complex III (1 μM antimycin A). Four baseline measurements and three response rates (after the addition of each compound) were obtained, and average rates were used for data analysis. Titration with 0.5–3.0 μM of FCCP was performed and 1.5 μM FCCP gave the maximum oxygen consumption rate, so this concentration was used for the experiments. The non-mitochondrial oxygen consumption rate was determined after adding antimycin A and subtracted from all other values before calculating the respiratory parameters [49].

Basal respiration was the baseline measurement obtained before the addition of oligomycin. Respiration driving proton leak was determined after oligomycin injection. Respiration driving ATP synthesis was the difference between basal respiration and respiration driving proton leak. Maximum respiratory capacity was the maximal rate measured after FCCP injection. Respiratory control ratio (RCR) was calculated as the relation between the maximum respiration and the respiration driving proton leak. Coupling efficiency was the ratio between respiration driving ATP synthesis and basal respiration. Spare respiratory capacity ratio is the relationship between maximum respiratory capacity and basal respiration.

Mitochondrial complex I- and complex II-dependent respiration was determined by measuring oxygen consumption in digitonin-permeabilized cells according to Salabei et al. [50].

Data analysis

Experiments were performed at least three times on independent days. All data are given as means ± SEM unless otherwise stated. Means were compared using Student's t-test and a value of P < 0.05 was considered significant. Flow cytometry data were analyzed using the Kolmogorov–Smirnov test.

Results

Highly purified alpha-synuclein oligomers and fibrils were obtained and characterized

We prepared and purified α-syn monomers, oligomers and fibrils, and characterized the morphological features and size distribution using different techniques (Figure 1). In native PAGE electrophoresis, oligomers are identified as a broad, high molecular mass band near the top of the gel, while the monomers migrate as a lower molecular mass band (Figure 1A). DLS studies indicated that monomeric species have a hydrodynamic radius (RH) smaller than α-syn oligomers (4 versus 12 nm) (Figure 1B), in agreement with previous reports [36]. α-Syn fibril and oligomer preparations were pure and structurally different as revealed by transmission electron microscopy (TEM). Oligomeric species exhibited rounded shapes of heterogeneous size ranging from 18 to 73 nm in width. α-Syn fibrils showed the characteristic features of fibrillar structures being ∼12–16 nm wide and 600 nm long (Figure 1C). Some authors include a sonication step when preparing α-syn fibrils [51,52]. We avoided this procedure to keep the original structure of the fibrils, which is more representative of fibrils found in the CNS [53,54]. TEM images were consistent with previous studies [45,5557].

Characterization of α-syn species.

Figure 1.
Characterization of α-syn species.

(A) Native gradient PAGE 7.5% stained with silver nitrate. Sample after a 20 h incubation (lane 1), after initial centrifugation (lane 2), monomeric α-syn (lanes 3 and 4), third wash (lane 6), purified α-syn oligomers (lane 7). (B) DLS showing the size distribution of monomeric (gray) and oligomeric (red) α-syn. (C) TEM images of uranyl acetate-stained α-syn oligomers (left) and fibrils (right).

Figure 1.
Characterization of α-syn species.

(A) Native gradient PAGE 7.5% stained with silver nitrate. Sample after a 20 h incubation (lane 1), after initial centrifugation (lane 2), monomeric α-syn (lanes 3 and 4), third wash (lane 6), purified α-syn oligomers (lane 7). (B) DLS showing the size distribution of monomeric (gray) and oligomeric (red) α-syn. (C) TEM images of uranyl acetate-stained α-syn oligomers (left) and fibrils (right).

α-Syn species induced primary astrocyte activation

Primary cultures of rat cortical astrocytes were incubated with monomeric, oligomeric and fibrillar α-syn (0.02 mg/ml for 16 h). After incubation with the three different species, α-syn could be detected on astrocytes monolayers by immunofluorescence (Figure 2A, inset) confirming that, at least part of the protein, remained in contact with the cells.

Treatment with α-syn species determines morphological changes in cortical astrocytes.

Figure 2.
Treatment with α-syn species determines morphological changes in cortical astrocytes.

(A) Confocal immunofluorescence photomicrographs of cultured cortical astrocytes showing GFAP expression (green). Astrocyte monolayers were treated with monomeric, oligomeric and fibrillar α-syn, or left untreated (control). Nuclei were stained with DAPI (blue). The inset shows representative epifluorescence images from immunofluorescence against α-syn (red) and GFAP (green). Scale bar is the same for all images. (B) Quantification of the ratio of GFAP fluorescence intensity and (C) ratio of processes bearing astrocyte to flat astrocytes. Data are expressed as means of three experiments, ±SEM. **P < 0.001; *** P < 0.0001.

Figure 2.
Treatment with α-syn species determines morphological changes in cortical astrocytes.

(A) Confocal immunofluorescence photomicrographs of cultured cortical astrocytes showing GFAP expression (green). Astrocyte monolayers were treated with monomeric, oligomeric and fibrillar α-syn, or left untreated (control). Nuclei were stained with DAPI (blue). The inset shows representative epifluorescence images from immunofluorescence against α-syn (red) and GFAP (green). Scale bar is the same for all images. (B) Quantification of the ratio of GFAP fluorescence intensity and (C) ratio of processes bearing astrocyte to flat astrocytes. Data are expressed as means of three experiments, ±SEM. **P < 0.001; *** P < 0.0001.

Incubation of astrocyte cultures with the different α-syn species did not affect the cell monolayer but induced changes in astrocyte cytoarchitecture. Exposed cells exhibited a retraction of the soma and nuclei, as well as thin and long processes, and their morphology clearly differed from the flat and polyhedral appearance of control cells (Figure 2A). The ratio of process bearing to flat astrocytes increased in cells treated with monomeric, oligomeric and fibrillar α-syn species, when compared with control cells. In particular, in the fibril-treated group, a 6-fold increase could be appreciated (Figure 2C). In addition, increased GFAP immunoreactivity was found in fibril- and oligomer-treated astrocytes (Figure 2B). The observed changes in cell morphology and the increase in GFAP immunofluorescence levels induced by α-syn were consistent with astrocyte activation [28].

Astrocytes incubated with α-syn promoted neuronal toxicity

Astrocyte monolayers were incubated with α-syn species as described before the culture media were changed to eliminate free α-syn and hippocampal neurons were seeded on top of the astrocyte monolayers. After 72 h of incubation, co-cultures were fixed and immunocytochemistry against β-III tubulin was performed to determine the number of surviving neurons (Figure 3). In control conditions, astrocyte monolayers provided sufficient trophic support to enable neurons to survive and develop neuritic processes without the addition of neurotrophic factors. However, co-culture with astrocytes pretreated with different forms of α-syn significantly reduced neuron survival with respect to control co-cultures, resulting in 58%, 55% and 15% survival for monomeric, oligomeric and fibrillar α-syn, respectively (Figure 3). These results indicate that astrocytes activated by α-syn treatment (monomeric, oligomeric and mainly fibrillar forms) can induce neuronal death or are less efficient at providing trophic support.

Pretreatment with different species of α-syn in astrocytes reduced neuronal survival in co-culture.

Figure 3.
Pretreatment with different species of α-syn in astrocytes reduced neuronal survival in co-culture.

(A) Representative images from the indicated co-cultures showing βIII tubulin-labeled hippocampal neurons (green) on top of astrocyte monolayer immunopositive to GFAP (red). Only viable neurons displaying neurites longer than four cell diameters (white arrows) were counted, while neurons exhibiting short neurites (open arrows) were not. Note the appearance of βIII tubulin immunoreactive detritus in the co-cultures. Scale bar: 25 µm. (B) Survival of hippocampal neurons after 72 h of co-culture on top of confluent monolayer of astrocytes pretreated for 16 h with the indicated α-syn species or untreated astrocytes (control). Data are expressed as means of three experiments, ±SEM. ***P < 0.0001.

Figure 3.
Pretreatment with different species of α-syn in astrocytes reduced neuronal survival in co-culture.

(A) Representative images from the indicated co-cultures showing βIII tubulin-labeled hippocampal neurons (green) on top of astrocyte monolayer immunopositive to GFAP (red). Only viable neurons displaying neurites longer than four cell diameters (white arrows) were counted, while neurons exhibiting short neurites (open arrows) were not. Note the appearance of βIII tubulin immunoreactive detritus in the co-cultures. Scale bar: 25 µm. (B) Survival of hippocampal neurons after 72 h of co-culture on top of confluent monolayer of astrocytes pretreated for 16 h with the indicated α-syn species or untreated astrocytes (control). Data are expressed as means of three experiments, ±SEM. ***P < 0.0001.

To assay whether soluble factors generated by the α-syn-activated astrocytes are mediators of neuronal death, we obtained conditioned media from astrocytes pretreated with the different α-syn species (monomeric, oligomeric and fibrillar α-syn). Supplementary Figure S2 shows the effect of the conditioned media on hippocampal neuron survival. There was no significant decrease in neuronal survival following incubation with astrocyte-conditioned media with any of the α-syn treatment (either monomeric, oligomeric or fibrillar forms).

Oligomeric forms of α-syn-induced mitochondrial dysfunction in primary astrocyte cultures

Since astrocyte-mediated toxicity to neurons has been associated with mitochondrial dysfunction [16], we investigated whether incubation with monomeric, oligomeric or fibrillar α-syn affected mitochondrial respiration. Primary rat cortical astrocytes were incubated for 16 h with the different forms of α-syn and oxygen consumption rate in intact cells was assessed. Basal respiration rates were not significantly affected by the treatment, but an increase in oligomycin-resistant respiration was observed after incubation with the oligomers resulting in a 17% reduction in coupling efficiency (Figure 4 and Table 1). Oligomer-treated cells also presented a decrease of 43% in the spare respiratory capacity and of 40% in the RCR (Figure 4 and Table 1), implying that in cells treated with oligomers mitochondria have lower capacity for substrate oxidation and ATP synthesis. Exposure to α-syn monomers or fibrils had milder effects on mitochondrial function, since only a small decrease in the spare respiratory capacity was detected in these conditions (Table 1).

Mitochondrial respiration of cortical astrocytes.

Figure 4.
Mitochondrial respiration of cortical astrocytes.

Cells were pretreated with α-syn fibrils (A), monomers (B) and oligomers (C). Oxygen consumption rate was assessed in a Seahorse XFe extracellular flux analyzer before and after the addition of oligomycin (0.5 µM) the uncoupler FCCP (1.5 µM) and antimycin A (1.0 µM). Data are expressed as means of three experiments, ±SEM. **P < 0.001.

Figure 4.
Mitochondrial respiration of cortical astrocytes.

Cells were pretreated with α-syn fibrils (A), monomers (B) and oligomers (C). Oxygen consumption rate was assessed in a Seahorse XFe extracellular flux analyzer before and after the addition of oligomycin (0.5 µM) the uncoupler FCCP (1.5 µM) and antimycin A (1.0 µM). Data are expressed as means of three experiments, ±SEM. **P < 0.001.

Table 1
Mitochondrial respiratory parameters of astrocytes incubated with monomeric, oligomeric and fibrillar α-syn
Parameter Control Monomer Oligomer Fibrils 
Basal respiration (pmol/min µg protein) 9.5 ± 0.7 8.5 ± 0.7 10.4 ± 0.7 10.2 ± 0.9 
Respiration driving ATP synthesis (pmol/min µg protein) 7.9 ± 0.4 6.9 ± 0.6 7.0 ± 0.4 8.4 ± 0.4 
Respiration driving proton leak (pmol/min µg protein) 1.7 ± 0.3 1.6 ± 0.2 3.4 ± 0.2* 1.8 ± 0.4 
Maximum respiratory capacity (pmol/min µg protein) 13.2 ± 1.1 10.3 ± 1.3 10.8 ± 2.7 11.3 ± 0.35 
Cell RCR 12.0 ± 2.0 8.7 ± 0.9 4.7 ± 1.2** 6.14 ± 0.9 
Coupling efficiency 0.83 ± 0.06 0.81 ± 0.01 0.69 ± 0.04** 0.82 ± 0.04 
Spare respiratory capacity ratio 1.6 ± 0.07 1.3 ± 0.04* 0.92 ± 0.2** 1.11 ± 0.06* 
Parameter Control Monomer Oligomer Fibrils 
Basal respiration (pmol/min µg protein) 9.5 ± 0.7 8.5 ± 0.7 10.4 ± 0.7 10.2 ± 0.9 
Respiration driving ATP synthesis (pmol/min µg protein) 7.9 ± 0.4 6.9 ± 0.6 7.0 ± 0.4 8.4 ± 0.4 
Respiration driving proton leak (pmol/min µg protein) 1.7 ± 0.3 1.6 ± 0.2 3.4 ± 0.2* 1.8 ± 0.4 
Maximum respiratory capacity (pmol/min µg protein) 13.2 ± 1.1 10.3 ± 1.3 10.8 ± 2.7 11.3 ± 0.35 
Cell RCR 12.0 ± 2.0 8.7 ± 0.9 4.7 ± 1.2** 6.14 ± 0.9 
Coupling efficiency 0.83 ± 0.06 0.81 ± 0.01 0.69 ± 0.04** 0.82 ± 0.04 
Spare respiratory capacity ratio 1.6 ± 0.07 1.3 ± 0.04* 0.92 ± 0.2** 1.11 ± 0.06* 

Respiratory parameters were calculated from the data shown in Figure 4 as decribed in Experimental. Results show the mean ± SEM (n = 5) (*P < 0.05, **P < 0.001).

We also assessed the extracellular acidification rate of the media, determined mainly by the excretion of metabolic acids such as lactic and carbonic acid [58]. Incubation of astrocytes with α-syn oligomers resulted in a nearly 3-fold increase in the extracellular acidification rate, suggestive of increased lactate formation (Supplementary Figure S3) and consistent with the decrease in mitochondrial function. Surprisingly, incubation with fibrils also resulted in an increase in extracellular acidification rate.

We then explored the molecular events behind the decrease in mitochondrial function after the exposure to α-syn oligomers. We first assessed mitochondrial respiratory chain activity in digitonin-permeabilized cells. No changes in oxygen consumption were observed between treatments and control conditions using saturating concentrations of substrates for complex I or II in the presence of ADP (Supplementary Figure S4). Secondly, mitochondrial morphology and number was assessed using confocal microscopy with MitoTrackerGreen and image processing (Figure 5A). These results showed a slight decrease in both mitochondrial number and mitochondrial length in astrocytes treated with the monomeric, oligomeric and fibrillar forms of α-syn (Figure 5B,C).

α-Syn-induced changes in mitochondrial dynamics in rat cortical astrocytes.

Figure 5.
α-Syn-induced changes in mitochondrial dynamics in rat cortical astrocytes.

(A) Fluorescence micrographs of cultured astrocytes with MitoTracker Green, treated with the different α-syn species or left untreated (control). (B) Analysis of mitochondrial length and (C) mitochondrial number per field. Data are expressed as means of three experiments, ±SEM. *P < 0.05; ***P < 0.0001.

Figure 5.
α-Syn-induced changes in mitochondrial dynamics in rat cortical astrocytes.

(A) Fluorescence micrographs of cultured astrocytes with MitoTracker Green, treated with the different α-syn species or left untreated (control). (B) Analysis of mitochondrial length and (C) mitochondrial number per field. Data are expressed as means of three experiments, ±SEM. *P < 0.05; ***P < 0.0001.

α-Syn species induced ROS production

Several reports show that mitochondrial dysfunction can be associated with an increase in oxidant species [59]. Thus, we measured oxidant species levels, following the oxidation of DCFH2DA to the fluorescent product DCF, and hydrogen peroxide (H2O2) formation with Amplex Red. DCF fluorescence significantly increased in astrocytes incubated with monomeric, oligomeric and fibrillar α-syn with respect to control condition, suggesting an increase in the intracellular levels of oxidant species (Figure 6A). However, in the same experimental conditions, we could only detect an increase in extracellular H2O2 production in α-syn oligomer-treated cultures, but not in monomeric or fibrillar α-syn-treated ones. The 1.5-fold increase in the rate of H2O2 production observed after incubation with the oligomers was comparable to that observed after treatment with PMA (Figure 6B).

Reactive species formation induced by α-syn monomers, oligomers and fibrils.

Figure 6.
Reactive species formation induced by α-syn monomers, oligomers and fibrils.

(A) Primary cortical astrocytes were incubated with 30 µM DCFH2DA during 15 min. The oxidation of DCFH2 was measured by flow cytometry in untreated astrocytes (black) or astrocytes incubated with α-syn oligomers (red). Quantification of the M2 population as the percentage of cells with higher fluorescence is shown at right. PMA (2 µg/ml) was used as a positive control. Statistical analysis of the data was performed using the Kolmogorov–Smirnov test with Cell Quest Pro software. (B) Amplex red fluorescence was measured over time after the 16 h treatment of astrocytes with α-syn monomers, oligomers and fibrils. Table at right: Quantification of the change in fluorescence over time. Only the oligomeric treatment induced a slight increase in H2O2 production. Data are expressed as means of three experiments, ±SEM. **P < 0.001; *P < 0.05.

Figure 6.
Reactive species formation induced by α-syn monomers, oligomers and fibrils.

(A) Primary cortical astrocytes were incubated with 30 µM DCFH2DA during 15 min. The oxidation of DCFH2 was measured by flow cytometry in untreated astrocytes (black) or astrocytes incubated with α-syn oligomers (red). Quantification of the M2 population as the percentage of cells with higher fluorescence is shown at right. PMA (2 µg/ml) was used as a positive control. Statistical analysis of the data was performed using the Kolmogorov–Smirnov test with Cell Quest Pro software. (B) Amplex red fluorescence was measured over time after the 16 h treatment of astrocytes with α-syn monomers, oligomers and fibrils. Table at right: Quantification of the change in fluorescence over time. Only the oligomeric treatment induced a slight increase in H2O2 production. Data are expressed as means of three experiments, ±SEM. **P < 0.001; *P < 0.05.

Last, to explore the formation of nitric oxide-derived oxidant species such as peroxynitre, nitrite levels were measured in astrocyte culture media, using the Griess assay. Nitrite levels were: control 6.22 ± 1.87 µM, monomers 8.91 ± 1.55 µM, oligomers 7.35 ± 4.04 µM and fibrils 7.51 ± 3.10 µM, respectively. No difference was detected between control and samples treated with α-syn species.

α-Syn species induced the expression of pro-inflammatory cytokines in astrocytes

Since neuroinflammation, which is mainly mediated by activated astrocytes and microglia, has been suggested to play a critical role in PD progression [2830], we measured the gene expression of pro-inflammatory cytokines previously reported to be involved in astrocyte-mediated toxicity [27]. Astrocytes treated with monomeric, oligomeric or fibrillar forms of α-syn presented different levels and kinetics for the expression of IL-1β and TNF-α (Figure 7).

α-Syn species induced TNF-α and IL-1ß production in cortical astrocytes.

Figure 7.
α-Syn species induced TNF-α and IL-1ß production in cortical astrocytes.

(A) TNF-α expression levels in astrocytes pretreated with α-syn monomers, oligomers and fibrils. (B) IL-1β expression levels in astrocytes pretreated as in (A). mRNA expression levels were quantified using qPCR and Rotor Gene Series 6000 software. Controls correspond to the untreated condition, and 1 µg of LPS was used as a positive control of cytokine production. Data are expressed relative to GAPDH mRNA expression. Data are expressed as means of three experiments, ±SEM. *P < 0.05.

Figure 7.
α-Syn species induced TNF-α and IL-1ß production in cortical astrocytes.

(A) TNF-α expression levels in astrocytes pretreated with α-syn monomers, oligomers and fibrils. (B) IL-1β expression levels in astrocytes pretreated as in (A). mRNA expression levels were quantified using qPCR and Rotor Gene Series 6000 software. Controls correspond to the untreated condition, and 1 µg of LPS was used as a positive control of cytokine production. Data are expressed relative to GAPDH mRNA expression. Data are expressed as means of three experiments, ±SEM. *P < 0.05.

Incubation with different α-syn species induced the expression of TNF-α to levels similar to those observed after the incubation with LPS. TNF-α expression peaked at 2 h after treatment with monomeric α-syn and was undetectable after 24 h, while in oligomer-treated cells the expression of this cytokine started at 16 h and increased furthermore at 24 h. Treatment with α-syn fibrils induced a high and constant TNF-α expression profile that continued from 2 to 24 h after exposure (Figure 7A).

Incubation with both oligomers and fibrils highly increased IL-1β expression, resulting in more than 100-fold increase after 24 h of treatment. Monomers instead produced a very modest but not significant increase in IL-1β expression (Figure 7B).

Discussion

In this work, we have obtained purified preparations of α-syn oligomers and fibrils and fully characterized them by native PAGE, DLS and TEM (Figure 1). The sizes exhibited by oligomers and fibrils are within the expected range for these species according to the literature [43,45,52].

Incubation of primary rat astrocytes with these preparations did not affect cell viability, but led to changes in cell morphology and in GFAP immunoreactivity, consistent with astrocyte activation [28]. These alterations were more pronounced in cultures exposed to α-syn oligomers and fibrils than in those treated with monomers (Figure 2). Moreover, the three different α-syn species induced a neurotoxic phenotype in astrocytes, as revealed by co-culture experiments (Figure 3). Since astrocyte monolayers were washed previous to neuronal seeding, a direct effect of α-syn species on neurons can be excluded.

Astrocyte toxicity has been associated with mitochondrial dysfunction, oxidative stress and pro-inflammatory cytokine secretion [16,27,41]. Thus, we assessed the effect of the α-syn preparations on these events, and to our surprise, our findings differed considerably depending on the α-syn species.

First of all, strong signs of mitochondrial dysfunction were identified following treatment with α-syn oligomers (Table 1), while fibrils and monomers had much milder effects. An increase in oligomycin-resistant respiration was observed after incubation with α-syn oligomers resulting in a reduction in coupling efficiency. These results are in agreement with increased mitochondrial uncoupling and might be due to an increase in proton leak or other proton-gradient consuming processes independent of ATP synthesis. The observed decrease in spare respiratory capacity after treatment with oligomers (and to a lesser extent with α-syn monomers and fibrils) could be due to the inhibition of one or more respiratory complexes, a decrease in mitochondrial number or alterations in mitochondrial dynamics (fission or fusion processes). Accordingly, a decrease in mitochondrial number and length was observed in astrocytes after the exposure to every α-syn species. Several reports point to a strong relationship between dynamics and oxidative phosphorylation in cells [60]. However, the decrease in mitochondrial number and length observed for cells treated with oligomers was not reflected in mitochondrial respiration in digitonin-permeabilized cells, where mitochondria were exposed to saturating concentrations of substrates for complexes I and II and ATP synthase. Although mitochondrial dysfunction has been widely reported to be involved in PD, most of the mitochondrial studies in PD have been performed in neuronal cells [18,20,21]. There is limited information concerning mitochondrial activity in astrocytes in this pathological setting, highlighting the value of our observations.

Secondly, in this work, we detected an increase in oxidant species measured by the oxidation of DCFH2, after treating cells with the different α-syn species. We also detected an increase in extracellular H2O2 levels after the incubation of astrocytes with α-syn oligomers (Figure 6). Mitochondria may function as a relevant source of ROS formation, such as superoxide anion () and H2O2 during astrocyte activation by α-syn. The formation of in astrocytes was reported before by Cassina et al. and others [16,61], and the dismutation of this anion leads to H2O2 production. LPS-activated astrocytes produce extracellular H2O2, and this process was shown to be dependent of glutathione levels and mitochondrial complex I activity [62]. Hydrogen peroxide may lead to the formation of hydroxyl radicals (·OH), via the Fenton reaction, that can, in turn, initiate lipid peroxidation, as well as protein and DNA oxidation, thus amplifying the damage. Since we could not find evidence of nitric oxide production during α-syn treatment, peroxynitrite-derived radicals are probably not involved in DCFH2 oxidation at this stage. However, there are other reports that show α-syn induction of the inducible nitric oxide synthase in astrocytes [63].

Finally, the different α-syn species induced pro-inflammatory cytokine expression, although the kinetics were different depending on the specie implicated (Figure 7). The role of TNF-α in PD has already been described. Levels of this pro-inflammatory cytokine are increased in the substantia nigra pars compacta, striatum, cerebrospinal fluid and peripheral blood mononuclear cells from patients with idiopathic PD [6466]. Serum IL-1β levels are also significantly increased in PD patients [67]. Our results suggest that α-syn activation of astrocytes might be behind this observed rise in pro-inflammatory cytokine levels and could promote neuronal death. The activation of astrocytes by α-syn species is similar to that seen with LPS treatment, in agreement with previous reports [68], and may suggest the involvement of Toll-like receptor-4 and nuclear factor-B (NF-κB) in induction of this pro-inflammatory response [63].

Table 2 summarizes and compares the effects of the different α-syn conformers on astrocyte activation profile. Monomeric α-syn presents minor to moderate effects on astrocyte activation, oligomers showed a strong impact on mitochondrial function and oxidants formation, while fibrillar α-syn induced the formation of high levels of pro-inflammatory cytokines. An interesting point is that none of the α-syn species is completely harmless; this can be visualized in terms of neuronal death in co-culture. However, the results obtained using conditioned media from astrocytes treated with different α-syn species demonstrate that soluble mediators generated by activated astrocytes are not sufficient to induce neuronal death. A direct contact between neurons and activated astrocytes is needed to decrease neuronal survival. As mentioned before, astrocytes are one of the main cells that provide metabolic and physiological support to neurons. In this scenario, α-syn oligomers and fibrils appear to be more potent astrocyte activators, inducing the formation of mediators, such as pro-inflammatory cytokines and oxidant species, that could amplify the ‘toxic’ environment and extend the damage to surrounding neurons, contributing to disease progression.

Table 2
Characterization of astrocyte response to distinct α-syn species respect to control condition
Effect Monomer Oligomer Fibrils 
Astrocyte activation 
 GFAP fluorescence ++ +++ 
 Ratio of processes bearing astrocyte to flat astrocytes ++ +++ 
Mitochondrial function 
 Decreased RCR ++ 
 Increased ECAR +++ ++ 
 Decreased mitochondrial length ++ ++ ++ 
 Decreased mitochondrial number 
Radical species formation 
 Oxidation of DCFH2 ++ +++ ++ 
 H2O2 formation +++ 
Pro-inflammatory cytokines (mRNA levels at 16 h) 
 TNF-α ++ +++ 
 IL-1β ++ 
Cellular toxicity 
 Neuronal death (co-cultures) ++ ++ +++ 
Effect Monomer Oligomer Fibrils 
Astrocyte activation 
 GFAP fluorescence ++ +++ 
 Ratio of processes bearing astrocyte to flat astrocytes ++ +++ 
Mitochondrial function 
 Decreased RCR ++ 
 Increased ECAR +++ ++ 
 Decreased mitochondrial length ++ ++ ++ 
 Decreased mitochondrial number 
Radical species formation 
 Oxidation of DCFH2 ++ +++ ++ 
 H2O2 formation +++ 
Pro-inflammatory cytokines (mRNA levels at 16 h) 
 TNF-α ++ +++ 
 IL-1β ++ 
Cellular toxicity 
 Neuronal death (co-cultures) ++ ++ +++ 

Conclusion

In this work, we dissect the activation effects of monomeric, oligomeric and fibrillar α-syn species on astrocytes. Our results help to understand the effect of α-syn species on astrocyte function and their potential impact on the pathogenesis of PD and related α-synucleinopathies. Besides, our findings indicate that astrocytes may contribute to spread α-syn pathology, revealing astrocytes as a potential target for therapeutic intervention.

Abbreviations

     
  • CNS

    central nervous system

  •  
  • DCF

    2′,7′-dichlorofluorescein

  •  
  • DCFH2

    2′,7′-dichlorodihydrofluorescein

  •  
  • DCFH2DA

    2′,7′-dichlorodihydrofluorescein diacetate

  •  
  • DLS

    dynamic light scattering

  •  
  • DMEM

    Dulbecco's modified Eagle's medium

  •  
  • ECAR

    extracellular acidification rate

  •  
  • FBS

    fetal bovine serum

  •  
  • FCCP

    carbonyl cyanide-p-trifluoromethoxyphenylhydrazone

  •  
  • GFAP

    glial fibrillary acidic protein

  •  
  • IL-1β

    interleukin 1β

  •  
  • PD

    Parkinson's disease

  •  
  • qPCR

    quantitative PCR

  •  
  • RCR

    respiratory control ratio

  •  
  • RH

    hydrodynamic radius

  •  
  • ROS

    reactive oxygen species

  •  
  • TEM

    transmission electron microscopy

  •  
  • Thio-T

    thioflavin T

  •  
  • TNF-α

    tumoral necrosis factor alpha

  •  
  • α-syn

    alpha-synuclein

Author Contribution

C.C., S.R.-B., C.Q., P.C. and J.M.S. conceived and designed the experiments. C.C. and S.R.-B. performed the experiments.C.C., S.R.-B., C.Q. and P.C. analyzed the data.C.Q., P.C. and J.M.S. contributed reagents/materials/analytic tools. C.C., S.R.B., C.Q., P.C. and J.M.S. wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

Funding

This work was supported by a scholarship provided by the Agencia Nacional de Investigación e Innovación to C.C. and also by Programa de Desarrollo de las Ciencias Básicas (PEDECIBA). This work was supported by grants from the Universidad de la República [CSIC GruposI+D #1104 and Núcleo Interdisciplinario IMAGINA, Espacio Interdisciplinario], and the Agencia Nacional de Investigación e Innovación (ANII), Uruguay [ANII-FCE 2011_1_6260 to J.M.S.]; a fellowship from the ANII, and Programa de Desarrollo de las Ciencias Básicas (PEDECIBA) to C.C.

Acknowledgments

We acknowledge Otto Pristch and Federico Carrión for their help in the experiments performed with the Dynamic Light Scattering equipment from the Institute Pasteur Montevideo; Rafael Radi for critical reading of the manuscript, Carolina Prolo for her help with flow cytometry experiments; Ernesto Miquel and Laura Martínez-Palma for helping in the astrocyte and neuron cells preparation and figures design, Gabriela Casanova and Gaby Martínez for their help in the experiments performed with the transmission electron microscope (Facultad de Ciencias), Claudio Benech for letting us use the Atomic Force Microscope at the Instituto Clemente Estable; and Mauricio Ramos and Federico Lecumberry from Núcleo Interdisciplinario-Imagina for the development of the mitochondria automatic segmentation plugin.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors contributed equally to this work.

Supplementary data