Molecular pathology endpoints useful for aging studies.

The first clinical trial aimed at targeting fundamental processes of aging will soon be launched (TAME: Targeting Aging with Metformin). In its wake is a robust pipeline of therapeutic interventions that have been demonstrated to extend lifespan or healthspan of preclinical models, including rapalogs, antioxidants, anti-inflammatory agents, and senolytics. This ensures that if the TAME trial is successful, numerous additional clinical trials are apt to follow. But a significant impediment to these trials remains the question of what endpoints should be measured? The design of the TAME trial very cleverly skirts around this based on the fact that there are decades of data on metformin in humans, providing unequaled clarity of what endpoints are most likely to yield a positive outcome. But for a new chemical entity, knowing what endpoints to measure remains a formidable challenge. For economy's sake, and to achieve results in a reasonable time frame, surrogate markers of lifespan and healthy aging are desperately needed. This review provides a comprehensive analysis of molecular endpoints that are currently being used as indices of age-related phenomena (e.g., morbidity, frailty, mortality) and proposes an approach for validating and prioritizing these endpoints.

Clinical relevance of transgenic mouse models for aging research.

Studies on transgenic mice have shown them to be useful models for human aging- and age-related diseases. Life span end points in yeast and Caenorhabditis elegans can identify highly conserved genes that promote longevity when their functions are lost and which can readily be manipulated in the mouse. Protein kinase A is an example of a highly conserved gene that has age-delaying effects when specific subunits are suppressed or removed in the mouse, suggesting that loss of function may be a rational pharmacologic target. Gain of function is also an attractive clinical approach because expression levels of some vital genes may decrease in an age-related manner. The antioxidant enzyme catalase can delay aging when the human gene is inserted into mitochondria of mice. Other antioxidant genes are of interest in this system, both individually and in combination with catalase. A challenging aspect is to determine how to deliver catalase, as well as other gene products, into the mitochondria in the clinical setting. A number of new and exciting genes will most likely be investigated as clinical antiaging targets as the result of a forward genetic life span screening approach in invertebrates and a reverse genetic life span approach in the mouse.

Mouse models of XRCC1 DNA repair polymorphisms and cancer.

DNA damage plays a major role in mutagenesis, carcinogenesis and aging. A gene that is emerging as an essential element in the repair of both damaged bases and single-strand breaks (SSB) is XRCC1. XRCC1 has been shown to have a large number of single-nucleotide polymorphisms (SNPs), several of which are being increasingly studied in cancer epidemiology investigations, in part because of their relative high frequency in the population. Although association trends with specific cancer types have occasionally been shown in a variety of ethnic backgrounds, there are often conflicting reports that weaken any substantial conclusions. The functional significance of these SNPs is still largely unknown. XRCC1 is an excellent prototype to provide a forum for determining how epidemiological cancer association studies with DNA repair gene polymorphisms can be validated or refuted. The focus is on the utilization of in silico data and biochemical studies in cell lines and existing mouse models to help provide a framework for the development of new mutant mouse lines that mimic human polymorphisms. These mouse lines will provide the next generation of mammalian tools for carcinogen exposure studies relevant to human cancer and variations in XRCC1, and provide the basis for investigating groups of genes and polymorphisms in an animal model.

Polymorphic variation in hOGG1 and risk of cancer: a review of the functional and epidemiologic literature.

The gene encoding human 8-oxoguanine glycosylase 1 (hOGG1) is involved in DNA base excision repair. The encoded DNA glycosylase excises 7,8-dihydro-8-oxoguanine (8-OHdG), a highly mutagenic base produced in DNA as a result of exposure to reactive oxygen species (ROS). Polymorphisms in this gene may alter glycosylase function and an individual's ability to repair damaged DNA, possibly resulting in genetic instability that can foster carcinogenesis. In order to elucidate the possible impact of polymorphisms in hOGG1, we performed a literature review of both functional and epidemiologic studies that assessed the effects of these polymorphisms on repair function, levels of oxidative DNA damage, or associations with cancer risk. Fourteen functional studies and 19 epidemiologic studies of breast, colon, esophageal, head and neck, lung, nasopharyngeal, orolaryngeal, prostate, squamous cell carcinoma of the head and neck (SCCHN), and stomach cancers were identified. Although the larger functional studies suggest reduced repair function with variant alleles in hOGG1, the evidence is generally inconclusive. There is some epidemiologic evidence that risk for esophageal, lung, nasopharyngeal, orolaryngeal, and prostate is related to hOGG1 genotype, whereas risk of breast cancer does not appear related. In studies that explored potential interactions with environmental factors, cancer risk for hOGG1 genotypes differed depending on exposure, especially for colon cancer. In summary, there is limited evidence that polymorphisms in hOGG1 affect repair function and carcinogenesis. Larger, well-designed functional and epidemiologic studies are needed to clarify these relationships, especially with respect to interactions with other DNA repair enzymes and interactions with environmental factors that increase carcinogenic load.

Generation of genetically altered mouse models for aging studies.

A number of mouse models have been identified and are being used for aging and age-associated disease research. However, the use of the genetically manipulated mouse model is still a relatively untapped resource for the study of the biology of aging. Genetically altered mice can be powerful tools for biology of aging research because gene expression can be controlled and correlated with established biomarkers. Standard transgene overexpression and gene targeting techniques were modified and used to generate 30 mouse lines during a 4-year period. These lines include models of Werner's syndrome (premature aging or progeria), Alzheimer's disease, other neurodegenerative condition, atherosclerosis, diabetes, immune dysfunction, musculoskeletal disorders, and oxidative stress. These new mouse models are providing additional insights into aging processes and will be useful for developing intervention strategies and collaborative interactions.

Transgenic and gene-targeted mouse lines for toxicology studies.

This unit contains an extensive tabular listing of transgenic and gene-targeted mice useful in toxicological studies. Each listing contains a brief description of the phenotype, relevant references, and where applicable, commercial sources.

Transgenic animals in toxicology.

Transgenic animals are a powerful tool in the exploration of the interaction of specific genes with the environment and/or toxins to understand the relation between biochemical and metabolic pathways and toxic effect. This overview discusses DNA construct design, methods for production of transgenic animals, including pronuclear fusion, gene targeting and chimera production, animal husbandry, and colony management.

Levels of DNA damage are unaltered in mice overexpressing human catalase in nuclei.

Two types of transgenic mice were generated to evaluate the role of hydrogen peroxide in the formation of nuclear DNA damage. One set of lines overexpresses wild-type human catalase cDNA, which is localized to peroxisomes. The other set overexpresses a human catalase construct that is targeted to the nucleus. Expression of the wild-type human catalase transgene was found in liver, kidney, skeletal muscle, heart, spleen, and brain with muscle and heart exhibiting the highest levels. Animals containing the nuclear-targeted construct had a similar pattern of expression with the highest levels in muscle and heart, but with lower levels in liver and spleen. In these animals, immunofluorescence detected catalase present in the nuclei of kidney, muscle, heart, and brain. Both types of transgenic animals had significant increases of catalase activities compared to littermate controls in most tissues examined. Despite enhanced activities of catalase, and its presence in the nucleus, there were no changes in levels of 8OHdG, a marker of oxidative damage to DNA. Nor were there differences in mutant frequencies at a Lac Z reporter transgene. This result suggests that in vivo levels of H(2)O(2) may not generate 8OHdG or other types of DNA damage. Alternatively, antioxidant defenses may be optimized such that additional catalase is unable to further protect nuclear DNA against oxidative damage.

Tissue specific expression of PKR protein kinase in aging B6D2F1 mice.

A decline in the rate of protein synthesis is a common biochemical change observed with aging in a wide variety of cells and organisms. The double stranded RNA-dependent protein kinase PKR has been shown to phosphorylate eukaryotic initiation factor 2 alpha (eIF-2alpha), a well-characterized factor for down-regulating protein synthesis, in response to environmental stress conditions. Therefore, we were interested in evaluating the role of PKR in the aging process. Tissues from 2- and 20-month-old B6D2F1 male mice were evaluated by Western blot analysis. PKR was detected in all tissues of aging mice confirming its ubiquitous nature. Tissues examined from young mice showed little evidence of PKR expression, suggesting an age-associated up-regulation. P58(IPK), a cellular inhibitor of PKR, was expressed in tissues from both age groups but to a greater extent in tissues of aging mice suggesting an up-regulation to control PKR activity. Hyperphosphorylated eIF-2alpha was increased in selected tissues from older mice compared with tissues from younger mice indicating a possible correlation between PKR expression and kinase function. The data suggest that translational activity is slowing down in a tissue specific manner during the aging process in mice, possibly as the result of increased levels of PKR, and could be a factor in the reduction of the rate of protein synthesis during senescence seen in specific tissues of many organisms.

Cellular Werner phenotypes in mice expressing a putative dominant-negative human WRN gene.

Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS). WS patients prematurely develop an aged appearance and various age-related disorders. We have generated transgenic mice expressing human WRN with a putative dominant-negative mutation (K577M-WRN). Primary tail fibroblast cultures from K577M-WRN mice showed three characteristics of WS cells: hypersensitivity to 4-nitroquinoline-1-oxide (4NQO), reduced replicative potential, and reduced expression of the endogenous WRN protein. These data suggest that K577M-WRN mice may provide a novel mouse model for the WS.

Predictive parameters of joint disease in DBA/1 transgenic mice.

We recently reported an accelerated onset of collagen-induced arthritis in DBAII mice overexpressing a T cell receptor Valpha11.1/Vbeta8.2 transgene as a preclinical animal model for age-associated T cell dysfunction. The accelerated onset is due to a transgenically sensitized T cell population that reacts to bovine type 11 collagen without prior in vivo sensitization. The model presents a readily observable distal joint phenotype that would allow preliminary aging and intervention studies to be evaluated by monitoring the presence or absence or degree of phenotypic expression of disease. In order to characterize clinical signs, we evaluated 69 transgenic mice in six different experiments for anticollagen antibody levels, and assigned each a modified arthritic score based on the degree of redness or swelling of the digital joints. We also correlated these parameters with signs of distress, including weight bearing, activity levels, and body posture. The average onset of disease was consistently within a 28 to 35-day period. The average arthritic score at the time of onset was 8. We found that none of the parameters predicted the onset of joint disease, but the modified scoring system reflected the severity of joint disease and predicted the degree of distress associated with the acute inflammation. The ability to determine the severity of joint disease by gross physical examination is a useful clinical feature because a numerical score is reflective of the degree of inflammation. Because the transgenic mouse model is a T cell-driven disease, the effect of aging on T cell activity can be monitored easily. In addition, the use of our modified arthritic scoring system makes it possible to conduct mouse experiments in a humane manner.

FVB/N (H2(q)) mouse is resistant to arthritis induction and exhibits a genomic deletion of T-cell receptor V beta gene segments.

Animal models of autoimmune diseases have been instrumental in advancing our understanding of autoimmunity in humans. Collagen-induced arthritis (CIA) in mice is an autoimmune disease model of rheumatoid arthritis. Susceptibility to CIA in mice is linked to genes of the major histocompatibility complex (MHC). CD4(+) T cells that express the T-cell receptor (TCR) Tcra-V11.1 and/or Tcrb-V8.2 play a key role in the pathogenesis of arthritis in the DBA/1 mouse (H2(q)). We identified an inbred mouse strain, FVB/NJ (H2(q)), that is resistant to arthritis induction and exhibits a genomic deletion of certain Tcrb-V gene segments. We report a novel polymerase chain reaction-based method for the rapid identification of new mouse strains that exhibit germline Tcrb-V gene deletions. We mapped for the first time both the 5' and 3' breakpoints of the Tcrb-V deletion in the FVB/NJ, SWR, SJL, C57L, and C57BR strains to within 1.1 kilobases. Since there is an association between a particular Tcra-V allele (Tcra-V11.1(d)) and arthritis susceptibility in H2(q) mouse strains, we examined the allelic polymorphisms of the Tcra-V11 gene subfamily members between the arthritis-susceptible DBA/1 mouse and the arthritis-resistant FVB/NJ mouse strain. The amino acid sequences of the Tcra-V11.1 alleles differ at two positions (codons 18 and 68). Therefore, the resistance of FVB/NJ mouse to arthritis induction may be due in part to Tcra-V11.1 coding sequence polymorphism and Tcrb-V8.2 gene segment deletion, as we have recently demonstrated in the case of SWR mouse strain.

T-cell receptor vbeta deletion and valpha polymorphism are responsible for the resistance of SWR mouse to arthritis induction.

Collagen type II-induced arthritis (CIA) develops in susceptible mouse strains after intradermal injections of type II collagen (CII) in complete Freund's adjuvant (CFA). Susceptibility to CIA in mice is linked to genes of the major histocompatibility complex (MHC). Although the SWR mouse has a susceptible MHC haplotype (H2q), it is resistant to CIA. SWR exhibits at least two known immunological defects: (1) it contains a germline deletion of about 50% of T-cell receptor (TCR) Vbeta-chain gene segments, and (2) SWR is deficient in complement component C5. It has been shown that T cells that express TCRValpha11.1 and TCRVbeta8.2 play a substantial role in the pathogenesis of arthritis in the DBA/1 mouse (H2q). We generated SWR transgenic (tg) mice to determine whether the expression of pathogenic Valpha11.1 and/or Vbeta8.2 transgenes would confer arthritis susceptibility. Arthritis was induced in the SWR TCRalphabeta tg mice, but not in SWR TCRbeta tg mice. To address the role of Valpha11.1 in arthritis susceptibility, we examined the allelic polymorphisms of the Tcra-V11-gene subfamily members between the arthritis susceptible DBA/1 mouse and the arthritis-resistant SWR mouse strain. The amino acid sequences of the Valpha11.1 alleles differ at two positions (codons 18 and 68). Accordingly, these two amino acid changes are sufficient to allow the production of pathogenic T cells in SWR mice. This is the first demonstration of the association of a particular Tcra-V allele and arthritis susceptibility in mice.

Expression of a type II collagen-specific TCR transgene accelerates the onset of arthritis in mice.

Animal models of autoimmune diseases have been instrumental in advancing our understanding of autoimmunity in humans. Collagen-induced arthritis in mice is an autoimmune disease model of rheumatoid arthritis, which is MHC class II restricted and CD4 T cell dependent. To better understand the fundamental role of T cells in arthritis, we have generated a transgenic mouse carrying the rearranged Valpha11.1 and Vbeta8.2 TCR chain genes isolated from a type II collagen (CII)-specific T cell hybridoma. Cell surface analysis indicated that Vbeta8.2 chain was expressed on the surface of nearly all peripheral T cells. Analysis of T cell subsets in transgenic mice revealed a profound skewing in peripheral T cells towards the CD4 population. Although peripheral T cells were not tolerant to CII and responded to CII stimulation in vitro, transgenic mice did not develop spontaneous arthritis. However, a rapid onset of arthritis with severe clinical signs was detected in transgenic mice after immunization with CII in complete Freund's adjuvant. Histological analysis of inflamed joints showed a great resemblance to arthritic joints in man. This unique transgenic mouse model provides valuable insights into the mechanism of arthritis and into potential specific immune interventions.

Transgenic mice over-expressing the C-99 fragment of betaPP with an alpha-secretase site mutation develop a myopathy similar to human inclusion body myositis.

Inclusion body myositis (IBM) is the most common muscle disease in the elderly. Amyloid-beta protein (A beta) has been shown to accumulate abnormally in the vacuolated fibers and to localize to amyloid-like fibrils in muscles from IBM patients. We studied the skeletal muscles from a line of transgenic mice over-expressing the carboxyl-terminal 99 amino acids (C99) of the beta-amyloid precursor protein (betaPP) with a substitution of lysine-612 to valine (K612V), intended to abolish alpha-secretase recognition and to preserve the A beta domain of C99. The majority (87%) of the 24-month-old transgenic mice showed myopathic changes, and approximately one-third of them had degenerating fibers with sarcoplasmic vacuoles and thioflavin-S-positive deposits. Ultrastructurally, the inclusions were aggregates of short thin amyloid-like fibrils, 6 to 8 nm in diameter. These features are similar to those of human IBM. Immunocytochemistry using an antibody against A beta showed membranous staining in most muscle fibers of transgenic mice, as well as granular or vacuolar cytoplasmic staining in the atrophic fibers. Western blots showed a high level of accumulation of carboxyl-terminal fragments of betaPP in the muscles of the transgenic mice with the most severe IBM-like lesions. The expression of IBM-like lesions was age dependent. These transgenic mice provide a model for the study of IBM and for the peripheral expression of a key element in the pathogenesis of Alzheimer disease.

Rapid detection of bovine type II collagen-specific T-cell hybridomas.

T-cell hybridomas are powerful tools in studying the fine specificities of antigen recognition by the T-cell receptor (TCR), the structure and genetic basis of the CD3-TCR complex, and the size of the TCR alpha/beta repertoire used in response to various antigens. A technical challenge in establishing T-cell hybridomas is the early identification of antigen-specific ones. We have established a rapid and efficient ELISA method for detecting antigen-specific T-cell hybridomas. Our ELISA technique significantly reduces the time and resources required for the primary screening of antigen-specific T-cell hybrids, eliminates the need of maintaining hundreds of rapidly growing nonspecific clones, and does not require the maintenance of IL-2/IL-4 dependent cell lines such as CTLL-2 or HT-2. In addition, the ELISA technique is designed to detect both types of CD4 T-cells: Th1 and Th2, by using a mixture of anti-IL-2 and anti-IL-4 monoclonal antibodies. Therefore, we believe that our ELISA technique provides a faster, less expensive, and higher throughput screening method for the early identification of antigen-specific T-cell hybridomas than the current bioassays.

SWR: an inbred strain suitable for generating transgenic mice.

Production of fertilized oocytes and generation of transgenic mice is generally more efficient using F2 hybrid embryos than embryos from inbred mice. Most F2 hybrids are of the C57BL/6 background because of its genetic and embryologic features. However, our goal of developing a transgenic mouse model for rheumatoid arthritis necessitated using a susceptible mouse strain such as DBA/1. We prepared alpha and beta T-cell receptor (TCR) chain gene constructs and microinjected them into embryos from DBA/1, SWR, (DBA/1 x SWR)F1, and (SWR x DBA/1)F1 strains. We found SWR female mice to be prolific ovulators in response to exogenous hormones, with oocyte numbers comparable to those produced by (C57BL/6 x C3H)F1 female mice. Embryos from the (SWR x DBA/1)F1 or SWR strain were large and had prominent pronuclei, whereas (DBA/1 x SWR)F1 embryos were smaller and had less visible pronuclei, similar to those of DBA/1 embryos. Therefore, the pronuclear size and visibility are features of the SWR female mice and are independent of the genotype of the fertilizing spermatozoa. Resistance to lysis after co-injection of alpha beta TCR constructs and the efficiency of generating DNA-positive founders were comparable in SWR, (SWR x DBA/1)F1, and (C57BL6 x C3H)F2 embryos. Thus, the SWR mouse is another inbred strain, in addition to the FVB inbred strain, found to be highly suitable for propagation of transgenes. Furthermore, the SWR mouse is well defined genetically, and SWR females have a high ovulation rate, comparable to that of F1 hybrid mice.

Characterization of the mouse Tcra-V22 gene subfamily.

T-cell receptors (Tcrs) of higher organisms play a key role in the specific recognition of self and non-self molecules in the immune system. The large number of Tcr variable (V) genes have been organized into V gene subfamilies according to their sequence similarity at the nucleotide and amino acid level. We cloned and characterized four new members of the Tcra-V22 gene subfamily at the genomic level using a simple and sensitive technique that can rapidly clone members of any multi-member gene family. Sequence analysis reveals that the four Tcra-V22 gene subfamily members have more than 98% sequence similarity in their coding regions, at the nucleotide and amino acid levels. However, the intron between the leader and the coding region varies up to 7% between members of the Tcra-V22 gene subfamily. Comparison of the multi-member Tcra-V22 gene subfamily with other multi-member Tcra-V gene subfamilies (V2, V8, and V11), shows that Tcra-V22 is unique in that it has multiple members with nearly identical amino acid sequence and which are not inherently pseudogenes. Sequence similarity analysis of the Tcra-V22 subfamily with the prototypes of all other Tcra-V subfamilies revealed that the Tcra-V22 subfamily has the closest sequence similarity to that of Tcra-V18 (77% at the nucleotide level and 71% at the amino acid level).