Passive immunization reduces behavioral and neuropathological deficits in an alpha-synuclein transgenic model of Lewy body disease

Abstract

Dementia with Lewy bodies (DLB) and Parkinson's Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (α-syn). This study investigated whether passive immunization with a novel monoclonal α-syn antibody (9E4) against the C-terminus (CT) of α-syn was able to cross into the CNS and ameliorate the deficits associated with α-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved α-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display α-syn accumulation and promotes α-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of α-syn may be of therapeutic relevance in patients with PD and DLB.

Figure 1. Plasma antibody titers and effects of passive immunization on motor behavior in passively immunized α-syn tg mice.

(A) Antibody titers determined by ELISA in non-tg or α-syn tg mice immunized with the C-terminal antibody (9E4) or IgG1 controls. Horizontal lines represent the mean of the data, whilst the points represent the spread of the individuals in each group. To examine the effects of immunization with the 9E4 antibody on motor behavior in the α-syn tg, mice were tested in the rotarod and pole tests. (B) Pole test performance (time taken to traverse pole) by non-tg mice or α-syn tg mice immunized with IgG1 or 9E4. N = 20 mice per group; 12 month old. Error bars represent mean ± SEM. (C) Rotarod performance (time spent on rotating rod) by non-tg mice or α-syn tg mice immunized with IgG1 or 9E4. Error bars represent mean ± SEM. When analyzing rotarod results (*) indicates p<0.05, when comparing α-syn tg immunized with IgG1 to non-tg group by repeated-measures two-way ANOVA and (#) indicates p<0.05, when comparing α-syn tg mice immunized with 9E4 to IgG1 immunized α-syn tg mice using repeated-measures two-way ANOVA. When analyzing pole test results (*) indicates p<0.05, when comparing α-syn tg immunized with IgG1 to non-tg group by one-way ANOVA with post hoc Dunnett's and (#) indicates p<0.05, when comparing α-syn tg immunized with 9E4 to IgG1 immunized α-syn tg mice by one-way ANOVA with post hoc Dunnett's.

Figure 2. Effects of passive immunization on behavioral performance in the water maze in passively immunized α-syn tg mice.

(A) Performance in the water maze (distance taken to locate platform) during training with the cued platform (days 1–3) and with the platform submerged (days 4–7) in non-tg mice or α-syn tg mice immunized with IgG1 or 9E4. (B) Performance in the water maze (time to locate the platform) during training with the cued platform (days 1–3) and with the platform submerged (days 4–7) in non-tg mice or α-syn tg mice immunized with IgG1 or 9E4. (C) Probe test performance (time spent in correct quadrant) (day 8) in non-tg mice or α-syn tg, mice immunized with IgG1 or 9E4. N = 20 mice per group; 12 month old. Error bars represent mean ± SEM. (D) Representative images of the swim paths of animals from each group when the platform was visible (Day 3, trial 4, red box indicates the location of the platform), hidden (Day 7, trial 4, red box indicates the location of the platform which is now submerged below the opaque surface) or absent (Day 8, Probe Test). When analyzing the water maze results (*) indicates p<0.05, when comparing α-syn tg immunized with IgG1 to non-tg IgG1 group by repeated-measures two-way ANOVA and (#) indicates p<0.05, when comparing α-syn tg immunized with 9E4 to IgG1 immunized α-syn tg mice by repeated-measures two-way ANOVA. When analyzing probe test results (*) indicates p<0.05, when comparing α-syn tg immunized with IgG1 to non-tg IgG1 group by one-way ANOVA with post hoc Dunnett's and (#) indicates p<0.05, when comparing α-syn tg immunized with 9E4 to IgG1 immunized α-syn tg mice by one-way ANOVA with post hoc Dunnett's.

Figure 3. Analysis of the effects of passive immunization on synaptic structure and markers in α-syn tg animals.

The effect of immunization with the 9E4 antibody on synaptic markers was evaluated in the temporal cortex of non-tg and α-syn tg mice by electron microscopy and immunoblot analysis. Representative electron micrographs are from the temporal cortex layers 5–6 obtained at 15,000 X. (A) non-tg mice immunized with IgG1 control. (B) non-tg mice immunized with 9E4. (C) α-syn tg mice immunized with the IgG control. (D) α-syn tg mice immunized with the 9E4 antibody. (E, F) Image analysis of the numbers of post-synaptic densities (PSD) and mean presynaptic terminal diameters respectively. (G) Representative immunoblot for PSD95, a postsynaptic marker and Synapsin I, a presynaptic marker, in non-tg mice or α-syn tg mice immunized with the IgG control or the 9E4 antibody. (H, I) Analysis of the levels of PSD95 and Synapsin I immunoreactive bands respectively. N = 20 mice per group; 12 month old. Error bars represent mean ± SEM. (*) indicates p<0.05, when comparing IgG1-immunized α-syn tg mice to IgG1-immunized non-tg mice and (#) indicates p<0.05 when comparing α-syn tg mice immunized with 9E4 to IgG1 immunized α-syn tg mice using one-way ANOVA with post hoc Dunnett's.

Figure 4. Comparative immunohistochemical analysis with antibodies against full length or calpain-cleaved a-syn in passively immunized a-syn tg mice.

To examine the effects of immunization on α-syn accumulation, immunohistochemical analysis using antibodies against FL-α-syn (layers 5–6) and CC-α-syn was conducted. Panels illustrate laser scanning confocal images of the temporal cortex and hippocampus (CA3) immunolabeled with antibodies against FL and CC α-syn immunoreactivity. (A, C) Temporal cortex of IgG1-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against FL-α-syn, respectively. (B, D) Temporal cortex of 9E4-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against FL-α-syn, respectively. (E, G) Hippocampus of IgG1-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against FL-α-syn, respectively. (F, H) Hippocampus of 9E4-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against FL-α-syn, respectively. (I) Image analysis of the numbers of neocortical α-syn immunoreactivity neurons with the FL α-syn antibody. (J) Analysis of the levels of α-syn immunoreactivity in the neuropil in the neocortex in sections labeled with the FL α-syn antibody. (K) Image analysis of the numbers of hippocampal α-syn immunoreactivity neurons with the FL α-syn antibody. (L) Analysis of the levels of α-syn immunoreactivity in the neuropil in the hippocampus in sections labeled with the FL α-syn antibody. (M, O) Temporal cortex of IgG1-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against CC α-syn, respectively. (N, P) Temporal cortex of 9E4-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against CC α-syn, respectively. (Q, S) Hippocampus of IgG1-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against CC α-syn, respectively. (R, T) Hippocampus of 9E4-immunized non-tg and α-syn tg mouse immunolabeled with an antibody against CC α-syn, respectively. (U) Image analysis of the numbers of neocortical α-syn immunoreactivity neurons with the CC α-syn antibody. (V) Analysis of the levels of α-syn immunoreactivity in the neuropil in the neocortex in sections labeled with the CC α-syn antibody. (W) Image analysis of the numbers of hippocampal α-syn immunoreactivity neurons with the CC α-syn antibody. (X) Analysis of the levels of α-syn immunoreactivity in the neuropil in the hippocampus in sections labeled with the CC α-syn antibody. Scale bar  = 30 µM. N = 20 mice per group; 12 month old. Error bars represent mean ± SEM. (*) indicates p<0.05, when comparing IgG1-immunized α-syn tg mice to IgG1-imunized non-tg mice by one-way ANOVA with post hoc Dunnett's. (#) Indicates p<0.05, when comparing α-syn tg mice immunized the 9E4 α-syn antibody to IgG1-treated α-syn tg mice by one-way ANOVA with post hoc Dunnett's.

Figure 5. Immunoblot analysis with antibodies against full length and calpain-cleaved a-syn in passively immunized α-syn tg mice.

To evaluate the effects of immunization on α-syn accumulation, immunoblot analysis using antibodies against FL-αsyn and CC-α-syn was conducted. (A) Representative immunoblot with anti-FL α-syn of the soluble fraction from non-tg and α-syn tg mice immunized with IgG1 control or 9E4. (B, C) Analysis of the levels of the α-syn immunoreactive bands corresponding to the monomer and oligomers respectively, as detected by the FL α-syn antibody in the soluble fraction. (D) Representative immunoblot with anti-FL α-syn of the insoluble fraction from non-tg and α-syn tg mice immunized with IgG1 control or 9E4. (E, F) Analysis of α-syn monomer or oligomer levels respectively, detected by the FL α-syn antibody in the insoluble fraction. (G) Representative immunoblot with anti-CC α-syn of the soluble fraction from non-tg and α-syn tg mice immunized with IgG1 control or 9E4. (H, I) Analysis of α-syn monomer or oligomer levels respectively detected by the CC α-syn antibody in the soluble fraction. (J) Representative immunoblot with anti-CC α-syn of the insoluble fraction from non-tg and α-syn tg mice immunized with IgG1 control or 9E4. (K, L) Analysis of α-syn monomer or oligomer levels respectively, detected by the CC α-syn antibody in the insoluble fraction. N = 20 mice per group; 12 month old. Error bars represent mean ± SEM. (*) indicates p<0.05, when comparing IgG1-immiunized α-syn tg mice with IgG1-immunized non-tg mice using one-way ANOVA with post hoc Dunnett's. (#) indicates p<0.05, when comparing α-syn tg mice immunized with 9E4 with IgG1 immunized α-syn tg mice using one-way ANOVA with post hoc Dunnett's.

Figure 6. Trafficking of the FITC-tagged α-syn 9E4 antibody in tg mice.

To investigate the distribution of the 9E4 antibody after passive immunization, the FITC tagged antibody was injected intravenously (IV) and analyzed by ELISA and confocal microscopy. (A) Antibody titers in the plasma and brain at 3, 14 and 30 days post-injection in mice immunized with the 9E4 antibody, determined by ELISA. (B) Image analysis of 9E4-FITC positive neurons in the α-syn tg mice at 3, 14 and 30 days post-injection. (C, D) Representative laser scanning confocal images of the signal in the FITC channel in the temporal cortex of α-syn tg mouse 30 days following intravenous IV injection with the FITC-tagged 9E4 antibody. Arrows highlight labeled intra-neuronal granular-like structures. (E) No signal is detected in the FITC channel in α-syn tg mouse 30 days following IV injection with the FITC-tagged IgG1 control antibody. (F) No signal in the FITC channel in non-tg mouse 30 days following IV injection with the FITC-tagged 9E4 antibody. (G) Confocal image of a section from an antibody-naive α-syn tg mouse immunolabeled with cerebrospinal fluid (CSF) from a mouse immunized with 9E4-FITC. (H) Confocal image of a section from non-tg mouse immunolabeled 9E4-FITC antibody. Scale bar (C, E–H)  = 50 µM; (D)  = 10 µM. N = 20 per group, 12 months of age. Error bars represent mean ± SEM.

Figure 7. Co-localization of the FITC-tagged α-syn 9E4 antibody with lysosomal and autophagosomal markers.

To analyze the sub-cellular distribution of the 9E4 antibody immunohistochemical and ultrastructural analysis was conducted in 9E4-FITC immunized α-syn tg mice. (A–C) Representative confocal image of a brain section from an α-syn tg mouse immunized 9E4-FITC and co-labeled with an antibody against α-syn. Arrows indicate co-localization of the 9E4-FITC signal with α-syn in neuronal granular-like structures. (D–F) Confocal image from an α-syn tg mouse immunized 9E4-FITC and co-labeled with an antibody against LC3. Arrows indicate co-localization of the 9E4-FITC with LC3 in neuronal autophagosome-like structures. (G–I) Confocal image from an α-syn tg mouse immunized 9E4-FITC and co-labeled with an antibody against cathepsin D. Arrows indicate co-localization of the 9E4-FITC with cathepsin-D in neuronal lysosomal-like structuresLC3. (J, K) Representative electron micrographs of sections from an α-syn tg mouse immunized with the 9E4 antibody and immunolabeld with gold-tagged anti-mouse antibody. (L, M) Electron micrographs of sections from an α-syn tg mouse immunized with the control IgG1 antibody and immunolabeld with gold-tagged anti-mouse antibody. (N, O) Representative electron micrographs of sections from a non-tg mouse immunized with the 9E4 antibody and immunolabeld with gold-tagged anti-mouse antibody. No reactivity is observed in lysosomes or autophagosomes. Scale bar (A–I)  = 10 µM; (J–O) magnification 25,000x.

Figure 8. Immunocytochemical analysis of the effects of passive immunization with 9E4 in markers of lysosomes and autophagy in α-syn tg mice.

To examine the sub-cellular distribution of the 9E4 antibody immunohistochemical and ultrastructural analysis was conducted in 9E4 immunized α-syn tg mice. (A) LC3 immunoreactivity in α-syn tg mouse immunized with IgG1 antibody. (B) LC3 immunoreactivity in α-syn tg mouse immunized with 9E4 antibody. (C) Analysis of LC3 immunoreactivity in α-syn tg mice immunized with IgG1 or 9E4 antibody. (D) Cathepsin-D immunoreactivity in α-syn tg mouse immunized with IgG1 antibody. (E) Cathepsin-D immunoreactivity in α-syn tg mouse immunized with 9E4 antibody. (F) Analysis of cathepsin-D immunoreactivity in α-syn tg mice immunized with IgG1 or 9E4 antibody. (G) CC α-syn immunoreactivity in α-syn tg mouse immunized with IgG1 antibody. (H) CC α-syn immunoreactivity in α-syn tg mouse immunized with 9E4 antibody. (I) Analysis of % area of CC α-syn immunoreactive neuropil in α-syn tg mice immunized with IgG1 or 9E4 antibody. (J–L) Co-localization of α-syn and cathepsin-D immunoreactivity in α-syn tg mouse immunized with 9E4 antibody. (M–O) Co-localization of α-syn and LC3 immunoreactivity in α-syn tg mouse immunized with 9E4 antibody. Scale bar (A, B)  = 30 µM (D, E, G and H)  = 20 µM, (J–O)  = 10 µM. (*) Indicates p<0.05, when comparing IgG1 to 9E4 group by unpaired Student's t test. Error bars represent mean ± SEM.

Figure 9. Immunoblot analysis of the effects of passive immunization with 9E4 in molecular components of the autophagy pathway in α-syn tg mice.

(A) Immunoblot analysis of mTor, Beclin 1, LC3, Atg 5, Atg 7, and Atg 10 protein immunoreactivity in α-syn tg mice that had been immunized with either the IgG1 control or 9E4 antibody. (B) Analysis of mTor, Beclin 1, LC3, Atg 5, Atg 7, and Atg 10 protein levels in α-syn tg mice that had been immunized with either the IgG1 control or 9E4 antibody. (*) Indicates p<0.05, when comparing IgG1 to 9E4 group by unpaired Student's t test. Error bars represent mean ± SEM.

Figure 10. The effects of the 9E4 monoclonal antibody on promotion of α-syn clearance via autophagy in a neuronal cell model.

(A) Baseline co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-control and treated with the IgG1 control antibody. (B) Baseline co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn and treated with the IgG1 control antibody. (C) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn and treated with the 9E4 antibody. (D) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-control, treated with the IgG1 control antibody and rapamycin, an inducer of autophagy. (E) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin, an inducer of autophagy. (F) Co-localization of α-syn and LC3-GFP in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA, an inhibitor of autophagy. (G) Analysis of α-syn immunoreactivity in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin. (H) Analysis of LC3-GFP signal in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and rapamycin. (I) Analysis of α-syn immunoreactivity in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA. (J) Quantitative analysis of LC3-GFP signal in neuronal cells infected with LV-α-syn, treated with the 9E4 antibody and 3MA. Scale bar (A–F)  = 10 µM (*) Indicates p<0.05 compared to LV-control infected and vehicle-treated cultures by one-way ANOVA with post-hoc Dunnett's test. (#) Indicates p<0.05 compared to LV-control infected and vehicle-treated cultures by one-way ANOVA with post-hoc Tukey-Kramer test. Error bars represent mean ± SEM.