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Nicotinamide Mononucleotide Adenylyl Transferase- Mediated Axonal Protection Requires Enzymatic Activity but Not Increased Level

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Nicotinamide Mononucleotide Adenylyl Transferase- Mediated Axonal Protection Requires Enzymatic Activity but Not Increased Level The Journal of Neuroscience, April 29, 2009 • 29(17):5525–5535 • 5525 Development/Plasticity/Repair Nicotinamide Mononucleotide Adenylyl Transferase- Mediated Axonal Protection Requires Enzymatic Activity But Not Increased Levels of Neuronal Nicotinamide Adenine Dinucleotide Yo Sasaki,1* Bhupinder P. S. Vohra,1* Frances E. Lund,4 and Jeffrey Milbrandt1,2,3 Departments of 1Pathology and Immunology and 2Neurology and 3Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri 63110, and 4Division of Allergy, Immunology, and Rheumatology, University of Rochester, Rochester, New York 14642 Axonal degeneration is a hallmark of many neurological disorders. Studies in animal models of neurodegenerative diseases indicate that axonaldegenerationisanearlyeventinthediseaseprocess,anddelayingthisprocesscanleadtodecreasedprogressionofthediseaseand survival extension. Overexpression of the Wallerian degeneration slow (Wld s) protein can delay axonal degeneration initiated via axotomy, chemotherapeutic agents, or genetic mutations. The Wld s protein consists of the N-terminal portion of the ubiquitination factor Ube4b fused to the nicotinamide adenine dinucleotide (NAD ϩ) biosynthetic enzyme nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1). We previously showed that the Nmnat1 portion of this fusion protein was the critical moiety for Wlds-mediated axonalprotection.Here,wedescribethedevelopmentofanautomatedquantitativeassayforassessingaxonaldegeneration.Thismethod successfullyshowedthatNmnat1enzymaticactivityisimportantforaxonalprotectionasmutantswithreducedenzymaticactivitylacked axon protective activity. We also found that Nmnat enzymes with diverse sequences and structures from various species, including Drosophila melanogaster, Saccharomyces cerevisiae, and archaebacterium Methanocaldococcus jannaschii, which encodes a protein with no homology to eukaryotic Nmnat enzymes, all mediate robust axonal protection after axotomy. Besides the importance of Nmnat enzymatic activity, we did not observe changes in the steady-state NAD ϩ level, and we found that inhibition of nicotinamide phospho- ribosyltransferase (Nampt), which synthesizes substrate for Nmnat in mammalian cells, did not affect the protective activity of Nmnat1. These results provide the possibility of a role for new Nmnat enzymatic activity in axonal protection in addition to NADϩ synthesis. Introduction enylyl transferase 1 (Nmnat1) (Coleman et al., 1998). This Wld s Axonal degeneration occurs in many neurological disorders and fusion protein protects axons from degeneration initiated by a often precedes neuronal cell death. It appears to be a self- variety of insults both in vitro and in vivo (Mack et al., 2001; destructive process that is linked to, but independent from, apo- Conforti et al., 2007). ptosis (Raff et al., 2002; Coleman, 2005). Insight into the process Initial insights into the molecular mechanism of axonal pro- of axonal degeneration has been gained through studies of Wal- tection by Wld s were made using an in vitro DRG culture system lerian degeneration slow (wlds) mice (Lunn et al., 1989; Perry et in which overexpression of only the nicotinamide mononucle- al., 1991). The wlds mutation results in the overexpression of a otide adenylyl transferase 1 (Nmnat1) portion of Wld s was suffi- fusion protein (Wld s) composed of the N-terminal 70 aa of cient to prevent axonal degeneration initiated by axotomy or Ube4b linked to full-length nicotinamide mononucleotide ad- vincristine (Araki et al., 2004). The Nmnat enzymes catalyze the last step in nicotinamide adenine dinucleotide (NAD) biosynthe- sis, which is synthesized via three major pathways in both pro- Received Nov. 12, 2008; revised Jan. 7, 2009; accepted Jan. 26, 2009. karyotes and eukaryotes: the de novo, salvage, and Preiss-Handler This work was supported by National Institutes of Health Neuroscience Blueprint Center Core Grant P30 ϩ NS057105toWashingtonUniversity,theHopeCenterforNeurologicalDisorders,NationalInstitutesofHealthGrants independent. In the de novo pathway, NAD is synthesized from NS040745 (J.M.), AG13730 (J.M.), and AI063399 (F.E.L.), Muscular Dystrophy Association Grant 10040 (J.M.), and tryptophan, whereas it is generated from nicotinic acid and nic- Ministry of Health, Labour and Welfare of JAPAN (SHA4431). We thank Kelli Simburger, Amy Strickland, Amber otinamide in the salvage pathway, and from nicotinamide ribo- Neilson,andTimFahrnerforexperimentalassistanceandmembersoftheMilbrandtlaboratoryandRobertBalohfor side (NmR) in a recently discovered third route (Bieganowski their comments on this manuscript and helpful discussions. We also thank Dr. Gilbert de Murcia for providing ϩ PARP1-deficient mice. and Brenner, 2004). Most NAD is produced via the salvage Y.S., B.P.S.V., F.E.L., J.M., and Washington University may derive benefit from a licensing agreement with Sirtris pathway in mammals by the actions of nicotinamide phosphori- Pharmaceuticals, which did not provide any support for this work. bosyltransferase (Nampt), which converts nicotinamide to nico- *Y.S. and B.P.S.V. contributed equally to this work. tinamide mononucleotide (NMN), followed by the conversion of Correspondence should be addressed to Jeffrey Milbrandt at the above address. E-mail: [email protected]. ϩ DOI:10.1523/JNEUROSCI.5469-08.2009 NMN to NAD via Nmnat (Berger et al., 2005). Interestingly, the ϩ Copyright © 2009 Society for Neuroscience 0270-6474/09/295525-11$15.00/0 overexpression of Nampt, as well as NAD itself and intermedi- 5526 • J. Neurosci., April 29, 2009 • 29(17):5525–5535 Sasaki et al. • Nmnat Activity Is Crucial for Axonal Protection ϩ ates of the NAD synthetic pathway, including nicotinamide, ated using overlapping oligonucleotides and PCR (Prodromou and NMN, nicotinic acid mononucleotide, or NmR, can also delay Pearl, 1992). axonal degeneration (Wang et al., 2005; Kaneko et al., 2006; Mac- Nampt small interfering RNA knock down. Oligonucleotides were de- Donald et al., 2006; Sasaki et al., 2006). However, the axonal signed to target Nampt using parameters set by Dharmacon on its small protection provided by these treatments is less potent than that interfering RNA (siRNA) design website (Dharmacon) (Reynolds et al., 2004). Fifty-nine base pair oligonucleotides were designed containing provided by Nmnat overexpression. These studies indicate that ϩ both forward and reverse sequences for the 19 bp siRNA target sequences the NAD biosynthetic pathways are protective against axonal linked together with a hairpin loop, and ligated into AgeI–BamHI cut degeneration; however, the mechanism of axonal protection may lentivirus transfer vector pFUIV downstream of the U6 promoter (Ryu et differ in each case. al., 2008). The Nampt target sequences used were GGAAAGACCAT- Several studies of neuronal injury in Drosophila demonstrated GAGAAAGA (siRNA #1) and GCAAGAGACTGCTGGCATA (siRNA that the mechanisms of axonal degeneration as well as Nmnat- #2). mediated protection are evolutionarily conserved, as overexpres- DRG culture and in vitro axotomy. DRG drop cultures were performed sion of Wld s or Drosophila Nmnat (dNmnat) were found to pro- based on a previously described method (Chen et al., 2008). DRGs were tect transected axons of mushroom body neurons (MacDonald et collected from embryonic day 12.5 mouse embryos. The DRGs from five embryos (ϳ200 total DRGs) were collected in 500 ␮l of DMEM (Sigma al., 2006). Others have demonstrated that dNmnat is essential for ϫ ␮ maintaining neuronal integrity in Drosophila (Zhai et al., 2006). D5671), centrifuged at 2000 g for 1 min, and incubated in 500 lofa solution containing 0.05% trypsin and 0.02% EDTA at 37°C for 15 min. Interestingly, Nmnat enzymatic activity was not required for pre- An equal volume of DMEM containing 10% FBS was then added, the vention of neuronal cell death in this model; instead, a DRGs were triturated using a 1000 ␮l pipette, and the cells were centri- chaperone-like activity of dNmnat appeared to be important for fuged (2000 ϫ g, 1 min), washed in DMEM containing 10% FBS, and neuronal survival. The contradictory nature of the reports thus resuspended in 40 ␮l of Neurobasal media (Invitrogen) containing 2% far prompted us to undertake additional studies to examine the B27 (Invitrogen) and 50 ng/ml NGF (2.5S; Harlan Bioproducts) per one role of Nmnat enzymatic activity in axonal protection. We found dissected embryo. The suspended DRG cells were cultured on plates that Nmnat enzymatic activity is essential for axonal protection, coated with poly-D-lysine and laminin. The wells were initially coated as mutations in Nmnat1 that severely reduced its ability to syn- with 250 ␮l of poly-D-lysine (0.1 mg/ml) (Sigma) solution overnight. The ϩ ␮ thesize NAD compromise the ability to prevent degeneration wells were washed twice with water and dried for 15 min, and then 250 l ␮ (Araki et al., 2004; Jia et al., 2007). While Nmnat1 enzymatic of mouse laminin (2–5 g/ml) (Invitrogen) was added for 1 h. Before the ϩ cells were seeded, the laminin was removed from the wells, and the plates activity is crucial, increased NAD levels alone do not provide were dried. The suspended DRG neurons (2 ␮l) were placed as a drop in axonal protection. In addition, Nmnat-mediated protection is ϩ the center of each well of a 24-well plate and incubated at 37°C with 5% not abrogated in neurons with severely reduced levels of NAD . ␮ CO2 for 15 min. After cell attachment, 500 l of Neurobasal media con- Finally, we found
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