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Healthspan is a complex trait, influenced by many genes and environmental factors that accelerate or delay aging, reduce or increase disease risk, and extend or reduce lifespan. Thus, assessing the role of genetic variation in aging requires an experimental strategy capable of modeling the genetic and biological complexity of human populations. The goal of the The Jackson Laboratory Nathan Shock Center (JAX NSC) is to provide research resources and training for geroscience investigators that seek to understand the role of genetics and genetic diversity on the fundamental process of aging and diseases of human aging using the laboratory mouse as a model system. The JAX NSC has available novel, deeply characterized populations of aged mice, performs state-of-the-art phenotyping of age-relevant traits, provides systems genetics analysis of complex data sets, and provides all of these resources to the geroscience community. The aged animal resources, phenotyping capacity, and genetic expertise available through the JAX NSC benefit the geroscience community by fostering cutting-edge, novel lines of research that otherwise would not be possible. Over the past 15 years, the JAX NSC has transformed aging research across the geroscience community, providing aging mouse resources and tissues to researchers. All JAX NSC data and tools are publicly disseminated on the Mouse Phenome Database and the JAX NSC website, thus ensuring that the resources generated and expertise acquired through the Center are readily available to the aging research community. The JAX NSC will continue to enhance its ability to perform innovative research using a mammalian model to illuminate novel genotype–phenotype relationships and provide a rational basis for designing effective risk assessments and therapeutic interventions to boost longevity and disease-free healthspan. [ABSTRACT FROM AUTHOR]

RASA3 is a Ras GTPase activating protein that plays a critical role in blood formation. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a missense mutation in Rasa3. Homozygotes present with a phenotype characteristic of bone marrow failure that is accompanied by alternating episodes of crisis and remission. The mechanism leading to impaired erythropoiesis and peripheral cell destruction as evidenced by membrane fragmentation in scat is unclear, although we previously reported that the mislocalization of RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the disease. In this study, we further characterized the bone marrow failure in scat and found that RASA3 plays a central role in cell cycle progression and maintenance of reactive oxygen species (ROS) levels during terminal erythroid differentiation, without inducing apoptosis of the precursors. In scat mice undergoing crises, there is a consistent pattern of an increased proportion of cells in the G0/G1 phase at the basophilic and polychromatophilic stages of erythroid differentiation, suggesting that RASA3 is involved in the G1 checkpoint. However, this increase in G1 is transient, and either resolves or becomes indiscernible by the orthochromatic stage. In addition, while ROS levels are normal early in erythropoiesis, there is accumulation of superoxide levels at the reticulocyte stage (DHE increased 40% in scat; p = 0.02) even though mitochondria, a potential source for ROS, are eliminated normally. Surprisingly, apoptosis is significantly decreased in the scat bone marrow at the proerythroblastic (15.3%; p = 0.004), polychromatophilic (8.5%; p = 0.01), and orthochromatic (4.2%; p = 0.02) stages. Together, these data indicate that ROS accumulation at the reticulocyte stage, without apoptosis, contributes to the membrane fragmentation observed in scat. Finally, the cell cycle defect and increased levels of ROS suggest that scat is a model of bone marrow failure with characteristics of aplastic anemia. [ABSTRACT FROM AUTHOR]

Dysregulation of splicing factor expression and altered alternative splicing are associated with aging in humans and other species, and also with replicative senescence in cultured cells. Here, we assess whether expression changes of key splicing regulator genes and consequent effects on alternative splicing are also associated with strain longevity in old and young mice, across 6 different mouse strains with varying lifespan (A/J, NOD.B10Sn-H2b/J, PWD.Phj, 129S1/SvlmJ, C57BL/6J and WSB/EiJ). Splicing factor expression and changes to alternative splicing were associated with strain lifespan in spleen and to a lesser extent in muscle. These changes mainly involved hnRNP splicing inhibitor transcripts with most changes more marked in spleens of young animals from long-lived strains. Changes in spleen isoform expression were suggestive of reduced cellular senescence and retained cellular proliferative capacity in long-lived strains. Changes in muscle isoform expression were consistent with reduced pro-inflammatory signalling in longer-lived strains. Two splicing regulators, HNRNPA1 and HNRNPA2B1, were also associated with parental longevity in humans, in the InCHIANTI aging study. Splicing factors may represent a driver, mediator or early marker of lifespan in mouse, as expression differences were present in the young animals of long-lived strains. Changes to alternative splicing patterns of key senescence genes in spleen and key remodelling genes in muscle suggest that correct regulation of alternative splicing may enhance lifespan in mice. Expression of some splicing factors in humans was also associated with parental longevity, suggesting that splicing regulation may also influence lifespan in humans. [ABSTRACT FROM AUTHOR]

Understanding the source of genetic variation in aging and using this variation to define the molecular mechanisms of healthy aging require deep and broad quantification of a host of physiological, morphological, and behavioral endpoints. The murine model is a powerful system in which to understand the relations across age-related phenotypes and to identify research models with variation in life span and health span. The Jackson Laboratory Nathan Shock Center of Excellence in the Basic Biology of Aging has performed broad characterization of aging in genetically diverse laboratory mice and has placed these data, along with data from several other major aging initiatives, into the interactive Mouse Phenome Database. The data may be accessed and analyzed by researchers interested in finding mouse models for specific aging processes, age-related health and disease states, and for genetic analysis of aging variation and trait covariation. We expect that by placing these data in the hands of the aging community that there will be (a) accelerated genetic analyses of aging processes, (b) discovery of genetic loci regulating life span, (c) identification of compelling correlations between life span and susceptibility for age-related disorders, and (d) discovery of concordant genomic loci influencing life span and aging phenotypes between mouse and humans. [ABSTRACT FROM AUTHOR]

To evaluate the effect of increased mouse density in a cage, mice were housed at the density recommended by the 1996 Guide for the Care and Use of Laboratory Animals and at densities that were approximately 2, 2.6, and 3 times greater. Five strains of mice (129S1/SvImJ, A/J, BALB/cByJ, C57BL/6J, and DBA/2J) were evaluated throughout 3- and 8-month timeframes for health and well-being, including mortality, cardiac measures, plasma cholesterol, body weight, bone mineral density, organ weights, hematology, behavioral observations, and open field and light–dark tests. For 22 of the 27 traits measured, increased housing density had no significant effect. Kidney weight, adrenal weight, and heart rate decreased as mice were housed more densely, and some of the decreases were statistically significant. Reduced kidney weight, adrenal weight, and heart rate are not considered to be negative outcomes and may even indicate reduced stress. However, all measurements of these three traits were within normal physiological ranges. Percent fat increased slightly in strains 129S1/SvImJ, A/J, and DBA/2J, but did not increase in strains BALB/cByJ, and C57BL/6J. These results indicate that mice can be housed at higher densities than those currently recommended. [ABSTRACT FROM AUTHOR]

Phenotype-driven approaches to gene discovery using inbred mice have been instrumental in identifying genetic determinants of inherited blood dyscrasias. The recessive mutant scat (severe combined anemia and thrombocytopenia) alternates between crisis and remission episodes, indicating an aberrant regulatory feedback mechanism common to erythrocyte and platelet formation. Here, we identify a missense mutation (G125V) in the scat Rasa3 gene, encoding a Ras GTPase activating protein (RasGAP), and elucidate the mechanism producing crisis episodes. The mutation causes mislocalization of RASA3 to the cytosol in scat red cells where it is inactive, leading to increased GTP-bound Ras. Erythropoiesis is severely blocked in scat crisis mice, and ∼94% succumb during the second crisis (∼30 d of age) from catastrophic hematopoietic failure in the spleen and bone marrow. Megakaryopoiesis is also defective during crisis. Notably, the scat phenotype is recapitulated in zebrafish when rasa3 is silenced. These results highlight a critical, conserved, and nonredundant role for RASA3 in vertebrate hematopoiesis. [ABSTRACT FROM AUTHOR]

Summary Spherocytosis is one of the most common inherited disorders, yet presents with a wide range of clinical severity. While several genes have been found mutated in patients with spherocytosis, the molecular basis for the variability in severity of haemolytic anaemia is not entirely understood. To identify candidate proteins involved in haemolytic anaemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in red blood cells (RBCs) from normal and β-adducin ( Add2) knock-out mice. We detected seven proteins that were decreased and 48 proteins that were increased in β-adducin null RBC ghosts. Since haemolytic anaemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Among the 48 proteins increased in Add2 knockout RBCs, only 11 were also increased in reticulocytes. Of the proteins decreased in Add2 knockout RBCs, α-adducin showed the greatest intensity difference, followed by SLC9A1, the sodium-hydrogen exchanger previously termed NHE1. We verified these mass spectrometry results by immunoblot. This is the first example of SLC9A1deficiency in haemolytic anaemia and suggests new insights into the mechanisms leading to fragile RBCs. [ABSTRACT FROM AUTHOR]

The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain βIll spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in βlll spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3; Mice lacking intact βlll spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most Spnb3-/- animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1,. GluRδ, IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide V and EAAT3 and increased calpain proteolysis of all spectrin. It appears that δlll spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y. [ABSTRACT FROM AUTHOR]

The breadth of genetic and phenotypic variation among inured strains is often underappreciated because assessments include only a limited number of strains. Evaluation of a larger collection of inbred strains provides not only a greater understanding of this variation but collectively mimics much of the variation observed in human populations. We used a high-throughput phenotyping protocol to measure females and males of 43 inbred strains for body composition (weight, fat, lean tissue mass, and bone mineral density), plasma triglycerides, high-density lipoprotein and total cholesterol, glucose, insulin, and leptin levels while mice consumed a high-fat, high-cholesterol diet. Mice were fed a chow diet until they were 6-8 wk old and then fed the high-fat diet for an additional 18 wk. As expected, broad phenotypic diversity was observed among these strains. Significant variation between the sexes was also observed for most traits measured. Additionally, the response to the high-fat diet differed considerably among many strains. By the testing of such a large set of inbred strains for many traits, multiple phenotypes can be considered simultaneously and thereby aid in the selection of certain inbred strains as models for complex human diseases. These data are publicly available in the web-accessible Mouse Phenome Database (http://www.jax.org/phenome), an effort established to promote systematic characterization of biochemical and behavioral phenotypes of commonly used and genetically diverse inbred mouse strains. Data generated by this effort builds on the value of inbred mouse strains as a powerful tool for biomedical research. [ABSTRACT FROM AUTHOR]

The mouse has been a powerful force in elucidating the genetic basis of human physiology and pathophysiology. From its beginnings as the model organism for cancer research and transplantation biology to the present, when dissection of the genetic basis of complex disease is at the forefront of genomics research, an enormous and remarkable mouse resource infrastructure has accumulated. This review summarizes those resources and provides practical guidelines for their use, particularly in the analysis of quantitative traits. [ABSTRACT FROM AUTHOR]

Part II. Examines the immune response of C57BL/6 mice on spaceflight. Body and lymphoid organ masses; Activation marker expression; Cytokine expression; Erythrocyte and thrombocyte characteristics.

Part I. Examines the immune response of C57BL/6 mice on spaceflight. Levels of general circulating leukocyte proportions; Splenic lymphocyte level; Concentration of T and B cells.

Reports on mouse models of cardiovascular disease. Review of high-throughput screens of mutagenized and inbred strains; Need for animal models that closely represent human disease; Establishment of the Center for New Mouse Models of Heart, Lung, Blood and Sleep Disorders at the Jackson Laboratory in Maine.

Abstract: The resting membrane potential of the human erythrocyte is largely determined by a constitutive Cl− conductance ∼100-fold greater than the resting cation conductance. The 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive electroneutral Cl− transport mediated by the human erythroid Cl−/HCO3 − exchanger, AE1 (SLC4A1, band 3) is >10,000-fold greater than can be accounted for by the Cl− conductance of the red cell. The molecular identities of conductive anion pathways across the red cell membrane remain poorly defined. We have examined red cell Cl− conductance in the Ae1 −/− mouse as a genetic test of the hypothesis that Ae1 mediates DIDS-sensitive Cl− conductance in mouse red cells. We report here that wildtype mouse red cell membrane potential resembles that of human red cells in the predominance of its Cl− conductance. We show with four technical approaches that the DIDS-sensitive component of erythroid Cl− conductance is reduced or absent from Ae1 −/− red cells. These results are consistent with the hypothesis that the Ae1 anion exchanger polypeptide can operate infrequently in a conductive mode. However, the fragile red cell membrane of the Ae1 −/− mouse red cell exhibits reduced abundance or loss of multiple polypeptides. Thus, loss of one or more distinct, DIDS-sensitive anion channel polypeptide(s) from the Ae1 −/− red cell membrane cannot be ruled out as an explanation for the reduced DIDS-sensitive anion conductance. [Copyright &y& Elsevier]

Abstract: Hemolytic anemia is one of the most common inherited disorders. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in RBCs from normal and beta-adducin (Add2) knock-out mice. We detected 7 proteins that were decreased and 48 proteins that were increased in the beta-adducin knock-out RBC ghost. Since hemolytic anemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Label-free analysis identified 47 proteins that were increased in the reticulocyte-enriched samples and 21 proteins that were decreased. Among the proteins increased in Add2 knockout RBCs, only 11 were also found increased in reticulocytes. Among the proteins decreased in Add2 knockout RBCs, beta- and alpha-adducin showed the greatest intensity difference, followed by NHE-1 (Slc9a1), the sodium–hydrogen exchanger. We verified these mass spectrometry results by immunoblot. This is the first example of a deficiency of NHE-1 in hemolytic anemia and suggests new insights into the mechanisms leading to fragile RBCs. Our use of label-free comparative proteomics to make this discovery demonstrates the usefulness of this approach as opposed to metabolic or chemical isotopic labeling of mice. [Copyright &y& Elsevier]

N-Myristoyltransferase (NMT) transfers myristate to an amino-terminal glycine of many eukaryotic proteins. In yeast, worms, and flies, this enzyme is essential for viability of the organism. Humans and mice possess two distinct but structurally similar enzymes, NMT1 and NMT2. These two enzymes have similar peptide specificities, but no one has examined the functional importance of the enzymes in vivo. To address this issue, we performed both genetic and biochemical studies. Northern blots with RNA from adult mice and in situ hybridization studies of day 13.5 embryos revealed widespread expression of both Nmt1 and Nmt2. To determine whether the two enzymes are functionally redundant, we generated Nmt1-deficient mice carrying a β-galactosidase marker gene. β-Galactosidase staining of tissues from heterozygous Nmt1-deficient (Nmt1+/-) mice and embryos confirmed widespread expression of Nmt1. Intercrosses of Nmt1+/- mice yielded no viable homozygotes (Nmt1-/-), and heterozygotes were born at a less than predicted frequency. Nmt1-/- embryos died between embryonic days 3.5 and 7.5. Northern blots revealed lower levels of Nmt2 expression in early development than at later time points, a potential explanation for the demise of Nmt1-/- embryos. To explore this concept, we generated Nmt1-/- embryonic stem (ES) cells. The Nmt2 mRNA could be detected in Nmt1-/- ES cells, but the total NMT activity levels were reduced by ∼95%, suggesting that Nmt2 contributes little to total enzyme activity levels in these early embryo cells. The Nmt1-/- ES cells were functionally abnormal; they yielded small embryoid bodies in in vitro differentiation experiments and did not contribute normally to organogenesis in chimeric mice. We conclude that Nmt1 is not essential for the viability of mammalian cells but is required for development, likely because it is the principal N-myristoyltransferase in early embryogenesis. [ABSTRACT FROM AUTHOR]

Objective: Hereditary spherocytosis (HS) is a heterogeneous group of spontaneously arising and inherited red blood cell disorders ranging from very mild subclinical cases to severe and life-threatening cases, with symptoms linked directly to the severity of the mutation at the molecular level. We investigated a novel mouse model in which the heterozygotes present with the diagnostic hallmarks of mild HS and surviving homozygotes phenocopy severe hemolytic HS. Materials and Methods: We used N-ethyl-N-nitrosourea mutagenesis to generate random point mutations in the mouse genome and a dominant screen to identify mouse models of human hematopoietic disease. Gene mapping of the HS strain revealed a unique in-frame nonsense mutation arising from a single base transversion in exon 27 of Ank1 (strain designation: Ank1 E924X). Employing conventional hematopoietic, pathological, biochemical, and cell biology assays, we characterized heterozygous and homozygous Ank1 E924X mice at the biochemical, cellular, and pathophysiological levels. Results: Although Ank1 E924X/E924X red blood cell ghosts lack abundant full-length ankyrin-1 isoforms, N-terminal epitope ankyrin-1 antibodies reveal a band consistent with the theoretical size of a truncated mutant ankyrin-1. Using domain-specific antibodies, we further show that this protein lacks both a spectrin-binding domain and a C-terminal regulatory domain. Finally, using antisera that detect C-terminal residues of the products of alternative Ank1 transcripts, we find unique immunoreactive bands not observed in red blood cell ghosts from wild-type or Ank1 E924X heterozygous mice, including a band similar in size to full-length ankyrin-1. Conclusions: The Ank1 E924X strain provides a novel tool to study Ank1 and model HS. [ABSTRACT FROM AUTHOR]