From Down Syndrome to the “Human” in “Human Genetics”**Previously presented at the annual meeting of The American Society of Human Genetics, in San Diego, on October 15, 2001.
2002; Elsevier BV; Volume: 70; Issue: 2 Linguagem: Inglês
10.1086/338915
ISSN1537-6605
Autores Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoI am deeply honored to receive the Allan Award from the American Society of Human Genetics and to join the ranks of the 38 illustrious awardees who have preceded me. The last time Arno Motulsky and I were both together on this podium, I was introducing him on his receipt of the Society's Education Award. That was a great pleasure for me and quite appropriate, because he was the teacher and I was the student. Now, things are the other way around, and it is an even greater pleasure for the student to be introduced by his teacher. I have been present at nearly all of the Allan Award addresses, the first one being given by Oliver Smithies as an after-dinner talk at the Society banquet in 1964. Oliver's presentation was memorable for me because he gave us a little flute concert after concluding his talk. For a fleeting second I thought that I might emulate him and play my 'cello for you—but you will be happy to hear that I thought better of it. There have been many other memorable talks over the years, and one that is still particularly vivid in my mind was given by Jerome Lejeune in 1969, ten years after the discovery of trisomy 21 (Lejeune Lejeune, 1970Lejeune J The William Allan Memorial Award lecture: on the nature of man.Am J Hum Genet. 1970; 22: 121-128PubMed Google Scholar). The 1969 meeting, which was in San Francisco, was notable for more that Lejeune's talk, since it had the added attraction of a San Francisco special—a gentle earthquake! More about all of this later. My first real exposure to human genetics came in conversations with Kurt Benirschke while I was in medical school. Kurt has been the legendary geneticist of the San Diego Zoo. It was from him that I learned about the sex life of nine-banded armadillos, which he allegedly kept in his basement, and heard about Tjio and Levan's work on the human karyotype and about the early discoveries of the chromosomal basis of Down syndrome and other disorders. However, my turn toward genetics as a career came about in a somewhat unusual way. When I appeared for the first time in Christian Anfinsen's laboratory in the National Heart Institute, I asked him what he wanted me to do. All that he said to me was, "Refold trypsin," and he then promptly went on a trip to Israel. What he meant was that I was supposed to reduce the six disulfide bonds of trypsin with mercaptoethanol, denature it completely in urea, and then see whether I could get it to reacquire enzymatic activity and, by inference, its native structure (fig. 1). All of this was by way of adding further support to Anfinsen's theory, for which he subsequently got the Nobel Prize, that the three-dimensional structure of a protein is determined by the linear sequence of amino acids in the polypeptide chain (Anfinsen Anfinsen, 1973Anfinsen CB Principles that govern the folding of protein chains.Science. 1973; 181: 223-230Crossref PubMed Scopus (4824) Google Scholar). While now part of the dogma of molecular biology, this was a very controversial issue at the time. Anfinsen's challenge to me and the work that ensued from it were my first introduction to genetics in action, since what was really being studied was the genetic control of protein structure. During the middle of my tenure at the NIH [the National Institutes of Health], I spent a year in Seattle as a fellow with Arno Motulsky. This was to be my only formal training in human and medical genetics. It was during this time that we began our work together—along with George Martin—on Werner syndrome. This work was stimulated by the woman in figure 2, who was 48 years old when the picture was taken. As hard as we tried to think about what the genetic defect might be (Epstein et al. Epstein et al., 1966Epstein CJ Martin GM Schultz AL Motulsky AG Werner's syndrome: a review of its symptomatology, natural history, pathologic features, genetics and relationship to the natural aging process.Medicine. 1966; 45: 177-221Crossref PubMed Scopus (716) Google Scholar), it took another thirty years before it was identified, by the Seattle group, as a mutation in a helicase gene (Yu et al. Yu et al., 1996Yu CE Oshima J Fu YH Wijsman EM Hisama F Alisch R Matthews S Nakura J Miki T Ouais S Martin GM Mulligan J Schellenberg GD Positional cloning of the Werner's syndrome gene.Science. 1996; 272: 258-262Crossref PubMed Scopus (1439) Google Scholar). Arno's mentorship has been instrumental in much that I have been able to accomplish since then. Both Chris Anfinsen and Arno Motulsky had a similar style of supervising their trainees—they led by example and by establishing expectations and standards of excellence. It was up to the trainees to figure out how to get things done. My time in Seattle convinced me that I did not want to be a protein chemist—even a genetic protein chemist—so, eventually, my wife, Lois, our two and a half children, and I headed west to San Francisco where I started down two parallel tracks. The first track involved a considerable gamble on my part. Without any prior experience or training, I switched to the mouse as an experimental system. To be more precise, I chose to work on the preimplantation mouse embryo (fig. 3). While still at the NIH, I had been very much taken by the work of many of the mouse developmental geneticists of the time—Salome Waelsch, Beatrice Mintz, Tibby Russell, and Dorothea Bennett were among the leaders. Of these, Beatrice Mintz was particularly influential in that she showed how both genetics and embryo manipulation could be used in combination to study various aspects of development. I was quite impressed by her ability to generate chimeric mice by the aggregation of early embryos, a technique that I was to use later in my own work (Epstein et al. Epstein et al., 1982bEpstein CJ Smith SA Zamora T Sawicki JA Magnuson TR Cox DR Production of viable adult trisomy 17 ↔ diploid mouse chimeras.Proc Natl Acad Sci USA. 1982b; 79: 4376-4380Crossref PubMed Scopus (24) Google Scholar) (fig. 4).Figure 4A trisomy 17 ↔ diploid chimera generated by preimplantation embryo aggregation, by the method developed by Beatrice Mintz (Mintz, 1972Mintz B Allophenic mice of multi-embryo origin.in: Daniel Jr, JC Methods in mammalian embryology. Freeman, San Francisco1972: 186-214Google Scholar). Reprinted, by permission, from Epstein et al. (Epstein et al., 1982bEpstein CJ Smith SA Zamora T Sawicki JA Magnuson TR Cox DR Production of viable adult trisomy 17 ↔ diploid mouse chimeras.Proc Natl Acad Sci USA. 1982b; 79: 4376-4380Crossref PubMed Scopus (24) Google Scholar). (© 1982 by the National Academy of Sciences, U.S.A.)View Large Image Figure ViewerDownload Hi-res image Download (PPT) To get into the preimplantation mouse system, I followed the lead of Ralph Brinster, who had been able to measure enzyme activities in just one or a few preimplantation mouse embryos (Brinster Brinster, 1965Brinster RL Lactate dehydrogenase activity in the preimplanted mouse embryo.Biochim Biophys Acta. 1965; 110: 439-441Crossref PubMed Google Scholar, Brinster, 1966Brinster RL Glucose-6-phosphate dehydrogenase activity in the preimplanted mouse embryo.Biochem J. 1966; 101: 161-163PubMed Google Scholar). Since I knew, from my earlier protein chemistry days, how to measure enzyme activities, this was a comfortable way to begin. The trick was to find really sensitive assays, and two that turned out to be particularly useful were for the X-linked enzymes: glucose-6-phosphate dehydrogenase and hypoxanthine-guanine phosphoribosyltransferase. Using these assays, we were able to show that embryonic X-inactivation starts at about the time of implantation, initially with the formation of the trophectoderm, and is reversed during oogenesis (fig. 5) (Epstein Epstein, 1969Epstein CJ Mammalian oocytes: X-chromosome activity.Science. 1969; 163: 1078-1079Crossref PubMed Scopus (101) Google Scholar, Epstein, 1972Epstein CJ Expression of the mammalian X-chromosome before and after fertilization.Science. 1972; 175: 1467-1468Crossref PubMed Scopus (80) Google Scholar, Epstein, 1981Epstein CJ Inactivation of the X-chromosome.in: Ritzen M Hall K Aperia A Larsson A Zetterburg A Zetterstrom R Biology of normal human growth. Raven, New York1981: 79-90Google Scholar; Epstein et al. Epstein et al., 1978Epstein CJ Smith S Travis B Tucker G Both X-chromosomes function prior to visible X-chromosome inactivation in female mouse embryos.Nature. 1978; 274: 500-503Crossref PubMed Scopus (146) Google Scholar). The effect of the lack of inactivation during oogenesis is to make the eggs from XO females functionally aneuploid and haploinsufficient, which, I believe, accounts for their premature degeneration. This work on the X chromosome was my first foray into matters of gene dosage. While all of this was going on, I was engaged in the second track of my academic life, as a clinical geneticist. In this capacity, I encountered the entire gamut of genetic disorders and birth defects and became involved in the establishment of a prenatal-diagnosis service and of several satellite genetic-counseling clinics. Of the many clinical disorders that I dealt with, the one that most captured my research attention was Down syndrome, and Edward Schneider, a postdoctoral fellow at the time, prepared matched pairs of trisomic and sibling fibroblasts for some early experiments (Schneider and Epstein Schneider and Epstein, 1972Schneider EL Epstein CJ Replication rate and lifespan of cultured fibroblasts in Down's syndrome.Proc Soc Exp Biol Med. 1972; 141: 1092-1094Crossref PubMed Scopus (77) Google Scholar). With these cells in hand, we were in a position to begin to look at the effects of a change in gene dosage in Down syndrome when Chris Tan, Jay Tischfield, and Frank Ruddle mapped the first two genes to human chromosome 21 in 1973 (Tan et al. Tan et al., 1973Tan YH Tischfield J Ruddle FH The linkage of genes for the human interferon-induced antiviral protein and indophenoloxidase-B traits to chromosome G-21.J Exp Med. 1973; 137: 317-330Crossref PubMed Scopus (211) Google Scholar). Although they had different names at the time, we now know these two genes to be SOD1, the gene for CuZn superoxide dismutase (SOD), and IFNAR1, the gene for the binding subunit of the interferon-alpha receptor. Because my wife, Lois, had by then become an internationally recognized expert on interferon and knew how to carry out the appropriate interferon-response assays, we chose to pursue the interferon-receptor gene first. Our investigations revealed that trisomy 21 cells bind more interferon—just about 50% more, in fact (fig. 6) (Epstein et al. Epstein et al., 1982aEpstein CJ McManus NH Epstein LB Branca AA D'Alessandro SB Baglioni C Direct evidence that the gene product of the human chromosome 21 locus, IFRC, is the interferon-α receptor.Biochem Biophys Res Commun. 1982a; 107: 1060-1066Crossref PubMed Scopus (40) Google Scholar), as was expected—and, as a result, are more sensitive to it (Epstein and Epstein Epstein and Epstein, 1976Epstein LB Epstein CJ Localization of the gene AVG for the antiviral expression of immune and classical interferon to the distal portion of the long arm of chromosome 21.J Infect Dis Suppl. 1976; 133: A56-A62Crossref PubMed Scopus (71) Google Scholar; Epstein et al. Epstein et al., 1980Epstein LB Lee SHS Epstein CJ Enhanced sensitivity of trisomy 21 monocytes to the maturation-inhibiting effects of interferon.Cell Immunol. 1980; 50: 191-194Crossref PubMed Scopus (25) Google Scholar). In fact, the trisomic cells could be as much as 10 times more sensitive to interferon (Weil et al. Weil et al., 1980Weil J Epstein LB Epstein CJ Synthesis of interferon-induced polypeptides in normal and chromosome 21-aneuploid human fibroblasts: relationship to relative sensitivities in antiviral assays.J Interferon Res. 1980; 1: 111-124Crossref PubMed Scopus (28) Google Scholar). This was taken to be a concrete demonstration of how a change in the dosage of a single gene of the degree occurring in trisomy 21 could have demonstrable functional consequences. (The actual situation may be somewhat more complicated because both the interferon-binding and signal-transducing subunits of the interferon-α receptor are encoded by contiguous loci on chromosome 21.) It was legitimate, therefore, to think in terms of the effects of the increased dosage of specific genes in producing the aneuploid phenotype. At this point, the two lines of research—mouse genetics and human trisomy 21—converged, with the bridge between the two being provided by the work of Alfred Gropp, a German pathologist interested in hunting and cytogenetics. These interests brought him to an isolated valley in the Italian Alps in which lives Mus poschiavinus, the so-called "tobacco mouse," which has only 26 chromosomes, rather than the usual 40, with seven pairs of Robertsonian fusion chromosomes (fig. 7). Gropp transferred many of these Robertsonians to regular laboratory mice and then devised a method for generating each of the mouse trisomies at a relatively high frequency (Gropp et al. Gropp et al., 1975Gropp A Kolbus U Giers D Systematic approach to the study of trisomy in the mouse. II.Cytogenet Cell Genet. 1975; 14: 42-62Crossref PubMed Scopus (163) Google Scholar). This made it possible to consider, for the first time, the development of a mouse model of human trisomy 21. There were several reasons for wanting to have such a model. For both practical and ethical reasons, we were restricted in our ability to study humans with Down syndrome. Only a few types of cells could be looked at, and there was no real access to the CNS. By contrast, all cells and tissues—including, in particular, the brain—could be studied in the mouse, and we would be able to exercise tight genetic control to reduce the effects of a variable background. And, although we did not know it at the time, we would eventually have the ability to manipulate the mouse genome almost at will. Finally, and perhaps of greatest importance, an appropriate model would permit potential therapeutic approaches based on a detailed knowledge of relationship between genotype and phenotype to be rationally designed and tested. We were ready to start on the development of a model, but there were two questions that had to be answered. First, given the number of chromosomal rearrangements that have occurred since the evolutionary divergence of humans and mice, would enough human chromosome 21 genes be syntenic in the mouse to make any single mouse trisomy a useful model? And, if so, which mouse chromosome most closely resembles human chromosome 21? The answers to both questions required the mapping of known human chromosome 21 genes into the mouse, and, when David Cox (then a fellow in my laboratory), Lois Epstein, and I began this work, we still knew of only two genes—the same SOD and interferon-receptor genes I mentioned earlier. However, good fortune smiled on our efforts, and it turned out that these two genes are both on mouse chromosome 16 (Cox et al. Cox et al., 1980Cox DR Epstein LB Epstein CJ Genes coding for sensitivity to interferon (IfRec) and soluble superoxide dismutase (SOD-1) are linked in mouse and man and map to mouse chromosome 16.Proc Natl Acad Sci USA. 1980; 77: 2168-2172Crossref PubMed Scopus (89) Google Scholar). With time, it has been shown that all of the genes on the long arm of human chromosome 21 down to the Mx locus in mid-21q22.3 are syntenic on mouse chromosome 16 (fig. 8). From examining the complete sequence of human chromosome 21, we now know that this corresponds to more than half of the ∼225 known and inferred genes on this chromosome (Roger Reeves, personal communication). The remaining human chromosome 21 genes are present on mouse chromosomes 10 and 17. However, from the work of Julie Korenberg, who had been analyzing the relationships between phenotype and genotype in the rare persons with segmental trisomy 21, we learned that many aspects of the Down syndrome phenotype—including dysmorphic changes in the craniofacies, hands, and feet; duodenal stenosis; hypotonia; lax ligaments; and mental retardation—can also occur with segmental trisomy—as in patients JS and KJ, in fig. 9—for just that region of human chromosome 21 that is in common with mouse chromosome 16.Figure 9Components of Down syndrome phenotype present in persons with segmental trisomy 21. The regions of trisomy in JS and KJ, indicated by the vertical bars, are quite similar to that present in the Ts65Dn mouse. Modified and reprinted, by permission, from Korenberg et al. (Korenberg et al., 1994Korenberg JR Chen X-N Schipper R Sun Z Gonsky R Gerwehr S Carpenter N Daumer C Dignan P Disteche C Graham Jr, JM Hudgins L McGillivray B Miyazaki K Ogasawara N Park JP Pagon R Pueschel S Sack G Say B Schuffenhauer S Soukup S Yamanaka T Down syndrome phenotypes: the consequences of chromosome imbalance.Proc Natl Acad Sci USA. 1994; 91: 4997-5001Crossref PubMed Scopus (561) Google Scholar). (© 1994 by the National Academy of Sciences, U.S.A.)View Large Image Figure ViewerDownload Hi-res image Download (PPT) All of this suggested that mouse trisomy 16 would be a valid genetic model for human trisomy 21, and the phenotype appeared to confirm this—congenital heart disease with an atrioventricular canal and conotruncal anomalies, thymic hypoplasia, delayed maturation of the immune system, craniofacial abnormalities, and midgestational edema (fig. 10) (Epstein et al. Epstein et al., 1985Epstein CJ Hofmeister BG Yee D Smith SA Philip R Cox DR Epstein LB Stem cell deficiencies and thymic abnormalities in fetal mouse trisomy 16.J Exp Med. 1985; 162: 695-712Crossref PubMed Scopus (24) Google Scholar; Epstein Epstein, 1986Epstein CJ The consequences of chromosome imbalance: principles, mechanisms, and models. Cambridge University Press, New York1986Crossref Google Scholar; Berger and Epstein Berger and Epstein, 1989Berger CN Epstein CJ Delayed thymocyte maturation in the trisomy 16 mouse fetus.J Immunol. 1989; 143: 389-396PubMed Google Scholar). If anything, the defects in the trisomic mouse were even more severe than in Down syndrome, and it was eventually realized that the trisomy 16 mouse was too good to be true—not because the human chromosome 21 genes were not there, but because too many other genes were! The principal problem is that mouse chromosome 16, in addition to having a large proportion of the human chromosome 21q genes, also has appreciable amounts of sequence homologous to regions of four other human chromosomes in its proximal 80% (fig. 8). Therefore, while still quite useful for studying various aspects of the effects of aneuploidy, complete mouse trisomy 16 was not the best model of human trisomy 21. What would be better would be a mouse with segmental trisomy 16—trisomic for just that region of mouse chromosome 16 that is homologous to human chromosome 21—and this is just what Muriel Davisson, at the Jackson Laboratory, was able to produce. The new mouse, designated "Ts65Dn" ("Ts65" for short), was the result of a radiation-induced translocation between parts of chromosomes 16 and 17 that gave rise to a tertiary trisomy (Reeves et al. Reeves et al., 1995Reeves RH Irving N Moran T Wohn A Kitt C Sisodia S Schmidt C Bronson R Davisson M A mouse model for Down syndrome exhibits learning and behavior deficits.Nat Genet. 1995; 11: 177-184Crossref PubMed Scopus (700) Google Scholar; Akeson et al. Akeson et al., 2001Akeson EC Lambert JP Narayanswami S Gardiner K Bechtel LJ Davisson MT Ts65Dn: localization of the translocation breakpoint and trisomic gene content in a mouse model for Down syndrome.Cytogenet Cell Genet. 2001; 93: 270-276Crossref PubMed Scopus (79) Google Scholar). This mouse is viable but tends to be small. Its immune system is in reasonably good shape, and there are no gross congenital malformations—in particular, no heart disease. As in human trisomy 21, the males are sterile, but it is possible to breed Ts65 mice, with some difficulty, from trisomic females, although not on an inbred background. How good is Ts65 as a model for Down syndrome? Quite good, I would say, and, in at least two areas, the CNS and the craniofacial skeleton, the Ts65 mouse exhibits very interesting and highly relevant abnormalities. Among the several abnormalities of the nervous system that have been found, we have been examining, in a longtime collaboration with William Mobley (formerly of UCSF [University of California–San Francisco] and now at Stanford), the status of the basal forebrain cholinergic neurons in the medial septal nucleus. These neurons are of particular interest because of their vulnerability in Alzheimer disease, a late consequence of trisomy 21. Mobley and his colleagues have shown that, in the trisomic brains, there is a decrease, with time, in the relative number and size of functional cholinergic neurons (Holtzman et al. Holtzman et al., 1993Holtzman DM Li YW Chen K Gage FH Epstein CJ Mobley WC Nerve growth factor reverses neuronal atrophy in a Down syndrome model of age-related neurodegeneration.Neurology. 1993; 43: 2668-2673Crossref PubMed Google Scholar, Holtzman et al., 1996Holtzman DM Santucci D Kilbridge J Chua-Couzens J Fontana DJ Daniels SE Johnson RM Chen K Sung Y Carlson E Alleva E Epstein CJ Mobley WC Developmental abnormalities and age-related neurodegeneration in a mouse model of Down syndrome.Proc Natl Acad Sci USA. 1996; 93: 13333-13338Crossref PubMed Scopus (368) Google Scholar; Cooper et al. Cooper et al., 2001Cooper J Salehi A Delcroix J-D Howe CL Belichenko PV Chua-Couzens J Kilbridge JK Carlson EJ Epstein CJ Mobley WC Failed retrograde transport of NGF in a mouse model of Down's syndrome: reversal of cholinergic neurodegenerative phenotypes following NGF infusion.Proc Natl Acad Sci USA. 2001; 98: 10439-10444Crossref PubMed Scopus (287) Google Scholar). Nerve growth factor (NGF) is a trophic factor made in the hippocampus that is transported by retrograde axonal transport to the cell bodies in the basal forebrain. When the rate of this retrograde transport was determined, it was found to be significantly decreased in the trisomic brain (Cooper et al. Cooper et al., 2001Cooper J Salehi A Delcroix J-D Howe CL Belichenko PV Chua-Couzens J Kilbridge JK Carlson EJ Epstein CJ Mobley WC Failed retrograde transport of NGF in a mouse model of Down's syndrome: reversal of cholinergic neurodegenerative phenotypes following NGF infusion.Proc Natl Acad Sci USA. 2001; 98: 10439-10444Crossref PubMed Scopus (287) Google Scholar). This finding suggested an experiment with obvious therapeutic implications. If the loss of cholinergic neurons is indeed the result of a deficiency of NGF, what effect would treatment with NGF have? This experiment was carried out by introducing NGF into the ventricles of the Ts65 mice through a cannula attached to an osmotic minipump, and the effect was quite gratifying (Cooper et al. Cooper et al., 2001Cooper J Salehi A Delcroix J-D Howe CL Belichenko PV Chua-Couzens J Kilbridge JK Carlson EJ Epstein CJ Mobley WC Failed retrograde transport of NGF in a mouse model of Down's syndrome: reversal of cholinergic neurodegenerative phenotypes following NGF infusion.Proc Natl Acad Sci USA. 2001; 98: 10439-10444Crossref PubMed Scopus (287) Google Scholar). The atrophy of the cholinergic neurons was partially reversed, as is indicated by the arrow in figure 11, and the number of neurons actually increased, indicating that, although they had disappeared functionally, they had not really disappeared anatomically. Dr. Mobley and his group are continuing these studies. In our studies of the interferon-response system that I described earlier, it was possible to demonstrate specific functional consequences of the increased dosage of just a single gene. Furthermore, an analysis of the specificity and reproducibility of the patterns of abnormalities in the various trisomies and monosomies, even in the face of individual variability, has convinced me that the aneuploid phenotypes that we see are truly the result of the particular genes that are genetically unbalanced when a duplication or deletion is present and are not the result of some kind of random noise or general loosening up of developmental homeostasis (Epstein Epstein, 1986Epstein CJ The consequences of chromosome imbalance: principles, mechanisms, and models. Cambridge University Press, New York1986Crossref Google Scholar). Therefore, although a phenotype is undoubtedly produced by the interaction of the effects—some large, some small—of many of the unbalanced genes, it is valid to investigate the contributions of individual loci to the phenotype. This conclusion suggested a second approach to the modeling of Down syndrome, and that was by investigating the imbalance of genes individually by the generation of transgenic mice. When Yoram Groner and his colleagues at the Weizmann Institute in Israel reported the cloning of the first human chromosome 21 gene—our old friend, SOD1—it became possible to do just that, and we collaborated to produce the first series of mice transgenic for CuZn SOD (Epstein et al. Epstein et al., 1987Epstein CJ Avraham KB Lovett M Smith S Elroy-Stein O Rotman G Bry C Groner Y Transgenic mice with increased CuZn-superoxide dismutase activity: an animal model of dosage effects in Down syndrome.Proc Natl Acad Sci USA. 1987; 84: 8044-8048Crossref PubMed Scopus (394) Google Scholar). Groner used these mice to look for abnormalities that might resemble those found in persons with Down syndrome, and he found several—abnormalities of the myoneural junctions in the tongue, an impairment in the uptake of serotonin by platelets, and a decreased synthesis of prostaglandins D2 and E2 (Groner Groner, 1995Groner Y Transgenic models for chromosome 21 gene dosage effects.Prog Clin Biol Res. 1995; 393: 193-212PubMed Google Scholar). We, on the other hand, set out to test Pierre-Marie Sinet's hypothesis that an increase in CuZn SOD activity might have deleterious effects on the nervous system because of an enhancement of the production of hydrogen peroxide from the superoxide that is generated within cells, the conversion of O2− to H2O2 being the function of the enzyme (Sinet Sinet, 1982Sinet P-M Metabolism of oxygen derivatives in Down's syndrome.Ann NY Acad Sci. 1982; 396: 83-94Crossref PubMed Scopus (166) Google Scholar). This proved not to be the case, at least in the mouse brain, but these studies led us in a direction entirely different from Down syndrome. Having these transgenic animals in hand, we became quite interested in the role of the SODs themselves—I use the plural because there are actually three different dismutases in mammals (CuZn SOD, Mn SOD, and EC SOD)—in longevity and in protecting against various forms of oxidative stress, and what we found is that, contrary to what was observed in fruit flies and proclaimed in the headlines a few years back (Kotulak and Gorner Kotulak and Gorner, 1992Kotulak R Gorner P Fruit flies provide hint on aging.Chicago Tribune. 1992; (February 9)Google Scholar), an increase in CuZn SOD activity by three- to fivefold did not prolong life span in transgenic mice (fig. 12) (Huang et al. Huang et al., 2000Huang TT Carlson EJ Gillespie AM Shi Y Epstein CJ Ubiquitous overexpression of CuZn superoxide dismutase does not extend life span in mice.J Gerontol A Biol Sci Med Sci. 2000; 55: B5-B9Crossref PubMed Google Scholar). However, studies carried out in collaboration with many different groups have indicated that, with just one exception (Fullerton et al. Fullerton et al., 1998Fullerton HJ Ditelberg JS Chen SF Sarco DP Chan PH Epstein CJ Ferriero DM Copper/zinc superoxide dismutase transgenic brain accumulates hydrogen peroxide after perinatal hypoxia ischemia.Ann Neurol. 1998; 44: 357-364Crossref PubMed Scopus (163) Google Scholar), increased CuZn SOD activity does protect, in a number of different experimental paradigms, against acute oxidative stress induced by a wide variety of physical and chemical agents (Huang et al. Huang et al., 1999Huang TT Carlson EJ Raineri I Gillespie AM Kozy H Epstein CJ The use of transgenic and mutant mice to study oxygen free radical metabolism.Ann NY Acad Sci. 1999; 893: 95-112Crossref PubMed Scopus (80) Google Scholar). To further this line of investigation, we decided to knock out Sod1, the gene for CuZn SOD. It should have been easy—and the technical aspects of the homologous recombination were—but when it came to generating a Sod1 null mouse from our knockout heterozygotes, we just could not seem to do it. Something was very wrong, but, fortunately, Haru Sago, a visiting fellow from Japan, found out what it was. In the course of generating the knockout, my colleague Ting-Ting Huang had inadvertently produced a reciprocal translocation between the end of chromosome 12 and the distal part of chromosome 16 just proximal to the knocked-out Sod1 gene (Sago et al. Sago et al., 1998Sago H Carlson EJ Smith DJ Kilbridge J Rubin EM Mobley WC Epstein CJ Huang T-T Ts1Cje, a new partial trisomy mouse model for Down syndrome, exhibits learning and behavioral abnormalities.Proc Natl Acad Sci USA. 1998; 95: 6256-6261Crossref PubMed Scopus (303) Google Scholar). This can be seen in the left panel of figure 13 in which the Gart gene, which is distal to Sod1, has moved—as the yellow arrow indicates—from end of chromosome 16 to the end of chromosome 12, whereas the more proximal App gene (fig. 13, middle panel), is still on chromosome 16, where it belongs. This was bad news for the SOD research—although Dr. Hu
Referência(s)