The role of the coactivators PGC-1α and PGC-1β in retina and skeletal muscle

Peroxisome proliferator-activated receptor γ coactivator- 1 (PGC-1) designates a family of coactivators consisting of PGC-1α, PGC-1β and PRC. By associating with transcription factors like nuclear receptors, they induce transcription of target genes that are responsible for mitochondrial biogenesis, glucose uptake, gluconeogenesis, oxidative phosphorylation and β-oxidation of fatty acids. Originally identified in brown adipose tissue, these coactivators are predominately expressed in tissues with high energy requirements (skeletal muscle, heart, liver, brain, kidney) upon stimulation with physiological stressors (exercise, cold, fasting). PGC-1’s expression and function is therefore highly tissue specific. PGC-1α is to date the best studied of all family members.
One of the tissues high in energy demand is the retina; its photoreceptors convert light into a signal that can be interpreted by the brain. However, thus far the physiological role of PGC-1α has never been studied there. In the first project of this thesis, we consequently analyzed PGC-1α and to a certain extent also PGC-1β expression and function in mouse retina: We first assessed expression patterns of PGC-1α and PGC-1β. Second, we subjected mice with a global deletion of the PGC-1α gene (PGC-1α knockout = KO) and wildtype (WT) control mice to high intensity light compared to a dark setting and studied their retinae’s morphology (histology), function (electroretinogram) and gene expression levels (microarray, real time PCR). We found the PGC-1α KO mice to display increased apoptosis and disrupted retinal photoreceptor structure compared to the wildtype (WT) control animals upon light exposure. The corresponding light damage could also be confirmed by ERG in some animals. Microarray analysis revealed downregulation of DNA repair and phototransduction as well as an upregulation of inflammatory and apoptotic pathways, respectively in the KO animals. Finally, we confirmed in an in vitro setting that overexpression of PGC-1α helped alleviate apoptosis.
The role of PGC-1β in skeletal muscle has not been thoroughly studied either: More specifically, we wanted to address its role in glucose metabolism/ insulin sensitivity and fiber type composition as well as to identify exclusive target genes of PGC-1β in this tissue in the second project. For this purpose, we aimed at generating a skeletal muscle specific knockdown mouse: Different small hairpin RNAs (shRNAs) and control, scrambled shRNAs against mouse PGC-1β were designed according to different design principles (shRNA vs shRNA-mir) and tested for their knockdown efficiency and specificity in cell culture. The most promising one was then inserted into a vector backbone used to generate adeno-associated virus (AAV). We decided to produce the AAV serotype AAV2/6, as it displays specific tropism for skeletal muscle (AAV2/6). Injection quantity, route and duration until onset of shRNA expression were optimized with a virus expressing green fluorescent protein (GFP). The virus carrying shRNA was then injected into the mouse tibialis anterior muscle and after 3 weeks of incubation time analyzed for inflammatory gene expression. Semi- quantitative real time PCR analysis revealed that expression of PGC-1β as well as of its downstream target genes was significantly reduced. In vivo testing of a skeletal muscle specific promoter, however, did not lead to a significant reduction of PGC-1β levels.
In summary, we showed that PGC-1α has a protective role in light induced apoptosis in the retina (project1).
In project 2, we showed that a reduction of PGC-1β in skeletal muscle leads to diminished expression of genes implicated in OXPHOS.