Background
Cellular inclusions in the brain, referred to as Lewy bodies and Lewy neurites, are pathological hallmarks of Parkinson’s disease (PD) [
1]. The inclusions predominantly consist of α-synuclein (α-syn) [
2], a protein which aggregates into insoluble fibrils via the formation of soluble intermediates [
3]. Such α-syn oligomers are particularly harmful [
4] and have for example been shown to disrupt cellular membranes [
5,
6] and induce mitochondrial dysfunction [
7,
8].
Targeting pathological α-syn by either active or passive immunization has been shown to reduce α-syn levels and ameliorate behavioral symptoms in transgenic mouse models of synucleinopathy [
9‐
13]. Due to their toxic nature, immunization against soluble α-syn oligomers is an especially attractive therapeutic target. Lewy body formation is believed to be protective rather than neurotoxic, and immunotherapy directed against fibrillar α-syn is therefore less likely to be effective. We have previously shown that treatment with antibodies directed against oligomeric α-syn lowers the CNS levels of oligomers in a transgenic α-syn-expressing mouse model [
9]. However, the cellular mechanisms by which the antibodies act and which cell types are targeted remain unknown.
Although α-syn deposits are primarily found in neurons in the PD brain, they also appear frequently in astrocytes [
14‐
19]. Being the most numerous glial cell type in the CNS, astrocytes have great impact on the brain environment and may constitute a very potent treatment target. Astrocytes play an important role in maintaining brain homeostasis [
20], and their functions include metabolic support of neurons, modification of synapse signaling, recycling of neurotransmitters, blood brain barrier regulation, and glymphatic clearance [
20‐
22]. In addition, astrocytes respond to neurodegenerative disorders, including PD, through astrogliosis, a process in which they convert to a reactive inflammatory state [
23,
24]. Yet, the role of astrocytes in the development and progression of α-syn pathology has been infrequently studied. In a recent study, we investigated uptake, degradation, and toxic effects of soluble oligomeric α-syn in a co-culture system consisting of predominant astrocytes and, to a lesser extent, neurons and oligodendrocytes [
25]. In contrast to neurons, the astrocytes were found to rapidly ingest large amounts of α-syn. Due to incomplete digestion, intracellular α-syn deposits remained in the astrocytes, which resulted in a mitochondrial impairment [
25].
The aim of the present study was to investigate whether α-syn oligomer-selective antibodies can affect astrocytic accumulation of α-syn oligomers in vitro. The oligomer-selective antibodies used in this study have approximately 500-times higher affinity for oligomers compared to monomeric protein [
26]. Interestingly, we found that oligomer-selective antibodies effectively prevented astrocytic accumulation of exogenously added α-syn oligomers. Moreover, the antibodies rescued the astrocytes from mitochondrial fragmentation. Our results suggest that immunotherapeutic approaches involving antibodies selective for soluble α-syn oligomers could improve astrocyte functioning, including their neuroprotective effects in alpha-synucleinopathies.
Methods
Animals
All animal experiments were approved by the Uppsala County Animal Ethics Board, following the rules and regulations of the Swedish Animal Welfare Agency and were in compliance with the European Communities Council Directive (2010/63/EU). The animals were housed at the Uppsala University Hospital in a 12:12 dark:light cycle. The mice were kept in an enriched environment and given water and food ad libitum. Embryonic C57BL/6 mice were used for the cell culture experiments.
Neural cell culture
Cortices from 14-day-old mouse embryos (E14) were dissected, and neurospheres were prepared and differentiated as previously described [
27]. The cells were expanded in DMEM/F12-GlutaMAX supplemented with 1 × B27, 50 U/ml penicillin, 50 mg/ml streptomycin and 8 mM Hepes buffer; 10 ng/ml bFGF (all from Invitrogen); and 20 mg/ml EGF (VWR). The neurospheres were passaged every second or third day by mechanical dissociation. Prior to experiments, the cells were plated as a monolayer, at a concentration of 1.5 × 10
4 cells/ml on cell culture dishes or coverslips coated with poly-
l-ornithine (0.0025%, Sigma-Aldrich) and laminin (1 μg/ml, Invitrogen). After 24 h, the media was replaced with mitogen-free media (DMEM/F12-GlutaMAX supplemented with 1 × B27, 50 U/ml penicillin, 50 mg/ml streptomycin, and 8 mM Hepes buffer (all from Invitrogen)) to initiate neural stem cell differentiation to a mixed population of neurons, astrocytes, and oligodendrocytes, but not microglia. This is a well-characterized cell culture system, based on the lineage-restricted differentiation of embryonic, cortical stem cells [
28‐
30]. By using immunocytochemistry, we have previously confirmed that the culture (after 1 week of differentiation) consists of a mixed population of neurons (20%), astrocytes (75%), and oligodendrocytes (5%), but importantly no microglia [
25]. Neurospheres from passages 1–3 were used for the experiments.
Generation and labeling of HNE-induced α-syn oligomers
Recombinant α-syn was produced as previously described [
31]. Monomeric α-syn (140 μM) was incubated with 4-hydroxy-2-nonenal (HNE) (Cayman Chemicals) in a HNE:α-syn molar ratio of 30:1 at 37 °C for 72 h. To control the ratio of oligomerization, samples were analyzed with SEC-HPLC, previously described in [
25]. The characteristics of the oligomers have been thoroughly described [
31]. 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. The α-syn oligomers were labeled with Cy3 using Amersham Cy3 Antibody Labeling Kit (GE Healthcare (cat. # PA33000)). Unbound excess Cy3 was removed by filtration in a Zeba spin desalting column (Thermo Scientific).
Alpha-synuclein and antibody exposure
Alpha-synuclein oligomers were pre-incubated with the α-syn oligomer-selective antibodies, mAb47 (IgG1) [
9], mAb49G (IgG1) [
32], or mAb38E2 (IgG2b), generated by the research group [
26] at a 1:1 ratio (0.05 μM) in regular growth media at 37 °C for 1 h. The co-cultures were then exposed to 0.05 μM oligomers or to oligomers pre-incubated with antibodies. To analyze the effect of mAb47 on α-syn accumulation further, mAb47 was added after the 24-h α-syn oligomer exposure. The cultures were thoroughly washed in cell culture media prior to the antibody addition and cultured with the antibody for 24 h. In another control experiment, the co-cultures were incubated with mAb47 for 24 h and then extensively washed in cell culture media, prior to 24-h Aβ
42 protofibril exposure. As negative control antibodies, the irrelevant antibody mAb-Ly128 (IgG
1, Mabtech), recognizing flagellin in bacteria, were used. For visualization of mitochondria, the cultures were transfected with Cell light Mitochondria-GFP (BacMam 2.0, Life Technologies) during the oligomer exposure. After 24 h of oligomer exposure, conditioned media was collected and the cells were washed in fresh media and fixed or lysed for further analyses. Cells were also recorded using a time-lapse microscope (Nikon Biostation IM Cell Recorder), during exposure to α-syn oligomers (0.05 μM) or to oligomers pre-incubated with mAb47 (0.05 μM, 1:1 ratio). Images (40X) were captured every 10 min for 24 h.
Lysosomal inhibition
In order to investigate the influence of the endosomal/lysosomal pathway on the antibody-mediated α-syn reduction in astrocytes, cells were pre-incubated with two different lysosomal inhibitors. Co-cultures were incubated with the lysosomal inhibitors bafilomycin (Baf; Calbiochem, Millipore 196000, 0.5 μM) or chloroquine (Chq; Sigma, C6628, 10 μM) for 30 min prior to addition of α-syn oligomers or oligomers pre-incubated with mAb47. The inhibitors remained in the media during the 24-h exposure. Following fixation, the accumulation of α-syn oligomers was assessed by immunofluorescence. The inhibitors did not have any apparent toxic effects in the concentration and exposure times used.
Immunocytochemistry
Cells were cultured on coverslips and fixed with 4% PFA for 15 min at room temperature (RT). The cells were then permeabilized and blocked for 30 min at RT in 0.1% Triton X-100 + 5% normal goat serum (NGS) in PBS and incubated for 1 h at RT with primary antibodies diluted in 0.1% Triton X-100 + 0.5% NGS in PBS. The primary antibodies used were polyclonal rabbit anti-GFAP (DAKO, 1:400) or monoclonal mouse anti-GFAP (Sigma, 1:400), specific for astrocytes; mAb38F, (1:100), selective for α-syn oligomers [
26]; and rabbit polyclonal anti-Lamp-1 (Abcam, 1:200), a lysosomal marker. Following washing 3× in PBS, the samples were incubated with secondary antibodies: goat anti-rabbit IgG (H+L), Alexa Fluor® 488 conjugate antibody; goat anti-mouse IgG (H+L), Alexa Fluor® 488 conjugate antibody; or goat anti-mouse IgG (H+L), Alexa Fluor® 647 conjugate antibody (Life Technologies, 1:200) in 0.1% Triton X-100 + 0.5% NGS in PBS at RT for 1 h. Then, the coverslips were washed and mounted using Vectashield Hard Set with DAPI (Vector Laboratories). Internalization of the α-syn oligomer-selective antibodies was detected by staining of permeabilized samples for mouse IgG with a secondary goat anti-mouse 488 antibody (Alexa, Life Technologies, 1:200).
ELISA
To assess Ab:α-syn oligomer complex formation, an α-syn oligomer-selective ELISA was used as previously described [
26]. A reduced ELISA signal was interpreted as interference due to mAb47 binding to oligomers and obstruction of mAb38F binding, in either the capture or detection steps. In short, oligomers were incubated with or without mAb47 (0.05 μM, 1:1 ratio) 1 h in regular media for 37 °C. High-binding 96-well polystyrene plates (Costar 3590) were coated with the oligomer-selective antibody mAb38F (100 ng/well) diluted in PBS overnight (4 °C). The plate was blocked with 1% bovine serum albumin for 2 h before adding the α-syn 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 (100 ng/well) for 1 h and Streptavidin-HRP (1:5000, Mabtech) for 45 min. Finally, TMB (Neogen) was used as a substrate and the plates were analyzed at 450 nm (Infinite M1000, Tecan). In addition, a sandwich ELISA was used to quantify the total α-syn levels in the cultures. Conditioned media from three different experiments (
n = 3) was collected after 24 h of exposure to α-syn oligomers or oligomers pre-incubated with antibody. The ELISA was performed as described above, but using Syn-1 (1 μg/ml, 610,787, BD Biosciences) as capture antibody and FL-140 (1 μg/ml, sc-10717, Santa Cruz Biotechnology) for detection. The samples were diluted 1:200 prior to loading on ELISA wells.
Immunoprecipitation and Western blot
Conditioned media were collected after 24 h exposure to mAb47+α-syn oligomer and stored at − 70 °C. The media were thawed on ice and incubated with 3-mg magnetic beads (Dynabeads, Novex, Life Technologies) with rotation for 2 h at 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 electrophoresis was carried out with the eluate 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). Chameleon kit pre-stained protein ladder (Li-Cor) was used as a standard. Transfer onto a PVDF membrane was performed for 1 h at 20 V (Novex, Life Technologies). The membrane was blocked with Odyssey TBS blocking buffer (Li-Cor) for 1 h at RT. After brief washing in TBS-T, the membrane was incubated with the mouse-anti-α-syn antibody mAb211 (Santa Cruz, sc-12,767, 1:500) in 0.5X Odyssey blocking buffer in TBS-T at 4 °C overnight. Washing (3 × 10 min in TBS-T) was followed by incubation with secondary antibody DyLight 680 goat anti-mouse (Li-Cor, 1:10,000) in 0.5X Odyssey blocking buffer in TBS-T for 1 h (RT). Washing 3 × 10 min in TBS-T was followed by imaging in an Odyssey SA infrared imaging system (Li-Cor). Regular media was used as a negative control, and media containing 0.05 μM α-syn oligomers pre-incubated with mAb47 for 1 h at 37 °C was used as a positive (0 h) control.
Analyses and statistics
When applicable, the results are presented as mean ± standard deviation. For statistical analyses, two-tailed Mann-Whitney U test (GraphPad Prism) was used and the levels of significance were set to *P < 0.05, **P < 0.01, and ***P < 0.001. The total number of observations was used for the statistical analyses. All of the experiments were performed on independent cell cultures, derived from mouse embryos of three different pregnant females. A wide-field microscope (Zeiss AxioImager Z1) was used for quantifications of α-syn accumulation (×40 magnification). The area and intensity of the Cy3-α-syn fluorescence signal was analyzed using ZEN 2012 software. In total, 45 images for each treatment (15 images per sample) were included in the statistical analysis. Values were normalized against the number of viable nuclei. Confocal micrographs were taken with a Zeiss LSM 700 confocal microscope (×63 magnification).
Mitochondrial morphology in cell light mitochondria-GFP transfected cells was quantified manually. The number of cells with disrupted mitochondrial networks was counted and normalized against the total number of transfected, GFP-expressing cells. Cells with mitochondria displaying a low degree of branching, signs of swelling, and a dotted pattern were addressed as cells with a “disrupted mitochondrial network.” In total, 45 images for each treatment (15 images per sample) were included in the analysis that was performed in a blinded fashion. At total of 250 cells or more were counted per sample.
Discussion
Immunotherapy has emerged as a promising method to target α-syn pathology, but the underlying mechanisms and the role of different cell types are poorly understood. Whether astrocytes are involved in the cellular responses to α-syn immunotherapy has not been previously addressed. Here, we demonstrate for the first time that oligomer-selective antibodies can increase clearance of pathological α-syn and maintain healthy mitochondria in astrocytes.
We have previously shown that treatment with the oligomer-selective antibody mAb47 leads to lowered α-syn oligomer levels in the spinal cord of transgenic A30P α-syn mice, correlating with ameliorated motor symptoms [
9]. In the present study, we investigated the effects of three different oligomer-selective antibodies: mAb49G, mab38E2, and mAb47, on α-syn-induced pathology in a co-culture system of astrocytes, neurons, and oligodendrocytes. The astrocytes were found to ingest large amounts of oligomeric α-syn that accumulated as intracellular deposits and caused mitochondrial impairment, similar to previous observations [
25]. Interestingly, the three oligomer-selective antibodies displayed different capacities in reducing the intracellular α-syn deposits, with mAb47 being the most effective. The varying effects of the antibodies could be due to differences in their interaction with α-syn oligomers in the extracellular milieu or to differences in the interaction of the antibodies to extracellular or intracellular receptors, depending on their different IgG sub-classes [
34,
35]. Our pull-down Western blot analysis demonstrated that although the oligomers of the Ab:oligomer complexes were cleared from the culture, the antibodies remained. It could therefore be speculated that the astrocytes actively recycled and secreted the antibodies, whereas the oligomers in the complex were effectively degraded by the cells. Recycling of antibodies has been suggested to be crucial in immunotherapy, by enabling each antibody to bind to its antigen multiple times and thereby increase the clearance of the target protein [
36].
In contrast to the α-syn oligomer-selective mAb47, the irrelevant antibody of the same IgG isotype only had a modest effect on the α-syn accumulation. This result indicates that the mechanism of action includes a direct interaction between α-syn and α-syn-oligomer-selective antibodies and is not only the result of a general glial activation (due to antibody binding to the astrocytes’ Fc-receptors). Moreover, addition of the oligomer-selective antibodies before the α-syn oligomer exposure had only a minor effect on the intracellular deposition whereas addition of the antibodies after the oligomer exposure had no effect at all. Taken together, these experiments demonstrated that immune complex formation needs to occur in the extracellular space prior to internalization in order to reduce intracellular α-syn accumulation.
Although the mechanisms responsible for the effectiveness of passive immunization against α-syn pathology are incompletely understood, its beneficial effects have been suggested to include increased clearance of toxic α-syn species from the extracellular space [
10], steric hindrance of α-syn aggregation [
32], and reduced α-syn propagation between cells [
37,
38]. These previous investigations have focused on processes in neurons [
38], neuron-like cell lines, or microglia [
10], but have not involved astrocytes. Being the most abundant glial cell type in the central nervous system, astrocytes play an important role in maintaining brain homeostasis [
20]. In addition, astrocytes respond to neurodegenerative disorders, including PD, through astrogliosis, a process in which they convert to a reactive inflammatory state [
23,
39]. The complex role of astrocytes in the pathological brain is largely dependent upon their release and uptake of substances from the micro-environment that they share with the neurons [
20]. Reactive astrocytes effectively engulf dead cells, synapses, and protein aggregates of amyloid beta (Aβ) and α-syn [
27,
40‐
46], and we and others have shown that astrocytes may play an important role in spreading of pathogenic proteins [
47].
Studies on transgenic mice and cell models have further indicated that anti-α-syn antibody treatment can mediate an upregulation of autophagy [
13] and promote lysosomal degradation [
10,
48]. In our immunocytochemical analyses, we detected low levels of intracellular α-syn after Ab:oligomer complex exposure. However, by enhancing the fluorescence signal, we could demonstrate that there were still low levels of Ab:oligomer complexes present within Lamp-1-positive structures in the astrocytes, indicating that at least some of the ingested complexes were directed to the endosomal-lysosomal pathway. However, since inhibition of the lysosomal pathway did not have an impact on the reduced accumulation, we speculate that the majority of the Ab:oligomer complexes may be degraded by another route, such as the proteasome pathway or another not yet described mechanism, but additional investigations will be needed to sort that out. Irrespective of degradation pathway, our data indicate that the astrocytic clearance of already ingested Ab:oligomer complexes is rapid. Another possible explanation for the reduced intracellular deposits of α-syn could be a lower uptake or an increased recycling and secretion of the engulfed Ab:oligomer complexes. However, our ELISA and Western blot data of conditioned media showed that both the intra- and extracellular levels of α-syn were lowered in the antibody-treated cultures. In accordance, the time-lapse recordings indicated a treatment-related decrease in the astrocytic accumulation and absence of free-floating Ab:oligomer complexes. Altogether, our data demonstrate that the Ab:oligomer complexes do not accumulate in the astrocytes to the same extent as α-syn oligomers alone, but are rather cleared from the culture.
There have been several reports about how α-syn aggregates induce mitochondrial dysfunction in neurons, including disturbance of the mitochondria fission-fusion homeostasis, autophagosome synthesis, and mitophagy function [
49‐
51]. Our previous results demonstrated a widespread mitochondrial fragmentation and dysfunction in α-syn oligomer treated astrocytes, indicating that the accumulation of α-syn disrupts the autophagosomal and mitophagy machinery [
25].
Conclusions
The present study demonstrates that α-syn oligomer-selective antibodies effectively prevent accumulation of toxic α-syn oligomers in the astrocytes and thereby rescue them from mitochondrial impairment. Neurodegenerative diseases, including PD, are defined by loss of brain homeostasis [
22,
52‐
55], which at least partly could be due to the severely stressed astrocytes that are unable to fulfill their normal tasks [
20‐
22]. Hence, maintaining astrocytic functionality is probably a crucial component for immunotherapy to be successful. In addition, increased astrocytic clearance of soluble α-syn oligomers may reduce PD progression, by preventing further spreading of the α-syn pathology. In order to optimize the design of future antibody-based therapies, we believe that more studies are needed, focusing on various cell types, including astrocytes.
Acknowledgements
This study was supported by grants from the Swedish Research Council, the Swedish Parkinson Foundation, the Swedish Alzheimer Foundation, Åhlén Foundation, The Swedish Dementia Association Foundation, Hedlund’s Foundation, Lennart and Christina Kalén, William N. & Bernice E. Bumpus Foundation Innovation Award, Marianne and Marcus Wallenberg Foundation, the Swedish Brain Foundation, Parkinson Research Foundation, King Gustaf V and Queen Victoria’s Foundation of Freemasons, the U4 Ageing Brain Network, and the Uppsala Berzelii Center for Neurodiagnostics.