Amphetamine-induced rotational behavior and HPLC analysis of striatal DA. A.

<p>Mean number of ipsilateral and contralateral turns made by each group over the 30-minute test period following an IP injection of 2 mg/kg amphetamine. The data for average number of ipsilateral and contralateral turns made per group were compared using a 2-tailed t-test (mCherry/saline n = 12, mCherry/MPTP n = 9, Pgc-1α/saline n = 12, Pgc-1α/MPTP n = 10). All relevant statistically significant comparisons are indicated on the graph. <b>B.</b> Box and whiskers plot of net ipsilateral turns per minute per group averaged across each group. Data were analyzed by one-way ANOVA, all relevant statistically significant comparisons are shown on the graph. <b>C.</b> Mean levels of striatal DA per mg of protein in the ipsilateral and contralateral (cntrl) striatum of each treatment group. Data were analyzed by paired t-test. (mCherry/saline n = 14, mCherry/MPTP n = 15, Pgc-1α/saline n = 15, Pgc-1α/MPTP n = 16) <b>D.</b> DA turnover in each treatment group. Data were analyzed by paired t-test for within group comparisons and Student’s t-test for between group comparisons.</p

Analysis of markers of the dopaminergic phenotype. A.

<p>SYBR-green PCR analysis of mRNA levels of <i>Th, Dat</i> and <i>Vmat2</i> in mCherry and Pgc-1α overexpressing SN samples (mCherry n = 6, Pgc-1α n = 5). <b>B.</b> Upper panel. SYBR-green PCR analysis of mRNA levels of <i>Gdnf</i> and <i>Bdnf</i> in Pgc-1α SN samples. Fold-change in gene expression was calculated with respect to the contralateral SN using the ΔΔCt method with normalization to 18S. Data were analyzed using a one-sample t-test to determine if fold-change differed from the baseline value of 1 (n = 6 per group). Lower panel. Bdnf expression in mCherry and Pgc-1α overexpressing SN samples. Fold-change was calculated as before and the data were compared using Student’s t-test (n = 6 each group). <b>C.</b> SYBR-green PCR analysis of mRNA levels of <i>Pitx3, Nurr1</i> and <i>Lmx1b</i> in mCherry and Pgc-1α overexpressing SN samples. Fold-change in gene expression relative to the contralateral side was calculated as before and data were analyzed by comparing the mean fold change between groups using a Student’s t-test (mCherry n = 6, Pgc-1α n = 5, except for <i>Lmx1b</i> where the analysis was performed on a separate set of samples; mCherry n = 6, Pgc-1α n = 4). <b>D.</b> Western blot analysis of Pitx3 protein levels in pooled SN whole-cell lysate (same samples as 1D). “Cntrl” refers to the contralateral control side. <b>E.</b> SYBR-green PCR analysis of mRNA levels of <i>Th, Dat</i> and <i>Vmat2</i> in the contralateral (cntrl) SN from mCherry and Pgc-1α overexpressing mice. Fold-change gene expression was calculated in relation to the overall mean gene expression level for the contralateral SN of all mCherry overexpressing animals (n = 6 for both groups). Data were analyzed using a one-sample <i>t</i>-test to determine if fold-change gene expression differed from 1. <b>F.</b> Schematic showing convergence of the genes found to be down-regulated after <i>Pgc-1α</i> overexpression, on Pitx3.</p

Th and Dat immunohistochemical staining and stereological cell counts. A.

<p>Representative images of the striatum used for Th densitometry with line graphs showing the integrated density of Th immunoreactivity for each animal analyzed. In the line graphs, for each animal a line connects the results for the uninjected side (contralateral, cntrl) and the microinjected side (mCherry or PGC-1a) (mCherry/saline n = 9, mCherry/MPTP n = 8, Pgc-1α/saline n = 9, Pgc-1α/MPTP n = 8). <b>B.</b> Representative images of the striatum used for Dat densitometry with line graphs showing the integrated density of Dat immunoreactivity for each animal analyzed (mCherry/saline n = 8, mCherry/MPTP n = 10, Pgc-1α/saline n = 11, Pgc-1α/MPTP n = 10). <b>C.</b> Representative images of the SN used for Th+ cell stereological analysis with line graphs showing the number of Th+ cells for each animal analyzed (mCherry/saline n = 10, mCherry/MPTP n = 8, Pgc-1α/saline n = 11, Pgc-1α/MPTP n = 10). <b>D.</b> Striatal Th immunoreactivity data for all treatment groups. Ipsilateral and contralateral striatal Th immunoreactivity within each treatment group was analyzed using a paired Student’s <i>t</i>-test (mCherry/saline n = 9, mCherry/MPTP n = 8, Pgc-1α/saline n = 9, Pgc-1α/MPTP n = 8). All significant comparisons are indicated on the graph. <b>E.</b> SN Th+ stereological cell count data for all treatment groups. The number of Th+ cells in the ipsilateral vs contralateral SNs within each treatment group were analyzed using a paired t-test. (mCherry/saline n = 10, Pgc-1α/saline n = 11). All significant comparisons are indicated on the graph. <b>F.</b> SN stereology data from MPTP-treated animals (mCherry and Pgc-1α) showing the number of Th+ neurons, Th- neurons (thionin staining) and total SN neurons (combined data). Data were analyzed using paired t-tests to compare the contralateral and ipsilateral (microinjected) SNs in each treatment group and Student’s t-test for comparing between groups. (mCherry/MPTP n = 8, Pgc-1α/MPTP n = 10). <b>G.</b> Western blot analysis of Th immunoreactivity in gross-dissected whole cell lysate SN and striatal samples (saline groups only). Band densitometry data was analyzed using a paired t-test to compare Th-band intensity between the contralateral and ipsilateral (microinjected) SN and striatum (n = 6 per group).</p

AAV2/10 expresses functional Pgc-1α in the nigro-striatal system. A.

<p>SYBR-green PCR analysis of <i>Pgc-1α</i> and <i>Pgc-1α</i>-target gene mRNA levels after saline treatment in mice microinjected unilaterally into the right SN with AAV2/10-FHA-<i>Pgc-1α</i> or AAV2/10-<i>mCherry</i>. mRNA levels were normalized to 18S and fold-change gene expression was calculated relative to the contralateral SN using the ΔΔCt method. Data were analyzed using a paired <i>t</i>-test (n = 6). <b>B.</b> The same analysis was performed after MPTP treatment (n = 6). <b>C.</b> Western blot analysis of pooled SN samples. 40 µg of protein was run on a 4–20% SDS-PAGE gel and the blot was probed with anti-Pgc1α antibody. Lanes 1 & 2 are positive controls for the 37-kD and 120 kD (full-length; FL) Pgc-1α isoforms respectively. Lanes 3 & 4 contain pooled whole-protein lysates from 3 contralateral SNs and mCherry-overexpressing (ipsilateral) SNs respectively. Lanes 5 and 6 lanes contain lysates from 3 contralateral SNs and Pgc-1α-overexpressing SNs respectively. <b>D.</b> SYBR-green PCR analysis of a separate set of SN samples for the mRNAs giving rise to the 120 kD (FL) and 35–38 kD (N-terminal; NT) <i>Pgc-1α</i> isoforms (n = 6). <b>E.</b> The left panel shows native EGFP fluorescence after injection of AAV2/10-EGFP into the SN. The right panel is a merged image of native EGPF (green) fluorescence and Th immunostaining (red). EGFP expressing Th+ cells appear orange/yellow. <b>F.</b> Immunofluorescence staining for the HA-tag (green), Th (red) and DAPI (blue; Magnification: × 40). The right panel shows the same immunofluorescently stained section at higher magnification (Magnification: × 200). G. Immunofluorescence staining of the same section shown in F shown here at higher magnification (Magnification: × 400). Panels from left to right: anti-HA, anti-TH, DAPI, merged image.</p

Western blot data for the mitochondrial marker CoxIV.

<p>5 µg of whole-cell lysate from SN or striatal samples from saline or MPTP-treated Pgc-1α-microinjected mice was run on a 4–20% gradient gel and probed with anti-CoxIV and anti-b-actin antibodies. Each pair of lanes represents the contralateral (L) and ipsilateral (microinjected, R) SN respectively. Band densitometry data were analyzed using a paired Student’s <i>t</i>-test (n = 6 per group).</p

Data from lower-titer injections. A.

<p>SYBR-green PCR analysis of Pgc-1α and Pgc-1α target gene mRNA levels after saline treatment in mice microinjected unilaterally into the right SN with AAV2/10-FHA-Pgc-1α or AAV2/10-mCherry. mRNA levels were normalized to 18S and fold-change gene expression was calculated relative to the contralateral SN using the ΔΔCt method. Data were analyzed using a paired <i>t</i>-test (n = 6). <b>B.</b> Striatal Th immunoreactivity data for the contralateral side (Cntrl) compared to the ipsilateral side microinjected in the SN with AAV2/10-FHA-PGC-1α. Ipsilateral and contralateral striatal Th immunoreactivity within each treatment group was analyzed using a paired t-test (n = 7). <b>C.</b> Mean levels of striatal DA per mg of protein in the ipsilateral and contralateral (cntrl) striatum of mice microinjected unilaterally with a lower titer of AAV2/10-FHA-PGC-1α. Data were analyzed by paired t-test (n = 5). <b>D.</b> DA turnover in each treatment group. Data were analyzed by paired t-test for within group comparisons and Student’s t-test for between group comparisons. <b>E.</b> SYBR-green PCR analysis of dopaminergic markers. Fold change in gene expression relative to the contralateral side was calculated as before. Data were analyzed using a one-sample t-test to determine if fold-change gene expression differed from 1 (n = 5). <b>F.</b> SYBR-green PCR analysis of <i>Pitx3</i> and <i>Nurr1</i> expression (n = 5). Fold change was calculated and data were analyzed as in E.</p