Publications by authors named "Diego De Stefani"

45 Publications

A Novel Loss of Function Melanocortin-4-Receptor Mutation (MC4R-F313Sfs*29) in Morbid Obesity.

J Clin Endocrinol Metab 2020 Nov 28. Epub 2020 Nov 28.

Department of Medicine - DIMED, University of Padua, Padua, Italy.

Context: Melanocortin receptor-4 (MC4R) gene mutations are associated with early-onset severe obesity and the identification of potential pathological variants is crucial for the clinical management of patients with obesity.

Objective: To explore whether and how a novel heterozygous MC4R variant (MC4R-F313Sfs*29), identified in a young boy (BMI 38.8 kg/m 2) during a mutation analysis conducted in a cohort of patients with obesity, plays a determinant pathophysiological role in the obesity development.

Design Setting And Patients: The genetic screening was carried out in a total of 209 unrelated patients with obesity (BMI ≥ 35 kg/m 2). Structural and functional characterization of the F313Sfs*29-mutated MC4R was performed using computational approaches and in vitro, using HEK293 cells transfected with genetically-encoded biosensors for cAMP and Ca 2+.

Results: The F313Sfs*29 was the only variant identified. In vitro experiments showed that HEK293 cells transfected with the mutated form of MC4R did not increase intracellular cAMP or Ca 2+ levels after the stimulation with a specific agonist in comparison with HEK293 cells transfected with the wild type form of MC4R (∆R/R0= -90%±8%; p<0,001). In silico modelling showed that the F313Sfs*29 mutation causes a major reorganization cytosolic domain of the MC4R, thus reducing the affinity of the putative GalphaS binding site.

Conclusions: The newly discovered F313Sfs*29 variant of MC4R may be involved in the impairment of α-MSH-induced cAMP and Ca 2+ signaling, blunting intracellular G protein mediated signal transduction. This alteration might have led to the dysregulation of satiety signaling, resulting in hyperphagia and early onset of obesity.
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http://dx.doi.org/10.1210/clinem/dgaa885DOI Listing
November 2020

A new target for an old DUB: UCH-L1 regulates mitofusin-2 levels, altering mitochondrial morphology, function and calcium uptake.

Redox Biol 2020 10 7;37:101676. Epub 2020 Aug 7.

Obesity Research Center, Molecular Medicine, Boston University School of Medicine, Boston, MA, 02111, USA; UCLA Section of Endocrinology, Department of Medicine, David Geffen School of Medicine, UCLA, CA, 9095-7073, USA. Electronic address:

UCH-L1 is a deubiquitinating enzyme (DUB), highly abundant in neurons, with a sub-cellular localization dependent on its farnesylation state. Despite UCH-L1's association with familial Parkinson's Disease (PD), the effects on mitochondrial bioenergetics and quality control remain unexplored. Here we investigated the role of UCHL-1 in mitochondrial dynamics and bioenergetics. We demonstrate that knock-down (KD) of UCH-L1 in different cell lines reduces the levels of the mitochondrial fusion protein Mitofusin-2, but not Mitofusin-1, resulting in mitochondrial enlargement and disruption of the tubular network. This was associated with lower tethering between mitochondria and the endoplasmic reticulum, consequently altering mitochondrial calcium uptake. Respiratory function was also altered, as UCH-L1 KD cells displayed higher proton leak and maximum respiratory capacity. Conversely, overexpression of UCH-L1 increased Mfn2 levels, an effect dramatically enhanced by the mutation of the farnesylation site (C220S), which drives UCH-L1 binding to membranes. These data indicate that the soluble cytosolic form of UCH-L1 regulates Mitofusin-2 levels and mitochondrial function. These effects are biologically conserved, since knock-down of the corresponding UCH-L1 ortholog in D. melanogaster reduces levels of the mitofusin ortholog Marf and also increases mitochondrial respiratory capacity. We thus show that Mfn-2 levels are directly affected by UCH-L1, demonstrating that the mitochondrial roles of DUBs go beyond controlling mitophagy rates.
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http://dx.doi.org/10.1016/j.redox.2020.101676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509235PMC
October 2020

Modulation of TRPV-1 by prostaglandin-E and bradykinin changes cough sensitivity and autonomic regulation of cardiac rhythm in healthy subjects.

Sci Rep 2020 09 16;10(1):15163. Epub 2020 Sep 16.

Department of Cardiac, Thoracic, Vascular Sciences and Public Health, University of Padova, Via Giustiniani 2, 35128, Padua, Italy.

A neurogenic pathway, involving airway TRPV-1, has been implicated in acute cardiovascular events occurring after peaks of air pollution. We tested whether inhaled prostaglandin-E (PGE) and bradykinin (BK) regulate TRPV-1 activity in vivo by changing cough response to capsaicin (CPS) and affecting heart rate variability (HRV), while also taking into account the influence of TRPV-1 polymorphisms (SNPs). Moreover, we assessed the molecular mechanism of TRPV-1 modulation in vitro. Seventeen healthy volunteers inhaled 100 μg PGE, 200 μg BK or diluent in a randomized double-blind fashion. Subsequently, the response to CPS was assessed by cough challenge and the sympathetic activity by HRV, expressed by low (nLF) and high (nHF) normalized frequency components, as well as nLF/nHF ratio. Intracellular [Ca] was measured in HeLa cells, transfected with wild-type TRPV-1, pre-treated with increasing doses of PGE, BK or diesel exhaust particulate (DEP), after CPS stimulation. Six functional TRPV-1 SNPs were characterized in DNA from each subject. Inhalation of PGE and BK was associated with significant increases in cough response induced by 30 μM of CPS (cough number after PGE = 4.20 ± 0.42; p < 0.001, and after BK = 3.64 ± 0.37; p < 0.01), compared to diluent (2.77 ± 0.29) and in sympathetic activity (nLF/nHF ratio after PGE = 6.1; p < 0.01, and after BK = 4.2; p < 0.05), compared to diluent (2.5-3.3). No influence of SNPs was observed on autonomic regulation and cough sensitivity. Unlike PGE and BK, DEP directly activated TRPV-1. Inhalation of PGE and BK sensitizes TRPV-1 and is associated with autonomic dysregulation of cardiac rhythm in healthy subjects.
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http://dx.doi.org/10.1038/s41598-020-72062-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494872PMC
September 2020

Altered MICOS Morphology and Mitochondrial Ion Homeostasis Contribute to Poly(GR) Toxicity Associated with C9-ALS/FTD.

Cell Rep 2020 08;32(5):107989

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. Electronic address:

Amyotrophic lateral sclerosis (ALS) manifests pathological changes in motor neurons and various other cell types. Compared to motor neurons, the contribution of the other cell types to the ALS phenotypes is understudied. G4C2 repeat expansion in C9ORF72 is the most common genetic cause of ALS along with frontotemporal dementia (C9-ALS/FTD), with increasing evidence supporting repeat-encoded poly(GR) in disease pathogenesis. Here, we show in Drosophila muscle that poly(GR) enters mitochondria and interacts with components of the Mitochondrial Contact Site and Cristae Organizing System (MICOS), altering MICOS dynamics and intra-subunit interactions. This impairs mitochondrial inner membrane structure, ion homeostasis, mitochondrial metabolism, and muscle integrity. Similar mitochondrial defects are observed in patient fibroblasts. Genetic manipulation of MICOS components or pharmacological restoration of ion homeostasis with nigericin effectively rescue the mitochondrial pathology and disease phenotypes in both systems. These results implicate MICOS-regulated ion homeostasis in C9-ALS pathogenesis and suggest potential new therapeutic strategies.
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http://dx.doi.org/10.1016/j.celrep.2020.107989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7433775PMC
August 2020

Polyphenols as Caloric Restriction Mimetics Regulating Mitochondrial Biogenesis and Mitophagy.

Trends Endocrinol Metab 2020 07 17;31(7):536-550. Epub 2020 Mar 17.

Department of Medicine and Health Sciences 'V. Tiberio', University of Molise, Campobasso, Italy.

The tight coordination between mitochondrial biogenesis and mitophagy can be dysregulated during aging, critically influencing whole-body metabolism, health, and lifespan. To date, caloric restriction (CR) appears to be the most effective intervention strategy to improve mitochondrial turnover in aging organisms. The development of pharmacological mimetics of CR has gained attention as an attractive and potentially feasible approach to mimic the CR phenotype. Polyphenols, ubiquitously present in fruits and vegetables, have emerged as well-tolerated CR mimetics that target mitochondrial turnover. Here, we discuss the molecular mechanisms that orchestrate mitochondrial biogenesis and mitophagy, and we summarize the current knowledge of how CR promotes mitochondrial maintenance and to what extent different polyphenols may mimic CR and coordinate mitochondrial biogenesis and clearance.
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http://dx.doi.org/10.1016/j.tem.2020.02.011DOI Listing
July 2020

Mitochondrial ion channels as targets for cardioprotection.

J Cell Mol Med 2020 07 3;24(13):7102-7114. Epub 2020 Jun 3.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

Acute myocardial infarction (AMI) and the heart failure (HF) that often result remain the leading causes of death and disability worldwide. As such, new therapeutic targets need to be discovered to protect the myocardium against acute ischaemia/reperfusion (I/R) injury in order to reduce myocardial infarct (MI) size, preserve left ventricular function and prevent the onset of HF. Mitochondrial dysfunction during acute I/R injury is a critical determinant of cell death following AMI, and therefore, ion channels in the inner mitochondrial membrane, which are known to influence cell death and survival, provide potential therapeutic targets for cardioprotection. In this article, we review the role of mitochondrial ion channels, which are known to modulate susceptibility to acute myocardial I/R injury, and we explore their potential roles as therapeutic targets for reducing MI size and preventing HF following AMI.
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http://dx.doi.org/10.1111/jcmm.15341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339171PMC
July 2020

Biosensors for detection of calcium.

Methods Cell Biol 2020 22;155:337-368. Epub 2019 Nov 22.

Department of Biomedical Sciences, University of Padova, Padova, Italy. Electronic address:

Calcium (Ca) is a universal intracellular messenger capable of governing a plethora of different biological functions. Its versatility is guaranteed on the one hand by a cell type-specific Ca signaling toolkit. On the other hand, the fine compartmentalization of changes in Ca concentration ([Ca]) into specific subcellular domains adds a level of complexity, thus generating a variety of signals that can be differentially decoded into specific cellular events. In this context, mitochondrial Ca dynamics plays a central role, by regulating both specific organelle functions (e.g., regulation of substrate oxidation, release of caspase cofactors) and global cellular events (e.g., shaping of cytoplasmic Ca waves). Here we describe a general method for the detection of intramitochondrial [Ca] using bioluminescent and fluorescent genetically-encoded Ca indicators (GECIs). We will discuss the characteristics of different GECIs, as well as their strengths, limitations and applications.
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http://dx.doi.org/10.1016/bs.mcb.2019.11.001DOI Listing
December 2020

A High-Throughput Screening Identifies MICU1 Targeting Compounds.

Cell Rep 2020 02;30(7):2321-2331.e6

Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy. Electronic address:

Mitochondrial Ca 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 Ca uptake. Two of them, named MCU-i4 and MCU-i11, are confirmed to reliably decrease mitochondrial Ca 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 Ca 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 Ca uptake in muscle growth. Overall, MCU-i4 and MCU-i11 represent leading molecules for the development of MICU1-targeting drugs.
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http://dx.doi.org/10.1016/j.celrep.2020.01.081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034061PMC
February 2020

Overexpression of Mitochondrial Calcium Uniporter Causes Neuronal Death.

Oxid Med Cell Longev 2019 16;2019:1681254. Epub 2019 Oct 16.

Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58B, Padova, Italy.

Neurodegenerative diseases are a large and heterogeneous group of disorders characterized by selective and progressive death of specific neuronal subtypes. In most of the cases, the pathophysiology is still poorly understood, although a number of hypotheses have been proposed. Among these, dysregulation of Ca homeostasis and mitochondrial dysfunction represent two broadly recognized early events associated with neurodegeneration. However, a direct link between these two hypotheses can be drawn. Mitochondria actively participate to global Ca signaling, and increases of [Ca] inside organelle matrix are known to sustain energy production to modulate apoptosis and remodel cytosolic Ca waves. Most importantly, while mitochondrial Ca overload has been proposed as the no-return signal, triggering apoptotic or necrotic neuronal death, until now direct evidences supporting this hypothesis, especially , are limited. Here, we took advantage of the identification of the mitochondrial Ca uniporter (MCU) and tested whether mitochondrial Ca signaling controls neuronal cell fate. We overexpressed MCU both , in mouse primary cortical neurons, and , through stereotaxic injection of MCU-coding adenoviral particles in the brain cortex. We first measured mitochondrial Ca uptake using quantitative genetically encoded Ca probes, and we observed that the overexpression of MCU causes a dramatic increase of mitochondrial Ca uptake both at resting and after membrane depolarization. MCU-mediated mitochondrial Ca overload causes alteration of organelle morphology and dysregulation of global Ca homeostasis. Most importantly, MCU overexpression is sufficient to trigger gliosis and neuronal loss. Overall, we demonstrated that mitochondrial Ca overload is sufficient to cause neuronal cell death both and , thus highlighting a potential key step in neurodegeneration.
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http://dx.doi.org/10.1155/2019/1681254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816006PMC
May 2020

Identification of an ATP-sensitive potassium channel in mitochondria.

Nature 2019 08 21;572(7771):609-613. Epub 2019 Aug 21.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

Mitochondria provide chemical energy for endoergonic reactions in the form of ATP, and their activity must meet cellular energy requirements, but the mechanisms that link organelle performance to ATP levels are poorly understood. Here we confirm the existence of a protein complex localized in mitochondria that mediates ATP-dependent potassium currents (that is, mitoK). We show that-similar to their plasma membrane counterparts-mitoK channels are composed of pore-forming and ATP-binding subunits, which we term MITOK and MITOSUR, respectively. In vitro reconstitution of MITOK together with MITOSUR recapitulates the main properties of mitoK. Overexpression of MITOK triggers marked organelle swelling, whereas the genetic ablation of this subunit causes instability in the mitochondrial membrane potential, widening of the intracristal space and decreased oxidative phosphorylation. In a mouse model, the loss of MITOK suppresses the cardioprotection that is elicited by pharmacological preconditioning induced by diazoxide. Our results indicate that mitoK channels respond to the cellular energetic status by regulating organelle volume and function, and thereby have a key role in mitochondrial physiology and potential effects on several pathological processes.
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http://dx.doi.org/10.1038/s41586-019-1498-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726485PMC
August 2019

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

Nat Commun 2019 06 12;10(1):2576. Epub 2019 Jun 12.

Venetian Institute of Molecular Medicine, via Orus 2, 35129, Padova, Italy.

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 Ca uptake and myofiber death. Our findings reveal that morphology of mitochondrial network is critical for several biological processes that control nuclear programs and Ca handling.
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http://dx.doi.org/10.1038/s41467-019-10226-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561930PMC
June 2019

MFN2 mutations in Charcot-Marie-Tooth disease alter mitochondria-associated ER membrane function but do not impair bioenergetics.

Hum Mol Genet 2019 06;28(11):1782-1800

Department of Biology, University of Padova 35131, Italy.

Charcot-Marie-Tooth disease (CMT) type 2A is a form of peripheral neuropathy, due almost exclusively to dominant mutations in the nuclear gene encoding the mitochondrial protein mitofusin-2 (MFN2). However, there is no understanding of the relationship of clinical phenotype to genotype. MFN2 has two functions: it promotes inter-mitochondrial fusion and mediates endoplasmic reticulum (ER)-mitochondrial tethering at mitochondria-associated ER membranes (MAM). MAM regulates a number of key cellular functions, including lipid and calcium homeostasis, and mitochondrial behavior. To date, no studies have been performed to address whether mutations in MFN2 in CMT2A patient cells affect MAM function, which might provide insight into pathogenesis. Using fibroblasts from three CMT2AMFN2 patients with different mutations in MFN2, we found that some, but not all, examined aspects of ER-mitochondrial connectivity and of MAM function were indeed altered, and correlated with disease severity. Notably, however, respiratory chain function in those cells was unimpaired. Our results suggest that CMT2AMFN2 is a MAM-related disorder but is not a respiratory chain-deficiency disease. The alterations in MAM function described here could also provide insight into the pathogenesis of other forms of CMT.
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http://dx.doi.org/10.1093/hmg/ddz008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522073PMC
June 2019

Loss of EMILIN-1 Enhances Arteriolar Myogenic Tone Through TGF-β (Transforming Growth Factor-β)-Dependent Transactivation of EGFR (Epidermal Growth Factor Receptor) and Is Relevant for Hypertension in Mice and Humans.

Arterioscler Thromb Vasc Biol 2018 10;38(10):2484-2497

Department of Molecular Medicine (N.F., D.B., F.D.R., P. Bonaldo, P. Braghetta, G.M.B.), University of Padova, Italy.

Objective- EMILIN-1 (elastin microfibrils interface located protein-1) protein inhibits pro-TGF-β (transforming growth factor-β) proteolysis and limits TGF-β bioavailability in vascular extracellular matrix. Emilin1 null mice display increased vascular TGF-β signaling and are hypertensive. Because EMILIN-1 is expressed in vessels from embryonic life to adulthood, we aimed at unravelling whether the hypertensive phenotype of Emilin1 null mice results from a developmental defect or lack of homeostatic role in the adult. Approach and Results- By using a conditional gene targeting inactivating EMILIN-1 in smooth muscle cells of adult mice, we show that increased blood pressure in mice with selective smooth muscle cell ablation of EMILIN-1 depends on enhanced myogenic tone. Mechanistically, we unveil that higher TGF-β signaling in smooth muscle cells stimulates HB-EGF (heparin-binding epidermal growth factor) expression and subsequent transactivation of EGFR (epidermal growth factor receptor). With increasing intraluminal pressure in resistance arteries, the cross talk established by TGF-β and EGFR signals recruits TRPC6 (TRP [transient receptor potential] classical type 6) and TRPM4 (TRP melastatin type 4) channels, lastly stimulating voltage-dependent calcium channels and potentiating myogenic tone. We found reduced EMILIN-1 and enhanced myogenic tone, dependent on increased TGF-β-EGFR signaling, in resistance arteries from hypertensive patients. Conclusions- Taken together, our findings implicate an unexpected role of the TGF-β-EGFR pathway in hypertension with current translational perspectives.
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http://dx.doi.org/10.1161/ATVBAHA.118.311115DOI Listing
October 2018

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

Cell Death Differ 2019 01 19;26(2):362-381. Epub 2018 Sep 19.

Department of Biomedical Sciences, University of Padova, 35131, Padova, Italy.

Skeletal muscle mitochondria readily accumulate Ca in response to SR store-releasing stimuli thanks to the activity of the mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca 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 Ca uptake, impairs muscle force and exercise performance, and determines a slow to fast switch in MHC expression. Mitochondrial Ca 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 MCU 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.
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http://dx.doi.org/10.1038/s41418-018-0191-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329801PMC
January 2019

Tau localises within mitochondrial sub-compartments and its caspase cleavage affects ER-mitochondria interactions and cellular Ca handling.

Biochim Biophys Acta Mol Basis Dis 2018 10 11;1864(10):3247-3256. Epub 2018 Jul 11.

Department of Biomedical Sciences, University of Padova, Padova, Italy; Padova Neuroscience Center (PNC), University of Padova, Padova, Italy. Electronic address:

Intracellular neurofibrillary tangles (NFT) composed by tau and extracellular amyloid beta (Aβ) plaques accumulate in Alzheimer's disease (AD) and contribute to neuronal dysfunction. Mitochondrial dysfunction and neurodegeneration are increasingly considered two faces of the same coin and an early pathological event in AD. Compelling evidence indicates that tau and mitochondria are closely linked and suggests that tau-dependent modulation of mitochondrial functions might be a trigger for the neurodegeneration process; however, whether this occurs either directly or indirectly is not clear. Furthermore, whether tau influences cellular Ca handling and ER-mitochondria cross-talk is yet to be explored. Here, by focusing on wt tau, either full-length (2N4R) or the caspase 3-cleaved form truncated at the C-terminus (2N4RΔC), we examined the above-mentioned aspects. Using new genetically encoded split-GFP-based tools and organelle-targeted aequorin probes, we assessed: i) tau distribution within the mitochondrial sub-compartments; ii) the effect of tau on the short- (8-10 nm) and the long- (40-50 nm) range ER-mitochondria interactions; and iii) the effect of tau on cytosolic, ER and mitochondrial Ca homeostasis. Our results indicate that a fraction of tau is found at the outer mitochondrial membrane (OMM) and within the inner mitochondrial space (IMS), suggesting a potential tau-dependent regulation of mitochondrial functions. The ER Ca content and the short-range ER-mitochondria interactions were selectively affected by the expression of the caspase 3-cleaved 2N4RΔC tau, indicating that Ca mis-handling and defects in the ER-mitochondria communications might be an important pathological event in tau-related dysfunction and thereby contributing to neurodegeneration. Finally, our data provide new insights into the molecular mechanisms underlying tauopathies.
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http://dx.doi.org/10.1016/j.bbadis.2018.07.011DOI Listing
October 2018

Mitochondrial Calcium Increase Induced by RyR1 and IP3R Channel Activation After Membrane Depolarization Regulates Skeletal Muscle Metabolism.

Front Physiol 2018 25;9:791. Epub 2018 Jun 25.

Muscle Physiology Laboratory, Center of Studies in Exercise, Metabolism and Cancer, Institute of Biomedical Sciences, Universidad de Chile, Santiago, Chile.

We hypothesize that both type-1 ryanodine receptor (RyR1) and IP-receptor (IPR) calcium channels are necessary for the mitochondrial Ca increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling). Mitochondria matrix and cytoplasmic Ca levels were evaluated in fibers isolated from muscle using plasmids for the expression of a mitochondrial Ca sensor (CEPIA3) or a cytoplasmic Ca sensor (RCaMP). The role of intracellular Ca channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IPR and Dantrolene for RyR1) and a genetic approach (shIPR1-RFP). O consumption was detected using Seahorse Extracellular Flux Analyzer. In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca levels. Mitochondrial Ca uptake required functional inositol IPR and RyR1 channels. Inhibition of either channel decreased basal O consumption rate but only RyR1 inhibition decreased ATP-linked O consumption. Cell membrane depolarization-induced Ca signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber. Ca-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an "excitation-metabolism" coupling process in skeletal muscle fibers.
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http://dx.doi.org/10.3389/fphys.2018.00791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026899PMC
June 2018

MICU3 is a tissue-specific enhancer of mitochondrial calcium uptake.

Cell Death Differ 2019 01 3;26(1):179-195. Epub 2018 May 3.

Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58B, 35131, Padova, Italy.

The versatility and universality of Ca as intracellular messenger is guaranteed by the compartmentalization of changes in [Ca]. In this context, mitochondrial Ca plays a central role, by regulating both specific organelle functions and global cellular events. This versatility is also guaranteed by a cell type-specific Ca signaling toolkit controlling specific cellular functions. Accordingly, mitochondrial Ca uptake is mediated by a multimolecular structure, the MCU complex, which differs among various tissues. Its activity is indeed controlled by different components that cooperate to modulate specific channeling properties. We here investigate the role of MICU3, an EF-hand containing protein expressed at high levels, especially in brain. We show that MICU3 forms a disulfide bond-mediated dimer with MICU1, but not with MICU2, and it acts as enhancer of MCU-dependent mitochondrial Ca uptake. Silencing of MICU3 in primary cortical neurons impairs Ca signals elicited by synaptic activity, thus suggesting a specific role in regulating neuronal function.
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http://dx.doi.org/10.1038/s41418-018-0113-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124646PMC
January 2019

LETM1-Mediated K and Na Homeostasis Regulates Mitochondrial Ca Efflux.

Front Physiol 2017 17;8:839. Epub 2017 Nov 17.

Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria.

Ca transport across the inner membrane of mitochondria (IMM) is of major importance for their functions in bioenergetics, cell death and signaling. It is therefore tightly regulated. It has been recently proposed that LETM1—an IMM protein with a crucial role in mitochondrial K/H exchange and volume homeostasis—also acts as a Ca/H exchanger. Here we show for the first time that lowering LETM1 gene expression by shRNA hampers mitochondrial K/H and Na/H exchange. Decreased exchange activity resulted in matrix K accumulation in these mitochondria. Furthermore, LETM1 depletion selectively decreased Na/Ca exchange mediated by NCLX, as observed in the presence of ruthenium red, a blocker of the Mitochondrial Ca Uniporter (MCU). These data confirm a key role of LETM1 in monovalent cation homeostasis, and suggest that the effects of its modulation on mitochondrial transmembrane Ca fluxes may reflect those on Na/H exchange activity.
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http://dx.doi.org/10.3389/fphys.2017.00839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698270PMC
November 2017

Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy.

Proc Natl Acad Sci U S A 2017 10 9;114(43):E9006-E9015. Epub 2017 Oct 9.

Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy;

The mitochondrial Ca uniporter complex (MCUC) is a multimeric ion channel which, by tuning Ca influx into the mitochondrial matrix, finely regulates metabolic energy production. In the heart, this dynamic control of mitochondrial Ca uptake is fundamental for cardiomyocytes to adapt to either physiologic or pathologic stresses. Mitochondrial calcium uniporter (MCU), which is the core channel subunit of MCUC, has been shown to play a critical role in the response to β-adrenoreceptor stimulation occurring during acute exercise. The molecular mechanisms underlying the regulation of MCU, in conditions requiring chronic increase in energy production, such as physiologic or pathologic cardiac growth, remain elusive. Here, we show that microRNA-1 (miR-1), a member of the muscle-specific microRNA (myomiR) family, is responsible for direct and selective targeting of MCU and inhibition of its translation, thereby affecting the capacity of the mitochondrial Ca uptake machinery. Consistent with the role of miR-1 in heart development and cardiomyocyte hypertrophic remodeling, we additionally found that MCU levels are inversely related with the myomiR content, in murine and, remarkably, human hearts from both physiologic (i.e., postnatal development and exercise) and pathologic (i.e., pressure overload) myocardial hypertrophy. Interestingly, the persistent activation of β-adrenoreceptors is likely one of the upstream repressors of miR-1 as treatment with β-blockers in pressure-overloaded mouse hearts prevented its down-regulation and the consequent increase in MCU content. Altogether, these findings identify the miR-1/MCU axis as a factor in the dynamic adaptation of cardiac cells to hypertrophy.
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http://dx.doi.org/10.1073/pnas.1708772114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664523PMC
October 2017

The MCU complex in cell death.

Cell Calcium 2018 01 25;69:73-80. Epub 2017 Aug 25.

Department of Biomedical Sciences, University of Padova, Italy. Electronic address:

During the 60s, the notion that positively charged Ca ions are rapidly accumulated in energized mitochondria has been first established. In the following decades, mitochondrial Ca homeostasis was shown to control cell metabolism, cell survival and other cell-specific functions through different mechanism. However, the molecular identity of the molecules controlling this process remained a mystery until just few years ago, when both mitochondrial Ca uptake and release systems were genetically dissected. This finally opened the possibility to develop genetic model to directly test the contribution of mitochondrial Ca homeostasis to cellular functions. Although the picture is still far from being clear, we here summarize and critically evaluate the current knowledge on how mitochondrial Ca handling controls cell death.
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http://dx.doi.org/10.1016/j.ceca.2017.08.008DOI Listing
January 2018

Mitochondrial Calcium Handling in Physiology and Disease.

Adv Exp Med Biol 2017 ;982:25-47

Department of Biomedical Sciences, University of Padova, Padua, Italy.

Calcium (Ca) accumulation inside mitochondria represents a pleiotropic signal controlling a wide range of cellular functions, including key metabolic pathways and life/death decisions. This phenomenon has been first described in the 1960s, but the identity of the molecules controlling this process remained a mystery until just few years ago, when both mitochondrial Ca uptake and release systems were genetically dissected. This finally opened the possibility to develop genetic models to directly test the contribution of mitochondrial Ca homeostasis to cellular functions. Here we summarize our current understanding of the molecular machinery that controls mitochondrial Ca handling and critically evaluate the physiopathological role of mitochondrial Ca signaling, based on recent evidences obtained through in vitro and in vivo models.
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http://dx.doi.org/10.1007/978-3-319-55330-6_2DOI Listing
September 2017

Critical reappraisal confirms that Mitofusin 2 is an endoplasmic reticulum-mitochondria tether.

Proc Natl Acad Sci U S A 2016 10 19;113(40):11249-11254. Epub 2016 Sep 19.

Department of Biology, University of Padua, 35121 Padua, Italy; Dulbecco-Telethon Institute, Venetian Institute of Molecular Medicine, 35129 Padua, Italy;

The discovery of the multiple roles of mitochondria-endoplasmic reticulum (ER) juxtaposition in cell biology often relied upon the exploitation of Mitofusin (Mfn) 2 as an ER-mitochondria tether. However, this established Mfn2 function was recently questioned, calling for a critical re-evaluation of Mfn2's role in ER-mitochondria cross-talk. Electron microscopy and fluorescence-based probes of organelle proximity confirmed that ER-mitochondria juxtaposition was reduced by constitutive or acute Mfn2 deletion. Functionally, mitochondrial uptake of Ca released from the ER was reduced following acute Mfn2 ablation, as well as in Mfn2 cells overexpressing the mitochondrial calcium uniporter. Mitochondrial Ca uptake rate and extent were normal in isolated Mfn2 liver mitochondria, consistent with the finding that acute or chronic Mfn2 ablation or overexpression did not alter mitochondrial calcium uniporter complex component levels. Hence, Mfn2 stands as a bona fide ER-mitochondria tether whose ablation decreases interorganellar juxtaposition and communication.
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http://dx.doi.org/10.1073/pnas.1606786113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056088PMC
October 2016

The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria.

Mol Cell 2016 10 15;64(1):148-162. Epub 2016 Sep 15.

Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Center for Molecular Medicine (CMMC), University of Cologne, 50931 Cologne, Germany; Max Planck Institute for Biology of Aging, 50931 Cologne, Germany. Electronic address:

Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca homeostasis.
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http://dx.doi.org/10.1016/j.molcel.2016.08.020DOI Listing
October 2016

Enjoy the Trip: Calcium in Mitochondria Back and Forth.

Annu Rev Biochem 2016 Jun 4;85:161-92. Epub 2016 May 4.

Department of Biomedical Sciences, University of Padova, 35121 Padova, Italy; email: , ,

In the last 5 years, most of the molecules that control mitochondrial Ca(2+) homeostasis have been finally identified. Mitochondrial Ca(2+) uptake is mediated by the Mitochondrial Calcium Uniporter (MCU) complex, a macromolecular structure that guarantees Ca(2+) accumulation inside mitochondrial matrix upon increases in cytosolic Ca(2+). Conversely, Ca(2+) release is under the control of the Na(+)/Ca(2+) exchanger, encoded by the NCLX gene, and of a H(+)/Ca(2+) antiporter, whose identity is still debated. The low affinity of the MCU complex, coupled to the activity of the efflux systems, protects cells from continuous futile cycles of Ca(2+) across the inner mitochondrial membrane and consequent massive energy dissipation. In this review, we discuss the basic principles that govern mitochondrial Ca(2+) homeostasis and the methods used to investigate the dynamics of Ca(2+) concentration within the organelles. We discuss the functional and structural role of the different molecules involved in mitochondrial Ca(2+) handling and their pathophysiological role.
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http://dx.doi.org/10.1146/annurev-biochem-060614-034216DOI Listing
June 2016

Structure and function of the mitochondrial calcium uniporter complex.

Biochim Biophys Acta 2015 Sep 18;1853(9):2006-11. Epub 2015 Apr 18.

Department of Biomedical Sciences, University of Padova, Italy.

The mitochondrial calcium uniporter (MCU) is the critical protein of the inner mitochondrial membrane mediating the electrophoretic Ca²⁺ uptake into the matrix. It plays a fundamental role in the shaping of global calcium signaling and in the control of aerobic metabolism as well as apoptosis. Two features of mitochondrial calcium signaling have been known for a long time: i) mitochondrial Ca²⁺ uptake widely varies among cells and tissues, and ii) channel opening strongly relies on the extramitochondrial Ca²⁺ concentration, with low activity at resting [Ca²⁺] and high capacity as soon as calcium signaling is activated. Such complexity requires a specialized molecular machinery, with several primary components can be variably gathered together in order to match energy demands and protect from toxic stimuli. In line with this, MCU is now recognized to be part of a macromolecular complex known as the MCU complex. Our understanding of the structure and function of the MCU complex is now growing promptly, revealing an unexpected complexity that highlights the pleiotropic role of mitochondrial Ca²⁺ signals. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
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http://dx.doi.org/10.1016/j.bbamcr.2015.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522341PMC
September 2015

The mitochondrial calcium uniporter controls skeletal muscle trophism in vivo.

Cell Rep 2015 Mar 26;10(8):1269-79. Epub 2015 Feb 26.

Department of Biomedical Sciences, University of Padua, Padua 35131, Italy; Neuroscience Institute, National Research Council, Padua 35131, Italy. Electronic address:

Muscle atrophy contributes to the poor prognosis of many pathophysiological conditions, but pharmacological therapies are still limited. Muscle activity leads to major swings in mitochondrial [Ca(2+)], which control aerobic metabolism, cell death, and survival pathways. We investigated in vivo the effects of mitochondrial Ca(2+) homeostasis in skeletal muscle function and trophism by overexpressing or silencing the mitochondrial calcium uniporter (MCU). The results demonstrate that in both developing and adult muscles, MCU-dependent mitochondrial Ca(2+) uptake has a marked trophic effect that does not depend on aerobic control but impinges on two major hypertrophic pathways of skeletal muscle, PGC-1α4 and IGF1-Akt/PKB. In addition, MCU overexpression protects from denervation-induced atrophy. These data reveal a novel Ca(2+)-dependent organelle-to-nucleus signaling route that links mitochondrial function to the control of muscle mass and may represent a possible pharmacological target in conditions of muscle loss.
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http://dx.doi.org/10.1016/j.celrep.2015.01.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351162PMC
March 2015

Lysosomal calcium signalling regulates autophagy through calcineurin and ​TFEB.

Nat Cell Biol 2015 Mar;17(3):288-99

The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signalling mechanism controls the activities of the phosphatase calcineurin and of its substrate ​TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release through ​mucolipin 1 (​MCOLN1) activates calcineurin, which binds and dephosphorylates ​TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed ​TFEB activity during starvation and physical exercise, while calcineurin overexpression and constitutive activation had the opposite effect. Induction of autophagy and lysosomal biogenesis through ​TFEB required ​MCOLN1-mediated calcineurin activation. These data link lysosomal calcium signalling to both calcineurin regulation and autophagy induction and identify the lysosome as a hub for the signalling pathways that regulate cellular homeostasis.
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http://dx.doi.org/10.1038/ncb3114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801004PMC
March 2015

Measuring baseline Ca(2+) levels in subcellular compartments using genetically engineered fluorescent indicators.

Methods Enzymol 2014 ;543:47-72

Department of Cell and Developmental Biology, Consortium for Mitochondrial Research, University College London, London, United Kingdom; Department of Biomedical Sciences, CNR Neuroscience Institute, University of Padua, Padua, Italy. Electronic address:

Intracellular Ca(2+) signaling is involved in a series of physiological and pathological processes. In particular, an intimate crosstalk between bioenergetic metabolism and Ca(2+) homeostasis has been shown to determine cell fate in resting conditions as well as in response to stress. The endoplasmic reticulum and mitochondria represent key hubs of cellular metabolism and Ca(2+) signaling. However, it has been challenging to specifically detect highly localized Ca(2+) fluxes such as those bridging these two organelles. To circumvent this issue, various recombinant Ca(2+) indicators that can be targeted to specific subcellular compartments have been developed over the past two decades. While the use of these probes for measuring agonist-induced Ca(2+) signals in various organelles has been extensively described, the assessment of basal Ca(2+) concentrations within specific organelles is often disregarded, in spite of the fact that this parameter is vital for several metabolic functions, including the enzymatic activity of mitochondrial dehydrogenases of the Krebs cycle and protein folding in the endoplasmic reticulum. Here, we provide an overview on genetically engineered, organelle-targeted fluorescent Ca(2+) probes and outline their evolution. Moreover, we describe recently developed protocols to quantify baseline Ca(2+) concentrations in specific subcellular compartments. Among several applications, this method is suitable for assessing how changes in basal Ca(2+) levels affect the metabolic profile of cancer cells.
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http://dx.doi.org/10.1016/B978-0-12-801329-8.00003-9DOI Listing
February 2015

Molecular control of mitochondrial calcium uptake.

Biochem Biophys Res Commun 2014 Jul 30;449(4):373-6. Epub 2014 Apr 30.

Department of Biomedical Sciences, University of Padova and CNR Neuroscience Institute, Padova, Italy. Electronic address:

The recently identified Mitochondrial Calcium Uniporter (MCU) is the protein of the inner mitochondrial membrane responsible for Ca(2+) uptake into the matrix, which plays a role in the control of cellular signaling, aerobic metabolism and apoptosis. At least two properties of mitochondrial calcium signaling are well defined: (i) mitochondrial Ca(2+) uptake varies greatly among different cells and tissues, and (ii) channel opening is strongly affected by extramitochondrial Ca(2+) concentration, with low activity at resting and high capacity after cellular stimulation. It is now becoming clear that these features of the mitochondrial Ca(2+) uptake machinery are not embedded in the MCU protein itself, but are rather due to the contribution of several MCU interactors. The list of the components of the MCU complex is indeed rapidly growing, thus revealing an unexpected complexity that highlights the pleiotropic role of mitochondrial calcium signaling.
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http://dx.doi.org/10.1016/j.bbrc.2014.04.142DOI Listing
July 2014