Large-scale RNA-seq mining reveals ciclopirox triggers TDP-43 cryptic exons

Abstract

Nuclear clearance and cytoplasmic aggregation of TDP-43 in neurons, initially identified in ALS-FTD, are hallmark pathological features observed across a spectrum of neurodegenerative diseases. We previously found that TDP-43 loss-of-function leads to the transcriptome-wide inclusion of deleterious cryptic exons in brains and biofluids post-mortem as well as during the presymptomatic stage of ALS-FTD, but upstream mechanisms that lead to TDP-43 dysregulation remain unclear. Here, we developed a web-based resource (SnapMine) to determine the levels of TDP-43 cryptic exon inclusion across hundreds of thousands of publicly available RNA sequencing datasets. We established cryptic exon inclusion across a variety of human cells and tissues to provide ground truth references for future studies on TDP-43 dysregulation. We then explored studies that were entirely unrelated to TDP-43 or neurodegeneration and found that ciclopirox olamine (CPX), an FDA-approved antifungal, can trigger the inclusion of TDP-43-associated cryptic exons in a variety of mouse and human primary cells. CPX induction of cryptic exon occurs via heavy metal toxicity and oxidative stress, suggesting that similar vulnerabilities could play a role in neurodegeneration. Our work demonstrates how diverse datasets can be linked through common biological features and underscores that public archives of sequencing data represent a vastly underutilized resource with tremendous potential for uncovering novel insights into complex biological mechanisms and diseases.

Figure 1:. SnapMine application mines Snaptron database for exon-exon junction usage.

(A) Schematic of SnapMine algorithm. (B) Example usage of SnapMine to identify one junction of the HDGFL2 cryptic exon in the Sequencing Read Archive (SRA). (C) Only a few samples in the SRA include the HDGFL2 cryptic exon at rates higher than 5%. Most of these are from ALS-associated or TDP-43-depletion associated studies.

Figure 2.. GTEx and SRA data compilations can be mined for cryptic exons of interest using SnapMine.

(A) The average PSI of the cryptic exons in AGRN, ATG4B, G3BP1, HDGFL2, MYO18A, PFKP, RANBP1, STMN2, UNC13A, and UNC13B per sample is not always 0 in the GTEx dataset. The cryptic exon in UNC13B has particularly high inclusion rates. (B) Tissue-specific average PSI of each cryptic exon in GTEx samples. The average PSI of the AGRN, ATG4B, G3BP1, HDGFL2, MYO18A, PFKP, RANBP1, STMN2, UNC13A, and UNC13B cryptic exons differs by tissue sampled. Some cryptic exons have generally low inclusion in all samples while others have tissue-specific enrichment. (C) A heatmap representation of the percentage of samples from each tissue which have an average PSI of greater than 5% for the cryptic exon. MYO18A, RANBP1, and UNC13B have tissue-specific enrichment in GTEx samples compared to the other cryptic exons. (D) Visualization of the STMN2 cryptic exon in some untreated neuroblastoma cell lines. (E) Visualization of MDA-MD-231 cells after TDP-43 and/or SRSF3 knockdown on UCSC Genome Browser (62). The PSI of HDGFL2, PFKP, and TRRAP cryptic exons appears to increase compared to a TDP-43 knockdown after co-knockdown of SRSF3. The HDGFL2 cryptic exon is present after SRSF3 knockdown only.

Figure 3.. The SRA can be mined to identify novel biological contexts of alternative splicing events.

(A) The average PSI of cryptic exons in ACTL6B, AGRN, ATG4B, G3BP1, MYO18A, PFKP, RANBP1, STMN2, UNC13A, and UNC13B can be extracted and quantified from human SRA datasets using SnapMine. Similar to the GTEx results, UNC13B is the most leaky event but some of the other cryptic exons also have greater than 5% inclusion in many samples. (B) The top 20 human samples from SRA with overall high inclusion of TDP-43-associated cryptic exons are associated with TDP-43 knockdown. TDP-43- samples are specifically depleted of nuclear TDP-43. (C) The average PSI of cryptic exons in Adnp2, Bud23, Crem, Fam135a, Hdac4, Ift81, Pnpla6, Slc7a6, Smg6, Spata7, Synj2bp, Tbc1d1, and UNC13B can be extracted and quantified from mouse SRA datasets using SnapMine. (D) Although the majority of the top 20 mouse samples from SRA with overall high inclusion of TDP-43-associated cryptic exons are associated with TDP-43 knockdown, two samples stand out. The two samples correspond to CD4+ Th1 cells treated with CPX in vitro. (E) CD4+ Th1 cells treated with CPX incorporate cryptic exons in Adnp2, Synj2bp, Tbc1d1, Tecpr1, and Usp15 at levels comparable to mouse neuronal and muscle cells with TDP-43 knocked down. (F) Treatment of a mouse splenocyte culture with 20 μM CPX for four hours leads to cryptic exon inclusion as measured by RT-PCR. The black arrow points to the cryptic band. (G) Treatment of a mouse splenocyte culture with 20 μM CPX for four hours leads to TDP-43 depletion as measured by immunoblot. TDP-43 protein levels are normalized to GAPDH protein levels.

Figure 4.. CPX treatment causes TDP-43 protein depletion in various primary cell types.

(A) Treatment of a mouse brain culture with 20 μM CPX for four hours leads to cryptic exon inclusion as measured by RT-PCR. The black arrow points to the cryptic band. The primers used to measure Ift81 and Unc13a cryptic exon inclusion target the cryptic exon directly and so no WT band is measured. (B) Treatment of a human PBMC culture with 20 μM CPX for four hours leads to cryptic exon inclusion in PFKP, EBP41L4A, and AGRN as measured by RT-PCR. The black arrow points to the cryptic band. (C) Treatment of a human i3N culture with 20 μM CPX for four hours leads to cryptic exon inclusion as measured by RT-PCR. The black arrow points to the cryptic band. (D) Treatment of a mouse brain, human PBMC, and human i3N cultures with 20 μM CPX for four hours leads to TDP-43 depletion as measured by immunoblot. TDP-43 protein levels are normalized to GAPDH protein levels. TDP-43 depletion varies by cell type. (E) Ex vivo treatment of mouse brain with 20 μM CPX for four hours leads to cryptic exon inclusion as measured by BaseScope probes targeting cryptic Ift81 and Unc13a. (F) Treatment of a human i3N culture with 20 μM CPX for four hours in combination with different small compound inhibitors can change cryptic exon inclusion rates. Treatment with MG-132, Carfilzomib, and MLN4924 appear to increase cryptic exon inclusion rates consistently while treatment with NAC attenuates cryptic exon inclusion. (G) Volcano plots from RNA-Seq data comparing CPX-treated to untreated cells across i3Neurons, PBMCs, Microglia, and Fibroblasts (n=2 for each condition) show upregulation of distinct gene signatures that are specific to i3Neurons and PBMCs, cell types that exhibit cryptic exons after treatment with CPX. By contrast, microglia and fibroblasts do not exhibit cryptic exons after CPX treatment and likewise do not exhibit strong gene upregulation of metallothioneins (highlighted in magenta). Further analysis of genes upregulated in i3Neurons (Fig. S3) confirms that heat shock response and oxidative stress pathways are upregulated, suggesting that heavy metal toxicity may be mediating CPX’s effect on TDP-43.