Development of a specific live-cell assay for native autophagic flux.
Development of a specific live-cell assay for native autophagic flux.
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一种针对内源性自噬流的特异性活细胞检测方法的开发。
DOI：
10.1016/j.jbc.2021.101003
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发表时间：
2021-09
期刊：
影响因子：
--
通讯作者：
Barmada SJ
中科院分区：
文献类型：
--
作者：
Safren N;Tank EM;Malik AM;Chua JP;Santoro N;Barmada SJ
自噬是一种进化上保守的介导细胞蛋白质和细胞器分解的途径。强调其关键性质，自噬功能障碍有助于许多疾病;然而，有效的自噬调节药物的开发受到测量自噬活性或通量的可用方法的根本缺陷的阻碍。为了克服这些局限性，我们通过CRISPR/Cas9基因组编辑将光转化蛋白Dendra 2引入人类细胞的MAP 1 LC 3B位点，从而通过光脉冲标记准确和灵敏地评估活细胞中的自噬。我们使用这种方法对四种化学文库进行高通量药物筛选，其中包括超过30，000种不同的化合物，鉴定了几种临床相关药物和新型自噬调节剂。一系列选定的候选化合物也调节了通过CRISPR/Cas9修饰以表达GFP标记的LC 3的人类运动神经元中的自噬通量。使用自动显微镜，我们在肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）的几个不同的神经元模型中测试了自噬诱导的治疗潜力。在这样做的过程中，我们发现自噬诱导表现出不一致的效果，提高了涉及RNA结合蛋白TDP-43的疾病模型的生存率，同时加剧了表达与家族性ALS/FTD相关的UBQLN 2和C9 ORF 72突变形式的神经元的毒性。这些研究证实了Dendra 2-LC 3检测的实用性，同时说明了不同ALS/FTD亚型中自噬诱导的矛盾效应。
Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction contributes to many diseases; nevertheless, development of effective autophagy modulating drugs is hampered by fundamental deficiencies in available methods for measuring autophagic activity or flux. To overcome these limitations, we introduced the photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of autophagy in living cells by optical pulse labeling. We used this assay to perform high-throughput drug screens of four chemical libraries comprising over 30,000 diverse compounds, identifying several clinically relevant drugs and novel autophagy modulators. A select series of candidate compounds also modulated autophagy flux in human motor neurons modified by CRISPR/Cas9 to express GFP-labeled LC3. Using automated microscopy, we tested the therapeutic potential of autophagy induction in several distinct neuronal models of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In doing so, we found that autophagy induction exhibited discordant effects, improving survival in disease models involving the RNA binding protein TDP-43, while exacerbating toxicity in neurons expressing mutant forms of UBQLN2 and C9ORF72 associated with familial ALS/FTD. These studies confirm the utility of the Dendra2-LC3 assay, while illustrating the contradictory effects of autophagy induction in different ALS/FTD subtypes.
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影响因子：
8.8
作者：
Flores, Brittany N.;Li, Xingli;Barmada, Sami J.
通讯作者：
Barmada, Sami J.
DOI：
10.1126/science.aaa3650
发表时间：
2015-03-27
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Cirulli ET;Lasseigne BN;Petrovski S;Sapp PC;Dion PA;Leblond CS;Couthouis J;Lu YF;Wang Q;Krueger BJ;Ren Z;Keebler J;Han Y;Levy SE;Boone BE;Wimbish JR;Waite LL;Jones AL;Carulli JP;Day-Williams AG;Staropoli JF;Xin WW;Chesi A;Raphael AR;McKenna-Yasek D;Cady J;Vianney de Jong JM;Kenna KP;Smith BN;Topp S;Miller J;Gkazi A;FALS Sequencing Consortium;Al-Chalabi A;van den Berg LH;Veldink J;Silani V;Ticozzi N;Shaw CE;Baloh RH;Appel S;Simpson E;Lagier-Tourenne C;Pulst SM;Gibson S;Trojanowski JQ;Elman L;McCluskey L;Grossman M;Shneider NA;Chung WK;Ravits JM;Glass JD;Sims KB;Van Deerlin VM;Maniatis T;Hayes SD;Ordureau A;Swarup S;Landers J;Baas F;Allen AS;Bedlack RS;Harper JW;Gitler AD;Rouleau GA;Brown R;Harms MB;Cooper GM;Harris T;Myers RM;Goldstein DB
通讯作者：
Goldstein DB
影响因子：
6.1
作者：
Evans CS;Holzbaur ELF
通讯作者：
Holzbaur ELF
影响因子：
14.8
作者：
Barmada, Sami J.;Serio, Andrea;Arjun, Arpana;Bilican, Bilada;Daub, Aaron;Ando, D. Michael;Tsvetkov, Andrey;Pleiss, Michael;Li, Xingli;Peisach, Daniel;Shaw, Christopher;Chandran, Siddharthan;Finkbeiner, Steven
通讯作者：
Finkbeiner, Steven
影响因子：
16.6
作者：
Chauhan S;Ahmed Z;Bradfute SB;Arko-Mensah J;Mandell MA;Won Choi S;Kimura T;Blanchet F;Waller A;Mudd MH;Jiang S;Sklar L;Timmins GS;Maphis N;Bhaskar K;Piguet V;Deretic V
通讯作者：
Deretic V