Figure 4.

Pharmacological and Genetic Inhibition of NMN Accumulation Promotes Growth and Survival of NMNAT2-Deficient Neurites

(A) Representative images of neurites of Sarm1^−/− or Nmnat2^gtE/gtE;Sarm1^−/− SCG neurons 24 hr after injection with 6.25 μg/μl Texas red dextran (for rapidly labeling neurites of injected neurons) together with 40 ng/μl GFP-NMN deamidase expression construct and 10 ng/μl pEGFP-C1 (GFP-NMNd (& GFP)), 10 ng/μl SARM1-GFP expression construct and 40 ng/μl pEGFP-C1 (SARM1-GFP (& GFP)), or 10 ng/μl SARM1-GFP and 40 ng/μl NMN deamidase-GFP expression constructs (SARM1-GFP & GFP-NMNd). SARM1-GFP induces degeneration of the proximal ∼1–2 mm of neurites, presumably shortly after exiting the soma.

(B) Quantification of neurite survival in experiments described in (A). Means ± SEM are plotted (n = 3–7 each treatment). Neurite survival after GFP-NMNd (& GFP) expression is not significantly different from GFP alone (not shown).

(C) Quantification of neurite outgrowth over 7 days for explant cultures of DRGs taken from E18.5 Nmnat2^gtE/gtE embryos (left) or wild-type embryos (right) treated with FK866 and/or NaAD after 2 days in vitro. Means ± SEM are plotted (n = 3–6 each treatment, statistical significance shown relative to untreated).

(D) Representative phase contrast images showing the physical appearance of treated Nmnat2^gtE/gtE DRG neurites (if present) at 5 days in vitro (3 days after compound addition). See also Figures S2 and S3.