a Department of Neurology , University of California at Los Angeles , Los Angeles , CA , USA.
b Molecular Toxicology Program , University of California at Los Angeles , Los Angeles , CA , USA.
Autophagy. 2019 Aug;15(8):1322-1332. doi: 10.1080/15548627.2019.1580511. Epub 2019 Mar 1.
The pathophysiology of most neurodegenerative diseases includes aberrant accumulation of protein aggregates. Recent evidence highlights the role of protein degradation pathways in neurodegeneration. Concurrently, genetic tools have been generated to enable zebrafish, Danio rerio, to be used as an animal model to study neurodegenerative processes. In addition to optical clarity and fast ex utero development, the zebrafish brain is relatively small and has conserved structures with its mammalian counterparts. To take advantage of this model organism and to aid further studies on autophagy and neurodegeneration, we created a stable transgenic zebrafish line that expresses eGFP-Map1lc3b specifically in post-mitotic neurons under the elavl3 promoter. This line is useful for indirectly monitoring autophagic activity in neurons in vivo and screening for macroautophagy/autophagy-modulating compounds. We determined the applicability of this transgenic line by modulating and quantifying the number of autophagosomes via treatment with a known autophagy inducer (rapamycin) and inhibitors (3-methyladenine, protease inhibitors). Additionally, we proposed an in vivo method for quantifying rates of autophagosome accumulation, which can be used to infer occurrence of autophagic flux. Last, we tested two FDA-approved drugs currently undergoing clinical studies for Parkinson disease, isradipine and nilotinib, and found that isradipine did not modulate autophagy, whereas nilotinib induced both autophagosome number and autophagic flux. It is hoped that others will find this line useful as an in vivo vertebrate model to find or validate autophagy modulators that might be used to halt the progression of neurodegenerative diseases. 3MA: 3-methyladenine; BafA: bafilomycin A1; dd: dorsal diencephalon; dpf: days post fertilization; e: eye; eGFP: enhanced green fluorescent protein; Elavl3: ELAV like neuron-specific RNA binding protein 3; FDA: Food and Drug Administration; hb: habenula; hpt, hours post treatment; Map1lc3b: microtubule-associated protein 1 light chain 3 beta; nt: neural tube; ot, optic tectum; P/E: pepstatin A and E64d; PD: Parkinson disease; PMTs: photomultiplier tubes; PTU: 1-phenyl-2-thiourea; Ta: annealing temperature; Tel, telencephalon.
大多数神经退行性疾病的病理生理学包括蛋白质聚集物的异常积累。最近的证据强调了蛋白质降解途径在神经退行性变中的作用。同时，遗传工具已经被开发出来，使斑马鱼（Danio rerio）能够被用作研究神经退行性过程的动物模型。除了光学清晰度和快速的体外发育外，斑马鱼的大脑相对较小，并且与哺乳动物具有保守的结构。为了利用这个模式生物，并进一步研究自噬和神经退行性变，我们创建了一个稳定的转基因斑马鱼系，该系在 elavl3 启动子下特异性地在有丝分裂后神经元中表达 eGFP-Map1lc3b。该系可用于在体内间接监测神经元中的自噬活性，并筛选巨自噬/自噬调节化合物。我们通过用已知的自噬诱导剂（雷帕霉素）和抑制剂（3-甲基腺嘌呤、蛋白酶抑制剂）处理来调节和量化自噬体的数量，从而确定了该转基因系的适用性。此外，我们提出了一种体内方法来量化自噬体积累的速率，该速率可用于推断自噬流的发生。最后，我们测试了两种正在进行帕金森病临床研究的 FDA 批准药物，即异搏定和尼洛替尼，并发现异搏定不能调节自噬，而尼洛替尼诱导自噬体数量和自噬流。希望其他人会发现该系作为一种体内脊椎动物模型有用，以找到或验证可能用于阻止神经退行性疾病进展的自噬调节剂。 3MA：3-甲基腺嘌呤；BafA：巴弗霉素 A1；dd：背侧间脑；dpf：受精后天数；e：眼；eGFP：增强型绿色荧光蛋白；Elavl3：ELAV 样神经元特异性 RNA 结合蛋白 3；FDA：美国食品和药物管理局；hb：缰核；hpt：处理后小时；Map1lc3b：微管相关蛋白 1 轻链 3β；nt：神经管；ot：视顶盖；P/E：胃蛋白酶抑制剂 A 和 E64d；PD：帕金森病；PMTs：光电倍增管；PTU：1-苯基-2-硫脲；Ta：退火温度；Tel：端脑。
