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.

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.

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.

Safety assessment of Urolithin A, a metabolite produced by the human gut microbiota upon dietary intake of plant derived ellagitannins and ellagic acid.

Urolithins are metabolites produced in the gut following consumption of ellagitannins and ellagic acid rich foods such as pomegranates, nuts and certain berries. Urolithin A (UA) is one of the predominant isoforms of urolithins in humans and has demonstrated compelling biological activities, suggesting potential benefits of direct consumption of UA. However, an evaluation of the safety of direct administration of UA has not yet been published. The aim of this study was to investigate for the first time the genotoxicity, toxicokinetics, and repeated dose safety of orally administered synthetic UA in rats. The battery of genotoxicity assays demonstrated that UA is not genotoxic. The ADME study showed that glucuronidated and sulfonated forms of UA are the predominant metabolites following both oral and i.v. administration. The 28-day (0, 0.175, 1.75, and 5.0% UA mixed in diet) and 90-day studies (0, 1.25, 2.5, and 5.0% UA mixed in diet) showed no alterations in clinical parameters, blood chemistry, or hematology, and did not indicate any target organs, or any specific toxic mechanisms. The NOAEL was the highest dose tested, 5% UA by weight in the diet, or 3451 mg/kg bw/day in males and 3826 mg/kg bw/day in females in the 90-day oral study.

Pten loss in the bone marrow leads to G-CSF-mediated HSC mobilization.

The phosphatase and tumor suppressor PTEN inhibits the phosphoinositol-3-kinase (PI3K) signaling pathway and plays a key role in cell growth, proliferation, survival, and migration. Pten conditional deletion using MxCre or Scl-CreER(T) leads to splenomegaly and leukemia formation, which occurs after the relocation of normal hematopoietic stem cells (HSCs) from the bone marrow to the spleen. Unexpectedly, dormant HSCs in the bone marrow do not enter the cell cycle upon Pten loss, they do not lose self-renewal activity, and they are not exhausted. Instead, Pten deficiency causes an up-regulation of the PI3K pathway in myeloid cells, but not in HSCs. Strikingly, myeloid cells secrete high levels of G-CSF upon Pten loss, leading to the mobilization of HSCs from the bone marrow and accumulation in the spleen. After deletion of Pten in mice lacking G-CSF, the splenomegaly, myeloproliferative disease, and splenic HSC accumulation are rescued. Our data show that although PTEN has little if any role in normal HSCs, it is essential to prevent overt G-CSF production by myeloid and stromal cells which otherwise causes HSCs to relocate to the spleen followed by lethal leukemia initiation.

IFNalpha activates dormant haematopoietic stem cells in vivo.

Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs) with long-term self-renewal capacity. After injury these cells are induced to proliferate to quickly re-establish homeostasis. The signalling molecules promoting the exit of HSCs out of the dormant stage remain largely unknown. Here we show that in response to treatment of mice with interferon-alpha (IFNalpha), HSCs efficiently exit G(0) and enter an active cell cycle. HSCs respond to IFNalpha treatment by the increased phosphorylation of STAT1 and PKB/Akt (also known as AKT1), the expression of IFNalpha target genes, and the upregulation of stem cell antigen-1 (Sca-1, also known as LY6A). HSCs lacking the IFNalpha/beta receptor (IFNAR), STAT1 (ref. 3) or Sca-1 (ref. 4) are insensitive to IFNalpha stimulation, demonstrating that STAT1 and Sca-1 mediate IFNalpha-induced HSC proliferation. Although dormant HSCs are resistant to the anti-proliferative chemotherapeutic agent 5-fluoro-uracil, HSCs pre-treated (primed) with IFNalpha and thus induced to proliferate are efficiently eliminated by 5-fluoro-uracil exposure in vivo. Conversely, HSCs chronically activated by IFNalpha are functionally compromised and are rapidly out-competed by non-activatable Ifnar(-/-) cells in competitive repopulation assays. Whereas chronic activation of the IFNalpha pathway in HSCs impairs their function, acute IFNalpha treatment promotes the proliferation of dormant HSCs in vivo. These data may help to clarify the so far unexplained clinical effects of IFNalpha on leukaemic cells, and raise the possibility for new applications of type I interferons to target cancer stem cells.

Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.

Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity.

Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Although conditional deletion of N-myc in the bone marrow does not affect hematopoiesis, combined deficiency of c-Myc and N-Myc (dKO) results in pancytopenia and rapid lethality. Interestingly, proliferation of HSCs depends on both myc genes during homeostasis, but is c-Myc/N-Myc independent during bone marrow repair after injury. Strikingly, while most dKO hematopoietic cells undergo apoptosis, only self-renewing HSCs accumulate the cytotoxic molecule Granzyme B, normally employed by the innate immune system, thereby revealing an unexpected mechanism of stem cell apoptosis. Collectively, Myc activity (c-Myc and N-Myc) controls crucial aspects of HSC function including proliferation, differentiation, and survival.

C-Myc and its target FoxM1 are critical downstream effectors of constitutive androstane receptor (CAR) mediated direct liver hyperplasia.

UNLABELLED: In the adult liver, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of the constitutive androstane receptor (CAR, NR1I3), produces rapid hepatomegaly in the absence of injury. In this study, we identify c-Myc as a gene induced by CAR and demonstrate that TCPOBOP-induced proliferation of hepatocytes depends on c-Myc function. Moreover, the TCPOBOP-induced cell cycle program (Cdc2, cyclins, MCM proteins, Cdc20, and genes implicated in the spindle assembly checkpoint) is severely impaired in c-Myc mutant livers. Strikingly, many of these genes overlap with a program controlled by the forkhead transcription factor FoxM1, known to control progression through S-phase and mitosis. Indeed, FoxM1 is also induced by TCPOBOP. Moreover, we show that c-Myc binds to the FoxM1 promoter in a TCPOBOP-dependent manner, suggesting a CAR --> c-Myc --> FoxM1 pathway downstream of TCPOBOP. CONCLUSION: Collectively, this study identifies c-Myc and FoxM1 mediated proliferative programs as key mediators of TCPOBOP-CAR induced direct liver hyperplasia.

Dormant and self-renewing hematopoietic stem cells and their niches.

In the mouse, over the last 20 years, a set of cell-surface markers and activities have been identified, enabling the isolation of bone marrow (BM) populations highly enriched in hematopoietic stem cells (HSCs). These HSCs have the ability to generate multiple lineages and are capable of long-term self-renewal activity such that they are able to reconstitute and maintain a functional hematopoietic system after transplantation into lethally irradiated recipients. Using single-cell reconstitution assays, various marker combinations can be used to achieve a functional HSC purity of almost 50%. Here we have used the differential expression of six of these markers (Sca1, c-Kit, CD135, CD48, CD150, and CD34) on lineage-depleted BM to refine cell hierarchies within the HSC population. At the top of the hierarchy, we propose a dormant HSC population (Lin(-)Sca1(+)c-Kit(+) CD48(-)CD150(+)CD34(-)) that gives rise to an active self-renewing CD34(+) HSC population. HSC dormancy, as well as the balance between self-renewal and differentiation activity, is at least, in part, controlled by the stem cell niches individual HSCs are attached to. Here we review the current knowledge about HSC niches and propose that dormant HSCs are located in niches at the endosteum, whereas activated HSCs are in close contact to sinusoids of the BM microvasculature.

Inducing tolerance to a soluble foreign antigen by encapsulated cell transplants.

The immune response to soluble antigens constitutes a current clinical problem impeding the development of protein therapeutics. We have developed an encapsulated-cell delivery system, which, transiently combined with an anti-CD154 antibody treatment, allows for the suppression of this immune response and the establishment of long-term secretion of a foreign antigen, human erythropoietin (huEPO). The chronic presence of antigen appears to be required to maintain this tolerance, as a 21-day gap in the exposure to huEPO is sufficient to restore the ability of mice to mount an antibody response. In contrast, chronic huEPO expression maintains tolerance even in the absence of further anti-CD154 treatment. These results suggest that a soluble antigenic protein can be continuously released, without inducing an antibody response, using encapsulated allogeneic cells. The temporary anti-CD154 treatment induces immune unresponsiveness to the delivered antigen, while the immunoprotected cell implant allows for chronic antigen release, favoring the establishment of tolerance.

Lentiviral-mediated RNA interference.

RNA interference (RNAi) is a form of posttranscriptional gene silencing mediated by short double-stranded RNA, known as small interfering RNA (siRNA). These siRNAs are capable of binding to a specific mRNA sequence and causing its degradation. The recent demonstration of a plasmid vector that directs siRNA synthesis in mammalian cells prompted us to examine the ability of lentiviral vectors to encode siRNA as a means of providing long-term gene silencing in mammalian cells. The RNA-polymerase III dependent promoter (H1-RNA promoter) was inserted in the lentiviral genome to drive the expression of a small hairpin RNA (shRNA) against enhanced green fluorescent protein (EGFP). This construct successfully silenced EGFP expression in two stable cell lines expressing this protein, as analyzed by fluorescence microscopy, flow cytometry, and Western blotting. The silencing, which is dose dependent, occurs as early as 72 hr postinfection and persists for at least 25 days postinfection. The ability of lentiviruses encoding siRNA to silence genes specifically makes it possible to take full advantage of the possibilities offered by the lentiviral vector and provides a powerful tool for gene therapy and gene function studies.