Mitochondrial DNA Integrity is Essential for Mitochondrial 2010
10
TISSUE-SPECIFIC STEMCELLS 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 differen- tiation 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 dam- age s in ogg1 2/2 cells inh ibi t the nor mal matu ration ofmitochondria 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 mi- tochondria. Aerobic metabolism increases during differen- tiation 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 mitochon- drial 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 ali- zation 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 impor- tant signaling roles in both oxygen sensing and redox balance [9, 10]. The generation of ROS is an inevitable byproduct ofelec 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 mitochon- drial 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 mitochon- drial 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 phosphodiesterbond 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
