This paper indicates an intriguing, but as yet unknown, essential function of Nmnat that is distinct from NAD synthesis. The activity-independent function is required for neuronal survival under normal physiological conditions and for resistance to neurodegeneration of excessively stimulated photoreceptors, caused either by constitutive phototransduction or exposure to intense light.

These results add to a growing debate as to whether Nmnat and its mammalian isoforms can delay the degeneration of injured axons (Wallerian degeneration). The relevance of Wallerian degeneration to Alzheimer disease is described below. Nmnat1 is part of the slow Wallerian degeneration protein (Wlds), an unusual chimeric protein which delays Wallerian degeneration for 14 days or more in mice and rats and also partially protects synapses [1-3]. The same gene can delay axon degeneration in some neurodegenerative diseases where there is no physical injury, suggesting that other insults such as a block of axonal transport can trigger a similar degenerative pathway to injury [4-6]. Data from primary...  Read more

This paper indicates an intriguing, but as yet unknown, essential function of Nmnat that is distinct from NAD synthesis. The activity-independent function is required for neuronal survival under normal physiological conditions and for resistance to neurodegeneration of excessively stimulated photoreceptors, caused either by constitutive phototransduction or exposure to intense light.

These results add to a growing debate as to whether Nmnat and its mammalian isoforms can delay the degeneration of injured axons (Wallerian degeneration). The relevance of Wallerian degeneration to Alzheimer disease is described below. Nmnat1 is part of the slow Wallerian degeneration protein (Wlds), an unusual chimeric protein which delays Wallerian degeneration for 14 days or more in mice and rats and also partially protects synapses [1-3]. The same gene can delay axon degeneration in some neurodegenerative diseases where there is no physical injury, suggesting that other insults such as a block of axonal transport can trigger a similar degenerative pathway to injury [4-6]. Data from primary neuronal culture experiments suggest that Nmnat1 is sufficient for this protective activity [7,8]. In contrast, overexpression of Nmnat1 in transgenic mice at levels similar to Wlds has no protective effect at all [9]. Ultimately, the protective agent must work in vivo, both to be sure that we fully understand the mechanism and because any future therapeutic application of this knowledge will be in vivo. Interestingly, Nmnat does partially protect injured axons in Drosophila, although its efficacy remains uncertain, as so far this protective effect of Nmnat is reported only for 5 days as opposed to 30 days for Wlds [10].

Critically, Nmnat, and NAD metabolites and precursors, may have several protective functions that can alter neurodegeneration, and it is essential not to confuse these with one another. The key test for protection against Wallerian degeneration is to ask whether transected axons are preserved. Without such a test, we cannot truly say that something protects against Wallerian degeneration, because Waller himself defined this process by cutting axons [11]. However, this is not just a matter of semantics. The stringency imposed by testing for survival of injured axons for 14 days in vivo is important to prevent confusion between different pathways of degeneration and neuroprotection.

This test was not applied to the enzyme-dead Nmnat in Zhai et al. Considering that the protective function of Nmnat described here is independent of NAD synthesis activity, in contrast to protection from Wallerian degeneration where enzyme activity is required, at least in primary culture [8,9], this is an essential gap to close before we can say that enzyme-dead Nmnat protects from Wallerian degeneration.

Regarding the implications for Alzheimer disease, the importance of axon degeneration in AD is becoming more and more clear. Dystrophic axons occur in the immediate vicinity of amyloid plaques [12,13], impairing axonal transport worsens plaque deposition [14], and shifting the site of Aβ synthesis away from axons and synapses reduces the amyloid burden [15]. Whether axon degeneration in AD is Wallerian-like, as it clearly is in at least some other neurodegenerative diseases [4,5], is an important next step to clarify. If and when this can be shown, factors that delay Wallerian degeneration in vivo may have therapeutic value in AD. So far, no other gene or drug, including Nmnat, has come close to the efficacy of Wlds in doing this.

References:
[1] Lunn ER, Perry VH, Brown MC, Rosen H, Gordon S: Absence of Wallerian degeneration does not hinder regeneration in peripheral nerve. Eur J Neurosci 1989, 1: 27-33. Abstract

[2] Mack TG, Reiner M, Beirowski B, Mi W, Emanuelli M, Wagner D, Thomson D, Gillingwater T, Court F, Conforti L, Fernando FS, Tarlton A, Andressen C, Addicks K, Magni G, Ribchester RR, Perry VH, Coleman MP: Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene. Nat Neurosci 2001, 4: 1199-1206. Abstract

[3] Gillingwater TH, Ingham CA, Parry KE, Wright AK, Haley JE, Wishart TM, Arbuthnott GW, Ribchester RR: Delayed synaptic degeneration in the CNS of Wlds mice after cortical lesion. Brain 2006, 129: 1546-1556. Abstract

[4] Ferri A, Sanes JR, Coleman MP, Cunningham JM, Kato AC: Inhibiting axon degeneration and synapse loss attenuates apoptosis and disease progression in a mouse model of motoneuron disease. Curr Biol 2003, 13: 669-673. Abstract

[5] Samsam M, Mi W, Wessig C, Zielasek J, Toyka KV, Coleman MP, Martini R: The Wlds mutation delays robust loss of motor and sensory axons in a genetic model for myelin-related axonopathy. J Neurosci 2003, 23: 2833-2839. Abstract

[6] Mi W, Beirowski B, Gillingwater TH, Adalbert R, Wagner D, Grumme D, Osaka H, Conforti L, Arnhold S, Addicks K, Wada K, Ribchester RR, Coleman MP: The slow Wallerian degeneration gene, WldS, inhibits axonal spheroid pathology in gracile axonal dystrophy mice. Brain 2005, 128: 405-416. Abstract

[7] Wang J, Zhai Q, Chen Y, Lin E, Gu W, McBurney MW, He Z: A local mechanism mediates NAD-dependent protection of axon degeneration. J Cell Biol 2005, 170: 349-355. Abstract

[8] Araki T, Sasaki Y, Milbrandt J: Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science 2004, 305: 1010-1013. Abstract

[9] Conforti L, Fang G, Beirowski B, Wang MS, Sorci L, Asress S, Adalbert R, Silva A, Bridge K, Huang XP, Magni G, Glass JD, Coleman MP: NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration. Cell Death Differ 2006, EPub before print. Abstract

[10] Macdonald JM, Beach MG, Porpiglia E, Sheehan AE, Watts RJ, Freeman MR: The Drosophila cell corpse engulfment receptor draper mediates glial clearance of severed axons. Neuron 2006, 50: 869-881. Abstract

[11] Waller A: Experiments on the section of glossopharyngeal and hypoglossal nerves of the frog and observations uf the alternatives produced thereby in the structure of their primitive fibres. Philos Trans R Soc Lond B Biol Sci 1850, 140: 423-429.

[12] Tsai J, Grutzendler J, Duff K, Gan WB: Fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches. Nat Neurosci 2004, 7: 1181-1183. Abstract

[13] Spires TL, Meyer-Luehmann M, Stern EA, McLean PJ, Skoch J, Nguyen PT, Bacskai BJ, Hyman BT: Dendritic spine abnormalities in amyloid precursor protein transgenic mice demonstrated by gene transfer and intravital multiphoton microscopy. J Neurosci 2005, 25: 7278-7287. Abstract

[14] Stokin GB, Lillo C, Falzone TL, Brusch RG, Rockenstein E, Mount SL, Raman R, Davies P, Masliah E, Williams DS, Goldstein LS: Axonopathy and transport deficits early in the pathogenesis of Alzheimer's disease. Science 2005, 307: 1282-1288. Abstract

[15] Lee EB, Zhang B, Liu K, Greenbaum EA, Doms RW, Trojanowski JQ, Lee VM: BACE overexpression alters the subcellular processing of APP and inhibits Abeta deposition in vivo. J Cell Biol 2005, 168: 291-302. Abstract

View all comments by Michael Coleman

This new study by Verghese et al. is a huge step in the right direction concerning mechanisms of neurodegeneration in neonatal hypoxia-induced encephalopathy (HIE). The field has been side-tracked, maybe even lost, for quite some time with the resurrection of apoptosis in the developing nervous system and its possible role in brain injury. It seems to have been forgotten that the overwhelming majority of the neurodegeneration seen in neonatal human HIE and in animal model HIE has more of a necrotic phenotype (see Northington et al., 2011; Martin, 2001). The work by Verghese et al. on Nmnat1 is consistent with this idea. A problem, though, with neonatal mouse models of HIE is the troublesome inherent variability in the damage, even among gender-matched littermates, and the robust strain effects, especially in investigations of cerebral ischemia and excitotoxicity. It will be extraordinarily important to get this work translated to large animal models of neonatal HIE to fully determine its...  Read more

This new study by Verghese et al. is a huge step in the right direction concerning mechanisms of neurodegeneration in neonatal hypoxia-induced encephalopathy (HIE). The field has been side-tracked, maybe even lost, for quite some time with the resurrection of apoptosis in the developing nervous system and its possible role in brain injury. It seems to have been forgotten that the overwhelming majority of the neurodegeneration seen in neonatal human HIE and in animal model HIE has more of a necrotic phenotype (see Northington et al., 2011; Martin, 2001). The work by Verghese et al. on Nmnat1 is consistent with this idea. A problem, though, with neonatal mouse models of HIE is the troublesome inherent variability in the damage, even among gender-matched littermates, and the robust strain effects, especially in investigations of cerebral ischemia and excitotoxicity. It will be extraordinarily important to get this work translated to large animal models of neonatal HIE to fully determine its relevance as a mechanism of brain injury.

View all comments by Lee J. Martin

The paper makes some interesting findings, but also raises some questions. Figures 1 and 2 are particularly impressive, showing major protection from tissue loss in the cytNmnat1 neonates. This result is limited to magnetic resonance imaging and low-resolution sections, so it would be useful to know what is happening at the cellular level, in particular, whether cell death is actually prevented in vivo.

The reduced NAD loss is also intriguing. Interestingly, there was an earlier suggestion that synthesis of NAD was not the reason why cytNmnat1 projects injured axons (Sasaki et al., 2009). I wonder whether there are other changes happening, and whether any are more causatively connected with tissue damage.

There is a striking reduction in lactate dehydrogenase release as an indirect measure of cell death. It would be nice to see the protected cell bodies and a direct quantification of their numbers, or propidium iodide staining, for example. Cell survival in vivo seems not to be assessed (unless I missed something in the supplementary figures), but if it occurs, it could...  Read more

The paper makes some interesting findings, but also raises some questions. Figures 1 and 2 are particularly impressive, showing major protection from tissue loss in the cytNmnat1 neonates. This result is limited to magnetic resonance imaging and low-resolution sections, so it would be useful to know what is happening at the cellular level, in particular, whether cell death is actually prevented in vivo.

The reduced NAD loss is also intriguing. Interestingly, there was an earlier suggestion that synthesis of NAD was not the reason why cytNmnat1 projects injured axons (Sasaki et al., 2009). I wonder whether there are other changes happening, and whether any are more causatively connected with tissue damage.

There is a striking reduction in lactate dehydrogenase release as an indirect measure of cell death. It would be nice to see the protected cell bodies and a direct quantification of their numbers, or propidium iodide staining, for example. Cell survival in vivo seems not to be assessed (unless I missed something in the supplementary figures), but if it occurs, it could still be secondary to axon survival. A similar finding is likely to underlie the preservation of both motor axons and cell bodies in progressive motor neuronopathy mice by Wlds (Ferri et al., 2003).

A study of Wlds in transient ischemia in adults also found protection of cell bodies (Gillingwater et al, 2004). From the point of view of Alzheimer's disease, this could be important because adult nervous systems should better model a disorder of the aging brain. Nevertheless, as the authors point out, hypoxia-ischemia is also very important in newborns.

I am intrigued by the fact that cytNmnat1 tissues show a huge increase in caspase-3 activity but still survive (Fig 4). I think this actually raises the question of whether cytNmnat1 is blocking a step downstream of caspase-3. It's hard to imagine that this increase in caspase-3 activity makes no contribution to cell death.

Regarding the implications for various neurodegenerative disorders, a lot depends on the pathogenic mechanisms in each disease. If ischemia is involved, which, in AD could be the case, this, together with the study in adult animals mentioned above, could be a useful step forward.

In summary, there is clearly strong protection from ischemic damage by an Nmnat enzyme, which in neonates at least is the first example. CtyNmnat1 may well be working downstream of NMDA excitotoxicity, but it will be important to learn more about the cell survival, including a direct demonstration and its causative relationship to axon survival.

References:
Sasaki Y, Vohra BP, Lund FE, Milbrandt J. Nicotinamide mononucleotide adenylyl transferase-mediated axonal protection requires enzymatic activity but not increased levels of neuronal nicotinamide adenine dinucleotide. J Neurosci. 2009 Apr 29;29(17):5525-35. Abstract

Ferri A, Sanes JR, Coleman MP, Cunningham JM, Kato AC. Inhibiting axon degeneration and synapse loss attenuates apoptosis and disease progression in a mouse model of motoneuron disease. Curr Biol. 2003 Apr 15;13(8):669-73. Abstract

Gillingwater TH, Haley JE, Ribchester RR, Horsburgh K. Neuroprotection after transient global cerebral ischemia in Wld(s) mutant mice. J Cereb Blood Flow Metab. 2004 Jan;24(1):62-6. Abstract

View all comments by Michael Coleman

An interesting paper extending the literature on axonal and neuronal protection by Nmnat1 to another model of neurological injury. An intriguing aspect of this study is that Nmnat1 protects against hypoxic-ischemia injury, but does not seem to affect the activation of caspase-3 or standard apoptosis pathways. This finding further emphasizes the fact that neuronal and axonal death may proceed by independent pathways, and identification of the relevant pathways will be important for designing and developing new therapeutics.

View all comments by Jonathan D. Glass