Simone Patergnani

Mitochondria-ros crosstalk in the control of cell death and aging.

Reactive oxygen species (ROS) are highly reactive molecules, mainly generated inside mitochondria that can oxidize DNA, proteins, and lipids. At physiological levels, ROS function as “redox messengers” in intracellular signalling and regulation, whereas excess ROS induce cell death by promoting the intrinsic apoptotic pathway. Recent work has pointed to a further role of ROS in activation of autophagy and their importance in the regulation of aging. This review will focus on mitochondria as producers and targets of ROS and will summarize different proteins that modulate the redox state of the cell. Moreover, the involvement of ROS and mitochondria in different molecular pathways controlling lifespan will be reported, pointing out the role of ROS as a “balance of power,” directing the cell towards life or death.

Role of the c subunit of the FO ATP synthase in mitochondrial permeability transition

The term “mitochondrial permeability transition” (MPT) refers to an abrupt increase in the permeability of the inner mitochondrial membrane to low molecular weight solutes. Due to osmotic forces, MPT is paralleled by a massive influx of water into the mitochondrial matrix, eventually leading to the structural collapse of the organelle. Thus, MPT can initiate mitochondrial outer membrane permeabilization (MOMP), promoting the activation of the apoptotic caspase cascade as well as of caspase-independent cell death mechanisms. MPT appears to be mediated by the opening of the so-called “permeability transition pore complex” (PTPC), a poorly characterized and versatile supramolecular entity assembled at the junctions between the inner and outer mitochondrial membranes. In spite of considerable experimental efforts, the precise molecular composition of the PTPC remains obscure and only one of its constituents, cyclophilin D (CYPD), has been ascribed with a crucial role in the regulation of cell death. Conversely, the results of genetic experiments indicate that other major components of the PTPC, such as voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT), are dispensable for MPT-driven MOMP. Here, we demonstrate that the c subunit of the FO ATP synthase is required for MPT, mitochondrial fragmentation and cell death as induced by cytosolic calcium overload and oxidative stress in both glycolytic and respiratory cell models. Our results strongly suggest that, similar to CYPD, the c subunit of the FO ATP synthase constitutes a critical component of the PTPC.

Mitochondrial Ca2+ and apoptosis

Mitochondria are key decoding stations of the apoptotic process. In support of this view, a large body of experimental evidence has unambiguously revealed that, in addition to the well-established function of producing most of the cellular ATP, mitochondria play a fundamental role in triggering apoptotic cell death. Various apoptotic stimuli cause the release of specific mitochondrial pro-apoptotic factors into the cytosol. The molecular mechanism of this release is still controversial, but there is no doubt that mitochondrial calcium (Ca2+) overload is one of the pro-apoptotic ways to induce the swelling of mitochondria, with perturbation or rupture of the outer membrane, and in turn the release of mitochondrial apoptotic factors into the cytosol. Here, we review as different proteins that participate in mitochondrial Ca2+ homeostasis and in turn modulate the effectiveness of Ca2+-dependent apoptotic stimuli. Strikingly, the final outcome at the cellular level is similar, albeit through completely different molecular mechanisms: a reduced mitochondrial Ca2+ overload upon pro-apoptotic stimuli that dramatically blunts the apoptotic response.

Calcium signaling around Mitochondria Associated Membranes (MAMs)

Calcium (Ca2+) homeostasis is fundamental for cell metabolism, proliferation, differentiation, and cell death. Elevation in intracellular Ca2+ concentration is dependent either on Ca2+ influx from the extracellular space through the plasma membrane, or on Ca2+ release from intracellular Ca2+ stores, such as the endoplasmic/sarcoplasmic reticulum (ER/SR). Mitochondria are also major components of calcium signalling, capable of modulating both the amplitude and the spatio-temporal patterns of Ca2+ signals. Recent studies revealed zones of close contact between the ER and mitochondria called MAMs (Mitochondria Associated Membranes) crucial for a correct communication between the two organelles, including the selective transmission of physiological and pathological Ca2+ signals from the ER to mitochondria. In this review, we summarize the most up-to-date findings on the modulation of intracellular Ca2+ release and Ca2+ uptake mechanisms. We also explore the tight interplay between ER- and mitochondria-mediated Ca2+ signalling, covering the structural and molecular properties of the zones of close contact between these two networks.

Protein Kinases and Phosphatases in the Control of Cell Fate

Protein phosphorylation controls many aspects of cell fate and is often deregulated in pathological conditions. Several recent findings have provided an intriguing insight into the spatial regulation of protein phosphorylation across different subcellular compartments and how this can be finely orchestrated by specific kinases and phosphatases. In this review, the focus will be placed on (i) the phosphoinositide 3-kinase (PI3K) pathway, specifically on the kinases Akt and mTOR and on the phosphatases PP2a and PTEN, and on (ii) the PKC family of serine/threonine kinases. We will look at general aspects of cell physiology controlled by these kinases and phosphatases, highlighting the signalling pathways that drive cell division, proliferation, and apoptosis.

Downregulation of the Mitochondrial Calcium Uniporter by Cancer-Related miR-25

Summary The recently discovered mitochondrial calcium uniporter (MCU) promotes Ca2+ accumulation into the mitochondrial matrix [1, 2]. We identified in silico miR-25 as a cancer-related MCU-targeting microRNA family and demonstrate that its overexpression in HeLa cells drastically reduces MCU levels and mitochondrial Ca2+ uptake, while leaving other mitochondrial parameters and cytosolic Ca2+ signals unaffected. In human colon cancers and cancer-derived cells, miR-25 is overexpressed and MCU accordingly silenced. miR-25-dependent reduction of mitochondrial Ca2+ uptake correlates with resistance to apoptotic challenges and can be reversed by anti-miR-25 overexpression. Overall, the data demonstrate that microRNA targeting of mitochondrial Ca2+ signaling favors cancer cell survival, thus providing mechanistic insight into the role of mitochondria in tumorigenesis and identifying a novel therapeutic target in neoplasia.

Redox control of protein kinase C: cell- and disease-specific aspects.

Hormones, growth factors, electrical stimulation, and cell-cell interactions regulate numerous cellular processes by altering the levels of second messengers, thus influencing biochemical reactions inside the cells. The Protein Kinase C family (PKCs) is a group of serine/threonine kinases that are dependent on calcium (Ca(2+)), diacylglycerol, and phospholipids. Signaling pathways that induce variations on the levels of PKC activators have been implicated in the regulation of diverse cellular functions and, in turn, PKCs are key regulators of a plethora of cellular processes, including proliferation, differentiation, and tumorigenesis. Importantly, PKCs contain regions, both in the N-terminal regulatory domain and in the C-terminal catalytic domain, that are susceptible to redox modifications. In several pathophysiological conditions when the balance between oxidants, antioxidants, and alkylants is compromised, cells undergo redox stress. PKCs are cell-signaling proteins that are particularly sensitive to redox stress because modification of their redox-sensitive regions interferes with their activity and, thus, with their biological effects. In this review, we summarize the involvement of PKCs in health and disease and the importance of redox signaling in the regulation of this family of kinases.

Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes

The jellyfish Aequorea victoria produces a 22-kDa protein named aequorin that has had an important role in the study of calcium (Ca2+) signaling. Aequorin reacts with Ca2+ via oxidation of the prosthetic group, coelenterazine, which results in emission of light. This signal can be detected by using a special luminescence reader (called aequorinometer) or luminescence plate readers. Here we describe the main characteristics of aequorin as a Ca2+ probe and how to measure Ca2+ in different intracellular compartments of animal cells (cytosol, different mitochondrial districts, nucleus, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes and subplasma-membrane cytosol), ranging from single-well analyses to high-throughput screening by transfecting animal cells using DNA vectors carrying recombinant aequorin chimeras. The use of aequorin mutants and modified versions of coelenterazione increases the range of calcium concentrations that can be recorded. Cell culture and transfection takes ∼3 d. An experiment including signal calibration and the subsequent analyses will take ∼1 d.

Mitochondrial calcium homeostasis as potential target for mitochondrial medicine

Mitochondria are crucial in different intracellular pathways of signal transduction. Mitochondria are capable of decoding a variety of extracellular stimuli into markedly different intracellular actions, ranging from energy production to cell death. The fine modulation of mitochondrial calcium (Ca2+) homeostasis plays a fundamental role in many of the processes involving this organelle. When mitochondrial Ca2+ homeostasis is compromised, different pathological conditions can occur, depending on the cell type involved. Recent data have shed light on the molecular identity of the main proteins involved in the handling of mitochondrial Ca2+ traffic, opening fascinating and ambitious new avenues for mitochondria-based pharmacological strategies.

Syndromic parkinsonism and dementia associated with OPA1 missense mutations.

Mounting evidence links neurodegenerative disorders such as Parkinson disease and Alzheimer disease with mitochondrial dysfunction, and recent emphasis has focused on mitochondrial dynamics and quality control. Mitochondrial dynamics and mtDNA maintenance is another link recently emerged, implicating mutations in the mitochondrial fusion genes OPA1 and MFN2 in the pathogenesis of multisystem syndromes characterized by neurodegeneration and accumulation of mtDNA multiple deletions in postmitotic tissues. Here, we report 2 Italian families affected by dominant chronic progressive external ophthalmoplegia (CPEO) complicated by parkinsonism and dementia.