Nicotinamide Mononucleotide Adenylyltransferase Expression in Mitochondrial Matrix Delays Wallerian Degeneration

“We showed that exogenously applied NAD results in activation of Sirt1, an NADdependent protein deacetylase, and discussed that this is the key mechanism that connects increased NMNAT activity and axonal protection (Araki et al., 2004).”

“Since NMNAT2 is constitutively expressed highly in neurons (Raffaelli et al., 2002), we decided to generate mice that overexpress mammalian NMNAT orthologs other than NMNAT2 (i.e., NMNAT1 and NMNAT3, either of which are unexpressed in neurons) (Emanuelli et al., 2001; Zhang et al., 2003).”

“Previous reports suggest that NMNAT3 is located mainly, but not exclusively, in mitochondria when it is transiently overexpressed in cultured cells (Zhang et al., 2003; Berger et al., 2005); however, we found that NMNAT3 was located almost exclusively in mitochondria of NMNAT3-Tg mice.”

“However, it was not clear how NMNAT overexpression affects Sirt1 activity, because, whereas Sirt1 activity is strictly dependent on intracellular NAD levels (Yamamoto et al., 2007), overexpression of NMNAT activity does not cause major changes in NAD levels (Mack et al.”

“anion-exchange column as described previously (Suter et al., 2006).”

“Mitochondria were isolated from cerebrum as described previously with minor modifications (Sims, 1990; Rajapakse et al., 2001) (also shown in supplemental Fig.”

“The mitochondrially localized mammalian isoform Nmnat3 has been shown to phenocopy the axonal degeneration-protective activity of the Nmnat1 wlds mutant, whereas simple overexpression of the nuclear-localized Nmnat1 or the golgi-localized Nmnat2 does not protect from axonal degeneration.(8,41) The axonal-protective ph…”

“Nmnat plays an essential role in maintaining axonal integrity by modifying mitochondrial function, and Nmnat expression in mitochondrial matrix delays Wallerian degeneration (Yahata et al., 2009; Fang et al., 2012).”

“1B and D) consistent with the prior observation demonstrating that NMNAT3 is mainly localized to the mitochondrial compartment in these mice.(15)”

“Although initially found to be most highly abundant in the nucleus, theWldS fusion protein was later noted to also be enriched in mitochondria, cytosol, peroxisomes/lysosomes, endoplasmic reticulum, and axons (Yahata et al. 2009; Avery et al. 2012; Wang et al. 2015).”

“…of action of Wld S have highlighted several potential effector pathways and sub-cellular targets that may be suitable as therapeutic targets [28], including; Nmnat-dependent pathways [18,45–46], mitochondria [31,47–48], VCP-dependent pathways [49,50], and cell cycle/cell stress pathways [32,51].”

“It has been shown that over-expression of the mitochondrially localized form of Nicotinamide mononucleotide adenylyltransferase, Nmnat3, can protect axons from injury in both mouse and drosophila models (Avery et al., 2012; Berger et al., 2005; Yahata et al., 2009).”

“to be enhanced in Wld mice and in transgenic flies expressing Wld [35,36].”

“1F), which localises to the nucleus (Yahata et al, 2009).”

“This change in bioenergetics, coupled with increased mitochondrial motility in WldS+ axons, might enhance distribution of ATP or other mitochondriallyderived metabolites.”

“Earlier reports revealed the ability of NMNAT3 in preventing axonal degeneration where its ectopic expression delayed degeneration of transected sciatic nerves of mice, an effect not reproduced by NMNAT1 expression (22).”

“In the mitochondria, both WLD/ NMNAT1 and NMNAT3 promote mitochondrial motility and increase the Ca(21)-buffering activity of mitochondria and this may contribute to its ability to delay axonal degeneration following injury.(20) Genetic studies have identified other pathways that also regulate axonal survival.”

“To test this possibility, we examined whether OD plasticity was reduced in a transgenic mouse line overexpressing nuclear NMNAT1 (Yahata et al., 2009).”

“Concurrent with this, is an increased ATP generation in isolated mitochondria with no alteration in ETC components (Yahata et al. 2009).”

“It was shown that the N-terminal 70-amino-acid stretch of WldS protein may contribute to association with other proteins and/or affect the conformation and subcellular localization of WldS protein (Wilbrey et al., 2008; Yahata et al., 2009).”

“These data demonstrate that downstream consequences of WldS expression, incorporating pathways including NAD biosynthesis, ubiquitination, the mitochondrial proteome, cell cycle status and cell stress [17,26-28], do not have any major adverse effects on non-neuronal organs.”

“15 Research on NMNAT1 has revealed intriguing findings, including the observation that homozygous null mutations in this gene, seen in both Drosophila melanogaster and murine models, lead to early lethality.”

“Substantial protective effects have also been observed by overexpressing NMNAT3 in transgenic mice and Drosophila [56,57].”

“It was also reported that mitochondrial localization of NMNAT activity results in increased ATP synthetic capacity without affecting the expression of the mitochondrial enzymes profile of the respiratory chain (Yahata et al., 2009), and increased NMNAT activity delayed WD (Araki et al., 2004).”

“The axon protection conferred by NMNAT1 seems to be closely related to mitochondria, which is a critical organelle for the maintenance of redox state (59, 60).”

“Overexpression of NMNAT in the mitochondrial matrix resulted in suppression of axonal degeneration seen in neurodenegeration (Sasaki & Milbrandt, 2010; Sasaki et al., 2009; Yahata et al., 2009).”

“Tissue processing for histological analysis Histological analysis of nerve tissues on semi-thin sections and by electron microscopy was performed as described previously (Yahata et al., 2009).”

“Another in vivo study shows that neuroprotection is observed only in mNMNAT3-transgenic mice, not in mNMNAT1-transgenic mice [108].”

“In flies and mice, NMNAT exerts neuronal protection via its chaperone activity (25–27).”

“Studies in transgenic mice overexpressing NMNAT3 or WLD showed that localization of the enzymatic activity to the mitochondrial matrix is necessary to provide axonal protection by increasing adenosine triphosphate (ATP) production.(25) However, NMNAT2 is the main isoform required for maintenance of healthy axons(26) a…”

“The WldS mutation is a gain-of-function mutation that results in increased nicotinamide mononucleotide adenylyltransferase (NMNAT) activity (Yahata et al., 2009; Sasaki et al., 2009a; Avery et al., 2009).”

“In mammals, there are 3 homologs of Nmnat: Nmnat1, which is in the nucleus; Nmnat2, which is in the golgi; and Nmnat3, which is mitochondrial (Berger et al., 2005; Lau et al., 2010).”

“Similar results are true for Nmnat3, a mitochondrially localized isoform of Nmnat [158].”