Author response: Neurotoxin-mediated potent activation of the axon degeneration regulator SARM1

Editor's evaluation: Nuclear NAD+-biosynthetic enzyme NMNAT1 facilitates development and early survival of retinal neurons

Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin-proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction.

BackgroundBioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer’s, Parkinson’s, and Huntington’s disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function.MethodsWe generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos.ResultsWe provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide “on-board” ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons.ConclusionNMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

BackgroundBioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer’s, Parkinson’s, and Huntington’s disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function.MethodsWe generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos.ResultsWe provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide “on-board” ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons.ConclusionNMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme

Identification and characterization of a second NMN adenylyltransferase gene in Saccharomyces cerevisiae

Neurotoxicity is one of the major toxicities of platinum-based anticancer drugs, especially oxaliplatin and ormaplatin. It has been postulated that biotransformation products are likely to be responsible for the toxicity of platinum drugs. In our preceding pharmacokinetic study, both oxaliplatin and ormaplatin were observed to produce the same types of major plasma biotransformation products. However, while the plasma concentration of ormaplatin was much lower than that of oxaliplatin at an equimolar dose, one of their common biotransformation products, Pt(dach)Cl2, was present at 29-fold higher concentrations in the plasma following the i.v. injection of ormaplatin than of oxaliplatin. Because ormaplatin has severe neurotoxicity and Pt(dach)Cl2 is very cytotoxic, we have postulated that Pt(dach)Cl2 is likely to be responsible for the differences in neurotoxicity between ormaplatin and oxaliplatin. In order to test this hypothesis, we compared the neurotoxicity of oxaliplatin, ormaplatin, and their biotransformation products. Since the dorsal root ganglia (DRGs) have been suggested to be the likely targtet for platinum drugs and in vitro DRG explant cultures have been suggested to be a valid model for studying cisplatin-associated neurotoxicity, our comparative neurotoxicity study was conducted with DRG explant cultures in vitro. Based on the previous studies of cisplatin neurotoxicity, we established our in vitro DRG explant culture utilizing DRGs dissected from E-19 embryonic rats. Rat DRGs were incubated for 30 min with different platinum compounds to mimic in vivo exposure conditions; this was by followed by a 48-h incubation in culture medium at 37 degrees C. At the end of the incubation, the neurites were fixed and stained with toluidine blue, and neurite outgrowth was quantitated by phase-contrast microscopy. The inhibition of neurite outgrowth by platinum compounds was used as an indicator of in vitro neurotoxicity. Since an in vivo study has indicated that the order of neurotoxicity is ormaplatin > cisplatin > oxaliplatin > carboplatin as measured by morphometric changes to rat DRGs, we initially validated our DRG explant culture model by comparing the in vitro neurotoxicity of ormaplatin, cisplatin, oxaliplatin, and carboplatin. After observing the same neurotoxicity rank between this study and a previous in vivo study, we further compared the neurotoxicity of oxaliplatin, ormaplatin, and their biotransformation products including Pt(dach)Cl2, Pt(dach)(H2O)Cl, Pt(dach)(H2O)2, Pt(dach)(Met), and Pt(dach)(GSH) utilizing the DRG explant culture model. Our study indicated that Pt(dach)Cl2 and its hydrolysis products were more potent at inhibiting neurite outgrowth than the parent drugs oxaliplatin and ormaplatin. In contrast, no detectable inhibition of neurite outgrowth was observed for DRGs dosed with Pt(dach)(Met) and Pt(dach)(GSH). This study suggests that biotransformation products such as Pt(dach)Cl2 and its hydrolysis products are more neurotoxic than the parent drugs oxaliplatin and ormaplatin. The different neurotoxicity profiles of oxaliplatin and ormaplatin are more likely due to the different plasma concentrations of their common biotransformation product Pt(dach)Cl2 than to differences in their intrinsic neurotoxicity.

When a nerve is injured, axons beyond the site of injury die through a process called Wallerian degeneration. This degeneration is delayed in mice that have a mutation called Wallerian degeneration slow (Wlds); these mice have three copies of a particular stretch of their DNA. Because this piece of DNA includes the gene for nicotinamide mononucleotide adenylyltransferase (NMNAT), which synthesizes a molecule called NAD, there has been a great deal of interest in whether NMNAT or NAD can protect against axonal degeneration. Hugo Bellen and colleagues show that NMNAT can, at least in the fruitfly Drosophila—and that its protective ability is independent of its function as a NAD synthase. Investigations into the putative protective role of NMNAT have produced conflicting results. In vitro evidence supports the idea that NMNAT protects against degeneration, as do studies in which NMNAT was overexpressed in Drosophila. But mice in which NMNAT is overexpressed show no protection. The authors used a screening system that allowed them to look for flies that are homozygous for mutations—meaning that both copies of a gene are mutated—that result in death in cells of the visual system but are heterozygous, with one normal copy and one mutated copy, in the rest of the body. This means that mutations that would normally be lethal can be investigated in living flies. To look for mutations that affect synaptic function or development, the authors screened mutated Drosophila for abnormalities in phototaxis (a test of vision in which the organism moves toward or away from light; normal flies move toward light). They then tested the visual responses of the identified flies and looked for abnormal synapse structure in the eyes. This screening process identified two “nonsense” mutations in the gene for Drosophila NMNAT that prevents the gene from generating the correct protein. When they characterized the protein, the authors found that it was highly homologous to NMNAT proteins in humans and mice. Staining with an antibody against NMNAT showed that the protein is highly enriched in the fly nervous system, mainly in the cell nuclei and nerve terminals. Flies carrying the NMNAT mutation had abnormal photoreceptors that became progressively damaged with age, indicating a degenerative process. Mutant photoreceptors appeared to develop normally but did not survive. NMNAT therefore seems to be required for the maintenance of mature neurons. What is the mechanism of degeneration in nmnat mutant photoreceptors? When mutant flies were raised in the dark, their degeneration was much less severe than when they were raised in the light, indicating that photoreceptor activity contributes to the degenerative process. Flies with double mutations that lacked both NMNAT and functioning photoreceptors also showed reduced neurodegeneration. This led the authors to conclude that the normal function of NMNAT is to protect photoreceptors against light-induced degeneration. They also showed that overexpression of NMNAT could protect photoreceptors against the degeneration caused by excessive activity in flies with mutations that cause continuous activation of photoreceptors. To investigate the importance of NAD synthesis in neuroprotection by NMNAT, the authors generated an enzymatically inactive form of NMNAT. To their surprise, they found that expression of this protein could prevent neurodegeneration in nmnat mutants. However, this inactive NMNAT could not stop a full, homozygous nmnat mutation from being lethal to flies. These results indicate that NMNAT has two functions: its NAD synthase activity is essential for survival, but another activity is responsible for neuroprotection. This study sheds new light on the mechanisms of neural degeneration and the functions of NMNAT. Attention will now turn to the identification of its second, non–NAD-dependent activity, as well as to continued attempts to reconcile the apparently conflicting results of earlier overexpression experiments.

Objective To investigate the effect and mechanism of interfering the nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) gene in Parkinson′s disease (PD) mouse models. Methods Thirty mice were randomly assigned to three groups: the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) group, the small interfering nicotinamide mononucleotide adenylyltransferase 1 (siNMNAT1)+ MPTP group, and the control group, with 10 mice in each group. After injecting siRNA-green fluorescent protein (GFP) lentivirals directly into substantia nigra (SN), mice received intraperitoneal injections of MPTP, which was the siNMNAT1+ MPTP group. While the MPTP group was only with injections of MPTP, and the control group was with neither siRNA nor MPTP. Then we assessed the motor coordination ability firstly. To observe the variation of nigrostriatal pathway, the counts of dopamanergic neurons in SN were measured by tyrosine hydroxylase (TH) immunofluorescence staining. And the expression of TH in striatum, which was used to estimate the dopaminergic neurons axonal variation, was analyzed by RT-PCR. Then the expression of TH, SOD1, Bcl2, Bax, Bcl2/Bax in SN was estimated through Western blotting. Results Compared with the control group, the siNMNAT1+ MPTP group and the MPTP group decreased significantly in motor coordination ability (creep down time: siNMNAT1+ MPTP group (62.8±15.7) s, MPTP group (77.9±13.5) s, control group (122.0±25.2) s), dopamanergic neuron counts (siNMNAT1+ MPTP group 45.0±6.7, MPTP group 68.0±11.3, control group 93.0±12.8) and the striatal TH expression (Creep down time: t=-6.291, P=0.000; t=-4.865, P=0.000. Dopamanergic neuron counts: t=-10.482, P=0.000; t=-4.624, P=0.000. TH expression: t=-9.117, P=0.000; t=-5.716, P=0.000). Although the siNMNAT1+ MPTP group showed lower coordination ability than the MPTP group, there was no statistically significant difference. Whereas the counts of dopamanergic neurons in SN (t=-5.487, P=0.000), the expression of TH in striatum (t=-5.146, P=0.003), SOD1 (t=-4.143, P=0.001) and Bcl2/Bax (t=-6.303, P=0.000) were obviously decreased in the siNMNAT1+ MPTP group, in which Bax increased significantly (t=3.550, P=0.002). Conclusions Interfering the expression of NMNAT1 aggravated the neurodegeneration in PD, and the mechanism might be related to oxidative stress and programmed cell death. Key words: Nicotinamide mononucleotide adenylyltransferase 1; Gene expression; Parkinson disease; 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine

The Trembler-J (TrJ) mouse, containing a point mutation in the peripheral myelin protein 22 gene, is characterized by severe hypomyelination and is a representative model of Charcot-Marie-Tooth 1A disease/Dejerine-Sottas Syndrome. Previous studies have shown that protein kinase inhibitor K252a enhances wild-type Schwann cell myelination in culture. We used a dorsal root ganglion (DRG) explant culture system from the heterozygous TrJ/+ mouse to investigate if myelination could be enhanced by K252a. The TrJ/+ DRG explant cultures replicated some important features of the TrJ/+ mouse, showing reduced myelin protein accumulation, thinner myelin sheaths, and shortened myelin internodes. K252a increased myelin protein accumulation and myelin sheath thickness but did not substantially increase myelin internode length. Furthermore, the TrJ/+ DRG explant culture and sciatic nerves continued to respond to K252a during the stage when myelination is complete in the wild type. A general tyrosine kinase inhibitor, genistein, but not inhibitors of serine/threonine protein kinase inhibitors, had a similar effect to K252a. K252a is therefore able to partially overcome hypomyelination by enhancing mutant Schwann cell myelin formation in the TrJ/+ mouse.

Peripheral nerves conducting motor and somatosensory signals in vertebrate consist of myelinated and unmyelinated axons. In vitro myelination culture, generated by co-culturing Schwann cells (SCs) and dorsal root ganglion (DRG) neurons, is an indispensable tool for modeling physiological and pathological conditions of the peripheral nervous system (PNS). This technique allows researchers to overexpress or downregulate molecules investigated in neurons or SCs to evaluate the effect of such molecules on myelination. In vitro myelination experiments are usually time-consuming and labor-intensive to perform. Here we report an optimized protocol for in vitro myelination using DRG explant culture. We found that our in vitro myelination using DRG explant (IVMDE) culture not only achieves myelination with higher efficiency than conventional in vitro myelination methods, but also can be used to observe Remak bundle and non-myelinating SCs, which were unrecognizable in conventional methods. Because of these characteristics, IVMDE may be useful in modeling PNS diseases, including Charcot Marie Tooth disease (CMT), in vitro. These results suggest that IVMDE may achieve a condition more similar to peripheral nerve myelination observed during physiological development.

Background: Nicotinamide adenine dinucleotide (NAD) participates in redox reactions as an electron-transferring molecule, thereby serving as a cofactor for metabolism. In the NAD biosynthetic salvage pathway, nicotinamide phosphoribosyltransferase (Nampt) which forms nicotinamide mononucleotide (NMN) is the rate-limiting enzyme and exhibits the protective effect against ischemia/ reperfusion (I/R) injury in the hearts of mice with cardiac-specific overexpression of Nampt. In addition, administration of NMN is reported to reverse the pancreatic beta-cell dysfunction observed in Nampt heterozygous knockout mice. Methods: To elucidate the protective effect of NMN against cardiac I/R injury, NMN was administered to mice subjected to I/R injury either before ischemia or immediately before reperfusion. Area at risk (AAR) and infarct area (IA) were evaluated 24 hours after reperfusion. Results: To confirm the distribution of NMN in the heart, NAD and NADH, into which NMN is quickly converted in the heart, were measured. One hour after NMN administration (500mg/ kg body weight, i.p.), the NAD and NADH contents of the NMN group (n=3) were significantly increased, whereas the NAD/NADH ratio was unchanged compared to the vehicle group (n=3) (NAD: NMN=483±42 pmole/mg tissue, vehicle=183±46 pmole/mg tissue, p<0.01; NADH: NMN=262±25 pmole/mg tissue, vehicle=96±17 pmole/mg tissue, p<0.01; NAD/NADH ratio: NMN=1.85±0.09, vehicle=1.82±0.18, n.s.). In the mice administered NMN or vehicle 30 min before ischemia, IA/AAR in the NMN group was smaller than that in the vehicle group (AAR: NMN=26±0.5%, vehicle=27±0.4%, n.s.; IA/AAR: NMN=23±1.8%, vehicle=42±1.5%, p<0.01, n=4). On the other hand, in the mice administered NMN or vehicle immediately before reperfusion, IA/AAR in the NMN group was not different from that in the vehicle group (AAR: NMN=27±0.5%, vehicle=26±1.0%, n.s.; IA/AAR: NMN=37±1.4%, vehicle=34±2.2%, n.s., n=4). Conclusions: NMN administration significantly increased the NAD content in the heart and exhibited a protective effect against cardiac I/R injury. Increasing NAD content before ischemia, rather than after reperfusion, may minimize I/R injury in clinical settings, such as in acute coronary syndrome before coronary intervention.

The mRNA expression of MT-encoded genes of OXPHS in ovarian GCS of infertile women and the effect of NMN on expression of MT-encoded genes in SVOG cells

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 1 (NMNAT-1) constitute a nuclear NAD(+) salvage pathway which regulates the functions of NAD(+)-dependent enzymes such as the protein deacetylase SIRT1. One of the major functions of SIRT1 is to regulate target gene transcription through modification of chromatin-associated proteins. However, little is known about the molecular mechanisms by which NAD(+) biosynthetic enzymes regulate SIRT1 activity to control gene transcription in the nucleus. In this study we show that stable short hairpin RNA-mediated knockdown of NAMPT or NMNAT-1 in MCF-7 breast cancer cells reduces total cellular NAD(+) levels and alters global patterns of gene expression. Furthermore, we show that SIRT1 plays a key role in mediating the gene regulatory effects of NAMPT and NMNAT-1. Specifically, we found that SIRT1 binds to the promoters of genes commonly regulated by NAMPT, NMNAT-1, and SIRT1 and that SIRT1 histone deacetylase activity is regulated by NAMPT and NMNAT-1 at these promoters. Most significantly, NMNAT-1 interacts with, and is recruited to target gene promoters by SIRT1. Collectively, our results reveal a mechanism for the direct control of SIRT1 deacetylase activity at a set of target gene promoters by NMNAT-1. This mechanism, in collaboration with NAMPT-dependent regulation of nuclear NAD(+) production, establishes an important pathway for transcription regulation by NAD(+).

Background: Nicotinamide adenine dinucleotide (NAD) participates in metabolic reactions as an electron-transferring molecule. In the NAD biosynthetic salvage pathway, nicotinamide phosphoribosyltransferase (Nampt) which forms nicotinamide mononucleotide (NMN) is the rate-limiting enzyme and exhibits the protective effect against ischemia/ reperfusion (I/R) injury in the hearts of mice with cardiac-specific overexpression of Nampt. In addition, administration of NMN is reported to reverse the pancreatic beta-cell dysfunction observed in Nampt heterozygous knockout mice. Methods: To elucidate the protective effect of NMN against cardiac I/R injury, NMN was administered to mice subjected to I/R injury either before ischemia or immediately before reperfusion. Area at risk (AAR) and infarct area (IA) were evaluated 24 hours after reperfusion. Results: To confirm the distribution of NMN in the heart, NAD and NADH, into which NMN is quickly converted in the heart, were measured. One hour after NMN administration (500mg/ kg body weight, i.p.), the NAD and NADH contents of the NMN group were significantly increased, whereas the NAD/NADH ratio was unchanged compared to the vehicle group(NAD: NMN=483±42 pmol/mg tissue, vehicle=183±46 pmol/mg tissue, p<0.01; NADH: NMN=262±25 pmol/mg tissue, vehicle=96±17 pmol/mg tissue, p<0.01; NAD/NADH ratio: NMN=1.85±0.09, vehicle=1.82±0.18, n.s.; n=3). In the mice administered NMN or vehicle 30 min before ischemia, IA/AAR in the NMN group was smaller than that in the vehicle group (AAR: NMN=30±1.9%, vehicle=29±1.7%, n.s.; IA/AAR: NMN=23±2.9%, vehicle=41±2.6%, p<0.01, n=6-7). On the other hand, in the mice administered NMN or vehicle immediately before reperfusion, IA/AAR in the NMN group was not different from that in the vehicle group (AAR: NMN=27±0.5%, vehicle=26±1.0%, n.s.; IA/AAR: NMN=37±1.4%, vehicle=34±2.2%, n.s., n=4). Conclusions: NMN administration significantly increased the NAD content in the heart and exhibited a protective effect against cardiac I/R injury. Increasing NAD content before ischemia, rather than after reperfusion, may minimize I/R injury in clinical settings, such as in acute coronary syndrome before coronary intervention.