Journal of Cell and Molecular Biology 10(2):21-30, 2012 Research Article 21 Haliç University, Printed in Turkey. http://jcmb.halic.edu.tr COX5B and COX2 gene expressions in Multiple Sclerosis Naeimeh SAFAVIZADEH1, Seyed Ali RAHMANI 1, Mohamad ZAEFIZADEH2* 1 Department of Science, Ahar Branch, Islamic Azad University- Ahar- Iran. 2 Department of Science, Ardabil Branch, Islamic Azad University- Ardabil- Iran. (* author for correspondence; [email protected]) Received: 8 June 2012; Accepted: 14 August 2012 Abstract Multiple Sclerosis (MS) is an autoimmune inflammatory disease which affects the Central Nervous System (CNS) and leads to the destruction of myelin and atrophy of the axons. Genetic factors, in addition to environmental ones, seem to play a role in MS. Numerous studies have reported mitochondrial defects including a reduction in COX complex function related to the decrease of mitochondrial gene expression in the cortex tissue of MS patients. This study aimed to assess COX5B and COX2 gene expression in MS patients and controls. By using Real-Time PCR method, expression levels of the COX5B and COX2 were determined, with reference to ß-actin and GAPDH. The results showed that COX5B gene expression is significantly reduced in MS patients compared to control (P<0.05), whereas there was no significant difference in the COX2 gene expression. Thus, it can be claimed that down-regulation of mitochondrial electron transport chain genes supported the hypothesis that hypoxia-like tissue injury in MS may be due to mitochondrial gene expression impairments. Keywords: Multiple Sclerosis, Mitochondria, COX5B, COX2, Real-Time PCR Özet Multipl Sklerozda COX5B and COX2 gen ekspresyonu Multipl Skleroz (MS) Merkezi Sinir Sistemini (MSS) etkileyen, akson atrofisi ve miyelin yıkılımına neden olan otoimmün bir hastalıktır. Çevresel faktörlere ek olarak genetik faktörlerin MS’de rol oynadığı görülmektedir. Pek çok çalışma MS’li hastaların korteks dokularındaki mitokondriyal gen ekspresyonunun azalmasıyla ilişkili COX kompleks fonksiyonundaki azalmayı kapsayan mitokondriyal defektleri bildirmiştir. Bu çalışma MS hastalarında COX5B ve COX2 gen ekspresyonlarını değerlendirmeyi ve onları kontrollerle karşılaştırmayı amaçlamıştır. Real-Time PCR metodu kullanılarak COX5B, COX2, genlerinin ve ß aktin ve GAPDH referans genlerinin ekspresyon düzeylerini belirledik. Elde edilen veriler referans genlerle standardize edilmiş ve Excel ve SPSS programlarıyla bağımsız örnek t-test kullanılarak analiz edilmiştir. Sonuç olarak kontrol ve hastalar arasında COX2 gen ekspresyonunda anlamlı farklılık bulunmazken MS hastalarında kontrole kıyasla anlamlı olarak azalmış COX5B gen ekspresyonunu gösterilmiştir (P<0,05). Böylece; mitokondriyel elektron transport zincir genlerinin aşağı düzenlenmesinin, MS’de hipoksi benzeri doku hasarının mitokondriyel genler ve farklı ekspresyon azalmalarındaki farklılıklar nedeniyle olabileceği hipotezini desteklediği iddia edilmektedir. Anahtar Kelimeler: Multipl Skleroz, Mitokondri, COX5B, COX2, Real-Time PCR. 22 Naeimeh SAFAVIZADEH et al. Introduction through Cu (A) and heme as cofactors, to the binuclear center buried inside subunit I Multiple Sclerosis (MS) is an inflammatory and composed of heme a and Cu (B), where demyelinating disease of the central nervous system 3 the incoming four electrons together with with axonal degeneration (Frohman et al., 2006). four protons from the matrix are sequentially The loss of myelin in MS may be the result of used for oxygen reduction. This exergonic direct damage to myelin through immune mediated redox reaction is coupled with proton processes and dysfunction of oligodendrocytes pumping across the inner mitochondrial (Vercellino et al., 2009). Approximately 15–20% membrane, but the coupling of the two of MS patients have a family history of MS, but processes (H+/e- stoichiometry) can be large extended pedigrees are uncommon, with most modulated. In addition to Mitchell’s MS families having no more than two or three chemiosmotic theory, a “second mechanism affected individuals. Studies in twins (Calabresi, of respiratory control” has been proposed 2007) and conjugal pairs indicate that much of this that involves the binding of adenine familial clustering was the result of shared genetic nucleotides to nuclear-encoded COX risk factors, while studies of migrants subunits. The key event is the (Ramagopalan et al., 2010) and apparent epidemics phosphorylation of subunit IV. Activity of (Koch et al., 2008) indicated a clear role for phosphorylated COX is regulated by environmental factors. Mitochondrial defects are ATP/ADP ratio and respiratory rate is known to occur during aging, cancer, heart disease, precisely controlled according to the ATP and a wide variety of degenerative diseases, such as utilization. The membrane potential is kept Alzheimer’s disease (AD), Parkinson’s disease low (100-150 mV) and COX works at high (PD), Huntington’s Disease (HD) and MS efficiency of proton translocation (H+/e-= 1). (Henchcliffe et al., 2008). Mitochondria contain the The COX biosynthesis and assembly is respiratory chain where energy in the form of ATP timely and complicated process involving is most efficiently produced (Damiano et al., 2010). several rate-limiting steps reflecting the The mitochondrial respiratory chain is located in sequential incorporation of the subunits the inner mitochondrial membrane and consists of from either the cytosol (nuclearly coded four complexes (complexes I–IV), whilst complex subunits) or from the mitochondrial matrix V is directly involved in ATP synthesis. The (subunits I, II and III). The majority of COX complexes of the mitochondrial respiratory chain defects thus originate from mutations in include multiple subunits; all but complex II (which nuclear genes (Acin-Perez et al., 2009). is entirely encoded by nuclear DNA) contain Mutations in the genes encoding several proteins encoded by nuclear and mitochondrial COX assembly factors have been described DNA (mtDNA). The final respiratory chain as a frequent cause of mitochondrial complex [complex IV or cytochrome-c oxidase diseases and have been assigned with (COX)] is the site at which over 90% of oxygen is specific clinical symptoms. The dysfunction consumed. This complex is also involved in proton of COX in these cases is mostly caused by pumping, essential for ATP synthesis (Alston et al., structural changes rather than by the changes 2011). The mammalian COX is composed of 13 in amount of the enzyme (Galati et al., subunits, of which the three largest are encoded by 2009). Cytochrome-c oxidase subunit II, the mtDNA and form the catalytic core of the abbreviated as CoxII, is the second subunit enzyme. The remaining ten, evolutionary younger, of cytochrome-c oxidase subunit 2 (CO II) nuclear-encoded subunits are involved in assembly transfers the electrons from cytochrome-c to and regulation of the enzyme (Zee et al., 2006). the catalytic subunit 1. It contains two The function of mammalian COX can be adjacent transmembrane regions in its N- physiologically modulated and the enzyme terminus and the major part of the protein is represents one of the key regulatory sites of energy exposed to the periplasmic or to the metabolism (Fernandes-Vizarra et al., 2009). COX mitochondrial intermembrane space, transfers electrons from cytochrome-c to molecular respectively. CO II provides the substrate- oxygen, which is reduced to water. The electrons binding site and contains a Cu centre called pass from cytochrome-c, binding at subunit II, Cox Expression in MS 23 Cu(A), probably the primary acceptor in RNA Extraction cytochrome-c oxidase. An exception is the Total RNA was isolated by using a corresponding subunit of the cbb3-type oxidase RNAeasy kit from Roche (Germany), which lacks the Cu(A) redox-centre. Several according to the manufacturer’s bacterial CO II have a C-terminal extension that recommendations. The RNAsamples were contains a covalently bound heme c (Barrientos et incubated with RNAse-free DNAse I at al., 2009). Subunit Vb of mammalian cytochrome c 37°C for 15 min. RNA samples were oxidase is encoded by a nuclear gene and purified with a RNAeasy kit. It is possible to assembled with the other 12 COX subunits encoded preserve extracted RNA for months under in both mitochondrial and nuclear DNA. This gene −80 °C. Total RNA quality and quantity located on chromosome 2, region cen-q13 were assessed in 2 ways. In the first method, (Williams et al., 2005). There is a common we estimated the RNA concentration by symptom of MS with mitochondrial diseases such ultraviolet absorbance at 260 nm (1 as AD and PD, and also the point mutations in absorbance unit at 260 nm = 40 ng/μL RNA) mtDNA can cause damage to the myelin (DiMauro and the RNA purity by measuring the ratio et al., 2008). In this study, we compared COX5B of absorbance at 260 nm and 280 nm (1.8 < and COX2 gene expression among MS patients A /A <2.1 for pure RNA). Total RNA with controls. In regard to the importance of 260 280 was run on 1% agarose gels to check size mitochondria in MS, we mainly centralized our and integrity. The quality of RNA was study on quantifying the expressions of COX5B confirmed by the detection of 18S and 28S and COX2 using Real-Time PCR. bands after 1% agarose gel electrophoresis. Materials and Methods Primer synthesis Subjects Primer sequences were designed using Thirty-six patients (16 male, 20 female) and 30 Primer3 software for COX5B controls (14 male, 16 female) took part in this (NM_001862.2), COX2 (NC_012920.1) and