Transglutaminase Type 2-MITF axis regulates phenotype switching in skin cutaneous melanoma.

Skin cutaneous melanoma (SKCM) is the deadliest form of skin cancer due to its high heterogeneity that drives tumor aggressiveness. Melanoma plasticity consists of two distinct phenotypic states that co-exist in the tumor niche, the proliferative and the invasive, respectively associated with a high and low expression of MITF, the master regulator of melanocyte lineage. However, despite efforts, melanoma research is still far from exhaustively dissecting this phenomenon. Here, we discovered a key function of Transglutaminase Type-2 (TG2) in regulating melanogenesis by modulating MITF transcription factor expression and its transcriptional activity. Importantly, we demonstrated that TG2 expression affects melanoma invasiveness, highlighting its positive value in SKCM. These results suggest that TG2 may have implications in the regulation of the phenotype switching by promoting melanoma differentiation and impairing its metastatic potential. Our findings offer potential perspectives to unravel melanoma vulnerabilities via tuning intra-tumor heterogeneity.

High-Throughput Microscopy Analysis of Mitochondrial Membrane Potential in 2D and 3D Models.

Recent proteomic, metabolomic, and transcriptomic studies have highlighted a connection between changes in mitochondria physiology and cellular pathophysiological mechanisms. Secondary assays to assess the function of these organelles appear fundamental to validate these -omics findings. Although mitochondrial membrane potential is widely recognized as an indicator of mitochondrial activity, high-content imaging-based approaches coupled to multiparametric to measure it have not been established yet. In this paper, we describe a methodology for the unbiased high-throughput quantification of mitochondrial membrane potential in vitro, which is suitable for 2D to 3D models. We successfully used our method to analyze mitochondrial membrane potential in monolayers of human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers. Moreover, by combining automated image analysis and machine learning, we were able to discriminate melanoma cells from macrophages in co-culture and to analyze the subpopulations separately. Our data demonstrated that our method is a widely applicable strategy for large-scale profiling of mitochondrial activity.

Comparison among Neuroblastoma Stages Suggests the Involvement of Mitochondria in Tumor Progression.

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.

Dysfunctional mitochondria accumulate in a skeletal muscle knockout model of Smn1, the causal gene of spinal muscular atrophy.

The approved gene therapies for spinal muscular atrophy (SMA), caused by loss of survival motor neuron 1 (SMN1), greatly ameliorate SMA natural history but are not curative. These therapies primarily target motor neurons, but SMN1 loss has detrimental effects beyond motor neurons and especially in muscle. Here we show that SMN loss in mouse skeletal muscle leads to accumulation of dysfunctional mitochondria. Expression profiling of single myofibers from a muscle specific Smn1 knockout mouse model revealed down-regulation of mitochondrial and lysosomal genes. Albeit levels of proteins that mark mitochondria for mitophagy were increased, morphologically deranged mitochondria with impaired complex I and IV activity and respiration and that produced excess reactive oxygen species accumulated in Smn1 knockout muscles, because of the lysosomal dysfunction highlighted by the transcriptional profiling. Amniotic fluid stem cells transplantation that corrects the SMN knockout mouse myopathic phenotype restored mitochondrial morphology and expression of mitochondrial genes. Thus, targeting muscle mitochondrial dysfunction in SMA may complement the current gene therapy.

Cell cycle alterations due to perfluoroalkyl substances PFOS, PFOA, PFBS, PFBA and the new PFAS C6O4 on bottlenose dolphin (Tursiops truncatus) skin cell.

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants in aquatic ecosystems worldwide. Marine mammals, as top predators, are constantly exposed to several PFAS compounds that accumulate in different tissues. As a proxy to assess cytotoxicity of PFAS in the bottlenose dolphin (Tursiops truncatus), we generated a new immortalized cell line derived from skin samples of bottlenose dolphin. Using high content imaging, we assessed the effects of increasing concentrations of PFOS, PFOA, PFBS, PFBA and C6O4 on cell viability and cell cycle phases. In particular, we classified all cells based on multiple morphometric differences of the nucleus in three populations, named respectively "Normal" (nuclei in G0, S and M phase); "Large" (nuclei showing characteristics of senescence) and "Small" (nuclei with fragmentation and condensed chromatin). Combining this approach with cell cycle analysis we determined which phases of the cell cycle were influenced by PFAS. The results revealed that the presence of PFOS, PFBS and PFBA could increase the number of cells in G0+G1 phase and decrease the number of those in the S phase. Moreover, PFOS and PFBS lowered the fraction of cells in the M phase. Interestingly PFOS, PFBS and PFBA reduced the prevalence of the senescence phenotype ("large" nuclei), suggesting a potential tumorigenic effect. Besides, the presence of PFOS and PFBS correlated also with a significant decrease in the number of "small" nuclei. The C6O4 exposure did not highlighted morphometric alteration or cell cycle modification bottlenose dolphin skin cell nuclei. While the effects of PFAS on cell cycle was clear, no significant change was detected either in term of cell proliferation or of viability. This study fosters the overall knowledge on the cellular effects of perfluoroalkyl substances in marine mammals.

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton.

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

The Interplay of Microtubules with Mitochondria-ER Contact Sites (MERCs) in Glioblastoma.

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.

Cisplatin resistance can be curtailed by blunting Bnip3-mediated mitochondrial autophagy.

Cisplatin (CDDP) is commonly used to treat a multitude of tumors including sarcomas, ovarian and cervical cancers. Despite recent investigations allowed to improve chemotherapy effectiveness, the molecular mechanisms underlying the development of CDDP resistance remain a major goal in cancer research. Here, we show that mitochondrial morphology and autophagy are altered in different CDDP resistant cancer cell lines. In CDDP resistant osteosarcoma and ovarian carcinoma, mitochondria are fragmented and closely juxtaposed to the endoplasmic reticulum; rates of mitophagy are also increased. Specifically, levels of the mitophagy receptor BNIP3 are higher both in resistant cells and in ovarian cancer patient samples resistant to platinum-based treatments. Genetic BNIP3 silencing or pharmacological inhibition of autophagosome formation re-sensitizes these cells to CDDP. Our study identifies inhibition of BNIP3-driven mitophagy as a potential therapeutic strategy to counteract CDDP resistance in ovarian carcinoma and osteosarcoma.

Inhibition of the mitochondrial protein Opa1 curtails breast cancer growth.

BACKGROUND: Mitochondrial fusion and fission proteins have been nominated as druggable targets in cancer. Whether their inhibition is efficacious in triple negative breast cancer (TNBC) that almost invariably develops chemoresistance is unknown. METHODS: We used a combination of bioinformatics analyses of cancer genomic databases, genetic and pharmacological Optic Atrophy 1 (OPA1) inhibition, mitochondrial function and morphology measurements, micro-RNA (miRNA) profiling and formal epistatic analyses to address the role of OPA1 in TNBC proliferation, migration, and invasion in vitro and in vivo. RESULTS: We identified a signature of OPA1 upregulation in breast cancer that correlates with worse prognosis. Accordingly, OPA1 inhibition could reduce breast cancer cells proliferation, migration, and invasion in vitro and in vivo. Mechanistically, while OPA1 silencing did not reduce mitochondrial respiration, it increased levels of miRNAs of the 148/152 family known to inhibit tumor growth and invasiveness. Indeed, these miRNAs were epistatic to OPA1 in the regulation of TNBC cells growth and invasiveness. CONCLUSIONS: Our data show that targeted inhibition of the mitochondrial fusion protein OPA1 curtails TNBC growth and nominate OPA1 as a druggable target in TNBC.

Mitochondria modulate ameloblast Ca2+ signaling.

The role of mitochondria in enamel, the most mineralized tissue in the body, is poorly defined. Enamel is formed by ameloblast cells in two main sequential stages known as secretory and maturation. Defining the physiological features of each stage is essential to understand mineralization. Here, we analyzed functional features of mitochondria in rat primary secretory and maturation-stage ameloblasts focusing on their role in Ca2+ signaling. Quantification of the Ca2+ stored in the mitochondria by trifluoromethoxy carbonylcyanide phenylhydrazone stimulation was comparable in both stages. The release of endoplasmic reticulum Ca2+ pools by adenosine triphosphate in rhod2AM-loaded cells showed similar mitochondrial Ca2+ (m Ca2+ ) uptake. However, m Ca2+ extrusion via Na+ -Li+ -Ca2+ exchanger was more prominent in maturation. To address if m Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) played a role in cytosolic Ca2+ (c Ca2+ ) buffering, we stimulated Ca2+ influx via the store-operated Ca2+ entry (SOCE) and blocked MCU with the inhibitor Ru265. This inhibitor was first tested using the enamel cell line LS8 cells. Ru265 prevented c Ca2+ clearance in permeabilized LS8 cells like ruthenium red, and it did not affect ΔΨm in intact cells. In primary ameloblasts, SOCE stimulation elicited a significantly higher m Ca2+ uptake in maturation ameloblasts. The uptake of Ca2+ into the mitochondria was dramatically decreased in the presence of Ru265. Combined, these results suggest an increased mitochondrial Ca2+ handling in maturation but only upon stimulation of Ca2+ influx via SOCE. These functional studies provide insights not only on the role of mitochondria in ameloblast Ca2+ physiology, but also advance the concept that SOCE and m Ca2+ uptake are complementary processes in biological mineralization.

The mitochondrial protein Opa1 promotes adipocyte browning that is dependent on urea cycle metabolites.

White to brown/beige adipocytes conversion is a possible therapeutic strategy to tackle the current obesity epidemics. While mitochondria are key for energy dissipation in brown fat, it is unknown if they can drive adipocyte browning. Here, we show that the mitochondrial cristae biogenesis protein optic atrophy 1 (Opa1) facilitates cell-autonomous adipocyte browning. In two cohorts of patients with obesity, including weight discordant monozygotic twin pairs, adipose tissue OPA1 levels are reduced. In the mouse, Opa1 overexpression favours white adipose tissue expandability as well as browning, ultimately improving glucose tolerance and insulin sensitivity. Transcriptomics and metabolomics analyses identify the Jumanji family chromatin remodelling protein Kdm3a and urea cycle metabolites, including fumarate, as effectors of Opa1-dependent browning. Mechanistically, the higher cyclic adenosine monophosphate (cAMP) levels in Opa1 pre-adipocytes activate cAMP-responsive element binding protein (CREB), which transcribes urea cycle enzymes. Flux analyses in pre-adipocytes indicate that Opa1-dependent fumarate accumulation depends on the urea cycle. Conversely, adipocyte-specific Opa1 deletion curtails urea cycle and beige differentiation of pre-adipocytes, and is rescued by fumarate supplementation. Thus, the urea cycle links the mitochondrial dynamics protein Opa1 to white adipocyte browning.

Chemical Modulation of Mitochondria-Endoplasmic Reticulum Contact Sites.

Contact sites between mitochondria and endoplasmic reticulum (ER) are points in which the two organelles are in close proximity. Due to their structural and functional complexity, their exploitation as pharmacological targets has never been considered so far. Notwithstanding, the number of compounds described to target proteins residing at these interfaces either directly or indirectly is rising. Here we provide original insight into mitochondria-ER contact sites (MERCs), with a comprehensive overview of the current MERCs pharmacology. Importantly, we discuss the considerable potential of MERCs to become a druggable target for the development of novel therapeutic strategies.

Interaction Between Mitochondrial DNA Variants and Mitochondria/Endoplasmic Reticulum Contact Sites: A Perspective Review.

Mitochondria contain their own genome, mitochondrial DNA (mtDNA), essential to support their fundamental intracellular role in ATP production and other key metabolic and homeostatic pathways. Mitochondria are highly dynamic organelles that communicate with all the other cellular compartments, through sites of high physical proximity. Among all, their crosstalk with the endoplasmic reticulum (ER) appears particularly important as its derangement is tightly implicated with several human disorders. Population-specific mtDNA variants clustered in defining the haplogroups have been shown to exacerbate or mitigate these pathological conditions. The exact mechanisms of the mtDNA background-modifying effect are not completely clear and a possible explanation is the outcome of mitochondrial efficiency on retrograde signaling to the nucleus. However, the possibility that different haplogroups shape the proximity and crosstalk between mitochondria and the ER has never been proposed neither investigated. In this study, we pose and discuss this question and provide preliminary data to answer it. Besides, we also address the possibility that single, disease-causing mtDNA point mutations may act also by reshaping organelle communication. Overall, this perspective review provides a theoretical platform for future studies on the interaction between mtDNA variants and organelle contact sites.

Developmental and Tumor Angiogenesis Requires the Mitochondria-Shaping Protein Opa1.

While endothelial cell (EC) function is influenced by mitochondrial metabolism, the role of mitochondrial dynamics in angiogenesis, the formation of new blood vessels from existing vasculature, is unknown. Here we show that the inner mitochondrial membrane mitochondrial fusion protein optic atrophy 1 (OPA1) is required for angiogenesis. In response to angiogenic stimuli, OPA1 levels rapidly increase to limit nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) signaling, ultimately allowing angiogenic genes expression and angiogenesis. Endothelial Opa1 is indeed required in an NFκB-dependent pathway essential for developmental and tumor angiogenesis, impacting tumor growth and metastatization. A first-in-class small molecule-specific OPA1 inhibitor confirms that EC Opa1 can be pharmacologically targeted to curtail tumor growth. Our data identify Opa1 as a crucial component of physiological and tumor angiogenesis.

(Neuro)degenerated Mitochondria-ER contacts.

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.

Nicotine mediates oxidative stress and apoptosis through cross talk between NOX1 and Bcl-2 in lung epithelial cells.

Nicotine contributes to the onset and progression of several pulmonary diseases. Among the various pathophysiological mechanisms triggered by nicotine, oxidative stress and cell death are reported in several cell types. We found that chronic exposure to nicotine (48h) induced NOX1-dependent oxidative stress and apoptosis in primary pulmonary cells. In murine (MLE-12) and human (BEAS-2B) lung epithelial cell lines, nicotine acted as a sensitizer to cell death and synergistically enhanced apoptosis when cells were concomitantly exposed to hyperoxia. The precise signaling pathway was investigated in MLE-12 cells in which NOX1 was abrogated by a specific inhibitor or stably silenced by shRNA. In the early phase of exposure (1h), nicotine mediated intracellular Ca(2+) fluxes and activation of protein kinase C, which in its turn activated NOX1, leading to cellular and mitochondrial oxidative stress. The latter triggered the intrinsic apoptotic machinery by modulating the expression of Bcl-2 and Bax. Overexpression of Bcl-2 completely prevented nicotine's detrimental effects, suggesting Bcl-2as a downstream key regulator in nicotine/NOX1-induced cell damage. These results suggest that NOX1 is a major contributor to the generation of intracellular oxidative stress induced by nicotine and might be an important molecule to target in nicotine-related lung pathologies.

The plasma membrane calcium pump: new ways to look at an old enzyme.

The three-dimensional structure of the PMCA pump has not been solved, but its basic mechanistic properties are known to repeat those of the other Ca(2+) pumps. However, the pump also has unique properties. They concern essentially its numerous regulatory mechanisms, the most important of which is the autoinhibition by its C-terminal tail. Other regulatory mechanisms involve protein kinases and the phospholipids of the membrane in which the pump is embedded. Permanent activation of the pump, e.g. by calmodulin, is physiologically as harmful to cells as its absence. The concept is now emerging that the global control of cell Ca(2+) may not be the main function of the pump; in some cell types, it could even be irrelevant. The main pump role would be the regulation of Ca(2+) in cell microdomains in which the pump co-segregates with partners that modulate the Ca(2+) message and transduce it to important cell functions.

Mitochondrial Ca2+-handling in fast skeletal muscle fibers from wild type and calsequestrin-null mice.

Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca(2+)-binding protein inside SR, calsequestrin. Mitochondrial free Ca(2+)-concentration ([Ca(2+)]mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca(2+) release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca(2+)]mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca(2+) concentration. The rise in [Ca(2+)]mito during electrical stimulation occurred in 20-30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s(-1). Accordingly, frequency-dependent increase in [Ca(2+)]mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca(2+)]mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca(2+)]mito transients were increased by inhibition of mitochondrial Na(+)/Ca(2+) exchanger (mNCX). These results provide direct evidence for fast Ca(2+) accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca(2+)-handling and a dependence of mitochondrial Ca(2+)-handling on intracellular (SR) and external Ca(2+) stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca(2+)-leakage.

Plasma membrane calcium ATPases and related disorders.

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.

Estradiol effects on intracellular Ca(2+) homeostasis in bovine brain-derived endothelial cells.

Estrogens diversely affect various physiological processes by genomic or non-genomic mechanisms, in both excitable and non-excitable cells. Additional to the trophic effects of estrogens promoting cell growth and differentiation, recent experimental evidence highlights their involvement in the regulation of intracellular Ca(2+) homeostasis. The effects of estrogens on excitable cells are well documented. However, these steroids also influence numerous physiological events in non-excitable cells, such as fibroblasts or vascular endothelial cells. We have focused our attention on an immortalized endothelial-like cell line derived from fetal bovine cerebellum. Estradiol (E(2)) effects on intracellular Ca(2+) homeostasis were tested by varying the exposure time to the hormone (8, 24, 48 h). Calcium measurements were performed with genetically encoded Ca(2+) probes (Cameleons) targeted to the main subcellular compartments involved in intracellular Ca(2+) homeostasis (cytosol, endoplasmic reticulum, mitochondria). Mitochondrial Ca(2+) uptake significantly decreased after 48-h exposure to E(2), whereas cytosolic and endoplasmic reticulum responses were unaffected. The effect of E(2) on mitochondrial Ca(2+) handling was blocked by ICI 182,780, a pure estrogen receptor antagonist, suggesting that the effect was estrogen-receptor-mediated. To evaluate whether the decrease of Ca(2+) uptake affected mitochondrial membrane potential (ΔΨm), cells were monitored in the presence of tetra-methyl-rhodamine-methylester; no significant changes were seen between cells treated with E(2) and controls. To investigate a mechanism of action, we assessed the possibile involvement of the permeability transition pore (PTP), an inner mitochondrial membrane channel influencing energy metabolism and cell viability. We treated cells with CyclosporinA (CsA), which binds to the matrix chaperone cyclophilin-D and regulates PTP opening. CsA reversed the effects of a 48-h treatment with E(2), suggesting a possible transcriptional modulation of proteins involved in the mitochondrial permeability transition process.