저자는 기술 지원에 대 한 연구소의 휴대 및 Organismic 생물학, 학계 Sinica의 핵심 시설을 감사합니다. 이 연구는 과학과 기술, 대만 (Y. f 가장 103-2320-B-001-016-MY3에 의해 지원 되었다 L.), 의약품 개발-기술 플랫폼 축 지원의 변환 혁신 프로그램 (Y. 플로리다에 NP7) 체계, 그리고 학계 Sinica (하 영-플로리다).

1. 해당 애플 리 케이 션-C99 또는 NΔE의 γ-Secretase 분열 하는 Luciferase 기자 신호 측정
참고: CG과 셀20,21의 세대에 대 한 자세한 설명에 대 한 이전 게시를 참조 하십시오.
<ol><li>
NG 또는 CG 셀 씨앗 (20, 000 셀/잘) 200 μ/잘 Dulbecco의 수정이 글 중간 (DMEM) 플러스 10% 태아 둔감 한 혈 청 (FBS), 5 μ g/mL blasticidin, 250 μ g/mL 항생제 (예: 구성 된 성장 매체에서의 마지막 볼륨에 96-잘 microplates에 zeocin), 그리고 200 μ g/mL hygromycin B.
	<ol>
<li>hemocytometer 셀을 계산 합니다.</li>
		<li>습도 CO2 배양 기에는 microplates를 전송 하 고 37 ° C에서 밤새 품 어.</li>
	</ol>
</li>
	<li>각 우물에서 성장 매체의 100 μ를 제거 합니다.</li>
	<li>성장의 100 μ/잘 추가 중 0.2% 디 메 틸 sulfoxide (DMSO), 2 μ M와 2 μ g/mL 항생물질을 포함 하 고 중간 CL-387,785, 2 μ M N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT), 또는 기타 관련 ErbB1/ErbB2 억제제.</li>
	<li>치료 CG 나 니 셀 24 h에 대 한 37 ° C에서 microplates에 품 어.</li>
	<li>Microplates에서 150 μ/잘 성장 매체를 제거 합니다.</li>
	<li>추가 50 μ/잘 luciferase 분석 시 약 ( 테이블의 자료를 참조 하시기 바랍니다).</li>
	<li>
발광 microplate 리더에는 microplates를 전송.
	<ol>
<li>부드러운 동요와 5 분 동안 실 온에서 microplates을 유지 관리 합니다.</li>
	</ol>
</li>
	<li>
반딧불 luciferase (FL) 결정-발광 발광 microplate 리더에 저장 된 미리 정의 된 프로그램을 사용 하 여 방출.
	<ol>
<li>인스트루먼트 컨트롤 소프트웨어를 열고 (악기 자세한 재료의 표 참조).</li>
		<li>'도구' 메뉴에서 'Luminometry'를 선택 합니다.<ol>
<li>'매개 변수' 설정에서 세 시간에 대 한 방출 필터, 방출 조리개에 대 한 진드기 '정상' 입력 '1' '필터'를 선택 하 고 발광 읽기를 설정 하려면 '확인'을 클릭 합니다.</li>
		</ol>
</li>
		<li>'악기 제어' 탭에서 활성 프로토콜 목록 하 고 발광을 읽기 위한 사전 저장된 프로토콜을 선택 합니다.</li>
		<li>'라이브 디스플레이' 탭 아래 풀 다운 목록에서 확장을 정의 하 고 형식에 대 한 '로그' 또는 '선'을 선택 합니다.</li>
		<li>'온도' 탭에서 접시 난방에 대 한 '끄기' 선택 합니다.</li>
		<li>읽고 발광을 수행 하려면 '시작' 버튼을 클릭 합니다.</li>
		<li>필요에 따라, 제어 소프트웨어 내에 포함 된 '탐색기'를 사용 하 여 스프레드시트 형식으로 결과 내보냅니다.</li>
	</ol>
</li>
</ol>2입니다. γ-Secretase 활동의 정량화
<ol><li>
CG에 의해 방출 하는 발광 신호 또는 NG 셀 100% 상대 γ-secretase 활동으로 혼자 1 μ g/mL 항생물질을 포함 하는 성장 매체에 0.1 %DMSO 치료를 정의 합니다.
	<ol>
<li>성장 매체 항생물질을 포함 하지 않는 세포를 치료 하 여 cg로 또는 NG 셀에서 배경 발광을 견적 한다.</li>
	</ol>
</li>
	<li>Luciferase 신호 1 μ g/mL 항생물질을 포함 하는 성장 매체와 교양 하는 CG 나 NG 셀에서 정상화 하 고 셀 DMSO 치료 CG 또는 NG에 의해 방출 된 luciferase 신호 DAPT의 어느 1 μ M CL-387,785 또는 1 μ M (100% 상대 γ-secretase 활동으로 2.1에서 정의).</li>
</ol>

<ol>
	<li>Selkoe, D. J., Hardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease+at+25+years.">The amyloid hypothesis of Alzheimer's disease at 25 years.</a> EMBO Mol Med. 8 (6), 595-608 (2016).</li><li>Wolfe, M. S., Guenette, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+at+a+glance.">APP at a glance.</a> J Cell Sci. 120 (Pt 18), 3157-3161 (2007).</li><li>Karran, E., Mercken, M., De Strooper, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+cascade+hypothesis+for+Alzheimer's+disease:+an+appraisal+for+the+development+of+therapeutics.">The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics.</a> Nat Rev Drug Discov. 10 (9), 698-712 (2011).</li><li>Citron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+strategies+for+disease+modification.">Alzheimer's disease: strategies for disease modification.</a> Nat Rev Drug Discov. 9 (5), 387-398 (2010).</li><li>Vassar, R., Kovacs, D. M., Yan, R., Wong, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-secretase+enzyme+BACE+in+health+and+Alzheimer's+disease:+regulation,+cell+biology,+function,+and+therapeutic+potential.">The beta-secretase enzyme BACE in health and Alzheimer's disease: regulation, cell biology, function, and therapeutic potential.</a> J Neurosci. 29 (41), 12787-12794 (2009).</li><li>Parks, A. L., Curtis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presenilin+diversifies+its+portfolio.">Presenilin diversifies its portfolio.</a> Trends Genet. 23 (3), 140-150 (2007).</li><li>Kukar, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-targeting+gamma-secretase+modulators.">Substrate-targeting gamma-secretase modulators.</a> Nature. 453 (7197), 925-929 (2008).</li><li>Kounnas, M. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+gamma-secretase+reduces+beta-amyloid+deposition+in+a+transgenic+mouse+model+of+Alzheimer's+disease.">Modulation of gamma-secretase reduces beta-amyloid deposition in a transgenic mouse model of Alzheimer's disease.</a> Neuron. 67 (5), 769-780 (2010).</li><li>Thathiah, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orphan+G+protein-coupled+receptor+3+modulates+amyloid-beta+peptide+generation+in+neurons.">The orphan G protein-coupled receptor 3 modulates amyloid-beta peptide generation in neurons.</a> Science. 323 (5916), 946-951 (2009).</li><li>Flajolet, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Alzheimer's+disease+amyloid-beta+formation+by+casein+kinase+I.">Regulation of Alzheimer's disease amyloid-beta formation by casein kinase I.</a> Proc Natl Acad Sci U S A. 104 (10), 4159-4164 (2007).</li><li>Eisele, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gleevec+increases+levels+of+the+amyloid+precursor+protein+intracellular+domain+and+of+the+amyloid-beta+degrading+enzyme+neprilysin.">Gleevec increases levels of the amyloid precursor protein intracellular domain and of the amyloid-beta degrading enzyme neprilysin.</a> Mol Biol Cell. 18 (9), 3591-3600 (2007).</li><li>He, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-secretase+activating+protein+is+a+therapeutic+target+for+Alzheimer's+disease.">Gamma-secretase activating protein is a therapeutic target for Alzheimer's disease.</a> Nature. 467 (7311), 95-98 (2010).</li><li>Haapasalo, A., Kovacs, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+substrates+of+presenilin/gamma-secretase.">The many substrates of presenilin/gamma-secretase.</a> J Alzheimers Dis. 25 (1), 3-28 (2011).</li><li>Gillman, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+Evaluation+of+BMS-708163,+a+Potent,+Selective+and+Orally+Bioavailable+gamma-Secretase+Inhibitor.">Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable gamma-Secretase Inhibitor.</a> ACS Med Chem Lett. 1 (3), 120-124 (2010).</li><li>Hopkins, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACS+chemical+neuroscience+molecule+spotlight+on+Begacestat+(GSI-953).">ACS chemical neuroscience molecule spotlight on Begacestat (GSI-953).</a> ACS Chem Neurosci. 3 (1), 3-4 (2012).</li><li>Yang, T., Arslanova, D., Gu, Y., Augelli-Szafran, C., Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+gamma-secretase+modulation+differentiates+inhibitor+compound+selectivity+between+two+substrates+Notch+and+amyloid+precursor+protein.">Quantification of gamma-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein.</a> Mol Brain. 1, 15 (2008).</li><li>McKee, T. D., Loureiro, R. M., Dumin, J. A., Zarayskiy, V., Tate, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+cell-based+method+for+determining+the+gamma-secretase+enzyme+activity+against+both+Notch+and+APP+substrates.">An improved cell-based method for determining the gamma-secretase enzyme activity against both Notch and APP substrates.</a> J Neurosci Methods. 213 (1), 14-21 (2013).</li><li>Basi, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+selective+gamma-secretase+inhibitors+for+treatment+of+Alzheimer's+disease.">Amyloid precursor protein selective gamma-secretase inhibitors for treatment of Alzheimer's disease.</a> Alzheimers Res Ther. 2 (6), 36 (2010).</li><li>Liao, Y. F., Tang, Y. C., Chang, M. Y., Wang, B. J., Hu, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+small+molecular+(D)-leucinamides+as+potent,+Notch-sparing+gamma-secretase+modulators.">Discovery of small molecular (D)-leucinamides as potent, Notch-sparing gamma-secretase modulators.</a> Eur J Med Chem. 79, 143-151 (2014).</li><li>Liao, Y. F., Wang, B. J., Cheng, H. T., Kuo, L. H., Wolfe, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-alpha,+interleukin-1beta,+and+interferon-gamma+stimulate+gamma-secretase-mediated+cleavage+of+amyloid+precursor+protein+through+a+JNK-dependent+MAPK+pathway.">Tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma stimulate gamma-secretase-mediated cleavage of amyloid precursor protein through a JNK-dependent MAPK pathway.</a> J Biol Chem. 279 (47), 49523-49532 (2004).</li><li>Wang, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ErbB2+regulates+autophagic+flux+to+modulate+the+proteostasis+of+APP-CTFs+in+Alzheimer's+disease.">ErbB2 regulates autophagic flux to modulate the proteostasis of APP-CTFs in Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 114 (15), E3129-E3138 (2017).</li><li>Sevigny, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibody+aducanumab+reduces+Abeta+plaques+in+Alzheimer's+disease.">The antibody aducanumab reduces Abeta plaques in Alzheimer's disease.</a> Nature. 537 (7618), 50-56 (2016).</li><li>Golde, T. E., Koo, E. H., Felsenstein, K. M., Osborne, B. A., Miele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Secretase+inhibitors+and+modulators.">gamma-Secretase inhibitors and modulators.</a> Biochim Biophys Acta. 1828 (12), 2898-2907 (2013).</li><li>Coric, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Prodromal+Alzheimer+Disease+With+Avagacestat:+A+Randomized+Clinical+Trial.">Targeting Prodromal Alzheimer Disease With Avagacestat: A Randomized Clinical Trial.</a> JAMA Neurol. 72 (11), 1324-1333 (2015).</li><li>Mitani, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+between+gamma-secretase+inhibitors+and+modulators+on+cognitive+function+in+amyloid+precursor+protein-transgenic+and+nontransgenic+mice.">Differential effects between gamma-secretase inhibitors and modulators on cognitive function in amyloid precursor protein-transgenic and nontransgenic mice.</a> J Neurosci. 32 (6), 2037-2050 (2012).</li><li>Penninkilampi, R., Brothers, H. M., Eslick, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Agents+Targeting+gamma-Secretase+Increase+Risk+of+Cancer+and+Cognitive+Decline+in+Alzheimer's+Disease+Patients:+A+Systematic+Review+and+Meta-Analysis.">Pharmacological Agents Targeting gamma-Secretase Increase Risk of Cancer and Cognitive Decline in Alzheimer's Disease Patients: A Systematic Review and Meta-Analysis.</a> J Alzheimers Dis. 53 (4), 1395-1404 (2016).</li></ol>

저자는 공개 없다.

광고에 대 한 aducanumab immunotherapy의 단계 1b 재판에서 유망한 결과 단단히 광고22 의 병 인에 Aβ의 중요 한 역할을 설립 하 고 안티-Aβ 접근은 여전히 안티 광고 약물의 개발에 대 한 실행 가능한 전략을 제안. 여기, 우리는 애플 리 케이 션-C99 및 반딧불 luciferase 기자 시스템을 사용 하 여 NΔE의 γ-secretase 촉매 베이스 측정을 위한 세포 기반 분석 설명 합니다. 응용 프로그램-C99 및 NΔE?...

아 밀 로이드 β (Aβ)의 oligomeric 모양은 neurodegeneration Alzheimer의 질병 (광고)1에서 고통 받는 환자의 두뇌에서의 주요 원인이 될 것으로 추정 된다. Aβ 펩 티 드 그리고 γ-secretase2β-secretase에 의해 처음으로 녹말 체 선구자 단백질 (APP)의 stepwise 분열에 의해 생산 됩니다. 지난 10 년간에서 광고의 치료로 치료 접근 Aβ 생산3의 예방에 집중 했다. 연구의 대부분은 애플 리 케이 션의 γ-secretase 분열을 배제 하 고 그로 인하여 Aβ의 생산 또는 β-및/또는 γ-secretase 활동4의 금지를 감소 수 있는 α-secretase 활동의 확대에 집중 했다. 불행히도, 피할 수 없는 부작용에 β 또는 γ secretases 결과의 비 선택적인 억제는 β와 γ-secretase5,6의 다른 생리 적인 기판의 기능 방해 예정 이다. Γ-secretase 억제제에 관한 최근 연구 화학 및 유전 변조기 노치7의 필수적인 γ-secretase 중재 처리에 사소한 영향을 발휘 하는 동안 Aβ 생산을 통제할 수 있는 수의 발견 보고 그러나 ,,89,10,11,12,, 성공적인 치료 아직 개발 하지 않은. 따라서, 더 체계적인 스크린은 보증 소설 유전과 화학 한정자 선택적으로 γ-secretase 중재 애플 리 케이 션 처리를 조절 수 있습니다 발견.
Γ-Secretase 이상의 90 다른 막 정박 단백질을 쪼개로 알려져 있다. 이러한 기판 중 그 활동은 세포 운명 결정과 분화 개발13과정에 대 한 중요 한 단계가입니다. 선택적으로 변조 γ-secretase 촉매 응용 프로그램 부재에 처리에 영향을 미치는 노치의 처리 부작용에 대 한 최소화 노치 관련 γ-secretase 억제제의 필수적인 되며이 선택도 기본 요소가 될 것으로 생각 됩니다. 광고의 만성 치료에 대 한 잠재적인 γ-secretase 변조기의 생물학적 효능을 지정 합니다. 다양 한 arylsulfonamide 유도체, GSI-953 (begacestat) 등 BMS-708163 (avagacestat), γ-secretase14,15의 강력 하 고 선택적인 억제를 전시 발견 되었습니다 되었습니다. 이전 연구는 APP와 노치의 γ-secretase 분열의 차동 저해 조합 vivo에서 유효성 검사 zebrafish16를 사용 하 여 양이 많은 ELISA 기반 분석 결과 사용 하 여 관찰 될 수 있다 설명 했다. 또한, 세포 기반 분석 결과 패러다임 γ-secretase 애플 리 케이 션 및 노치17,18대의 잠재적인 기판 선택도 해결 하기 위해 설립 되었습니다. 여기 설명한 비슷한 프로토콜을 사용 하 여, 우리는 최근에 발견 (D)의 소설 클래스-potently 노치를 살려주는 선택도19APP의 γ-secretase 분열을 조절 하는 leucinamides. 함께,이 연구의 제공의 증거-개념 γ-secretase의 기판 선택/가용성 화학 변조 및 실험 검증 대상이 될 수 있는 개념을 지 원하는 됩니다. 그러나, 이러한 분석 결과 플랫폼 종종 상대적으로 낮은 처리량 판독 및 약물 발견 프로그램에 대 한 산업 표준에 맞지 수 있습니다 노동 집약적인 접근에 의존 합니다.
우리는 최근 셀 기반 luciferase 취재 원 유전자 분석 실험 양적 γ-secretase 두 가지 기판, APP (응용 프로그램-C99)의 99 아미노 산 성 C 터미널 단편과는 세포 외의 촉매 활동을 확인할 수 있는 생성 된 도메인 삭제 노치 펩 티 드 (NΔE)입니다. 애플 리 케이 션-C99 및 NΔE γ-secretase 다양 한 분석 실험에서의 직접 기판으로 널리 사용 되었습니다 있다. 균일 하 고 일관 된 luciferase 리포터 유전자 분석 애플 리 케이 션-C99 또는 NΔE의 γ-secretase 분열에 대 한 생성, 우리 생성 한 HEK 파생 안정적인 셀 라인 (CG) constitutively Gal4 발기인 구동 반딧불 luciferase 기자 (표현 하 Gal4-Luc)와 Gal4/VP16 태그 애플 리 케이 션-C99 융합 단백질 (C99-GV). 또한, constitutively Gal4 루크와 Gal4/VP16 태그 NΔE (NΔE-GV)를 표현 하는 또 다른 HEK 파생 안정적인 셀 라인 (NG)를 생성 했습니다. 이러한 새로운 기판 전용 γ-secretase 분석 실험을 사용 하 여, 우리 이제 계량 하 고 γ-secretase 별개 기판 (애플 리 케이 션-C99 CG 세포에서) 또는 NG 셀에 NΔE로의 촉매 활동을 구별 하는 방법을 제공 합니다. 또한, 기판 전용 γ-secretase 분석 실험이 콘텐츠 심사 플랫폼에 도움이 되도록 설계 되었습니다. 이 새로 생성 된 γ-secretase 분석 도출 다음-세대 치료에 대 한 바람직하지 않은 노치 저해 하지 않고 Aβ 생산을 억제 하 여 인지 기능을 향상 시킬 수 있습니다. 있는 소설 γ-secretase 변조기의 발견에 대 한 길을 닦은 수 있습니다. 광고.

<table>
	<tbody>
		<tr>
			<td>VictorLight luminescence plate reader</td>
			<td>PerkinElmer</td>
			<td>2030-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II, Type 2 Biological Safety Cabinet</td>
			<td>Thermo Scientific</td>
			<td>1300 Series A2</td>
			<td></td>
		</tr>
		<tr>
			<td>CL-387,785</td>
			<td>EMD Calbiochem</td>
			<td>233100-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Merck</td>
			<td>565770</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher</td>
			<td>12100046</td>
			<td>main compnent of growth medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Thermo Fisher</td>
			<td>A1113903</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>Thermo Fisher</td>
			<td>R25005</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Gibco</td>
			<td>10687010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytomer</td>
			<td>Sigma-Aldrich</td>
			<td>BR717805 Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>Nunc</td>
			<td>156545</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Sigma</td>
			<td>T7660</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Steady-Glo luciferase assay reagent</td>
			<td>Promega</td>
			<td>E2510</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified CO2 incubator</td>
			<td>Revco</td>
			<td>Ultima II</td>
			<td></td>
		</tr>
		<tr>
			<td>T-REx293 cell line</td>
			<td>Invitrogen</td>
			<td>R71007</td>
			<td></td>
		</tr>
		<tr>
			<td>Wallac 1420 software version 3.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>instrument control software of the luminescence microplate reader</td>
		</tr>
	</tbody>
</table>

quantitative measurement of &#947;-secretase-mediated amyloid precursor protein and notch cleavage in cell-based luciferase reporter assay platforms

우리는 양적 측정 별개 기판의 γ-secretase 분열 하 셀 기반 리포터 유전자 분석 실험의 한 쌍을 개발 했습니다. 이 원고는 애플 리 케이 션-C99 또는 Gal4 발기인 구동 반딧불 luciferase 기자 시스템을 사용 하 여 노치의 γ-secretase 중재 분열을 모니터링 하는 데 사용할 수 있는 절차를 설명 합니다. 이 분석 실험 안정적으로 공동 Gal4 구동 luciferase 취재 원 유전자와 응용 프로그램 (애플 리 케이 션-C99;의 C-터미널 Gal4/VP16 태그 조각 HEK293 세포 transfecting에 의해 설립 되었다 Cg로 셀), 또는 Gal4/VP16 태그 노치-ΔE (NΔE; NG 셀)입니다. 동시에 이러한 기자 분석 실험을 사용 하 여, 우리는 증명 하고있다 ErbB2 억제제, CL-387,785, 우선적으로 CG 셀에 애플 리 케이 션-C99 하지만 NG 셀에 NΔE 하지의 γ-secretase 분열을 억제 하실 수 있습니다. CL-387,785로 치료 하면 CG과 세포에 의해 전시 하는 차동 응답 γ-secretase 변조기에 대 한 선호 특성 나타내고 이러한 응답 γ-secretase DAPT에 의해 유도 된 팬 억제에 강한 대조에 있다. 우리의 연구는 다른 기판으로 γ-secretase 활동 세포 맥락에서 분화 될 수 있다 직접적인 증거를 제공 합니다. 이러한 새로운 분석 따라서 향상 된 광고 치료를 위한 약물 발견에 유용한 도구를 수 있습니다.

CL 387,785에 의해 ErbB2 저해에 의해 애플 리 케이 션-C99의 처리 홍보 차동 수 있습니다. Γ -secretase 노치 분열에 영향을 주지 않고 양적 특정 γ-secretase 기판의 분해 분열을 측정할 수 있는 세포 기반 분석 결과 설정 하는 항생물질을 유도할 수 있는 C 말기 Gal4/VP16 태그의 안정적인 공동 transfection에 의해 HEK293 파생 CG 셀 라인 생성 응용 프?...

Watch this Scientific Journal Video about Quantitative Measurement of ÃŽÂ³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

우리는 성공적으로 두 기판 전용 γ-secretase 분석 생성 된 있다. 여기에 제시 된 두 세포 기반 분석은 계량 반딧불 luciferase 기자 들의 출력을 통해 γ-secretase 효소 활동 설계 되었습니다.

Γ-Secretase 중재 녹말 체 선구자 단백질 및 세포 기반 Luciferase 기자 시험 플랫폼에 노치 분열의 정량적 측정

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript ...

quantitative-measurement-secretase-mediated-amyloid-precursor-protein

Research

JoVE Journal

Neuroscience

6.7K 조회수.  Academia Sinica. 우리는 성공적으로 두 기판 전용 γ-secretase 분석 생성 된 있다. 여기에 제시 된 두 세포 기반 분석은 계량 반딧불 luciferase 기자 들의 출력을 통해 γ-secretase 효소 활동 설계 되었습니다.

비디오: Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

아밀로이드 β 단백질, 알츠하이머 병의 원인이되는 에이전트에 대한 Aptamers의 선택

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

연구

신경과학

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

손상의 감각 오르간 전구체 세포 라이브 셀 이미징 Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

외부 응용 직류 전기 분야에 신경 전구체 세포 마이그레이션 속도론 분석을위한 Galvanotaxis 분석

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

루시 페라 제 활동을 통해 수용체 활성화의 측정 : 포유류의 후각 수용체의 높은 처리량 분석

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

검출<em&gt; 현장에서</em상기&gt; 단백질 - 단백질 복합체<em&gt; 초파리</em근접 내고 분석을 사용하기&gt; 애벌레 신경 근육 접합부

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

마우스 해마에 올리고머 아밀로이드 - 베타의 정위 주입

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

F9를 RARE-LacZ를 셀 기반 리포터 분석을 사용하여 E8.5 배아 및 Neurosphere 문화에서 상대 레티노 산 수준의 정량적 측정

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

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

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

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 ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

생체 내 뇌 단백질 합성의 지역 비율 측정을 위한 정량적 자가 방사선 측정 방법

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening

The tau aggregation assay described in this manuscript mimics the anticipated features of in vivo tau misfolding and aggregation.

생체 외에서 타우 단백질 및 마약 검사의 집계를 공부에 대 한 분석 결과

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer&#39;s disease and other tauopathies. Compared to other ...