The mitochondrial genome, a growing interest inside an organelle

Mitochondria are semi-autonomously reproductive organelles within eukaryotic cells carrying their own genetic material, called the mitochondrial genome (mtDNA). Until some years ago, mtDNA had primarily been used as a tool in population genetics. As scientists began associating mtDNA mutations with dozens of mysterious disorders, as well as the aging process and a variety of chronic degenerative diseases, it became increasingly evident that the information contained in this genome had substantial potential applications to improve human health. Today, mitochondria research covers a wide range of disciplines, including clinical medicine, biochemistry, genetics, molecular cell biology, bioinformatics, plant sciences and physiology. The present review intends to present a summary of the most exiting fields of the mitochondrial research bringing together several contributes in terms of original prospective and future applications

Evaluation of brain activity changes occurring in an animal model for multiple sclerosis: a functional Magnetic Resonance Imaging study

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Magnetic Resonance Imaging (MRI), showing the extent of the involvement of CNS, plays a major role in the assessment of patients with MS. Further information can be obtained with functional MRI (fMRI) which may be used in MS patients to investigate the functional reorganization of cortical areas. fMRI observations in MS are already available in humans, but deeper knowledge on its usefulness might be gained using reliable animal models. We investigated by means of fMRI the brain plasticity in a chronic model of MS, i.e. Experimental Autoimmune Encephalomyelitis (EAE) in the Dark Agouti (DA) rat strain. Serial fMRI acquisitions were performed before, 30 and 60 days after EAE induction. fMRI with somatosensory stimulation was performed according to ref [1]. Briefly electrical stimulation (a train of squared pulses with frequency=3Hz, current=2mA, duration=0.5ms) was delivered to the left forepaw during acquisition of MR images sensitive to Blood-Volume. A single stimulation protocol was composed of 30 images under rest condition and 10 images acquired during stimulation. After appropriate image analysis, performed using the FSL software package [2], the brain region activated by the applied stimulus was determined. Prior to EAE induction, electrical stimulation resulted in a localized response in the contralateral sensory motor cortex according to previously reported results [1]. Thirty and 60 days after EAE Induction, the activated area was greatly increased covering large regions of both contra and ipsilateral somatosensory cortex and extending also to extra-cortical regions. Our results show that the experimental model of EAE in DA rats reproduces a remarkable findings observed in MS patients, i.e. the functional reorganization of motor cortex. It remains to be investigated whether this effect could represent an innovative platform for testing new therapeutic approaches for MS

Functional Magnetic Resonance Imaging of Rats with Experimental Autoimmune Encephalomyelitis Reveals Brain Cortex Remodeling.

UNLABELLED: Cortical reorganization occurring in multiple sclerosis (MS) patients is thought to play a key role in limiting the effect of structural tissue damage. Conversely, its exhaustion may contribute to the irreversible disability that accumulates with disease progression. Several aspects of MS-related cortical reorganization, including the overall functional effect and likely modulation by therapies, still remain to be elucidated. The aim of this work was to assess the extent of functional cortical reorganization and its brain structural/pathological correlates in Dark Agouti rats with experimental autoimmune encephalomyelitis (EAE), a widely accepted preclinical model of chronic MS. Morphological and functional MRI (fMRI) were performed before disease induction and during the relapsing and chronic phases of EAE. During somatosensory stimulation of the right forepaw, fMRI demonstrated that cortical reorganization occurs in both relapsing and chronic phases of EAE with increased activated volume and decreased laterality index versus baseline values. Voxel-based morphometry demonstrated gray matter (GM) atrophy in the cerebral cortex, and both GM and white matter atrophy were assessed by ex vivo pathology of the sensorimotor cortex and corpus callosum. Neuroinflammation persisted in the relapsing and chronic phases, with dendritic spine density in the layer IV sensory neurons inversely correlating with the number of cluster of differentiation 45-positive inflammatory lesions. Our work provides an innovative experimental platform that may be pivotal for the comprehension of key mechanisms responsible for the accumulation of irreversible brain damage and for the development of innovative therapies to reduce disability in EAE/MS. SIGNIFICANCE STATEMENT: Since the early 2000s, functional MRI (fMRI) has demonstrated profound modifications in the recruitment of cortical areas during motor, cognitive, and sensory tasks in multiple sclerosis (MS) patients. Experimental autoimmune encephalomyelitis (EAE) represents a reliable model of the chronic-progressive variant of MS. fMRI studies in EAE have not been performed extensively up to now. This paper reports fMRI studies in a rat model of MS with somatosensory stimulation of the forepaw. We demonstrated modifications in the recruitment of cortical areas consistent with data from MS patients. To the best of our knowledge, this is the first report of cortical remodeling in a preclinical in vivo model of MS.This work was supported by grants from the National Multiple Sclerosis Society (NMSS; RG-4001-A1 to SP), the Italian Multiple Sclerosis Foundation (FISM; RG 2010/R/31 to SP and FISM Grant 10/12/F14/2011 to PM), the Italian Ministry of Health (GR08/7 to SP), the European Research Council (ERC) 2010-SIG (RG 260511-SEM_SEM to SP), the European Community (EC) 7th Framework Programme (FP7/2007-2013; RG 280772-iONE to SP), The Evelyn Trust (RG 69865 to SP), The Bascule Charitable Trust (RG 75149 to SP). LPJ is supported by a Wellcome Trust Research Training Fellowship (RRZA/057).This is the final version of the article. It first appeared from Society for Neuroscience via http://dx.doi.org/10.1523/JNEUROSCI.0540-15.201

Human Mesenchymal Stem Cells and Endothelial Progenitor Cells exert a neuroprotective effect on rat cortical neurons injured by oxygen and glucose deprivation

Oxygen and glucose deprivation (OGD) due to ischemic events or trauma in the brain result in neuronal loss. The therapeutic approaches available inadequate and often the outcome is unfavorable for the patient or at least unpredictable. Stem cells could be useful for the treatment of OGD injured-neurons. Mesenchymal Stem Cells (MSCs), isolated from bone marrow as well as from various tissues, have poor immunogenicity and neuroprotective properties being able to alleviate ischemic brain injuries in animal models. The Endothelial Progenitor Cells (EPCs) are present at low frequencies both in the bone marrow and in the peripheral blood. They are thought to play a role in the recovery of cerebrovasculature integrity after stroke. In the present study we evaluated the potential neuroprotective effect of human MSCs and human EPCs on rat embryonic cortical neurons injured by OGD. OGD was induced by incubating the cortical neurons in a hypoxia chamber in a 95% N2 + 5% CO2 atmosphere at 37°C without glucose. To set up the experimental protocol, OGD was maintained for 1, 2 and 3 hours. The neurons were returned in normoxic atmosphere and after 2 and 5 days neuronal survival was evaluated by MTT assay, LDH assay and viable cellular counting. The 2 hours OGD was able to reduce neuronal viability by 50% and was chosen for the subsequent experiments. To assess MSCs and EPCs neuroprotective action, after 2 hours-long OGD the neurons were 1) co-cultured with either MSCs or EPCs seeded on a cell culture insert avoiding direct contact while sharing the same medium, or 2) cultured in a medium previously conditioned by either MSCs or EPCs. Neuronal survival was evaluated by MTT assay after 2 and 5 days. Both MSCs and EPCs increased neuronal survival after ODG. The effect was observed in absence of a direct contact between MSCs or EPCs and the injured neurons, suggesting that the release of soluble factors may be involved in their neuroprotective action. In conclusion both MSCs and EPCs could represent a potential therapeutic approach for the treatment of brain ischemic injury. Further studies are needed to identify the specific molecules and pathways that play a role in the neuroprotective effect of MSCs and EPCs

Positive effect of Mesenchymal Stem Cells therapeutic administration on chronic Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a crippling chronic disease of the Central Nervous System caused by the presence of self-antibodies which progressively damage axonal myelin sheath, leading to axonal transmission impairment and to the development of neurological symptoms. MS is characterized by a Relapsing-Remitting course, and current therapies rely only on the use of immunosuppressive drugs, which are however unable to reverse disease progression. Encouraging results have been obtained in preclinical studies with the administration of Mesenchymal Stem Cells (MSCs) before disease onset (Zappia et al., 2005). Here, we investigate the therapeutic potential of MSC administration after disease onset into an animal model of MS, represented by Dark Agouti rats affected by chronic Relapsing-Remitting Experimental Autoimmune Encephalomyelitis (EAE) (Cavaletti et al., 2004). 106 MSC were intravenously injected in EAE rats after disease onset. Clinical score was assessed daily, and after 45 days rats were sacrificed and histological analysis of spinal cords performed to evaluate the demyelinating lesions. After the first peak of disease, no further relapses were observed in EAE rats treated with MSCs, differently from what observed in EAE group. Histological analysis demonstrated the presence of demyelinated plaques in spinal cords of EAE rats, (Luxol fast Blue staining and anti-MBP immunohystochemistry). On the contrary the therapeutic schedule with MSCs significantly reduces the number and the extension of demyelinated areas in the spinal cords, confirming clinical score evaluations. These results demonstrated that MSCs ameliorate the clinical course of EAE and hamper the disease relapsing by reducing the areas of demyelinated lesions. Granted by MIUR – FIRB Futuro in Ricerca 2008 Prot. N° RBFR08VSVI_001

Excess of NPM-ALK oncogenic signaling promotes cellular apoptosis and drug dependency.

Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A 'drug holiday' where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.We thank Maria Stella Scalzo for technical support, Dr Emanuela Colombo for kindly providing MEFs that lack NPM1 (MEF NPM−/−p53−/−) and control fibroblasts (MEF p53−/−), Dr Guido Serini for the use of his confocal microscopy unit at the Candiolo Cancer Institute—IRCCS, Torino, Italy. We also thank Ariad Pharmaceutical, Pfizer, Astellas and Novartis that kindly provided all drugs used in this study. This work was supported by the Regione Lombardia (ID14546A) and Fondazione Berlucchi Onlus Grant 2014 (to CGP), and by grants FP7 ERC-2009-StG (Proposal No. 242965—‘Lunely’); Associazione Italiana per la Ricerca sul Cancro (AIRC) Grant IG-12023; Koch Institute/DFCC Bridge Project Fund; Ellison Foundation Boston; Worldwide Cancer Research Association (former AICR) grant 12-0216; the Grant for Oncology Innovation by Merck-Serono and R01 CA196703-01 (to RC).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/onc.2015.45

De novo UBE2A mutations are recurrently acquired during chronic myeloid leukemia progression and interfere with myeloid differentiation pathways

Despite the advent of tyrosine kinase inhibitors, a proportion of chronic myeloid leukemia patients in chronic phase fails to respond to Imatinib or to second generation inhibitors and progress to blast crisis. Limited improvements in the understanding of the molecular mechanisms responsible for chronic myeloid leukemia transformation from chronic phase to the aggressive blast crisis were achieved until now. We present here a massive parallel sequencing analysis of 10 blast crisis samples and of the corresponding autologous chronic phase controls which reveals, for the first time, recurrent mutations affecting the ubiquitin-conjugating enzyme E2A gene (UBE2A, formerly RAD6A). Additional analyses on a cohort of 24 blast crisis, 41 chronic phase as well as 40 acute myeloid leukemia and 38 atypical chronic myeloid leukemia patients at onset confirmed that UBE2A mutations are specifically acquired during chronic myeloid leukemia progression with a frequency of 16.7% in advanced phases. In vitro studies show that the mutations here described cause a decrease in UBE2A activity, leading to an impairment of myeloid differentiation in chronic myeloid leukemia cells

Another Brick to Confirm the Efficacy of Rigosertib as Anticancer Agent

Rigosertib is a small molecule in preclinical development that, due to its characteristics as a dual PLK1 and PI3K inhibitor, is particularly effective in counteracting the advance of different types of tumors. In this work, we evaluated the efficacy of Rigosertib and the expression of p53 in five different human tumor cell lines in vitro, A549 (lung adenocarcinoma), MCF-7 and MDA-MB231 (breast cancer cells), RPMI 8226 (multiple myeloma), and U87-MG (glioblastoma). We demonstrated that in all cell lines, the effect was dose- and time-dependent, but A549 cells were the most sensible to the treatment while higher concentrations were required for the most resistant cell line U87-MG. Moreover, the highest and lowest p53 levels have been observed, respectively, in A459 and U87-MG cells. The alterations in the cell cycle and in cell-cycle-related proteins were observed in A549 at lower concentrations than U87-MG. In conclusion, with this article we have demonstrated that Rigosertib has different efficacy depending on the cell line considered and that it could be a potential antineoplastic agent against lung cancer in humans