Pgc-1α Overexpression Downregulates Pitx3 and Increases Susceptibility to MPTP Toxicity Associated with Decreased Bdnf

Multiple mechanisms likely contribute to neuronal death in Parkinson’s disease (PD), including mitochondrial dysfunction and oxidative stress. Peroxisome proliferator-activated receptor gamma co-activator-1 alpha (PGC-1α) positively regulates the expression of genes required for mitochondrial biogenesis and the cell’s antioxidant responses. Also, expression of PGC-1α-regulated genes is low in substantia nigra (SN) neurons in early PD. Thus upregulation of PGC-1α is a candidate neuroprotective strategy in PD. Here, an adeno-associated virus (AAV) was used to induce unilateral overexpression of Pgc-1α, or a control gene, in the SN of wild-type C57BL/6CR mice. Three weeks after AAV administration, mice were treated with saline or MPTP. Overexpression of Pgc-1α in the SN induced expression of target genes, but unexpectedly it also greatly reduced the expression of tyrosine hydroxylase (Th) and other markers of the dopaminergic phenotype with resultant severe loss of striatal dopamine. Reduced Th expression was associated with loss of Pitx3, a transcription factor that is critical for the development and maintenance of dopaminergic cells. Expression of the neurotrophic factor Bdnf, which also is regulated by Pitx3, similarly was reduced. Overexpression of Pgc-1α also led to increased sensitivity to MPTP-induced death of Th+ neurons. Pgc-1α overexpression alone, in the absence of MPTP treatment, did not lead to cell loss in the SN or to loss of dopaminergic terminals. These data demonstrate that overexpression of Pgc-1α results in dopamine depletion associated with lower levels of Pitx3 and enhances susceptibility to MPTP. These data may have ramifications for neuroprotective strategies targeting overexpression of PGC-1α in PD

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

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

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

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

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

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