Proteomic and transcriptomic profiling reveal different aspects of aging in the kidney.

Little is known about the molecular changes that take place in the kidney during the aging process. In order to better understand these changes, we measured mRNA and protein levels in genetically diverse mice at different ages. We observed distinctive change in mRNA and protein levels as a function of age. Changes in both mRNA and protein are associated with increased immune infiltration and decreases in mitochondrial function. Proteins show a greater extent of change and reveal changes in a wide array of biological processes including unique, organ-specific features of aging in kidney. Most importantly, we observed functionally important age-related changes in protein that occur in the absence of corresponding changes in mRNA. Our findings suggest that mRNA profiling alone provides an incomplete picture of molecular aging in the kidney and that examination of changes in proteins is essential to understand aging processes that are not transcriptionally regulated

Genome-wide transcript and protein analysis highlights the role of protein homeostasis in the aging mouse heart.

Investigation of the molecular mechanisms of aging in the human heart is challenging because of confounding factors, such as diet and medications, as well as limited access to tissues from healthy aging individuals. The laboratory mouse provides an ideal model to study aging in healthy individuals in a controlled environment. However, previous mouse studies have examined only a narrow range of the genetic variation that shapes individual differences during aging. Here, we analyze transcriptome and proteome data from 185 genetically diverse male and female mice at ages 6, 12, and 18 mo to characterize molecular changes that occur in the aging heart. Transcripts and proteins reveal activation of pathways related to exocytosis and cellular transport with age, whereas processes involved in protein folding decrease with age. Additional changes are apparent only in the protein data including reduced fatty acid oxidation and increased autophagy. For proteins that form complexes, we see a decline in correlation between their component subunits with age, suggesting age-related loss of stoichiometry. The most affected complexes are themselves involved in protein homeostasis, which potentially contributes to a cycle of progressive breakdown in protein quality control with age. Our findings highlight the important role of post-transcriptional regulation in aging. In addition, we identify genetic loci that modulate age-related changes in protein homeostasis, suggesting that genetic variation can alter the molecular aging process

Multi-omics analysis identifies drivers of protein phosphorylation.

BACKGROUND: Phosphorylation of proteins is a key step in the regulation of many cellular processes including activation of enzymes and signaling cascades. The abundance of a phosphorylated peptide (phosphopeptide) is determined by the abundance of its parent protein and the proportion of target sites that are phosphorylated. RESULTS: We quantified phosphopeptides, proteins, and transcripts in heart, liver, and kidney tissue samples of mice from 58 strains of the Collaborative Cross strain panel. We mapped ~700 phosphorylation quantitative trait loci (phQTL) across the three tissues and applied genetic mediation analysis to identify causal drivers of phosphorylation. We identified kinases, phosphatases, cytokines, and other factors, including both known and potentially novel interactions between target proteins and genes that regulate site-specific phosphorylation. Our analysis highlights multiple targets of pyruvate dehydrogenase kinase 1 (PDK1), a regulator of mitochondrial function that shows reduced activity in the NZO/HILtJ mouse, a polygenic model of obesity and type 2 diabetes. CONCLUSIONS: Together, this integrative multi-omics analysis in genetically diverse CC strains provides a powerful tool to identify regulators of protein phosphorylation. The data generated in this study provides a resource for further exploration

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, eluci- dating mechanisms that may underlie genetic susceptibility to heart failure in human populations

Analysis of the role of Hspg2 gene in the variability of skeletal and vascular phenotypes in Martan Syndrome

A Síndrome de Marfan (SMF) é uma doença que afeta o tecido conjuntivo, de caráter autossômico dominante. Acomete cerca de 1 em 5.000 indivíduos e é causada por mutações no gene FBN1, que codifica a proteína de matriz extracelular fibrilina-1. As principais manifestações clínicas incluem aneurismas e dissecções da aorta, e deformidades esqueléticas, como crescimento excessivo dos ossos longos e escoliose. Apesar de ter penetrância completa, a SMF apresenta uma grande variabilidade clínica, inclusive entre indivíduos com a mesma mutação, sugerindo a existência de genes modificadores do fenótipo. Recentemente nosso grupo identificou loci associados a variabilidade fenotípica esquelética e vascular em um modelo murino da SMF. Dentre os genes localizados em um dos loci associado ao fenótipo vascular, identificamos o gene Hspg2, que codifica a proteína perlecan. O perlecan é um proteoglicano heparano-sulfato associado ao controle da composição da matriz extracelular, regulação da proliferação de células vasculares e da cartilagem, e que interage fisicamente com a fibrilina-1 na formação das microfibrilas e fibras elásticas. Assim, neste trabalho testamos a hipótese de que Hspg2 pode atuar como gene modificador da SMF. Para isso, foi analisada a expressão de Hspg2 em camundongos SMF em diferentes fundos genéticos, avaliando a associação entre níveis de expressão do gene e gravidade dos fenótipos; foi realizado RNA-seq de coluna espinal de camundongos graves e leves com o intuito de analisar a expressão diferencial de outros possíveis genes modificadores, bem como as vias moleculares envolvidas na doença. Além disso, nós geramos um modelo de camundongo SMF haploinsuficiente para o gene Hspg2 como ferramenta para investigações futuras mais aprofundadas do papel deste gene na fisiopatologia da SMF. Nós observamos que animais considerados graves para o fenótipo esquelético e vascular expressam menores quantidades de Hspg2 e que esta expressão é correlacionada a expressão de Fbn1. Isto sugere que Hspg2 pode apresentar uma função protetora dos fenótipos esquelético e vascular e evidencia a importante relação entre Fbn1 e Hspg2 na manutenção destes tecidos. A função de Hspg2 na modulação dos fenótipos da SMF poderá ser mais bem estudada no novo modelo de camundongo gerado neste trabalho, o que aumentará o conhecimento sobre a fisiopatologia da doença. Isso por sua vez, poderá levar ao desenvolvimento de novas estratégias terapêuticas para a SMF e outras doenças que envolvam os mesmos sistemasMarfan syndrome (MFS) is an autosomal dominant disease of the connective tissue which affects about 1 in 5,000 individuals, and is caused by mutations in FBN1 gene, which encodes the extracellular matrix protein fibrillin-1. The main clinical manifestations include aneurysms and aortic rupture and skeletal phenotypes, such as excessive growth of bones and scoliosis. Despite complete penetrance, MFS has a large clinical variability, even between individuals with the same mutation, indicating the existence of modifier genes. Recently, our group has identified loci associated with skeletal and vascular variability in a murine model of the syndrome. Among the genes within one of the loci associated with the vascular phenotype, we identified the Hspg2 gene that encodes perlecan protein. Perlecan is a proteoglycan heparan sulfate associated with the control of the composition of the extracellular matrix, proliferation of vascular and cartilage cells, and that physically interacts with fibrillin-1 during microfibril and elastic fibers assembly. Thus, in this project we tested the hypothesis that Hspg2 can play a role as a modifier gene of MFS. To this, we measured Hspg2 expression in MFS mice with different genetic backgrounds, evaluating the association between gene expression levels and severity of phenotypes; we obtained RNA-seq data from spinal column from mildly and severely affected mice to analyze the differential expression of other candidate modifier genes, as well as the molecular pathways involved in the disease. Besides that, we generate a SMF mouse model carrying a haploinsufficient mutation in Hspg2 gene as a tool for future investigations about the role of this gene in the SMF physiopathology. We found that animals severely affected for both skeletal and vascular phenotypes showed lower Hspg2 levels and that Hspg2 expression is positively correlated to Fbn1 expression. This suggests that Hspg2 gene can play a protective role in skeletal and vascular phenotypes and highlights the important relation between Fbn1 and Hspg2 in the maintenance of these tissues. The function of Hspg2 in the modulation of MFS phenotypes will be further studied in the new mouse model generated in this work, which will increase our knowledge about the physiopathology of the disease. This in turn may lead to the development of new therapeutic strategies for MFS and other diseases involving the same system

Analysis of the role of Hspg2 gene in the variability of skeletal and vascular phenotypes in Martan Syndrome

A Síndrome de Marfan (SMF) é uma doença que afeta o tecido conjuntivo, de caráter autossômico dominante. Acomete cerca de 1 em 5.000 indivíduos e é causada por mutações no gene FBN1, que codifica a proteína de matriz extracelular fibrilina-1. As principais manifestações clínicas incluem aneurismas e dissecções da aorta, e deformidades esqueléticas, como crescimento excessivo dos ossos longos e escoliose. Apesar de ter penetrância completa, a SMF apresenta uma grande variabilidade clínica, inclusive entre indivíduos com a mesma mutação, sugerindo a existência de genes modificadores do fenótipo. Recentemente nosso grupo identificou loci associados a variabilidade fenotípica esquelética e vascular em um modelo murino da SMF. Dentre os genes localizados em um dos loci associado ao fenótipo vascular, identificamos o gene Hspg2, que codifica a proteína perlecan. O perlecan é um proteoglicano heparano-sulfato associado ao controle da composição da matriz extracelular, regulação da proliferação de células vasculares e da cartilagem, e que interage fisicamente com a fibrilina-1 na formação das microfibrilas e fibras elásticas. Assim, neste trabalho testamos a hipótese de que Hspg2 pode atuar como gene modificador da SMF. Para isso, foi analisada a expressão de Hspg2 em camundongos SMF em diferentes fundos genéticos, avaliando a associação entre níveis de expressão do gene e gravidade dos fenótipos; foi realizado RNA-seq de coluna espinal de camundongos graves e leves com o intuito de analisar a expressão diferencial de outros possíveis genes modificadores, bem como as vias moleculares envolvidas na doença. Além disso, nós geramos um modelo de camundongo SMF haploinsuficiente para o gene Hspg2 como ferramenta para investigações futuras mais aprofundadas do papel deste gene na fisiopatologia da SMF. Nós observamos que animais considerados graves para o fenótipo esquelético e vascular expressam menores quantidades de Hspg2 e que esta expressão é correlacionada a expressão de Fbn1. Isto sugere que Hspg2 pode apresentar uma função protetora dos fenótipos esquelético e vascular e evidencia a importante relação entre Fbn1 e Hspg2 na manutenção destes tecidos. A função de Hspg2 na modulação dos fenótipos da SMF poderá ser mais bem estudada no novo modelo de camundongo gerado neste trabalho, o que aumentará o conhecimento sobre a fisiopatologia da doença. Isso por sua vez, poderá levar ao desenvolvimento de novas estratégias terapêuticas para a SMF e outras doenças que envolvam os mesmos sistemasMarfan syndrome (MFS) is an autosomal dominant disease of the connective tissue which affects about 1 in 5,000 individuals, and is caused by mutations in FBN1 gene, which encodes the extracellular matrix protein fibrillin-1. The main clinical manifestations include aneurysms and aortic rupture and skeletal phenotypes, such as excessive growth of bones and scoliosis. Despite complete penetrance, MFS has a large clinical variability, even between individuals with the same mutation, indicating the existence of modifier genes. Recently, our group has identified loci associated with skeletal and vascular variability in a murine model of the syndrome. Among the genes within one of the loci associated with the vascular phenotype, we identified the Hspg2 gene that encodes perlecan protein. Perlecan is a proteoglycan heparan sulfate associated with the control of the composition of the extracellular matrix, proliferation of vascular and cartilage cells, and that physically interacts with fibrillin-1 during microfibril and elastic fibers assembly. Thus, in this project we tested the hypothesis that Hspg2 can play a role as a modifier gene of MFS. To this, we measured Hspg2 expression in MFS mice with different genetic backgrounds, evaluating the association between gene expression levels and severity of phenotypes; we obtained RNA-seq data from spinal column from mildly and severely affected mice to analyze the differential expression of other candidate modifier genes, as well as the molecular pathways involved in the disease. Besides that, we generate a SMF mouse model carrying a haploinsufficient mutation in Hspg2 gene as a tool for future investigations about the role of this gene in the SMF physiopathology. We found that animals severely affected for both skeletal and vascular phenotypes showed lower Hspg2 levels and that Hspg2 expression is positively correlated to Fbn1 expression. This suggests that Hspg2 gene can play a protective role in skeletal and vascular phenotypes and highlights the important relation between Fbn1 and Hspg2 in the maintenance of these tissues. The function of Hspg2 in the modulation of MFS phenotypes will be further studied in the new mouse model generated in this work, which will increase our knowledge about the physiopathology of the disease. This in turn may lead to the development of new therapeutic strategies for MFS and other diseases involving the same system

Proteomic and transcriptomic profiling reveal different aspects of aging in the kidney

Little is known about the molecular changes that take place in the kidney during the aging process. In order to better understand these changes, we measured mRNA and protein levels in genetically diverse mice at different ages. We observed distinctive change in mRNA and protein levels as a function of age. Changes in both mRNA and protein are associated with increased immune infiltration and decreases in mitochondrial function. Proteins show a greater extent of change and reveal changes in a wide array of biological processes including unique, organ-specific features of aging in kidney. Most importantly, we observed functionally important age-related changes in protein that occur in the absence of corresponding changes in mRNA. Our findings suggest that mRNA profiling alone provides an incomplete picture of molecular aging in the kidney and that examination of changes in proteins is essential to understand aging processes that are not transcriptionally regulated

Induced Pluripotent Stem Cell for the Study and Treatment of Sickle Cell Anemia

Sickle cell anemia (SCA) is a monogenic disease of high mortality, affecting millions of people worldwide. There is no broad, effective, and safe definitive treatment for SCA, so the palliative treatments are the most used. The establishment of an in vitro model allows better understanding of how the disease occurs, besides allowing the development of more effective tests and treatments. In this context, iPSC technology is a powerful tool for basic research and disease modeling, and a promise for finding and screening more effective and safe drugs, besides the possibility of use in regenerative medicine. This work obtained a model for study and treatment of SCA using iPSC. Then, episomal vectors were used for reprogramming peripheral blood mononuclear cells to obtain integration-free iPSC. Cells were collected from patients treated with hydroxyurea and without treatment. The iPSCP Bscd lines were characterized for pluripotent and differentiation potential. The iPSC lines were differentiated into HSC, so that we obtained a dynamic and efficient protocol of CD34+CD45+ cells production. We offer a valuable tool for a better understanding of how SCA occurs, in addition to making possible the development of more effective drugs and treatments and providing better understanding of widely used treatments, such as hydroxyurea

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