원저 인간신경줄기세포 HB1.F3 에서 alpha-synuclein 과 synphilin-1 유전자전이를통한봉입체형성과세포자멸사 동아대학교의과대학신경과, 아주대학교뇌질환연구센터 a 천상명이광 a 김재우김승업 a Inclusion Body Formation and Apoptotic Cell Death in the Human Neural Stem Cells HB1.F3 Following Gene Transfection of Alpha-Synuclein and Synphilin-1 Sang-Myung Cheon, M.D., Gwang Lee, Ph.D. a, Jae Woo Kim, M.D., Seung Up Kim, M.D. a Department of Neurology, College of Medicine, Dong-A University, Busan; Brain Disease Research Center, Ajou University a, Suwon, Korea Background: The etiology of Parkinson s disease (PD) has not been established, but familial forms of the disease have some clues for its pathogenesis. Autosomal dominantly inherited familial PD induced by aberrations of the alpha-synucein gene has been known as a genetic model of PD and sheds light to the understanding of PD pathogenesis. Synphilin-1 is a protein which interacts with alpha-synuclein and constitutes the Lewy body. Methods: Immortalized human neural stem cells were transfected with the alpha-synuclein gene and synphilin-1 gene, to define the role of Lewy body inclusions in neuronal cell death. Results: Human neural stem cells with Lewy body-like inclusions showed an increased apoptotic cell death compared to those with diffuse alpha-synuclein-positive and synphilin-1-positive reaction after transfection with the alpha-synuclein gene and synphilin-1 gene. Tyrosine hydroxylase over-expressing cells produced a high level of levodopa and showed a higher rate of the apoptotic marker. Conclusions: These results suggest that the formation of Lewy body-like inclusions by the over-expression of alpha-synuclein and synphilin-1 could be an underlying cause of apoptotic neuronal cell death and the dopaminergic cell might be more susceptible. J Korean Neurol Assoc 25(3):344-352, 2007 Key Words: Parkinson s disease, Alpha-synuclein, Lewybody, Levodopa, Apoptosis 서론 파킨슨병 (Parkinson s disease) 은대표적인신경계퇴행성 Received January 19, 2007 Accepted March 9, 2007 *Jae Woo Kim, M.D. Department of Neurology, College of Medicine, Dong-A University 1, 3-ga, Dongdaesin-dong, Seo-gu, Busan, 602-715, Korea Tel: +82-51-240-5266 Fax: +82-51-244-8338 E-mail: jwkim@mail.donga.ac.kr * 이논문은 2004 학년도동아대학교학술연구비 ( 공모과제 ) 에의하여연구되었음. 질환의하나로서동, 강직, 진전, 보행장애등의운동증상을주증상으로하고중뇌흑색질의도파민성신경세포의변성과소실, 신경세포질내의루이체 (Lewy body) 가특징적인병리소견이다. 1 대부분특발성으로발병하게되고다양한유전적, 환경적요인등이원인으로추정되고있으나현재까지뚜렷한병인이밝혀지지는않았다. 2 특발성발병이대부분이지만일부는유전자이상으로인해가족성으로발병하게되는데원인유전자로는 alpha-synuclein, parkin, ubiquitin carboxy-terminal hydrolase-l1, DJ-1 등이알려져있다. 3-6 이가운데 alpha-synuclein 유전자이상 344
인간신경줄기세포 HB1.F3 에서 alpha-synuclein 과 synphilin-1 유전자전이를통한봉입체형성과세포자멸사 으로인한가족성파킨슨병은병리소견을포함하여대부분의특발성질환의특징을가지고있어파킨슨병의병태생리를이해하는중요한병리기전으로주목받고있다. 7 루이체를구성하는중요성분이 alpha-synuclein 이고이단백질의이상이루이체를형성하고신경세포손상과연관된다는기존의가정에반해서, 최근에는세포보호를위한기제로루이체가만들어진다는연구결과가보고되고있다. 8,9 즉정상적으로세포질내에용해되어존재하는 alpha-synuclein 등의단백질이과발현되거나돌연변이, 혹은산화손상등에의해응집된형태로변하게되고이들의축적으로인한세포독성을막기위해불필요한단백질제거기제인 ubiquitine-proteosome system 이작동하여이들을격리혹은제거시키고, 이러한과정의산물이루이체라는것이다. 10,11 그러나이에대한뚜렷한결론은아직내려지지않았다. 또한파킨슨병에서루이체가중뇌흑색질의도파민성신경세포뿐아니라뇌간, 대뇌피질, 척수그리고말초신경에서도발견되기는하지만질환초기에선택적이며우선적으로중뇌에서발견되고중뇌의도파민성신경세포의소실정도가운동증상의발현을예측할수있다고알려져있는데, 12 모든신경세포에존재하는 alpha-synuclein 이유독중뇌의도파민성세포에루이체를형성하고세포자멸사를유발하는이유에대해서는명확히밝혀져있지않다. 비록도파민이대사과정에서산화되어다양한반응성산소화합물을생성해내고이에의한산화손상이도파민성신경세포를먼저손상시킨다는가설이있고도파민으로인한직접적인세포손상을확인한보고도있으나, 13-15 이는다양한손상기전에대한일반적인설명일뿐 alphasynuclein 이라는특정단백질과의연관성을설명하기에는미흡하다. alpha-synuclein 과반응하는단백질로밝혀진 synphilin-1 은 alpha-synuclein 과같이시냅스전신경말단에존재하며루이체를구성하는한요소이다. 16,17 alpha-synclein 과 synphilin-1 유전자를동시에전이시켜과발현시킨경우세포내두단백질이같은위치에존재하는것이면역염색을통해밝혀졌고루이체와같은세포내봉입체생성이증가한다고알려져있다. 18,19 이에아직확립되지않은루이체병리소견과세포자멸사와의연관성을밝히고도파민성신경세포에서이러한연관성이어떻게변화하는지알아보고자 alpha-synuclein 과 synphilin-1 유전자를이용하여본연구를수행하였다. 사용된인간신경세포주는 v-myc 로불사화된인간신경줄기세포주이며여기에 human tyrosine hydroxylase 유전자로형질전환된세포주에서도파민을생성하는지확인하여도파민성세포로 간주하고이두세포주를비교하여도파민성세포에서세포자멸사의변화를확인하고자하였다. 그리고 alpha-synuclein 과 synphilin-1 유전자를전이시킨뒤면역염색을통하여유전자발현을확인하고루이체와같은봉입체형성과세포자멸사를관찰하여연관성을살펴보고, 두세포주를비교하여도파민성세포에서봉입체형성과세포자멸사의관계를확인하고자하였다. 대상과방법 1. 세포배양 이연구에서는 15주된인간태아종뇌 (human embryonic telencephalon) 에서얻은일차세포를배양하여 v-myc oncogene 으로형질전환하여불사화된신경줄기세포 (neural stem cell) 를이용하였다. 19-21 이세포주가운데하나의클론인 HB1. F3는신경줄기세포에특이적인 nestin 과 vimentin 을발현하고신경세포 (nerve cell) 와신경교세포 (glial cell) 의표지도포함하고있다. 19,22 여기에 human tyrosine hydroxylase gene (hth) 으로형질전환시킨 HB1.F3.TH 를 HB1.F3 와함께사용하였다. 23 두세포주를 10% fetal bovine serum이포함된 Dulbecco s Modified Eagle Medium (DMEM, Gibco-BRL, Gathersberg, MD, USA) 에서배양하였고 10 μm의 5,6,7,8,- Tetrahydrobiopterin (BH 4, Sigma, St. Louis, MO, USA) 을첨가하여세포성장과형태, 배양시간등이두세포사이에서차이가없음을확인한후이를이용하였다. 2. 유전자전이사용한유전자는 Lee 등의연구에서제작한인간 alphasynuclein 유전자와 N-termminal FLAG tag synphilin-1 유전자를사용하였다. 24 HB1.F3 와 HB1.F3.TH 두세포에정상인간 alpha-synuclein 유전자와 synphilin-1 유전자를 Super- Fect Transfection Reagent (Qiagen, Valencia, CA, USA) 를이용하여제조사의방법에따라일시적유전자전이 (transient transfection) 를수행하였다. 3. 레보도파 (Levodopa) 와도파민의측정두세포주에서 BH 4 를첨가하기전과첨가한후의레보도파와도파민을측정하였다. 배양액은세포를 2일동안배양한뒤 alumina (180 mg/ml, Sigma) 와섞어 4 에서하루동안반응 J Korean Neurol Assoc Volume 25 No. 3, 2007 345
천상명이광김재우김승업 시켰다. Sample buffer (0.1 M perchloric acid, 0.1 mm EDTA) 400 μl으로세척분리하고 0.22 μm filter 로걸러냈다. 세포부분의분석을위해세포를긁어모은뒤, 400 μl의 sample buffer 와혼합하여분쇄시켰다. 이를원심분리한뒤상층액을 0.22 μm filter 로걸러냈다. 이렇게정제된배양액과세포부분을 Reverse-Phase HPLC (Waters, Milford, MA, USA) system 에각각 10 μl 주사하고, 0.5 ml/min 으로 mobile phase (0.07 M sodium phosphate monobasic, 1 mm sodium octanesulfonic acid, 0.1 mm EDTA, 8% acetonitrile, ph3.4) 를흘려주었다. Column 은 HR-80 RP-C18 (Esa, Chelmsford, MA, USA) 을이용하였으며, 400 mv에서 coulchem II ecletrochemical detector (Esa) 로분석하였다. 4. Western blot analysis 배양된세포를 Phosphate buffered saline (PBS) 로세척하고 lysis buffer (0.1% SDS, 1.0% Triton X-100 and 1.0% Deoxycholate in PBS containing 1 mm DTT, 1% protease inhibitor cocktail (Sigma), 1 mm PMSF, 1 mm Na 3VO 4, 1 mm NaF and 1 mm -glycerophosphat) 를첨가하여용해시켰다. 이용해물을 4 에 30 분간정치시킨후 13000 rpm 으로 20분동안원심분리하여상층액을취한다. 취해진상층액을 Protein assay kit (Bio-Rad) 를이용하여단백질농도를측정한다음동일한양의단백질을사용하여실험을수행하였다. 100 에서 8분동안 denaturation 시킨뒤 SDS-plyacrylamid gel (SDS-PAGE) 을사용하여분리하고 Polyvinylidene fluoride (PVDF) membrane (Millipore, Boston, MA, USA) 로옮겼다. 이를 1시간동안상온에서 blocking buffer (5% skim milk, and 0.1% Tween 20 in PBS) 를처리한뒤 4 에서 rabbit anti-tyrosine hydroxylase antibody (Chemicon, Temecula, CA, USA), monoclonal mouse anti-alpha-synuclein antibody (Sigma), monoclonal rabbit anti-flag antibody (Sigma) 의일차항체로각각 16시간반응시켰다. 반응이끝난 membrane 은 wash buffer (PBS with 0.1% Tween 20) 로세척한다음상온에서 1시간동안 Hoarseradish peroxidaseconjugated anti-mouse, anti-rabbit immunoglobulin G (Amersham Pharmacia) 의 2차항체와각각반응시킨후 ECL kit (Amersham Pharmacia, Uppsala, Sweden) 에반응시켜 Luminescent image analyzer system (Fujifilm, Tokyo, Japan) 으로관찰하였다. 5. 면역염색 세포를 ECM gel (Sigma) 이도포된 Aclar plastic coverslip 에서배양한다음 4% formaldehyde 로상온에서 30분간고정하였다. PBS 로세척한뒤 0.3% Triton X-100 와 0.1% bovine serum albumin, 10% normal goat serum으로 permeabilization 과 blocking 한다음다시 PBS 로세척하고 rabbit antityrosine hydroxylase antibody (Chemicon), monoclonal mouse anti-alpha-synuclein antibody (Sigma), monoclonal rabbit anti-flag antibody (Sigma) 의일차항체로 4 에서 16시간동안각각반응시켰다. 핵을확인하기위해 4,6-diamino-2-phenylindole (DAPI, Sigma) 로 5분동안염색하였고세포자멸사를확인하기위해서는 TUNNEL kit (Takara, Otsu, Japan) 을사용하여제조사의방법에따라염색하였다. 다시세척한후상온에서 1시간동안 fluorescein-conjugated goat anti-mouse, anti-rabbit immunoglobulin G (Molecular Probe, Eugene, OR, USA) 로반응시킨후 Crystal-mount (Biomeda) 를사용하여 glass silde 에올린다음형광현미경 (Carl Zeiss) 으로관찰하였다. 결과 1. Tyrosin hydroxylase 의발현 이연구에서불멸화된인간신경줄기세포주 HB1.F3 와여기에 human tyrosin hydroxylase (TH) 유전자를삽입시켜형질전환시킨 HB1.F3.TH 를실험에사용하였다. 두세포주가성장속도와형태에있어차이가없음을확인하고 HB1.F3.TH 에서 TH가발현되는가를확인하기위한실험을수행하였다. 단백질수준의발현을확인하기위해 western blot assay 를시행한결과 60-62 kda 의띠가 HB1.F3.TH 에서확인되었고 HB1.F3 에서는관찰되지않았다 (Fig. 1-A). TH 항체를이용하여면역세포화학염색을시행한결과 HB1.F3 에서는염색이되지않았고 HB1.F3.TH 에서세포질에고르게 TH 항체로염색이되는것을확인할수있었다 (Fig. 1-B). 2. 레보도파와도파민의측정두세포주에서 HPLC 를시행하여배양액과세포내에서레보도파와도파민의양을측정하였다. 세포내에서와배양액에서도파민의양은측정하기힘들정도로소량이었고두세포주사이의차이도없었다. 생성되는레보도파의양은 HB1.F3 와 HB1.F3.TH 에서 24 시간배양액에서는 10 6 개세포에 363 pg과 921 pg이었고세포 346
인간신경줄기세포 HB1.F3 에서 alpha-synuclein 과 synphilin-1 유전자전이를통한봉입체형성과세포자멸사 Figure 1. Expression of tyrosine hydroxylase (TH) in HB1.F3.TH. (A) Western blotting analysis showed 60 kda band in HB1.F3.TH. (B) Immunostaining with anti-th antibody showed homogenous expression of TH (red) in cytoplasm of HB1.F3.TH. 내에서는 10 6 개세포에 5.8 pg 과 8.5 pg 이었다. 세포내보다배양액에서높은농도로확인되었는데이는세포내에서생산된후배양액에분비되어축적되었기때문으로보인다. 두세포주에서큰차이가없다고판단하고카테콜아민생성의보조인자인 5,6,7,8,-Tetrahydrobiopterin (BH 4) 를배양액에첨가하여보았다. 세포독성이있는 BH 4 를농도에따라배양액에첨가하여본결과 10 μm의 BH 4 가첨가된경우두세포가성장과형태에있어차이가없는것을확인하고 2시간배양액에서레보도파농도를측정하였다. HB1.F3 와 HB1.F3.TH 에서 10 6 개세포에 1.6 ng과 20.3 ng으로 10배이상차이를보여, Table 1. Inclusion body formation in human neural stem cells following gene transfection of a-synuclein and synphilin-1 Inclusion body formation a-synuclein (+) a-synuclein (+) synphilin-1 (+) & synphilin-1 (+) HB1.F3 13.6±9.2% ND 20.0±11.8% HB1.F3.TH 16.2±12.4% ND 23.5±18.5% HB1.F3.TH-BH 4* 24.4±14.8% ND 42.3±16.1% *HB1.F3.TH cells grown in BH 4-containing media, ND; Not detectable. 의미있게차이나는것으로판단하고 BH 4 를배양액에첨가하여실험을수행하고첨가하지않은경우와비교하였다. 3. Alpha-synuclein과 synphilin-1의발현두세포주를배양하여 alpha-synuclein과 synphilin-1 유전자가일시적유전자전이를통해발현되는지단백질수준에서확인하기위해시행한 western blotting assay 에서유전자삽입을하지않은대조군에서나타나지않는 40 kda 의 alphasynuclein 띠를유전자전이시킨 HB1.F3 와 HB1.F3.TH 두세포주에서관찰할수있었다 (Fig. 2-A). 두단백질에특이적인항체를이용하여면역화학적염색을시행한결과각각 10% 이하의발현율을보였으며그중 20-30% 정도에서이중으로염색되는것을확인하였다 (Fig. 2-B). 4. 봉입체 (Inclusion body) 와세포자멸사 (Apoptosis) 의확인 Alpha-synuclein 과 synphilin-1 을모두발현하여면역세 J Korean Neurol Assoc Volume 25 No. 3, 2007 347
천상명이광김재우김승업 Figure 2. Epxression of a-synuclein and synphilin-1 in HB1.F3 following gene transfection of a-synuclein and synphilin-1. (A) Western blotting analysis showed 40 kda alpha-synuclein band following gene transfection. (B) Immuostaining with anti-a-synuclein (a), antisynphilin-1 (b) antibody showed diffuse expression of alpha-synclein (green) and synphilin-1 (red), and merged image (d) showed colocalization of two proteins in cytoplasm of transfected cells. 포화학적염색에서이중으로염색되는세포가운데루이체와유사한봉입체를가진세포는 30% 미만으로적게관찰되었다. 그러나 alpha-synuclein 만을발현하는세포에비해봉입체가관찰되는비율은약 2배정도증가되어관찰되었고, synphilin-1 만발현하는세포에서는봉입체를관찰할수없었다 (Table 1). 그리고 BH 4 를배지에첨가하여레보도파를생산하는 HB1.F3.TH (HB1.F3.TH-BH 4) 와 HB1.F3 를비교해보았을때봉입체가나타나는비율이더증가되는양상이었으나통계적유의성은없었다. DAPI 염색으로핵이분절되거나응축되는염색질 (fragmented or condensed chromatin) 이관찰되거나 TUNNEL assay 를통해발색되는것을형광현미경을통해확인한경우를자멸사로보고확인한결과각세포군에서 alpha-synuclein Table 2. Apoptotic cell death in human neural stem cell lines as shown by DAPI staining and TUNNEL assay following gene transfection of a-synuclein and synphilin-1 Apoptotic cells control a-synuclein (+) a-synuclein (+) & synphilin-1 (+) HB1.F3 10.7±2.7% 9.8±1.3% 40.0±22.5% HB1.F3.TH 12.9±3.4% 8.8±2.1% 33.3±15.2% HB1.F3.TH-BH 4* 10.7±0.8% 29.4±12.2% 53.3±18.1% *HB1.F3.TH cells grown in BH 4-containing media. 과 synphilin-1 을모두발현하는경우는 alpha-synuclein 만을발현하는경우보다세포자멸사비율이높게관찰되었으며 HB1.F3 와 HB1.F3.TH 두세포주사이에는큰차이가없었으나 HB1.F3.TH-BH 4 에서는 alpha-synuclein 만을발현하는경우에도세포자멸사가관찰되는비율이높았다 (Table 2, Fig. 3) 봉입체가관찰되는세포와세포질에미만성으로염색이되는세포두군에서세포자멸사를 DAPI 염색과 TUNNEL assay 를통해비교한결과 HB1.F3.TH 와 HB1.F3, 그리고 HB1.F3.TH- BH 4 모두에서봉입체가관찰되는세포가미만성으로발현되는세포에비해세포자멸사의비율이더높았다 (Fig. 4). 고찰 도파민성신경세포의자멸사를관찰하기위해본연구에서는불사화한인간신경줄기세포 (immortalized human neural stem cell) 를사용하였다. 이세포주는계대 (passage) 및배양조건에따라서신경줄기세포 (neural stem cell) 와신경세포 (nerve cell), 신경교세표 (glial cell) 의비가달라지고조건에따른정량적인지표가정립되어있지않다는점등이단점으로지적될수있다. 그러나이러한모든세포들이포함되어있고무엇보다도한세포에서클론으로분리된세포주이므로유전적으로나세포생리학적으로균질성을가지고있어쉽게유전학 348
인간신경줄기세포 HB1.F3 에서 alpha-synuclein 과 synphilin-1 유전자전이를통한봉입체형성과세포자멸사 Figure 3. Apoptotic cell death in HB1.F3.TH-BH 4 as shown by DAPI staining and TUNNEL assay following gene transfection of a-synuclein and synphilin-1. Alpha-synuclein over-expressing cell (red in a) showed nuclear fragmentation and chromatin condensation (arrow in b) which was TUNNELpositive (green in c) and co-localized in merged image (d). 적, 생화학적조작을할수있는인간신경세포라는점에서큰장점이있다. 불사화시키기위해 v-myc 유전자로형질전환되었으나신경세포의특이적인흥분성 (excitability) 을가지고있으며여러종류의신경계세포지표를포함하는것이확인되었다. 20-22 도파민성신경세포로형질전환을위해 human tyrosine hydroxylase gene (TH) 을삽입시킨 HB1.F3.TH Figure 4. Apoptotic cell death as shown by DAPI staining and TUNNEL assay in human neural stem cells with or without inclusions following gene transfection of a-synuclein and synphilin-1. Lines indicate standard deviation. 세포주에서단백질수준과그리고면역염색에서고르게발현되는 tyrosine hydroxylase (TH) 를확인하였다. 그리고도파민과그전구물질인레보도파를측정해본결과두세포모두도파민은측정할수없을정도로생산량이적었고레보도파의경우에도극히소량이었다. TH는도파민을비롯한카테콜아민의생성에필요한속도조절효소 (rate-limiting enzyme) 이지만여러보조인자를필요로한다. 그가운데하나가 5,6, 7,8,-Tetrahydrobiopterin (BH 4) 이다. 이실험에서는 HB1.F3 와 HB1.F3.TH 두세포주의배양액에농도에따라 BH 4 를첨가하여두세포주의성장과형태가차이가없는농도를확인하고도파민과레보도파를측정한결과도파민은미량이었으나레보도파는 HB1.F3.TH 에서 HB1.F3 보다 10배이상증가되는것을확인하였다. 여기서도파민생성이적었다는것은레보도파이후필요한단계인 aromatic acid decarboxylase 효소의결핍때문으로생각한다. 그러나레보도파양은연구방법과단위의차이가있어기존의보고들과비교하기는힘들지만 TH로형질전환된다른세포주와유사한결과를보여주었다. 25,26 레보도파는도파민의직접적인전구물질로혈액-뇌장벽을통과하기때문에파킨슨병의가장중요한치료제로사용된다. 도파민이분해되면서산화되는과정중에생기는산화부산물이세포독성을유발한다는보고와함께레보도파역시세포독성을가지고세포의산화손상을유발시킬수있는것으로알려져있다. 27,28 이에본연구에서는 BH 4 을보충해주면레보도파생산이증가되는 HB1.F3.TH (HB1.F3.TH-BH 4) 를도파민성세포로간주하고이를 HB1.F3 와비교하여각세포주에서 alpha- J Korean Neurol Assoc Volume 25 No. 3, 2007 349
천상명이광김재우김승업 synuclein 과 synphilin-1 유전자에의한봉입체생성과세포자멸사를관찰하였다. Synuclein 은신경세포에용해되어존재하는단백질로정확한생리적기능은알려지지않았으나시냅스낭 (synaptic vesicle) 의재순환에기여한다는가설이지지를받고있다. 29 사람에서발견되는 synuclein 은 alpha-, beta-, gamma- 세종류가확인되었는데그중 alpha-synuclein 은알쯔하이머병환자에서발견되는 senile plaque 을이루는성분의하나로 non-amyloid component (NAC) 로도알려져있다. 3,29 이유전자는염색체 4q21.3-q22 에존재하며 2종류의점돌연변이에의해발병된가족성파킨슨병가계가확인되었다. 7 최근에는정상 alphasynuclein 의발현이증가된 triplet 으로발병된가계가발견되어돌연변이가아닌정상단백질의과발현으로도파킨슨병이발병된다는것이확인되었다. 30,31 이러한가족성파킨슨병의원인유전자라는이유외에도파킨슨병에서 alpha-synuclein 이주목받고있는이유는이질환의가장중요한병리소견인루이체 (Lewy body) 를구성하는주요성분이기때문이다. 루이체는세포질내에존재하는지름 8-30 μm 정도의봉입체로일반적인 Hematoxylin-Eosin 염색에서는분홍색으로보인다. 2 면역염색을통해루이체를구성하는성분을확인할수있게되어 neurofilament, ubiquitine, alpha-synuclein, synphilin-1, torsin A, heat shock protein 등의단백질이밝혀졌다. 17,32-34 또한 ubiquitination과 deubiquitination 효소, proteosomal subunit, proteosome activator 등진핵세포에서불필요한단백질을제거하는중요한기제인 ubiquitine-proteosome system 의구성요소도포함하고있는것이확인되었다. 35,36 전자현미경으로관찰한소견에서중앙에는과립형물질이존재하고그주변으로방사형으로 fibrillar alpha-synuclein 과신경미세섬유가구성하고있는데중앙의과립형물질은 ubiquitin 화된단백질로추정되고있다. 37 Synphilin-1 은 alpha-synuclein 과작용하며루이체를구성하는한성분으로알려져있고 alpha-synuclein 과같은제거기제인 ubiquitine-proteosome system 을통해분해된다. 16,17,38 자세한생리적역할은알려져있지않으나시냅스전신경말단에존재하여시냅스낭과결합하는역할을하며 alpha-synuclein 을시냅스낭에연결시키는고리역할을한다고보고되었고 alphasynuclein 과같이과발현시킨경우봉입체생성이증가한다고알려져있다. 16,18,39 본연구에서는루이체와유사한병리소견을얻기위해 alpha-synuclein 과 synphilin-1 두유전자를동시에과발현시켰는데전반적인유전자전이 (transfection) 효율은 10% 이하였고이가운데 alpha-synuclein 과 synphilin-1 두단백질모 두를발현하는것은 20-30% 정도로전체세포의 2-3% 였다. 두단백질모두를발현하는세포가운데세포질전체에걸쳐미만성으로염색되는경우가많고루이체와같은봉입체가확인된경우는 20% 이하로소수인데이에대한명확한설명을하기어렵지만유전자삽입후의배양시간에따른차이즉세포질에서발현된두단백질이다시봉입체를형성할만한충분한기제나시간이경과하지않았을가능성이크다. 이결과는 HEK293 세포를사용하여유전자전이효율은 30-40% 로, 그리고두단백질모두과발현되어봉입체가생성되는것은 15-20% 로보고한타연구에비해전이효율은상당히낮다고보인다. 18 그러나이러한차이는세포성상의차이로인한결과로보이며, 태아신장기원인 HEK293 세포에비해본연구에사용된뇌신경세포기원의 HB1.F3 가파킨슨병의병태생리모델로적절할것임은더언급할필요가없을것이다. 생성된봉입체는 alpha-synuclein 과 synphilin-1 모두에염색되는경우가 alpha-synuclein 만발현되는경우보다더많은비율로관찰되었고 synphilin-1 만염색되는경우에는봉입체를관찰할수없었다. 그리고유의하지는않으나 HB1. F3.TH-BH 4 에서봉입체생성비율이더증가되는양상이었다. 면역염색에서 alpha-synuclein 과 synphilin-1 에염색이되는봉입체는대부분작고다발성이었고극히일부에서핵크기의봉입체를관찰할수있었다. 그리고세포의미세구조가 HB1.F3 과 HB1.F3.TH 두세포주에서차이를보이지않았으므로배양하면서관찰하였던두세포의유사성을확인할수있었는데이는유전자전이를통한형질전환후에도큰변화없이기존의신경줄기세포의특성을유지하고있는것으로판단할수있는근거라생각한다. 봉입체가확인되는경우와미만성으로두단백질을발현하는경우에있어세포자멸사의표지를확인한결과봉입체가있는세포에서세포자멸사비율이높게관찰되었다. 루이체를 proteosome 혹은 aggresome 으로보고이는세포보호를위한기제라는것이최근지배적인견해들인데, 공통적으로언급하고있는것은 alpha-synuclein 이봉입체를이루기전인 protofibril 형태가세포에더독성이있다는것이다. 본연구에서는같은조건의두세포주를비교하였을때도파민성세포로형질전환시킨 HB1.F3.TH-BH 4 에서봉입체생성비율이더증가되고봉입체가생성된세포에서세포자멸사역시증가되었다는것은봉입체가세포보호기전이라는견해에반대되는결과로판단되나세포사비율에유의한차이가없다는결과에서본다면아직결론을내리기힘들다고보인다. 봉입체를생성하는비율이전반적으로매우낮기때문에드물게관찰되는세포자멸사표지가과장되었을가능성을배제할수는없으나일관 350
인간신경줄기세포 HB1.F3 에서 alpha-synuclein 과 synphilin-1 유전자전이를통한봉입체형성과세포자멸사 되게레보도파를생성하는세포주에서발현이증가된소견이관찰되고있어이소견에대한계속적인연구가필요하다고본다. 배양시간이나봉입체를생성하는기전 ( 예컨대, 도파민을분비하고순환시키기위한 dopamine transporter 나시냅스낭 ) 이부족하기때문일가능성이있지만궁극적으로도파민을생성하지는못하기때문에이러한결과를설명하기위해서는추가적인연구가필요하다. 그리고정상 alpha-synuclein 단백질의양적인증가가세포자멸사와연관이있고가족성파킨슨병을유발하는원인으로보고되었으나본실험에서유전자삽입후일시적으로발현되는 alpha-synuclein 단백질의양을세포수준에서확인할수는없으므로정량적으로과발현을조절하여확인하는과정도필요하리라생각한다. HB1.F3.TH-BH 4 에서 HB1.F3와 HB1.F3.TH 두세포주에비해세포자멸사표지가더많이관찰되었는데이는 alphasynuclein 에의한도파민성세포의선택적세포자멸사기전을설명할수있는또하나의근거로생각한다. 도파민의세포독성에관해서는이미더이상언급할필요가없으나현재까지의실험결과들은배양액에도파민을첨가하여세포독성을관찰한것으로세포내에서생성되는도파민으로인한세포독성과같은양상으로설명하기곤란하다고판단된다. 그러므로본연구와같이유전자삽입으로형질전환시킨도파민성세포는안정적으로세포내에서레보도파를생성하고있다는점과인간기원의신경세포에서현재까지보고된결과를확인할수있었다는점은파킨슨병의병태생리를이해하는데보다효과적인실험방법이라고볼수있을것이다. 세포자멸사를확인하기위한방법으로 DAPI 염색과 TUN- NEL assay 를이용하였는데유전자전이를통한세포손상, 특히두유전자가동시에전이된경우손상이클것으로추정되어, 두단백질이과발현된경우와하나의단백질이과발현된경우의세포자멸사를비교하는것은무리가있을것으로생각된다. 그러나본연구에서는두단백질이동시에과발현된경우를비교하였으므로유전자전이과정자체로인한세포손상이봉입체가형성된경우나, 각각의세포주에서차이가없다고보인다. 그러므로루이체와같은봉입체의생성이세포자멸사에기여하고도파민성세포가이러한봉입체생성과세포자멸사에대해감수성이더높다고판단할수있는근거가된다고생각한다. 하지만 TH로형질전환시킨세포주가도파민을생성하지못하므로필요한효소를보완하여도파민을생성하는세포주에서확인을하는것이더욱좋았으리라는점과정량적으로유전자의발현을조절할수있는기법을이용하여보다정밀한검증을할필요가있다고생각된다. 그리고신경줄기세포, 신경세포, 신경교세포등이모두포함된이세포주를이용하여같은 synuclein 에의한기전으로알려져있으나다른병리소견과임상양상을보이는 multiple system atrophy, 루이체치매 (Lewy body dementia) 등의신경계변성질환의모델을확인하는연구도의미있을것으로생각한다. 도파민성세포에서봉입체생성과세포자멸사를연관성을알고자인간줄기세포주에서 alpha-synuclein 과 synphilin-1 유전자를이용하여수행한본연구에서저자들은다음과같은결론을얻었다. 첫째, 레보도파생산이루이체와같은봉입체형성에필수적인것은아니나레보도파가증가된환경에서봉입체형성이증가되었다. 둘째, Synphilin-1 은 alpha-synuclein 에의한봉입체형성과세포자멸사를증가시키나단독으로발현된경우의미있는결과를확인할수없었다. 셋째, 봉입체가관찰되는세포에서세포자멸사가증가하였다. 넷째, 레보도파가증가한세포에서 alpha-synuclein 에의한세포자멸사가증가되어도파민성신경세포는 alpha-synuclein 에대해선택적으로감수성이높은것으로보인다. 이상과같은결론으로루이체와같은봉입체형성이 alphasynuclein 에의한세포자멸사에기여한다는근거가될수있다고판단되며향후도파민을분비하는신경세포에서 alphasynuclein 유전자의발현을정량적으로조절하여 alphasynuclein 과봉입체, 도파민, 그리고세포자멸사의관계를더명확히밝힐수있는후속연구가진행되었으면한다. REFERENCES 1. Forno LS. Neuropathology of Parkinson s disease. J Neuropathol Exp Neurol 1996;55:259-272. 2. Olanow CW, Watts RL, Koller WC. An algorithm for the management of Parkinson s disease: treatment guidelines. Neurology 2001; 56:S1-S88 3. Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Durta A, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson s disease. Science 1997;276:2045-2047. 4. Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, Kosel S, et al. Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson s disease. Nat Genet 1998;18:106-108. 5. Matsumine H, Saito M, Shimoda-Matsubayashi S, Takara H, Ishikawa A, Nakagawa-Hattori Y, et al. Localization of a gene for an autosomal recessive form of juvenile Parkinsonism to chromosome 6q25.2-27. Am J Hum Genet 1997;60:588-596. 6. Leroy E, Boyer R, Auburger G, Leube B, Ulm G, Mezey E. The ubiquitin pathway in Parkinson s disease. Nature 1998;395:451-452. 7. Savitt JM, Dawson VL, Dawson TM. Diagnosis and treatment of Parkinson disease: molecules to medicine. J Clin Invest 2006;116:1744-1754. 8. Tompkins MM, Hill WD. Contribution of somal Lewy bodies to neuronal death. Brain Res 1997;775:24-29. J Korean Neurol Assoc Volume 25 No. 3, 2007 351
천상명이광김재우김승업 9. Volles MJ, Lansbury PT. Zeroing in on the pathogenic form of alpha-synucleinand its mechanism of neurotoxicity in Parkinson s disease. Biochemistry 2003;42:7871-7878. 10. Kopito RR. Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 2000;10:524-530. 11. Taylor JP, Tanaka F, Robitschek J, Sandoval CM, Taye A, Markovic-Plese S, et al. Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein. Hum Mol Genet 2003;12:749-757. 12. Braak H, Del Tredici KF, Rub U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson s disease. Neurobiol Aging 2003;24:197-211. 13. Xu J, Kao SY, Lee FJ, Song W, Jin LW, Yankner BA. Dopamine-dependent neurotoxicity of alpha-synuclein: a mechanism for selective neurodegeneration in Parkinson s disease. Nat Med 2002; 8:600-606. 14. Volles MJ, Landsbury PT. Vesicle permeabilization by protofibrillar alpha-synuclein is sensitive to Parkinson s disease-linked mutations and occurs by a pore-like mechanism. Biochemistry 2002; 41:4595-4602. 15. Cooper AA, Gitler AD, Cashikar A, Haynes CM, Hill KJ, Bhullar B, et al. Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson s models. Science 2006;313:324-328. 16. Kruger R. The role of synphilin-1 in synaptic function and protein degradation. Cell Tissue Res 2004;318:195-199. 17. Wakabayashi K, Engelender S, Yoshimoto M, Tsuji S, Ross CA, Takahashi H. Synphilin-1 is present in Lewy bodies in Parkinson s disease. Ann Neurol 2000;47:521-523. 18. Engelender S, Kaminsky Z, Guo X, Sharp AH, Amaravi RK, Kleiderlein JJ, et al. Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions. Nat Genet 1999; 22:110-114. 19. Kawamata H, McLean PJ, Sharma N, Hyman BT. Interaction of alpha-synuclein and synphilin-1: effect of Parkinson s disease-associated mutations. J Neurochem 2001;77:929-934. 20. Ryu JK, Kim SU, McLarnon JG, Neuroprotective effects of pyruvate in the quinolinic acid rat model of Huntington s disease. Exp Neurol 2003;183:700-704. 21. Chu K, Kim M, Jeong SW, Kim SU, Yoon BW. Human neural stem cells can migrate, differentiate, and integrate after intravenous transplantateion in adult rats with transient forebrain ischemia. Neurosci Lett 2003;343:129-133. 22. Cho T, Bae JH, Choi HB, Kim SS, McLarnon JG, Suh-Kim H, et al. Human neural stem cells: electrophysiological properties of voltage-gated ion channels. Neuroreport 2002;13:1447-1452. 23. Kim SU. Human neural stem cells genetically modified for brain repair in neurological disorders. Neuropathology 2004;24:159-171. 24. Lee G, Tanaka M, Park K, Lee SS, Kim YM, Junn E, et al. Casein kinase II-mediated phosphorylation regulates alpha-synuclein/ synphilin-1 interaction and inclusion body formation. J Biol Chem 2004;279:6834-6839. 25. Anton R, Kordower JH, Maidment NT, Manaster JS, Kane DT, Rabizadeh S, et al. Neural-targeted gene therapy for rodent and primate hemiparkinsonism. Exp Neurol 1994;127:207-218. 26. Jiao S, Gurevich V, Wolff JA. Long-term correction of rat model of Parkinson s disease by gene therapy. Nature 1993;362:450-453. 27. Fahn S; and the Parkinson Study Group. Does levodopa slow or hasten the rate of progression of Parkinson s disease? J Neurol 2005; 252(Suppl 4):37-42. 28. Gille G, Hung S, Reichmann H, Rausch W. Oxidative stress to dopaminergic neuron as models of Parkinson s disease. Ann N Y Acad Sci 2004;1018:533-540. 29. Clayton DF, George JM. The synucleins: a family of proteins involved in synaptic function, plasticity, neurodegeneration and disease. Trends Neurosci 1998;21:249-254. 30. Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J. Alpha-synuclein locus triplication causes Parkinson s disease. Science 2003;302:841. 31. Farrer M, Kachergus J, Forno L, Lincoln S, Wang DS, Hulihan M, et al. Comparison of kindreds with parkinsonism and alphasynuclein genomic multiplications. Ann Neurol 2004;55:174-179. 32. Schmidt ML, Murray J, Lee VM, Hill WD, Wertkin A, Trojanowski JQ. Epitope map of neurofilament protein domains in cortical and peripheral nervous system Lewy bodies. Am J Pathol 1991;139: 53-65. 33. Lennox G, Lowe J, Morrell K, Landon M, Mayer RJ. Antiubiquitin immunocytochemistry is more sensitive than conventional technique in the detection of diffuse Lewy body disease. J Neurol Neurosurg Psychiatry 1989;52:67-71. 34. Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert R. Alpha-synuclein in Lewy bodies. Nature 1997;388:839-840. 35. Shashidharan P, Good PF, Hsu A, Perl DP, Brin MF, Olanow CW. Torsin A accumulation in Lewy bodies in sporadic Parkinson s disease. Brain Res 2000;877:379-381. 36. Sherman MY, Goldberg AL. Cellular defenses against unfolded proteins: a cell biologist thinks about neurodegenerative diseases. Neuron 2001;29:15-32. 37. Ii K, Ito H, Tanaka K, Hirano A. Immunocytochemical co-localization of the proteosome in ubiquitinated structures in neurodegenerative diseases and the elderly. J Neuropathol Exp Neurol 1997; 56:125-131. 38. Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M. Alpha-synuclein in filamentous inclusions of Lewy bodies from Parkinson s disease and dementia with Lewy bodies. Proc Natl Acad Sci USA 1998;95:6469-6473. 39. Lee G, Junn E, Tanaka M, Kim YM, Mouradian MM. Synphilin-1 degradation by ubiquitin-proteosome pathway and effects on cell survival. J Neurochem 2002;83:346-352. 352