Leader: Carlo Viscomi (UNIPD); Other collaborator(s):
We aim at studying how the impairment of oxidative phosphorylation impacts on the aging process. We will exploit mouse models with mild defects in the oxidative phosphorylation system for the development of age-related features, including: autophagy alterations and telemore length. In addition, the role of altered mitochondrial autophagy will be evaluated in the context of ageing.
Brief description of the activities and of the intermediate results: We started the analysis of the methylome in the skeletal muscle of the Ndufs4-/- mouse model of Leigh disease. The use of a seevre model gave us the possibility to optimize the techniques and the analysis pipeline. Preliminary evidence indicate that indeed there is hypermethylation of pathways related to genomic instability. We also run a RNAseq experiment on the same tissues and the analysis is ongoing.
We analyzed the methylome in the skeletal muscle of the Ndufs4 KO mouse model of Leigh disease. The use of a severe model gave us the possibility to optimize the techniques and the analysis pipeline. By using Reduced-representation bisulfite sequencing (RRBS-Seq) we observed a global hypomethylation of DNA in KO vs WT animals, with no difference in markers of epigenetic aging.
RNAseq analysis highlighted that differentially expressed genes between KO and WT animals affect several metabolic and signaling pathways, including adipocytokines, arginine and proline metabolism, ribosomal subunits, inflammatory and coagulation cascades and circadian rhythms. Importantly, a significant number of pro-inflammatory genes were downregulated, while anti-inflammatory genes were upregulated. Additionally, multiple genes involved in the DNA damage response and cell cycle regulation exhibited significant changes in their expression profiles. These include Cdkn1a, Btg2, Gadd45g, and Cebpd, all of which showed markedly increased expression.
Western blot and immunofluorescence analysis identified significant upregulation of gH2AX and 53BP1, as well as increased cleavage of histone H3.
These data suggest that Ndufs4 KO mice do not display an aging epigenome, but show signs of genomic instability in the nucleus.
We also started the analysis of RNAseq and DNA methylation in the mutator mice harboring a point mutation in the proofreading domain of mitochondrial DNA polymerase POLg, leading to accumulation of mutations in mtDNA. This model has been proposed as a model of accelerated aging. However, the impact on the nuclear genome has been investigated.
Our preliminary analysis indicates that the Polg model also shows increased DNA damage, evidenced by a significant increase in the phosphorylation of histone H2A.x and a recurring pattern of enhanced histone H3 cleavage. Further analysis of the Polg methylome and RNAseq is underway to investigate the impact of a mild phenotype on accelerated aging.
We have now completed the analysis of the mutator mouse skeletal muscle. We confirmed the no differences in the methylome were detected in mutator vs control animals. However, we experienced some problems wiht our model, which does not accurately predict the age of the 12-month-old animals. We are currently trying to sort out this problem, by better calibrating the model with new data.
We also run RNAseq of skeletal muscles from mutator and control mice. We just had the data and started the analysis. Additiional experiments confirmed that the Polg mutator model also shows increased DNA damage, ie a significant increase in the phosphorylation of histone H2A.x and a recurring pattern of enhanced histone H3 cleavage.
We started drafting the manuscript reporting our results on the Ndufs4 KO mice.
Coming soon31