Beclin 1 Gene Transfer Activates Autophagy and Ameliorates the Neurodegenerative Pathology in α-Synuclein Models of Parkinson's and Lewy Body Diseases

Accumulation of α-syn in autophagic structures in neuronal cells infected with LV-α-syn. A, B103 cells were infected with the LV-α-syn and examined by immunofluorescence for α-syn (red, arrows) and tubulin (green). Scale bar, 5 μm. B–D, Neuronal cells infected with LV-α-syn (B, C) or LV-β-syn (D) were examined by electron microscopy showing electrodense autophagic vesicles (arrows). n, Nucleus; m, mitochondria. Scale bars, 1 μm (B–D). E, The total number of electrodense AVs was counted per cell and compared with control-infected B103 cells. F, Total protein from LV-infected B103 cells was extracted and examined for total α-syn accumulation by Western blot.

Confocal analysis of the alterations in autophagy in neuronal cells infected with LV-α-syn and LC3-GFP. The B103 cells were infected with lentivirus expressing the LC3-GFP fusion protein and kept under baseline (serum fed) (A, D) or starvation (serum starved) (B, E) for 12 h or treated with rapamycin (C, F) for 6 h and then visualized for GFP by confocal microscopy. A–F, Neuronal cells were infected with LV-LC3-GFP and LV-control (A–C) or LV-α-syn (D–F). G, H, Image analysis of serial confocal frames to determine the average number of LC3-GFP grains below or above 1 μm in diameter. Serum fed (baseline) LC3-GFP granules (arrows) were quantified and compared with serum starved (starvation) or rapamycin treated. Scale bar, 10 μm. *Statistical significance compared with baseline. ^#Statistical significance compared with control-treated cells (p < 0.05, 1-way ANOVA, post hoc Fisher test).

Overexpression of beclin 1 from the lentivirus reduces levels of α-syn and restores neurite outgrowth. B103 neuronal cells were infected with LV-α-syn alone or in combination with LV-Beclin 1 and analyzed by immunoblot, PCR, and confocal analysis. A, Total protein was extracted and examined by Western blot for α-syn, phosphorylated (Ser129) α-syn, and beclin 1. B, Analysis of levels of α-syn immunoreactivity. C, Total RNA was extracted and examined by real-time PCR for expression of α-syn mRNA. D–G, Neuronal cells were infected with LV-α-syn alone or in combination with LV-Beclin 1 and double labeled with antibodies against α-syn (red) and tubulin III (green). H, I, Analysis of the levels of α-syn and tubulin III immunoreactivity from digital images acquired by confocal microscopy. J, Computer-aided image analysis of neurite length. Scale bar, 10 μm. *Statistical significance (p < 0.05, 1-way ANOVA, post hoc Fisher test).

Laser-scanning confocal imaging of the effects LV-Beclin 1 expression in neuronal cells expressing LC3-GFP and α-synuclein and treated with modulators of the autophagy pathway. B103 neuronal cells were infected with LV-α-syn alone or with LV-Beclin 1 and then further infected with LV-LC3-GFP and treated with blockers or activators of the autophagy pathway. A–D, Confocal images of vehicle control group (A); effects of 3-MA (10 mm) (B); effects of bafilomycin A1 (200 nm) (C); and effects of rapamycin (200 nm) (D). Average numbers of LC3-GFP grains per cell and levels of α-syn immunofluorescence were analyzed using confocal images and the ImageQuant system. E–H, Vehicle control group (E); effects of 3-MA (10 mm) (F); effects of bafilomycin A1 (200 nm) (G); and effects of rapamycin (200 nm) (H).*Statistical significance compared with uninfected control cultures; ^#statistical significance compared with untreated LV-α-syn-infected cultures (p < 0.05, 1-way ANOVA, post hoc Fisher test).

Immunoblot analysis of the effects of LV-Beclin 1 expression on LC3 II formation and α-syn accumulation in cells treated with modulators of autophagy. B103 neuronal cells were infected with LV-α-syn alone or with LV-Beclin 1 and then were treated with blockers or activators of the autophagy pathway. A–C, Representative Western blots with antibodies against LC3, α-syn, and actin. A–I, Effects of 3-MA (A, D, G), bafilomycin-A1 (B, E, H), or rapamycin (C, F, I). D–F, Levels of LC3 protein were estimated as a ratio of LC3-II to actin levels. G–I, Levels of α-syn immunoreactivity were estimated as a ratio to levels of actin. *Statistical significance compared with uninfected control cultures; ^#statistical significance compared with untreated LV-α-syn-infected cultures (p < 0.05, 1-way ANOVA, post hoc Fisher test).

Lentivirus-mediated delivery of beclin 1 to α-syn tg mice reduces α-syn accumulation. Immunocytochemical and confocal analysis 3 months after injection. A, B, Levels of beclin 1 immunoreactivity in α-syn tg mice (line D) that were injected with LV-control. C, D, Beclin 1 immunoreactivity in α-syn tg mice treated with LV-Beclin 1. Arrows (A, C) indicate injection tract in the neocortex. Low-power images of the brain (A, C) were taken with a 5× objective, and higher-magnification images (B, D) were taken with a 40× objective. E, Immunoblot analysis of brain tissue from the injection site to confirm expression of beclin 1. F–N, Confocal analysis of sections double immunolabeled with antibodies against α-syn (red) and beclin 1 (green). F–H, α-syn tg mice that were injected with LV-control. I–N, Colocalization of α-syn and beclin 1 in discrete granular structures in tg mice injected with LV-Beclin 1. Samples were further stained with DAPI to visualize nuclei (n) (blue). Arrows indicate areas of beclin 1 and α-syn colocalization. Scale bar, 5 μm.