Journal: bioRxiv
Article Title: Astrocytes control motor neuronal mitochondrial axonal transport deficits in C9ORF72 ALS
doi: 10.1101/2025.06.10.658820
Figure Lengend Snippet: C9 astrocytes impair mitochondrial bioenergetics in MNs. (a) & (b) Quantification of the OCR as measured by the Seahorse Analyzer denoting basal and maximal FCCP-uncoupled mitochondrial respiration, involving co-cultures of astrocytes and MNs derived from patient-specific hiPSC lines. The astrocytes were obtained from three C9 hiPSC lines and their corresponding isogenic gene-corrected counterparts created via CRISPR/Cas9. MNs were generated from one pair of C9 mutant and isogenic gene-corrected control lines. The open triangles represent technical replicates and the filled triangles represent independent biological replicates (mean of technical replicates). Data are represented as the mean of biological replicates ± SEM of the means of biological replicates. p-values (*<0.05; **<0.01; ***<0.001; ****<0.0001) determined by fitting generalised linear mixed models, followed by post-hoc tests with Bonferroni correction. (c) Quantification of the mean fluorescence intensity of MitoTracker Red CMXROS in hiPSC-derived C9 and C9Δ MNs. The open triangles represent technical replicates and the filled triangles represent independent biological replicates (mean of technical replicates). In total, over 20 MNs per well were analysed to evaluate intensity per cell body area (automated analysis). Data are represented as the mean of biological replicates ± SEM of the means of biological replicates . (d) Quantification of the mean fluorescence intensity of MitoTracker Red CMXROS in hiPSC- derived C9 and C9Δ MNs co-cultured with hiPSC-derived C9 A and C9A astrocytes (from the three-independent patient-derived C9ORF72 lines). The open triangles represent technical replicates and the filled triangles represent independent biological replicates (mean of technical replicates). In total, over 20 MNs per well were analysed to evaluate intensity per cell body area (automated analysis). Data are represented as the mean of biological replicates ± SEM of the means of biological replicates. p-values (*<0.05; **<0.01; ***<0.001; ****<0.0001) determined by fitting generalised linear mixed models, followed by post-hoc tests with Bonferroni correction. (e) C9 astrocytes, when compared to their corresponding gene-edited controls, display defective bioenergetics (diminished capacity to produce ATP). OCR as measured by the Seahorse Analyzer, normalised to the amount of total protein, denoting basal, ATP-linked, and maximal FCCP-uncoupled mitochondrial respiration for C9 astrocytes and gene-corrected paired controls across the three independent pairs. The open triangles represent technical replicates, and the filled triangles represent independent biological replicates (mean of technical replicates). Data are represented as the mean of biological replicates ± SEM of the means of biological replicates. p-values (*<0.05; **<0.01; ***<0.001; ****<0.0001) determined by fitting generalised linear mixed models, followed by post-hoc tests with Bonferroni correction.
Article Snippet: After 21 days of MN plate down, astrocyte-MN co-cultures and MN monocultures were incubated with 100 nM of potentiometric dye MitoTracker Red CMXRos (Thermo Fisher) for 45 minutes at 37°C ( ).
Techniques: Derivative Assay, CRISPR, Generated, Mutagenesis, Control, Fluorescence, Cell Culture
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