Pilot projects
One of the purposes of the Center was to stimulate interest in aging research at The Jackson Laboratory. This goal has been very successful. Before the Center, there was one scientist whose primary research was aging, and 4 others with projects in aging. Now, out of 32 staff, 25% are directly involved in the Shock/Ellison center and another 25% have aging related projects funded by the pilot project funds or funds from the Animal Core for dispersed colonies. Thus, fully 50% of the staff have some involvement with aging research projects.
The following pilot projects have been awarded: some of these extend over two years.
Mapping Bone and IGF-1 Quantitative Trait Loci Using the Collaborative Cross
Dr. Cheryl Ackert-Bicknell’s project goal is to genetically map quantitative trait loci (QTL) for two phenotypes that may be predictive of lifespan: serum IGF-1 and peak bone mineral density (BMD). It has been shown in a variety of species that low levels of IGF-1 are associated with increased lifespan, but low serum IGF-1 levels are also known to be associated with low BMD. While it is clear that low BMD in the elderly is associated with increased mortality due to fracture associated modalities, it is presumed that this low BMD is achieved after a period of prolonged bone loss. In mice, peak BMD is achieved at 16 weeks of age and out to 6 months of age, there is little drift in BMD from peak values. What is unclear is the relationship between peak BMD, serum IGF-1 at the age of peak BMD (i.e. BMD and IGF-1 in early adulthood) and the ability of these two measures to predict total lifespan. QTL for the phenotypes of BMD and serum IGF-1 have been roughly mapped using traditional F2 mapping crosses, actual gene identification using this approach has been less than fruitful. Studies in congenic mice have suggested that common genetic elements may co-regulate these two phenotypes. Furthermore, pilot studies conducted here at the Jackson Aging Center have suggested that QTL for median lifespan may overlap with these same BMD and IGF-1 QTL. The Collaborative Cross is a set of Recombinant Inbred (RI) strains derived from an eight-way mouse cross and is designed to maximized genetic diversity. Using innovative analysis techniques, we will be able to map QTL for peak BMD and serum IGF-1 with improved peak resolution over what was possible using traditional F2 mapping approaches, thus improving our future ability to identify the underlying genes. Hence, the central hypotheses of this proposal are that the improved analyses possible using The Collaborative Cross will allow for significant breakthroughs in: 1) the mapping of BMD and IGF-1 QTL, 2) the identification of common genetic elements controlling these two phenotypes and 3) may aid in the identification genes associated with lifespan.
Investigating Systematic IGF1 Isoform Variability
The objective of Joel H Graber’s pilot project is to investigate the regulation of IGF1, a critical regulatory molecule with demonstrated roles in diverse processes including, but not limited to, metabolism, protein synthesis, cell growth, cell differentiation, carcinogenesis, and aging. Despite many years of intensive study, the regulation of IGF1 has defied easy characterization. Measurement of IGF1 expression typically focuses on mature IGF1 peptide, a product of a complex expression process, including selection among multiple distinct transcripts, along with peptide cleavage of a progenitor protein. Previous studies have shown that expression of the mature, circulating IGF1 protein is negatively correlated with median lifespan in the mice under study. This correlation is imperfect however, and we hypothesize that differences in the isoforms of IGF1, and specifically differences in the balance between isoforms, may be contributing factor to the discrepancy. We will use a combination of qPCR and Northern assays to test for systematic variability in the distribution of isoforms across a selection of mouse strains, tissue types, and specimen ages, focusing on strains and tissues with relevance to aging. We will correlate these results with the phenotypic data already collected by the Jackson Aging Center to draw new hypotheses regarding the role of alternative isoforms in the expression of IGF1, and its role in aging.
Senescence Associated beta Galactosidase (SA-beta-gal) and the cell biology and demographics of cellular aging
Dr. Ken Paigen propose to use SA-beta-gal to address several issues in aging research: (a) is the increase in SA-beta-gal seen in senescent cells unique to this enzyme or is it an indirect marker of an expanding lysosomal space as cells age; (b) to what extent do organs and tissues age synchronously v. independently; (c) are rates of tissue aging measured by SA-beta-gal increases correlated with longevity, and (d) how are these rates subject to genetic variation. Our results will complement the work of Dr. Rong Yuan at The Jackson Laboratory who is examining SA-beta-gal histochemically in various tissues as a function of age. The enzyme assay approach we are taking provides the ability to evaluate SA-beta-gal responses quantitatively in large numbers of tissue samples, providing a common metric for evaluating aging across tissues, organs and inbred strains of mice. In a complementary manner, the histochemical approach examines individual cells and can determine whether tissue differences result from synchronous aging in all cells of the tissue, or from stochastic loss of cells resulting in diminished function.
The effects of physiologically elevated IGF-1 level on aging
The IGF-1 signaling pathway plays a key role in regulating longevity from yeast to mammals. To study the genetic regulation of IGF-1 in mouse, Dr. Rong Yuan surveyed IGF-1 levels at 6-, 12- and 18-month in 30 inbred strains, and found an inverse relationship between median life spans and 6-month plasma IGF-1 levels. C3H/HeJ (C3H), in particular, has a noticeably higher plasma IGF-1 level than C57BL/6J (B6) (366 vs. 262 ng/ml). Survival study shows C3H mice, both females and males, have relatively shorter life span than B6. Quantitative trait loci (QTL) analysis identified a significant QTL (Igfs1) located on chromosome (Chr) 10 that regulates plasma IGF-1 level. Cliff Rosen, at the Jackson laboratory, generated a congenic strain, B6.C3H-Igfs1, which contains the C3H alleles of this QTL on the B6 background. IGF-1 level is found significantly higher in B6.C3H-Igfs1 than B6. We propose to use B6.C3H-Igfs1 to test the hypothesis that elevated physiological IGF-1 will accelerate biological processes of aging and reduce life span.
Genetic regulation of IGF-1 and aging
Drs. Cliff Rosen and Wes Beamer are testing relationships between IGF-1 and aging, which have been suggested by studies in short-lived organisms. They have identified quantitative trait loci (QTL) that regulate serum IGF-I levels in F2 progeny of a cross between B6 and C3H/HeJ (C3H), and generated a congenic strain (B6.C3H-6T; hereafter 6T) by backcrossing a region from Chromosome (Chr) 6 of C3H onto a B6 background for 10 generations. In comparison with the B6 controls, the congenic 6T mice have a) 20% reduction in serum IGF-I; b) 50% less IGF-I mRNA transcripts in liver, bone, fat and muscle: c) reduced bone mineral density; d) enhanced adipose tissue within marrow and liver; and e) marked resistance to a high fat diet in respect to insulin production, glucose intolerance, and free fatty acid/ triglyceride levels. Drs. Rosen and Beamer will test the hypothesis that, in the congenic 6T mice, the modest reduction in circulating and tissue IGF-I over a lifetime will result in enhanced longevity in comparison to B6 controls. This will determine if the genetic determinant on Chr 6 that regulates IGF-I may also be an important anti-aging gene or genes. These researchers are aging groups of 6T and B6 female mice to obtain life span data in concert with comprehensive IGF-I phenotyping data at 6-month intervals to determine the relationship between IGF-I and longevity.
Anemia in aging mice
Dr. Luanne Peters is modeling genetic regulation of anemia in the elderly, a major health problem, focusing on models for the ~30% of cases in which the underlying cause is unknown. QTL analysis provides an unbiased approach to the identification of loci participating in interacting pathwaysthat may result in anemias of age. Inbred mice provide powerful tools for complex trait analysis. Moreover, a significant advantage of QTL mapping is that it focuses on genes that are likely to control key regulatory steps that provide attractive targets for therapeutic intervention. Erythropoiesis proceeds in a similar manner in humans and mice, and morphologically identical cell types are produced. The growth and transcription factors required for the establishment of the hematopoietic lineage and the structural components of red cells in mice and humans share striking similarities. Mice are prone to the same types of anemias as are humans, so anemias of aging may be regulated in some cases by homologous regulatory mechanisms. Strain characterization in the 32 Shock/Ellison center strains over a 2-year interval will provide the information required to dissect QTL involved in the idiopathic anemia of aging. Therefore, Dr. Peters’s goal is to identify strains that exhibit changes in key parameters related to anemia of unknown origin and its associated complications.Ten males and 10 females from each of the high priority MPP strains will be analyzed for a 24-month period for complete blood counts (CBCs) and serum chemistries. Because of their relatively high cost, cytokines will be measured on the top 10 MPP strains only, using quantitative ELISA assays. These analyses will provide the information required to choose the appropriate strains with which to establish F2 intercrosses for future QTL studies. The 2 strains showing the most divergent data for the largest number of phenotypes will be used as the parental strains to maximize the efficiency of the analysis. The QTL studies will identify chromosomal segments and, ultimately, genes influencing the phenotypes associated with anemia in aging.
Modeling motor neuron loss with inducible Choline acetyltransferase knockout mice
Dr. Rob Burgess is undertaking a study of motor performance following "genetic denervation" using a conditional allele of choline acetyltransferase (ChAT) that he made. ChAT is the enzyme responsible for the synthesis of the neurotransmitter acetylcholine (ACh), and its loss leads to the silencing of cholinerigic neurons, including motor neurons. Studies to date have used these mice to look at the developmental role of cholinergic activity at the neuromuscular junction (NMJ). He is now proposing to use these mice in combination with an inducible cre system (Cre-ER that responds to tamoxifen) to silence cholinergic transmission in subsets of motor neurons in adult animals. The tamoxifen responsive Cre is dose dependent, allowing a percentage of motor neurons to be silenced with each dose, and repeated doses will create an escalating severity of "denervation." The motor unit properties of these mice will be analyzed to determine how the remaining motor neurons compensate for the denervation and at what point that compensation fails. This is relevant to aging because the same phenomenon of happens in humans with age. This analysis will combine electrophysiological measures with an analysis of gross motor performance, measured using an automated gait analysis device. This study provides 3 major benefits: 1) It allows age-dependent motor neuron loss, and the accompanying compensation and changes in motor performance, to be efficiently modeled in younger mice. 2) It provides a "clinical" measure to go with the neuromuscular physiology. Third, using the ChAT mice as the proof of principle, it will determine the clinical presentation of motor neuron loss, which will enable gait analysis to be used as a screen to identify new models for neuromuscular dysfunction.
BCR-ABL cancer retarded by age-retarding procedures
Dr. Shaoguang Li is exploring relationships of aging and cancer using a mouse model of human Philadelphia chromosome-positive (Ph +) leukemias induced by the BCR-ABL oncogene. The model is established by transducing bone marrow cells with BCR-ABL retrovirus, and transplanting this marrow into lethally irradiated syngeneic recipient mice. Dr. Li will compare the disease latency and survival of the B-cell acute lymphocytic leukemic (B-ALL) mice between recipients that are lit/lit (little) or Snell dwarf mutants or that are treated with diet restriction. Preliminary results show that little mice (which fail to produce circulating growth hormone) receiving BCR-ABL-transduced wild-type bone marrow cells survive significantly longer than do normal control lit/+ mice receiving portions of the same BCR-ABL transduced cells. The development of leukemia appears to be delayed by the little mutation. Importantly, this mutation also increases life spans in C57BL/6J (B6) mice. The same mechanism that regulates rates of aging in vital biological systems may also regulate leukemia latency and growth rates. This hypothesis is supported when the little mutation that increases maximum life spans also increases leukemia survival. Interestingly, diet restriction may not retard leukemia in this model. If confirmed, these results distinguish diet restriction from the little mutation, help to focus work relating aging and cancer on pathways affected by growth hormone.
Effect of insulin resistance on life span in two new models of obesity
Dr. Ed Leiter is testing whether insulin sensitivity rather than absolute fat mass is the critical determinant driving life span extension. He uses a panel of recombinant congenic strains on the Non-Obese Non-Diabetic (NON/Lt) inbred background, developed by selective introgression of quantitative trait loci from the unrelated New Zealand Obese (NZO/HlLt) strain (a model of polygenic obesity produced by many additive or codominant genes). Two of these new strains, NONcNZO5 (Line 5) and NONcNZO10 (Line 10) are the focus. Line 10 males develop diabetes but neither Line 5 males nor females of either line do. Both males and females of Line 10 share the phenotype of glucose intolerance when young. The difference in Line 5 male diabetes resistance vs. Line 10 male susceptibility correlates with differential age-associated insulin secretory responses. Dr. Leiter’s project has 2 major goals: 1) To do endocrinologic profiling of Line 5 vs. Line 10 mice, analyzing changes in both insulin and IGF-1. 2) To compare survivorship within a 2-year aging period. Over the course of the aging period, mice will be compared for aging biomarkers tested in the Shock/Ellison center. This project tests the relative importance of insulin sensitivity and adiposity in aging. We predict that females of both strains will remain comparably adipose as they age, but that only Line 5 females will retain normal insulin sensitivity. Should life span differences be obtained, the results would argue that life span extension reflects induction of increased insulin sensitivity rather than a reduction in insulin signaling.
The role of chronic oxidative stress in neurodegenerative disease
Dr. Sue Ackerman is interested in the identification of novel genes that are necessary for survival of neurons in the aging brain (i.e., cause neurodegeneration when disrupted) and recently determined that the neurodegeneration observed in the cerebellum and retina of aging Harlequin (Hq) mutant mice is due to a mutation in the gene encoding the mitochondrial oxidoreductase, apoptosis inducing factor (AIF). The Hq mutation causes a dramatic downregulation of Aif transcripts and protein which in turn results in oxidative stress, a process known to occur in many late-onset neurodegenerative diseases including Parkinson’s disease and Alzheimer’s disease, demonstrating that oxidative stress can be causal in age-related neurodegeneration. Dr Ackerman will use Hq mutants to investigate the role of AIF and oxidative stress in the survival of dopaminergic neurons, the target neurons in Parkinson’s disease, by (1) testing whether the downregulation of AIF causes loss of dopaminergic neurons in aging Hq mice, and (2) whether oxidatively stressed dopaminergic neurons are more sensitive to cell death from MPTP. Finally, she will test whether transgenic mice overexpressing AIF will protect dopaminergic neurons from MPTP. The mouse mutant harlequin provides the first animal model of chronic oxidative stress that is accompanied by neurodegeneration; thus these studies are likely to suggest new opportunities for therapeutic interventions.
