Parvalbumin deficiency and GABAergic dysfunction in mice lacking PGC-1alpha

Abstract

The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) is a master regulator of metabolism in peripheral tissues, and it has been proposed that PGC-1alpha plays a similar role in the brain. Recent evidence suggests that PGC-1alpha is concentrated in GABAergic interneurons, so we investigated whether male and female PGC-1alpha -/- mice exhibit abnormalities in interneuron gene expression and/or function. We found a striking reduction in the expression of the Ca(2+)-binding protein parvalbumin (PV), but not other GABAergic markers, throughout the cerebrum in PGC-1alpha +/- and -/- mice. Furthermore, PGC-1alpha overexpression in cell culture was sufficient to robustly induce PV expression. Consistent with a reduction in PV rather than a loss of PV-expressing interneurons, spontaneous synaptic inhibition was not altered in PGC-1alpha -/- mice. However, evoked synaptic responses displayed less paired-pulse depression and dramatic facilitation in response to repetitive stimulation at the gamma frequency. PV transcript expression was also significantly reduced in retina and heart of PGC-1alpha -/- animals, suggesting that PGC-1alpha is required for proper expression of PV in multiple tissues. Together these findings indicate that PGC-1alpha is a novel regulator of interneuron gene expression and function and a potential therapeutic target for neurological disorders associated with interneuron dysfunction.

Figure 1.

Loss of PV immunoreactivity throughout the cerebrum in PGC-1α −/− mice. Immunohistochemistry with an antibody specific to PV was conducted on 20 μm sagittal and coronal brain sections from PGC-1α +/+ and −/− littermates. A, Low-magnification photos in the sagittal plane demonstrate a striking absence of PV immunoreactivity in −/− animals compared to +/+ littermates throughout the cortex (arrowheads), hippocampus, and basal ganglia (nuclei shown by arrows). B, Low-magnification photos in the coronal plane reveal that loss of PV immunoreactivity was not uniform across the cerebrum. Motor cortex (arrows) was more affected than parietal cortex (arrowheads). This differential loss was more pronounced in posterior (bottom panels) than anterior (top panels) brain regions. C, Representative higher-magnification photos show that the dentate gyrus of −/− animals was devoid of PV-labeled cell bodies (arrowheads). Some preservation of labeled cell bodies was observed in other areas of hippocampus such as the CA4 region (arrows). n = 3/group. Scale bars, 1 mm.

Figure 2.

PGC-1α is necessary and sufficient for neural expression of PV. A, Region-specific q-RT-PCR was conducted on homogenates of hippocampus, cortex, striatum, and cerebellum of PGC-1α +/+, +/−, and −/− mice. Significant decreases in PV transcript expression were found in PGC-1α +/− and −/− compared to +/+ mice in the hippocampus, cortex, and striatum but not the cerebellum. In the hippocampus and cortex, PV expression in −/− animals was significantly decreased compared to +/− animals, indicating a dose-dependent relationship between PGC-1α and PV. B, The dose-dependent decreases in PGC-1α and PV transcript expression were replicated in the hippocampus of a separate line of PGC-1α-deficient animals (Leone et al., 2005). Values for mRNA abundance were normalized to actin and presented as fold +/+ ± SEM. Group sizes (n) are indicated on the bars. C, PV expression was robustly induced in neuroblastoma cells transfected with an adenovirus (AdV) for PGC-1α (multiplicity of infection 10:1 and 20:1, n = 4/group) as compared to cells transfected with GFP alone. Values for mRNA abundance were normalized to 18S and presented as percentage maximal expression ± SEM. One-way ANOVA was used, followed by two-tailed t tests. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 3.

Other GABAergic markers are unaffected by loss of PGC-1α in the hippocampus. A, Immunohistochemistry with an antibody specific to GAD67 was conducted on 20 μm sagittal and coronal brain sections from PGC-1α +/+ and −/− littermates. Representative high-magnification photos of the dentate gyrus in the coronal plane demonstrate that immunoreactivity for GAD67 (arrowheads) was not observably different in intensity or distribution between +/+ and −/− animals. n = 3/group. B, Region-specific q-RT-PCR was conducted on homogenates of gross anatomical dissections of hippocampus of PGC-1α +/+, +/−, and −/− mice. Transcript expression of GAD67, GAD65, and the PV-interneuron-specific potassium transporter KV3.1 was not significantly different among genotypes. C, Expression of other interneuron-specific transcripts was not significantly altered among genotypes. Values for mRNA abundance were normalized to actin and presented as fold +/+ ± SEM. Group sizes (n) are indicated on the bars. One-way ANOVAs were used, and no significant differences were found.

Figure 4.

Metabolic gene targets are not affected in the hippocampus of PGC-1α −/− animals. PGC-1α metabolic gene targets MnSOD, Mfn2, Tfam, and Glut4 were analyzed in homogenates of gross anatomical dissections of the hippocampus and cerebellum by region-specific q-RT-PCR in PGC-1α +/+, +/−, and −/− littermates. A, No significant differences were found for these targets in the hippocampus. B, Transcript expression of MnSOD and Mfn2 was significantly decreased in −/− compared to +/+ mice in the cerebellum. Values for mRNA abundance were normalized to actin and presented as fold +/+ ± SEM. Group sizes (n) are indicated on the bars. One-way ANOVA was used, followed by two-tailed t tests. *p < 0.05, **p < 0.01.

Figure 5.

Ablation of PGC-1α alters short-term plasticity of evoked IPSCs but not basal GABA release. A, Representative traces of sIPSCs (blocked by the GABA_A receptor antagonist PTX) in +/+ and −/− animals. Quantification of spontaneous IPSCs revealed that the IPSC frequency and amplitude were not different between genotypes. B, Paired-pulse depression was reduced in PGC-1α −/− mice. Example IPSCs are the average of 15 traces with stimulus artifacts blanked for clarity (left). Reduced paired-pulse depression was seen at the 20 and 30 ms interstimulus intervals (right, n = 12 per interval). Mann–Whitney U tests. *p < 0.05.

Figure 6.

Repetitive stimulation at the gamma frequency enhances GABA release. Stimulation trains (500 ms duration) at 40 Hz delivered to axons surrounding the somas of mature granule cells resulted in robust increases in GABA release in PGC-1α −/− mice. A, The amplitude of the last IPSC in the train (gray dotted lines) was significantly larger in −/− compared to +/+ animals. Reponses were normalized to the peak amplitude of the first IPSC (black dotted line). B, Total charge during the train (gray area) was also significantly increased in −/− compared to +/+ mice. Traces shown in A and B are the average normalized response from all 12 cells in +/+ and −/− mice. C, Asynchronous release was apparent in individual IPSCs during and after the 40 Hz train in recordings from −/− mice. Seven individual episodes from one cell in a +/+ and −/− mouse are overlaid.

Figure 7.

PGC-1α regulates PV in a tissue-specific manner. Transcript expression was analyzed by q-RT-PCR in homogenates of retina, heart, and skeletal muscle taken from PGC-1α +/+, +/−, and −/− mice. A, Transcript expression of PGC-1α significantly differed among all genotypes in the retina and heart, but only −/− mice differed from +/+ and +/− mice in skeletal muscle. B, Decreased PV transcript expression was found in −/− mice compared with +/+ and +/− mice in the retina and heart. C, Cardiac expression of MnSOD and Tfam was decreased in −/− mice compared to +/+ and +/− mice. Values for mRNA abundance were normalized to actin and presented as fold +/+ ± SEM. Group sizes (n) are indicated on the bars. One-way ANOVA was used, followed by two-tailed t tests. *p < 0.05, **p < 0.01, ***p < 0.005.