Abstract
Mutations in the RNA/DNA-binding proteins FUS and TDP-43 cause the fatal disease amyotrophic lateral sclerosis (ALS). The precise mechanisms behind the selective motor neuron degeneration remain unclear and it is uncertain if ALS-causative mutations trigger motor neuron death through shared or distinct pathogenic pathways. To address these two questions, we performed single-cell RNA sequencing across neuron types derived from isogenic induced pluripotent stem cell lines, harbouring FUS P525L, FUS R495X, TARDBP M337V mutations or FUS knockout. The mutations elicited 5- to 15-fold greater transcriptional responses in motor neurons than interneurons. Approximately 20% of transcripts uniquely dysregulated in motor neurons were shared across FUS mutations, with half being driven by FUS gain-of-function. Among these, a majority pointed towards mitochondrial impairments, with attenuated pathways shared with the TARDBP M337V mutation. Meta-analysis demonstrated convergence on mitochondrial dysfunction with C9orf72-ALS patient-derived motor neurons. We observed impaired mitochondrial motility across ALS motor axons, even in isogenic FUS R244C motor neurons, which retain FUS in the nucleus, demonstrating shared toxic gain-of-function mechanisms across FUS- and TARDBP-ALS, uncoupled from protein mislocalization. These early signs of mitochondrial dysfunction unique to motor neurons could have profound implications for their survival and represent promising therapeutic targets across multiple ALS forms.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We quantified the degree of mislocalization of mutant FUS using quantitative high-throughput immunofluorescence microscopy (Fig. 2b,c). We show that the similar level of protein mislocalization in the R495X and the P525L homozygous lines indicates that the number of DEGs identified across lines (Fig. 1i) does not correlate with the degree of mislocalization. We demonstrate that the localization of TDP-43 did not vary between control and TARDBP M337V motor neurons, and neither was there a significant difference in phosphorylated TDP-43 levels (Fig. 3d,e). We identified potential upstream regulators which could explain the dysregulation elicited by mutant TARDBP. Among the significant regulators, we found TARDBP itself. We interrogated its functional status further by calculating its activation z-score. At −3.13, it indicates that a significant inhibition of TARDBP function occurs in TARDBP M337V motor neurons (Fig. 3i) despite its stable expression level. We also conducted an upstream regulator analysis on our full dataset to identify master regulators that agree with the observed differential expression in each motor neuron line (Extended Data Fig. 7a). We identified three significant protein-coding regulators shared across ALS-lines, which are all mitochondria-associated factors (Extended Data Fig. 7b). We performed immunofluorescent staining against MT-CO1, MT-CO-2, MTCO-3 and NDUFA12 in combination with antibody-staining against TOM22, as a marker of mitochondria, and neurofilament intermediate chain (NEFM) (Fig. 5h). Systemic quantification of these markers within mitochondria in motor axons demonstrated a reduction of mitochondrial MT-CO1 (Fig. 5i), and MT-CO2 (Fig. 5j) in the FUS R495X line. MT-CO3 was downregulated in mitochondria in TARDBP M337V motor axons (Fig. 5k). Mitochondrial levels of NDUFA12 were also reduced in the FUS R495X line (Fig. 5l). All Seahorse data was moved to Extended Data Figure 9. We observed a significantly increased spreading of mitochondria in all ALS motor axons compared to the control line (Fig. 6h). The largest differences were observed in the FUS R495X and TARDBP M337V motor axons (Fig. 6h). We also interrogated how regular mitochondria are spaced out by calculating the difference of the distance from each mitochondria to its anterograde and retrograde neighbor and found significant deviations in the distribution of mitochondria in the ALS motor axons (Fig. 6h).
Abbreviations
- ALS
- amyotrophic lateral sclerosis
- ANOVA
- analysis of variance
- ATP
- adenosine triphosphate
- 2-DG
- 2-deoxyglucose
- CCA
- canonical correlation analysis
- DEA
- differential (gene) expression analysis
- DEG
- differentially expressed genes
- DNP
- 2,4-dinitrophenol
- DTT
- dithiothreitol
- ECAR
- extracellular acidification rate
- FDR
- false discovery rate
- GO
- gene ontology
- GOF
- gain of function
- GSEA
- gene set enrichment analysis
- HRE
- hexanucleotide repeat expansion (in C9orf72)
- IPA
- Ingenuity pathway analysis
- iPSC
- induced pluripotent stem cells
- KO
- knockout
- LOF
- loss of function
- NLS
- nuclear localization signal
- OCR
- oxygen consumption rate
- PCR
- polymerase chain reaction
- qPCR
- quantitative PCR
- RPKM
- reads per kilobase of exon per million reads mapped
- SEM
- standard error of the mean
- UMAP
- uniform manifold approximation and projection
