A Genome-Wide Longitudinal Transcriptome Analysis of the Aging Model Podospora anserine

et al. (2013) A Genome-Wide Longitudinal Transcriptome Analysis of the Aging Model Podospora anserine. PLoS ONE 8(12): e83109. doi:10.1371/journal.pone.0083109 A Genome-Wide Longitudinal Transcriptome Analysis of the Aging Model Podospora anserine Oliver Philipp 0 Andrea Hamann 0 Jo rg Servos 0 Alexandra Werner 0 Ina Koch 0 Heinz D. Osiewacz 0 Gustavo Henrique Goldman, Universidade de Sao Paulo, Brazil 0 1 Molecular Developmental Biology, Institute of Molecular Biosciences, Faculty for Biosciences & Cluster of Excellence 'Macromolecular Complexes', Johann Wolfgang Goethe University , Frankfurt am Main, Germany, 2 Molecular Bioinformatics , Institute of Computer Science, Faculty of Computer Science and Mathematics & Cluster of Excellence 'Macromolecular Complexes', Johann Wolfgang Goethe University , Frankfurt am Main , Germany Aging of biological systems is controlled by various processes which have a potential impact on gene expression. Here we report a genome-wide transcriptome analysis of the fungal aging model Podospora anserina. Total RNA of three individuals of defined age were pooled and analyzed by SuperSAGE (serial analysis of gene expression). A bioinformatics analysis identified different molecular pathways to be affected during aging. While the abundance of transcripts linked to ribosomes and to the proteasome quality control system were found to decrease during aging, those associated with autophagy increase, suggesting that autophagy may act as a compensatory quality control pathway. Transcript profiles associated with the energy metabolism including mitochondrial functions were identified to fluctuate during aging. Comparison of wildtype transcripts, which are continuously down-regulated during aging, with those down-regulated in the long-lived, copper-uptake mutant grisea, validated the relevance of age-related changes in cellular copper metabolism. Overall, we (i) present a unique age-related data set of a longitudinal study of the experimental aging model P. anserina which represents a reference resource for future investigations in a variety of organisms, (ii) suggest autophagy to be a key quality control pathway that becomes active once other pathways fail, and (iii) present testable predictions for subsequent experimental investigations. - Biological aging is a complex process leading to physiological impairments, the degeneration of cellular and organ functions, the development of disease and finally death of the system [13]. The underlying molecular mechanisms are multifactorial and only partially defined. It is clear that aging is accompanied by changes in gene expression. The available data, however, are mainly derived from single gene analyses and the comparison of only a few age stages (e.g., young vs. old). The comprehensive and systematic analyses of changes over the lifetime of individuals can identify new key pathways and regulatory circuits involved in aging and lifespan control and can open the field for the development of strategies to intervene into aging and age-related diseases (e.g., cancer, dementia, Parkinsons disease, cardiovascular impairments). Nowadays, the availability of efficient highthroughput techniques makes such studies possible, in particular when the study is performed with experimentally accessible shortlived systems. Podospora anserina is such a system [46]. In contrast to most filamentous fungi this ascomycete is characterized by a welldefined aging process that is under the control of genetic and environmental traits. After germination of an ascospore, a mycelium develops which grows at the periphery until it reaches a phase where the growth rate first decreases until it comes to a complete growth stop [7]. Finally, the hyphal tips burst and die. This process occurs under nutrient-replete growth conditions and thus clearly differs from those described as aging in fungi grown under nutrient starvation [8] and as chronological aging in the yeast Saccharomyces cerevisiae [9,10]. In P. anserina, various molecular pathways have been identified to be involved in the control of aging and development [1113]. The lifespan of the fungus is short (typically two to four weeks for wild-type strain s) and depends on the growth medium and on cultivation conditions [5,14]. The vegetation body of P. anserina is simply consisting of branched filamentous cellsforming a mycelium. For sexual reproduction specialized organs, protoperithecia and spermogonia, are formed in dikaryotic as well as in monokaryotic strains. P. anserina is accessible to experimentation [4,5]. Biomolecules like DNA, RNA or proteins as well as whole mitochondria can be isolated and analyzed from individuals of well-defined age [5]. The complete genome of P. anserina is sequenced and consists of about 36 MBp coding for more than 10,600 putative proteins [15,16]. P. anserina can be genetically manipulated by classical genetic approaches and by genetic engineering [5,17,18]. Here we describe a genome-wide transcriptome profiling of three P. anserina individuals from which total RNA was isolated after 6, 9, 10, 11, 12, 13 and 14 days of cultivation. Quantitative transcript profiles were generated by serial analysis of gene expression (SuperSAGE) and analyzed by bioinformatical and statistical approaches [1921]. Previously we used SuperSAGE Figure 1. Transcript analysis of Pa_3_10440 (PaCtr3), Pa_2_4460 (PaSod2) and Pa_3_1710 (PaAox). Upper row: RNA of the three biological replicates (individuals) was isolated, pooled and analyzed by SuperSAGE. Gene expression was quantified as tags per million (tpm). Lower row: The same RNA was used for qRT-PCR analysis. RNA samples from each individual were individually analyzed for relative gene expression. Error bars represent the standard error. In both analyses, x-axes indicate the age of the individuals at which total RNA was isolated. doi:10.1371/journal.pone.0083109.g001 successfully to characterize the transcriptome of a specific longlived mutant of P. anserina and compared it to the transcriptome of the wild type. Validation by qRT-PCR demonstrated the reliability of this method [22]. The data of the current longitudinal study, in which RNA was isolated from the same fungal individuals after a defined period of growth and subjected to a genome-wide SuperSAGE analyses, identified autophagy as a quality control pathway up-regulated late in the life of P. anserina at a time when transcripts, encoding components of other pathways (e.g., proteasome), are down-regulated. Materials and Methods Podospora anserina Strains and Cultivation For all experiments, three independent monokaryotic spore isolates (mating type minus) of the wild-type strain s [7] were used. Cultivation was essentially performed as described previously [23]. Briefly, single ascospores were germinated for 2 days on germination medium. Pieces of mycelium of this two day old culture were either directly transferred to a fresh PASM [24] plate overlaid (...truncated)