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Nicotinamide Mononucleotide Adenylyltransferase Expression in Mitochondrial Matrix Delays Wallerian Degeneration

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Nicotinamide Mononucleotide Adenylyltransferase Expression in Mitochondrial Matrix Delays Wallerian Degeneration 6276 • The Journal of Neuroscience, May 13, 2009 • 29(19):6276–6284 Cellular/Molecular Nicotinamide Mononucleotide Adenylyltransferase Expression in Mitochondrial Matrix Delays Wallerian Degeneration Naoki Yahata,1 Shigeki Yuasa,2 and Toshiyuki Araki1 Departments of 1Peripheral Nervous System Research and 2Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan Studies of naturally occurring mutant mice, wlds, showing delayed Wallerian degeneration phenotype, suggest that axonal degeneration is an active process. We previously showed that increased nicotinamide adenine dinucleotide (NAD)-synthesizing activity by overexpres- sion of nicotinamide mononucleotide adenylyltransferase (NMNAT) is the essential component of the Wld s protein, the expression of whichisresponsibleforthedelayedWalleriandegenerationphenotypeinwlds mice.Indeed,NMNAToverexpressioninculturedneurons providesrobustprotectiontoneurites,aswell.ToexaminetheeffectofNMNAToverexpressioninvivoandtoanalyzethemechanismthat causes axonal protection, we generated transgenic mice (Tg) overexpressing NMNAT1 (nuclear isoform), NMNAT3 (mitochondrial isoform), or the Wld s protein bearing a W258A mutation, which disrupts NAD-synthesizing activity of the Wld s protein. Wallerian degeneration delay in NMNAT3-Tg was similar to that in wlds mice, whereas axonal protection in NMNAT1-Tg or Wld s(W258A)-Tg was not detectable. Detailed analysis of subcellular localization of the overexpressed proteins revealed that the axonal protection phenotype was correlated with localization of NMNAT enzymatic activity to mitochondrial matrix. Furthermore, we found that isolated mitochon- dria from mice showing axonal protection expressed unchanged levels of respiratory chain components, but were capable of increased ATP production. These results suggest that axonal protection by NMNAT expression in neurons is provided by modifying mitochondrial function. Alteration of mitochondrial function may constitute a novel tool for axonal protection, as well as a possible treatment of diseases involving axonopathy. Introduction to the complete sequence of nicotinamide mononucleotide ad- Axonal degeneration is now regarded as a major component of enylyltransferase 1 (NMNAT1), an enzyme in the NAD biosyn- many neurodegenerative diseases, including Parkinson’s disease thetic pathway that generates NAD in the nucleus (Conforti et al., and Alzheimer’s disease (Raff et al., 2002). Therefore, the axonal 2000; Mack et al., 2001). We previously showed that overexpres- degeneration process could well be a therapeutic target against sion of NMNAT enzymatic activity is the essential mechanism s these diseases. The discovery of a spontaneous dominant muta- that causes the axonal protection phenotype in wld mice (Araki tion in mice that results in delayed axonal degeneration, the Wal- et al., 2004). NMNAT is a family of proteins that mediates NAD lerian degeneration slow (wlds) mice, strongly suggests that ax- synthesis by using nicotinamide mononucleotide (NMN) and onal degeneration is an active process of self-destruction (Lunn et ATP as substrates. In addition to NMNAT1, NMNAT2 (located al., 1989). The wlds mutation comprises an 85 kb tandem tripli- mostly in Golgi apparatus and expressed most highly in mamma- cation, which causes overexpression of a chimeric molecule lian brain) and NMNAT3 (located mostly in mitochondria) have (Wld s protein). This protein is composed of the N-terminal 70 aa been identified in mammals (Berger et al., 2005). We previously of Ufd2a (ubiquitin fusion degradation protein 2a)/Ube4b (ubiq- showed that not only NMNAT1 but also NMNAT3 overexpres- uitination factor E4b), a ubiquitin-chain assembly factor, fused sion in neurons is protective in an in vitro Wallerian degeneration model, which supports our hypothesis that NMNAT enzymatic activity is the key for axonal protection (Sasaki et al., 2006). Received Sept. 9, 2008; revised April 1, 2009; accepted April 13, 2009. There are conflicting results against our hypothesis. Coleman This work was supported by grants from Ministry of Health, Labour, and Welfare (Research on Psychiatric and and colleagues showed that the NAD content in wlds mouse tissue Neurological Diseases and Mental Health), National Institute of Biomedical Innovation (Program for Promotion of is similar to that in wild-type despite the increased NMNAT ac- FundamentalStudiesinHealthSciences),Kakenhi(19650089),TerumoLifeScienceFoundation,SuzukenMemorial s Foundation,KowaLifeScienceFoundation,SankyoFoundationforLifeScience,AstellasFoundationforResearchon tivity in wld mice (Mack et al., 2001). Furthermore, they recently Metabolic Disorders, Mishima Kaiun Memorial Foundation, and Takeda Science Foundation (T.A.). pCAGGS is a gift showed that NMNAT1-overexpressing transgenic mice do not from Dr. J. Miyazaki (Osaka University, Osaka, Japan). show delayed Wallerian degeneration observed in wlds mice Correspondence should be addressed to Toshiyuki Araki, Department of Peripheral Nervous System Research, (Conforti et al., 2007), which supports their hypothesis that National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-higashi, Kodaira, s Tokyo 187-8502, Japan. E-mail: [email protected]. N-terminal Ufd2a region of the Wld protein plays a role (Laser et DOI:10.1523/JNEUROSCI.4304-08.2009 al., 2006). These reports suggest that NMNAT activity may not be Copyright © 2009 Society for Neuroscience 0270-6474/09/296276-09$15.00/0 crucial for axonal protection. Yahata et al. • Delayed Wallerian Degeneration in NMNAT3-Tg Mice J. Neurosci., May 13, 2009 • 29(19):6276–6284 • 6277 In this study, to analyze the requirement of NMNAT activity 1-␮m-thick semithin sections were cut and stained with toluidine blue for axonal protection in vivo, we generated and analyzed the phe- for initial light-microscopic analysis. Subsequently, ultrathin sections notype of mice that overexpressed either NMNAT1 (NMNAT1- were prepared, stained with lead citrate and uranyl acetate, and analyzed Tg), NMNAT3 (NMNAT3-Tg), or the Wld s protein bearing a by a transmission electron microscope (Hitachi H-7000). W258A mutation that disrupts NAD producing activity of the For counting protected axons in injured nerve, the numbers of mor- Wld s protein [Wld s(W258A)-Tg]. We found that NMNAT3-Tg phologically intact axons (designated by preserved myelin sheath struc- ϭ mice demonstrated delayed Wallerian degeneration, similar to tures) in transected sciatic nerves of indicated genotypes (n 7 for each s s genotype) were determined on semithin transverse sections at 7 d after wld mice; whereas NMNAT1-Tg or Wld (W258A)-Tg mice did ϫ ␮ not show any axonal protection. In both NMNAT3-Tg and wlds injury. The entire axons in a 100 100 m square field were counted mice, significant amounts of NMNAT activity were found to be under a light microscopy for each sample. Assays of NMNAT enzymatic activities. Whole cerebrum was lysed in a located in the mitochondrial matrix. We further found that mi- buffer containing 50 mM Tris-HCl at pH 7.4, 300 mM NaCl, 5 mM EDTA, tochondrial localization of NMNAT activity resulted in increased and 1% Triton X-100 and protease inhibitor mixture EDTA-free ATP synthetic capacity without affecting the expression profile of (Roche). NMNAT activity was measured by SpectraMax M2 (Molecular mitochondrial enzymes of the respiratory chain. These data suggest Devices) at 37°C on 96-well plates, based on the procedure as previously that mitochondrial localization of NMNAT activity plays an impor- described (Balducci et al., 1995). Briefly, a 100 ␮l mixture containing 28 tant role in NMNAT expression-mediated axonal protection. mM HEPES at pH 7.4, 46 mM ethanol, 16 mM semicarbazide-HCl, 10 mM MgCl2,2mM ATP,6Uofalcohol dehydrogenase (Sigma-Aldrich), and Materials and Methods lysate supernatant (30 ␮g protein) was made. The reaction was started by ␮ ␧ Generation of transgenic mice. The transgene was constructed by inserting adding 10 lof20mM NMN. Optical density at 340 nm ( 340 of NADH: ␮ the cDNA 3Ј downstream of the chicken ␤-actin promoter in the 3.0 mM/100 l in 96-well plate, one-half area) was measured for 1 h. One pCAGGS plasmid (a kind gift from Dr. J. Miyazaki, Osaka University, unit of activity was defined as the amount of protein that catalyzes the Osaka, Japan) (Niwa et al., 1991). The cDNAs for mouse NMNAT1 and synthesis of 1 mmol of NAD per hour. NMNAT3 used as transgenes were obtained from expressed sequence- Subcellular fractionation. Fractionation of adult mouse cerebrum to tagged clones (BC038133 for murine NMNAT1; BC005737 for murine obtain nucleus, mitochondria, lysosome/peroxysome, microsome, and NMNAT3). cDNA for Wld s protein bearing W258A mutation was gen- cytosol fractions was performed by sequential centrifugation as described erated as previously described (Araki et al., 2004). The integrity of each previously (Tateno et al., 2004) (also shown in Fig. 4A). Ten percent of each clone was verified by nucleotide sequence analysis. Transgenic constructs fraction was subjected to SDS-PAGE and immunoblotting with antibodies were microinjected into fertilized BDF1 mouse oocytes by following stan- against His tag, Wld-18, Nucleoporin p62 (BD Biosciences), Prohibitin dard procedure (Conner, 2004). Genomic integrations of transgenes were (NeoMarkers), Adaptin ␥ (BD Biosciences), PMP70 (Affinity Bioreagents), examined by Southern blot analysis and/or PCR with the following prim- and superoxide
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