Genome-wide CRISPR screen reveals v-ATPase as a drug target to lower levels of ALS protein ataxin-2

Garam Kim, Lisa Nakayama, Jacob A Blum, Tetsuya Akiyama, Steven Boeynaems, Meenakshi Chakraborty, Julien Couthouis, Eduardo Tassoni-Tsuchida, Caitlin M Rodriguez, Michael C Bassik, Aaron D Gitler, Garam Kim, Lisa Nakayama, Jacob A Blum, Tetsuya Akiyama, Steven Boeynaems, Meenakshi Chakraborty, Julien Couthouis, Eduardo Tassoni-Tsuchida, Caitlin M Rodriguez, Michael C Bassik, Aaron D Gitler

Abstract

Mutations in the ataxin-2 gene (ATXN2) cause the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). A therapeutic strategy using antisense oligonucleotides targeting ATXN2 has entered clinical trial in humans. Additional ways to decrease ataxin-2 levels could lead to cheaper or less invasive therapies and elucidate how ataxin-2 is normally regulated. Here, we perform a genome-wide fluorescence-activated cell sorting (FACS)-based CRISPR-Cas9 screen in human cells and identify genes encoding components of the lysosomal vacuolar ATPase (v-ATPase) as modifiers of endogenous ataxin-2 protein levels. Multiple FDA-approved small molecule v-ATPase inhibitors lower ataxin-2 protein levels in mouse and human neurons, and oral administration of at least one of these drugs-etidronate-is sufficient to decrease ataxin-2 in the brains of mice. Together, we propose v-ATPase as a drug target for ALS and SCA2 and demonstrate the value of FACS-based screens in identifying genetic-and potentially druggable-modifiers of human disease proteins.

Trial registration: ClinicalTrials.gov NCT04494256.

Keywords: ALS; CP: Neuroscience; FACS; SCA2; TDP-43; ataxin-2; bisphosphonate; etidronate; genetic screens; small-molecule therapy; v-ATPase.

Conflict of interest statement

Declaration of interests A.D.G. is a scientific founder of Maze Therapeutics. Stanford University has filed a provisional patent (63/286,436) on methods described in this manuscript for treatment of neurodegenerative diseases through the inhibition of ataxin-2.

Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. Genome-wide CRISPR-Cas9 KO screens in…
Figure 1.. Genome-wide CRISPR-Cas9 KO screens in human cells identify regulators of ataxin-2 protein levels
(A) Pooled CRISPR-Cas9 screening paradigm. After transducing HeLa cells expressing Cas9 with a lentiviral whole-genome sgRNA library, we fixed and co-immunostained the cells for ataxin-2 and a control protein (β-actin or GAPDH). We then used FACS to sort top and bottom 20% ataxin-2 expressors relative to control protein levels (duplicate sorts per each control). After isolating genomic DNA from these populations, as well as the unsorted control population, we performed NGS to read sgRNA barcodes. (B) Volcano plots based on effect and confidence scores summarizing genes that modify ataxin-2 protein levels relative to β-actin (left) or GAPDH (right) levels when knocked out (FDR PNISR, PAXBP1, or ATXN2 siRNAs in HeLa cells.
Figure 2.. Schematic of proteins encoded by…
Figure 2.. Schematic of proteins encoded by selected hits (5% FDR), categorized by function and subcellular localization
Figure 3.. Genetically and pharmacologically perturbing lysosomal…
Figure 3.. Genetically and pharmacologically perturbing lysosomal v-ATPase leads to decreased ataxin-2 protein levels in vitro
(A) Left, volcano plot shows confidence score on y axis and effect score on x axis, with gene hits encoding lysosomal v-ATPase subunits highlighted in red. Right, a representation of the lysosomal v-ATPase, with its V0 and V1 domains, as well as individual subunits. (B and C) Immunoblot (B) and quantification (C) of ataxin-2 protein levels after HeLa cells were transfected with siRNAs against various v-ATPase subunits. (D) Quantification of ATXN2 RNA levels after siRNA knockdown of v-ATPase subunits in HeLa cells. Values normalized to β-actin RNA levels. Quantifications for (C) and (D) are normalized to the NT siRNA condition (mean ± SD; analyzed using one-way ANOVA with post hoc Dunnett’s multiple comparisons tests; ****p ATP6V1A Cas9-edited HeLa cell lines. (F) Representative microscopy images of WT or ATP6V1A Cas9-edited HeLa cells, immunostained for ataxin-2 or β-actin (scale bar: 20 μm). Ataxin-2 fluorescence quantifications are shown on the right (lines denote mean ± SD; analyzed using unpaired t test; ****p < 0.0001).
Figure 4.. Small-molecule drug etidronate lowers ataxin-2…
Figure 4.. Small-molecule drug etidronate lowers ataxin-2 protein levels in human iPSC-derived neurons, mouse primary neurons, and in vivo in mice
(A) Timeline of induced neuron differentiation in a human iPSC line with NGN2 stably integrated and drug treatment. (B) Immunoblot on lysates from human iPSC-derived neurons treated with various doses of etidronate. (C) Quantification of Figure 4B, with ataxin-2 protein levels normalized to H2O-treated condition (mean ± SD; analyzed using one-way ANOVA with post hoc Dunnett’s multiple comparisons tests; *p < 0.05). (D) Timeline of primary neuron plating from embryonic mouse cortex and drug treatment. (E) Immunoblot on lysates from mouse primary neurons treated with various doses of etidronate. (F) Quantification of the dose-dependent effect of etidronate on ataxin-2 (normalized to control condition). (G) Representative microscopy images of mouse cortical neurons treated with sham (H2O) or 10 μM etidronate for 24 h, stained for MAP2, ataxin-2, and DAPI (scale bar: 10 μm). (H) Representative microscopy images of mouse cortical neurons treated with H2O or 10 μM etidronate for 24 h, with 0.5 mM sodium arsenite treatment for the final hour. The neurons were stained for PABP, MAP2, and DAPI (scale bar: 10 μm). (I) Quantifications of cells containing PABP-positive stress granules (SGs) (mean ± SEM; analyzed using unpaired t test; ****p 2O-treated condition) using lysates from cortices of mice that received water or drug treatment (mean ± SEM; analyzed using Welch’s t test; **p < 0.01). We performed the experiment two independent times, for a total of n = 16 in the control group and n = 14 in the drug treatment group.

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