이 작품은 한국 정부 (MSIP) (NRF-2014K1A4A7A01074642), 한국 국가 연구 재단 (NRF) 개별 과학자 지원 프로그램 (NRF-2013M3A9B5076486/NRF-2015R1D1A1A09057239)에 의해 투자 된 교부 금에 의해 지원 되었다.

1. lentivirus 생산 참고: 생산과 lentiviral 벡터의 조작 재조합 관련 된 연구를 위한 국가 학회 (NIH) 지침에 따라 실시 DNA입니다. 플라스 미드는 야생 형 및 A4V 돌연변이 체 SOD1 (SOD1WT-YFP 및 SOD1A4V YFP) 향상 된 YFP 태그로 인코딩 김 외에 설명 되어 있습니다. 11 두 유전자 융합 제품 PCR 뇌관 쌍 5를 사용 하 여 증폭 했다 &#8242;-ATCGTCTAGACACCATGGCGACGAAGGTCGTGTGC-3 &#8242; 5 &#8242;-TAGCGG CCGCTACTTGTACAGCTCGTCCATGCC-3 &#8242; pTRIP 델타 U3 CMV에 삽입 플라스 미드 19 XhoI 및 BsrGI 금지 사이트를 사용 하 여. 이전 실험에서 HEK 293T 세포는 매 2 일 1:6 1: 4의 비율로 분할 했다. 20 개 이상의 통로 적은 60 %confluency 하는 데 도움이 좋은 transfection 효율 보장에서 세포의 유지 보수의 회피.
<ol><li>에 1 일, 바이러스 생산을 위한 세포 배양 매체 (10 %FBS 4 m L-글루타민 m 높은 포도 당 DMEM) 10 mL에 10 cm 직경 조직 문화 접시 (3 x 10 6 셀/접시)에 30-40% 합류에서 HEK 293T 세포 분할.</li>
	<li>유지 10 cm 직경 조직 문화는 CO 2 배양 기 (37 ° C, 5% CO 2)에서 하룻밤 접시.</li>
	<li>주 2에 준비 lentiviral 벡터 10 µ g를 포함 하는 DNA transfection 솔루션 (pTRIP-델타 U3 CMV-SOD1WT-YFP 또는-SOD1A4V-YFP)는 lentiviral 함께 플라스 미드 (5 µ g의 pVSVg, pCMV dR8.71의 10 µ g) (포장 그림 2A) 20. 1 M CaCl 2 125 µ L을 추가 하 고 증류수를 사용 하 여 500 µ L에 양을 맞추십시오. 부드럽게 혼합물에 0.05 M HEPES의 500 µ L을 추가 하 고 실 온에서 10 분 동안 품 어.</li>
	<li>바꾸기 HEK 293T 세포 10 mL가 미리 데워 진 신선한 문화 매체 항생제 무료 매체 1 mL DNA transfection 솔루션 혼합 하 고 37 ° C, 5% CO 2에서 밤새 품 어.</li>
	<li>3 일에서 신선한 문화 매체와 셀 표면에 뜨는 바꿉니다.</li>
	<li>에 4 일, 수확 15 mL 튜브와 죽은 세포와 파편 제거 하 x 500g에 5 분 동안 원심 분리기에 상쾌한. 더 큰 60 mL 주사기를 사용 하 여 0.45 μ m 필터를 통해 그것을 전달 하 여는 상쾌한 정화. 즉시 단일 사용 aliquots (300 µ L)으로 분배 하 고-80 ° c.에 저장 최대 제품 활동을 유지 하기 위해 피하 동결-해 동 주기.</li>
</ol> 2. Lentiviral 변환 <ol><li>분할 셀 라인 60% 합류에 감염 (HEK 293 세포와 SH-SY5Y; 잘 당 5 x 10 5 셀 및 U2OS; 잘 당 1 x 10 5 셀) 보충 DMEM 미디어의 2 mL에는 6 잘 조직 문화 접시 10 %FBS.</li>
	<li>6 잘 조직 배양 플레이트 37 ° C 배양 기에서 5% CO 2에서 하룻밤 유지.</li>
	<li>-80 ° C 냉장고에서 냉동된 lentivirus를 제거 하 고 각 사용 하기 전에 얼음에 약 수를 녹여; refreeze 하지.</li>
	<li>동안 바이러스 녹고 따뜻한 세포 배양 포함 하는 혈 청의 셀 라인와 호환. 일단 바이러스가 완전히 해 동 준비 희석의 범위 (1:3, 1:10, 1:30, 그리고 1: 100) 1.5 mL 신선한 microfuge 관에서 DMEM에.</li>
	<li>감소 된 혈 청 매체 1 ml 튜브에 볼륨을 키울.</li>
	<li>바이러스/미디어의 1 ml (4 µ g / µ L의 재고)에서 Polybrene의 추가 2 µ L. Pipetting으로 잘 혼합 하 고 셀에 혼합물의 1 mL를 추가 합니다. 24 헤에 대 한 바이러스 세포를 품 어</li>
	<li>바이러스 미디어를 제거 하 고 정상적인 DMEM 미디어 10% 보충 바꿉니다 FBS. 37 ° C, 5% CO 2에서 셀 유지.</li>
	<li>셀의 성장 모니터링 하 고 변경 문화 미디어 매 2 일. 에 합류, 10 cm 직경 조직 문화 접시로 6-잘 접시 확장.</li>
	<li>일단 셀 충분히 확장 되었으며, 384-잘 분석 결과 접시는 YFP 식 수준 확인 하기에 DMEM 미디어의 50 µ L에 잘 당 씨 1.5 x 10 4 셀 현미경 검사 법에 의해 단백질을 태그. 신호 대 잡음 비율 표시는 YFP 태그 단백질 셀 식 &#8805; 3, 해당 셀 라인 셀 재고 준비.</li>
</ol> 3. 시간 프로테아좀 억제제의 단백질 집계에 종속 효과 참고: 자동된 현미경을 사용 하 여 이미지 수집을 수행 (자료 참조).
<ol><li>특 면 100 %DMSO (ALLN, 2 mM)에 녹아 있는 384-잘 플랫 바닥 접시에 DMSO 또는 프로테아좀 억제제의 0.25 µ L.</li>
	<li>DMEM의 50 µ L에 잘 당 수동으로 씨 1.5 x 10 4 셀 미디어 같은에 시험 접시. 프로테아좀 억제제 (ALLN) 최종 농도 10 µ M 이어야 합니다.</li>
	<li>37 ° C, 5% CO 2 현미경 시스템 환경 제어 장치를 설정합니다. 실험적인 체제의 화면 그림 3에 표시 됩니다.</li>
	<li>운영 소프트웨어를 사용 하 여 다음 설정을 가진 넓은 필드 형광 모드로 현미경: 시간 캡처 간격 목표, 비 confocal 모드, 4 필드/글쎄, LWD 20. 각 실행의 끝에, 캡처된 이미지는 서버에 자동으로 업로드 한다.</li>
</ol> 4. 시간 경과 영상 살아있는 세포에 YFP 식에 대 한 및 이미지 분석 (단일 채널) 참고: 다음 단계에서는 응용 프로그램 (예를 들어, 콜럼버스) 소프트웨어의 설명.
기본 개체 (cytosol 및 집계) 세그먼트 <ol><li>선택 적절 한 알고리즘입니다. 필요한 경우 조정 배경 임계값 및 대비 매개 변수 ( 그림 4).</li>
	<li>계산 셀을 사용 하 여 &#39; 셀을 찾을 &#39; 0.1 YFP 강도 채널에 대 한 개별 임계값. 집계를 사용 하 여 결정 한 &#39; 장소를 찾을 &#39; 상대 YFP 자리 강도 알고리즘 &#62; 0.2와 1.0의 분할 계수.</li>
</ol> 5. 복용량 응답의 효과 프로테아좀 억제제 단백질 집계에 살아있는 세포의 핵 (2 채널)에 대 한 스테인드 참고: 자동된 현미경을 사용 하 여 이미지 수집을 수행. 최적의 곡선 적합 되도록 예상된 IC 50 값에 따라 프로테아좀 억제제 (ALLN, Epoxomicin, 및 MG132)의 농도 범위 결정.
384 잘 저장 폴 리 프로필 렌에 화합물의 <ol><li>준비 직렬 희석 1:3 희석 시리즈 표준 접시. 복합 희석 혼합 확인 잘 되도록 화합물 농도 정확.</li>
	<li>빈 플랫-하단 검은 384-잘 시험 접시에 프로테아좀 억제제의 0.25 µ L 분배. 384-모 세관 머리 볼륨을 전송할 수를 갖춘 액체 처리기 사용.</li>
	50에 잘 당 10 4 셀 x <li>씨 1.5 µ L DMEM 미디어의 분석 결과 접시에. 분석 결과 접시 24 h. 위해 37 ° C, 5% CO 2를 품 어</li>
	<li>솔루션 (10 mg/mL의 재고에 Hoechst 33342) 얼룩과 DMEM 미디어 (37 ° C에서 미리 예 열)의 10 µ L을 추가 하 고 10 분에 대 한 실 온에서 품 어</li>
	<li>발사는 마이크로운영 소프트웨어 대처. 실험 설정의 화면 캡처는 그림 6에 표시 됩니다. 선택은 &#34; 구성 &#34; 탭 하 고 객관적이 고 정확한 판형 X 20을 선택 합니다. 확인 &#34; 칼라 &#34; 다른 접시 종류와 적절 한 초점을 수 있도록 하는 목적에 올바른 값으로 설정 됩니다.</li>
	<li>선택은 &#34; 현미경 &#34; 탭 정의 노출 YFP로 1 (488 레이저) 및 노출 2 Hoechst (405 레이저). 모두 노출에 필터를 활성화 하 고 카메라 1 카메라 2 노출 2 노출 1을 할당 합니다. 노출 1 ~ 800 ms 노출 시간 설정 노출 2 ~ 40 ms.</li>
	<li>선택 노출 1입니다. 0 µ m. 선택 초점 높이 설정 &#34; 초점 &#34;. 일단 초점, 노출 카메라 1. 최적화 노출 평면을 클릭 초점 높이 조정 &#34; 높이 걸릴 &#34;. 노출 시간을 변경 하 고 ~ 3, 000의 최대 픽셀 강도 게 전원 레이저. 노출 매개 변수를 저장 합니다. 반복 노출 2.</li>
	<li>선택 &#34; 실험 정의 &#34; 탭 만들기 레이아웃 및 sublayout. 드래그 앤 드롭 관련 레이아웃, 노출, 참조 이미지, skewcrop 파일 및 sublayout. 실험 저장.</li>
	<li>선택 &#34; 자동 실험 &#34; 탭 및 이미지. 각 실행의 끝에, 캡처된 이미지는 서버에 자동으로 업로드 한다.</li>
</ol> 6. 두 이미지의 분석 이미지 <ol><li>세그먼트는 기본 개체 (핵, cytosol, 및 집계) 소프트웨어 알고리즘을 선택 합니다 ( 그림 7).</li>
	<li>세그먼트 개체의 검사에 의해 정확 하 게 핵을 세그먼트 방법을 선택 합니다. Hoechst 스테인드 개체 1 채널에서 (Ch1) 셀의 수를 결정 하기 위해 사용 됩니다. 돌연변이 체 SOD1-채널 2 (Ch2) A4V의 양을 결정 하는 데 사용 됩니다에서 YFP 얼룩.</li>
	<li>계산 셀을 사용 하는 &#39; 핵을 찾을 &#39; 알고리즘 Hoechst 얼룩 영역으로 &#62; 20 µ m 2, 7.0, 0.40, 그리고 대조의 개별 임계값의 분할 계수와 &#62; 0.10.</li>
	<li>데이터 테이블을 만들고 사용 하 여 화합물의 각각에 대 한 EC 50를 결정는 &#39; 비 선형 회귀 &#39; 방정식 그래프 소프트웨어에서.</li>
</ol>

<ol><li>Schulte, J., Sepp, K. J., Wu, C., Hong, P., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-content+chemical+and+rnai+screens+for+suppressors+of+neurotoxicity+in+a+huntington%27s+disease+model%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">High-content chemical and rnai screens for suppressors of neurotoxicity in a huntington's disease model.</a> PLoS ONE. 6 (8), e23841(2011).</li>
<li>Honarnejad, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+implementation+of+a+high-throughput+compound+screening+assay+for+targeting+disrupted+ER+calcium+homeostasis+in+Alzheimer%27s+disease%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Development and implementation of a high-throughput compound screening assay for targeting disrupted ER calcium homeostasis in Alzheimer's disease.</a> PLoS ONE. 8 (11), 1-12 (2013).</li>
<li>Burkhardt, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cellular+model+for+sporadic+ALS+using+patient-derived+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22Mol+Cell+Neurosci%22%5BJournal%5D)">A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells.</a> Mol Cell Neurosci. 56, 355-364 (2013).</li>
<li>Mattiazzi, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Content+Screening+for+Quantitative+Cell+Biology%5BTitle%5D)+AND+%22Trends+Cell+Biol%22%5BJournal%5D)">High-Content Screening for Quantitative Cell Biology.</a> Trends Cell Biol. 26 (8), 598-611 (2016).</li>
<li>Kato, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amyotrophic+lateral+sclerosis+models+and+human+neuropathology%3A+Similarities+and+differences%5BTitle%5D)+AND+%22Acta+Neuropathologica%22%5BJournal%5D)">Amyotrophic lateral sclerosis models and human neuropathology: Similarities and differences.</a> Acta Neuropathologica. 115 (1), 97-114 (2008).</li>
<li>Strong, M. J., Kesavapany, S., Pant, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pathobiology+of+amyotrophic+lateral+sclerosis%3A+a+proteinopathy%3F%5BTitle%5D)+AND+%22J+neuropathol+exp+neurol%22%5BJournal%5D)">The pathobiology of amyotrophic lateral sclerosis: a proteinopathy?</a> J neuropathol exp neurol. 64 (8), 649-664 (2005).</li>
<li>Cleveland, D. W., Rothstein, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+Charcot+to+Lou+Gehrig%3A+deciphering+selective+motor+neuron+death+in+ALS%5BTitle%5D)+AND+%22Nat+rev+Neurosci%22%5BJournal%5D)">From Charcot to Lou Gehrig: deciphering selective motor neuron death in ALS.</a> Nat rev Neurosci. 2 (11), 806-819 (2001).</li>
<li>Bruijn, L. I., Miller, T. M., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unraveling+the+Mechanisms+Involved+in+Motor+Neuron+Degeneration+in+Als%5BTitle%5D)+AND+%22Annu+Rev+Neurosci%22%5BJournal%5D)">Unraveling the Mechanisms Involved in Motor Neuron Degeneration in Als.</a> Annu Rev Neurosci. 27 (1), 723-749 (2004).</li>
<li>Vassall, K. a, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreased+stability+and+increased+formation+of+soluble+aggregates+by+immature+superoxide+dismutase+do+not+account+for+disease+severity+in+ALS%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS.</a> Proc Natl Acad Sci USA. 108 (6), 2210-2215 (2011).</li>
<li>Hwang, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonamyloid+aggregates+arising+from+mature+copper%2Fzinc+superoxide+dismutases+resemble+those+observed+in+amyotrophic+lateral+sclerosis%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Nonamyloid aggregates arising from mature copper/zinc superoxide dismutases resemble those observed in amyotrophic lateral sclerosis.</a> J Biol Chem. 285 (53), 41701-41711 (2010).</li>
<li>Kim, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dimerization%2C+Oligomerization%2C+and+Aggregation+of+Human+Amyotrophic+Lateral+Sclerosis+Copper%2FZinc+Superoxide+Dismutase+1+Protein+Mutant+Forms+in+Live+Cells%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Dimerization, Oligomerization, and Aggregation of Human Amyotrophic Lateral Sclerosis Copper/Zinc Superoxide Dismutase 1 Protein Mutant Forms in Live Cells.</a> J Biol Chem. 289 (21), 15094-15103 (2014).</li>
<li>Shaw, B. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detergent-insoluble+aggregates+associated+with+amyotrophic+lateral+sclerosis+in+transgenic+mice+contain+primarily+full-length%2C+unmodified+superoxide+dismutase-1%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Detergent-insoluble aggregates associated with amyotrophic lateral sclerosis in transgenic mice contain primarily full-length, unmodified superoxide dismutase-1.</a> J Biol Chem. 283 (13), 8340-8350 (2008).</li>
<li>Banci, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SOD1+and+amyotrophic+lateral+sclerosis%3A+Mutations+and+oligomerization%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">SOD1 and amyotrophic lateral sclerosis: Mutations and oligomerization.</a> PLoS ONE. 3 (2), 1-8 (2008).</li>
<li>Johnston, J. a, Dalton, M. J., Gurney, M. E., Kopito, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+of+high+molecular+weight+complexes+of+mutant+Cu%2C+Zn-superoxide+dismutase+in+a+mouse+model+for+familial+amyotrophic+lateral+sclerosis%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci%22%5BJournal%5D)">Formation of high molecular weight complexes of mutant Cu, Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis.</a> Proc Natl Acad Sci. 97 (23), 12571-12576 (2000).</li>
<li>Niwa, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disulfide+bond+mediates+aggregation%2C+toxicity%2C+and+ubiquitylation+of+familial+amyotrophic+lateral+sclerosis-linked+mutant+SOD1%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Disulfide bond mediates aggregation, toxicity, and ubiquitylation of familial amyotrophic lateral sclerosis-linked mutant SOD1.</a> J Biol Chem. 282 (38), 28087-28095 (2007).</li>
<li>Juneja, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prognosis+in+familial+amyotrophic+lateral+sclerosis%3A+progression+and+survival+in+patients+with+glu100gly+and+ala4val+mutations+in+Cu%2CZn+superoxide+dismutase%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">Prognosis in familial amyotrophic lateral sclerosis: progression and survival in patients with glu100gly and ala4val mutations in Cu,Zn superoxide dismutase.</a> Neurology. 48 (1), 55-57 (1997).</li>
<li>Kaur, S. J., McKeown, S. R., Rashid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutant+SOD1+mediated+pathogenesis+of+Amyotrophic+Lateral+Sclerosis%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">Mutant SOD1 mediated pathogenesis of Amyotrophic Lateral Sclerosis.</a> Gene. 577 (2), 109-118 (2016).</li>
<li>de Bruyns, A., Geiling, B., Dankort, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Construction+of+Modular+Lentiviral+Vectors+for+Effective+Gene+Expression+and+Knockdown%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown.</a> Methods Mol Biol. 1448, 3-21 (2016).</li>
<li>Sirven, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+Transgene+Expression+in+Cord+Blood+CD34%2B-Derived+Hematopoietic+Cells%2C+Including+Developing+T+Cells+and+NOD%2FSCID+Mouse+Repopulating+Cells%2C+Following+Transduction+with+Modified+TRIP+Lentiviral+Vectors%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Enhanced Transgene Expression in Cord Blood CD34+-Derived Hematopoietic Cells, Including Developing T Cells and NOD/SCID Mouse Repopulating Cells, Following Transduction with Modified TRIP Lentiviral Vectors.</a> Mol Ther. 3 (4), 438-448 (2001).</li>
<li>Wu, C., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-titre+retroviral+vector+system+for+efficient+gene+delivery+into+human+and+mouse+cells+of+haematopoietic+and+lymphocytic+lineages%5BTitle%5D)+AND+%22J+gen+virol%22%5BJournal%5D)">High-titre retroviral vector system for efficient gene delivery into human and mouse cells of haematopoietic and lymphocytic lineages.</a> J gen virol. 91 (Pt 8), 1909-1918 (2010).</li>
<li>Irshad, H., Veillard, A., Roux, L., Racoceanu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+nuclei+detection%2C+segmentation%2C+and+classification+in+digital+histopathology%3A+A+review-current+status+and+future+potential%5BTitle%5D)+AND+%22IEEE+Rev+Biomed+Eng%22%5BJournal%5D)">Methods for nuclei detection, segmentation, and classification in digital histopathology: A review-current status and future potential.</a> IEEE Rev Biomed Eng. 7, 97-114 (2014).</li>
<li>Purschke, M., Rubio, N., Held, K. D., Redmond, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phototoxicity+of+Hoechst+33342+in+time-lapse+fluorescence+microscopy%5BTitle%5D)+AND+%22Photochem+Photobiol+Sci%22%5BJournal%5D)">Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy.</a> Photochem Photobiol Sci. 9 (12), 1634-1639 (2010).</li>
<li>Jaskova, K., Pavlovicova, M., Jurkovicova, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+transporters+and+their+role+in+the+development+of+neuronal+disease+and+neuronal+damage%5BTitle%5D)+AND+%22Gen+physiol+biophys%22%5BJournal%5D)">Calcium transporters and their role in the development of neuronal disease and neuronal damage.</a> Gen physiol biophys. 31 (4), 375-382 (2012).</li>
<li>Kieran, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treatment+with+arimoclomol%2C+a+coinducer+of+heat+shock+proteins%2C+delays+disease+progression+in+ALS+mice%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice.</a> Nat Med. 10 (4), 402-405 (2004).</li>
</ol>

저자는 공개 없다.

안정적인 세포를 생성 하기 위한 두 가지 주요 방법 있다. 처음 몇 주를 소요 하며 게놈 통합된 플라스 미드 DNA 벡터의 과도 transfection 및 저항 선택. 두 번째는 제한 노력으로 여러 셀 라인에서이 프로토콜을 대상 단백질의 효과적인 표현 의무가 만들기 lentivirus, 사용 시간의 문제를 걸립니다. 여행-CMV 벡터19 지속적인된 벡터 보존된 변환 효율과 안정적인 transgene 식 사용 했다. 놀?...

단백질 집계는 생물학 과정을 단백질 그룹을 misfolded 및 신경 퇴행 성 질환 (amyloidosis)에 원인이 되는 에이전트 역할을 할 수는 있습니다. 단백질 집계 특성화 촉진은 병의 발병에 영향을 주는 새로운 요인의 발견에서 세포 장애 뿐만 집계의 역할 이해에 필수적 이다. 살아있는 세포에 형광 태그 단백질의 시각화 높은 콘텐츠 심사 (HCS)1,2,,34에 적용 가능한 분석 실험의 개발에 도움이 수 있는 강력한 방법입니다.
루 경화 증 (ALS) 집계 가족 ALS (fALS)에 산발적 ALS (염)5,6 모터 신경에서 축적 하는 성향으로 misfolded 단백질의 존재로 인 한 proteopathic 질병으로 간주 됩니다. . FALS 경우의 ~ 20%의 하위 집합 관련 된 인코딩 cytosolic 항 산화 효소 유전자에 지배적인 돌연변이와 구리-아연 superoxide dismutase 입력 1 (SOD1)7,8. SOD1 변형, 탈 선 안정성, 증가 전개 속도, 집계9, 성향 등의 기능과 구조에 변화를 포함 하 여이 유전자 파생된 전에 대 한 몇 가지 잠재적인 원인 제안 되었습니다. 10. 특히, 두 ALS 연결 된 SOD1 이체에 의해 공유 하는 유일한 확인 하 고 잠재적으로 유독한 속성 이며 야생-타입 (WT) SOD1 compartmentalized 단백질 집계 또는 배치할 포함11,12 형태로 증가 성향 . Misfolded 돌연변이 체 SOD1, 지속적으로 polyubiquitinated 이며, 유비퀴틴-프로테아좀 시스템에 의해 타락. 그 결과, 프로테아좀의 활동의 억제의 저수준 SOD1 집계13,14, 수용 성 돌연변이 체 SOD1와 교환할 수 있는 수용 성 구성 요소 형태로 비정 질 구조를 구성 하는 돌연변이의 축적에 리드 cytosol15. 특히, SOD1 돌연변이 A4V에서 (알라닌 codon 4에서 valine 변경) 가장 일반적인 ALS를 일으키는 돌연변이 이며 2 년 미만 질병 발병16후의 평균 생존 시간 빠른 neurodegeneration을 리드. 화학적, SOD1 A4V monomerize, 집계, 녹말 체 모;를 형성 하는 증가 경향이 있다 그것의 기 공 같은 집계 다른 질병에 연결 된 돌연변이 형태, α-synuclein와 β 아 밀 로이드 단백질17등의 녹말 체 모와 비슷합니다. SOD1 집계 축적의 역학 연구, 가용성과 불용 성 SOD1 집계 형태를 모니터링 하기 위한 방법 개발 유지.
우리는 이전, 라이브 셀 이미징 사용 하 여 및 HEK 293 세포 형광 단백질 태그 SOD1, ALS 관련 돌연변이 SOD1 이합체 화 및 집계11손상으로 뚜렷이 페. 과도 식 시스템 단기 유전자 overexpression의 생물 학적 결과 대 한 유용한 정보를 제공할 수 있습니다, 방법 원하는 유전자의 안정적인 통합을 제공 하는 분석 결과 개발에 대 한 선호 있을 수 있습니다. 따라서, lentiviral 벡터 포유류 세포 18에 장기 및 규제 유전자 발현을 부여 하는 기능을 제공 합니다. 이 연구에서 우리가 재조합 lentivirus WT 베어링으로 불리고 안정적인 셀 라인의 세대에 집중 하 고 돌연변이 체 SOD1 태그 노란 형광 성 단백질 (YFP)와. 라이브 셀 이미징 현미경 SOD1 집계의 자동화 된 정량화에 사용 하 여, 우리는 실행 하 고 SOD1 집계 이벤트는 프로테아좀의 억제 시 계량.

<table><tbody><tr><td>ALLN (C&lt;sub&gt;20&lt;/sub&gt;H&lt;sub&gt;37&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;)</td><td>Millipore</td><td>208719</td><td></td></tr><tr><td>MG132 (C&lt;sub&gt;26&lt;/sub&gt;H&lt;sub&gt;41&lt;/sub&gt;N&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;5&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C2211</td><td></td></tr><tr><td>Epoxomicin (C&lt;sub&gt;28&lt;/sub&gt;H&lt;sub&gt;50&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>E3652</td><td></td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>H-3570</td><td></td></tr><tr><td>Opera</td><td>Perkin Elmer</td><td>OP-QEHS-01</td><td></td></tr><tr><td>Opera EvoShell software</td><td>Perkin Elmer</td><td>Ver 1.8.1</td><td></td></tr><tr><td>Operetta</td><td>Perkin Elmer</td><td>OPRT1288</td><td></td></tr><tr><td>Harmony Imaging software</td><td>Perkin Elmer</td><td>Ver 3.0.0</td><td></td></tr><tr><td>Columbus Image analysis software</td><td>Perkin Elmer</td><td>Ver 2.3.2</td><td></td></tr><tr><td>CyBi Hummingwell liquid handling</td><td>CyBio AG</td><td>OL 3387 3 0110</td><td></td></tr></tbody></table>

assay development for high content quantification of sod1 mutant protein aggregate formation in living cells

루 경화 증 (ALS) 유전자 인코딩 구리-아연 superoxide dismutase 1 (SOD1)에서 상속 된 돌연변이 의해 발생할 수 있는 치명적인 신경 질환 이다. SOD1의 구조적 불안정 및 가족 ALS 환자에 SOD1 긍정적인 포함 검색 misfolded 또는 집계 SOD1 ALS 병 리에 대 한 잠재적인 원인 역할을 지원합니다. 이 연구에서 우리는 높은 콘텐츠 접근을 차단 하 여 살아있는 세포에 SOD1 집계의 역학을 계량 하도록 세포 기반 분석 결과의 개발을 설명 합니다. 안정적인 셀 라인 표현 야생-타입 및 돌연변이 A4V SOD1 태그로 노란 형광 성 단백질 및 두 단백질 집단의 흔적 없이 cytosol에 표현 되었다 발견 lentiviral 벡터를 사용 하 여 생성 되었습니다. 흥미롭게도,만 SOD1 A4V HEK 293 안정적으로 표현 하지만 하지 U2OS 또는 SH SY5Y 셀 라인에 프로테아좀 억제제 처리 시 집계를 형성 했다. 우리는 다양 한 프로테아좀 억제제의 복용량 응답 분석을 기반으로 집계를 계량 하 고 시간 경과 현미경 검사 법에 의해 집계 형성 속도 론을 추적 가능 하다는 것을 보여줍니다. 우리의 접근 ALS 돌연변이의 역할에 SOD1 집계 형성에 효과 측정 SOD1 A4V 집계를 방지 하는 작은 분자에 대 한 심사의 가능성을 소개 합니다.

Lentivirus를 사용 하 여 생성 안정적인 셀 라인: SOD1 단백질 집계 모니터링에 대 한 전반적인 전략은 그림 1에 나와 있습니다. 첫 번째 단계에서 셀 라인 (1 단계)에 SOD1 안정적인 유전자 배달에 대 한 식 lentiviral 벡터를 생성 했습니다. 인코딩 SOD1 WT YFP 태그 및 SOD1 A4V 두 lentiviral 벡터 (SOD1WT-YFP 및 SOD1A4V-YFP, 각각) 포장 및 봉투 플라스 미드 (<stro...

Watch this Scientific Journal Video about Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells at JoVE.com

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

계량 misfolded 단백질의 집계 하는 방법을 설명 합니다. 우리의 프로토콜 세부 사항을 lentiviral 안정적인 셀 라인 생성, 자동된 confocal 영상, 및 단백질의 이미지 분석을 유도 한다. 설명 응용 프로그램으로 우리는 SOD1 집계 시간 복용량에 따라 방식에서 홍보에 작은 분자의 효과 공부 했습니다.

살아있는 세포에 Sod1 돌연변이 단백질 집계 형성의 높은 콘텐츠 정량화에 대 한 분석 결과 개발

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

assay-development-for-high-content-quantification-sod1-mutant-protein

Research

JoVE Journal

Neuroscience

7.2K 조회수.  Institut Pasteur Korea. 계량 misfolded 단백질의 집계 하는 방법을 설명 합니다. 우리의 프로토콜 세부 사항을 lentiviral 안정적인 셀 라인 생성, 자동된 confocal 영상, 및 단백질의 이미지 분석을 유도 한다. 설명 응용 프로그램으로 우리는 SOD1 집계 시간 복용량에 따라 방식에서 홍보에 작은 분자의 효과 공부 했습니다.

비디오: Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

이 콘텐츠 이미징 분석 Rhodopsin 전송 분석 결과

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

연구

화학

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

모터 뉴런과 유사한 NSC-34 세포 내에서 실시간으로 핵 세포질 수송을 측정하는 분석

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

신경과학

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

초파리 Melanogaster 에서 프리온과 같은 단백질의 셀 전송 모니터링

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the study of &#945;S aggregation in Parkinson&#39;s Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased &#945;S transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of &#945;S aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed &#945;S fibrils.
We have developed a method to induce the formation of intracellular &#945;S inclusions in primary neurons through the exogenous administration of native microsomes-associated &#945;S aggregates, a highly toxic &#945;S species isolated from diseased areas of transgenic mice. This fraction of &#945;S aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated &#945;S. Compared to in vitro-preformed fibrils which are made from recombinant &#945;S, our method is faster and guarantees that the pathogenic seeding is made with authentic &#945;S aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in &#945;S inclusions is mandatory.
We believe that this method will provide a versatile cell-based model to study the microscopic aspects of &#945;S aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

The goal of this protocol is to provide a cell-based system that replicates the formation of alpha-synuclein aggregates in vivo. Intracellular alpha-synuclein inclusions are seeded in primary neurons by the internalization and propagation of exogenous administered native microsomes-associated alpha-synuclein aggregates isolated from diseased alpha-synuclein transgenic mice.

소 대형의 강력한 셀 모델로 기본 신경 하 Microsomes 관련 알파 synuclein 집계의 외 인 관리

For years, the inability of replicating formation of insoluble alpha-synuclein (&#945;S) inclusions in cell cultures has been a great limitation in the ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

1 차 배아 마우스 중뇌 도파민 뉴런에서 사전 형성 된 피브릴 유도 α-시뉴클레인 축적 연구

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

쥐 성상 세포와 미세 아교 세포의 1 차 배양과 근 위축성 측삭 경화증 연구에서의 사용

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

세포의 잘못 폴딩 된 단백질의 분해에 대한 분석

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

생물학

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

형광 상관 분광법을 사용하여 단백질 응집 감지

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated &#945;-synuclein. &#945;-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of &#945;-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

An in vivo physiological model of &#945;-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of &#945;-synuclein using a humanized yeast model.

인간화 효모 모델을 이용한 α-시뉴클레인 독성 및 응집을 연구하는 방법

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

Protein Aggregate Formation Assay: A Method to Detect and Quantify Protein Aggregation in Cultured Cells upon Induction by Proteasome Inhibitor

Source: Lee, H. et al. <a href="https://www.jove.com/v/56425">Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells.</a> J. Vis. Exp. (2017)
In this video, we demonstrate a cell-based protein aggregation assay using proteasome inhibitors, which block proteasome activity, preventing misfolded, mutant proteins, fused to a fluorescent label, from undergoing ubiquitin-dependent proteasomal degradation, leading to their accumulation within the cell cytoplasm. The protein aggregates are then visualized and quantified by fluorescence microscopy.

Protein Aggregate Formation Assay: 프로테아솜 억제제에 의한 유도 시 배양된 세포에서 단백질 응집을 검출하고 정량화하는 방법

Source: Lee, H. et al. Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells. J. Vis. Exp. (2017)
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