Fig. 6: Expression of dominant negative SARM1 (dnSARM1) protects LUHMES from AIND and Vacor toxicity.

LUHMES cell were lentivirally transduced to stably express an inducible construct controlling the expression of dominant negative SARM1 (dnSARM1) and turbo red fluorescent protein (tRFP). Because SARM1 functions as an octamer, certain point mutations in SARM1 disturb the formation and function of the SARM1 octamer. This mutated SARM1 isoform thereby acts as a dominant negative suppressor of endogenous SARM1 function [59]. A Expression of dnSARM1 was induced with tamoxifen (TAM) in plated spheroids of LUHMES cells for 6 days. On DoD15, axotomy was performed to obtain isolated neurites. Lysates were produced and analyzed by western blots for the total amount of SARM1 (endogenous SARM1 + dnSARM1) (cf. Fig S6B). The triangle symbolizes the increasing TAM concentrations used: 0 nM, 2 nM, 20 nM. B Inducibility of the construct by TAM was also tested as follows: On DoD15 (after exposure to TAM for 6 days), images of neurites were recorded by epifluorescence microscopy to monitor the expression of tRFP from the dnSARM1/tRFP construct (cf. Fig S6C). Scale bar = 200 µm. C LUHMES cells with the dnSARM1 construct were treated with Vacor on DoD6 for 18 h. Expression of dnSARM1 was induced with 0 nM or 20 nM TAM on DoD2. Cells were stained with calcein-AM and Hoechst-33342 and imaged by epifluorescence imaging. The neurite area and cell viability (Fig. S6D) were quantified. D The expression of dnSARM1 was induced in plated spheroids on DoD9. On DoD14, axotomy was performed. After 18 h, neurites were stained with calcein-AM and imaged by epifluorescence imaging. Representative images are shown. Scale bar = 200 µm. E Neurite integrity was quantified by an image analysis algorithm in neurites with or without dnSARM1 induction. Data was normalized to intact, uncut neurites. Images were obtained as described in (D). Each data point represents a biological replicate with 10 field recorded from 3-5 technical replicates. Neurite fragmentation is shown in Fig. S6D. **p < 0.01 by Student’s t test. F Isolated neurites were obtained as above, but they were grown on glass coverslips on minimal Matrigel coating. 12 h after axotomy, samples were fixed and processed before imaging by scanning electron microscopy. Representative images are shown.