Rossana Domenis

Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function

The multifunctional APE1 protein is required for tumor progression and is associated with cancer resistance. It is shown that APE1 presents structural elements that function in distinct cellular roles, highlighting the molecular determinants of the multifunctional nature of this protein and providing the basis for a new role of the C65 residue.

Skeletal muscle oxidative function in vivo and ex vivo in athletes with marked hypertrophy from resistance training

Oxidative function during exercise was evaluated in 11 young athletes with marked skeletal muscle hypertrophy induced by long-term resistance training (RTA; body mass 102.6 ± 7.3 kg, mean ± SD) and 11 controls (CTRL; body mass 77.8 ± 6.0 kg). Pulmonary O2 uptake (Vo2) and vastus lateralis muscle fractional O2 extraction (by near-infrared spectroscopy) were determined during an incremental cycle ergometer (CE) and one-leg knee-extension (KE) exercise. Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in permeabilized vastus lateralis fibers obtained by biopsy. Quadriceps femoris muscle cross-sectional area, volume (determined by magnetic resonance imaging), and strength were greater in RTA vs. CTRL (by ∼40%, ∼33%, and ∼20%, respectively). Vo2peak during CE was higher in RTA vs. CTRL (4.05 ± 0.64 vs. 3.56 ± 0.30 l/min); no difference between groups was observed during KE. The O2 cost of CE exercise was not different between groups. When divided per muscle mass (for CE) or quadriceps muscle mass (for KE), Vo2 peak was lower (by 15-20%) in RTA vs. CTRL. Vastus lateralis fractional O2 extraction was lower in RTA vs. CTRL at all work rates, during both CE and KE. RTA had higher ADP-stimulated mitochondrial respiration (56.7 ± 23.7 pmol O2·s(-1)·mg(-1) ww) vs. CTRL (35.7 ± 10.2 pmol O2·s(-1)·mg(-1) ww) and a tighter coupling of oxidative phosphorylation. In RTA, the greater muscle mass and maximal force and the enhanced mitochondrial respiration seem to compensate for the hypertrophy-induced impaired peripheral O2 diffusion. The net results are an enhanced whole body oxidative function at peak exercise and unchanged efficiency and O2 cost at submaximal exercise, despite a much greater body mass.

Cardiac differentiation promotes mitochondria development and ameliorates oxidative capacity in H9c2 cardiomyoblasts.

H9c2 undergoing cardiac differentiation induced by all-trans-retinoic acid were investigated for mitochondria structural features together with the implied functional changes, as a model for the study of mitochondrial development in cardiogenic progenitor cells. As the expression of cardiac markers became detectable, mitochondrial mass increased and mitochondrial morphology and ultrastructure changed. Reticular network organization developed and more bulky mitochondria with greater numbers of closely packed cristae and more electron-dense matrix were detected. Increased expression of PGC-1α proved the occurrence of mitochondrial biogenesis. Improvements in mitochondrial energetic competence were also documented, linked to better assembly between F(0) and F(1) sectors of the F(0)F(1)ATPsynthase enzyme complex.

Glucose-Modulated Mitochondria Adaptation in Tumor Cells: A Focus on ATP Synthase and Inhibitor Factor 1

Warburg’s hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking “positive” (activation/biogenesis) or “negative” (silencing) mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase β subunit and Inhibitor Factor 1 (IF1). Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on β-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation.

Mitochondrial bioenergetic profile and responses to metabolic inhibition in human hepatocarcinoma cell lines with distinct differentiation characteristics.

The classical view of tumour cell bioenergetics has been recently revised. Then, the definition of the mitochondrial profile is considered of fundamental importance for the development of anti-cancer therapies, but it still needs to be clarified. We investigated two human hepatocellular carcinoma cell lines: the partially differentiated HepG2 and the undifferentiated JHH-6. High resolution respirometry revealed a marked impairment/uncoupling of OXPHOS in JHH-6 compared with HepG2, with the phosphorylation system limiting the capacity for electron transport much more in JHH-6. Blocking glycolysis or mitochondrial ATP synthase we demonstrated that in JHH-6 ATP synthase functions in reverse and consumes glycolytic ATP, thereby sustaining ΔΨm. A higher expression level of ATP synthase Inhibitor Factor 1 (IF1), a higher extent of IF1 bound to ATP synthase and a lower ATPase/synthase capacity were documented in JHH-6. Thus, here IF1 appears to down-regulate the reverse mode of ATPsynthase activity, thereby playing a crucial role in controlling energy waste and ΔΨm. These results, while confirming the over-expression of IF1 in cancer cells, are the first to indicate an inverse link between cell differentiation status and IF1 (expression level and regulatory function).

The redox function of APE1 is involved in the differentiation process of stem cells toward a neuronal cell fate.

Low-to-moderate levels of reactive oxygen species (ROS) govern different steps of neurogenesis via molecular pathways that have been decrypted only partially. Although it has been postulated that redox-sensitive molecules are involved in neuronal differentiation, the molecular bases for this process have not been elucidated yet. The aim of this work was therefore to study the role played by the redox-sensitive, multifunctional protein APE1/Ref-1 (APE1) in the differentiation process of human adipose tissue-derived multipotent adult stem cells (hAT-MASC) and embryonic carcinoma stem cells (EC) towards a neuronal phenotype. Methods and results: Applying a definite protocol, hAT-MASC can adopt a neural fate. During this maturation process, differentiating cells significantly increase their intracellular Reactive Oxygen Species (ROS) levels and increase the APE1 nuclear fraction bound to chromatin. This latter event is paralleled by the increase of nuclear NF-κB, a transcription factor regulated by APE1 in a redox-dependent fashion. Importantly, the addition of the antioxidant N-acetyl cysteine (NAC) to the differentiation medium partially prevents the nuclear accumulation of APE1, increasing the neuronal differentiation of hAT-MASC. To investigate the involvement of APE1 in the differentiation process, we employed E3330, a specific inhibitor of the APE1 redox function. The addition of E3330, either to the neurogenic embryonic carcinoma cell line NT2-D1or to hAT-MASC, increases the differentiation of stem cells towards a neural phenotype, biasing the differentiation towards specific subtypes, such as dopaminergic cells. In conclusion, during the differentiation process of stem cells towards a neuroectodermic phenotype, APE1 is recruited, in a ROS-dependent manner, to the chromatin. This event is associated with an inhibitory effect of APE1 on neurogenesis that may be reversed by E3330. Therefore, E3330 may be employed both to boost neural differentiation and to bias the differentiation potential of stem cells towards specific neuronal subtypes. These findings provide a molecular basis for the redox-mediated hypothesis of neuronal differentiation program.

A human neuronal model of Niemann Pick C disease developed from stem cells isolated from patient’s skin

BackgroundNiemann Pick C (NPC) disease is a neurovisceral lysosomal storage disorder due to mutations in NPC1 or NPC2 genes, characterized by the accumulation of endocytosed unesterified cholesterol, gangliosides and other lipids within the lysosomes/late endosomes. Even if the neurodegeneration is the main feature of the disease, the analysis of the molecular pathways linking the lipid accumulation and cellular damage in the brain has been challenging due to the limited availability of human neuronal models.ObjectiveThe aim of this study was to develop a human neuronal model of NPC disease by inducing neuronal differentiation of multipotent adult stem cells (MASC) isolated from NPC patients.MethodsStem cells were isolated from 3 NPC patients and 3 controls both from skin biopsies and previously established skin fibroblast cultures. Cells were induced to differentiate along a neuronal fate adapting methods previously described by Beltrami et al, 2007. The surface immunophenotype of stem cells was analyzed by FACS. Stem cell and neuronal markers expression were evaluated by immunofluorescence. Intracellular accumulation of cholesterol and gangliosides were assessed by filipin staining and immunofluorescence, respectively. A morphometric analysis was performed using a Neurite outgrowth image program.ResultsAfter 3 passages in selective medium, MASC isolated either from skin biopsies or previously established skin fibroblast cultures displayed an antigenic pattern characteristic of mesenchymal stem cells and expressed the stem cell markers Oct-4, Nanog, Sox-2 and nestin. A massive lysosomal accumulation of cholesterol was observed only in cells isolated from NPC patients. After the induction of neural differentiation, remarkable morphologic changes were observed and cells became positive to markers of the neuronal lineage NeuN and MAP2. Differentiated cells from NPC patients displayed characteristic features of NPC disease, they showed intracellular accumulation of unesterified cholesterol and GM2 ganglioside and presented morphological differences with respect to cells derived from healthy donors.In conclusion, we generated a human neuronal model of NPC disease through the induction of differentiation of stem cells obtained from patient’s easily accessible sources. The strategy described here may be applied to easily generate human neuronal models of other neurodegenerative diseases.

A novel method to generate and culture human mast cells: Peripheral CD34 + stem cell-derived mast cells (PSCMCs)

The identification and characterization of human mast cell (MC) functions are hindered by the shortage of MC populations suitable for investigation. Here, we present a novel technique for generating large numbers of well differentiated and functional human MCs from peripheral stem cells (=peripheral stem cell-derived MCs, PSCMCs). Innovative and key features of this technique include 1) the use of stem cell concentrates, which are routinely discarded by blood banks, as the source of CD34+ stem cells, 2) cell culture in serum-free medium and 3) the addition of LDL as well as selected cytokines. In contrast to established and published protocols that use CD34+ or CD133+ progenitor cells from full blood, we used a pre-enriched cell population obtained from stem cell concentrates, which yielded up to 10(8) differentiated human MCs per batch after only three weeks of culture starting with 10(6) total CD34+ cells. The total purity on MCs (CD117+, FcεR1+) generated by this method varied between 55 and 90%, of which 4-20% were mature MCs that contain tryptase and chymase and show expression of FcεRI and CD117 in immunohistochemistry. PSCMCs showed robust histamine release in response to stimulation with anti-FcεR1 or IgE/anti-IgE, and increased proliferation and differentiation in response to IL-1β or IFN-γ. Taken together, this new protocol of the generation of large numbers of human MCs provides for an innovative and suitable option to investigate the biology of human MCs.

Systemic T cells immunosuppression of glioma stem cell-derived exosomes is mediated by monocytic myeloid-derived suppressor cells

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression.

Separate and combined effects of a 10-d exposure to hypoxia and inactivity on oxidative function in vivo and mitochondrial respiration ex vivo in humans

An integrative evaluation of oxidative metabolism was carried out in 9 healthy young men (age, 24.1 ± 1.7 yr mean ± SD) before (CTRL) and after a 10-day horizontal bed rest carried out in normoxia (N-BR) or hypoxia (H-BR, FiO2 = 0.147). H-BR was designed to simulate planetary habitats. Pulmonary O2 uptake (V̇o2) and vastus lateralis fractional O2 extraction (changes in deoxygenated hemoglobin+myoglobin concentration, Δ[deoxy(Hb+Mb)] evaluated using near-infrared spectroscopy) were evaluated in normoxia and during an incremental cycle ergometer (CE) and one-leg knee extension (KE) exercise (aimed at reducing cardiovascular constraints to oxidative function). Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in permeabilized vastus lateralis fibers. During CE V̇o2peak and Δ[deoxy(Hb+Mb)]peak were lower (P < 0.05) after both N-BR and H-BR than during CTRL; during KE the variables were lower after N-BR but not after H-BR. During CE the overshoot of Δ[deoxy(Hb+Mb)] during constant work rate exercise was greater in N-BR and H-BR than CTRL, whereas during KE a significant difference vs. CTRL was observed only after N-BR. Maximal mitochondrial respiration determined ex vivo was not affected by either intervention. In N-BR, a significant impairment of oxidative metabolism occurred downstream of central cardiovascular O2 delivery and upstream of mitochondrial function, possibly at the level of the intramuscular matching between O2 supply and utilization and peripheral O2 diffusion. Superposition of hypoxia on bed rest did not aggravate, and partially reversed, the impairment of muscle oxidative function in vivo induced by bed rest. The effects of longer exposures will have to be determined.