Fig. 3.

(a–e) ALS variants suppress Hspa8 mRNA levels in motor neurons. Hspa8 mRNA was quantified by smFISH 3 days after microinjection of TDP-43^G348C, FUS^R521G, or SOD1^G93A plus eGFP vectors. Microinjected motor neurons were identified by double-immunolabeling with antibodies to eGFP and TDP-43, FUS, or SOD1. (a) Examples of Hspa8 smFISH. Significant decrease in RNA spot counts in (b) somata and (c) dendrites of motor neurons expressing ALS variants compared to eGFP control. (d) and (e) Reduction in Hspa8 mRNA only in somata of neurons expressing wild-type (WT) TDP-43. Data presented as mean spot counts ± SD, n = 11–61 neurons. (f) and (g) Reduction in HSPA8 protein in somata of motor neurons expressing ALS variants, compared to noninjected motor neurons in the same culture (CTL). (f) Representative micrographs of spinal cord-DRG culture double-labeled with anti-HSPA8 and anti-TDP-43. Arrow points to a neuron expressing TDP-43^G348C. (g) Graphed is mean pixel intensity of immunolabeling signal ± SD, n = 9–24 neurons. Statistical significance was evaluated through one-way ANOVA followed by Bonferroni post hoc analysis. *P < 0.05, ****P < 0.0001. Scale bar = 15 μm unless stated otherwise. Abbreviations used: ALS, amyotrophic lateral sclerosis; FUS, fused in sarcoma; smFISH, single molecule fluorescence in situ hybridization; SD, standard deviation; SOD1, superoxide dismutase I; TDP-43, TAR DNA binding protein 43 kDa. eGFP; enhanced green fluorescent protein.