Fig. 5: Dysregulation in key mitochondrial genes and pathways points to mitochondrial impairment in motor neurons across isogenic ALS lines. | Nature Communications

Fig. 5: Dysregulation in key mitochondrial genes and pathways points to mitochondrial impairment in motor neurons across isogenic ALS lines.

From: Single-cell RNA-sequencing reveals early mitochondrial dysfunction unique to motor neurons shared across FUS- and TARDBP-ALS

Fig. 5

a The mean expression changes of respiratory genes in ALS motor neurons (as mean log2 foldchange of FUS R495X, FUS P525L heterozygous, FUS P525L homozygous, and TARDBP M337V compared to control) were mapped to the protein structures of the mammalian mitochondrial respiratory complexes I–V (PDB entries 5lc5, 1zoy, 1bgy, 1occ, and 5ara) and rendered in UCSF Chimera. The composition was inspired by Sousa et al. The location of genes from expression plots in (c, e, g) are indicated with pointers. b, d, f Modified GSEA plots showing the rank of all genes within this specific GO-term for all mutant motor neurons generated using the homogeneous protocol, according to the GSEA test statistic. The most significantly upregulated gene is on the top and the most significantly downregulated gene on the bottom. c, e, g Violin plots showing normalized expression values (RPKM) of three example genes of the pathway from (b, d, f) in motor neurons generated with the homogeneous protocol. Error bars show mean ± SEM. * p  <  0.05, ** p  <  0.01, *** p  <  0.001 vs. control, derived from two-sided DEA testing in DESeq2 corrected for an FDR of 0.05. The p-values for COX6A1 are 9.5E-3 (FUS KO), 4.3E-7 (FUS R495X), 6.4E-10 (FUS P525L heterozygous), 2.7E-7 (FUS P525L homozygous), and 6.8E-2 (n.s., TARDBP M337V). The p-values for NDUFS6 are 9.1E-1 (n.s., FUS KO), 5.3E-5 (FUS R495X), 1.7E-4 (FUS P525L heterozygous), 5.5E-3 (FUS P525L homozygous), and 3.4E-1 (n.s., TARDBP M337V). The p-values for NDUFA12 are 6.6E-1 (n.s., FUS KO), 4.9E-3 (FUS R495X), 4.4E-4 (FUS P525L heterozygous), 4.5E-6 (FUS P525L homozygous), and 2.6E-3 (TARDBP M337V). The p-values for ATP5MF are 7.8E-1 (n.s., FUS KO), 2.7E-6 (FUS R495X), 4.6E-5 (FUS P525L heterozygous), 1.2E-3 (FUS P525L homozygous), and 2.1E-1 (n.s., TARDBP M337V). The p-values for COX7C are 9.6E-1 (n.s., FUS KO), 1.5E-5 (FUS R495X), 7.6E-6 (FUS P525L heterozygous), 2.0E-3 (FUS P525L homozygous), and 6.1E-1 (n.s., TARDBP M337V). The p-values for NDUFB9 are 3.5E-1 (n.s., FUS KO), 1.3E-4 (FUS R495X), 3.8E-6 (FUS P525L heterozygous), 1.2E-8 (FUS P525L homozygous), and 1.2E-4 (TARDBP M337V).The p-values for MT-CO3 are 2.5E-3 (FUS KO), 2.4E-7 (FUS R495X), 8.5E-4 (FUS P525L heterozygous), 5.1E-9 (FUS P525L homozygous), and 6.0E-7 (TARDBP M337V). The p-values for MT-ND3 are 7.5E-1 (n.s., FUS KO), 5.3E-9 (FUS R495X), 6.2E-10 (FUS P525L heterozygous), 1.8E-1 (n.s., FUS P525L homozygous), and 1.2E-11 (TARDBP M337V). The p-values for MT-ATP6 are 5.3E-1 (n.s., FUS KO), 3.2E-4 (FUS R495X), 1.9E-2 (FUS P525L heterozygous), 3.3E-5 (FUS P525L homozygous), and 1.4E-13 (TARDBP M337V). h Confocal fluorescence microscopy with immunostaining of mitochondrial complex I (NDUFA12) or complex IV (MT-CO1, MT-CO2, MT-CO3) markers was performed in several wells per motor neuron line and in three independent differentiations each. The axons and mitochondria were stained using NEFM and TOM22 markers respectively. Scale bars are 30 µm. The location of example images for panels i-l are indicated by boxes. i–l Quantitative immunofluorescence of the expression levels of MT-CO1, MT-CO2, MT-CO3, and NDUFA12 in TOM22+ mitochondria in NEFM+ neurites. The violin plots represent the expression levels in individual mitochondria, the dots are the mean in replicate regions, and the colors are three independent motor neuron differentiation with the homogenous protocol. The p-values were calculated with two-sided Wilcoxon rank sum test between mutant and control motor neurons and significance levels are ns p  > = 0.05, * p  <  0.05, ** p  <  0.01, *** p  <  0.001. The p-values for MT-CO1 expressions are 0.0023 (FUS R495X), 0.49 (FUS P525L homozygous), 0.19 (TARDBP M337V). The p-values for MT-CO2 expressions are 0.0014 (FUS R495X), 0.94 (FUS P525L homozygous), 0.085 (TARDBP M337V). The p-values for MT-CO3 expressions are 0.12 (FUS R495X), 0.064 (FUS P525L homozygous), 0.0014 (TARDBP M337V). The p-values for NDUFA12 expressions are 0.0013 (FUS R495X), 0.15 (FUS P525L homozygous), 0.89 (TARDBP M337V). m, n Mitochondria in motor neurons were quantified by the copy number of mitochondrial genomes per nuclear genomes by qRT-PCR on total DNA extracted from cultures at the indicated time-points in motor neurons from the DF6-9-9T.B (m, our lines) and KOLF2.1 J background (l).

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