Aged Mice Research Articles

Multi-Protective Effects of Petunidin-3-O-(trans-p-coumaroylrutinoside)-5-O-glucoside on D-Gal-Induced Aging Mice

Petunidin-3-O-(trans-p-coumaroylrutinoside)-5-O-glucoside (PtCG), the primary anthocyanin ingredient in Lycium ruthenicum Murr., possesses a range of biological activities, including antioxidative properties and melanin inhibition. This study aimed to investigate the protective effect of PtCG on D-galactose (D-gal)-induced aging in female mice and elucidate the underlying molecular pathways. Behavioral experiments, including the MWW and Y-maze tests, revealed that PtCG significantly ameliorated cognitive decline and enhanced learning and memory abilities in aging mice. Regarding biochemical indicators, PtCG considerably improved superoxide dismutase (SOD) and glutathione (GSH) activity while reducing malondialdehyde (MDA) and acetylcholinesterase (AChE) levels in the hippocampus and serum. Furthermore, PtCG ingestion alleviated liver injury by decreasing alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (AKP) levels, and attenuated renal damage by reducing blood urea nitrogen (BUN) and uric acid (UA) levels. Transmission electron microscopy (TEM) results demonstrated that PtCG restored the function and quantity of synapses in the hippocampus. Hematoxylin and eosin (H&E), Masson’s trichrome, and Nissl staining revealed that PtCG significantly improved the relevant pathological characteristics of liver and hippocampal tissues in aging mice. The molecular mechanism investigation showed that PtCG downregulated the protein expression of microglial marker ionized calcium-binding adapter molecule 1 (Iba1), astrocytic marker glial fibrillary acidic protein (GFAP), β-secretase 1 (BACE-1), and amyloid-beta1–42 (Aβ1–42) in the hippocampus of aging mice. The protein expression of inflammatory pathway components, including nuclear factor-kappa B (NF-κB), cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), and interleukin-1 beta (IL-1β), was also suppressed. These findings suggest that PtCG may possess anti-aging properties, with its mechanism of action potentially linked to the attenuation of neuroinflammation, oxidative stress, and liver and kidney damage. PtCG may have future applications as a functional food for the treatment of aging-related disorders.

Nerve-associated macrophages control adipose homeostasis across lifespan and restrain age-related inflammation.

Age-related inflammation or inflammaging is a key mechanism that increases disease burden and may control lifespan. How adipose tissue macrophages (ATMs) control inflammaging is not well understood in part because the molecular identities of niche-specific ATMs are incompletely known. Using intravascular labeling to exclude circulating myeloid cells and subsequent single-cell sequencing with orthogonal validation, we define the diversity and alterations in niche resident ATMs through lifespan. Aging led to depletion of vessel-associated macrophages (VAMs), expansion of lipid-associated macrophages (LAMs), and emergence of a unique subset of CD38+ age-associated macrophages (AAMs) in visceral white adipose tissue (VAT). Interestingly, CD169+CD11c- ATMs are enriched in a subpopulation of nerve-associated macrophages (NAMs) that declines with age. Depletion of CD169+ NAMs in aged mice increases inflammaging and impairs lipolysis suggesting that they are necessary for preventing catecholamine resistance in VAT. These findings reveal specialized ATMs control adipose homeostasis and link inflammation to tissue dysfunction during aging.

Balance performance in aged mice is dependent on unipolar brush cells.

The vestibular processing regions of the cerebellum integrate vestibular information with other sensory modalities and motor signals to regulate balance, gaze stability, and spatial orientation. A class of excitatory glutamatergic interneurons known as unipolar brush cells (UBCs) are highly concentrated within the granule cell layer of these regions. UBCs receive vestibular signals directly from primary vestibular afferents and indirectly from mossy fibers. Each UBC excites numerous granule cells and could contribute to computations necessary for balance-related motor function. Prior research has implicated UBCs in motor function, but their influence on balance performance remains unclear, especially in aged mice that have age-related impairment. Here we tested whether UBCs contribute to motor coordination and balance by disrupting their activity with chemogenetics in aged and young mice. Age-related balance deficits were apparent in mice > 6 months old. Disrupting the activity of a subpopulation of UBCs caused aged mice to fall off a balance beam more frequently and altered swimming behaviors that are sensitive to vestibular dysfunction. These effects were not seen in young (7-week-old) mice. Thus, disrupting the activity of UBCs impairs mice with age-related balance issues and suggest that UBCs are essential for balance and vestibular function in aged mice.

Spinster homolog 2 (SPNS2) deficiency drives endothelial cell senescence and vascular aging via promoting pyruvate metabolism mediated mitochondrial dysfunction

Endothelial cell (EC) senescence and vascular aging are important hallmarks of chronic metabolic diseases. An improved understanding of the precise regulation of EC senescence may provide novel therapeutic strategies for EC and vascular aging-related diseases. This study examined the potential functions of Spinster homolog 2 (SPNS2) in EC senescence and vascular aging. We discovered that the expression of SPNS2 was significantly lower in older adults, aged mice, hydrogen peroxide-induced EC senescence models and EC replicative senescence model, and was correlated with the expression of aging-related factors. in vivo experiments showed that the EC-specific knockout of SPNS2 markedly aggravated vascular aging by substantially, impairing vascular structure and function, as evidenced by the abnormal expression of aging factors, increased inflammation, reduced blood flow, pathological vessel dilation, and elevated collagen levels in a naturally aging mouse model. Moreover, RNA sequencing and molecular biology analyses revealed that the loss of SPNS2 in ECs increased cellular senescence biomarkers, aggravated the senescence-associated secretory phenotype (SASP), and inhibited cell proliferation. Mechanistically, silencing SPNS2 disrupts pyruvate metabolism homeostasis via pyruvate kinase M (PKM), resulting in mitochondrial dysfunction and EC senescence. Overall, SPNS2 expression and its functions in the mitochondria are crucial regulators of EC senescence and vascular aging.

Inhibition of CILP2 Improves Glucose Metabolism and Mitochondrial Dysfunction in Sarcopenia via the Wnt Signalling Pathway.

Skeletal muscle is the primary organ involved in insulin-mediated glucose metabolism. Elevated levels of CILP2 are a significant indicator of impaired glucose tolerance and are predominantly expressed in skeletal muscle. It remains unclear whether CILP2 contributes to age-related muscle atrophy through regulating the glucose homeostasis and insulin sensitivity. Initially, the expression levels of CILP2 were assessed in elderly mice and patients with sarcopenia. Lentiviral vectors were used to induce either silencing or overexpression of CILP2 in C2C12 myoblast cells. The effects of CILP2 on proliferation, myogenic differentiation, insulin sensitivity and glucose uptake were evaluated using immunofluorescence, western blotting, real-time quantitative polymerase chain reaction, RNA sequencing, glucose uptake experiments, dual-luciferase reporter assays and co-immunoprecipitation (CO-IP). An adeno-associated virus-9 containing a muscle-specific promoter was injected into SAMP8 senile mice to observe the efficacy of CILP2 knockout. We found that there was more CLIP2 expressed in the skeletal muscle of ageing mice (+1.1-fold, p < 0.01) and in patients with sarcopenia (+2.5-fold, p < 0.01) compared to the control group. Following the overexpression of CILP2, Ki67 (-65%, p < 0.01), PCNA (-32%, p < 0.05), MyoD1 (-89%, p < 0.001), MyoG (-31%, p < 0.05) and MyHC (-85%, p < 0.001), which indicate proliferation and differentiation potential, were significantly reduced. In contrast, MuRF-1 (+59%, p < 0.05), atrogin-1 (+43%, p < 0.05) and myostatin (+31%, p < 0.05), the markers of muscular atrophy, were significantly increased. Overexpression of CILP2 decreased insulin sensitivity, glucose uptake (-18%, p < 0.001), GLUT4 translocation to the membrane and the maximum respiratory capacity of mitochondria. Canonical Wnt signalling was identified through RNA sequencing as a potential pathway for CILP2 regulation in C2C12, and Wnt3a was confirmed as an interacting protein of CILP2 in the CO-IP assay. The addition of recombinant Wnt3a protein reversed the inhibitory effects on myogenesis and glucose metabolism caused by CILP2 overexpression. Conversely, CILP2 knockdown promoted myogenesis and glucose metabolism. CILP2 knockdown improved muscle atrophy in mice, characterized by significant increases in time to exhaustion (+42%, p < 0.001), grip strength (+19%, p < 0.01), muscle mass (+15%, p < 0.001) and mean muscle cross-sectional area (+37%, p < 0.01). CILP2 knockdown enhanced glycogen synthesis (+83%, p < 0.001) and the regeneration of oxidative and glycolytic muscle fibres in SAMP8 ageing mice via the Wnt/β-catenin signalling pathway. Our results indicate that CILP2 interacts with Wnt3a to suppress the Wnt/β-catenin signalling pathway and its downstream cascade, leading to impaired insulin sensitivity and glucose metabolism in skeletal muscle. Targeting CILP2 inhibition could offer potential therapeutic benefits for sarcopenia.

Sirt6 Mono-ADP-Ribosylates YY1 to Promote Dystrophin Expression for Neuromuscular Transmission.

The degeneration of the neuromuscular junction (NMJ) and the decline in motor function are common features of aging, but the underlying mechanisms have remained largely unclear. This study reveals that Sirt6 is reduced in aged mouse muscles. Ablation of Sirt6 in skeletal muscle causes a reduction of Dystrophin levels, resulting in premature NMJ degeneration, compromised neuromuscular transmission, and a deterioration in motor performance. Mechanistic studies show that Sirt6 negatively regulates the stability of the Dystrophin repressor YY1 (Yin Yang 1). Specifically, Sirt6 mono-ADP-ribosylates YY1, causing its disassociation from the Dystrophin promoter and allowing YY1 to bind to the SMURF2 E3 ligase, leading to its degradation. Importantly, supplementation with nicotinamide mononucleotide (NMN) enhances the mono-ADP-ribosylation of YY1 and effectively delays NMJ degeneration and the decline in motor function in elderly mice. These findings provide valuable insights into the intricate mechanisms underlying NMJ degeneration during aging. Targeting Sirt6 could be a potential therapeutic approach to mitigate the detrimental effects on NMJ degeneration and improve motor function in the elderly population.

Acetylcholine receptor-β inhibition by interleukin-6 in skeletal muscles contributes to modulating neuromuscular junction during aging

BackgroundAging-related strength decline contributes to physiological deterioration and is a good predictor of poor prognosis. However, the mechanisms underlying neuromuscular junction disorders affecting contraction in aging are not well described. We hypothesized that the autocrine effect of interleukin (IL)-6 secreted by skeletal muscle inhibits acetylcholine receptor (AChR) expression, potentially causing aging-related strength decline. Therefore, we investigated IL-6 and AChR β-subunit (AChR-β) expression in the muscles and sera of aging C57BL/6J mice and verified the effect of IL-6 on AChR-β expression.MethodsAnimal experiments, in vitro studies, bioinformatics, gene manipulation, dual luciferase reporter gene assays, and chromatin immunoprecipitation experiments were used to explore the role of the transcription cofactor peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) and its interacting transcription factors in the IL-6-mediated regulation of AChR-β expression.ResultsIL-6 expression gradually increased during aging, inhibiting AChR-β expression, which was reversed by tocilizumab. Both tocilizumab and the PGC1α agonist reversed the inhibiting effect of IL-6 expression on AChR-β. Compared to inhibition of signal transducer and activator of transcription 3, extracellular signal-regulated kinases 1/2 (ERK1/2) inhibition suppressed the effects of IL-6 on AChR-β and PGC1α. In aging mouse muscles and myotubes, myocyte enhancer factor 2 C (MEF2C) was recruited by PGC1α, which directly binds to the AChR-β promoter to regulate its expression.ConclusionsThis study verifies AChR-β regulation by the IL-6/IL-6R-ERK1/2-PGC1α/MEF2C pathway. Hence, evaluating muscle secretion, myokines, and AChRs at an earlier stage to determine pathological progression is important. Moreover, developing intervention strategies for monitoring, maintaining, and improving muscle structure and function is necessary.

Single-cell analysis reveals alternations between the aged and young mice prostates

BackgroundAging of the male prostate is an inevitable process in which the prostate undergoes hyperplasia, and this growth may lead to compression of the urethra, resulting in voiding dysfunction and associated symptoms, and an increased risk of prostate cancer. Despite the significance of prostate aging, the molecular mechanisms involved are still not fully understood.MethodsProstate split by lobes from young (2 months) and aged (24 months) mice were collected for single-cell RNA sequencing (scRNA-seq) analysis. Tissues from both anterior prostate (AP) and ventral/dorsal/lateral prostate (VDLP) were included in the study. Data analysis included unsupervised clustering using the uniform manifold approximation and projection (UMAP) algorithm to identify distinct cell types based on marker gene expression. Differential gene expression analysis was performed to identify age-related changes in gene expression across different cell types. Functional enrichment analysis was conducted to elucidate biological pathways associated with differentially expressed genes. Additionally, cellular interactions and developmental trajectories were analyzed to characterize cellular dynamics during prostate aging.ResultsThe single-cell transcriptome analysis of the mouse prostate during aging revealed heterogeneity across various cell types and their changes during the aging process. We found a significant increase in the proportion of mesenchymal and immune cells in aged mice. Our study unveiled alterations in genes and pathways associated with cellular senescence, oxidative stress, and regeneration in epithelial cells. Furthermore, we observed that basal cells may undergo epithelial-mesenchymal transition (EMT) to become mesenchymal cells, particularly prominent in aged mice. Additionally, immune cells, notably macrophages and T cells, exhibited a heightened inflammatory response in aged mice.ConclusionIn summary, our study provides a comparative analysis of the single-cell transcriptome of the aged and young mice prostates, elucidating cellular and molecular changes between the aged and young mice prostates.

Intestinal barrier, immunity and gut microbiota-based protective effects of Lactococcus lactis HF08 and its postbiotic derivative on aging and aging colitis mice

The prevalence and severity of gastrointestinal diseases were increased with age. In this study, the intestinal protective effects of Lactococcus lactis HF08 (HF08) and its derived postbiotic (P-HF08) on D-gal-induced aging mice and D-gal/DSS-induced aging colitis mice were investigated. In D-gal-induced aging mice, both HF08 and P-HF08 alleviated aging-related intestinal barrier dysfunction, inflammatory status, and gut microbiota disorder. The effects of probiotic HF08 were superior to those of postbiotic P-HF08, attributed to ability of HF08 to regulate the gut microbiota. However, in D-gal/DSS-induced aging colitis mice, the effects of P-HF08 on colitis surpassed that of HF08. Specifically, both HF08 and P-HF08 could reduce symptoms of age-related colitis, including reduction of lose weight, the DAI score, colonic shortening, and colon tissue damage. The inhibitory effects of P-HF08 on intestinal inflammation surpassed those of HF08, as evidenced by the levels of colon IL-6, IL-1β, and IL-10. Western blot results demonstrated that the anti-inflammatory effects of P-HF08 were attributed to the downregulation of key proteins in the TLR4/NF-κB pathway. And four potential TLR4 inhibitors were identified from HF08 metabolites (eplerenone, genistein, indoleacrylic acid, and turanose) by molecular docking. Nevertheless, HF08 could better regulate gut microbiota and metabolite in aging-related colitis than P-HF08, which was consistent with the results on aging mice. Overall, our finding revealed that when the intestinal barrier was intact (aging), probiotics showed superior regulation of intestinal microbiota, while postbiotics offered greater safety in case of intestinal barrier damage (aging colitis). This study offered a novel perspective into the applications of probiotics and their derivatives in the aging related gastrointestinal diseases adjuvant therapy.

CaMKIIα-TARPγ8 signaling mediates hippocampal synaptic impairment in aging.

Aging-related decline in memory and synaptic function are associated with the dysregulation of calcium homeostasis, attributed to the overexpression of voltage-gated calcium channels (VGCC). The membrane insertion of AMPAR governed by the AMPAR auxiliary proteins is essential for synaptic transmission and plasticity (LTP). In this study, we demonstrated the hippocampal expression of the transmembrane AMPAR regulatory proteins γ-8 (TARPγ8) was reduced in aged mice along with the reduced CaMKIIα activity and memory impairment. We further showed that TARPγ8 expression was dependent on CaMKIIα activity. Inhibition of CaMKIIα activity significantly reduced the hippocampal TARPγ8 expression and CA3-CA1 LTP in young mice to a similar level to that of the aged mice. Furthermore, the knockdown of hippocampal TARPγ8 impaired LTP and memory in young mice, which mimicked the aging-related changes. We confirmed the enhanced hippocampal VGCC (Cav-1.3) expression in aged mice and found that inhibition of VGCC activity largely increased both p-CaMKIIα and TARPγ8 expression in aged mice, whereas inhibition of NMDAR or Calpains had no effect. In addition, we found that the exogenous expression of human TARPγ8 in the hippocampus in aged mice restored LTP and memory function. Collectively, these results indicate that the synaptic and cognitive impairment in aging is associated with the downregulation of CaMKIIα-TARPγ8 signaling caused by VGCC activation. Our results suggest that TARPγ8 may be a key molecular biomarker for brain aging and that boosting CaMKIIα-TARPγ8 signaling may be critical for the restoration of synaptic plasticity of aging and aging-related diseases.

Inactivation of Tnf-α/Tnfr signaling attenuates progression of intervertebral disc degeneration in mice.

Intervertebral disc degeneration (IVDD) is a major cause of low back pain (LBP), worsened by chronic inflammatory processes associated with aging. Tumor necrosis factor alpha (Tnf-α) and its receptors, Tnf receptor type 1 (Tnfr1) and Tnf receptor type 2 (Tnfr2), are upregulated in IVDD. However, its pathologic mechanisms remain poorly defined. To investigate the role of Tnfr in IVDD, we generated global Tnfr1/2 double knockout (KO) mice and age-matched control C57BL/6 male mice, and analyzed intervertebral disc (IVD)-related phenotypes of both genotypes under physiological conditions, aging, and lumbar spine instability (LSI) model through histological and immunofluorescence analyses and μCT imaging. Expression levels of key extracellular matrix (ECM) proteins in aged and LSI mice, especially markers of cell proliferation and apoptosis, were evaluated in aged (21-month-old) mice. At 4 months, KO and control mice showed no marked differences of IVDD-related parameters. However, at 21 months of age, the loss of Tnfr expression significantly alleviated IVDD-like phenotypes, including a significant increase in height of the nucleus pulposus (NPs) and reductions of endplates (EPs) porosity and histopathological scores, when compared to controls. Tnfr deficiency promoted anabolic metabolism of the ECM proteins and suppressed ECM catabolism. Tnfr loss largely inhibited hypertrophic differentiation, and, in the meantime, suppressed cell apoptosis and cellular senescence in the annulus fibrosis, NP, and EP tissues without affecting cell proliferation. Similar results were observed in the LSI model, where Tnfr deficiency significantly alleviated IVDD and enhanced ECM anabolic metabolism while suppressing catabolism. The deletion of Tnfr mitigates age-related and LSI-induced IVDD, as evidenced by preserved IVD structure, and improved ECM integrity. These findings suggest a crucial role of Tnf-α/Tnfr signaling in IVDD pathogenesis in mice. Targeting this pathway may be a novel strategy for IVDD prevention and treatment.

Increased levels of extracellular matrix proteins associated with extracellular vesicles from brains of aged mice.

Extracellular vesicles (EVs) are secreted by all major cell types of the brain, providing a mode of intercellular communication and a pathway for disposal of cellular debris. EVs help maintain healthy brain function, but may also contribute to diseases affecting the brain. EVs might contribute to aging of the brain, as aging-related processes such as inflammation and cellular senescence may alter EV cargo, promoting further inflammation and senescence. However, the effects of aging on brain EVs and the function of EVs in the aging brain remain poorly understood. To address this question, we measured the levels and protein cargo of EVs isolated from the brains of 4-, 12-, and 22-month-old C57BL/6J mice. We detected no changes in EV levels, but observed age-dependent changes in EV proteins. EV fractions from aged (22 month old) brains contained higher levels of extracellular matrix proteins than EV fractions from young (4 month old) brains, with intermediate levels in 12-month-old brains. Specifically, EV fractions from aged mice contained elevated levels of hyaluronan and proteoglycan link proteins 1 and 2 and several chondroitin sulfate proteoglycans (CSPGs). Analysis of extracellular matrix in several brain regions of aged mice revealed increased immunolabeling for the CSPG aggrecan, but reduced labeling with Wisteria floribunda agglutinin, which binds to chondroitin sulfate side chains of CSPGs. These data are consistent with prior studies showing changes to the composition of extracellular matrix in aged brains, and indicate a novel association of EVs with changes in the extracellular matrix of the aging brain.

Hippocampal rejuvenation by a single intracerebral injection of one-carbon metabolites in C57BL6 old wild-type mice.

The Izpisua-Belmonte group identified a cocktail of metabolites that promote partial reprogramming in cultured muscle cells. We tested the effect of brain injection of these metabolites in the dentate gyrus of aged wild-type mice. The dentate gyrus is a brain region essential for memory function and is extremely vulnerable to aging. A single injection of the cocktail containing four compounds (putrescine, glycine, methionine and threonine) partially reversed brain aging phenotypes and epigenetic alterations in age-associated genes. Our analysis revealed three levels: chromatin methylation, RNA sequencing, and protein expression. Functional studies complemented the previous ones, showing cognitive improvement. In summary, we report the reversal of various age-associated epigenetic changes, such as the transcription factor Zic4, and several changes related to cellular rejuvenation in the dentate gyrus (DG). These changes include increased expression of the Sox2 protein. Finally, the increases in the survival of newly generated neurons and the levels of the NMDA receptor subunit GluN2B were accompanied by improvements in both short-term and long-term memory performance. Based on these results, we propose the use of these metabolites to explore new strategies for the development of potential treatments for age-related brain diseases.

Alzheimer's disease-associated genotypes differentially influence chronic evoked seizure outcomes and antiseizure medicine activity in aged mice.

Alzheimer's disease (AD) patients are at greater risk of focal seizures than similarly aged adults; these seizures, left untreated, may worsen functional decline. Older people with epilepsy generally respond well to antiseizure medications (ASMs). However, whether specific ASMs can differentially control seizures in AD is unknown. The corneal kindled mouse model of acquired chronic secondarily generalized focal seizures allows for precisely timed drug administration studies to quantify the efficacy and tolerability of ASMs in an AD-associated genetic model. We hypothesized that mechanistically distinct ASMs would exert differential anticonvulsant activity and tolerability in aged AD mice (8-15 months) to define whether rational ASM selection may benefit specific AD genotypes. Aged male and female PSEN2-N141I versus age-matched non-transgenic control (PSEN2 control) C57Bl/6J mice, and APP swe /PS1 dE9 versus transgene negative (APP control) littermates underwent corneal kindling to quantify latency to fully kindled criterion. Dose-related ASM efficacy was then compared in each AD model versus matched control over 1-2 months using ASMs commonly prescribed in older adults with epilepsy: valproic acid, levetiracetam, lamotrigine, phenobarbital, and gabapentin. Sex and AD genotype differentially impacted seizure susceptibility. Male PSEN2-N141I mice required more stimulations to attain kindling criterion (X 2 =5.521; p<0.05). Male APP/PS1 mice did not differ in kindling rate versus APP control mice, but they did have more severe seizures. There were significant ASM class-specific differences in acute seizure control and dose-related tolerability. APP/PS1 mice were more sensitive than APP controls to valproic acid, levetiracetam, and gabapentin. PSEN2-N141I mice were more sensitive than PSEN2 controls to valproic acid and lamotrigine. AD genotypes may differentially impact ASMs activity and tolerability in vivo with advanced biological age. These findings highlight the heterogeneity of seizure risk in AD and suggest that precisely selected ASMs may beneficially control seizures in AD, thus reducing functional decline.

Senescent cell transplantation into the skin induces age-related peripheral dysfunction and cognitive decline.

Cellular senescence is an established cause of cell and tissue aging. Senescent cells have been shown to increase in multiple organs during aging, including the skin. Here we hypothesized that senescent cells residing in the skin can spread senescence to distant organs, thereby accelerating systemic aging processes. To explore this hypothesis, we initially observed an increase in several markers of senescence in the skin of aging mice. Subsequently, we conducted experiments wherein senescent fibroblasts were transplanted into the dermis of young mice and assessed various age-associated parameters. Our findings reveal that the presence of senescent cells in the dermal layer of young mice leads to increased senescence in both proximal and distal host tissues, alongside increased frailty, and impaired musculoskeletal function. Additionally, there was a significant decline in cognitive function, concomitant with increased expression of senescence-associated markers within the hippocampus brain area. These results support the concept that the accumulation of senescent cells in the skin can exert remote effects on other organs including the brain, potentially explaining links between skin and brain disorders and diseases and, contributing to physical and cognitive decline associated with aging.

The senolytic drug ABT-263 accelerates ovarian aging in older female mice

Previous studies have reported that senolytic drugs can reverse obesity-mediated accumulation of senescent cells in the ovary and protect against cisplatin-induced ovarian injury by removing senescent cells. Early intervention with ABT-263 has been shown to mitigate ovarian aging. However, it remains unknown whether treatment with ABT-263 could rejuvenate the aged ovary in reproductively old females. Therefore, the current study was aimed to investigate whether advanced age intervention with ABT-263 could ameliorate age-related decline in ovarian function. Fourteen 16-month-old mice with a C57/BL6 background were treated with ABT-263 (N = 7) or vehicle (N = 7) for two weeks. Mice were initially treated with ABT-263 (60 mg/kg/d) or vehicle for 7 consecutive days. After a 7-day break, the treatment was repeated for another 7 consecutive days. Six 2-month-old mice with C57BL/6 were used as a young control. The hormonal levels, estrus cycles, ovarian reserve, ovarian cell proliferation and apoptosis, ovarian fibrosis, and steroidogenic gene expression of ovarian stromal cells were evaluated. ABT-263 treatment did not rescue abnormal estrus cycles and sex hormonal levels, or inhibit the formation of multinucleated giant cells and ovarian stromal cell apoptosis in aged ovaries. However, it reduced ovarian fibrosis and preserved the steroidogenic gene expression of ovarian stromal cells in aged ovaries. Importantly, ABT-263 treatment further depleted ovarian follicles in aged mice. In conclusion, ABT-263 treatment accelerated the depletion of ovarian follicles in aged mice, suggesting that senolytic drugs for reproductively old female may adversely affect female fertility.

The senolytic agent ABT263 ameliorates osteoporosis caused by active vitamin D insufficiency through selective clearance of senescent skeletal cells

Background/ObjectiveActive vitamin D insufficiency accelerates the development of osteoporosis, with senescent bone cells and the senescence-associated secretory phenotype (SASP) playing crucial roles. This study aimed to investigate whether the senolytic agent ABT263 could correct osteoporosis caused by active vitamin D insufficiency by selectively clearing senescent cells. MethodsBone marrow mesenchymal stem cells (BM-MSCs) from young and aged mice were treated with ABT263 in vitro, and 1,25(OH)2D-insufficient (Cyp27b1+/−) mice were administered ABT263 in vivo. Cellular, molecular, imaging, and histopathological analyses were performed to compare treated cells and mice with control groups. ResultsABT263 induced apoptosis in senescent BM-MSCs by downregulating Bcl2 and upregulating Bax expression. It also induced apoptosis in senescent BM-MSCs from 1,25(OH)2D-insufficient mice. ABT263 administration corrected bone loss caused by 1,25(OH)2D insufficiency by increasing bone density, bone volume, trabecular number, trabecular thickness, and collagen synthesis. It also enhanced osteoblastic bone formation and reduced osteoclastic bone resorption in vivo. ABT263 treatment corrected the impaired osteogenic action of BM-MSCs by promoting their proliferation and osteogenic differentiation. Furthermore, it corrected oxidative stress and DNA damage caused by 1,25(OH)2D insufficiency by increasing SOD-2 and decreasing γ-H2A.X expression. Finally, ABT263 corrected bone cell senescence and SASP caused by 1,25(OH)2D insufficiency by reducing the expression of senescence and SASP-related genes and proteins. ConclusionABT263 can correct osteoporosis caused by active vitamin D insufficiency by selectively clearing senescent skeletal cells, reducing oxidative stress, DNA damage, and SASP, and promoting bone formation while inhibiting bone resorption. These findings provide new insights into the potential therapeutic application of senolytic agents in the treatment of osteoporosis associated with active vitamin D insufficiency. The translational potential of this articleThis study highlights the therapeutic potential of ABT263, a senolytic compound, in treating osteoporosis caused by active vitamin D insufficiency. By selectively eliminating senescent bone cells and their associated SASP, ABT263 intervention demonstrates the ability to restore bone homeostasis, prevent further bone loss, and promote bone formation. These findings contribute to the growing body of research supporting the use of senolytic therapies for the prevention and treatment of age-related bone disorders. The translational potential of this study lies in the development of novel therapeutic strategies targeting cellular senescence to combat osteoporosis, particularly in cases where vitamin D insufficiency is a contributing factor. Further clinical studies are warranted to validate the efficacy and safety of ABT263 and other senolytic agents in the treatment of osteoporosis in humans.

8500 Fvb/Ant Mice Carrying the Thr92Ala-Dio2 Polymorphism Have a Goiter

Abstract Disclosure: A. Batistuzzo: None. B. Bocco: None. E. McAninch: None. M.O. Ribeiro: None. A.C. Bianco: Consulting Fee; Self; Abbvie. Fvb/Ant Mice Carrying the Thr92Ala-Dio2 Polymorphism Have a Goiter Introduction: The Thr92Ala-DIO2 polymorphism is prevalent worldwide, with about 50% of the population carrying at least one polymorphic allele. Ala-D2 is ectopically located in the Golgi apparatus and causes endoplasmic reticulum (ER) stress. It is also about 40 % less active than Thr92-D2, thus T3 production could be compromised in carriers of the polymorphism. Objective: C57BL/6J (B6) mice carrying the Ala92-DIO2 polymorphism exhibit no significant thyroid phenotype as compared to control Thr92-Dio2 mice. Here we wished to test whether changing the genetic background of mice can influence the thyroidal impact of the Thr92Ala-DIO2 polymorphism. Therefore, we backcrossed B6-Thr92Ala-Dio2 mice to the FVB/Ant background for 8 generations. Methods: Mice were killed by asphyxiation in a CO2 chamber and blood collected from 7-18 weeks old female and male FVB/Ant Ala-Dio2 mice to measure plasma levels of TSH, T4, T3, and rT3. The thyroid gland was dissected, weighed and processed for histological analysis. The weight of the thyroid gland was normalized by femoral length. The QuPath Software was used for measuring follicular area, cell height, and the number of internalized follicular cells. Thr92-Dio2 mice of the same age and sex were used as controls. Data were analyzed using PRISM software and expressed as mean±SE (TH plasma levels and thyroid weight) or mean±SD (thyroid histology). A Student t-test was used to compare 2 groups. Results: Male and female Ala mice had significantly higher levels of TSH (males: 0.36±0.04 vs 1.1±0.21 ng/ml; p&amp;lt;0.01 and females: 0.017±0.006 vs 0.12±0.06 ng/ml; p&amp;lt;0.01), lower levels of plasmatic T4 (males: 2.9±0.2 vs 1.3±0.25 µg/dl; p&amp;lt;0.01 and females: 2.1±0.16 vs 1.15±0.16 µg/dl; p&amp;lt;0.001) and lower levels of rT3 (males: 23.8±2 vs 14.2±1.3 ng/dl; p&amp;lt;0.001 and females: 16.45±1.4 vs 10.4±1.17 ng/dl; p&amp;lt;0.01). Additionally, the thyroid gland of female polymorphic mice was heavier (4.3±0.39 vs 1.8±0.17 mg/cm; p&amp;lt;0.001) and both sexes presented enlarged thyroid follicle area (males: 1464±1464 vs 2055±2443 µm2; p&amp;lt;0.0001 and females: 1519±1364 vs 2597±2624 µm2; p&amp;lt;0.0001) and a higher number of internalized follicular cells (females: 0.06±0.007 vs 0.17±0.008 internalized cells/follicle; p&amp;lt;0.0001). No differences in the follicular cell height were observed. Discussion: In contrast to B6, FVB mice carrying the Thr92Ala-D2 polymorphism exhibit goiter, increased follicular area, slightly reduced serum T4 levels, and elevated serum TSH levels. These findings suggest that the impact of the Thr92Ala polymorphism varies according to the genetic background. They may explain why clinical studies of patients carrying the Thr92Ala-DIO2 polymorphism have yielded inconsistent results when different populations are analyzed. Presentation: 6/3/2024

7427 Cellular Senescence As A Pro-Tumorigenic Process And Therapeutic Target In Adrenocortical Carcinoma

Abstract Disclosure: L. Smith: None. K. Warde: None. S. Haston: None. J. Martinez-Barbera: None. K. Basham: None. Adrenocortical carcinoma (ACC) is a rare, yet highly aggressive malignancy that arises in the adrenal cortex. Advanced age is a major risk factor for ACC, with the peak age incidence between 50 and 60 years old. Older individuals above age 50 also have significantly worse survival outcomes. A better understanding of how aging contributes to the development and progression of ACC will uncover new treatment strategies. A major hallmark of aging is the systemic accumulation of senescent cells. While senescent cells have been implicated in various age-related diseases, including cancer, their role in ACC remains poorly understood. ACC tumors from older patients have an enrichment of senescence-associated gene signatures. Moreover, advanced ACC patients are treated with etoposide, doxorubicin, and cisplatin (EDP), which are chemotherapies that induce senescence. While EDP offers short-term benefit in some patients, more than half will progress within 6-months. We hypothesize that the accumulation of senescent cells that arise after EDP treatment are ultimately tumor-supportive long-term. As a result, we postulate that the use of senolytic compounds that clear away senescent cells after EDP treatment will lead to improved survival outcomes for ACC patients. We developed a new ACC mouse model that provides a window to study the role of senescence in ACC tumor progression and treatment. Our model is based on adrenal specific loss of ZNRF3, which is a Wnt inhibitor that is frequently inactivated in human ACC tumors. Despite early adrenal hyperplasia, we showed that Znrf3 conditional knockout (cKO) mice do not develop adrenal tumors until 18-months of age, which corresponds to the peak age incidence in humans. Additionally, the adrenals of Znrf3 cKO mice show a robust senescence activation phenotype that precedes adrenal tumorigenesis. We hypothesize that the accumulation of senescent cells is pro-tumorigenic long-term. To test our hypothesis, we combined Znrf3 cKO mice with a newly established mouse model that enables selective ablation of senescent cells (ZKO-FDR mice). Ongoing aging studies will determine whether the removal of early and late senescent cells in ZKO-FDR mice reduces adrenal tumorigenesis. Further, we established new primary tumor cell lines from aged ZKO-FDR mice. These cell lines will facilitate testing of the pro-tumor mechanisms of senescence. Additionally, they will permit the removal of senescent cells, both genetically and pharmacologically with senolytics, as a new treatment strategy in ACC. Overall, this project aims to investigate the pro-tumor biology of age- and therapy-induced senescence in ACC. Results from these studies will advance our understanding of senescence in cancer and aid in the development of new therapeutic strategies for ACC, including senolytics. Presentation: 6/2/2024

6681 Age and Sex Disparity in NASH and the Potential Role of Estradiol Treatment for NASH

Abstract Disclosure: M. Tripathi: None. S. Sakthivel: None. A. Suzuki: None. B.K. Singh: None. P.M. Yen: None. Introduction: NAFLD (nonalcoholic fatty liver disease) and its more severe form, NASH (nonalcoholic steatohepatitis) are sexually dimorphic conditions that exhibit higher prevalence in men than women during reproductive ages but have similar prevalence after menopause. Reproductive age women are protected from hepatic fibrosis relative to men, although they lose this protection after menopause. These sex differences in NASH have been attributed, at least partly, to different circulating estrogen levels. However, the underlying mechanism(s) is not fully understood. We thus conducted animal experiments to investigate: 1) sex differences in the histologic severity of NASH, necroinflammatory and fibrogenic activities, and other relevant pathways in young and old male and female mice and 2) the effects of estradiol administration on these parameters in old male and female mice to evaluate the therapeutic potential of estradiol in NASH. Methodology: Young/reproductive age (12-weeks old) and old/post-reproductive age female and male (48∼50-weeks weeks old) C57BL/6J mice were fed control chow diet (NCD) or high-fat methionine/choline-deficient (MCD) diet for six weeks to induce NASH. The old mice were administered 200nM 17β-estradiol (physiological) in drinking water. We analyzed serum and hepatic NASH parameters, ER stress, and lysosome-autophagy defects. Results and Conclusion: Among reproductive age mice with NASH, females developed less severe histologic inflammation and fibrosis and decreased gene expression of Il1b, Il6, Tgfb, Col1a1, Ccl2, and Ccl5, and lower hepatic collagen than males. Females with NASH also had less inhibition of hepatic autophagy and lysosomal protein expression than males with NASH. Among old mice, post-reproductive age females fed NCD or MCD exhibited more severe hepatic inflammation and fibrosis and had higher gene expression of inflammatory and fibrogenic markers with comparable autophagic inhibition and lysosomal defects as similar age males. Physiological doses of estradiol significantly reduced hepatic gene expression of inflammatory and fibrogenic markers, ER stress and lysosomal-autophagic defects in post-reproductive age females. In conclusion, similar to clinical NASH, we observed marked sex and age-related differences in the severity of NASH. Furthermore, physiological doses of estradiol treatment improved the hepatic histology, lysosome-autophagy, inflammation, and fibrosis in post-reproductive age female mice with NASH and suggests that estradiol supplementation may decrease or reverse NASH progression in post-menopausal women with NASH. Presentation: 6/2/2024