Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice.

BACKGROUND: Primary osteoarthritis (OA) occurs without identifiable underlying causes such as previous injuries or specific medical conditions. Age is a major contributing factor to OA, and as one ages, various joint tissues undergo gradual change, including degeneration of the articular cartilage, alterations in subchondral bone (SCB) morphology, and inflammation of the synovium. METHODS: We investigated the prevalence of primary OA in aged, genetically diverse UM-HET3 mice. Articular cartilage (AC) integrity and SCB morphology were assessed in 182 knee joints of 22-25 months old mice using the Osteoarthritis Research Society International (OARSI) scoring system and micro-CT, respectively. Additionally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. RESULTS: Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13, inducible nitric oxide synthase, and the NLR family pyrin domain containing-3 inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and ß-galactosidase, also correlated with AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Notably, MB and MitoQ treatments influenced the disease's progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. CONCLUSIONS: Our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

Lifespan effects in male UM-HET3 mice treated with sodium thiosulfate, 16-hydroxyestriol, and late-start canagliflozin.

Genetically heterogeneous UM-HET3 mice born in 2020 were used to test possible lifespan effects of alpha-ketoglutarate (AKG), 2,4-dinitrophenol (DNP), hydralazine (HYD), nebivolol (NEBI), 16α-hydroxyestriol (OH_Est), and sodium thiosulfate (THIO), and to evaluate the effects of canagliflozin (Cana) when started at 16 months of age. OH_Est produced a 15% increase (p = 0.0001) in median lifespan in males but led to a significant (7%) decline in female lifespan. Cana, started at 16 months, also led to a significant increase (14%, p = 0.004) in males and a significant decline (6%, p = 0.03) in females. Cana given to mice at 6 months led, as in our previous study, to an increase in male lifespan without any change in female lifespan, suggesting that this agent may lead to female-specific late-life harm. We found that blood levels of Cana were approximately 20-fold higher in aged females than in young males, suggesting a possible mechanism for the sex-specific disparities in its effects. NEBI was also found to produce a female-specific decline (4%, p = 0.03) in lifespan. None of the other tested drugs provided a lifespan benefit in either sex. These data bring to 7 the list of ITP-tested drugs that induce at least a 10% lifespan increase in one or both sexes, add a fourth drug with demonstrated mid-life benefits on lifespan, and provide a testable hypothesis that might explain the sexual dimorphism in lifespan effects of the SGLT2 inhibitor Cana.

The Gehan test identifies life-extending compounds overlooked by the log-rank test in the NIA Interventions Testing Program: Metformin, Enalapril, caffeic acid phenethyl ester, green tea extract, and 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride.

The National Institute on Aging Interventions Testing Program (ITP) has so far identified 12 compounds that extend the lifespan of genetically heterogeneous mice using the log-rank test. However, the log-rank test is relatively insensitive to any compound that does not uniformly reduce mortality across the lifespan. This test may thus miss compounds that only reduce mortality before midlife, for example, a plausible outcome if a compound only mitigates risk factors before midlife or if its efficacy is reduced at later ages. We therefore reanalyzed all data collected by the ITP from 2004-2022 using the Gehan test, which is more sensitive to mortality differences earlier in the life course and does not assume a uniformly reduced mortality hazard across the lifespan. The Gehan test identified 5 additional compounds, metformin, enalapril, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), caffeic acid phenethyl ester (CAPE), and green tea extract (GTE), which significantly increased survival but were previously missed by the log-rank test. Three (metformin, enalapril, and 17-DMAG) were only effective in males and two (CAPE and GTE) were only effective in females. In addition, 1,3-butanediol, which by log-rank analysis increased survival in females but not males, increased survival in males by the Gehan test. These results suggest that statistical tests sensitive to non-uniformity of drug efficacy across the lifespan should be included in the standard statistical testing protocol to minimize overlooking geroprotective interventions.

Deciphering the Timing and Impact of Life-extending Drugs: A Novel Analytic Approach that Differentiates Early, Midlife, and Senescence Phase Efficacies.

Evidence that life-extending interventions are not uniformly effective across the lifespan calls for an analytic tool that can estimate age-specific treatment effects on mortality hazards. Here we report such a tool, applying it to mouse data from 42 agents tested in the NIA Interventions Testing Program. This tool identified agents that either reduced (22) or increased (16) mortality hazards or did both (6), all with marked variation in the duration of efficacy and magnitude of effect size. Only 7 reduced mortality hazards after the 90% mortality, when the burden of senescence is greatest. Sex differences were apparent in all parameters. This new analytic tool complements the commonly used log-rank test. It detects more potential life-extending candidates (22 versus 10) and indicates when during the life course they are effective. It also uncovers adverse effects. Most importantly, it identifies agents that specifically reduce mortality hazards during the senescent phase of life.

Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice.

Background: Primary osteoarthritis (OA) occurs without identifiable underlying causes such as previous injuries or specific medical conditions. Age is a major contributing factor to OA, and as one ages, various joint tissues undergo gradual change, including degeneration of the articular cartilage, alterations in subchondral bone (SCB) morphology, and inflammation of the synovium. Methods: We investigated the prevalence of primary OA in aged, genetically diverse UM-HET3 mice. Articular cartilage (AC) integrity and SCB morphology were assessed in 182 knee joints of 22-25 months old mice using the Osteoarthritis Research Society International (OARSI) scoring system and micro-CT, respectively. Additionally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. Results: Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13, inducible nitric oxide synthase, and the NLR family pyrin domain containing-3 inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and ß-galactosidase, also correlated with AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Notably, MB and MitoQ treatments influenced the disease's progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. Conclusions: Our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice.

This study investigated the prevalence and progression of primary osteoarthritis (OA) in aged UM-HET3 mice. Using the Osteoarthritis Research Society International (OARSI) scoring system, we assessed articular cartilage (AC) integrity in 182 knee joints of 22-25 months old mice. Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS), and the NLR family pyrin domain containing-3 (NLRP3) inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and ß-galactosidase, also correlated with AC scores. Using micro-CT, we examined the correlations between subchondral bone (SCB) morphology traits and AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Finally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. Notably, MB and MitoQ treatments influenced the disease's progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. In conclusion, our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

Castration reduces mortality and increases resilience in male mice: what is next?

This commentary concerns our recent report that prepubertal castration rescued the shorter lifespan of males, using the first mouse line that robustly shows the same shorter longevity with a similar age-variable mortality disadvantage as human males. This model provides a unique opportunity for research to uncover the basis for this clinically important sex difference in aging. Researchers can now identify the hormones involved, the duration of exposure required, and, most important, the cellular and molecular targets, with the ultimate goal of developing therapeutic interventions to enhance health and reduce mortality without castration-compromising reproductive function.

Astaxanthin and meclizine extend lifespan in UM-HET3 male mice; fisetin, SG1002 (hydrogen sulfide donor), dimethyl fumarate, mycophenolic acid, and 4-phenylbutyrate do not significantly affect lifespan in either sex at the doses and schedules used.

In genetically heterogeneous (UM-HET3) mice produced by the CByB6F1 × C3D2F1 cross, the Nrf2 activator astaxanthin (Asta) extended the median male lifespan by 12% (p = 0.003, log-rank test), while meclizine (Mec), an mTORC1 inhibitor, extended the male lifespan by 8% (p = 0.03). Asta was fed at 1840 ± 520 (9) ppm and Mec at 544 ± 48 (9) ppm, stated as mean ± SE (n) of independent diet preparations. Both were started at 12 months of age. The 90th percentile lifespan for both treatments was extended in absolute value by 6% in males, but neither was significant by the Wang-Allison test. Five other new agents were also tested as follows: fisetin, SG1002 (hydrogen sulfide donor), dimethyl fumarate, mycophenolic acid, and 4-phenylbutyrate. None of these increased lifespan significantly at the dose and method of administration tested in either sex. Amounts of dimethyl fumarate in the diet averaged 35% of the target dose, which may explain the absence of lifespan effects. Body weight was not significantly affected in males by any of the test agents. Late life weights were lower in females fed Asta and Mec, but lifespan was not significantly affected in these females. The male-specific lifespan benefits from Asta and Mec may provide insights into sex-specific aspects of aging.

Impaired aldehyde detoxification exacerbates motor deficits in an alpha-synuclein mouse model of Parkinson's disease.

INTRODUCTION: The discovery of biogenic aldehydes in the postmortem parkinsonian brain and the ability of these aldehydes to modify and cross-link proteins has called attention to their possible role in Parkinson's disease. For example, many in vitro studies have found that the aldehyde metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL), induces the formation of stable, neurotoxic alpha-synuclein oligomers. METHODS: To study this in vivo, mice deficient in the two aldehyde dehydrogenase enzymes (Aldh1a1 and Aldh2, DKO) primarily responsible for detoxification of DOPAL in the nigrostriatal pathway were crossed with mice that overexpress human wild-type alpha-synuclein. DKO overexpressing human wild-type alpha-synuclein (DKO/ASO) offspring were evaluated for impairment on motor tasks associated with Parkinsonism. RESULTS: DKO/ASO mice developed severe motor deficits greater than that of mice overexpressing human wild-type alpha-synuclein alone. CONCLUSION: These results provide evidence to support the idea that biogenic aldehydes such as DOPAL interact with human wild-type alpha-synuclein, directly or indirectly, in vivo to exacerbate locomotor deficits in Parkinson's disease.

Prepubertal castration eliminates sex differences in lifespan and growth trajectories in genetically heterogeneous mice.

Sex differences in aging and longevity have been widely observed, with females consistently outliving males across human populations. However, the mechanisms driving these disparities remain poorly understood. In this study, we explored the influence of post-pubertal testicular effects on sex differences in aging by prepubertally castrating genetically heterogeneous (UM-HET3) mice, a unique mouse model that emulates human sex differences in age-related mortality. Prepubertal castration eliminated the longevity disparity between sexes by reducing the elevated early- to mid-life mortality rate observed in males and extending their median lifespan to match that of females. Additionally, castration extended the duration of body weight growth and attenuated the inverse correlation between early-age body weight and lifespan in males, aligning their growth trajectories with those of females. Our findings suggest that post-pubertal testicular actions in genetically diverse mice are primarily responsible for sex differences in longevity as well as growth trajectories. These findings offer a foundation for further investigation into the fundamental mechanisms driving sex-specific aging patterns and the development of potential pro-longevity interventions.

Long-term effects of canagliflozin treatment on the skeleton of aged UM-HET3 mice.

Sodium glucose cotransporter-2 inhibitors (SGLT2is) promote urinary glucose excretion and decrease plasma glucose levels independent of insulin. Canagliflozin (CANA) is an SGLT2i, which is widely prescribed, to reduce cardiovascular complications, and as a second-line therapy after metformin in the treatment of type 2 diabetes mellitus. Despite the robust metabolic benefits, reductions in bone mineral density (BMD) and cortical fractures were reported for CANA-treated subjects. In collaboration with the National Institute on Aging (NIA)-sponsored Interventions Testing Program (ITP), we tested skeletal integrity of UM-HET3 mice fed control (137 mice) or CANA-containing diet (180 ppm, 156 mice) from 7 to 22 months of age. Micro-computed tomography (micro-CT) revealed that CANA treatment caused significant thinning of the femur mid-diaphyseal cortex in both male and female mice, did not affect trabecular bone architecture in the distal femur or the lumbar vertebra-5 in male mice, but was associated with thinning of the trabeculae at the distal femur in CANA-treated female mice. In male mice, CANA treatment is associated with significant reductions in cortical bone volumetric BMD by micro-CT, and by quantitative backscattered scanning electron microscopy. Raman microspectroscopy, taken at the femur mid-diaphyseal posterior cortex, showed significant reductions in the mineral/matrix ratio and an increased carbonate/phosphate ratio in CANA-treated male mice. These data were supported by thermogravimetric assay (TGA) showing significantly decreased mineral and increased carbonate content in CANA-treated male mice. Finally, the sintered remains of TGA were subjected to X-ray diffraction and showed significantly higher fraction of whitlockite, a calcium orthophosphate mineral, which has higher resorbability than hydroxyapatite. Overall, long-term CANA treatment compromised bone morphology and mineral composition of bones, which likely contribute to increased fracture risk seen with this drug.

DOPAL initiates αSynuclein-dependent impaired proteostasis and degeneration of neuronal projections in Parkinson's disease.

Dopamine dyshomeostasis has been acknowledged among the determinants of nigrostriatal neuron degeneration in Parkinson's disease (PD). Several studies in experimental models and postmortem PD patients underlined increasing levels of the dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is highly reactive towards proteins. DOPAL has been shown to covalently modify the presynaptic protein αSynuclein (αSyn), whose misfolding and aggregation represent a major trait of PD pathology, triggering αSyn oligomerization in dopaminergic neurons. Here, we demonstrated that DOPAL elicits αSyn accumulation and hampers αSyn clearance in primary neurons. DOPAL-induced αSyn buildup lessens neuronal resilience, compromises synaptic integrity, and overwhelms protein quality control pathways in neurites. The progressive decline of neuronal homeostasis further leads to dopaminergic neuron loss and motor impairment, as showed in in vivo models. Finally, we developed a specific antibody which detected increased DOPAL-modified αSyn in human striatal tissues from idiopathic PD patients, corroborating the translational relevance of αSyn-DOPAL interplay in PD neurodegeneration.

Canagliflozin retards age-related lesions in heart, kidney, liver, and adrenal gland in genetically heterogenous male mice.

Canagliflozin (Cana), a clinically important anti-diabetes drug, leads to a 14% increase in median lifespan and a 9% increase in the 90th percentile age when given to genetically heterogeneous male mice from 7 months of age, but does not increase lifespan in female mice. A histopathological study was conducted on 22-month-old mice to see if Cana retarded diverse forms of age-dependent pathology. This agent was found to diminish incidence or severity, in male mice only, of cardiomyopathy, glomerulonephropathy, arteriosclerosis, hepatic microvesicular cytoplasmic vacuolation (lipidosis), and adrenal cortical neoplasms. Protection against atrophy of the exocrine pancreas was seen in both males and females. Thus, the extension of lifespan in Cana-treated male mice, which is likely to reflect host- or tumor-mediated delay in lethal neoplasms, is accompanied by parallel retardation of lesions, in multiple tissues, that seldom if ever lead to death in these mice. Canagliflozin thus can be considered a drug that acts to slow the aging process and should be evaluated for potential protective effects against many other late-life conditions.

Lifespan benefits for the combination of rapamycin plus acarbose and for captopril in genetically heterogeneous mice.

Mice bred in 2017 and entered into the C2017 cohort were tested for possible lifespan benefits of (R/S)-1,3-butanediol (BD), captopril (Capt), leucine (Leu), the Nrf2-activating botanical mixture PB125, sulindac, syringaresinol, or the combination of rapamycin and acarbose started at 9 or 16 months of age (RaAc9, RaAc16). In male mice, the combination of Rapa and Aca started at 9 months and led to a longer lifespan than in either of the two prior cohorts of mice treated with Rapa only, suggesting that this drug combination was more potent than either of its components used alone. In females, lifespan in mice receiving both drugs was neither higher nor lower than that seen previously in Rapa only, perhaps reflecting the limited survival benefits seen in prior cohorts of females receiving Aca alone. Capt led to a significant, though small (4% or 5%), increase in female lifespan. Capt also showed some possible benefits in male mice, but the interpretation was complicated by the unusually low survival of controls at one of the three test sites. BD seemed to produce a small (2%) increase in females, but only if the analysis included data from the site with unusually short-lived controls. None of the other 4 tested agents led to any lifespan benefit. The C2017 ITP dataset shows that combinations of anti-aging drugs may have effects that surpass the benefits produced by either drug used alone, and that additional studies of captopril, over a wider range of doses, are likely to be rewarding.

Sex- and age-dependent genetics of longevity in a heterogeneous mouse population.

DNA variants that modulate life span provide insight into determinants of health, disease, and aging. Through analyses in the UM-HET3 mice of the Interventions Testing Program (ITP), we detected a sex-independent quantitative trait locus (QTL) on chromosome 12 and identified sex-specific QTLs, some of which we detected only in older mice. Similar relations between life history and longevity were uncovered in mice and humans, underscoring the importance of early access to nutrients and early growth. We identified common age- and sex-specific genetic effects on gene expression that we integrated with model organism and human data to create a hypothesis-building interactive resource of prioritized longevity and body weight genes. Finally, we validated Hipk1, Ddost, Hspg2, Fgd6, and Pdk1 as conserved longevity genes using Caenorhabditis elegans life-span experiments.

San Antonio Nathan Shock Center: your one-stop shop for aging research.

With evolving cores, enrichment and training programs, and supported research projects, the San Antonio (SA) Nathan Shock Center has for 26 years provided critical support to investigators locally, nationally, and abroad. With its existing and growing intellectual capital, the SA Nathan Shock Center provides to local and external investigators an enhanced platform to conduct horizontally integrated (lifespan, healthspan, pathology, pharmacology) transformative research in the biology of aging, and serves as a springboard for advanced educational and training activities in aging research. The SA Nathan Shock Center consists of six cores: Administrative/Program Enrichment Core, Research Development Core, Aging Animal Models and Longevity Assessment Core, Pathology Core, Analytical Pharmacology and Drug Evaluation Core, and Integrated Physiology of Aging Core. The overarching goal of the SA Nathan Shock Center is to advance knowledge in the basic biology of aging and to identify molecular and cellular mechanisms that will facilitate the development of pharmacologic interventions and other strategies to extend healthy lifespan. In pursuit of this goal, we provide an innovative "one-stop shop" venue to accelerate transformative research in the biology of aging through our integrated research cores. Moreover, we aim to foster and promote career development of early-stage investigators in aging biology through our research development programs, to serve as a resource and partner to investigators from other Shock Centers, and to disseminate scientific knowledge and enhanced awareness about aging research. Overall, the SA Nathan Shock Center aims to be a leader in research that advances our understanding of the biology of aging and development of approaches to improve longevity and healthy aging.

17-a-estradiol late in life extends lifespan in aging UM-HET3 male mice; nicotinamide riboside and three other drugs do not affect lifespan in either sex.

In genetically heterogeneous mice produced by the CByB6F1 x C3D2F1 cross, the "non-feminizing" estrogen, 17-α-estradiol (17aE2), extended median male lifespan by 19% (p < 0.0001, log-rank test) and 11% (p = 0.007) when fed at 14.4 ppm starting at 16 and 20 months, respectively. 90th percentile lifespans were extended 7% (p = 0.004, Wang-Allison test) and 5% (p = 0.17). Body weights were reduced about 20% after starting the 17aE2 diets. Four other interventions were tested in males and females: nicotinamide riboside, candesartan cilexetil, geranylgeranylacetone, and MIF098. Despite some data suggesting that nicotinamide riboside would be effective, neither it nor the other three increased lifespans significantly at the doses tested. The 17aE2 results confirm and extend our original reports, with very similar results when started at 16 months compared with mice started at 10 months of age in a prior study. The consistently large lifespan benefit in males, even when treatment is started late in life, may provide information on sex-specific aspects of aging.