Unveiling the molecular landscape of cognitive aging: insights from polygenic risk scores, DNA methylation, and gene expression.

BACKGROUND: Aging represents a significant risk factor for the occurrence of cerebral small vessel disease, associated with white matter (WM) lesions, and to age-related cognitive alterations, though the precise mechanisms remain largely unknown. This study aimed to investigate the impact of polygenic risk scores (PRS) for WM integrity, together with age-related DNA methylation, and gene expression alterations, on cognitive aging in a cross-sectional healthy aging cohort. The PRSs were calculated using genome-wide association study (GWAS) summary statistics for magnetic resonance imaging (MRI) markers of WM integrity, including WM hyperintensities, fractional anisotropy (FA), and mean diffusivity (MD). These scores were utilized to predict age-related cognitive changes and evaluate their correlation with structural brain changes, which distinguish individuals with higher and lower cognitive scores. To reduce the dimensionality of the data and identify age-related DNA methylation and transcriptomic alterations, Sparse Partial Least Squares-Discriminant Analysis (sPLS-DA) was used. Subsequently, a canonical correlation algorithm was used to integrate the three types of omics data (PRS, DNA methylation, and gene expression data) and identify an individual "omics" signature that distinguishes subjects with varying cognitive profiles. RESULTS: We found a positive association between MD-PRS and long-term memory, as well as a correlation between MD-PRS and structural brain changes, effectively discriminating between individuals with lower and higher memory scores. Furthermore, we observed an enrichment of polygenic signals in genes related to both vascular and non-vascular factors. Age-related alterations in DNA methylation and gene expression indicated dysregulation of critical molecular features and signaling pathways involved in aging and lifespan regulation. The integration of multi-omics data underscored the involvement of synaptic dysfunction, axonal degeneration, microtubule organization, and glycosylation in the process of cognitive aging. CONCLUSIONS: These findings provide valuable insights into the biological mechanisms underlying the association between WM coherence and cognitive aging. Additionally, they highlight how age-associated DNA methylation and gene expression changes contribute to cognitive aging.

Emerging Roles of tRNAs in RNA Virus Infections.

Viruses rely on the host cell translation machinery for efficient synthesis of their own proteins. Emerging evidence highlights different roles for host transfer RNAs (tRNAs) in the process of virus replication. For instance, different RNA viruses manipulate host tRNA pools to favor viral protein translation. Interestingly, specific host tRNAs are used as reverse transcription primers and are packaged into retroviral virions. Recent data also demonstrate the formation of tRNA-derived fragments (tRFs) upon infection to facilitate viral replication. Here, we comprehensively discuss how RNA viruses exploit distinct aspects of the host tRNA biology for their benefit. In light of the recent advances in the field, we propose that host tRNA-related pathways and mechanisms represent promising cellular targets for the development of novel antiviral strategies.

Activation of Laminin γ2 by Helicobacter pylori Promotes Invasion and Survival of Gastric Cancer Cells With E-Cadherin Defects.

BACKGROUND: Helicobacter pylori infection induces cellular phenotypes relevant for cancer progression, namely cell motility and invasion. We hypothesized that the extracellular matrix (ECM) could be involved in these deleterious effects. METHODS: Microarrays were used to uncover ECM interactors in cells infected with H. pylori. LAMC2, encoding laminin γ2, was selected as a candidate gene and its expression was assessed in vitro and in vivo. The role of LAMC2 was investigated by small interference RNA (siRNA) combined with a set of functional assays. Laminin γ2 and E-cadherin expression patterns were evaluated in gastric cancer cases. RESULTS: Laminin γ2 was found significantly overexpressed in gastric cancer cells infected with H. pylori. This finding was validated in vitro by infection with clinical isolates and in vivo by using gastric biopsies of infected and noninfected individuals. We showed that laminin γ2 overexpression is dependent on the bacterial type IV secretion system and on the CagA. Functionally, laminin γ2 promotes cell invasion and resistance to apoptosis, through modulation of Src, JNK, and AKT activity. These effects were abrogated in cells with functional E-cadherin. CONCLUSIONS: These data highlight laminin γ2 and its downstream effectors as potential therapeutic targets, and the value of H. pylori eradication to delay gastric cancer onset and progression.

MicroRNA-186-5p controls GluA2 surface expression and synaptic scaling in hippocampal neurons.

Homeostatic synaptic scaling is a negative feedback response to fluctuations in synaptic strength induced by developmental or learning-related processes, which maintains neuronal activity stable. Although several components of the synaptic scaling apparatus have been characterized, the intrinsic regulatory mechanisms promoting scaling remain largely unknown. MicroRNAs may contribute to posttranscriptional control of mRNAs implicated in different stages of synaptic scaling, but their role in these mechanisms is still undervalued. Here, we report that chronic blockade of glutamate receptors of the AMPA and NMDA types in hippocampal neurons in culture induces changes in the neuronal mRNA and miRNA transcriptomes, leading to synaptic upscaling. Specifically, we show that synaptic activity blockade persistently down-regulates miR-186-5p. Moreover, we describe a conserved miR-186-5p-binding site within the 3'UTR of the mRNA encoding the AMPA receptor GluA2 subunit, and demonstrate that GluA2 is a direct target of miR-186-5p. Overexpression of miR-186 decreased GluA2 surface levels, increased synaptic expression of GluA2-lacking AMPA receptors, and blocked synaptic scaling, whereas inhibition of miR-186-5p increased GluA2 surface levels and the amplitude and frequency of AMPA receptor-mediated currents, and mimicked excitatory synaptic scaling induced by synaptic inactivity. Our findings elucidate an activity-dependent miRNA-mediated mechanism for regulation of AMPA receptor expression.

The Role of MicroRNAs in Proteostasis Decline and Protein Aggregation during Brain and Skeletal Muscle Aging.

Aging can be defined as the progressive deterioration of cellular, tissue, and organismal function over time. Alterations in protein homeostasis, also known as proteostasis, are a hallmark of aging that lead to proteome imbalances and protein aggregation, phenomena that also occur in age-related diseases. Among the various proteostasis regulators, microRNAs (miRNAs) have been reported to play important roles in the post-transcriptional control of genes involved in maintaining proteostasis during the lifespan in several organismal tissues. In this review, we consolidate recently published reports that demonstrate how miRNAs regulate fundamental proteostasis-related processes relevant to tissue aging, with emphasis on the two most studied tissues, brain tissue and skeletal muscle. We also explore an emerging perspective on the role of miRNA regulatory networks in age-related protein aggregation, a known hallmark of aging and age-related diseases, to elucidate potential miRNA candidates for anti-aging diagnostic and therapeutic targets.

Protein synthesis inhibition and GADD34 control IFN-ß heterogeneous expression in response to dsRNA.

In innate immune responses, induction of type-I interferons (IFNs) prevents virus spreading while viral replication is delayed by protein synthesis inhibition. We asked how cells perform these apparently contradictory activities. Using single fibroblast monitoring by flow cytometry and mathematical modeling, we demonstrate that type-I IFN production is linked to cell's ability to enter dsRNA-activated PKR-dependent translational arrest and then overcome this inhibition by decreasing eIF2α phosphorylation through phosphatase 1c cofactor GADD34 (Ppp1r15a) expression. GADD34 expression, shown here to be dependent on the IRF3 transcription factor, is responsible for a biochemical cycle permitting pulse of IFN synthesis to occur in cells undergoing protein synthesis inhibition. Translation arrest is further demonstrated to be key for anti-viral response by acting synergistically with MAVS activation to amplify TBK1 signaling and IFN-ß mRNA transcription, while GADD34-dependent protein synthesis recovery contributes to the heterogeneous expression of IFN observed in dsRNA-activated cells.

The role of non-standard translation in Candida albicans pathogenesis.

Candida albicans typically resides in the human gastrointestinal tract and mucosal membranes as a commensal organism. To adapt and cope with the host immune system, it has evolved a variety of mechanisms of adaptation such as stress-induced mutagenesis and epigenetic regulation. Niche-specific patterns of gene expression also allow the fungus to fine-tune its response to specific microenvironments in the host and switch from harmless commensal to invasive pathogen. Proteome plasticity produced by CUG ambiguity, on the other hand is emerging as a new layer of complexity in C. albicans adaptation, pathogenesis, and drug resistance. Such proteome plasticity is the result of a genetic code alteration where the leucine CUG codon is translated mainly as serine (97%), but maintains some level of leucine (3%) assignment. In this review, we dissect the link between C. albicans non-standard CUG translation, proteome plasticity, host adaptation and pathogenesis. We discuss published work showing how this pathogen uses the fidelity of protein synthesis to spawn novel virulence traits.

De novo sequencing of proteins by mass spectrometry.

INTRODUCTION: Proteins are crucial for every cellular activity and unraveling their sequence and structure is a crucial step to fully understand their biology. Early methods of protein sequencing were mainly based on the use of enzymatic or chemical degradation of peptide chains. With the completion of the human genome project and with the expansion of the information available for each protein, various databases containing this sequence information were formed. AREAS COVERED: De novo protein sequencing, shotgun proteomics and other mass-spectrometric techniques, along with the various software are currently available for proteogenomic analysis. Emphasis is placed on the methods for de novo sequencing, together with potential and shortcomings using databases for interpretation of protein sequence data. EXPERT OPINION: As mass-spectrometry sequencing performance is improving with better software and hardware optimizations, combined with user-friendly interfaces, de-novo protein sequencing becomes imperative in shotgun proteomic studies. Issues regarding unknown or mutated peptide sequences, as well as, unexpected post-translational modifications (PTMs) and their identification through false discovery rate searches using the target/decoy strategy need to be addressed. Ideally, it should become integrated in standard proteomic workflows as an add-on to conventional database search engines, which then would be able to provide improved identification.

Phenotypic heterogeneity promotes adaptive evolution.

Genetically identical cells frequently display substantial heterogeneity in gene expression, cellular morphology and physiology. It has been suggested that by rapidly generating a subpopulation with novel phenotypic traits, phenotypic heterogeneity (or plasticity) accelerates the rate of adaptive evolution in populations facing extreme environmental challenges. This issue is important as cell-to-cell phenotypic heterogeneity may initiate key steps in microbial evolution of drug resistance and cancer progression. Here, we study how stochastic transitions between cellular states influence evolutionary adaptation to a stressful environment in yeast Saccharomyces cerevisiae. We developed inducible synthetic gene circuits that generate varying degrees of expression stochasticity of an antifungal resistance gene. We initiated laboratory evolutionary experiments with genotypes carrying different versions of the genetic circuit by exposing the corresponding populations to gradually increasing antifungal stress. Phenotypic heterogeneity altered the evolutionary dynamics by transforming the adaptive landscape that relates genotype to fitness. Specifically, it enhanced the adaptive value of beneficial mutations through synergism between cell-to-cell variability and genetic variation. Our work demonstrates that phenotypic heterogeneity is an evolving trait when populations face a chronic selection pressure. It shapes evolutionary trajectories at the genomic level and facilitates evolutionary rescue from a deteriorating environmental stress.

Correction: Phenotypic heterogeneity promotes adaptive evolution.

[This corrects the article DOI: 10.1371/journal.pbio.2000644.].

Human cells adapt to translational errors by modulating protein synthesis rate and protein turnover.

Deregulation of tRNAs, aminoacyl-tRNA synthetases (aaRS) or tRNA modifying enzymes, increase the level of protein synthesis errors (PSE) and are associated with several diseases, but the cause-effect mechanisms of these pathologies remain elusive. To clarify the role of PSE in human biology, we have engineered a HEK293 cell line to overexpress a wild type (Wt) tRNASer and two tRNASer mutants that misincorporate serine at non-cognate codon sites. Then, we followed long-term adaptation to PSE of such recombinant cells by analysing cell viability, protein synthesis rate and activation of protein quality control mechanisms (PQC). Engineered cells showed higher level of misfolded and aggregated proteins; activated the ubiquitin-proteasome system (UPS) and the unfolded protein response (UPR), indicative of proteotoxic stress. Adaptation to PSE involved increased protein turnover, UPR up-regulation and altered protein synthesis rate. Gene expression analysis showed that engineered cells presented recurrent alterations in the endoplasmic reticulum, cell adhesion and calcium homeostasis. Herein, we unveil new phenotypic consequences of protein synthesis errors in human cells and identify the protein quality control processes that are necessary for long-term adaptation to PSE and proteotoxic stress. Our data provide important insight on how chronic proteotoxic stress may cause disease and highlight potential biological pathways that support the association of PSE with disease.

Protein mistranslation: friend or foe?

The translation of genes into functional proteins involves error. Mistranslation is a known cause of disease, but, surprisingly, recent studies suggest that certain organisms from all domains of life have evolved diverse pathways that increase their tolerance of translational error. Although the reason for these high error rates are not yet clear, evidence suggests that increased mistranslation may have a role in the generation of diversity within the proteome and other adaptive functions. Error rates are regulated, and there appears to be an optimal mistranslation rate that varies by organism and environmental condition. Advances in unbiased interrogation of error types and experiments involving wild organisms may help our understanding of the potentially adaptive roles for protein translation errors.

Evolution of Robustness to Protein Mistranslation by Accelerated Protein Turnover.

Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome-mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that translational fidelity and the ubiquitin-proteasome system are functionally linked to each other and may, therefore, co-evolve in nature.

Codon misreading tRNAs promote tumor growth in mice.

Deregulation of tRNAs, aminoacyl-tRNA synthetases and tRNA modifying enzymes are common in cancer, raising the hypothesis that protein synthesis efficiency and accuracy (mistranslation) are compromised in tumors. We show here that human colon tumors and xenograft tumors produced in mice by two epithelial cancer cell lines mistranslate 2- to 4-fold more frequently than normal tissue. To clarify if protein mistranslation plays a role in tumor biology, we expressed mutant Ser-tRNAs that misincorporate Ser-at-Ala (frequent error) and Ser-at-Leu (infrequent error) in NIH3T3 cells and investigated how they responded to the proteome instability generated by the amino acid misincorporations. There was high tolerance to both misreading tRNAs, but the Ser-to-Ala misreading tRNA was a more potent inducer of cell transformation, stimulated angiogenesis and produced faster growing tumors in mice than the Ser-to-Leu misincorporating tRNA. Upregulation of the Akt pathway and the UPR were also observed. Most surprisingly, the relative expression of both misreading tRNAs increased during tumor growth, suggesting that protein mistranslation is advantageous in cancer contexts. These data highlight new features of protein synthesis deregulation in tumor biology.

Impact of tRNA Modifications and tRNA-Modifying Enzymes on Proteostasis and Human Disease.

Transfer RNAs (tRNAs) are key players of protein synthesis, as they decode the genetic information organized in mRNA codons, translating them into the code of 20 amino acids. To be fully active, tRNAs undergo extensive post-transcriptional modifications, catalyzed by different tRNA-modifying enzymes. Lack of these modifications increases the level of missense errors and affects codon decoding rate, contributing to protein aggregation with deleterious consequences to the cell. Recent works show that tRNA hypomodification and tRNA-modifying-enzyme deregulation occur in several diseases where proteostasis is affected, namely, neurodegenerative and metabolic diseases. In this review, we discuss the recent findings that correlate aberrant tRNA modification with proteostasis imbalances, in particular in neurological and metabolic disorders, and highlight the association between tRNAs, their modifying enzymes, translational decoding, and disease onset.

Fungal Chitin Induces Trained Immunity in Human Monocytes during Cross-talk of the Host with Saccharomyces cerevisiae.

The immune system is essential to maintain the mutualistic homeostatic interaction between the host and its micro- and mycobiota. Living as a commensal,Saccharomyces cerevisiaecould potentially shape the immune response in a significant way. We observed thatS. cerevisiaecells induce trained immunity in monocytes in a strain-dependent manner through enhanced TNFα and IL-6 production upon secondary stimulation with TLR ligands, as well as bacterial and fungal commensals. Differential chitin content accounts for the differences in training properties observed among strains, driving induction of trained immunity by increasing cytokine production and direct antimicrobial activity bothin vitroandin vivo These chitin-induced protective properties are intimately associated with its internalization, identifying a critical role of phagosome acidification to facilitate microbial digestion. This study reveals how commensal and passenger microorganisms could be important in promoting health and preventing mucosal diseases by modulating host defense toward pathogens and thus influencing the host microbiota-immune system interactions.

Helicobacter pylori Activates Matrix Metalloproteinase 10 in Gastric Epithelial Cells via EGFR and ERK-mediated Pathways.

Helicobacter pylori colonizes the human stomach and increases the risk for peptic ulcer disease and gastric carcinoma. H. pylori upregulates the expression and activity of several matrix metalloproteinases (MMPs) in cell lines and in the gastric mucosa. The aim of this study was to explore the mechanisms leading to upregulation of MMP10 in gastric epithelial cells induced by H. pylori Infection of gastric cells with H. pylori led to an increase in levels of MMP-10 messenger RNA, protein secretion, and activity. cagA knockout mutants or CagA phosphorylation-defective mutants failed to increase MMP10 expression. These results were confirmed in infection experiments with clinical isolates with known cagA status and in human gastric biopsy specimens. Treatment of cells with chemical inhibitors of the receptor tyrosine kinase EGFR and the kinase Src abrogated H. pylori-induced MMP10 expression. Inhibitors of ERK1/2 and JNK kinases abolished and significantly decreased H. pylori-induced MMP10 expression, respectively, whereas inhibition of the kinase p38 had no effect. Finally, inhibition of MMP10 expression by small interfering RNA led to a decrease in the gastric cell-invasive phenotype mediated by the infection. In conclusion, CagA-positive H. pylori strains stimulate MMP10 expression. MMP-10 modulation occurs via EGFR activation in a process that involves Src, ERK, and JNK pathways. MMP-10 may be implicated in H. pylori-mediated extracellular matrix remodeling.

Comparison of the compatible solute pool of two slightly halophilic planctomycetes species, Gimesia maris and Rubinisphaera brasiliensis.

Gimesia maris and Rubinisphaera brasiliensis are slightly halophilic representatives of the deep-branching phylum Planctomycetes. For osmoadaptation both species accumulated α-glutamate, sucrose, ectoine and hydroxyectoine. A major role was found for ectoine, hydroxyectoine as well as sucrose under hyper-osmotic shock conditions. Nevertheless, the levels of sucrose were up-regulated by the increased salinity levels and also by low nitrogen availability. Additionally, G. maris accumulated glucosylglycerate (GG) as major solute specifically under low nitrogen levels, which prompted us to analyse the transcript abundance of two homologues genes known for the biosynthesis of GG, namely glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP). By qPCR using a suitable reference gene selected in this study, the transcript abundance of the biosynthetic genes was quantified in G. maris cells under hyper-osmotic shock or under low nitrogen conditions. The gpgS gene was induced under nitrogen-limiting conditions suggesting that GG synthesis is regulated primarily at the transcription level. Moreover, the expression of a gene coding for a putative sucrose-phosphorylase (Spase) located upstream the gpgS and gpgP genes was up-regulated, predicting a metabolic role of Spase probably related to GG synthesis.

Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification.

Many fungi restructured their proteomes through incorporation of serine (Ser) at thousands of protein sites coded by the leucine (Leu) CUG codon. How these fungi survived this potentially lethal genetic code alteration and its relevance for their biology are not understood. Interestingly, the human pathogen Candida albicans maintains variable Ser and Leu incorporation levels at CUG sites, suggesting that this atypical codon assignment flexibility provided an effective mechanism to alter the genetic code. To test this hypothesis, we have engineered C. albicans strains to misincorporate increasing levels of Leu at protein CUG sites. Tolerance to the misincorporations was very high, and one strain accommodated the complete reversion of CUG identity from Ser back to Leu. Increasing levels of Leu misincorporation decreased growth rate, but production of phenotypic diversity on a phenotypic array probing various metabolic networks, drug resistance, and host immune cell responses was impressive. Genome resequencing revealed an increasing number of genotype changes at polymorphic sites compared with the control strain, and 80% of Leu misincorporation resulted in complete loss of heterozygosity in a large region of chromosome V. The data unveil unanticipated links between gene translational fidelity, proteome instability and variability, genome diversification, and adaptive phenotypic diversity. They also explain the high heterozygosity of the C. albicans genome and open the door to produce microorganisms with genetic code alterations for basic and applied research.

Hyperpolarization-activated cyclic nucleotide-gated channels and cAMP-dependent modulation of exocytosis in cultured rat lactotrophs.

Hormone and neurotransmitter release from vesicles is mediated by regulated exocytosis, where an aqueous channel-like structure, termed a fusion pore, is formed. It was recently shown that second messenger cAMP modulates the fusion pore, but the detailed mechanisms remain elusive. In this study, we asked whether the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are activated by cAMP, are involved in the regulation of unitary exocytic events. By using the Western blot technique, a real-time PCR, immunocytochemistry in combination with confocal microscopy, and voltage-clamp measurements of hyperpolarizing currents, we show that HCN channels are present in the plasma membrane and in the membrane of secretory vesicles of isolated rat lactotrophs. Single vesicle membrane capacitance measurements of lactotrophs, where HCN channels were either augmented by transfection or blocked with an HCN channel blocker (ZD7288), show modulated fusion pore properties. We suggest that the changes in local cation concentration, mediated through HCN channels, which are located on or near secretory vesicles, have an important role in modulating exocytosis.