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*Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2
Homeostasis, Autophagy, and CLN3 Protein Function*S
Received for publication, October 28, 2014, and in revised form, April 7, 2015 Published, JBC Papers in Press, April 15, 2015, DOI 10.1074/jbc.M114.621706
Uma Chandrachud‡, Mathew W. Walker§, Alexandra M. Simas‡, Sasja Heetveld‡, Anton Petcherski‡1, Madeleine Klein‡, Hyejin Oh‡, Pavlina Wolf‡, Wen-Ning Zhao‡, Stephanie Norton‡, Stephen J. Haggarty‡, Emyr Lloyd-Evans§, and Susan L. Cotman‡2
From the ‡Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 and the §Sir Martin Evans Building, School of Biosciences, Cardiff University, Cardiff CF10 3AX, United Kingdom
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.
Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neu- rodegenerative 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 juve- nile 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 com- mon disease mutation in CLN3. Thapsigargin, a sarco/endoplas- mic 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, mitochon-
drial, 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.
The neuronal ceroid lipofuscinoses (NCLs)3 are a group of inherited neurodegenerative diseases characterized by motor and cognitive decline, seizures, and typically vision loss. The NCLs are further typified by the occurrence of abnormal accu- mulations of protein- and lipid-containing autofluorescent storage material in both neuronal and non-neuronal cells, which most often contains abundant levels of the pore-forming subunit of the mitochondrial ATP synthase (subunit c of the F0-ATPase complex; hereafter referred to as subunit c) (1). Cur- rently, 13 genes are known to cause different subtypes of NCL, encoding proteins of varied, often incompletely understood functions within the secretory and endosomal-lysosomal sys- tems (2– 4). In juvenile onset NCL (JNCL), autosomal recessive inheritance of loss-of-function mutations in the CLN3 gene lead to disease, with most patients carrying at least one copy of a common 1-kb deletion in CLN3 (5).
CLN3 encodes a multipass transmembrane protein, referred to as CLN3, CLN3p, or battenin (5), that has been demon- strated to localize to multiple membrane compartments, including within the endosomal, lysosomal, and autophago- somal pathways (6). Although the primary function of CLN3 has not yet been fully uncovered it is proposed to play a role in vesicular trafficking because its deficiency leads to altered dis-
* This work was supported, in whole or in part, by National Institutes of Health Grants R01NS073813 from NINDS (to S. L. C.) and R33MH087896 from National Institute of Mental Health (to S. J. H.). This work was also sup- ported by the Batten Disease Support and Research Association (to S. L. C.), the National Tay-Sachs and Allied Diseases Association, Inc. (to S. L. C.), the Tau Consortium (to S. J. H.), National Contest for Life Foundation (NCL Stif- tung) fellowship (to A .P.), the Finnish Foundation for JNCL Research fel- lowship (to A. P.), a Knowledge Economy Skills Scholarship (to M. W. W.), a Research Councils UK Fellowship (to E. L. E.), a Basil O’Connor Starter Schol- arship (to E. L. E.), and a Royal Society project grant (to E. L. E.) S. L. C. is a member of the Batten Disease Support and Research Association Advisory Board. P. W. is currently an employee of Sanofi.
S This article contains supplemental Fig. 1 and Tables 1 and 2. 1 Present address: NeuroToponomics Group, Center for Membrane Proteom-
ics, Goethe Universität Frankfurt am Main, 60438 Frankfurt am Main, Germany.
2 To whom correspondence should be addressed: Center for Human Genetic Research, Dept. of Neurology, 185 Cambridge St., Boston, MA. Tel.: 617- 726-9180; Fax: 617-643-3203; E-mail: [email protected].
3 The abbreviations used are: NCL, neuronal ceroid lipofuscinose; JNCL, juve- nile neuronal ceroid lipofuscinosis; LC3, microtubule-associated protein 1 light chain 3; SERCA, sarco/endoplasmic reticulum Ca2-ATPase; ER, endo- plasmic reticulum; BAPTA, 1,2-bis(o-aminophenoxy ethane-N,N,N,N-tet- raacetic acid; NPC, neural progenitor cell; Bistris, 2-[bis(2-hydroxyethyl)- amino]-2-(hydroxymethyl)propane-1,3-diol; ANOVA, analysis of variance; iPSC, induced pluripotent stem cell; mTOR, mammalian target of rapamycin.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 23, pp. 14361–14380, June 5, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
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tribution of endosomal and lysosomal proteins and phospho- lipids (7–12), abnormal morphology of endocytic and lyso- somal organelles (7, 11), lysosomal pH dyshomeostasis (13–15), and amino acid transport defects (16). The hallmark JNCL stor- age material containing subunit c accumulates in both autopha- gosomes and lysosomes implicating further impact of CLN3 deficiency on the autophagy pathway (17).
In previous work employing a genetically accurate neuronal progenitor cell model of JNCL that bears a homozygous 1-kb deletion in the murine Cln3 gene, recapitulating the most com- mon genetic defect found in JNCL patients (7), we further dem- onstrated that Cln3 mutation leads to LC3-II-positive autopha- gosome accumulation, even preceding the onset of detectable storage material (17). To further dissect the autophagy pathway abnormalities caused by Cln3 mutation, here we have devel- oped 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 Cln3 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 Cln3 disease mutations, compared with the wild type cells, we have identified specific intracellular Ca2
handling alterations that impact JNCL pathophysiological pathways in vitro, supporting further investigation of CLN3 in Ca2 homeostasis and directed targeting of Ca2 handling defects as potential JNCL therapies.
Experimental Procedures
Reagents and Cell Lines
Establishment and Maintenance of CbCln3/ and CbCln3ex7/8/ex7/8 Cell Lines Expressing GFP-LC3— CbCln3/ and CbCln3ex7/8/ex7/8 cell lines were generated as described previously (7). To establish stably expressing GFP- LC3 derivative cell lines from these, cells were first transiently transfected with the pCAG-EGFP-LC3 expression plasmid (a generous gift from Dr. Noboru Mizushima) using Lipo- fectamine 2000 (Invitrogen), according to the manufactur- er’s protocol. Stable transfectant subclonal lines were then established by replating for limiting dilution subcloning 72 h post-transfection, to expand from single cells. Positive sub- clones were identified by visual scoring for GFP fluores- cence. Initially, 6 subclones per genotype were established and screened for relative GFP cytosolic and vesicular signal, and representative subclones for each genotype were subse- quently chosen for use in the primary screen and in fol- low-up experiments.
For maintenance, cells were grown at 33 °C, with 5% CO2
atmosphere control, in Cbc media (Dulbecco’s modified Eagle’s medium (DMEM; Gibco catalog no. 11995-065), 10% heat-in- activated FBS (Sigma catalog no. F4135), 24 mM KCl, 1 peni- cillin/streptomycin/glutamine (Corning Cellgro catalog no. 0-009-CI), and 200 g/ml G418 (Gibco catalog no. 11811-098)). Unless otherwise indicated, cells were maintained between 30 and 90% confluency on 100-mm plastic tissue culture dishes, as described previously (7).
Compounds (Not Including the Screening Library) Used in This Study—The following compounds were used: thapsigargin (Enzo catalog no. BML-PE180); BAPTA-AM (Life Technolo- gies, Inc., catalog no. B-6769); bafilomycin B1 (AG Scientific Inc., catalog no. B-1185); tunicamycin (Sigma, catalog no. T7765); chelerythrine (Sigma, catalog no. C-2932); arvanil (Biomol catalog no. VR-101); nifedipine (Biomol catalog no. CA-210); A-23187 (Biomol catalog no. CA-100); ikarugamycin (Biomol catalog no. EI-313); and CA-074 (Biomol catalog no. PI-126). All compounds were reconstituted in DMSO.
Cell-based Screening Assay
Compound Library Used for Screening—Plate 1 from the ICCB Known Bioactives Library (Biomol catalog no. 2840- 0001) was used in our primary cell screen; this library is a col- lection of diverse biologically active compounds with defined biological activity. Briefly, plate 1 contained 320 test com- pounds, suspended in DMSO, and 64 “vehicle” wells containing only DMSO, randomly positioned throughout a 384-well plate. Note that rapamycin, a well known autophagy inducer (18, 19), was not present in this library.
Primary Screen—CbCln3/ and CbCln3ex7/8/ex7/8 cells expressing GFP-LC3 were plated into clear-bottomed 384-well plates at a density of 2 103 cells/well and allowed to attach and recover from plating overnight. The following morning, test compounds and DMSO negative control were added to the wells by robotic pin-transfer from the library plate. Dupli- cate plates were prepared each for the CbCln3/ and CbCln3ex7/8/ex7/8 cells expressing GFP-LC3. Cells were then incubated with compounds for 24 h (33 °C, 5% CO2). At the 24-h time point, cells were fixed with freshly prepared 4% form- aldehyde in phosphate-buffered saline (PBS, pH 7.4) for 20 min at room temperature, followed by PBS wash (two 10-min washes) and Hoechst nuclear counterstaining (Life Technolo- gies, Inc., catalog no. H3570). Following nuclear counterstain- ing, PBS containing 0.3% sodium azide was added to each well, and plates were sealed, wrapped in foil, and stored at 4 °C until they were imaged.
Imaging—Automated imaging of plates was performed at ambient temperature with a 10 objective, using an ImageX- press Micro high content imaging system (Molecular Devices Inc., Sunnyvale, CA). MetaXpress software, version 2.0.1.28, was used to acquire and analyze the images. For DAPI, laser- and image-based focusing was used, and images were acquired with a 100-ms exposure time. For GFP, laser- and image-based focusing was used, and images were acquired with a 1500-ms exposure time. Nuclei and vesicle compartments were identi- fied on the appropriate channels using the “nuclei” and “vesi- cles” parameters in the Transfluor module of MetaXpress. For vesicles, parameters were set to a 5-pixel minimum, a 14-pixel maximum, and an intensity of at least 80 gray levels above local background. For nuclei, parameters were set to a 12-pixel minimum, a 50-pixel maximum, and an intensity of at least 150 gray levels above local background. One image per well (typically containing 250 cells) was analyzed for each compound treatment to obtain per well and per cell image mea- surements. The end point measurement for the primary screening assay was “vesicle count per cell.”
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Data Processing and Analysis—To account for plate-to-plate variation in signal, each compound treatment well was com- pared with the collection of DMSO treatment well measure- ments performed in the same plate. First, the values of DMSO treatment wells in a given plate were compiled, and values fall- ing above or below twice the standard deviation for this overall distribution were discarded as outliers (this accounted for 5% of the total DMSO wells in the plate and typically represented edge wells). The remaining DMSO treatment measurements were then used to arrive at a “mean vesicle count per cell” value for DMSO treatment. To obtain a z score for each compound well, the mean DMSO treatment value () was subtracted from the individual compound value (xi), and this value was then divided by twice the standard deviation of the DMSO treatment wells (2), using the equation z (xi )/2. Finally, to arrive at our “hit list,” a mean z score was calculated from each of the replicate plates for each compound, and a cutoff was then applied to define hits as those compounds that had a mean z score of 2 or 2. To facilitate the identification of “geno- type-sensitive” compounds among the compounds displaying activity in the primary screen, i.e. compounds that tended to display more or less activity in the CbCln3ex7/8/ex7/8 cells compared with the CbCln3/ cells, a “z score ratio” was cal- culated. Briefly, for each hit compound, the absolute value of the mean z score in CbCln3ex7/8/ex7/8 cells divided by the mean z score in CbCln3/ cells was calculated, and genotype- sensitive activities were hypothesized if a hit compound’s z score ratio was 2 or 0.5. The complete list of significant hits, with corresponding z score values and z score ratios, is provided in supplemental Table 1. z scores for all 320 compounds in the primary screen are provided in supplemental Table 2.
Secondary Dose-Response Analysis of Selected Hit Com- pounds—Seven of the hypothesized genotype-sensitive com- pounds from the primary screen data analysis (supplemental Table 1) were selected for follow-up dose-response experi- ments. These were selected based on putative mechanism-of- action, which was Ca2-related in five cases (e.g. Ca2 channels, Ca2-responsive signaling) and was otherwise in a pathway of established interest in CLN3 research (e.g. lysosomal proteoly- sis and endocytosis). A secondary screening plate was prepared containing each of the selected compounds at a total of 11 con- centrations per compound (in most cases, this included the original screening dose, one dose 2-fold more concentrated, and nine 2-fold serial dilutions), with DMSO-only control wells included in the plate. A single plate each for CbCln3/ and CbCln3ex7/8/ex7/8 cells was screened with the dilution series plate, exactly as described for the primary screening assay. End- point again was vesicle count per cell. “Nuclei count per image” was used as an additional end-point to assess relative toxicity of the compound treatments in the dose-response experiment. These data are provided in supplemental Fig. 1.
Cell Treatment in Follow-up Pharmacological Studies
For epifluorescence and confocal microscopy studies, CbCln3/ and CbCln3ex7/8/ex7/8 cells were cultured on 18-mm diameter number 1 glass coverslips (Fisher). Cells were typically seeded at a density of 5 104 and were grown over- night at 33 °C, 5% CO2 in Cbc media. For preparation of lysates,
CbCln3/ and CbCln3ex7/8/ex7/8 cells were seeded at den- sity of 9 105 into 100-mm dishes and were grown overnight at 33 °C, 5% CO2 in Cbc media. Cells were treated 24 h post- plating as follows.
Treatment with Thapsigargin—Unless otherwise indicated, cells were treated with 0.1 M thapsigargin for 24 h.
Treatment with Tunicamycin—Cells were treated with 0.1 and 1 g/ml tunicamycin for 24 h.
Treatment with Bafilomycin—To determine the saturating dose/incubation time for bafilomycin treatment, cells were treated with 0.1 or 1 M bafilomycin. Lysates were collected at 6- and 24-h time points for immunoblot analysis. For micros- copy experiments, cells were treated with 1 M bafilomycin for 24 h.
Co-treatment with Bafilomycin and Thapsigargin—Cells were co-treated with 0.1 M thapsigargin together with 1 M
bafilomycin for 24 h. Treatment with BAPTA-AM—Cells were treated with or
without (DMSO only) 0.1 M thapsigargin for 23 h, and for the last hour, 5 M BAPTA-AM was added to the media at a final concentration of 5 M.
Starvation—Complete media were aspirated away and exchanged for Hanks’ balanced salt solution (HBSS) (Invitro- gen, catalog no. 14025092), following a brief wash with warmed HBSS. Cells were incubated in HBSS for 1.5 h prior to fixation.
Treatment with Lysosomal Protease Inhibitors—For treat- ments with lysosomal protease inhibitors, the mouse cerebellar cells were treated with E64 (10 g/ml) (E3132, Sigma) and pep- statin A (100 g/ml) (P5318, Sigma) for 16 h. The NPCs were treated with E64 (10 g/ml) and pepstatin A (50 g/ml) for 16 h. For both sets of cells, the indicated doses were predeter- mined to be the saturating doses (data not shown), as recom- mended (19). For treatment with thapsigargin in the presence of lysosomal protease inhibitors, a 16-h pretreatment was per- formed with the protease inhibitors and then thapsigargin was added for 24 h.
Treatment with Known Autophagy Inducers—Cells were treated with 2 M rapamycin (BML-A275, Enzo Life Sciences) and 10 M torin (S2827, Selleck Chemical) for the indicated treatment times.
Human Neural Progenitor Cell (NPC) Culture
Human NPCs were derived and maintained as described pre- viously (11). The control line was derived from GM8330-8 patient fibroblasts. The patient NPCs (CLN3ex7/8/ex7/8 and CLN3IVS13/E15) were derived from de-identified fibroblasts from JNCL individuals. NPCs were maintained on poly-L-orni- thine- and laminin-coated plates, in media containing 97% DMEM/F-12 (Life Technologies, Inc., catalog no. 11330-032), 2% 50 B-27 Supplement (Life Technologies, Inc., catalog no. 17504-044) and 1% 1 penicillin/streptomycin/glutamine (Corning Cellgro catalog no. 30-009-CI) and supplemented with 20 ng/ml of the growth factors EGF (Sigma catalog no. E9644) and FGF (Millipore catalog no. GF003). Cells were plated on either 6-well plates or on 14-mm diameter number 1 glass coverslips (Fisher) and left to attach overnight before treatment. For Western blotting, one well of a 6-well plate was harvested, and cell lysates were made using the proce-
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dure described below (see under “Immunoblotting and Densitometry”).
Immunostaining
Cells (CbCln3 cells or human iPSC-derived NPCs) grown on coverslips were fixed with one of two methods, depending on the primary antibody. For Rab7 antibody (Cell Signaling catalog no. 9367, used at 1:100 dilution), p62/SQSTM1 antibody (Enzo catalog no. BML-PW9860, used at 1:100 dilution in Cb cells and 1:400 in NPCs), and LAMP-2a antibody (Abcam catalog no. ab18528, used at 1:500 dilution), cells were fixed in freshly pre- pared 4% paraformaldehyde diluted in PBS, pH 7.4, at room temperature, for 20 min. Alternatively, for LAMP-1 antibody (Santa Cruz Biotechnology catalog no. sc-19992, used at 1:200 dilution) and in some experiments for LC3 antibody (Abcam catalog no. ab51520, used at 1:500 dilution), cells were fixed in an ice-cold 50:50 (v/v) mixture of methanol/acetone for 10 min. The immunostaining procedure was performed as described previously (11, 20). Briefly, 0.05% Triton X-100 diluted in PBS, pH 7.4, was used for cell permeabilization, and 5% bovine serum albumin (BSA) diluted in PBS, pH 7.4, was used for blocking. However, permeabilization was different for the following, as indicated: for LAMP-2a antibody, cell permeabilization was with 0.5% saponin, diluted in blocking buffer; SERCA immuno- staining (SERCA 2 antibody, catalog no. ab3625, Abcam) used 20 g/ml digitonin; and p62 staining in NPCs instead utilized a 1-h 3% normal horse serum, 0.2% Triton X-100 permeabiliza- tion step. Notably, the different fixation and staining proce- dures required for the different primary antibodies resulted in slight differences in the overall fluorescent signal of GFP-LC3. It is also noteworthy that our staining conditions are expected to allow for visualization of GFP fluorescence in the late endo- somal and lysosomal acidic compartments, because it is well documented that the permeabilization and neutral pH of fixa- tion and staining solutions eliminates the quenching of GFP in these compartments (19, 21). Anti-rabbit Alexa Fluor-555 (Life Technologies, Inc.) and anti-rat Alexa Fluor-568 (Life Technologies, Inc.) were used as secondary antibody (1:800). Coverslips were semi-permanently mounted onto microscope slides using ProLong Gold antifade reagent with DAPI (Invit-…