Nicotinamide riboside, a form of vitamin B3, protects against excitotoxicity-induced axonal degeneration
ABSTRACT: NAD+ depletion is a common phenomenon in neurodegenerative pathologies. Excitotoxicity occurs in multiple neurologic disorders and NAD+ was shown to prevent neuronal degeneration in this process through mechanisms that remained to be determined. The activity of nicotinamide riboside (NR) in neuroprotective models and the recent description of extracellular conversion of NAD+ to NR prompted us to probe the effects of NAD+ and NR in protection against excitotoxicity. Here, we show that intracortical administration of NR but not NAD+ reduces brain damage induced by NMDA injection. Using cortical neurons, we found that provision of extracellular NR delays NMDA-induced axonal degeneration (AxD) much more strongly than extracellular NAD+. Moreover, the stronger effect of NR compared to NAD+ depends of axonal stress since in AxD induced by pharmacological inhibition of nicotinamide salvage, both NAD+ and NR prevent neuronal death and AxD in a manner that depends on internalization of NR. Taken together, our findings demonstrate that NR is a better neuroprotective agent than NAD+ in excitotoxicity-induced AxD and that axonal protection involves defending intracellular NAD+ homeostasis.—Vaur, P., Brugg, B., Mericskay, M., Li, Z., Schmidt, M. S., Vivien, D., Orset, C., Jacotot, E., Brenner, C., Duplus, E. Nicotinamide riboside, a form of vitamin B3, protects against excitotoxicity-induced axonal degeneration. FASEB J. 31, 000–000 (2017). www.fasebj.org
Axonal degeneration (AxD) is an early major event in acute cerebral injury and in chronic neurodegenerative diseases, including Alzheimer and Parkinson diseases (1–4). Preserving axonal integrity by stopping neurode- generative expansion and preventing irreversible neuro-nal damage could represent a powerful therapeutic strategy in prevention of brain disorders. Several lines of reasoning support AxD proceeding via a mechanism associated with dysregulated NAD+ metabolism that is distinct from neuronal cell body death (5–8). Notably, a spontaneous mutation termed Wlds, which delays axotomy-induced AxD in neurons derived from theperipheral nervous system (PNS), has been discovered in mice (9). Wlds encodes a fusion protein that over-expresses and stabilizes the complete coding sequence of nicotinamide mononucleotide adenylyltransferase (Nmnat)-1, an enzyme that converts nicotinamide mononucleotide (NMN) to NAD+ (10). Moreover, sev- eral studies revealed a protective effect of Nmnat over- expression or application of NAD+ or NAD+ precursors in axotomy-induced AxD in the PNS (5, 7, 8, 11). One such precursor, nicotinamide riboside (NR), protects against diabetic peripheral neuropathy in vivo (12).The molecular cascade and the time course of events in AxD have been explored intensively. Axonal lesion in dorsal root ganglia induces a loss of Nmnat2 protein and activation of sterile a and TIR motif–containing (Sarm)-1 protein, thereby triggering the MAPK signaling pathway, NAD+ depletion and subsequent AxD (13–15).
More re- cently, it has been shown that Nmnat1 expression can inhibit Sarm1-mediated NAD+ depletion (16). These results highlight the essential role of NAD+ metabo- lism in AxD of PNS-derived neurons. Whether a sim- ilar relationship exists between NAD+ metabolism and AxD in the CNS has not been well explored.A strong NAD+ depletion in neurons has been revealed during excitotoxicity (17–20). Excitotoxicity is a common process taking place in most neurodegenerative disorders affecting the CNS and is the main cell death mechanism in ischemia and hypoxia. Moreover, excitotoxicity has beenshown to induce AxD in CNS-derived neurons (21, 22). The involvement of NAD+ in excitotoxic stress was con- firmed by studies demonstrating a neuroprotective effect of NAD+ repletion via overexpression of NAD+ bio- synthetic enzymes, application of extracellular NAD+ or its precursors (17, 19, 23).In the CNS, it has not been determined whether NAD+ functions as a neurotransmitter or as a source of intracellular NAD+ in protecting against excitotoxicity-induced AxD. This mechanistic understanding is needed in pursuing therapeutic strategies with either NR or NAD+. Because NAD+ contains 2 phosphate groups, transport across the plasma membrane is not expected, such that the protectiveeffect of extracellular NAD+ may be related to extracellular conversion to NR, one of its biosynthetic precursors (24–28).
This extracellular conversion pathway is mediated by ecto- nucleotide pyrophosphatase/phosphodiesterase (ENPPase) and ectonucleotidase (ENTase) activities, previously char-acterized for extracellular ATP conversion (29). After NAD+ and NMN are converted to NR extracellularly, NR is used intracellularly through the NR kinase pathway (28, 30).NR is a recently described vitamin precursor of NAD+ that is found in milk (31, 32) and is orally available in humans (33). It enters cells through nucleoside trans- porters (NTs) and is converted to NMN and NAD+ through the successive action of NR kinases (Nmrk1/2) and Nmnat isozymes (28). In vitro, NR delays AxD after axotomy in dorsal root ganglion neurons (7). In mice, NR prevents cognitive decline and amyloid-b peptide aggre- gation (34), protects against noise-induced neurite re- traction of inner hair cells and hearing loss (35), and protects against diabetic peripheral neuropathy (12). In rats, NR both protects and reverses chemotherapeutic peripheral neuropathy (36). We performed a comparative study of NAD+ and NR and examined NAD+ extracellular conversion in cortical neurons during excitotoxic stress. We found that NR has a strong protective effect on NMDA-induced neurodegeneration both in vitro and in vivo. More specifically, NR, but not NAD+, had a strong effect on NMDA-induced AxD, involving a local NR me- tabolism within the axon. Finally, we showed that NAD+ efficiency in cortical AxD protection is limited by conver- sion to NR.The 3-compartment chip master was constructed as described in Kanaan et al. (37). For chip production, polydimethylsiloxane (Sylgard 184, PDMS; Dow Corning, Midland, MI, USA) was mixed with a curing agent (9:1 ratio) and degassed under a vacuum. The resulting preparation was poured onto a polyester resin replicate and reticulated at 70°C for at least 2 h. The elas- tomeric polymer print was detached, and 2 reservoirs were punched for each macrochannel.
The polymer print and a glass coverslip, cleaned with isopropanol and dried, were treated for 3 min in an air plasma generator (98% power, 0.6 mbar; Diener Electronic, Ebhausen, Germany) and bonded together. The chips were placed under UV for 20 min and then coated with a solution of poly D-lysine (10 mg/ml, P7280; Millipore-Sigma, St. Louis, MO, USA) overnight and washed with Dulbecco-PBS (D-PBS) (14190169; Thermo Fisher Scientific, Waltham, MA, USA) before cell seeding. Alternatively, 3-compartment chips were purchased from Microbrain Biotech (Paris, France).All animals were ethically maintained and used in compliance with the European Policy on Ethics. Cortices were microdissected from E14 embryos of Swiss mice (Janvier, Le Genest Saint Isle, France) in D-PBS supplemented with 0.1% (w/v) glucose (Thermo Fisher Scientific). C57BL/6N mice were used in Nmrk2- knockout (KO) experiments. Dissected structures were digested with papain (15 U/ml in DMEM; 76220; Millipore-Sigma) for 5 min at 37°C. After papain inactivation with 10% (v/v) fetal bo- vine serum (GE Healthcare, Little Chalfont, United Kingdom), structures were mechanically dissociated with a pipette in DMEM Glutamax-1 (31966; Thermo Fisher Scientific) containing DNAse-I (D5025, Millipore-Sigma). After 2 7 min centrifugations at 700 g, cells were resuspended in a medium containing DMEM Glutamax-1, penicillin (100 U/ml)/streptomycin (100 mg/ml) (15140; Thermo Fisher Scientific), 5% (v/v) fetal bovine serum, N2 supplement (17502048; Thermo Fisher Scientific), and B-27 supplement (17504-044; Thermo Fisher Scientific) to a final den- sity of 50 million cells/ml. Cortical cells were then seeded in the somatic compartment. Cell culture medium was added equally to the 4 reservoirs. Microfluidic chips were placed in Petri dishes containing 10% EDTA (Millipore-Sigma) and incubated at 37°C in a 5% CO2 atmosphere. The culture medium was renewed every 6 d. Upon differentiation, cortical axons entered themicrochannels and reached the second chambers after 4–5 d. Cortical axons continued growing thereafter.Nmrk2-KO mice were obtained by the injection of embryonic stem cells (Nmrk2tm1(KOMP)Vlcg allele) from the Knockout Mouse Project (KOMP) into C57BL/6N blastocysts at Centre National de la Recherche Scientifique–Transgenesis, Archiving andAnimal Models (CNRS–TAAM) Transgenic Animal Facility(SEAT; Villejuif, France). All exons and introns of the Nmrk2 geneare replaced by a lacZ reporter gene to create a null allele in em- bryonic stem cells (University of California, Davis, Davis, CA, USA; https://www.komp.org/geneinfo.php?geneid=65082).
Chimeric male mice were used to obtain heterozygous Nmrk2-mutant mice on a C57BL/6N background. Homozygous Nmrk2-KO mice were viable and fertile. We validated the deletion by sequencing the genomic DNA from the KO mice.Cells were pretreated and/or treated between 11 and 13 d in vitro. To ensure fluidic isolation, a differential hydrostatic pressure between compartments was maintained.The following compounds were used: NMDA, (100 mM; M3262; Millipore-Sigma), FK866 (10 mM; F8557; Millipore- Sigma), NAD+ (N3014; Millipore-Sigma), NMN (N3501; Millipore-Sigma), NR (ChromaDex, Irvine, CA, USA), NAM (N0636; Millipore-Sigma), dipyridamole (DP, 50 mM; D9766; Millipore-Sigma), cytidine monophosphate (CMP; 25 mM; C1006; Millipore-Sigma), and (+)MK-801-maleate (10/50 mM; 0924; Tocris Bioscience, Bristol, United Kingdom).Immunofluorescence detection and image acquisitionAt various times, cultures were fixed with 4% (w/v) para- formaldehyde (15714-S; Euromedex, Souffelweyersheim, France) +4% (w/v) sucrose (S0389; Millipore-Sigma) for 20 min at room temperature. Cells were then washed once with PBS for 10 min and permeabilized for 30 min with D-PBS containing 0.2% (v/v) TritonX-100 (Millipore-Sigma) and 1% (w/v) BSA (Millipore-Sigma). Immunostaining was performed as de- scribed in Magnifico et al. (6) using the following conjugated antibodies diluted in D-PBS: anti-bIII tubulin-Alexa Fluor 488 (1:500, AB15708A4; Millipore-Sigma) and anti-microtubule- associated protein 2 (MAP2)-Alexa 555 (1:500, MAB3418A5; Millipore-Sigma). Cell nuclei were stained by using Hoechst 33342 (2 mg/ml). The chips were then rinsed once with PBS and filled with PBS+0.1% sodium azide.Images were acquired with an Axio-observer Z1 microscope (Zeiss, Wetzlar, Germany) fitted with a cooled CCD camera (CoolsnapHQ2; Roper Scientific, Trenton, NJ, USA). The micro- scope was controlled with Metamorph (Berlin, Germany) soft- ware, and images were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).Axonal fragmentation was assessed with a macro developed in ImageJ software that contained the following steps: 1) subtractbackground, 2) plug in tubeness (s = 1), 3) plug in Otsu thresholding, and 4) analyze particles by measuring total parti- sample.
Total RNA was isolated with an RNeasy kit (74104; Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. From the total RNA, 1 mg was RNase free-DNase treated and retrotranscribed with a SuperScript II reverse tran- scriptase kit (18064-022; Thermo Fisher Scientific) according to the manufacturer’s instruction. The remaining RNA was hy- drolyzed using RNase H from E. coli (AM2293; Thermo Fisher Scientific).PCR amplification was performed in a thermocycler by mix- ing 2 ml of cDNA with 0.5 mM of forward and reverse nucleotide sequences (Table 1) in a PCR buffer containing dNTP mix (200 mM each), MgCl2 (1.5 mM), and 0.5 U of Taq DNA poly- merase (18038; Thermo Fisher Scientific). Initially, the template was denatured by heating to 95°C for 5 min, followed by 30 amplification cycles. Each cycle consisted of 3 steps: the first for 45 s at 94°C, the second for 30 s at annealing temperature (depending of the amplified mRNA), and the third for 1 min at 72°C for polymerization. The final step was an extension at 72°C for 10 min. PCR products were electrophoresed on 1.5% (w/v) agarose gel and stained with ethidium bromide (0.5 mg/ml).Quantitative PCR was performed with a LightCycler480 (Roche Applied Science, Penzberg, Germany), by mixing 1:50 diluted cDNA with the target-specific primers and Light Cycler 480 SYBRGreen I Master mix (4707516001; Roche, Basel, Swit- zerland) according to the manufacturer’s instructions. Amplifi-cation conditions were initial denaturation for 5 min at 95°Cfollowed by 40 cycles of 10 s at 95°C, 15 s at annealing tempera- ture and 10 s at 72°C. Results were analyzed with LightCycler 480 SW 1.5 software. Individual PCR products were analyzed by melting-point analysis. The expression level of a gene was nor- malized relative to that of mouse b-actin or hypoxanthine- guanine phosphoribosyltransferase (HPRT).NAD+ was measured using EnzyChrom NAD+/NADH Assay Kit (E2ND-100; Euromedex) according to the manufacturer’s higher than 0.2 (A0.2).
The fragmentation index is obtained as the ratio A0.2/ATOTAL. This index is almost linearly correlated with fragmented axon percentages: indices under 0.2, ;0.5, and up to0.8 correspond to ,10, 50, and more than 90% of fragmented axons, respectively. Neuronal death was quantified by calcu- lating the ratio of condensing nuclei to total nuclei.Primary cortical neurons were lysed at DIV 11, and tissues were lysed from 8-wk-old mice. Three wells were pooled to obtain 1 formed as described in Trammell and Brenner (38).Experiments were performed on male C57/BL6 mice (23 6 3 g, produced and provided by GIP Cyce´ron animal facilities) in ac- cordance with European Union Directives (210/63/UE) and French Ethics laws (R214; 87-137; Ministe`re de l’Agriculture) onanimal experimentation and were approved by the Universite´Caen Normandie Animal Welfare Committee. Mice were deeply anesthetized with isoflurane 5% and maintained under anesthesia with 2% isoflurane in a 70/30% gas mixture (N2O/O2) during surgery. The rectal temperature was maintained at 37 6 0.5°C throughout the surgical procedure using a feedback- regulated heating system.A cortical unilateral injection [coordinates: 0.5 mm posterior,2.0 mm lateral, 20.5 mm ventral to the bregma (39)] of NMDA (5 nmol; in 0.33 ml), coinjected or not with NAD+ or NR (50 nmol) was performed after placing the animals in a stereotaxic frame. Solutions were injected by the use of a micropipette made with hematologic micropipettes (calibrated at 15 mm/ml, assistant ref 555/5; Hecht, Sondheim-Rhoen, Germany). The micropipette was removed 3 min later. The volume of the lesion was analyzed 48 h later.Experiments were performed on a Pharmascan 7T (Bruker, Bre- men, Germany). T2-weighted images were acquired with a multislice multiecho sequence: TE/TR 51.3/2500 ms 48 h after injection. Lesion volumes were quantified on MRI with ImageJ software.Differences were assessed by a Mann-Whitney U test to compare 2 conditions or a Kruskal-Wallis test to compare .2 conditions.
RESULTS
To dissect the protective effects of NAD+ and NR on AxD, we cultured cortical neurons in microfluidic devices containing 3 chambers connected to each other (37). When seeded in the left chamber, cortical neurons projected their axons from the proximal to the distal chamber through asymmetric filtering microchannels, whereas the somata and dendrites were confined in the left chamber. As a consequence of such compartmen- talization, the right chamber of the microfluidic device contained only axons. When depressurized, the central chamber acts as siphon and ensures optimal fluidic isolation of the distal axonal and somatodendritic chambers (Fig. 1A, B).
Analysis of dendrites (MAP2, red), nuclear chromatin (Hoechst 33342, blue), and axons (b3-tubulin) indicated that the seeded cortical neurons were healthy after 2 wk in culture (Fig. 1C). Somatodendritic application of 100 mM NMDA in the proximal chamber induced dendrite de- generation marked by a loss of MAP2 immunostaining and nuclear condensation associated with a severe AxD visualized by punctiform b3-tubulin immunostaining in the distal chamber (Fig. 1C–G). By contrast, local ap- plication of NMDA to axons in the distal chamber had no effect on neurons, consistent with the fact that NMDA receptors (NMDARs) are exclusively localized to the somatodendritic compartment (22) (Fig. 1E–G). NMDAR involvement in NMDA-induced neurodegeneration was confirmed with the noncompetitive NMDAR antagonist MK-801. Nuclear condensation and AxD after 8 h NMDA treatment were fully blocked by coaddition of 10 or 50 mM MK-801 (Fig. 1H, I).
To evaluate and compare the potential neuro- protective properties of NAD+ and NR, cortical neu- rons were treated with 100 mM NMDA in the presence or absence of 5 mM NAD+ or 1 mM NR. Nuclear con- densation and AxD were then quantified over time. As shown in Fig. 2A, after 24 h of treatment, no inhibition of dendrite degeneration (MAP2) nor inhibition of nuclear condensation was observed after NAD+ or NR
treatment, whereas both molecules delayed AxD (Fig. 2A–C). NAD+ protection was weak (67% of protection compared to NMDA effect after 8 h of treatment, 22% after 24 h), whereas NR fully protected axons after 8 h and still provided 72% protection 24 h after NMDA treatment (Fig. 2C). NAD+ and NR efficiencies were also compared in a dose–range assay in cortical neu- rons cotreated with 100 mM NMDA for 24 h (Fig. 2D).
NAD+ started to delay AxD from 5 mM. In contrast, NR was efficient at a 10-fold lower dose (0.5 mM; 33 vs. 43% of protection) with a maximum protective activity at 1 mM. We asked whether the absence of protection of NAD+ and NR on NMDA-induced nuclear con- densation was due to the high-dose of NMDA (100 mM) leading to irretrievable degeneration. Accord- ingly, we challenged 5 mM NAD+ or 1 mM NR with a lower 10 mM NMDA concentration during 24 h (Sup- plemental Fig. S1). A lower nuclear condensation was observed with 10 mM NMDA (30%) compared with that obtained with 100 mM NMDA (53%) but nei- ther NAD+ nor NR had any protective effect on this phenomenon.Considering the different neuroprotective activities of NAD+ and NR in our in vitro model of excitotoxicity, we asked if NAD+ and NR could be neuroprotective in an in vivo setting of excitotoxicity. For this purpose, we coin- jected NMDA (5 nmol) and NAD+ or NR (50 nmol each) into mouse cortical structures as published (40). Strikingly, the volume of the NMDA-induced lesion as measured by MRI was significantly reduced after NR coinjection, whereas NAD+ did not reveal a significant protection (Fig. 2E, F). Thus, NR has a more potent neuroprotective effect than NAD+ in NMDA-induced neurodegeneration, both in vitro and in vivo.
We performed targeted quantitative NAD+ metabolomics to evaluate the effect of NR and NAD+ on NMDA-treated mouse brains (38). As shown in Fig. 2G, NR significantly elevated brain NAD+ from ,400 pmol/mg to nearly 500 pmol/mg, whereas the increase in brain NAD+ from NAD+ injection was not significant. Consistent with an elevated NAD+ metabolome with NR as a neuroprotective compound, NR tended to elevate brain NAM and adenosine diphosphate receptor (ADPR), whereas no changes were evident in brains treated with NAD+ (Fig. 2H, I). application did not prevent it (Fig. 3A–F). Local NAD+ treatment revealed no significant protective effects (Fig. 3G), but for both molecules NR and NAD+, the pro-tection was increased or detected when applied on both compartments together. These findings indicate that lo- cal axonal treatment with NR is required for axonal protection and that somatic NAD+ or NR treatment is insufficient to fully prevent AxD. The data indicate that somatic application of these molecules helps to in- crease axonal protection induced by axonal applica- tion of the same compounds.NR is converted to NAD+ in cortical neuronsNot all models of WldS-dependent neuroprotection in- voke a requirement for NAD+ biosynthesis (42). Accord- ingly, we tested the hypothesis that the protective effect of NR depends upon its intracellular conversion to NAD+ (Fig. 4A). First, we asked whether NR enters cortical neu- rons for further conversion to NAD+. NR is a nucleoside reported to cross plasma membranes through NT (25, 27). Among NT isoforms, expression analysis in our neuronal cell culture model indicated that all equilibrative nucleo- side transporter (ENT1-4) transcripts were expressed (Fig. 4B). However, none of the concentrative nucleoside transporter (CNT1-3) mRNAs could be detected. DP, a potent pharmacological inhibitor of ENT1 and -2 activities, blocks NR cell entry in human cell lines (25, 43). Cotreat- ment of cortical neurons with 100 mM NMDA, 1 mM NR, and 50 mM DP almost completely prevented NR-induced axonal protection (axonal protection decreased by 68%; from 88 to 20%), establishing that the neuroprotective ef- fect of NR requires DP-sensitive transport through the plasma membrane (Fig. 4C).
Intracellular NR conversion to NAD+ is initiated by Nmrk1/2, which generates the NAD+-proximal pre- cursor, NMN (Fig.4A). We analyzed Nmrk1 and -2 mRNA expression by quantitative RT-PCR from both specific mouse brain areas and from cortical neurons in culture. We discovered that Nmrk1 mRNA is constitutively expressed in all tissues, whereas Nmrk2 mRNA was absent in all brain areas (cortex, hippocampus, and cere- bellum) (Fig. 4D, E). However, Nmrk2 transcript was highly expressed in heart and striated skeletal muscles, as previously described (30, 44). Nmrk1 but not Nmrk2 mRNA was also expressed in cultured cortical neurons (Fig. 4E). Nmrk2 mRNA was reported to be induced in PNS neurons after sciatic nerve axotomy (7). To investigate a potential induction of Nmrk2 after an excitotoxic insult in cortical neurons, Nmrk2 mRNA was quantified after 100 mM NMDA treatment for 24 h. We found a low induction of Nmrk2 transcript, but a 4-fold increase of Nmrk1 mRNA. NR kinase activity leads to formation of NMN, which is adenylylated to NAD+ via Nmnat enzymes. We analyzed Nmnat1-3 mRNA ex- pression in cortical neurons treated or not with NMDA 100 mM for 3 or 24 h. We showed that all the transcripts are expressed, with the highest level for the cytosolic isoform Nmnat2, followed by the mitochondrial en- zyme Nmnat3 (Supplemental Fig. S2A). Each of the mRNAs was induced by 24 h NMDA treatment such that all components of the NR kinase pathway to NAD+ biosynthesis were upregulated during neuronal insult. NR conversion to NAD+ can be initiated by nucleoside phosphorylase, which converts NR to NAM, which is further converted to NAD+ (45). However, cotreatment of cortical neurons with NMDA 100 mM and NAM 5 mM for 24 h demonstrated that NAM is unable to prevent NMDA-induced AxD (Supplemental Fig. S2B).
We next evaluated NAD+ levels in neurons during an excitotoxic challenge. Treatment of cortical neurons with 100 mM NMDA for 24 h induced a 36% decrease in intracellular NAD+. This effect was reverted by cotreatment with 1 mM NR (Fig. 4F). Together, these results establish that conversion of NR to intracellular NAD+ via the transcriptionally induced NR kinase pathway is critical for maintaining axoplasmic NAD+ in opposition to excitotoxicity-induced AxD.Extracellular NAD+ involves a potentially unique mechanism to prevent excitotoxicity-induced AxDExtracellular NAD+ has a lower axon-protective effect than NR in our model. As reported previously in cell lines and PNS-derived neuronal cells, NAD+ can be converted extracellularly to NMN by ENPPase and then to NR by 59-ENTase (Fig. 5A). We asked whether extracellular conversion of NAD+ to NR is necessary for NAD+- mediated axonal protection or if this effect is independent of NR action. For this purpose, we probed 59-ENTase ac- tivity in cortical neurons, using a high concentration of CMP, a competitive inhibitor of this enzyme (29). First, we treated cortical neurons with 100 mM NMDA, with or without 5 mM NMN for 24 h. Extracellular NMN signifi- cantly reduced NMDA-induced AxD and 25 mM CMP reversed the protective effect of NMN (Fig. 5B). Consistent with data in other tissues (28, 30), these data indicate that NMN must be converted to NR to mediate axonal pro- tection. The beneficial effect of NMN was also prevented when cortical neurons were cotreated with NMDA and the ENT1/2 inhibitor DP, confirming that NMN is converted to the nucleoside NR before plasma membrane transport to prevent NMDA-induced AxD. Consistent with a de- pendence on conversion to NR, NR was more protective at a lower concentration than NMN. Surprisingly, 5 mM NAD+ was slightly but significantly protective, yet nei- ther CMP nor DP blocked the axonal protection con- ferred by this high concentration of NAD+ (Fig. 5C).
NAD+ and NR protect neurons with the same efficiency after FK866-induced NAD+ depletionBecause excitotoxicity and SARM1 activation induce NAD+ depletion in a manner that can be protected by NR and NAD+, we tested whether pharmacological inhibition of NAD+ homeostasis causes AxD. FK866, a specific inhibitor of Nampt (46), which salvages NAM for resynthesis of NAD+ (Fig. 6A), was used to induce NAD+ depletion. Addition of 10 mM FK866 to the somatodendritic com- partment of cortical neurons induced a rapid decrease of NAD+ levels after 24 h of treatment (Fig. 6B). At 72 h, this drop in NAD+ induced somatodendritic degeneration, nuclear condensation, and AxD (Fig. 6C, D). Cotreatment of cortical neurons with 10 mM FK866 and 50 mM NAD+or NR fully prevented these effects (Fig. 6D–F). Notably, the protective concentrations of NAD+ and NR were lower than those used in the NMDA-induced neuro- degeneration model (50 mM for both in the FK866 model and 5 mM NAD+ or 1 mM NR in the NMDA model) (Figs. 2A–D and 6D–F). NAD+ and NR remained fully pro-tective when applied 24 h after treatment, at a time whenNAD+ depletion was already observed, but dropped to 20% protection when applied 48 h later (Fig. 6E, F). These findings highlight the existence of a temporal window in which neuronal integrity can be rescued after a neurotoxic insult. Taken together, these data show that NAD+ and NR have the same neuroprotective effect against FK866- induced NAD+ depletion.We next evaluated the metabolic pathway from extracel- lular NAD+/NMN/NR to intracellular NAD+ in FK866- induced neurodegeneration. First, cortical neurons were cotreated with 50 mM FK866, 50 mM NR, and 50 mM DP for 72 h, and axonal fragmentation was investigated. DP only partially limited the NR-protective effect against FK866 (axonal protection reduced by 40%), whereas the pro- tective effect of NR was totally inhibited under NMDA stress (Figs. 4C and 7A).
These data suggest that FK866 treatment induces expression of NT that is relatively re- sistant to DP. We analyzed ENT1-4 mRNA in cortical neurons treated or not with 10 mM FK866 for 72 h. ENT2 and -4 mRNA were induced 2.2- and 1.7-fold, respectively, by FK866 (Supplemental Fig. S3). NAD+ and NMN also prevented FK866-induced neurodegeneration in a manner that was partially prevented by DP (Fig. 7B, C). As NAD+ may depend on NT in protection against FK866 neuro- degeneration, we tested whether extracellular NAD+ and NMN need to be converted to NR before transport through plasma membrane. Indeed, CMP fully blocked the axon-protective effect of NAD+ and NMN, indicating that both molecules require 59-ENTase activity to be con- verted to NR and mediate their axon-protective effect (Fig. 7B, C). We also analyzed Nmrk1 and -2 mRNA expression in cortical neurons treated or not with FK866 for 24–72 h. As expected, Nmrk1 mRNA was highly expressed com-pared with the Nmrk2 transcript, which was not detectable (Supplemental Fig. S4B, C). As previously described with NMDA, FK866 treatment for 48 or 72 h induced Nmrk2 (3- fold at 72 h). As Nmrk2 induction was a common phe- nomenon observed in our experiments, we used cortical neurons from wild-type or Nmrk2-KO mice. These neurons were treated with 10 mM FK866 for 72h before condensed nuclei and axonal fragmentation were analyzed. Even in the absence of the Nmrk2 gene, NAD+ and NR provided full protection against FK866-induced neurodegeneration, indicating that Nmrk2 is not required for these effects (Supplemental Fig. S4D, E). These results demonstrate that NAD+, NMN, and NR usea similar pathway, likely independent of Nmrk2, to prevent FK866-induced neurodegeneration.
DISCUSSION
Excitotoxicity is a process commonly described in several brain disorders, including stroke, traumatic brain injuries, and the major neurodegenerative diseases. High gluta- mate release activates ionotropic glutamate receptor, as NMDAR, and triggers disruption of ion homeostasis, leading to energetic dysfunction and oxidative stress.Major events related to excitotoxicity are therefore mitochondrial dysfunction, NAD+ depletion and metabolic dysregulation. Moreover, because neuronal stress induces DNA damages, poly (ADP-ribose) poly- merase (PARP) activation, and NAD+ depletion, the role of NAD+ metabolism in excitotoxicity requires careful characterization (47).Several in vitro studies examined the effect of NAD+, its precursors, and biosynthetic enzymes in prevention of excitotoxicity-induced neuronal degeneration (17, 19, 23, 48–50). However, the results appeared to depend on par- ticulars of the excitotoxicity models. For instance, exoge-nous NAM protects against neuronal degeneration after 6 or 12 h of glutamate/NMDA treatment, but not after 24 h (19). Furthermore, only weak neuroprotective effects of NAD+ were observed after 100 mM glutamate application, even with NAD+ doses up to 15 mM (23, 50). Our results showing that neither NAD+ nor NR protects against NMDA-induced somatic degeneration are consistent with these studies. We discovered unexpectedly that NR and NAD+ have differential effects on NMDA-induced AxD in vitro and in vivo. In vitro, 1 mM NR was highly efficient in preventing AxD, whereas 5 mM NAD+ was partially protective. In vivo, injection of NR, but not NAD+, signif- icantly reduced NMDA-induced lesions (Fig. 2E, F) and elevated brain NAD+ (Fig. 2G). We considered the possi- bility that NR coinjection altered the severity of the initial NMDA-triggered insult.
One of the major initial neuro- toxic events involved in NMDAreceptor activation is a rise in intracellular calcium (51). Metabolomic analyses have shown that NR coinjection leads to ADPR induction (Fig. 2H, I). Actually, ADPR is known to enhance cytoplasmic calcium concentration, indicating that NR should enhance NMDA neurotoxicity, but this was not the case in this study, suggesting that NR injection reduces NMDA- induced lesion by acting downstream of the initial insult. In PNS-derived neurons injured by axotomy, NR is protective reportedly because Nmrk2 is transcription- ally up-regulated by the injury (7). In the current study we showed that NR enters neurons via DP-sensitive transporters. Although Nmrk2 was slightly induced by NMDA or FK866 treatment (Fig. 4E and Supplemental Fig. S4B, C), Nmrk1 was more highly induced and NR prevented nuclear condensation and AxD in neurons from Nmrk2-KO mice treated with FK866 (Supplemental Fig. S4D, E). Thus, this result suggests that Nmrk1 is the key mediator of the neuroprotective activity of NR. Al- though NAD+, NMN, or NR can break down to NAM, we found that NMDA-induced AxD is not prevented by NAM (Supplemental Fig. S2B).In PNS-derived neurons injured by axotomy, the cyto- plasmic NAD+ biosynthetic enzyme Nmnat2 is reportedly unstable, leading to axonal NMN accumulation, which has been described as deleterious and accelerating AxD (27). However, NMN toxicity is not universally accepted, as it was reported that high levels of NMN are not suffi- cient to induce AxD in DRG neurons (16).
In our model, no NMN or NR toxicity was observed. On the contrary, we report strong axonal protection with both molecules and an NR-identical intracellular mechanism for NMN con- sistent with inhibition by CMP and DP (Fig. 5). Moreover,we discovered that transcripts encoding Nmnat1–3 are all induced after 24 h of NMDA treatment (Supplemental Fig.S2A). Thus, the 2-step NR kinase pathway to NAD+ (31) is induced by excitotoxic stress in cortical neurons. Nmnat2 protein subcellular localization and stability have been described to modulate the axon-protective capacity of this enzyme after axotomy in PNS-derived neurons (52), sug- gesting that enzyme regulation at the protein level is an- other important component of the response to NR.Optimal NAD+ and NR axon-protective effects were observed when the compounds were applied to both compartments, suggesting axonal protection can be assisted by a somatic signal. Mitochondria trans- port within the axon was recently shown to partici- pate in axonal integrity (53). It will therefore be interesting to test whether NR promotes an increase in mitochondria biogenesis, the axonal transport of mi- tochondria, or both as a component of its neuro- protective effects.
Finally, we note that the effective concentrations of NR and NAD+ for protection against two different models of cortical neurotoxicity were quite distinct. Whereas 1 mM or greater concentrations of NAD+, NMN, and NR were necessary to protect against NMDA-induced AxD, 50 mM was sufficient to prevent FK866-induced neuronal degeneration (Figs. 2D, 6, and 7). We propose that the NMDA excitotoxic model in- duces SARM1 activation (13, 16, 54, 55) in a manner that leads to active NAD+ depletion, whereas FK866 simply produces a block of the NAD+ salvage pathway. The mechanisms involved in NAD+ depletion are different, and some distinct pathways could be required to me- diate both NAD+ and NR protective effects. We have shown that NR-induced axonal protection was com- pletely or partially prevented by DP in NMDA excito- toxicity and FK866 models, respectively (Figs. 4C and 7A). DP specifically inhibits ENT1/2 activities, but we have shown that all the transcripts coding the different ENT isoforms are expressed in cortical neurons (Fig. 4B). ENT3 is known to be an intracellular transporter predominantly localized in lysosome or mitochondria, whereas ENT4 is expressed at the plasma membrane and was described to be very abundant in brain and heart (56). FK866 treatment of cortical neurons in- duced ENT2 and -4 mRNA, suggesting that ENT4, which is not inhibited by DP, is involved in nucleoside transport when NAD+ is highly depleted in neurons by FK866 (Supplemental Fig. S2A). Further mechanistic studies are under way to reveal the differences between FK866 and NMDA-induced cortical neurotoxicity. The human oral availability of NR (33) suggests the potential for testing NR in diseases and conditions of Nicotinamide Riboside central neuro-degeneration including Alzheimer’s disease, Parkin- son’s disease, and traumatic brain injury.