Functional Genomic Analyses of Mendelian and Sporadic Disease Identify Impaired eIF2α Signaling as a Generalizable Mechanism for Dystonia

Abstract

Dystonia is a brain disorder causing involuntary, often painful movements. Apart from a role for dopamine deficiency in some forms, the cellular mechanisms underlying most dystonias are currently unknown. Here, we discover a role for deficient eIF2α signaling in DYT1 dystonia, a rare inherited generalized form, through a genome-wide RNAi screen. Subsequent experiments including patient-derived cells and a mouse model support both a pathogenic role and therapeutic potential for eIF2α pathway perturbations. We further find genetic and functional evidence supporting similar pathway impairment in patients with sporadic cervical dystonia, due to rare coding variation in the eIF2α effector ATF4. Considering also that another dystonia, DYT16, involves a gene upstream of the eIF2α pathway, these results mechanistically link multiple forms of dystonia and put forth a new overall cellular mechanism for dystonia pathogenesis, impairment of eIF2α signaling, a pathway known for its roles in cellular stress responses and synaptic plasticity.

Keywords: dystonia; regulation of translation; stress signaling.

Copyright © 2016 Elsevier Inc. All rights reserved.

Figures

Figure 1. Torsin1a mislocalization assay and genome-wide siRNA screen

(A and B) Flp-In T-REx 293T stable cell lines expressing ΔE (A) and WT (B) EGFP-hTorsin1a following 72 h tetracycline induction. Green – EGFP-hTorsin1a. Blue – Hoescht nuclear stain. Scale bars = 10 μm. (C) Percent of cells with ≥ 1 EGFP puncta (“Percent Selected Cells” – see Figure S2) after siRNA silencing of Torsin1b. n = 16 wells for control siRNA and 32 wells for TOR1B siRNAs. (D) Percent of cells with ≥ 1 EGFP puncta after treatment with the protesome inhibitor MG132 (10 μM) or the chemical chaperone phenylbutyric acid (PBA, 20 mM). n = 4 DMSO-treated wells and 8 MG132/PBA-treated wells. (E-G) Cell lines under high-throughput screening conditions after transfection with non-silencing control siRNA (E and F) or positive control siRNA (PC, panel G). Scale bars = 50 μm. (H) Whole genome siRNA (WGS) screen controls and siRNA pooling strategy. Inset – Four independent siRNAs targeting each gene were split into two unique pools of two siRNAs. (I) Results of WGS screen. Dots represent data for individual gene targets, with results from independent siRNA pools plotted on orthogonal axes. Yellow shaded area – genes with concordant >3SD normalizing effects. (J) Schematic depicting WGS workflow for analyzing primary hits. (K) Luciferase secretion in DYT1 patient-derived fibroblasts after lentiviral delivery of shRNAs targeting 4 top WGS hits. n = 4 DYT1 lines and 3 control lines, 5 independent replicates each (except SCD – 3 replicates). *, p < 0.05; ***, p < 0.0005 by unpaired t test. All data are presented as means ± S.E.M.

Figure 2. Enhancing eIF2α signaling corrects ΔE Torsin1a mislocalization

(A) eIF2α signaling pathway diagram. Actions of compounds tested in panels (C) through (F) are indicated in blue. (B) WGS results relevant to the eIF2α pathway. Blue – gene hits bioinformatically implicating the eIF2α pathway. Red – eIF2α kinases. Dashed lines indicate +/−3SD from mean. (C-H) Left – Effects of the indicated compounds on Torsin1a localization (black), cell count (grey), and EGFP-Torsin1a expression (green) in the ΔE (C-E, G, H) or WT (F) assay cell lines. Percent Selected Cells was normalized such that vehicle-treated ΔE cells = 100 and vehicle-treated WT cells = 0. GFP Intensity and Cell Count were normalized such that vehicle-treated ΔE cells = 100. Right – Representative images from the respective treatments. Scale bars = 20 μm. n = 4 per compound dose for dose response data or 24 for untreated control data (used for normalization). All data are presented as means ± S.E.M.

Figure 3. ATF4 overexpression is sufficient to correct ΔE Torsin1a mislocalization

(A) Torsin1a localization in the ΔE and WT assay cell lines after transfection with the indicated FLAG-tagged ATF4 constructs. White arrows – FLAG-positive cells. Scale bars = 20 μm. (B) Quantification of conditions shown in (A). Percent Selected Cells was normalized such that empty vector-transfected ΔE cells = 100 and vector-transfected WT cells = 0. *, p

Figure 4. Enhancing eIF2α signaling rescues deficient corticostriatal LTD in heterozygous DYT1 knockin mice and improves neonatal survival of homozygous DYT1 mice

(A) Long-term depression (LTD) of corticostriatal synapses from WT mice in the presence of 5 nM ISRIB (black dots) or vehicle control (white dots). LTD was induced by 4 trains of 100 Hz stimulation in cortex layer V (HFS, see Experimental Procedures). n = 8 slices/4 mice (vehicle) or 9 slices/4 mice (ISRIB). Traces above graph – Representative excitatory postsynaptic potentials (EPSPs) recorded before HFS (black) or 21 min post-HFS (gray). (B) LTD of corticostriatal synapses from DYT1 mice in the presence of 20 μM Sal-003 (red dots) or vehicle control (white dots). n = 12 slices/7 mice (vehicle) or 12 slices/8 mice (Sal-003). (C) Mean magnitude of LTD in (A) and (B). Data in blue shaded boxes in panels (A) and (B) (minutes 21-31) were averaged. *, p Top – Schematic depicting experimental workflow. Bottom – Perinatal survival after in utero salubrinal exposure. n = 29 vehicle- and 28 salubrinal-treated homozygous pups and 65 vehicle- and 91 salubrinal-treated pooled heterozygous/WT pups. No effect on mortality was observed for WT or heterozygous genotypes, so results were combined to simplify presentation. **, p

Figure 5. Cells from human DYT1 dystonia patients show eIF2α pathway dysfunction

(A) ATF4 expression in control and DYT1 patient fibroblasts after treatment with 1 μM thapsigargin. Green – anti-ATF4. Red – anti-β-actin. (B) Quantification of conditions shown in (A). ATF4 expression was normalized to β-actin expression. n = 3 control cell lines and 4 DYT1 cell lines, 3 replicates each. (C) CReP expression with and without 4 hours exposure to 1 μM thapsigargin. (D and E) Quantification of conditions shown in (C). CReP expression was normalized to β-actin expression. n = 3 control cells lines and 4 DYT1 cell lines, 3 replicates each. *, p

Figure 6. Identification of loss-of-function ATF4 mutations in sporadic dystonia patients

(A) Exons bearing missense coding mutations in at least two of 20 unrelated sporadic dystonia patients and none of 571 matched controls, as determined by whole exome sequencing. (B) Top – Rare and common variants in ATF4 exon 1 from 239 additional sporadic cervical dystonia patients; frequency, enrichment and predicted mutation severity shown at right (see also Table S5). Bottom – ATF4 protein schematic showing rare variant locations (red dots). (C) Transcriptional activation activity of mutant ATF4 constructs, as measured by a luciferase reporter under transcriptional control of an ATF4-sensitive response element (AARE-RLuc). Data was normalized such that luciferase activity after WT ATF4 transfection = 1. *, p www.cbs.dtu.dk/services/NetPhos).

Figure 7

Model depicting shared cellular mechanism of eIF2α pathway dysfunction across multiple forms of dystonia.