title: 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.
authors: From the Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and.
authors: Uma
authors: U
authors: Chandrachud
authors: the Sir Martin Evans Building, School of Biosciences, Cardiff University, Cardiff CF10 3AX, United Kingdom.
authors: Mathew W
authors: MW
authors: Walker
authors: From the Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and.
authors: Alexandra M
authors: AM
authors: Simas
authors: From the Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and.
authors: Sasja
authors: S
authors: Heetveld
authors: From the Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and.
authors: Anton
authors: A
authors: Petcherski
authors: From the Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and.
authors: Madeleine
authors: M
authors: Klein
authors: From the Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and.
authors: Hyejin
authors: H
authors: Oh
authors: From the Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and.
authors: Pavlina
authors: P
authors: Wolf
authors: From the Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and.
authors: Wen-Ning
authors: WN
authors: Zhao
authors: From the Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and.
authors: Stephanie
authors: S
authors: Norton
authors: From the Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and.
authors: Stephen J
authors: SJ
authors: Haggarty
authors: the Sir Martin Evans Building, School of Biosciences, Cardiff University, Cardiff CF10 3AX, United Kingdom.
authors: Emyr
authors: E
authors: Lloyd-Evans
authors: From the Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and cotman@helix.mgh.harvard.edu.
authors: Susan L
authors: SL
authors: Cotman
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D058953:Neural Stem Cells / Q000378:metabolism / Q000473:pathology*; D009472:Neuronal Ceroid-Lipofuscinoses / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C105199:CLN3 protein, human; C102229:CLN3 protein, mouse; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D002118:Calcium
keywords: CLN3; GFP-LC3; autophagy; calcium; chemical biology; endoplasmic reticulum (ER); juvenile neuronal ceroid lipofuscinosis (JNCL); lysosomal storage disease; lysosome; thapsigargin