Biological and genetic determinants of glycolysis: Phosphofructokinase isoforms boost energy status of stored red blood cells and transfusion outcomes.

Mature red blood cells (RBCs) lack mitochondria and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine (HYPX) levels-and the genetic traits linked to them-were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes.

Quantitative trait loci mapping provides insights into the genetic regulation of dendritic cell numbers in mouse tissues.

To explore the influence of genetics on homeostatic regulation of dendritic cell (DC) numbers, we present a screen of DCs and their progenitors in lymphoid and non-lymphoid tissues in Collaborative Cross (CC) and Diversity Outbred (DO) mice. We report 30 and 71 loci with logarithm of the odds (LOD) scores >8.18 and ranging from 6.67 to 8.19, respectively. The analysis reveals the highly polygenic and pleiotropic architecture of this complex trait, including many of the previously identified genetic regulators of DC development and maturation. Two SNPs in genes potentially underlying variation in DC homeostasis, a splice variant in Gramd4 (rs235532740) and a missense variant in Orai3 (rs216659754), are confirmed by gene editing using CRISPR-Cas9. Gramd4 is a central regulator of DC homeostasis that impacts the entire DC lineage, and Orai3 regulates cDC2 numbers in tissues. Overall, the data reveal a large number of candidate genes regulating DC homeostasis in vivo.

Discovery of genomic loci for liver health and steatosis reveals overlap with glutathione redox genetics.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver condition in the United States, encompassing a wide spectrum of liver pathologies including steatosis, steatohepatitis, fibrosis, and cirrhosis. Despite its high prevalence, there are no medications currently approved by the Food and Drug Administration for the treatment of NAFLD. Recent work has suggested that NAFLD has a strong genetic component and identifying causative genes will improve our understanding of the molecular mechanisms contributing to NAFLD and yield targets for future therapeutic investigations. Oxidative stress is known to play an important role in NAFLD pathogenesis, yet the underlying mechanisms accounting for disturbances in redox status are not entirely understood. To better understand the relationship between the glutathione redox system and signs of NAFLD in a genetically-diverse population, we measured liver weight, serum biomarkers aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and graded liver pathology in a large cohort of Diversity Outbred mice. We compared hepatic endpoints to those of the glutathione redox system previously measured in the livers and kidneys of the same mice, and we screened for statistical and genetic associations using the R/qtl2 software. We discovered several novel genetic loci associated with markers of liver health, including loci that were associated with both liver steatosis and glutathione redox status. Candidate genes within each locus point to possible new mechanisms underlying the complex relationship between NAFLD and the glutathione redox system, which could have translational implications for future studies targeting NAFLD pathology.

Longitudinal fragility phenotyping contributes to the prediction of lifespan and age-associated morbidity in C57BL/6 and Diversity Outbred mice.

Aging studies in mammalian models often depend on natural lifespan data as a primary outcome. Tools for lifespan prediction could accelerate these studies and reduce the need for veterinary intervention. Here, we leveraged large-scale longitudinal frailty and lifespan data on two genetically distinct mouse cohorts to evaluate noninvasive strategies to predict life expectancy in mice. We applied a modified frailty assessment, the Fragility Index, derived from existing frailty indices with additional deficits selected by veterinarians. We developed an ensemble machine learning classifier to predict imminent mortality (95% proportion of life lived [95PLL]). Our algorithm represented improvement over previous predictive criteria but fell short of the level of reliability that would be needed to make advanced prediction of lifespan and thus accelerate lifespan studies. Highly sensitive and specific frailty-based predictive endpoint criteria for aged mice remain elusive. While frailty-based prediction falls short as a surrogate for lifespan, it did demonstrate significant predictive power and as such must contain information that could be used to inform the conclusion of aging experiments. We propose a frailty-based measure of healthspan as an alternative target for aging research and demonstrate that lifespan and healthspan criteria reveal distinct aspects of aging in mice.

Systems genetics uncover new loci containing functional gene candidates in Mycobacterium tuberculosis-infected Diversity Outbred mice.

Mycobacterium tuberculosis infects two billion people across the globe, and results in 8-9 million new tuberculosis (TB) cases and 1-1.5 million deaths each year. Most patients have no known genetic basis that predisposes them to disease. Here, we investigate the complex genetic basis of pulmonary TB by modelling human genetic diversity with the Diversity Outbred mouse population. When infected with M. tuberculosis, one-third develop early onset, rapidly progressive, necrotizing granulomas and succumb within 60 days. The remaining develop non-necrotizing granulomas and survive longer than 60 days. Genetic mapping using immune and inflammatory mediators; and clinical, microbiological, and granuloma correlates of disease identified five new loci on mouse chromosomes 1, 2, 4, 16; and three known loci on chromosomes 3 and 17. Further, multiple positively correlated traits shared loci on chromosomes 1, 16, and 17 and had similar patterns of allele effects, suggesting these loci contain critical genetic regulators of inflammatory responses to M. tuberculosis. To narrow the list of candidate genes, we used a machine learning strategy that integrated gene expression signatures from lungs of M. tuberculosis-infected Diversity Outbred mice with gene interaction networks to generate scores representing functional relationships. The scores were used to rank candidates for each mapped trait, resulting in 11 candidate genes: Ncf2, Fam20b, S100a8, S100a9, Itgb5, Fstl1, Zbtb20, Ddr1, Ier3, Vegfa, and Zfp318. Although all candidates have roles in infection, inflammation, cell migration, extracellular matrix remodeling, or intracellular signaling, and all contain single nucleotide polymorphisms (SNPs), SNPs in only four genes (S100a8, Itgb5, Fstl1, Zfp318) are predicted to have deleterious effects on protein functions. We performed methodological and candidate validations to (i) assess biological relevance of predicted allele effects by showing that Diversity Outbred mice carrying PWK/PhJ alleles at the H-2 locus on chromosome 17 QTL have shorter survival; (ii) confirm accuracy of predicted allele effects by quantifying S100A8 protein in inbred founder strains; and (iii) infection of C57BL/6 mice deficient for the S100a8 gene. Overall, this body of work demonstrates that systems genetics using Diversity Outbred mice can identify new (and known) QTLs and functionally relevant gene candidates that may be major regulators of complex host-pathogens interactions contributing to granuloma necrosis and acute inflammation in pulmonary TB.

Ferroptosis regulates hemolysis in stored murine and human red blood cells.

Red blood cell (RBC) metabolism regulates hemolysis during aging in vivo and in the blood bank. Here, we leveraged a diversity outbred mouse population to map the genetic drivers of fresh/stored RBC metabolism and extravascular hemolysis upon storage and transfusion in 350 mice. We identify the ferrireductase Steap3 as a critical regulator of a ferroptosis-like process of lipid peroxidation. Steap3 polymorphisms were associated with RBC iron content, in vitro hemolysis, and in vivo extravascular hemolysis both in mice and 13,091 blood donors from the Recipient Epidemiology and Donor evaluation Study. Using metabolite Quantitative Trait Loci analyses, we identified a network of gene products (FADS1/2, EPHX2 and LPCAT3) - enriched in donors of African descent - associated with oxylipin metabolism in stored human RBCs and related to Steap3 or its transcriptional regulator, the tumor protein TP53. Genetic variants were associated with lower in vivo hemolysis in thousands of single-unit transfusion recipients. Highlights: Steap3 regulates lipid peroxidation and extravascular hemolysis in 350 diversity outbred miceSteap3 SNPs are linked to RBC iron, hemolysis, vesiculation in 13,091 blood donorsmQTL analyses of oxylipins identified ferroptosis-related gene products FADS1/2, EPHX2, LPCAT3Ferroptosis markers are linked to hemoglobin increments in transfusion recipients.

Into the Wild: A novel wild-derived inbred strain resource expands the genomic and phenotypic diversity of laboratory mouse models.

The laboratory mouse has served as the premier animal model system for both basic and preclinical investigations for over a century. However, laboratory mice capture only a subset of the genetic variation found in wild mouse populations, ultimately limiting the potential of classical inbred strains to uncover phenotype-associated variants and pathways. Wild mouse populations are reservoirs of genetic diversity that could facilitate the discovery of new functional and disease-associated alleles, but the scarcity of commercially available, well-characterized wild mouse strains limits their broader adoption in biomedical research. To overcome this barrier, we have recently developed, sequenced, and phenotyped a set of 11 inbred strains derived from wild-caught Mus musculus domesticus. Each of these "Nachman strains" immortalizes a unique wild haplotype sampled from one of five environmentally distinct locations across North and South America. Whole genome sequence analysis reveals that each strain carries between 4.73-6.54 million single nucleotide differences relative to the GRCm39 mouse reference, with 42.5% of variants in the Nachman strain genomes absent from current classical inbred mouse strain panels. We phenotyped the Nachman strains on a customized pipeline to assess the scope of disease-relevant neurobehavioral, biochemical, physiological, metabolic, and morphological trait variation. The Nachman strains exhibit significant inter-strain variation in >90% of 1119 surveyed traits and expand the range of phenotypic diversity captured in classical inbred strain panels. These novel wild-derived inbred mouse strain resources are set to empower new discoveries in both basic and preclinical research.

Unraveling the genetics of arsenic toxicity with cellular morphology QTL.

The health risks that arise from environmental exposures vary widely within and across human populations, and these differences are largely determined by genetic variation and gene-by-environment (gene-environment) interactions. However, risk assessment in laboratory mice typically involves isogenic strains and therefore, does not account for these known genetic effects. In this context, genetically heterogenous cell lines from laboratory mice are promising tools for population-based screening because they provide a way to introduce genetic variation in risk assessment without increasing animal use. Cell lines from genetic reference populations of laboratory mice offer genetic diversity, power for genetic mapping, and potentially, predictive value for in vivo experimentation in genetically matched individuals. To explore this further, we derived a panel of fibroblast lines from a genetic reference population of laboratory mice (the Diversity Outbred, DO). We then used high-content imaging to capture hundreds of cell morphology traits in cells exposed to the oxidative stress-inducing arsenic metabolite monomethylarsonous acid (MMAIII). We employed dose-response modeling to capture latent parameters of response and we then used these parameters to identify several hundred cell morphology quantitative trait loci (cmQTL). Response cmQTL encompass genes with established associations with cellular responses to arsenic exposure, including Abcc4 and Txnrd1, as well as novel gene candidates like Xrcc2. Moreover, baseline trait cmQTL highlight the influence of natural variation on fundamental aspects of nuclear morphology. We show that the natural variants influencing response include both coding and non-coding variation, and that cmQTL haplotypes can be used to predict response in orthogonal cell lines. Our study sheds light on the major molecular initiating events of oxidative stress that are under genetic regulation, including the NRF2-mediated antioxidant response, cellular detoxification pathways, DNA damage repair response, and cell death trajectories.

Genetic regulation of carnitine metabolism controls lipid damage repair and aging RBC hemolysis in vivo and in vitro.

ABSTRACT: Recent large-scale multiomics studies suggest that genetic factors influence the chemical individuality of donated blood. To examine this concept, we performed metabolomics analyses of 643 blood units from volunteers who donated units of packed red blood cells (RBCs) on 2 separate occasions. These analyses identified carnitine metabolism as the most reproducible pathway across multiple donations from the same donor. We also measured l-carnitine and acyl-carnitines in 13 091 packed RBC units from donors in the Recipient Epidemiology and Donor Evaluation study. Genome-wide association studies against 879 000 polymorphisms identified critical genetic factors contributing to interdonor heterogeneity in end-of-storage carnitine levels, including common nonsynonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, and SLC16A9); carnitine synthesis (FLVCR1 and MTDH) and metabolism (CPT1A, CPT2, CRAT, and ACSS2), and carnitine-dependent repair of lipids oxidized by ALOX5. Significant associations between genetic polymorphisms on SLC22 transporters and carnitine pools in stored RBCs were validated in 525 Diversity Outbred mice. Donors carrying 2 alleles of the rs12210538 SLC22A16 single-nucleotide polymorphism exhibited the lowest l-carnitine levels, significant elevations of in vitro hemolysis, and the highest degree of vesiculation, accompanied by increases in lipid peroxidation markers. Separation of RBCs by age, via in vivo biotinylation in mice, and Percoll density gradients of human RBCs, showed age-dependent depletions of l-carnitine and acyl-carnitine pools, accompanied by progressive failure of the reacylation process after chemically induced membrane lipid damage. Supplementation of stored murine RBCs with l-carnitine boosted posttransfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality.

Longitudinal Fragility Phenotyping Predicts Lifespan and Age-Associated Morbidity in C57BL/6 and Diversity Outbred Mice.

Aging studies in mammalian models often depend on natural lifespan data as a primary outcome. Tools for lifespan prediction could accelerate these studies and reduce the need for veterinary intervention. Here, we leveraged large-scale longitudinal frailty and lifespan data on two genetically distinct mouse cohorts to evaluate noninvasive strategies to predict life expectancy in mice. We applied a modified frailty assessment, the Fragility Index, derived from existing frailty indices with additional deficits selected by veterinarians. We developed an ensemble machine learning classifier to predict imminent mortality (95% proportion of life lived [95PLL]). Our algorithm represented improvement over previous predictive criteria but fell short of the level of reliability that would be needed to make advanced prediction of lifespan and thus accelerate lifespan studies. Highly sensitive and specific frailty-based predictive endpoint criteria for aged mice remain elusive. While frailty-based prediction falls short as a surrogate for lifespan, it did demonstrate significant predictive power and as such must contain information that could be used to inform the conclusion of aging experiments. We propose a frailty-based measure of healthspan as an alternative target for aging research and demonstrate that lifespan and healthspan criteria reveal distinct aspects of aging in mice.

GenomeMUSter mouse genetic variation service enables multitrait, multipopulation data integration and analysis.

Hundreds of inbred mouse strains and intercross populations have been used to characterize the function of genetic variants that contribute to disease. Thousands of disease-relevant traits have been characterized in mice and made publicly available. New strains and populations including consomics, the collaborative cross, expanded BXD, and inbred wild-derived strains add to existing complex disease mouse models, mapping populations, and sensitized backgrounds for engineered mutations. The genome sequences of inbred strains, along with dense genotypes from others, enable integrated analysis of trait-variant associations across populations, but these analyses are hampered by the sparsity of genotypes available. Moreover, the data are not readily interoperable with other resources. To address these limitations, we created a uniformly dense variant resource by harmonizing multiple data sets. Missing genotypes were imputed using the Viterbi algorithm with a data-driven technique that incorporates local phylogenetic information, an approach that is extendable to other model organisms. The result is a web- and programmatically accessible data service called GenomeMUSter, comprising single-nucleotide variants covering 657 strains at 106.8 million segregating sites. Interoperation with phenotype databases, analytic tools, and other resources enable a wealth of applications, including multitrait, multipopulation meta-analysis. We show this in cross-species comparisons of type 2 diabetes and substance use disorder meta-analyses, leveraging mouse data to characterize the likely role of human variant effects in disease. Other applications include refinement of mapped loci and prioritization of strain backgrounds for disease modeling to further unlock extant mouse diversity for genetic and genomic studies in health and disease.

Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes.

Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in the blood bank. Here we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify an association between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs), hexokinase 1, ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice, and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine levels - and the genetic traits linked to them - were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes. Highlights: Blood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers;Ancestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis;Modeled PFKP effects relate to preventing loss of the total AXP pool in stored RBCs;ATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo.

Cross-sectional association between blood cholesterol and calcium levels in genetically diverse strains of mice.

Genetically diverse outbred mice allow for the study of genetic variation in the context of high dietary and environmental control. Using a machine learning approach, we investigated clinical and morphometric factors that associate with serum cholesterol levels in 840 genetically unique Diversity Outbred mice of both sexes (n = 417 male and 423 female), and on both a control chow (% kcals in diet: protein 22%, carbohydrate 62%, fat 16%, no cholesterol) and high fat high sucrose (% kcals in diet: protein 15%, carbohydrate 41%, fat 45%, 0.05% cholesterol). We find expected elevations of cholesterol in male mice, as well as in mice with elevated serum triglycerides and/or fed a high fat high sucrose diet. The third strongest predictor was serum calcium which correlated with serum cholesterol across both diets and sexes (r = 0.39-0.48) in both Diversity Outbred (P = 3.0 × 10-43 ) and BXD (P = 0.005) mice. This is in-line with several human cohort studies which show associations between calcium and cholesterol, and calcium as an independent predictor of cardiovascular events.

Cell morphology QTL reveal gene by environment interactions in a genetically diverse cell population.

Genetically heterogenous cell lines from laboratory mice are promising tools for population-based screening as they offer power for genetic mapping, and potentially, predictive value for in vivo experimentation in genetically matched individuals. To explore this further, we derived a panel of fibroblast lines from a genetic reference population of laboratory mice (the Diversity Outbred, DO). We then used high-content imaging to capture hundreds of cell morphology traits in cells exposed to the oxidative stress-inducing arsenic metabolite monomethylarsonous acid (MMAIII). We employed dose-response modeling to capture latent parameters of response and we then used these parameters to identify several hundred cell morphology quantitative trait loci (cmQTL). Response cmQTL encompass genes with established associations with cellular responses to arsenic exposure, including Abcc4 and Txnrd1, as well as novel gene candidates like Xrcc2. Moreover, baseline trait cmQTL highlight the influence of natural variation on fundamental aspects of nuclear morphology. We show that the natural variants influencing response include both coding and non-coding variation, and that cmQTL haplotypes can be used to predict response in orthogonal cell lines. Our study sheds light on the major molecular initiating events of oxidative stress that are under genetic regulation, including the NRF2-mediated antioxidant response, cellular detoxification pathways, DNA damage repair response, and cell death trajectories.

Into the Wild: A novel wild-derived inbred strain resource expands the genomic and phenotypic diversity of laboratory mouse models.

The laboratory mouse has served as the premier animal model system for both basic and preclinical investigations for a century. However, laboratory mice capture a narrow subset of the genetic variation found in wild mouse populations. This consideration inherently restricts the scope of potential discovery in laboratory models and narrows the pool of potentially identified phenotype-associated variants and pathways. Wild mouse populations are reservoirs of predicted functional and disease-associated alleles, but the sparsity of commercially available, well-characterized wild mouse strains limits their broader adoption in biomedical research. To overcome this barrier, we have recently imported, sequenced, and phenotyped a set of 11 wild-derived inbred strains developed from wild-caught Mus musculus domesticus. Each of these "Nachman strains" immortalizes a unique wild haplotype sampled from five environmentally diverse locations across North and South America: Saratoga Springs, New York, USA; Gainesville, Florida, USA; Manaus, Brazil; Tucson, Arizona, USA; and Edmonton, Alberta, Canada. Whole genome sequence analysis reveals that each strain carries between 4.73-6.54 million single nucleotide differences relative to the mouse reference assembly, with 42.5% of variants in the Nachman strain genomes absent from classical inbred mouse strains. We phenotyped the Nachman strains on a customized pipeline to assess the scope of disease-relevant neurobehavioral, biochemical, physiological, metabolic, and morphological trait variation. The Nachman strains exhibit significant inter-strain variation in >90% of 1119 surveyed traits and expand the range of phenotypic diversity captured in classical inbred strain panels alone. Taken together, our work introduces a novel wild-derived inbred mouse strain resource that will enable new discoveries in basic and preclinical research. These strains are currently available through The Jackson Laboratory Repository under laboratory code NachJ.

GenomeMUSter mouse genetic variation service enables multi-trait, multi-population data integration and analyses.

Hundreds of inbred laboratory mouse strains and intercross populations have been used to functionalize genetic variants that contribute to disease. Thousands of disease relevant traits have been characterized in mice and made publicly available. New strains and populations including the Collaborative Cross, expanded BXD and inbred wild-derived strains add to set of complex disease mouse models, genetic mapping resources and sensitized backgrounds against which to evaluate engineered mutations. The genome sequences of many inbred strains, along with dense genotypes from others could allow integrated analysis of trait - variant associations across populations, but these analyses are not feasible due to the sparsity of genotypes available. Moreover, the data are not readily interoperable with other resources. To address these limitations, we created a uniformly dense data resource by harmonizing multiple variant datasets. Missing genotypes were imputed using the Viterbi algorithm with a data-driven technique that incorporates local phylogenetic information, an approach that is extensible to other model organism species. The result is a web- and programmatically-accessible data service called GenomeMUSter ( https://muster.jax.org ), comprising allelic data covering 657 strains at 106.8M segregating sites. Interoperation with phenotype databases, analytic tools and other resources enable a wealth of applications including multi-trait, multi-population meta-analysis. We demonstrate this in a cross-species comparison of the meta-analysis of Type 2 Diabetes and of substance use disorders, resulting in the more specific characterization of the role of human variant effects in light of mouse phenotype data. Other applications include refinement of mapped loci and prioritization of strain backgrounds for disease modeling to further unlock extant mouse diversity for genetic and genomic studies in health and disease.

SEESAW: detecting isoform-level allelic imbalance accounting for inferential uncertainty.

Detecting allelic imbalance at the isoform level requires accounting for inferential uncertainty, caused by multi-mapping of RNA-seq reads. Our proposed method, SEESAW, uses Salmon and Swish to offer analysis at various levels of resolution, including gene, isoform, and aggregating isoforms to groups by transcription start site. The aggregation strategies strengthen the signal for transcripts with high uncertainty. The SEESAW suite of methods is shown to have higher power than other allelic imbalance methods when there is isoform-level allelic imbalance. We also introduce a new test for detecting imbalance that varies across a covariate, such as time.

Lipidomic QTL in Diversity Outbred mice identifies a novel function for α/ß hydrolase domain 2 (Abhd2) as an enzyme that metabolizes phosphatidylcholine and cardiolipin.

We and others have previously shown that genetic association can be used to make causal connections between gene loci and small molecules measured by mass spectrometry in the bloodstream and in tissues. We identified a locus on mouse chromosome 7 where several phospholipids in liver showed strong genetic association to distinct gene loci. In this study, we integrated gene expression data with genetic association data to identify a single gene at the chromosome 7 locus as the driver of the phospholipid phenotypes. The gene encodes α/ß-hydrolase domain 2 (Abhd2), one of 23 members of the ABHD gene family. We validated this observation by measuring lipids in a mouse with a whole-body deletion of Abhd2. The Abhd2KO mice had a significant increase in liver levels of phosphatidylcholine and phosphatidylethanolamine. Unexpectedly, we also found a decrease in two key mitochondrial lipids, cardiolipin and phosphatidylglycerol, in male Abhd2KO mice. These data suggest that Abhd2 plays a role in the synthesis, turnover, or remodeling of liver phospholipids.

Global and tissue-specific aging effects on murine proteomes.

Maintenance of protein homeostasis degrades with age, contributing to aging-related decline and disease. Previous studies have primarily surveyed transcriptional aging changes. To define the effects of age directly at the protein level, we perform discovery-based proteomics in 10 tissues from 20 C57BL/6J mice, representing both sexes at adult and late midlife ages (8 and 18 months). Consistent with previous studies, age-related changes in protein abundance often have no corresponding transcriptional change. Aging results in increases in immune proteins across all tissues, consistent with a global pattern of immune infiltration with age. Our protein-centric data reveal tissue-specific aging changes with functional consequences, including altered endoplasmic reticulum and protein trafficking in the spleen. We further observe changes in the stoichiometry of protein complexes with important roles in protein homeostasis, including the CCT/TriC complex and large ribosomal subunit. These data provide a foundation for understanding how proteins contribute to systemic aging across tissues.

Genetic architecture of heart mitochondrial proteome influencing cardiac hypertrophy.

Mitochondria play an important role in both normal heart function and disease etiology. We report analysis of common genetic variations contributing to mitochondrial and heart functions using an integrative proteomics approach in a panel of inbred mouse strains called the Hybrid Mouse Diversity Panel (HMDP). We performed a whole heart proteome study in the HMDP (72 strains, n=2-3 mice) and retrieved 848 mitochondrial proteins (quantified in ≥50 strains). High-resolution association mapping on their relative abundance levels revealed three trans-acting genetic loci on chromosomes (chr) 7, 13 and 17 that regulate distinct classes of mitochondrial proteins as well as cardiac hypertrophy. DAVID enrichment analyses of genes regulated by each of the loci revealed that the chr13 locus was highly enriched for complex-I proteins (24 proteins, P=2.2E-61), the chr17 locus for mitochondrial ribonucleoprotein complex (17 proteins, P=3.1E-25) and the chr7 locus for ubiquinone biosynthesis (3 proteins, P=6.9E-05). Follow-up high resolution regional mapping identified NDUFS4, LRPPRC and COQ7 as the candidate genes for chr13, chr17 and chr7 loci, respectively, and both experimental and statistical analyses supported their causal roles. Furthermore, a large cohort of Diversity Outbred mice was used to corroborate Lrpprc gene as a driver of mitochondrial DNA (mtDNA)-encoded gene regulation, and to show that the chr17 locus is specific to heart. Variations in all three loci were associated with heart mass in at least one of two independent heart stress models, namely, isoproterenol-induced heart failure and diet-induced obesity. These findings suggest that common variations in certain mitochondrial proteins can act in trans to influence tissue-specific mitochondrial functions and contribute to heart hypertrophy, elucidating mechanisms that may underlie genetic susceptibility to heart failure in human populations.