The Grillari LABS - Research Focus
One of the major challenges that modern medicine and biology are facing today is the continuously increasing life expectance of the population and in consequence the increase in age-related pathologies. Since the changes in physiology and morphology of organisms, tissues, and cells during their life span are still poorly understood, it is of vital importance to gain insight into the molecular mechanisms of aging and aging associated pathologies in order to design strategies that allow for maximizing the human health span.
Therefore, our focus of research at the University of Natural Resources and Life Sciences, Vienna (BOKU-VIBT) is on understanding the cellular and molecular changes that occur during aging, how these changes affect tissue functionality and on where and how repair and regeneration needs to counteract these changes.
Overview on how cellular senescence might contribute to aging of tissues and organisms.
(1) Cells are exposed to DNA damage, reactive oxygen species (ROS), high oncogenic signalling or to telomere shortening due to multiple replications. (2) If no repair or cell cycle arrest checkpoints are operative, cells might undergo immortalization and transformation as first steps of tumorigenesis or (3) cells might undergo cellular senescence or apoptosis. The senescent cells then (4) show an altered secretory phenotype and thus influence signalling, or (5a) they might be removed after undergoing senescence by apoptosis or by the immune system. This in turn (5b) leads to replication/transdifferentiation of neighbouring cells or of replication/differentiation of adult stem and progenitor cells, decreasing their proliferative potential. Finally, (6) the senescent cells display an altered behaviour and physiology in regard to their ‘daily’ tasks within a tissue. All this in turn leads to (7) changes in the microenvironment of tissues and to their functional decline, which in turn (8) enhances the risk of tumor development and (9) accelerates senescence, thus largely contributing to aging of organisms.
Specialized Ribosomes in Aging
Our lab recently discovered that NSUN5, one of the few highly conserved genes modulating longevity, is able to influence the aging process of different organisms . Schosserer et al. revealed that protein synthesis in cells can be “reprogrammed” by reducing NSUN5 levels, which in turn extends the lifespans of flies, worms and baker’s yeast. This study was carried out in cooperation with international partners and was recently published in Nature Communications.
Ribosomes are molecular machines carrying out cellular protein synthesis. In the last few years it became clear that this is not a pure mechanical process, but that it is actively regulated by differentially composed ribosomes. By building these “specialized” ribosomes, cells are better able to react to different environmental conditions, such as heat, starvation and other types of stress –.
NSUN5 adds a single methyl group to ribosomal RNA, one of the most important building blocks of ribosomes , , . If NSUN5 and thereby the methyl group are missing, these “specialized” ribosomes synthesize proteins, which render flies, worms and yeast more resistant to stress and thereby allow them to live longer. Thereby we could show for the first time that modification of ribosomal RNA can directly modulate animal lifespan and stress resistance. The perspective of altering the function of a huge molecular machine, like the ribosome, just by a small modification of a single highly conserved building block (in this case an RNA nucleotide) and thereby adapting cells to better counteract stress is of high interest, especially as several mechanisms controlling the life span of organisms seem to converge on the ribosome.
So far only methylations of DNA were considered to be important for the regulation of gene expression. However, this study and others emphasize that methylation of rRNAs, tRNAs and other ncRNAs can impact on protein translation and might therefore be involved in post-transcriptional regulatory processes , . The analysis of RNA methylations at different nucleotides is not as well established yet as the detection of DNA methylations, but new techniques and protocols, such as bisulfite sequencing of RNA for the analysis of m5C methylation, are constantly emerging .
Thus, we aim to identify other ribosomal RNA-modifying enzymes that are also able to modulate aging and stress resistance in the nematode C. elegans and in various human cellular aging models. Although the gap between simple model organisms and potential applications in humans is still large, we believe that our findings will contribute to a better understanding of evolutionary conserved ageing processes and related diseases and thereby promote a healthier life at old age. Aging is one of the major challenges of our modern society. Therefore, the discovery of novel mechanisms to improve life- and healthspan, like specialized ribosomes, might lead to the development of prognostic markers that help to design intervention steps against the early onset of age-associated diseases.
 M. Schosserer, N. Minois, T. B. Angerer, M. Amring, H. Dellago, E. Harreither, A. Calle-Perez, A. Pircher, M. P. Gerstl, S. Pfeifenberger, C. Brandl, T. Mohr, M. Sonntagbauer, A. Kriegner, A. Linder, A. Weinhäusel, M. Steiger, D. Mattanovich, M. Rinnerthaler, T. Karl, S. Sharma, K. Entian, M. Kos, M. Breitenbach, I. B. H. Wilson, N. Polacek, R. Grillari-Voglauer, L. Breitenbach-Koller, and J. Grillari, “Methylation of ribosomal RNA by NSUN5 is a conserved mechanism modulating organismal lifespan,” Nat. Commun., vol. 6, p. 6158, 2015.
 S. Xue and M. Barna, “Specialized ribosomes: a new frontier in gene regulation and organismal biology.,” Nat. Rev. Mol. Cell Biol., vol. 13, no. 6, pp. 355–69, Jun. 2012.
 A. Filipovska and O. Rackham, “Specialization from synthesis: how ribosome diversity can customize protein function,” FEBS Lett., Feb. 2013.
 J. W. Bauer, C. Brandl, O. Haubenreisser, B. Wimmer, M. Weber, T. Karl, A. Klausegger, M. Breitenbach, H. Hintner, T. von der Haar, M. F. Tuite, and L. Breitenbach-Koller, “Specialized Yeast Ribosomes: A Customized Tool for Selective mRNA Translation,” PLoS One, vol. 8, no. 7, p. e67609, Jul. 2013.
 A. Gigova, S. Duggimpudi, T. Pollex, M. Schaefer, and M. Koš, “A cluster of methylations in the domain IV of 25S rRNA is required for ribosome stability.,” RNA, Aug. 2014.
 S. Sharma, J. Yang, P. Watzinger, P. Kötter, and K.-D. Entian, “Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively.,” Nucleic Acids Res., pp. 1–15, Aug. 2013.
 C. S. Chow, T. N. Lamichhane, and S. K. Mahto, “Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications,” ACS Chem Biol, vol. 2, no. 9, pp. 610–619, 2007.
 W. A. Decatur and M. J. Fournier, “rRNA modifications and ribosome function,” Trends Biochem Sci, vol. 27, no. 7, pp. 344–351, 2002.
 M. Schaefer, T. Pollex, K. Hanna, and F. Lyko, “RNA cytosine methylation analysis by bisulfite sequencing,” Nucleic Acids Res, vol. 37, no. 2, p. e12, Feb. 2009.
The Christian Doppler laboratory on Biotechnology of Skin Aging
Influence of plant extracts on senescence and skin aging
In this project we characterize the effect of plant extracts on cellular senescence and how this might relate to skin functionality in aging. Thereby, we specifically focus on the regulatory role of miRNAs in this context.
SNEV and skin functionality
SNEVhPrp19/hPso4 is an essential splicing factor (Grillari et al. 2005) and well-studied DNA repair factor (Mahajan&Mitchell 2003; Zhang et al. 2005; Abbas et al. 2014;) that additionally plays critical roles in adipogenesis (Khan et al, in preparation) and neuronal differentiation (Yamada et al. 2013). SNEVhPrp19/hPso4 possesses E3 ubiquitin ligase activity, which is required for its function in splicing (Song et al. 2010) as well as in at least one of the DNA repair pathways it is involved in (Wan & Huang 2014; Marechal et al. 2014). SNEVhPrp19/hPso4 is involved in cancer, but its expression level correlate with either good (Benjamin et al. 2014) or bad prognosis (Yin et al. 2016), depending on the cancer type.
We have focussed on the contribution of the protein SNEVhPrp19/hPso4 to cellular and organismal aging in generally with a focus on skin functionality and skin aging.
So far, we found that SNEVhPrp19/hPso4 is down-regulated in a variety of senescent cells and its over-expression postpones entry into replicative senescence in human endothelial cells and increases stress resistance (Voglauer et al., 2006), and this effect depends in part on phosphorylation of SNEVhPrp19/hPso4 at serin149 catalyzed by ATM, one DNA damage master regulator (Dellago et al. 2012). Mouse embryonic fibroblasts (MEFs) from heterozygous knock-out mice enter senescence earlier than wild type litter mate controls (Fortschegger et al., 2007), and those mice exhibit premature senescence of skin cells in response to PUVA (Monteforte et al. 2016).
Currently, we are focussing on the role of SNEVhPrp19/hPso4 in skin functionality and skin aging. Our studies rely on the generation of stable SNEVhPrp19/hPso4-overexpressing fibroblasts and keratinocytes and their functional characterization with regard to cell morphology, stress resistance, growth characteristics and senescence. We study how the cellular properties modified by SNEVhPrp19/hPso4 overexpression impacts on wound healing and functionality of 3-dimensional skin models.
Major challenges that complicate our efforts to nail down the precise molecular mechanisms modulated by SNEVhPrp19/hPso4 comprise the high stability of the protein, its essentiality, the high degree of post-transcriptional regulation and the narrow range of expression level that is tolerated by cells.
3-dimensional skin models
The human skin is the most exposed organ to factors which accelerate the aging process. One factor, cellular senescence, has recently been identified to be of crucial importance in the functional decline of tissues during aging. It is well established that up to 20 % of the skin cells are senescent in the elderly, however, the influence of senescent cells on the microenvironment in the human skin is not fully understood yet. Senescent cells acquire a senescence-associated secretory phenotype (SASP) which leads to the secretion of soluble signaling factors and thus changing the microenvironment drastically. Additionally, recent studies suggest that microRNAs are also part of the SASP and can influence the surrounding cells thus promoting skin aging.
The complex interaction between fibroblasts, extracellular matrix and keratinocytes cannot be investigated in a two-dimensional cell cultivation system. Thus, the development of a robust, three-dimensional "aged" skin equivalent model is necessary. Since fibroblasts in the dermis rarely divide, it is more likely that the cells reach senescence by stress-induced events rather than by replication. Hence, we decided to use a stress-induced premature senescent (SIPS) model by stressing the cells repeatedly with H2O2. The efficacy of the treatment was verified by two different senescence markers, measurement of growth arrest as well as by staining for senescence-associated beta-galactosidase. The "aged" skin equivalents were then built by adding increasing concentrations of SIPS fibroblasts into the collagen matrix that resembles the dermis. Preliminary results suggest that senescent fibroblasts indeed interfere with the differentiation capacity of keratinocytes in the skin equivalent and resemble an "aged" phenotype with thinning of epidermis and aberrant differentiation of keratinocytes. Additionally, we could show that the senescent cells withstand the stress of being embedded into collagen as good as their non-stressed counterparts and can influence the keratinocytes over the duration of the whole experiment.
These first results are promising for the further development of a skin equivalent model system with a fibroblast-driven aging phenotype thus allowing us to study the crosstalk between senescent fibroblasts and keratinocytes in detail.