Aged Wild-type Mice Research Articles

Disease-associated astrocytes in Alzheimer's disease and aging.

The role of non-neuronal cells in Alzheimer’s disease (AD) progression has not been fully elucidated. Using single-nucleus RNA-seq, we identified a population of disease associated astrocytes (DAAs) in an AD mouse model. The DAA population appeared at early disease stages and increased in abundance with age. We discovered that similar astrocytes appeared in aged wild-type mice and in aging human brains, suggesting their linkage to genetic and age-related factors.

Age-Dependent Progression in Lung Pathophysiology can be Prevented by Restoring Fatty Acid and Ceramide Imbalance in Cystic Fibrosis.

Cystic fibrosis (CF) is a progressive disease which causes a continuous decline in lung capacity with age. Our study aimed to investigate the age-dependent deterioration in lung function and the effects of treatment with Fenretinide formulation (LAU-7b) in Cftr knockout (KO) mice. Non-invasive whole-body plethysmography (WBP) was done to measure the baseline lung functions of KO and wild-type (WT) mice at the ages of 2 and 4months. Mice were then treated for 21days with PBS or 10mg/kg/day LAU-7b initiated at 4 and 7months. Standard airway resistance measurements, haematoxylin and eosin staining, and analysis of lipids, and markers of oxidation were performed. The 4- and 7-month-old KO mice had significantly higher lung enhanced pause (Penh) and resistance values than age-matched WT mice and 2-month-old KO mice. Likewise, analysis of ceramides showed that PBS-treated mice had higher levels of long-chain ceramides (LCCs; C14-C18) and lower levels of very-long-chain ceramides (VLCCs; C24-C26) compared to LAU-7b-treated mice. Cftr KO mice displayed markedly greater inflammatory cell infiltration and epithelial hyperplasia at the ages of 2, 4, and 7months compared to WT. LAU-7b treatment significantly diminished this cellular infiltration and epithelial hyperplasia compared to PBS-treated mice. Our results demonstrate a progressive age-dependent decline in lung function in Cftr KO mice. Treatment with LAU-7b corrects the lipid imbalance observed in the aging KO and WT mice and, more importantly, inhibits the age-dependent deterioration in lung physiology and histopathology.

Lifelong SIRT‐1 Overexpression Attenuates Aortic Stiffening with Advancing Age

Advanced age is accompanied by aortic stiffening that is associated with decreased vascular expression of the cellular deacetylase, sirtuin‐1 (SIRT‐1). Interventions, such as caloric restriction, that increase SIRT‐1 expression also lower age‐related aortic stiffness. Thus, we sought to determine if lifelong whole body SIRT‐1 overexpression attenuates age‐related aortic stiffening. Aortic stiffness, as determined by aortic pulse wave velocity (PWV), was assessed in SIRT‐1 transgenic overexpressing (SIRTTG) and littermate wild type (WT) control mice at 3‐mo intervals from 3–24 mo of age. To determine the role of aortic structural changes on age‐related aortic stiffening, histological assessment of aortic wall characteristics was performed in young (~5 mo), middle‐age (~12 mo), and old (~24 mo) mice. Aortic SIRT‐1 mRNA gene expression, measured by RT‐PCR, was ~3‐fold higher in young SIRTTG vs. WT mice (P<0.05). Advanced age resulted in a ~6 fold decrease in SIRT‐1 mRNA expression in both old SIRTTG and WT mice (P<0.05), still, SIRT‐1 expression remained >3‐fold higher in old SIRTTG vs. WT mice (P<0.05). Although there was a similar increase in aortic lumen diameter with advancing age in both WT and SIRTTG mice, an age‐related increase in media‐to‐lumen ratio was only present in WT mice (Table 1, P<0.05). Across the age range (3–24 mo), aortic PWV was 8–17% lower in SIRTTG vs. WT mice (P<0.05). Moreover, the slope of age‐related aortic stiffening was 45% lower in SIRTTG vs. WT mice (2.1±0.2 vs. 3.8±0.3 cm/sec/mo, respectively; Figure 1). Aortic elastin content decreased with advancing age in WT mice (Table 1, P<05 old vs. young WT), whereas elastin content was maintained across the lifespan in SIRTTG mice (Table 1, P>0.05). There was an age‐related increase in aortic collagen content, advanced glycation end products (AGEs), and calcification in WT mice (Table 1, P<0.05 old vs. young WT). However, age‐related increases in aortic collagen content, AGEs, or calcification did not occur in SIRTTG mice (Table 1, P>0.05). These findings indicate that lifelong SIRT‐1 overexpression attenuates aortic stiffening with advancing age. These functional data are complemented by histological assessment of aortic wall characteristics, demonstrating that the deleterious changes to the aorta that normally occur with advancing age, such as medial wall hypertrophy, reduced elastin, accumulation of collagen, AGEs, and aortic calcification are all prevented in SIRTTG mice. Thus, the use of SIRT‐1 activators to prevent or reverse the age‐related aortic stiffness may be a viable approach compared to other lifestyle interventions, such as caloric restriction.Support or Funding InformationThis study was funded in part by grants from the National Institutes of Health (R01 AG060395, R01 AG050238, R01 AG048366, K02 AG045339, K99 AT010017) and US Department of Veterans Affairs (I01 BX002151).Scatter plot of aortic stiffening, as determined by pulse wave velocity (PWV), with advancing age in wild type (WT) and sirtuin‐1 transgenic overexpressing (SIRTTG) mice. Separate regression lines are given for WT and SIRTTG mice (dotted lines define 95% confidence intervals).Figure 1 Characteristics of animals used for aortic histology. Table 1 ‐ Animal Characteristics WT SIRTTG Young Middle‐Age Old Young Middle‐Age Old Male:female, n 7:7 7:8 6:4 3:2 7:4 4:6 Age, mo 5.6±0.2 12.2±0.1 † 23.8±0.2 † ‡ 6.0±0.4 12.4±0.0 † 23.9±0.2 † ‡ Body mass, g 26.8±0.9 32.8±1.6 † 30.8±1.5 † ‡ 28.0±1.8 33.4±1.9 † 28.6±0.8 ‡ Lumen Diameter, μm 611±20 634±21 707±31 † ‡ 649±±8 644±18 737±26 † ‡ Medial CSA, mm2 0.81±0.09 0.92±0.08 1.32±0.14 † ‡ 0.90±0.09 0.92±0.12 1.01 ±0.07 * Media‐to‐Lumen Ratio, U 0.27±0.02 0.29±0.03 0.33±0.01 † 0.27±0.03 0.28±0.02 0.24±0.01 * Elastin, AU 1.00±0.06 1.02 ±0.04 0.85±0.05 † ‡ 1.04±0.06 1.06±0.03 1.04±0.04 * Collagen, AU 1.00±0.21 1.60±0.38 2.27±0.50 † 0.78±0.28 1.21±0.35 1.24±0.32 * AGEs, AU 1.00±0.09 1.17±0.16 1.62±0.29 † 1.00±0.21 1.17±0.16 1.21±0.18 Calcified Area, AU 1.00±0.27 1.56±0.15 † 1.47±0.20 † 0.79±0.16 0.47±0.11 * 0.67±0.19 * *P<0.05 vs. WT. †P<0.05 vs. Young. ‡P<0.05 vs. Middle‐Age. Data are mean±SEM. AGEs, advanced glycation endproducts; CSA, cross‐sectional area; SIRTTG, sirtuin‐1 transgenic overexpressing; WT, wild type.

Probing the crosslinking independent roles of tissue transglutaminase in vascular aging using a novel transamidation‐deficient TGM2‐C277S mouse model

IntroductionTissue transglutaminase (TG2) is a multifunctional protein of the transglutaminase family with putative transamidation‐independent functions in aging associated vascular stiffening and dysfunction. The development of preclinical models is critical to fully understand the physiological relevance of transamidation independent functions of TG2 and to identify the specific function of TG2 for therapeutic targeting. Therefore, In this study we harnessed the CRISPR‐Cas9 gene editing technology to introduce a mutation at the active site cysteine (C277) in the mouse TGM2 gene.Methods and resultsWe investigated the contributions of the transamidation‐dependent vs. transamidaiton‐independent functions of TG2 in vascular aging using TGM2‐C277S mice, littermate WT mice, and age‐matched TG2−/− mice. Heterozygous and homozygous TGM2‐C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein abundance was similar to wild‐type (WT) in the TGM2‐C277S mutant mice in the liver, lung, heart, aorta, and kidney. GTP binding, and interactions with fibronectin and integrin beta1 were preserved in the TG2‐C277S mouse protein. Transglutaminase function was absent in the TG2 protein from TGM2‐C277S mice when compared with wild‐type (WT) littermates. Loss of transamidation function resulted in a marked loss in serotonin induced vasoconstriction and in vitro remodeling of the carotid artery in response to chronic vasoconstriction. On the other hand, cell adhesion and proliferation were comparable to that of WT in the vascular smooth muscle cells from TGM2‐C277S but not in those from TG2−/− mice. Vascular stiffness increased with age in WT mice, as measured by pulse wave velocity, tensile testing, and pressure myography. TGM2‐C277S mice were markedly protected from age‐associated vascular stiffening, but not to the same extent as TG2−/− mice.ConclusionsTogether, these studies show that the TGM2‐C277S mice ubiquitously express a TG2 protein that lacks transamidation function and retains its other functions and can be used a tool for in vivo studies to examine the transamidation‐independent roles of TG2 in physiology and pathophysiology. While TG2‐mediated ECM crosslinking and VSMC dysfunction, both propagate vascular stiffening in aging, the transamidation function is the primary shareholder in aging associated mechanical decline of the large vessels.Support or Funding InformationThis work was funded by a StAAR Investigator Award (to LS) and a MedImmune Research Funds Award (to LS).

Pregnancy Associated Plasma Protein A (PappA) Knockout Mice: In Vivo Disc Study

INTRODUCTIONPast research in disc biology has highlighted the beneficial effects of growth factors, such as BMPs and IGF‐1, in vitro. Based on this research, it was proposed that exogenous IGF‐1 be used as a therapy for intervertebral disc degeneration (IDD). In the aging research field, suppressing the IGF‐1‐mediated signaling pathway has consistently decreased the incidence of age‐related disorders and increased lifespan of mammals and rodents. One model using PappA knockout mice has shown decreasing IGF‐1 signaling leads to positive effects on aging and longevity. Active PappA protease cleaves IGF binding protein 4 (IGFBP4), releasing active IGF‐1 which can bind to its receptor and exert downstream signaling effects. When you genetically remove PappA, IGF‐1 stays bound to IGFBP4. Here, we examined the discs of aged PAPPA−/− mice versus wild type (WT) to determine whether there was an effect on age‐associated IDD.METHODSThis study was conducted according to IACUC approved protocols at University of Pittsburgh. Genetic deletion of PAPP‐A protease in mice with a mixed C57BL/6‐129SV/E background was achieved by deletion of both alleles of PAPP‐A. Spine segments were harvested from WT and PAPPA−/− mice at 23 months. Disc proteoglycan content was assessed by safranin‐O‐histology and DMMB assay was used to quantify sulfated glycosaminoglycans (GAG) in lumbar NP tissue. Western blotting was performed to measure the disc aggrecan proteolytic fragments, and the catabolic markers MMP‐3 and ADAMTS‐4. Levels of disc cellular senescence (loss of lamin B1expression) was assessed by immunohistochemistry. Student’s t‐test was used to test significance between groups (p<0.05). Average values (n=4 mice) were shown with SEM.RESULTSCompared to WT, PAPPA−/− mice had decreased proteoglycan content and two times less disc GAG content (Figure 1). PAPPA−/− mice also had significantly decreased aggrecan fragmentation and decreased protein levels of MMP‐3 and ADAMTS‐4 in their discs compared to WT mice (Figure 2). Additionally, greater level of disc lamin B1 was seen in PAPPA−/− compared to WT mice, suggesting reduced disc cellular senescence in PAPPA−/− mice.DISCUSSIONThe role of IGF‐1 in disc health and aging is currently unresolved. PAPPA−/− mice have reduced matrix content but the aggrecan matrix present after aging appears to be less fragmented based on our findings. Lowered disc fragmentation corresponded to a decrease in disc catabolic factors MMP‐3 and ADAMTS‐4 protein in PAPPA−/− mice. Lamin B1 staining suggests there are less disc senescent cells in old PAPPA−/− mice compared to normally aged mice. Further studies are needed to determine the mechanism for the decreased disc catabolism and more full length aggrecan in aged PAPPA−/− mice compared to aged WT mice. Additionally, further studies should confirm whether young PAPPA−/− mice also have decreased GAG content or this loss occurs during the aging process.SIGNIFICANCEThe results of this study suggest the net effect of IGF‐1 regarding both anabolic and catabolic effects must be examined to fully understand if IGF‐1 is beneficial for disc homeostasis during age‐associated IDD.Support or Funding InformationR01‐AG044376‐01 & Ferguson Foundation GrantQualitative and quantitative results show decreased disc proteoglycan content, including aggrecan, in aged PAPPA −/− mice.Figure 1Despite less proteoglycan content, aged PAPPA −/− mice had less fragmented aggrecan and lower levels of catabolic markers, MMP‐3 and ADAMTS‐4 compared to aged WT mice.Figure 2

Epigenetic Regulation of KL (Klotho) via H3K27me3 (Histone 3 Lysine [K] 27 Trimethylation) in Renal Tubule Cells.

KL (klotho) levels decline with age, which is an important mechanistic driver of aging. KL gene deficiency is associated with hypertension. The purpose of this study is to investigate the potential role of H3K27me3 (histone 3 lysine [K] 27 trimethylation) in the regulation of KL gene expression and examine the related molecular pathways that may drive kidney cell aging. Kidneys were collected from 6-month-old WT (wild type; young WT), 30-month-old WT (aged WT), and 6- (young) and 20-month-old (aged) KL mutant mice, respectively. We demonstrated that the H3K27me3 level was increased in kidneys of aged WT and KL mutant mice versus young WT mice. Elevation of H3K27me3 levels was likely due to downregulation of the H3K27 (histone H3 Lys 27)-specific demethylase JMJD3 (the Jumonji domain containing-3) in the aged kidneys. Inhibition of PRC2 (polycomb repressive complex C2; histone trimethyltransferase) decreased the H3K27me3 levels leading to an increase in the expression of KL in cultured primary renal tubule cells assessed by Western blot and KL promoter activity assays. The chromatin immunoprecipitation qPCR assay revealed that H3K27me3 was physically associated with the KL promoter region. Furthermore, aging impaired the SGK1 (serum- and glucocorticoid-induced protein kinase 1)/FOXO3a (the forkhead box class O 3a) signaling leading to upregulation of p53 and p16 (aging markers) in the kidney of aged WT mice. KL may regulate the SGK1/FOXO3 signaling, which was decreased due to KL deficiency. Thus, aging-associated downregulation of KL gene expression may be partly attributed to upregulation of H3K27me3 levels. Downregulation of KL may impair the SGK1/FOXO3 signaling contributing to kidney cell aging.

Three-dimensional visualization of opacifications in the murine crystalline lens by in vivo optical coherence tomography.

Diagnostic classification techniques used to diagnose cataracts, the world's leading cause of blindness, are currently based on subjective methods. Here, we present optical coherence tomography as a noninvasive tool for volumetric visualization of lesions formed in the crystalline lens. A custom-made swept-source optical coherence tomography (SS-OCT) system was utilized to investigate the murine crystalline lens. In addition to imaging cataractous lesions in aged wildtype mice, we studied the structure and shape of cataracts in a mouse model of Alzheimer's disease. Hyperscattering opacifications in the crystalline lens were observed in both groups. Post mortem histological analysis were performed to correlate findings in the anterior and posterior part of the lens to 3D OCT in vivo imaging. Our results showcase the capability of OCT to rapidly visualize cataractous lesions in the murine lens and suggest that OCT might be a valuable tool that provides additional insight for preclinical studies of cataract formation.

Age and Sex Differences in Hearts of Soluble Epoxide Hydrolase Null Mice.

Biological aging is an inevitable part of life that has intrigued individuals for millennia. The progressive decline in biological systems impacts cardiac function and increases vulnerability to stress contributing to morbidity and mortality in aged individuals. Yet, our understanding of the molecular, biochemical and physiological mechanisms of aging as well as sex differences is limited. There is growing evidence indicating CYP450 epoxygenase-mediated metabolites of n–3 and n–6 polyunsaturated fatty acids (PUFAs) are active lipid mediators regulating cardiac homeostasis. These epoxy metabolites are rapidly hydrolyzed and inactivated by the soluble epoxide hydrolase (sEH). The current study characterized cardiac function in young and aged sEH null mice compared to the corresponding wild-type (WT) mice. All aged mice had significantly increased cardiac hypertrophy, except in aged female sEH null mice. Cardiac function as assessed by echocardiography demonstrated a marked decline in aged WT mice, notably significant decreases in ejection fraction and fractional shortening in both sexes. Interestingly, aged female sEH null mice had preserved systolic function, while aged male sEH null mice had preserved diastolic function compared to aged WT mice. Assessment of cardiac mitochondria demonstrated an increased expression of acetyl Mn-SOD levels that correlated with decreased Sirt-3 activity in aged WT males and females. Conversely, aged sEH null mice had preserved Sirt-3 activity and better mitochondrial ultrastructure compared to WT mice. Consistent with these changes, the activity level of SOD significantly decreased in WT animals but was preserved in aged sEH null animals. Markers of oxidative stress demonstrated age-related increase in protein carbonyl levels in WT and sEH null male mice. Together, these data highlight novel cardiac phenotypes from sEH null mice demonstrating a sexual dimorphic pattern of aging in the heart.

Hydroxytyrosol stimulates neurogenesis in aged dentate gyrus by enhancing stem and progenitor cell proliferation and neuron survival.

The dentate gyrus of the hippocampus is one of two brain areas generating throughout life new neurons, which contribute to the formation of episodic/associative memories. During aging, the production of new neurons decreases and a cognitive decline occurs. Dietary factors influence neuronal function and synaptic plasticity; among them the phenolic compound hydroxytyrosol (HTyr), present in olive oil, displays neuroprotective effects. As age impacts primarily on the hippocampus-dependent cognitive processes, we wondered whether HTyr could stimulate hippocampal neurogenesis in vivo in adult and aged wild-type mice as well as in the B-cell translocation 1 gene (Btg1) knockout mouse model of accelerated neural aging. We found that treatment with HTyr activates neurogenesis in the dentate gyrus of adult, aged, and Btg1-null mice, by increasing survival of new neurons and decreasing apoptosis. Notably, however, in the aged and Btg1-null dentate gyrus, HTyr treatment also stimulates the proliferation of stem and progenitor cells, whereas in the adult dentate gyrus HTyr lacks any proliferative effect. Moreover, the new neurons generated in aged mice after HTyr treatment are recruited to existing circuits, as shown by the increase of BrdU+ /c-fos+ neurons. Finally, HTyr treatment also reduces the markers of aging lipofuscin and Iba1. Overall, our findings indicate that HTyr treatment counteracts neurogenesis decline during aging.

Abstract WP263: Increased Cardiac Inflammation and Atrial Fibrillation Inducibility in Aged Females: The Niche for Cardio-Embolic Strokes?

Background: Elderly women have higher morbidity, mortality and disability after stroke compared to age-matched men. Cardio embolism from atrial fibrillation (AF) remains the primary cause of severe strokes in elderly women. Why females have higher rates of atrial arrhythmias and embolism as they age is unknown. Studying differences in atrial substrate inflammation and arrhythmogenesis may help us understand these sex differences. Methods: Aged male and female C57BL6 wild type mice (18-20 months old) were injected with carbachol 50μg/kg for AF induction and ambulatory telemetry monitors were implanted. AF events were analyzed using Ponemah software and LabChart software for one day. Another cohort of naïve aged wild type mice were sacrificed and intracardiac perfusion was performed. Hearts were collected and prepared into a single cell suspension using series of mechanical and enzymatic digestion steps. The cells were stained with antibodies for F4/80, Ly6C, CD11b, CD11c, and Gr1 antibodies. Sample data was acquired on a Cytoflex and analyzed using FlowJo software. Results: Aged females had significantly higher AF events, (110.7± 22.6, n=6), as compared to aged males (13.7±10.8 events, n=4), p=0.05. Flow cytometry showed that aged females have significantly more macrophages (CD11b + /F480 + ) (102.6±18.5) as compared to aged males, 26.6±3.4, p=.01, n=3/group. There were also significantly more monocytes (CD11b + /Ly6c + ) in aged wild type females (176.6±50.8) vs. aged males (44.3±13.2), p=0.05, n=3/group. No significant difference was seen in dendritic cell numbers (CD11c + ), p=0.3, n=3/ group. Conclusion: Our study shows that aged females have higher rates of AF after chemical induction as compared to males. They also have higher monocyte and macrophage populations in cardiac tissue as compared to aged males. This suggests that there is increased baseline cardiac inflammation in aged females, which may be the niche for arrhythmia generation in females. This in itself may lead to increased stroke risk. Anti-inflammatory therapies that control atrial substrate inflammation may be an important potential target for stroke prevention in women. Quantification of brain embolic events using MRI and Fluoro-jade staining will also be presented.

Environmental Enrichment: Disentangling the Influence of Novelty, Social, and Physical Activity on Cerebral Amyloid Angiopathy in a Transgenic Mouse Model.

Cerebral amyloid angiopathy (CAA) is the deposition of amyloid protein in the cerebral vasculature, a common feature in both aging and Alzheimer’s disease (AD). However, the effects of environmental factors, particularly cognitive stimulation, social stimulation, and physical activity, on CAA pathology are poorly understood. These factors, delivered in the form of the environmental enrichment (EE) paradigm in rodents, have been shown to have beneficial effects on the brain and behavior in healthy aging and AD models. However, the relative importance of these subcomponents on CAA pathology has not been investigated. Therefore, we assessed the effects of EE, social enrichment (SOC), and cognitive enrichment (COG) compared to a control group that was single housed without enrichment (SIN) from 4 to 8 months of age in wild-type mice (WT) and Tg-SwDI mice, a transgenic mouse model of CAA that exhibits cognitive/behavioral deficits. The results show that individual facets of enrichment can affect an animal model of CAA, though the SOC and combined EE conditions are generally the most effective at producing physiological, cognitive/behavioral, and neuropathological changes, adding to a growing literature supporting the benefits of lifestyle interventions.

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice.

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence‐associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1‐XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16Ink4a and p21Cip1 mRNA were increased ~15‐fold in peripheral lymphocytes from 4‐ to 5‐month‐old Ercc1 −/∆ and 2.5‐year‐old wild‐type (WT) mice, suggesting that these animals exhibit a similar biological age. p16Ink4a and p21Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4‐ to 5‐month‐old Ercc1 −/∆ mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16Ink4a and p21Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence‐associated β–galactosidase activity and p21Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1 −/Δ mice with a p16Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1 −/∆ and aged WT mice support the conclusion that the DNA repair‐deficient mice accurately model the age‐related accumulation of senescent cells, albeit six‐times faster.

PAS kinase deficiency reduces aging effects in mice.

Several signaling pathways may be affected during aging. All are regulated by nutrient levels leading to a decline in mitochondrial function and autophagy and to an increase in oxidative stress. PAS Domain Kinase (PASK) is a nutrient and bioenergetic sensor. We have previously found that PASK plays a role in the control of hepatic metabolic balance and mitochondrial homeostasis. To investigate PASK’s role in hepatic oxidative stress during aging, we analyzed the mitochondrial function, glucose tolerance, insulin resistance, and lipid-related parameters in aged PASK-deficient mice. Hepatic Pask mRNA decreased in step with aging, being undetectable in aged wild-type (WT) mice. Aged PASK-deficient mice recorded lower levels of ROS/RNS compared to aged WT. The regulators of mitochondrial biogenesis, PGC1a, SIRT1 and NRF2, decreased in aged WT, while aged PASK-deficient mice recorded a higher expression of NRF2, GCLm and HO1 proteins and CS activity under fasted conditions. Additionally, aged PASK-deficient mice recorded an overexpression of the longevity gene FoxO3a, and maintained elevated PCNA protein, suggesting that hepatic cell repair mechanisms might be functional. PASK-deficient mice have better insulin sensitivity and no glucose intolerance, as confirmed by a normal HOMA-IR index. PASK may be a good target for reducing damage during aging.

Declining BRCA-Mediated DNA Repair in Sperm Aging and its Prevention by Sphingosine-1-Phosphate.

Recent data suggest that paternal age can have major impact on reproductive outcomes, and with increased age, there is increased likelihood of chromosomal abnormalities in the sperm. Here, we studied DNA damage and repair as a function of male aging and assessed whether sphingosine-1-phosphate (S1P), a ceramide-induced death inhibitor, can prevent sperm aging by enhancing DNA double-strand breaks (DSB) repair. We observed a significant increase in DNA damage with age and this increase was associated with a decline in the expression of key DNA DSB repair genes in mouse sperm. The haploinsufficiency of BRCA1 male mice sperm showed significantly increased DNA damage and apoptosis, along with decreased chromatin integrity when compared to similar age wild type (WT) mice. Furthermore, haploinsufficiency of BRCA1 male mice had lower sperm count and smaller litter size when crossed with WT females. The resulting embryos had a higher probability of growth arrest and reduced implantation. S1P treatment decreased genotoxic-stress-induced DNA damage in sperm and enhanced the expressions of key DNA repair genes such as BRCA1. Co-treatment with an ATM inhibitor reversed the effects of S1P, implying that the impact of S1P on DNA repair is via the ATM-mediated pathway. Our findings indicate a key role for DNA damage repair mechanism in the maintenance of sperm integrity and suggest that S1P can improve DNA repair in sperm. Further translational studies are warranted to determine the clinical significance of these findings and whether S1P can delay male reproductive aging. There is mounting evidence that sperm quality declines with age, similar to that of the oocyte. However, the reasons behind this decline are poorly understood and there is no medical intervention to improve sperm quality. Our study suggests a strong role for DNA damage repair in maintenance of sperm quality, and for the first time, a potential pharmaceutical approach to prevent sperm aging.

Deletion of NKX3.1 via CRISPR/Cas9 Induces Prostatic Intraepithelial Neoplasia in C57BL/6 Mice.

Several techniques have been employed for deletion of the NKX3.1 gene, resulting in developmental defects of the prostate, including alterations in ductal branching morphogenesis and prostatic secretions as well as epithelial hyperplasia and dysplasia. To investigate whether the CRISPR/Cas9-mediated technique can be applied to study prostate carcinogenesis through exon I deletion of NKX3.1 gene, alterations in the prostatic intraepithelial neoplasia (PIN) and their regulatory mechanism were observed in the prostate of NKX3.1 knockout (KO) mice produced by the CRISPR/Cas9-mediated NKX3.1 mutant gene, at the ages of 16 and 24 weeks. The weight of dorsal-lateral prostate (DLP) and anterior prostate (AP) were observed to be increased in only the 24 weeks KO mice, although morphogenesis was constant in all groups. Obvious PIN 1 and 2 lesions were frequently detected in prostate of the 24 weeks KO mice, as compared with the same age wild type (WT) mice. Ki67, a key indicator for PIN, was densely stained in the epithelium of prostate in the 24 weeks KO mice, while the expression of p53 protein was suppressed in the same group. Also, both the 16 and 24 weeks KO mice reveal inhibition of the PI3K/AKT/mTOR pathway in the prostate. However, prostate specific antigen (PSA) levels and Bax/Bcl-2 expressions were decreased in the prostate of 16 weeks KO mice, and were increased in only the 24 weeks KO mice. Taken together, the results of the present study provide additional evidence that CRISPR/Cas9-mediated exon 1 deletion of the NKX3.1 gene successfully induces PIN lesions, along with significant alterations of Ki67 expression, EGFR signaling pathway, and cancer-regulated proteins.

Angiotensin type 2 receptors: Role in aging and neuroinflammation in the substantia nigra

Overactivity of the angiotensin-type-1 receptor (AT1)/NADPH-oxidase axis enhances aging processes, neuroinflammation and neurodegeneration. The role of AT2 receptors in the above-mentioned AT1-related effects in the aged brain, particularly substantia nigra, was investigated in this study. In the nigra, we observed a progressive decrease in AT2 mRNA expression with aging, and AT2 deletion led to changes in spontaneous motor behavior, dopamine receptors, renin-angiotensin system, and pro-oxidative and pro-inflammatory markers similar to those observed in aged wild type (WT) mice. Both aged WT mice and young AT2 KO mice showed an increased AT1, decreased MAS receptor and increased angiotensinogen mRNA and/or protein expression, as well as upregulation of pro-oxidative and pro-inflammatory markers. In cultures of microglial cells, activation of AT2 receptors inhibited the LPS-induced increase in AT1 mRNA and protein expression and neuroinflammatory markers. Both in AT2 KO microglial cultures and microglia obtained from adult AT2 KO mice, an increase in AT1 mRNA expression was observed. In cultured dopaminergic neurons, AT2 activation down-regulated AT1 mRNA and protein, and dopaminergic neurons from adult AT2 KO mice showed upregulation of AT1 mRNA expression. Both in microglia and dopaminergic neurons the pathway AT2/nitric oxide/cyclic guanosine monophosphate mediates the regulation of the AT1 mRNA and protein expression through downregulation of the Sp1 transcription factor. MAS receptors are also involved in the regulation of AT1 mRNA and protein expression by AT2. The results suggest that an aging-related decrease in AT2 expression plays a major role in the aging-related AT1 overexpression and AT1-related pro-inflammatory pro-oxidative effects.

Granisetron Alleviates Alzheimer's Disease Pathology in TgSwDI Mice Through Calmodulin-Dependent Protein Kinase II/cAMP-Response Element Binding Protein Pathway.

Alzheimer's disease (AD) is characterized by a compromised blood-brain barrier (BBB) and disrupted intracellular calcium homeostasis in the brain. Therefore, rectifying the BBB integrity and restoring calcium homeostasis could provide an effective strategy to treat AD. Recently, we developed a high throughput-screening assay to screen for compounds that enhance a cell-based BBB model integrity, which identified multiple hits among which is granisetron, a Food and Drug Administration approved drug. Here, we evaluated the therapeutic potential of granisetron against AD. Granisetron was tested in C57Bl/6J young and aged wild-type mice, and in a transgenic mouse model of AD namely TgSwDI for its effect on BBB intactness and amyloid-β (Aβ)-related pathology. Our study findings showed that granisetron enhanced BBB integrity in both aged and TgSwDI mice. This effect was associated with an overall reduction in Aβ load and neuroinflammation in TgSwDI mice brains. In addition, and supported by proteomics analysis, granisetron significantly reduced Aβ induced calcium influx in vitro, and rectified calcium dyshomeostasis in TgSwDI mice brains by restoring calmodulin-dependent protein kinase II/cAMP-response element binding protein pathway, which was associated with cognitive improvement. These results support granisetron repurposing as a potential drug to hold, slow, and/or treat AD.

Age-Associated Mitochondrial Dysfunction Accelerates Atherogenesis.

Rationale: Aging is one of the strongest risk factors for atherosclerosis. Yet whether aging increases the risk of atherosclerosis independently of chronic hyperlipidemia is not known. Objective: To determine if vascular aging before the induction of hyperlipidemia enhances atherogenesis. Methods and Results: We analyzed the aortas of young and aged normolipidemic wild type, disease-free mice and found that aging led to elevated IL (interleukin)-6 levels and mitochondrial dysfunction, associated with increased mitophagy and the associated protein Parkin. In aortic tissue culture, we found evidence that with aging mitochondrial dysfunction and IL-6 exist in a positive feedback loop. We triggered acute hyperlipidemia in aged and young mice by inducing liver-specific degradation of the LDL (low-density lipoprotein) receptor combined with a 10-week western diet and found that atherogenesis was enhanced in aged wild-type mice. Hyperlipidemia further reduced mitochondrial function and increased the levels of Parkin in the aortas of aged mice but not young mice. Genetic disruption of autophagy in smooth muscle cells of young mice exposed to hyperlipidemia led to increased aortic Parkin and IL-6 levels, impaired mitochondrial function, and enhanced atherogenesis. Importantly, enhancing mitophagy in aged, hyperlipidemic mice via oral administration of spermidine prevented the increase in aortic IL-6 and Parkin, attenuated mitochondrial dysfunction, and reduced atherogenesis. Conclusions: Before hyperlipidemia, aging elevates IL-6 and impairs mitochondrial function within the aorta, associated with enhanced mitophagy and increased Parkin levels. These age-associated changes prime the vasculature to exacerbate atherogenesis upon acute hyperlipidemia. Our work implies that novel therapeutics aimed at improving vascular mitochondrial bioenergetics or reducing inflammation before hyperlipidemia may reduce age-related atherosclerosis.

Centella Asiatica Improves Memory and Promotes Antioxidative Signaling in 5XFAD Mice.

Centella asiatica (CA) herb is a traditional medicine, long reputed to provide cognitive benefits. We have reported that CA water extract (CAW) treatment improves cognitive function of aged Alzheimer’s disease (AD) model Tg2576 and wild-type (WT) mice, and induces an NRF2-regulated antioxidant response in aged WT mice. Here, CAW was administered to AD model 5XFAD female and male mice and WT littermates (age: 7.6 +/ 0.6 months), and object recall and contextual fear memory were tested after three weeks treatment. CAW’s impact on amyloid-β plaque burden, and markers of neuronal oxidative stress and synaptic density, was assessed after five weeks treatment. CAW antioxidant activity was evaluated via nuclear transcription factor (erythroid-derived 2)-like 2 (NRF2) and NRF2-regulated antioxidant response element gene expression. Memory improvement in both genders and genotypes was associated with dose-dependent CAW treatment without affecting plaque burden, and marginally increased synaptic density markers in the hippocampus and prefrontal cortex. CAW treatment increased Nrf2 in hippocampus and other NRF2 targets (heme oxygenase-1, NAD(P)H quinone dehydrogenase 1, glutamate-cysteine ligase catalytic subunit). Reduced plaque-associated SOD1, an indicator of oxidative stress, was observed in the hippocampi and cortices of CAW-treated 5XFAD mice. We postulate that CAW treatment leads to reduced oxidative stress, contributing to improved neuronal health and cognition.

Cathepsin S-Mediated Negative Regulation of Wnt5a/SC35 Activation Contributes to Ischemia-Induced Neovascularization in Aged Mice.

Given that cathepsin S (CatS) gained attention due to its enzymatic and non-enzymatic functions in signaling, the role of CatS in ischemia-induced angiogenesis of aged mice was explored.Methods and Results:To study the role of CatS in the decline in aging-related vascular regeneration capacity, a hindlimb ischemia model was applied to aged wild-type (CatS+/+) and CatS-deficient (CatS-/-) mice. CatS-/-mice exhibited impaired blood flow recovery and capillary formation and increased levels of p-insulin receptor substrate-1, Wnt5a, and SC35 proteins and decreased levels of phospho-endothelial nitric oxide synthase (p-eNOS), p-mTOR, p-Akt, p-ERK1/2, p-glycogen synthase kinase-3α/β, and galatin-3 proteins, as well as decreased macrophage infiltration and matrix metalloproteinase-2/-9 activities in the ischemic muscles. In vitro, CatS knockdown altered the levels of these targeted essential molecules for angiogenesis. Together, the results suggested that CatS-/-leads to defective endothelial cell functions and that CatS-/-is associated with decreased circulating endothelial progenitor cell (EPC)-like CD31+/c-Kit+cells. This notion was reinforced by the study finding that pharmacological CatS inhibition led to a declined angiogenic capacity accompanied by increased Wnt5a and SC35 levels and decreased eNOS/Akt-ERK1/2 signaling in response to ischemia. These findings demonstrated that the impairment of ischemia-induced neovascularization in aged CatS-/-mice is due, at least in part, to the attenuation of endothelial cell/EPC functions and/or mobilization associated with Wnt5a/SC35 activation in advanced age.