Neurobiology of Aging 20 (1999) 555–564 ␣MUPA mice: a transgenic model for increased life span૾ Ruth Miskina,*, Tamar Masosa, Shlomo Yahavb, Dimitri Shinderb, Amiela Globersona a Departments of Biological Chemistry and Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel b Institute of Animal Science, Agricultural Research Organization, Bet Dagan, Israel Abstract ␣MUPA is a line of transgenic mice that, compared with their wild type (WT) counterparts, spontaneously eat less (ϳ20%) and live longer (average ϳ20%), thus resembling dietary-restricted (DR) mice. Here, we show that body temperature was significantly reduced in ␣MUPA compared with WT throughout a wide range of ages. Plasma corticosterone was significantly higher in young ␣MUPA compared to young WT; however, it significantly declined in aged ␣MUPA, but not in aged WT. In addition, age-associated thymus involution occurred in ␣MUPA as it did in WT. Thus ␣MUPA mice appear to largely resemble, but also to somewhat differ from diet-restricted animals. We also report on four new transgenic lines that, like ␣MUPA, produced in the brain the mRNA that encodes the extracellular protease urokinase (uPA); however, transgenic uPA expression was most extensive and widespread in the ␣MUPA brain, where it also occurred in the hypothalamus. ␣MUPA was also the only line that ate less, but also showed another characteristic, high frequency leg muscle tremor seen only at unstable body states. We hypothesize that transgenic uPA in the brain could have caused the ␣MUPA phenotypic alterations. Thus ␣MUPA offers a unique transgenic model of inherently reduced eating to investigate the homeostatic state of delayed aging at the systemic and single-cell levels. © 1999 Elsevier Science Inc. All rights reserved. Keywords: ␣MUPA transgenic mice; Urokinase-type plasminogen activator; uPA; Longevity; Dietary restriction; Body temperature; Corticosterone; Muscle tremor; Hypothalamus 1. Introduction Life span can be prolonged in experimental animals by restricting food intake. This phenomenon, first discovered by McCay et al. [31], has been reproduced in a wide range of animal species and has been extensively studied in mice and rats. In these species, dietary restriction retards the decline of multiple, age-associated physiological processes and delays the onset of age-related diseases (for reviews, see 27,51,58,59,61). Ongoing experiments in nonhuman primates have also indicated several beneficial effects of dietary restriction [6,11,19,23]. However, the basic mechanisms underlying the anti-aging effect of dietary restriction are not clear. We previously described a line of transgenic mice, des- ૾ This research was funded by The Leo and Julia Forchheimer Center for Molecular Genetics and The Belle S. and Irving E. Meller Center for the Biology of Aging at the Weizmann Institute of Science (to R.M. and A.G.). * Corresponding author. Tel.: ϩ972-8-934-3150; fax: ϩ972-8-9344118. E-mail address: Ruth.Miskin@weizmann.ac.il (R. Miskin) ignated ␣MUPA, which phenotypically resemble dietaryrestricted (DR) mice [35]. Thus, compared with their wild type (WT) control, ␣MUPA mice spontaneously ate less when fed ad libitum (ϳ20%) and had an increased average life span (ϳ20%, the maximal life span has not been determined; however, the age of 10th percentile survivors was ϳ15% longer in ␣MUPA). In addition, ␣MUPA mice showed a reduction of body weight, body length, blood sugar, litter sizes, and birth frequencies. ␣MUPA mice also maintain an overall young look at advanced ages (see Fig. 1). ␣MUPA mice carry the entire cDNA of the murine urokinase-type plasminogen activator (uPA), including the coding sequence and ϳ1 kb 3ЈUTR, linked downstream from the promoter of the lens-specific ␣A-crystallin gene [34]. In situ hybridization experiments showed that, in addition to the expected transgenic expression in the lens, ␣MUPA mice produced uPA mRNA in neuronal cells in multiple brain regions [32,35], most of which were devoid of endogenous uPA mRNA [29], including the hypothalamus, which plays a central role in the control of feeding and energy homeostasis [2,13,36]. uPA is an ϳ48 kDa, secreted serine protease that specifically converts the abundant inactive zymogen plasmin- 0197-4580/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 7 - 4 2 8 0 ( 9 9 ) 0 0 0 9 3 - 7 556 R. Miskin et al. / Neurobiology of Aging 20 (1999) 555–564 Fig. 1. 30-month-old WT and ␣MUPA mice. The old ␣MUPA mouse maintained an overall young look and was more active, whereas the age-matched WT mouse looked old, as also reflected by the state of the hair. ogen into plasmin, the ultimate blood clot-dissolving enzyme [4]. Plasmin is a nonspecific, trypsin-like protease that can also directly degrade diverse extracellular components and can activate proenzymes of matrix degrading metalloproteases, thereby mediating extracellular proteolysis and cell adhesion and migration. Accordingly, the PA/plasmin system has been implicated in various normal and pathological events involving extracellular proteolysis and tissue remodeling (for reviews, see 1,5,7,42,44,57). Although plasminogen is the primary physiological substrate for uPA, the enzyme can also directly cleave fibronectin [40] or activate growth factors from their inactive precursors through extracellular proteolytic cleavage [25,39]. uPA is encoded by an inducible gene that can respond to diverse hormone-like, biological modulators in various cell types in vivo and in vitro. We have recently shown that in the normal murine brain, uPA mRNA could be detected in neuronal cells in just a few sites, primarily the subiculum and entorhinal cortex communicating with the hippocampus, which functions in learning and memory [29]. Kainate, an analog of the neurotransmitter glutamate, has markedly induced uPA mRNA in neuronal cells in many sites in the murine brain, indicating that the uPA gene can respond to neuronal triggering [30]. Previously, we also reported that young homozygous ␣MUPA mice over expressing uPA in the brain showed learning deficits, while displaying normal motor and sensory capabilities [32]. Thus, these and the aforementioned results of uPA in the normal brain led us to hypothesize that uPA is involved in learning-related brain plasticity [29,30,32]. Here, we have examined ␣MUPA for several physiological parameters previously shown to be altered in DR animals, i.e., body temperature, plasma corticosterone level, and age-associated thymus involution. Although our results indicated mainly a similarity between ␣MUPA and DR animals, there were also some differences. In addition, we recently investigated, in the homozygous state, four new transgenic lines carrying the ␣MUPA construct. All of these lines overproduced uPA mRNA in the brain, however, in spatial patterns different and less widespread than that of ␣MUPA, and notably, with no detectable transgenic expression in the hypothalamus. All new lines ate normally and had normal weight, but none showed another ␣MUPA phenotypic change, a high frequency leg muscle tremor seen only when the mouse is found in a nonstable position. We discuss the possibility that transgenic uPA in the brain causes this reduced eating and increased longevity in ␣MUPA. 2. Materials and methods 2.1. Mice Transgenic homozygous mice FVB/N-TgN(␣MUPA) 524West (␣MUPA) [34], and parental WT mice (the NIH inbred mouse line FVB/N [53]) were propagated and maintained at the Weizmann Institute Transgenic Mouse Facilities according to the National Institutes of Health Guide for Care and Use of Laboratory Animals. The mice were housed either five in small cages or 10 in large cages, at 23°C, under a 12-h light/12-h dark cycle (light changes occurring at 0600 h and 1800 h) with water and food ad libitum. Mice were fed with Experimental Animal Center Mice and Rat Breeding Diet, as previously described [35], and were continuously monitored for health. Female mice at the indicated ages were used for the experiments. 2.2. New transgenic lines New transgenic lines were generated as previously described for ␣MUPA [34] with respect to the transgenic construct, parental mice, and microinjection procedure. These lines were brought to the homozygous state as previously described for ␣MUPA [34] and were maintained as above. Data are reported for homozygous mice. Results are presented for lines FVB/N-TgN(␣MUPA)C6915West R. Miskin et al. / Neurobiology of Aging 20 (1999) 555–564 557 (␣MUPA/15) and FVB/N-TgN(␣MUPA)54Mis (␣MUPA/ 54). 2.3. In situ hybridization Preparation of eye and brain sections, hybridization protocol, and uPA-specific riboprobes at the sense and antisense orientations were as previously described [29,32]. 2.4. Body temperature Rectal body temperature was measured in 1–16-monthold WT and ␣MUPA mice, at the indicated hours, with several-days intervals between measurements, by using a cooper constatane thermocouple connected to digital thermometer (Newton thermometer 5005 Ϫ single 1/P Ϯ 0.1°C, Taiwan). 2.5. Plasma corticosterone Blood samples were collected at 0800 h into tubes containing EDTA (ethylenediaminetetraacetic acid) (15 mg/100 mL). Plasma samples were collected after centrifugation (1814 ϫ g, 10 min) and stored at Ϫ20° for 4 days. Corticosterone was determined by radioimmunoassay by using Pharmatrade-Veterinary application kits (Diagnostic Products Corporation, Los Angeles, CA) for Rat Corticosterone (Coat-A-Count, tubes coated with the antibodies), characterized by an intraassay and interassay variations (cv) of 4.3% and 5.8%, respectively. The kit was validated for mice by using spiking recovery technique. 2.6. Flow cytometry analysis Analysis was done on thymocytes prepared from each mouse separately (2 ϫ 106 cells/pellet). Single and double color staining were as previously described [66]. For CD4/ CD8 double color analysis we used phycoerythrin conjugated anti-CD4 and fluorescein isothiocyanate anti-CD8 (Serotec, Oxford, UK). Anti-CD62L (MEL-14, L-Selectin) supernatant fluid from MEL-14-5 hybridoma cell line and anti-CD44 (pgp-1) supernatant fluid from IM-781 cell line (CD44) (courtesy of IL Weissman, Stanford, CA) were used with fluorescin isothiocyanate-goat-anti-rat immunoglobulin (Jackson Laboratories, West Grove, PA) as the second antibody. Staining with the second antibody only was used as negative control. Flow cytometry was done on a FACScan, or FACSort (Becton Dickinson, Mountain View, CA) by using LYSYS software for analysis of the data. 2.7. Statistical analysis Unless otherwise stated, the results were subjected to one-way ANOVA [50] and to Duncan’s multiple range test [10]. The means were considered significantly different at p Յ 0.05. Fig. 2. Body temperature of ␣MUPA and WT mice at different ages and hours. Body temperature was measured at the indicated mouse ages at 1500 h (a), 2000 h (b), and 0800 h (c). Values are presented as mean Ϯ SE. The number of mice is shown next to each value. Values with stars (between mouse types) or different letters (within each mouse type) were significantly different (p Յ 0.05). For each time, numbers in parentheses represent the mean Ϯ SE temperature of the entire range of ages (excluding 1 month) of each mouse type (p Յ 0.001 between mouse types). Within WT, the mean temperature at 0800 h was significantly lower (p Յ 0.001). Within ␣MUPA, all mean values differed significantly (p Յ 0.001). 3. Results 3.1. Body temperature and plasma corticosterone in ␣MUPA and WT mice Dietary restriction was reported to reduce the body temperature of several species [8,9,23,64]. We, therefore, compared ␣MUPA and WT for body temperature throughout a wide range of ages, three times during the day (Fig. 2). The results indicated a significantly lower body temperature in ␣MUPA at all three times, at all ages, with the exception of one month, which was shortly after weaning. It appeared that the largest difference (mean ϳ1°C) occurred at 1500 h. 558 R. Miskin et al. / Neurobiology of Aging 20 (1999) 555–564 Table 1 Plasma corticosterone and food intake in young and old ␣MUPA and WT mice Age (months) Parameter ␣MUPAa WTa 3 15 3 15 168.5 Ϯ 24.2* 65.5 Ϯ 9.9** 2.03 Ϯ 0.04*** 2.20 Ϯ 0.05*** 114.3 Ϯ 24.2** 100.2 Ϯ 20.9** 2.74 Ϯ 0.07** 3.36 Ϯ 0.15* Corticosterone (ng/mL)b Food intakec (g/mouse/day) a Mean Ϯ SE. Within parameters, mean values with different superscripts are significantly different ( p Յ 0.01, n ϭ 10). b At 0800 h. c Last 13 days. At that time, within ␣MUPA the mean body temperature (37.07 Ϯ 0.07°C) declined significantly (p Յ 0.001), compared to the other hours, whereas within WT the mean temperature (38.03 Ϯ 0.06°C) increased, compared to 0800 h (p Յ 0.001). We also measured at 1500 h the body temperature of mice (2.5–3-months old, n ϭ 15) from the new transgenic line ␣MUPA/15. The result (37.95 Ϯ 0.18°C) was similar to the WT value, and it was significantly different (p Յ 0.001) from the ␣MUPA temperature. We examined ␣MUPA and WT for plasma corticosterone levels, another parameter reported to be altered in DR, compared to ad libitum-fed, animals [43,52,65]. The hormone was significantly higher in young ␣MUPA, compared to that in young WT (Table 1). Furthermore in ␣MUPA, corticosterone level significantly declined at the old age, whereas in aged WT the corticosterone level found in young mice was maintained. Clearly, ␣MUPA ate significantly less (26 –35%) than did WT at the two ages (Table 1), thus confirming our previous report [35]. Notably, ␣MUPA did not differ from WT in the amount of drinking (unpublished observations). Table 2 Thymocyte parameters in young and old ␣MUPA and WT micea Age (months) Parameter ␣MUPAb WTb 2 17–26 2 17–26 2 17–26 Thymocytec Thymocyte CD4ϪCD8Ϫd CD4ϪCD8Ϫ CD4ϩCD8ϩd CD4ϩCD8ϩ 86.0 Ϯ 5.6 53.7 Ϯ 7.3 2.6 Ϯ 0.2 9.3 Ϯ 1.8 79.3 Ϯ 1.1 46.5 Ϯ 5.0 88.7 Ϯ 3.8 53.2 Ϯ 4.3 2.4 Ϯ 0.4 8.5 Ϯ 0.9 78.9 Ϯ 2.0 58.1 Ϯ 3.1 a Results of three independent experiments, examining each mouse individually (total three old and six young ␣MUPA; four old and five young WT). b Mean Ϯ SE. Within parameters, values of ␣MUPA and WT at the same age group are not significantly different, whereas values for young versus old are significantly different ( p Յ 0.016; student’s t-test). c Cells ϫ 106 per thymus. d Percent. 3.2. Thymocyte parameters in ␣MUPA and WT mice Dietary restriction retards age-associated changes of variable immunological parameters in the peripheral lymphoid tissues [12,61,62] and in the thymus [60]. In the latter, it was observed that dietary restriction delayed the agerelated decrease in the frequency of reactive T cell precursors [33], suggesting some effects on developmental mechanisms in the T cell compartment. To determine whether such a delay occurred in ␣MUPA, we examined several basic parameters of T cell development in age-matched WT and ␣MUPA mice. As shown in Table 2, age-related decline in thymic cellularity was similar in the aged WT and ␣MUPA mice. In addition, we tested two markers characteristic of aging in the thymus, i.e., decreased proportion of CD4ϩCD8ϩ (double positive) and increased CD4ϪCD8Ϫ (double negative) subsets [14,54,66]. Such age-related changes were also observed in both WT and ␣MUPA mice, with no significant difference between them (Table 2). Further analysis focused on the expression of CD44, which characterizes an early developmental stage in the thymus [47], and in the aging thymus was reported to occur in an altered fraction of cells [54,66]. Our preliminary data have indicated an increased percent of cells expressing CD44 in aged WT and ␣MUPA mice, with no substantial difference between them (data not shown). Furthermore, a preliminary determination of cells expressing CD62L, which characterizes the early mature T cells in the thymus [48], has shown a similar decrease in both WT and ␣MUPA mice (data not shown). It is thus concluded that age-related changes in these parameters were not affected by the reduced eating of ␣MUPA, and these changes apparently did not contribute to the increased life span of these mice. 3.3. Comparison of ␣MUPA and new transgenic lines for transgenic expression Recently, to test whether transgenic expression is causing the ␣MUPA phenotype, we have generated new transgenic lines carrying the ␣MUPA construct. We used in situ hybridization analysis to localize uPA mRNA in the eyes and brains of ␣MUPA mice and in the new transgenic lines. Cryostat, thin eye sections derived from these mice were tested with a uPA mRNA-specific 35S-labeled antisense riboprobe. Fig. 3 shows the results for the ␣MUPA and WT eyes. Strong hybridization signals were localized in the ␣MUPA lens (Fig. 3a and b), but not in the WT lens (Fig. 3c and d). uPA mRNA was first detected in the lens at Embryonic Day 10.5, which was in agreement with previous data on the ␣A-crystallin promoter activity in transgenic and normal mice [38]. In the ␣MUPA eye, hybridization signals were also seen in the ganglion cell layer and the inner nuclear layer of the retina (Fig. 3b), which were devoid of signals in the WT eye. Hybridization signals in the retina appeared to be associated with neuronal cells; they were much lower compared to the lens, and they were R. Miskin et al. / Neurobiology of Aging 20 (1999) 555–564 559 Fig. 3. uPA mRNA hybridization signals in WT and ␣MUPA eyes. Cryostat eye sections (12 m) were prepared from 4-day-old ␣MUPA (a) and WT (c) mice. Sections were hybridized with uPA-specific 35S-labeled antisense riboprobe. (b) and (d) are the same sections as (a) and (c), respectively, only microphotographed in dark field illumination. Symbols: L, lens; R, retina. Bar ϭ 0.2 mm. No hybridization signals were detected with the uPA sense riboprobe, indicting signal specificity for uPA mRNA. first detected only at Day 17.5 of gestation. All new transgenic lines exhibited uPA mRNA signals in the lens, but not in the retina (results not shown). Notably, we previously reported on trangenic uPA expression in the retina of transgenic ␣HUPA mice carrying a construct similar to ␣MUPA, except that the cDNA encoded human uPA [34]. Fig. 4 shows the results of in situ hybridization analysis of brain sections. Fig. 4a– c present WT (Fig. 4a) and ␣MUPA (Fig. 4b and c) sections containing the hypothal- amus. In ␣MUPA, but not in WT, uPA mRNA signals were seen in the suprachiasmatic nucleus (SCN; Fig. 4b) and paraventricular nucleus (PVN; Fig. 4c). In ␣MUPA, uPA mRNA signals were also seen throughout the neocortex, where WT signals were low and restricted to layers IV and VI, as previously reported [29]. In ␣MUPA, signals were also seen in a few other regions, including the hippocampus, where they appeared to be associated with interneurons rather than with pyramidal or granular cells. Fig. 4. uPA mRNA hybridization signals in brain sections of WT, ␣MUPA, and new line ␣MUPA/54. Coronal brain sections (12 m) through the anterior (a– d) or posterior (e,f) hippocampus were prepared from WT (a), ␣MUPA (b, c, and e) or a new transgenic line ␣MUPA/54 (d and f). Sections were hybridized as in Fig. 3. a– b, c– d, and e–f are parallel or close sections. Microphotography was in dark field illumination. Symbols: short arrows, hippocampus; long arrows, neocortex; full arrowheads, presubiculum; empty arrowheads, retrosplenial cortex. SCN, suprachiasmatic nucleus; PVN, paraventricular nucleus; IP, interpeduncular nucleus. Bar ϭ 0.9 mm. 560 R. Miskin et al. / Neurobiology of Aging 20 (1999) 555–564 strained upon strong muscle contraction. The tremor was seen in homozygous, but not in heterozygous, ␣MUPA mice. It was first observed on Day 15 postnatally, was consolidated on Day 17, and somewhat declined at ϳ30 months. However, the motor behavior of ␣MUPA mice was normal [32] and did not appear to be disturbed by the tremor. ␣MUPA mice were also distinguished from WT mice by the overall young appearance that they maintained at old ages (Fig. 1) consistently with their prolonged longevity [35]. 4. Discussion 4.1. Physiological phenotypic alterations of ␣MUPA mice Fig. 5. High frequency leg muscle tremor of ␣MUPA mice. The ␣MUPA mouse (3-month-old) showed high frequency muscle tremor in all legs; however, only when found in a nonstable position, such as sustained by the tail. The WT mouse did not show this tremor. Brain analysis of the four new transgenic lines tested in the homozygous state also showed transgenic uPA mRNA signals in neuronal cells, however, with spatial patterns different and less widespread, compared to ␣MUPA. An example is shown in Fig. 4, presenting sections through the hypothalamus and the anterior hippocampus of WT (Fig. 4a), ␣MUPA (Fig. 4b and c), and the new line ␣MUPA/54 (Fig. 4d). Parallel sections through the posterior hippocampus of ␣MUPA (Fig. 4e) and ␣MUPA/54 (Fig. 4f) are also shown. It is evident that in ␣MUPA/54 the hypothalamus is devoid of signals (Fig. 4d), whereas signals were particularly strong in the presubiculum and retrosplenial cortex, as well as in cells scattered throughout the sections shown in Fig. 4e and f. Interestingly, in some transgenic lines uPA mRNA was specifically intense in brain regions normally producing uPA mRNA, especially the subicular area and layers IV and VI of the neocortex; the latter was also seen in line ␣MUPA/54 (Fig. 4d and f). In this line, as well as in ␣MUPA, hybridization signals were largely confined to the grey matter and were associated with cells recognized as neurons according to their morphological appearance at high magnifications (see Fig. 4c, insert). Like ␣MUPA/54, none of the other new transgenic lines exhibited uPA mRNA in the hypothalamus. 3.4. ␣MUPA mice show a high frequency leg tremor throughout life and a young look at old ages ␣MUPA mice, but not WT or the new transgenic lines, showed a high frequency muscle tremor in all legs; however, only when the mouse was found in a nonstable position, such as sustained by the tail (Fig. 5). The tremor was reminiscent of “cold” shivering and appeared to be re- Data presented here and previously [35] showed mostly similarities between ␣MUPA and DR animals; however, there were also some differences. In comparing the two, it should be taken into account that ␣MUPA, having free access to food, are not affected by hunger-induced stress nor by abrupt feeding, which in DR animals shifts the circadian profile of parameters, such as body temperature and plasma corticosterone. Thus in ad libitum-fed rodents, the levels of both parameters rise close to the onset of darkness and are maximal shortly thereafter, whereas in DR animals the rise and maximal levels are shifted to the time of prefeedingfeeding [8,9,37,52,63]. In addition, food reduction in ␣MUPA is milder (15–35%), compared to most DR regimens (Ͼ30%). Accordingly, less severe physiological changes are expected in ␣MUPA. Also, as ␣MUPA mice were derived from the FVB/N strain, it is of interest to note that in this strain 60% survival was reported at 24 months [24], in excellent agreement with our WT mice that showed 60% survival at 25 months and a median life span of 28.5 months [35]. Furthermore, FVB/N mice showed some agerelated brain disorder, which was accelerated in transgenic mice over expressing the Alzheimer amyloid precursor protein [18]; aged FVB/N mice showed a higher than usual rate of lung tumors and a lower rate of liver tumors and lymphomas [24], and FVB/N mice also differed from other mouse strains in the profile of chemically induced tumors [16]. In ␣MUPA, the spontaneously reduced food consumption coincided with a significant reduction of body temperature throughout a wide range of ages. This is in agreement with previous reports on DR mice [8,9,55], rats [8,9], monkeys [23], and domestic fowls [64]. Interestingly, reduced body temperature was also demonstrated in desert homeotherm animals at periods of lower energy metabolism [46]. Reduction of body temperature, as well as oxygen consumption, are known to result from the depression of the rate of energy metabolism upon dietary restriction [46]. It is, however, not clear if and how this temperature reduction contributes to the anti-aging effect; possibilities, such as decreased DNA damage and protooncogene expression, R. Miskin et al. / Neurobiology of Aging 20 (1999) 555–564 have been discussed [23,37]. Our data also show that, within WT, the mean body temperature was lowest at 0800 h and was similarly higher at 1500 and 2000, as if the daily temperature rise occurred in WT earlier than reported in the literature [8,9,37]. Notably, FVB/N mice are blind because of the rd (retinal degeneration) mutation causing photoreceptor deterioration shortly after birth [53]. We wonder whether this mutation could have affected the temperature circadian rhythm. In any case, the ␣MUPA body temperature was significantly lower than that of WT at all times tested. The plasma corticosterone levels that we measured differed between ␣MUPA and WT in two respects: first, at the young age it was higher in ␣MUPA than in WT; second, within ␣MUPA the level declined in aged mice, whereas it remained unchanged within WT. Corticosterone levels were determined here only at one daily hour, i.e., 0800 h, when, according to the circadian rhythm reported for the hormone, the level is low [52,63]. Our limited data on corticosterone levels appear to agree with those of Stewart et al. [52], who reported that in young DR rats the overall level of corticosterone was somewhat elevated over a 24-h period; however, it was reduced in aged DR rats compared to ad libitum-fed animals. In this context it is of interest that, in desert rodents, corticosterone reduction, as an adaptation to a chronic reduction in metabolic rate, was observed [17,56]. Our findings on ␣MUPA and those of Stewart et al. [52] suggest that corticosterone reduction with age could be beneficial. This possibility thus supports the hypothesis that normal aging increases the susceptability to corticosteronemediated neurotoxicity, which may be caused by dysregulation of neuronal calcium homeostasis [22]. A different view, though, was raised by Masoro [28], who suggested that moderately increased levels of corticosterone may act to extend the life span. This was based on the observation that the maximal daily corticosterone levels in DR rats was higher throughout their life time than in ad libitum-fed animals [43]. Interestingly, it was noted that dietary restriction did not affect the age-related changes of adrenal catecholamines in rats [20]. All age-related changes in thymocyte parameters tested here occurred similarly in old WT and ␣MUPA, indicating normal thymus involution in ␣MUPA. These mice thus differ from DR animals, where the latter process was attenuated [60]. Thus is appears that increased ␣MUPA life span is not supported by maintaining the youthful state of the thymus. Several types of transgenic or knock out mice showing reduced survival or premature death have been reported (for example, see 5,18,21). To our knowledge, ␣MUPA are the first genetically modified experimental mice showing increased survival. It is, therefore, of interest to study ␣MUPA as a model for increased life span and to test these mice for additional metabolic, physiological, and immunological parameters; for hypothalamic neuropeptides; as well as for other factors suggested to contribute to aging and cellular scenescence, such as DNA damage, and the levels 561 of oncogenes and tumor suppressors [3,15,37,59]. It is also of interest to follow the ␣MUPA response to various environmental stressores [26]. 4.2. Possible causal factors of the ␣MUPA phenotype Based on the well established inverse relation between longevity and food intake, it is conceivable that ␣MUPA mice live longer because they eat less. The biological mechanisms underlying the ␣MUPA behavior are not known. Furthermore, because reduced eating was detected in only one among several lines carrying the ␣MUPA transgene, we cannot yet exclude as a causal factor an insertional mutation in a yet undefined gene, resulting from the randomly occurring transgenic insertion. However, the unusual nature of ␣MUPA transgenic expression supports the possibility that uPA over expression causes the ␣MUPA phenotype, as will be discussed. The ␣MUPA construct was consistently expressed in nonlens sites [34], specifically in the brain, as shown here and previously [32,35], and also in the developing oral cavity and teeth, as recently found in four transgenic lines (unpublished data). In the latter site, we detected uPA mRNA also in WT mice, but the transgenic mRNA preceded it and was more intense. Strikingly, a phenotype of imperfect pigmentation of the incisor enamel was detected in all three transgenic lines that were tested for this effect, including ␣MUPA. Thus, although the WT enamel was homogeneously dark yellow, the transgenic enamel was largely colorless. Clearly, this phenotypic change is caused by nonlens transgenic expression. These results also indicate that the reproducible transgenic expression in the developing teeth and oral cavity is a genuine inherent feature of the transgenic construct rather than resulting from the position of transgenic insertion. We assume that a similar situation has occurred in the brain, i.e., the ectopic transgenic expression in the brain could have led to the phenotypic alteration of feeding behavior and muscle tremor. The restriction of these changes to only one transgenic line could have resulted from the substantially more extensive and spatially widespread transgenic expression occurring in ␣MUPA, compared to the other lines. Such differences could be caused by factors like site of transgene insertion, or the number and organization of transgene copies. Only in the ␣MUPA line was uPA mRNA detected in brain regions known to control feeding behavior, i.e., the hypothalamic PVN and SCN, the latter being involved in circadian behavior [13,41]. We, therefore, hypothesize that hypothalamic transgenic expression could have led to the reduced eating in ␣MUPA. We think that the nonlens tissue-specific expression of transgenic uPA is determine primarily by the 3Ј UTR of the uPA gene that is included in the transgenic construct. Thus, we have recently shown that the 3Ј UTR contains sequences that can exert variable effects on chimeric gene expression, including transcriptional enhancement. In addition, we have 562 R. Miskin et al. / Neurobiology of Aging 20 (1999) 555–564 generated transgenic mice that carry a chimeric transgene, where the luciferase reporter gene was inserted in the ␣MUPA construct between the promoter and the cDNA. These transgenic mice exhibited transgenic expression similar to that of the lines carrying the ␣MUPA construct; i.e., luciferase activity was detected specifically in the lens and brain, and luciferase mRNA was found in many brain sites, but with variable spatial patterns among the different transgenic lines [49]. These findings support a model in which the 3Ј UTR of the uPA gene is involved in the expression of the endogenous uPA gene, at least in the brain and developing oral cavity, and the same 3Ј UTR is overactive where the ␣A-crystallin promoter substituted for the homologous uPA promoter. Accordingly, the nonlens transgenic expression is clearly ectopic with respect to the ␣A-crystallin promoter, but with respect to the uPA gene it represents a state of over expression. We assume that such over expression could have caused the ␣MUPA phenotypic alterations of reduced eating, reduced learning abilities, tremor, and imperfect enamel. The possibility that transgenic uPA could be influential in the ␣MUPA hypothalamus is supported by the biochemical capacity of the enzyme. We could not detect uPA mRNA in the WT hypothalamus, nor was it induced in the hypothalamus upon administration of kainate into mice [29, 30]. However, plasminogen activator activity probably plays a role in the hypothalamus. This is indicated by the hypothalamic localization of two elements of the PA/plasmin system: high activity of tissue-type PA (tPA), the second PA type that is also abundant in other brain sites [45], and the mRNA encoding plasminogen activator inhibitor-1, the physiological inhibitor of tPA and uPA [30]. It is thus possible that transgenic uPA could exert in the hypothalamus some influence, such as uncontrolled proteolytic processing or degradation of PVN or SCN neuropeptides controlling eating, or their cognate surface receptors. Yet, we cannot at present exclude other possible uPA-related causes, such as the muscle tremor, or as yet undetected developmental anatomic impairment in the oral cavity occurring specifically in the ␣MUPA line. In conclusion, our model implicating hypothalamic transgenic uPA in the reduced eating and prolonged life span of ␣MUPA is thus far supported by circumstantial rather than direct evidence. 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