PMC

The substantia nigra of three rats was transduced with AAV-sh[SNCA] on one side and AAV-sh[control] on the other (cohort 1). Twenty-one days later, contiguous midbrain sections were labeled by RNA in situ hybridization using antisense cRNA probes to Snca, Sncb, and Sncg; by a sense control probe; or by immunofluorescence for TH to label dopaminergic neurons. (A) Low-power micrographs of the midbrain. Arrows indicate the position of the substantia nigra, and arrowheads indicate punch marks labeling the side that received AAV-sh[SNCA]. Scale bar (for all panels): 1 mm. (B) The substantia nigra on each side of the corresponding section is shown at higher magnification. Specific loss of the Snca transcript is seen after transduction with AAV-sh[SNCA] but not AAV-sh[control]. Scale bar (for all panels): 100 μm.

(A) siRNAs targeting Snca were identified and tested in vitro (). The most effective of these, siRNA526, is shown in red. Multiple base mismatches between siRNA526 and the corresponding sequences of Sncb and Sncg (encoding β- and γ-synucleins, respectively) are colored green. (B) A variety of viral vectors was evaluated for in vivo gene transfer to the rat substantia nigra (). An adeno-associated virus serotype 2 (AAV2) vector expressing a GFP reporter gene showed extensive transduction of TH⁺ nigral dopaminergic neurons (upper panels), with resulting GFP expression in their striatal terminals (lower panels). Scale bars: 100 μm. (C) An AAV2 vector, AAV-sh[SNCA], was constructed to express shRNA526 targeting Snca from the U6 promoter and a GFP reporter from a separate expression cassette. The control vector AAV-sh[control] was isogenic to AAV-sh[SNCA], except that it expressed a nontargeting shRNA instead of shRNA526.

Animals from cohort 6 received either AAV-sh[SNCA] (black squares) or AAV-sh[control] (gray circles) unilaterally in the substantia nigra or no vector (white triangles). Starting at 21 days after transduction, rats were administered rotenone 2.8 mg/kg/d via intraperitoneal injection for 6 days, after which brains were harvested for analysis. (A) A postural instability test was used to evaluate forelimb motor function during rotenone administration. Mean ± SEM distance to trigger a compensatory forelimb movement is shown for the right forepaw (controlled by nontransduced side of brain; left graph) and left forepaw (controlled by vector-transduced side of brain; right graph). ***P < 0.001, ****P < 0.0000001, left forepaw of AAV-sh[SNCA] group versus left forepaw of AAV-sh[control] or non-vector groups, one-way ANOVA. (B and C) Once motor asymmetry was clearly established in the AAV-sh[SNCA] group after 6 days of rotenone exposure, brains were analyzed for striatal dopaminergic terminal integrity. Quantitative near-infrared immunofluorescence was used to measure dorsolateral striatal TH expression on each side of 5–6 sections per animal. Small markers show the mean for each animal (+, vector side; –, nontransduced control side; lines join the means for the two sides of each brain); large markers show mean ± SEM for all eight animals in each group.

Animals from cohorts 2 (A–E) and 3 (F and G) were analyzed for changes in the nigrostriatal system following unilateral α-synuclein knockdown. Measurements from the nontransduced (white triangles), AAV-sh[control]–transduced (gray circles), and AAV-sh[SNCA]–transduced (black squares) sides of each brain are shown. In A, C, and E–G, small markers show data points from each individual animal (the two sides of each brain are connected by a line); large markers show the group mean ± SEM. (A) Unbiased stereology was employed to quantify the number of nigral dopaminergic neurons on each side of the brain. (B and C) TH expression was measured in nigral dopaminergic neurons by confocal microscopy. In B, measurements for individual neurons are shown normalized to nontransduced cells in the same sections (small markers). Large markers show mean ± SEM for each animal. (D and E) Striatal TH expression was measured using quantitative near-infrared immunofluorescence. In D, measurements in individual sections are shown normalized to the nontransduced side of the same section (small markers). Large markers show mean ± SEM for each animal. (F and G) HPLC was employed to quantify dopamine levels (F) and the ratio of dopamine metabolites to dopamine (G) as an index of dopamine turnover. Data were analyzed using 2-tailed paired t tests comparing the vector-transduced and control sides of each brain; *P < 0.05.

The integrity of striatal dopaminergic terminals was analyzed after rotenone exposure in cohort 4 (unilateral vector transduction, E and F) and cohort 5 (bilateral vector transduction, A–D). (A) Near-infrared immunofluorescence scan, showing TH expression (green) in a coronal section of the forebrain; the plane of the section is indicated in the inset. The white arrow shows loss of TH signal in the dorsolateral striatum on the AAV-sh[control] side; no lesion was seen on the AAV-sh[SNCA] side. Scale bar: 1 mm. (B) Forebrain sections were immunolabeled for TH expression (brown). The micrographs show the dorsolateral striatum on each side of brains from cohort 1 (upper panels; bilateral vector transduction, no rotenone) and cohort 5 (lower panels; bilateral vector transduction, post-rotenone). Scale bar (for all four panels): 100 μm. (C–F) Dorsolateral striatal TH signal was measured by quantitative near-infrared immunofluorescence. (C) Cohort 5: striatal TH signal on the AAV-sh[SNCA] side of each section is shown as a percentage of the signal measured on the AAV-sh[control] side of the same section. Small markers show individual sections; large markers show mean ± SEM for each animal. (D, E, and F) Mean striatal TH signal is shown for cohorts 5 (D) and 4 (E and F). The two sides from each animal are shown as small markers connected by lines; large markers show group mean ± SEM. **P < 0.01, ***P < 0.001, AAV-sh[SNCA] side versus contralateral side, 2-tailed paired t test.

The number of substantia nigra dopaminergic neurons remaining after chronic rotenone exposure was determine in cohorts 4 (unilateral vector transduction) and 5 (bilateral vector transduction). (A) Confocal micrographs show the substantia nigra from a representative midbrain section derived from a rat that received AAV-sh[control] on one side (left panel) and AAV-sh[SNCA] on the other (right panel) prior to chronic rotenone exposure. There was striking loss of dopaminergic neurons on the AAV-sh[control] side, as expected following chronic rotenone exposure using this regimen. In contrast, the AAV-sh[SNCA] side showed evidence of neuroprotection. Scale bar: 200 μm. (B–D) Unbiased stereology was used to determine the total number of remaining substantia nigra dopaminergic neurons on each side of the brain in cohorts 4 (unilateral vector transduction; C and D) and 5 (bilateral vector transduction; B). The stereological count for each individual animal is shown (small markers; data for the two sides of each brain are connected by a line). Large markers show the group mean ± SEM. White triangles, nontransduced side; gray circles, AAV-sh[control]–transduced side; black squares, AAV-sh[SNCA]–transduced side. **P < 0.01, 2-tailed paired t test, AAV-sh[SNCA] side versus contralateral side.

The processes of substantia nigra dopaminergic neurons remaining after chronic rotenone exposure were evaluated in cohorts 4 (unilateral vector transduction; C and D) and 5 (bilateral vector transduction; A and B). (A) An automated tracing algorithm was used to identify TH-immunoreactive processes (white) and cell bodies (red). The pictures show two sides of the same section, from an animal that received AAV-sh[control] on one side (upper panel) and AAV-sh[SNCA] on the other (lower panel). The dendritic arbors of surviving dopaminergic neurons appeared compromised on the AAV-sh[control] side and relatively preserved on the AAV-sh[SNCA] side. Scale bar: 100 μm. (B–D) The total length of TH-immunoreactive processes per surviving TH⁺ neuron was measured on each side of the brain in animals from cohorts 5 (B) and 4 (C and D). Small markers show the mean for each individual animal (data for the two sides of each brain are connected by a line); large markers show group mean ± SEM. **P < 0.01, 2-tailed paired t test, AAV-sh[SNCA] side versus contralateral side.

Two groups of 5 rats received either AAV-sh[SNCA] or AAV-sh[control] unilaterally in the substantia nigra (cohort 2). Forty-two days later, midbrain sections were immunolabeled for TH (green), α-synuclein (red), and DAPI (blue). Measurements of α-synuclein immunoreactivity were taken from nontransduced (white triangles), AAV-sh[control]–transduced (gray circles), or AAV-sh[SNCA]–transduced (black squares) substantia nigra dopaminergic neurons. (A and B) High-magnification confocal images of the substantia nigra from (A) no-vector and (B) AAV-sh[SNCA]–transduced sides of a single section. The positions of dopaminergic neuronal outlines revealed by TH labeling are shown as white dotted lines in the α-synuclein channel. Scale bars: 10 μm. (C) Cytoplasmic α-synuclein immunoreactivity was quantified in 60–100 nigral dopaminergic neurons on each side of each section by confocal imaging. The small markers show α-synuclein immunofluorescence signal for each cell on the vector-transduced side expressed as percentage of the mean value for dopaminergic neurons on the nontransduced side of the same section. The large markers show mean ± SEM for each animal. (D) Mean dopaminergic neuron α-synuclein immunofluorescence is shown for each side of the brain in each animal (+, vector side; –, nontransduced side; lines join the means for the two sides of each brain). The left graph shows measurements from animals that were transduced with AAV-sh[control] and the right AAV-sh[SNCA]. Large markers show mean ± SEM for all five animals in each group.*P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed paired t test, vector transduced side versus control side.

Animals from cohort 2 (unilateral AAV-sh[SNCA] or AAV-sh[control] into the substantia nigra) were analyzed for phenotypes attributable to α-synuclein knockdown. (A) The rats’ weights were measured 7 days prior to stereotactic vector inoculation and then daily for 42 days thereafter. Data points show mean ± SEM. (B) A postural instability test was used to evaluate forelimb motor function. For each forelimb, the displacement of the rat necessary to provoke a compensatory forelimb movement was measured (see Methods). The ratio of this distance between the left and right forelimbs was determined at each time point shown. Data points show mean ± SEM for the AAV-sh[control] (gray circles) and AAV-sh[SNCA] (black squares) groups; the line shows ratio = 1 that would indicate perfectly symmetric motor function. Pre, before surgery. (C and D) Spontaneous exploratory behavior in a transparent cylinder was evaluated as a second test for forelimb motor function. (C) Rearing movements resulting in a unilateral forelimb contact with the cylinder wall were counted in each 5-minute time period. (D) The number of wall contacts made by the left forepaw was calculated as a percentage of the total unilateral forepaw contacts. Data points show mean ± SEM for the AAV-sh[control] (gray circles) and AAV-sh[SNCA] (black squares) groups. The line in D shows a value of 50% that would indicate perfectly symmetric motor function.

Animals in cohort 4 received either AAV-sh[SNCA] (black squares) or AAV-sh[control] (gray circles) unilaterally in the substantia nigra, or no vector (white triangles). Starting at 21 days after transduction, rats were administered rotenone 2.8 mg/kg/d via intraperitoneal injection. (A) Weights were measured daily during rotenone administration. Data points show mean ± SEM expressed as percentage of initial starting weight for each animal. There were no significant differences in weight loss between the three groups. (B) Survival curve for time to predefined end points in the three experimental groups. (C) The postural instability test was used to evaluate forelimb motor function during rotenone administration. Mean ± SEM distance to trigger a compensatory forelimb movement is shown for the right forepaw (controlled by nontransduced side of brain; left graph) and left forepaw (controlled by vector-transduced side of brain; right graph). (D) Spontaneous exploratory behavior in a transparent cylinder was evaluated as a second test of forelimb motor function during rotenone administration. Mean ± standard error number of rearing movements is shown for each group. The inset panel shows rearing movements with unilateral wall contacts made by the right or left forelimbs after 8 days of rotenone exposure. **P < 0.01, ***P < 0.005, ****P < 0.000001, AAV-sh[SNCA] animals versus other groups, 1-way ANOVA with Tukey’s post hoc test.