Sara Leo

Ca2+ transfer from the ER to mitochondria: when, how and why

The heterogenous subcellular distribution of a wide array of channels, pumps and exchangers allows extracellular stimuli to induce increases in cytoplasmic Ca(2+) concentration ([Ca(2+)]c) with highly defined spatial and temporal patterns, that in turn induce specific cellular responses (e.g. contraction, secretion, proliferation or cell death). In this extreme complexity, the role of mitochondria was considered marginal, till the direct measurement with targeted indicators allowed to appreciate that rapid and large increases of the [Ca(2+)] in the mitochondrial matrix ([Ca(2+)]m) invariably follow the cytosolic rises. Given the low affinity of the mitochondrial Ca(2+) transporters, the close proximity to the endoplasmic reticulum (ER) Ca(2+)-releasing channels was shown to be responsible for the prompt responsiveness of mitochondria. In this review, we will summarize the current knowledge of: i) the mitochondrial and ER Ca(2+) channels mediating the ion transfer, ii) the structural and molecular foundations of the signaling contacts between the two organelles, iii) the functional consequences of the [Ca(2+)]m increases, and iv) the effects of oncogene-mediated signals on mitochondrial Ca(2+) homeostasis. Despite the rapid progress carried out in the latest years, a deeper molecular understanding is still needed to unlock the secrets of Ca(2+) signaling machinery.

High glucose induces adipogenic differentiation of muscle-derived stem cells.

Regeneration of mesenchymal tissues depends on a resident stem cell population, that in most cases remains elusive in terms of cellular identity and differentiation signals. We here show that primary cell cultures derived from adipose tissue or skeletal muscle differentiate into adipocytes when cultured in high glucose. High glucose induces ROS production and PKCβ activation. These two events appear crucial steps in this differentiation process that can be directly induced by oxidizing agents and inhibited by PKCβ siRNA silencing. The differentiated adipocytes, when implanted in vivo, form viable and vascularized adipose tissue. Overall, the data highlight a previously uncharacterized differentiation route triggered by high glucose that drives not only resident stem cells of the adipose tissue but also uncommitted precursors present in muscle cells to form adipose depots. This process may represent a feed-forward cycle between the regional increase in adiposity and insulin resistance that plays a key role in the pathogenesis of diabetes mellitus.

Mitochondria, calcium and cell death: A deadly triad in neurodegeneration

Mitochondrial Ca(2+) accumulation is a tightly controlled process, in turn regulating functions as diverse as aerobic metabolism and induction of cell death. The link between Ca(2+) (dys)regulation, mitochondria and cellular derangement is particularly evident in neurodegenerative disorders, in which genetic models and environmental factors allowed to identify common traits in the pathogenic routes. We will here summarize: i) the current view of mechanisms and functions of mitochondrial Ca(2+) homeostasis, ii) the basic principles of organelle Ca(2+) transport, iii) the role of Ca(2+) in neuronal cell death, and iv) the new information on the pathogenesis of Alzheimers, Huntingtons and Parkinsons diseases, highlighting the role of Ca(2+) and mitochondria.

Long-term modulation of mitochondrial Ca2+ signals by protein kinase C isozymes.

The modulation of Ca2+ signaling patterns during repetitive stimulations represents an important mechanism for integrating through time the inputs received by a cell. By either overexpressing the isoforms of protein kinase C (PKC) or inhibiting them with specific blockers, we investigated the role of this family of proteins in regulating the dynamic interplay of the intracellular Ca2+ pools. The effects of the different isoforms spanned from the reduction of ER Ca2+ release (PKCα) to the increase or reduction of mitochondrial Ca2+ uptake (PKCζ and PKCβ/PKCδ, respectively). This PKC-dependent regulatory mechanism underlies the process of mitochondrial Ca2+ desensitization, which in turn modulates cellular responses (e.g., insulin secretion). These results demonstrate that organelle Ca2+ homeostasis (and in particular mitochondrial processing of Ca2+ signals) is tuned through the wide molecular repertoire of intracellular Ca2+ transducers.

The Mitochondrial Antioxidants MitoE2 and MitoQ10 Increase Mitochondrial Ca2+ Load upon Cell Stimulation by Inhibiting Ca2+ Efflux from the Organelle

Mitochondrial reactive oxygen species (ROS) production is recognized as a major pathogenic event in a number of human diseases, and mitochondrial scavenging of ROS appears a promising therapeutic approach. Recently, two mitochondrial antioxidants have been developed; conjugating α‐tocopherol and the ubiquinol moiety of coenzyme Q to the lipophilic triphenylphosphonium cation (TPP+), denominated MitoE2 and MitoQ10, respectively. We have investigated the effect of these compounds on mitochondrial Ca2+ homeostasis, which controls processes as diverse as activation of mitochondrial dehydrogenases and pro‐apoptotic morphological changes of the organelle. We demonstrate that treatment of HeLa cells with both MitoE2 and MitoQ10 induces (albeit with different efficacy) a major enhancement of the increase in matrix Ca2+ concentration triggered by cell stimulation with the inositol 1,4,5‐trisphosphate‐generating agonist histamine. The effect is a result of the inhibition of Ca2+ efflux from the organelle and depends on the TPP+ moiety of these compounds. Overall, the data identify an effect independent of their antioxidant activity, that on the one hand may be useful in addressing disorders in which mitochondrial Ca2+ handling is impaired (e.g., mitochondrial diseases) and on the other may favor mitochondrial Ca2+ overload and thus increase cell sensitivity to apoptosis (thus possibly counteracting the benefits of the antioxidant activity).

Mitochondrial calcium signalling in cell death

The development of targeted probes (based on the molecular engineering of luminescent or fluorescent proteins) has allowed the specific measurement of [Ca2+] in intracellular organelles or cytoplasmic subdomains. This approach gave novel information on different aspects of cellular Ca2+ homeostasis. Regarding mitochondria, it was possible to demonstrate that, upon physiological stimulation of cells, Ca2+ is rapidly accumulated in the matrix. We will discuss the basic characteristics of this process, its role in modulating physiological and pathological events, such as the regulation of aerobic metabolism and the induction of cell death, and new insight into the regulatory mechanisms operating in vivo.

Mitochondrial calcium signalling in cell death.

The development of targeted probes (based on the molecular engineering of luminescent or fluorescent proteins) has allowed the specific measurement of [Ca2+] in intracellular organelles or cytoplasmic subdomains. This approach gave novel information on different aspects of cellular Ca2+ homeostasis. Regarding mitochondria, it was possible to demonstrate that, upon physiological stimulation of cells, Ca2+ is rapidly accumulated in the matrix. We will discuss the basic characteristics of this process, its role in modulating physiological and pathological events, such as the regulation of aerobic metabolism and the induction of cell death, and new insight into the regulatory mechanisms operating in vivo.

The selective inhibition of nuclear PKCζ restores the effectiveness of chemotherapeutic agents in chemoresistant cells

The atypical protein kinase C (PKC) isoform zeta (PKCζ) has been implicated in the intracellular transduction of mitogenic and apoptotic signals by acting on different signaling pathways. The key role of these processes in tumorigenesis suggests a possible involvement of PKCζ in this event. PKCζ is activated by cytotoxic treatments, inhibits apoptotic cell death and reduces the sensitivity of cancer cells to chemotherapeutic agents. Here, using pharmacological and DNA recombinant approaches, we show that oxidative stress triggers nuclear translocation of PKCζ and induces resistance to apoptotic agents. Accordingly, chemoresistant cells show accumulation of PKCζ within the nucleus, and a nuclear-targeted PKCζ transfected in tumor cells decreases sensitivity to apoptosis. We thus developed a novel recombinant protein capable of selectively inhibiting the nuclear fraction of PKCζ that restored the susceptibility to apoptosis in cells in which PKCζ was enriched in the nuclear fraction, including chemoresistant cells. These findings establish the importance of PKCζ as a possible target to increase the effectiveness of anticancer therapies and highlight potential sites of intervention.