Supplementary material
Supplementary Figures
Suppl. Fig. 1. FK866 treated neurites remain healthy for at least three days. (A) Neurites
of SCG explants cultured for 7 days, then left untreated (a-c and g-j), or treated with 100nM
FK866 (d-f and k-m) for 3 days or for 4 days as indicated. Neurites were imaged at a
proximal (a-f) and at a distal (g-m) site from the cell body mass. Neurites started to
degenerate 4 days after FK866 addition to the culture medium. This result indicates that 3-4
fold depletion of NAD is not sufficient to induce axon degeneration within this timeframe.
1nM FK866 confers axon protection after injury. (B) Bright field images of SCG
untreated (top panels) or treated with 1nM FK866 for 1 day (bottom panels), then cut and
imaged at the indicated time points. (C) Bright field images of SCG untreated (top panels) or
treated with 1nM FK866 for 3 days (bottom panels), then cut and imaged at the indicated
time points showing a still detectable protective effect on neurites 8h after cut. FK866 does
not affect NMNAT2 rapid turnover. (D) Western blot of cut neurites, treated with 100nM
FK866 or left untreated as indicated, and collected at the indicated time points shows that
FK866 fails to prevent NMNAT2 turnover (n=3, one representative experiment shown).
Suppl. Fig. 2. FK866 delays axon degeneration of injured DRG neurites by fourfold. (A)
DRG explants were treated for the indicated time with 100nM FK866, then whole explants
(left panel), or the cell bodies (middle panel) and neurite fractions (right panel) were
separately collected. NAD was determined with an HPLC-based method (see methods; n=3,
Mean and SD are shown). (B) DRG neurites were cut and left untreated or treated with
100nM FK866 just after cut. Images were acquired at the indicated timepoints. (C)
Degeneration index of experiments in (B) was calculated from 3 independent experiments
and three to four fields per experiment were quantified. The results show that there is a time
effect (P < 0.0001) and the effect of treatment is highly significant (n = 10, Mean ±SEM,
two-way repeated measures ANOVA followed by Bonferroni post-hoc test, ****p<0.0001).
Suppl. Fig. 3. CHS-828 delays axon degeneration of injured SCG neurites. (A) SCG
neurites were left untreated or treated with CHS-828 at the indicated concentrations the day
before or immediately after transecting them. Images were acquired at the indicated time
points after transection. Degeneration index was calculated from three or four fields in two or
three different experiments for CHS-828 preincubated (B) or added just after cut (C) (Mean
±SEM, n = 8-14, one-way ANOVA followed by Bonferroni post-hoc test, *, p<0.05, **,
p<0.01, ***p<0.001). FK866 and CHS-828 pharmacological target NAMPT is present
within axons and it is stable for at least 24h after injury. The presence of NAMPT in
neurites was confirmed both by immunocytochemistry and western blot analysis. (D)
NAMPT immunofluorescence (green) of dissociated SCG neurites. Nuclei are stained with
DAPI (blue). Scale bar, 10 m. (E) Western blot of wild-type and WldS SCG neurites and cell
body fractions collected 0h or 24h after a cut, probed with NAMPT antibody. Wild-type SCG
neurites were kept in the presence of FK866 for 24h collection to avoid degeneration. (F)
Integrated NAMPT band intensity of SCG neurites normalised to -actin control (Mean and
SD shown, n = 3) shows that NAMPT declines by only 40% during 24h of isolation from its
site of synthesis in cell bodies.
Suppl. Fig. 4. Blocking the extracellular degradation of NAD and NMN or blocking the
intracellular uptake of NR robustly reduces their effect on axon degeneration. (A) NAD
and NMN extracellular degradation pathways convert them to NR which is then transported
inside the cell and re-converted in NMN by the action of NRK (adapted from(33)). In cut
axons NMN cannot be converted to NAD due to the loss of NMNAT2. Some direct uptake of
NMN remains possible. (B, C) SCG neurites were left untreated or treated immediately after
cut with the compounds indicated. NMN (B) and NAD (C) were used at 1 mM. CMP and
UMP at 25 mM, dipyridamole at 50 µM and Ap4A (diadenosine tetraphosphate) at 10 mM.
Images were acquired at the indicated time points. Degeneration index was calculated in three
fields of three independent experiments (Mean ±SEM, n = 9, 2-way ANOVA followed by
Bonferroni post-hoc test, ***p<0.001).
Suppl. Fig. 5. NAD precursors from Na salvage pathway do not affect axon
degeneration and maintain cell bodies in the presence of FK866. (A) Dissociated SCG
untreated [(a) and (b)], or treated with 100nM FK866 [(c) and (d)], with 100nM FK866 and
1mM NMN [(e) and (f)], or with 100nM FK866 and 1mM NAD [(g) and (h)]. FK866induced NAD depletion causes neurodegeneration at 5 days but NMN or NAD prevent
neurodegeneration, presumably by restoring NAD levels. (B) DRG neurites were separated
by their cell bodies and treated with 100nM FK866 and with various NAD precursors (1mM)
as indicated. Neurites were imaged at 0h, 24h and 48h as indicated. Cell body explants were
imaged at 0, 6 and 13 days. NAD depletion by FK866 causes degeneration of the cell body
compartment which is detectable 6 days after FK866 addition and 2 weeks later all cell
bodies have degenerated and detached (b). Cell bodies are maintained if NAD synthesis is
restored by the addition of NMN (c) or all precursors of the Na salvage pathway (d, e, f) (see
also Fig. 1A). FK866 axon protection is affected only by NMN (c) which restores rapid
degeneration. In contrast all other precursors which are converted to NAD via the Na salvage
(Preiss-Handler) pathway do not affect axon degeneration (see also Fig. 1A).
Suppl. Fig. 6. FK866 strongly delays Vincristine-induced axon degeneration and NMN
abolishes FK866 protection. (A) Bright field images of wild-type SCG distal axons cultured
for 7 days and then treated with 0.02μM Vincristine, with 0.02μM Vincristine and 100nM
FK866 or with 0.02μM Vincristine, 100nM FK866 and 1mM NMN as indicated. Images of
an untreated explant are also shown. (B) Degeneration index was calculated from three
independent experiments (n=3, single measurements indicated, two-way repeated measures
ANOVA followed by Bonferroni post-hoc test, **, p<0.01, ****p<0.0001).
Suppl. Fig. 7. S. oneidensis NMN deamidase confers robust protection to injured SCG
axons. Shewanella oneidensis NMN deamidase has a similar activity to the E. coli enzyme
despite its divergent amino acid sequence. (A) Western blot of HEK293T cells transfected
with S. oneidensis NMN deamidase expression vector and harvested two days after
transfection, probed with an anti-his antibody. (B) Extracts of HEK293T cells transfected
with NMN deamidase cDNA in pcDNA3.1/HISA or with empty pcDNA3.1/HISA were used
for NMN deamidase activity determination (see methods). (C) S. oneidensis NMN deamidase
expressing vector or the empty vector was microinjected into SGC nuclei along with
fluorescent marker DsRed2 to visualise transfected neurons. Fluorescent neurites were cut
and imaged at the indicated time points. SCG neurons expressing S. oneidensis NMN
deamidase are resistant to degeneration after cut. NAMPTG217R partially resists FK866
inhibition. (D) Western blot of cell extracts of HEK293T cells transfected with empty vector
or with plasmid cDNA vectors encoding NAMPTG217R or NAMPT wild-type (WT) and
probed with NAMPT antibody or with a -acting loading control confirms overexpression of
NAMPT constructs. (E) Integrated NAMPT band intensity normalised to -actin control
(Mean ±SEM, n = 10). (F) HEK293T untransfected or transfected with empty vector or with
plasmid cDNA vectors encoding NAMPTG217R or NAMPT WT were homogenised and NAD
levels were determined with an HPLC based method as a measure of NAMPT enzymatic
activity in the absence or presence of 100nM FK866. NAMPTG217R enzyme activity is in part
retained in the presence of the drug (n=3, Mean and SD shown, one-way ANOVA followed
by Bonferroni post-hoc test, ***p<0.001).
Suppl. Fig. 8. NR, ATP and other nucleotides are stable in sciatic nerves after a 30h
lesion. (A) (a) HPLC chromatograms of pooled sciatic nerves 30h after a lesion and of
unlesioned contralateral nerves. The peaks of interest are labelled; among them, NMN and
nicotinamide (Nam) are partially overlapped with other interfering peaks or below the
detection limit. Wild-type (b) uncut and (c) 30h-cut nerve extracts (see above and “Methods”)
were subjected to derivatization with acetophenone(41) and analysed by reverse-phase C18HPLC. The image shows chromatographic fluorescence profiles of the above nerve extracts
after derivatization, before and after addition of 7 pmol NMN spike. Black arrows indicate
the position of an NR standard showing absence in both nerve extracts (before and after
cutting) under these experimental conditions. (B) NMN and NR levels in subcellular fractions
of explanted pooled mouse sciatic nerves after 0h and 30h ex vivo, as determined by liquid
chromatography electrospray ionization–tandem mass spectrometry.
Supplementary Videos
Suppl. video 1. Wallerian degeneration in axotomised larval zebrafish neurons treated
with vehicle. Time-lapse movie corresponding to Fig. 6C (top panels). Each frame is a
projection of a confocal image stack. Frames every 20 minutes.
Suppl. video 2. FK866 robustly delays Wallerian degeneration in axotomised larval
zebrafish neurons. Time-lapse movie corresponding to Fig. 6C (centre panels). Each frame
is a projection of a confocal image stack. Frames every 22 minutes.