Mitophagy Activation by Urolithin A to Target Muscle Aging.

The age-related loss of skeletal muscle function starts from midlife and if left unaddressed can lead to an impaired quality of life. A growing body of evidence indicates that mitochondrial dysfunction is causally involved with muscle aging. Muscles are tissues with high metabolic requirements, and contain rich mitochondria supply to support their continual energy needs. Cellular mitochondrial health is maintained by expansing of the mitochondrial pool though mitochondrial biogenesis, by preserving the natural mitochondrial dynamic process, via fusion and fission, and by ensuring the removal of damaged mitochondria through mitophagy. During aging, mitophagy levels decline and negatively impact skeletal muscle performance. Nutritional and pharmacological approaches have been proposed to manage the decline in muscle function due to impaired mitochondria bioenergetics. The natural postbiotic Urolithin A has been shown to promote mitophagy, mitochondrial function and improved muscle function across species in different experimental models and across multiple clinical studies. In this review, we explore the biology of Urolithin A and the clinical evidence of its impact on promoting healthy skeletal muscles during age-associated muscle decline.

Mitophagy in human health, ageing and disease.

Maintaining optimal mitochondrial function is a feature of health. Mitophagy removes and recycles damaged mitochondria and regulates the biogenesis of new, fully functional ones preserving healthy mitochondrial functions and activities. Preclinical and clinical studies have shown that impaired mitophagy negatively affects cellular health and contributes to age-related chronic diseases. Strategies to boost mitophagy have been successfully tested in model organisms, and, recently, some have been translated into clinics. In this Review, we describe the basic mechanisms of mitophagy and how mitophagy can be assessed in human blood, the immune system and tissues, including muscle, brain and liver. We outline mitophagy's role in specific diseases and describe mitophagy-activating approaches successfully tested in humans, including exercise and nutritional and pharmacological interventions. We describe how mitophagy is connected to other features of ageing through general mechanisms such as inflammation and oxidative stress and forecast how strengthening research on mitophagy and mitophagy interventions may strongly support human health.

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Scopo di questa trattazione è quello di fare una disamina conoscitiva dei personaggi, dei fatti e dei tempi in cui la chirurgia di questo ultimo secolo si è sviluppata ed ha acquisito la dignità di essere considerata "scienza" fino a raggiungere quella bellezza esecutiva che si riscontra solo nell'arte. Maestri del bisturi dunque e del sapere, non più praticanti come erano nei secoli dell'alto Medioevo. Infatti la tecnica e la tecnologia Chirurgica di oggi con le loro grandi conquiste hanno improntato, o meglio rivoluzionato, tutta la storia di questa disciplina entrando alla metà del XX secolo come ausilio del chirurgo e ed oggi diventate protagoniste assolute della Chirurgia stessa. Ed i Chirurghi? Da grandi interpreti "solisti" sono diventati "dipendenti" dalle tecnologie sempre più imperanti e co-gestori della disciplina nella sua complessità.

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.

Tetracycline-induced mitohormesis mediates disease tolerance against influenza.

Mitohormesis defines the increase in fitness mediated by adaptive responses to mild mitochondrial stress. Tetracyclines inhibit not only bacterial but also mitochondrial translation, thus imposing a low level of mitochondrial stress on eukaryotic cells. We demonstrate in cell and germ-free mouse models that tetracyclines induce a mild adaptive mitochondrial stress response (MSR), involving both the ATF4-mediated integrative stress response and type I interferon (IFN) signaling. To overcome the interferences of tetracyclines with the host microbiome, we identify tetracycline derivatives that have minimal antimicrobial activity, yet retain full capacity to induce the MSR, such as the lead compound, 9-tert-butyl doxycycline (9-TB). The MSR induced by doxycycline (Dox) and 9-TB improves survival and disease tolerance against lethal influenza virus (IFV) infection when given preventively. 9-TB, unlike Dox, did not affect the gut microbiome and also showed encouraging results against IFV when given in a therapeutic setting. Tolerance to IFV infection is associated with the induction of genes involved in lung epithelial cell and cilia function, and with downregulation of inflammatory and immune gene sets in lungs, liver, and kidneys. Mitohormesis induced by non-antimicrobial tetracyclines and the ensuing IFN response may dampen excessive inflammation and tissue damage during viral infections, opening innovative therapeutic avenues.

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.

Effect of Urolithin A Supplementation on Muscle Endurance and Mitochondrial Health in Older Adults: A Randomized Clinical Trial.

Importance: Aging is associated with a decline in mitochondrial function and reduced exercise capacity. Urolithin A is a natural gut microbiome-derived food metabolite that has been shown to stimulate mitophagy and improve muscle function in older animals and to induce mitochondrial gene expression in older humans. Objective: To investigate whether oral administration of urolithin A improved the 6-minute walk distance, muscle endurance in hand and leg muscles, and biomarkers associated with mitochondrial and cellular health. Design, Setting, and Participants: This double-blind, placebo-controlled randomized clinical trial in adults aged 65 to 90 years was conducted at a medical center and a cancer research center in Seattle, Washington, from March 1, 2018, to July 30, 2020. Muscle fatigue tests and plasma analysis of biomarkers were assessed at baseline, 2 months, and 4 months. Six-minute walk distance and maximal ATP production were assessed using magnetic resonance spectroscopy at baseline and at the end of study at 4 months. The analysis used an intention-to-treat approach. Interventions: Participants were randomized to receive daily oral supplementation with either 1000 mg urolithin A or placebo for 4 months. Main Outcomes and Measures: The primary end point was change from baseline in the 6-minute walk distance and change from baseline to 4 months in maximal ATP production in the hand skeletal muscle. The secondary end points were change in muscle endurance of 2 skeletal muscles (tibialis anterior [TA] in the leg and first dorsal interosseus [FDI] in the hand). Cellular health biomarkers were investigated via plasma metabolomics. Adverse events were recorded and compared between the 2 groups during the intervention period. Results: A total of 66 participants were randomized to either the urolithin A (n = 33) or the placebo (n = 33) intervention group. These participants had a mean (SD) age of 71.7 (4.94) years, were predominantly women (50 [75.8%]), and were all White individuals. Urolithin A, compared with placebo, significantly improved muscle endurance (ie, increase in the number of muscle contractions until fatigue from baseline) in the FDI and TA at 2 months (urolithin A: FDI, 95.3 [115.5] and TA, 41.4 [65.5]; placebo: FDI, 11.6 [147.4] and TA, 5.7 [127.1]). Plasma levels of several acylcarnitines, ceramides, and C-reactive protein were decreased by urolithin A, compared with placebo, at 4 months (baseline vs 4 mo: urolithin A, 2.14 [2.15] vs 2.07 [1.46]; placebo, 2.17 [2.52] vs 2.65 [1.86]). The mean (SD) increase from baseline in the 6-minute walk distance was 60.8 (67.2) m in the urolithin A group and 42.5 (73.3) m in the placebo group. The mean (SD) change from baseline to 4 months in maximal ATP production in the FDI was 0.07 (0.23) mM/s in the urolithin A group and 0.06 (0.20) mM/s in the placebo group; for the TA, it was -0.03 (0.10) mM/s in the urolithin A group and 0.03 (0.10) mM/s in the placebo group. These results showed no significant improvement with urolithin A supplementation compared with placebo. No statistical differences in adverse events were observed between the 2 groups. Conclusions and Relevance: This randomized clinical trial found that urolithin A supplementation was safe and well tolerated in the assessed population. Although the improvements in the 6-minute walk distance and maximal ATP production in the hand muscle were not significant in the urolithin A group vs the placebo group, long-term urolithin A supplementation was beneficial for muscle endurance and plasma biomarkers, suggesting that urolithin A may counteract age-associated muscle decline; however, future work is needed to confirm this finding. Trial Registration: ClinicalTrials.gov Identifier: NCT03283462.

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.

Sphingolipids accumulate in aged muscle, and their reduction counteracts sarcopenia.

Age-related muscle dysfunction and sarcopenia are major causes of physical incapacitation in older adults and currently lack viable treatment strategies. Here we find that sphingolipids accumulate in mouse skeletal muscle upon aging and that both genetic and pharmacological inhibition of sphingolipid synthesis prevent age-related decline in muscle mass while enhancing strength and exercise capacity. Inhibition of sphingolipid synthesis confers increased myogenic potential and promotes protein synthesis. Within the sphingolipid pathway, we show that accumulation of dihydroceramides is the culprit disturbing myofibrillar homeostasis. The relevance of sphingolipid pathways in human aging is demonstrated in two cohorts, the UK Biobank and Helsinki Birth Cohort Study in which gene expression-reducing variants of SPTLC1 and DEGS1 are associated with improved and reduced fitness of older individuals, respectively. These findings identify sphingolipid synthesis inhibition as an attractive therapeutic strategy for age-related sarcopenia and co-occurring pathologies.

SMA-miRs (miR-181a-5p, -324-5p, and -451a) are overexpressed in spinal muscular atrophy skeletal muscle and serum samples.

Background: Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the degeneration of the second motor neuron. The phenotype ranges from very severe to very mild forms. All patients have the homozygous loss of the SMN1 gene and a variable number of SMN2 (generally 2-4 copies), inversely related to the severity. The amazing results of the available treatments have made compelling the need of prognostic biomarkers to predict the progression trajectories of patients. Besides the SMN2 products, few other biomarkers have been evaluated so far, including some miRs. Methods: We performed whole miRNome analysis of muscle samples of patients and controls (14 biopsies and 9 cultures). The levels of muscle differentially expressed miRs were evaluated in serum samples (51 patients and 37 controls) and integrated with SMN2 copies, SMN2 full-length transcript levels in blood and age (SMA-score). Results: Over 100 miRs were differentially expressed in SMA muscle; 3 of them (hsa-miR-181a-5p, -324-5p, -451a; SMA-miRs) were significantly upregulated in the serum of patients. The severity predicted by the SMA-score was related to that of the clinical classification at a correlation coefficient of 0.87 (p<10-5). Conclusions: miRNome analyses suggest the primary involvement of skeletal muscle in SMA pathogenesis. The SMA-miRs are likely actively released in the blood flow; their function and target cells require to be elucidated. The accuracy of the SMA-score needs to be verified in replicative studies: if confirmed, its use could be crucial for the routine prognostic assessment, also in presymptomatic patients. Funding: Telethon Italia (grant #GGP12116).

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.

The TRAPP complex mediates secretion arrest induced by stress granule assembly.

The TRAnsport Protein Particle (TRAPP) complex controls multiple membrane trafficking steps and is strategically positioned to mediate cell adaptation to diverse environmental conditions, including acute stress. We have identified the TRAPP complex as a component of a branch of the integrated stress response that impinges on the early secretory pathway. The TRAPP complex associates with and drives the recruitment of the COPII coat to stress granules (SGs) leading to vesiculation of the Golgi complex and arrest of ER export. The relocation of the TRAPP complex and COPII to SGs only occurs in cycling cells and is CDK1/2-dependent, being driven by the interaction of TRAPP with hnRNPK, a CDK substrate that associates with SGs when phosphorylated. In addition, CDK1/2 inhibition impairs TRAPP complex/COPII relocation to SGs while stabilizing them at ER exit sites. Importantly, the TRAPP complex controls the maturation of SGs. SGs that assemble in TRAPP-depleted cells are smaller and are no longer able to recruit RACK1 and Raptor, two TRAPP-interactive signaling proteins, sensitizing cells to stress-induced apoptosis.

The RNA-Binding Protein PUM2 Impairs Mitochondrial Dynamics and Mitophagy During Aging.

Little information is available about how post-transcriptional mechanisms regulate the aging process. Here, we show that the RNA-binding protein Pumilio2 (PUM2), which is a translation repressor, is induced upon aging and acts as a negative regulator of lifespan and mitochondrial homeostasis. Multi-omics and cross-species analyses of PUM2 function show that it inhibits the translation of the mRNA encoding for the mitochondrial fission factor (Mff), thereby impairing mitochondrial fission and mitophagy. This mechanism is conserved in C. elegans by the PUM2 ortholog PUF-8. puf-8 knock-down in old nematodes and Pum2 CRISPR/Cas9-mediated knockout in the muscles of elderly mice enhances mitochondrial fission and mitophagy in both models, hence improving mitochondrial quality control and tissue homeostasis. Our data reveal how a PUM2-mediated layer of post-transcriptional regulation links altered Mff translation to mitochondrial dynamics and mitophagy, thereby mediating age-related mitochondrial dysfunctions.

Mitogen-activated kinase kinase kinase 1 inhibits hedgehog signaling and medulloblastoma growth through GLI1 phosphorylation.

The aberrant activation of hedgehog (HH) signaling is a leading cause of the development of medulloblastoma, a pediatric tumor of the cerebellum. The FDA­approved HH inhibitor, Vismodegib, which targets the transmembrane transducer SMO, has shown limited efficacy in patients with medulloblastoma, due to compensatory mechanisms that maintain an active HH­GLI signaling status. Thus, the identification of novel actionable mechanisms, directly affecting the activity of the HH­regulated GLI transcription factors is an important goal for these malignancies. In this study, using gene expression and reporter assays, combined with biochemical and cellular analyses, we demonstrate that mitogen­activated kinase kinase kinase 1 (MEKK1), the most upstream kinase of the mitogen­activated protein kinase (MAPK) phosphorylation modules, suppresses HH signaling by associating and phosphorylating GLI1, the most potent HH­regulated transcription factor. Phosphorylation occurred at multiple residues in the C­terminal region of GLI1 and was followed by an increased association with the cytoplasmic proteins 14­3­3. Of note, the enforced expression of MEKK1 or the exposure of medulloblastoma cells to the MEKK1 activator, Nocodazole, resulted in a marked inhibitory effect on GLI1 activity and tumor cell proliferation and viability. Taken together, the results of this study shed light on a novel regulatory mechanism of HH signaling, with potentially relevant implications in cancer therapy.

Enhancing mitochondrial proteostasis reduces amyloid-ß proteotoxicity.

Alzheimer's disease is a common and devastating disease characterized by aggregation of the amyloid-ß peptide. However, we know relatively little about the underlying molecular mechanisms or how to treat patients with Alzheimer's disease. Here we provide bioinformatic and experimental evidence of a conserved mitochondrial stress response signature present in diseases involving amyloid-ß proteotoxicity in human, mouse and Caenorhabditis elegans that involves the mitochondrial unfolded protein response and mitophagy pathways. Using a worm model of amyloid-ß proteotoxicity, GMC101, we recapitulated mitochondrial features and confirmed that the induction of this mitochondrial stress response was essential for the maintenance of mitochondrial proteostasis and health. Notably, increasing mitochondrial proteostasis by pharmacologically and genetically targeting mitochondrial translation and mitophagy increases the fitness and lifespan of GMC101 worms and reduces amyloid aggregation in cells, worms and in transgenic mouse models of Alzheimer's disease. Our data support the relevance of enhancing mitochondrial proteostasis to delay amyloid-ß proteotoxic diseases, such as Alzheimer's disease.

Cytosolic Proteostasis Networks of the Mitochondrial Stress Response.

Mitochondrial stress requires timely intervention to prevent mitochondrial and cellular dysfunction. Re-establishing the correct protein homeostasis is crucial for coping with mitochondrial stress and maintaining cellular homeostasis. The best-characterized adaptive pathways for mitochondrial stress involve a signal originating from stressed mitochondria that triggers a nuclear response. However, recent findings have shown that mitochondrial stress also affects a complex network of protein homeostasis pathways in the cytosol. We review how mitochondrial dysregulation affects cytosolic proteostasis by regulating the quantity and quality of protein synthesis, protein stability, and protein degradation, leading to an integrated regulation of cellular metabolism and proliferation. This mitochondria to cytosol network extends the current model of the mitochondrial stress response, with potential applications in the treatment of mitochondrial disease.

Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals.

Mitochondrial stress activates a mitonuclear response to safeguard and repair mitochondrial function and to adapt cellular metabolism to stress. Using a multiomics approach in mammalian cells treated with four types of mitochondrial stressors, we identify activating transcription factor 4 (ATF4) as the main regulator of the stress response. Surprisingly, canonical mitochondrial unfolded protein response genes mediated by ATF5 are not activated. Instead, ATF4 activates the expression of cytoprotective genes, which reprogram cellular metabolism through activation of the integrated stress response (ISR). Mitochondrial stress promotes a local proteostatic response by reducing mitochondrial ribosomal proteins, inhibiting mitochondrial translation, and coupling the activation of the ISR with the attenuation of mitochondrial function. Through a trans-expression quantitative trait locus analysis, we provide genetic evidence supporting a role for Fh1 in the control of Atf4 expression in mammals. Using gene expression data from mice and humans with mitochondrial diseases, we show that the ATF4 pathway is activated in vivo upon mitochondrial stress. Our data illustrate the value of a multiomics approach to characterize complex cellular networks and provide a versatile resource to identify new regulators of mitochondrial-related diseases.

ZeGlobalTox: An Innovative Approach to Address Organ Drug Toxicity Using Zebrafish.

Toxicity is one of the major attrition causes during the drug development process. In that line, cardio-, neuro-, and hepatotoxicities are among the main reasons behind the retirement of drugs in clinical phases and post market withdrawal. Zebrafish exploitation in high-throughput drug screening is becoming an important tool to assess the toxicity and efficacy of novel drugs. This animal model has, from early developmental stages, fully functional organs from a physiological point of view. Thus, drug-induced organ-toxicity can be detected in larval stages, allowing a high predictive power on possible human drug-induced liabilities. Hence, zebrafish can bridge the gap between preclinical in vitro safety assays and rodent models in a fast and cost-effective manner. ZeGlobalTox is an innovative assay that sequentially integrates in vivo cardio-, neuro-, and hepatotoxicity assessment in the same animal, thus impacting strongly in the 3Rs principles. It Reduces, by up to a third, the number of animals required to assess toxicity in those organs. It Refines the drug toxicity evaluation through novel physiological parameters. Finally, it might allow the Replacement of classical species, such as rodents and larger mammals, thanks to its high predictivity (Specificity: 89%, Sensitivity: 68% and Accuracy: 78%).