Emory Mouse Research Articles

Ubiquitination of Ku70 by Parkin promotes apoptosis of lens epithelial cells.

Oxidative damage-triggered apoptosis in lens epithelial cells is considered as a main risk factor in the pathogenesis of age-related cataracts. Ku70 is a key factor in the DNA repair process of double-strand breaks. In the present study, we aimed to investigate the role of Ku70 and its related E3 ubiquitin ligase in lens epithelial cell apoptosis. The levels of Ku70 in the anterior lens capsules of human cataracts and Emory mice were lower compared to controls. H2 O2 treatment resulted in decreased expression of Ku70 through accelerating Ku70 ubiquitination. Parkin, an E3 ubiquitin ligase, could interact with Ku70 and promote the ubiquitination and degradation of this protein. In addition, ubiquitinated Ku70 was regulated by ubiquitin-proteasome, autophagy-lysosome and mitophagy pathways. Ectopic expression of Ku70 protected SRA01/04 cells from H2 O2 -induced apoptosis, whereas silencing Ku70 exhibited the opposite trend. Co-transfected with Parkin non-ubiquitinatable Ku70 mutant could maintain its anti-apoptosis ability, whereas wild-type Ku70 failed. Moreover, Ku70 might facilitate mitochondrial fusion by increasing the expression of Mitofusin 1/2. The present study revealed that Parkin-mediated Ku70 ubiquitination facilitated H2 O2 -induced lens epithelial cell apoptosis through alleviating mitochondrial fusion, which could provide potential targets for age-related cataract treatment.

Whole-exome sequencing prioritizes candidate genes for hereditary cataract in the Emory mouse mutant.

The Emory cataract (Em) mouse mutant has long been proposed as an animal model for age-related or senile cataract in humans-a leading cause of visual impairment. However, the genetic defect(s) underlying the autosomal dominant Em phenotype remains elusive. Here, we confirmed development of the cataract phenotype in commercially available Em/J mice [but not ancestral Carworth Farms White (CFW) mice] at 6-8 months of age and undertook whole-exome sequencing of candidate genes for Em. Analysis of coding and splice-site variants did not identify any disease-causing/associated mutations in over 450 genes known to underlie inherited and age-related forms of cataract and other lens disorders in humans and mice, including genes for lens crystallins, membrane/cytoskeleton proteins, DNA/RNA-binding proteins, and those associated with syndromic/systemic forms of cataract. However, we identified three cataract/lens-associated genes each with one novel homozygous variant including predicted missense substitutions in Prx (p.R167C) and Adamts10 (p.P761L) and a disruptive in-frame deletion variant (predicted missense) in Abhd12 (p.L30_A32delinsS) that were absent in CFW and over 35 other mouse strains. In silico analysis predicted that the missense substitutions in Prx and Adamts10 were borderline neutral/damaging and neutral, respectively, at the protein function level, whereas, that in Abhd12 was functionally damaging. Both the human counterparts of Adamts10 and Abhd12 are clinically associated with syndromic forms of cataract known as Weil-Marchesani syndrome 1 and polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract syndrome, respectively. Overall, while we cannot exclude Prx and Adamts10, our data suggest that Abhd12 is a promising candidate gene for cataract in the Em/J mouse.

Age-related cataract: GSTP1 ubiquitination and degradation by Parkin inhibits its anti-apoptosis in lens epithelial cells

PurposeOxidative stress-induced apoptosis of lens epithelial cells (LECs) contributes to the pathogenesis of age-related cataract (ARC). The purpose of this research is to underlie the potential mechanism of E3 ligase Parkin and its oxidative stress-associated substrate in cataractogenesis. MethodsThe central anterior capsules were obtained from patients with ARC, Emory mice, and corresponding controls. SRA01/04 cells were exposed to H2O2 combined with cycloheximide (a translational inhibitor), MG-132 (a proteasome inhibitor), chloroquine (an autophagy inhibitor), Mdivi-1 (a mitochondrial division inhibitor), respectively. Co-immunoprecipitation was employed to detect protein-protein interactions and ubiquitin-tagged protein products. Levels of proteins and mRNA were evaluated by western blotting and quantitative RT-PCR assays. ResultsGlutathione-S-transferase P1 (GSTP1) was identified as a novel Parkin substrate. Compared with corresponding controls, GSTP1 was significantly decreased in the anterior lens capsules obtained from human cataracts and Emory mice. Similarly, GSTP1 was declined in H2O2-stimulated SRA01/04 cells. Ectopic expression of GSTP1 mitigated H2O2-induced apoptosis, whereas silencing GSTP1 aggregated apoptosis. In addition, H2O2 stimulation and Parkin overexpression could promote the degradation of GSTP1 through the ubiquitin-proteasome system, autophagy-lysosome pathway, and mitophagy. After co-transfection with Parkin, the non-ubiquitinatable GSTP1 mutant maintained its anti-apoptotic function, while wildtype GSTP1 failed. Mechanistically, GSTP1 might promote mitochondrial fusion through upregulating Mitofusins 1/2 (MFN1/2). ConclusionOxidative stress induces LECs apoptosis via Parkin-regulated degradation of GSTP1, which may provide potential targets for ARC therapy.

Genome-Wide Repertoire of Transfer RNA-Derived Fragments in a Mouse Model of Age-Related Cataract

Purpose To investigate the roles of tRNA-derived small RNAs (tsRNAs) containing transfer RNA-derived fragments (tRFs) and tRNA halves in age-related cataracts (ARCs). Methods Lens capsule tissue from Emory mice at 3 months and 8 months of age were dissected for integrated tsRNA and gene transcriptome sequencing. A quantitative real-time PCR assay (qRT-PCR) was performed for validating sequencing results. Bioinformatics analysis was constructed to reveal the roles of tsRNAs Results A total of 422 differential expression (DE) tsRNAs were changed, in which 156 were elevated while 266 were declined in 8-month-old mice. Subsequently, the gene sequencing data exhibited 375 upregulated and 456 downregulated DE genes. Validation by qRT-PCR in 5 selected upregulated tRFs was consistent with tsRNAs sequencing results. Moreover, bioinformatics analysis identified 25 downregulated target genes of the 5 validated tRFs. Furthermore, GO analysis revealed that these target genes were mainly enriched in camera-type eye development, sensory organ development, and so on. Conclusion Our study provides a novel perspective on the role of tsRNAs in the pathogenesis of ARC, and thus therapeutic potential targets for ARC.

Regional changes of AQP0-dependent square array junction and gap junction associated with cortical cataract formation in the Emory mutant mouse

The Emory mutant mouse has been widely used as an animal model for human senile cataract since it develops late-onset hereditary cataract. Here, we focus on the regional changes of aquaporin-0 (AQP0) and connexins that are associated with the cortical cataract formation in the Emory mutant mice. Emory mutant and CFW wild-type mice at age 1–16 months were used in this study. By using an established photography system with dissecting microscopy, the opacities were first detected at the anterior or posterior lens center surface in Emory mice at age 7 months, and gradually extended toward the equator during the 16 months examined. Scanning EM verified that disorganized and fragmented fiber cells were associated with the areas of opacities within approximately 200 μm from the lens surface, indicating that Emory mouse cataracts belong to the cortical cataracts. Freeze-fracture TEM further confirmed that cortical cataracts exhibited extensive wavy square array junctions, small gap junctions and globules. Immunofluorescence analysis showed that in contrast to the high labeling intensity of AQP0-loop antibody, the labeling of AQP0 C-terminus antibody was decreased considerably in superficial fibers in Emory cataracts. Similarly, a significant decrease in the labeling of the antibody against Cx50 C-terminus, but not Cx46 C-terminus, occurred in superficial and outer cortical fibers in Emory cataracts. Western blotting further revealed that the C-termini of both AQP0 and Cx50 in Emory cataracts were decreased to over 50% to that of the wild-type. Thus, this systematic study concludes that the Emory mouse cataract belongs to the cortical cataract which is due to regional breakdown of superficial fibers associated with formation of AQP0-dependent wavy square array junctions, small gap junctions and globules. The marked decreases of the C-termini of both AQP0 and Cx50 in the superficial fibers may disturb the needed interaction between these two proteins during fiber cell differentiation and thus play a role in the cortical cataract formation in Emory mutant mice.

Mutant Huntingtin in Glial Cells Exacerbates Neurological Symptoms of Huntington Disease Mice

Huntington disease (HD) is caused by an expansion of the polyglutamine (polyQ) repeat (>37Q) in huntingtin (htt), and age of onset is inversely correlated with the length of the polyQ repeat. Mutant htt with expanded polyQ is ubiquitously expressed in various types of cells, including glia, but causes selective neurodegeneration. Our recent study demonstrated that expression of the N-terminal mutant htt with a large polyQ repeat (160Q) in astrocytes is sufficient to induce neurological symptoms in mice (Bradford, J., Shin, J. Y., Roberts, M., Wang, C. E., Li, X.-J., and Li, S. H. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 22480-22485). Because glia-neuron interactions are critical for maintaining the normal function and survival of neurons in the brain and because mutant htt is more abundant in neurons than in glial cells, it is important to investigate whether glial htt can still contribute to HD pathology when mutant htt is abundantly expressed in neuronal cells. We generated transgenic mice that express mutant htt with 98Q in astrocytes. Unlike our recently generated htt-160Q transgenic mice, htt-98Q mice do not show obvious neurological phenotypes, suggesting that the length of the polyQ repeat determines the severity of glial dysfunction. However, htt-98Q mice show increased susceptibility to glutamate-induced seizure. Mice expressing mutant htt in astrocytes were mated with N171-82Q mice that express mutant htt primarily in neuronal cells. Double transgenic mice expressing mutant htt in both neuronal and glial cells display more severe neurological symptoms and earlier death than N171-82Q mice. These findings indicate a role of glial mutant htt in exacerbating HD neuropathology and underscore the importance of improving glial function in treating HD.

The effect of pantethine and ultraviolet-B radiation on the development of lenticular opacity in the emory mouse

Purpose. Few studies have examined the impact of long-term treatments or exposures on the development of cataract in maturity-onset animal models. We studied the effect of treatment with D-pantethine and exposure to ultraviolet-B (UVB) radiation on the development of lenticular opacity in the Emory mouse. Methods. A total of 164 Emory mice were randomized by litter at weaning to exposure to UVB light at 12 mJ/cm 2 for 6 hr/day (UV) or usual room light (A), and within litter, were further randomized to bi-weekly intra-peritoneal injections of 0.8 g/kg pantethine (T) or no treatment (C). Retro illumination lens photos were taken at 2, 4, 6, 8, and 10 months after weaning, and graded in masked fashion. The animals were sacrificed at 10 months and the lenses analyzed for total pantethine and total cysteamine. Results. Lens pantethine and cysteamine levels were significantly (P < 0.001) higher for the T as compared to C litters. Mean cataract grade increased monotonically over time for all four groups. Unadjusted mean grade for the AT group at 8 (1.32) and 10 (1.86) months appeared lower than for the other groups (AC: 2.17, 2.39; UVC: 1.77, 2.40; UVT: 1.88, 2.37). However, the mean grade for the pantethine-treated litters did not differ significantly from the untreated litters except at 2 months (when untreated litters had significantly lower grades), when adjusting for UV treatment, gender and litter effect. No significant difference in cataract score existed between UV-exposed and ambient litters. Mortality was higher among pantethine-treated (hazard ratio = 1.8, p = 0.05) and UV-exposed animals (hazard ratio = 1.8, p = 0.03) than among the untreated and unexposed litters. Conclusion. Significantly increased lens levels of pantethine are achieved with long-term intra-peritoneal dosing. The impact of pantethine on the progression of lenticular opacity in the Emory mouse is less than has been reported in other models. This level of chronic UVB exposure appeared to have no effect on the development of cataract in this model.

The effect of pantethine and ultraviolet-B radiation on the development of lenticular opacity in the emory mouse

Few studies have examined the impact of long-term treatments or exposures on the development of cataract in maturity-onset animal models. We studied the effect of treatment with D-pantethine and exposure to ultraviolet-B (UVB) radiation on the development of lenticular opacity in the Emory mouse. A total of 164 Emory mice were randomized by litter at weaning to exposure to UVB light at 12 mJ/cm(2) for 6 hr/day (UV) or usual room light (A), and within litter, were further randomized to bi-weekly intra-peritoneal injections of 0.8 g/kg pantethine (T) or no treatment (C). Retro illumination lens photos were taken at 2, 4, 6, 8, and 10 months after weaning, and graded in masked fashion. The animals were sacrificed at 10 months and the lenses analyzed for total pantethine and total cysteamine. Lens pantethine and cysteamine levels were significantly (P < 0.001) higher for the T as compared to C litters. Mean cataract grade increased monotonically over time for all four groups. Unadjusted mean grade for the AT group at 8 (1.32) and 10 (1.86) months appeared lower than for the other groups (AC: 2.17, 2.39; UVC: 1.77, 2.40; UVT: 1.88, 2.37). However, the mean grade for the pantethine-treated litters did not differ significantly from the untreated litters except at 2 months (when untreated litters had significantly lower grades), when adjusting for UV treatment, gender and litter effect. No significant difference in cataract score existed between UV-exposed and ambient litters. Mortality was higher among pantethine-treated (hazard ratio = 1.8, p = 0.05) and UV-exposed animals (hazard ratio = 1.8, p = 0. 03) than among the untreated and unexposed litters. Significantly increased lens levels of pantethine are achieved with long-term intra-peritoneal dosing. The impact of pantethine on the progression of lenticular opacity in the Emory mouse is less than has been reported in other models. This level of chronic UVB exposure appeared to have no effect on the development of cataract in this model.

Lens-Specific Regulation of the Thioredoxin-1 Gene, but Not Thioredoxin-2, upon in Vivo Photochemical Oxidative Stress in the Emory Mouse

Thioredoxin (TRX)-1 and TRX-2 redox-regulatory genes were analyzed in the lens and some other tissues of the Emory mouse, a model for age-related human cataract. The cDNA transcripts of mRNAs encoding TRX-1 and TRX-2 genes were isolated and cloned by RT-PCR from the lens, liver, kidney, and tail, and the cDNA sequences were similar to the reported sequences of murine TRX-1 and TRX-2 genes. In vivo photochemical oxidative stress to the Emory mice resulted in fivefold upregulation of the lens TRX-1 gene at 3 weeks and declined thereafter. Western blot analysis revealed a fourfold increase of TRX-1 protein in the lens at 3 weeks after oxidative stress. The TRX-2 gene in the lens was not changed up to 5 weeks and decreased by 50% thereafter. However, the expressions of these genes in the liver, kidney, and tail were not changed. Fluorescent light or riboflavin alone did not affect the expressions of TRX-1 and TRX-2 genes in the lens. Thus, we show the expressions of TRX-1 and TRX-2 genes in the lens, liver, kidney, and tail and lens-specific upregulation of the TRX-1 gene and protein expressions, possibly as a protective response to the altered redox state of the lens after in vivo oxidative stress to the Emory mouse.

Calorie restriction, stress and the ubiquitin-dependent pathway in mouse livers

Calorie restriction (R) is the only known method to delay the aging process and extend mean and maximal lifespan in rodents. R has been shown to delay the age-related accumulation of damaged proteins and to protect organisms from various stresses which can produce damaged proteins. Such stresses include irradiation, heat shock, and oxidative stress. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) has been associated with the degradation of abnormal and/or damaged proteins. We examined the effect of diet and oxidative stress on activities of the UPP in supernatants from livers taken from 23-month-old Emory mice which had been exposed to an in-vivo injection of paraquat. Paraquat induces oxidative stress by generating superoxide radicals. In livers from non-stressed animals, steady-state levels of endogenous ubiquitin conjugates, de novo conjugate formation, and E1 and E2 activities were significantly lower in R animals than in control (C) animals. However, after exposure to paraquat, levels of endogenous ubiquitin conjugates were significantly higher in R versus C animals, and de novo conjugate formation and E1 and E2 activities in R animals rose to levels which were indistinguishable from levels of these activities noted in C animals. R was associated with an increased ability to degrade β-lactoglobulin by the UPP after an oxidative stress was imposed. Ability to degrade β-lactoglobulin by the C or R livers in non-stressed animals was not significantly different. Taken together, these data indicate that oxidative stress in R animals is associated with enhanced levels of ubiquitin conjugates and that this enhancement may be due to an increase in UPP activity. These data also indicate that the ability to form ubiquitin conjugates and the UPP system does not change with oxidative stress in C animals. The latter is consistent with prior reports that suggests that older C animals may already be in a state of enhanced oxidative stress and that activities of the UPP provide a sensitive indicator of levels of cellular redox status.

The effects of aging and calorie restriction on plasma nutrient levels in male and female Emory mice

We examined the effect of diet, age (4.5, 13 and 23 months), and sex on plasma levels of retinol, tocopherol, ascorbate, cholesterol, glucose and glycohemoglobin in male and female Emory mice which were fed control (C) and 50% calorie restricted (R) diets. Results showed that C fed animals tended to have higher levels of plasma ascorbate (50–71%), cholesterol (23–71%), glucose (38–81%) and glycohemoglobin (50%). However, these diet differences varied with the age and sex of the animals. Plasma retinol levels were lower only in R males vs. C males (50%). Novel sex-related differences in levels of plasma retinol (2-fold higher in C male mice than in C or R female mice) are described. Aging was associated with trends towards lower levels of plasma ascorbate (14–25%), glucose (34–36%) and glycohemoglobin (47–57%) from 4.5 to 23 months of age. However, these age differences depended upon the diet and sex of the animals. These data suggest that lower plasma levels of glucose, glycosylated hemoglobin and cholesterol may be causally related to the life extension noted in R animals since elevated levels of these moieties have been related to aging. Since oxidative stress is thought to be causally related to aging it appears unlikely that retinol, tocopherol and ascorbate are causally related to R-induced life-extension.

Aging, calorie restriction and ubiquitin-dependent proteolysis in the livers of Emory mice

Calorie restriction (R), the only known method to delay the aging process and extend mean and maximal lifespan, has been shown to delay the age-related decline in protein degradation. There are several proteolytic pathways. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) is frequently associated with degradation of damaged abnormal and/or regulatory proteins. We examined the effect of aging and R on supernatants of livers taken from young (4.5 months) and old (23 months) Emory mice. Aging was associated with increased levels of endogenous ubiquitin conjugates, enhanced ability to form high molecular weight conjugates and ubiquitin activating (E1) and ubiquitin conjugating (E2) activity in the control (C) liver supernatants. The age-related increase in levels of endogenous ubiquitin conjugates in liver appears to be primarily due to increased E1 and E2 activities. R prevented the age-related increase in E1 and E2 activity, and thus prevented the age-related increase in levels of ubiquitin conjugates. In spite of the age-related increase in ubiquitin conjugates, no age-related changes in ubiquitin-dependent proteolytic pathway were observed in the C animals. R was associated with an enhanced ability (130%) to degrade β-lactoglobulin by the ubiquitin-dependent proteolytic pathway in livers from 4.5-month-old animals relative to age-matched C livers. However, rates of the ubiquitin-dependent degradation of β-lactoglobulin in the 23-month-old C and R animals were indistinguishable. There were no age- or diet-related differences in the ability to degrade another substrate, oxidized ribonuclease (RNase).

Antioxidant enzyme activities in lens, liver and kidney of calorie restricted Emory mice

Dietary calorie restriction extends both mean and maximum life span and retards age-related diseases, including eye lens cataract in Emory mice. The beneficial effects of calorie restriction have been hypothesized to reflect enhanced tissue antioxidant capacity. As a test of this hypothesis, we reared male and female Emory mice on control (C) or 40% calorie-restricted (R) diets. We then determined activities of total superoxide dismutase (T-SOD), Cu/Zn-SOD, Mn-SOD, glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT) in eye lens, liver and kidney of young (4.5 or 6 months), mature (11 or 12 months) and old (22 months) animals. Effects of diet, age and sex were evaluated by multi-factor ANOVA. Only kidney GR activities (mean±S.E.M.) were significantly enhanced with the R diet (R, 61±2 vs. C, 54±3 U/mg protein; P=0.03). More frequently, we noted reduced antioxidant enzyme activity in R as compared with C animals, including reduced activities of T-SOD in lens, liver and kidney, Cu/Zn-SOD in liver and kidney, liver Mn-SOD and liver CAT ( P<0.05). Effects of age on antioxidant enzyme activity in C mice included age-dependent decreases in lens and kidney CAT and in liver Mn-SOD. There was also an age-dependent increases in liver and kidney Cu/Zn-SOD and liver GR. None of these age-dependent alterations in antioxidant enzyme function were attenuated in tissues of mice fed the R diet. Values for liver CAT were significantly lower in females than in males ( P=0.05). These results indicate that antioxidant enzyme activities in Emory mouse tissues are influenced by diet, age and sex. However, it is unlikely that increased lifespan and attenuation of cataract (and perhaps other age-dependent debilities), which are associated with the R diet in the Emory mouse, are due to enhanced antioxidant enzyme capabilities.

Confocal Microscopy of Cataracts from Animal Model Systems: Relevance to Human Nuclear Cataract

A recent study demonstrated that cytosolic lipid membrane structures, independent of the plasma membrane, preferentially occurred in human cataractous lenses. Animal model systems of cataractogensis (selenite treated rats; galactose fed rats; buthionine-sulfoxime treated mice; Emory mice) were screened for possible relevant structures using the lipid membrane probe DiI and confocal microscopy. Well delineated plasma membranes of lens fiber cells with independent cytosolic staining structures were only observed in the selenite model system. These cytosolic structures were not observed in aged matched control lenses or within the transparent cortical regions of selenite treated animals with intense nuclear opacification. These results suggested that the morphological changes in DiI staining structures seen in the nucleus of the human cataractous lens were best approximated by those seen in the selenite model system.

Menadione-induced oxidative stress accelerates onset of Emory mouse cataract in vivo

PURPOSE. We evaluated the effect of the free-radical generator, menadione, on the time of occurrence of cataract in the Emory mouse, a model for human cataract. Concomitant experiments were done to compare production and level of reactive metabolites of oxygen in the Emory mouse and its cataract resistant (CR) genetic control. METHODS. Test and control mice were fed both a normal diet and a diet supplemented with menadione, and the lenses were evaluated for the time of occurrence of cataract and the level of membrane ATPases. Effects of menadione were determined on incubated lenses of Emory and CR mice, assaying reactive species of oxygen, levels of antioxidant enzymes, and formation of the lipid peroxidation product, malondialdehyde. RESULTS. Systemic administration of menadione markedly accelerated the onset of Emory mouse cataract, and decreased ATPase activities suggested oxidative damage to membrane proteins. Cumulative levels of O 2 · -, H 2 O 2, ·OH and malondial-dehyde were significantly higher than controls in the lenses incubated in the presence of menadione, showing that it generates oxidative stress. However, [GSH] in lenses decreased equally in test and control mice. The observed increases in catalase and glutathione peroxidase activities in the test lenses indicated an early protective response to oxidative insult. CONCLUSIONS. Acceleration by menadione of the appearance of cataract in the Emory mouse demonstrates that oxidative stress is a risk factor in late-onset cataract. This quinone caused a greater increase in the production and levels of reactive metabolites of oxygen in Emory mice than in CR mice, indicative of a higher susceptibility of the former to oxidative insult.

Effects of calorie restriction and aging on the expression of antioxidant enzymes and ubiquitin in the liver of Emory mice

We studied the effect of age and calorie restriction on the expression of genes involved in antioxidant defenses in livers of young (4.5–6 months) and old (22 months) Emory mice fed a control (C) or restricted (R) diet. Specifically examined were catalase (CAT), glutathione peroxidase (Gpx), Cu Zn and Mn superoxide dismutase ( Cu ZnSOD ). As an indicator of oxidative damage to the tissues we measured lipid peroxidation. As indicators of oxidative stress we determined ubiquitin mRNA levels and endogenous high molecular weight (HMW) ubiquitin conjugates. Lower mRNA levels of ubiquitin ( P < 0.05), CAT ( P < 0.01) and Gpx ( P < 0.01) were observed in tissues from young R versus C animals. The old C group had a lower CAT mRNA level ( P < 0.0001) compared with young C. In the R group, age did not affect the CAT mRNA levels or Gpx mRNA levels; however, ubiquitin mRNA levels were higher ( P < 0.05). No significant changes in Cu Zn or MnSOD mRNA were observed with age or diet. Cu ZnSOD protein levels were lower in the young R at 4.5 months ( P < 0.05) than young C, and higher in the old R group versus old C ( P < 0.05). CAT protein levels were higher in the old C versus old R ( P < 0.05). Changes of HMW ubiquitin conjugates with age or diet were not significant. Of the four groups, the old R group whowed the highest levels of lipid peroxidation.

Dietary restriction delays cataract and reduces ascorbate levels in emory mice

Dietary restriction can effectively extend lifespan and retard many age-related debilities. One hypothesis to explain the beneficial effects of dietary restriction is that it prolongs maintenance of cellular homeostasis by limiting endogenous oxidative stress and preserves oxidative defense mechanisms during aging. Ascorbate, a primary antioxidant, may play a major role in preventing oxidative damage. Ascorbate levels were determined in dietary restricted (R) and control (C) Emory mice, a strain which develops age-related cataract due in part to oxidative damage to lens proteins. Mice which consumed a diet restricted by 40% in calories had lower ascorbate concentrations in plasma, liver and kidney. Nevertheless, R animals showed significantly delayed progression of cataract which extended over the entire second half of life. The R diet did not result in different ascorbate levels in this lens. Aging was associated with a decrease in ascorbate in all the examined tissues except lens of both the R and C groups. It is not clear from these data that ascorbate is a prominent factor in the delay of cataract formation or other debilities in R Emory mice. However, it also appears unlikely that lens ascorbate is cataractogenic.

Effect of calorie restriction on liver and kidney glutathione in aging emory mice

Increases in antioxidant defense capacity have been associated with increases in the health and life span of calorie restricted animals. Emory mice develop late-life cataract, a lesion associated with oxidative damage and loss of lens glutathione (GSH). The effect of calorie restriction on GSH in liver and kidney in this model has not been explored. GSH and oxidized GSH (GSSG) were measured by HPLC in liver and kidney of Emory mice fed a control diet (C; 85% calories of ad-lib fed mice) or 60% calorie intake of C (R; 40% calorie restriction relative to C mice) for up to 22 mo age. Liver GSH concentration increased significantly in C and R mice from 4.5 to 12 mo old with no difference observed between the two groups. At 22 mo of age, liver GSH was lower than that of 12 mo old in both groups. As compared with GSH at 12 mo old, this decrease was almost twice as greater in C (70%, p=0.001) than in R mice (36%, p=0.02), so that R mice had a significantly higher concentration of GSH in liver than C mice at 22 mo of age (R = 32.8+5.1, C= 22.1+8.3 imol GSH/g protein, p<0.01). Liver GSSG was similar in C and R mice at 12 mo of age (4.45+1.35 vs. 4.75+1.83 imol GSSG/g protein), but increased in R mice at 22 mo (R=5:43±1.48; C=3.22±1.02, p<0.01). Therefore, at 22 mo old, total liver glutathione (GSH+GSSG) was higher in R than in C mice. There was no significant difference in GSH, GSSG and total GSH in kidney from C and R mice at these ages. Thus, calorie restriction reduces the age-related loss of GSH antioxidant capacity in liver but not kidney of Emory mice.

Dietary calorie restriction in the Emory mouse: effects on lifespan, eye lens cataract prevalence and progression, levels of ascorbate, glutathione, glucose, and glycohemoglobin, tail collagen breaktime, DNA and RNA oxidation, skin integrity, fecundity, and cancer

The Emory mouse is the best model for age-related cataract. In this work we compare the effects of feeding a control diet (C) with a diet restricted (R) by 40% relative to C animals. In the R animals, median lifespan was extended by 40%. The proportion of R mice with advanced cataract was lower than C mice as early as 5 months of age. The mean grade of cataract was lower in R animals, beginning at 11 months and continuing until the end of the study. Ascorbate levels in R plasma and liver were 41–56% of C animals. There was no difference between diet groups with respect to lens ascorbate. Aging was associated with a decrease in ascorbate in lenses and kidneys in C and R mice. By 22 months, R animals had 48% higher liver glutathione levels than C mice. Liver glutathione levels were maximal at 12 months. Plasma glucose levels were > 27% lower in R animals at 6.5 and 22 months, and there was a 14% increase in glucose levels upon aging for both diet groups. In R mice, glycohemoglobin levels were 51% lower and tail collagen breaktime was decreased by 40%, even in younger animals. Collagen breaktime increased > 360% upon aging for both diet groups. Rates of production of urinary oxo 8dG and oxo 8G were higher in R animals compared with C animals, and increased upon aging. C animals exhibited more cancer and dermatological lesions, but less tail tip necrosis and inflamed genitals than R mice. These data allow evaluation of several theories of aging.

Studies on Emory Mouse Cataracts: Oxidative Factors

Emory mouse cataracts were analyzed for amino acids, protein carbonyls and fatty acids. The tissue membrane integrity was assessed by studying chromium-51 efflux. An effect of vitamin E-free diet on cataract progression was also studied. Chromium leakage was faster from the cataractous lenses, indicating a generalized membrane damage. This was also apparent from the loss of amino acids. The damage involves oxidation of proteins, as well as of lipids. Protein oxidation was apparent by a hydrazone formation with 2,4-dinitrophenyl hydrazine. The lipid oxidation was apparent from a decrease in oleic acid and appearance of the corresponding ketoacids. Lipid oxidation was also apparent by an attenuating effect of vitamin E.