Fig. 4: rAAV vector-mediated expression of hu-α-Syn in the dorsal hippocampus leads to sustained overexpression of pK-resistant α-Synuclein.
a Schematic representation of the experimental design for the short-term and long-term experiments, including behavioral testing starting 4- or 24-weeks after intra-brain injection and lasting for about 4 weeks. After behavioral testing, animals were sacrificed for immunohistochemical, biochemical and ex-vivo electrophysiological analysis (for the short-term time point). b Representative sagittal brain section (mosaic reconstruction) of the injection placement of rAAV-GFP (left panel) and rAAV-hu-α-Syn (right panel) vectors, showing a wide distribution of GFP and hu-α-Syn in the whole hippocampus (Scale bar: 1 mm). c Representative confocal images showing α-Syn (green) and p-α-Syn (magenta) expression in the CA1 of the HP injected with rAAV-hu-α-Syn or r-AAV-GFP, in basal immunostaining conditions (pK-) and after the treatment with proteinase K (pK+), for mice sacrificed at 8 weeks (ST) or 28 weeks (LT) after intra-brain injection (Scale bar: 50 μm). Confocal images at the bottom show undetectable α-Syn (green) and p-α-syn (magenta) expression in the CA1 of the HP of an rAAV-GFP mouse sacrificed at 28 weeks after injection, under basal conditions and after treatment with pK (Scale bar: 50 μm). Quantitation of the number of p-α-Syn positive cells (p-α-Syn+ cell/mm2) showed a significant effect for vector injection in the ST experiment (Injection, F(1,14) = 29.244, P < 0.0001, pK treatment, F(1,14) = 0.890, P = 0.3615, Injection × pK treatment, F(1,14) = 0.811, P = 0.3830), and a significant difference between rAAV-GFP and rAAV hu-α-Syn pK- (unpaired t test, t = 2.978, P = 0.0247, rAAV-GFP, n = 3; rAAV-hu-α-Syn, n = 5) and pK+ (unpaired t test, t = 6.789, P = 0.0001, rAAV-GFP, n = 5; rAAV-hu-α-Syn, n = 6). A similar effect was found in the LT experiment (Injection, F(1,16) = 12.433, P = 0.0028, pK treatment, F(1,16) = 0.728, P = 0.4062, Injection × pK treatment, F(1,16) = 0.022, P = 0.8846), again with a significant difference between rAAV-GFP and rAAV hu-α-Syn pK- (unpaired t test, t = 2.448, P = 0.0401, rAAV-GFP, n = 4; rAAV-hu-α-Syn, n = 6) and pK+ (Mann–Whitney test, P = 0.0095, rAAV-GFP, n = 4; rAAV-hu-α-Syn, n = 6). *P < 0.05 vs. HP-GFP pK-. $P < 0.05 vs. HP-GFP pK+. d, e Dot-blot analysis of hu-α-Syn (unpaired t test, t = −4.81, P = 0.0004, rAAV-GFP, n = 7; rAAV-hu-α-Syn, n = 7) and pan-α-Syn expression (unpaired t test, t = −2.58, P = 0.023, rAAV-GFP, n = 7; rAAV-hu-α-Syn, n = 7) in hippocampal tissue of HP-hu-α-Syn and HP-GFP mice. Data are presented as mean ± SEM. *P < 0.01 different from HP-GFP.
