Validation of microarray data in human lymphoblasts shows a role of the ubiquitin-proteasome system and NF-kB in the pathogenesis of Down syndrome

BMC Medical Genomics Validation of microarray data in human lymphoblasts shows a role of the ubiquitin- proteasome system and NF-kB in the pathogenesis of Down syndrome Barbara Granese 0 Iris Scala 0 Carmen Spatuzza 3 Anna Valentino 0 Marcella Coletta 0 Rosa Anna Vacca 2 Pasquale De Luca 4 Generoso Andria 0 1 0 Department of Pediatrics, Federico II University , Naples 80131 , Italy 1 % of genes in category 2 Institute of Biomembranes and Bioenergetics, National Council of Research , Bari 70126 , Italy 3 Department of Biotechnological Sciences, Federico II University , Naples 80131 , Italy 4 Stazione Zoologica A. Dohrn, c/o BioGeM , Via Camporeale, Ariano Irpino 83031 , Italy Background: Down syndrome (DS) is a complex disorder caused by the trisomy of either the entire, or a critical region of chromosome 21 (21q22.1-22.3). Despite representing the most common cause of mental retardation, the molecular bases of the syndrome are still largely unknown. Methods: To better understand the pathogenesis of DS, we analyzed the genome-wide transcription profiles of lymphoblastoid cell lines (LCLs) from six DS and six euploid individuals and investigated differential gene expression and pathway deregulation associated with trisomy 21. Connectivity map and PASS-assisted exploration were used to identify compounds whose molecular signatures counteracted those of DS lymphoblasts and to predict their therapeutic potential. An experimental validation in DS LCLs and fetal fibroblasts was performed for the most deregulated GO categories, i.e. the ubiquitin mediated proteolysis and the NF-kB cascade. Results: We show, for the first time, that the level of protein ubiquitination is reduced in human DS cell lines and that proteasome activity is increased in both basal conditions and oxidative microenvironment. We also provide the first evidence that NF-kB transcription levels, a paradigm of gene expression control by ubiquitin-mediated degradation, is impaired in DS due to reduced IkB-alfa ubiquitination, increased NF-kB inhibitor (IkB-alfa) and reduced p65 nuclear fraction. Finally, the DSCR1/DYRK1A/NFAT genes were analysed. In human DS LCLs, we confirmed the presence of increased protein levels of DSCR1 and DYRK1A, and showed that the levels of the transcription factor NFATc2 were decreased in DS along with a reduction of its nuclear translocation upon induction of calcium fluxes. Conclusions: The present work offers new perspectives to better understand the pathogenesis of DS and suggests a rationale for innovative approaches to treat some pathological conditions associated to DS. Down syndrome; Trisomy 21; Expression; Ubiquitin-proteasome system; NF-kB - Background Down syndrome (DS) (MIM 190685) is a human complex disorder caused by the trisomy of either the entire, or a critical region of chromosome (chr) 21 (21q22.1-22.3). DS phenotypes are often variable. Intellectual disability and hypotonia are the hallmarks of the syndrome, while a wealth of distinct clinical manifestations, including congenital malformations, increased incidence of cancer, immune and endocrine abnormalities occur only in subsets of DS subjects. DS is also characterized by premature aging and dementia with neurological features that mimic those found in Alzheimers disease. The underlying molecular mechanisms of DS are largely unknown. Several genome-wide expression studies have been performed both in mouse and human trisomic tissues. While DS mice models have unravelled a generalized overexpression of triplicated genes [1-4], the analysis of human DS tissues showed contradictory results. In fact, some studies reported the selective over-expression of a limited subset of chr 21 genes [5-9], and others described subtle upregulations of chr 21 genes associated to a secondary, generalized and more extreme transcriptional deregulation of genes mapping on other chromosomes [10-15]. An additional level of complexity comes from the observation that gene expression differs extensively among unaffected individuals [16-19], including the expression of a number of chr 21 genes [20,21]. Therefore, some authors suggested to regard as poor candidates for DS pathogenesis those genes with high expression variation among controls and as reliable candidates those genes over-expressed in DS and tightly regulated in euploid cells [21-23]. Gene expression studies failed to provide definitive results; however, evidence in human DS cells point to the presence of abnormalities of extracellular matrix, of mitochondrial function and other metabolic pathways, including purine metabolism, in fetal specimens [5,11,13], and changes in transcriptional regulation, oxidative stress and immunerelated genes in adult tissues [6,12,14,24,25]. Recently, the effect of single chr 21 genes on the trisomic trancriptome was established by comparing the genome-wide expression from mouse ES cells, engineered to host the whole human chr 21, with those overexpressing only single chr 21 genes. A subset of genes, including Runx1, Erg, Nrip, Olig2, PdxK and Aire, produced the strongest transcriptional response when overexpressed [26]. More recently, Vilardell et al. [27] performed a meta-analysis from 45 publicly available DS data sets, from both human and mouse transcriptome and proteome. The identified biological functions were mainly related to nervous system development, neurodegenerative disorders (e.g. Huntingtons disease, Alzheimers disease and Parkinsons disease) and defects in synapsis function (e.g. axon guidance, NGF signalling). Seventy distinct transcription factors, including RelA, NFATc1, NFATc2 and NFATc3, were identified as being affected by dosage imbalance. Besides genome-wide expression analysis, few studies attempted to identify selected gene networks associated to specific DS features and to characterize molecular and biochemical functions disrupted in DS. In mice, Arron et al. [28] found that two critical chr 21 genes (DYRK1A and DSCR1, also known as RCAN1) act synergistically to control the nuclear localization of NFAT family of transcription factors and that knock-out mice for NFATc1, NFATc2, NFATc3 and NFATc4 display cardiovascular, neurological, skeletal and immune phenotypes strikingly similar to DS. In addition, DSCR1 promotes neurotoxicity [29] and attenuates the inflammatory response by stabilizing IkB-alfa [30]. The extra copy of DYRK1A in DS has been also associated to early onset of Alzheimers disease [31] and to defective neuronal development mediated by the reduction of REST, a key regulator of pluripotency and neuronal differentiation [32]. DSCR1/NFAT pathway was also associated with neuronal susceptibility to oxidative stress [33], a biochemical feature of DS. Finally, a limited number of functional studies in human DS cells have unravelled a disruption of mitochondrial function as a pathogenetic trigger [34-37]. In this study we analyzed the genome-wide transcription profile of lymphoblastoid cell lines (LCLs) from (...truncated)