CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity

Abstract

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases.

Figure 1. Genome-wide CRISPR-Cas9 knockout screens in human cells identify modifiers of C9orf72 DPR toxicity

FLAG-tagged synthetic DPRs (PR₂₀ or GR₂₀) were added to the culture media of K562 cells, and dose-dependent cytotoxicity was measured using flow cytometry (a, c). The DPRs were internalized and localized to the nucleus of cells as visualized by immunocytochemistry (b, d, blue = DAPI, green = anti-FLAG, scale bar = 10μm). (e) Pooled CRISPR-Cas9 screening paradigm. K562 cells stably expressing Cas9 were infected with a lentiviral sgRNA library (10 sgRNAs/gene), the population was split and half the cells were treated with PR₂₀ or GR₂₀ for 4 pulses (at concentrations sufficient to kill 50% of cells each pulse), and then the resulting populations were subjected to deep sequencing and analysis. Screens were repeated 2 independent times for each DPR. (f) Volcano plot for all human genes in the GR₂₀ screen. Colored in blue are all the gene conferring resistance to GR₂₀ when knocked out (10% FDR) and colored in red are all the genes conferring sensitivity to GR₂₀ when knocked out (10% FDR). (g) Correlation of signed confidence scores of significant hits between GR₂₀ and PR₂₀ screens (R² = 0.61). (h) Validation of hits using independently generated single gene knockout K562 lines in co-culture competitive growth assays. Equal numbers of knockout cells (GFP⁺) and WT control cells (GFP⁻) were co-cultured in equal starting numbers and subsequently treated with PR₂₀ (10 μM). The abundance of knockout cells (GFP⁺ cells) was quantified by flow-cytometry 48 hours after PR₂₀ treatment and compared to the untreated population (n = 3, two-tailed t-test; *** p < 0.001, ** p < 0.01; error bars are ± S.D.) Each gene was validated using two distinct sgRNAs.

Figure 2. Summary of modifiers identified from genome-wide knockout screens in K562 cells

Schematic of selected hits (10% FDR) from both PR₂₀ and GR₂₀ screens colored coded by significance (p-value) and depicted at subcellular localizations based on gene ontology.

Figure 3. CRISPR-Cas9 knockout screens in primary mouse neurons

(a) Synthetic PR₂₀ added to primary neuron cultures caused dose-dependent cytotoxicity within 24 hours measured by lactose dehydrogenase (LDH) release or counts of surviving NeuN⁺ neuronal nuclei by immunocytochemistry (two-tailed t-test; **** p < 0.0001, *** p < 0.001, ** p < 0.01; error bars are ± S.E.M). (b) Expression of PR₅₀ using lentiviral transductions caused time dependent cytotoxicity measured by LDH release or counts of surviving NeuN⁺ neuronal nuclei by immunocytochemistry (two-tailed t-test; **** p < 0.0001, *** p < 0.001, ** p < 0.01; error bars are ± S.E.M). (c) CRISPR-Cas9 screening paradigm in primary mouse neuron cultures. Cortical neurons were plated and infected with sgRNA libraries (~200 genes passing a 5% FDR cutoff from the genome-wide PR screen with 10 sgRNAs/gene + ~1000 negative control sgRNAs). Four independent screening conditions were conducted, each in replicate: synthetic PR₂₀ treated neurons (1.5μM, overnight), untreated neurons, lentiviral expression of PR₅₀ (5 days)_, and lentiviral expression of GFP (5 days) as a control. The abundance of sgRNAs in the surviving cells was measured by sgRNA deep sequencing. PR₅₀ localized to the nucleus of cultured neurons (blue = DAPI, red = anti-FLAG (PR50), green = anti-MAP2). (d, e) Volcano plots of effect scores and confidence scores of all genes for each screen. Effect and confidence scores are calculated using casTLE . The effect score is a gene-level summary of how protective or sensitizing the knockout is, while the confidence score is a log-likelihood ratio describing the significance of the effect. Genes were designated hits if their 95% credible intervals derived from casTLE did not contain zero; 13 genes in the synthetic PR₂₀ screen and 17 genes in the lentiviral PR₅₀ screen passed this criteria (Supplementary Table 2).

Figure 4. Validation of RAB7 and TMX2 as modifiers of PR toxicity in an independent neuronal culture system

(a) Dorsal root ganglion (DRG) axon degeneration was evaluated by Tuj1 immunocytochemistry in the presence of 0.5 μM PR₂₀ in cultures transduced with sgRNAs targeting a control genomic region, Tmx2, or Rab7. DRG axon degeneration was further quantified by caspase 3/7 activity (b), ATP levels (c), and an apoptotic index (d), which is the ratio of active caspase 3/7:ATP levels (one-way ANOVA, Dunnett’s multiple comparisons test, *** p < 0.001, ** p < 0.01; error bars are ± S.E.M).

Figure 5. Transcriptional analysis of PR treated primary neurons and K562 cells reveals ER stress signatures

(a) RNA sequencing of primary mouse neurons treated with lentiviruses expressing either PR₅₀ or GFP under control of the synapsin promoter for 4 days. Differentially expressed genes were determined using DESeq2. (b) MA plot of differential gene expression of PR₅₀ expressing neurons compared to control GFP expressing neurons (red = significantly upregulated genes, adjusted p-value < 0.05; blue = significantly down regulated genes, adjusted p-value < 0.05). (c) Selected gene ontology (GO) terms enriched within significantly upregulated genes (red) or significantly downregulated genes (blue) determined using DAVID 6.8. (d) Fold change in expression of select genes that were found to be significantly differentially expressed by DESeq2 (n = 3, adjusted p-value < 0.001; error bars are ± S.E.M) grouped by GO category. (e) qRT-PCR validation of Atf4, Atf5, Chac1, Ddit3, and Bbc3 upregulation in neurons expressing PR₅₀ for 3, 4, or 5 days compared to control neurons. (f) RNA sequencing in K562 cells treated for 24 hours with synthetic PR₂₀ also show upregulation of ER stress related genes. Relative expression of significantly differentially expressed genes determined by DESeq2 (n = 3, adjusted p-value < 0.001; error bars are ± S.D.) plotted using FPKMs for each gene relative to the untreated population of cells. (g) K562 cells pre-incubated with 15nM ISRIB or equal amounts of DMSO were treated with 15 μM PR₂₀ (red) or untreated (black) for 24 hours, then measured for viability by flow cytometry (FSC/SSC). Each measurement was performed in triplicate with mean and SD plotted by error bars (** p-value < 0.005, two-tailed t-test). (h) Reverse transcriptase PCR analysis to measure amounts of spliced XBP1 mRNA (relative to GAPDH levels) in cortical neurons transduced with PR₅₀ for 3, 4, or 5 days (+ Thaps. = 200 nM thapsigargin treatment, 2hr.).

Figure 6. Tmx2 reduction is protective against PR mediated toxicity by modulating the ER stress response and improves the survival of C9orf72 ALS patient derived motor neurons

(a) Tmx2 protein levels measured in primary Cas9⁺ cortical neurons by immunoblot 10 days post sgRNA transduction (uncropped blot images are shown in Supplementary Data Set 1). (b) Quantification of lactose dehydrogenase levels (LDH) in primary cortical neurons 5 days post-transduction with PR₅₀ or GFP lentiviruses as a measurement of cytoxicity. Tmx2 reduction by sgRNA lentivirus transduction on DIV1 (TMX2 sgRNA) protected primary neurons against PR₅₀ induced cytotoxicity relative to control infected neurons (Safe sgRNA) (one-way ANOVA; n = 3; * p < 0.05; error bars are ± S.E.M). (c) Quantification of activated caspase 3/7 (DEVDase activity; RLU = relative light units) in primary neurons 4 days post-transduction with PR₅₀ or GFP lentiviruses (n = 3–4, one-way ANOVA, Dunnett’s multiple comparisons test, ** p < 0.01). (d) RNA-seq analysis of neurons infected with Safe or TMX2 targeting sgRNAs 4 days post-transduction with PR₅₀ or GFP lentiviruses. Log₂ fold change (TMX2 sgRNA/Safe sgRNA in PR₅₀ treated conditions) of differentially expressed genes determined using DESeq2 (n = 3; adjusted p-value < 0.001; error bars are ± S.E.M.) enriched in select GO categories (e) Select GO term enrichment determined using DAVID 6.8 (red = upregulated genes, blue = downregulated genes) of differentially expressed genes (adjusted p-value < 0.001) determined by comparing RNA-seq results between control (Safe sgRNA) and Tmx2 KO (TMX2 sgRNA) neuron cultures expressing PR₅₀. (f) qRT-PCR validation of Atf3 expression changes after PR₅₀ expression in control (Safe sgRNA) and Tmx2 KO (TMX2 sgRNA) neurons (n = 3; p < 0.0001; one-way ANOVA; error bars are ± S.E.M.). (g) V5-tagged ATF3 overexpression by lentiviral transduction of primary cortical neurons confers protection of PR₅₀ induced dendrite degeneration, as measured by immunocytochemistry (blue = DAPI, green = anti-MAP2 (dendrite marker), magenta = anti-V5 (ATF3-V5 overexpression), SYN = lentiviral synapsin promoter, scale bar = 20μm). (h) Quantification of % area of MAP2 immunofluorescence (n=5, one-way ANOVA, Dunnett’s multiple comparisons test, *** p < 0.001). (i) Quantification of active caspase 3/7 (DEVDase) in PR₅₀ or GFP expressing cortical neurons with or without ATF3 overexpression (n=5, one-way ANOVA, Dunnett’s multiple comparisons test, (**** p < 0.0001).

Figure 7. Reduction of TMX2 improves the survival of C9-ALS iPSC derived induced motor neurons

Quantification of surviving motor neurons with or without TMX2 reduction by lentiviral shRNA transduction in two independent C9-ALS iPSC lines (a = C9-ALS line 1, b = C9-ALS line 2). Induced motor neurons were generated from iPSCs using a seven factor (7F) differentiation system. The survival of HB9-RFP⁺/shRNA-GFP⁺ iMNs was tracked by imaging after the addition of 10 μM glutamate to the cultures. All iMN survival experiments were analyzed by log-rank test, and statistical significance was calculated using the entire survival time course. * p < 0.05, ** p < 0.01, **** p < 0.0001 (survival assays were performed in triplicate with the indicated number of iMNs analyzed for each group, Supplementary Table 4).