Figure 4. SARM1-dependent injury-induced NAD⁺ consumption in the axon is the critical determinant of axon degeneration.

(a) Axonal NAD⁺ consumption and synthesis rates were measured by using stable isotope Nam (D4-Nam; 2,4,5,6-deutrium Nam). Culture medium was supplemented with 300 µM of D4-Nam at time 0 and then deuterium-labeled (heavy) NAD⁺ (to follow synthesis) and non-labeled (light) NAD⁺ (to follow consumption) were measured from axonal metabolites using LC-MS/MS. Representative graphs of NAD⁺ synthesis and consumption before and after axotomy in wt and SARM1 KO are shown (mean ± s.d., n=3 for each data point). (b) Quantification of axonal NAD⁺ consumption rate under various manipulations of NAD⁺ biosynthesis pathways before and 4 hr after axotomy (NR concentration was 1 mM for cytNMNAT1 and 5 mM for NMN DD NMN SN expressing cells); data show the first and third quartile (box height) and median (line in the box) ± 1.5 time interquartile (details in the method), one-way ANOVA F(13,118) = 17.19 p<2 × 10^–16. *p<0.005 denotes significant difference from the NAD⁺ consumption rate of control before axotomy with Holm-Bonferroni multiple comparison (Figure 4—source data 1, n=9 except NAMPT where n = 12). (c) Fold increase of axonal NAD⁺ synthesis at 2 hr after axotomy compared with the uncut control under various manipulations of the NAD⁺ biosynthesis pathway. The NAD⁺ synthesis rate was normalized to that of corresponding uncut axons; data show the first and third quartile (box height) and median (line in the box) ± 1.5 time interquartile (details in the method), one-way ANOVA F(4, 43) = 21.41, p=9 × 10^–10. *p<4 × 10^–5 denotes a significant difference form fold increase of NAD⁺ synthesis in control with Holm-Bonferroni multiple comparison (Figure 4—source data 1, n=9 except for control where n = 12).

DOI: http://dx.doi.org/10.7554/eLife.19749.013

Figure 4—figure supplement 1. Axonal degeneration profiles after axotomy were not altered by the addition of 1 mM Nam to the culture medium at the time of axotomy (Figure 4—source data 2, n=4).

Both control and 1 mM Nam treated axons degenerate (DI > 0.3) in 9 hr.

DOI: http://dx.doi.org/10.7554/eLife.19749.017

Figure 4—figure supplement 2. Representative NAD⁺ consumption and synthesis in DRG neurons.

D4-Nam (300 µM) is added to the DRG culture medium at 0 hr and then whole cell metabolites are extracted at the indicated time and light NAD⁺ and heavy NAD⁺ are quantified using LC-MS/MS. Net NAD⁺ consumption and synthesis are measured as a decrease of light NAD and an increase of heavy NAD⁺, respectively. Note that total NAD⁺ is increased about 20% at 30 hr after the addition of D4-Nam (mean ± s.d., n=3 for each data point).

DOI: http://dx.doi.org/10.7554/eLife.19749.018

Figure 4—figure supplement 3. Absolute axonal NAD⁺ consumption rates in neurons expressing various NAD⁺ biosynthesis enzymes or in control neurons.

D4-Nam was added for 4 hr and axonal metabolites were extracted. ATP level and light and heavy NAD⁺ levels were measured using LC-MSMS. NAD⁺concentrations were normalized to ATP, which is stable in uninjured axons, and NAD⁺ consumption rates were calculated as described in the methods. Data show the mean ± s.d., one-way ANOVA F(4, 10) = 28.99, p=1.77 10^–5. * p < 0.03 denotes significant difference from the control NAD⁺ consumption rate with Holm-Bonferroni multiple comparison (Figure 4—source data 3, n=3).

DOI: http://dx.doi.org/10.7554/eLife.19749.019

Figure 4—figure supplement 4. Representative axonal NAD⁺ consumption under various manipulations of NAD⁺ biosynthesis pathways are shown (mean ± s.d., n=3 for each data point).

DOI: http://dx.doi.org/10.7554/eLife.19749.020