Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function
Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function
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DOI：
10.1074/jbc.m114.621706
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发表时间：
2015-06-05
影响因子：
4.8
通讯作者：
Cotman, Susan L.
中科院分区：
文献类型：
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作者：
Chandrachud, Uma;Walker, Mathew W.;Cotman, Susan L.
未消化大分子的异常积累（通常是疾病特异性的）是溶酶体和神经退行性疾病的主要特征，通常归因于自噬缺陷。自噬缺陷的机制基础是深入研究的主题，并得到遗传疾病模型的帮助。为了更好地了解调节缺陷自噬的途径，特别是在幼年神经元蜡样质脂褐素沉着症（JNCL 或 Batten 病）（一种儿童神经退行性疾病）中，我们开发并试验了 GFP 微管相关蛋白 1 轻链 3 (GFP-LC3) 筛选试验，以公正的方式识别基因型敏感的小细胞。 分子自噬调节剂，采用带有 CLN3 中最常见疾病突变的 JNCL 神经元细胞模型。 Thapsigargin 是一种肌浆/内质网 Ca2+-ATP 酶 (SERCA) Ca2+ 泵抑制剂，在小鼠 JNCL 细胞中可重复地表现出显着更高的活性，在人诱导的多能干细胞衍生的 JNCL 神经祖细胞中也观察到了这种效应。毒胡萝卜素敏感性的机制是Ca2+介导的，JNCL细胞中自噬体的积累可以通过Ca2+螯合来逆转。对细胞内 Ca2+ 处理的研究突出显示了 JNCL 细胞中内质网、线粒体和溶酶体 Ca2+ 池以及储存操作的 Ca2+ 摄取的变化。这些结果进一步支持了 CLN3 蛋白在细胞内 Ca2+ 处理和自噬途径通量中的重要作用，并为治疗筛选建立了一个强大的新平台。
Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca2+-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca2+ chelation. Interrogation of intracellular Ca2+ handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca2+ pools and in store-operated Ca2+ uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca2+ handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening.