Image_1_Region-Specific Effects of Immunotherapy With Antibodies Targeting α-synuclein in a Transgenic Model of Synucleinopathy.JPEG

<p>Synucleinopathies represent a group of neurodegenerative disorders which are characterized by intracellular accumulation of aggregated α-synuclein. α-synuclein misfolding and oligomer formation is considered a major pathogenic trigger in these disorders. Therefore, targeting α-synuclein species represents an important candidate therapeutic approach. Our aim was to analyze the biological effects of passive immunization targeting α-synuclein and to identify the possible underlying mechanisms in a transgenic mouse model of oligodendroglial α-synucleinopathy. We used PLP-α-synuclein mice overexpressing human α-synuclein in oligodendrocytes. The animals received either antibodies that recognize α-synuclein or vehicle. Passive immunization mitigated α-synuclein pathology and resulted in reduction of total α-synuclein in the hippocampus, reduction of intracellular accumulation of aggregated α-synuclein, particularly significant in the spinal cord. Lowering of the extracellular oligomeric α-synuclein was associated with reduction of the density of activated iba1-positive microglia profiles. However, a shift toward phagocytic microglia was seen after passive immunization of PLP-α-synuclein mice. Lowering of intracellular α-synuclein was mediated by autophagy degradation triggered after passive immunization in PLP-α-synuclein mice. In summary, the study provides evidence for the biological efficacy of immunotherapy in a transgenic mouse model of oligodendroglial synucleinopathy. The different availability of the therapeutic antibodies and the variable load of α-synuclein pathology in selected brain regions resulted in differential effects of the immunotherapy that allowed us to propose a model of the underlying mechanisms of antibody-aided α-synuclein clearance.</p

Immunocytochemistry with anti-mouse secondary antibodies (Cy3) displays internalization of the α-synuclein monoclonal antibodies (+mAb).

<p>Forty-eight hours after transfection with the two α-synuclein hemi:GFP constructs, cells displayed GFP fluorescence in the whole cell soma (green) (Fig. A). Cells transfected with the constructs and treated with the α-synuclein antibodies mAb49/G and mAb211 displayed less diffuse GFP fluorescence but more localized GFP-punctae (Fig. D, E, arrows). These signals occasionally co-occurred with signals from an anti-mouse secondary antibody, indicating internalization (yellow) of the treatment antibodies (Fig. D, E, arrows). Cells treated with the mAb5C2 antibody only displayed diffuse GFP-fluorescence in the whole cell soma (Fig. F). After staining with an anti-mouse secondary antibody red punctae could be detected in the cells indicating no co-occurrence with GFP (Fig. F, arrows). The blue signal represents DAPI nuclear staining (40x magnification. Scale bar 20 µm).</p

Confocal microscopy showing internalization of the α-synuclein antibodies.

<p>Forty-eight hours after addition of the mAb49/G, mAb211 and mAb5C2 α-synuclein antibodies, red punctate structures were detected within the cells after staining with anti-mouse secondary antibodies (Fig A, B and C, arrows). For comparison, immunocytochemistry was carried out using mAb49/G, mAb211 and mAb5C2 as primary antibodies (Fig. D,E and F). The blue signal represents DAPI nuclear staining (63x magnification. Scale bar 20 µm).</p

ELISA measurement of α-synuclein levels in lysates and media from BiFC expressing cells.

<p>In lysate from BiFC expressing cells (-mAb), the levels were 5.6 ng/ml (Fig. A), with mAb49/G treatment (+mAb) 2.8 ng/ml (*p<0.04), mAb211 treatment 3.9 ng/ml (*p<0.05) and with mAb5C2 treatment 3.3 ng/ml (*p<0.05) showing a reduction in protein content (Fig. A). In conditioned media from untreated cells (-mAb) the α-synuclein levels were 6.9 ng/ml (Fig. B). In media from antibody treated cells (+mAb), the levels were 3.2 ng/ml for mAb49/G (*p<0.05), 2.2 ng/ml for mAb211 (*p<0.05) and 2.3 ng/ml (*p<0.05) with mAb5C2 (Fig. B).</p

Characterization of mAb49/G by inhibition ELISA.

<p>Binding of mAb49/G to α-synuclein monomers (□) or α-synuclein oligomers (•) was analyzed on HNE stabilized α-synuclein oligomer coated plates. On the x-axis, the molar concentration of α-synuclein is displayed. The IC50 values are calculated as the concentration of either α-synuclein monomers or α-synuclein oligomers needed to quench half of the signal in the ELISA. Note that, due to uncertainties concerning the size of the α-synuclein oligomers used in this assay, the concentration in pM for both species is based on the molecular weight of one α-synuclein monomer. These data are representative of at least three independent experiments.</p