Fig. 4: C9orf72 deficiency alters lysosomal fitness in iPSC-derived microglia.
From: C9orf72 hexanucleotide repeat expansions impair microglial response in ALS
a, CTSD protein levels in cortex (top) and spinal cord (bottom) of C9-ALS and sALS. Western blot quantification of cortex (b) and spinal cord (c). Data were normalized to GAPDH. ANOVA with Tukeyʼs post hoc multiple correction testing (n = 2 healthy controls, n = 5 C9-ALS and n = 5 sALS). Western blot was repeated two independent times, and the technical replicates were averaged. d, Quantification showing increased abundance of lysosomal (CTSD) clusters in C9-HRE iPSC microglia versus C9-ISO. Unpaired t-test (C9-ISO n = 4 independent replicates; C9-HRE n = 5 independent replicates). e, High-resolution expansion microscopy images and quantification showing increased abundance of lysosomal clusters in C9KO microglia. Scale bar, corrected for a 4× expansion of the sample, 2.5 µm. f, Quantification showing increased abundance of CTSD clusters in C9KO microglia versus controls. Unpaired t-test (CTRL n = 5 independent replicates; C9KO n = 5 independent replicates). g,h, TEM images and quantification showing increased abundance of late endosomes/lysosomes in C9KO compared to control microglia. Unpaired t-test (CTRL n = 5 independent replicates; C9KO n = 5 independent replicates). Scale bar, upper images, 10 µm; lower images, 20 µm. i, Microscopy images showing RAB7-positive and LAMP1-positive structures (that is, late endosomes) in C9KO and control iPSC microglia. Scale bar, 5 µm. Box plots in b−d, f and h show the median, interquartile range and full data range. Live-cell imaging experiment showing the kinetics of pHrodo-conjugated E. coli particles accumulation in C9KO versus control microglia over 15 hours (j). Data points represent log10-transformed median pHrodo intensities, aggregated via a two-step median process (Methods). LOESS curves with 95% confidence intervals (gray area) were plotted to illustrate temporal trends (n = 6 independent replicates per genotype). k, Quantification of AUC and pHrodo median intensity at 30 minutes (l) and 900 minutes (m) as a proxy for uptake and cargo degradation processes, respectively. Ratio paired t-test (n = 6 independent replicates per group).
