Alterations in peroxisomal function induced by genomic instability and their relevance for aging

Abstract

Peroxisomes are single-membrane, multipurpose, cytoplasmic organelles playing a crucial role in numerous anabolic and catabolic functions, mainly related to lipid and reactive oxygen species (ROS) metabolism. Defects in either their biogenesis or metabolic aspects result in severe developmental disorders, involving multiple organs, markedly liver and brain. Peroxisomes are remarkably heterogeneous in the different cell types and are capable of modifying their own shape and functions, according to the cellular needs, to meet prevailing environmental conditions. Because of their essential role in cell homeostasis and preservation, peroxisomes' contribution to the ageing process is receiving increasing attention. Indeed, it has been shown that in normal ageing the inner organization of these organelles is compromised and upset. In addition, when catalase activity is chronically reduced, cells respond in a dramatic manner, displaying a cascade of accelerated ageing reactions. Ageing has been intrinsically associated with accumulation of macromolecular damage, particularly in DNA. Consistently, defects in DNA repair mechanisms result in accelerated ageing. Nucleotide excision repair (NER) is a versatile pathway responsible for repairing helixdistorting DNA lesions - including UV radiation-induced pyrimidine dimers - chemical adducts, and oxidative lesions. NER progresses along two subpathways: the global-genome NER (GG-NER) and the transcriptioncoupled NER (TC-NER). GG-NER removes lesions genomewide, while TC-NER repairs DNA damage that hampers the progression of the RNA polymerase II complex (RNAPIIo) and therefore concerns transcriptionally active genes. Both sub-pathways converge into a common mechanism that involves DNA unwinding, lesion verification and dual incision, followed by DNA re-synthesis and ligation. Null and hypomorphic Ercc1 mutant mice display accelerated ageing phenotype accurately recapitulating that observed in progeroid human syndromes - which are caused by inherited defective NER - such as xeroderma pigmentosum and Cockayne syndrome. Particularly, previous investigations on hepatic tissue of Ercc1 mutant mice have revealed alterations in the expression of peroxisome proliferator-activated receptor (PPAR)-α and –γ. Changes in these genes, which are not only crucial ii regulators of peroxisomal biogenesis and function, but more generally of cell metabolism, were framed in a context of bioenergetics. Collectively, these findings emphasize the involvement of peroxisomes in ageing processes and provided the rationale for my PhD project. This aimed at exploring mutual relationship between peroxisomes and DNA damage, clarifying whether: (i) genomic instability affects peroxisomal function and/or biogenesis, and (ii) modulating peroxisomal function/biogenesis may influence cellular response to exogenous genotoxic insult. The first part of my PhD project focussed on possible peroxisomal alterations in a progeroid condition generated by Ercc1 deficiency. To this end, we performed molecular and morphological experiments in the liver tissue of wild type and Ercc1Δ/- to evaluate the expression of peroxisomal membrane and matrix markers. Relevantly, several important pathways seem to be dysregulated, in particular those related to ROS and lipid metabolism, and the variation of specific peroxisomal proteins emphasized that some alterations actually involved peroxisomes. Additionally, ultrastructural analyses highlighted abnormalities in the cellular organization of mutants in respect to wild type, especially concerning mitochondrial membrane system. This interesting finding is in line with the compelling evidence establishing a close evolutionary, functional, and biogenetic link between peroxisomes and mitochondria. Furthermore, peroxisomal changes were investigated even in the brain, where the expression of peroxisomal proteins varies among specific neuronal populations. Defective DNA repair was demonstrated to also influence brain peroxisomes, in a non-linear fashion, since changes observed in mild vs. severe mutants can consist in either induction or repression of peroxisomal functions, also depending on the considered cerebral area. As a conclusion for this part of my work, we may assert that defects in NER impact peroxisomal metabolism not only in the liver of mutants mice, but even in their brain, where their importance is still dimly understood and that we were able to shed light on the unexplored role of peroxisomes in accelerated ageing process. iii The second part of the study further explored the link between DNA damage accumulation and peroxisomes, shifting to cell models, including human dermal fibroblasts (CHDF) and neuroblastoma cells (SH-SY5Y). In view of a flipped experimental procedure, we have begun modulating the dynamic feature of peroxisomes, by pre-treating cells with fenofibrate (FF), a peroxisome proliferator mainly employed as a hypolipidemic drug. Subsequently, cells underwent exogenous DNA injury (UVC radiation). FF is able to influence the expression levels of peroxisomal proteins. This effect was observed in all cell types considered, and generally dosedependent. Interestingly, UV radiation enhances the response to the drug, probably exacerbating microenvironmental stress conditions, which promote cellular response to the damaging insult. More specifically, FF impacts NER capacity of the cells modulating both the unscheduled DNA synthesis (UDS) and transcription recovery. Noteworthy, cells, because of the combination of a double induced-stress condition, responded varying their own redox status according to the dose of the drug administered. Definitively, our data provide a consistent depiction of how treatment with FF induces changes in damaged cells. Yet FF affects cellular metabolism depending on cell type considered and dose administered. Particularly, in non-neuronal, dividing, primary and secondary cells FF improves DNA repair; conversely in differentiated post-mitotic neuronal cells (retinoic acid treated SH-SY5Y) FF impairs DNA repair. The effect is dichotomous and may, or may not, involve PPARα activation, thus further studies will address the reasons of such differences. In conclusion, data collected during my three-year PhD project strongly argue for a reciprocal modulation of the organelles and DNA damage repair mechanisms, particularly in accelerated ageing, either endogenously generated, or exogenously produced. These findings, shedding new light into the relationship linking cell senescence with peroxisomal functions and biogenesis, open the way to future studies aiming at investigating potential protective strategies against age-related processes.