Tissue-specific effects of exercise as NAD+-boosting strategy: Current knowledge and future perspectives

Nicotinamide adenine dinucleotide (NAD⁺) is an evolutionarily highly conserved coenzyme with multi-faceted cell functions, including energy metabolism, molecular signaling processes, epigenetic regulation, and DNA repair. Since the discovery that lower NAD⁺ levels are a shared characteristic of various diseases and aging per se, several NAD⁺-boosting strategies have emerged. Other than pharmacological and nutritional approaches, exercise is thought to restore NAD⁺ homeostasis through metabolic adaption to chronically recurring states of increased energy demand. In this review we discuss the impact of acute exercise and exercise training on tissue-specific NAD⁺ metabolism of rodents and humans to highlight the potential value as NAD⁺-boosting strategy. By interconnecting results from different investigations, we aim to draw attention to tissue-specific alterations in NAD⁺ metabolism and the associated implications for whole-body NAD⁺ homeostasis. Acute exercise led to profound alterations of intracellular NAD⁺ metabolism in various investigations, with the magnitude and direction of changes being strongly dependent on the applied exercise modality, cell type, and investigated animal model or human population. Exercise training elevated NAD⁺ levels and NAD⁺ metabolism enzymes in various tissues. Based on these results, we discuss molecular mechanisms that might connect acute exercise-induced disruptions of NAD⁺/NADH homeostasis to chronic exercise adaptions in NAD⁺ metabolism. Taking this hypothesis-driven approach, we hope to inspire future research on the molecular mechanisms of exercise as NAD⁺-modifying lifestyle intervention, thereby elucidating the potential therapeutic value in NAD⁺-related pathologies.     

An NAD+ biosynthetic pathway enzyme functions cell non‐autonomously in C. elegans development

Background: Disruption of cellular metabolite levels can adversely impact development. Specifically, loss‐of‐function of the C. elegans NAD⁺ salvage biosynthesis gene PNC‐1 results in an array of developmental phenotypes. Intriguingly, PNC‐1 and its functional equivalent in vertebrates are secreted, but the contributions of the extracellular enzymes are poorly understood. We sought to study the tissue‐specific requirements for PNC‐1 expression and to examine the role of the secreted isoform. Results: A thorough analysis of PNC‐1 expression did not detect expression in tissues that require PNC‐1 function. Limited expression of both the secreted and intracellular PNC‐1 isoforms provided function at a distance from the tissues with phenotypes. We also find that the secreted isoform contributes to in vivo PNC‐1 activity. Furthermore, uv1 cell survival has the most stringent requirements in terms of PNC‐1 expression pattern or level. Conclusions: Using careful promoter analysis and a restricted expression approach, we have shown that both the secreted and the intracellular PNC‐1 isoforms function cell non‐autonomously, and that the PNC‐1a isoform is functionally relevant in vivo. Our work suggests a model where PNC‐1 function is provided cell non‐autonomously by a mix of intra and extracellular activity, most likely requiring NAD⁺ salvage metabolite transport between tissues. Developmental Dynamics 243:965–976, 2014. © 2014 Wiley Periodicals, Inc.     

High-affinity Na-dependent dicarboxylate cotransporter promotes cellular senescence by inhibiting SIRT1

High-affinity Na⁺-dependent dicarboxylate cotransporter (NaDC3) can transport Krebs cycle intermediates into cells. Our previous study has shown that NaDC3 promotes cellular senescence, but its mechanism is not clear. It is known that when the concentration of intermediates in Krebs cycle is increased, NAD⁺/NADH ratio will be decreased. NAD⁺-dependent histone deacetylase sirtuin1 (SIRT1) prolongs mammalian cellular lifespan. Therefore, we propose that NaDC3 accelerates cellular aging by inhibiting SIRT1. After NaDC3 was overexpressed in two human embryo lung fibroblastic cell lines, WI38 and MRC-5, we found that the cells displayed aging-related phenotypes in advance. Meanwhile, the level of SIRT1 activity was down-regulated. In WI38/hNaDC3 cells treated with the activators of SIRT1, aging-related phenotypes induced by NaDC3 were obviously improved. The NAD⁺/NADH ratio in WI38/hNaDC3 cells was also decreased. Further study found that enhanced intracellular NAD⁺ level could attenuate the aging phenotypes induced by NaDC3. Thus, NaDC3 promotes cellular senescence probably by inhibiting NAD⁺-dependent SIRT1.     

Nicotinamide phosphoribosyltransferase delays cellular senescence by upregulating SIRT1 activity and antioxidant gene expression in mouse cells

Senescent cells accumulate in tissues of aged animals and deteriorate tissue functions. The elimination of senescent cells from aged mice not only attenuates progression of already established age‐related disorders, but also extends median lifespan. Nicotinamide phosphoribosyltransferase (NAMPT), the rate‐limiting enzyme in mammalian NAD⁺ salvage pathway, has shown a protective effect on cellular senescence of human primary cells. However, it still remains unclear how NAMPT has a protective impact on aging in vitro and in vivo. In this study, we found that primary mouse embryonic fibroblast (MEF) cells undergo progressive decline of NAMPT and NAD⁺ contents during serial passaging before becoming senescent. Furthermore, we showed that constitutive Nampt over‐expression increases cellular NAD⁺ content and delays cellular senescence of MEF cells in vitro. We further found that constitutive Nampt over‐expression increases SIRT1 activity, increases the expression of antioxidant genes, superoxide dismutase 2 and catalase and promotes resistance against oxidative stress. These findings suggest that Nampt over‐expression in MEF cells delays cellular senescence by the mitigation of oxidative stress via the upregulation of superoxide dismutase 2 and catalase gene expressions by SIRT1 activation.     

Over‐expression of Nicotinamide phosphoribosyltransferase in mouse cells confers protective effect against oxidative and ER stress‐induced premature senescence

A main feature of aged organisms is the accumulation of senescent cells. Accumulated senescent cells, especially stress‐induced premature senescent cells, in aged organisms lead to the decline of the regenerative potential and function of tissues. We recently reported that the over‐expression of NAMPT, which is the rate‐limiting enzyme in mammalian NAD⁺ salvage pathway, delays replicative senescence in vitro. However, whether Nampt‐overexpressing cells are tolerant of stress‐induced premature senescence remains unknown. Here, we show that primary mouse embryonic fibroblasts derived from Nampt‐overexpressing transgenic mice (Nampt Tg‐MEF cells) possess resistance against stress‐induced premature senescence in vitro. We found that higher oxidative or endoplasmic reticulum (ER) stress is required to induce premature senescence in Nampt Tg‐MEF cells compared to wild‐type cells. Moreover, we found that Nampt Tg‐MEF cells show acute expression of unfolded protein response (UPR)‐related genes, which in turn would have helped to restore proteostasis and avoid cellular senescence. Our results demonstrate that NAMPT/NAD⁺ axis functions to protect cells not only from replicative senescence, but also from stress‐induced premature senescence in vitro. We anticipate that in vivo activation of NAMPT activity or increment of NAD⁺ would protect tissues from the accumulation of premature senescent cells, thereby maintaining healthy aging.     

Detection of cerebral NAD+ by in vivo 1H NMR spectroscopy

Nicotinamide adenine dinucleotide (NAD⁺) plays a central role in cellular metabolism both as a coenzyme for electron‐transfer enzymes as well as a substrate for a wide range of metabolic pathways. In the current study NAD⁺ was detected on rat brain in vivo at 11.7T by 3D localized ¹H MRS of the NAD⁺ nicotinamide protons in the 8.7–9.5 ppm spectral region. Avoiding water perturbation was critical to the detection of NAD⁺ as strong, possibly indirect cross‐relaxation between NAD⁺ and water would lead to a several‐fold reduction of the NAD⁺ intensity in the presence of water suppression. Water perturbation was minimized through the use of localization by adiabatic spin‐echo refocusing (LASER) in combination with frequency‐selective excitation. The NAD⁺ concentration in the rat cerebral cortex was determined at 296 ± 28 μm , which is in good agreement with recently published ³¹P NMR‐based results as well as results from brain extracts in vitro (355 ± 34 μm ). The T₁ relaxation time constants of the NAD⁺ nicotinamide protons as measured by inversion recovery were 280 ± 65 and 1136 ± 122 ms in the absence and presence of water inversion, respectively. This confirms the strong interaction between NAD⁺ nicotinamide and water protons as observed during water suppression. The T₂ relaxation time constants of the NAD⁺ nicotinamide protons were determined at 60 ± 13 ms after confounding effects of scalar coupling evolution were taken into account. The simplicity of the MR sequence together with the robustness of NAD⁺ signal detection and quantification makes the presented method a convenient choice for studies on NAD⁺ metabolism and function. As the method does not critically rely on magnetic field homogeneity and spectral resolution it should find immediate applications in rodents and humans even at lower magnetic fields. Copyright © 2014 John Wiley & Sons, Ltd.     

Sirtuins in mitochondrial stress: Indispensable helpers behind the scenes

Mitochondria play an essential part in guaranteeing normal cellular physiological functions through providing ATP and participating in diverse processes and signaling pathways. Recently, more and more studies have revealed the vital roles of mitochondria in coping with stressors in the aging process, metabolic disturbances and neurological disorders. Mitochondrial stress responses, including the mitochondrial unfolded protein response (UPR^mt), antioxidant defense, mitochondrial fission, mitochondrial fusion and mitophagy, are induced to maintain cellular integrity in response to stress. The sirtuin family, a group of NAD⁺-dependent deacetylases, has been the focus of much attention in recent years for their multiple regulatory functions, especially in aging and metabolism. Recent reports validated the significant link between mitochondrial stress responses and the sirtuin family, which may help to elucidate the pathogenesis and therapies for diseases such as Alzheimer’s disease or Parkinson’s disease. This review will summarize recent related studies and illuminate the interplay between sirtuins and mitochondrial stress.     

SIRT6/NF-κB signaling axis in ginsenoside Rg1-delayed hematopoietic stem/progenitor cell senescence

Objective: To investigate the role of SIRT6/NF-κB signaling axis in ginsenoside Rg1-delayed hematopoietic stem/progenitor cell senescence and to provide theoretical and experimental evidence for delaying HSC/HPC senescence pathway. Methods: After the separation and purification by immunomagnetic sorting, Sca-1⁺HSC/HPC was divided into: normal control group; aging group; positive control group; Rg1 delaying group and Rg1 treatment group. Senescence-associated β-galactosidase (SA-β-Gal) staining, flow cytometry analysis of cell cycle and hematopoietic progenitor cells mixed colony (CFU-Mix) culture were performed to determine the delaying or curing roles of Rg1 in Sca-1⁺HSC/HPC senescence. Quantitative PCR and Western blotting were used to detect the mRNA and protein expression of senescence regulatory molecules, such as SIRT6 and NF-κB. Results: Compared with the aging group, the positive rate of SA-β-gal staining cells and the proportion of cells in G1 phase decreased; the number of CFU-Mix increased; mRNA and protein expression of SIRT6 increased; mRNA and protein expression of NF-κB was down-regulated in Rg1 delaying and treatment groups; the changes of the indicators in Rg1 delaying group were more significant than those in Rg1 treatment group. Conclusion: Rg1 may fight against Sca-1⁺HSC/HPC senescence induced by t-BHP through regulating SIRT6-NF-κB signaling pathway.     

Over-expression of nicotinamide phosphoribosyltransferase in ovarian cancers

Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide (NAD⁺) synthesis and is required for cell growth, survival, DNA replication and repair, and angiogenesis. Nampt expression increases gene expression which promotes cell survival and increases SirT1 activity, promoting angiogenesis, and it is increased in several human malignancies. Recently, others have shown that ovarian serous adenocarcinomas (OSAs) express high levels of activated Stat3. Since Nampt expression is increased by Stat3, we hypothesized that Nampt protein might be highly expressed in OSAs. Using tissue microarray (TMA) and the avidin-biotin complex immunohistochemical technique we examined Nampt expression in 47 samples of benign ovarian tissue and 49 samples of ovarian serous adenoacarcinomas. Our data show that Nampt protein expression is significantly increased in OSAs as compared to benign ovarian tissue (0.49+/-0.12 benign vs. 4.78+/-0.46 malignant; +/-standard error of the mean). This is the first report demonstrating Nampt overexpression in OSA, which may shed light on the pathogenesis of OSA. Further studies of the role of Nampt overexpresion in OSA may shed light on the prognosis and clinical course of OSA. Last, since an effective pharmacologic Nampt inhibitor is currently in clinical use, further studies of Nampt overexpression in OSA may be used in selecting patients for Nampt inhibitor therapy.     

Comparison between developmental and senescent changes in enzyme activities linked to energy metabolism in rat heart

The developmental and senescent patterns of a number of heart enzyme activities linked to energy metabolism have been studied in rats aged between 4 days and 21 months. A morphometric study of mitochondrial volume fractions and numbers has been also carried out. Developmental changes result in a rise of most mitochondrial enzymes (NADP⁺-isocitrate dehydrogenase, malic enzyme, succinate dehydrogenase, citrate synthase) and mitochondrial volume fractions. Exceptions are NAD⁺-isocitarte dehydrogenase, which declines from 4 days onwards, and NAD⁺-malate dehydrogenase, which declines and then rises over the same period. Senescent changes follow two different trends. While pyruvate kinase and those mitochondrial enzymes lying between citrate formation and isocitrate oxidation (citrate synthase, NADP⁺- and NAD⁺-isocitrate dehydrogenases) decline to some degree, mitochondrial succinate dehydrogenase and NAD⁺-malate dehydrogenase activities increase over the same period. This could point towards a partial impairment of Krebs cycle function, and a reduced energy-producing capacity in the aged rat heart.     

In vivo 31P MRS assessment of intracellular NAD metabolites and NAD+/NADH redox state in human brain at 4 T

NAD⁺ and NADH play key roles in cellular respiration. Intracellular redox state defined by the NAD⁺/NADH ratio (RX) reflects the cellular metabolic and physiopathological status. By taking advantage of high/ultrahigh magnetic field strengths, we have recently established a novel in vivo ³¹P MRS‐based NAD assay for noninvasive and quantitative measurements of intracellular NAD concentrations and redox state in animal and human brains at 16.4 T, 9.4 T and 7 T. To explore its potential for clinical application, in this study we investigated the feasibility of assessing the NAD metabolism and redox state in human brain at a lower field of 4 T by incorporating the ¹H‐decoupling technique with the in vivo ³¹P NAD assay. The use of ¹H decoupling significantly narrowed the linewidths of NAD and α‐ATP resonances, resulting in higher sensitivity and better spectral resolution as compared with the ¹H‐coupled ³¹P spectrum. These improvements made it possible to reliably quantify cerebral NAD concentrations and RX, consistent with previously reported results obtained from similar age human subjects at 7 T. In summary, this work demonstrates the capability and utility of the ¹H‐decoupled ³¹P MRS‐based NAD assay at lower field strength; thus, it opens new opportunities for studying intracellular NAD metabolism and redox state in human brain at clinical settings. This conclusion is supported by the simulation results, indicating that similar performance and reliability as observed at 4T can be achieved at 3 T with the same signal‐to‐noise ratio. Copyright © 2016 John Wiley & Sons, Ltd.     

Acetate metabolism and aging: An emerging connection

Sirtuins are NAD⁺-dependent protein deacetylases that regulate gene silencing, energy metabolism and aging from bacteria to mammals. SIRT3, a mammalian mitochondrial sirtuin, deacetylates acetyl-CoA synthetase (AceCS2) in the mitochondria. AceCS2 is conserved from bacteria to humans, catalyzes the conversion of acetate to acetyl-CoA and enables peripheral tissues to utilize acetate during fasting conditions. Here, we review the regulation of acetate metabolism by sirtuins, the remarkable conservation of this metabolic regulatory pathway and its emerging role in the regulation of aging and longevity.     

CHIP E3 ligase regulates mammalian senescence by modulating the levels of oxidized proteins

Senescence can be induced by various stressors including oxidative stress. It has been reported that CHIP (C-terminus of Hsp70-interacting protein) ligase is induced during senescence while CHIP^−/− mice exhibit accelerated aging. Here, we explore the effects of modulating CHIP expression on mammalian senescence. We demonstrate that CHIP silencing induces premature senescence that is accompanied by elevated levels of oxidized proteins. On the contrary, ectopic expression of CHIP leads to oxidized proteins levels reduction. Moreover, we reveal that CHIP^−/− mouse fibroblasts have an impaired ubiquitin proteasome system. Taken together, we propose that CHIP influences cellular senescence by modulating the oxidative load.     

Divergent mechanisms of metabolic dysfunction drive fibroblast and T-cell senescence

The impact of cellular senescence during ageing is well established, however senescence is now recognised to play a role in a variety of age related and metabolic diseases, such as cancer, autoimmune and cardiovascular diseases. It is therefore crucial to gain a better understanding of the mechanisms that control cellular senescence. In recent years our understanding of the intimate relationship between cell metabolism, cell signalling and cellular senescence has greatly improved. In this review we discuss the differing roles of glucose and protein metabolism in both senescent fibroblast and CD8⁺ T-cells, and explore the impact cellular metabolism has on the senescence-associated secretory phenotype (SASP) of these cell types.     

BIOCHEMISTRY, LOCALIZATION AND FUNCTIONAL ROLES OF ECTO-NUCLEOTIDASES IN THE NERVOUS SYSTEM

Nucleotides such as ATP, ADP, UTP or the diadenosine polyphosphates and possibly even NAD⁺ are extracellular signaling substances in the brain and in other tissues. Enzymes located on the cell surface catalyze the hydrolysis of these compounds and thus limit their spatio-temporal activity. As a final hydrolysis product they generate the nucleoside and phosphate. The paper discusses the biochemical properties, cellular localization and functional properties of surface-located enzymes that hydrolyse nucleotides released from nervous tissue. This is preceded by a brief discussion of nucleotide receptors, cellular storage and mechanisms of nucleotide release. In nervous tissue nucleoside 5′-triphosphates are hydrolysed by ecto-ATP-diphosphohydrolase and possibly in addition also by ecto-nucleoside triphosphatase and ecto-nucleoside diphosphatase. The molecular identity of the ATP-diphosphohydrolase has now been revealed. The hydrolysis of nucleoside 5′-monophosphates is catalysed by 5′-nucleotidase whose biochemical properties and molecular structure have been studied in detail. Little is known about the molecular properties of the diadenosine polyphosphatases. Surface located enzymes for the extracellular hydrolysis of NAD⁺ and also ecto-protein kinases are discussed briefly. The cellular localization of the ecto-nucleotidases is only partly defined. Whereas in adult mammalian brain activity for hydrolysis of ATP and ADP may be associated with nerve cells or glial cells 5′-nucleotidase appears to have a preferential glial allocation in the adult mammal. The extracellular hydrolysis of the nucleotides is of functional importance not only during synaptic transmission where it functions in signal elimination. It plays a crucial role also for the survival and differentiation of neural cells in vitro and presumably during neuronal development in vivo. Copyright © 1996 Elsevier Science Ltd.     

Neurotransmitter signalling via NMDA receptors leads to decreased T helper type 1‐like and enhanced T helper type 2‐like immune balance in humans

Given the pivotal roles that CD4⁺ T cell imbalance plays in human immune disorders, much interest centres on better understanding influences that regulate human helper T‐cell subset dominance in vivo. Here, using primary CD4⁺ T cells and short‐term T helper type 1 (Th1) and Th2‐like lines, we investigated roles and mechanisms by which neurotransmitter receptors may influence human type 1 versus type 2 immunity. We hypothesized that N‐methyl‐d ‐aspartate receptors (NMDA‐R), which play key roles in memory and learning, can also regulate human CD4⁺ T cell function through induction of excitotoxicity. Fresh primary CD4⁺ T cells from healthy donors express functional NMDA‐R that are strongly up‐regulated upon T cell receptor (TCR) mediated activation. Synthetic and physiological NMDA‐R agonists elicited Ca²⁺ flux and led to marked inhibition of type 1 but not type 2 or interleukin‐10 cytokine responses. Among CD4⁺ lines, NMDA and quinolinic acid preferentially reduced cytokine production, Ca²⁺ flux, proliferation and survival of Th1‐like cells through increased induction of cell death whereas Th2‐like cells were largely spared. Collectively, the findings demonstrate that (i) NMDA‐R is rapidly up‐regulated upon CD4⁺ T cell activation in humans and (ii) Th1 versus Th2 cell functions such as proliferation, cytokine production and cell survival are differentially affected by NMDA‐R agonists. Differential cytokine production and proliferative capacity of Th1 versus Th2 cells is attributable in part to increased physiological cell death among fully committed Th1 versus Th2 cells, leading to increased Th2‐like dominance. Hence, excitotoxicity, beyond its roles in neuronal plasticity, may contribute to ongoing modulation of human T cell responses.     

Profiling molecular targets of TGF-beta1 in prostate fibroblast-to-myofibroblast transdifferentiation

The development of age-related proliferative disorders of the prostate gland is supported by transdifferentiation and cellular senescence processes in the stroma. Both processes are involved in remodeling of stromal tissue, as observed in benign prostatic hyperplasia (BPH), and in “reactive stroma” adjacent to prostate cancer (PCa). It has been assumed that TGF-β1 plays a key role in the aging prostate by inducing premature senescence and favoring myofibroblast differentiation. Therefore, we evaluated the stromal cell phenotypes of human primary adult prostatic fibroblasts (n = 3) and the molecular and cellular mechanisms of growth arrest after treatment with TGF-β1 and of in vitro cellular senescence. Microarray analysis, quantitative PCR, immunofluorescence and western blot revealed that cellular senescence and transdifferentiation of fibroblasts have distinct underlying mechanisms, pathways and gene and protein expression profiles in human PrSCs. In clear contrast to senescent cells, TGF-β1-treated cells morphologically transdifferentiated into myofibroblasts with dense cytoskeletal fibers and increased expression of smooth muscle cell α-actin, calponin and tenascin. TGF-β1 induced neither expression of senescence-associated markers nor genes involved in terminal growth arrest, such as senescence-associated beta-galactosidase and cyclin-dependent kinase (cdk) inhibitors p16^Ink4A and p21^Cip1 but increased p15^Ink4B protein expression. Differentiation inhibitor (Id-1) protein level down-regulation was observed under both conditions. Genes specifically up-regulated by transdifferentiation but not by cellular senescence of PrSCs were metalloproteinase 1 tissue inhibitor (Timp1), transgelin (Tagln), gamma 2 actin (Actg2), plasminogen activator inhibitor 1 (Serpinel), insulin-like growth factor binding protein 3 (Igfbp3), parathyroid hormone-like hormone (Pthlp), Tgfb-1, four and a half LIM domains 2 (Fhl-2), hydrogen peroxide-inducible clone 5 (Hic5) and cartilage oligomeric matrix protein (Comp). Other genes, such as Cdc28 protein kinase 1 (Cks1b), v-myb myeloblastosis viral oncogene homolog (MybL2), pyruvate kinase, muscle 2 (Pkm2) and Forkhead box M1 (FoxM1), were down-regulated only upon TGF-β1 treatment but not by cellular senescence. Pyruvate dehydrogenase kinase 3 (Pdk3) and connective tissue growth factor (Ctgf) were up-regulated and hyaluronan synthase 3 (Has3) down-regulated under both conditions. Moreover, GageC1, a prostate/testis-specific protein overexpressed in symptomatic BPH and PCa was induced in transdifferentiated stromal cells. Genes such as GageC1 could be promising targets for therapeutic inhibitors of stromal tissue remodeling and progression of BPH and PCa.     

Regulators of Glucose Metabolism in CD4+ and CD8+ T Cells

Much like cancer cells, activated T cells undergo various metabolic changes that allow them to grow and proliferate rapidly. By adopting aerobic glycolysis upon activation, T cells effectively prioritize efficiency in biosynthesis over energy generation. There are distinct differences in the way CD4⁺ and CD8⁺ T cells process activation signals. CD8⁺ effector T cells are less dependent on Glut1 and oxygen levels compared to their CD4⁺ counterparts. Similarly the downstream signaling by TCR also differs in both effector T cell types. Recent studies have explored PI3K/Akt, mTORC, HIF1α, p70S6K and Bcl-6 signaling in depth providing definition of the crucial roles of these regulators in glucose metabolism. These new insights may allow improved therapeutic manipulation against inflammatory conditions that are associated with dysfunctional T-cell metabolism such as autoimmune disorders, metabolic syndrome, HIV, and cancers.     

Defective induction of the proteasome associated with T‐cell receptor signaling underlies T‐cell senescence

The proteasome degradation machinery is essential for a variety of cellular processes including senescence and T‐cell immunity. Decreased proteasome activity is associated with the aging process; however, the regulation of the proteasome in CD4⁺ T cells in relation to aging is unclear. Here, we show that defects in the induction of the proteasome in CD4⁺ T cells upon T‐cell receptor (TCR) stimulation underlie T‐cell senescence. Proteasome dysfunction promotes senescence‐associated phenotypes, including defective proliferation, cytokine production and increased levels of PD‐1⁺ CD44^High CD4⁺ T cells. Proteasome induction by TCR signaling via MEK‐, IKK‐ and calcineurin‐dependent pathways is attenuated with age and decreased in PD‐1⁺ CD44^High CD4⁺ T cells, the proportion of which increases with age. Our results indicate that defective induction of the proteasome is a hallmark of CD4⁺ T‐cell senescence.