Fig. 7: Expression of WLD(s) protects neurites from AIND.

LUHMES cells were lentivirally transduced to express the “Wallerian degeneration slow” (WLD(s)) fusion protein [58]. Spheroids were generated from WT and WLD(s)-expressing cells. Spheroids were plated on DoD9. On DoD15, axotomy was induced and neurites were isolated. A, B Neurites of WT and WLD(s)-expressing LUHMES cells were left intact, or axotomy was induced. 18 h later, neurites were stained with (A) calcein-AM and (B) TMRE and were imaged by epifluorescence imaging. 10 fields (0.32 mm² each) each were recorded per condition in three biological replicates. Representative images are shown. (A) Scale bar = 200 µm, (B) scale bar = 50 µm. C Neurite integrity and fragmentation was evaluated from calcein stained cut neurites by an image analysis algorithm. Images were obtained as in (A). Data of WLD(s) neurites was normalized to neurite parameters of intact (100% integrity) WLD(s) neurites or fully degenerated (100% fragmentation) WT neurites. ***p < 0.001 by ANOVA with Dunnet’s post hoc test. D The number of TMRE+ mitochondria was quantified by an image analysis algorithm from images recorded as described in (B). Data from each image was normalized to the neurite area in each image. The relative number of TMRE+ mitochondria was normalized to respective intact controls. *p < 0.05 by Student’s t test. E The total pool of NAD+ and NADH (NAD(H)) was measured in WT or WLD(s) neurites after lysis of all neurites in a well. Data was normalized to NAD+ measured in freshly isolated neurites of the respective genotype (t = 0 h) (cf. Fig S7A). **p < 0.01 by Student’s t test. F ATP was measured and normalized analogously to the NAD(H) measurements described in (E) (cf. Fig S7B). **p < 0.01 by Student’s t test.