No abstract

SummaryMitochondrial calcium accumulation was recently shown to depend on a complex composed of an inner-membrane channel (MCU and MCUb) and regulatory subunits (MICU1, MCUR1, and EMRE). A fundamental property of MCU is low activity at resting cytosolic Ca2+ concentrations, preventing deleterious Ca2+ cycling and organelle overload. Here we demonstrate that these properties are ensured by a regulatory heterodimer composed of two proteins with opposite effects, MICU1 and MICU2, which, both in purified lipid bilayers and in intact cells, stimulate and inhibit MCU activity, respectively. Both MICU1 and MICU2 are regulated by calcium through their EF-hand domains, thus accounting for the sigmoidal response of MCU to [Ca2+] in situ and allowing tight physiological control. At low [Ca2+], the dominant effect of MICU2 largely shuts down MCU activity; at higher [Ca2+], the stimulatory effect of MICU1 allows the prompt response of mitochondria to Ca2+ signals generated in the cytoplasm.

The versatility and universality of Ca2+ as intracellular messenger is guaranteed by the compartmentalization of changes in [Ca2+]. In this context, mitochondrial Ca2+ plays a central role, by regulating both specific organelle functions and global cellular events. This versatility is also guaranteed by a cell type-specific Ca2+ signaling toolkit controlling specific cellular functions. Accordingly, mitochondrial Ca2+ 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 Ca2+ uptake. Silencing of MICU3 in primary cortical neurons impairs Ca2+ signals elicited by synaptic activity, thus suggesting a specific role in regulating neuronal function.

Several stimuli induce programmed cell death by increasing Ca 2+transfer from the endoplasmic reticulum (ER) to mitochondria. Perturbation of this process has a special relevance in pathologies as cancer and neurodegenerative disorders. Mitochondrial Ca 2+ uptake mainly takes place in correspondence of mitochondria-associated ER membranes (MAM), specialized contact sites between the two organelles. Here, we show the important role of FATE1, a cancer-testis antigen, in the regulation of ER-mitochondria distance and Ca 2+ uptake by mitochondria.FATE1 is localized at the interface between ER and mitochondria, fractionating into MAM. FATE1 expression in adrenocortical carcinoma (ACC) cells under the control of the transcription factor SF-1 decreases ER-mitochondria contact and mitochondrial Ca 2+ uptake, while its knockdown has an opposite effect. FATE1 also decreases sensitivity to mitochondrial Ca 2+-dependent proapoptotic stimuli and to the chemotherapeutic drug mitotane. In patients with ACC, FATE1 expression in their tumor is inversely correlated with their overall survival. These results show that the ER-mitochondria uncoupling activity of FATE1 is harnessed by cancer cells to escape apoptotic death and resist the action of chemotherapeutic drugs.

The direct measurement of mitochondrial [Ca 2؉ ] with highly specific probes demonstrated that major swings in organellar [Ca 2؉ ] parallel the changes occurring in the cytosol and regulate processes as diverse as aerobic metabolism and cell death by necrosis and apoptosis. Despite great biological relevance, insight was limited by the complete lack of molecular understanding. The situation has changed, and new perspectives have emerged following the very recent identification of the mitochondrial Ca 2؉ uniporter, the channel allowing rapid Ca 2؉ accumulation across the inner mitochondrial membrane.

Mutant Cu/Zn superoxide dismutase (SOD1) causes mitochondrial alterations that contribute to motor neuron demise in amyotrophic lateral sclerosis (ALS). When mitochondria are damaged, cells activate mitochondria quality control (MQC) mechanisms leading to mitophagy. Here, we show that in the spinal cord of G93A mutant SOD1 transgenic mice (SOD1‐G93A mice), the autophagy receptor p62 is recruited to mitochondria and mitophagy is activated. Furthermore, the mitochondrial ubiquitin ligase Parkin and mitochondrial dynamics proteins, such as Miro1, and Mfn2, which are ubiquitinated by Parkin, and the mitochondrial biogenesis regulator PGC1α are depleted. Unexpectedly, Parkin genetic ablation delays disease progression and prolongs survival in SOD1‐G93A mice, as it slows down motor neuron loss and muscle denervation and attenuates the depletion of mitochondrial dynamics proteins and PGC1α. Our results indicate that Parkin is a disease modifier in ALS, because chronic Parkin‐mediated MQC activation depletes mitochondrial dynamics‐related proteins, inhibits mitochondrial biogenesis, and worsens mitochondrial dysfunction.

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.

Mitochondrial disorders are a group of pathologies characterized by impairment of mitochondrial function mainly due to defects of the respiratory chain and consequent organellar energetics. This affects organs and tissues that require an efficient energy supply, such as brain and skeletal muscle. They are caused by mutations in both nuclear-and mitochondrial DNA (mtDNA)-encoded genes and their clinical manifestations show a great heterogeneity in terms of age of onset and severity, suggesting that patient-specific features are key determinants of the pathogenic process. In order to correlate the genetic defect to the clinical phenotype, we used a cell culture model consisting of fibroblasts derived from patients with different mutations in the mtDNA-encoded ND5 complex I subunit and with different severities of the illness. Interestingly, we found that cells from patients with the 13514A4G mutation, who manifested a relatively late onset and slower progression of the disease, display an increased autophagic flux when compared with fibroblasts from other patients or healthy donors. We characterized their mitochondrial phenotype by investigating organelle turnover, morphology, membrane potential and Ca 2+ homeostasis, demonstrating that mitochondrial quality control through mitophagy is upregulated in 13514A4G cells. This is due to a specific downregulation of mitochondrial Ca 2+ uptake that causes the stimulation of the autophagic machinery through the AMPK signaling axis. Genetic and pharmacological manipulation of mitochondrial Ca 2+ homeostasis can revert this phenotype, but concurrently decreases cell viability. This indicates that the higher mitochondrial turnover in complex I deficient cells with this specific mutation is a pro-survival compensatory mechanism that could contribute to the mild clinical phenotype of this patient. Mitochondrial disorders include a wide range of pathological conditions characterized by defects in organelle homoeostasis and energy metabolism, in particular in the electron transport chain (ETC) complexes. They are mostly caused by mutations in nuclear-or mtDNA-encoded genes of the respiratory chain complexes leading to a variety of clinical manifestations, ranging from lesions in specific tissues, such as in Leber's hereditary optic neuropathy, to complex multisystem syndromes, such as myoclonic epilepsy with ragged-red fibers, Leigh syndrome or the mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). 1,2 Despite the detailed knowledge of the molecular defects in these diseases, their pathogenesis remains poorly understood. The heterogeneity of signs and symptoms depends on the diversity of the genetic background and on patient-specific compensatory mechanisms. Several studies investigated the consequences of nuclear DNA mutations on intracellular organelle physiology and Ca 2+ homeostasis. 3,4 Here we analyzed a cohort of patients with mutations in the mtDNA-encoded ND5 subunit of NADH dehydrogenase in order to correlate the clinical phenot...