Absence of SARM1 rescues development and survival of NMNAT2-deficient axons

Abstract

SARM1 function and nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) loss both promote axon degeneration, but their relative relationship in the process is unknown. Here, we show that NMNAT2 loss and resultant changes to NMNAT metabolites occur in injured SARM1-deficient axons despite their delayed degeneration and that axon degeneration specifically induced by NMNAT2 depletion requires SARM1. Strikingly, SARM1 deficiency also corrects axon outgrowth in mice lacking NMNAT2, independently of NMNAT metabolites, preventing perinatal lethality. Furthermore, NAMPT inhibition partially restores outgrowth of NMNAT2-deficient axons, suggesting that the NMNAT substrate, NMN, contributes to this phenotype. NMNAT2-depletion-dependent degeneration of established axons and restricted extension of developing axons are thus both SARM1 dependent, and SARM1 acts either downstream of NMNAT2 loss and NMN accumulation in a linear pathway or in a parallel branch of a convergent pathway. Understanding the pathway will help establish relationships with other modulators of axon survival and facilitate the development of effective therapies for axonopathies.

Graphical abstract

Figure 1

SARM1 Acts Downstream of NMNAT2 Loss during Axon Degeneration (A) Representative immunoblot of uncut (0 hr) and 4-hr-cut wild-type and Sarm1^−/− SCG neurite extracts probed for NMNAT2 and β-actin (sample control). NMNAT2 migrates at ∼32 kDa. Quantification of normalized NMNAT2 levels (to β-actin), is shown below with 4-hr data presented relative to uncut (set at 1). Means ± SEM are plotted (n = 3; p = 0.86 wild-type versus Sarm1^−/− at 4 hr). (B) NMN and NAD levels in uncut, and 30-hr- or 120-hr-lesioned wild-type and Sarm1^−/− sciatic nerves. Means ± SEM are plotted (n = 3–5). Levels in 120-hr-cut wild-type nerves were not determined (ND) as their axons are degenerated. (C) Representative images of distal neurites of wild-type and Sarm1^−/− SCG neurons 24 and 72 hr after injection with 100 ng/μl Nmnat2 siRNA (siNmnat2) and 10 ng/μl pEGFP-C1 (expressed EGFP allows visualization of the neurites of injected neurons). (D) Quantification of survival of wild-type (WT) and Sarm1^−/− SCG neurites (left) and cell bodies (right) for 3 days after injection with pEGFP-C1 and siNmnat2 or non-targeting siRNA (siNT) as in (C). Means ± SEM are plotted (n = 3–4).

Figure 2

Normal Development and Survival of Nmnat2^gtE/gtE;Sarm1^−/− Mice (A) Neurofilament-L immunostaining of diaphragms from E18.5 Nmnat2^gtE/gtE and Nmnat2^gtE/gtE;Sarm1^−/− embryos (montages representative of n = 3 for each genotype). Boxed areas are magnified (right). Distal branches of phrenic nerves were present in wild-type, Nmnat2^+/gtE, Sarm1^−/−, and Nmnat2^+/gtE;Sarm1^−/− embryos as expected (not shown). (B) DiI-labeled intercostal nerves in ribcages from P0 Nmnat2^gtE/gtE and Nmnat2^gtE/gtE;Sarm1^−/− pups (montages, representative of n = 3 for each genotype). Nerve truncation was seen in all Nmnat2^gtE/gtE pups but was rescued in all Nmnat2^gtE/gtE;Sarm1^−/− pups. (C) Quantification of radial neurite outgrowth over 7 days for E18.5 SCG explant cultures of the genotypes listed. Means ± SEM are plotted (n = 3–6 embryos of each genotype, average of both ganglia). Extension of the majority of Nmnat2^gtE/gtE neurites (mass) and a more robust sub-population (max) are both shown. (D) Representative images of Nmnat2^gtE/gtE and Nmnat2^gtE/gtE;Sarm1^−/− neurite outgrowth at 7 days in vitro in E18.5 SCG explant cultures. Mass and max extension of Nmnat2^gtE/gtE neurites are marked (continuous and dashed lines). (E) Viability of offspring from Nmnat2^+/gtE or Nmnat2^+/gtE;Sarm1^−/− crosses. (F) Weights of 10-week-old male or female Nmnat2^gtE/gtE;Sarm1^−/− mice relative to indicated control groups. Both individual weights and means ± SEM are plotted. The weights of different genotypes in control groups do not vary significantly. (G) Representative RT-PCR analysis of Nmnat2, Sarm1, and Actb (sample control) mRNA levels in E18.5 embryo brains of the indicated genotypes. Quantification of normalized Nmnat2 levels (to Actb), relative to wild-type levels, is shown below. Means ± SEM are plotted (n = 3). (H) Representative immunoblots (of n = 3) showing NMNAT2, SARM1, and ßIII-tubulin (sample control) expression in brains from E18.5 embryo of the indicated genotypes.

Figure 3

NMN and NAD Changes Resulting from a Lack of NMNAT2 during Development Occur in the Absence of SARM1 (A) NMN and NAD levels (nmol/g tissue) and NMN/NAD ratios in brains from E18.5 embryos/P0 pups of the indicated genotypes. Means ± SEM are plotted (n = 5–7 each genotype). Data for samples wild-type for Sarm1 (red tones) have been presented previously in a different format (Di Stefano et al., 2014). Re-use of the same dataset here allows direct comparison with the Sarm1^−/− background data (blue tones), which was obtained in parallel. (B) NMN and NAD levels (nmol/mg extracted protein) and NMN/NAD ratios in separate neurite or ganglia fractions from 7-days-in-vitro explant cultures of DRGs from E13.5/14.5 embryos of the indicated genotypes. Ganglia fractions contain short proximal neurite stumps as well as cell bodies. Means ± SEM are plotted (n = 3–5 each genotype). See also Figure S1, where data in (B) are presented as relative changes.

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.