Article Figures & Data
Figures
- Figure 1.
New antibodies directed against the CT of α-syn recognized α-syn by immunoblot and immunohistochemistry in α-syn tg mice. A, Schematic representation of the α-syn molecule and binding sites of CT α-syn antibodies 1H7 (aa 91–99), 9E4 (aa 118–126), 5C1 (aa 118–126), and 5D12 (aa 118–126). The protease cleavage site (118–122) is also depicted. B, Immunoblot analysis of non-tg, PDGF-α-syn tg, and mThy1-α-syn tg brain homogenates probed with 1H7, 9E4, 5C1, or 5D12 antibodies. The 14 kDa band is monomeric α-syn and the 12 kDa band is CT-α-syn. Higher-molecular-weight bands correspond to different α-syn oligomers. Actin was used as a loading control. C, mThy1-α-syn tg mouse brain sections were immunostained with the CT α-syn antibodies 1H7, 9E4, 5C1, or 5D12. Antibody binding was detected using a FITC-tagged secondary antibody. Scale bar, 10 μm.
- Figure 2.
Plasma antibody titers in passively immunized α-syn tg mice. Antibody titers (μg/ml) determined by ELISA in α-syn tg mice immunized with the CT α-syn antibodies 9E4, 1H7, 5C1, or 5D12. For each antibody, different groups of mice were used and individual animals within each group are represented by different colors. Antibody titers were measured every 3 weeks.
- Figure 3.
Immunohistochemical analysis of passively immunized α-syn tg mice with antibodies against FL α-syn or CT-truncated α-syn. To determine the effects of passive immunization on FL-α-syn (A–C) or CT-α-syn (D–F) accumulation, immunohistochemical analysis using species-specific antibodies was conducted. A, Top, Images of the neocortex and hippocampus of non-tg mice immunized with control antibody 27-1 and α-syn tg mice immunized with antibodies 27-1, 9E4, 1H7, 5C1, or 5D12. Sections were immunolabeled with an antibody against FL-α-syn. Bottom, Higher-magnification images of the neocortex of the sections above. Scale bar, 30 μm. B, Analysis of the levels of α-syn immunoreactivity in the neocortex, measured as optical density in sections labeled with the FL-α-syn antibody. C, Stereological analysis of the numbers of neocortical α-syn-immunoreactive neurons stained with the FL-α-syn antibody. D, Top, Images of the neocortex, hippocampus, and striatum of non-tg mice immunized with control antibody 27-1 and α-syn tg mice immunized with antibodies 27-1, 9E4, 1H7, 5C1, or 5D12. Sections were immunolabeled with the antibody SYN105 against CT-α-syn. Bottom, Higher-magnification images of the striatum of the sections showed above. Arrows highlight α-syn-positive granular-like structures. Scale bar, 5 μm. E, Analysis of the levels of α-syn immunoreactivity in the neocortex, measured as the percentage of neuropil area stained in sections labeled with the CT-α-syn antibody. F, Analysis of the levels of α-syn immunoreactivity in the striatum, measured as the percentage of neuropil area stained in sections labeled with the CT-α-syn antibody. Error bars represent ± SEM. ***p < 0.001 when comparing non-tg 27-1-immunized mice to α-syn tg 27-1-immunized mice; #p < 0.05; ##p < 0.01; ###p < 0.001 comparing α-syn tg mice immunized with 27-1 with α-syn tg mice immunized with 9E4, 1H7, or 5C1.
- Figure 4.
Binding of CT α-syn antibodies did not affect SYN105 immunoreactivity in α-syn tg mice. To determine whether CT α-syn antibody binding blocked the binding of the CT-truncated α-syn antibody SYN105, brain sections of non-immunized non-tg or α-syn tg mice were preincubated with 27-1, 9E4, 1H7, 5C1, or 5D12 antibodies (1:100) for 1 h and then immunostained with the CT-α-syn antibody SYN105. Representative images from the neocortex and striatum are shown. Scale bar, 15 μm.
- Figure 5.
Immunoblot analysis of brain extracts of passively immunized α-syn tg mice with antibodies against full-length and CT-truncated α-syn. To examine the effects of immunization on α-syn protein levels, immunoblot analysis of non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12 was performed. A, B, Protein immunoblots were probed with antibodies against total α-syn (BD), FL-α-syn, CT-α-syn (SYN105), and actin as a loading control. The 14 kDa band is monomeric α-syn and the 12 kDa band is CT-α-syn. Higher molecular weight bands correspond to different α-syn oligomers. C, Densitometric analysis of the α-syn immunoreactive bands as detected by the total α-syn antibody (BD). D, Densitometric analysis of the α-syn immunoreactive bands as detected by the FL-α-syn antibody. E, Densitometric analysis of the α-syn immunoreactive bands as detected by the CT-α-syn antibody (SYN105). Error bars represent ± SEM. (#) indicates p < 0.05 when comparing α-syn tg mice immunized with 27-1 to α-syn tg mice immunized with 9E4, 1H7, or 5C1.
- Figure 6.
Colocalization of the neurofilament marker SMI312 and the CT-α-syn antibody SYN105 was reduced in passively immunized α-syn tg mice. To determine whether passive immunization altered the subcellular distribution of CT-α-syn, double labeling of brain sections of non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12 was performed using a neurofilament marker (SMI312) and the CT-α-syn antibody SYN105. A, Representative confocal images of the neuropil in brain sections from non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12. Arrows indicate colocalization of the SMI312 signal with CT-α-syn in neurofilament-like structures. Scale bar, 10 μm. B, Analysis of the percentage of colocalization between the neurofilament marker SMI312 and the CT-α-syn antibody SYN105. Error bars represent ± SEM. ***p < 0.001 comparing non-tg 27-1-immunized mice with α-syn tg 27-1-immunized mice. ###p < 0.001 comparing α-syn tg mice immunized with 27-1 with α-syn tg mice immunized with 9E4, 1H7, or 5C1.
- Figure 7.
Passive immunization with CT α-syn antibodies improved synaptic pathology in α-syn tg mice. The effect of passive immunization with CT α-syn antibodies on synaptic markers was evaluated in the neocortex, striatum, and hippocampus of non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12. A, Brain sections were stained with an antibody against synaptophysin (red) or an antibody against MAP2 (green), and representative fluorescence images are shown. Arrows highlight synaptic loss in the hippocampus of α-syn tg mice. Scale bar, 250 μm. B, Analysis in neocortex of the percentage of area of the neuropil stained by the synaptophysin antibody. C, Analysis in striatum of the percentage of area of the neuropil stained by the synaptophysin antibody. D, Analysis in neocortex of the percentage of area of the neuropil stained by the MAP2 antibody. E, Analysis of the percentage of neuropil stained by the MAP2 antibody in the CA3 area of the hippocampus. F, Immunoblot analysis of the synaptic markers PSD-95 and synaptophysin. Actin was used as a loading control. G, Densitometric analysis of the PSD-95-immunoreactive bands. H, Densitometric analysis of the synaptophysin-immunoreactive bands. Error bars represent ± SEM. *p < 0.05 and ***p < 0.001 comparing non-tg 27-1-immunized mice with α-syn tg 27-1-immunized mice; #p < 0.05, ##p < 0.01, ###p < 0.001 comparing α-syn tg mice immunized with 27-1 to α-syn tg mice immunized with 9E4, 1H7, or 5C1.
- Figure 8.
Passive immunization with CT α-syn antibodies improved TH pathology and neuroinflammation in the striatum of α-syn tg mice. To determine whether immunization with CT α-syn antibodies rescued the loss of TH fibers in α-syn tg mice, brain sections of non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12 were immunostained with an antibody against TH. A, Representative images of the substantia nigra and the striatum of non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12 immunostained with an antibody against TH. Scale bar, 50 μm. B, Cell counts of TH-immunoreactive neurons in substantia nigra. C, Optical density analysis of TH immunoreactivity in striatum. D, To determine whether immunization with CT α-syn antibodies reduced neuroinflammation in α-syn tg mice, brain sections of non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12 were immunostained with an antibody against GFAP (astrocytes) or Iba1 (microglia). Representative images of the striatum are shown. Scale bar, 250 μm. E, Cell counts of GFAP-immunoreactive cells in striatum. F, Cell counts of Iba1-immunoreactive cells in striatum. Error bars represent ± SEM. **p < 0.01 and ***p < 0.001 comparing non-tg 27-1-immunized mice with α-syn tg 27-1-immunized mice; #p < 0.05, ##p < 0.01, ###p < 0.001 comparing α-syn tg mice immunized with 27-1 with α-syn tg mice immunized with 9E4, 1H7, or 5C1.
- Figure 9.
Passive immunization with CT α-syn antibodies improved behavioral deficits on α-syn tg mice. The effect of immunization with CT α-syn antibodies in learning and memory and motor performance was analyzed by water maze and the round beam test. A, Performance in the water maze (distance to the platform) with the platform submerged in non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12. B, Probe test performance, measured as the time spent in correct quadrant by non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12. C, Performance in the transversal round beam test, measured as slips per 10 cm in non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12. D, Average body weight in grams of non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12. Error bars represent ± SEM. *p < 0.05 and ***p < 0.001 comparing non-tg 27-1-immunized mice with α-syn tg 27-1-immunized mice; ##p < 0.01; ###p < 0.001 comparing α-syn tg mice immunized with 27-1 to α-syn tg mice immunized with 9E4, 1H7, or 5C1; &p < 0.05 comparing α-syn tg 9E4-immunized mice with α-syn tg 5D12-immunized mice.
- Figure 10.
Effect of CT α-syn antibodies on calpain-1 levels and activity and α-syn-dependent release of calpain-1 to the extracellular compartment. To investigate whether changes in CT-α-syn levels were associated with changes in enzymatic α-syn CT truncation, calpain-1 levels and activity were analyzed. A, Calpain-1 levels were measured by immunoblot in brains of non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12. Error bars represent ± SEM. B, As a control, calpain-1-mediated spectrin degradation was measured in brains of non-tg mice immunized with 27-1 or α-syn tg mice immunized with 27-1, 9E4, 1H7, 5C1, or 5D12. Results are expressed as the densitometry ratio between degraded spectrin (≈150 kDa) and nondegraded spectrin (240 kDa). Error bars represent ± SEM. C, Activity of calpain-1 was measured by analyzing the digestion rate of 1 μg of recombinant FL-α-syn using 0.2 U of calpain-1 and results were visualized by immunoblot. Control experiments included incubation with the calpain-1 inhibitor Calpeptin at 40 μm. The 14 kDa band is monomeric α-syn and the 12 kDa band is CT-α-syn. D, Location of α-syn and calpain-1 and their release to the extracellular medium was analyzed in B103 cells 0, 24, 48, and 72 h after infection with a lentiviral construct expressing human FL-α-syn. Protein immunoblots were probed with antibodies against FL-α-syn, CT-α-syn (SYN105), and calpain-1 and actin was used as a loading control. The 14 kDa band is monomeric FL-α-syn and the 12 kDa band is CT-α-syn.
- Figure 11.
Passive immunization with CT α-syn antibodies reduced propagation of FL-α-syn and CT-α-syn in vitro. A, Schematic representation of FL-α-syn, CT-α-syn, and the expression vectors used for lentiviral transfection. The calpain-1 cleavage site is also depicted. B, To analyze the correct expression of FL-α-syn or CT-α-syn by transfected B103 cells, α-syn levels were measured by immunoblot. Digestion of recombinant α-syn by calpain-1 was used as a molecular weight control. The higher-molecular-weight band represents FL-α-syn and the lower-molecular-weight band represents CT-α-syn. Both proteins are expressed at comparable levels. C, Schematic representation of the in vitro α-syn propagation assay, where red represents B103 cells expressing α-syn and growing in the insert (donors) and green represents B103 cells expressing GFP and growing in coverslips (recipients). α-syn propagation can be analyzed after 24 h of coincubation by measuring the colocalization between red and green fluorescence in recipient cells. D–I, To analyze the effect of CT antibodies on α-syn propagation, B103 cells expressing FL-α-syn or CT-α-syn were plated in cell culture inserts and B103 cells expressing GFP were plated in coverslips. Cultures were combined so that the α-syn-expressing cells (donor) were growing on an insert separated from the GFP-expressing cells (recipient) plated on a coverslip next to the 0.4 μm membrane. The antibodies 27-1, 9E4, 1H7, 5C1, or 5D12 were added to the incubation medium (1:100) and propagation was measured 24 h later. D, Schematic representation of FL-α-syn and the epitope location of antibodies. E, Colocalization images of FL-α-syn (red) with GFP (green) in recipient B103 cells. Scale bar, 10 μm. F, Percentage of colocalization between FL-α-syn and GFP. G, Schematic representation of CT-α-syn and the epitope location of antibodies. The recognition site of 9E4, 5C1, and 5D12 is located downstream of the truncation site of α-syn. H, Colocalization images of CT-α-syn (red) with GFP (green) in recipient B103 cells. Scale bar, 10 μm. I, Percentage of colocalization between CT-α-syn and GFP. Error bars represent ± SEM. *p < 0.05 and ***p < 0.001 comparing 27-1-treated cells with cells treated with 9E4, 1H7, or 5C1 antibodies.
- Figure 12.
Biological consequences of blocking α-syn CT truncation by passive immunotherapy. A, Schematic representation of a neuron overexpressing α-syn in the α-syn tg mouse model. α-syn oligomers are released to the extracellular environment, together with calpain-1. Extracellular α-syn oligomers can propagate to other neurons and glial cells. Furthermore, α-syn oligomers can also be cleaved by calpain-1, generating extracellular CT-α-syn, which is more prone to aggregation and subsequent neurotoxicity. B, In tg animals immunized with CT α-syn antibodies, propagation of extracellular α-syn oligomers is inhibited and α-syn is protected from CT truncation. The rate of α-syn aggregation is thus diminished and α-syn oligomers can be effectively directed toward clearance pathways.
