Exposure to an acute stress impaired the metabolic plasticity of resilient rats by enhancing fatty acid ß-oxidation in the ventral hippocampus.

The concept of resilience has changed over time and nowadays it refers to the positive adaptation to life adversities, rather than to the absence of a pathological response normally occurring in susceptible people. Based on our previous data showing that the exposure to the chronic mild stress (CMS) paradigm differently affected bioenergetics in the ventral hippocampus of vulnerable and resilient animals, here we investigated whether resilience is a stable trait or if the energetic strategy set in motion to sustain resilience unveils a vulnerability feature in a more dynamic situation. To this aim, vulnerable and resilient rats after 6 weeks of CMS were subjected to a further acute, unfamiliar restraint stress (ARS) and metabolomic studies were conducted in the ventral hippocampus. We observed that exposure to a single novel challenge negatively affects the fuel utilization of resilient animals. Indeed, while they increase glycolysis to sustain the non-hedonic phenotype when exposed to CMS, they shift to fatty acid ß-oxidation after ARS, as vulnerable animals following CMS, suggesting that the energy strategy that guarantees resilience is fragile and can be negatively modified by a different environmental condition. These results suggest that strengthening resilience to foster individuals to bounce back from stressful life events may represent a strategy to decrease vulnerability or prevent the risk of relapsing to a pathological state.

Discovery and characterization of a new class of NAD+-independent SIRT1 activators.

The activity of sirtuin 1 (SIRT1, a member of the NAD+-dependent deacetylases family) decreases during aging as NAD+ levels naturally decline, thus increasing the risk of several age-associated diseases. Several sirtuin-activating compounds (STACs) have been developed to counteract the age-associated reduction in SIRT1 activity, and some of them are currently under development in clinical trials. STACs induce SIRT1 activation, either through allosteric activation of the enzyme in the presence of NAD+, or by increasing NAD+ levels by inhibiting its degradation or by supplying a key precursor in biosynthesis. In this study, we have identified (E)-2'-des-methyl sulindac analogues as a novel class of STACs that act also in the absence of NAD+, a peculiar behavior demonstrated through enzymatic and mass spectrometry experiments, both in vitro and in cell lines. The activation of the SIRT1 pathway was confirmed in vivo through gene expression and metabolomics analysis. Our data suggest that these compounds could serve as candidate leads for a novel therapeutic strategy aimed at addressing a key metabolic deficiency that may contribute to metabolic and age-associated diseases.

Frataxin deficiency shifts metabolism to promote reactive microglia via glucose catabolism.

Immunometabolism investigates the intricate relationship between the immune system and cellular metabolism. This study delves into the consequences of mitochondrial frataxin (FXN) depletion, the primary cause of Friedreich's ataxia (FRDA), a debilitating neurodegenerative condition characterized by impaired coordination and muscle control. By using single-cell RNA sequencing, we have identified distinct cellular clusters within the cerebellum of an FRDA mouse model, emphasizing a significant loss in the homeostatic response of microglial cells lacking FXN. Remarkably, these microglia deficient in FXN display heightened reactive responses to inflammatory stimuli. Furthermore, our metabolomic analyses reveal a shift towards glycolysis and itaconate production in these cells. Remarkably, treatment with butyrate counteracts these immunometabolic changes, triggering an antioxidant response via the itaconate-Nrf2-GSH pathways and suppressing the expression of inflammatory genes. Furthermore, we identify Hcar2 (GPR109A) as a mediator involved in restoring the homeostasis of microglia without FXN. Motor function tests conducted on FRDA mice underscore the neuroprotective attributes of butyrate supplementation, enhancing neuromotor performance. In conclusion, our findings elucidate the role of disrupted homeostatic function in cerebellar microglia in the pathogenesis of FRDA. Moreover, they underscore the potential of butyrate to mitigate inflammatory gene expression, correct metabolic imbalances, and improve neuromotor capabilities in FRDA.

Prostaglandin D2 synthase controls Schwann cells metabolism.

We previously reported that in the absence of Prostaglandin D2 synthase (L-PGDS) peripheral nerves are hypomyelinated in development and that with aging they present aberrant myelin sheaths. We now demonstrate that L-PGDS expressed in Schwann cells is part of a coordinated program aiming at preserving myelin integrity. In vivo and in vitro lipidomic, metabolomic and transcriptomic analyses confirmed that myelin lipids composition, Schwann cells energetic metabolism and key enzymes controlling these processes are altered in the absence of L-PGDS. Moreover, Schwann cells undergo a metabolic rewiring and turn to acetate as the main energetic source. Further, they produce ketone bodies to ensure glial cell and neuronal survival. Importantly, we demonstrate that all these changes correlate with morphological myelin alterations and describe the first physiological pathway implicated in preserving PNS myelin. Collectively, we posit that myelin lipids serve as a reservoir to provide ketone bodies, which together with acetate represent the adaptive substrates Schwann cells can rely on to sustain the axo-glial unit and preserve the integrity of the PNS.

Activation of endogenous retroviruses and induction of viral mimicry by MEK1/2 inhibition in pancreatic cancer.

While pancreatic ductal adenocarcinomas (PDACs) are addicted to KRAS-activating mutations, inhibitors of downstream KRAS effectors, such as the MEK1/2 kinase inhibitor trametinib, are devoid of therapeutic effects. However, the extensive rewiring of regulatory circuits driven by the attenuation of the KRAS pathway may induce vulnerabilities of therapeutic relevance. An in-depth molecular analysis of the transcriptional and epigenomic alterations occurring in PDAC cells in the initial hours after MEK1/2 inhibition by trametinib unveiled the induction of endogenous retroviruses (ERVs) escaping epigenetic silencing, leading to the production of double-stranded RNAs and the increased expression of interferon (IFN) genes. We tracked ERV activation to the early induction of the transcription factor ELF3, which extensively bound and activated nonsilenced retroelements and synergized with IRF1 (interferon regulatory factor 1) in the activation of IFNs and IFN-stimulated genes. Trametinib-induced viral mimicry in PDAC may be exploited in the rational design of combination therapies in immuno-oncology.

Acetyl-CoA production by Mediator-bound 2-ketoacid dehydrogenases boosts de novo histone acetylation and is regulated by nitric oxide.

Histone-modifying enzymes depend on the availability of cofactors, with acetyl-coenzyme A (CoA) being required for histone acetyltransferase (HAT) activity. The discovery that mitochondrial acyl-CoA-producing enzymes translocate to the nucleus suggests that high concentrations of locally synthesized metabolites may impact acylation of histones and other nuclear substrates, thereby controlling gene expression. Here, we show that 2-ketoacid dehydrogenases are stably associated with the Mediator complex, thus providing a local supply of acetyl-CoA and increasing the generation of hyper-acetylated histone tails. Nitric oxide (NO), which is produced in large amounts in lipopolysaccharide-stimulated macrophages, inhibited the activity of Mediator-associated 2-ketoacid dehydrogenases. Elevation of NO levels and the disruption of Mediator complex integrity both affected de novo histone acetylation within a shared set of genomic regions. Our findings indicate that the local supply of acetyl-CoA generated by 2-ketoacid dehydrogenases bound to Mediator is required to maximize acetylation of histone tails at sites of elevated HAT activity.

Glucose-derived glutamate drives neuronal terminal differentiation in vitro.

Neuronal maturation is the phase during which neurons acquire their final characteristics in terms of morphology, electrical activity, and metabolism. However, little is known about the metabolic pathways governing neuronal maturation. Here, we investigate the contribution of the main metabolic pathways, namely glucose, glutamine, and fatty acid oxidation, during the maturation of primary rat hippocampal neurons. Blunting glucose oxidation through the genetic and chemical inhibition of the mitochondrial pyruvate transporter reveals that this protein is critical for the production of glutamate, which is required for neuronal arborization, proper dendritic elongation, and spine formation. Glutamate supplementation in the early phase of differentiation restores morphological defects and synaptic function in mitochondrial pyruvate transporter-inhibited cells. Furthermore, the selective activation of metabotropic glutamate receptors restores the impairment of neuronal differentiation due to the reduced generation of glucose-derived glutamate and rescues synaptic local translation. Fatty acid oxidation does not impact neuronal maturation. Whereas glutamine metabolism is important for mitochondria, it is not for endogenous glutamate production. Our results provide insights into the role of glucose-derived glutamate as a key player in neuronal terminal differentiation.

Exploring rat corpus cavernosum alterations induced by finasteride treatment and withdrawal.

Despite its efficacy for treating androgenetic alopecia, finasteride, an inhibitor of 5α-reductase (i.e., the enzyme converting testosterone, T, into dihydrotestosterone, DHT), is associated with several side effects including sexual dysfunction (e.g., erectile dysfunction). These side effects may persist after drug suspension, inducing the so-called post-finasteride syndrome (PFS). The effects of subchronic treatment with finasteride (i.e., 20 days) and its withdrawal (i.e., 1 month) in rat corpus cavernosum have been explored here. Data obtained show that the treatment was able to decrease the levels of the enzyme 5α-reductase type II in the rat corpus cavernosum with increased T and decreased DHT levels. This local change in T metabolism was linked to mechanisms associated with erectile dysfunction. Indeed, by targeted metabolomics, we reported a decrease in the nitric oxide synthase (NOS) activity, measured by the citrulline/arginine ratio and confirmed by the decrease in NO2 levels, and a decrease in ornithine transcarbamylase (OTC) activity, measured by citrulline/ornithine ratio. Interestingly, the T levels are negatively correlated with NOS activity, while those of DHT are positively correlated with OTC activity. Finasteride treatment also induced alterations in the levels of other molecules involved in the control of penile erection, such as norepinephrine and its metabolite, epinephrine. Indeed, plasma levels of norepinephrine and epinephrine were significantly increased and decreased, respectively, suggesting an impairment of these mediators. Interestingly, these modifications were restored by suspension of the drug. Altogether, the results reported here indicate that finasteride treatment, but not its withdrawal, affects T metabolism in the rat corpus cavernosum, and this alteration was linked to mechanisms associated with erectile dysfunction. Data here reported could also suggest that the PFS sexual side effects are more related to dysfunction in a sexual central control rather than peripheral compromised condition.

The inhibition of inner mitochondrial fusion in hepatocytes reduces non-alcoholic fatty liver and improves metabolic profile during obesity by modulating bile acid conjugation.

AIMS: Mitochondria are plastic organelles that continuously undergo biogenesis, fusion, fission, and mitophagy to control cellular energy metabolism, calcium homeostasis, hormones, sterols, and bile acids (BAs) synthesis. Here, we evaluated how the impairment of mitochondrial fusion in hepatocytes affects diet-induced liver steatosis and obesity. METHODS AND RESULTS: Male mice selectively lacking the key protein involved in inner mitochondrial fusion, optic atrophy 1 (OPA1) (OPA1ΔHep) were fed a high fat diet (HFD) for 20 weeks. OPA1ΔHep mice were protected from the development of hepatic steatosis and obesity because of reduced lipid absorption; a profile which was accompanied by increased respiratory exchange ratio in vivo, suggesting a preference for carbohydrates in OPA1ΔHep compared to controls. At the molecular level, this phenotype emerged as a consequence of poor mitochondria-peroxisome- endoplasmic reticulum (ER) tethering in OPA1 deficient hepatocytes, which impaired BAs conjugation and release in the bile, thus impacting lipid absorption from the diet. Concordantly, the liver of subjects with non-alcoholic fatty liver disease (NAFLD) presented an increased expression of OPA1 and of the network of proteins involved in mitochondrial function when compared with controls. CONCLUSION: Patients with NAFLD present increased expression of proteins involved in mitochondrial fusion in the liver. The selective deficency of OPA1 in hepatocytes protects mice from HFD-induced metabolic dysfunction by reducing BAs secretion and dietary lipids absorption as a consequence of reduced liver mitochondria-peroxisome-ER tethering.

Activation of a non-neuronal cholinergic system in visceral white adipose tissue of obese mice and humans.

BACKGROUND AND OBJECTIVES: Since white adipose tissue (WAT) lacks parasympathetic cholinergic innervation, the source of the acetylcholine (ACh) acting on white adipocyte cholinergic receptors is unknown. This study was designed to identify ACh-producing cells in mouse and human visceral WAT and to determine whether a non-neuronal cholinergic system becomes activated in obese inflamed WAT. METHODS: Mouse epididymal WAT (eWAT) and human omental fat were studied in normal and obese subjects. The expression of the key molecules involved in cholinergic signaling was evaluated by qRT-PCR and western blotting whereas their tissue distribution and cellular localization were investigated by immunohistochemistry, confocal microscopy and in situ hybridization. ACh levels were measured by liquid chromatography/tandem mass spectrometry. The cellular effects of ACh were assessed in cultured human multipotent adipose-derived stem cell (hMADS) adipocytes. RESULTS: In mouse eWAT, diet-induced obesity modulated the expression of key cholinergic molecular components and, especially, raised the expression of choline acetyltransferase (ChAT), the ACh-synthesizing enzyme, which was chiefly detected in interstitial macrophages, in macrophages forming crown-like structures (CLSs), and in multinucleated giant cells (MGCs). The stromal vascular fraction of obese mouse eWAT contained significantly higher ACh and choline levels than that of control mice. ChAT was undetectable in omental fat from healthy subjects, whereas it was expressed in a number of interstitial macrophages, CLSs, and MGCs from some obese individuals. In hMADS adipocytes stressed with tumor necrosis factor α, ACh, alone or combined with rivastigmine, significantly blunted monocyte chemoattractant protein 1 and interleukin 6 expression, it partially but significantly, restored adiponectin and GLUT4 expression, and promoted glucose uptake. CONCLUSIONS: In mouse and human visceral WAT, obesity induces activation of a macrophage-dependent non-neuronal cholinergic system that is capable of exerting anti-inflammatory and insulin-sensitizing effects on white adipocytes.

Mutant p53 sustains serine-glycine synthesis and essential amino acids intake promoting breast cancer growth.

Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent growth. These findings reveal vulnerabilities potentially exploitable for tackling breast tumors bearing missense TP53 mutations.

Butyrate prevents visceral adipose tissue inflammation and metabolic alterations in a Friedreich's ataxia mouse model.

Friedreich's ataxia (FA) is a neurodegenerative disease resulting from a mutation in the FXN gene, leading to mitochondrial frataxin deficiency. FA patients exhibit increased visceral adiposity, inflammation, and heightened diabetes risk, negatively affecting prognosis. We investigated visceral white adipose tissue (vWAT) in a murine model (KIKO) to understand its role in FA-related metabolic complications. RNA-seq analysis revealed altered expression of inflammation, angiogenesis, and fibrosis genes. Diabetes-like traits, including larger adipocytes, immune cell infiltration, and increased lactate production, were observed in vWAT. FXN downregulation in cultured adipocytes mirrored vWAT diabetes-like features, showing metabolic shifts toward glycolysis and lactate production. Metagenomic analysis indicated a reduction in fecal butyrate-producing bacteria, known to exert antidiabetic effects. A butyrate-enriched diet restrained vWAT abnormalities and mitigated diabetes features in KIKO mice. Our work emphasizes the role of vWAT in FA-related metabolic issues and suggests butyrate as a safe and promising adjunct for FA management.

Venlafaxine's effect on resilience to stress is associated with a shift in the balance between glucose and fatty acid utilization.

Brain metabolism is a fundamental process involved in the proper development of the central nervous system and in the maintenance of the main higher functions in humans. As consequence, energy metabolism imbalance has been commonly associated to several mental disorders, including depression. Here, by employing a metabolomic approach, we aimed to establish if differences in energy metabolite concentration may underlie the vulnerability and resilience in an animal model of mood disorder named chronic mild stress (CMS) paradigm. In addition, we have investigated the possibility that modulation of metabolite concentration may represent a pharmacological target for depression by testing whether repeated treatment with the antidepressant venlafaxine may normalize the pathological phenotype by acting at metabolic level. The analyses were conducted in the ventral hippocampus (vHip) for its key role in the modulation of anhedonia, a core symptom of patients affected by depression. Interestingly, we showed that a shift from glycolysis to beta oxidation seems to be responsible for the vulnerability to chronic stress and that vHip metabolism contributes to the ability of the antidepressant venlafaxine to normalize the pathological phenotype, as shown by the reversal of the changes observed in specific metabolites. These findings may provide novel perspectives on metabolic changes that could serve as diagnostic markers and preventive strategies for the early detection and treatment of depression as well as for the identification of potential drug targets.

Oxidative stress enhances the therapeutic action of a respiratory inhibitor in MYC-driven lymphoma.

MYC is a key oncogenic driver in multiple tumor types, but concomitantly endows cancer cells with a series of vulnerabilities that provide opportunities for targeted pharmacological intervention. For example, drugs that suppress mitochondrial respiration selectively kill MYC-overexpressing cells. Here, we unravel the mechanistic basis for this synthetic lethal interaction and exploit it to improve the anticancer effects of the respiratory complex I inhibitor IACS-010759. In a B-lymphoid cell line, ectopic MYC activity and treatment with IACS-010759 added up to induce oxidative stress, with consequent depletion of reduced glutathione and lethal disruption of redox homeostasis. This effect could be enhanced either with inhibitors of NADPH production through the pentose phosphate pathway, or with ascorbate (vitamin C), known to act as a pro-oxidant at high doses. In these conditions, ascorbate synergized with IACS-010759 to kill MYC-overexpressing cells in vitro and reinforced its therapeutic action against human B-cell lymphoma xenografts. Hence, complex I inhibition and high-dose ascorbate might improve the outcome of patients affected by high-grade lymphomas and potentially other MYC-driven cancers.

Spinocerebellar ataxia 38: structure-function analysis shows ELOVL5 G230V is proteotoxic, conformationally altered and a mutational hotspot.

Fatty acid elongase ELOVL5 is part of a protein family of multipass transmembrane proteins that reside in the endoplasmic reticulum where they regulate long-chain fatty acid elongation. A missense variant (c.689G>T p.Gly230Val) in ELOVL5 causes Spinocerebellar Ataxia subtype 38 (SCA38), a neurodegenerative disorder characterized by autosomal dominant inheritance, cerebellar Purkinje cell demise and adult-onset ataxia. Having previously showed aberrant accumulation of p.G230V in the Golgi complex, here we further investigated the pathogenic mechanisms triggered by p.G230V, integrating functional studies with bioinformatic analyses of protein sequence and structure. Biochemical analysis showed that p.G230V enzymatic activity was normal. In contrast, SCA38-derived fibroblasts showed reduced expression of ELOVL5, Golgi complex enlargement and increased proteasomal degradation with respect to controls. By heterologous overexpression, p.G230V was significantly more active than wild-type ELOVL5 in triggering the unfolded protein response and in decreasing viability in mouse cortical neurons. By homology modelling, we generated native and p.G230V protein structures whose superposition revealed a shift in Loop 6 in p.G230V that altered a highly conserved intramolecular disulphide bond. The conformation of this bond, connecting Loop 2 and Loop 6, appears to be elongase-specific. Alteration of this intramolecular interaction was also observed when comparing wild-type ELOVL4 and the p.W246G variant which causes SCA34. We demonstrate by sequence and structure analyses that ELOVL5 p.G230V and ELOVL4 p.W246G are position-equivalent missense variants. We conclude that SCA38 is a conformational disease and propose combined loss of function by mislocalization and gain of toxic function by ER/Golgi stress as early events in SCA38 pathogenesis.

Sustained IP3-linked Ca2+ signaling promotes progression of triple negative breast cancer cells by regulating fatty acid metabolism.

Rewiring of mitochondrial metabolism has been described in different cancers as a key step for their progression. Calcium (Ca2+) signaling regulates mitochondrial function and is known to be altered in several malignancies, including triple negative breast cancer (TNBC). However, whether and how the alterations in Ca2+ signaling contribute to metabolic changes in TNBC has not been elucidated. Here, we found that TNBC cells display frequent, spontaneous inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations, which are sensed by mitochondria. By combining genetic, pharmacologic and metabolomics approaches, we associated this pathway with the regulation of fatty acid (FA) metabolism. Moreover, we demonstrated that these signaling routes promote TNBC cell migration in vitro, suggesting they might be explored to identify potential therapeutic targets.

Bicalutamide and Trehalose Ameliorate Spinal and Bulbar Muscular Atrophy Pathology in Mice.

Spinal and bulbar muscular atrophy (SBMA) is characterized by motor neuron (MN) degeneration that leads to slowly progressive muscle weakness. It is considered a neuromuscular disease since muscle has a primary role in disease onset and progression. SBMA is caused by a CAG triplet repeat expansion in the androgen receptor (AR) gene. The translated poly-glutamine (polyQ) tract confers a toxic gain of function to the mutant AR altering its folding, causing its aggregation into intracellular inclusions, and impairing the autophagic flux. In an in vitro SBMA neuronal model, we previously showed that the antiandrogen bicalutamide and trehalose, a natural disaccharide stimulating autophagy, block ARpolyQ activation, reduce its nuclear translocation and toxicity and facilitate the autophagic degradation of cytoplasmic AR aggregates. Here, in a knock-in SBMA mouse model (KI AR113Q), we show that bicalutamide and trehalose ameliorated SBMA pathology. Bicalutamide reversed the formation of the AR insoluble forms in KI AR113Q muscle, preventing autophagic flux blockage. We demonstrated that apoptosis is activated in KI AR113Q muscle, and that both compounds prevented its activation. We detected a decrease of mtDNA and an increase of OXPHOS enzymes, already at early symptomatic stages; these alterations were reverted by trehalose. Overall, bicalutamide and/or trehalose led to a partial recovery of muscle morphology and function, and improved SBMA mouse motor behavior, inducing an extension of their survival. Thus, bicalutamide and trehalose, by counteracting ARpolyQ toxicity in skeletal muscle, are valuable candidates for future clinical trials in SBMA patients.

Liver-directed gene therapy for ornithine aminotransferase deficiency.

Gyrate atrophy of choroid and retina (GACR) is a chorioretinal degeneration caused by pathogenic variants in the gene encoding ornithine aminotransferase (OAT), an enzyme mainly expressed in liver. Affected patients have increased ornithine concentrations in blood and other body fluids and develop progressive constriction of vision fields leading to blindness. Current therapies are unsatisfactory and better treatments are highly needed. In two mouse models of OAT deficiency that recapitulates biochemical and retinal changes of GACR, we investigated the efficacy of an intravenously injected serotype 8 adeno-associated (AAV8) vector expressing OAT under the control of a hepatocyte-specific promoter. Following injections, OAT-deficient mice showed reductions of ornithine concentrations in blood and eye cups compared with control mice injected with a vector expressing green fluorescent protein. AAV-injected mice showed improved electroretinogram response and partial restoration of retinal structure up to one-year post-injection. In summary, hepatic OAT expression by AAV8 vector was effective at correction of hyperornithinemia and improved function and structure of the retina. In conclusion, this study provides proof-of-concept of efficacy of liver-directed AAV-mediated gene therapy of GACR.