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Original Article J Korean Geriatr Soc 2009;13(3):142-151 사람신경모세포종에서아밀로이드베타 (Aß 25-35 ) 투여후사립체기능장애와세포자멸사관련유전자들의발현 가톨릭대학교의과대학병리학교실 최영숙 김상호 Mitochondrial Dysfunction and Apoptosis Related Gene Expression in Aβ 25-35 -Treated Human Neuroblastoma Cell Line, SK-N-SH Young-Sook Choi, MS, Sang-Ho Kim, MD Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea Background: Mitochondrial dysfunction plays an important role in Aβ-induced neuronal toxicity in Alzheimer`s disease (AD). We measured the membrane potentials of mitochondria ( Ψm) and assessed the genetic expressions of Aβ 25-35 - induced neurotoxicity in the human neuroblastoma cell line, SK-N-SH cell. Methods: SK-N-SH cells were incubated with a single dose of 25 μm Aβ 25-35 for 0-24 hours, and kinetic study was done. Ψm was measured by flow cytometry. Messenger RNA expressions of cytochrome c oxidase (COX), cytochrome c, succinate dehydrogenase (SDH), amyloid-β alcohol dehydrogenase (ABAD), caspase 9, and Bcl-2 were measured by quantitative real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). Cell death rate was measured by MTT reduction assay. Results: Ψm was reduced at 24 hours. mrna expression for COX gradually decreased by about 29% (p<0.05) whileexpressions for cytochrome c, SDH, ABAD, and caspase 9 increased (p<0.05) progressively during the 24-hour time period. Bcl-2 expression decreased (p<0.05) gradually; and apoptotic cell death rate was about 24% (p<0.01) by 24 hours. Conclusion: Extracellular administration of Aβ 25-35 contributes directly to mitochondrial dysfunction in SK-N-SH cells with the enzymatic impairment of the tricarboxylic acid cycle and electron transport chain, and eventually leading to apoptotic cell death. Key Words: Amyloid beta-protein, Apoptosis, Caspase 9, Cytochrome c, Electron transport complex IV, Membrane potentials, Mitochondria 서 론 Received: July 9, 2009 Revised: Sep 23, 2009 Accepted: Sep 25, 2009 Address for correspondence: Sang-Ho Kim, MD Department of Pathology, College of Medicine, The Catholic University of Korea, Banpo-dong, Seocho-gu, Seoul 137-701 Korea Tel: Tel: 82-2-2258-7306, Fax: 82-2-537-6586 E-mail: complt@catholic.ac.kr 알츠하이머 (Alzheimer`s disease, AD) 환자의뇌는관자- 마루엽및이마엽피질이위축되어있고, 세포밖의아밀로이드판, 세포내에과인산화된 tau 단백에의한신경섬유농축체형성및신경세포의소실, 신경세포의돌기와연접의소실등의조직병리학적특징을보인다 1). AD에서 142 J Korean Geriatr Soc 13(3) September 2009

Young-Sook Choi, et al: Amyloid β-protein, Apoptosis, Caspase 9, Cytochrome c, Electron transport complex IV 신경세포사의기전은아직불분명하지만, 주로사립체의산화성인산화 (oxidative phosphorylation, OXPHOS) 의결함으로그원인이설명되어왔다. 신경변성은사립체기능이상과산화스트레스에의해발생한다 2). AD병소에서뇌신경세포의삼카르복실산회로 (tricarboxylic acid cycle, TCA cycle) 내효소활성의변화와함께사립체수가감소되고, 아밀로이드베타펩티드 (Aβ) 가직접이기능이상을유발한다 3). 아밀로이드판이형성되기전에이미세포내의 Aβ 응집물이나타나며, Aβ는세포표면, 세포질세망의내강쪽, 사립체및골지체막등에서도발견된다 1). 사립체내 Aβ가축적됨으로인하여종말전자운반쇄복합체효소인 cytochrome c oxidase (COX) 활성의억제 4), 막전위의저하, 사립체의팽창및 permeability transition pore (PTP) 의열림등이일어난다 5). 또한 Aβ는사립체에있는 ABAD에결합하면사립체효소활성을억제시켜서신경세포의세포자멸사를유도한다 6). 저산소증, 저혈당등의대사장애로인한산화스트레스는 AD의초기현상으로 AD의시작, 진행, 병인론에중요한매개자이며, 활성산소종생성에의한세포손상이나, 세포반응은신경세포에서볼수있는 AD 병리의이정표역할을담당한다 7). 사립체외막의투과성증가로사립체내막에저장되어있던시토크롬 c (cytochrome c), death-promoting protein 등이세포액내로유리된후스트레스유도세포자멸사경로 8,9) 를따라이들 cytotoxin이 apoptotic protease activating factor (Apaf) 와결합한후 caspase 9를동원하여 apoptosome을형성한다. Bcl-2는사립체외막, 세포질세망, 핵막에존재하며 bcl-xl, bcl-w, A1, Mcl-1들과함께세포자멸사억제기능을가진다고알려져있다 10). AD에서 Aβ는신경변성 cascade( 연쇄반응 ) 의핵심분자이다. 그러나 Aβ의신경세포독성에의한세포자멸사기전에관하여아직완전한설명이없다. 따라서본실험의목적은 Aβ에의하여손상받은신경세포에서 24시간이내일어나는사립체의기능이상과세포자멸사관련몇가지중요유전자들의발현정도와그발현시기를알아냄으로서신경세포소실기전을구체적으로관찰하고자하였다. 즉, 사람신경모세포종인 SK-N-SH 세포에 Aβ 25-35 를투여하여 Aβ에의한세포내사립체기능부전연쇄반응에미 치는영향을알기위하여먼저사립체막전위의변동을확인한후, 사립체의시토크롬 c와그효소인 COX, TCA cycle 내효소 SDH, 아밀로이드베타결합효소 ABAD 및세포자멸사에관여하는 caspase 9와 Bcl-2 유전자를선택하여, 이들의시간별발현정도를실시간역전사중합효소반응 (real-time RT-PCR) 으로정량화하였다. 또한이때세포생존율도검사하였다. 그결과를토대로궁극적으로 AD 발생초기에세포밖 Aβ매개신경세포독성에의한신경세포사의기전에관련된정보를얻고자하였다. 대상및방법 1. 세포배양 세포는사람신경모세포종인 SK-N-SH (ATCC; HTB-11) 세포주 (Korea Research Institute of Bioscience and Biotechnology, Taejeon, Korea) 를사용하였다. 배지는 2mM L-glutamine, 1.5 g/l sodium bicarbonate, 0.1 mm non-essential amino acid, 1 mm sodium pyruvate를포함한 Eagle's Minimum Essential Medium (Whittaker, Walkersville, MD, USA) 에 100 IU/mL penicillin-streptomycin (Gibco BRL, Grand Island, NY, USA) 과불활성화시킨 10% 우태아혈청 (Gibco BRL) 을첨가하여사용하였고, 세포는 37, 5% CO 2 배양기에서배양하였다. 2. Aβ 25-35 투여 SK-N-SH 세포의농도를 6 well culture plate에서 well 당 3 10 5 개로유지한후 Aβ 25-35 (Bachem, Bubendorf, Switzerland) 는최종 25 μm 농도로세포배양액에첨가한다음 0-24시간까지각시간별로배양하였다. 이세포들로부터 total RNA를분리하여해당유전자발현의증감을정량하였다. 3. 사립체의막전위측정사립체막전위 ( Ψm) 변동은 flow cytometer로측정하였다. SK-N-SH세포에 Aβ 25-35 를첨가하여 24시간세포배양을한후, 제조사의지침서에따라 JC-1 (10 μg /ml, Mole- J Korean Geriatr Soc 13(3) September 2009 143

최영숙외 : 아밀로이드베타 (Aβ 25-35) 투여후사립체기능장애와관련유전자들의발현 cular Probes, Eugene, OR, USA) 로 37 에서 10분간염색한다음 flow cytometry FASCan (BD Bioscience, San Jose, CA, USA) 으로 Ψm 을측정하였다. Excitation과 emission 파장의조건은각각 544 nm와 590 nm이었다. JC-1은막전위에의해사립체에축적되는양이온염료이며, 사립체로들어가면단량체의형태 ( 녹색형광, R4) 에서집합체의형태 ( 적색형광, R2) 로변화된다. 정상사립체에서는막전위가높아서적색형광이증가하여 R2구역에주로신호가나타나지만, 손상된사립체에서는막전위가감소하여 R2구역에서녹색형광을나타내는 R4구역으로신호가이동한다. Flow cytometer로분석한 4구역은 Ψm 의양에기초하여정하였다. 양성대조군은 valinomycin (Sigma, St. Louis, CA, USA) 을최종농도 0.5 μm로 24시간반응하여측정하였다. 4. 각종유전자 mrna 발현과실시간역전사중합효소반응 (real-time reverse transcriptase polymerase chain reaction, realtime RT-PCR) 6가지유전자들, 즉 COX, 시토크롬 c, SDH, ABAD, caspase 9 및 Bcl-2 각각의 mrna 발현을정량하였다. 먼저 24 시간까지각유전자의발현정도를비교한다음 0-24시간까지시간별로유전자발현정도를정량분석하였다. Real-time RT-PCR을위하여 Aβ 25-35 와반응한 SK-N-SH 세포에서 Trizol (Invitrogen, Carlsbad, CA, USA) 을사용하여 total RNA를분리하였다. Transciptor first strand cdna syn- Table 1. Primers and conditions used in the real-time reverse transcriptase PCR Gene Primer sequence (5' to 3') COX II S-AAC TAT CCT GCC CGC CAT CAT C AS-ATG GTA AGG GAG GGA TCG TTG AC Cytochrome c S-GCT CTT TTG GCA ATC CGT CAT CAG AS-TGG GAG ACA AAG TTT CTG GTC AGG SDH S-AAC ACT GTT GTT GCC ACA GG AS-AGG TCC TGG CAA GGA AGG ABAD S-GCC CAA GCC AAG AAG TTA GGA AAC AS-ACA GTT GAC AGC TAC ATC CAC ACG Caspase 9 S-TCA GGC CCC ATA TGA TCG AS-GAC TCC CTC GAG TCT CCA GAT Bcl-2 S-GTC TGG GAA TCG ATC TGG AAA TCC AS-TTT GAA ACT TCC CAA TGA ATC AGG AG GAPDH S-CCA TGT TCG TCA TGG GTG TGA ACC A AS-GCC AGT AGA GGC AGG GAT GAT GTT C S, sense primer; AS, antisense primer. Product size (bp) GeneBank accession# Anneal ( ) 96 X55654 60 136 NM018947 60 119 NM004168 55.7 129 NM004493 60 99 NM032996 55 78 NM000657 60 251 NM008084 144 J Korean Geriatr Soc 13(3) September 2009

Young-Sook Choi, et al: Amyloid β-protein, Apoptosis, Caspase 9, Cytochrome c, Electron transport complex IV thesis kit (Roche, Mannheim, Germany) 로 cdna를합성한후, 50 ng cdna를 iq5 SYBR Green supermix (Bio-Rad, Hercule, CA, USA) 와각각의 primer ( 최종농도 5pmol/uL) 와함께혼합하여 real-time PCR machine (iq5, Bio-Rad) 을이용하여각각의유전자 mrna 발현정도를측정하였다. Primer는 Beacon designer 프로그램 (Bio-Rad) 을사용하여설계 하였고, 코스모진텍 (Seoul, Korea) 에의뢰하여합성하였다. Primer의반응조건은 Table 1에제시하였다. 5. 세포생존율검사 SK-N-SH세포의세포생존율검사는 tetrazolium salt (MTT) Fig. 1. Effects of Aβ 25-35 on mitochondrial inner transmembrane potential ( Ψm). Ψm was measured in human neuroblastoma cell line, SK-N-SH cells (3 10 5 /well), treated with 25 μm of Aβ 25-35 at 37 for 0-24 hours, followed by staining with the fluorochrome, JC-1 (10 μg /ml), at 37 for 10 min. 0.5 μm of valinomycin, a K + ionophore, was used as positive control. Flow cytometric analysis was done with FACScan. Four regions depicted were differentiated by color based on magnitude of Ψm. Briefly, cells with high membrane potentials indicated R2 and cells with low membrane potentials indicated R4. Note the inhibition of mitochondrial respiration by Aβ 25-35. J Korean Geriatr Soc 13(3) September 2009 145

최영숙외 : 아밀로이드베타 (Aβ 25-35) 투여후사립체기능장애와관련유전자들의발현 quantitative colorimeteric assay 방법으로하였다. 즉, 5 10 4 개세포에 Aβ를첨가한후, 0-24시간배양한다음배양액을제거하고, 0.5 mg/ml MTT (Sigma) 를첨가하여 37 에서 2시간방치하였다. MTT 시약을제거하고, 세포를 DMSO 에녹인후, microplate reader (molecular device, Sunnydale, CA, USA) 로파장 570 nm에서흡광도를측정하여세포생 존율을얻었다. 6. 통계처리 Kinetic study는 4회반복실험을한후그값을평균 ± 표준오차로표기하였고, unpaired Student t-test를사용하여통 Fig. 2. The kinetics of mitochondrial function-related genetic expression in Aβ 25-35 -treated SK-N-SH cells. Cells (3 10 5 /well) were incubated with 25 μm of Aβ 25-35 or medium alone at 37 for 0-24 hours. Total RNA was prepared. Messenger RNA expression of mitochondrial chain complex activity-related genes including COX, cytochrome c, SDH, ABAD, and apoptosis-related genes including caspase 9 and Bcl-2 were measured by real-time RT-PCR. Each column represents mean ±SE for four independent experiments compared with zero hour (control). COX, cytochrome c oxidase; SDH, succinate dehydrogenase; ABAD, amyloid-β alcohol dehydrogenase. *p<0.05 versus control. Statistical analysis was performed by Student's t-test. 146 J Korean Geriatr Soc 13(3) September 2009

Young-Sook Choi, et al: Amyloid β-protein, Apoptosis, Caspase 9, Cytochrome c, Electron transport complex IV 계분석을하였으며, p value는 0.05 이하를의의있다고판정하였다. 결 과 1. 사립체의막전위측정 SK-N-SH 세포에 25 μm의 Aβ 25-35 투여후 24시간째의 Ψm 을검사한결과, 대조군에비하여 R2는전체세포수의 70% 에서 56.7% 로감소하였고, R3과 R4는각각전체세포수의 3.9% 에서 16.9% 로 0.3% 에서 1.2% 로의증가를보여 Aβ 25-35 의사립체호흡억제현상을확인할수있었다 (Fig. 1). 양성대조군으로 K + 이온운반체인 valinomycin을사용하여사립체의막전위가완전히감소되는것을확인하였다. 2. 각유전자의발현정도 SK-N-SH 세포에 25 μm의 Aβ 25-35 투여후 kinetic study로 0-24시간까지각시간별유전자발현정도를정량하였다. 그결과 COX는대조군 (1을기준 ) 에비해 4, 8, 12, 24시간배양시각각 0.85, 0.81, 0.76, 0.71로 24시간까지지속적으로감소하여최대 29% 감소 (p<0.05) 하였고, 시토크롬 c는 4, 8, 12, 24시간배양시대조군에비해각각 1.1, 1.21, 1.23, 1.05로 12시간에서는 23% 의증가 (p<0.05) 를보였으나 24시간에서는회복하였다. SDH는 4, 8, 12, 24시간배양시대조군에비해각각 1.09, 1.16, 1.22, 1.31로 24시간에서최대 31% (p<0.05) 증가하였고, ABAD는 4, 8, 12, 24시간배양시대조군에비해각각 1.16, 1.25, 1.28, 1.36으로 24시간까지최대 36% 의증가 (p<0.05) 를보였다. 세포자멸사관련유전자중 caspase 9는 4, 8, 12, 24시간배양시각각 1.14, 1.35, 1.39, 1.43으로대조군에비해발현이증가하여 24시간에서최대 43% 의증가 (p<0.05) 를보였으나, Bcl-2는 4, 8, 12, 24시간배양시각각 0.93, 0.92, 0.91, 0.81로대조군에비해 24시간까지지속적으로감소하여최대 19% 의감소 (p<0.05) 를보였다 (Fig. 2). 3. 세포생존율 SK-N-SH세포에 25 μm의 Aβ 25-35 투여후 kinetic study로 Fig. 3. Cytotoxic effects of Aβ 25-35 on SK-N-SH cells. Cells (5 10 4 /well) were incubated with 25 μm of Aβ 25-35 or medium alone for 24 hours. Cell viability was estimated by MTT reduction assay. Each column represents mean±se for four independent experiments compared with zero hour (control). *p< 0.01 versus control. Statistical analysis was performed by Student's t-test. 0-24시간까지각시간별로세포독성효과를 MTT reduction assay로검사한결과최대 24%(p<0.01) 의세포사를확인하였다 (Fig. 3). 고 찰 불용성섬유형태의 Aβ 1-42, Aβ 1-43 은 Aβ 전구단백이 β- secretase와 γ-secretase에의하여분해되어생긴다. 이 Aβ는뇌신경세포에산화스트레스, 사립체기능이상, 연접전달의억제, 세포막통합성 (integrity) 의파괴및축삭운반장애등에직접관여한다고알려져있다 3). 사립체는 Krebs cycle-호흡쇄-산화성인산화경로를통하여 ATP를생산한다. 호흡쇄는사립체내막에위치한 4 개전자운반쇄 I, II, III, IV (COX) 효소복합체와복합체 V (H + ATP synthase) 로구성되어있고, 전자의이동을통하여에너지를생산한다. 작은전자운반체인시토크롬 c의 85 % 는 tubular cristae라고부르는사립체내막에있고, 15-20 % 는 inter membrane space에존재한다 11). 전자운반쇄는활성산소종생성에의한산화스트레스의중요한원천이며, 동시에세포자멸사경로의중요조절자이기때문에신경세포의생존또는세포사에중추적역할을한다. J Korean Geriatr Soc 13(3) September 2009 147

최영숙외 : 아밀로이드베타 (Aβ 25-35) 투여후사립체기능장애와관련유전자들의발현 지연발현산발형 AD의발생기전으로최근에사립체연쇄반응가설이제시되었다. 이는 AD 환자에서사립체기능이상이먼저오고, 이것이신경세포연접의변성및신경섬유농축체형성의원인이된다는설이다 12,13). 따라서 AD의초기는사립체내호흡쇄복합체의기능결핍을동반한사립체기능이상과산화스트레스에따른세포자멸사가중요병인이며, 노화는이를악화시키고, AD의말기에는아밀로이드판과세포내신경섬유농축체형성의병변을보인다 2). 사립체는 AD 신경세포에서 Aβ의축적장소이며, 이 Aβ 때문에활성산소종생성과그에의한산화손상이초래된다고하였다 14). 사립체에서 Aβ 단량체와소중합체 (oligomer) 의위치는내막또는바탕질에있으며, 세포밖보다도세포내의 Aβ가먼저관찰되고, 병변이진행되면서세포밖으로축적되며, 이들 Aβ들은모두외부에서유입된것이아닌가추측된다 15). 동물실험에서세포내로들어간 Aβ 25-35 나 Aβ 1-42 는사립체내막을손상시켜 COX가감소하였고, 호흡쇄에서계속적인전자유출로활성산소종이생산된다. 이활성산소종은다시사립체를표적으로 ATP 생산저하와사립체내, 외막을연결하는 channel 인 PTP 형성을통하여결국세포자멸사가온다고하였다 16,17). 세포내칼슘항상성유지는사립체의중요기능중하나이며, 신경세포내 Aβ 응집자체또는사립체내로과량의칼슘의유입은사립체기능부전과세포자멸사를초래한다 18). 본실험에서 Aβ 25-35 를사용한이유는 Aβ 25-35 는 Aβ 1-42 의대체물질로, 숙성이필요없이바로응집되어 β-pleated sheet 구조로변하며, 값도싸고, 독성도 Aβ 1-42 와매우흡사하기때문이다 19). SK-N-SH 세포주에 Aβ 25-35 를투여후 12-72시간배양한결과 COX I, II, III 유전자발현이모두저하되었고 20), 사람신경모세포종의하나인 SH-SY5Y에서 Aβ 1-42 를 0.1-12 μm 농도로 24시간배양 21) 또는 Aβ 25-35 를 10 μm, 4시간배양 22) 시세포자멸사가온다고알려져있다. 본실험결과, Aβ 25-35 에의해배양 24시간이내에사립체막전위의저하, 시간별로 COX의발현저하및 TCA cycle 내효소시토크롬 c와 SDH의발현증가등의사립체기능을직접적으로억제하는결과를보여 Aβ 25-35 가투여초기부터신경독성이있음을확인하였다. 산화스트레스 7) 나 ATP 수준이낮을경우사립체외막의 투과성증가로사립체의팽창, tubular cristae의변형및 PTP 가열려시토크롬 c, apoptosis-inducing factor와같은작은분자가사립체내막에서세포액내로빠져나가며, 특히시토크롬 c (apoptotic protease activating factor-2, Apaf-2) 는 Apaf-1과결합하여 caspase 연쇄반응을활성화시킨다 10). 그러나시토크롬 c가어떻게사립체외막을관통하는지그기전은아직불분명하다 23). Aβ는사립체내막에있는시토크롬 c의 heme과직접결합하여시토크롬 c의결핍을초래한다 1). AD 환자뇌에서 COX의소단위중 mitochondrial DNA-encoded subunit II mrna가감소되어있다 24). 본실험에서도 COX의소단위중 COX II를선택하여관찰한결과이들보고들과동일한성적을얻었다. AD환자에서는 Aβ의신경세포사립체기능에대한직접억제효과로 COX 이외에 pyruvate dehydrogenase, α-ketoglutarate dehydrogenase의감소, malate dehydrogenase와 SDH의증가및 AD 환자뇌의 PET 소견에서국소포도당섭취와이용이감소되었음이알려졌고, 이러한 TCA cycle 활성도는 AD환자의임상상태와상관관계가있다 25,26). 또한 Aβ 처리기전의실패역시사립체손상과포도당저대사증및에너지결핍을초래하며, 따라서사립체는활성산소종의주된공급처이면서동시에주된표적이라고하였다 27). 사립체경로 ( 스트레스유도 ) 세포자멸사는 Apaf-1/cytochrome c 복합체에의하여 procaspase 9의동원과활성화로 caspase 3, 6, 7의활성화가일어나는경로로서, Bcl-2 family 단백에의하여조절되며, apoptosis-inducing factor (AIF) 가사립체에서나와서핵으로이동하여 DNA nuclease의활성화를일으킨다 8,9). Aβ 1-42 를사람신경세포내로 microinjection하면 p53, Bax 세포사경로에의하여신경세포의세포자멸사가일어났고, 세포밖 Aβ 1-42 보다세포내 Aβ 1-42 가 10만배더신경독성을보였으며, Bcl-2는이세포자멸사를억제하였다 28). 신경세포의세포질세망에서 Aβ를과량생산할경우세포액내에 Aβ가축적되며, 이들의일부는핵에들어가서 p53 promoter를활성화시켜 p53-의존세포자멸사를촉진하고, 이는 AD 신경변성의경로가된다고하였다 29). Follicular B-cell lymphoma (Bcl-2) family는세포자멸사내부경로의중요한세포내 checkpoint이며시토크롬 c 유리의억제 30) 와시토크롬 c의탈출조절 23) 기능이있어서 A 148 J Korean Geriatr Soc 13(3) September 2009

Young-Sook Choi, et al: Amyloid β-protein, Apoptosis, Caspase 9, Cytochrome c, Electron transport complex IV β의신경독성효과에의한세포자멸사를억제시킨다 28). 본실험에서 SK-N-SH 세포에 Aβ 25-35 투여는세포자멸사를보여서 caspase 9의발현이특히 8시간이후부터현저히증가하여 24시간째최대 43% 의증가를보였다. 이는사립체기능부전시기와비례하였다. 이때세포사망률은 MTT 검사에서배양 24시간에최대 24% (p<0.01) 에이르러사립체기능부전을동반한 Aβ의세포독성이확인되었다. 그러나 Bcl-2는서서히감소하여배양 24시간에는최대 19% 정도의발현감소를보일뿐이어서, 세포자멸사를효과적으로억제하지는못하였다. Aβ 25-35 투여후신경세포사의한가지기전으로 c-jun N-terminal kinase의활성화와 Bcl-w의 downregulation 및사립체의 second mitochondrion-derived activator of caspase 유리에의한다고보고되었다 31). Amyloid-β binding alcohol dehydrogenase (ABAD, 일명 ERAB, HSD-10) 는 dehydrogenase reductase family의하나로사립체에위치하여 Aβ와결합한다. Aβ-ABAD 복합체는사립체바탕질에서형성된후, 그자체가산화스트레스로또는직접사립체기능이상을유도하여결국신경세포에세포자멸사를초래한다고알려져있다 32). 본실험에서 Aβ 투여후 24시간까지 ABAD 유전자 mrna의지속적인발현증가가 caspase 9의발현증가와비례하는모양을보였으며, 그러나이현상이세포자멸사를직접유발하는원인 32) 으로작용할지또는궁극적으로는 Aβ의처리를위한세포의방어반응인지는알수없다. AD 환자의신경세포에서 ABAD가 up-regulation 되어있다는보고가있다 33). AD 환자의대뇌피질에서 Aβ-ABAD 복합체가검출되며, 신경세포의사립체에서전자현미경으로확인하였다. Age-matching non-demented brain에서는이복합체가매우적었으며, Aβ-ABAD 끼리의결합을막으면 AD의새로운치료전략이될수있다고하였다 6). 이상의실험결과를종합하여볼때, 세포밖 Aβ 25-35 투여자체는신경세포독성을지녀 24시간까지에서 SK-N- SH세포의 TCA cycle과전자운반쇄복합체내효소기능을방해하여사립체기능을억제함으로써세포자멸사를유도함을알수있었다. 요약 연구배경 : 사립체기능부전은알츠하이머병에서 Aβ 에 의하여유도되는신경세포독성에중요한역할을담당한다. 본연구에서는사람신경모세포종세포주인 SK-H-SH에서 Aβ 투여에의한사립체기능과관련유전자및세포자멸사유전자들의발현정도를측정하였다. 방법 : SK-N-SH에 25 μm의 Aβ 25-35 를투여한후 24시간째에사립체의막전위 ( Ψm) 변동을 flow cytometry로측정하였고, COX, cytochrome c, SDH, ABAD, caspase 9 및 Bcl- 2 유전자의발현정도를실시간역전사중합효소반응으로 0-24시간까지 kinetic study로정량하였다. MTT reduction assay로세포생존율을측정하였다. 결과 : Ψm은 24시간째에현저히감소하였다. COX mrna는 24시간까지지속적으로감소하였으며 (p<0.05), cytochrome c, SDH, ABAD 및 caspase 9 mrna는 24시간까지지속적으로그발현이각각증가 (p<0.05) 하였다. Bcl-2 의발현은 24시간까지서서히감소 (p<0.05) 하였다. 세포사망률은최대 24% 이었다 (p<0.01). 결론 : 세포밖 Aβ 25-35 투여는 SK-N-SH 세포내사립체기능을직접방해하여세포자멸사를유발함을확인할수있었다. REFERENCES 1. Vińa J, Lloret A, Vallés SL, Borrás C, Badìa MC, Pallardó FV, et al. Effect of gender on mitochondrial toxicity of Alzheimer's Aβ peptide. Antioxid Redox Signal 2007;9: 1677-90. 2. Leuner K, Hauptmann S, Abdel-Kader R, Scherping I, Keil U, Strosznajder JB, et al. Mitochondrial dysfunction: the first domino in brain aging and Alzheimer's disease? Antioxid Redox Signal 2007;9:1659-75. 3. Crouch PJ, Harding SM, White AR, Camakaris J, Bush AI, Masters CL. Mechanisms of Aβ mediated neurodegeneration in Alzheimer's disease. Int J Biochem Cell Biol 2008;40:181-98. 4. Chagnon P, Bétard C, Robitaille Y, Cholette A, Gauvreau D. Distribution of brain cytochrome oxidase activity in various neurodegenerative diseases. Neuroreport 1995;6:711-5. 5. Aleardi AM, Benard G, Augereau O, Malgat M, Talbot JC, Mazat JP, et al. Gradual alteration of mitochondrial J Korean Geriatr Soc 13(3) September 2009 149

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