+ Pharmacological bypass of NAD salvage pathway protects from chemotherapy- induced degeneration

Hui-wen Liua, Chadwick B. Smitha, Mark S. Schmidtb, Xiaolu A. Cambronnea,1, Michael S. Cohenc, Marie E. Migaudd, Charles Brennerb,2, and Richard H. Goodmana,2

aVollum Institute, Oregon Health & Science University, Portland, OR 97239; bDepartment of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 55242; cDepartment of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR 97239; and dMitchell Cancer Institute, University of South Alabama, Mobile, AL 33604

Contributed by Richard H. Goodman, August 22, 2018 (sent for review June 4, 2018; reviewed by Michael P. Coleman and William Lee Kraus) degeneration, a hallmark of chemotherapy-induced periph- (NMNAT1) linked to a sequence that mislocalizes this normally eral neuropathy (CIPN), is thought to be caused by a loss of the nuclear protein to the cytoplasm (5, 6). The rescue engendered by essential metabolite nicotinamide adenine dinucleotide (NAD+) via Wlds is consistent with the observation that NMNAT2, the pre- the prodegenerative protein SARM1. Some studies challenge this dominant cytoplasmic isoform of the , becomes depleted in notion, however, and suggest that an aberrant increase in a direct after transection because of its short half-life (7). This de- + precursor of NAD+, nicotinamide mononucleotide (NMN), rather pletion was predicted to result in decreased levels of axonal NAD than loss of NAD+, is responsible. In support of this idea, blocking (8), an idea supported by the findings that protection from de- + NMN accumulation in neurons by expressing a bacterial NMN dea- generation could be achieved by providing the NAD precursors, midase protected axons from degeneration. We hypothesized that nicotinamide mononucleotide (NMN) or nicotinamide riboside protection could similarly be achieved by reducing NMN produc- (NR), augmenting activity of nicotinamide phosphoribosyl- tion pharmacologically. To achieve this, we took advantage of an transferase (NAMPT) (9), or increasing NMNAT expression + alternative pathway for NAD generation that goes through the (3) (Fig. 1A). intermediate nicotinic acid mononucleotide (NAMN), rather than A second key observation was the finding that an intrinsic axon NMN. We discovered that nicotinic acid riboside (NAR), a precursor degeneration pathway depended on the prodegenerative protein, of NAMN, administered in combination with FK866, an inhibitor of SARM1 (10). Initially identified in , the SARM1 the enzyme nicotinamide phosphoribosyltransferase that produces ortholog in mammalian neurons is both necessary and sufficient NMN, protected dorsal root ganglion (DRG) axons against vincristine- for axonal degeneration (11). Moreover, CIPN, as well as other induced degeneration as well as NMN deamidase. Introducing a dif- models of such as posttraumatic brain ferent bacterial enzyme that converts NAMN to NMN reversed injury, can be rescued by the loss of SARM1 (12). The finding that + this protection. Collectively, our data indicate that maintaining SARM1 contains an intrinsic NAD hydrolase activity sup- + + NAD is not sufficient to protect DRG neurons from vincristine- ported the idea that NAD plays a central role in maintaining induced axon degeneration, and elevating NMN, by itself, is not sufficient to cause degeneration. Nonetheless, the combination Significance of FK866 and NAR, which bypasses NMN formation, may provide a therapeutic strategy for neuroprotection. Axon degeneration is caused by multiple chemotherapeutic agents and is a hallmark of chemotherapy-induced peripheral chemotherapy-induced peripheral neuropathy | axon degeneration | + neuropathy (CIPN). CIPN is associated with the loss of the es- NAD | nicotinamide mononucleotide | nicotinic acid riboside sential metabolite nicotinamide adenine dinucleotide (NAD+), a vital metabolite involved in energy homeostasis, but whether + + xon degeneration is an early feature of chemotherapy- NAD loss or the accumulation of an NAD precursor, nicotin- Ainduced peripheral neuropathy (CIPN), a severe and often amide mononucleotide (NMN), is responsible for axon degen- permanent condition caused by anticancer agents that perturb eration is controversial. We found that axon degeneration microtubule depolymerization, such as taxanes (paclitaxel), vinca caused by vincristine, a widely used chemotherapeutic agent, alkaloids (vincristine, vinblastine), and proteasome inhibitors could be ameliorated by forcing peripheral neurons to use an + (bortezomib) (1). The asymmetrical shape of peripheral neurons, NAD biosynthetic pathway that bypasses NMN formation. with axons that can extend for meters, renders these cells par- This strategy provides an approach for preventing CIPN. ticularly sensitive to insults that impair fast axonal transport. The numbness, pain, and heat/cold hyperalgesia associated with Author contributions: H.-w.L., C.B., and R.H.G. designed research; H.-w.L., C.B.S., and CIPN can be extremely troubling to patients, potentially limiting M.S.S. performed research; X.A.C., M.S.C., and M.E.M. contributed new reagents/analytic the use of otherwise effective chemotherapeutic agents (2). tools; H.-w.L., M.S.S., and R.H.G. analyzed data; and H.-w.L., C.B., and R.H.G. wrote the paper. In many ways, CIPN is reminiscent of Wallerian degeneration, Reviewers: M.P.C., Babraham Institute and Cambridge Centre for Brain Repair; and a self-destructive process involving the distal portion of trans- W.L.K., The University of Texas Southwestern Medical Center at Dallas. ected axons characterized by a beaded appearance, retraction, Conflict of interest statement: C.B. owns stock in and consults for ChromaDex, Inc., and and axonal disintegration (3). Initially attributed to a failure to consults for Cytokinetics, Inc. M.E.M. has consulted for ChromaDex, Inc. transport essential nutrients, Wallerian degeneration is now Published under the PNAS license. thought to result from an intrinsic axon destruction pathway. A 1Present address: Department of Molecular Biosciences, University of Texas at Austin, key finding in this more recent understanding was the discovery of Austin, TX 78712. a spontaneously occurring mouse mutation, Wallerian degeneration 2To whom correspondence may be addressed. Email: [email protected] or s slow (Wld ), which prevented degeneration of transected axons [email protected]. s (4). Molecular characterization of Wld revealed a fusion protein This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. + containing the nicotinamide adenine dinucleotide (NAD )biosyn- 1073/pnas.1809392115/-/DCSupplemental. thetic enzyme nicotinamide mononucleotide adenylyltransferase 1 Published online September 26, 2018.

10654–10659 | PNAS | October 16, 2018 | vol. 115 | no. 42 www.pnas.org/cgi/doi/10.1073/pnas.1809392115 Downloaded by guest on September 28, 2021 A NMN that triggers degeneration. Support for this idea includes the findings that a degree of protection was afforded by FK866, an NAMPT inhibitor, and that reducing NMN levels in neurons by expressing Escherichia coli NMN deamidase (NMNd), a bac- terial enzyme that converts NMN to its deamidated form, nic- otinic acid mononucleotide (NAMN), protected axons as well as Wlds or eliminating SARM1 (16, 17). Reconciling these two models has been difficult because elevating NMN levels by increasing NAMPT expression has also been shown to be protective (9). Although inducing expression of genes such as Wlds and NMNd were effective in preventing Wallerian degeneration, these ap- proaches have limited potential for clinical translation. We sought + BCto take advantage of the well-characterized pathways for NAD synthesis to develop a pharmacological approach for CIPN treatment, aiming to identify a small molecule or combination of small molecules that would eliminate NMN accumulation while + + maintaining NAD levels. The three classical pathways for NAD biosynthesis use tryptophan (the de novo pathway), nicotinic acid (NA; the Preiss-Handler pathway), and nicotinamide (NAM; the NAM salvage pathway). The de novo and Preiss-Handler path- ways converge at the level of NAMN, whereas the NAM salvage pathway and the recently identified NR kinase pathway (18) go through NMN. We hypothesized, therefore, that use of the former two pathways, which do not produce NMN, might be beneficial for preventing CIPN. As a model for CIPN, we examined axon de- generation in dorsal root ganglion (DRG) neurons treated with D the chemotherapeutic agent, vincristine. We found that the de novo and Preiss-Handler pathways are not functional in DRGs, and that these neurons relied on the NAM salvage pathway for + + NAD synthesis. To reroute the pathway for NAD synthesis to one that avoided NMN, we employed a deamidated form of NR, nicotinic acid riboside (NAR) (19). Similar to NR, NAR is con- verted to its product, in this instance NAMN, through the actions of the widely expressed NR kinases, NRK1 and NRK2 (18, 20). Of note, nerve injury has been shown to induce expression of the NRK gene products (9, 20), potentially increasing the efficacy of + NAR in supporting NAD synthesis. + Fig. 1. NAR maintains NAD synthesis in DRG neurons that lack NAPRT. (A) Although NAR treatment delayed the axon degeneration + Diagram of NAD synthesis pathways. The de novo pathway converts tryp- caused by vincristine, it was only marginally more effective than tophan (Trp) to quinolinic acid (QA) and then, via quinolinic acid phos- NR and did not reduce NMN levels below those in DRGs treated phoribosyltransferase (QPRT), to NAMN. NAMN is converted to NAAD and, + + with vincristine alone. To decrease NMN production further, we ultimately, NAD , via NMNAT and NAD synthetase (NADS). NAMN can also blocked NAMPT activity with the inhibitor FK866. This combi- be produced from nicotinic acid and NAR via NAPRT and NRK, respectively. NAM and NR are converted to NMN by NAMPT and NRK1 and NRK2, as nation of NAR and FK866 reduced NMN levels and protected the + neurons as well as NMNd. In addition, we showed that expressing indicated, and NMNAT converts NMN to NAD . FK866 is a specific inhibitor Francisella tularensis of NAMPT. NMNd and NMNsyn are bacterial that convert NMN to the NMN synthetase (NMNsyn), an enzyme NAMN and NAMN to NMN, respectively. Axon injury activates SARM1, which that converts NAMN to NMN, reversed this protection. The ef- hydrolyzes NAD+ to produce ADPR, cADPR, and NAM. (B) Relative levels of fects of FK866 and NAR administered in combination provide the + NAD biosynthetic enzymes in DRG neurons determined by RT-qPCR rationale for a therapeutic strategy for preventing CIPN. (mean ± SD; n = 3; P < 0.005 for each gene compared with NAMPT). (C) + Levels of NAD in DRG neurons 16 h after treatment with 100 nM FK866 and Results μ ± = < + 500 M nicotinic acid or NAR (mean SD; n 3; **P 0.005). (D) Repre- NAR Can Maintain NAD Synthesis in DRG Neurons. The principal sentative images (Left) and quantitation of tetramethylrhodamine methyl + routes for NAD synthesis in mammalian cells are depicted in ester staining (Right) 24 h after exposure to the indicated agents. F0 repre- A + sents the signal intensity after addition of the uncoupling agent FCCP. Fig. 1 . To determine which NAD biosynthetic pathways are functional in DRG neurons, we performed RT-qPCR analysis of mRNA isolated from rat E15 DRGs maintained for 6 d in culture + axonal integrity after injury (13). Whether NAD loss and the (DIV6) and treated with deoxyfluorouridine to minimize glial associated defects in energy are the primary effec- contamination. We found that NA phosphoribosyltransferases + tors of axonal degeneration is controversial, however. NAD (NAPRT) and quinolinic acid phosphoribosyltransferases were

\expressed at low levels compared with NAMPT, with NRK1 and BIOCHEMISTRY hydrolysis mediated by in vitro translated SARM1 generates + NAD synthetase at intermediate levels (Fig. 1B). Treatment of other bioactive molecules, namely, ADP ribose (ADPR) and cells with NA, NR, or FK866 did not change the expression pat- cyclic ADP ribose (cADPR) (14), both of which can cause po- + tern (SI Appendix,Fig.S1A). These results suggested that most 2 + tentially deleterious elevations in intracellular Ca (15). NAD production in DRG neurons occurs through the NAM Although there is considerable evidence supporting the idea + salvage pathway. Moreover, because of the relatively low expres- that loss of NAD contributes to axon degeneration, other sion of NAPRT, we predicted that NA would not be able to serve + studies suggest a different model; namely, that axonal injury and as an effective NAD precursor. In contrast, NAR, which re- the subsequent loss of NMNAT2 activity increases levels of the quires only NRK to produce NAMN, should be able to produce + NMNAT2 substrate NMN, and that it is the elevated level of NAD in the absence of NAPRT. To test this hypothesis, we

Liu et al. PNAS | October 16, 2018 | vol. 115 | no. 42 | 10655 Downloaded by guest on September 28, 2021 + + used quantitative targeted NAD metabolomics (21) to ascertain possible to follow NMN or NAD levels at later times because + whether NA or NAR could support NAD levels in DRG neu- the axons displayed significant degeneration. rons treated with the NAMPT inhibitor, FK866. As expected, To examine how these treatments affected axon degeneration, + NAD levels fell in DRGs treated with FK866 and were not we treated cells with NR or NAR at the time of vincristine ex- + restored by NA (Fig. 1C). In contrast, NAR restored NAD levels posure and monitored axon degeneration at various times after to normal even when NAMPT was inhibited. To test whether this treatment, shown in representative images 72 h after treatment + pharmacological rerouting of NAD synthesis pathways could (Fig. 2B) and in determination of the degeneration index (DI; sustain mitochondrial function, we examined mitochondrial mem- Fig. 2C and SI Appendix, SI Materials and Methods). Both treat- brane potential using tetramethylrhodamine methyl ester. No ments delayed degeneration compared with vincristine alone, but diminution in tetramethylrhodamine methyl ester staining was NAR was only slightly more effective than NR despite the dif- evident in DRGs treated with NAR and FK866 (Fig. 1D), and ference in NMN accumulation. By 96 h, no protection was evi- SI Appendix B dent after either agent. Taken together, these studies showed axon integrity was maintained ( ,Fig.S1 ). Taken + + together, these experiments indicate that although NAD syn- that restoration of NAD levels afforded by NR and NAR only + thesis in DRG neurons normally occurs via NAM salvage, NAD partially protected against axon degeneration. Consistent with the production can be redirected to a deamidated pathway by com- idea that NMN appears to be a result of NMNAT2 depletion (17), bining NAR and FK866, bypassing the putatively neurotoxic NMN NAR treatment did not reduce the vincristine-induced accu- intermediate. mulation of NMN, possibly accounting for the limited degree of protection. In addition, consistent with the reversibility of the NR and NAR Delay Vincristine-Induced Axon Degeneration. Chemo- NMNAT reaction (22), some NMN accumulation was also seen therapeutic drugs are thought to mediate axon degeneration by in DRGs treated with NAR and FK866 in the absence of vin- + SI Appendix activating SARM1, leading to a loss of NAD and production of cristine ( ,Fig.S2). cADPR and ADPR (Fig. 1). Many of these drugs, particularly Combining NAR with FK866 Prevents Axon Degeneration. Previous those that interfere with microtubule polymerization and de- studies showed that E. coli NMNd protected primary polymerization, reduce levels of axonal NMNAT2, which is cultures from Wallerian degeneration, presumably by converting predicted to increase NMN accumulation in addition to contrib- + NMN to NAMN (17). This protection was reminiscent of that uting to the depression of NAD . We confirmed these changes s + provided by Wld , which has been shown to lower NMN levels in by measuring NAD and NMN levels in E15 DRG neurons injured axons (16). In an effort to recapitulate the actions of treated with 50 nM vincristine at DIV6. After 16 h of vincristine NMNd and Wlds pharmacologically, we tested whether rerouting treatment, NMN levels increased from undetectable to 14.5 ± + 6 + NAD synthesis from the NAM salvage pathway to the deami- 6.8 pmol/10 cells and NAD fell to about 40% of control levels dated pathway could protect axons from degeneration as well as A (Fig. 2 ). The observation that the alternative NR kinase sub- these genetic approaches. We first examined effects of FK866, strate, NAR, produces NAMN rather than NMN (19) led us to which blocks formation of NMN from NAM. FK866 eliminated test whether NAR might be more effective than NR in protecting vincristine-induced NMN production, but at the expense of + axons from the effects of vincristine. Further, we thought these drastically reducing levels of NAD (Fig. 3A). To augment + + agents might help sort out whether the NMN increase or NAD NAD production, we added 500 μM NAR to the regimen. In- + decrease is responsible for degeneration. For these experiments, terestingly, NAD levels after treatment with vincristine, FK866, we treated DRG neurons with 500 μM NR or NAR at the same and NAR were approximately the same as after vincristine alone, time that vincristine was administered and assayed for NMN and and still substantially lower than levels in untreated neurons. + NAD 16 h later. As expected, NMN levels in DRGs treated with However, this combination significantly depressed NMN accu- vincristine were significantly higher after NR than NAR, whereas mulation compared with vincristine alone (note difference in + both treatments restored NAD to the basal level. It was not scale between Figs. 2 and 3). For comparison, we showed that a

AB C

Fig. 2. NR and NAR delay but do not prevent vincristine-induced axon degeneration. (A) Levels of NMN (Top) and NAD+ (Bottom) in DRG neurons treated with 50 nM vincristine. At the same time as vincristine on DIV6, 500 μM NR or NAR were added, and cells were collected for LC-MS 16 h later. (B) Repre- sentative images of DRG axons 72 h after vincristine treatment. NAR or NR were added at same time as vincristine. (C)DI(Methods) at various times after − treatment. For A, statistics were determined using one-way ANOVA. ns, not significant; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 10 4 denote differences using Bonferroni’s multiple comparisons test (mean ± SD; n ≥ 3 for each treatment). For C, statistics were determined using two-way ANOVA; asterisks denote P values between indicated treatments, using Bonferroni’s multiple comparisons test (n = 12 for each time).

10656 | www.pnas.org/cgi/doi/10.1073/pnas.1809392115 Liu et al. Downloaded by guest on September 28, 2021 AB C

+ Fig. 3. Combination of FK866 and NAR protects axons from degeneration. (A) NMN (Top) and NAD (Bottom) levels in DRGs exposed to 50 nM vincristine and treated with the indicated compounds or transduced at DIV2 with a lentivirus expressing NMNd. NAR and FK866 were added at the same time as vincristine on DIV6, and cells were collected for LC-MS analysis 16 h later (mean ± SD; n ≥ 3 for each treatment). (B) Representative images of DRG axons at 72 h posttreatment. Control represents neurons treated with vincristine alone. (C) Quantitation of axon degeneration at various times after vincristine administration. Asterisks denote P values as earlier (n = 12 for each time).

lentivirus expressing E. coli NMNd completely eliminated NMN F. tularensis NMNsyn Reverses Protection Afforded by NAR and FK866. accumulation. To test the idea that the combination of NAR plus FK866 pro- We then tested the combination of NAR and FK866 in axonal tected against axon degeneration by depressing NMN levels, we protection assays, treating DRG neurons with both agents at the infected DRGs with a lentivirus expressing NMNsyn. This en- time of vincristine administration. As shown in representative zyme functions in a manner opposite to NMNd, converting images (Fig. 3B) and DI assays (Fig. 3C), the combination of NAR NAMN to NMN. In vincristine-treated neurons, the depression and FK866 protected neurons as well as NMNd expression. Taken in NMN accumulation achieved by treatment with FK866 and together, these experiments indicate that substantial protection NAR (compare lanes 1 and 2 of Fig. 4A) was reversed by ex- + from vincristine-induced axon degeneration can be achieved by pression of NMNsyn, and NAD levels were restored (Fig. 4A, simultaneously blocking NMN accumulation and providing an lane 3). NMNsyn, when combined with NAR, increased NMN to + alternative source for NAD . The FK866 and NAR combination the level detected in DRG neurons treated with vincristine and + + did not increase NAD levels back to normal, however, suggesting elevated NAD to levels above normal. In degeneration assays of that the key determinant for axon protection is reducing NMN DRGs treated with vincristine, NMNsyn largely reversed the + accumulation. Thus, a depressed level of NAD , when combined protective effect of NAR plus FK866 (Fig. 4 B and D), whereas with low levels of NMN, is not sufficient for axon degeneration. NMNsyn administered with NAR did not cause degeneration

A B C

D BIOCHEMISTRY

Fig. 4. NMNsyn reverses protection afforded by FK866 plus NAR. (A) NMN (Top), and NAD+ (Bottom) levels in DRGs transduced with a lentivirus expressing F. tularensis NMNsyn or control on DIV2. Cells were cultured for 5 d after infection and treated with 50 nM vincristine, FK866, and NAR, as indicated (mean ± SD; n ≥ 3 for each treatment). (B) Representative images of DRG neurons at 72 h posttreatment with the agents indicated. (C) Representative images of NMNSyn- expressing DRG neurons at 72 h post-NAR treatment. (Axons were not treated with vincristine.) (D) Quantitation of axon degeneration. Asterisks denote P values as earlier (n = 12 for each time).

Liu et al. PNAS | October 16, 2018 | vol. 115 | no. 42 | 10657 Downloaded by guest on September 28, 2021 when vincristine was absent (Fig. 4C). Taken together, these reconcile the two models and provide a pharmacologic approach + experiments indicate that shunting NAD synthesis from a to preventing CIPN. We call this approach NAR addiction be- pathway through NAMN to one that involves NMN eliminates cause it renders DRG neurons dependent on an exogenous + the protection from vincristine-induced degeneration. In the ab- precursor not normally used in NAD synthesis that feeds into sence of vincristine, however, the rise in NMN was insufficient to the normally vestigial deamidated pathway. + induce degeneration if NAD levels were elevated. Consistent with Consistent with the second model, using quantitative targeted a previous study (23), NR alone similarly increased NMN levels, metabolomics, we observed a persistent elevation of NMN levels + elevated NAD , and did not cause degeneration (SI Appendix,Fig. after vincristine treatment. Unlike a previous study that showed S3). In contrast, blocking the salvage pathway via FK866 treatment the elevated NMN levels decreasing to baseline at 6 h after caused degeneration after 72 h (SI Appendix,Fig.S3B). axotomy (23), we found that the levels remained elevated for 24 The idea behind using NMNd was to reduce levels of the h. This discrepancy could be a result of different types of injury, potentially neurotoxic NMN, but this manipulation should also axotomy versus vincristine, improved internal standards, or that + increase flux through the deamidated NAD synthesis pathway we quantify whole-cell NMN levels as opposed to those in axons (Fig. 1A). NAR uses this pathway as well, and should similarly alone. NR, commonly used to prevent conditions associated with + + increase levels of deamidated NAD precursors. It is possible NAD loss (20, 25, 26), protected against the vincristine-induced + that some of these deamidated intermediates could contribute to decrease in NAD , but generated even higher levels of NMN. In the axon protective effects of NMNd or the FK866 plus NAR an effort to reduce NMN formation pharmacologically, we combination by blocking SARM1 activity. To test whether flux treated DRGs with NAR, a precursor of NAMN that feeds into through the deamidated pathway was augmented, we quantified the deamidated pathway (Fig. 1). The Preiss-Handler pathway levels of nicotinic acid adenine dinucleotide (NAAD), the final normally depends on NAPRT, the NA-dependent equivalent of + step in the deamidated pathway before NAD production (24). NAMPT. NAPRT mRNA levels in DRG neurons were found to As anticipated, levels of NAAD were increased in DRGs treated be extremely low, however, rendering this pathway nonfunctional. with vincristine and NAR, NMNd, or the FK866 plus NAR NAR, similar to NR, requires only phosphorylation by NRK1/ + combination (SI Appendix, Fig. S4). NMNsyn eliminated the NRK2 to generate the next intermediate in the NAD biosynthesis + NAAD accumulation mediated by FK866 plus NAR. pathway. Both precursors restored NAD levels to normal, but SARM1 contains an amino-terminal autoinhibitory domain NAR caused a substantially smaller increase in NMN. Still, NAR that prevents its activity under basal conditions. Removal of this did not eliminate the vincristine-induced increase in NMN, which inhibitory domain reveals an enzymatic activity that allows the might account for the residual propensity for degeneration. We + hydrolysis of NAD to produce ADPR and cADPR (14). To test suggest that this NMN is generated from the activity of NAMPT, + whether a deamidated NAD precursor such as NAAD might which produces NMN from nicotinamide, and from the bidirec- interfere with SARM1 activity, we purified epitope-tagged SARM1, tional activity of NMNAT. Notably, our results indicated that re- + either full-length or the active SARM1 fragment, from HEK293 storing NAD levels to normal was not sufficient for full protection. cells and tested its ADPR cyclase activity in vitro. No interference Consistent with observations of others (17), we found that E. + with NAD hydrolysis or cADPR generation was detected (SI Ap- coli NMNd greatly depressed NMN levels in neurons treated pendix,Fig.S5) in the presence of NAAD, suggesting that NAAD with vincristine and protected neurons from degeneration. In does not function as a SARM1 inhibitor. an effort to reduce NMN levels pharmacologically, we used a combination of agents that simultaneously blocked the NAM + Discussion salvage pathway while supporting NAD production through the Wallerian degeneration has served as a model of multiple neu- deamidated pathway. In the presence of vincristine, FK866 + rological conditions, but perhaps its most common manifestation greatly depressed NAD levels, as expected from the block of is in CIPN, a frequent adverse effect of many commonly used two salvage pathway enzymes, and this decrease was partially chemotherapeutic agents. Insights into the mechanisms under- rescued by adding NAR. The combination of FK866 and NAR lying Wallerian degeneration provide the hope of developing was as protective as E. coli NMNd in axon degeneration assays, + agents capable of preventing or reversing this serious condition. however. NAD levels in neurons treated with vincristine, NAR, Genetic studies in Drosophila and mouse models led to the dis- and FK866 were no greater than those observed after vincristine + covery of two key determinants of Wallerian degeneration; alone. This suggests that the loss of NAD does not fully account namely, Wlds (4) and SARM1 (10). The finding that loss of for vincristine-induced axon degeneration. s SARM1 phenocopies Wld suggested that the two might intersect As a further test of the idea that NAR plus FK866 protected on a common pathway, and this idea was supported by the ob- axons by eliminating production of NMN, we examined the ef- + servations that Wlds contains an NAD biosynthetic enzyme and fects of F. tularensis NMNsyn, whose activity is the opposite of + SARM1 has an NAD hydrolase activity. Regardless of whether the E. coli NMNd. NMNsyn shunts the Preiss-Handler in- + SARM1 promotes axon degeneration by depleting NAD or by termediate NAMN to NMN, allowing NAR to feed into the generating ADPR and cADPR, pharmacologic agents capable of salvage pathway at the level of NMN. NMNsyn severely limited inhibiting SARM1 would be good candidates for CIPN pre- the axonal protection afforded by FK866 and NAR, apparently + vention. Unfortunately, no such agents have been developed. by reestablishing neurotoxic levels of NMN. Because NAD Our approach was to address the consequences of Wlds and levels in neurons expressing NMNsyn returned to baseline, these + + SARM1 action; namely, effects on the NAD metabolome. studies suggest that the NMN increase, rather than NAD loss, There is general agreement that axon injury leads to a loss of was responsible for degeneration. One caveat of our studies is + NMNAT2 and a decrease in NAD (3). How this relates to axon that in addition to depressing NMN levels, NAR increased levels + degeneration is controversial, however. Although considerable of the deaminated NAD precursor, NAAD. However, we found + evidence supports the idea that NAD loss, and perhaps the that NAAD did not reduce cADPR generation by purified, full- + generation of bioactive NAD metabolites, are major factors (9, length SARM1 or a SARM1 fragment lacking the autoinhibitory 13), some studies have suggested that build-up of the NMNAT2 domain, suggesting that NAAD inhibition of SARM1 activity substrate NMN is more important (16, 17). Obviously, these two is unlikely. models lead to opposing predictions; in the first, it should be It has been suggested that SARM1 inhibitors might be useful + + beneficial to increase NAD production by providing NAD for axon protection, but to date, no SARM1 inhibitors have been precursors, whereas in the second, efforts should be devoted to developed. The agents we have used target pathways known to depressing elevated NMN levels. In this report, we attempt to be affected by SARM1 and are readily available. It could be

10658 | www.pnas.org/cgi/doi/10.1073/pnas.1809392115 Liu et al. Downloaded by guest on September 28, 2021 + imagined that elevating cellular NAD content might promote derived through the deamidated pathway. The final step in this + growth of transformed cells, but the combination of NAR with pathway depends on NAD synthetase, an enzyme that is ex- + + FK866 did not lead to supraphysiological NAD levels, and cluded from mitochondria (27). Thus, all NAD in the NMN- neurons treated with this combination appeared normal (SI depleted cells must be produced in the cytoplasm, and as a + Appendix, Fig. S1) and maintained normal mitochondrial ion consequence, mitochondrial NAD must be transported from gradients. the cytoplasm rather than generated in the matrix via NMNAT3. Taken together, our results largely support the idea that NMN This finding supports our observation that knockdown of NMNAT2 + promotes axon degeneration. Depressing levels of NMN confers in other cell types reduced mitochondrial NAD levels (28). Mi- + + axon protection even in the face of lower NAD levels. However, tochondrial NAD transporters have been identified in yeast and elevating NMN was not sufficient to cause axon degeneration, as bacteria, but have not been identified in mammals. neurons treated with NAR and NMNsyn displayed an elevation in NMN but did not degenerate. It is possible that the aug- Materials and Methods + mented level of NAD in this setting was protective (17). In Animal use followed NIH guidelines and complied with Oregon Health and addition, how NMN relates mechanistically to axon degenera- Science University Institutional Animal Care and Use Committee (IACUC) under an approved protocol. Methods of cell culture, lentiviral packaging, tion, and presumably SARM1 activation, remains unresolved. There + is no evidence supporting the notion that NMN regulates SARM1 NAD metabolite extraction, LC-MS/MS analysis, axon degeneration as- directly, raising the possibility that some other NMN-binding pro- say, and statistical analyses are provided in SI Appendix, SI Materials and Methods. tein is required in SARM1 regulation. Finally, the viability of NAR-addicted neurons has implica- + ACKNOWLEDGMENTS. This work was supported by NIH Grants 1R01AG055431- tions for how mitochondrial NAD levels are maintained. In + 01 (to R.H.G.) and DP2GM126897 (to X.A.C.), Roy J. Carver Trust (C.B.), and Pew these neurons, there is no cytoplasmic NMN, and all NAD is Biomedical Scholar (M.S.C.).

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