Publications by authors named "Rodolfo Negri"

50 Publications

Genome-Wide Gene Expression Analysis of Mtb-Infected DC Highlights the Rapamycin-Driven Modulation of Regulatory Cytokines the mTOR/GSK-3β Axis.

Front Immunol 2021 16;12:649475. Epub 2021 Apr 16.

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy.

In human primary dendritic cells (DC) rapamycin-an autophagy inducer and protein synthesis inhibitor-overcomes the autophagy block induced by (Mtb) and promotes a Th1 response IL-12 secretion. Here, the immunostimulatory activity of rapamycin in Mtb-infected DC was further investigated by analyzing both transcriptome and translatome gene profiles. Hundreds of differentially expressed genes (DEGs) were identified by transcriptome and translatome analyses of Mtb-infected DC, and some of these genes were found further modulated by rapamycin. The majority of transcriptome-associated DEGs overlapped with those present in the translatome, suggesting that transcriptionally stimulated mRNAs are also actively translated. analysis of DEGs revealed significant changes in intracellular cascades related to cytokine production, cytokine-induced signaling and immune response to pathogens. In particular, rapamycin treatment of Mtb-infected DC caused an enrichment of IFN-β, IFN-λ and IFN-stimulated gene transcripts in the polysome-associated RNA fraction. In addition, rapamycin led to an increase of IL-12, IL-23, IL-1β, IL-6, and TNF-α but to a reduction of IL-10. Interestingly, upon silencing or pharmacological inhibition of GSK-3β, the rapamycin-driven modulation of the pro- and anti-inflammatory cytokine balance was lost, indicating that, in Mtb-infected DC, GSK-3β acts as molecular switch for the regulation of the cytokine milieu. In conclusion, our study sheds light on the molecular mechanism by which autophagy induction contributes to DC activation during Mtb infection and points to rapamycin and GSK-3β modulators as promising compounds for host-directed therapy in the control of Mtb infection.
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http://dx.doi.org/10.3389/fimmu.2021.649475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086600PMC
April 2021

The Role of Histone Lysine Methylation in the Response of Mammalian Cells to Ionizing Radiation.

Front Genet 2021 30;12:639602. Epub 2021 Mar 30.

Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy.

Eukaryotic genomes are wrapped around nucleosomes and organized into different levels of chromatin structure. Chromatin organization has a crucial role in regulating all cellular processes involving DNA-protein interactions, such as DNA transcription, replication, recombination and repair. Histone post-translational modifications (HPTMs) have a prominent role in chromatin regulation, acting as a sophisticated molecular code, which is interpreted by HPTM-specific effectors. Here, we review the role of histone lysine methylation changes in regulating the response to radiation-induced genotoxic damage in mammalian cells. We also discuss the role of histone methyltransferases (HMTs) and histone demethylases (HDMs) and the effects of the modulation of their expression and/or the pharmacological inhibition of their activity on the radio-sensitivity of different cell lines. Finally, we provide a bioinformatic analysis of published datasets showing how the mRNA levels of known HMTs and HDMs are modulated in different cell lines by exposure to different irradiation conditions.
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http://dx.doi.org/10.3389/fgene.2021.639602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042281PMC
March 2021

Active microbial ecosystem in Iron-Age tombs of the Etruscan civilization.

Environ Microbiol 2021 Jul 2;23(7):3957-3969. Epub 2020 Dec 2.

Department of Biology and Biotechnology, Sapienza University of Rome, Rome, Italy.

Earth's microbial biosphere extends down through the crust and much of the subsurface, including those microbial ecosystems located within cave systems. Here, we elucidate the microbial ecosystems within anthropogenic 'caves'; the Iron-Age, subterranean tombs of central Italy. The interior walls of the rock (calcium-rich macco) were painted ~2500 years ago and are covered with CaCO needles (known as moonmilk). The aims of the current study were to: identify biological/geochemical/biophysical determinants of and characterize bacterial communities involved in CaCO precipitation; challenge the maxim that biogenic activity necessarily degrades surfaces; locate the bacterial cells that are the source of the CaCO precipitate; and gain insight into the kinetics of moonmilk formation. We reveal that this environment hosts communities that consist primarily of bacteria that are mesophilic for temperature and xerotolerance (including Actinobacteria, Bacteroidetes and Proteobacteria); is populated by photosynthetic Cyanobacteria exhibiting heterotrophic nutrition (Calothrix and Chroococcidiopsis); and has CaCO precipitating on the rock surfaces (confirmation that this process is biogenic) that acts to preserve rather than damage the painted surface. We also identified that some community members are psychrotolerant (Polaromonas), acidotolerant or acidophilic (members of the Acidobacteria), or resistant to ionizing radiation (Brevundimonas and Truepera); elucidate the ways in which microbiology impacts mineralogy and vice versa; and reveal that biogenic formation of moonmilk can occur rapidly, that is, over a period of 10 to 56 years. We discuss the paradox that these ecosystems, that are for the most part in the dark and lack primary production, are apparently highly active, biodiverse and biomass-rich.
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http://dx.doi.org/10.1111/1462-2920.15327DOI Listing
July 2021

Artemisinin Derivatives with Antimelanoma Activity Show Inhibitory Effect against Human DNA Topoisomerase 1.

ACS Med Chem Lett 2020 May 10;11(5):1035-1040. Epub 2020 Apr 10.

Dipartimento di Biologia, Università di Padova Distaccato presso il "Centro Linceo Beniamino Segre" Accademia Nazionale dei Lincei, Palazzo Corsini, Via della Lungara 10, 00165 Rome, Italy.

Artesunic acid and artemisinin are natural substances with promiscuous anticancer activity against different types of cancer cell lines. The mechanism of action of these compounds is associated with the formation of reactive radical species by cleavage of the sesquiterpene pharmacophore endoperoxide bridge. Here we suggested topoisomerase 1 as a possible molecular target for the improvement of the anticancer activity of these compounds. In this context, we report that novel hybrid and dimer derivatives of artesunic acid and artemisinin, bearing camptothecin and SN38 as side-chain biological effectors, can inhibit growth of yeast cells overexpressing human topoisomerase 1 and its enzymatic activity . These derivatives showed also anticancer activity in melanoma cell lines higher than camptothecin and paclitaxel. molecular docking calculations highlighted a common binding mode for the novel derivatives, with the sesquiterpene lactone scaffold being located near the traditional recognition site for camptothecin, while the bioactive side-chain effector laid in the camptothecin cleft.
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http://dx.doi.org/10.1021/acsmedchemlett.0c00131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236541PMC
May 2020

A Self-Organized PLT/Auxin/ARR-B Network Controls the Dynamics of Root Zonation Development in Arabidopsis thaliana.

Dev Cell 2020 05 7;53(4):431-443.e23. Epub 2020 May 7.

Computational Developmental Biology Group, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, the Netherlands. Electronic address:

During organogenesis, coherent organ growth arises from spatiotemporally coordinated decisions of individual cells. In the root of Arabidopsis thaliana, this coordination results in the establishment of a division and a differentiation zone. Cells continuously move through these zones; thus, a major question is how the boundary between these domains, the transition zone, is formed and maintained. By combining molecular genetics with computational modeling, we reveal how an auxin/PLETHORA/ARR-B network controls these dynamic patterning processes. We show that after germination, cell division causes a drop in distal PLT2 levels that enables transition zone formation and ARR12 activation. The resulting PLT2-ARR12 antagonism controls expansion of the division zone (the meristem). The successive ARR1 activation antagonizes PLT2 through inducing the cell-cycle repressor KRP2, thus setting final meristem size. Our work indicates a key role for the interplay between cell division dynamics and regulatory networks in root zonation and transition zone patterning.
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http://dx.doi.org/10.1016/j.devcel.2020.04.004DOI Listing
May 2020

X-ray irradiated cultures of mouse cortical neural stem/progenitor cells recover cell viability and proliferation with dose-dependent kinetics.

Sci Rep 2020 04 16;10(1):6562. Epub 2020 Apr 16.

Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Rome, Italy.

Exposure of the developing or adult brain to ionizing radiation (IR) can cause cognitive impairment and/or brain cancer, by targeting neural stem/progenitor cells (NSPCs). IR effects on NSPCs include transient cell cycle arrest, permanent cell cycle exit/differentiation, or cell death, depending on the experimental conditions. In vivo studies suggest that brain age influences NSPC response to IR, but whether this is due to intrinsic NSPC changes or to niche environment modifications remains unclear. Here, we describe the dose-dependent, time-dependent effects of X-ray IR in NSPC cultures derived from the mouse foetal cerebral cortex. We show that, although cortical NSPCs are resistant to low/moderate IR doses, high level IR exposure causes cell death, accumulation of DNA double-strand breaks, activation of p53-related molecular pathways and cell cycle alterations. Irradiated NSPC cultures transiently upregulate differentiation markers, but recover control levels of proliferation, viability and gene expression in the second week post-irradiation. These results are consistent with previously described in vivo effects of IR in the developing mouse cortex, and distinct from those observed in adult NSPC niches or in vitro adult NSPC cultures, suggesting that intrinsic differences in NSPCs of different origins might determine, at least in part, their response to IR.
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http://dx.doi.org/10.1038/s41598-020-63348-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162981PMC
April 2020

The COP9 signalosome mediates the Spt23 regulated fatty acid desaturation and ergosterol biosynthesis.

FASEB J 2020 04 19;34(4):4870-4889. Epub 2020 Feb 19.

Department of Biology and Environment, Faculty of Natural Sciences, University of Haifa, Oranim, Israel.

The COP9 signalosome (CSN) is a conserved eukaryotic complex, essential for vitality in all multicellular organisms and critical for the turnover of key cellular proteins through catalytic and non-catalytic activities. Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of the CSN complex, since it includes a conserved enzymatic core but lacks non-catalytic activities, probably explaining its non-essentiality for life. A previous transcriptomic analysis of an S. cerevisiae strain deleted in the CSN5/RRI1 gene, encoding to the CSN catalytic subunit, revealed a downregulation of genes involved in lipid metabolism. We now show that the S. cerevisiae CSN holocomplex is essential for cellular lipid homeostasis. Defects in CSN assembly or activity lead to decreased quantities of ergosterol and unsaturated fatty acids (UFA); vacuole defects; diminished lipid droplets (LDs) size; and to accumulation of endoplasmic reticulum (ER) stress. The molecular mechanism behind these findings depends on CSN involvement in upregulating mRNA expression of SPT23. Spt23 is a novel activator of lipid desaturation and ergosterol biosynthesis. Our data reveal for the first time a functional link between the CSN holocomplex and Spt23. Moreover, CSN-dependent upregulation of SPT23 transcription is necessary for the fine-tuning of lipid homeostasis and for cellular health.
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http://dx.doi.org/10.1096/fj.201902487RDOI Listing
April 2020

MiR-146a is over-expressed and controls IL-6 production in cystic fibrosis macrophages.

Sci Rep 2019 11 7;9(1):16259. Epub 2019 Nov 7.

Department of Biology and Biotechnology, "C. Darwin" Sapienza University, Rome, Italy.

Cystic fibrosis (CF) is an inherited disease that is characterised by susceptibility to bacterial infections and chronic lung inflammation. Recently, it was suggested that macrophages contribute to impaired host defence and excessive inflammatory responses in CF. Indeed, dysfunction attributed to CF macrophages includes decreased bacterial killing and exaggerated inflammatory responses. However, the mechanisms behind such defects have only been partially defined. MicroRNAs (miRNAs) have emerged as key regulators of several macrophage functions, including their activation, differentiation and polarisation. The goal of this study was to investigate whether miRNA dysregulation underlies the functional abnormalities of CF macrophages. MiRNA profiling of macrophages was performed, with 22 miRNAs identified as differentially expressed between CF and non-CF individuals. Among these, miR-146a was associated with significant enrichment of validated target genes involved in responses to microorganisms and inflammation. As miR-146a dysregulation has been reported in several human inflammatory diseases, we analysed the impact of increased miR-146a expression on inflammatory responses of CF macrophages. These data show that inhibition of miR-146a in lipopolysaccharide-stimulated CF macrophages results in increased interleukin-6 production, which suggests that miR-146a overexpression in CF is functional, to restrict inflammatory responses.
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http://dx.doi.org/10.1038/s41598-019-52770-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838115PMC
November 2019

Statins interfere with the attachment of mtDNA to the inner mitochondrial membrane.

J Enzyme Inhib Med Chem 2020 Dec;35(1):129-137

Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome, Rome, Italy.

The 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme of the mevalonate pathway for the synthesis of cholesterol in mammals (ergosterol in fungi), is inhibited by statins, a class of cholesterol lowering drugs. Indeed, statins are in a wide medical use, yet statins treatment could induce side effects as hepatotoxicity and myopathy in patients. We used as a model to investigate the effects of statins on mitochondria. We demonstrate that statins are active in by lowering the ergosterol content in cells and interfering with the attachment of mitochondrial DNA to the inner mitochondrial membrane. Experiments on murine myoblasts confirmed these results in mammals. We propose that the instability of mitochondrial DNA is an early indirect target of statins.
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http://dx.doi.org/10.1080/14756366.2019.1687461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844431PMC
December 2020

Changes in the microsomal proteome of tomato fruit during ripening.

Sci Rep 2019 10 4;9(1):14350. Epub 2019 Oct 4.

Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.

The variations in the membrane proteome of tomato fruit pericarp during ripening have been investigated by mass spectrometry-based label-free proteomics. Mature green (MG30) and red ripe (R45) stages were chosen because they are pivotal in the ripening process: MG30 corresponds to the end of cellular expansion, when fruit growth has stopped and fruit starts ripening, whereas R45 corresponds to the mature fruit. Protein patterns were markedly different: among the 1315 proteins identified with at least two unique peptides, 145 significantly varied in abundance in the process of fruit ripening. The subcellular and biochemical fractionation resulted in GO term enrichment for organelle proteins in our dataset, and allowed the detection of low-abundance proteins that were not detected in previous proteomic studies on tomato fruits. Functional annotation showed that the largest proportion of identified proteins were involved in cell wall metabolism, vesicle-mediated transport, hormone biosynthesis, secondary metabolism, lipid metabolism, protein synthesis and degradation, carbohydrate metabolic processes, signalling and response to stress.
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http://dx.doi.org/10.1038/s41598-019-50575-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778153PMC
October 2019

Molecular Signatures of the Aging Brain: Finding the Links Between Genes and Phenotypes.

Neurotherapeutics 2019 07;16(3):543-553

Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Piazzale A. Moro, 00185, Rome, Italy.

Aging is associated with cognitive decline and increased vulnerability to neurodegenerative diseases. The progressive extension of the average human lifespan is bound to lead to a corresponding increase in the fraction of cognitively impaired elderly individuals among the human population, with an enormous societal and economic burden. At the cellular and tissue levels, cognitive decline is linked to a reduction in specific neuronal subpopulations, a widespread decrease in synaptic plasticity and an increase in neuroinflammation due to an enhanced activation of astrocytes and microglia, but the molecular mechanisms underlying these functional changes during normal aging and in neuropathological conditions remain poorly understood. In this review, we summarize very recent and outstanding progress in elucidating the molecular changes associated with cognitive decline through the genome-wide profiling of aging brain cells at different molecular levels (genomic, epigenomic, transcriptomic, proteomic). We discuss how the correlation of different molecular and phenotypic traits driven by mathematical and computational analyses of large datasets has led to the prediction of key molecular nodes of neurodegenerative pathways, and provide a few examples of candidate regulators of cognitive decline identified with these approaches. Furthermore, we highlight the dysregulation of the synaptic transcriptome in neuronal cells and of the inflammatory transcriptome in glial cells as some of the key events during normal and neuropathological human brain aging.
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http://dx.doi.org/10.1007/s13311-019-00743-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694319PMC
July 2019

Small Molecule Inhibitors of KDM5 Histone Demethylases Increase the Radiosensitivity of Breast Cancer Cells Overexpressing JARID1B.

Molecules 2019 May 4;24(9). Epub 2019 May 4.

Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, 00185 Rome, Italy.

: KDM5 enzymes are H3K4 specific histone demethylases involved in transcriptional regulation and DNA repair. These proteins are overexpressed in different kinds of cancer, including breast, prostate and bladder carcinomas, with positive effects on cancer proliferation and chemoresistance. For these reasons, these enzymes are potential therapeutic targets. : In the present study, we analyzed the effects of three different inhibitors of KDM5 enzymes in MCF-7 breast cancer cells over-expressing one of them, namely KDM5B/JARID1B. In particular we tested H3K4 demethylation (western blot); radio-sensitivity (cytoxicity and clonogenic assays) and damage accumulation (COMET assay and kinetics of H2AX phosphorylation). : we show that all three compounds with completely different chemical structures can selectively inhibit KDM5 enzymes and are capable of increasing sensitivity of breast cancer cells to ionizing radiation and radiation-induced damage. : These findings confirm the involvement of H3K4 specific demethylases in the response to DNA damage, show a requirement of the catalytic function and suggest new strategies for the therapeutic use of their inhibitors.
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http://dx.doi.org/10.3390/molecules24091739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6540222PMC
May 2019

Depletion of ATP-Citrate Lyase (ATPCL) Affects Chromosome Integrity Without Altering Histone Acetylation in Mitotic Cells.

Front Physiol 2019 4;10:383. Epub 2019 Apr 4.

Dipartimento di Biologia e Biotecnologie "Charles Darwin", Sapienza - Università di Roma, Rome, Italy.

The Citrate Lyase (ACL) is the main cytosolic enzyme that converts the citrate exported from mitochondria by the SLC25A1 carrier in Acetyl Coenzyme A (acetyl-CoA) and oxaloacetate. Acetyl-CoA is a high-energy intermediate common to a large number of metabolic processes including protein acetylation reactions. This renders ACL a key regulator of histone acetylation levels and gene expression in diverse organisms including humans. We have found that depletion of ATPCL, the Drosophila ortholog of human ACL, reduced levels of Acetyl CoA but, unlike its human counterpart, does not affect global histone acetylation and gene expression. Nevertheless, reduced ATPCL levels caused evident, although moderate, mitotic chromosome breakage suggesting that this enzyme plays a partial role in chromosome stability. These defects did not increase upon X-ray irradiation, indicating that they are not dependent on an impairment of DNA repair. Interestingly, depletion of ATPCL drastically increased the frequency of chromosome breaks (CBs) associated to mutations in , which encodes the ortholog of the mitochondrial citrate carrier SLC25A1 that is also required for chromosome integrity and histone acetylation. Our results indicate that ATPCL has a dispensable role in histone acetylation and prevents massive chromosome fragmentation when citrate efflux is altered.
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http://dx.doi.org/10.3389/fphys.2019.00383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458238PMC
April 2019

JARID1B expression and its function in DNA damage repair are tightly regulated by miRNAs in breast cancer.

Cancer Sci 2019 Apr 18;110(4):1232-1243. Epub 2019 Mar 18.

Department of Biology and Biotechnology "C. Darwin,", "Sapienza" - University of Rome, Rome, Italy.

JARID1B/KDM5B histone demethylase's mRNA is markedly overexpressed in breast cancer tissues and cell lines and the protein has been shown to have a prominent role in cancer cell proliferation and DNA repair. However, the mechanism of its post-transcriptional regulation in cancer cells remains elusive. We performed a computational analysis of transcriptomic data from a set of 103 breast cancer patients, which, along with JARID1B upregulation, showed a strong downregulation of 2 microRNAs (miRNAs), mir-381 and mir-486, potentially targeting its mRNA. We showed that both miRNAs can target JARID1B 3'UTR and reduce luciferase's activity in a complementarity-driven repression assay. Moreover, MCF7 breast cancer cells overexpressing JARID1B showed a strong protein reduction when transfected with mir-486. This protein's decrease is accompanied by accumulation of DNA damage, enhanced radiosensitivity and increase of BRCA1 mRNA, 3 features previously correlated with JARID1B silencing. These results enlighten an important role of a miRNA's circuit in regulating JARID1B's activity and suggest new perspectives for epigenetic therapies.
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http://dx.doi.org/10.1111/cas.13925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6447846PMC
April 2019

Ergosterol reduction impairs mitochondrial DNA maintenance in S. cerevisiae.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 03 12;1864(3):290-303. Epub 2018 Dec 12.

Department of Biology and Biotechnology "Charles Darwin", La Sapienza University of Rome, Piazzale A. Moro 5, 00185 Rome, Italy.; Pasteur Institute-Cenci Bolognetti Foundation, Viale Regina Elena, 291, 00100 Rome, Italy.. Electronic address:

Sterols are essential lipids, involved in many biological processes. In Saccharomyces cerevisiae, the enzymes of the ergosterol biosynthetic pathway (Erg proteins) are localized in different cellular compartments. With the aim of studying organelle interactions, we discovered that Erg27p resides mainly in Lipid Droplets (LDs) in respiratory competent cells, while in absence of respiration, is found mostly in the ER. The results presented in this paper demonstrate an interplay between the mitochondrial respiration and ergosterol production: on the one hand, rho° cells show lower ergosterol content when compared with wild type respiratory competent cells, on the other hand, the ergosterol biosynthetic pathway influences the mitochondrial status, since treatment with ketoconazole, which blocks the ergosterol pathway, or the absence of the ERG27 gene, induced rho° production in S. cerevisiae. The loss of mitochondrial DNA in the ∆erg27 strain is fully suppressed by exogenous addition of ergosterol. These data suggest the notion that ergosterol is essential for maintaining the mitochondrial DNA attached to the inner mitochondrial membrane.
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http://dx.doi.org/10.1016/j.bbalip.2018.12.002DOI Listing
March 2019

Calcite moonmilk of microbial origin in the Etruscan Tomba degli Scudi in Tarquinia, Italy.

Sci Rep 2018 10 26;8(1):15839. Epub 2018 Oct 26.

La Sapienza University of Rome, Dept. of Biology and Biotechnology Charles Darwin, Rome, 00185, Italy.

A white deposit covering the walls in the Stanza degli Scudi of the Tomba degli Scudi, Tarquinia, Italy, has been investigated. In this chamber, which is still preserved from any kind of intervention such as cleaning and sanitization, ancient Etruscans painted shields to celebrate the military power of the Velcha family. Scanning electron microscopy analysis has revealed the presence of characteristic nanostructures corresponding to a calcite secondary mineral deposit called moonmilk. Analysis of the microbial community identified Proteobacteria, Acidobacteria and Actinobacteria as the most common phyla in strong association with the moonmilk needle fibre calcite and nanofibers of calcium carbonate. Employing classical microbiological analysis, we isolated from moonmilk a Streptomyces strain able to deposit gypsum and calcium carbonate on plates, supporting the hypothesis of an essential contribution of microorganisms to the formation of moonmilk.
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http://dx.doi.org/10.1038/s41598-018-34134-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203712PMC
October 2018

A role for microbial selection in frescoes' deterioration in Tomba degli Scudi in Tarquinia, Italy.

Sci Rep 2017 07 20;7(1):6027. Epub 2017 Jul 20.

La Sapienza University of Rome, Departement of Biology and Biotechnology "Charles Darwin", Piazzale Aldo Moro, 5, 00185, Rome, Italy.

Mural paintings in the hypogeal environment of the Tomba degli Scudi in Tarquinia, Italy, show a quite dramatic condition: the plaster mortar lost his cohesion and a white layer coating is spread over almost all the wall surfaces. The aim of this research is to verify if the activity of microorganisms could be one of the main causes of deterioration and if the adopted countermeasures (conventional biocide treatments) are sufficient to stop it. A biocide treatment of the whole environment has been carried out before the conservative intervention and the tomb has been closed for one month. When the tomb was opened again, we sampled the microorganisms present on the frescoes and we identified four Bacillus species and one mould survived to the biocide treatment. These organisms are able to produce spores, a highly resistant biological form, which has permitted the survival despite the biocide treatment. We show that these Bacillus strains are able to produce calcium carbonate and could be responsible for the white deposition that was damaging and covering the entire surface of the frescoes. Our results confirm that the sanitation intervention is non always resolutive and could even be deleterious in selecting harmful microbial communities.
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http://dx.doi.org/10.1038/s41598-017-06169-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519700PMC
July 2017

Antitumor Effects of Epidrug/IFNα Combination Driven by Modulated Gene Signatures in Both Colorectal Cancer and Dendritic Cells.

Cancer Immunol Res 2017 07 14;5(7):604-616. Epub 2017 Jun 14.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy.

Colorectal cancer results from the progressive accumulation of genetic and epigenetic alterations. IFN signaling defects play an important role in the carcinogenesis process, in which the inability of IFN transcription regulatory factors (IRF) to access regulatory sequences in IFN-stimulated genes (ISG) in tumors and in immune cells may be pivotal. We reported that low-dose combination of two FDA-approved epidrugs, azacytidine (A) and romidepsin (R), with IFNα2 (ARI) hampers the aggressiveness of both colorectal cancer metastatic and stem cells and triggers immunogenic cell death signals that stimulate dendritic cell (DC) function. Here, we investigated the molecular signals induced by ARI treatment and found that this drug combination increased the accessibility to regulatory sequences of ISGs and IRFs that were epigenetically silenced in both colorectal cancer cells and DCs. Likewise, specific ARI-induced histone methylation and acetylation changes marked epigenetically affected ISG promoters in both metastatic cancer cells and DCs. Analysis by ChIP-seq confirmed such ARI-induced epigenetically regulated IFN signature. The activation of this signal endowed DCs with a marked migratory capability. Our results establish a direct correlation between reexpression of silenced ISGs by epigenetic control and ARI anticancer activity and provide new knowledge for the development of innovative combined therapeutic strategies for colorectal cancer. .
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http://dx.doi.org/10.1158/2326-6066.CIR-17-0080DOI Listing
July 2017

Leptin induction following irradiation is a conserved feature in mammalian epithelial cells and tissues.

Int J Radiat Biol 2017 09 10;93(9):947-957. Epub 2017 Jul 10.

c Dipartimento di Biologia e Biotecnologie C. Darwin , Sapienza Università di Roma , Rome , Italy.

Purpose: Leptin (LEP) is a peptide hormone with multiple physiological functions. Besides its systemic actions, it has important peripheral roles such as a mitogen action on keratinocytes following skin lesions. We previously showed that LEP mRNA is significantly induced in response to neutron irradiation in mouse skin and that the protein increases in the irradiated epidermis and in the related subcutaneous adipose tissue. In this work, we investigated the post-transcriptional regulation of LEP by miRNAs and the conservation of LEP's role in radiation response in human cells.

Methods: We used microarray analysis and real-time polymerase chain reaction (RT-PCR) to analyze modulation of miRNAs potentially targeting LEP in mouse skin following irradiation and bioinformatic analysis of transcriptome of irradiated human cell lines and cancer tissues from radiotherapy-treated patients to evaluate LEP expression.

Results And Conclusions: We show that a network of miRNAs potentially targeting LEP mRNA is modulated in irradiated mouse skin and that LEP itself is significantly modulated by irradiation in human epithelial cell lines and in breast cancer tissues from radiotherapy-treated patients. These results confirm and extend the previous evidence that LEP has a general and important role in the response of mammalian cells to irradiation.
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http://dx.doi.org/10.1080/09553002.2017.1339918DOI Listing
September 2017

Transcriptional response of genes to retinoid signalling is regionally restricted along the neural tube rostrocaudal axis.

R Soc Open Sci 2017 Apr 5;4(4):160913. Epub 2017 Apr 5.

Istituto Pasteur- Fondazione Cenci Bolognetti, Sapienza University of Rome, 00185 Rome, Italy.

During vertebrate neural development, positional information is largely specified by extracellular morphogens. Their distribution, however, is very dynamic due to the multiple roles played by the same signals in the developing and adult neural tissue. This suggests that neural progenitors are able to modify their competence to respond to morphogen signalling and autonomously maintain positional identities after their initial specification. In this work, we take advantage of culture systems of mouse neural stem/progenitor cells (NSPCs) to show that NSPCs isolated from rostral or caudal regions of the mouse neural tube are differentially responsive to retinoic acid (RA), a pivotal morphogen for the specification of posterior neural fates. genes are among the best known RA direct targets in the neural tissue, yet we found that RA could promote their transcription only in caudal but not in rostral NSPCs. Correlating with these effects, key RA-responsive regulatory regions in the cluster displayed opposite enrichment of activating or repressing histone marks in rostral and caudal NSPCs. Finally, RA was able to strengthen chromatin activation in caudal NSPCs, but was ineffective on the repressed chromatin of rostral NSPCs. These results suggest that the response of NSPCs to morphogen signalling across the rostrocaudal axis of the neural tube may be gated by the epigenetic configuration of target patterning genes, allowing long-term maintenance of intrinsic positional values in spite of continuously changing extrinsic signals.
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http://dx.doi.org/10.1098/rsos.160913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414248PMC
April 2017

Neuroserpin polymers cause oxidative stress in a neuronal model of the dementia FENIB.

Neurobiol Dis 2017 Jul 28;103:32-44. Epub 2017 Mar 28.

Dpt. of Biology and Biotechnologies 'Charles Darwin', Sapienza University of Rome, Italy; Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Italy. Electronic address:

The serpinopathies are human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity. The dementia FENIB is caused by polymerisation of the neuronal serpin neuroserpin (NS) within the endoplasmic reticulum (ER) of neurons. With the aim of understanding the toxicity due to intracellular accumulation of neuroserpin polymers, we have generated transgenic neural progenitor cell (NPC) cultures from mouse foetal cerebral cortex, stably expressing the control protein GFP (green fluorescent protein), or human wild type, G392E or delta NS. We have characterised these cell lines in the proliferative state and after differentiation to neurons. Our results show that G392E NS formed polymers that were mostly retained within the ER, while wild type NS was correctly secreted as a monomeric protein into the culture medium. Delta NS was absent at steady state due to its rapid degradation, but it was easily detected upon proteasomal block. Looking at their intracellular distribution, wild type NS was found in partial co-localisation with ER and Golgi markers, while G392E NS was localised within the ER only. Furthermore, polymers of NS were detected by ELISA and immunofluorescence in neurons expressing the mutant but not the wild type protein. We used control GFP and G392E NPCs differentiated to neurons to investigate which cellular pathways were modulated by intracellular polymers by performing RNA sequencing. We identified 747 genes with a significant upregulation (623) or downregulation (124) in G392E NS-expressing cells, and we focused our attention on several genes involved in the defence against oxidative stress that were up-regulated in cells expressing G392E NS (Aldh1b1, Apoe, Gpx1, Gstm1, Prdx6, Scara3, Sod2). Inhibition of intracellular anti-oxidants by specific pharmacological reagents uncovered the damaging effects of NS polymers. Our results support a role for oxidative stress in the cellular toxicity underlying the neurodegenerative dementia FENIB.
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http://dx.doi.org/10.1016/j.nbd.2017.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439028PMC
July 2017

Histone Methylation and microRNA-dependent Regulation of Epigenetic Activities in Neural Progenitor Self-Renewal and Differentiation.

Curr Top Med Chem 2017 ;17(7):794-807

Department of Biology and Biotechnology "Charles Darwin", Sapienza University of Rome, Rome, Italy.

Neural stem/progenitor cell (NSPC) self-renewal and differentiation in the developing and the adult brain are controlled by extra-cellular signals and by the inherent competence of NSPCs to produce appropriate responses. Stage-dependent responsiveness of NSPCs to extrinsic cues is orchestrated at the epigenetic level. Epigenetic mechanisms such as DNA methylation, histone modifications and non-coding RNA-mediated regulation control crucial aspects of NSPC development and function, and are also implicated in pathological conditions. While their roles in the regulation of stem cell fate have been largely explored in pluripotent stem cell models, the epigenetic signature of NSPCs is also key to determine their multipotency as well as their progressive bias towards specific differentiation outcomes. Here we review recent developments in this field, focusing on the roles of histone methylation marks and the protein complexes controlling their deposition in NSPCs of the developing cerebral cortex and the adult subventricular zone. In this context, we describe how bivalent promoters, carrying antagonistic epigenetic modifications, feature during multiple steps of neural development, from neural lineage specification to neuronal differentiation. Furthermore, we discuss the emerging cross-talk between epigenetic regulators and microRNAs, and how the interplay between these different layers of regulation can finely tune the expression of genes controlling NSPC maintenance and differentiation. In particular, we highlight recent advances in the identification of astrocyte-enriched microRNAs and their function in cell fate choices of NSPCs differentiating towards glial lineages.
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http://dx.doi.org/10.2174/1568026616666160414124456DOI Listing
February 2017

Yeast as a tool to select inhibitors of the cullin deneddylating enzyme Csn5.

J Enzyme Inhib Med Chem 2016 Dec 30;31(6):1632-7. Epub 2016 Mar 30.

a Istituto Pasteur Fondazione Cenci Bolognetti, Department of Biology and Biotechnology, Sapienza University of Rome , Rome , Italy .

The CSN complex plays a key role in various cellular pathways: through a metalloprotease activity of its Csn5 deneddylating enzyme, it regulates the activity of Cullin-RING ligases (CRLs). Indeed, Csn5 has been found amplified in many tumors, but, due to its pleiotropic effects, it is difficult to dissect its function and the involvement in cancer progression. Moreover, while growing evidences point to the neddylation function as a good target for drug development; specific inhibitors have not yet been developed for the CSN. Here, we propose the yeast Saccharomyces cerevisiae as a model system to screen libraries of small molecules as inhibitors of cullins deneddylation, taking advantage of the unique feature of this organism to survive without a functional CSN5 gene and to accumulate a fully neddylated cullin substrate. By combining molecular modeling and simple genetic tools, we were able to identify two small molecular fragments as selective inhibitors of Csn5 deneddylation function.
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http://dx.doi.org/10.3109/14756366.2016.1160901DOI Listing
December 2016

Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups.

PLoS One 2015 24;10(7):e0133607. Epub 2015 Jul 24.

Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Rome, Italy; Pasteur Institute, Cenci Bolognetti Foundation, Rome, Italy; Institute of Molecular Biology and Pathology, CNR, Rome, Italy.

Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD) canine adenovirus type 2 vectors (CAV-2) are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HD-CAV-2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133607PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4514711PMC
May 2016

An high-throughput in vivo screening system to select H3K4-specific histone demethylase inhibitors.

PLoS One 2014 29;9(1):e86002. Epub 2014 Jan 29.

Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza Università di Roma, Rome, Italy.

Background: Histone demethylases (HDMs) have a prominent role in epigenetic regulation and are emerging as potential therapeutic cancer targets. The search for small molecules able to inhibit HDMs in vivo is very active but at the present few compounds were found to be specific for defined classes of these enzymes.

Methodology/principal Findings: In order to discover inhibitors specific for H3K4 histone demethylation we set up a screening system which tests the effects of candidate small molecule inhibitors on a S.cerevisiae strain which requires Jhd2 demethylase activity to efficiently grow in the presence of rapamycin. In order to validate the system we screened a library of 45 structurally different compounds designed as competitive inhibitors of α -ketoglutarate (α-KG) cofactor of the enzyme, and found that one of them inhibited Jhd2 activity in vitro and in vivo. The same compound effectively inhibits human Jumonji AT-Rich Interactive Domain (JARID) 1B and 1D in vitro and increases H3K4 tri-methylation in HeLa cell nuclear extracts (NEs). When added in vivo to HeLa cells, the compound leads to an increase of tri-methyl-H3K4 (H3K4me3) but does not affect H3K9 tri-methylation. We describe the cytostatic and toxic effects of the compound on HeLa cells at concentrations compatible with its inhibitory activity.

Conclusions/significance: Our screening system is proved to be very useful in testing putative H3K4-specific HDM inhibitors for the capacity of acting in vivo without significantly altering the activity of other important 2-oxoglutarate oxygenases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0086002PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3906020PMC
September 2014

The COP9 signalosome is involved in the regulation of lipid metabolism and of transition metals uptake in Saccharomyces cerevisiae.

FEBS J 2014 Jan 25;281(1):175-90. Epub 2013 Nov 25.

Istituto Pasteur - Fondazione Cenci Bolognetti, Department of Biology and Biotechnology 'C. Darwin', Sapienza University of Rome, Italy.

The COP9 signalosome (CSN) is a highly conserved eukaryotic protein complex which regulates the cullin RING family of ubiquitin ligases and carries out a deneddylase activity that resides in subunit 5 (CSN5). Whereas CSN activity is essential for the development of higher eukaryotes, several unicellular fungi including the budding yeast Saccharomyces cerevisiae can survive without a functional CSN. Nevertheless, the budding yeast CSN is biochemically active and deletion mutants of each of its subunits exhibit deficiency in cullins deneddylation, although the biological context of this activity is still unknown in this organism. To further characterize CSN function in budding yeast, we present here a transcriptomic and proteomic analysis of a S. cerevisiae strain deleted in the CSN5/RRI1 gene (hereafter referred to as CSN5), coding for the only canonical subunit of the complex. We show that Csn5 is involved in modulation of the genes controlling amino acid and lipid metabolism and especially ergosterol biosynthesis. These alterations in gene expression correlate with the lower ergosterol levels and increased intracellular zinc content which we observed in csn5 null mutant cells. We show that some of these regulatory effects of Csn5, in particular the control of isoprenoid biosynthesis, are conserved through evolution, since similar transcriptomic and/or proteomic effects of csn5 mutation were previously observed in other eukaryotic organisms such as Aspergillus nidulans, Arabidopsis thaliana and Drosophila melanogaster. Our results suggest that the diverged budding yeast CSN is more conserved than was previously thought.
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http://dx.doi.org/10.1111/febs.12584DOI Listing
January 2014

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles.

PLoS One 2013 26;8(7):e69808. Epub 2013 Jul 26.

Dipartimento di Biologia e Biotecnologie Chrales Darwin, Sapienza, Università di Roma, Roma, Italy.

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways--but in opposite directions--suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069808PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724896PMC
April 2014

HMGB1 induces the overexpression of miR-222 and miR-221 and increases growth and motility in papillary thyroid cancer cells.

Oncol Rep 2012 Dec 26;28(6):2285-9. Epub 2012 Sep 26.

Department of Experimental Medicine, Sapienza University of Rome, I-00161 Rome, Italy.

Experimental and epidemiological studies have revealed that chronic inflammation contributes to cancer progression and even predisposes to cellular transformation. Inflammatory infiltrates in papillary thyroid cancer include lymphocytes, macrophages and cytokines. High-mobility group box 1 protein (HMGB1) is a late inflammatory cytokine that signals danger to the immune system through the receptor for advanced glycation end-products (RAGE) and Toll-like receptor. The activation of the above receptors results in the secretion of growth, chemotactic and angiogenic factors that contribute to chronic inflammation. In this study, we suggest that apart from the activation of signal transduction pathways by the activation of RAGE, the indirect inhibition of cell cycle regulators [such as phosphatase and tensin homolog (PTEN)] may also cause an increase in cell growth and motility. MicroRNAs (miRNAs) have increasingly been implicated in regulating the malignant progression of cancer. MiR-221 and miR-222 have been found to be deregulated in human papillary thyroid carcinomas. They are involved in cell proliferation through the inhibition of the cell cycle regulator, p27kip1, in human papillary carcinomas. In this study, we show that HMGB1 increases the expression of miR-221 and miR-222 in primary cultures of excised papillary lesions and in an established papillary cancer cell line (BC PAP). The overexpression of oncogenic miR-221 and miR-222 caused by HMGB1 is associated with an increase in malignancy scores, namely cell growth and motility.
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http://dx.doi.org/10.3892/or.2012.2058DOI Listing
December 2012

The transcriptional response of mammalian cancer cells to irradiation is dominated by a cell cycle signature which is strongly attenuated in non-cancer cells and tissues.

Int J Radiat Biol 2012 Nov 26;88(11):822-9. Epub 2012 Apr 26.

Laboratory of Functional Genomics and Proteomics of Model Systems, Department of Biology and Biotechnology Charles Darwin, University of Rome, La Sapienza.

Purpose: Our goal was to identify genes showing a general transcriptional response to irradiation in mammalian cells and to analyze their response in function of dose, time and quality of irradiation and of cell type.

Materials And Methods: We used a modified MIAME (Minimal Information About Microarray Experiments) protocol to import microarray data from 177 different irradiation conditions in the Radiation Genes database and performed cut-off-based selections and hierarchical gene clustering.

Results: We identified a set of 29 genes which respond to a wide range of irradiation conditions in different cell types and tissues. Functional analysis of the negatively modulated genes revealed a dominant signature of mitotic cell cycle regulation which appears both dose and time-dependent. This signature is prominent in cancer cells and highly proliferating tissues but it is strongly attenuated in non cancer cells.

Conclusions: The transcriptional response of mammalian cancer cells to irradiation is dominated by a mitotic cell cycle signature both dose and time-dependent. This core response, which is present in cancer cells and highly proliferating tissues such as skin, blood and lymph node, is weaker or absent in non-cancer cells and in liver and spleen. CDKN1A (cyclin-dependent kinase inhibitor 1A) appears as the most generally induced mammalian gene and its response (mostly dose- and time-independent) seems to go beyond the typical DNA damage response.
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http://dx.doi.org/10.3109/09553002.2012.676230DOI Listing
November 2012

Lack of dystrophin in mdx mice modulates the expression of genes involved in neuron survival and differentiation.

Eur J Neurosci 2012 Mar 7;35(5):691-701. Epub 2012 Feb 7.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie Charles Darwin, Sapienza Università di Roma, P.le A. Moro 5 - 00185, Roma, Italy.

Duchenne muscular dystrophy is an X-linked disease characterized by progressive and lethal muscular wasting. Dystrophic patients, however, are also afflicted by several neurological disorders, the importance of which is generally underestimated. As promising therapies for muscles are currently in clinical trial stages, with the potential to provide an increase in the lifespan of young patients, determination of the genetic and molecular aspects characterizing this complex disease is crucial in order to allow the development of therapeutic approaches specifically designed for the nervous system. In this study, differences in gene expression in the superior cervical ganglion of postnatal day (P)5, P10 and 6-7-week-old wild-type and genetically dystrophic mdx mice were evaluated by DNA microarray analysis. The main aim was to verify whether the lack of dystrophin affected the transcript levels of genes related to different aspects of neuron development and differentiation. Ontological analysis of more than 500 modulated genes showed significant differences in genetic class enrichment at each postnatal date. Upregulated genes mainly fell in the categories of vesicular trafficking, and cytoskeletal and synaptic organization, whereas downregulated genes were associated with axon development, growth factors, intracellular signal transduction, metabolic processes, gene expression regulation, synapse morphogenesis, and nicotinic receptor clustering. These data strongly suggest that the structural and functional alterations previously described in both the autonomic and central nervous systems of mdx mice with respect to wild-type mice and related to crucial aspects of neuron life (i.e. postnatal development, differentiation, and plasticity) result not only from protein post-translational modifications, but also from direct and/or indirect modulation of gene expression.
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http://dx.doi.org/10.1111/j.1460-9568.2011.07984.xDOI Listing
March 2012
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