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

Abstract 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.

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

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

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.

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. : 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. Keywords: mitochondrial calcium uptake, MICU1, mitochondrial calcium uniporter, MCU, high-throughput screening, HTS, molecular modeling, active compounds, small molecules

Abstract A finely tuned Ca2+ homeostasis in restricted cell domains is of fundamental importance for neurons, where transient Ca2+ 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 Ca2+ 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 Ca2+ dynamics in MNs. Here, we generated and functionally validated adeno-associated viral vectors for the expression of gene-encoded fluorescent Ca2+ 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 Ca2+ 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 Ca2+ 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.

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.