Genome-wide expression profiling and functional characterization of SCA28 lymphoblastoid cell lines reveal impairment in cell growth and activation of apoptotic pathways

BMC Medical Genomics

Alfredo Brusco^1,10  Stefano Gustincich³  Giuseppe Gasparre⁷  Ada Funaro⁶  Anna Maria Porcelli⁵  Giorgio Casari⁸  Dario Ghigo²  Alexis Brice⁹  Alexandra Durr⁹  Isabelle Le Ber¹  Sylvie Forlani¹  Claudia Cagnoli⁶  Luisa Iommarini⁵  Maria Antonietta Calvaruso^1,10  Anna Bartoletti Stella⁷  Elena Gazzano²  Francesca Maltecca⁸  Helena Krmac³  Nicola Lo Buono⁶  Giovanni Stevanin⁴  Alessandro Brussino⁶  Paola Roncaglia³  Cecilia Mancini⁶ 

[1] Centre de Recherche de l’Institut du Cerveau et de la Moelle épinière (INSERM / UPMC Univ. Paris 6, UMR_S975 ; CNRS 7225), Pitié-Salpêtrière Hospital, Paris, France;Department of Oncology, University of Torino, Candiolo, Italy;Neurobiology Sector, SISSA/ISAS, Trieste, Italy;Neurogenetics team, Ecole Pratique des Hautes Etudes, Institut du Cerveau et de la Moelle épinière, CHU Pitié-Salpêtrière, Paris, France;Department of Pharmacy and Biotechnologies (FABIT), University of Bologna, Bologna, Italy;Department of Medical Sciences, University of Torino, via Santena 19, 10126 Torino, Italy;Department Medical and Surgical Sciences, Medical Genetics, University of Bologna, Bologna, Italy;San Raffaele Scientific Institute, Vita-Salute San Raffaele University and Center for Translational Genomics and Bioinformatics, Milan-I, Italy;APHP, Fédération de génétique, Pitié-Salpêtrière Hospital, Paris, France;Medical Genetics Unit, “Città della Salute e della Scienza” Hospital, Torino, Italy

关键词: LCLs;    Genome-wide expression;    AFG3L2;    SCA28;    Spinocerebellar ataxia;    Autosomal dominant cerebellar ataxia;   

Others  :  1092351
DOI  :  10.1186/1755-8794-6-22

 received in 2013-02-11, accepted in 2013-06-05,  发布年份 2013

【 摘 要 】

Background

SCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We performed a whole genome expression profiling using lymphoblastoid cell lines (LCLs) from four SCA28 patients and six unrelated healthy controls matched for sex and age.

Methods

Gene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR.

Results

We found 66 genes whose expression was statistically different in SCA28 LCLs, 35 of which were up-regulated and 31 down-regulated. The differentially expressed genes were clustered in five functional categories: (1) regulation of cell proliferation; (2) regulation of programmed cell death; (3) response to oxidative stress; (4) cell adhesion, and (5) chemical homeostasis. To validate these data, we performed functional experiments that proved an impaired SCA28 LCLs growth compared to controls (p < 0.005), an increased number of cells in the G0/G1 phase (p < 0.001), and an increased mortality because of apoptosis (p < 0.05). We also showed that respiratory chain activity and reactive oxygen species levels was not altered, although lipid peroxidation in SCA28 LCLs was increased in basal conditions (p < 0.05). We did not detect mitochondrial DNA large deletions. An increase of TFAM, a crucial protein for mtDNA maintenance, and of DRP1, a key regulator of mitochondrial dynamic mechanism, suggested an alteration of fission/fusion pathways.

Conclusions

Whole genome expression profiling, performed on SCA28 LCLs, allowed us to identify five altered functional categories that characterize the SCA28 LCLs phenotype, the first reported in human cells to our knowledge.

【 授权许可】

   
2013 Mancini et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Durr A: Autosomal dominant cerebellar ataxias: polyglutamine expansions and beyond. Lancet Neurol 2010, 9(9):885-894.
• [2]Cagnoli C, Mariotti C, Taroni F, Seri M, Brussino A, Michielotto C, Grisoli M, Di Bella D, Migone N, Gellera C, et al.: SCA28, a novel form of autosomal dominant cerebellar ataxia on chromosome 18p11.22-q11.2. Brain 2006, 129(Pt 1):235-242.
• [3]Di Bella D, Lazzaro F, Brusco A, Plumari M, Battaglia G, Pastore A, Finardi A, Cagnoli C, Tempia F, Frontali M, et al.: Mutations in the mitochondrial protease gene AFG3L2 cause dominant hereditary ataxia SCA28. Nat Genet 2010, 42(4):313-321.
• [4]Cagnoli C, Stevanin G, Brussino A, Barberis M, Mancini C, Margolis RL, Holmes SE, Nobili M, Forlani S, Padovan S, et al.: Missense mutations in the AFG3L2 proteolytic domain account for approximately 1.5% of European autosomal dominant cerebellar ataxias. Hum Mutat 2010, 31(10):1117-1124.
• [5]Edener U, Wollner J, Hehr U, Kohl Z, Schilling S, Kreuz F, Bauer P, Bernard V, Gillessen-Kaesbach G, Zuhlke C: Early onset and slow progression of SCA28, a rare dominant ataxia in a large four-generation family with a novel AFG3L2 mutation. Eur J Hum Genet 2010, 18(8):965-968.
• [6]Banfi S, Bassi MT, Andolfi G, Marchitiello A, Zanotta S, Ballabio A, Casari G, Franco B: Identification and characterization of AFG3L2, a novel paraplegin-related gene. Genomics 1999, 59(1):51-58.
• [7]Leonhard K, Stiegler A, Neupert W, Langer T: Chaperone-like activity of the AAA domain of the yeast Yme1 AAA protease. Nature 1999, 398(6725):348-351.
• [8]Arlt H, Tauer R, Feldmann H, Neupert W, Langer T: The YTA10-12 complex, an AAA protease with chaperone-like activity in the inner membrane of mitochondria. Cell 1996, 85(6):875-885.
• [9]Leonhard K, Guiard B, Pellecchia G, Tzagoloff A, Neupert W, Langer T: Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface. Mol Cell 2000, 5(4):629-638.
• [10]Nolden M, Ehses S, Koppen M, Bernacchia A, Rugarli EI, Langer T: The m-AAA protease defective in hereditary spastic paraplegia controls ribosome assembly in mitochondria. Cell 2005, 123(2):277-289.
• [11]Koppen M, Bonn F, Ehses S, Langer T: Autocatalytic processing of m-AAA protease subunits in mitochondria. Mol Biol Cell 2009, 20(19):4216-4224.
• [12]Gerdes F, Tatsuta T, Langer T: Mitochondrial AAA proteases - Towards a molecular understanding of membrane-bound proteolytic machines. Biochim Biophys Acta 2011, 1823(1):49-55.
• [13]Casari G, De Fusco M, Ciarmatori S, Zeviani M, Mora M, Fernandez P, De Michele G, Filla A, Cocozza S, Marconi R, et al.: Spastic paraplegia and OXPHOS impairment caused by mutations in paraplegin, a nuclear-encoded mitochondrial metalloprotease. Cell 1998, 93(6):973-983.
• [14]Pierson TM, Adams D, Bonn F, Martinelli P, Cherukuri PF, Teer JK, Hansen NF, Cruz P, Blakesley RW, Mullikin For The Nisc Comparative Sequencing Program JC, et al.: Whole-exome sequencing identifies homozygous AFG3L2 mutations in a spastic ataxia-neuropathy syndrome linked to mitochondrial m-AAA proteases. PLoS Genet 2011, 7(10):e1002325.
• [15]Klebe S, Depienne C, Gerber S, Challe G, Anheim M, Charles P, Fedirko E, Lejeune E, Cottineau J, Brusco A, et al.: Spastic paraplegia gene 7 in patients with spasticity and/or optic neuropathy. Brain 2012, 135(Pt 10):2980-2993.
• [16]Maltecca F, Aghaie A, Schroeder DG, Cassina L, Taylor BA, Phillips SJ, Malaguti M, Previtali S, Guenet JL, Quattrini A, et al.: The mitochondrial protease AFG3L2 is essential for axonal development. J Neurosci 2008, 28(11):2827-2836.
• [17]Maltecca F, Magnoni R, Cerri F, Cox GA, Quattrini A, Casari G: Haploinsufficiency of AFG3L2, the gene responsible for spinocerebellar ataxia type 28, causes mitochondria-mediated Purkinje cell dark degeneration. J Neurosci 2009, 29(29):9244-9254.
• [18]Bioconductor. http://www.bioconductor.org webcite
• [19]MultiExperiment Viewer. http://www.tm4.org/mev/ webcite
• [20]Breitling R, Armengaud P, Amtmann A, Herzyk P: Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments. FEBS Lett 2004, 573(1–3):83-92.
• [21]Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 2001, 98(9):5116-5121.
• [22]Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al.: Gene ontology: tool for the unification of biology, The Gene Ontology Consortium. Nat Genet 2000, 25(1):25-29.
• [23]da Huang W, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009, 4(1):44-57.
• [24]da Huang W, Sherman BT, Lempicki RA: Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 2009, 37(1):1-13.
• [25]Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA: DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 2003, 4(5):P3. BioMed Central Full Text
• [26]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25(4):402-408.
• [27]Mosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983, 65(1–2):55-63.
• [28]Cunningham RE: Overview of flow cytometry and fluorescent probes for flow cytometry. Methods Mol Biol 2010, 588:319-326.
• [29]Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C: A novel assay for apoptosis, Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995, 184(1):39-51.
• [30]Brumatti G, Sheridan C, Martin SJ: Expression and purification of recombinant annexin V for the detection of membrane alterations on apoptotic cells. Methods 2008, 44(3):235-240.
• [31]Robinson BH: Use of fibroblast and lymphoblast cultures for detection of respiratory chain defects. Methods Enzymol 1996, 264:454-464.
• [32]Ronner P, Friel E, Czerniawski K, Frankle S: Luminometric assays of ATP, phosphocreatine, and creatine for estimation of free ADP and free AMP. Anal Biochem 1999, 275(2):208-216.
• [33]Zanna C, Ghelli A, Porcelli AM, Martinuzzi A, Carelli V, Rugolo M: Caspase-independent death of Leber's hereditary optic neuropathy cybrids is driven by energetic failure and mediated by AIF and Endonuclease G. Apoptosis 2005, 10(5):997-1007.
• [34]Yano E: Mineral fiber-induced malondialdehyde formation and effects of oxidant scavengers in phagocytic cells. Int Arch Occup Environ Health 1988, 61(1–2):19-23.
• [35]Iommarini L, Maresca A, Caporali L, Valentino ML, Liguori R, Giordano C, Carelli V: Revisiting the issue of mitochondrial DNA content in optic mitochondriopathies. Neurology 2012, 79(14):1517-1519.
• [36]Kleinle S, Wiesmann U, Superti-Furga A, Krahenbuhl S, Boltshauser E, Reichen J, Liechti-Gallati S: Detection and characterization of mitochondrial DNA rearrangements in Pearson and Kearns-Sayre syndromes by long PCR. Hum Genet 1997, 100(5–6):643-650.
• [37]Sacco T, Boda E, Hoxha E, Pizzo R, Cagnoli C, Brusco A, Tempia F: Mouse brain expression patterns of Spg7, Afg3l1, and Afg3l2 transcripts, encoding for the mitochondrial m-AAA protease. BMC Neurosci 2010, 11:55. BioMed Central Full Text
• [38]National Center for Biotechnology Information. http://www.ncbi.nlm.nih.gov webcite
• [39]Yan H, Yuan W, Velculescu VE, Vogelstein B, Kinzler KW: Allelic variation in human gene expression. Science 2002, 297(5584):1143.
• [40]Cheung VG, Conlin LK, Weber TM, Arcaro M, Jen KY, Morley M, Spielman RS: Natural variation in human gene expression assessed in lymphoblastoid cells. Nat Genet 2003, 33(3):422-425.
• [41]Cooper-Knock J, Kirby J, Ferraiuolo L, Heath PR, Rattray M, Shaw PJ: Gene expression profiling in human neurodegenerative disease. Nat Rev Neurol 2012, 8(9):518-530.
• [42]Yang Y, Herrup K: Cell division in the CNS: protective response or lethal event in post-mitotic neurons? Biochim Biophys Acta 2007, 1772(4):457-466.
• [43]Margineantu DH, Gregory Cox W, Sundell L, Sherwood SW, Beechem JM, Capaldi RA: Cell cycle dependent morphology changes and associated mitochondrial DNA redistribution in mitochondria of human cell lines. Mitochondrion 2002, 1(5):425-435.
• [44]Parone PA, Da Cruz S, Tondera D, Mattenberger Y, James DI, Maechler P, Barja F, Martinou JC: Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA. PLoS One 2008, 3(9):e3257.
• [45]Utomo A, Jiang X, Furuta S, Yun J, Levin DS, Wang YC, Desai KV, Green JE, Chen PL, Lee WH: Identification of a novel putative non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx) essential for alleviating oxidative stress generated from polyunsaturated fatty acids in breast cancer cells. J Biol Chem 2004, 279(42):43522-43529.
• [46]Masmoudi-Kouki O, Douiri S, Hamdi Y, Kaddour H, Bahdoudi S, Vaudry D, Basille M, Leprince J, Fournier A, Vaudry H, et al.: Pituitary adenylate cyclase-activating polypeptide protects astroglial cells against oxidative stress-induced apoptosis. J Neurochem 2011, 117(3):403-411.
• [47]Ferber EC, Peck B, Delpuech O, Bell GP, East P, Schulze A: FOXO3a regulates reactive oxygen metabolism by inhibiting mitochondrial gene expression. Cell Death Differ 2012, 19(6):968-979.
• [48]Hruszkewycz AM: Evidence for mitochondrial DNA damage by lipid peroxidation. Biochem Biophys Res Commun 1988, 153(1):191-197.
• [49]Sultana R, Perluigi M, Allan Butterfield D: Lipid peroxidation triggers neurodegeneration: A redox proteomics view into the Alzheimer disease brain. Free Radic Biol Med 2012, S0891-5849(12):01161-01166.
• [50]Kang D, Kim SH, Hamasaki N: Mitochondrial transcription factor A (TFAM): roles in maintenance of mtDNA and cellular functions. Mitochondrion 2007, 7(1–2):39-44.
• [51]Noack H, Bednarek T, Heidler J, Ladig R, Holtz J, Szibor M: TFAM-dependent and independent dynamics of mtDNA levels in C2C12 myoblasts caused by redox stress. Biochim Biophys Acta 2006, 1760(2):141-150.