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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., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Enrico Costanzo, Luigi Nataloni and Giulia Cestonaro (Cereal Docks S.p.A.) has patent # IT102022000016812 pending to Assignee., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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 Ca 2+ signaling contributes to the progression of carcinoma of different origins. However, whether adipocyte-derived factors modulate mitochondrial Ca 2+ signaling in tumours is unknown. Here, we show that conditioned media derived from adipose tissue cultures (ADCM) enriched in precursor cells impinge on mitochondrial Ca 2+ homeostasis of target cells. Moreover, in modulating mitochondrial Ca 2+ 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 Ca 2+ uptake, migration and proliferation of esophageal and breast tumour cells, respectively., (© 2024. The Author(s).)

MitoK ATP 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 mitoK ATP were identified, allowing the genetic manipulation of the channel. Here, we analyzed the role of mitoK ATP 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 mitoK ATP activity is required for the maintenance of skeletal muscle homeostasis and function., (© 2024. The Author(s).)

Mitochondrial Ca 2+ (mitCa 2+ ) uptake controls both intraorganellar and cytosolic functions. Within the organelle, [Ca 2+ ] 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 Ca 2+ (cytCa 2+ ) increases, thus regulating Ca 2+ -dependent cellular processes. In pathological conditions, mitCa 2+ overload triggers the opening of the mitochondrial permeability transition pore (mPTP) and the release of apoptotic cofactors. MitCa 2+ uptake occurs in response of local [Ca 2+ ] increases in sites of proximity between the endoplasmic reticulum (ER) and the mitochondria and is mediated by the mitochondrial Ca 2+ 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 Ca 2+ conductance., Competing Interests: Declaration of Competing Interest The authors of the manuscript entitled “The Mitochondrial Calcium Uniporter (MCU) complex - A play in five acts” declare no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Rewiring of mitochondrial metabolism has been described in different cancers as a key step for their progression. Calcium (Ca 2+ ) 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 Ca 2+ 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 Ca 2+ 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Filadi, De Mario, Audano, Romani, Pedretti, Cardenas, Dupont, Mammucari, Mitro and Pizzo.)

Neuroblastoma (NB) is the most common extracranial tumor of early childhood and accounts for 15% of all pediatric cancer mortalities. However, the precise pathways and genes underlying its progression are unknown. Therefore, we performed a differential gene expression analysis of neuroblastoma stage 1 and stage 4 + 4S to discover biological processes associated with NB progression. From this preliminary analysis, we found that NB samples (stage 4 + 4S) are characterized by altered expression of some proteins involved in mitochondria function and mitochondria-ER contact sites (MERCS). Although further analyses remain necessary, this review may provide new hints to better understand NB molecular etiopathogenesis, by suggesting that MERCS alterations could be involved in the progression of NB.

Gliomas are heterogeneous neoplasms, classified into grade I to IV according to their malignancy and the presence of specific histological/molecular hallmarks. The higher grade of glioma is known as glioblastoma (GB). Although progress has been made in surgical and radiation treatments, its clinical outcome is still unfavorable. The invasive properties of GB cells and glioma aggressiveness are linked to the reshaping of the cytoskeleton. Recent works suggest that the different susceptibility of GB cells to antitumor immune response is also associated with the extent and function of mitochondria-ER contact sites (MERCs). The presence of MERCs alterations could also explain the mitochondrial defects observed in GB models, including abnormalities of energy metabolism and disruption of apoptotic and calcium signaling. Based on this evidence, the question arises as to whether a MERCs-cytoskeleton crosstalk exists, and whether GB progression is linked to an altered cytoskeleton-MERCs interaction. To address this possibility, in this review we performed a meta-analysis to compare grade I and grade IV GB patients. From this preliminary analysis, we found that GB samples (grade IV) are characterized by altered expression of cytoskeletal and MERCs related genes. Among them, the cytoskeleton-associated protein 4 (CKAP4 or CLIMP-63) appears particularly interesting as it encodes a MERCs protein controlling the ER anchoring to microtubules (MTs). Although further in-depth analyses remain necessary, this perspective review may provide new hints to better understand GB molecular etiopathogenesis, by suggesting that cytoskeletal and MERCs alterations cooperate to exacerbate the cellular phenotype of high-grade GB and that MERCs players can be exploited as novel biomarkers/targets to enhance the current therapy for GB.

The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca 2+ 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 Ca 2+ 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 Ca 2+ 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Skeletal muscle mitochondria are placed in close proximity of the sarcoplasmic reticulum (SR), the main intracellular Ca 2+ store. During muscle activity, excitation of sarcolemma and of T-tubule triggers the release of Ca 2+ from the SR initiating myofiber contraction. The rise in cytosolic Ca 2+ 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 Ca 2+ uptake. The Ca 2+ -dependent activation of TCA cycle enzymes increases the synthesis of ATP required for SERCA activity. Thus, Ca 2+ is transported back into the SR and cytosolic [Ca 2+ ] 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 Ca 2+ uptake in the pathophysiology of skeletal muscle has been uncovered. In this chapter, we will introduce the reader to a general overview of mitochondrial Ca 2+ accumulation. We will tackle the key molecular players and the cellular and pathophysiological consequences of mitochondrial Ca 2+ dyshomeostasis. In the second part of the chapter, we will discuss novel findings on the physiological role of mitochondrial Ca 2+ uptake in skeletal muscle. Finally, we will examine the involvement of mitochondrial Ca 2+ signaling in muscle diseases., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mitochondrial Ca 2+ 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 2+ uptake. Two of them, named MCU-i4 and MCU-i11, are confirmed to reliably decrease mitochondrial Ca 2+ 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 2+ 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 2+ uptake in muscle growth. Overall, MCU-i4 and MCU-i11 represent leading molecules for the development of MICU1-targeting drugs., Competing Interests: Declaration of Interests B.J., C.B., V.T.-E., D.H., B.O., S.S., G.M., and M.F. are employees of Novartis Pharma AG, and some are also shareholders of Novartis., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 Ca 2+ -dependent astrocyte-MN cross-talk. In this respect, it has been recently proposed that altered astrocytic store-operated Ca 2+ 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 Ca 2+ indicators, allowing the direct assessment of Ca 2+ fluctuations in different cell domains. We found increased SOCE, associated with decreased expression of the sarco-endoplasmic reticulum Ca 2+ -ATPase and lower ER resting Ca 2+ concentration in SOD1(G93A) astrocytes compared to control cells. Such findings add novel insights into the involvement of astrocytes in ALS MN damage., Competing Interests: The authors declare no conflict of interest.

The cellular prion protein (PrP C ) is an ubiquitous cell surface protein mostly expressed in neurons, where it localizes to both pre- and post-synaptic membranes. PrP C aberrant conformers are the major components of mammalian prions, the infectious agents responsible for incurable neurodegenerative disorders. PrP C 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 PrP C 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 PrP C in synaptic transmission and Ca 2+ homeostasis. Indeed, by controlling gene expression and signaling cascades, PrP C is able to optimize glutamate secretion and regulate Ca 2+ entry via store-operated channels and ionotropic glutamate receptors, thereby protecting neurons from threatening Ca 2+ overloads and excitotoxicity. We will also illustrate and discuss past and unpublished results demonstrating that Aβ oligomers perturb Ca 2+ homeostasis and cause abnormal mitochondrial accumulation of reactive oxygen species by possibly affecting the PrP-dependent downregulation of Fyn kinase activity.

Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca 2+ concentrations ([Ca 2+ ]) of specific intracellular organelles in intact cells. After the binding of Ca 2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca 2+ ]. While native aequorin is suitable for measuring cytosolic [Ca 2+ ] after cell stimulation in a range from 0.5 to 10 μM, it cannot be used in organelles where [Ca 2+ ] 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 Ca 2+ 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 Ca 2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.

Mitochondrial Ca 2+ is involved in heterogeneous functions, ranging from the control of metabolism and ATP production to the regulation of cell death. In addition, mitochondrial Ca 2+ uptake contributes to cytosolic [Ca 2+ ] shaping thus impinging on specific Ca 2+ -dependent events. Mitochondrial Ca 2+ concentration is controlled by influx and efflux pathways: the former controlled by the activity of the mitochondrial Ca 2+ uniporter (MCU), the latter by the Na + /Ca 2+ exchanger (NCLX) and the H + /Ca 2+ (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 Ca 2+ uptake, this review discusses the structure of the MCU complex and the regulation of its activity, the importance of mitochondrial Ca 2+ signaling in different physiological settings, and the consequences of MCU modulation on organ physiology.

The cellular prion protein (PrP C ) 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 PrP C limits Ca 2+ entry via store-operated Ca 2+ channels in neurons, we investigated whether the protein could also control the activity of ionotropic glutamate receptors (iGluRs). To this end, we compared local Ca 2+ movements in primary cerebellar granule neurons and cortical neurons transduced with genetically encoded Ca 2+ probes and expressing, or not expressing, PrP C Our investigation demonstrated that PrP C downregulates Ca 2+ 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 Ca 2+ uptake in PrP-KO neurons than in their PrP C -expressing counterpart. This was because the increased Ca 2+ entry through iGluRs in PrP-KO neurons led to a parallel increase in Ca 2+ -induced Ca 2+ release via ryanodine receptor channels. These data thus suggest that PrP C takes part in the cell apparatus controlling Ca 2+ homeostasis, and that PrP C is involved in protecting neurons from toxic Ca 2+ overloads., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

A finely tuned Ca 2+ homeostasis in restricted cell domains is of fundamental importance for neurons, where transient Ca 2+ oscillations direct the proper coordination of electro-chemical signals and overall neuronal metabolism. Once such a precise regulation is unbalanced, however, neuronal functions and viability are severely compromised. Accordingly, disturbed Ca 2+ metabolism has often been claimed as a major contributor to different neurodegenerative disorders, such as amyotrophic lateral sclerosis that is characterised by selective motor neuron (MN) damage. This notion highlights the need for probes for the specific and precise analysis of local Ca 2+ dynamics in MNs. Here, we generated and functionally validated adeno-associated viral vectors for the expression of gene-encoded fluorescent Ca 2+ indicators targeted to different cell domains, under the transcriptional control of a MN-specific promoter. We demonstrated that the probes are specifically expressed, and allow reliable local Ca 2+ measurements, in MNs from murine primary spinal cord cultures, and can also be expressed in spinal cord MNs in vivo, upon systemic administration to newborn mice. Preliminary analyses using these novel vectors have shown larger cytosolic Ca 2+ responses following stimulation of AMPA receptors in the cytosol of primary cultured MNs from a murine genetic model of ALS compared to the healthy counterpart.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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 PrP Sc , the β sheet-rich pathogenic isoform produced by the conformational conversion of the α-helix-rich physiological isoform PrP C . The peculiarity of PrP Sc is 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.

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 prion protein (PrP(C)) is a cell surface glycoprotein mainly expressed in neurons, whose misfolded isoforms generate the prion responsible for incurable neurodegenerative disorders. Whereas PrP(C) involvement in prion propagation is well established, PrP(C) physiological function is still enigmatic despite suggestions that it could act in cell signal transduction by modulating phosphorylation cascades and Ca(2+) homeostasis. Because PrP(C) binds neurotoxic protein aggregates with high-affinity, it has also been proposed that PrP(C) acts as receptor for amyloid-β (Aβ) oligomers associated with Alzheimer's disease (AD), and that PrP(C)-Aβ binding mediates AD-related synaptic dysfunctions following activation of the tyrosine kinase Fyn. Here, use of gene-encoded Ca(2+) probes targeting different cell domains in primary cerebellar granule neurons (CGN) expressing, or not, PrP(C), allowed us to investigate whether PrP(C) regulates store-operated Ca(2+) entry (SOCE) and the implication of Fyn in this control. Our findings show that PrP(C) attenuates SOCE, and Ca(2+) accumulation in the cytosol and mitochondria, by constitutively restraining Fyn activation and tyrosine phosphorylation of STIM1, a key molecular component of SOCE. This data establishes the existence of a PrP(C)-Fyn-SOCE triad in neurons. We also demonstrate that treating cerebellar granule and cortical neurons with soluble Aβ(1-42) oligomers abrogates the control of PrP(C) over Fyn and SOCE, suggesting a PrP(C)-dependent mechanizm for Aβ-induced neuronal Ca(2+) dyshomeostasis.

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.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)