A PET-Surrogate Signature for the Interrogation of the Metabolic Status of Breast Cancers.

Metabolic alterations in cancers can be exploited for diagnostic, prognostic, and therapeutic purposes. This is exemplified by 18F-fluorodeoxyglucose (FDG)-positron emission tomography (FDG-PET), an imaging tool that relies on enhanced glucose uptake by tumors for diagnosis and staging. By performing transcriptomic analysis of breast cancer (BC) samples from patients stratified by FDG-PET, a 54-gene signature (PETsign) is identified that recapitulates FDG uptake. PETsign is independently prognostic of clinical outcome in luminal BCs, the most common and heterogeneous BC molecular subtype, which requires improved stratification criteria to guide therapeutic decision-making. The prognostic power of PETsign is stable across independent BC cohorts and disease stages including the earliest BC stage, arguing that PETsign is an ab initio metabolic signature. Transcriptomic and metabolomic analysis of BC cells reveals that PETsign predicts enhanced glycolytic dependence and reduced reliance on fatty acid oxidation. Moreover, coamplification of PETsign genes occurs frequently in BC arguing for their causal role in pathogenesis. CXCL8 and EGFR signaling pathways feature strongly in PETsign, and their activation in BC cells causes a shift toward a glycolytic phenotype. Thus, PETsign serves as a molecular surrogate for FDG-PET that could inform clinical management strategies for BC patients.

Mitochondrial Ca2+ signaling is a hallmark of specific adipose tissue-cancer crosstalk.

Obesity is associated with increased risk and worse prognosis of many tumours including those of the breast and of the esophagus. Adipokines released from the peritumoural adipose tissue promote the metastatic potential of cancer cells, suggesting the existence of a crosstalk between the adipose tissue and the surrounding tumour. Mitochondrial Ca2+ signaling contributes to the progression of carcinoma of different origins. However, whether adipocyte-derived factors modulate mitochondrial Ca2+ signaling in tumours is unknown. Here, we show that conditioned media derived from adipose tissue cultures (ADCM) enriched in precursor cells impinge on mitochondrial Ca2+ homeostasis of target cells. Moreover, in modulating mitochondrial Ca2+ responses, a univocal crosstalk exists between visceral adipose tissue-derived preadipocytes and esophageal cancer cells, and between subcutaneous adipose tissue-derived preadipocytes and triple-negative breast cancer cells. An unbiased metabolomic analysis of ADCM identified creatine and creatinine for their ability to modulate mitochondrial Ca2+ uptake, migration and proliferation of esophageal and breast tumour cells, respectively.

The mitochondrial ATP-dependent potassium channel (mitoKATP) controls skeletal muscle structure and function.

MitoKATP is a channel of the inner mitochondrial membrane that controls mitochondrial K+ influx according to ATP availability. Recently, the genes encoding the pore-forming (MITOK) and the regulatory ATP-sensitive (MITOSUR) subunits of mitoKATP were identified, allowing the genetic manipulation of the channel. Here, we analyzed the role of mitoKATP in determining skeletal muscle structure and activity. Mitok-/- muscles were characterized by mitochondrial cristae remodeling and defective oxidative metabolism, with consequent impairment of exercise performance and altered response to damaging muscle contractions. On the other hand, constitutive mitochondrial K+ influx by MITOK overexpression in the skeletal muscle triggered overt mitochondrial dysfunction and energy default, increased protein polyubiquitination, aberrant autophagy flux, and induction of a stress response program. MITOK overexpressing muscles were therefore severely atrophic. Thus, the proper modulation of mitoKATP activity is required for the maintenance of skeletal muscle homeostasis and function.

Sunflower seed-derived bioactive peptides show antioxidant and anti-inflammatory activity: From in silico simulation to the animal model.

The evolving field of food technology is increasingly dedicated to developing functional foods. This study explored bioactive peptides from sunflower protein isolate (SPI), obtained from defatted flour, a by-product of the oil processing industry. SPI underwent simulated gastrointestinal digestion and the obtained peptide-enriched fraction (PEF) showed antioxidant properties in vivo, in zebrafish. Among the peptides present in PEF identified by mass spectrometry analysis, we selected those with antioxidant properties by in silico evaluation, considering their capability to interact with Keap1, key protein in the regulation of antioxidant response. The selected peptides were synthesized and evaluated in a cellular model. As a result, DVAMPVPK, VETGVIKPG, TTHTNPPPEAE, LTHPQHQQQGPSTG and PADVTPEEKPEV activated Keap1/Nrf2 pathway leading to Antioxidant Response Element-regulated enzymes upregulation. Since the crosstalk between Nrf2 and NF-κB is well known, the potential anti-inflammatory activity of the peptides was assessed and principally PADVTPEEKPEV showed good features both as antioxidant and anti-inflammatory molecule.

Negative modulation of mitochondrial calcium uniporter complex protects neurons against ferroptosis.

Ferroptosis is an iron- and reactive oxygen species (ROS)-dependent form of regulated cell death, that has been implicated in Alzheimer's disease and Parkinson's disease. Inhibition of cystine/glutamate antiporter could lead to mitochondrial fragmentation, mitochondrial calcium ([Ca2+]m) overload, increased mitochondrial ROS production, disruption of the mitochondrial membrane potential (ΔΨm), and ferroptotic cell death. The observation that mitochondrial dysfunction is a characteristic of ferroptosis makes preservation of mitochondrial function a potential therapeutic option for diseases associated with ferroptotic cell death. Mitochondrial calcium levels are controlled via the mitochondrial calcium uniporter (MCU), the main entry point of Ca2+ into the mitochondrial matrix. Therefore, we have hypothesized that negative modulation of MCU complex may confer protection against ferroptosis. Here we evaluated whether the known negative modulators of MCU complex, ruthenium red (RR), its derivative Ru265, mitoxantrone (MX), and MCU-i4 can prevent mitochondrial dysfunction and ferroptotic cell death. These compounds mediated protection in HT22 cells, in human dopaminergic neurons and mouse primary cortical neurons against ferroptotic cell death. Depletion of MICU1, a [Ca2+]m gatekeeper, demonstrated that MICU is protective against ferroptosis. Taken together, our results reveal that negative modulation of MCU complex represents a therapeutic option to prevent degenerative conditions, in which ferroptosis is central to the progression of these pathologies.

The mitochondrial calcium uniporter complex-A play in five acts.

Mitochondrial Ca2+ (mitCa2+) uptake controls both intraorganellar and cytosolic functions. Within the organelle, [Ca2+] increases regulate the activity of tricarboxylic acid (TCA) cycle enzymes, thus sustaining oxidative metabolism and ATP production. Reactive oxygen species (ROS) are also generated as side products of oxygen consumption. At the same time, mitochondria act as buffers of cytosolic Ca2+ (cytCa2+) increases, thus regulating Ca2+-dependent cellular processes. In pathological conditions, mitCa2+ overload triggers the opening of the mitochondrial permeability transition pore (mPTP) and the release of apoptotic cofactors. MitCa2+ uptake occurs in response of local [Ca2+] increases in sites of proximity between the endoplasmic reticulum (ER) and the mitochondria and is mediated by the mitochondrial Ca2+ uniporter (MCU), a highly selective channel of the inner mitochondrial membrane (IMM). Both channel and regulatory subunits form the MCU complex (MCUC). Cryogenic electron microscopy (Cryo-EM) and crystal structures revealed the correct assembly of MCUC and the function of critical residues for the regulation of Ca2+ conductance.

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.

Defective excitation-contraction coupling and mitochondrial respiration precede mitochondrial Ca2+ accumulation in spinobulbar muscular atrophy skeletal muscle.

Polyglutamine expansion in the androgen receptor (AR) causes spinobulbar muscular atrophy (SBMA). Skeletal muscle is a primary site of toxicity; however, the current understanding of the early pathological processes that occur and how they unfold during disease progression remains limited. Using transgenic and knock-in mice and patient-derived muscle biopsies, we show that SBMA mice in the presymptomatic stage develop a respiratory defect matching defective expression of genes involved in excitation-contraction coupling (ECC), altered contraction dynamics, and increased fatigue. These processes are followed by stimulus-dependent accumulation of calcium into mitochondria and structural disorganization of the muscle triads. Deregulation of expression of ECC genes is concomitant with sexual maturity and androgen raise in the serum. Consistent with the androgen-dependent nature of these alterations, surgical castration and AR silencing alleviate the early and late pathological processes. These observations show that ECC deregulation and defective mitochondrial respiration are early but reversible events followed by altered muscle force, calcium dyshomeostasis, and dismantling of triad structure.

The mitochondrial calcium homeostasis orchestra plays its symphony: Skeletal muscle is the guest of honor.

Skeletal muscle mitochondria are placed in close proximity of the sarcoplasmic reticulum (SR), the main intracellular Ca2+ store. During muscle activity, excitation of sarcolemma and of T-tubule triggers the release of Ca2+ from the SR initiating myofiber contraction. The rise in cytosolic Ca2+ determines the opening of the mitochondrial calcium uniporter (MCU), the highly selective channel of the inner mitochondrial membrane (IMM), causing a robust increase in mitochondrial Ca2+ uptake. The Ca2+-dependent activation of TCA cycle enzymes increases the synthesis of ATP required for SERCA activity. Thus, Ca2+ is transported back into the SR and cytosolic [Ca2+] returns to resting levels eventually leading to muscle relaxation. In recent years, thanks to the molecular identification of MCU complex components, the role of mitochondrial Ca2+ uptake in the pathophysiology of skeletal muscle has been uncovered. In this chapter, we will introduce the reader to a general overview of mitochondrial Ca2+ accumulation. We will tackle the key molecular players and the cellular and pathophysiological consequences of mitochondrial Ca2+ dyshomeostasis. In the second part of the chapter, we will discuss novel findings on the physiological role of mitochondrial Ca2+ uptake in skeletal muscle. Finally, we will examine the involvement of mitochondrial Ca2+ signaling in muscle diseases.

Identification and functional validation of FDA-approved positive and negative modulators of the mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ entry, plays important roles in physiology and pathology. However, only few pharmacological compounds directly and selectively modulate its activity. Here, we perform high-throughput screening on a US Food and Drug Administration (FDA)-approved drug library comprising 1,600 compounds to identify molecules modulating mitochondrial Ca2+ uptake. We find amorolfine and benzethonium to be positive and negative MCU modulators, respectively. In agreement with the positive effect of MCU in muscle trophism, amorolfine increases muscle size, and MCU silencing is sufficient to blunt amorolfine-induced hypertrophy. Conversely, in the triple-negative breast cancer cell line MDA-MB-231, benzethonium delays cell growth and migration in an MCU-dependent manner and protects from ceramide-induced apoptosis, in line with the role of mitochondrial Ca2+ uptake in cancer progression. Overall, we identify amorolfine and benzethonium as effective MCU-targeting drugs applicable to a wide array of experimental and disease conditions.

In the right place at the right time: ROS and Ca2+ are allies in the battle for survival.

Both Ca2+ and reactive oxygen species (ROS) are double face entities, acting as signaling messengers or cell fate determinants according to their concentration and to spatial temporal restrictions. Recently, Beretta and colleagues found that ROS generated at ER-mitochondria contact sites (MAMs) support cell survival in stress conditions by decreasing inter-organelle Ca2+ transfer.

Skeletal muscle mitochondria in health and disease.

Mitochondrial activity warrants energy supply to oxidative myofibres to sustain endurance workload. The maintenance of mitochondrial homeostasis is ensured by the control of fission and fusion processes and by the mitophagic removal of aberrant organelles. Many diseases are due to or characterized by dysfunctional mitochondria, and altered mitochondrial dynamics or turnover trigger myopathy per se. In this review, we will tackle the role of mitochondrial dynamics, turnover and metabolism in skeletal muscle, both in health and disease.

The Mitochondrial Ca2+ Uptake and the Fine-Tuning of Aerobic Metabolism.

Recently, the role of mitochondrial activity in high-energy demand organs and in the orchestration of whole-body metabolism has received renewed attention. In mitochondria, pyruvate oxidation, ensured by efficient mitochondrial pyruvate entry and matrix dehydrogenases activity, generates acetyl CoA that enters the TCA cycle. TCA cycle activity, in turn, provides reducing equivalents and electrons that feed the electron transport chain eventually producing ATP. Mitochondrial Ca2+ uptake plays an essential role in the control of aerobic metabolism. Mitochondrial Ca2+ accumulation stimulates aerobic metabolism by inducing the activity of three TCA cycle dehydrogenases. In detail, matrix Ca2+ indirectly modulates pyruvate dehydrogenase via pyruvate dehydrogenase phosphatase 1, and directly activates isocitrate and α-ketoglutarate dehydrogenases. Here, we will discuss the contribution of mitochondrial Ca2+ uptake to the metabolic homeostasis of organs involved in systemic metabolism, including liver, skeletal muscle, and adipose tissue. We will also tackle the role of mitochondrial Ca2+ uptake in the heart, a high-energy consuming organ whose function strictly depends on appropriate Ca2+ signaling.

A High-Throughput Screening Identifies MICU1 Targeting Compounds.

Mitochondrial Ca2+ uptake depends on the mitochondrial calcium uniporter (MCU) complex, a highly selective channel of the inner mitochondrial membrane (IMM). Here, we screen a library of 44,000 non-proprietary compounds for their ability to modulate mitochondrial Ca2+ uptake. Two of them, named MCU-i4 and MCU-i11, are confirmed to reliably decrease mitochondrial Ca2+ influx. Docking simulations reveal that these molecules directly bind a specific cleft in MICU1, a key element of the MCU complex that controls channel gating. Accordingly, in MICU1-silenced or deleted cells, the inhibitory effect of the two compounds is lost. Moreover, MCU-i4 and MCU-i11 fail to inhibit mitochondrial Ca2+ uptake in cells expressing a MICU1 mutated in the critical amino acids that forge the predicted binding cleft. Finally, these compounds are tested ex vivo, revealing a primary role for mitochondrial Ca2+ uptake in muscle growth. Overall, MCU-i4 and MCU-i11 represent leading molecules for the development of MICU1-targeting drugs.

Crosstalk between Mitochondrial Ca2+ Uptake and Autophagy in Skeletal Muscle.

Autophagy is responsible for the maintenance of skeletal muscle homeostasis, thanks to the removal of aberrant and dysfunctional macromolecules and organelles. During fasting, increased autophagy ensures the maintenance of the amino acid pool required for energy production. The activity of the mitochondrial Ca2+ uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ uptake, controls skeletal muscle size, force, and nutrient utilization. Thus, both autophagy and mitochondrial Ca2+ accumulation play a pivotal role to maintain muscle homeostasis and to sustain muscle function. Here, we address whether, in skeletal muscle, mitochondrial Ca2+ uptake and autophagy are mutually related. Muscle-restricted MCU silencing partially inhibits the autophagy flux. Moreover, skeletal muscle-specific deletion of the essential autophagy gene Atg7, known to cause the accumulation of dysfunctional mitochondria, drastically reduces mitochondrial Ca2+ accumulation. Thus, a vicious cycle takes place, in which reduced MCU activity hampers the autophagic flux, and loss of autophagy further impairs mitochondrial Ca2+ signaling.

DRP1-mediated mitochondrial shape controls calcium homeostasis and muscle mass.

Mitochondrial quality control is essential in highly structured cells such as neurons and muscles. In skeletal muscle the mitochondrial fission proteins are reduced in different physiopathological conditions including ageing sarcopenia, cancer cachexia and chemotherapy-induced muscle wasting. However, whether mitochondrial fission is essential for muscle homeostasis is still unclear. Here we show that muscle-specific loss of the pro-fission dynamin related protein (DRP) 1 induces muscle wasting and weakness. Constitutive Drp1 ablation in muscles reduces growth and causes animal death while inducible deletion results in atrophy and degeneration. Drp1 deficient mitochondria are morphologically bigger and functionally abnormal. The dysfunctional mitochondria signals to the nucleus to induce the ubiquitin-proteasome system and an Unfolded Protein Response while the change of mitochondrial volume results in an increase of mitochondrial Ca2+ uptake and myofiber death. Our findings reveal that morphology of mitochondrial network is critical for several biological processes that control nuclear programs and Ca2+ handling.

A Synthetic Fluorescent Mitochondria-Targeted Sensor for Ratiometric Imaging of Calcium in Live Cells.

Ca2+ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca2+ concentration in living cells is of pivotal interest. Genetically-encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria-targeted synthetic Ca2+ indicators are plagued by several drawbacks, for example, non-specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca2+ sensor, named mt-fura-2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca2+ indicator fura-2. Mt-fura-2 binds Ca2+ with a dissociation constant of ≈1.5 µm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca2+ ] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt-fura-2 to cell types where the delivery of genetically-encoded indicators is troublesome.

PSEN2 (presenilin 2) mutants linked to familial Alzheimer disease impair autophagy by altering Ca2+ homeostasis.

PSEN2 (presenilin 2) is one of the 3 proteins that, when mutated, causes early onset familial Alzheimer disease (FAD) cases. In addition to its well-known role within the γ-secretase complex (the enzyme ultimately responsible for Aß peptides formation), PSEN2 is endowed with some γ-secretase-independent functions in distinct cell signaling pathways, such as the modulation of intracellular Ca2+ homeostasis. Here, by using different FAD-PSEN2 cell models, we demonstrate that mutated PSEN2 impairs autophagy by causing a block in the degradative flux at the level of the autophagosome-lysosome fusion step. The defect does not depend on an altered lysosomal functionality but rather on a decreased recruitment of the small GTPase RAB7 to autophagosomes, a key event for normal autophagy progression. Importantly, FAD-PSEN2 action on autophagy is unrelated to its γ-secretase activity but depends on its previously reported ability to partially deplete ER Ca2+ content, thus reducing cytosolic Ca2+ response upon IP3-linked cell stimulations. Our data sustain the pivotal role for Ca2+ signaling in autophagy and reveal a novel mechanism by which FAD-linked presenilins alter the degradative process, reinforcing the view of a causative role for a dysfunctional quality control pathway in AD neurodegeneration.Abbreviations: Aß: amyloid ß; AD: Alzheimer disease; ACTB: actin beta; AMPK: AMP-activated protein kinase; APP: amyloid-beta precursor protein; BafA: bafilomycin A1; BAPTA-AM: 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; CFP: cyan fluorescent protein; EGTA-AM: ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester; ER: endoplasmic reticulum; EGFP-HDQ74: enhanced GFP-huntingtin exon 1 containing 74 polyglutamine repeats; FAD: familial Alzheimer disease; FCS: fetal calf serum; FRET: fluorescence/Förster resonance energy transfer; GFP: green fluorescent protein; IP3: inositol trisphosphate; KD: knockdown; LAMP1: lysosomal associated membrane protein 1; MAP1LC3-II/LC3-II: lipidated microtubule-associated protein 1 light chain 3; MCU: mitochondrial calcium uniporter; MICU1: mitochondrial calcium uptake 1; MEFs: mouse embryonic fibroblasts; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; SQSTM1/p62: sequestosome 1; PSEN1: presenilin 1; PSEN2: presenilin 2; RAB7: RAB7A: member RAS oncogene family; RFP: red fluorescent protein; ATP2A/SERCA: ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting; siRNA: small interference RNA; V-ATPase: vacuolar-type H+-ATPase; WT: wild type.

Ex Vivo Measurements of Ca2+ Transients in Intracellular Compartments of Skeletal Muscle Fibers by Means of Genetically Encoded Probes.

We report a method for ex vivo measurements of Ca2+ transients in skeletal muscle fibers, both in the sarcoplasma and into the mitochondria. These measurements are based on the use of genetically encoded probes. Addition of targeting DNA sequences, in frame with the probe encoding sequence, ensures protein expression in specific compartments. The use of probes with different excitation spectra allows the simultaneous determination of cytosolic and mitochondrial Ca2+ transients in the same fiber. Probe encoding plasmids are expressed in flexor digitorum brevis (FDB) muscles by means of the in vivo electroporation technique. Measurements are then performed ex vivo in isolated single myofibers.

Loss of mitochondrial calcium uniporter rewires skeletal muscle metabolism and substrate preference.

Skeletal muscle mitochondria readily accumulate Ca2+ in response to SR store-releasing stimuli thanks to the activity of the mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ uptake. MCU positively regulates myofiber size in physiological conditions and counteracts pathological loss of muscle mass. Here we show that skeletal muscle-specific MCU deletion inhibits myofiber mitochondrial Ca2+ uptake, impairs muscle force and exercise performance, and determines a slow to fast switch in MHC expression. Mitochondrial Ca2+ uptake is required for effective glucose oxidation, as demonstrated by the fact that in muscle-specific MCU-/- myofibers oxidative metabolism is impaired and glycolysis rate is increased. Although defective, mitochondrial activity is partially sustained by increased fatty acid (FA) oxidation. In MCU-/- myofibers, PDP2 overexpression drastically reduces FA dependency, demonstrating that decreased PDH activity is the main trigger of the metabolic rewiring of MCU-/- muscles. Accordingly, PDK4 overexpression in MCUfl/fl myofibers is sufficient to increase FA-dependent respiration. Finally, as a result of the muscle-specific MCU deletion, a systemic catabolic response impinging on both liver and adipose tissue metabolism occurs.