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

Uma 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

doi:10.1074/jbc.m114.621706

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.

Highlights

CLN3 protein function is still unknown, but its loss causes Batten disease
Cell-based Screening Assay Identifies Autophagy Modifiers in CbCln3⌬ex7/8 Cells—We previously established a panel of wild type and homozygous cerebellar neuronal progenitor cells derived from Cln3⌬ex7/8 mice, which were engineered to precisely model juvenile onset NCL (JNCL) by recapitulating the major genetic defect found in JNCL patients (CbCln3ϩ/ϩ and CbCln3⌬ex7/8/⌬ex7/8 cells) [7, 26]
The established autophagy-related phenotypes in CLN3 models and the relationship of these to neurodegeneration in JNCL are incompletely understood, we further reasoned that developing a screening assay around autophagy in an accurate genetic model would serve as a useful starting point because abnormalities in autophagy are detected early in the disease process [17]

Summary

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. To gain an improved understanding of the pathways regulating defective autophagy 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. To further dissect the autophagy pathway abnormalities caused by Cln mutation, here we have developed a high throughput, cell-based autophagy assay, employing the use of a green fluorescent protein-tagged LC3 transgene (GFP-LC3), stably expressed in our mouse Cln cell culture model of JNCL Using this cell system, we conducted a screen to identify small molecule modifiers of autophagy. By focusing on the hit compounds that showed differential sensitivities in the cells bearing the Cln disease mutations, compared with the wild type cells, we have identified specific intracellular Ca2ϩ handling alterations that impact JNCL pathophysiological pathways in vitro, supporting further investigation of CLN3 in Ca2ϩ homeostasis and directed targeting of Ca2ϩ handling defects as potential JNCL therapies

Experimental Procedures

Results

Discussion