Changes in gene expression with age: metabolism, repair, and signaling

Rok vydání:	2016
Předmět:	Transcriptomic and Metabolomics Analysis of Aging Skeletal Muscle Calorie Restriction Protects against Age-Related Dysregulation of Neural Stem Cells in the Murine Subventricular Zone Inhibitors of the intron debranching enzyme (DBR1) and amyotrophic lateral sclerosis Expanding the Landscape of Healthspan-Extending Drugs through Directed Drug Discovery congenital hereditary and neonatal diseases and abnormalities Higher levels of the small chaperone HSP25 contribute to longer lifespan Biologic aldehydes contribute to a Parkinson-related behavioral phenotype in alpha-synuclein overexpressing mice 17α-Estradiol Curtails Metabolic and Inflammatory Dysfunction in Male Mice NLRP3 inflammasome controls adipose tissue leukocytosis and inflammation during aging NF-κB-regulating Alzheimer's disease associated TAU protein in skeletal muscle and brain Session VII: Pharmacology-Based Metabolic Interventions in Effects of Transient High-Dose Rapamycin Treatment on Lifespan and Healthspan Abstracts Working Smarter Not Harder with High Intensity Interval Training Alterations in metabolic profiling of Leber's Hereditary Optic Neuropathy Inflammation in the aging V-SVZ neural stem cell niche Immunoproteasome Regulates Mitochondrial Function in the Retina Modulation of skeletal muscle IGF-1 production impairs glucose handling in mice Metformin Reduces Rapamycin-Induced Glucose Intolerance in Female but Not Male Mice Mir-155 reduces polymerase beta (pol β) and may promote cellular senescence in Down syndrome The metabolic characterization of the interleukin-10tm1Cgn mouse Role of tyrosine in longevity insulin-like signaling and innate immunity in Caenorhabditis elegans Loss of Gpx4 in forebrain neurons leads to neurodegeneration gliosis and cognitive decline A Novel Mitochondrial Retrograde Signaling Pathway Modulates Longevity Chronic Inhibition of mTOR improves BBB function in vitro and restores vascular function and improves cognitive outcomes and in a mouse model of atherosclerosis Non-cell autonomous control of metabolism by neuronal mTOR signaling nutritional and metabolic diseases Naked Mole-Rat Cardiac Function and Myofilament Proteins Respond Uniquely to Oxidative Stress Session I: Metabolic Signatures of Aging and Age-Related Diseases eye diseases Changes in Gene Expression with Age: Metabolism Repair and Signaling Insulin signaling in the CM9 motor neuron negatively regulates synaptic vesicle exocytosis via the FOXO-dependent transcription of the eif4e binding protein Loss of TMEM127 protects against obesity-induced insulin resistance A key role of TOR in cognitive and brain vascular aging 2015 Barshop Symposium on Aging Chronic rapamycin treatment attenuates age-related motor deficits in sex-dependent manner in UM-HET3 mice Protective effects of 17α estradiol against AD-type catecholaminergic neuron loss in dtg APP/PS1 mice Efficacy and Metabolic Impacts of Risperidone in an Insulin-Resistant Mouse Model of Autistic Behaviors
Zdroj:	Pathobiology of Aging & Age Related Diseases
ISSN:	2001-0001
Popis:	Leber's Hereditary Optic Neuropathy (LHON) is a neurodegenerative mitochondrial disease characterized by retinal ganglionic cell death and eventual loss of central vision. Specific mtDNA mutations in respiratory complex I subunits (ND4/ND6/ND1) coding genes have been identified in most of LHON patients, and these mutations have been shown to cause mitochondrial dysfunctions leading to increased oxidative stress and bioenergetics failure in cell models and LHON patients. In this study, we investigated the alterations in metabolic profiling of various pathways converging to mitochondria. We aim to identify metabolic signatures of LHON associated with complex I defects. We utilized trans-mitochondrial hybrids (cybrids) of LHON carrying ND4/ND6/ND1 mutations grown in regular and mitochondrial challenged conditions. We investigated polar metabolites profiling in the cell extracts using LC-MS approach and found alterations in various metabolic intermediates of pentose phosphate pathway, amino acid metabolism, fatty acid metabolism, purine metabolism, glycolysis, and TCA cycle. Our studies indicated downregulations of some important metabolites involved in the antioxidant defense and neuro-protective mechanism, and accumulations of other metabolites involved in tryptophan degradation and protein glycation. Further investigations would provide mechanistic insights on how mtDNA mutations lead to retinal ganglionic cell death in LHON patients and may help to develop metabolic markers for disease diagnostics and to explore novel intervention approaches for treatment. Circulating microRNAs (miRNAs) regulate a large number of genes and mediate a broad spectrum of biological processes in humans including aging and changes in metabolism. A total of 48 subjects from the Established Populations for the Epidemiologic Study of the Elderly (EPESE), aged 76–79 at the time of the blood draw, were randomly selected into eight bins of six subjects each crossed by gender, race, and dead within 2 years vs. dead in more than 10 years post-blood draw. We profiled plasma for 179 miRNA and multiple metabolites. MiRNA were correlated with metabolites and existing cytokine and function data. High quality miRNA with good yields and highly reliable metabolite data were able to be generated from plasma samples stored for 20 years. Twelve miRNA were differentially expressed according to longevity status: four in women and men (hsa-miR-26b-5p, -374a-5p, -374b-5p, -18b-5p, and -26a-5p); five exclusive to women (hsa-miR-374b-5p, -126-3p, -500a-5p, -23a-3p, and -421); and three exclusive to men (hsa-miR-190a, -331-3p, and -30e-3p). Except for hsa-miR-500a-5p, all the miRNA positively associated with longevity in women were also associated with function. Circulating miRNAs identified in this study also correlated with inflammatory markers and cardiovascular small-molecule. MiRNA may play a more critical role in regulating metabolites and functional performance in women rather than in men. Given that miRNA are being explored as pharmacologic therapies, these findings could stimulate development of novel interventions to enhance function and longevity in elders. Aging is associated with chronic inflammation and increased risk for degenerative disorders and diseases. In the elderly there is increased visceral adiposity and increased numbers of fat-associated lymphocyte clusters within adipose tissue (AT); however, the role for AT in age-induced immune defects is unclear. Our prior studies have identified that the canonical inflammasome, NLRP3, is required for diet-induced AT inflammation and age-related inflammation in multiple organs. We hypothesized that NLRP3 is required for age-induced changes in AT immune cells and metabolic dysfunction. We found that aging increases AT lymphocytes; compared to 24-month-old control mice, Nlrp3-/- animals were protected from increases in AT leukocytosis. RNA sequencing analysis of AT macrophages from aged mice revealed distinct inflammatory signatures that were regulated by NLRP3. AT displayed immunological properties, as during infection, a higher frequency of antigen-specific T cells are recruited to AT than to spleen. Our results show that NLRP3 is required for age-induced B cell infiltration into visceral AT. Furthermore, our data suggest that pharmacological inhibition of NLRP3 activation and IL-1β signaling may improve AT inflammation and insulin resistance in aged mice. These data demonstrates that macrophage-expressed NLRP3 inflammasome controls age-related inflammation and AT function. To define the transcriptional changes associated with aging in an emerging model system, we conducted RNA-Seq at five timepoints over the life cycle of the rotifer Brachionus manjavacas, from eggs to senescence. We identified genes and pathways differentially regulated between the five sequential life-stages using baySEQ and GSEA. Of the 22,064 total transcripts, 15,826 were significantly differentially expressed (SDE) in one or more pairwise time comparison. Three transcripts were SDE in all four life-stage transitions: an unannotated gene, homogentisate 1,2-dioxygenase and an ATP-binding cassette, suggesting involvement in metabolism. Pathways involved in metabolism, maintenance and repair, and metabolic function were upregulated in one or more of the first three transitions and downregulated in late life. Many such pathways, including those involved in Parkinson's, Huntington's, and Alzheimer's diseases, were united by genes involved in mitochondrial function and oxidative phosphorylation, including NDUFA, UQCRC, COX genes, CYC1, SDHA, VDAC2, and SLC25A31. Insulin signaling, tor, and adipocytokine pathways were all down regulated in the transition to senescence. An array of metabolism-altering interventions is known to increase lifespan in rotifers, including caloric restriction, decreased temperature, glycerol supplementation, and knockdown of jnk or tor. Whether these interventions work through separate or overlapping pathways, and how these differ between genotypes, is the subject of ongoing investigation. This study provides the first insights into genome-wide changes in gene expression over the lifespan of B. manjavacas and continues development of monogonont rotifers as a model system to study the biology of aging. Alzheimer's disease (AD) is estimated to have afflicted 5.3 million people in the U.S. in 2015. AD is characterized by neurodegeneration and crippling dementia. Age is the largest risk factor for AD; however, the etiology is poorly understood which is reflected by the very limited treatment options currently available. Mounting evidence suggests that oxidative stress plays a significant role in AD pathogenesis but the mechanism(s) remain unclear. Aging is associated with increased generation of reactive oxygen species (ROS) that is thought to explain the strong correlation between advancing age and AD threat. The brain is especially sensitive to increased ROS because of the relatively low antioxidant defense capabilities and large amount of oxygen consumed by the organ. Oxidative damage in the brain predominantly manifests as lipid peroxidation (LP) due to the high concentration of polyunsaturated fatty acids (PUFAs) present in its membrane rich architecture. LP accumulation can damage membrane structure and function which adversely effects healthy synaptic activity. Markers for LP damage are a hallmark of AD but it is unknown whether LP plays a causal role in pathogenesis or is merely a physiological consequence of pathogenesis. Interestingly, LP damage in the brain naturally accumulates with age. Further, the increase in LP damage markers in AD patients seems to mirror neurodegeneration and cognitive decline. Taken together, this suggests a causative or at the very least permissive role for LP in AD pathogenesis. A limiting factor in evaluating the role of LP in the pathophysiological sequelae of AD is the lack of in vivo tools to specifically manipulate LP in the cells most important to synaptic integrity: neurons. To address this question, we generated a novel transgenic mouse that allows inducible deletion of the essential lipid peroxide detoxifying protein glutathione peroxidase-4 (Gpx4) in neurons of the forebrain. Using this model, we report significant neurodegeneration, gliosis, and cognitive decline following Gpx4 knockout in adult mice. Gpx4 may thus be an essential component for neuronal health and survival in the context of brain aging and related neurodegenerative diseases. Mitochondrial dysfunction underlies numerous age-related human pathologies. In an effort to uncover how the detrimental effects of mitochondrial dysfunction might be mitigated, we examine how the nematode Caenorhabditis elegans not only adapts to disruption of the mitochondrial electron transport chain, but in some instances responds with extended lifespan. Recent studies have shown cellular surveillance mechanisms are activated in these worms, including the well-studied ATFS-1 dependent mitochondrial unfolded protein response (UPRmt). Here we identify a novel mitochondrial retrograde pathway activated in C. elegans independent of ATFS-1. This pathway incorporates a p38 MAPK cascade and acts in a complementary manner to ATFS-1/SKN-1 activation. Finally, we show that in those mutants which activate this pathway, it is required for their life extension. Herein we analyze how the aging process affects the structure and functionality of the lymphatic collectors (LCs) with reference to their ability to maintain pathogen clearance. Ultrastructural, biochemical and proteomic analysis indicated a loss of extracellular matrix proteins, an increase in protein oxidative modifications as well as activation of nuclear factor-κB signaling as a sign of ‘low grade’ inflammation in aged LCs. This resulted in a decrease in contractile and pumping activity of LCs, as measured in vivo. Functionally, this impairment also translated into a reduced ability for in vivo bacterial transport as determined by time-lapse microscopy. Ultrastructural and proteomic analysis also indicated a decrease in the thickness of the endothelial cell glycocalyx and loss of gap-junction proteins in aged LCs. Multiplex analysis data shows that in aged lymphatic tissues there is an elevation of NF-κB-dependent pro-inflammatory cytokines, which results in increase adhesion molecules expressions, delayed pathogen transport to lymph nodes and altered immune response to pathogen. Functionally, these modifications translated into higher ability of the pathogen to escape from aged LCs into the surrounding tissue. Altogether, our analysis mapped the complexity of the aging-related anatomical, biochemical, and functional changes in LCs. The decreased ability to transport bacteria to the draining nodes, associated with increased bacterial escape in the surrounding tissue can contribute to the decreased ability of the immune system to clear pathogens in the elderly, as observed in immunosenescence. The frail, Interleukin 10tm1Cgn (IL10tm) mice have been utilized as a model of frailty because of their propensity to develop skeletal muscle weakness and chronic activation in NFkB pathways. Previous findings in humans indicate that aging and frailty are associated with impaired insulin sensitivity and glucose homeostasis, decreased metabolic rate and locomotor activity, as well as altered respiratory quotient (RQ) and body composition. However, to date, little is known about the body composition and energy metabolism of this frail mouse model. To determine if old IL10tm frail mice have altered insulin sensitivity, glucose homeostasis, oxygen consumption (VO2), RQ, spontaneous locomotor activity, body composition, and plasma adipokine levels compared to age- and gender-matched C57Bl/6 control strain, we performed insulin tolerance tests, glucose tolerance tests, body composition analysis, indirect calorimetry with activity monitoring, and plasma adiponectin and leptin measurements in cohorts of 3-, 10-, and 20-month-old female mice. Insulin sensitivity, glucose homeostasis, locomotor activity, and RQ were not significantly altered. Interestingly, old IL10tm mice had markedly decreased VO2 when normalized by lean mass, but not when normalized by fat mass or the lean/fat mass ratio. NMR-based body composition analysis and dissection weights show that fat mass is decreased with age in IL10tm mice compared to controls. Further, plasma adiponectin and leptin were also decreased in IL10tm.These findings suggest that frailty observed in this mouse model of chronic inflammation may be driven by alterations in fat mass, hormone secretion, and energy metabolism. In age-related macular degeneration (AMD), the retinal pigment epithelium (RPE) cells are heavily exposed to oxidative stress which not only generates mitochondrial DNA damage, but has been shown to increase levels of the immunoproteasome. Evidence from our laboratory suggests that the immunoproteasome modulates a key cellular stress pathway (NF-κB) as well as modulating cell survival signaling (PTEN/AKT) after injury or disease in the retina. However, the ability of the immunoproteasome to modulate cell stress and survival pathways, but not completely control these signaling pathways provides a rationale look at interconnected pathways. Mitochondrial function and the NF-κB and PTEN/AKT signaling pathways are all affected by ROS and inflammation, supporting a role for the immunoproteasome in cell metabolic function. The purpose of this study is to determine the role of the immunoproteasome in the metabolic function of the retina after induced damage. We used RPE cells isolated from WT and KO mice lacking either one (lmp2-/-) or two (lmp7-/-/mecl-/-) catalytic subunits of the immunoproteasome and assessed differences after exogenous hydrogen peroxide or etoposide treatment. Lmp2-/- RPE cells show protection against DNA damage-induced cell death. Both lmp2-/- and lmp7-/-/mecl-/- RPE cells show altered temporal signaling of DNA damage response proteins compared to WT RPE cells. LMP2 deficiency strongly alters the metabolic phenotype of RPE cells and protects metabolic function after damage by hydrogen peroxide. The immunoproteasome is a promising therapeutic target in multiple diseases, and this current work is crucial to identify the signaling pathways affected by the immunoproteasome. Age-related sarcopenia predisposes to frailty and loss of independence. However, the molecular basis behind aging-induced muscle loss is not known. In this study we performed transcriptomic and metabolomics analyses in aged mice to identify molecular and cellular signatures that may play a role in the pathogenesis of sarcopenia of aging. Three groups of c57bl/6 mice were studied; young, mature, and old. The transcriptome in gastrocnemius muscle was assessed by whole genome RNA sequencing. Metabolomics profiling was performed by LC/MS and GC/MS. Compared to young, there was a 10.0% reduction in gastrocnemius muscle mass in mature mice, and a 20.4% reduction in old mice. A total of 143 genes differed between young and mature age (p Studies in long-lived animal models such as the naked mole-rat (NMR) show resistance to a broad spectrum of environmental stressors. Collectively these findings suggest that NMRs possess efficient mechanisms to maintain protein quality in multiple tissues. Our previous work indicated that this is attributed in part to altered changes in molecular chaperone levels that influence the transport and disposal of damaged proteins. We extend this study of chaperones and protein degradation mechanisms that compared NMRs and mice to include eight rodent species of various sizes and lifespans by examining key proteasome-related molecular chaperones, proteasome, and autophagy markers in the liver and quadriceps muscles. The small molecular weight chaperone heat-shock chaperone 25 kDa (HSP25) showed as significant correlation with longevity in both tissues, and was highest in the NMR, suggesting that HSP25 may play a key role in age-related maintenance of protein homeostasis in long-lived animals. To examine the role of the NMRHSP25 further, we constructed a transgenic Caenorhabditis elegans worms (HSP25NMR), which expresses HSP25 from the naked mole rat with the ubiquitously expressed sur-5 promoter. The HSP25NMR transgenic worms show both resistance to heat stress and an extended lifespan. This finding suggests that HSP25 could play a causal role in the improved proteostasis and lifespan of the NMR, and provides a genetically tractable system in which to examine the mechanisms involved in the effects of this chaperone on longevity, stress resistance, and healthspan. Aging is a major risk factor for increased susceptibility to damage from brain insults like stroke, inflammation, and disease. Calorie restriction (CR) improves physiological markers of health during aging, including extending lifespan and protecting against age-related damage to the brain. The largest source of neural stem cells in the adult brain is the subventricular zone (SVZ). We sought to determine the effect of long-term CR on neurogenesis and the neural stem cell niche in the SVZ of young and aged mice. Here, we show that aged mice fed standard control chow have fewer SVZ-derived neurons in the olfactory bulb, indicating that aging impairs neural stem cell function. Long-term CR preserved neural stem cell function and resulted in a significant increase in neurogenesis in aged mice compared with ad libitum-fed controls. Paradoxically, we have observed that proliferation of neural stem cells is decreased in aged CR mice. This is in the presence of increased neuroblast formation and increased neurogenesis. Confocal imaging and fluorescent staining of SVZ revealed an increase in both the total number and reactivity of microglia in the aged control mouse, suggesting increased inflammation in the neural stem cell niche during aging. Remarkably, these age-related inflammatory markers were not observed in the long-term CR aged mice, which appeared no different from young control and young CR mice. We have found that the neural stem cell chemoattractant mechanism CXCL12, secreted by endothelial cells, and its receptor expressed on neural stem cells, CXCR4, are dysregulated in the aged mouse fed ad libitum, but not in the aged CR mouse. Further, the recently identified rejuvenation factor GDF11 was found to be decreased in the aged SVZ, but not in the aged CR SVZ. These initial experiments have revealed a protective role for CR in the aging SVZ and are an important first step in understanding how CR may be an effective therapeutic intervention for the aging brain. Both cardiovascular disease and one of its greatest risk factors, aging, are commonly attributed to the accrual of oxidative damage. Unlike every other mammal studied, the naked mole-rat (NMR) resists age-related changes in cardiovascular structure and function for at least 75% of its 32-year maximum lifespan. We questioned if doxorubicin (DOX) could induce oxidative stress to cause cardiac dysfunction in NMRs. Echocardiography showed that 7 days after a 20 mg/kg single dose of DOX, NMR cardiac contractility was unchanged (28±1%). After a similar treatment, mice had a 24% decline in cardiac contractility (36±1% to 27±2%). We also subjected NMRs to dobutamine exercise-like stress and found that by day 7, DOX-treated NMRs had a significant reduction in their dobutamine response, signifying a diminished cardiac reserve. Interestingly, the phosphorylation of serines on cardiac myosin binding protein-C was not changed in DOX-treated NMRs. Conversely, the phosphorylation of these serines was increased with DOX treatment in mice. NMR ventricles are comprised primarily of the β-myosin heavy chain isoform, which is associated with slowed myocardial contraction and increased efficiency, and this does not change with DOX. This is in stark contrast to mouse ventricles, which express predominately the α-isoform, and switch to the β-isoform with DOX treatment. Overall the DOX-induced reduction in cardiac reserve and lack of change in myofilament phosphorylation in NMRs represents a unique way of handling enhanced cardiac oxidative stress, and may provide insights into the species’ outstanding cardiovascular health and longevity. The mechanistic target of rapamycin (mTOR) is a major regulator of cellular and organismal metabolism. Reduction of TOR signaling by rapamycin alters organismal metabolism and increases lifespan and healthspan. In invertebrate models, selective reduction of function of TOR in the nervous system is sufficient to extend life. We hypothesized that attenuating mTOR signaling in mature mammalian neurons would extend lifespan by altering critical aspects of metabolism non-autonomously. To test this hypothesis we knocked down the mTOR complex 1 (mTORC1)-specific protein, Raptor, in adult neurons in mice. Reduction of mTORC1 complex formation in neurons of mice by 35 or 60% did not affect body weight but increased lean mass while reducing metabolism. This was associated with enhanced exercise endurance and absent post-exercise hypoglycemia, even though glucose and insulin tolerance were unchanged. The 35% KD animals exhibit increased hepatic glucose production as determined by a pyruvate tolerance test. Wild-type muscle cells treated with serum from 35% KD mice have reduced glycogen content as compared those treated with WT mouse serum indicating a change in muscle glycogen production or utilization caused by a circulating factor in the serum. To determine cell-autonomous effects of mTORC1 knockdown in neurons we measured spatial learning and memory. While 60% mTORC1 knockdown impaired cognitive plasticity, 35% reduction in mTORC1 complex formation resulted in enhanced spatial memory. Consistent with these observations, 60% neuronal mTORC1 KD reduced brain glucose metabolism and cerebral blood flow, while a 35% decrease enhanced brain glucose uptake with no changes in cerebral blood flow (CBF). Taken together, our data suggest that reduction of neuronal mTORC1 may have significant non-cell autonomous effects on basal and exercise metabolism. Furthermore, the relationship between levels of mTORC1 in neurons and spatial memory is not linear. Rather, spatial memory maybe maximal when neuronal mTORC1 levels are slightly lower than WT, but decreases with further reductions in mTORC1. Background Tau proteins are infamously recognized for their accumulation in several neurodegenerative diseases, including Alzheimer's disease. Physiologically, Tau functions to stabilize microtubules within neuronal axons. Alterations in Tau expression, post-translational modification or subcellular localization contribute to neurodegeneration and dementia. Typically, Tau proteins are considered ‘brain specific’. However, alternative splicing of its coding gene, microtubule associated protein tau (MAPT), can produce over 30 unique human proteins that are differentially expressed dependent on age and tissue type. Though less is known about MAPT expression in tissues outside the nervous system, Tau protein aggregation occurs in skeletal muscle of patients with inclusion body myositis, an inflammatory myopathy characterized by chronic, progressive muscle inflammation accompanied by muscle weakness. Materials and methods Our laboratory utilizes a transgenic mouse with muscle-specific inhibition of NF-κB, or MISR (muscle-specific expression of IkBa superrepressor) to assess the impact of skeletal muscle inflammation on aging and exercise. We performed RNA sequence analysis (RNA-seq) on skeletal muscle from young (3–6 months), middle-aged (12–18 months) and old (33–36 months) MISR and wild type mice. Additionally, separate cohorts of young and middle-aged animals were exercised prior to tissue collection. We assessed differential gene and protein expression of Tau and NF-κB associated pathways with age and exercise between MISR and wild type mice. Results and conclusions MISR mice express significantly higher levels of Mapt transcript and Tau protein in skeletal muscle than wild type mice. Further, exercise significantly lowers Mapt levels in wild type, but not MISR mice, providing additional evidence for NF-κB's negative regulation of Mapt expression. Incredibly, decreased skeletal muscle NF-κB alters Tau protein in brain as well. To our knowledge, we are the first to expose NF-κB's regulation of Mapt expression and provide compelling evidence for cell non-autonomous effects of decreased skeletal muscle NF-κB on brain. Experiments are currently underway to elucidate the implications of life-long decreased levels of skeletal muscle NF-κB on muscle and brain aging. Cerebrovascular changes such as cerebral hypo-perfusion and blood–brain barrier dysfunction are ubiquitous attributes in diseases of aging and are often even present in ostensibly healthy aged subjects. Because endothelium-dependent vascular dysfunction is a universal feature of aging associated with susceptibility to neurodegenerative diseases such as Alzheimer's, and TOR inhibition may delay aging, we hypothesized that mTOR-dependent mechanisms of neurovascular dysfunction may be common to different models of age-associated neurological disease, and may contribute to associated cognitive deficits. Our lab recently showed that attenuation of the mechanistic target of rapamycin (mTOR) restores brain vascular integrity and cognitive function in mice modeling Alzheimer's disease. Here we investigate the mechanisms by which mTOR attenuation with rapamycin decreases BBB permeability and vascular leakage in vitro and examine cognitive outcomes in a well-characterized vascular mouse model, the low density lipoprotein receptor knock-out (LDLr-/-) mouse. Our findings indicate that acute rapamycin treatment is sufficient to decrease BBB permeability by facilitating expression of tight junction proteins and subsequently improving barrier function. Chronic TOR attenuation also rescues severe peripheral and central vascular deficits in HFD-fed LDLr-/- mice, abolishes neurovascular damage to restore cerebral blood flow (CBF) regardless of diet, and ameliorates significant memory deficits in HFD-fed LDLr-/- animals. Overall, these data suggest that mTOR contributes to vascular and neurovascular dysfunction that is associated with cognitive impairment in mice that model atherosclerosis. Taken together with our previous studies in a mouse model of AD, these data provide support for the hypothesis that mTOR-dependent vascular dysfunction associated with aging may be a mechanism shared by neurological diseases of aging, and suggest that rapamycin or other inhibitors of the mTOR pathway may have promise for the treatment of AD and atherosclerosis-associated cognitive impairment, and potentially other neurological diseases of aging. Parkinson's disease (PD) is the second leading neurodegenerative disease affecting ~1% of people over the age of 65 years. Clinical features include, resting tremor, bradykinesia, and muscular rigidity. Pathologically, PD is characterized by the progressive loss of dopaminergic neurons in the nigrostriatal pathway, ultimately leading to dopamine deficits in the striatum. Surviving dopamine neurons have been found to contain cytoplasmic Lewy bodies primarily composed of the pre-synaptic protein, alpha-synuclein (aSyn). In its native form, aSyn remains unfolded. However, under certain conditions of oxidative stress, aSyn may aggregate and alter the release of neurotransmitters and the activity of mitochondrial complex I. Toxic aldehydes, including the dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde, and the lipid peroxidation end product, 4-hydroxynonenal, promote aSyn aggregation. The two enzymes primarily responsible for the detoxification of aldehydes in dopamine neurons, are aldehyde dehydrogenase ALDH1a1 and ALDH2. Decreased expression of ALDH1a1 has been observed in the substantia nigra of patients who died with PD. Polymorphisms in ALDH2 have been found to increase the risk of PD in individuals exposed to pesticides. We previously reported that Aldh1a1-/-x Aldh2-/- knockout mice exhibit pathological manifestations of PD, including elevated biogenic aldehydes, motor deficits that are ameliorated by L-dopa, and the loss of TH-immunoreactive neurons. In the current study, we tested the hypothesis that elevated biogenic aldehydes exacerbate the behavioral and neurochemical deficits that are associated with increased expression of α-Syn. The results show that elevated biogenic aldehydes, in the presence of overexpressed human wild-type aSyn, exacerbates neurochemical and behavioral deficits observed in PD. Down syndrome (DS) is a chromosomal condition characterized by accelerated aging. This aging phenotype of DS has yet to be directly tied to a DNA repair defect. Reduced DNA polymerase beta (polB) abundance and unrepaired damage from oxidative stress observed in DS point toward defective base excision repair (BER), a process we have shown declines with age. We hypothesize that accelerated aging in DS is a consequence of early and continuous inhibition of BER via overexpression of miR-155, a miRNA located on chromosome 21. We evaluated senescence phenotypes and found that DS primary fibroblasts exposed to H2O2 exhibit a two-fold increase over non-DS fibroblasts in senescence-positive cells (p The ventricular-subventricular zone (V-SVZ) is the largest neural stem cell (NSC) reservoir in the adult murine brain, giving rise to new neurons and glia throughout life. A key mediator of proper NSC function is the underlying vascular plexus and the ependymal layer which houses NSC germinal pinwheels. However, NSC proliferation and neurogenesis is sharply reduced at mid-age and the mechanisms for this reduction are unknown. Here we show that microglia, the resident immune cells in the brain, are integral V-SVZ niche cells that are closely associated with NSCs, germinal pinwheels, and the microvasculature. During aging, microglia undergoes substantial positional changes within the niche, losing their close association to the vasculature while becoming increasingly associated with the ependymal and germinal pinwheels. We observed an early and chronic activation of V-SVZ microglia that was not seen in microglia outside of the niche during aging. This activation was accompanied by increased anti-neurogenic inflammatory mediators within the NSC compartment. Furthermore, we observed a substantial number of infiltrating monocytes within the V-SVZ niche that increased during aging, suggesting that the peripheral immune system is an important mediator of V-SVZ inflammation during aging. Using a chronic inflammatory model in young mice, we recapitulated microglia activation and reduced proliferation in the V-SVZ observed in old mice. Furthermore, in vitro studies revealed secreted factors from activated microglia reduced proliferation and neuron production compared to secreted factors from resting microglia. Our results suggest that age-associated chronic inflammation contributes to declines in NSC function within the aging neurogenic niche. The phenotype of the transgenic mouse expressing a single human mutation for amyloid precursor protein (APP) share several pathological features with Alzheimer's disease (AD). Among these AD-type neuropathological features are deposition of insoluble beta amyloid and neuroglial proliferation in cortical brain regions. The one characteristic that distinguishes AD from transgenic APP mice is neuronal loss. Conversely, the double transgenic mouse expressing both APP and presenilin 1 (PS1) exhibit neuronal loss within catecholaminergic regions. It has been reported that estrogen is a powerful and potentially valuable candidate for protection against neuroinflammation and neural damage in the brains of patients suffering from AD and other neurological diseases. 17α-estradiol, a non-feminizing enantiomer of estrogen lacks activity at classical estrogen receptors retaining activity in several models of neuroprotection. The goal of this study was to assess an age-dependent pattern of cell loss in the catecholaminergic population of the brainstem and determine whether 17α-estradiol can ameliorate this loss in the double transgenic model of AD. To more precisely define the time course of neuron loss in LC of female dtg APP/PS1 mice, we analyzed LC neuronal loss in young, middle, and aged dtg APP/PS1 mice and age-matched non-tg littermate controls. Stereological analysis revealed age-related increases in the extent of neuronal loss in dtg APP/PS1 females. Stereological analysis revealed that 60 days of 17aE2 treatment prevented neuronal cell loss in the LC. This study may expand our insight into neuroprotective therapies to block neurodegeneration in dtg APP/PS1 mice and, by analogy, in the brains of Alzheimer's patients. Human life expectancy has increased dramatically in the past several decades and no deceleration of this trend appears imminent. Healthspan extension and the compression of morbidity have failed to match enhancements in lifespan. As a consequence, the incidence of chronic diseases and onset of multimorbidity have become commonplace in older adults. Functional declines in white adipose tissue (WAT) contribute to the onset of chronic diseases by promoting visceral adiposity, increased inflammation, and metabolic dysfunction. Visceral adiposity and elevated pro-inflammatory mediators are linked with morbidity and all-cause mortality risk in aged mammals. The goal of this work was to determine if late-life administration of 17α-estradiol (17α-E2), a nonfeminizing enantiomer of 17β-estradiol, would curtail age-related changes in regional adiposity, inflammatory status, and metabolic homeostasis. We found that 17α-E2 beneficially alters body mass and adiposity in growing and weight-stable male mice. Improvements in body composition were primarily attributed to declines in visceral adiposity. The reduction in adiposity was also associated with concomitant declines in ectopic lipid deposition. 17α-E2 reduced several pro-inflammatory mediators in visceral WAT and plasma. These phenotypic alterations were associated with improvements in glucose homeostasis and systemic insulin sensitivity. Importantly, 17α-E2 also positively modulated nutrient-sensing pathways (AMPK and mTOR) in visceral WAT, which has previously been linked with systemic metabolic regulation. Chronic administration of 17α-E2 did not alter sex organ mass or circulating sex hormones, confirming previous reports of the nonfeminizing effects of 17α-E2. Collectively, our studies demonstrate that late-life administration of 17α-E2 alleviates age-related WAT dysfunction and improves systemic metabolic homeostasis and inflammatory status. Our findings suggest 17α-E2 may be an attractive therapeutic option for translation into older human populations with multimorbidity. Obese individuals have higher circulating levels of aromatic amino acids including tyrosine. High level of tyrosine is correlated with the development of diabetes and heart disease. One of the underlying pathologies of obesity, diabetes, and heart disease is chronic inflammation. Whether there is a causal relationship between increased tyrosine, inflammation, and diabetes is not known. Using the model organism Caenorhabditis elegans we have found that increased tyrosine levels in the worm can modulate the daf-2 insulin-like signaling pathway. Since this pathway shows strong homology to the human insulin signaling pathway, these findings suggest that the elevated tyrosine levels observed in obese and pre-diabetic people may impair insulin signaling and hence play a causal role in the progression to the development of diabetes. The daf-2 pathway has also been widely studied as an important regulator of longevity in worms, and from our studies we have found a mutant worm allele with high levels of tyrosine to have an increased lifespan as well. Our recent studies show that tyrosine also plays a role in the innate immune response of C. elegans to pathogenic bacteria, and this is mediated by the p38 MAP-kinase pathway. In humans, p38 MAPK pathway is involved in inflammation. Understanding the effect of tyrosine on insulin signaling and p38 pathway is thus important in the context of inflammation, aging, and the age-related disease of diabetes. Altered insulin signaling within the brain has been linked to cognitive dysfunction and neurodegenerative disease. Functional signaling downstream of the insulin/IGF-1 receptor has been linked to a number of cell processes that could contribute to the effects of insulin signaling on brain function including maintenance of neuronal health, reduced cell stress, neuron development, and synapse function. However, a role for insulin signaling during the regulation of neurotransmission has not been demonstrated. Using a novel synaptic preparation in adult Drosophila, we have found that cell autonomous insulin signaling negatively regulates the presynaptic release of neurotransmitter via the activity of the eif-4e binding protein (4eBP), a negative regulator of protein translation. In this context, the activity of 4eBP is regulated transcriptionally by the forkhead transcription factor Foxo and not the mammalian target of rapamycin (mTOR). Furthermore, the regulation of neurotransmission by insulin signaling requires the mRNA binding protein Staufen, which is known to localize mRNAs to distinct compartments within neurons, and is blocked by the protein synthesis inhibitor cycloheximide. Our data support the model that cell autonomous insulin signaling regulates the presynaptic release of neurotransmitter via the local translation of negative regulators of synaptic vesicle exocytosis. Insulin-like growth factor-1 (IGF-1) is a pleiotropic factor involved in developmental and metabolic processes on multiple tissues and particularly skeletal muscle. Skeletal muscle comprises approximately 40% of the body's mass and has a fundamental role in glucose homeostasis as the major site of glucose uptake and utilization. To address the effects of IGF-1 on metabolism, we generated a mouse model with inducible skeletal muscle-specific igf1 gene deletion (MID). With adult onset deletion, MID mice showed no reduction in serum IGF-1 levels and in total growth – as illustrated by unaltered body weight, tibia and femoral length – compared to wild-type littermates. Despite no loss of skeletal muscle mass and function, the MID mice displayed abnormal glucose tolerance. Overnight fasting induced fasting hypoglycemia, but interestingly they displayed diminished glucose clearance after glucose injection. This was accompanied by decreased glycogen content within the muscle as indicated by Periodic Acid Staining (PAS) staining. Furthermore, the diminished glucose clearance ability was strongly correlated with the degree of deletion of the igf1 gene. Thus, skeletal muscle-specific IGF-1,via its autocrine/paracrine mode of action, impairs whole body glucose tolerance. Mechanistic target of rapamycin (mTOR) controls cell growth and cancer development by coordinating the cellular response to nutrients and growth factors. The tumor suppressor gene TMEM127 is an endosomal protein that impinges on the mTOR pathway by yet unknown mechanisms. Here we investigated the effects of Tmem127 loss in vivo by targeted deletion of Tmem127 gene in mice (KO). Normal chow diet fed KO mice have lower body weight, decreased fat mass and adipocyte size, low insulin, glucose and leptin levels, and tolerant glucose and insulin curves. Livers of these mice show modest upregulation of mTOR signaling compared to WT. When challenged with a high-fat diet (HFD), KO mice developed obesity and glucose intolerance. However, in contrast to WT mice, Tmem127KO remained insulin-tolerant, and paradoxically liver mTOR signaling was decreased relative to WT. Histologically, KO liver had reduced areas of fat deposition, while white adipocytes had significantly more multilocular vesicles. Transcriptional profiling of KO liver revealed a decrease in expression of genes involved in lipid synthesis and storage and upregulation of gluconeogenesis mediators, both in chow and HFD. Taken together, these results suggest that loss of Tmem127 protects against obesity-induced insulin resistance by an imbalance in the lipid/glucose synthesis pathways. We propose that the favorable insulin metabolism of the Tmem127KO mice may be associated with reduced mTOR signaling under HFD stress. The mechanisms underlying a switch in mTOR status are under investigation. Sarcopenia, a condition marked by loss of muscle mass, is becoming increasingly prevalent in aging populations resulting in greater likelihood of falls, immobility, and loss of independence. Strength training exercise is currently the only known intervention against sarcopenia, yet only 14% of individuals over the age of 65 routinely participate. Participation may increase by creating exercise modalities providing similar or improved benefit with less time commitment. Towards this goal we investigate the benefits and deeper mechanistic impacts of a novel high intensity interval training (HIIT) program administered to a 24-month-old male c57BL/6J mice (equivalent to a ~70-year-old human). Our exercise program features a mix of alternating fast walks and sprints over a 10-min period, a total of three times a week, with increasing difficulty over the 4-month experimental period. At the conclusion of the program, HIIT trained mice had dramatically improved scores for grip strength, treadmill endurance, uphill sprint endurance, and gait speed. Additionally, HIIT trained mice exhibited greater muscle cell cross sectional area and mitochondrial potential (biomass x activity), reduced cerebral oxidative stress, and potentially an increase in anti-aging serum protein Klotho. These data demonstrate dramatic benefits of HIIT in aged mice, achieved using a shorter session time and fewer days per week than commonly used in endurance training programs reported in the literature. Ultimately, benefits identified using this mouse exercise model hold promise in leading to the development of a human equivalent version that promotes successful aging in our growing elderly population. Advanced age is the greatest risk factor for virtually every chronic disease state. Mutations in the target of rapamycin (TOR) pathway increase lifespan in invertebrates, and inhibition of TOR by chronic rapamycin (RAPA) treatment extends lifespan in mammals. We previously provided evidence that RAPA attenuates disease progression in established animal models of neurological and cerebrovascular disease. Here we sought to determine whether RAPA delays functional neurological deficits associated with aging in rats. Using behavioral approaches as well as MRI- and PET-based functional imaging tools we demonstrated that RAPA treatment abolished profound deficits in cerebral blood flow (CBF), evoked functional hyperemia, and learning and memory and also restored adult-like brain glucose metabolism in very old (34-month-old) rats. Initial morphometric and biochemical studies suggest increased autophagy markers and enlarged synaptic boutons without loss of total synaptic area in old rats treated with RAPA as compared to controls. A link between increased autophagy and synaptic bouton size, associated with improved cognitive outcomes, suggests that specific synaptic remodeling may compensate for age-associated learning and memory dysfunction in very old rats. Thus, our studies indicate that RAPA, the only drug known to prolong lifespan in mammals, also protects against age-associated cognitive, brain hemodynamic and metabolic changes in very old rats. Taken together with prior evidence that RAPA blocks pathological and functional changes associated with Alzheimer's disease and atherosclerosis animal models, our data suggest that mTOR-driven brain vascular dysfunction associated with aging may be a critical target for therapeutic intervention. Rapamycin is an FDA approved drug indicated for the prophylaxis of organ rejection in renal transplantation. Because single therapy with rapamycin has not been associated with immunosuppression, rapamycin or rapalogs may have immediate potential for the treatment of age-associated neurological diseases that have vascular dysfunction as a common mechanism, including but not limited to Alzheimer's and vascular dementia. The RNA-debranching enzyme (Dbr1) plays a fundamental role in RNA homeostasis in eukaryotic cells. Splicing of pre-mRNA generates RNA lariats with an unusual 2′,5′-phosphodiester bond, and Dbr1 is the only enzyme known to debranch these RNA lariats. Small nucleolar RNAs and approximately one third of micro RNAs are derived from lariat RNA. A recent paper has shown that accumulation of RNA lariats in ▵dbr1 cells protects against TDP-43 toxicity in models of amyotrophic lateral sclerosis (ALS), suggesting that inhibitors of Dbr1 might provide a therapeutic benefit for ALS patients. We have recently determined the first X-ray crystal structure of Dbr1 from Entamoeba histolytica and analyzed the effect of different metal substitutions in the active site as well as the interaction of an inactive Dbr1 variant with a bona fide branched RNA substrate. Through collaboration with Dr. Masad Damha (McGill University), we have developed a fluorogenic probe of Dbr1 activity and performed a preliminary assessment of the enzymatic properties of Dbr1. Results from these studies indicate that large gaps exist in our understanding of this unique, medically important enzyme, therefore we performed a comprehensive analysis of the debranching process using the tools of structural biology and mechanistic enzymology. To test the hypothesis that modulation of Dbr1 activity can provide a therapeutic benefit to patients suffering from ALS or FTLD, we have screened a library of 30,000 small molecules and identified several promising lead compounds. To better understand the basic role of Dbr1 in normal biology, we have also analyzed the tissue distribution of human Dbr1 expression. Together, these results illuminate the role of Dbr1 in biology and allow us to test the ability of Dbr1 inhibitors to ameliorate symptoms of ALS and related diseases. Our lab has reported that dietary supplementation with rapamycin increased lifespan in UM-HET3 mice (1,2) consistently more in females than males. However, whether this sex-dependent increase in lifespan by rapamycin is accompanied by an improvement in motor performance with age in a sex-specific manner is unknown. We hypothesized that if rapamycin treatment slows aging it should also prevent or delay age-related motor deficits in UM-HET3 mice of each sex. Our results showed that age-related decline in coordinated running was greater in old female mice. Interestingly, rapamycin treatment attenuated this decline in motor coordination significantly more in females compared to males. In females, rapamycin reduced basal anxiety levels and depressive-like behavior. Age-related decline in locomotor activity in females was also prevented by rapamycin. Rearing was found to be reduced with age in both sexes. However, rapamycin prevented this decline significantly in females than males. Using an FTC-based proteomic approach, we measured the protein carbonyl content in different brain regions. Protein carbonyl content was significantly increased with age in striatum, midbrain, hippocampus, cortex, and cerebellum regions of both sexes. More protein carbonylation was observed in the detergent-soluble protein fraction as compared to cytosolic fraction with age. Rapamycin reduced protein carbonyl content in both fractions in each sex. Interestingly, we found more protein carbonylation with age in the insoluble protein fraction of the striatum region in females compared to males. The decline in locomotor function in aged females compared to males suggests a possible association between age-related motor deficits and increase in protein carbonylation. Effect of rapamycin on glial fibrillary acidic protein (GFAP) expression with age in brain was also evaluated. Calorie restriction is known to reduce the expression of GFAP in the striatum. To elucidate whether rapamycin treatment mimics calorie restriction, we measured GFAP expression in the striatum of both sexes. Rapamycin treatment increased the GFAP expression in old mice of both sexes suggesting that rapamycin does not mimic calorie restriction. Altogether, our findings reveal that the increase in lifespan resulting from rapamycin supplementation is accompanied by improvements in age-sensitive behavioral traits, reduced protein carbonylation, and increased astrocyte proliferation. Inhibition of mechanistic target of rapamycin (mTOR) signaling with rapamycin extends longevity and delays the onset of many aging phenotypes in mice; however, the translational potential of this drug and its analogs may be limited due its potential for adverse risks. One example is a significant increase in the risk for new-onset diabetes or impaired glucose metabolism associated with rapamycin that has been shown in rodent models and human clinical administration. Working with the NIA-funded Interventions Testing Program, we tested whether these metabolic defects could be prevented by co-treatment with metformin, the most-commonly administered and first-line drug of choice for type 2 diabetes. Beginning at 4 months of age, genetically heterogeneous HET3 mice were treated with encapsulated rapamycin, metformin, or both simultaneously, with all treatments incorporated into their diet. Metformin did not alter the effect of rapamycin on mTOR inhibition in vivo, but did independently activate AMPK signaling in both male and female mice. Over 9 months of treatment, rapamycin significantly reduced body weight gain in both males and females, with metformin co-treatment eliminating this weight reduction in males only. Glucose metabolism was impaired by rapamycin beginning as early as 1 month after treatment and continued throughout the duration of the study. Metformin did not alter this outcome in males, but females treated with both metformin and rapamycin were indistinguishable from controls showing a clear beneficial effect of metformin treatment. At least partly, these beneficial effects of metformin could be attributed to reduction in hepatic gluconeogenesis caused by this drug, though we also found that metformin altered circulating concentrations of adipokines such as leptin and adiponectin. The extension of lifespan caused by rapamycin has been somewhat paradoxical when viewed in light of its potential negative effects on metabolism. In ongoing studies, the Interventions Testing Program will assess the lifespan of HET3 mice co-treated with rapamycin and metformin and, combined with our findings, will soon test whether there is further extension of rapamycin-mediated longevity by alleviation of its metabolic defects. Aging and age-related diseases are responsible for the greater part of healthcare expenses in industrialized societies, posing an ever increasing challenge to public health as the mean age of the population increases. The FDA approved drug rapamycin increases lifespan and healthspan in rodents and improves age-related immune decline in humans; thus, rapamycin has promising translational potential as an age-delaying intervention. A partial dose-response profile has been obtained in UMHET3 mice, where it was observed that continuous feeding throughout life of 42 ppm encapsulated rapamycin (eRAPA) in the diet extends lifespan to a greater extent than 14 ppm or 4 ppm; however, the optimal dose, treatment regimen, maximum efficacy, and safety of rapamycin administration remain to be ascertained in the context of healthy aging. Here we report the effects of 3 month treatment regimens using significantly higher doses of rapamcyin than previously reported in middle-aged C57BL/6 mice. Striking effects on lifespan, healthspan, disease burden, and microbiome were observed and will be discussed. Compounds that improve mammalian lifespan are being identified by the NIA Interventions Testing Program. The target molecules of these compounds are of high interest for drug discovery because repertoires of compounds active against critical aging-driver targets could be identified that have improved pharmacokinetic profiles, tolerability, or lifespan- and healthspan-improving properties. We will discuss one such effort focused on the mechanistic target of rapamycin (mTOR). TOR controls organismal lifespan in mammals. Chronic inhibition of mTOR by rapamycin extends lifespan in mice by delaying aging and ameliorates modeled age-associated diseases including Alzheimer's. These data provide proof-of-concept that diseases of aging can be treated with compounds that target molecules involved in the control of aging and suggest that rapamycin may have immediate therapeutic potential. Potential side effects associated with rapamycin, however, could be a concern or preclude its use to treat age-associated disease in some patients. To identify small molecule compounds that (1) specifically target the nutrient-stimulated mTORC1 pathway and (2) are devoid of undesirable side effects we designed and performed high-throughput screening against nutrient-activated mTORC1. We identified four novel small molecule leads currently being optimized through medicinal chemistry that could be broadly applied to the treatment of mTORC1-driven age-associated diseases. Thus, our studies (1) demonstrate that the nutrient-stimulated mTORC1 can be specifically targeted; (2) establish a systematic search for drugs that have the potential to optimally delay age-associated disease by slowing aging; and (3) have the potential to lead to novel insights into the mechanisms that regulate the mTORC1 pathway and organismal aging. Autism Spectrum Disorders are persistent developmental disorders classified by the symptoms of social impairments and restrictive repetitive behaviors. Approximately 1 in 68 children have autism, showing up four times more often in males. There is presently no comprehensive treatment for all autism symptoms, but patients are most commonly given risperidone, an atypical antipsychotic to control impulsivity and aggression. Risperidone has tranquilizing properties, acting as an antagonist at serotonin and dopamine receptors. Risperidone's long-term use comes with serious side effects including increased appetite, obesity, and early onset of type II diabetes. We hypothesize that because chronic risperidone treatment can accelerate onset of metabolic syndrome, this drug might ironically blunt its own effectiveness by inducing elevated insulin levels that modify the expression and functions of dopamine transporters and receptors. We sought to identify a mouse model in which to test this. Black-and-tan Brachyury Tuffed mice, crossed with C57BL/6 mice yield F1 hybrid offspring that are socially impaired and glucose deficient. F1 mice have increased abdominal adiposity, and elevated insulin levels relative to either parent strain by 10 weeks of age. Their social and repetitive behavior was not improved by risperidone administered acutely. F1 mice were more impulsive than either parent strain in a social dominance test. [3H] WIN 35,428 binding to dopamine transporters was similar among strains, but dopamine uptake was significantly increased in BTBR mice. Dopamine transmission may be suppressed in the F1 mice, which may alter their responsiveness to risperidone. As evidence of this, marble burying in F1 mice was unaffected by an acute risperidone treatment, while the same dose of risperidone reduced this in C57BL/6 mice. Studies in F1 mice will establish whether chronic risperidone use may not only promote type II diabetes, but also hamper the functional efficacy of the drug.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=pmid________::f6fb7d940d33d9cfc138d2f0fea33d0f https://pubmed.ncbi.nlm.nih.gov/26725924 Zobrazit plný text záznamu