Duchenne muscular dystrophy (DMD) is one of the most frequent and severe childhood muscle diseases. Its pathophysiology is multifaceted and still incompletely understood, but we and others have previously shown that oxidative stress plays an important role. In particular, we have demonstrated that inhibition of mitochondrial monoamine oxidases could improve some functional and biohumoral markers of the pathology. In the present study we report the use of dystrophic mdx mice to evaluate the efficacy of a dual monoamine oxidase B (MAO-B)/semicarbazide-sensitive amine oxidase (SSAO) inhibitor, PXS-5131, in reducing inflammation and fibrosis and improving muscle function. We found that a one-month treatment starting at three months of age was able to decrease reactive oxygen species (ROS) production, fibrosis, and inflammatory infiltrate in the tibialis anterior (TA) and diaphragm muscles. Importantly, we also observed a marked improvement in the capacity of the gastrocnemius muscle to maintain its force when challenged with eccentric contractions. Upon performing a bulk RNA-seq analysis, PXS-5131 treatment affected the expression of genes involved in inflammatory processes and tissue remodeling. We also studied the effect of prolonged treatment in older dystrophic mice, and found that a three-month administration of PXS-5131 was able to greatly reduce the progression of fibrosis not only in the diaphragm but also in the heart. Taken together, these results suggest that PXS-5131 is an effective inhibitor of fibrosis and inflammation in dystrophic muscles, a finding that could open a new therapeutic avenue for DMD patients.

Background: Cardiomyopathy is a major cause of death for Duchenne muscular dystrophy (DMD) patients. Current treatments cannot prevent cardiac tissue remodeling. Oxidative stress, inflammation and fibrosis are early events in DMD, preceding heart dysfunction. Monoamine Oxidase B (MAOB), which forms H 2 O 2 by oxidizing amines, is overactivated in inflammatory conditions and overcomes cell antioxidant defenses, thus altering redox homeostasis and eliciting harmful effects. We showed that targeting MAOB with inhibitors (iMAOB) improves skeletal muscle function in dystrophic mice by lowering oxidative stress, and reduces inflammation in murine models of sepsis and arthritis by dampening NF-kB, a redox-sensitive transcription factor. Aim: We explore the therapeutic potential of iMAOB to alleviate cardiomyopathy using dystrophin-deficient mdx mice. We hypothesize that iMAOB treatment could dampen oxidative stress, inflammation and fibrosis in dystrophic mdx hearts by modulating the phenotype of cells that are crucial in cardiac remodeling. Methods: Three-month-old mdx mice, that already show signs of fibrosis but no cardiac dysfunction, were orally treated with iMAOB or vehicle for one month (n≥ 6). Heart ventricular mononucleated cells were obtained by enzymatic digestion. Myeloid, endothelial and fibroblast cells were isolated by cell sorting by FACS and analysed by RT-PCR. Oxidative stress, inflammation and fibrosis were measured in whole tissue by immunohistochemistry. Results: The expression of proinflammatory and profibrotic genes was markedly increased in myeloid [Interleukin (Il)-1b, Il-6, Tgf-b and Spp1, the gene coding for osteopontin], endothelial [Il-1b, Il-6, mmp2 and nos3] and cardiac fibroblast cells [Tgf-b, Spp1, Timp1 and Col1] isolated from mdx hearts, as compared to wild type mice. All these genes were significantly dampened by iMAOB treatment. In parallel, once again iMAOB treatment blunted the increase in oxidative stress, inflammation and fibrosis observed in mdx cardiac sections. Of notice, the differences were not linked to changes in the percentage of the various cell types, as they were unmodified amongst wild type, mdx and iMAOB-treated mdx hearts. Conclusions: We show that iMAOB can positively affect the phenotype of cells that are important in cardiac tissue remodeling. Our data suggest that iMAOB can be a viable therapeutic tool in DMD cardiomyopathy. As iMAOB are already in clinical use, such approach could be easily translated to patients.

Objective Calcium pyrophosphate (CPP) crystal deposition in the joints is associated with a heterogeneous set of debilitating syndromes characterized by inflammation and pain, for which no effective therapies are currently available. Because we found that the mitochondrial enzyme monoamine oxidase B (MAO‐B) plays a fundamental role in promoting inflammatory pathways, this study aims at assessing the efficacy of two clinical‐grade inhibitors (iMAO‐Bs) in preclinical models of this disease to pave the way for a novel treatment. Methods We tested our hypothesis in two murine models of CPP‐induced arthritis, by measuring cytokine and chemokine levels, along with immune cell recruitment. iMAO‐Bs (rasagiline and safinamide) were administered either before or after crystal injection. To elucidate the molecular mechanism, we challenged in vitro primed macrophages with CPP crystals and assessed the impact of iMAO‐Bs in dampening proinflammatory cytokines and in preserving mitochondrial function. Results Both in preventive and therapeutic in vivo protocols, iMAO‐Bs blunted the release of proinflammatory cytokines (interleukin [IL]‐6 and IL1‐β) and chemokines (CXCL10, CXCL1, CCL2 and CCL5) (n > 6 mice/group). Importantly, they also significantly reduced ankle swelling (50.3% vs 17.1%; P < 0.001 and 23.1%; P = 0.005 for rasagiline and safinamide, respectively). Mechanistically, iMAO‐Bs dampened the burst of reactive oxygen species and the mitochondrial dysfunction triggered by CPP crystals in isolated macrophages. Moreover, iMAO‐Bs blunted cytokine secretion and NLRP3 inflammasome activation through inhibition of the NF‐κB and STAT3 pathways. Conclusion iMAO‐Bs dampen inflammation in murine models of crystal‐induced arthropathy, thereby uncovering MAO‐B as a promising target to treat these diseases. image

Macrophages are essential players for the host response against pathogens, regulation of inflammation and tissue regeneration. The wide range of macrophage functions rely on their heterogeneity and plasticity that enable a dynamic adaptation of their responses according to the surrounding environmental cues. Recent studies suggest that metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the metabolic pathways orchestrating macrophage activation are still under scrutiny. Optic atrophy 1 (OPA1) is a mitochondria-shaping protein controlling mitochondrial fusion, cristae biogenesis and respiration; clear evidence shows that the lack or dysfunctional activity of this protein triggers the accumulation of metabolic intermediates of the TCA cycle. In this study, we show that OPA1 has a crucial role in macrophage activation. Selective Opa1 deletion in myeloid cells impairs M1-macrophage commitment. Mechanistically, Opa1 deletion leads to TCA cycle metabolite accumulation and defective NF-κB signaling activation. In an in vivo model of muscle regeneration upon injury, Opa1 knockout macrophages persist within the damaged tissue, leading to excess collagen deposition and impairment in muscle regeneration. Collectively, our data indicate that OPA1 is a key metabolic driver of macrophage functions.

Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS-low, glutaminolysis-high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N-acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL-10, enforces GS expression in macrophages. In turn, GS-high macrophages acquire M2-like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2-like macrophage phenotype, IL-10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti-inflammatory, protumoral function of NAA. This study reveals a crosstalk between ovarian cancer cells and tumor associated macrophages. Glutamine addicted cancer cells release the signaling metabolite N-acetylaspartate (NAA), which in turn polarizes macrophages towards a GS-high, M2-like state. This study reveals a crosstalk between ovarian cancer cells and tumor associated macrophages. Glutamine addicted cancer cells release the signaling metabolite N-acetylaspartate (NAA), which in turn polarizes macrophages towards a GS-high, M2-like state.

Macrophages are immune cells that are important for the development of the defensive front line of the innate immune system. Following signal recognition, macrophages undergo activation toward specific functional states, consisting not only in the acquisition of specific features but also of peculiar metabolic programs associated with each function. For these reasons, macrophages are often isolated from mice to perform cellular assays to study the mechanisms mediating immune cell activation. This requires expensive and time-consuming breeding and housing of mice strains. To overcome this issue, we analyzed an in-house J2-generated immortalized macrophage cell line from BMDMs, both from a functional and metabolic point of view. By assaying the intracellular and extracellular metabolism coupled with the phenotypic features of immortalized versus primary BMDMs, we concluded that classically and alternatively immortalized macrophages display similar phenotypical, metabolic and functional features compared to primary cells polarized in the same way. Our study validates the use of this immortalized cell line as a suitable model with which to evaluate in vitro how perturbations can influence the phenotypical and functional features of murine macrophages.

Reactive oxygen species (ROS) are fundamental for macrophages to eliminate invasive microorganisms. However, as observed in nonphagocytic cells, ROS play essential roles in processes that are different from pathogen killing, as signal transduction, differentiation, and gene expression. The different outcomes of these events are likely to depend on the specific subcellular site of ROS formation, as well as the duration and extent of ROS production. While excessive accumulation of ROS has long been appreciated for its detrimental effects, there is now a deeper understanding of their roles as signaling molecules. This could explain the failure of the "all or none" pharmacologic approach with global antioxidants to treat several diseases. NADPH oxidase is the first source of ROS that has been identified in macrophages. However, growing evidence highlights mitochondria as a crucial site of ROS formation in these cells, mainly due to electron leakage of the respiratory chain or to enzymes, such as monoamine oxidases. Their role in redox signaling, together with their exact site of formation is only partially elucidated. Hence, it is essential to identify the specific intracellular sources of ROS and how they influence cellular processes in both physiological and pathological conditions to develop therapies targeting oxidative signaling networks. In this review, we will focus on the different sites of ROS formation in macrophages and how they impact on metabolic processes and inflammatory signaling, highlighting the role of mitochondrial as compared to non-mitochondrial ROS sources.

From advances in the knowledge of the immune system, it is emerging that the specialized functions displayed by macrophages during the course of an immune response are supported by specific and dynamically-connected metabolic programs. The study of immunometabolism is demonstrating that metabolic adaptations play a critical role in modulating inflammation and, conversely, inflammation deeply influences the acquisition of specific metabolic settings.This strict connection has been proven to be crucial for the execution of defined immune functional programs and it is now under investigation with respect to several human disorders, such as diabetes, sepsis, cancer, and autoimmunity. The abnormal remodelling of the metabolic pathways in macrophages is now emerging as both marker of disease and potential target of therapeutic intervention. By focusing on key pathological conditions, namely obesity and diabetes, rheumatoid arthritis, atherosclerosis and cancer, we will review the metabolic targets suitable for therapeutic intervention in macrophages. In addition, we will discuss the major obstacles and challenges related to the development of therapeutic strategies for a pharmacological targeting of macrophage’s metabolism.

Duchenne muscular dystrophy (DMD) is one of the most severe forms of inherited muscular dystrophies. The disease is caused by the lack of dystrophin, a structurally essential protein; hence, a definitive cure would necessarily have to pass through some form of gene and/or cell therapy. Cell- and genetic-based therapeutics for DMD have been explored since the 1990s; recently, two of the latter have been approved for clinical use, but their efficacy is still very low. In parallel, there have been great ongoing efforts aimed at targeting the downstream pathogenic effects of dystrophin deficiency using classical pharmacological approaches, with synthetic or biological molecules. However, as it is always the case with rare diseases, R&D costs for new drugs can represent a major hurdle for researchers and patients alike. This problem can be greatly alleviated by experimenting the use of molecules that had originally been developed for different conditions, a process known as drug repurposing or drug repositioning. In this review, we will describe the state of the art of such an approach for DMD, both in the context of clinical trials and pre-clinical studies.

Coenzyme Q (CoQ), a redox-active lipid, is comprised of a quinone group and a polyisoprenoid tail. It is an electron carrier in the mitochondrial respiratory chain, a cofactor of other mitochondrial dehydrogenases, and an essential antioxidant. CoQ requires a large set of enzymes for its biosynthesis; mutations in genes encoding these proteins cause primary CoQ deficiency, a clinically and genetically heterogeneous group of diseases. Patients with CoQ deficiency often respond to oral CoQ10supplementation. Treatment is however problematic because of the low bioavailability of CoQ10and the poor tissue delivery. In recent years, bypass therapy using analogues of the precursor of the aromatic ring of CoQ has been proposed as a promising alternative. We have previously shown using a yeast model that vanillic acid (VA) can bypass mutations ofCOQ6, a monooxygenase required for the hydroxylation of the C5 carbon of the ring. In this work, we have generated a human cell line lacking functionalCOQ6using CRISPR/Cas9 technology. We show that these cells cannot synthesize CoQ and display severe ATP deficiency. Treatment with VA can recover CoQ biosynthesis and ATP production. Moreover, these cells display increased ROS production, which is only partially corrected by exogenous CoQ, while VA restores ROS to normal levels. Furthermore, we show that these cells accumulate 3-decaprenyl-1,4-benzoquinone, suggesting that in mammals, the decarboxylation and C1 hydroxylation reactions occur before or independently of the C5 hydroxylation. Finally, we show thatCOQ6isoform c (transcript NM_182480) does not encode an active enzyme. VA can be produced in the liver by the oxidation of vanillin, a nontoxic compound commonly used as a food additive, and crosses the blood-brain barrier. These characteristics make it a promising compound for the treatment of patients with CoQ deficiency due toCOQ6mutations.

Monoamine oxidase (MAO), a mitochondrial enzyme that oxidizes biogenic amines generating hydrogen peroxide, is a major source of oxidative stress in cardiac injury. However, the molecular mechanisms underlying its overactivation in pathological conditions are still poorly characterized. Here, we investigated whether the enhanced MAO-dependent hydrogen peroxide production can be due to increased substrate availability using a metabolomic profiling method. We identified N1-methylhistamine -the main catabolite of histamine- as an important substrate fueling MAO in Langendorff mouse hearts, directly perfused with a buffer containing hydrogen peroxide or subjected to ischemia/reperfusion protocol. Indeed, when these hearts were pretreated with the MAO inhibitor pargyline we observed N1-methylhistamine accumulation along with reduced oxidative stress. Next, we showed that synaptic terminals are the major source of N1-methylhistamine. Indeed, in vivo sympathectomy caused a decrease of N1-methylhistamine levels, which was associated with a marked protection in post-ischemic reperfused hearts. As far as the mechanism is concerned, we demonstrate that exogenous histamine is transported into isolated cardiomyocytes and triggers a rise in the levels of reactive oxygen species (ROS). Once again, pargyline pretreatment induced intracellular accumulation of N1-methylhistamine along with decrease in ROS levels. These findings uncover a receptor-independent mechanism for histamine in cardiomyocytes. In summary, our study reveals a novel and important pathophysiological causative link between MAO activation and histamine availability during pathophysiological conditions such as oxidative stress/cardiac injury.

Analyses of cellular responses to fast oxygen dynamics are challenging and require ad hoc technological solutions, especially when decoupling from liquid media composition is required. In this work, we present a microfluidic device specifically designed for culture analyses with high resolution and magnification objectives, providing full optical access to the cell culture chamber. This feature allows fluorescence-based assays, photoactivated surface chemistry, and live cell imaging under tightly controlled pO2 environments. The device has a simple design, accommodates three independent cell cultures, and can be employed by users with basic cell culture training in studies requiring fast oxygen dynamics, defined media composition, and in-line data acquisition with optical molecular probes. We apply this technology to produce an oxygen/glucose deprived (OGD) environment and analyze cell mortality in murine and human cardiac cultures. Neonatal rat ventricular cardiomyocytes show an OGD time-dependent sensitivity, resulting in a robust and reproducible 66 ± 5% death rate after 3 h of stress. Applying an equivalent stress to human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) provides direct experimental evidence for fetal-like OGD-resistant phenotype. Investigation on the nature of such phenotype exposed large glycogen deposits. We propose a culture strategy aimed at depleting these intracellular energy stores and concurrently activate positive regulation of aerobic metabolic molecular markers. The observed process, however, is not sufficient to induce an OGD-sensitive phenotype in hiPS-CMs, highlighting defective development of mature aerobic metabolism in vitro.

Oxidative stress and mitochondrial dysfunction play a crucial role in the pathophysiology of muscular dystrophies. We previously reported that the mitochondrial enzyme monoamine oxidase (MAO) is a relevant source of reactive oxygen species (ROS) not only in murine models of muscular dystrophy, in which it directly contributes to contractile impairment, but also in muscle cells from collagen VI-deficient patients. Here, we now assessed the efficacy of a novel MAO-B inhibitor, safinamide, using in vivo and in vitro models of Duchenne muscular dystrophy (DMD). Specifically, we found that administration of safinamide in 3-month-old mdx mice reduced myofiber damage and oxidative stress and improved muscle functionality. In vitro studies with myogenic cultures from mdx mice and DMD patients showed that even cultured dystrophic myoblasts were more susceptible to oxidative stress than matching cells from healthy donors. Indeed, upon exposure to the MAO substrate tyramine or to hydrogen peroxide, DMD muscle cells displayed a rise in ROS levels and a consequent mitochondrial depolarization. Remarkably, both phenotypes normalized when cultures were treated with safinamide. Given that safinamide is already in clinical use for neurological disorders, our findings could pave the way toward a promising translation into clinical trials for DMD patients as a classic case of drug repurposing.

Parkinson’s disease (PD) is a neurodegenerative disorder bearing motor and nonmotor symptoms. The treatment today is symptomatical rather than preventive or curative and this leaves the field open for the search of both novel molecular targets and drug candidates. Interference with α-synuclein fibrillation, monoamine oxidase (MAO) inhibition, modulation of adenosine receptors and the inhibition of specific phosphodiesterase (PDE) isoforms are some of the currently pursued strategies. We synthesised and studied some semi-synthetic berberine derivatives using a set of in silico tools. We evaluated their drug-likeness and tested the compounds against a set of target proteins involved in the onset or progression of PD, with a particular attention to MAO-B. Preliminary in vitro assay on MAO-B confirmed our in silico predictions.

In the super-relaxed state of myosin, ATPase activity is strongly inhibited by binding of the myosin heads to the core of the thick filament in a structure known as the interacting-heads motif. In the disordered relaxed state myosin heads are not bound to the core of the thick filament and have an ATPase rate that is 10 fold greater. In the interacting-heads motif the two regulatory light chains appear to bind to each other. We have made single cysteine mutants of the regulatory light chain, placed both paramagnetic and fluorescent probes on them, and exchanged them into skinned skeletal muscle fibers. Many of the labeled light chains tended to disrupt the stability of the super-relaxed state, and showed spectral changes in the transition from the disordered relaxed state to the super-relaxed state. These data support the putative interface between the two regulatory light chains identified by cryo electron microscopy and show that both the divalent cation bound to the regulatory light chain and the N-terminus of the regulatory light chain play a role in the stability of the super-relaxed state. One probe showed a shift to shorter wavelengths in the super-relaxed state such that a ratio of intensities at 440nm to that at 520nm provided a measure of the population of the super-relaxed state amenable for high throughput screens for finding potential pharmaceuticals. The results provide a proof of concept that small molecules that bind to this region can destabilize the super-relaxed state and provide a method to search for small molecules that do so leading to a potentially effective treatment for Type 2 diabetes and obesity.

In resting skeletal muscle, myosin is found in 2 states, the super-relaxed state (SRX) and the disordered relaxed state (DRX). In the SRX the ATPase activity is strongly inhibited by binding of the myosin heads to the core of the thick filament; in the DRX the myosin heads are not bound to the core of the thick filament and have an ATPase rate that is an order of magnitude greater. We made a series of single cysteine mutants of the myosin regulatory light chain, placed fluorescent probes on them, and exchanged them into skinned rabbit fast skeletal muscle fibers. We found fluorescent probes that reported on the relative populations of the SRX and DRX, and used them to carry out a high throughput screen. The screen identified one compound, piperine, which destabilized the SRX as measured by probe fluorescence. The kd for inhibition was ∼3 μM with a maximum inhibition of ∼50% at high concentations. The compound was also shown to destabilize the SRX using single nucleotide turnover measurements. The effect was only seen in fast twitch fibers. Piperine was shown to increase the ATPase activity of relaxed fibers by 40%. It had no effect on the mechanics of either active or resting muscle fibers. Previous work has shown that piperine can attenuate both obesity and type 2 diabetes in rodent models of these conditions. We propose that these effects are due to the up-regulation of resting muscle metabolism. The results described here suggest that piperine would provide a useful lead compound for the development of therapies to treat obesity and type 2 diabetes in humans.

Significance We have developed a method for finding pharmaceuticals that would treat obesity and type 2 diabetes by increasing the metabolic rate of resting skeletal muscle. The metabolic rate is increased by shifting the motor protein myosin from a low activity state to a higher activity state. We devised an assay, screened for compounds, and found one molecule, piperine. Piperine increased the metabolic rate of resting muscle fibers. Piperine does not have the properties required to be a pharmaceutical in humans, but it would make a good lead compound for finding compounds that do. Our results provide proof of concept that these metabolic diseases can be treated by future pharmaceuticals that target myosin to increase the metabolism of excess calories.

Purpose: Oxidative stress plays a key role in cardiac diseases, although the sources of reactive oxygen species (ROS) have not been defined conclusively. Recent studies demonstrated that the mitochondrial enzymes monoamine oxidases (MAO) are a major source of ROS in reperfusion injury and decompensated hypertrophy. The present study characterized the molecular mechanisms responsible for the increased activity of MAO. Based upon available information, the activity of these enzymes depends mostly on substrate availability. Therefore, we aimed at identifying the major substrates of MAO in hearts undergoing oxidative stress. Mass spectrometry was used to identify and quantitate potential substrates by comparing their contents in the absence and the presence of MAO inhibition. Methods and Results: Firstly, we applied a metabolomic profiling method to investigate changes in amine contents in isolated mouse hearts, by means of a LC-MS/MS approach in the precursor ion scanning mode. Maximal oxidative stress was induced by perfusing isolated mouse hearts with 1 mM hydrogen peroxide for 15 min. Addition of 0.5 mM pargyline to the perfusion buffer 10 min before hydrogen peroxide resulted in a significant increased content of the typical MAO substrates serotonin and epinephrine, along with histamine and its product N1-methyl histamine. N1-methyl histamine was found to be the more aboundant metabolite and its content displayed a 180% increase in pargyline-treated hearts, as compared to the untreated ones. The accumulation of MAO substrates upon pargyline treatment correlated with a reduced MAO-dependent production of hydrogen peroxide. In fact we observed a decreased extent of (i) oxidation of myofibrillar proteins, as detected by disulfide bond formation in tropomyosin (Western blot under non reducing conditions), and (ii) ROS levels in tissue, as detected by dihydroethidine (DHE) staining. Surprisingly, these findings imply that the profound injury induced by H2O2 administration is not due to a direct action. Indeed, H2O2 perfusion appears to trigger an amplification pathway whereby the increase in MAO activity due to a larger substrate availability is the end-effector of the initial oxidative stress. Conclusions: This study provides the first information on endogenous substrates of MAO becoming available under conditions of oxidative stress that is then amplified by the increased MAO activity. The identification of histamine and N1-methyl histamine, that are involved in neurotransmission and immune response, suggests a significant trafficking of MAO substrates between myocytes and non-myocyte cells in the heart.