Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function

• Published in: The Journal of biological chemistry 2015-06, Vol.290 (23), p.14361-14380
• Main Authors: Chandrachud, Uma, Walker, Mathew W., Simas, Alexandra M., Heetveld, Sasja, Petcherski, Anton, Klein, Madeleine, Oh, Hyejin, Wolf, Pavlina, Zhao, Wen-Ning, Norton, Stephanie, Haggarty, Stephen J., Lloyd-Evans, Emyr, Cotman, Susan L.
• Format: Article
• Language: English
• Subjects: Animals; autophagy; Autophagy - drug effects; calcium; Calcium - metabolism; Cell Line; Cells, Cultured; chemical biology; CLN3; Drug Evaluation, Preclinical; endoplasmic reticulum (ER); GFP-LC3; Humans; juvenile neuronal ceroid lipofuscinosis (JNCL); lysosomal storage disease; lysosome; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Mice; Molecular Bases of Disease; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Mutation; Neural Stem Cells - metabolism; Neural Stem Cells - pathology; Neuronal Ceroid-Lipofuscinoses - drug therapy; Neuronal Ceroid-Lipofuscinoses - genetics; Neuronal Ceroid-Lipofuscinoses - metabolism; Neuronal Ceroid-Lipofuscinoses - pathology; Signal Transduction - drug effects; thapsigargin
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.621706

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. Background: CLN3 protein function is still unknown, but its loss causes Batten disease. Results: Drug screening in a Batten disease model was developed to identify modifiers of altered cellular pathways. Conclusion: Alterations in Ca2+ handling are implicated in Batten disease, which may negatively influence the intracellular pathways regulated by Ca2+. Significance: A proof-of-concept is established for the application of drug screening to Batten disease research.