Age-associated Changes Research Articles

Phasic/tonic glial GABA differentially transduce for olfactory adaptation and neuronal aging

Phasic (fast) and tonic (sustained) inhibition of γ-aminobutyric acid (GABA) are fundamental for regulating day-to-day activities, neuronal excitability, and plasticity. However, the mechanisms and physiological functions of glial GABA transductions remain poorly understood. Here, we report that the AMsh glia in Caenorhabditis elegans exhibit both phasic and tonic GABAergic signaling, which distinctively regulate olfactory adaptation and neuronal aging. Through genetic screening, we find that GABA permeates through bestrophin-9/-13/-14 anion channels from AMsh glia, which primarily activate the metabolic GABAB receptor GBB-1 in the neighboring ASH sensory neurons. This tonic action of glial GABA regulates the age-associated changes of ASH neurons and olfactory responses via a conserved signaling pathway, inducing neuroprotection. In addition, the calcium-evoked, vesicular glial GABA release acts upon the ionotropic GABAA receptor LGC-38 in ASH neurons to regulate olfactory adaptation. These findings underscore the fundamental significance of glial GABA in maintaining healthy aging and neuronal stability.

Bulk RNA sequencing of human pediatric lung cell populations reveals unique transcriptomic signature associated with postnatal pulmonary development.

Postnatal lung development results in an increasingly functional organ prepared for gas exchange and pathogenic challenges. It is achieved through cellular differentiation and migration. Changes in the tissue architecture during this development process are well-documented and increasing cellular diversity associated with it are reported in recent years. Despite recent progress, transcriptomic and molecular pathways associated with human postnatal lung development are yet to be fully understood. In this study, we investigated gene expression patterns associated with healthy pediatric lung development in four major enriched cell populations (epithelial, endothelial, and nonendothelial mesenchymal cells, along with lung leukocytes) from 1-day-old to 8-yr-old organ donors with no known lung disease. For analysis, we considered the donors in four age groups [less than 30 days old neonates, 30 days to < 1 yr old infants, toddlers (1 to < 2 yr), and children 2 yr and older] and assessed differentially expressed genes (DEG). We found increasing age-associated transcriptional changes in all four major cell types in pediatric lung. Transition from neonate to infant stage showed highest number of DEG compared with the number of DEG found during infant to toddler- or toddler to older children-transitions. Profiles of differential gene expression and further pathway enrichment analyses indicate functional epithelial cell maturation and increased capability of antigen presentation and chemokine-mediated communication. Our study provides a comprehensive reference of gene expression patterns during healthy pediatric lung development that will be useful in identifying and understanding aberrant gene expression patterns associated with early life respiratory diseases.NEW & NOTEWORTHY This study presents postnatal transcriptomic changes in major cell populations in human lung, namely endothelial, epithelial, mesenchymal cells, and leukocytes. Although human postnatal lung development continues through early adulthood, our results demonstrate that greatest transcriptional changes occur in first few months of life during neonate to infant transition. These early transcriptional changes in lung parenchyma are particularly notable for functional maturation and activation of alveolar type II cell genes.

On epigenetic stochasticity, entropy and cancer risk.

Epigenetic changes are known to accrue in normal cells as a result of ageing and cumulative exposure to cancer risk factors. Increasing evidence points towards age-related epigenetic changes being acquired in a quasi-stochastic manner, and that they may play a causal role in cancer development. Here, I describe the quasi-stochastic nature of DNA methylation (DNAm) changes in ageing cells as well as in normal cells at risk of neoplastic transformation, discussing the implications of this stochasticity for developing cancer risk prediction strategies, and in particular, how it may require a conceptual paradigm shift in how we select cancer risk markers. I also describe the mounting evidence that a significant proportion of DNAm changes in ageing and cancer development are related to cell proliferation, reflecting tissue-turnover and the opportunity this offers for predicting cancer risk via the development of epigenetic mitotic-like clocks. Finally, I describe how age-associated DNAm changes may be causally implicated in cancer development via an irreversible suppression of tissue-specific transcription factors that increases epigenetic and transcriptomic entropy, promoting a more plastic yet aberrant cancer stem-cell state. This article is part of a discussion meeting issue 'Causes and consequences of stochastic processes in development and disease'.

Senolytics prevent age-associated changes in female mice brain

PurposeConsidering that the combination of dasatinib and quercetin (D + Q) demonstrated a neuroprotective action, as well as that females experience a decline in hormonal levels during aging and this is linked to increased susceptibility to Alzheimer’s disease, in this study we evaluated the effect of D + Q on inflammatory and oxidative stress markers and on acetylcholinesterase and Na+, K+-ATPase activities in brain of female mice. MethodsFemale C57BL/6 mice were divided in Control and D (5 mg/kg) + Q (50 mg/kg) treated. Treatment was administered via gavage for three consecutive days every two weeks starting at 30 days of age. The animals were euthanized at 6 months of age and at 14 months of age. ResultsResults indicate an increase in reactive species (RS), thiol content and lipid peroxidation followed by a reduction in nitrite levels and superoxide dismutase, catalase and glutathione S-transferase activity in the brain of control animals with age. D+Q protected against age-associated increase in RS and catalase activity reduction. Acetylcholinesterase activity was increased, while Na+, K+-ATPase activity was reduced at 14 months of age and D+Q prevented this reduction. ConclusionThese data demonstrate that D+Q can protect against age-associated neurochemical alterations in the female brain.

The impact of age and sex on the inflammatory response during bone fracture healing.

Inflammation is thought to be dysregulated with age leading to impaired bone fracture healing. However, broad analyses of inflammatory processes during homeostatic bone aging and during repair are lacking. Here, we assessed changes in inflammatory cell and cytokine profiles in circulation and in bone tissue to identify age- and sex-dependent differences during homeostasis and repair. During homeostatic aging, male mice demonstrated accumulation of CD4+ helper T cells and CD8+ cytotoxic T cells within bone while both pro-inflammatory "M1" and anti-inflammatory "M2" macrophage numbers decreased. Female mice saw no age-associated changes in immune-cell population in homeostatic bone. Concentrations of IL-1β, IL-9, IFNγ, and CCL3/MIP-1α increased with age in both male and female mice, whereas concentrations of IL-2, TNFα, TNFR1, IL-4, and IL-10 increased only in female mice - thus we termed these "age-accumulated" cytokines. There were no notable changes in immune cell populations nor cytokines within circulation during aging. Sex-dependent analysis demonstrated slight changes in immune cell and cytokine levels within bone and circulation, which were lost upon fracture injury. Fracture in young male mice caused a sharp decrease in number of M1 macrophages; however, this was not seen in aged male mice nor in female mice of any age. Injury itself induced a decrease in the number of CD8+ T cells within the local tissue of aged male and of female mice but not of young mice. Cytokine analysis of fractured mice revealed that age-accumulated cytokines quickly dissipated after fracture injury, and did not re-accumulate in newly regenerated tissue. Conversely, CXCL1/KC-GRO, CXCL2/MIP-2, IL-6, and CCL2/MCP-1 acted as "fracture response" cytokines: increasing sharply after fracture, eventually returning to baseline. Collectively, we classify measured cytokines into three groups: (1) age-accumulated cytokines, (2) female-specific age-accumulated cytokines, and (3) fracture response cytokines. These inflammatory molecules represent potential points of intervention to improve fracture healing outcome.

Temporal dynamics of the fecal microbiome in female pigs from early life through estrus, parturition, and weaning of the first litter of piglets.

Age-associated changes in the gastrointestinal microbiome of young pigs have been robustly described; however, the temporal dynamics of the fecal microbiome of the female pig from early life to first parity are not well understood. Our objective was to describe microbiome and antimicrobial resistance dynamics of the fecal microbiome of breeding sows from early life through estrus, parturition and weaning of the first litter of piglets (i.e., from 3 to 53weeks of age). Our analysis revealed that fecal bacterial populations in developing gilts undergo changes consistent with major maturation milestones. As the pigs progressed towards first estrus, the fecal bacteriome shifted from Rikenellaceae RC9 gut group- and UCG-002-dominated enterotypes to Treponema- and Clostridium sensu stricto 1-dominated enterotypes. After first estrus, the fecal bacteriome stabilized, with minimal changes in enterotype transition and associated microbial diversity from estrus to parturition and subsequent weaning of first litter piglets. Unlike bacterial communities, fecal fungal communities exhibited low diversity with high inter- and intra-pig variability and an increased relative abundance of certain taxa at parturition, including Candida spp. Counts of resistant fecal bacteria also fluctuated over time, and were highest in early life and subsequently abated as the pigs progressed to adulthood. This study provides insights into how the fecal microbial community and antimicrobial resistance in female pigs change from three weeks of age throughout their first breeding lifetime. The fecal bacteriome enterotypes and diversity are found to be age-driven and established by the time of first estrus, with minimal changes observed during subsequent physiological stages, such as parturition and lactation, when compared to the earlier age-related shifts. The use of pigs as a model for humans is well-established, however, further studies are needed to understand how our results compare to the human microbiome dynamics. Our findings suggest that the fecal microbiome exhibited consistent changes across individual pigs and became more diverse with age, which is a beneficial characteristic for an animal model system.

Loss of chaperone-mediated autophagy does not alter age-related bone loss in male mice.

Chaperone-mediated autophagy (CMA) is a lysosome-dependent degradation pathway that eliminates proteins that are damaged, partially unfolded, or targeted for selective proteome remodeling. CMA contributes to several cellular processes, including stress response and proteostasis. Age-associated increase in cellular stressors and decrease in CMA contribute to pathologies associated with aging in various tissues. CMA contributes to bone homeostasis in young mice. An age-associated reduction in CMA was reported in osteoblast lineage cells; however, whether declining CMA contributes to skeletal aging is unknown. Herein we show that cellular stressors stimulate CMA in UAMS-32 osteoblastic cells. Moreover, the knockdown of an essential component of the CMA pathway, LAMP2A, sensitizes osteoblasts to cell death caused by DNA damage, ER stress, and oxidative stress. As elevations in these stressors are thought to contribute to age-related bone loss, we hypothesized that declining CMA contributes to the age-associated decline in bone formation by sensitizing osteoblast lineage cells to elevated stressors. To test this, we aged male CMA-deficient mice and controls up to 24 months of age and examined age-associated changes in bone mass and architecture. We showed that lack of CMA did not alter age-associated decline in bone mineral density as measured by dual x-ray absorptiometry (DXA). Moreover, microCT analysis performed at 24 months of age showed that vertebral cancellous bone volume, cortical thickness, and porosity of CMA-deficient and control mice were similar. Taken together, these results suggest that reduction of CMA does not contribute to age-related bone loss.

The pro-resolving mediator, annexin A1 regulates blood pressure, and age-associated changes in cardiovascular function and remodeling.

Aging is associated with chronic, low-level inflammation which may contribute to cardiovascular pathologies such as hypertension and atherosclerosis. This chronic inflammation may be opposed by endogenous mechanisms to limit inflammation, for example, by the actions of annexin A1 (ANXA1), an endogenous glucocorticoid-regulated protein that has anti-inflammatory and pro-resolving activity. We hypothesized the pro-resolving mediator ANXA1 protects against age-induced changes in blood pressure (BP), cardiovascular structure and function, and cardiac senescence. BP was measured monthly in conscious mature (4-month) and middle-aged (12-month) ANXA1-deficient (ANXA1-/- ) and wild-type C57BL/6 mice. Body composition was measured using EchoMRI, and both cardiac and vascular function using ultrasound imaging. Cardiac hypertrophy, fibrosis and senescence, vascular fibrosis, elastin, and calcification were assessed histologically. Gene expression relevant to structural remodeling, inflammation, and cardiomyocyte senescence were also quantified. In C57BL/6 mice, progression from 4 to 12 months of age did not affect the majority of cardiovascular parameters measured, with the exception of mild cardiac hypertrophy, vascular calcium, and collagen deposition. Interestingly, ANXA1-/- mice exhibited higher BP, regardless of age. Additionally, age progression had a marked impact in ANXA1-/- mice, with markedly augmented vascular remodeling, impaired vascular distensibility, and body composition. Consistent with vascular dysfunction, cardiac dysfunction, and hypertrophy were also evident, together with markers of senescence and inflammation. These findings suggest that endogenous ANXA1 plays a critical role in regulating BP, cardiovascular function, and remodeling and delays cardiac senescence. Our findings support the development of novel ANXA1-based therapies to prevent age-related cardiovascular pathologies.

Abstract B027: Exploring how aging-associated changes in autophagy dictate the development of leukemic disease

Abstract Old age leads to cellular maintenance decline, disrupting homeostasis and increasing disease risks, including cancer. Aging-related autophagy impairments contribute to harmful protein accumulation, reduced hematopoietic cell self-renewal, and impaired mitochondrial function. Autophagy plays a central role in maintaining cellular homeostasis and key cellular functions, such as cell death, cell cycle control, and differentiation. This research aims to investigate how aging-related microenvironmental changes influence Tet2 clonal hematopoiesis (CH) development, with a focus on understanding the roles of aging-associated autophagy changes. We hypothesize that the decline in cellular and tissue maintenance during aging, characterized by reduced autophagy function, contributes to changes in the bone marrow microenvironment. These changes, in turn, create a milieu conducive to the selection of oncogenic mutations. To test this hypothesis, we aimed to boost autophagy in older mice to investigate if it can mitigate oncogenesis, given that CH and cancers are less common in young individuals but prevalent in the elderly. 15-month-old mice were treated with a spermidine supplemented diet for 3 months, then LSK were transplanted with fluorescently tagged, CRISPR induced Tet2 mutations such that a small percent of HSPC bear mutations. We then monitored the competitive expansion of cells with Tet2. We found that old mice supplemented with spermidine show decreased Tet2 selection within the HSPC compartment similar to young (2mo) mice and compared to old mice on a control diet. We plan to test Tet2 competitive expansion in aged transgenic mice with high autophagy as well as the opposite experiment using transgenic young mice with decreased autophagy to further support our hypothesis. Our research emphasizes the role of autophagy in shaping the bone marrow microenvironment and influencing the development of CH. By examining how aging-associated changes in autophagy impact the selection of oncogenic mutations, our work contributes to a deeper understanding of the complex interplay between aging and cancer development. Our research has the potential to impact cancer treatment and the understanding of cancer evolution by shedding light on the mechanisms that drive oncogenic adaptation in the context of aging. Specifically, our findings suggest that enhancing autophagy in older individuals may mitigate the selection of oncogenic mutations, potentially reducing cancer risk. This insight could inform future therapeutic strategies for cancer prevention and treatment, especially in the elderly population. In summary, our research unveils the role of autophagy in modulating the aging bone marrow microenvironment's impact on CH development. By boosting autophagy in aged mice and observing reduced Tet2 selection, we provide evidence that autophagy plays a crucial role in shaping the oncogenic landscape. This underscores the potential of autophagy-enhancing interventions in countering age-related oncogenic effects. Citation Format: Amy N. Briggs. Exploring how aging-associated changes in autophagy dictate the development of leukemic disease [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B027.

Role of the gut microbiota in aging and maintenance of active longevity. Part 1

The role of intestinal microbiota in aging and maintaining active longevity is considered. The first part of the article describes the characteristics of a healthy microbiome, its changes during aging, and the composition of the intestinal microbiome of centenarians. The importance of high microbiome biodiversity in maintaining active longevity is emphasized. The role of intestinal permeability and new biomakers that assess its condition (including zonulin) in maintaining health and in the development of intestinal dysbiosis is revealed. Separate groups of microorganisms that are associated with longevity have been identified (such as Verrucomicrobia, which includes Akkermansia), and the important role of the Bacteroidetes – Firmicutes ratio has been noted. The influence of age-associated changes in the microbiome on health, the connection between changes in the microbiome and physical activity, the possibility of using microbiome analysis to predict biological age, and the correlation of microbiome composition with biomarkers of health and disease are discussed.

Long term methionine restriction: Influence on gut microbiome and metabolic characteristics.

The Methionine restriction (MR) diet has been shown to delay aging and extend lifespan in various model organisms. However, the long-term effects of MR diet on the gut microbiome composition remain unclear. To study this, male mice were started on MR and control diet regimens at 6 months and continued until 22 months of age. MR mice have reduced body weight, fat mass percentage, and bone mineral density while having increased lean mass percentage. MR mice also have increased insulin sensitivity along with increasing indirect calorimetry markers such as energy expenditure, oxygen consumption, carbon dioxide production, and glucose oxidation. Fecal samples were collected at 1 week, 18 weeks, and 57 weeks after the diet onset for 16S rRNA amplicon sequencing to study the gut microbiome composition. Alpha and beta diversity metrics detected changes occurring due to the timepoint variable, but no changes were detected due to the diet variable. The results from LEfSe analysis surprisingly showed that more bacterial taxa changes were linked to age rather than diet. Interestingly, we found that the long-term MR diet feeding induced smaller changes compared to short-term feeding. Specific taxa changes due to the diet were observed at the 1 or 18-week time points, including Ileibacterium, Odoribacter, Lachnoclostridium, Marinifilaceae, and Lactobacillaceae. Furthermore, there were consistent aging-associated changes across both groups, with an increase in Ileibacterium and Erysipelotrichaceae with age, while Eubacterium_coprostanoligenes_group, Ruminococcaceae, Peptococcaceae, and Peptococcus decreased with age.

Causality-enriched epigenetic age uncouples damage and adaptation.

Machine learning models based on DNA methylation data can predict biological age but often lack causal insights. By harnessing large-scale genetic data through epigenome-wide Mendelian randomization, we identified CpG sites potentially causal for aging-related traits. Neither the existing epigenetic clocks nor age-related differential DNA methylation are enriched in these sites. These CpGs include sites that contribute to aging and protect against it, yet their combined contribution negatively affects age-related traits. We established a new framework to introduce causal information into epigenetic clocks, resulting in DamAge and AdaptAge-clocks that track detrimental and adaptive methylation changes, respectively. DamAge correlates with adverse outcomes, including mortality, while AdaptAge is associated with beneficial adaptations. These causality-enriched clocks exhibit sensitivity to short-term interventions. Our findings provide a detailed landscape of CpG sites with putative causal links to lifespan and healthspan, facilitating the development of aging biomarkers, assessing interventions, and studying reversibility of age-associated changes.

Altered metabolism and DAM-signatures in female brains and microglia with aging

Despite Alzheimer’s disease (AD) disproportionately affecting women, the mechanisms remain elusive. In AD, microglia undergo ‘metabolic reprogramming’, which contributes to microglial dysfunction and AD pathology. However, how sex and age contribute to metabolic reprogramming in microglia is understudied. Here, we use metabolic imaging, transcriptomics, and metabolic assays to probe age- and sex-associated changes in brain and microglial metabolism. Glycolytic and oxidative metabolism in the whole brain was determined using Fluorescence Lifetime Imaging Microscopy (FLIM). Young female brains appeared less glycolytic than male brains, but with aging, the female brain became ‘male-like.’ Transcriptomic analysis revealed increased expression of disease-associated microglia (DAM) genes (e.g., ApoE, Trem2, LPL), and genes involved in glycolysis and oxidative metabolism in microglia from aged females compared to males. To determine whether estrogen can alter the expression of these genes, BV-2 microglia-like cell lines, which abundantly express DAM genes, were supplemented with 17β-estradiol (E2). E2 supplementation resulted in reduced expression of DAM genes, reduced lipid and cholesterol transport, and substrate-dependent changes in glycolysis and oxidative metabolism. Consistent with the notion that E2 may suppress DAM-associated factors, LPL activity was elevated in the brains of aged female mice. Similarly, DAM gene and protein expression was higher in monocyte-derived microglia-like (MDMi) cells derived from middle-aged females compared to age-matched males and was responsive to E2 supplementation. FLIM analysis of MDMi from young and middle-aged females revealed reduced oxidative metabolism and FAD+ with age. Overall, our findings show that altered metabolism defines age-associated changes in female microglia and suggest that estrogen may inhibit the expression and activity of DAM-associated factors, which may contribute to increased AD risk, especially in post-menopausal women.

Laxatives in Geriatric Practice: From Preoperative Bowel Preparation to Correction of Chronic Constipation

The first part of the article discusses the issues of preoperative bowel preparation in older and oldest-old patients, with a particular emphasis on the safest drugs. The second part of the article presents the problem of chronic constipation as a geriatric syndrome, including a description of age-associated changes in the structure and function of colon, causes of constipation and features of correction in geriatric practice.

Trop T, hand grip strength and waist circumference as markers of sarcopenic obesity in postmenopausal women: An analytical cross-sectional study

Objectives: Sarcopenic obesity is defined as age-associated histological changes and adiposity in muscles that impair muscle function. Asian Working Group for Sarcopenia suggested hand grip strength (HGS) and gait speed for screening sarcopenia. Troponin is a putative biomarker of muscle health. The study aims to evaluate Trop T, HGS, and waist circumference (WC) as markers of sarcopenic obesity in postmenopausal women. Materials and Methods: An analytical cross-sectional study was conducted in healthy postmenopausal women. Anthropometric measurements, HGS, and gait speed were documented. Postmenopausal women were identified as sarcopenic obesity with the following criteria: (a) The cut-points for weak handgrip strength (kg) for women as &lt;18 in Asians (b) WC ≥85 cm in women. Total cholesterol, triglycerides, high-density lipoprotein (HDLc), low-density lipoprotein (LDLc), and high sensitive troponin T levels were estimated in the automated biochemistry analyser. Results: About 33% sarcopenic obesity was observed in postmenopausal women aged 56 to 60 years. WC (93 cm) and body mass index (29.64) were increased in sarcopenic obese. Decreased HGS (9.44 kg) and increased gait speed (13.62 s) were observed in sarcopenic obese as compared to non-sarcopenic obese women. Increased troponin T (0.068 ng/mL), small dense LDLc (mmol/L), LDLc, non-HDLc, and decreased HDLc were observed in sarcopenic obese postmenopausal women. Receiver’s operating characteristic curve: sensitivity of waist circumference, HGS, gait, and troponin T were 97% (area under the curve [AUC] 0.76), 83% (AUC 0.84), 82% (AUC 0.76) and 67% (AUC 0.77), respectively. Conclusion: Measurement of waist circumference, hand grip strength, and gait are diagnostic tools of sarcopenic obesity that divulges the quality of postmenopausal life.

Squalamine reverses age-associated changes of firing patterns of myenteric sensory neurons and vagal fibres

Vagus nerve signaling is a key component of the gut-brain axis and regulates diverse physiological processes that decline with age. Gut to brain vagus firing patterns are regulated by myenteric intrinsic primary afferent neuron (IPAN) to vagus neurotransmission. It remains unclear how IPANs or the afferent vagus age functionally. Here we identified a distinct ageing code in gut to brain neurotransmission defined by consistent differences in firing rates, burst durations, interburst and intraburst firing intervals of IPANs and the vagus, when comparing young and aged neurons. The aminosterol squalamine changed aged neurons firing patterns to a young phenotype. In contrast to young neurons, sertraline failed to increase firing rates in the aged vagus whereas squalamine was effective. These results may have implications for improved treatments involving pharmacological and electrical stimulation of the vagus for age-related mood and other disorders. For example, oral squalamine might be substituted for or added to sertraline for the aged.

Dimension-based quantification of aging-associated cerebral microvasculature determined by optical coherence tomography and two-photon microscopy.

Cerebral microvascular health is a key biomarker for the study of natural aging and associated neurological diseases. Our aim is to quantify aging-associated change of microvasculature at diverse dimensions in mice brain. We used optical coherence tomography (OCT) and two-photon microscopy (TPM) to obtain nonaged and aged C57BL/6J mice cerebral microvascular images in vivo. Our results indicated that artery & vein, arteriole & venule, and capillary from nonaged and aged mice showed significant differences in density, diameter, complexity, perimeter, and tortuosity. OCT angiography and TPM provided the comprehensive quantification for arteriole and venule via compensating the limitation of each modality alone. We further demonstrated that arteriole and venule at specific dimensions exhibited negative correlations in most quantification analyses between nonaged and aged mice, which indicated that TPM and OCT were able to offer complementary vascular information to study the change of cerebral blood vessels in aging.

The impact of age on vitamin D receptor expression, vitamin D metabolism and cytokine production in ex vivo Rhodococcus equi infection of equine alveolar macrophages

Rhodococcus equi (R. equi), a pneumonia-causing intracellular bacterium, results in significant morbidity and mortality in young foals, while healthy adult horses rarely develop disease. Survival and replication within alveolar macrophages (AMφ) are the hallmarks of R. equi’s pathogenicity. The vitamin D receptor (VDR) and its ligand, the active vitamin D metabolite 1,25(OH)2D, are important in immune responses to intracellular bacteria. The vitamin D/VDR pathway regulates the downstream production of cytokines in infected human AMφ. The immunomodulatory role of the vitamin D/VDR pathway in equine leukocytes is unknown. The objective of the current study was to determine the impact of R. equi infection and age on synthesis of 1,25(OH)2D, VDR expression, and cytokine production in an ex vivo model of R. equi infection in equine AMφ. AMφ were collected from ten healthy foals at 2-, 4- and 8-weeks old and from nine healthy adult horses once via bronchoalveolar lavage. AMφ were mock infected (CONTROL) or infected with a virulent laboratory strain of R. equi for 7 days (INFECTED). VDR expression was determined via RT-qPCR from cell lysates. 1,25(OH)2D and cytokines were measured in cell supernatant by immunoassays. VDR expression was impacted by age (P = 0.001) with higher expression in AMφ from 8-week-old foals than from 2-week-old foals and adults. There was no significant effect of infection in foal AMφ, but in adults, relative VDR expression was significantly lower in INFECTED AMφ compared to CONTROL AMφ (P = 0.002). There was no effect of age or infection on 1,25(OH)2D concentration (P > 0.37). Mean TNFα production was significantly higher from INFECTED compared to CONTROL AMφ from 4- and 8-week-old foals and adults (P < 0.005). Mean IFNγ production was significantly higher from AMφ from foals at 8-weeks-old compared to 2-weeks-old (P = 0.013) and higher from INFECTED AMφ than from CONTROL AMφ in foals at 4-weeks-old and in adults (P < 0.027). The proportion of samples producing IL-1β and IL-10 was also significantly higher from INFECTED compared to CONTROL AMφ isolated from 4-week-old foals (P < 0.008). Similarly, in adult samples, IL-17 was produced from a greater proportion of INFECTED compared to CONTROL samples (P = 0.031). These data document age-associated changes in VDR expression and cytokine production in equine AMφ in response to R. equi infection. This preliminary investigation supports the need for further research to fully elucidate if the vitamin D pathway has an immunomodulatory role in the horse.

Age-associated sleep spindle characteristics in Duchenne muscular dystrophy.

Brain oscillations of non-rapid eye movement sleep, including slow oscillations (SO, 0.5-1.5 Hz) and spindles (10-16 Hz), mirror underlying brain maturation across development and are associated with cognition. Hence, age-associated emergence and changes in the electrophysiological properties of these rhythms can lend insight into cortical development, specifically in comparisons between pediatric populations and typically developing peers. We previously evaluated age-associated changes in SOs in male patients with Duchenne muscular dystrophy (DMD), finding a significant age-related decline between 4 and 18 years. While primarily a muscle disorder, male patients with DMD can also have sleep, cognitive, and cortical abnormalities, thought to be driven by altered dystrophin expression in the brain. In this follow-up study, we characterized the age-associated changes in sleep spindles. We found that age-dependent spindle characteristics in patients with DMD, including density, frequency, amplitude, and duration, were consistent with age-associated trends reported in the literature for typically developing controls. Combined with our prior finding of age-associated decline in SOs, our results suggest that SOs, but not spindles, are a candidate intervention target to enhance sleep in patients with DMD.

Changes in the Proteome of the Circle of Willis during Aging Reveal Signatures of Vascular Disease.

Approximately 70% of all strokes occur in patients over 65 years old, and stroke increases the risk of developing dementia. The circle of Willis (CoW), the ring of arteries at the base of the brain, links the intracerebral arteries to one another to maintain adequate cerebral perfusion. The CoW proteome is affected in cerebrovascular and neurodegenerative diseases, but changes related to aging have not been described. Here, we report on a quantitative proteomics analysis comparing the CoW from five young (2-3-month-old) and five aged male (18-20-month-old) mice using gene ontology (GO) enrichment, ingenuity pathway analysis (IPA), and iPathwayGuide tools. This revealed 242 proteins that were significantly dysregulated with aging, among which 189 were upregulated and 53 downregulated. GO enrichment-based analysis identified blood coagulation as the top biological function that changed with age and integrin binding and extracellular matrix constituents as the top molecular functions. Consistent with these findings, iPathwayGuide-based impact analysis revealed associations between aging and the complement and coagulation, platelet activation, ECM-receptor interaction, and metabolic process pathways. Furthermore, IPA analysis revealed the enrichment of 97 canonical pathways that contribute to inflammatory responses, as well as 59 inflammation-associated upstream regulators including 39 transcription factors and 20 cytokines. Thus, aging-associated changes in the CoW proteome in male mice demonstrate increases in metabolic, thrombotic, and inflammatory processes.