Nmnat Delays Axonal Degeneration Caused by Mitochondrial and Oxidative Stress

“Mitochondrial dysfunction caused by mutations or toxic agents has been implicated in many neurodegenerative diseases, including those associated with axonal degeneration (Lin and Beal, 2006).”

“, DJ-1 and PINK-1) (Martin, 2006) and ingestion of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP), an inhibitor of mitochondrial complex I (Langston et al., 1983; Betarbet et al., 2000).”

“…Parkinson’s disease (PD), which can be associated with mutations in mitochondria-associated proteins (e.g., DJ-1 and PINK-1) (Martin, 2006) and ingestion of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP), an inhibitor of mitochondrial complex I (Langston et al., 1983; Betarbet et al., 2000).”

“…chronic rotenone model of PD, which more closely mimics the degenerative progression observed in PD patients, because this model only works in rats (Hornykiewicz, 1966; Betarbet et al., 2000; Richter et al., 2007); however, our results suggest that increasing Nmnat expression in this model may…”

“The suppression of ROS accumulation by Nmnat proteins is reminiscent of that observed in cells treated with antioxidants, whose utility in preventing axonal degeneration secondary to mitochondrial inhibition has been suggested by others (Sherer et al., 2003, 2007; Testa et al., 2005).”

“…axonal degeneration resulting from damage caused by mechanical, genetic, or chemical injury (Gillingwater and Ribchester, 2001; Wang et al., 2001a,b; Ferri et al., 2003; Samsam et al., 2003; Araki et al., 2004; Gillingwater et al., 2004; Mi et al., 2005; Sasaki et al., 2006; Zhai et al., 2006).”

“These changes are followed by calpain activation and eventual axonal protein degradation (Coleman, 2005).”

“For example, complex I inhibition and oxidative damage to mitochondrial proteins have been observed in brains of PD patients, changes that can be replicated by treatment of isolated mitochondria with rotenone (Keeney et al., 2006).”

“…of PD (Sherer et al., 2002a); in particular, there is a strong association between PD and exposure to pesticides, including many that inhibit mitochondrial complex I (Semchuk et al., 1992; Gorell et al., 1998, 2004; Priyadarshi et al., 2000, 2001; Ascherio et al., 2006; Brown et al., 2006).”

“It has been hypothesized that increasing NAD concentration via NMNAT1 and the consequent SIRT1 activation might play a role (212, 218, 219).”

“…subcellular localization studies have found substantial amounts of WldS within the mitochondrial matrix where it may enhance ATP synthesis (Yahata et al., 2009), prevent free radical formation (Press and Milbrandt, 2008), and/or maintain mitochondrial membrane potential (Ikegami and Koike, 2003).”

“, 2001), fails to delay axon degeneration after axotomy (George et al., 1995; Press and Milbrandt, 2008), suggesting that Na entry is not the primary signal to trigger Ca influx, or does not lead to sufficient Ca entry to initiate Wallerian axon de­ generation.”

“One proposal is that Nmnat activity decreases reactive oxygen species in mitochondria because it protects axons from rotenone-induced damage without restoring normal ATP levels (Press & Milbrandt 2008).”

“, 2005), but the relevance of ATP depletion was questioned by other studies (Press and Milbrandt, 2008; Summers et al., 2014).”

“…with decreased enzymatic activity have reduced protective capacity, whereas other enzymatically active Nmnat isoforms like the mitochondrial Nmnat3 are protective (Sasaki et al., 2006; Press and Milbrandt, 2008), supporting the idea that Nmnat enzymatic activity is important for axonal protection.”

“Additionally, the WldS gene product has been shown to modulate energy metabolism within the axon to maintain ATP levels following injury [221] as well as to reduce ROS production [184,199].”

“We demonstrated previously that exposure to the ETC complex I inhibitor rotenone induces axon degeneration and cell death in neurons (Press and Milbrandt, 2008).”

“We and others showed previously that increase in oxidative stress is a key and early step in axonal degeneration (Press and Milbrandt, 2008; Fischer and Glass, 2010; Calixto et al., 2012; O’Donnell et al., 2013).”

“As oxidative stress was reported to be directly responsible for axonal degeneration in cultured primary neurons (15, 51) or in vivo in an experimental autoimmune encephalomyelitis (EAE) model (2), it seems reasonable to propose oxidative burden (direct consequence of peroxisomal metabolic impairment) as a first hit in…”

“Further evidence that Nmnat1 is the key component for Wld -mediated axonal protection was obtained from experiments showing that Nmnat enzymes localized to multiple cellular compartments or derived from species ranging from Archaebacterium Methanococcus jannaschii to Saccharomyces cerevisiae to Mus musculus all provid…”

“Finally, NMNATs overexpression and Sarm1 deletion in sensory neurons delay axon degeneration caused by rotenone, another mitochondrial complex-1 inhibitor, in sensory neurons (Press and Milbrandt, 2008; Summers et al., 2014).”

“Intriguingly, other reports show that mitochondrial dysfunctions activate a pathway of degeneration sensitive to regulators of Wallerian degeneration (Press and Milbrandt, 2008; Summers et al., 2014).”

“Prolonged exposure to hydrogen peroxide leads to axonal degeneration [21].”

“Consistent with previous studies [7–13], expression of Wlds, mNmnat1, mNmnat3, and Drosophila Nmnat (dNmnat) all dramatically delayed axon degeneration (p < 0.”

“Exposure to the mitochondrial toxin, rotenone (a complex I inhibitor), has also been shown to cause axonal degeneration in vitro (Press and Milbrandt, 2008).”

“Nmnat3, located in mitochondria, can prevent axonal degeneration triggered by ROS in dorsal root ganglia culture (Press and Milbrandt, 2008).”

“Interestingly, NMNAT over-expression can prevent axonal damage resulting from exogenous application of oxidants (28).”

“A recent in vitro study suggests an alternative mechanism based on Nmnat blocking production of reactive oxygen species (ROS) from mitochondria (Press and Milbrandt, 2008).”

“Interestingly, an increasing number of studies have indicated that cytotoxic-free radicals, such as superoxides, nitric oxide, and others may contribute to neural damage, including attenuation in axonal growth [46].”

“…is associated with aging and neurodegeneration (Balaban et al., 2005; Lin and Beal, 2006) and has been proposed to play a role in axon degeneration (Press and Milbrandt, 2008; Fischer and Glass, 2010; Calixto et al., 2012), we determined whether mitochondrial redox state was affected by axotomy,…”

“shown that NMNAT proteins (both NMNAT1 and NMNAT3) protects against axon degeneration induced by injury or oxidative stress [7, 11, 12, 59].”

“For example, the specific complex I inhibitor rotenone lowered intraneuritic ATP concentration and diminished neurite outgrowth in mouse adult sensory neurons (53) and embryonic rat neurons (54).”

“Using a mechanism that is still mysterious, NMNAT gain-offunction in the axon blocks axonal degeneration in Drosophila and mouse from a wide range of insults (Mack et al., 2001; Hoopfer et al., 2006; MacDonald et al., 2006; Press and Milbrandt, 2008; Zhai et al., 2008; Sasaki et al., 2009a).”

“…al., 1998; Mack et al., 2001) and subsequently found to be protective of axons in many animal models of neurodegenerative disease (Sajadi et al., 2004; Hasbani and O’Malley, 2006; Press and Milbrandt, 2008; Cheng and Burke, 2010). aSyn and WldS were co-expressed in peripheral sensory neurons (Fig.”

“The reduced form of this coenzyme, NADPH, has a crucial role in ROS buffering, so SARM1-dependent NADPase puts axons at considerable risk of ROS-induced degeneration, and indeed activation of Wallerian degeneration by vincristine does cause an increase in ROS (Press and Milbrandt, 2008).”

“Elevated ROS levels in rotenone-treated neurites In addition to impaired ATP production, rotenone has also been shown to cause elevated levels of ROS in neurites when administered to dissociated DRG cultures (Press and Milbrandt, 2008).”

“The mechanism underlying the axonal preservation is proposed to depend on overexpression of the Nmnat1 portion of the locus, and NMNAT may be serving as a chaperone to decrease protein misfolding or to decrease reactive oxygen species, preventing mitochondrial dysfunction (Press and Milbrandt, 2008; Zhai et al., 2006;…”

“This result is consistent with the idea that moderate mitochondrial dysfunction can cause axonopathy via excess ROS production rather than through abnormalities associated with energy deficits per se (Bao et al., 2005; Press and Milbrandt, 2008).”

“In vitro overexpression of Nmnat1 or addition of Nmnat1 products, substrates and precursors of these substrates protect axons (Araki et al., 2004; Press and Milbrandt, 2008; Sasaki et al., 2006, 2009b;Wang et al., 2005).”

“JCB • Volume 211 • NumBer 4 • 2015 892 for dopaminergic neuronal cell bodies in rodent Parkinson’s disease models induced by 6OHDA or 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (Sajadi et al., 2004; Press and Milbrandt, 2008).”

“Recent studies indicated the impairment of several genes concerned with mitochondrial function in vincristine induced peripheral neuropathy, which rendering the possible involvement of mitochondrial dysfunction and corresponding oxidative stress generation in the disease pathology [31].”

“…containing excess Nmnat, and the involvement of other metabolic substrates besides NAD+ (Wang et al., 2005; Suzuki and Koike, 2007a,b; Press and Milbrandt, 2008; Wishart et al., 2008; Conforti et al., 2009; Sasaki et al., 2009; Yahata et al., 2009; Barrientos et al., 2011; Avery et al., 2012;…”

“Several in vitro models of oxidative stress-mediated axonal degeneration have also been developed using exogenous agents, such as rotenone [49], H2O2 [41], and 4-hydroxynonenal [1].”

“Nmnat3 also provides axonal protection from mechanical injury and toxins in dorsal root ganglion (DRG) neuronal cultures (Sasaki et al., 2006; Press and Milbrandt, 2008).”

“Studies of the wld mutant mouse led to the discovery that overexpression of NMNAT1 delays the degeneration of axons in response to a wide variety of injuries including mechanical severing, mitochondrial inhibitors, and chemotherapeutic agents (12, 24, 25).”

“Finally, there is accumulation of ROS (reactive oxygen species) that occurs either before or after SARM1 activation whose origin and function in axon degeneration requires further study (Press and Milbrandt, 2008; Summers et al., 2014).”

“Nmnats have protective effects against activity- and injury-induced neurodegeneration and axonal degeneration [24–28].”

“We assessed neurite degeneration in these cells using phasecontrast microscopy; neurite segments were considered degenerated if they showed evidence of swellings and/or blebbing (see Materials and methods) (Press and Milbrandt, 2008).”

“Strikingly, NADand NAD precursors can also provide protection against axonal degeneration, suggesting that the effect of Nmnat1 is achieved through a function involving NAD synthesis (Sasaki et al., 2006, 2009; Press and Milbrandt, 2008; Wang and He, 2009; Yahata et al., 2009).”

“Nmnat1 and Nmnat3 also promote axonal protection caused by exogenous oxidants (Press and Milbrandt, 2008).”

“have reported that cytoplasmic Nmnat1 (cyto Nmnat1) and Nmnat3, which is primarily localized in the mitochondria, also confer a higher level of protection than Nmnat1 [35,36].”

“These deficits are also associated with numerous neurodegenerative disorders including Parkinson’s, Alzheimer’s, Charcot-Marie Tooth, Amyotrophic Lateral Sclerosis, and Huntington’s diseases (Wang et al., 2005; Press and Milbrandt, 2008; Chen and Chan, 2009; Vohra et al., 2010; Wen et al., 2011; Martin, 2012; Alobuia…”

“This hypothesis has received experimental support from another recent study showing that NMNAT1 can protect against mitochondrial and oxidative stress [69].”

“1c, using the phase-contrast microscope we observed that after 48 h of 30K treatment mouse MNs were largely degenerated (Press and Milbrandt 2008).”

“Similar to Nmnat1, Nmnat3 was also reported to protect axon against mitochondrial dysfunction induced by a variety of insults like injury or chemicals in culture DRG (Sasaki et al., 2006; Press and Milbrandt, 2008) (Table 2).”

“, 2002) and expressing the Wld protein confers neuronal protection in other species and in cultured neurons (Press and Milbrandt, 2008; Tokunaga and Araki, 2012; Wang et al., 2005).”

“A previous study showed that a reduction in ATP levels by inhibition of glycolysis does not cause axon degeneration (Press and Milbrandt 2008) questioning the importance of ATP generated by glycolysis for axons.”

“The previous finding that Nmnat can inhibit ROS accumulation (Press and Milbrandt, 2008), coupled with our data showing that Nmnat acts upstream of autophagy, together support the possibility that Nmnat may suppress autophagy by maintaining low levels of ROS under hypoxic conditions.”

“…with a prior report showing that, in NGF, embryonic sensory axons do not undergo degeneration on inhibition of glycolysis using 2-deoxyglucose (Press and Milbrandt, 2008), in our studies neither GIM nor pharmacological inhibition of GAPDH resulted in axon degeneration in the presence of NGF.”

“The role of mitochondria in Wallerian degeneration, which has long been disputed (Press & Milbrandt, 2008; Avery et al., 2012; Court & Coleman, 2012; Fang et al., 2012; Tokunaga & Araki, 2012; Felici et al., 2013; Kitaoka et al., 2013; Kitay et al., 2013; O’Donnell et al., 2013; Panneerselvam et…”

“In this case, ROS generation may be a necessary upstream step (Press and Milbrandt, 2008), although the mechanism linking excess ROS and NMNAT2 depletion has not been established.”

“Previous report has shown that a mitochondria-localized Nmnat isoform Nmnat3 could reduce oxidative or mitochondrial stress-induced ROS generation to delay axonal degeneration [35].”

“…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 is thus not surprising that impaired bioenergetic states have been shown to result in axonal injury, growth inhibition, and degeneration in vitro (Gibbons et al. 2010; Kitayama et al. 2008; Natera-Naranjo et al. 2012; Persson et al. 2013a; Press and Milbrandt 2008).”

“Other than the neuroprotective examples mentioned above, degeneration of dorsal root ganglion axons induced by rotenone, which inhibits mitochondrial electron transport and causes oxidative stresses, is delayed by NMNAT overexpression [5].”