Article Figures & Data
Figures
- Figure 1.
Axonal protection is mediated by enforced expression of NAD biosynthetic enzymes. In vitro Wallerian degeneration assays were performed using DRG neuronal explant cultures. A, DRG neurons were infected with lentivirus expressing the indicated enzyme (Nmnat1, NmPRT, or EGFP control) 5 d before axotomy. Representative pictures taken at 12, 24, 48, and 72 h after transection are shown. B, In vitro Wallerian degeneration assays were performed and axonal degeneration was quantified as outlined in Materials and Methods. The percentage ± SD of remaining axons relative to pretransection at 12, 24, 48, and 72 h after transection is displayed. *p < 0.0001 indicates a significant difference (n = 6) with EGFP-expressing cells. C, Protein expression of QPRT, NaPRT, NmPRT, Nmnat1, QNS, Nrk1, and Nrk2 by lentivirus was confirmed by immunoblot analysis of HEK293T cells infected with each virus. The enzymatic activity of each clone was confirmed as described in the supplemental data (available at www.jneurosci.org as supplemental material). D, The subcellular localization of the indicated NAD biosynthetic enzymes was examined using immunocytochemistry with an anti-hexahistidine tag antibody of transfected HEK293T cells. The cells were also stained with bis-benzimide to highlight the nucleus to determine the nuclear versus cytoplasmic distribution of each protein. Scale bar, 10 μm.
- Figure 2.
Nmnat enzymes located in the nucleus, cytoplasm, or mitochondria all promote axonal protection. A, Subcellular localization of Nmnat1, cytNmnat1, Nmnat3, or nucNmnat3 in HEK293T cells. Immunohistochemistry using antibody against the hexahistidine epitope tag was used to detect each protein. The cells were also stained with bis-benzimide. As expected, the cytNmnat1 mutant is located in the cytoplasm, and the nucNmnat3 mutant is located in the nucleus. Scale bar, 10 μm. B, In vitro Wallerian degeneration assay using lentivirus-infected DRG neuronal explant cultures expressing Nmnat1, cytNmnat1, Nmnat3, nucNmnat3, or EGFP control. Representative pictures taken at 12 and 72 h after transection show robust protection against axonal degeneration regardless of the subcellular distribution. C, Quantitative analysis of axonal degeneration in DRG explant cultures expressing Nmnat1, cytNmnat1, Nmnat3, or nucNmnat3 at 12, 24, 48, and 72 h after transection. Nmnat1-, cytNmnat1-, Nmnat3-, and nucNmnat3-expressing cells have a significant difference (p < 0.0001; n = 6) with EGFP-expressing cells at 24, 48, and 72 h after axotomy.
- Figure 3.
Treatment of cultured DRG neurons with NAD precursors delays axonal degeneration. A, In vitro Wallerian degeneration assay using DRG explant cultures after exogenous application of NAD or NmR. Representative pictures at 12, 24, 48, and 72 h after transection are shown. B, In vitro Wallerian degeneration assay using DRG neuronal explant cultures after exogenous application of Na, nicotinamide (Nam), NaMN, NMN, NaAD, NAD, and NmR. Quantitative analysis of axonal protection at 12, 24, 48, and 72 h after axotomy is shown. *p < 0.002 indicates a significant difference (n = 6) compared with control. C, Axonal degeneration in DRG explants infected with NaPRT-expressing lentivirus is delayed only when 1 mm nicotinic acid is added to the medium 24 h before axotomy. Percentage of remaining axons at 12, 24, 48, and 72 h after transection is shown. *p < 0.01 indicates a significant difference (n = 6) compared with control. D, NAD, NMN, or NmR (1 mm) was added either at the time of transection (control) or 24 h before axonal transection (24 h before incubation). Representative pictures taken 24 h after transection show axonal protection only in cultures in which substrates were added before injury.
- Figure 4.
Expression of NAD biosynthetic enzymes is regulated by injury in DRG neurons. Quantitative RT-PCR analysis of indicated NAD biosynthesis enzyme mRNA levels in rat DRGs 0, 1, 3, 7, and 14 d after sciatic nerve transection. The expression level was normalized to glyceraldehyde-3-phosphate dehydrogenase and is indicated relative to expression in uninjured DRGs.
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Figure S1. NAD biosynthetic pathway. Predicted mammalian NAD biosynthesis is based on studies from yeast and prokaryotes (Magni et al., 2004). Abbreviations used: QPRT, quinolinate phosphoribosyltransferase; NaPRT, nicotinic acid phosphoribosyltransferase; NmPRT, nicotinamide phosphoribosyltransferase; Nrk, nicotinamide riboside kinase; Nmnat, nicotinamide mononucleotide adenylyltransferase; QNS, NAD synthetase. Magni G, Amici A, Emanuelli M, Orsomando G, Raffaelli N, Ruggieri S (2004) Enzymology of NAD+ homeostasis in man. Cell Mol Life Sci 61:19-34.
- supplemental material - Figure S2. The hexahistidine tagged NAD biosynthetic enzymes are catalytically active. The enzymatic activity of the indicated enymes was examined qualitatively by incubating the purifed enzymes with their cognate substrate and monitoring product formation using HPLC (see Methods). In each case, reactions were analyzed in the absence (top) or presence (bottom) of the enzyme to be tested A, QPRT activity was assessed by conversion of Qa to NaMN followed by the conversion of NaMN to NaAD by Nmnat1. NaAD was observed in the presence of QPRT (bottom) but not in its absence (top). B, NaPRT activity was assessed by monitoring the production of NaMN from Na. NaMN was only observed in the presence of NaPRT (bottom). C, NmPRT activity was examined by monitoring NMN production from Nam. NMN was only observed in the presence of NmPRT (bottom). D, Nrk1 and Nrk2 activities were assessed by monitoring the phosphorylation of NmR to produce NMN. NMN was only observed in the presence of Nrk1 (middle) or Nrk2 (bottom). Materials and methods QPRT, NaPRT, NmPRT, Nrk1, and Nrk2 enzymatic activity measurement. The cDNAs for QPRT, NaPRT, NmPRT, Nrk1 and Nrk2 were cloned into the BamHI site of the pET3a bacterial expression vector (Novagen, San Diego, CA ). BL21(DE3)pLysS cells were transformed with these constructs and protein expression was induced by growth in 1mM IPTG according manufacturer�s instructions (Stratagene, Garden Grove, CA). The enzymes were purified using Nickel affinity chromatography according to manufacturer�s instructions (Sigma). The catalytic activity of QPRT, NaPRT, and NmPRT was examined by incubating 10 μg of purified protein with the appropriate substrates 4 mM Qa for QPRT, 5 mM Na for NaPRT, and 10 mM Nam for NmPRT in buffer containing 50 mM Tris-HCl (pH7.5), 10 mM MgCl2, 0.4 mM 5-Phospho-D-ribose 1-diphosphate (PRPP; Sigma P8296). Nrk activity was measured by incubation with 1 mM NmR in buffer containing 20 mM Tris-HCl (pH7.5), 5 mM 2-Mercaptoethanol, 1 mM ATP, 5m M MgCl2. All reactions were carried out for 30 min at 37�C, stopped by addition of 1 M HClO4 and incubation for 5 min at 4�C. The reactants were spun at 14,000 g in a microcentrifuge and neutralized with K2CO3. For QPRT measurement, 20 ul of reacted sample was treated with Nmnat1 by incubating with 15 μg of recombinant Nmnat1 in buffer containing 30 mM HEPES (pH7.4), 12 mM MgCl2, and 1 mM ATP for 30 min at 37�C. All control reactions were performed under the same conditions in the absence of the requisite enzyme. All neutralized reactants were cleared by centrifugation and diluted with 3 volumes of buffer containing 50 mM K2HPO4 and 50 mM KH2PO4 (pH 7.0). An aliquot of each reactant solution was analyzed by HPLC using LC-18T reverse phase column at flow rate of 1 ml/min and the absorbance at 254 nm was recorded. Each elution peak was compared with standards (obtained from Sigma) to determine its identity.
- supplemental material - Figure S3. Enzymatic activities of wild type and mutant Nmnat1 and Nmnat3. The enzymatic activities of Nmnat1, cytNmnat1, Nmnat3, and nucNmnat3 were examined as described bellow. For each enzyme, triplicate reactions were averaged and normalized to activity of Nmnat1. Control reactions were performed without recombinant protein. Materials and methods Nmnat protein overexpression and enzymatic assay. HEK293T cells were transfected with Nmnat1, cytNmnat1, Nmnat3, or nucNmnat3 expression plasmids. Cell lysates were prepared and Nmnat proteins were purified using Nickel affinity chromatography according to manufacturer�s instructions (Sigma). Proteins (0.2 μg) were assayed in 100 μl of reaction buffer containing 28 mM HEPES (pH 7.4), 10 mM MgCl2, 46 mM ethyl alcohol, 16 mM semicarbazide-HCl (pH 7.4), 0.5 unit of alcohol dehydrogenase (Sigma), 1 mM ATP, 1 mM NMN, and 5 μl solution consisting of MTS and phenazine methosulphate (CellTiter 96 AQueous cell proliferation assay: Promega). Reactions were carried out for 1 hour at 37�C. In this reaction, NMN is converted to NAD by Nmnat and then to NADH by alcohol dehydrogenase. The NADH reduces the MTS. The absorbance at 490 nm, which is specific for reduced MTS, is monitored to determine the relative amount of NADH (Cuzzocrea et al., 1999). Cuzzocrea S, Costantino G, Mazzon E, Caputi AP. (1999) Beneficial effects of raxofelast (IRFI 016), a new hydrophilic vitamin E-like antioxidant, in carrageenan-induced pleurisy. Br J Pharmacol 126:407-414.
