Mitochondrial DNA Integrity is Essential for Mitochondrial 2010

TISSUE-SPECIFIC STEM CELLS Mitochondrial DNA Integrity Is Essential for Mitochondrial Maturation During Differentiation of Neural Stem Cells WEI WANG, a PIA OSENBROCH, a RAGNHILD SKINNES, a YING ESBENSEN, b MAGNAR BJØRA ˚ S, a,c LARS EIDE a a Institute of Clinical Biochemistry, Oslo University Hospital and Centre for Molecular Biology and Neuroscience, Oslo Unive rsity Hospital, Oslo, Norway ; b Department of Clinical Molecular Biology (EpiGen), Akers hus Unive rsity Hospital, Universit y of Oslo, Norway; c Department of Microbiology, Oslo University Hospi tal, Oslo, Norway Key Words. mtDNA • 8-Oxoguanine • OGG1 • ETC • Supercomplex • DNA repair • Mitochondrial maturation ABSTRACT Dif fer ent iat ion of neu ral ste m cells (NS Cs) inv olv es the activ ation of aerobic metab olism, which is depen dent on mitoch ondrial functi on. Here, we show that the differentiation of NSCs involves robust increases in mitochondrial mas s, mitochond ria l DNA (mtD NA) cop y number , and resp iration capac ity. The incr eased resp irati on activ ity re nders mtDNA vulner able to oxi dative damage, and NSCs defectiv e for the mitochondrial 8-oxogu anine DNA glycos ylase (OGG1) function accumulate mtDNA damage during the differentiation. The accumulated mtDNA damage s in ogg1 2/2 cells inh ibi t the nor mal matu ration of mitochondria that is manifested by reduced cellular levels of mitochondria l enc oded comple x proteins (compl ex I [c I] , cIII , and cIV) wi th normal le vels of the nucl ear enc ode d cII pre sen t. The spe cifi c cI activi ty and inner membrane organization of respiratory complexes are sim- ilar in wt and ogg1 2/2 cel ls, inf err ing that mtDNA damage manife sts its elf as dimini she d mit ochond rial biogenesis rather than the generation of dysfunctional mitochondria. Aerobic metabolism increases during differentiation in wild-type cells and to a lesser extent in ogg1 2/2 cells, whereas anaerobic rates of metabolism are constant and simila r in bot h cell types. Our res ult s demons trate that mtDNA integr ity is essen tial for effec tive mitochondrial maturation duri ng NSC diffe renti ation . STEM CELLS 2010;28:2 195–220 4 Disclosure of potential conflicts of interest is found at the end of this article. INTRODUCTION Mitochondria are subcellular organelles with a heterogeneous compos ition that varies depend ing on tiss ue type [1]. Speci alization of cells in differing tissue, therefore, relies on the cor- respo nding speci alization of cell ular mitoc hondri a. The pro- cess by which mitochondrial compon ents become specialize d, known as mitochondrial maturation, is essential for stem cell differentiation [2]. Neural cells have a high aerobic metabolic rat e, which is hig her than that in neu ral stem cel ls (NS Cs) [3]. In neu rons , suf ficient res pir ato ry capaci ty pro vided by funct ional mitochondria is esse ntial to resto re resis tance to excitotoxic effects of neurotransmitter activation [4]. The het- erogeneity of mitochondria in the brain is important for ensur- ing normal neuron-as trocyt e inter actio ns by balanc ing nitro- gen met abo lis m [5]. Dif fer ent iat ion of NSCs to neu ron s, astro cytes, or oligodendrocytes is controlled by several factor s including oxygen availability and intracellular redox state [6– 8]. Mitochondri al react ive oxygen speci es (ROS) serve important signaling roles in both oxygen sensing and redox balance [9, 10]. The generation of ROS is an inevitable byproduct of elec tron trans port chain (ETC) activ ity; therefore, incre ased aerob ic acti vity can poten tiall y influe nce the differ entia tion proce ss and rende r mitoc hondrial macro molec ules vulner able to oxidation damage. Mitoc hondri al DNA (mtDNA) residing in the mitochondrial matrix is readily damaged by ROS. Damage and muta- tions to mtDNA have a critical effect on ETC activity because mtDNA encodes 13 essential subunits of ETC complex I [cI], cIII, cIV, and cV. cII (succinate dehydrogenase) is composed of nuclear encoded subunits. Damages to mtDNA potentially can interfere with transcription and replication. The mitochondrial tra nsc rip tion fac tor A (TFAM) is pre sent in suf fici ent quant ities to comple tely cover mtDNA, it plays an esse ntial role in bot h tra nsc ription and replic ati on of mtDNA, and it binds preferentially to damaged DNA [11, 12]. In the mito- chondrion, mtDNA damage is removed by the base excision repair (BER), an innermembrane-a ssociated mul tie nzy me DNA repair cascade [13]. The BER pathway is initiated by a DNA glycos yla se, lik e the 8-ox ogua nine DNA glycos yla se (OGG1), which recogniz es and remov es several base lesions including 8-oxoguanine. The resulting abasic site is processed by the Ape1 endonuclease, which cleaves the phosphodiester bond on the 5 0 side thereby producing a substrate for mtDNA polymerase c. Its nucleotide incorporation prior to ligation by Author contributions: W.W.: conception and design, data collection and analysis, final approval of manuscript; P.O., Y.E., and R.S.: data collection and analysis, final approval of manuscript; M.B.: conception and design, financial support, final approval of manuscript; L.E.: conception and design, financial support, manuscript writing and final approval. Correspondence: Lars Eide, Dr. Scient, Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet, Oslo 0027, Norway. Telephone: 47-23 0710 62; Fa x: 47-2 3070 902; e -mail: lars.eide@m edisi n.ui o.no Received July 28, 2010; accepted for publication September 28, 2010; first published online in STEM CELLS E XPRESS October 15, 2010. V C AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.542 STEM CELLS 2010;28:2195–2204 www.StemCells.com

Mitochondrial DNA Integrity is Essential for Mitochondrial 2010

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