The NAD synthesizing protein nicotinamide mononucleotide adenylyltransferase (Nmnat) got the attention of neuroscience researchers as one-half of a chimeric, neuroprotective protein responsible for slowing Wallerian degeneration in the Wlds mutant mouse (see ARF related news story). It was considered the lesser half, until experiments showed that overexpression of just the Nmnat enzyme was sufficient to protect axons from neurodegeneration (see ARF related news story). Now some new data secure Nmnat’s standing as a bona fide neuroprotective protein, but not precisely as expected.

In a paper published in the December issue of PLoS Biology, Hugo Bellen and colleagues at Baylor College of Medicine in Houston, Texas, demonstrate that Nmnat is crucial for saving photoreceptor neurons from activity-induced degeneration in Drosophila. The surprising finding is that, contrary to previous suggestions, the protective power of fly Nmnat does not rely on NAD synthesis activity.

The researchers, led by first author Grace Zhai and colleagues, isolated the Drosophila Nmnat gene in a forward genetic screen for synapse malfunction. They used mosaic flies that carry homozygous mutations restricted to the visual system, a strategy that allows for the isolation of mutants that would be lethal if absent completely from the animals. They first screened for loss of phototaxis, and then looked for mutants with normal gross eye morphology but abnormal photoreceptor axons, terminals, and synaptic function.

Two blind mutants with poor synapse structure and function turned out to have substitutions in the coding region of a gene that the researchers identified as the Drosophila version of human Nmnat. The mutations, which created null alleles, caused an age-dependent neurodegeneration of photoreceptor cells. Eye development appeared normal, and the degeneration was activity-dependent: rearing flies in the dark, or interrupting the phototransduction cascade with other mutations, retarded neuron loss.

To test if the phenotype was due to loss of NAD synthase activity, the researchers attempted to rescue the neurodegeneration using different Nmnat constructs. They found that two mutants, each one showing a 99 percent reduction in enzymatic activity, were just as effective at supporting neuronal survival as a wild-type human NMNAT3. However, the mutants could not reverse the lethality of a ubiquitous Nmnat homozygous deletion, indicating that the protein has at least two independent roles—one required for organismal viability and another for neuroprotection.

In the Wlds mouse, overexpression of the Nmnat fusion protein delays degeneration, and the researchers saw a similar effect after overexpression of Nmnat in flies. Introduction of either wild-type or mutant, catalytically inactive protein prevented degeneration due to two different retinal degeneration mutants, and also in response to excessive neuronal activity induced by constant intense light.

The demonstration that catalytically inactive Nmnat is just as effective at neuroprotection is at odds with earlier reports on overexpression of the proteins in vitro (see ARF related news story), which showed that enzymatic activity and NAD were required to protect axon degeneration. While the reasons for this discrepancy are not clear, the current results are consistent with findings in the original Wlds mice in that even as the levels of Nmnat were increased thanks to the chimera, NAD concentrations were not.

The study puts the question of how Nmnat protects neurons back to square one. But the authors suggest the protein might be “exploited to protect neurons against activity-induced neurodegeneration,” a prospect that might be of some use in Alzheimer and other neurodegenerative diseases.—Pat McCaffrey

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  1. 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:

    . Absence of Wallerian Degeneration does not Hinder Regeneration in Peripheral Nerve. Eur J Neurosci. 1989;1(1):27-33. PubMed.

    . Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene. Nat Neurosci. 2001 Dec;4(12):1199-206. PubMed.

    . Delayed synaptic degeneration in the CNS of Wlds mice after cortical lesion. Brain. 2006 Jun;129(Pt 6):1546-56. PubMed.

    . 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. PubMed.

    . The Wlds mutation delays robust loss of motor and sensory axons in a genetic model for myelin-related axonopathy. J Neurosci. 2003 Apr 1;23(7):2833-9. PubMed.

    . The slow Wallerian degeneration gene, WldS, inhibits axonal spheroid pathology in gracile axonal dystrophy mice. Brain. 2005 Feb;128(Pt 2):405-16. PubMed.

    . A local mechanism mediates NAD-dependent protection of axon degeneration. J Cell Biol. 2005 Aug 1;170(3):349-55. PubMed.

    . Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science. 2004 Aug 13;305(5686):1010-3. PubMed.

    . NAD(+) and axon degeneration revisited: Nmnat1 cannot substitute for Wld(S) to delay Wallerian degeneration. Cell Death Differ. 2007 Jan;14(1):116-27. PubMed.

    . The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron. 2006 Jun 15;50(6):869-81. PubMed.

    . 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.

    . Fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches. Nat Neurosci. 2004 Nov;7(11):1181-3. PubMed.

    . Dendritic spine abnormalities in amyloid precursor protein transgenic mice demonstrated by gene transfer and intravital multiphoton microscopy. J Neurosci. 2005 Aug 3;25(31):7278-87. PubMed.

    . Axonopathy and transport deficits early in the pathogenesis of Alzheimer's disease. Science. 2005 Feb 25;307(5713):1282-8. PubMed.

    . BACE overexpression alters the subcellular processing of APP and inhibits Abeta deposition in vivo. J Cell Biol. 2005 Jan 17;168(2):291-302. PubMed.

References

News Citations

  1. Protein Chimera Found to Protect Axons from Degeneration
  2. The Wine and Wherefore of Wallerian Degeneration

Further Reading

Primary Papers

  1. . Drosophila NMNAT maintains neural integrity independent of its NAD synthesis activity. PLoS Biol. 2006 Nov;4(12):e416. PubMed.