Additional file 1 of A toxic gain-of-function mechanism in C9orf72 ALS impairs the autophagy-lysosome pathway in neurons
Creators: name: Beckers, Jimmy
name: Tharkeshwar, Arun Kumar
name: Fumagalli, Laura
name: Contardo, Matilde
name: Van Schoor, Evelien
name: Fazal, Raheem
name: Thal, Dietmar Rudolf
name: Chandran, Siddharthan
name: Mancuso, Renzo
name: Van Den Bosch, Ludo
name: Van Damme, Philip
Publisher: name: figshare
Year: 2023
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Pharmacology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1: Generation and characterization of iPSC-derived MNs from C9orf72 ALS patients and isogenic controls. a Schematic representation of the protocol used to differentiate iPSCs to spinal motor neurons (MNs). Abbreviations: BDNF: brain-derived neurotrophic factor; CHIR: CHIR99021; CNTF: ciliary neurotrophic factor; DAPT: a γ-secretase inhibitor; GDNF: glial cell line-derived neurotrophic factor; iPSC: induced pluripotent stem cell; LDN: LDN-193189; NEP: neuroepithelial stem cell; NPC: neuronal progenitor cell; RA: retinoic acid; SAG: smoothened agonist; SB: SB 431542; Y: Y-27632. b, c Immunocytochemistry (ICC) of multiple (motor) neuron markers TUJ1 and ISL1 (b), ChAT and SMI32 (c) and DAPI in 40-day old C9orf72 and isogenic control MNs. Scale bar = 50 µm. d–f Quantification of the ISL-positive (d), ChAT-positive (e), and SMI-32 positive (f) cells relative to the total DAPI-labeled cell count; each dot represents one biological replicate. Data represent mean ± SEM. Statistical significance was assessed by one-way ANOVA (d–f) and Tukey’s multiple comparison test (b, c, t); ns = not significant. Figure S2: C9orf72 MNs have defects in lysosomal function and display reduced levels of mature lysosomes. a Flow cytometry analysis graphs of MNs stained with Lysotracker Red. b Quantification of the relative Lysotracker Red fluorescence shown in (a). c Flow cytometry graphs of MNs treated with DQ-BSA which generates fluorescence upon proteolytic cleavage by lysosomes. d Quantification of the relative DQ-BSA fluorescence shown in (c). e Representative Western blot detecting pro-cathepsin D (CTSD) and mature CTSD. β-actin was used to normalize data. f Quantifications of the Western blot shown in (e), measuring the relative levels of mature CTSD. g Quantification of the relative QTSD enzyme activity as measured by a fluorometric CTSD activity assay kit. Data represent mean ± SEM. Statistical significance was assessed by Kruskal–Wallis test and Dunn’s multiple comparison test (b, d, f, g); *p &lt; 0.05, **p &lt; 0.01. Figure S3: Mature C9orf72 MNs do not display mislocalization of TDP-43, nor increased phosphorylation of TDP-43 and only display a minor reduction in cell viability. a Immunocytochemistry (ICC) of 40-day old C9orf72 and isogenic control MNs stained for TDP-43 and phosphorylated TDP-43 (pTDP-43). The presence of a typical neuronal morphology (which includes a soma and elongated neurites was used to select the MNs for analysis. Scale bar = 10 µm. b, c Quantifications of the nuclear vs cytoplasmic (N/C) TDP-43 ratio (b) and the corrected total cell fluorescence (CTCF) of pTDP-43 (c) from the ICC images shown in (a). Each dot represents a cell that was measured (C9-1, n = 166; C9-1iso, n = 189; C9-2, n = 163; C9-2iso, n = 172). d Quantification of the relative cell viability in 40-day-old C9orf72 MNs relative to their isogenic controls as measured by the CellTiter-Glo® assay. For each independent differentiation, at least 6 technical replicates of 5000 cells were plated in 96-well plates the average intensity of each C9orf72 patient line was normalized to that of their respective isogenic control; each dot represents one biological replicate. e, Quantification of the percentage of apoptotic cells staining positive for cleaved caspase-3 in the TUJ1-positive 40-day-old MN population. Each dot represents one biological replicate in which between 107 and 224 TUJ1-positive cells were scored for cleaved caspase-3 staining. Data represent mean ± SEM; data are pooled from four-five independent differentiations. Statistical significance was assessed by Kruskal–Wallis test and Dunn’s multiple comparison test (b, c) or one-way ANOVA and Tukey’s multiple comparison test (d, e); *p &lt; 0.05, ns: not significant. Figure S4: Signs of dysregulations in the autophagy-lysosome pathway are present in post-mortem brain and spinal cord tissue of ALS patients. a Representative Western blot detecting LC3-I and LC3-II, LAMP1, GAPDH and total protein, used to measure levels of autophagosomes, lysosomes or for protein normalization respectively in post-mortem human central cortex tissue lysates from C9orf72 ALS patients (C9orf72), ALS patients tested negative for the C9orf72 HRE (Non-C9 ALS) and healthy controls (Control). b, c Quantifications of the Western blot shown in (a), measuring the ratio of LC3-II/LC3-I (b) and relative levels of LAMP1 (c); (C9orf72, n = 9; Non-C9 ALS, n = 8; Control, n = 7). GAPDH or total protein was used to normalize data. d, Representative Western blot detecting LC3-I and LC3-II, p62, LAMP1, GAPDH and total protein, used to measure levels of autophagosomes, aggregates, lysosomes or for protein normalization respectively in post-mortem human cervicothoracic spinal cord tissue lysates from C9orf72 ALS patients (C9orf72), ALS patients tested negative for the C9orf72 HRE (Non-C9 ALS) and healthy controls (Control). e–g, Quantifications of the Western blot shown in (d), measuring the ratio of LC3-II/LC3-I (e), the relative levels of LAMP1 (f) and the relative levels of p62 (g); each dot represents one tissue sample (C9orf72, n = 8; Non-C9 ALS, n = 8; Control, n = 8). GAPDH or total protein was used to normalize data. Data represent mean ± SEM. Statistical significance was assessed by Kruskal–Wallis test and Dunn’s multiple comparison test (b, c, e–g); *p &lt; 0.05. Figure S5: C9orf72 protein levels are downregulated in post-mortem brain and spinal cord samples, but not in C9orf72 MNs. a–c Representative Western blots detecting C9orf72, GAPDH or β3-tubulin and total protein in post-mortem human central cortex (a) or human cervicothoracic spinal cord tissue lysates (c) from C9orf72 ALS patients (C9orf72), ALS patients tested negative for the C9orf72 HRE (Non-C9 ALS) and healthy controls (Control) and C9orf72 patient iPSC-derived MNs and their respective isogenic controls (c). d–f Quantifications of the Western blots shown in (a–c), measuring the levels of C9orf72 in human central cortex (d), human cervicothoracic spinal cord (e) and C9orf72 patient iPSC-derived MNs and their respective isogenic controls (f); each dot represents one tissue sample (C9orf72, n = 8–9; Non-C9 ALS, n = 8; Control, n = 7–8) or one independent differentiation. GAPDH, β3-tubulin or total protein was used to normalize data. Data represent mean ± SEM; data are pooled from twelve-thirteen (f) independent differentiations. Statistical significance was assessed by Kruskal–Wallis test and Dunn’s multiple comparison test (d, e) or one-way ANOVA and Tukey’s multiple comparison test (f); *p &lt; 0.05. Figure S6: Generation of C9orf72 knockout line (C9-KO). a General overview of the C9-KO iPSC line generation. b, Schematic representation showing multi-guide RNAs targeting exon 2 of the C9orf72 gene (upper panel). Sanger sequencing traces of homozygous C9-KO and CTRL lines spanning the cut side of guide 1 (lower panel) c PCR product showing 130 bp deletion in the C9-KO line. d The C9-KO iPSC line retains pluripotency features as assessed by immunostaining of OCT4, SOX2, and NANOG. Scale bar = 100 µm. e, Representative Western blots detecting C9orf72 and total protein levels in iPSCs (left) or 40-day-old MNs derived from these iPSCs (right). Figure S7: Mature C9-KO iPSC-derived MNs do not display mislocalization of TDP-43, nor increased phosphorylation of TDP-43 and only a minor reduction in cell viability. a Immunocytochemistry (ICC) images of 40-day-old control and C9 KO MNs stained for TDP-43 and phosphorylated TDP-43 (pTDP-43). The presence of a typical neuronal morphology (which includes a soma and elongated neurites was used to select the MNs for analysis. Scale bar = 10 µm. b, c Quantifications of the nuclear vs cytoplasmic (N/C) TDP-43 ratio (b) and the corrected total cell fluorescence (CTCF) of pTDP-43 (c) from the ICC images shown in (a). Each dot represents a cell that was measured (CTRL, n = 168; C9 KO, n = 173). d Quantification of the relative cell viability in 40-day-old C9 KO MNs relative to its isogenic control line as measured by the CellTiter-Glo® assay. For each independent differentiation, at least 6 technical replicates of 5000 cells were plated in 96-well plates and the average intensity of the C9 KO line was normalized to that of its isogenic control line; each dot represents one biological replicate. e, Quantification of the percentage of apoptotic cells staining positive for cleaved caspase-3 in the TUJ1-positive 40-day-old MN population. Each dot represents one biological replicate in which between 191 and 279 TUJ1-positive cells were scored for cleaved caspase-3 staining. Data represent mean ± SEM; data are pooled from four-five independent differentiations. Statistical significance was assessed by unpaired t-test (b, c, e) or one sample t-test (d); *p &lt; 0.05, **p &lt; 0.01. Figure S8: C9orf72 patient, but not C9-KO MNs display an increased vulnerability to autophagic stress caused by Lys05. a, b Quantifications of the survival rate of 40-day-old C9orf72 patient (a) or C9-KO (b) iPSC-derived MNs and their respective isogenic control after 24 h treatment with 3-30 µM of Lys05. The survival rate of iPSC-derived MNs is calculated by normalizing the CellTiter-Glo® signal of Lys05-treated cells to DMSO control-treated wells; each dot represents the average of one biological replicate. Data represent mean ± SEM; data are pooled from three-six (a) or four (b) independent differentiations. Statistical significance was assessed by one-way ANOVA and Tukey’s multiple comparison test (a, b); *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001, ****p &lt; 0.0001.