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Skeletal muscle mitochondria are placed in close proximity of the sarcoplasmic reticulum (SR), the main intracellular Ca

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.

Summary 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., Graphical abstract, Highlights • We screen an FDA-approved drug library for mitochondrial Ca2+ uptake modulators • Amorolfine and benzethonium modulate MCU activity • Amorolfine increases MCU-dependent mitochondrial metabolism and muscle size • Benzethonium decreases MCU-dependent cancer cell growth and migration, In search of mitochondrial Ca2+ uptake modulators, De Mario et al. identify amorolfine and benzethonium as positive hits of a 1,600 FDA drug library high-throughput screen. Amorolfine increases mitochondrial metabolism and muscle size in an MCU-dependent manner. Benzethonium negatively regulates MCU activity, mROS formation, and cancer cell growth and migration.

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.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons (MNs), probably by a combination of cell- and non-cell-autonomous processes. The past decades have brought many important insights into the role of astrocytes in nervous system function and disease, including the implication in ALS pathogenesis possibly through the impairment of Ca2+-dependent astrocyte-MN cross-talk. In this respect, it has been recently proposed that altered astrocytic store-operated Ca2+ entry (SOCE) may underlie aberrant gliotransmitter release and astrocyte-mediated neurotoxicity in ALS. These observations prompted us to a thorough investigation of SOCE in primary astrocytes from the spinal cord of the SOD1(G93A) ALS mouse model in comparison with the SOD1(WT)-expressing controls. To this purpose, we employed, for the first time in the field, genetically-encoded Ca2+ indicators, allowing the direct assessment of Ca2+ fluctuations in different cell domains. We found increased SOCE, associated with decreased expression of the sarco-endoplasmic reticulum Ca2+-ATPase and lower ER resting Ca2+ concentration in SOD1(G93A) astrocytes compared to control cells. Such findings add novel insights into the involvement of astrocytes in ALS MN damage.

The cellular prion protein (PrPC) is an ubiquitous cell surface protein mostly expressed in neurons, where it localizes to both pre- and post-synaptic membranes. PrPC aberrant conformers are the major components of mammalian prions, the infectious agents responsible for incurable neurodegenerative disorders. PrPC was also proposed to bind aggregated misfolded proteins/peptides, and to mediate their neurotoxic signal. In spite of long-lasting research, a general consensus on the precise pathophysiologic mechanisms of PrPC has not yet been reached. Here we review our recent data, obtained by comparing primary neurons from PrP-expressing and PrP-knockout mice, indicating a central role of PrPC in synaptic transmission and Ca2+ homeostasis. Indeed, by controlling gene expression and signaling cascades, PrPC is able to optimize glutamate secretion and regulate Ca2+ entry via store-operated channels and ionotropic glutamate receptors, thereby protecting neurons from threatening Ca2+ overloads and excitotoxicity. We will also illustrate and discuss past and unpublished results demonstrating that Aβ oligomers perturb Ca2+ homeostasis and cause abnormal mitochondrial accumulation of reactive oxygen species by possibly affecting the PrP-dependent downregulation of Fyn kinase activity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons (MNs), probably by a combination of cell- and non-cell-autonomous processes. The past decades have brought many important insights into the role of astrocytes in nervous system function and disease, including the implication in ALS pathogenesis possibly through the impairment of Ca2+-dependent astrocyte-MN cross-talk. In this respect, it has been recently proposed that altered astrocytic store-operated Ca2+ entry (SOCE) may underlie aberrant gliotransmitter release and astrocyte-mediated neurotoxicity in ALS. These observations prompted us to a thorough investigation of SOCE in primary astrocytes from the spinal cord of the SOD1(G93A) ALS mouse model in comparison with the SOD1(WT)-expressing controls. To this purpose, we employed, for the first time in the field, genetically-encoded Ca2+ indicators, allowing the direct assessment of Ca2+ fluctuations in different cell domains. We found increased SOCE, associated with decreased expression of the sarco-endoplasmic reticulum Ca2+-ATPase and lower ER resting Ca2+ concentration in SOD1(G93A) astrocytes compared to control cells. Such findings add novel insights into the involvement of astrocytes in ALS MN damage.

Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca

Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10 μM, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.

Summary 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., Graphical Abstract, Highlights • An HTS identifies MCU-i4 and MCU-i11 as negative modulators of the MCU • MCU-i4 and MCU-i11 bind MICU1 • MICU1 is required for the activity of MCU-i4 and MCU-i11 • MCU-i4 and MCU-i11 impair muscle cell growth, Di Marco et al. report the discovery of MCU-i4 and MCU-i11, two negative modulators of the MCU that decrease mitochondrial Ca2+ uptake. MCU-i4 and MCU-i11 bind MICU1, the key MCU interactor that controls channel gating, and MICU1 is required for their activity. Thus, these small molecules represent lead MICU1-targeting compounds.

Mitochondrial Ca2+ is involved in heterogeneous functions, ranging from the control of metabolism and ATP production to the regulation of cell death. In addition, mitochondrial Ca2+ uptake contributes to cytosolic [Ca2+] shaping thus impinging on specific Ca2+-dependent events. Mitochondrial Ca2+ concentration is controlled by influx and efflux pathways: the former controlled by the activity of the mitochondrial Ca2+ uniporter (MCU), the latter by the Na+/Ca2+ exchanger (NCLX) and the H+/Ca2+ (mHCX) exchanger. The molecular identities of MCU and of NCLX have been recently unraveled, thus allowing genetic studies on their physiopathological relevance. After a general framework on the significance of mitochondrial Ca2+ uptake, this review discusses the structure of the MCU complex and the regulation of its activity, the importance of mitochondrial Ca2+ signaling in different physiological settings, and the consequences of MCU modulation on organ physiology.

Metal ions are key elements in organisms' life acting like cofactors of many enzymes but they can also be potentially dangerous for the cell participating in redox reactions that lead to the formation of reactive oxygen species (ROS). Any factor inducing or limiting a metal dyshomeostasis, ROS production and cell injury may contribute to the onset of neurodegenerative diseases or play a neuroprotective action. Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a group of fatal neurodegenerative disorders affecting the central nervous system (CNS) of human and other mammalian species. The causative agent of TSEs is believed to be the scrapie prion protein PrPSc, the β sheet-rich pathogenic isoform produced by the conformational conversion of the a-helix-rich physiological isoform PrPC. The peculiarity of PrPScis its ability to self-propagate in exponential fashion in cells and its tendency to precipitate in insoluble and protease-resistance amyloid aggregates leading to neuronal cell death. The expression "prion-like diseases" refers to a group of neurodegenerative diseases that share some neuropathological features with prion diseases such as the involvement of proteins (α-synuclein, amyloid β, and tau) able to precipitate producing amyloid deposits following conformational change. High social impact diseases such as Alzheimer's and Parkinson's belong to prion-like diseases. Accumulating evidence suggests that the exposure to environmental metals is a risk factor for the development of prion and prion-like diseases and that metal ions can directly bind to prion and prion-like proteins affecting the amount of amyloid aggregates. The diet, source of metal ions but also of natural antioxidant and chelating agents such as polyphenols, is an aspect to take into account in addressing the issue of neurodegeneration. Epidemiological data suggest that the Mediterranean diet, based on the abundant consumption of fresh vegetables and on low intake of meat, could play a preventive or delaying role in prion and prion-like neurodegenerative diseases. In this review, metal role in the onset of prion and prion-like diseases is dealt with from a nutritional, cellular, and molecular point of view.

The cellular prion protein (PrPC) whose conformational misfolding leads to the production of deadly prions, has a still-unclarified cellular function despite decades of intensive research. Following our recent finding that PrPC limits Ca2+ entry via store-operated Ca2+ channels in neurons, we investigated whether the protein could also control the activity of ionotropic glutamate receptors (iGluRs). To this end, we compared local Ca2+ movements in primary cerebellar granule neurons and cortical neurons transduced with genetically encoded Ca2+ probes and expressing, or not expressing, PrPC Our investigation demonstrated that PrPC downregulates Ca2+ entry through each specific agonist-stimulated iGluR and after stimulation by glutamate. We found that, although PrP-knockout (KO) mitochondria were displaced from the plasma membrane, glutamate addition resulted in a higher mitochondrial Ca2+ uptake in PrP-KO neurons than in their PrPC-expressing counterpart. This was because the increased Ca2+ entry through iGluRs in PrP-KO neurons led to a parallel increase in Ca2+-induced Ca2+ release via ryanodine receptor channels. These data thus suggest that PrPC takes part in the cell apparatus controlling Ca2+ homeostasis, and that PrPC is involved in protecting neurons from toxic Ca2+ overloads.

The cellular prion protein (PrP

In the last years, a considerable amount of experimental evidence has highlighted the association between neurodegenerative disorders (NDD) and the biology of mitochondria-Endoplasmic Reticulum contacts (MERCs). In this review, we summarize the most recent findings on this topic. We underline that dysregulation of MERCs can contribute to the neurodegenerative process either by altering directly the functionality of neurons and their response to stress stimuli and metabolic shifts or by indirectly influencing the neuroinflammatory response that accompanies NDD. Our overview of the current literature suggest that defective MERCs could be a common determinant to the "hypergeneration" and "neurodegeneration" programs, leading respectively to tumours and NDD.

Huntington disease (HD) is caused by the CAG (Q) expansion in exon 1 of the IT15 gene encoding a polyglutamine (poly-Q) stretch of the Huntingtin protein (Htt). In the wild type protein, the repeats specify a stretch of up 34 Q in the N-terminal portion of Htt. In the pathological protein (mHtt) the poly-Q tract is longer. Proteolytic cleavage of the protein liberates an N-terminal fragment containing the expanded poly-Q tract becomes harmful to cells, in particular to striatal neurons. The fragments cause the transcriptional dysfunction of genes that are essential for neuronal survival. Htt, however, could also have non-transcriptional effects, e.g. it could directly alter Ca2+ homeostasis and/or mitochondrial morphology and function. Ca2+ dyshomeostasis and mitochondrial dysfunction are considered important in the molecular aetiology of the disease. Here we have analyzed the effect of the overexpression of Htt fragments (18Q, wild type form, wtHtt and 150Q mutated form, mHtt) on Ca2+ homeostasis in striatal neuronal precursor cells (Q7/7). We have found that the transient overexpression of the Htt fragments increases Ca2+ transients in the mitochondria of cells stimulated with Ca2+-mobilizing agonists. The bulk Ca2+ transients in the cytosol were unaffected, but the Ca2+ content of the endoplasmic reticulum was significantly decreased in the case of mHtt expression. To rule out possible transcriptional effects due to the presence of mHtt, we have measured the mRNA level of a subunit of the respiratory chain complex II, whose expression is commonly altered in many HD models. No effects on the mRNA level was found suggesting that, in our experimental condition, transcriptional action of Htt is not occurring and that the effects on Ca2+ homeostasis were dependent to non-transcriptional mechanisms.

The plasma membrane Ca(2+) ATPases (PMCA pumps) cooperate with other transport systems in the plasma membrane and in the organelles in the regulation of cell Ca(2+). They have high Ca(2+) affinity and are thus the fine tuners of cytosolic Ca(2+). They belong to the superfamily of P-type ATPases: their four basic isoforms share the essential properties of the reaction cycle and the general membrane topography motif of 10 transmembrane domains and three large cytosolic units. However they also differ in other important properties, e.g., tissue distribution and regulatory mechanisms. Their chief regulator is calmodulin, that removes their C-terminal cytosolic tail from autoinhibitory binding sites next to the active site of the pump, restoring activity. The number of pump isoforms is increased to over 30 by alternative splicing of the transcripts at a N-terminal site (site A) and at site C within the C-terminal calmodulin binding domain: the splice variants are tissue specific and developmentally regulated. The importance of PMCAs in the maintenance of cellular Ca(2+) homeostasis is underlined by the disease phenotypes, genetic or acquired, caused by their malfunction. Non-genetic PMCA deficiencies have long been considered possible causative factors in disease conditions as important as cancer, hypertension, or neurodegeneration. Those of genetic origin are better characterized: some have now been discovered in humans as well. They concern all four PMCA isoforms, and range from cardiac dysfunctions, to deafness, to hypertension, to cerebellar ataxia.

Aims: Monoamine oxidases (MAOs) are mitochondrial flavoenzymes responsible for neurotransmitter and biogenic amines catabolism. MAO-A contributes to heart failure progression via enhanced norepinephrine catabolism and oxidative stress. The potential pathogenetic role of the isoenzyme MAO-B in cardiac diseases is currently unknown. Moreover, it is has not been determined yet whether MAO activation can directly affect mitochondrial function. Results: In wild type mice, pressure overload induced by transverse aortic constriction (TAC) resulted in enhanced dopamine catabolism, left ventricular (LV) remodeling, and dysfunction. Conversely, mice lacking MAO-B (MAO-B−/−) subjected to TAC maintained concentric hypertrophy accompanied by extracellular signal regulated kinase (ERK)1/2 activation, and preserved LV function, both at early (3 weeks) and late stages (9 weeks). Enhanced MAO activation triggered oxidative stress, and dropped mitochondrial membrane potential in the presence of ATP synthase inhibitor oligomycin both in neonatal and adult cardiomyocytes. The MAO-B inhibitor pargyline completely offset this change, suggesting that MAO activation induces a latent mitochondrial dysfunction, causing these organelles to hydrolyze ATP. Moreover, MAO-dependent aldehyde formation due to inhibition of aldehyde dehydrogenase 2 activity also contributed to alter mitochondrial bioenergetics. Innovation: Our study unravels a novel role for MAO-B in the pathogenesis of heart failure, showing that both MAO-driven reactive oxygen species production and impaired aldehyde metabolism affect mitochondrial function. Conclusion: Under conditions of chronic hemodynamic stress, enhanced MAO-B activity is a major determinant of cardiac structural and functional disarrangement. Both increased oxidative stress and the accumulation of aldehyde intermediates are likely liable for these adverse morphological and mechanical changes by directly targeting mitochondria. Antioxid. Redox Signal. 20, 267–280.

Background Abnormalities in brain cholesterol homeostasis have been reported in Huntington's disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein. Results Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality also influenced the detection of intracellular cholesterol. Conclusions Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ mutation in a constitutive or inducible manner. *Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it.

Hearing relies on the ability of the inner ear to convert sound waves into electrical signals. The main actors in this process are hair cells. Their stereocilia contain a number of specific proteins and a scaffold of actin molecules. They are organized in bundles by tip-link filaments composed of cadherin 23 and protocadherin 15. The bundle is deflected by sound waves leading to the opening of mechano-transduction channels and to the influx of K(+) and Ca(2+) into the stereocilia. Cadherin 23 and the plasma membrane calcium ATPase isoform 2 (PMCA2) are defective in human and murine cases of deafness. While the involvement of cadherin 23 in deafness/hearing could be expected due to its structural role in the tip-links, that of PMCA2 has been discovered only recently. This review will summarize the structural and functional characteristics of hair cells, focusing on the proteins whose mutations may lead to a deafness phenotype.

The inner ear converts sound waves into hearing signals through the mechanoelectrical transduction (MET) process. Deflection of the stereocilia bundle of hair cells causes the opening of channels that allow the entry of endolymph K(+) and Ca(2+). Ca(2+) that enters is crucial to the hearing process and is exported to the endolymph by the plasma membrane Ca(2+) pump (isoform PMCA2w/a): disturbances of the balance between Ca(2+) penetration and ejection, e.g. by pump mutations, generate deafness. Hearing loss caused by PMCA defects is frequently exacerbated by mutations in cadherin 23, a single pass stereociliar Ca(2+) binding protein that forms the tip links which permit the deflection of the stereocilia bundle and thus the opening of the MET channels. The PMCA2w/a pump ejects Ca(2+) to the endolymph even in the absence of the natural activator calmodulin. This satisfies the special Ca(2+) homeostasis requirements of the stereocilia/endolymph system. Here we have analyzed a mice and a human previously described pump mutant. The human mutant only exacerbated the deafness produced by a cadherin 23 mutation. The murine mutant overexpressed in model cells displayed an evident defect both in the basal activity of the pump and in the long range ejection of Ca(2+), the human mutant instead failed to impair the Ca(2+) ejection by the pump.