studies of wld , an aberrant fusion protein with critical nicotinamide mononucleotide adenylyltransferase ( nmnat ) activity responsible for the slow wallerian degeneration phenotype , indicate this often shares molecular features with injury - induced axon degeneration ( conforti et  al . , 2014 ) .
new , endogenous regulators of wallerian degeneration have emerged , so establishing interactions among them will be important for developing appropriate therapies ( conforti et  al .
, 2014 ; neukomm and freeman , 2014 ; pease and segal , 2014 ) .
while a conserved pathway is emerging , the relationship between nmnat2 and sterile alpha and tir motif containing 1 ( sarm1 ) , key regulators of axon degeneration with opposing influences , is still unknown ( conforti et  al . , 2014 ) .
each robustly delays wallerian degeneration when re - localized or sufficiently overexpressed ( mack et  al . , 2001 ; milde et  al . , 2013
2009 ) , but only depletion of nmnat2 , not nmnat1 or nmnat3 , triggers wld - sensitive neurite degeneration in neuronal cultures ( gilley and coleman , 2010 ) .
this suggests nmnat2 is the isoform that contributes most to axon survival in a physiological context .
drosophila nmnat ortholog , dnmnat , appears to have a similar role in flies ( fang et  al . , 2012 ) .
early loss of short - lived nmnat2 in injured axons , prior to frank degeneration , has thus been proposed as a trigger for wld - sensitive axon degeneration and the point of wld influence ( conforti et  al . , 2014 ; gilley and coleman , 2010 ) .
this is supported by studies that show that a critical requirement for phr1/highwire ubiquitin ligase during wallerian degeneration may be linked to its regulation of nmnat2/dnmnat turnover ( babetto et  al . , 2013 ;
xiong et  al . , 2012 ) and the proposal that accumulation of the nmnat substrate nmn , a predicted consequence of nmnat2 loss , acts as a pro - degenerative signal ( di stefano et  al . , 2014 ) .
severe truncation of peripheral nerve axons and cns axon tracts in nmnat2-deficient embryos , likely responsible for their perinatal lethality , is also consistent with a critical axon survival function for nmnat2 in  vivo ( gilley et  al . , 2013 ; hicks et  al . , 2012 )
importantly , though , this developmental phenotype appears to be primarily due to an initial outgrowth defect rather than dying - back degeneration of established axons ( gilley et  al . , 2013 ) .
it is required for the normal , rapid progression of degeneration after injury and other wld - sensitive insults ( gerdts et  al . , 2013 ; osterloh et  al . , 2012 ) .
multimerization , mediated by its sam ( sterile  motif ) domain , appears to be required for a pro - degenerative function dependent on its tir ( toll - interleukin-1 receptor ) domain ( gerdts et  al . , 2013 ) .
crucially , sarm1 depletion is currently the only known manipulation that delays wld - sensitive axon degeneration as robustly as expression of wld or other nmnats . in this study
, we used axon injury in nerves and neurons from sarm1 mice to establish the relationship between nmnat2 loss and a sarm1-dependent function during wld - sensitive degeneration .
remarkably , we also find that mice lacking both nmnat2 and sarm1 are viable , enabling us to examine the mechanistic origins of the developmental axon outgrowth defect associated with constitutive nmnat2 deficiency .
nmnat2 levels have previously been found to decline rapidly in the distal stumps of transected wild - type scg and drg neurites prior to their degeneration at 8  hr ( gilley and coleman , 2010 ; yang et  al . , 2013 ) . here , we find equivalent loss of nmnat2 in wild - type and sarm1 scg neurites by 4  hr after cut ( figure  1a ) , even though degeneration of transected sarm1 neurites is delayed for at least 3  days ( osterloh et  al . , 2012 ) .
levels of the nmnat substrate , nmn , and product , nad , have also been shown to respectively rise and fall in the distal stumps of transected wild - type sciatic nerves ( di stefano et  al . , 2014 ) prior to axon degeneration , which occurs at around 36  hr ( beirowski et  al . , 2005 ) .
this is consistent with a predicted loss of nmnat2 in injured axons in  vivo . here
, we find that these changes are not prevented in sarm1 nerves at 30  hr after lesion ( figure  1b ) , despite sarm1 axons being preserved for at least 14  days ( osterloh et  al . , 2012 ) .
in fact , by 30  hr , nmn has risen significantly more than in wild - type nerves , and by 120  hr , nad levels have dropped below those in wild - type nerves at 30  hr ( a short time before they degenerate ) .
together , these data suggest sarm1 promotes wallerian degeneration downstream or independently of nmnat2 loss and consequent changes in nmn and nad levels .
although nmnat2 loss has been strongly implicated as a trigger for wallerian degeneration , this is difficult to prove definitively , as injury induces many simultaneous changes .
we therefore assessed whether sarm1 is required during related , wld - sensitive axon degeneration induced specifically by small interfering rna ( sirna)-mediated depletion of nmnat2 ( gilley and coleman , 2010 ) .
we found distal neurites of sarm1 neurons are completely protected from nmnat2 sirna - induced degeneration for at least 72  hr , and also from the later , slower loss of cell viability ( figures 1c and 1d ) .
nmnat2 depletion can thus trigger sarm1-dependent axon degeneration in the absence of injury or other initiating stimuli supporting a situation in which either sarm1 itself , or a sarm1-dependent activity of a convergent pathway , promotes wld - sensitive axon degeneration downstream of nmnat2 loss .
we next bred mice doubly homozygous for the nmnat2 gene trap allele ( effectively a null allele ; gilley et  al .
, 2013 ) and a sarm1 knockout allele ( kim et  al . , 2007 ) to investigate whether sarm1 might also contribute to axon defects associated with constitutive nmnat2 deficiency during development .
intriguingly , whole - mount labeling of distal phrenic nerve branches in the diaphragm and of intercostal nerves in nmnat2;sarm1 embryos / newborn pups revealed clear and reproducible rescue of the peripheral nerve axon truncation defect associated with nmnat2 deficiency in nmnat2 animals ( figures 2a and 2b ) .
the underlying cause of this defect in nmnat2 embryos is restricted axon extension ( gilley et  al . , 2013 ) ; therefore , to obtain a quantitative measure of rescue by sarm1 deficiency , we compared neurite outgrowth in scg explant cultures from nmnat2 and nmnat2;sarm1 embryos with that of relevant controls ( figures 2c and 2d ) . strikingly , nmnat2;sarm1 neurite outgrowth matched that in sarm1 and nmnat2;sarm1 littermate cultures , as well as in wild - type and nmnat2 cultures assessed in parallel .
the axon extension defect associated with a lack of nmnat2 thus appears fully corrected in late - stage embryos and newborn  mice deficient for sarm1 .
consistent with this , while nmnat2 ( and nmnat2;sarm1 ) pups die at birth , nmnat2;sarm1 mice in contrast are viable and reach weaning at the expected mendelian ratios ( figure  2e ) .
adult nmnat2;sarm1 mice are also fertile and , despite subtle gender- and sarm1-genotype - specific differences , their weights at 10  weeks are within a healthy range and similar to control groups ( figure  2f ) .
crucially , rt - pcr and immunoblotting confirmed that the nmnat2 allele remains effectively silenced on a sarm1 background ( figures 2 g and 2h ) .
this rules out reduced efficacy of the gene trap as being responsible for rescue of the phenotype . as in injured nerves ,
sarm1 deficiency does not prevent expected changes to nmnat metabolites in the brains of embryos lacking nmnat2 ( figure  3a ) .
in fact , the relative increase in nmn in nmnat2;sarm1 brains is greater than in nmnat2 brains , presumably due to rescued outgrowth of cns axons , where changes , away from the influence of nuclear nmnat1 , should be greatest .
a small , but significant , reduction in nad is also evident in nmnat2;sarm1 brains .
in contrast , heterozygosity for the nmnat2 allele does not significantly alter brain nmn or nad levels on either sarm1 background .
interestingly , there is a trend toward higher nmn and nad levels in sarm1 brains , independent of nmnat2-related changes , and the total adenylate pool ( atp  + adp  + amp ) is increased ( 1,764  113  nmol / g tissue in sarm1 brains compared to 1,314  97 in sarm1 brains ; p  = 0.009 ) .
thus , sarm1 itself may have some influence over nad and/or energy metabolism . in case
the complexity of whole brain concealed more marked changes specifically in axons , we also assessed nucleotide levels separately in drg neurites and ganglia ( figures 3b and s1 ) . indeed , consistent with nmnat2 being the major axonal nmnat , we detected a greater relative increase in nmn and decrease in nad in nmnat2;sarm1 neurites than in brains .
in contrast , changes in ganglia fractions were far more modest , indicating nmnat2 has less influence in the cell body ( and associated proximal neurite stumps ) . surprisingly , accumulation of nmn was not evident in the limited neurite outgrowth in nmnat2 cultures
. however , the nmn / nad ratio is increased to the same extent as in nmnat2;sarm1 neurites , due to a much greater reduction in nad , consistent with comparable loss of nmnat activity .
we speculate that , even though stunted nmnat2 neurites do not show signs of overt degeneration , their integrity could still be compromised , resulting in nmn and nad efflux and the lower than expected levels of both nucleotides .
low nad in nmnat2 ganglia fractions suggests this may also extend to proximal neurite stumps and possibly even cell bodies . finally , as in brains , heterozygosity for the nmnat2 allele does not significantly alter nmn and nad levels in either drg fraction .
overall , these results indicate that sarm1 is critically involved in the manifestation of the phenotype of nmnat2 mice , with it , or a dependent activity , acting downstream of predominantly axonal alterations to nmn and nad resulting from the lack of nmnat2 .
crucially , this suggests significant mechanistic overlap between wld - sensitive axon degeneration and the axon outgrowth defect in these mice .
we have previously implicated rising nmn , rather than declining nad , as a critical pro - degenerative event in injured axons , in part by showing that blocking nmn synthesis with nampt inhibitor fk866 and scavenging nmn via ectopic expression of bacterial nmn deamidase ( converting it to namn ) both delay wallerian degeneration ( di stefano et  al . , 2014 ) .
we therefore used these reagents to investigate whether nmn also limits outgrowth and survival of axons lacking nmnat2 .
first , we tested the prediction that survival of nmnat2;sarm1 scg neurites should be impaired by ectopic re - expression of sarm1 due to re - activation of a sarm1-dependent degeneration pathway downstream of nmnat2 deficiency , and assessed whether these events are nmn - dependent using co - expression of nmn deamidase ( figures 4a and 4b ) .
while ectopic re - expression of sarm1 ( c - terminal gfp tagged ) in sarm1 neurons ( with normal nmnat2 ) unexpectedly induced some neurite degeneration by 24  hr , it induced much more extensive degeneration of nmnat2;sarm1 neurites ( lacking nmnat2 ) and , crucially , this was significantly reduced by co - expression of nmn deamidase ( n - terminal gfp tagged ) .
this suggests that , as well as promoting wallerian degeneration , nmn also drives sarm1-dependent degeneration in this context .
importantly , these experiments additionally confirm that rescue of nmnat2;sarm1 axons is due to sarm1 deficiency and not other , undefined changes resulting from mouse breeding .
next , we used fk866 to assess whether nmn accumulation also limits nmnat2 neurite outgrowth ( figures 4c and 4d ) .
interestingly , fk866 stimulated additional outgrowth of nmnat2 drg neurites , but only for about 24  hr , after which neurites underwent complete degeneration . because fk866 causes simultaneous reductions to both nmn and nad in neuronal cultures ( di stefano et  al .
, 2014 ) , we considered that this later degeneration could be due to an extreme reduction in nad in neurites now lacking both nampt and nmnat2 activities and/or secondary to neuronal death caused by reduced somal nad . to overcome this , we co - treated cultures with potential sources of nad .
nad itself was ineffective ( figure  s2 ) , likely due to inefficient entry into neurons and/or failed conversion of more readily internalized nad precursors ( generated by extracellular degradation ) back into nad when nmnat2 is absent ( di stefano et  al .
, 2014 ; nikiforov et  al . , 2011 ) , possibly even leading to further nmn accumulation in nmnat2 neurites . however , naad , an alternative source of nad via endogenous nad synthetase ( di stefano et  al . , 2014 )
, did promote additional outgrowth and survival of nmnat2 neurites when added with fk866 ( but , importantly , not when added alone ) .
together , these data suggest increased nmn does contribute to the restricted outgrowth of nmnat2 neurites , although rescue of nmnat2 neurite outgrowth by fk866 and naad still falls some way short of the outgrowth of wild - type neurites treated in the same way ( despite this also being slightly inhibitory ) ( figure  4c ) .
therefore , either inhibition of nampt by fk866 or clearance of pre - accumulated nmn is relatively ineffective and/or outgrowth is not exclusively limited by nmn - dependent events .
despite their shared ability to profoundly influence axon survival after injury , no link between nad metabolism and sarm1 function had previously been firmly established .
our studies show not only that either sarm1 itself , or a sarm1-dependent convergent activity , acts downstream of nmnat2 loss and resulting changes to key nmnat metabolites to promote wld - sensitive axon degeneration but also that this also occurs downstream of nmnat2 deficiency and dependent changes during development to impair axon outgrowth and limit mouse survival .
intriguingly , an nmnat2 depletion - dependent accumulation of nmnat substrate , nmn , also appears to be a critical pro - degenerative signal in both situations ( this study ; di stefano et  al . , 2014 ) , strongly suggesting they are each mediated by activation of the same nmn- and sarm1-dependent degeneration pathway .
importantly , our data appear to rule out any lasting stabilization of nad or reduced accumulation of nmn as part of the mechanism underlying protection of sarm1-deficient axons , although intriguing increases in total adenylate pool and steady - state levels of nmn and nad in sarm1 brains may yet be found to be involved .
overall , our studies suggest either that changes resulting from nmnat2 loss directly activate a pro - degenerative sarm1 function in axons in a linear pathway or that parallel sarm1-dependent and nmnat2 loss - dependent signals merge at a common downstream step in a convergent pathway ( figure  s3 ) .
importantly , the critical sarm1-dependent event in either model likely acts upstream of calpain activation ( yang et  al . , 2013 ) . given that nmnat2 depletion alone can activate sarm1-dependent neurite degeneration , the convergent pathway model would appear to require that sarm1 has constitutive pro - degenerative activity , with activation or disinhibition of a critical downstream step only occurring after nmnat2 loss .
while a proposed regulatory role for the n - terminal domain of sarm1 ( gerdts et  al . , 2013 ) appears more consistent with activation of a pro - degenerative function in a linear pathway , our finding that exogenous expression of sarm1(-gfp ) induces some degeneration even when nmnat2 is present ( in sarm1 neurons ) suggests it could have some constitutive pro - degenerative activity ( although conditions in these assays could also influence this outcome ) .
understanding precisely how endogenous sarm1 functions in the context of nmnat2 loss and nmn accumulation during axon degeneration , and how this relates to other cellular roles of sarm1 ( conforti et  al . ,
we had previously proposed that the early , limited extension of axons lacking nmnat2 reflects restricted supply of an nmnat - activity - dependent molecule required for axon growth , with nad being a prime candidate ( gilley et  al . , 2013 ) .
however , initially improved outgrowth of nmnat2 neurites further deprived of nad by fk866 challenges this idea , and the involvement of sarm1 instead suggests these neurites stop extending at the point where sarm1-dependent degeneration becomes active . in light of our findings
we predict that , due to a diminishing influence of nuclear nmnat1 , nmn in nmnat2-deficient axons will rise as a function of distance from the soma , with some axon outgrowth possible up to the point at which a critical threshold is exceeded and sarm1-dependent degeneration is triggered .
nad plays a key role in many diverse cellular functions ( di stefano and conforti , 2013 ) , so the fact that significant reductions in nad do not prevent the delayed degeneration of cut sarm1-deficient axons or limit the normal growth and survival of intact axons lacking both nmnat2 and sarm1 ( in culture and in mice ) was somewhat surprising .
this , together with the ability of fk866 to delay degeneration of cut axons ( di stefano et  al . , 2014 ) and temporarily rescue nmnat2 neurite outgrowth , despite further depletion of nad , clearly indicates that fairly substantial reductions in nad are not necessarily detrimental .
however , it would be surprising if greater nad depletion did not limit axon survival in some situations . on its own , survival of nmnat2;sarm1 mice for several months with no overt problems
suggests very strong correction of the defects associated with constitutive nmnat2 deficiency during early development .
however , it will still be critical to establish the precise extent and duration of this rescue in an aging study .
nevertheless , these mice already represent a unique resource for the identification or confirmation of other early pro - degenerative events triggered by nmnat2 loss and nmn accumulation , given that our data suggest an activated degeneration pathway is paused at a sarm1-dependent step in nmnat2;sarm1 axons .
for example , sarm1 appears to act downstream of reactive oxygen species accumulation , atp depletion , and ca influx when cell death and axon degeneration are induced by the mitochondrial toxin carbonyl cyanide m - chlorophenyl hydrazone ( cccp ) ( summers et  al . , 2014 ) , but it will be important to establish whether these changes also occur downstream of nmnat2 loss during wld - sensitive degeneration
. they should also be particularly useful for studying non - axonal phenotypes associated with nmnat2 deficiency that may be sarm1 independent , such as its role in cardiac hypertrophy ( cai et  al . , 2012 ) .
to conclude , these studies have now firmly established the order of two core events in an emerging wld - sensitive and sarm1-dependent axon degeneration pathway .
as additional steps are added , it will become increasingly apparent how this relates to other cell - death programs and how it might be most effectively targeted in axonal disorders .
crucially , the lack of any overt defects in adult nmnat2;sarm1 mice suggest that if effective targeting of this pathway can be achieved , it could potentially provide long - term relief from symptoms without major side effects .
animal work was carried out in accordance with the animals ( scientific procedures ) act , 1986 , under project license ppl 70/7620 .
explant cultures of superior cervical ganglia ( scgs ) from embryonic day 18.5 ( e18.5 ) mouse embryos and postnatal day 0 ( p0 ) to p2 pups and dorsal root ganglia ( drgs ) from e13.5/14.5 and e17.5/e18.5 embryos and p0 pups , and dissociated scg neuron cultures ( p0p2 ) , were set up as described previously ( gilley et  al .
neurite outgrowth assays were performed and quantified as described previously ( gilley et  al . , 2013 ) .
nampt inhibitor fk866 ( 100  nm , generously supplied by the national institute of mental health ) , naad ( 250  m , sigma - aldrich ) , nad ( 1  mm , sigma - aldrich ) , or nmn ( 1  mm , sigma - aldrich ) were added to e17.5/e18.5 nmnat2 drg explant cultures at 2  days in  vitro as described .
whole brains from e18.5 mouse embryos and neurite fractions from scg  explant cultures were processed as described previously for the generation of rna and/or protein extracts ( gilley et  al . , 2013 ; gilley and coleman , 2010 ) .
primers for rt - pcr , and primary antibodies and concentrations for immunoblotting , are listed in the supplemental experimental procedures .
nmn and nad measurements in whole brains from e18.5 embryos or newborn ( p0 ) pups , uncut or lesioned sciatic nerves from adult mice , and neurite and ganglia fractions from drg cultures ( prepared as for scg cultures in gilley and coleman , 2010 ) were performed using uv - c18 high - performance liquid chromatography ( hplc ) and/or spectrofluorometric hplc analysis essentially as described previously ( di stefano et  al . , 2014 ) .
injections and quantification of neurite / cell - body survival were performed essentially as described previously ( gilley and coleman , 2010 ) .
plasmids and sirnas injected are listed in the figure legends and in the supplemental experimental procedures .
anterograde labeling of intercostal nerve axons with the lipophilic dye dii ( 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate ) in fixed p0 mouse pups and whole - mount neurofilament light - chain immunostaining of fixed diaphragms from e17.5/e18.5 embryos were performed as described previously ( gilley et  al . , 2013 ) .
phase - contrast and fluorescence images of primary cultures and stained / labeled tissues were acquired as described previously ( gilley et  al . , 2013 ) .
the student s t test , anova with tukey s or dunnett s post hoc correction ( as applicable ) , and pearson s chi - square test were used in this  study .
a p value > 0.05 was considered not significant , and   indicates p  < 0.05 ,   indicates p  < 0.01 , and   indicates p  < 0.001 .
conceived the study . j.g . designed and performed all experiments with the exception of nucleotide measurements , obtained by g.o . ,
and in  vitro and in  vivo neurite and nerve injury experiments , designed and performed by i.n .- f . j.g . and m.p.c .
 summarysarm1 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 . 