Figure 4.
Sarm1 ASOs completely reverse the neurite outgrowth deficit in DRGs lacking NMNAT2
(A) Western blot shows that SARM1 levels are lower in Sarm1 ASO-treated DRGs cultured for 7 DIV than cASO-treated DRGs when normalized to β-actin; representative bands are shown below the quantification. Each data point corresponds to an individual mouse where three DRGs were cultured for 7 days in the presence of ASOs.
(B) Representative phase-contrast images of full-length and distal neurites (at 5× and 20× magnification, respectively) of DRGs from E13.5 embryos.
(C) Quantification of neurite outgrowth of Nmnat2+/+Sarm1+/+ in the presence of plain medium (naive), cASO, or ASOa across 7 days in vitro. The triangle indicates the point to which the majority of neurites grow, referred to as “mass,” and arrows indicate the point to which the longest neurites grow, referred to as “max.” Where only “max” is indicated, “mass” is the same. The scale bar represents 50 μm. A two-way ANOVA was performed, followed by Tukey post hoc analysis to determine whether there were significant differences between the groups indicated on the graph. An unpaired t test was used to determine whether there was a significant difference between the cASO- and ASOa-treated DRG explant SARM1 band intensity. ∗∗∗∗p < 0.0001. Sample sizes for outgrowth were from 4–6 embryos per condition, with each experimental condition being run on three separate occasions. Each data point represents average neurite length from three fields of view from three DRGs cultured together from one mouse.
Data are presented as mean SEM. See Figure S1 for the effects of Sarm1 ASOs on neurite outgrowth and SARM1 protein levels in Sarm1+/+ SCGs.