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

Abstract
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 Ca(2+)-ATPase (SERCA) Ca(2+) 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 Ca(2+)-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca(2+) chelation. Interrogation of intracellular Ca(2+) handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca(2+) pools and in store-operated Ca(2+) uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca(2+) handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening.
AuthorsUma Chandrachud, Mathew W Walker, Alexandra M Simas, Sasja Heetveld, Anton Petcherski, Madeleine Klein, Hyejin Oh, Pavlina Wolf, Wen-Ning Zhao, Stephanie Norton, Stephen J Haggarty, Emyr Lloyd-Evans, Susan L Cotman
JournalThe Journal of biological chemistry (J Biol Chem) Vol. 290 Issue 23 Pg. 14361-80 (Jun 05 2015) ISSN: 1083-351X [Electronic] United States
PMID25878248 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
Copyright© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Chemical References
  • CLN3 protein, human
  • CLN3 protein, mouse
  • Membrane Glycoproteins
  • Molecular Chaperones
  • Calcium
Topics
  • Animals
  • Autophagy (drug effects)
  • Calcium (metabolism)
  • Cell Line
  • Cells, Cultured
  • Drug Evaluation, Preclinical
  • Humans
  • Membrane Glycoproteins (genetics, metabolism)
  • Mice
  • Molecular Chaperones (genetics, metabolism)
  • Mutation
  • Neural Stem Cells (metabolism, pathology)
  • Neuronal Ceroid-Lipofuscinoses (drug therapy, genetics, metabolism, pathology)
  • Signal Transduction (drug effects)

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