Aberrant Adenosine Triphosphate Release and Impairment of P2Y2-Mediated Signaling in Sarcoglycanopathies.

Sarcoglycanopathies, limb-girdle muscular dystrophies (LGMD) caused by genetic loss-of-function of the membrane proteins sarcoglycans (SGs), are characterized by progressive degeneration of skeletal muscle. In these disorders, muscle necrosis is associated with immune-mediated damage, whose triggering and perpetuating molecular mechanisms are not fully elucidated yet. Extracellular adenosine triphosphate (eATP) seems to represent a crucial factor, with eATP activating purinergic receptors. Indeed, in vivo blockade of the eATP/P2X7 purinergic pathway ameliorated muscle disease progression. P2X7 inhibition improved the dystrophic process by restraining the activity of P2X7 receptors on immune cells. Whether P2X7 blockade can display a direct action on muscle cells is not known yet. In this study, we investigated eATP effects in primary cultures of myoblasts isolated from patients with LGMDR3 (α-sarcoglycanopathy) and in immortalized cells isolated from a patient with LGMDR5 (γ-sarcoglycanopathy). Our results demonstrated that, owing to a reduced ecto-ATPase activity and/or an enhanced release of ATP, patient cells are exposed to increased juxtamembrane concentrations of eATP and display a higher susceptivity to eATP signals. The purinoceptor P2Y2, which proved to be overexpressed in patient cells, was identified as a pivotal receptor responsible for the enhanced ATP-induced or UTP-induced Ca2+ increase in affected myoblasts. Moreover, P2Y2 stimulation in LDMDR3 muscle cells induced chemotaxis of immune cells and release of interleukin-8. In conclusion, a higher eATP concentration and sensitivity in primary human muscle cells carrying different α-SG or γ-SG loss-of-function mutations indicate that eATP/P2Y2 is an enhanced signaling axis in cells from patients with α-/γ-sarcoglycanopathy. Understanding the basis of the innate immune-mediated damage associated with the dystrophic process may be critical in overcoming the immunologic hurdles associated with emerging gene therapies for these disorders.

Sirtuin 6 Regulates the Activation of the ATP/Purinergic Axis in Endothelial Cells.

Sirtuin 6 (SIRT6) is a member of the mammalian NAD+-dependent deac(et)ylase sirtuin family. SIRT6's anti-inflammatory roles are emerging increasingly often in different diseases and cell types, including endothelial cells. In this study, the role of SIRT6 in pro-inflammatory conditions was investigated by engineering human umbilical vein endothelial cells to overexpress SIRT6 (SIRT6+ HUVECs). Our results showed that SIRT6 overexpression affected the levels of adhesion molecules and sustained megakaryocyte proliferation and proplatelet formation. Interestingly, the pro-inflammatory activation of the ATP/purinergic axis was reduced in SIRT6+ HUVECs. Specifically, the TNFα-induced release of ATP in the extracellular space and the increase in pannexin-1 hemichannel expression, which mediates ATP efflux, were hampered in SIRT6+ cells. Instead, NAD+ release and Connexin43 expression were not modified by SIRT6 levels. Moreover, the Ca2+ influx in response to ATP and the expression of the purinergic receptor P2X7 were decreased in SIRT6+ HUVECs. Contrary to extracellular ATP, extracellular NAD+ did not evoke pro-inflammatory responses in HUVECs. Instead, NAD+ administration reduced endothelial cell proliferation and motility and counteracted the TNFα-induced angiogenesis. Altogether, our data reinforce the view of SIRT6 activation as an anti-inflammatory approach in vascular endothelium.

Neuroprotective Potential of Dendritic Cells and Sirtuins in Multiple Sclerosis.

Myeloid cells, including parenchymal microglia, perivascular and meningeal macrophages, and dendritic cells (DCs), are present in the central nervous system (CNS) and establish an intricate relationship with other cells, playing a crucial role both in health and in neurological diseases. In this context, DCs are critical to orchestrating the immune response linking the innate and adaptive immune systems. Under steady-state conditions, DCs patrol the CNS, sampling their local environment and acting as sentinels. During neuroinflammation, the resulting activation of DCs is a critical step that drives the inflammatory response or the resolution of inflammation with the participation of different cell types of the immune system (macrophages, mast cells, T and B lymphocytes), resident cells of the CNS and soluble factors. Although the importance of DCs is clearly recognized, their exact function in CNS disease is still debated. In this review, we will discuss modern concepts of DC biology in steady-state and during autoimmune neuroinflammation. Here, we will also address some key aspects involving DCs in CNS patrolling, highlighting the neuroprotective nature of DCs and emphasizing their therapeutic potential for the treatment of neurological conditions. Recently, inhibition of the NAD+-dependent deac(et)ylase sirtuin 6 was demonstrated to delay the onset of experimental autoimmune encephalomyelitis, by dampening DC trafficking towards inflamed LNs. Thus, a special focus will be dedicated to sirtuins' role in DCs functions.

Sirt6 inhibition delays the onset of experimental autoimmune encephalomyelitis by reducing dendritic cell migration.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of multiple sclerosis (MS), a neuroinflammatory and demyelinating disease characterized by multifocal perivascular infiltrates of immune cells. Although EAE is predominantly considered a T helper 1-driven autoimmune disease, mounting evidence suggests that activated dendritic cells (DC), which are the bridge between innate and adaptive immunity, also contribute to its pathogenesis. Sirtuin 6 (SIRT6), a NAD+-dependent deacetylase involved in genome maintenance and in metabolic homeostasis, regulates DC activation, and its pharmacological inhibition could, therefore, play a role in EAE development. METHODS: EAE was induced in female C57bl/6 mice by MOG35-55 injection. The effect of treatment with a small compound SIRT6 inhibitor, administered according to therapeutic and preventive protocols, was assessed by evaluating the clinical EAE score. SIRT6 inhibition was confirmed by Western blot analysis by assessing the acetylation of histone 3 lysine 9, a known SIRT6 substrate. The expression of DC activation and migration markers was evaluated by FACS in mouse lymph nodes. In addition, the expression of inflammatory and anti-inflammatory cytokines in the spinal cord were assessed by qPCR. T cell infiltration in spinal cords was evaluated by immunofluorescence imaging. The effect of Sirt6 inhibition on the migration of resting and activated bone marrow-derived dendritic cells was investigated in in vitro chemotaxis assays. RESULTS: Preventive pharmacological Sirt6 inhibition effectively delayed EAE disease onset through a novel regulatory mechanism, i.e., by reducing the representation of CXCR4-positive and of CXCR4/CCR7-double-positive DC in lymph nodes. The delay in EAE onset correlated with the early downregulation in the expression of CD40 on activated lymph node DC, with increased level of the anti-inflammatory cytokine IL-10, and with a reduced encephalitogenic T cell infiltration in the central nervous system. Consistent with the in vivo data, in vitro pharmacological Sirt6 inhibition in LPS-stimulated, bone marrow-derived DC reduced CCL19/CCL21- and SDF-1-induced DC migration. CONCLUSIONS: Our findings indicate the ability of Sirt6 inhibition to impair DC migration, to downregulate pathogenic T cell inflammatory responses and to delay EAE onset. Therefore, Sirt6 might represent a valuable target for developing novel therapeutic agents for the treatment of early stages of MS, or of other autoimmune disorders.

Differential modulation of SIRT6 deacetylase and deacylase activities by lysine-based small molecules.

Sirtuin 6 (SIRT6) is an NAD+-dependent deacetylase regulating important functions: modulators of its enzymatic activity have been considered as possible therapeutic agents. Besides the deacetylase activity, SIRT6 also has NAD+-dependent deacylase activity, whereby it regulates the secretion of cytokines and proteins. We identified novel SIRT6 modulators with a lysine-based structure: compound 1 enhances SIRT6 deacylase while inhibiting the deacetylase activity. As expected based on the biological effects of SIRT6 deacetylase activity, compound 1 increased histone 3 lysine 9 acetylation and the activity of glycolytic enzymes. Moreover, the fact that compound 1 enhanced SIRT6 deacylase activity was accompanied by an increased TNF-α release. In conclusion, new SIRT6 modulators with a lysine-like structure were identified, with differential effects on specific SIRT6 activities. The novel SIRT6 modulator concomitantly inhibits deacetylase and enhances deacylase activity.

Survivorship and clinical outcomes of 147 consecutive isolated or combined arthroscopic bone plug free meniscal allograft transplantation.

PURPOSE: To present the results of a survival analysis of a series of 147 arthroscopic MAT procedures. METHODS: One-hundred and forty-seven patients (117 males and 30 females) underwent arthroscopic MAT without bone plugs (82 medial MAT and 65 lateral MAT) using fresh-frozen, non-irradiated grafts. They were retrospectively reviewed at a mean of 4.0 ± 1.9-year follow-up. Mean age at surgery was 40.9 ± 11.2 (range 16.7-68.8) years; 70 patients (48 %) underwent combined procedures. Clinical evaluation was performed with KOOS, Lysholm and a 0-100 VAS for pain. Survival analysis was performed using two endpoints: surgical failure (revision procedure with direct relation to MAT) and clinical failure (revision procedure or poor Lysholm score, <65). RESULTS: There was a significant (p < 0.05) and clinically relevant decrease in the VAS and increase in KOOS and Lysholm from pre-operative mean score to post-operative mean score. Seven (5 %) patients (two medial and five lateral) experienced surgical failure (five meniscectomies, one lateral graft peripheral suture and one unicompartmental knee arthroplasty). The mean overall survival time was 9.7 years (CI 9.1-10.3). As 16 (11 %) patients presented poor Lysholm score, a total of 23 (16 %) patients were considered clinical failures. The mean overall survival time was 8 years (CI 7.1-8.8). No statistically significant differences in failure and survival rate were present between medial and lateral MAT, isolated or combined MAT, patients >50 or <50 years old and patients with body mass index <25 or >25. CONCLUSIONS: MAT, eventually associated with other needed procedures, was able to significantly relieve pain and improve function of the knee joint at midterm follow-up, with a survival rate from 9.7 to 8.0 years based on failure criteria. Most additional procedures were done in the first 2 post-operative years. MAT eventually associated with other needed procedures could represent an effective treatment for post-meniscectomy syndrome. LEVEL OF EVIDENCE: Therapeutic study, retrospective case series, Level IV.

Long-term clinical outcomes of combined BPTB ACL reconstruction and popliteus tendon plasty.

PURPOSE: A deficiency of posterolateral structures significantly increases the varus load on the ACL, while a chronic ACL lesion, the increased tibial rotation and the repetitive non-physiological knee motion, could affect and damage the integrity of the popliteus tendon. Therefore, the aim of the present study was to report the very long clinical outcomes of a combined single-bundle BPTB ACL reconstruction and popliteus plasty according to Bousquets technique, for the treatment of combined chronic anterior and posterolateral laxities. METHODS: Fifteen patients that underwent combined ACL reconstruction and popliteal plasty according to Bousquets technique were available at mean 26.8 ± 1.0 years (range 25.4-28.0 years). All the patients were evaluated clinically and 13 by means of KT-1000 Arthrometer as well. Subjective evaluation was performed with the subjective IKDC, WOMAC and a 0-10 VAS for pain scales. RESULTS: At clinical evaluation, 10 patients (67 %) presented a negative anterior drawer test; Lachman test was negative in nine patients (60 %); the varus stress test was negative in eight (53 %); and the dial test was negative in all but one patient (93 %). Only two patients (15 %) presented a side-to-side difference >5 mm at the instrumented laxity evaluation. CONCLUSION: The combined single-bundle BPTB ACL reconstruction and popliteal plasty according to Bousquets technique were able to produce very good long-term results, in terms of knee stability, subjective outcomes, functional results and return to sport activity, in case of chronic anterior and posterolateral laxities. LEVEL OF EVIDENCE: Retrospective case series, Level IV.

Medial patellotibial ligament (MPTL) reconstruction for patellar instability.

PURPOSE: To evaluate mid-term clinical and radiographic outcomes after an original medial patellotibial ligament reconstruction in patients with patellar dislocation. METHODS: Twenty-nine knees (27 patients, 8 males and 19 females) treated for patellar dislocation with medialization of the patellar tendon medial third combined with medial and lateral release were evaluated clinically and radiographically at a mean follow-up of 6.1±2.5 years. Trochleoplasty was performed in case of severe flat trochlea (6 knees, 21%). Aetiology of patellofemoral instability was traumatic in 6 (21%) and atraumatic in 23 (79%) knees. The mean age at first dislocation was 19.2±10.1 years. WOMAC, subjective and objective IKDC, Kujala, VAS for pain, Tegner activity and EQ-5D scores were used. Anteroposterior, lateral and 30° axial views were performed for radiographic monitoring. RESULTS: There was a significant improvement of all clinical scores and significant reduction in knee pain. Twenty-four knees (83%) were normal or nearly normal by objective IKDC score at final follow-up. Radiographs showed a higher incidence of patella alta and flat trochlea in the atraumatic group. Severe signs of patellar osteoarthritis were found in 1 knee (3%). A higher body mass index (BMI) was correlated with worse pre-operative scores. Four knees (14%) were considered failures (2 further dislocations, 2 revision surgeries). The overall survival rate at 6 years was 0.811. CONCLUSIONS: The presented techniques produced good clinical and radiographic results at mean 6.1 years follow-up, with 14 % failures. Signs of patellofemoral dysplasia were found in patients with atraumatic patellar dislocation. BMI was related to worse pre-operative clinical status. LEVEL OF EVIDENCE: Retrospective study, Level IV.

Clinical outcomes and complications of a collagen meniscus implant: a systematic review.

PURPOSE: The purpose of this systematic review was to summarise and evaluate the clinical outcomes of the collagen meniscus implant (CMI) and its complication and failure rates. These data were then used to evaluate the results of the CMI at different follow-up time periods and investigate possible differences in the behaviour of lateral and medial CMI. METHODS: A comprehensive search was performed in PubMed, MEDLINE, CINAHL, Cochrane, EMBASE and Google Scholar databases using various combinations of the following keywords: "collagen meniscus implant" or "collagen meniscal implant". All studies evaluating medial or lateral CMI using the Lysholm score, visual analogue scale (VAS) for pain, Tegner activity scale and subjective or objective International Knee Documentation Committee (IKDC) scores were included in the systematic review. RESULTS: Eleven studies were included in the systematic review. The pooled number of patients involved in CMI surgery were 396 (90.2 % medial, 9.8 % lateral), with a mean age at surgery of 37.8 years. Concomitant procedures were present in 48.8 % of patients; most of them were anterior cruciate ligament (ACL) reconstruction, high tibial osteotomy (HTO) and microfractures. The Lysholm score and VAS for pain showed an improvement at six months up to ten years. No noticeable differences were present comparing short-term values of Lysholm score between medial and lateral CMI. The Tegner activity level reached its peak at 12 months after surgery and showed a progressive decrease through five and ten years post CMI implantation, however always remaining above the pre-operative level. Only a few knees were rated as "nearly abnormal" or "abnormal" at IKDC grading at all follow-up evaluations. CONCLUSIONS: The CMI could produce good and stable clinical results, particularly regarding knee function and pain, with low rates of complications and reoperations.

New Analogues of the Nicotinamide Phosphoribosyltransferase Inhibitor FK866 as Potential Anti-Pancreatic Cancer Agents.

BACKGROUND: During the past two decades, many nicotinamide phosphoribosyltransferase (NAMPT) inhibitors were prepared and tested because this enzyme is overexpressed in pancreatic cancer. Although FK866 is a well-known, strong NAMPT inhibitor, it suffers severe drawbacks. OBJECTIVE: Our work aimed to synthesize efficient NAMPT inhibitors featuring better pharmacokinetic properties than the pyridine-containing FK866. To this aim, the new anticancer agents were based on benzene, pyridazine, or benzothiazole moieties as a cap group instead of the pyridine unit found in FK866 and other NAMPT inhibitors. METHODS: The new compounds, prepared exploiting standard heterocycle chemistry and coupling reactions (e.g., formation of amides, ureas, and cyanoguanidines, copper-mediated azide-alkyne cycloaddition), have been fully characterized using NMR and HRMS analyses. Their activity has been evaluated using cytotoxicity and intracellular NAD depletion assays in the human pancreatic cancer cell line MiaPaCa-2. RESULTS: Among the 14 products obtained, compound 28, bearing a pyridazine unit as the cap group and a thiophene moiety as the tail group, showed 6.7 nanomolar inhibition activity in the intracellular NAD depletion assay and 43 nanomolar inhibition in the MiaPaCa-2 cells cytotoxicity assay, comparable to that observed for FK866. CONCLUSION: The positive results observed for some newly synthesized molecules, particularly those carrying a thiophene unit as a tail group, indicate that they could act as in vivo anti-pancreatic cancer agents.

The TRPM2 ion channel regulates metabolic and thermogenic adaptations in adipose tissue of cold-exposed mice.

Introduction: During thermogenesis, adipose tissue (AT) becomes more active and enhances oxidative metabolism. The promotion of this process in white AT (WAT) is called "browning" and, together with the brown AT (BAT) activation, is considered as a promising approach to counteract obesity and metabolic diseases. Transient receptor potential cation channel, subfamily M, member 2 (TRPM2), is an ion channel that allows extracellular Ca2+ influx into the cytosol, and is gated by adenosine diphosphate ribose (ADPR), produced from NAD+ degradation. The aim of this study was to investigate the relevance of TRPM2 in the regulation of energy metabolism in BAT, WAT, and liver during thermogenesis. Methods: Wild type (WT) and Trpm2-/- mice were exposed to 6°C and BAT, WAT and liver were collected to evaluate mRNA, protein levels and ADPR content. Furthermore, O2 consumption, CO2 production and energy expenditure were measured in these mice upon thermogenic stimulation. Finally, the effect of the pharmacological inhibition of TRPM2 was assessed in primary adipocytes, evaluating the response upon stimulation with the ß-adrenergic receptor agonist CL316,243. Results: Trpm2-/- mice displayed lower expression of browning markers in AT and lower energy expenditure in response to thermogenic stimulus, compared to WT animals. Trpm2 gene overexpression was observed in WAT, BAT and liver upon cold exposure. In addition, ADPR levels and mono/poly-ADPR hydrolases expression were higher in mice exposed to cold, compared to control mice, likely mediating ADPR generation. Discussion: Our data indicate TRPM2 as a fundamental player in BAT activation and WAT browning. TRPM2 agonists may represent new pharmacological strategies to fight obesity.

Iminosugar-Based Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors as Potential Anti-Pancreatic Cancer Agents.

The nicotinamide phosphoribosyltransferase (NAMPT) is considered a very promising therapeutic target because it is overexpressed in pancreatic cancer. Although many inhibitors have been prepared and tested, clinical trials have shown that NAMPT inhibition may result in severe haematological toxicity. Therefore, the development of conceptually new inhibitors is an important and challenging task. We synthesized ten ß-d-iminoribofuranosides bearing various heterocycle-based chains carbon-linked to the anomeric position starting from non-carbohydrate derivatives. They were then submitted to NAMPT inhibition assays, as well as to pancreatic tumor cells viability and intracellular NAD+ depletion evaluation. The biological activity of the compounds was compared to that of the corresponding analogues lacking the carbohydrate unit to assess, for the first time, the contribution of the iminosugar moiety to the properties of these potential antitumor agents.

PLX4032 resistance of patient-derived melanoma cells: crucial role of oxidative metabolism.

Background: Malignant melanoma is the most lethal form of skin cancer which shows BRAF mutation in 50% of patients. In this context, the identification of BRAFV600E mutation led to the development of specific inhibitors like PLX4032. Nevertheless, although its initial success, its clinical efficacy is reduced after six-months of therapy leading to cancer relapse due to the onset of drug resistance. Therefore, investigating the mechanisms underlying PLX4032 resistance is fundamental to improve therapy efficacy. In this context, several models of PLX4032 resistance have been developed, but the discrepancy between in vitro and in vivo results often limits their clinical translation. Methods: The herein reported model has been realized by treating with PLX4032, for six months, patient-derived BRAF-mutated melanoma cells in order to obtain a reliable model of acquired PLX4032 resistance that could be predictive of patient's treatment responses. Metabolic analyses were performed by evaluating glucose consumption, ATP synthesis, oxygen consumption rate, P/O ratio, ATP/AMP ratio, lactate release, lactate dehydrogenase activity, NAD+/NADH ratio and pyruvate dehydrogenase activity in parental and drug resistant melanoma cells. The intracellular oxidative state was analyzed in terms of reactive oxygen species production, glutathione levels and NADPH/NADP+ ratio. In addition, a principal component analysis was conducted in order to identify the variables responsible for the acquisition of targeted therapy resistance. Results: Collectively, our results demonstrate, for the first time in patient-derived melanoma cells, that the rewiring of oxidative phosphorylation and the maintenance of pyruvate dehydrogenase activity and of high glutathione levels contribute to trigger the onset of PLX4032 resistance. Conclusion: Therefore, it is possible to hypothesize that inhibitors of glutathione biosynthesis and/or pyruvate dehydrogenase activity could be used in combination with PLX4032 to overcome drug resistance of BRAF-mutated melanoma patients. However, the identification of new adjuvant targets related to drug-induced metabolic reprogramming could be crucial to counteract the failure of targeted therapy in metastatic melanoma.

CD38-Induced Metabolic Dysfunction Primes Multiple Myeloma Cells for NAD+-Lowering Agents.

Cancer cells fuel growth and energy demands by increasing their NAD+ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD+-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the backbone for treatment of newly diagnosed and relapsed multiple myeloma patients. Nevertheless, further steps are needed to enable a full exploitation of these strategies, including deeper insights of the mechanisms by which CD38 promotes tumorigenesis and its metabolic additions that could be selectively targeted by therapeutic strategies. Here, we present evidence that CD38 upregulation produces a pervasive intracellular-NAD+ depletion, which impairs mitochondrial fitness and enhances oxidative stress; as result, genetic or pharmacologic approaches that aim to modify CD38 surface-level prime MM cells to NAD+-lowering agents. The molecular mechanism underlying this event is an alteration in mitochondrial dynamics, which decreases mitochondria efficiency and triggers energetic remodeling. Overall, we found that CD38 handling represents an innovative strategy to improve the outcomes of NAD+-lowering agents and provides the rationale for testing these very promising agents in clinical studies involving MM patients.

Paracrine ADP Ribosyl Cyclase-Mediated Regulation of Biological Processes.

ADP-ribosyl cyclases (ADPRCs) catalyze the synthesis of the Ca2+-active second messengers Cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from NAD+ as well as nicotinic acid adenine dinucleotide phosphate (NAADP+) from NADP+. The best characterized ADPRC in mammals is CD38, a single-pass transmembrane protein with two opposite membrane orientations. The first identified form, type II CD38, is a glycosylated ectoenzyme, while type III CD38 has its active site in the cytosol. The ectoenzymatic nature of type II CD38 raised long ago the question of a topological paradox concerning the access of the intracellular NAD+ substrate to the extracellular active site and of extracellular cADPR product to its intracellular receptors, ryanodine (RyR) channels. Two different transporters, equilibrative connexin 43 (Cx43) hemichannels for NAD+ and concentrative nucleoside transporters (CNTs) for cADPR, proved to mediate cell-autonomous trafficking of both nucleotides. Here, we discussed how type II CD38, Cx43 and CNTs also play a role in mediating several paracrine processes where an ADPRC+ cell supplies a neighboring CNT-and RyR-expressing cell with cADPR. Recently, type II CD38 was shown to start an ectoenzymatic sequence of reactions from NAD+/ADPR to the strong immunosuppressant adenosine; this paracrine effect represents a major mechanism of acquired resistance of several tumors to immune checkpoint therapy.

Structure-Based Identification and Biological Characterization of New NAPRT Inhibitors.

NAPRT, the rate-limiting enzyme of the Preiss-Handler NAD biosynthetic pathway, has emerged as a key biomarker for the clinical success of NAMPT inhibitors in cancer treatment. Previous studies found that high protein levels of NAPRT conferred resistance to NAMPT inhibition in several tumor types whereas the simultaneous blockade of NAMPT and NAPRT results in marked anti-tumor effects. While research has mainly focused on NAMPT inhibitors, the few available NAPRT inhibitors (NAPRTi) have a low affinity for the enzyme and have been scarcely characterized. In this work, a collection of diverse compounds was screened in silico against the NAPRT structure, and the selected hits were tested through cell-based assays in the NAPRT-proficient OVCAR-5 ovarian cell line and on the recombinant hNAPRT. We found different chemotypes that efficiently inhibit the enzyme in the micromolar range concentration and for which direct engagement with the target was verified by differential scanning fluorimetry. Of note, the therapeutic potential of these compounds was evidenced by a synergistic interaction between the NAMPT inhibitor FK866 and the new NAPRTi in terms of decreasing OVCAR-5 intracellular NAD levels and cell viability. For example, compound IM29 can potentiate the effect of FK866 of more than two-fold in reducing intracellular NAD levels. These results pave the way for the development of a new generation of human NAPRTi with anticancer activity.

P2X7 Receptor Antagonist Reduces Fibrosis and Inflammation in a Mouse Model of Alpha-Sarcoglycan Muscular Dystrophy.

Limb-girdle muscular dystrophy R3, a rare genetic disorder affecting the limb proximal muscles, is caused by mutations in the α-sarcoglycan gene (Sgca) and aggravated by an immune-mediated damage, finely modulated by the extracellular (e)ATP/purinoceptors axis. Currently, no specific drugs are available. The aim of this study was to evaluate the therapeutic effectiveness of a selective P2X7 purinoreceptor antagonist, A438079. Sgca knockout mice were treated with A438079 every two days at 3 mg/Kg for 24 weeks. The P2X7 antagonist improved clinical parameters by ameliorating mice motor function and decreasing serum creatine kinase levels. Histological analysis of muscle morphology indicated a significant reduction of the percentage of central nuclei, of fiber size variability and of the extent of local fibrosis and inflammation. A cytometric characterization of the muscle inflammatory infiltrates showed that A438079 significantly decreased innate immune cells and upregulated the immunosuppressive regulatory T cell subpopulation. In α-sarcoglycan null mice, the selective P2X7 antagonist A438079 has been shown to be effective to counteract the progression of the dystrophic phenotype and to reduce the inflammatory response. P2X7 antagonism via selective inhibitors could be included in the immunosuppressant strategies aimed to dampen the basal immune-mediated damage and to favor a better engraftment of gene-cell therapies.

Role of Liver CD38 in the Regulation of Metabolic Pathways during Cold-Induced Thermogenesis in Mice.

Boosting NAD+ levels are considered a promising means to promote healthy aging and ameliorate dysfunctional metabolism. The expression of CD38, the major NAD+-consuming enzyme, is downregulated during thermogenesis in both brown and white adipose tissues (BAT and WAT). Moreover, BAT activation and WAT "browning" were enhanced in Cd38-/- mice. In this study, the role of CD38 in the liver during thermogenesis was investigated, with the liver being the central organ controlling systemic energy metabolism. Wild-type mice and Cd38-/- mice were exposed to cold temperatures, and levels of metabolites and enzymes were measured in the livers and plasma. During cold exposure, CD38 expression was downregulated in the liver, as in BAT and WAT, with a concomitant increase in NAD(H) and a marked decrease in NADPH levels. Glucose-6-phosphate dehydrogenase and the malic enzyme, along with enzymes in the glycolytic pathway, were downregulated, which is in line with glucose-6-P being re-directed towards glucose release. In Cd38-/- mice, the cross-regulation between glycolysis and glucose release was lost, although this did not impair the glucose release from glycogen. Glycerol levels were decreased in the liver from Cd38-/- animals upon cold exposure, suggesting that glyceroneogenesis, as gluconeogenesis, was not properly activated in the absence of CD38. SIRT3 activity, regulating mitochondrial metabolism, was enhanced by cold exposure, whereas its activity was already high at a warm temperature in Cd38-/- mice and was not further increased by the cold. Notably, FGF21 and bile acid release was enhanced in the liver of Cd38-/- mice, which might contribute to enhanced BAT activation in Cd38-/- mice. These results demonstrate that CD38 inhibition can be suggested as a strategy to boost NAD+ and would not negatively affect hepatic functions during thermogenesis.

The ABA-LANCL1/2 Hormone-Receptors System Protects H9c2 Cardiomyocytes from Hypoxia-Induced Mitochondrial Injury via an AMPK- and NO-Mediated Mechanism.

Abscisic acid (ABA) regulates plant responses to stress, partly via NO. In mammals, ABA stimulates NO production by innate immune cells and keratinocytes, glucose uptake and mitochondrial respiration by skeletal myocytes and improves blood glucose homeostasis through its receptors LANCL1 and LANCL2. We hypothesized a role for the ABA-LANCL1/2 system in cardiomyocyte protection from hypoxia via NO. The effect of ABA and of the silencing or overexpression of LANCL1 and LANCL2 were investigated in H9c2 rat cardiomyoblasts under normoxia or hypoxia/reoxygenation. In H9c2, hypoxia induced ABA release, and ABA stimulated NO production. ABA increased the survival of H9c2 to hypoxia, and L-NAME, an inhibitor of NO synthase (NOS), abrogated this effect. ABA also increased glucose uptake and NADPH levels and increased phosphorylation of Akt, AMPK and eNOS. Overexpression or silencing of LANCL1/2 significantly increased or decreased, respectively, transcription, expression and phosphorylation of AMPK, Akt and eNOS; transcription of NAMPT, Sirt1 and the arginine transporter. The mitochondrial proton gradient and cell vitality increased in LANCL1/2-overexpressing vs. -silenced cells after hypoxia/reoxygenation, and L-NAME abrogated this difference. These results implicate the ABA-LANCL1/2 hormone-receptor system in NO-mediated cardiomyocyte protection against hypoxia.

Role of CD38 in Adipose Tissue: Tuning Coenzyme Availability?

Nicotinamide adenine dinucleotide (NAD+) is a fundamental molecule in the regulation of energy metabolism, representing both a coenzyme and a substrate for different NAD+ degrading enzymes. Among these enzymes, CD38 can be seen under two perspectives: as the enzyme synthesizing Ca2+-mobilizing second messenger, starting from NAD+, and as the major NAD+-consumer, to be inhibited to increase NAD+ levels. Indeed, the regulation of NAD+ availability is a key event during different processes. In this review, we examine the recent studies related to the modulation of CD38 expression and activity, and the consequent changes in NAD(P)(H), in adipose tissue, during inflammation and cold-induced thermogenesis.