iPSC motor neurons, but not other derived cell types, capture gene expression changes in postmortem sporadic ALS motor neurons

Abstract

Motor neuron degeneration, the defining feature of amyotrophic lateral sclerosis (ALS), is a primary example of cell-type specificity in neurodegenerative diseases. Using isogenic pairs of induced pluripotent stem cells (iPSCs) harboring different familial ALS mutations, we assess the capacity of iPSC-derived lower motor neurons, sensory neurons, astrocytes, and superficial cortical neurons to capture disease features including transcriptional and splicing dysregulation observed in human postmortem neurons. At early time points, differentially regulated genes in iPSC-derived lower motor neurons, but not other cell types, overlap with one-third of the differentially regulated genes in laser-dissected motor neurons from ALS compared with control postmortem spinal cords. For genes altered in both the iPSC model and bona fide human lower motor neurons, expression changes correlate between the two populations. In iPSC-derived lower motor neurons, but not other derived cell types, we detect the downregulation of genes affected by TDP-43-dependent splicing. This reduction takes place exclusively within genotypes known to involve TDP-43 pathology.

Keywords: ALS; CP: Neuroscience; CP: Stem cell research; TDP-43; cell-type specificity; disease modeling; nonsense-mediated decay; splicing.

Figure 1.. Differentiation of LMNs, cortical neurons, and sensory neurons

(A) Schematic for tet-inducible differentiation. Timeline summarizes the differentiation protocol. (B) Masking of neurons using BFP2 and TUJ1. Lighter, darker, and gray colors indicate nuclei, cytoplasm, and neurites, respectively. (C–H) Immunofluorescence and quantification of LMNs (C and D), cortical neurons (E and F), and sensory neurons (G and H). Scale bars: 100 μM; n = 8 wells for all groups. Arrows indicate magnified cells in insets.

Figure 2.. RNA sequencing across cell types and fALS mutations

CRISPR editing strategy to generate heterozygous fALS mutations sharing the same genetic background. (B) Differentiation of edited iPSCs. (C) Heatmap for selected cell markers (n = 3 libraries per condition). (D) Volcano plots comparing all control lines vs. all ALS lines. (E) Differentially expressed genes due to fALS mutations across different cell types. (F) Comparison of differentially expressed genes in fALS iPSC LMNs with laser-captured LMNs from postmortem sALS samples (n = 8 postmortem control and n = 13 postmortem sALS spinal cords).

Figure 3.. Alternatively spliced genes in TDP-43-depleted FTD-ALS postmortem neurons are downregulated in iPSC LMNs

(A) 61 genes are differentially spliced in postmortem neurons that are depleted for TDP-43^, and have sufficient read counts to analyze in iPSC LMNs. Those genes are plotted for each cell type and fALS mutation combination. p values were calculated using gene set enrichment analysis (GSEA). (B) STMN2 splicing in the TARDBP LMN isogenic pair, TDP-43 knockdown, and sALS postmortem LMNs. Black arrows indicate exon 2a. (C) Expression of splicing genes in C9ORF72 iPSC LMNs. Significant genes are colored in red; n = 6 libraries per condition. (D) STMN2 splicing in C9ORF72 iPSC LMNs. (E) Expression of splicing genes in SOD1^A4V/+ iPSC LMNs. Significant genes are colored in red; n = 3 control libraries and n = 2 SOD1^A4V/+ libraries.

Figure 4.. Transcripts containing cryptic exons are rapidly degraded

qRT-PCR for full-length transcripts and cryptic exon transcripts in STMN2 and UNC13A; n = 3 wells per condition. (B and C) Western blots for TDP-43 in the RIPA-soluble (S) fraction from LMNs. (D) qRT-PCR for cryptic exon transcripts after inhibiting translation with CHX. Black circles and gray squares indicate separate experiments. (E) Expression of NMD-sensitive transcripts in postmortem sALS LMNs and TARDBP^G298S/+ iPSC LMNs. Two-sided t tests or GSEA were used to compare between groups. ***p < 0.001, **p < 0.01, and *p < 0.05.