Expansion of T memory stem cells with superior anti-tumor immunity by Urolithin A-induced mitophagy.

T memory stem cells (TSCM) display increased self-renewal and prolonged survival capabilities, thus preventing T cell exhaustion and promoting effective anti-tumor T cell responses. TSCM cells can be expanded by Urolithin A (UA), which is produced by the commensal gut microbiome from foods rich in ellagitannins and is known to improve mitochondrial health. Oral UA administration to tumor-bearing mice conferred strong anti-tumor CD8+ T cell immunity, whereas ex vivo UA pre-treated T cells displayed improved anti-tumor function upon adoptive cell transfer. UA-induced TSCM formation depended on Pink1-mediated mitophagy triggering cytosolic release of the mitochondrial phosphatase Pgam5. Cytosolic Pgam5 dephosphorylated ß-catenin, which drove Wnt signaling and compensatory mitochondrial biogenesis. Collectively, we unravel a critical signaling pathway linking mitophagy to TSCM formation and suggest that the well-tolerated metabolic compound UA represents an attractive option to improve immune therapy.

Systems genetics of metabolism: the use of the BXD murine reference panel for multiscalar integration of traits.

Metabolic homeostasis is achieved by complex molecular and cellular networks that differ significantly among individuals and are difficult to model with genetically engineered lines of mice optimized to study single gene function. Here, we systematically acquired metabolic phenotypes by using the EUMODIC EMPReSS protocols across a large panel of isogenic but diverse strains of mice (BXD type) to study the genetic control of metabolism. We generated and analyzed 140 classical phenotypes and deposited these in an open-access web service for systems genetics (www.genenetwork.org). Heritability, influence of sex, and genetic modifiers of traits were examined singly and jointly by using quantitative-trait locus (QTL) and expression QTL-mapping methods. Traits and networks were linked to loci encompassing both known variants and novel candidate genes, including alkaline phosphatase (ALPL), here linked to hypophosphatasia. The assembled and curated phenotypes provide key resources and exemplars that can be used to dissect complex metabolic traits and disorders.

An evolutionarily conserved role for the aryl hydrocarbon receptor in the regulation of movement.

The BXD genetic reference population is a recombinant inbred panel descended from crosses between the C57BL/6 (B6) and DBA/2 (D2) strains of mice, which segregate for about 5 million sequence variants. Recently, some of these variants have been established with effects on general metabolic phenotypes such as glucose response and bone strength. Here we phenotype 43 BXD strains and observe they have large variation (-5-fold) in their spontaneous activity during waking hours. QTL analyses indicate that -40% of this variance is attributable to a narrow locus containing the aryl hydrocarbon receptor (Ahr), a basic helix-loop-helix transcription factor with well-established roles in development and xenobiotic metabolism. Strains with the D2 allele of Ahr have reduced gene expression compared to those with the B6 allele, and have significantly higher spontaneous activity. This effect was also observed in B6 mice with a congenic D2 Ahr interval, and in B6 mice with a humanized AHR allele which, like the D2 allele, is expressed much less and has less enzymatic activity than the B6 allele. Ahr is highly conserved in invertebrates, and strikingly inhibition of its orthologs in D. melanogaster and C. elegans (spineless and ahr-1) leads to marked increases in basal activity. In mammals, Ahr has numerous ligands, but most are either non-selective (e.g. resveratrol) or highly toxic (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)). Thus, we chose to examine a major environmental influence--long term feeding with high fat diet (HFD)--to see if the effects of Ahr are dependent on major metabolic differences. Interestingly, while HFD robustly halved movement across all strains, the QTL position and effects of Ahr remained unchanged, indicating that the effects are independent. The highly consistent effects of Ahr on movement indicate that changes in its constitutive activity have a role on spontaneous movement and may influence human behavior.

Urolithin A improves muscle strength, exercise performance, and biomarkers of mitochondrial health in a randomized trial in middle-aged adults.

Targeting mitophagy to activate the recycling of faulty mitochondria during aging is a strategy to mitigate muscle decline. We present results from a randomized, placebo-controlled trial in middle-aged adults where we administer a postbiotic compound Urolithin A (Mitopure), a known mitophagy activator, at two doses for 4 months (NCT03464500). The data show significant improvements in muscle strength (∼12%) with intake of Urolithin A. We observe clinically meaningful improvements with Urolithin A on aerobic endurance (peak oxygen oxygen consumption [VO2]) and physical performance (6 min walk test) but do not notice a significant improvement on peak power output (primary endpoint). Levels of plasma acylcarnitines and C-reactive proteins are significantly lower with Urolithin A, indicating higher mitochondrial efficiency and reduced inflammation. We also examine expression of proteins linked to mitophagy and mitochondrial metabolism in skeletal muscle and find a significant increase with Urolithin A administration. This study highlights the benefit of Urolithin A to improve muscle performance.

Urolithin A improves mitochondrial health, reduces cartilage degeneration, and alleviates pain in osteoarthritis.

Osteoarthritis (OA) is the most common age-related joint disorder with no effective therapy. According to the World Health Organization, OA affects over 500 million people and is characterized by degradation of cartilage and other joint tissues, severe pain, and impaired mobility. Mitochondrial dysfunction contributes to OA pathology. However, interventions to rescue mitochondrial defects in human OA are not available. Urolithin A (Mitopure) is a natural postbiotic compound that promotes mitophagy and mitochondrial function and beneficially impacts muscle health in preclinical models of aging and in elderly and middle-aged humans. Here, we showed that Urolithin A improved mitophagy and mitochondrial respiration in primary chondrocytes from joints of both healthy donors and OA patients. Furthermore, Urolithin A reduced disease progression in a mouse model of OA, decreasing cartilage degeneration, synovial inflammation, and pain. These improvements were associated with increased mitophagy and mitochondrial content, in joints of OA mice. These findings indicate that UA promotes joint mitochondrial health, alleviates OA pathology, and supports Urolithin A's potential to improve mobility with beneficial effects on structural damage in joints.

Direct supplementation with Urolithin A overcomes limitations of dietary exposure and gut microbiome variability in healthy adults to achieve consistent levels across the population.

BACKGROUND: Urolithin A (UA) is produced by gut microflora from foods rich in ellagitannins. UA has been shown to improve mitochondrial health preclinically and in humans. Not everyone has a microbiome capable of producing UA, making supplementation with UA an appealing strategy. OBJECTIVE: This is the first detailed investigation of the prevalence of UA producers in a healthy population and the ability of direct UA supplementation to overcome both microbiome and dietary variability. Dietary intake of a glass of pomegranate juice (PJ) was used to assess UA producer status (n = 100 participants) and to characterize differences in gut microbiome between UA producers from non-producers. METHODS: Subjects were randomized (1:1) to either PJ or a food product containing UA (500 mg). Prevalence of UA producers and non-producers were determined in the PJ group. Diet questionnaires and fecal samples were collected to compare differences between UA producers and non-producers along with plasma samples at different time points to assess levels of UA and its conjugates between the interventions. RESULTS: Only 12% of subjects had detectable levels of UA at baseline. Following PJ intake ~40% of the subjects converted significantly the precursor compounds into UA. UA producers were distinguished by a significantly higher gut microbiome diversity and ratio of Firmicutes to Bacteroides. Direct supplementation with UA significantly increased plasma levels and provided a >6-fold exposure to UA vs. PJ (p < 0.0001). CONCLUSIONS: Differences in gut microbiome and diet that dictate natural exposure to UA can be overcome via direct dietary UA supplementation.

Impact of the Natural Compound Urolithin A on Health, Disease, and Aging.

Urolithin A (UA) is a natural compound produced by gut bacteria from ingested ellagitannins (ETs) and ellagic acid (EA), complex polyphenols abundant in foods such as pomegranate, berries, and nuts. UA was discovered 40 years ago, but only recently has its impact on aging and disease been explored. UA enhances cellular health by increasing mitophagy and mitochondrial function and reducing detrimental inflammation. Several preclinical studies show how UA protects against aging and age-related conditions affecting muscle, brain, joints, and other organs. In humans, benefits of UA supplementation in the muscle are supported by recent clinical trials in elderly people. Here, we review the state of the art of UA's biology and its translational potential as a nutritional intervention in humans.

Urolithin A improves muscle function by inducing mitophagy in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy, and despite advances in genetic and pharmacological disease-modifying treatments, its management remains a major challenge. Mitochondrial dysfunction contributes to DMD, yet the mechanisms by which this occurs remain elusive. Our data in experimental models and patients with DMD show that reduced expression of genes involved in mitochondrial autophagy, or mitophagy, contributes to mitochondrial dysfunction. Mitophagy markers were reduced in skeletal muscle and in muscle stem cells (MuSCs) of a mouse model of DMD. Administration of the mitophagy activator urolithin A (UA) rescued mitophagy in DMD worms and mice and in primary myoblasts from patients with DMD, increased skeletal muscle respiratory capacity, and improved MuSCs' regenerative ability, resulting in the recovery of muscle function and increased survival in DMD mouse models. These data indicate that restoration of mitophagy alleviates symptoms of DMD and suggest that UA may have potential therapeutic applications for muscular dystrophies.

Publisher Correction: Mitochondrial function is impaired in the skeletal muscle of pre-frail elderly.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The mitophagy activator urolithin A is safe and induces a molecular signature of improved mitochondrial and cellular health in humans.

Urolithin A (UA) is a natural dietary, microflora-derived metabolite shown to stimulate mitophagy and improve muscle health in old animals and in preclinical models of aging1. Here, we report the results of a first-in-human clinical trial in which we administered UA, either as a single dose or as multiple doses over a 4-week period, to healthy, sedentary elderly individuals. We show that UA has a favourable safety profile (primary outcome). UA was bioavailable in plasma at all doses tested, and 4 weeks of treatment with UA at doses of 500 mg and 1,000 mg modulated plasma acylcarnitines and skeletal muscle mitochondrial gene expression in elderly individuals (secondary outcomes). These observed effects on mitochondrial biomarkers show that UA induces a molecular signature of improved mitochondrial and cellular health following regular oral consumption in humans.

Mitochondrial function is impaired in the skeletal muscle of pre-frail elderly.

Aging is accompanied by a gradual decline in both muscle mass and strength over time, which can eventually lead to pathologies, such as frailty and sarcopenia. While these two conditions are well characterized, further investigation of the early biological signs present in pre-frail elderly is still needed to help identify strategies for preventative therapeutic intervention. The goal of the present clinical study was to evaluate the level of mitochondrial (dys)function in a well-defined population of pre-frail elderly (>60 years of age). Pre-frail elderly were compared with an age-matched population of active elderly. Muscle mitochondrial function was assessed in vivo using phosphorus magnetic resonance spectroscopy (31P-MRS) and a comprehensive set of biological biomarkers were measured ex vivo in vastus lateralis muscle biopsies. In pre-frail subjects, phosphocreatine recovery was impaired and mitochondrial respiratory complex protein and activity levels were significantly lower when compared with active elderly. Analysis of microarray data showed that mitochondrial genes were also significantly down-regulated in muscle of pre-frail compared to active elderly. These results show that mitochondrial impairment is a hallmark of pre-frailty development and the onset of decline in muscle function in the elderly.

Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents.

The biological effects of urolithins remain poorly characterized, despite wide-spread human exposure via the dietary consumption of their metabolic precursors, the ellagitannins, which are found in the pomegranate fruit, as well as in nuts and berries. We identified urolithin A (UA) as a first-in-class natural compound that induces mitophagy both in vitro and in vivo following oral consumption. In C. elegans, UA prevented the accumulation of dysfunctional mitochondria with age and extended lifespan. Likewise, UA prolonged normal activity during aging in C. elegans, including mobility and pharyngeal pumping, while maintaining mitochondrial respiratory capacity. These effects translated to rodents, where UA improved exercise capacity in two different mouse models of age-related decline of muscle function, as well as in young rats. Our findings highlight the health benefits of urolithin A and its potential application in strategies to improve mitochondrial and muscle function.

A method to identify and validate mitochondrial modulators using mammalian cells and the worm C. elegans.

Mitochondria are semi-autonomous organelles regulated by a complex network of proteins that are vital for many cellular functions. Because mitochondrial modulators can impact many aspects of cellular homeostasis, their identification and validation has proven challenging. It requires the measurement of multiple parameters in parallel to understand the exact nature of the changes induced by such compounds. We developed a platform of assays scoring for mitochondrial function in two complementary models systems, mammalian cells and C. elegans. We first optimized cell culture conditions and established the mitochondrial signature of 1,200 FDA-approved drugs in liver cells. Using cell-based and C. elegans assays, we further defined the metabolic effects of two pharmacological classes that emerged from our hit list, i.e. imidazoles and statins. We found that these two drug classes affect respiration through different and cholesterol-independent mechanisms in both models. Our screening strategy enabled us to unequivocally identify compounds that have toxic or beneficial effects on mitochondrial activity. Furthermore, the cross-species approach provided novel mechanistic insight and allowed early validation of hits that act on mitochondrial function.

Pharmacological approaches to restore mitochondrial function.

Mitochondrial dysfunction is not only a hallmark of rare inherited mitochondrial disorders but also implicated in age-related diseases, including those that affect the metabolic and nervous system, such as type 2 diabetes and Parkinson's disease. Numerous pathways maintain and/or restore proper mitochondrial function, including mitochondrial biogenesis, mitochondrial dynamics, mitophagy and the mitochondrial unfolded protein response. New and powerful phenotypic assays in cell-based models as well as multicellular organisms have been developed to explore these different aspects of mitochondrial function. Modulating mitochondrial function has therefore emerged as an attractive therapeutic strategy for several diseases, which has spurred active drug discovery efforts in this area.

The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity.

As NAD(+) is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38-both NAD(+) consumers-increases NAD(+) bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD(+) precursor with the ability to increase NAD(+) levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD(+) levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function.

Murine gut microbiota is defined by host genetics and modulates variation of metabolic traits.

The gastrointestinal tract harbors a complex and diverse microbiota that has an important role in host metabolism. Microbial diversity is influenced by a combination of environmental and host genetic factors and is associated with several polygenic diseases. In this study we combined next-generation sequencing, genetic mapping, and a set of physiological traits of the BXD mouse population to explore genetic factors that explain differences in gut microbiota and its impact on metabolic traits. Molecular profiling of the gut microbiota revealed important quantitative differences in microbial composition among BXD strains. These differences in gut microbial composition are influenced by host-genetics, which is complex and involves many loci. Linkage analysis defined Quantitative Trait Loci (QTLs) restricted to a particular taxon, branch or that influenced the variation of taxa across phyla. Gene expression within the gastrointestinal tract and sequence analysis of the parental genomes in the QTL regions uncovered candidate genes with potential to alter gut immunological profiles and impact the balance between gut microbial communities. A QTL region on Chr 4 that overlaps several interferon genes modulates the population of Bacteroides, and potentially Bacteroidetes and Firmicutes-the predominant BXD gut phyla. Irak4, a signaling molecule in the Toll-like receptor pathways is a candidate for the QTL on Chr15 that modulates Rikenellaceae, whereas Tgfb3, a cytokine modulating the barrier function of the intestine and tolerance to commensal bacteria, overlaps a QTL on Chr 12 that influence Prevotellaceae. Relationships between gut microflora, morphological and metabolic traits were uncovered, some potentially a result of common genetic sources of variation.

The metabolic footprint of aging in mice.

Aging is characterized by a general decline in cellular function, which ultimately will affect whole body homeostasis. Although DNA damage and oxidative stress all contribute to aging, metabolic dysfunction is a common hallmark of aging at least in invertebrates. Since a comprehensive overview of metabolic changes in otherwise healthy aging mammals is lacking, we here compared metabolic parameters of young and 2 year old mice. We systemically integrated in vivo phenotyping with gene expression, biochemical analysis, and metabolomics, thereby identifying a distinguishing metabolic footprint of aging. Among the affected pathways in both liver and muscle we found glucose and fatty acid metabolism, and redox homeostasis. These alterations translated in decreased long chain acylcarnitines and increased free fatty acid levels and a marked reduction in various amino acids in the plasma of aged mice. As such, these metabolites serve as biomarkers for aging and healthspan.