Downregulation of Hsp90 and the antimicrobial peptide Mtk suppresses poly(GR)-induced neurotoxicity in C9ORF72-ALS/FTD

Abstract

GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat RNAs can be translated into dipeptide repeat proteins, including poly(GR), whose mechanisms of action remain largely unknown. In an RNA-seq analysis of poly(GR) toxicity in Drosophila, we found that several antimicrobial peptide genes, such as metchnikowin (Mtk), and heat shock protein (Hsp) genes are activated. Mtk knockdown in the fly eye or in all neurons suppresses poly(GR) neurotoxicity. These findings suggest a cell-autonomous role of Mtk in neurodegeneration. Hsp90 knockdown partially rescues both poly(GR) toxicity in flies and neurodegeneration in C9ORF72 motor neurons derived from induced pluripotent stem cells (iPSCs). Topoisomerase II (TopoII) regulates poly(GR)-induced upregulation of Hsp90 and Mtk. TopoII knockdown also suppresses poly(GR) toxicity in Drosophila and improves survival of C9ORF72 iPSC-derived motor neurons. These results suggest potential novel therapeutic targets for C9ORF72-ALS/FTD.

Keywords: ALS; C9ORF72; DPR; FTD; Hsp90; Mtk; TopoII; antimicrobial peptide; neurodegeneration; poly(GR).

Figure 1.. RNA-Seq analysis identifies overactivated AMP gene Mtk as a contributing factor to poly(GR) toxicity in Drosophila

(A) Hierarchical clustering heatmap showing the relative expression of differentially expressed genes in the heads of 3-week-old flies expressing Cont-(GR)₈₀ or (GR)₈₀ driven by elav-Gal4. UAS-Cont-(GR)₈₀ expresses (GR)₈₀ mRNA but the start codon AUG is changed into the stop codon UAA. Each sample is from an independent cross, n=3, 4 independent crosses. (B) Volcano plot depicting the −log10 p-value vs log2 fold change of genes between controls and poly(GR). Genes with FDR <0.05 and log2 FC >1 are shown in red. Genes with FDR <0.05 and log2 FC <−1 are shown in blue. Genes of particular interest are labeled. (C) Top enriched Gene Ontology (GO) terms of biological processes for upregulated differentially expressed genes in poly(GR)-expressing flies compared to control. (D) Protein-protein interactions for upregulated genes identified through the STRING database (https://string-db.org/). Only proteins with connections are shown. (E) Table showing upregulated antimicrobial peptide (AMP) genes in a poly(GR)-expressing fly model. (F) Validation of RNA-seq data of Mtk, DptB, AttC, DptA, Drosocin, and CecA2 genes expression by RT-qPCR in the head of 3-week-old male flies expressing poly(GR) under elav-GAL4. The P value was determined by two-tailed Student-t test. n=4, each from an independent genetic cross and mRNA measurement. (G) Representative images of adult eye phenotypes of different genotypes showing partial inhibition of poly(GR) toxicity by decreasing Mtk activity but not DptB. Flies were crossed to a w¹¹¹⁸ control strain. (H) External and internal eye phenotypes of poly(GR) flies are partially blocked by Mtk depletion. Toluidine blue staining of eye cross-sections shows the structure of rhabdomeres. UAS-GFP was used for control for genetic crosses with other UAS elements. Scale bar 25 uM. (I) Quantification of the genetic effects of Mtk and DptA/B on the rough eye phenotype caused by poly(GR). The number of flies of each genotype is shown in each column. The P value was determined by chi-square test. (J) Control and pan-neuronal poly(GR)-expressing male flies with or without Mtk modulation were analyzed by negative geotaxis climbing assay. For each genotype, the climbing assay was performed in at least eight cohorts consisting of 14-21 flies per vial grown at 27 °C for 18 days (n=10, 10, 10, 8 and 10 vials respectively, Tukey–Kramer test). All data values are mean ± s.e.m. **** p < 0.0001, *** P < 0.001, **P < 0.01, * P < 0.05, ns, not significant. See also Figure S1.

Figure 2.. The AMP gene Mtk is activated by Hsp90, and inhibition of Hsp90 alleviates neurodegeneration in poly(GR)-expressing flies and C9ORF72 iPSC-derived motor neurons

(A) Table showing upregulated HSPs in a pan-neuronal poly(GR)-expressing fly model. (B) External and internal eye phenotypes of poly(GR) flies are dramatically rescued by Hsp90 knockdown. Sectioned adult eyes stained with toluidine blue. UAS-GFP was used for control for genetic crosses with other UAS elements. Scale bar 25 uM. (C) Representative images of adult eye phenotypes in flies of different genotypes showing suppression of poly(GR) toxicity by genetic reduction or overexpression of Hsp90 activity. Flies were crossed to a w¹¹¹⁸ control strain. (D) Quantification of the suppressor effects of Hsp90 on the eye degeneration phenotype caused by poly(GR). The number of flies of each genotype is presented in each column. The P value was determined by chi-square test. (E) Hsp90 knockdown does not change poly(GR) protein levels in the fly head. One-way ANOVA with Tukey post-hoc test for multiple comparisons. (F) Elevated Mtk expression level is dramatically attenuated by Hsp90 knockdown in poly(GR)-expressing flies. One-way ANOVA with Tukey post-hoc test for multiple comparisons. UAS-GFP was used as control for genetic crosses with other UAS elements. (G and H) Western blot analysis (G) and quantification (H) of HSP90 level in 3-month-old control and C9ORF72 iPSC-derived motor neurons. n = 4, two-way ANOVA and Bonferroni post-hoc test. (I) Quantification of the efficiency of HSP90AA1 ASOs knockdown in iPSC-derived motor neurons. One-way ANOVA with Dunnett’s post-hoc test for multiple comparisons. (J) Decreased survival of C9ORF72-ALS patient iPSC-derived motor neurons upon withdrawal of neurotrophic factors is partially rescued by HSP90AA1 ASO treatment. Three biologically independent iPSC lines were differentiated (n = 100 neurons per condition). All motor neuron survival experiments were analyzed by two-sided log-rank test, and statistical significance was calculated from the entire survival time course. All data values are mean ± s.e.m. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns, not significant. See also Figure S2.

Figure 3.. Partial loss of TopoII activity suppresses poly(GR) toxicity in flies and neurodegeneration in C9ORF72 iPSC-derived motor neurons

(A) Genetic knockdown of TopoII strongly suppresses poly(GR) toxicity in Drosophila photoreceptor neuron. Representative images of adult external and internal eye phenotypes of flies with different genotype. UAS-GFP was used as control for genetic crosses with other UAS elements. Scale bar 25 uM. (B) Quantification of the effects of TopoII knockdown on poly(GR)-induced external eye degeneration phenotype. The number of flies of each genotype is presented in each column. The P value was determined by chi-square test. (C) Doxorubicin suppresses poly(GR)-induced toxicity in Drosophila eye. Representative images of eye phenotypes of control and (GR)₈₀ flies fed vehicle or doxorubicin. (D) Quantification of the eye degeneration phenotype. The number of flies of each genotype is shown in each column. The P value was determined by chi-square test. (E) Pan-neuronal downregulation of TopoII or Hsp90 rescues the locomotor defect of (GR)₈₀ flies in negative geotaxis climbing assay. For each genotype, climbing assay was performed at least five cohorts consisting of 16-20 flies grown at 27 °C at 3 weeks (n=5, 7, 8, and 8, Tukey–Kramer test). UAS-mCherry RNAi was used as control for genetic crosses with other UAS-RNAi elements. (F) Quantification of the efficiency of TopoIIβ ASOs knockdown in iPSC-derived motor neurons. One-way ANOVA with Dunnett’s post-hoc test for multiple comparisons. (G) Decreased survival of C9ORF72 ALS patient motor neurons. Three control and three C9ORF72 iPSC lines were differentiated (n = 100 neurons per line). (H) Decreased survival of C9ORF72 motor neurons was significantly increased by TopoIIβ ASOs treatment. Three C9ORF72 iPSC lines were differentiated (n = 100 neurons per line). (I) Survival of control iPSC-derived motor neurons treated with TopoIIβ ASOs or control ASO. Three iPSC lines were differentiated (n = 100 neurons per line). All motor neuron survival experiments were analyzed by two-sided log-rank test, and statistical significance was calculated from the entire survival time course. All data values are mean ± s.e.m. **** P < 0.0001, *** P < 0.001, **P < 0.01, * P < 0.05, ns, not significant. See also Figure S3.

Figure 4.. Upregulation of Mtk and Hsp90 in poly(GR)-expressing flies is controlled by TopoII

(A) RT-qPCR analysis of TopoII expression in 10-day-old fly heads expressing poly(GR) under GMR-Gal4 driver, n = 4, the P value was determined by two-tailed Student-t test. (B and C) Western blot analysis (B) and quantification (C) of TopoII protein level in 10-day-old control and poly(GR)-expressing flies. UAS-GFP was used as control for genetic crosses with other UAS elements. n = 4, the P value was determined by two-tailed Student-t test. (D and E) Western blot analysis (D) and quantification (E) of TopoIIβ protein level in 3-month-old control and C9ORF72 iPS-derived motor neurons. n = 4, Two-way ANOVA and Bonferroni post-hoc test for multiple comparisons. (F-H) RT-qPCR analysis of TopoII (F), Mtk (G), and DptB (H) mRNA levels in the heads of control and (GR)₈₀ flies with or without TopoII RNAi. UAS-GFP was used as control for genetic crosses with other UAS elements. n ≥ 3, One-way ANOVA with Tukey post-hoc test for multiple comparisons. (I and J) TopoII knockdown does not change poly(GR) mRNA level (I) and protein levels (J) in the fly head. One-way ANOVA with Tukey post-hoc test for multiple comparisons. (K and L) RT-qPCR analysis of Hsp90 (K) and Hsp27 (L) mRNA levels in the heads of control and (GR)₈₀ flies with or without TopoII RNAi. UAS-GFP was used as control for genetic crosses with other UAS elements. n ≥ 4, One-way ANOVA with Tukey post-hoc test for multiple comparisons. (M) TopoIIβ ChIP assay of the human HSP90AA1 gene. Schematic diagram of the human HSP90AA1 promoter and amplicons used in the ChIP assay (upper). ChIP-qPCR showing TopoIIβ binding to the indicated sites in HEK293T Cells (bottom). (N) Quantification of TopoIIβ binding to regions 1 to 3. One-way ANOVA with Tukey post-hoc test for multiple comparisons. All data values are mean ± s.e.m. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns, not significant. See also Figure S4.