Análise de Co-Expressão: AgingNet

The second objective of this work is to identify transcriptional programs that exist throughout life and, mainly, to understand how their behaviour can be related to immunosenescence. Two large modules consisting of highly co-expressed transcripts throughout life were identified, with each module, M1 and M2, having 4 transcript subgroups with distinct expression profiles.

‌ Like the AgingGenes, the profile of both modules is marked by two major phases, where the first changes in their behaviour occur around the age of 30, followed by more intense disturbances around the age of 50, corresponding to the inflexion phase. of the aforementioned expression.

M1 Module

The M1 module has two major pathway structures, the first related to gene expression and TP53 regulation and the second related to immune system signalling pathways, both from the adaptive system (such as signalling pathways downstream of B cell receptors and T) and the innate system, represented by cytokine signalling pathways and Toll-Like Receptors (TLRs) cascades. In general, gene expression pathways have a negative correlation with age, especially those of transcription by RNA Polymerase II (RNA-Pol II). Transcription pathways by RNA polymerase I (RNA-Pol I) were also enriched, however it did not show any correlation with age. Although the gene expression pathway has a negative correlation, the expression of epigenetic regulatory pathways increases with age, especially the regulation of ribosomal RNA expression. RNA-Pol II transcribes mainly messenger and/or non-protein-coding RNAs (miRNAs, snRNAs and snoRNAs). In this same large structure, there are also enriched pathways for regulating TP53 activity, both by phosphorylation and acetylation, and are respectively positively and negatively correlated with age. Phosphorylation of p53 inhibits its interaction with Mdm2 and therefore prevents Mdm2-mediated ubiquitination and its degradation (REED; QUELLE, 2014). Once stabilized, p53 can undergo post-translational changes, such as acetylation, leading to an increase in p53 binding to DNA and p53-specific promoters (REED; QUELLE, 2014). Furthermore, the p53 acetylation pathway is elevated in responses to stress and its expression level correlates with the activation and stabilization of p53 (REED; QUELLE, 2014). The possible consequences of p53 modulation in a cell are cell cycle arrest and DNA repair, senescence and apoptosis, depending on the amount of p53 activated and which sites were acetylated (REED; QUELLE, 2014). The fact that RNA Polymerase II activity decreases with age and the TP53 regulation pathways by phosphorylation and acetylation have discrepant correlations, may suggest that there is a disturbance in the signal transduction for the stress response and DNA repair pathways.

‌ As mentioned earlier, the second large enrichment structure found in the M1 module suggests a strong enrichment of pathways for downstream signalling from B-cell receptors (BCRs), T (TCRs), C-type lectin (CLRs), Fcε and TLRs. Although the general pathways of the immune system have a positive correlation with age, more specific signalling pathways have a negative correlation and are mostly related to NF-kB modulation. In the adaptive system pathways, it is noticed that there is a lifetime increase in the expression of genes related to degradation in the processing of MHC class I group antigens and negative correlations with age both in the modulation of NF-kB by the BCRs and in the signalling by TCRs, which may suggest a decrease in the regulation of NF-kB responsive genes and an increase in T cell proliferation, through the TCR-AKT-mTOR pathway. Furthermore, the non-activation of the TCR allows an increase in p53 activity, described above, and consequently in p21 activity, resulting in cell cycle arrest and/or apoptosis.

‌ Analyzing the innate system pathways, it can be seen that the NF-kB modulation pathways by TLRs (except TLR3 and TLR4) have negative correlations with age, either by MyD88 and TRAF6 pathways or by non-signalling pathways. Canonical of Dectin-1. All TLRs, except TLR3, can initiate downstream signalling through MyD88 or MyD88:MAL. In this MyD88-dependent pathway, TLR activation (such as TLR7 and TLR8) leads to the recruitment of IL-1 receptors associated with the IRAK family, which is followed by activation of TRAF6. TRAF6 induces TAK1 to auto-phosphorylate and, once activated, can mediate the induction of transcription factor NF-kB or MAPK, such as JNK, p38 and ERK. This results in the translocation of active NF-kB and MAPKs to the nucleus and the initiation of appropriate gene transcription, leading to the production of many pro-inflammatory cytokines and antimicrobial peptides. Another adapter, known as MyD88-adaptor-like (MAL), also participates in TLR2 and 4 signallings. Activation of TLR1:TLR2 or TLR6:TLR2 receptors, through MyD88 and TIRAP/MAL, activates IRAK and TRAF6, which by therefore they increase the expression of pro-inflammatory cytokines through NF-kB (early stage). In the adaptive system signalling pathways, B and T cell receptors also have negative linear correlations with age, suggesting that there are unfavourable changes in signalling between the innate and adaptive immune systems and possible changes in the ability to mount immune responses through processes inflammatory facing a challenge.

‌ At the same time, there is an increase in the signalling of Fcε receptors (FcεRI), which in humans control the activation of mastoid and basophil cells, participating in the presentation of antigens mediated by Immunoglobulin E (IgE). Aggregation of this receptor induces multiple signalling pathways that control several effector responses, including the secretion of allergic mediators and induction of transcription of cytokine genes, resulting in the secretion of molecules such as IL-6, TNF-alpha and granulocyte-colony-stimulating factors. macrophages (TURNER; KINET, 1999). FcεRI is therefore central to the induction and maintenance of allergic responses and may provide physiological protection from parasitic infections. However, signalling by this receptor significantly increases with life, which can exacerbate serum concentrations of IL-6, a cytokine that is closely related to ageing and dubbed the “Gerontologists' Cytocide”. There is strong evidence to suggest that serological concentrations of IL-6 increase with age (MAGGIO et al., 2006) and also that excessive production or reduced scavenging of oxygen free radicals, which stimulates IL-6 production, may be important for this increase (MAGGIO et al., 2006). Furthermore, it remains a fundamental question whether high levels of IL-6 are intended to resolve an inflammatory response that is inappropriately long or whether a primary dysregulation in IL-6 production is responsible for the chronic pro-inflammatory state, which has an impact negative on the quality of health.

Módulo M2

Module M2 has smaller enrichment structures than M1, so the presence of signalling pathways with few enriched components was allowed. But, like the M1 module, the median expression of the general pathways of the immune system (innate and adaptive) have a positive correlation with age. In this module, two major structures of signalling pathways were also detected, related to diseases of signal transduction in the immune system and DNA repair, in a way analogous to the mechanisms highlighted by the AgingGenes,

‌ Apesar de não estarem correlacionadas com a idade, há diversas vias altamente enriquecidas para maquinarias de reparo de DNA, especialmente reparo por excisão de base e dipirimidinação, podendo indicar que mecanismos de reparo possam estar sendo ativados em diversos momento durante vida, não sendo necessariamente algo progressivo, detectável por correlações lineares.

‌ The signal transduction disease pathway has a strong positive correlation with age, while MyD88 and TNFR1 deficiency pathways in TLR2 and/or TLR4 decrease throughout life, in agreement with the findings in the M1 module on TLR2. This fact suggests that there is a possible decrease in the signal transduction of these two TLRs due to possible problems of accumulation of mutations in genes related to the production and/or modulation of MyD88, IRAK6 and TRAF6, members of the signalling axis of these two TLRs since that signalling-related disease pathways through MyD88 and IRAK6 have been enriched and have a negative correlation with age. Likewise, both TICAM1 and TRAF6 genes, which are associated with the NF-kB activation cascade, have a negative expression trend throughout life. Another option would be that possible mutations/repressions to the Ly96 gene, encoding the MD2 protein and associated with the TLR4 receptor, lead to disturbances in the perfect configuration of TLR4, since its expression profile tends to decrease with age, with a correlation value Rho = -0.82.

‌ Furthermore, enriched pathways of TNFR1-mediated ceramide production have negative correlations with age. This pathway intervenes both in cell survival through NF-kB and in cell death through the activation of caspases. Since the TRAF2 gene, which participates in the modulation of NF-kB in the TNFR1-IKK axis, has a negative correlation with age and the activation pathway of caspases by ligands was enriched, with a positive correlation with age, it can presuppose a possible increase in the modulation of programmed cell death. Finally, there were also enriched pathways of gene expression regulation by enriched PERK and with a positive correlation. PERK, encoded by the EIF2AK3 gene (Rho = 0.225), is a protein located in the membrane of the endoplasmic reticulum (ER) and kept inactive by linking the portion located in the lumen with an ER chaperone called BiP. This chaperone also binds to unfolded proteins and therefore dissociates from PERK when unfolded proteins accumulate in the ER. When activated, PERK can induce translation arrest, causing the translation of proteins targeted to the ER to be down-regulated, triggering the depletion of Cyclin D1, which in turn triggers the arrest of the cell cycle in the G1 phase. Based on the expression profile of submodule 3 (M2S3), which is related to this pathway, it can be suggested that imbalances in proteostasis, one of the hallmarks of ageing, can happen early in life, around 35 years old, and tend to intensify continuously even in older ages, around 80 years old, where they become extremely disturbed.