Alpha-synuclein oligomer-selective antibodies reduce intracellular accumulation and mitochondrial impairment in alpha-synuclein exposed astrocytes

Abstract

Background: Due to its neurotoxic properties, oligomeric alpha-synuclein (α-syn) has been suggested as an attractive target for passive immunization against Parkinson's disease (PD). In mouse models of PD, antibody treatment has been shown to lower the levels of pathogenic α-syn species, including oligomers, although the mechanisms of action remain unknown. We have previously shown that astrocytes rapidly engulf α-syn oligomers that are intracellularly stored, rather than degraded, resulting in impaired mitochondria.

Methods: The aim of the present study was to investigate if the accumulation of α-syn in astrocytes can be affected by α-syn oligomer-selective antibodies. Co-cultures of astrocytes, neurons, and oligodendrocytes were derived from embryonic mouse cortex and exposed to α-syn oligomers or oligomers pre-incubated with oligomer-selective antibodies.

Results: In the presence of antibodies, the astrocytes displayed an increased clearance of the exogenously added α-syn, and consequently, the α-syn accumulation in the culture was markedly reduced. Moreover, the addition of antibodies rescued the astrocytes from the oligomer-induced mitochondrial impairment.

Conclusions: Our results demonstrate that oligomer-selective antibodies can prevent α-syn accumulation and mitochondrial dysfunction in cultured astrocytes.

Keywords: Antibodies; Astrocytes; Lysosomal degradation; Mitochondria; Parkinson’s disease; α-synuclein oligomers.

Fig. 1

Oligomer-selective antibodies reduce α-syn oligomer accumulation in astrocytes. a α-syn oligomers incubated with mAb47 (0.05 μM, 1:1 ratio) 1 h at 37 °C were analyzed with mab38F/biot-mab38F sandwich ELISA. The addition of mAb47 resulted in a strong reduction of the α-syn oligomer signal, implying steric hindrance by mAb47 due to high Ab:α-syn oligomer complex formation. b Cells were exposed to fluorescent α-syn oligomers or to oligomers pre-incubated with the α-syn oligomer-selective antibodies: mAb47, mAb49G, or mAb38E2, for 24 h. The α-syn accumulation was strongly reduced when oligomers had been pre-incubated with antibodies. The mAb47 had the most pronounced reducing effect on α-syn oligomer accumulation. Measurements of the intracellular Cy3-labeled α-syn, using the Zen-software, confirmed that the c area/cell and d intensity/cell were significantly decreased in the antibody-treated cultures. Scale bars = 10 μm. Data are presented as mean ± SD from three independent experiments and levels of significance were set to *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 2

Extracellular antibody binding is critical for the effect on α-syn oligomer accumulation. Cells were exposed to Cy3-α-syn oligomers and mAb47 in three different combinations. a The cells were either exposed to α-syn oligomers for 24 h (α-syn), resulting in high intracellular accumulation, or to mAb47:oligomer complex (α-syn+47). A clear reduction in α-syn fluorescence signal was detected. Parallel cultures received mAb47 24 h prior to α-syn oligomer exposure (α-syn+47 pre), resulting in a minor reduction of the fluorescence signal. When mAb47 was added 24 h after the oligomer exposure (α-syn+47 post), there was no effect on α-syn accumulation. b Fluorescence quantification of Cy3-α-syn area and c intensity demonstrated that there was only a significant decrease of α-syn accumulation when mAb47 was added at the same time as the oligomers (α-syn+47) or added before the α-syn exposure (α-syn+47 pre). Scale bars = 10 μm. Data are presented as mean ± SD from three independent experiments and levels of significance were set to *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 3

Treatment with irrelevant antibodies has no effect on α-syn accumulation. To investigate the specificity of the antibody-mediated reduction of α-syn accumulation, cells were exposed to Cy3-oligomers pre-incubated with an irrelevant antibody of the same IgG subclass as mAb47 (IgG1). The IgG1 LY-128 has no affinity for α-syn and therefore served as an irrelevant isotype control. Alpha-synuclein oligomers were pre-incubated with mAb47 or LY-128 prior to the 24-h cell exposure. a The cultures that were treated with mAb47 (α-syn+47) displayed a reduced α-syn accumulation. This effect was significantly stronger than in cultures treated with LY-128 (α-syn+LY128). b Quantification of Cy3-α-syn fluorescence area and (c) intensity confirmed a significant decrease of α-syn signal in mAb47-treated cultures compared to LY-128-treated cultures. Scale bars = 10 μm. Data are presented as mean ± SD from three independent experiments and levels of significance were set to *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 4

Intracellular mAb47 co-localizes with α-syn oligomers in astrocytes. Co-cultures were exposed to Cy3-α-syn oligomers or to Cy3-oligomers pre-incubated with mAb47. a Immunofluorescence with secondary antibodies directed towards mAb47 displayed the antibody internalization (green). The Cy3-α-syn signal (red) was weak, indicating low oligomer accumulation. Enhancing the Cy3-α-syn signal revealed intracellular co-localization of oligomers and mAb47 (blue = DAPI). b As a control, cells were exposed to non-fluorescent α-syn oligomers that had been pre-incubated with mAb47. Co-staining with antibodies to GFAP (white), mAb47 (green), and oligomeric α-syn (detected by mAb38F, red) revealed co-localization of mAb47 and oligomeric α-syn inside astrocytes. Scale bars = 10 μm

Fig. 5

Internalized α-syn oligomers co-localize with lysosomal markers, irrespective of antibody treatment. Co-cultures were exposed to either Cy3-α-syn oligomers or Cy3-oligomers pre-incubated with mAb47. a After 24 h, oligomers were localized in LAMP-1-positive endosomal-lysosomal compartments. b After pre-incubation with mAb47, the levels of intracellular α-syn were very low. However, when the signal was enhanced the Cy3-α-syn still displayed co-localization with LAMP-1 (arrow) (blue = DAPI). Scale bar = 10 μm

Fig. 6

Treatment with mAb47 prevents α-syn oligomer-mediated mitochondrial stress effects. During the 24 h exposure to α-syn oligomers or Ab:oligomer complexes, cells were transfected with Cell light Mitochondria-GFP to label the mitochondria. a Astrocytes exposed to α-syn oligomers frequently displayed a disrupted mitochondrial network and mitochondrial swelling. b The astrocytes in cultures exposed to α-syn oligomers pre-incubated with mAb47 displayed an elongated, branched mitochondrial network throughout the cells, similar to that of c non-treated cells. d Cells with disrupted mitochondrial networks were counted and normalized against the number of transfected cells. Oligomer exposure led to a clear increase in cells with mitochondrial fragmentation, whereas cultures exposed to oligomers pre-incubated with mAb47 did not significantly differ from the untreated control cells. Scale bars = 10 μm. Data are presented as mean ± SD from three independent experiments and levels of significance were set to *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 7

Lysosomal-endosomal inhibition does not alter the antibody-mediated effect on oligomer accumulation. a Co-cultures were exposed to either Cy3-α-syn oligomers or to mAb47-treated oligomers for 24 h, in combination with the lysosomal inhibitors bafilomycin (+Baf) or chloroquine (+Chq). The α-syn accumulation was unchanged, both in the absence and presence of mAb47. Hence, chemical inhibition of the lysosomal pathway did not alter the mAb47-mediated reduction of α-syn accumulation. b Fluorescence quantification of Cy3-α-syn area and (c) intensity confirmed that the inhibitors did not reduce the antibody-mediated effect on α-syn accumulation. Scale bars = 10 μm. Data are presented as mean ± SD from three independent experiments and levels of significance were set to *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 8

The presence of mAb47 slows down astrocytic accumulation of α-syn oligomers. Co-cultures were exposed to either Cy3-α-syn oligomers or to mAb47-treated oligomers and monitored with time-lapse imaging for 24 h. Areas highlighted with white rectangles to the left (20x) are displayed at higher magnification to the right (40x). When cells were exposed to oligomers alone, the astrocytic accumulation of fluorescent α-syn (red) was rapid, with deposits detectable already after 15 min that were increasing throughout the experiment (a). Exposure to mAb47-treated oligomers led to a much lower accumulation in astrocytes. At 24 h, a weak Cy3-α-syn signal was visible in the astrocytes (b). Moreover, no free-floating fluorescent aggregates were observed in the cell media, suggesting an overall increased clearance of α-syn oligomers (scale bars = 10 μm)

Fig. 9

Antibody treatment results in lower extracellular levels of α-syn. Extracellular levels of total α-syn were measured with a sandwich ELISA and immunoprecipitation followed by Western blot. a Co-cultures were exposed to α-syn oligomers or to oligomers pre-incubated with mAb47, mAb49G, or 38E2. Conditioned media analyzed with sandwich ELISA demonstrated that the α-syn levels were dramatically decreased when any of the antibodies were present. The largest reduction in extracellular α-syn levels was seen with mAb47. Data are presented as mean ± SD, and the levels of significance were set to *P < 0.05, **P < 0.01, and ***P < 0.001 (n = 3). b To detect the fraction of α-syn oligomers bound to mAb47, immunoprecipitation was performed on conditioned media from the mAb47-treated sample (24 h). As a positive control, media containing mAb47:oligomer complex that had not been in contact with cells was used (0 h). Western blot on eluted immune complexes displayed a smear of high molecular weight α-syn species, dimer (35 kDa), and monomer bands (14 kDa) in the 0 h control. The 24 h samples showed reduced α-syn signals, as compared to the 0 h control. In addition, the heavy (50 kDa) and light (25 kDa) IgG chains from mAb47 were detected. c Quantification of α-syn bands revealed a clear decrease after 24 h, as compared to 0 h. d The heavy and light chain bands of mAb47 were quantified and displayed only a modest reduction after 24 h (n = 3)