Older Mice Research Articles

CNSC-47. SENOLYTICS ERADICATE SENESCENT MICROGLIA IN THE BRAIN PARENCHYMA AND IMPROVE SURVIVAL IN OLDER ADULT MICE WITH GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most common aggressive primary malignant brain tumor in adults with a poor median survival rate. Despite the aggressive standard of care treatment combining surgical resection, chemo-, and radio-therapy, the median overall survival (OS) is only ~15-18 months. Progressively increasing subject age is inversely associated with GBM patient OS such that older adults tend to have significantly worse survival outcomes compared to younger counterparts. Senescent cells accumulate in the body during progressive aging and contribute to worse outcomes in older adult mice with GBM. Senolytics are pharmacologics that cause senescent cells to undergo cell death. OBJECTIVE To comprehensively profile the treatment effect of senolytics combined with or without immunotherapy in mice with GBM across the lifespan. METHODS Young 7-9- and older adult 97-104-week-old wild-type (WT; C57BL/6) and 110-130-week-old INK-ATTAC mice with or without intracranial SB28 cells were treated with or without brain radiation (RT), PD-1 mAb, and an IDO enzyme inhibitor with the senolytics, dasatinib + quercetin, or the AP compound that induces p16INK4A+ senescent cell death in INK-ATTAC mice (n=5/group). The quantification of beta-galactosidase positive cells by flow cytometry was performed on brain-resident neurons, astrocytes, oligodendrocytes, microglia, as well as dendritic cells, macrophages, neutrophils, T cells in the tumor and non-tumor bulk mass. Overall survival was also evaluated. RESULTS Senolytics or AP compound treatment both decrease beta-galactosidase positive microglia in the older adult brain with GBM (p<0.05). Combining radio- and immuno-therapy with senolytics improves overall survival of older adult mice with intracranial GBM (p<0.05). CONCLUSIONS Our ongoing work aims to understand how senescent microglia affect neuronal health and function in the older adult brain with GBM. KEYWORDS Glioma, senescence, senolytic.

CNSC-38. INFLUENCE OF AGE, IMMUNOREGULATORY IDO, AND IMMUNOTHERAPY ON THE MICROBIOME IN MICE WITH AN EXPERIMENTAL BRAIN TUMOR

Abstract OBJECTIVE This study aimed to determine how IDO and subject age impact the gut microbiome and microbial metabolites during immunotherapy for glioblastoma (GBM). METHODS Serum and colon contents were collected from young 16-20- or older adult 92-118-week-old wild-type (WT; C57BL/6), IDO knockout (IDOKO), and IDO enzyme null (H350A) mice analyzed for 16S rRNA gut microbiome composition and microbial-derived aromatic amino acid metabolites via LC/MS/MS. Fecal samples from young 7-8 or older adult 81-85 week-old WT mice with intracranial GL261 brain tumors treated with or without brain radiation (RT) and PD-1 mAb were also analyzed. RESULTS Young and old IDOKO and H350A mice demonstrated a unique gut microbiome signature with an elevated abundance of Bifidobacterium and Helicobacter (p<0.05) and a reduced abundance of Ventriosum and Sireaum compared to WT mice (p<0.001). Prevotellaceae, Muribaculum, Alistipes, Enterohabdus, Clostridia, and Akkermansia were different in IDOKO and H350A mice compared to WT - but only in old mice. Serum aryl-lactates including phenyllactate, indolelacatate, and 4-hydroxyphenyllactate were increased in IDOKO mice compared to WT and H350A mice (p<0.001) – again, only in old mice. Older adult WT mice with GL261 showed lower fecal aryl-lactates compared to younger counterparts. Treatment with RT + PD-1 mAb decreased phenyllactate levels in old but not young mice with GL261 (p<0.05). CONCLUSIONS There are IDO enzyme- and non-enzyme-dependent effects on gut microbiota composition and bioactive microbial aromatic amino acid metabolites. These data warrant further investigation into how microbial metabolism affects immunotherapy efficacy and brain tumor survival across the lifespan.

Oligodendrocyte Slc48a1 (Hrg1) encodes a functional heme transporter required for myelin integrity.

Oligodendrocytes (OLs) of the central nervous system require iron for proteolipid biosynthesis during the myelination process. Although most heme is found complexed to hemoglobin in red blood cells, surprisingly, we found that Slc48a1, encoding the heme transporter Hrg1, is expressed at higher levels in OLs than any other cell type in rodent and humans. We confirmed in situ that Hrg1 is expressed in OLs but not their precursors (OPCs) and found that Hrg1 proteins in CNS white matter co-localized within myelin sheaths. In older Hrg1 null mutant mice we observed reduced expression of myelin associated glycoprotein (Mag) and ultrastructural myelin defects reminiscent of Mag-null animals, suggesting myelin adhesion deficiency. Further, we confirmed reduced myelin iron levels in Hrg1 null animals in vivo, and show that OLs in vitro can directly import both the fluorescent heme analogue ZnMP and heme itself, which rescued iron deficiency induced inhibition of OL differentiation in a heme-oxidase-dependent manner. Together these findings indicate OL Hrg1 encodes a functional heme transporter required for myelin integrity.

Fertilization and developmental competence of in vitro fertilized embryos from C57BL/6J mice of different ages and the impact of vitrification.

Prepubertal animals are often preferred as sources of oocytes for assisted reproductive technologies (ARTs) in laboratory mice, but the normality and developmental competence of these oocytes remain controversial. This study systematically examined in vitro fertilization competence, embryo development, and fetal development after embryo transfer (ET) using oocytes from C57BL/6J mice aged 3 to 10 weeks. Oocytes were collected from superovulated females, fertilized, and cultured in vitro for 96 h or transferred at 2-cell stage to recipient females. Additionally, fetal development was compared between unfrozen and frozen-thawed in vitro-fertilized 2-cell embryos across different age groups. The number of ovulated oocytes per animal decreased with age, while the percentage of morphologically normal oocytes was highest in 3-week-old mice (99%) compared to older ages (70-86%, P < 0.05). Although fertilization percentages were consistently high (≥ 97%), blastocyst development in vitro, the nuclear counts of blastocysts and fetal development after ET were lowest for embryos from 3-week-old mice. Development of frozen-thawed embryos to fetuses was significantly reduced compared to unfrozen embryos in all age groups, except for those from 10-week-old mice. These findings suggest that oocytes from prepubertal mice, particularly from 3-week-old mice, are less developmentally competent than those from older mice. Therefore, the age of animals for oocyte source should be carefully considered based on the specific requirements of the research or ART applications.

Developing forebrain synapses are uniquely vulnerable to sleep loss

Sleep is an essential behavior that supports lifelong brain health and cognition. Neuronal synapses are a major target for restorative sleep function and a locus of dysfunction in response to sleep deprivation (SD). Synapse density is highly dynamic during development, becoming stabilized with maturation to adulthood, suggesting sleep exerts distinct synaptic functions between development and adulthood. Importantly, problems with sleep are common in neurodevelopmental disorders including autism spectrum disorder (ASD). Moreover, early life sleep disruption in animal models causes long-lasting changes in adult behavior. Divergent plasticity engaged during sleep necessarily implies that developing and adult synapses will show differential vulnerability to SD. To investigate distinct sleep functions and mechanisms of vulnerability to SD across development, we systematically examined the behavioral and molecular responses to acute SD between juvenile (P21 to P28), adolescent (P42 to P49), and adult (P70 to P100) mice of both sexes. Compared to adults, juveniles lack robust adaptations to SD, precipitating cognitive deficits in the novel object recognition task. Subcellular fractionation, combined with proteome and phosphoproteome analysis revealed the developing synapse is profoundly vulnerable to SD, whereas adults exhibit comparative resilience. SD in juveniles, and not older mice, aberrantly drives induction of synapse potentiation, synaptogenesis, and expression of perineuronal nets. Our analysis further reveals the developing synapse as a putative node of convergence between vulnerability to SD and ASD genetic risk. Together, our systematic analysis supports a distinct developmental function of sleep and reveals how sleep disruption impacts key aspects of brain development, providing insights for ASD susceptibility.

Optimisation of bronchoalveolar lavage technique for isolating alveolar macrophages in mice

Bronchoalveolar lavage (BAL) is a widely used technique to collect immune cells from the lungs, with alveolar macrophages (AMs) being the most prevalent cells in BAL fluid. AMs are vital for maintaining lung homeostasis and providing immune defence against airborne pathogens. However, in murine models, BAL procedures usually yield low numbers of AMs, thus limiting experimental design, especially when high cell counts are needed. Objective — to optimise the BAL technique in mice to maximise the collection of AMs. Materials and methods. Young and older BALB/c mice were used in the study. Bronchoalveolar lavage was performed following the method of Luckow and Lehmann (2021) with modifications. Statistical analysis was done using the Mann‑Whitney U test, with a significance level set at p&lt;0.05. Results. Female BALB/c mice of different ages were chosen due to the frequency of their use as models of pulmonary diseases. A simplified method described by Luckow and Lehmann (2021), which avoids tracheotomy by using peroral cannula insertion, was employed. The protocol was modified by securing the cannula with a ligature to prevent BAL fluid leakage in older mice. To reduce mechanical stress on alveoli, a buffer volume of 0.6 mL was used, and the study compared two buffer variants: one at room temperature without EDTA, and another heated to 37°C with EDTA. The pre‑heated buffer with EDTA significantly increased BAL cell yields in all mice groups, confirming the importance of these optimisations for higher cell recovery. Conclusions. Our modified bronchoalveolar lavage protocol includes securing the trachea with a ligature to prevent BAL fluid leakage, reducing lavage volume to 0.6 mL to minimise lung damage, and using a 37°C solution with EDTA for improved AM recovery rates. Further studies are needed to explore the significance of other buffer components for BAL protocol optimisation, the possible age‑related differences in AM isolation in male BALB/c mice, and the strain‑specific features of the BAL technique.

Crucial role of Snf7-3 in synaptic function and cognitive behavior revealed by conventional and conditional knockout mouse models

Snf7-3 is a crucial component of the endosomal sorting complexes required for transport (ESCRT) pathway, playing a vital role in endolysosomal functions. To elucidate the role of Snf7-3 in vivo, we developed conventional-like and conditional Snf7-3 knockout (KO) mouse models using a “Knockout-first” strategy. Conventional-like Snf7-3 KO mice showed significantly reduced Snf7-3 mRNA expression, and older mice (25–40 weeks) exhibited impaired social recognition and increased miniature excitatory postsynaptic currents (mEPSCs). Similarly, conditional KO mice aged 8–24 weeks, with Snf7-3 specifically deleted in forebrain excitatory neurons, displayed impaired object location memory and elevated mEPSC frequency. Consistently, Snf7-3 knockdown in cultured mouse hippocampal neurons led to increased densities of pre- and postsynaptic puncta, supporting the observed increase in mEPSC frequency. In addition, enhanced dendritic complexity was observed in the medial prefrontal cortex of these mice, indicating early synaptic disturbances. Our findings underscore the critical role of Snf7-3 in maintaining normal cognitive functions and social behaviors. The observed synaptic and behavioral deficits in both conventional-like and conditional KO mice highlight the importance of Snf7-3 in specific neuronal populations, suggesting that early synaptic changes could precede more pronounced cognitive impairments.

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.

Hyperactive Dendritc Cells Redirect Aged Antitumor Immunity.

Aging is one of the biggest risk factors for cancer development. More than 85% of all cancers occur in individuals above 55 years old, often accompanied by age-associated immune defects. Previous studies on the tumor microenvironment during aging have identified several factors, such as the roles of fibroblasts, immunosuppression, and metastasis. However, the aging-associated defects in antitumor immunity, particularly with regard to T cells, remain underexplored. Recent findings by Zhivaki and colleagues suggest that age-related immune defects affecting antitumor responses involve reduced levels of CD8+ T cells and compromised dendritic cell (DC) functions such as antigen presentation and migration. Their study demonstrates that a hyperactive DC vaccine can restore DC functions in older mice. Furthermore, these hyperactive DCs, characterized by increased IL1β production and better migratory capability to the lymph node, promote the development of cytolytic CD4+ T cells exhibiting Th1-like phenotypes. This research reveals mechanisms underlying the response to hyperactive DC vaccines in older mice and highlights the critical role of cytolytic CD4+ T cells as substitutes for CD8+ T cells in driving antitumor immunity and achieving long-term tumor control in older mice.

Increased hydrogen sulfide turnover serves a cytoprotective role during the development of replicative senescence

The mammalian gasotransmitter hydrogen sulfide (H2S) is produced by enzymes such as cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), 3-mercaptopyruvate sulfurtransferase (3-MST). Prior studies suggest that H2S may have cytoprotective and anti-aging effects. This project explores the regulation and role of endogenous H2S in a murine model of replicative senescence. H2S and polysulfide levels in RAW 264.7 murine macrophages (control cells: passage 5–10; senescent cells: passage 30–40) were measured using fluorescent probes. The expression of H2S-related enzymes and the activity of senescence marker beta-galactosidase (SA-β-Gal) were also analyzed. CBS, CSE, and 3-MST were inhibited using selective pharmacological inhibitors. Senescence led to a moderate upregulation of CBS and in a significant increase in CSE and 3-MST. H2S degradation enzymes were also elevated in senescence. Inhibition of H2S-producing enzymes reduced H2S levels but increased polysulfides. Inhibition of H2S production during senescence suppressed cell proliferation, and elevated SA-β-Gal and p21 levels. Comparing young and old mice spleens revealed downregulation of CBS and ETHE1 and upregulation of rhodanese and SUOX in older mice. The results demonstrate that increased reactive sulfur turnover occurs in senescent macrophages and that reactive sulfur species supports cell proliferation and regulate cellular senescence

Increased inflammation in older high-pressure glaucoma mice

Besides an elevated intraocular pressure (IOP), advanced age is one of the most crucial risk factors for developing glaucoma. βB1-Connective Tissue Growth Factor (βB1-CTGF) high-pressure glaucoma mice were used in this study to assess whether glaucoma mice display more inflammatory and aging processes than age-matched controls. Therefore, 20-month-old βB1-CTGF and corresponding wildtype (WT) controls were examined. After IOP measurements, retinas were processed for (immuno-)histological and quantitative real-time PCR analyses. A significantly higher IOP and diminished retinal ganglion cell numbers were noted in βB1-CTGF mice compared to WT. An enhanced macrogliosis as well as an increased number of microglia/macrophages and microglia was detected in retinas of old glaucoma mice. Interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and transforming growth factor-β2 were upregulated, suggesting an ongoing inflammation. Moreover, βB1-CTGF retinas displayed an increased senescence-associated β-galactosidase staining accompanied by a downregulation of Lmnb1 (laminin-B1) mRNA levels. Our results provide a deeper insight into the association between inflammation and high-pressure glaucoma and thus might help to develop new therapy strategies.

Melatonin facilitates oocyte growth in goats and mice through increased nutrient reserves and enhanced mitochondrial function.

Oogenesis involves two phases: initial volumetric growth driven by nutrient accumulation and subsequent nuclear maturation. While melatonin (MLT) has been employed as a supplement to enhance the quality of fully grown oocytes during nuclear maturation phase, its impact on oocyte growth remains poorly studied. Here, we provide invivo evidence demonstrating that follicle-stimulating hormone increases MLT content in ovary. Administration of MLT improves oocyte growth and quality in mice and goats by enhancing nutrient reserves and mitochondrial function. Conversely, MLT-deficient mice have smaller oocytes and dysfunctional mitochondria. Exploring the clinical implications of MLT in promoting oocyte growth, we observe that a brief 2-day MLT treatment enhances oocyte quality and reproductive performance in older mice. These findings highlight the role of MLT in regulating oocyte growth and provide a specific treatment window for optimizing oocyte quality and reproductive performance in female animals.

A novel pulmonary fibrosis NOD/SCID murine model with natural aging

BackgroundIdiopathic pulmonary fibrosis (IPF) is an age-related disease severely affecting life quality with its prevalence rising as the population ages, yet there is still no effective treatment available. Cell therapy has emerged as a promising option for IPF, however, the absence of mature and stable animal models for IPF immunodeficiency hampers preclinical evaluations of human cell therapies, primarily due to rapid immune clearance of administered cells. This study aims to establish a reliable pulmonary fibrosis (PF) model in immunodeficient mice that supports autologous cell therapy and to investigate underlying mechanism.MethodsWe utilized thirty 5-week-old male NOD/SCID mice, categorizing them into three age groups: 12weeks, 32 weeks and 43 weeks, with 6 mice euthanized randomly from each cohort for lung tissue analysis. We assessed fibrosis using HE staining, Masson’s trichrome staining, α-SMA immunohistochemistry and hydroxyproline content measurement. Further, β-galactosidase staining and gene expression analysis of MMP9, TGF-β1, TNF-α, IL-1β, IL-6, IL-8, SOD1, SOD2, NRF2, SIRT1, and SIRT3 were performed. ELISA was employed to quantify protein levels of TNF-α, TGF-β1, and IL-8.ResultsWhen comparing lung tissues from 32-week-old and 43-week-old mice to those from 12-week-old mice, we noted a marked increase in inflammatory infiltration, fibrosis severity, and hydroxyproline content, alongside elevated expression levels of α-SMA and MMP9. Notably, the degree of fibrosis intensified with age. Additionally, β-galactosidase staining became more pronounced in older mice. Quantitative PCR analyses revealed age-related, increases in the expression of senescence markers (GLB1, P16, P21), and proinflammatory genes (TGF-β1, TNF-α, IL-1β, IL-6, and IL-8). Conversely, the expression of anti-oxidative stress-related genes (SOD1, SOD2, NRF2, SIRT1, and SIRT3) declined, showing statistically significant differences (*P < 0.05, **P < 0.01, ***P < 0.001). ELISA results corroborated these findings, indicating a progressive rise in the protein levels of TGF-β1, TNF-α, and IL-8 as the mice aged.ConclusionsThe findings suggest that NOD/SCID mice aged 32 weeks and 43 weeks effectively model pulmonary fibrosis in an elderly context, with the disease pathogenesis likely driven by age-associated inflammation and oxidative stress.

Abstract A069: Development of an AAV-mediated GEM model to investigate the impact of age on pancreatic tumorigenesis

Abstract Much of our understanding of PDAC initiation comes from Genetically Engineered Mouse (GEM) models where a pancreas-specific Cre (such as Pdx- or p48-Cre) drives expression of specific mutations. While these GEM models generally recapitulate the key histopathological features of human PDAC, they have limitations when it comes to studying the impact of age on PDAC initiation since the Cre drivers induce recombination at the embryonic stage. This is an important consideration since in humans the disease manifests later in life, with the median age at diagnosis being 70 years old. Here, we describe the development of an intrapancreatic injection model using adeno-associated virus (AAV) to study how age influences PDAC initiation and the dynamics of tumor progression. This new viral transduction-based mouse model employs AAV-Cre to induce expression of an oncogenic KRasG12D allele together with a Trp53R172H tumor suppressor mutation within the pancreata of mice at different ages. Our approach utilizes mice with the genotype LSL-KrasG12D; LSL-Trp53R172H that are injected with different AAV-Cre directly into the parenchyma of the pancreas. In our initial studies, we used rAAV vectors expressing Cre recombinase and EGFP (AAV-Cre-EGFP) into young mice (10-14 weeks old) and older mice (32 weeks old) and analyzed EGFP expression using IVIS imaging and immunohistochemistry (IHC) at various timepoints post-injection. To evaluate tumor initiation, we assessed the extent of PanIN lesion formation using H&amp;E staining. Our preliminary findings indicate that AAV-Cre-EGFP effectively transduces cells in vivo and triggers tumor development in the pancreas. To account for potential immunogenicity of the fluorescent protein, we then used rAAV vectors lacking EGFP expression. As controls, we administered identical rAAV vectors to wild-type cohorts matched for age and sex, along with mock injections. Transduction efficiency within the pancreas was determined by evaluating Cre and p53 expression levels, given that the R172H mutation stabilizes the protein. Notably, we found that EGFP is not necessary for either successful transduction or tumor initiation. We are currently evaluating PDAC initiation using a wide spectrum of ages, but our preliminary data using this model suggest that increased age may impact tumor progression, and that this effect may be sex-specific. Our novel AAV-Cre injection model holds promise for investigating age-related aspects of PDAC initiation, and in the future, such approaches could be leveraged to investigate how age impacts the effectiveness of different therapeutic modalities. Citation Format: Karen XD Duong-Polk, Madelaine Neff, Cosimo Commisso. Development of an AAV-mediated GEM model to investigate the impact of age on pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A069.

Challenging Sarcopenia: Exploring AdipoRon in Aging Skeletal Muscle as a Healthspan-Extending Shield.

Sarcopenia, characterized by loss of muscle mass, quality, and function, poses significant risks in aging. We previously demonstrated that long-term treatment with AdipoRon (AR), an adiponectin receptor agonist, alleviated myosteatosis and muscle degeneration in middle-aged obese mice. This study aimed to determine if a shorter AR treatment could effectively offset sarcopenia in older mice. Two groups of old mice (20-23 months) were studied, one untreated (O) and one orally-treated with AR (O-AR) at 50 mg/kg/day for three months, compared with control 3-month-old young mice (Y) or 10-month-old young-adult mice (C-10). Results showed that AR remarkably inversed the loss of muscle mass by restoring the sarcopenia index and fiber count, which were greatly diminished with age. Additionally, AR successfully saved muscle quality of O mice by halving the accumulation of tubular aggregates and aberrant mitochondria, through AMPK pathway activation and enhanced autophagy. AR also bolstered muscle function by rescuing mitochondrial activity and improving exercise endurance. Finally, AR markedly curbed muscle fibrosis and mitigated local/systemic inflammation. Thus, a late three-month AR treatment successfully opposed sarcopenia and counteracted various hallmarks of aging, suggesting AR as a promising anti-aging therapy for skeletal muscles, potentially extending healthspan.

Decreased Amino Acid Transporter LAT2 Is the Main Determinant of Impaired Protein Utilization During Aging

As the global demographic shifts toward an aging population, understanding the efficiency of protein utilization in older adults becomes crucial. Our study explores the intricate relationship between protein intake and aging, with a focus on precision nutrition for older people. Through a meta-analysis, we confirm a decline in protein-utilization capacity in older individuals and examine the different contributions of plant and animal protein. In experiments involving mice of different ages, older mice exhibited decreases in the biological utilization of four proteins (casein, beef protein, soy protein, and gluten), particularly casein. In subsequent research, casein was studied as a key protein. A decline in gastric digestion function was observed through peptidomics and the examination of pepsin levels using casein. Nevertheless, this decline did not significantly affect the overall protein digestion during the aging process. The combined application of targeted amino acid metabolomics identified abnormal absorption of amino acids as the underlying cause of decreased protein utilization during aging, particularly emphasizing a reduction in branched-chain amino acids (BCAAs) in older mice. Delving deeper into the proteomics of the intestinal protein digestion and absorption pathway, a reduction of over 60% in large neutral amino acid transporter 2 (LAT2) protein expression was observed in both older humans and aged mice. The reduction in LAT2 protein was found to be a key factor influencing the diminished BCAA availability. Overall, our study establishes the significance of amino acid absorption through LAT2 in protein utilization during aging and offers a new theoretical foundation for improving protein utilization in the older adults.

Modeling aging and retinal degeneration with mitochondrial DNA mutation burden.

Somatic mitochondrial DNA (mtDNA) mutation accumulation has been observed in individuals with retinal degenerative disorders. To study the effects of aging and mtDNA mutation accumulation in the retina, a polymerase gamma (POLG) exonuclease-deficient model, the PolgD257A mutator mice (D257A), was used. POLG is an enzyme responsible for regulating mtDNA replication and repair. Retinas of young and older mice with this mutation were analyzed invivo and exvivo to provide new insights into the contribution of age-related mitochondrial (mt) dysfunction due to mtDNA damage. Optical coherence tomography (OCT) image analysis revealed a decrease in retinal and photoreceptor thickness starting at 6 months of age in mice with the D257A mutation compared to wild-type (WT) mice. Electroretinography (ERG) testing showed a significant decrease in all recorded responses at 6 months of age. Sections labeled with markers of different types of retinal cells, including cones, rods, and bipolar cells, exhibited decreased labeling starting at 6 months. However, electron microscopy analysis revealed differences in retinal pigment epithelium (RPE) mt morphology beginning at 3 months. Interestingly, there was no increase in oxidative stress and parkin-mediated mitophagy in the ages analyzed in the retina or RPE of D257A mice. Additionally, D257A RPE exhibited an accelerated rate of autofluorescence cytoplasmic granule formation and accumulation. Mt markers displayed different abundance in protein lysates obtained from retina and RPE samples. These findings suggest that the accumulation of mtDNA mutations leads to impaired mt function and accelerated aging, resulting in retinal degeneration.

Lumican is Both a Novel Risk Factor and Potential Plasma Biomarker for Vascular Aging, Capable of Promoting VSMCs Senescence through Interacting with Integrin α2β1.

Vascular aging, a common pathogenesis of senile chronic diseases, significantly increases morbidity and mortality in older adults; its intricate cellular and molecular mechanisms necessitate further investigation. Lumican (LUM) and integrin α2β1(ITGα2β1) are profibrotic extracellular matrix proteins and vital cell regulatory receptors, respectively. However, their roles in vascular aging remain unclear. This study sought to elucidate the connection between LUM and vascular aging as well as the biological mechanism of LUM/ITGα2β1 in this process. Using an enzyme-linked immunosorbent assay, we discovered that plasma LUM was elevated in vascular aging individuals and was positively correlated with brachial-ankle pulse wave velocity. Additionally, immunohistochemical and western blot analyses confirmed LUM upregulation in arteries of older adults and aged mice, as well as in senescent vascular smooth cells (VSMCs). Wild-type and LUM semiknockout (Lum-/+) mice, along with primary VSMCs extracted from these mice, were exposed to angiotensin II (Ang II) to induce stress-induced senescence model. LUM semiknockout mitigated Ang Ⅱ-induced arteriosclerosis, hypertension, vascular aging and remodeling in mice. Both in vitro and in vivo studies revealed that LUM deficiency suppressed p53, p21, collagen 1 and collagen 3 upregulation and synthetic phenotype formation in VSMCs stimulated by Ang Ⅱ. Treating VSMCs with a ITGα2β1 antagonist reversed the aforementioned changes triggered by LUM proteins. Briefly, LUM functions as a potential marker and risk factor for vascular aging and promotes pathological changes by affecting ITGα2β1 in VSMCs. This study introduces a novel molecular target for the early diagnosis and treatment of vascular aging and age-related vascular diseases.

Autophagy Regulates Age-Related Jawbone Loss via LepR+ Stromal Cells

Bone aging and decreased autophagic activity are related but poorly explored in the jawbone. This study aimed to characterize the aging jawbones and jawbone-derived stromal cells (JBSCs) and determine the role of autophagy in jawbone mass decline. We observed that the jawbones of older individuals and mice exhibited similar age-related bone loss. Furthermore, leptin receptor (LepR)–lineage cells served as the primary source for in vitro cultured and expanded JBSCs, referred to as LepR-Cre+/JBSCs. RNA-sequencing data from the jawbones and LepR-Cre+/JBSCs showed the upregulated expression of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway during aging. Through single-cell transcriptomics, we identified a decrease in the proportion of osteogenic lineage cells and the activation of the PI3K/AKT pathway in LepR-lineage cells in aging bone tissues. Reduced basal autophagic activity, diminished autophagic flux, and decreased osteogenesis occurred in the jawbones and LepR-Cre+/JBSCs from older mice (O-mice; O-JBSCs). Pharmacologic and constitutive autophagy activation alleviated the impaired osteogenesis in O-JBSCs. In addition, the suppression of mTOR-induced autophagy improved the aging phenotype of O-JBSCs. The activation of autophagy in LepR-Cre+/JBSCs using chemical autophagic activators reduced the alveolar bone resorption in O-mice. Therefore, our study demonstrated that ATG molecules and pathways are crucial in jawbone aging, providing novel approaches to understanding age-related jawbone loss.

Sex- and Age-Specific Differences in Mice Fed a Ketogenic Diet.

The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that results in the elevation of serum ketone bodies, known as ketosis. This metabolic consequence has been suggested as a method for treating neurological conditions, improving exercise performance, and facilitating weight loss for overweight individuals. However, since most research primarily uses male populations, little is known about the potential sex differences during the consumption of the KD. In addition, the effects of the KD on aging are relatively unexplored. Therefore, the purpose of this study was to explore sex- and age-specific differences in mice fed the KD. Male and female C57BL/6N mice at either 12 wks or 24 wks of age were randomly assigned to a KD (90% fat, 1% carbohydrate) or chow (13% fat, 60% carbohydrate) group for 6 wks. KD induced weight gain, increased adiposity, induced hyperlipidemia, caused lipid accumulation in the heart and liver, and led to glycogen depletion in the heart, liver, and muscle with varying degrees of changes depending on age and sex. While younger and older male mice on the KD were prone to glucose intolerance, the KD acutely improved rotarod performance in younger females. Overall, this study highlights potential sex and aging differences in the adaptation to the KD.