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 \u3c 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

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

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

Senolytic therapy in mild Alzheimer’s disease: a phase 1 feasibility trial

Cellular senescence contributes to Alzheimer’s disease (AD) pathogenesis. An open-label, proof-of-concept, phase I clinical trial of orally delivered senolytic therapy, dasatinib (D) and quercetin (Q), was conducted in early-stage symptomatic patients with AD to assess central nervous system (CNS) penetrance, safety, feasibility and efficacy. Five participants (mean age = 76 + 5 years; 40% female) completed the 12-week pilot study. D and Q levels in blood increased in all participants (12.7–73.5 ng ml−1 for D and 3.29–26.3 ng ml−1 for Q). In cerebrospinal fluid (CSF), D levels were detected in four participants (80%) ranging from 0.281 to 0.536 ml−1 with a CSF to plasma ratio of 0.422–0.919%; Q was not detected. The treatment was well-tolerated, with no early discontinuation. Secondary cognitive and neuroimaging endpoints did not significantly differ from baseline to post-treatment further supporting a favorable safety profile. CSF levels of interleukin-6 (IL-6) and glial fibrillary acidic protein (GFAP) increased (t(4) = 3.913, P = 0.008 and t(4) = 3.354, P = 0.028, respectively) with trending decreases in senescence-related cytokines and chemokines, and a trend toward higher Aβ42 levels (t(4) = −2.338, P = 0.079). In summary, CNS penetrance of D was observed with outcomes supporting safety, tolerability and feasibility in patients with AD. Biomarker data provided mechanistic insights of senolytic effects that need to be confirmed in fully powered, placebo-controlled studies. ClinicalTrials.gov identifier: NCT04063124

Caspase-2 maintains bone homeostasis by inducing apoptosis of oxidatively-damaged osteoclasts.

Osteoporosis is a silent disease, characterized by a porous bone micro-structure that enhances risk for fractures and associated disabilities. Senile, or age-related osteoporosis (SO), affects both men and women, resulting in increased morbidity and mortality. However, cellular and molecular mechanisms underlying senile osteoporosis are not fully known. Recent studies implicate the accumulation of reactive oxygen species (ROS) and increased oxidative stress as key factors in SO. Herein, we show that loss of caspase-2, a cysteine aspartate protease involved in oxidative stress-induced apoptosis, results in total body and femoral bone loss in aged mice (20% decrease in bone mineral density), and an increase in bone fragility (30% decrease in fracture strength). Importantly, we demonstrate that genetic ablation or selective inhibition of caspase-2 using zVDVAD-fmk results in increased numbers of bone-resorbing osteoclasts and enhanced tartrate-resistant acid phosphatase (TRAP) activity. Conversely, transfection of osteoclast precursors with wild type caspase-2 but not an enzymatic mutant, results in a decrease in TRAP activity. We demonstrate that caspase-2 expression is induced in osteoclasts treated with oxidants such as hydrogen peroxide and that loss of caspase-2 enhances resistance to oxidants, as measured by TRAP activity, and decreases oxidative stress-induced apoptosis of osteoclasts. Moreover, oxidative stress, quantified by assessment of the lipid peroxidation marker, 4-HNE, is increased in Casp2-/- bone, perhaps due to a decrease in antioxidant enzymes such as SOD2. Taken together, our data point to a critical and novel role for caspase-2 in maintaining bone homeostasis by modulating ROS levels and osteoclast apoptosis during conditions of enhanced oxidative stress that occur during aging

Caspase-2 regulates osteoclast numbers via apoptosis.

<p>(A) Primary cultures of <i>in vitro</i> differentiated <i>Casp2^+/+</i> and <i>Casp2^−/−</i> osteoclasts grown in 96-well plates were TRAP-stained. (B) TRAP-positive, multinucleated osteoclasts were counted (<i>*</i>, <i>p</i><0.05). (C) TRAP activity of in vitro cultured primary osteoclasts from <i>Casp2^+/+</i> and <i>Casp2^−/−</i> mice was measured and normalized to cell viability as determined by MTT assay (<i>*</i>, <i>p</i><0.0005). (D) Osteoclasts were cultured in the presence of a caspase-2-selective inhibitor, zVDVAD and, (E) Intact TRAP-positive osteoclasts were counted (<i>*</i>, <i>p</i><0.0001). (F) Osteoclasts cultured with or without zVDVAD were stained with TUNEL and counted (<i>*</i>, <i>p</i><0.001). (G) Overexpression of wild type caspase-2 but not its enzymatic mutant (Casp2-m) in the osteoclast precursor cell line, RAW264.7, significantly reduced TRAP activity (<i>*</i>, <i>p</i>< 0.005). Scale bar, 100 μm.</p

Caspase-2 is induced in osteoclasts on treatment with oxidative stress-inducing agents.

<p>(A) Primary cultures of <i>Casp2^+/+</i> osteoclasts treated with 500 μM H₂O₂ for the indicated time points (n = 3), were fixed and probed for caspase-2 (green); nuclei were stained blue. <i>Casp2^−/−</i> osteoclasts treated with 500 μM H₂O₂ for 4 h served as a negative control. Scale bar, 50 μm. (B) RAW 264.7 macrophages were differentiated into osteoclasts, treated with 500 μM H₂O₂ for the indicated time periods, lysed and probed for caspase-2 (n = 3). Stripped blot was probed for total actin as loading control.</p

Loss of caspase-2 increases oxidative stress <i>in vivo</i>.

<p>(A) Bone sections from 27-month old mice (n = 3) were immunostained for 4-HNE; <i>Casp2^−/−</i> section was used as a no-primary control to determine antibody specificity. Data shown is representative of two experiments. Scale bar, 100 μm. (B) Percentage of cells stained (<i>*</i>, <i>p</i> = 0.0007) and relative intensities of staining were measured using Image J and plotted (<i>*</i>, <i>p</i><0.0001). (C) Representative western blots of osteoclast lysates probed for the anti-oxidant enzyme, SOD2; GAPDH was used as a loading control.</p

Loss of caspase-2 results in altered biomechanical properties of bone.

<p>Parameters measured using micro-indentation analyses (n = 3) include (A) Total indentation distance (TID) (B) Indentation distance increase (IDI), (C) Energy dissipated (ED), (D) Average Loading Slope (Avg LS) and (E) Average Unloading Slope (Avg US). Three-point bending tests (n = 3) were used to measure (F) Load to Yield and (G) Yield Stress. Data are represented as mean ± SEM. <i>*</i>, <i>p</i><0.05.</p