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Acute myeloid leukemia (AML) is an aggressive hematologic malignancy that continues to have poor prognosis despite recent therapeutic advances. Venetoclax (Ven), a BCL2-inhibitor has shown a high response rate in AML; however, relapse is invariable due to mitochondrial dysregulation that includes upregulation of the antiapoptotic protein MCL1, a central mechanism of Ven resistance (Ven-res). We have previously demonstrated that the transcription factor STAT3 is upregulated in AML hematopoietic stem and progenitor cells (HSPCs) and can be effectively targeted to induce apoptosis of these aberrant cells. We now show that overexpression of STAT3 alone is sufficient to initiate a strong AML phenotype in a transgenic murine model. Phospho-proteomic data from Ven treated AML patients show a strong correlation of high total STAT3 and phospho-STAT3 [both p-STAT3(Y705) and p-STAT3(S727)] expression with worse survival and reduced remission duration. Additionally, significant upregulation of STAT3 was observed in Ven-res cell lines, in vivo models and primary patient samples. A novel and specific degrader of STAT3 demonstrated targeted reduction of total STAT3 and resulting inhibition of its active p-STAT3(Y705) and p-STAT3(S727) forms. Treatment with the STAT3 degrader induced apoptosis in parental and Ven-res AML cell lines and decreased mitochondrial depolarisation, and thereby dependency on MCL1 in Ven-res AML cell line, as observed by BH3 profiling assay. STAT3 degrader treatment also enhanced differentiation of myeloid and erythroid colonies in Ven-res peripheral blood mononuclear cells (PBMNCs). Upregulation of p-STAT3(S727) was also associated with pronounced mitochondrial structural and functional dysfunction in Ven-res cell lines, that were restored by STAT3 degradation. Treatment with a clinical-stage STAT3 degrader, KT-333 resulted in a significant reduction in STAT3 and MCL1 protein levels within two weeks of treatment in a cell derived xenograft model of Ven-res AML. Additionally, this treatment significant improvement in the survival of a Ven-res patient-derived xenograft in-vivo study. Degradation of STAT3 resulting in downregulation of MCL1 and improvements in global mitochondrial dysfunction suggests a novel mechanism of overcoming Ven-res in AML., Statement of Purpose: Five-year survival from AML is dismal at 30%. Our prior research demonstrated STAT3 over-expression in AML HSPC's to be associated with inferior survival. We now explore STAT3 over-expression in Ven-res AML, explain STAT3 mediated mitochondrial perturbations and describe a novel therapeutic strategy, STAT3 degradation to overcome Ven-res.

Mitochondrial metabolism plays a central role in the regulation of hematopoietic stem cell (HSC) biology. Mitochondrial fatty acid oxidation (FAO) is pivotal in controlling HSC self-renewal and differentiation. Herein, we discuss recent evidence suggesting that NADPH generated in the mitochondria can influence the fate of HSCs. Although NADPH has multiple functions, HSCs show high levels of NADPH that are preferentially used for cholesterol biosynthesis. Endogenous cholesterol supports the biogenesis of extracellular vesicles (EVs), which are essential for maintaining HSC properties. We also highlight the significance of EVs in hematopoiesis through autocrine signaling. Elucidating the mitochondrial NADPH-cholesterol axis as part of the metabolic requirements of healthy HSCs will facilitate the development of new therapies for hematological disorders., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mitochondrial fatty acid oxidation (FAO) is essential for hematopoietic stem cell (HSC) self-renewal; however, the mechanism by which mitochondrial metabolism controls HSC fate remains unknown. Here, we show that within the hematopoietic lineage, HSCs have the largest mitochondrial NADPH pools, which are required for proper HSC cell fate and homeostasis. Bioinformatic analysis of the HSC transcriptome, biochemical assays, and genetic inactivation of FAO all indicate that FAO-generated NADPH fuels cholesterol synthesis in HSCs. Interference with FAO disturbs the segregation of mitochondrial NADPH toward corresponding daughter cells upon single HSC division. Importantly, we have found that the FAO-NADPH-cholesterol axis drives extracellular vesicle (EV) biogenesis and release in HSCs, while inhibition of EV signaling impairs HSC self-renewal. These data reveal the existence of a mitochondrial NADPH-cholesterol axis for EV biogenesis that is required for hematopoietic homeostasis and highlight the non-stochastic nature of HSC fate determination., Competing Interests: Declaration of interests T.F. is a founder and equity holder in Generian Pharmaceuticals and Coloma Therapeutics. D.T.S. is a director and equity holder of Agios Pharmaceuticals, Magenta Therapeutics, Editas Medicines, Clear Creek Bio, and LifeVaultBio; he is a founder of Fate Therapeutics and Magenta Therapeutics and a consultant to FOG Pharma, VcanBio, and Flagship Pioneering., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

There are scarce data regarding influenza vaccination among people with HIV infection (PWHIV). The goal of this explorative study is to assess hesitancy toward influenza vaccination in a group of PWHIV during the COVID-19 pandemic. A questionnaire was administered to 219 patients vaccinated at our clinic during the 2020-2021 campaign. It evaluated subjects' adherence to influenza vaccine over the last three seasonal vaccination campaigns, vaccine confidence, complacency and convenience, and the effect of the pandemic on the choice to become vaccinated. The population was divided into two groups: fully adherent to influenza vaccine (all three campaigns, 117 patients) and non-fully adherent (one or two campaigns, 102 patients). Adherence increased in the non-fully adherent group in 2020-2021, but the pandemic did not affect the choice. Misbeliefs emerged: the influenza vaccine was considered protective against SARS-CoV-2 (22.8% of the total population); almost half of all patients thought the influenza vaccine could improve their CD4 T cell level (57.3% in fully adherent, 40.2% in non-fully adherent, p < .05). In 2020-2021 campaign, three quarters of the non-fully adherent group would not have been vaccinated in a location other than our clinic (75.5% vs. 88.9% in the fully adherent group, p < .05). Conclusively, offering a secure and private space for vaccination against influenza seems to encourage vaccination; healthcare professionals should improve counseling to increase adherence and correct misbeliefs.

Cellular metabolism is a key regulator of hematopoietic stem cell (HSC) maintenance. HSCs rely on anaerobic glycolysis for energy production to minimize the production of reactive oxygen species and shift toward mitochondrial oxidative phosphorylation upon differentiation. However, increasing evidence has shown that HSCs still maintain a certain level of mitochondrial activity in quiescence, and exhibit high mitochondrial membrane potential, which both support proper HSC function. Since glycolysis and the tricarboxylic acid (TCA) cycle are not directly connected in HSCs, other nutrient pathways, such as amino acid and fatty acid metabolism, generate acetyl-CoA and provide it to the TCA cycle. In this review, we discuss recent insights into the regulatory roles of cellular metabolism in HSCs. Understanding the metabolic requirements of healthy HSCs is of critical importance to the development of new therapies for hematological disorders., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis with morphologic dysplasia and a propensity to transform into overt acute myeloid leukemia (AML). Our analysis of two cohorts of 20 MDS and 49 AML with multi-lineage dysplasia patients shows a reduction in Nucleophosmin 1 (NPM1) expression in 70% and 90% of cases, respectively. A mouse model of Npm1 conditional knockout (cKO) in hematopoietic cells reveals that Npm1 loss causes premature aging of hematopoietic stem cells (HSCs). Mitochondrial activation in Npm1-deficient HSCs leads to aberrant activation of the NLRP3 inflammasome, which correlates with a developing MDS-like phenotype. Npm1 cKO mice exhibit shortened survival times, and expansion of both the intra- and extra-medullary myeloid populations, while evoking a p53-dependent response. After transfer into a p53 mutant background, the resulting Npm1/p53 double KO mice develop fatal leukemia within 6 months. Our findings identify NPM1 as a regulator of HSC aging and inflammation and highlight the role of p53 in MDS progression to leukemia., (© 2022 The Authors.)

Hematopoietic stem cells (HSCs) rely on complex regulatory networks to preserve stemness. Due to the scarcity of HSCs, technical challenges have limited our insights into the interplay between metabolites, transcription, and the epigenome. In this study, we generated low-input metabolomics, transcriptomics, chromatin accessibility, and chromatin immunoprecipitation data, revealing distinct metabolic hubs that are enriched in HSCs and their downstream multipotent progenitors. Mechanistically, we uncover a non-classical retinoic acid (RA) signaling axis that regulates HSC function. We show that HSCs rely on Cyp26b1, an enzyme conventionally considered to limit RA effects in the cell. In contrast to the traditional view, we demonstrate that Cyp26b1 is indispensable for production of the active metabolite 4-oxo-RA. Further, RA receptor beta (Rarb) is required for complete transmission of 4-oxo-RA-mediated signaling to maintain stem cells. Our findings emphasize that a single metabolite controls stem cell fate by instructing epigenetic and transcriptional attributes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) pathogenesis often involves a mutation in the NPM1 nucleolar chaperone, but the bases for its transforming properties and overall association with favorable therapeutic responses remain incompletely understood. Here we demonstrate that an oncogenic mutant form of NPM1 (NPM1c) impairs mitochondrial function. NPM1c also hampers formation of promyelocytic leukemia (PML) nuclear bodies (NB), which are regulators of mitochondrial fitness and key senescence effectors. Actinomycin D (ActD), an antibiotic with unambiguous clinical efficacy in relapsed/refractory NPM1c-AMLs, targets these primed mitochondria, releasing mitochondrial DNA, activating cyclic GMP-AMP synthase signaling, and boosting reactive oxygen species (ROS) production. The latter restore PML NB formation to drive TP53 activation and senescence of NPM1c-AML cells. In several models, dual targeting of mitochondria by venetoclax and ActD synergized to clear AML and prolong survival through targeting of PML. Our studies reveal an unexpected role for mitochondria downstream of NPM1c and implicate a mitochondrial/ROS/PML/TP53 senescence pathway as an effector of ActD-based therapies., Significance: ActD induces complete remissions in NPM1-mutant AMLs. We found that NPM1c affects mitochondrial biogenesis and PML NBs. ActD targets mitochondria, yielding ROS which enforce PML NB biogenesis and restore senescence. Dual targeting of mitochondria with ActD and venetoclax sharply potentiates their anti-AML activities in vivo. This article is highlighted in the In This Issue feature, p. 2945., (©2021 The Authors; Published by the American Association for Cancer Research.)

Mitochondrial dysfunction and stem cell exhaustion are two hallmarks of aging. In the hematopoietic system, aging is linked to imbalanced immune response and reduced regenerative capacity in hematopoietic stem cells (HSCs), as well as an increased predisposition to a spectrum of diseases, including myelodysplastic syndrome and acute myeloid leukemia. Myeloid-biased differentiation and loss of polarity are distinct features of aged HSCs, which generally exhibit enhanced mitochondrial oxidative phosphorylation and increased production of reactive oxygen species (ROS), suggesting a direct role for mitochondria in the degenerative process. Here, we provide an overview of current knowledge of the mitochondrial mechanisms that contribute to age-related phenotypes in HSCs. These include mitochondrial ROS production, alteration/activation of mitochondrial metabolism, the quality control pathway of mitochondria, and inflammation. Greater understanding of the key machineries of HSC aging will allow us to identify new therapeutic targets for preventing, delaying, or even reversing aspects of this process.

Citrate, generated in the mitochondria, is a key metabolite that might link metabolism with signaling, chromatin structure and transcription to orchestrate mesenchymal stem cells (MSCs) fate determination. Based on a detailed morphological analysis of 3D reconstruction of mitochondria and nuclei in single cells, we identified contact sites between these organelles that drastically increase in volume and number during the early stage of mesenchymal stem cell differentiation. These contact sites create a microdomain that facilitates exchange of signals from mitochondria to the nucleus. Interestingly, we found that the citrate derived from mitochondria is necessary for osteogenic lineage determination. Indeed, inhibition of the citrate transporter system dramatically affected osteogenesis, reduced citrate levels that could be converted in α-ketoglutarate, and consequently affected epigenetic marker H3K9me3 associated with the osteogenesis differentiation process. These findings highlight that mitochondrial metabolites play key regulatory roles in the MSCs differentiation process. Further in-depth investigation is needed to provide novel therapeutic strategies in the field of regenerative medicine .

Presenilin 1 (PS1) mutations are the most common cause of familial Alzheimer's disease (FAD). PS1 also plays a role in cellular processes such as calcium homeostasis and autophagy. We hypothesized that mutant presenilins increase cellular vulnerability to stress. We stably expressed human PS1, mutant PS1E280A and mutant PS1Δ9 in mouse neuroblastoma N2a cells. We examined early signs of stress in different conditions: endoplasmic reticulum (ER) stress, calcium overload, oxidative stress, and Aβ 1-42 oligomers toxicity. Additionally, we induced autophagy via serum starvation. PS1 mutations did not have an effect in ER stress but PS1E280A mutation affected autophagy. PS1 overexpression influenced calcium homeostasis and generated mitochondrial calcium overload modifying mitochondrial function. However, the opening of the mitochondrial permeability transition pore (MPTP) was affected in PS1 mutants, being accelerated in PS1E280A and inhibited in PS1Δ9 cells. Altered autophagy in PS1E280A cells was neither modified by inhibition of γ-secretase, nor by ER calcium retention. MPTP opening was directly regulated by γ-secretase inhibitors independent on organelle calcium modulation, suggesting a novel direct role for PS1 and γ-secretase in mitochondrial stress. We identified intrinsic cellular vulnerability to stress in PS1 mutants associated simultaneously with both, autophagic and mitochondrial function, independent of Aβ pathology.

The role of mitochondria in the fate determination of hematopoietic stem and progenitor cells (HSPCs) is not solely limited to the switch from glycolysis to oxidative phosphorylation, but also involves alterations in mitochondrial features and properties, including mitochondrial membrane potential (ΔΨ mt ). HSPCs have a high ΔΨ mt even when the rates of respiration and phosphorylation are low, and we have previously shown that the minimum proton flow through ATP synthesis (or complex V) enables high ΔΨ mt in HSPCs. Here we show that HSPCs sustain a unique equilibrium between electron transport chain (ETC) complexes and ATP production. HSPCs exhibit high expression of ETC complex II, which sustains complex III in proton pumping, although the expression levels of complex I or V are relatively low. Complex II inhibition by TTFA caused a substantial decrease of ΔΨ mt , particularly in HSPCs, while the inhibition of complex I by Rotenone mainly affected mature populations. Functionally, pharmacological inhibition of complex II reduced in vitro colony-replating capacity but this was not observed when complex I was inhibited, which supports the distinct roles of complex I and II in HSPCs. Taken together, these data highlight complex II as a key regulator of ΔΨ mt in HSPCs and open new and interesting questions regarding the precise mechanisms that regulate mitochondrial control to maintain hematopoietic stem cell self-renewal., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (ΔΨm), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure ΔΨm across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when ΔΨm-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Background/objectives: Obesity is a complex disease characterized by the accumulation of excess body fat, which is caused by an increase in adipose cell size and number. The major source of adipocytes comes from mesenchymal stem cells (MSCs), although their roles in obesity remain unclear. An understanding of the mechanisms, regulation, and outcomes of adipogenesis is crucial for the development of new treatments for obesity-related diseases. Recently an unexpected role for the tumor suppressor promyelocytic leukemia protein (PML) in hematopoietic stem cell biology and metabolism regulation has come to light, but its role in MSC biology remains unknown. Here, we investigated the molecular pathway underlying the role of PML in the control of adipogenic MSC differentiation., Subjects/methods: Muscle-derived stem cells (MDSCs) and adipose-derived stem cells (ADSCs) obtained from mice and voluntary patients (as a source of MSCs) were cultured in the presence of high glucose (HG) concentration, a nutrient stress condition known to promote MSCs differentiation into mature adipocytes and the adipogenic potential of PML was assessed., Results: PML is essential for a correct HG-dependent adipogenic differentiation, and the enhancement of PML levels is fundamental during adipogenesis. Increased PML expression enables the upregulation of protein kinase Cβ (PKCβ), which, in turn, by controlling autophagy levels permits an increase in peroxisome proliferator-activated receptor γ (PPARγ) that leads the adipogenic differentiation. Therefore, genetic and pharmacological depletion of PML prevents PKCβ expression, and by increasing autophagy levels, impairs the MSCs adipogenic differentiation. Human ADSCs isolated from overweight patients displayed increased PML and PKCβ levels compared to those found in normal weight individuals, indicating that the PML-PKCβ pathway is directly involved in the enhancement of adipogenesis and human metabolism., Conclusions: The new link found among PML, PKCβ, and autophagy opens new therapeutic avenues for diseases characterized by an imbalance in the MSCs differentiation process, such as metabolic syndromes and cancer.

Proper control of mitochondrial function is a key factor in the maintenance of hematopoietic stem cells (HSCs). Mitochondrial content is commonly measured by staining with fluorescent cationic dyes. However, dye staining can be affected, not only by xenobiotic efflux pumps, but also by dye intake, which is dependent on the negative charge of mitochondria. Therefore, mitochondrial membrane potential (ΔΨ mt ) must be considered in these measurements because a high ΔΨ mt due to respiratory chain activity can enhance dye intake, leading to the overestimation of mitochondrial volume. Here, we show that HSCs exhibit the highest ΔΨ mt of the hematopoietic lineages and, as a result, ΔΨ mt -independent methods most accurately assess the relatively low mitochondrial volumes and DNA amounts of HSC mitochondria. Multipotent progenitor stage or active HSCs display expanded mitochondrial volumes, which decline again with further maturation. Further characterization of the controlled remodeling of the mitochondrial landscape at each hematopoietic stage will contribute to a deeper understanding of the mitochondrial role in HSC homeostasis., (Copyright © 2018. Published by Elsevier Inc.)

Recent cardiology research studies have reported the role, function, and structure of the mitochondrial permeability transition pore (mPTP) and have shown that its opening plays a key role in the progression of myocardial cell death secondary to reperfusion. In this manuscript, we validated a new pharmacological approach as an adjunct to reperfusion in myocardial infarction (MI) treatment and describe the discovery, optimization, and structure-activity relationship (SAR) studies of the first small-molecule mPTP opening inhibitors based on a 1,3,8-triazaspiro[4.5]decane scaffold that targets the c subunit of the F 1 /F O -ATP synthase complex. We identified three potential compounds with good mPTP inhibitory activity and beneficial effects in a model of MI, including a decreased apoptotic rate in the whole heart and overall improvement of cardiac function upon administration during reperfusion. The selected compounds did not show off-target effects at the cellular and mitochondrial levels. Moreover, the compounds preserved the mitochondrial ATP content despite interacting with the ATP synthase complex.

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are highly specialized subcellular compartments that are shaped by ER subdomains juxtaposed to mitochondria but are biochemically distinct from pure ER and pure mitochondria. MAMs are enriched in enzymes involved in lipid synthesis and transport, channels for calcium transfer, and proteins with oncogenic/oncosuppressive functions that modulate cell signaling pathways involved in physiological and pathophysiological processes. The term "cancer" denotes a group of disorders that result from uncontrolled cell growth driven by a mixture of genetic and environmental components. Alterations in MAMs are thought to account for the onset as well as the progression and metastasis of cancer and have been a focus of investigation in recent years. In this review, we present the current state of the art regarding MAM-resident proteins and their relevance, alterations, and deregulating functions in different types of cancer from a cell biology and clinical perspective., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease (PD) and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1) Activated Kinase 6 (PAK6). Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.

The impact of the mitochondrial permeability transition (MPT) on cellular physiology is well characterized. In contrast, the composition and mode of action of the permeability transition pore complex (PTPC), the supramolecular entity that initiates MPT, remain to be elucidated. Specifically, the precise contribution of the mitochondrial F 1 F O ATP synthase (or subunits thereof) to MPT is a matter of debate. We demonstrate that F 1 F O ATP synthase dimers dissociate as the PTPC opens upon MPT induction. Stabilizing F 1 F O ATP synthase dimers by genetic approaches inhibits PTPC opening and MPT Specific mutations in the F 1 F O ATP synthase c subunit that alter C-ring conformation sensitize cells to MPT induction, which can be reverted by stabilizing F 1 F O ATP synthase dimers. Destabilizing F 1 F O ATP synthase dimers fails to trigger PTPC opening in the presence of mutants of the c subunit that inhibit MPT The current study does not provide direct evidence that the C-ring is the long-sought pore-forming subunit of the PTPC, but reveals that PTPC opening requires the dissociation of F 1 F O ATP synthase dimers and involves the C-ring., (© 2017 The Authors.)

Antipsychotic drugs are currently used in clinical practice for a variety of mental disorders. Among them, clozapine is the most effective medication for treatment-resistant schizophrenia and is most helpful in controlling aggression and the suicidal behavior in schizophrenia and schizoaffective disorder. Although clozapine is associated with a low likelihood of extrapyramidal symptoms and other neurological side effects, it is well known for the weight gain and metabolic side effects, which expose the patient to a greater risk of cardiovascular disorders and premature death, as well as psychosocial issues, leading to non-adherence to therapy. The mechanisms underlying these iatrogenic metabolic disorders are still controversial. We have therefore investigated the in vivo effects of the selective PKCβ inhibitor, ruboxistaurin (LY-333531), in a preclinical model of long-term clozapine-induced weight gain. Cell biology, biochemistry, and behavioral tests have been performed in wild-type and PKCβ knockout mice to investigate the contribution of endogenous PKCβ and its pharmacological inhibition to the psychomotor effects of clozapine. Finally, we also shed light on a novel aspect of the mechanism underlying the clozapine-induced weight gain, demonstrating that the clozapine-dependent PKCβ activation promotes the inhibition of the lipid droplet-selective autophagy process. This paves the way to new therapeutic approaches to this serious complication of clozapine therapy.

The execution of proper Ca 2+ signaling requires close apposition between the endoplasmic reticulum (ER) and mitochondria. Hence, Ca 2+ released from the ER is "quasi-synaptically" transferred to mitochondrial matrix, where Ca 2+ stimulates mitochondrial ATP synthesis by activating the tricarboxylic acid (TCA) cycle. However, when the Ca 2+ transfer is excessive and sustained, mitochondrial Ca 2+ overload induces apoptosis by opening the mitochondrial permeability transition pore. A large number of regulatory proteins reside at mitochondria-associated ER membranes (MAMs) to maintain the optimal distance between the organelles and to coordinate the functionality of both ER and mitochondrial Ca 2+ transporters or channels. In this chapter, we discuss the different pathways involved in the regulation of ER-mitochondria Ca 2+ flux and describe the activities of the various Ca 2+ players based on their primary intra-organelle localization.

Mitochondrial permeability transition (mPT) refers to a sudden increase in the permeability of the inner mitochondrial membrane. Long-term studies of mPT revealed that this phenomenon has a critical role in multiple pathophysiological processes. mPT is mediated by the opening of a complex termed the mPT pore (mPTP), which is responsible for the osmotic influx of water into the mitochondrial matrix, resulting in swelling of mitochondria and dissipation of the mitochondrial membrane potential. Here we provide three independent optimized protocols for monitoring mPT in living cells: (i) measurement using a calcein-cobalt technique, (ii) measurement of the mPTP-dependent alteration of the mitochondrial membrane potential, and (iii) measurement of mitochondrial swelling. These procedures can easily be modified and adapted to different cell types. Cell culture and preparation of the samples are estimated to take ∼1 d for methods (i) and (ii), and ∼3 d for method (iii). The entire experiment, including analyses, takes ∼2 h.

The p53 protein is probably the most important tumor suppressor, acting as a nuclear transcription factor primarily through the modulation of cell death. However, currently, it is well accepted that p53 can also exert important transcription-independent pro-cell death actions. Indeed, cytosolic localization of endogenous wild-type or transactivation-deficient p53 is necessary and sufficient for the induction of apoptosis and autophagy. Here, we present the extra-nuclear activities of p53 associated with the mitochondria and the endoplasmic reticulum, highlighting the activities of the p53 mutants on these compartments. These two intracellular organelles play crucial roles in the regulation of cell death, and it is now well established that they also represent sites where p53 can accumulate.

Although PTEN has been widely described as a nuclear and cytosolic protein, in the last 2 years, alternative organelles, such as the endoplasmic reticulum (ER), pure mitochondria, and mitochondria-associated membranes (MAMs), have been recognized as pivotal targets of PTEN activity.Here, we describe different methods that have been used to highlight PTEN subcellular localization.First, a protocol to extract nuclear and cytosolic fractions has been described to assess the "canonical" PTEN localization. Moreover, we describe a protocol for mitochondria isolation with proteinase K (PK) to further discriminate whether PTEN associates with the outer mitochondrial membrane (OMM) or resides within the mitochondria. Finally, we focus our attention on a subcellular fractionation protocol of cells that permits the isolation of MAMs containing unique regions of ER membranes attached to the outer mitochondrial membrane (OMM) and mitochondria without contamination from other organelles. In addition to biochemical fractionations, immunostaining can be used to determine the subcellular localization of proteins; thus, a detailed protocol to obtain good immunofluorescence (IF) is described. The employment of these methodological approaches could facilitate the identification of different PTEN localizations in several physiopathological contexts.

A substantial body of evidence indicates that mitochondrial morphology and function change during osteogenic differentiation. However, molecular mechanisms linking mitochondrial dynamics with the regulation of osteoblast functions are poorly understood. Amongst the molecules that influence the decision of human mesenchymal stem cells (hMSCs) to become osteoblasts are Slug and NFATc1 transcription factors (TFs). These molecules also interfere with different mitochondria-dependent pathways in response to a variety of cellular demands. The present study investigated the recruitment of Slug and NFATc1 at the D-loop regulatory region of mitochondrial DNA (mtDNA) in osteogenic differentiated hMSCs with the aim of exploring whether Slug and NFATc1 also act as mitoTFs in the mitochondrial pool of nuclear TFs. The results demonstrate that NFATc1, but not Slug, is localized in the mitochondria. Using chromatin immunoprecipitation assay, we found that NFATc1 is recruited at mtDNA, but this occurs only when the calcification process is at its highest in osteo-induced MSC and the maximum level of differentiation is reached. Occupancy of the mtDNA by NFATc1 is associated with a decreased expression of crucial mitochondrial genes such as Cytochrome B and NADH dehydrogenase 1. This suggests that NFATc1 acts as a negative regulator of mtDNA transcription during the calcification process and interruption of aerobic energy demand. The finding of NFATc1 participation in osteogenic differentiation through its direct involvement in the regulatory machinery of mitochondria suggests a new role for this TF and adds information on communication between mitochondrial and nuclear genomes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

One challenge in biology is signal transduction monitoring in a physiological context. Intravital imaging techniques are revolutionizing our understanding of tumor and host cell behaviors in the tumor environment. However, these deep tissue imaging techniques have not yet been adopted to investigate the second messenger calcium (Ca²⁺). In the present study, we established conditions that allow the in vivo detection of Ca²⁺ signaling in three-dimensional tumor masses in mouse models. By combining intravital imaging and a skinfold chamber technique, we determined the ability of photodynamic cancer therapy to induce an increase in intracellular Ca²⁺ concentrations and, consequently, an increase in cell death in a p53-dependent pathway.

In this study we have inhibited the expression of two negative regulators of chondrogenesis, Slug transcription factor (TF) and the small non-coding single stranded RNA microRNA-221 (miR-221), in human mesenchymal stem cells (MSCs). Our aim was test a new approach to guide the cells toward a chondrocyte - like phenotype, without the employment of differentiating agents, in the prospect of their clinical applications for cell-based cartilage tissue engineering. We have characterized these manipulated cells by gene expression analysis at the RNA and protein levels. We demonstrated that decreased miR-221 or Slug induced an increase of chondrogenic markers, including collagen type II (Col2A1), and the positive chondrogenic TFs Sox9 and TRPS1. Slug and TRPS1 are not direct targets of miR-221 since their expression was not affected by miR-221 content. Further, we showed by gene expression and Chromatin Immunoprecipitation analyses that i. miR-221 is positively regulated by Slug in hMSCs, where Slug and miR-221 high levels hamper cell differentiation, and ii. TRPS1 contributes to maintaining low levels of miR-221, both in hMSCs committed toward chondrogenesis by Slug depletion and in chondrocytes, where the low levels of miR-221 and Slug allow a chondrogenic phenotype.Taken together, our data may be relevant both to understand yet unknown miRNA - TF regulatory loops in cartilage biology and to establish new strategies based on a siRNA approach for cartilage tissue engineering.