Excess Glutamate, Low Oxygen: Can Common Enzyme Mask Effects?

Drought and deluges are rarely good. In the brain, too little oxygen or too much of the neurotransmitter glutamate are both bad news. But there is a potential savior. The enzyme nicotinamide mononucleotide adenylyl transferase (Nmnat) is known to protect axons in the peripheral nervous system. Writing in this week’s Proceedings of the National Academy of Sciences, researchers at Washington University, St. Louis, Missouri, extend that protective role to cell bodies as well as axons, and to the central as well as peripheral nervous system. The authors, at Washington University in St. Louis, Missouri, discovered that Nmnat prevents neuron loss in a mouse model of neonatal oxygen deprivation, or hypoxia, and protects against the toxic effects of excess glutamate, a major neurotransmitter in the brain. The researchers are now looking for clues to how the protection works in the hopes of turning their discovery into a treatment for not only oxygen loss, but perhaps other disorders as well. Glutamate excitotoxicity is thought to be a factor in neurodegenerative diseases such as amyotrophic lateral sclerosis (see ARF related news story on Aizawa et al., 2010 and Cucchiaroni et al., 2010) and Alzheimer’s (see ARF related news story on Li et al., 2009 and ARF related news story on Shankar et al., 2007 and De Felice et al., 2007).

Nmnat1, which is normally found in the nucleus, helps make the metabolic coenzyme nicotinamide adenine dinucleotide (NAD). Its potential neuroprotective role emerged when researchers discovered it was part of the mutation in the slow Wallerian degeneration mouse (Wlds). Wallerian degeneration, or the dying back of severed axons, normally takes 48 hours, but stretches for weeks in Wlds mice (see ARF related news story on Mack et al., 2001 and Wang et al., 2001).

Study coauthor Jeffrey Milbrandt, at Washington University, has been researching axonal degeneration for several years, looking for ways to interfere with the process, which is often an early step in neurodegenerative disease, he told ARF. He and lab member Yo Sasaki developed a mouse that overexpresses a cytoplasmic-localized version of Nmnat1, which strongly protects peripheral axons from injury (Sasaki et al., 2009). They wondered if the transgene would also be protective in the hypoxia model system of Milbrandt’s close friend and colleague David Holtzman, senior author on the paper. First author Philip Verghese led the work in Holtzman’s lab.

Hypoxia can occur in newborns—for example, if the umbilical cord cuts off blood flow or the placenta detaches from the uterus too early—and can cause brain damage or cerebral palsy. Similarly, in older people, strokes cause temporary loss of oxygenated blood flow in the brain, causing neurodegeneration. Holtzman’s model entails tying off the left carotid artery in seven-day-old pups and placing the mice in low oxygen (8 percent) for 45 minutes. The thin air, combined with reduced blood flow, causes brain damage on the left side of the brain; the right side receives sufficient oxygen to remain undamaged and provides an internal control.

Normally, this treatment causes swelling in the brain, which appears on magnetic resonance images as a hyper-intense signal. But in mice overexpressing Nmnat, there was little swelling. “That was very striking,” said Holtzman, who has worked with the model for several years and rarely seen such extensive protection.

A week after inducing hypoxia, the researchers sacrificed the mice to measure the volume of different areas of their brains. They calculated the tissue loss in the damaged left side as a percentage of the volume of the undamaged right side. In wild-type mice without Nmnat overexpression, hypoxia shrank the brain. The volume of the hippocampus was reduced by more than one-third, the striatum was 15 percent smaller, and the cortex and thalamus lost 6 and 5 percent of their normal volume, respectively. While the Nmnat transgenic mice suffered tissue loss as well, it was attenuated. Their hippocampus loss was less than one-fifth, their striata lost only 12 percent, their cortexes less than 1 percent, and their thalami 3 percent. The researchers are now planning to test the mice when they are older for behavioral deficits.

In several previous studies, Nmnat has been shown to prevent axon decay (e.g., Sasaki and Milbrandt, 2010). But Milbrandt, impressed by the “dramatic” tissue preservation, hypothesized that cell bodies, too, must be spared. The researchers used an in-vitro system to look for protective effects. They cultured embryonic cortical neurons from the mice and measured release of lactate dehydrogenase (LDH) into the culture media, a common test of neurodegeneration.

The researchers exposed these cultures to toxic concentrations of N-methyl-D-aspartic acid (NMDA), which binds glutamate receptors and mimics glutamate excitotoxicity. Twelve hours later, control non-transgenic cells had spewed out LDH, indicating they were dying. Cells from the Nmnat mice produced only one-third as much LDH, suggesting whole cells, as well as the axons, were protected from the excitotoxic effects.

How does Nmnat do it? Overexpression of the enzyme might generate more NAD—but the researchers found that levels were similar between transgenic mice and their wild-type littermates, suggesting more of the metabolic factor is not the protective mechanism. Plus, in their earlier experiments with these mice, Milbrandt found that increasing NAD levels does not replicate Nmnat’s protective effects (Sasaki et al., 2009).

The researchers also considered that Nmnat could somehow block apoptosis. But in the hippocampus, the activity of caspase-3, which initiates cell death pathways and ramps up after hypoxic injury, was also similar between the Nmnat and wild-type mice, suggesting Nmnat did not stop apoptosis from starting. But it might keep it from proceeding. “I am intrigued by the fact that [Nmnat] tissues show a huge increase in caspase-3 activity but still survive,” wrote Michael Coleman of the Babraham Institute in Cambridge, U.K., in an e-mail to ARF. “I think this actually raises the question of whether [Nmnat] is blocking a step downstream of caspase-3. It is hard to imagine that this increase in caspase-3 activity makes no contribution to cell death.” Coleman was not involved in the PNAS study. As of yet, it is unclear how Nmnat halts apoptosis, said Milbrandt.

Another option, raised by Hugo Bellen of the Baylor College of Medicine in Houston, Texas, is that Nmnat acts like a chaperone to prevent neurodegeneration (Zhai et el., 2008). Nmnat “seems to bind non-selectively to many different proteins and ensures that the proteins are properly folded,” said Bellen, who did not participate in the current work. Work in flies predicted that Nmnat protects cell bodies and central nervous system cells in vertebrates, Bellen added (see ARF related news story on Zhai et al., 2006; Zhai et el., 2008).

Could the finding have any practical benefit? The only approved treatment for neonatal oxygen loss is therapeutic hypothermia. The effect of Nmnat is at least as good as hypothermia, Holtzman said. However, neonatal mouse models often suffer variable damage that is highly strain dependent, noted Lee Martin of Johns Hopkins University in Baltimore, Maryland, in an e-mail to ARF. “It will be extraordinarily important to get this work translated to large animal models of neonatal [hypoxic injury] to fully determine its relevance,” wrote Martin, who was not involved in the study.

Since glutamate excitotoxicity is broadly tied to neurodegeneration, Nmnat-based treatments might be useful in diseases of aging, too. For example, Nmnat overexpression protects neurons in a mouse model of tauopathy (Ljungberg et al., 2011). Milbrandt’s group is performing high-throughput screens to look for small molecules that might mimic the effects of excess Nmnat, or at least identify the pathways involved.—Amber Dance

Comments

    • User Profile Image Lee J. Martin
      Johns Hopkins University School of Medicine
    • Posted:

    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.

    References:

    Northington FJ, Chavez-Valdez R, Martin LJ. Neuronal cell death in neonatal hypoxia-ischemia. Ann Neurol. 2011 May;69(5):743-58. PubMed.

    Martin LJ. Neuronal cell death in nervous system development, disease, and injury (Review). Int J Mol Med. 2001 May;7(5):455-78. PubMed.

    View all comments by Lee J. Martin

  2. 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. PubMed.

    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. PubMed.

    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. PubMed.

    View all comments by Michael Coleman

  3. 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

Make a Comment

To make a comment you must login or register.

References

News Citations

  1. Glutamate Gums Up Motor, Dopaminergic Neurons
  2. Neuronal Glutamate Fuels Aβ-induced LTD
  3. Aβ Oligomers and NMDA Receptors—One Target, Two Toxicities
  4. Protein Chimera Found to Protect Axons from Degeneration
  5. Blind Flies Reveal Novel Neuroprotective Role for NAD Synthase

Paper Citations

  1. Aizawa H, Sawada J, Hideyama T, Yamashita T, Katayama T, Hasebe N, Kimura T, Yahara O, Kwak S. TDP-43 pathology in sporadic ALS occurs in motor neurons lacking the RNA editing enzyme ADAR2. Acta Neuropathol. 2010 Jul;120(1):75-84. PubMed.
  2. Cucchiaroni ML, Viscomi MT, Bernardi G, Molinari M, Guatteo E, Mercuri NB. Metabotropic glutamate receptor 1 mediates the electrophysiological and toxic actions of the cycad derivative beta-N-Methylamino-L-alanine on substantia nigra pars compacta DAergic neurons. J Neurosci. 2010 Apr 14;30(15):5176-88. PubMed.
  3. Li S, Hong S, Shepardson NE, Walsh DM, Shankar GM, Selkoe D. Soluble oligomers of amyloid Beta protein facilitate hippocampal long-term depression by disrupting neuronal glutamate uptake. Neuron. 2009 Jun 25;62(6):788-801. PubMed.
  4. Shankar GM, Bloodgood BL, Townsend M, Walsh DM, Selkoe DJ, Sabatini BL. Natural oligomers of the Alzheimer amyloid-beta protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway. J Neurosci. 2007 Mar 14;27(11):2866-75. PubMed.
  5. De Felice FG, Velasco PT, Lambert MP, Viola K, Fernandez SJ, Ferreira ST, Klein WL. Abeta oligomers induce neuronal oxidative stress through an N-methyl-D-aspartate receptor-dependent mechanism that is blocked by the Alzheimer drug memantine. J Biol Chem. 2007 Apr 13;282(15):11590-601. PubMed.
  6. 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 Dec;4(12):1199-206. PubMed.
  7. Wang MS, Fang G, Culver DG, Davis AA, Rich MM, Glass JD. The WldS protein protects against axonal degeneration: a model of gene therapy for peripheral neuropathy. Ann Neurol. 2001 Dec;50(6):773-9. PubMed.
  8. Sasaki Y, Vohra BP, Baloh RH, Milbrandt J. Transgenic mice expressing the Nmnat1 protein manifest robust delay in axonal degeneration in vivo. J Neurosci. 2009 May 20;29(20):6526-34. PubMed.
  9. Sasaki Y, Milbrandt J. Axonal degeneration is blocked by nicotinamide mononucleotide adenylyltransferase (Nmnat) protein transduction into transected axons. J Biol Chem. 2010 Dec 31;285(53):41211-5. PubMed.
  11. Zhai RG, Cao Y, Hiesinger PR, Zhou Y, Mehta SQ, Schulze KL, Verstreken P, Bellen HJ. Drosophila NMNAT maintains neural integrity independent of its NAD synthesis activity. PLoS Biol. 2006 Nov;4(12):e416. PubMed.
  12. Ljungberg MC, Ali YO, Zhu J, Wu CS, Oka K, Zhai RG, Lu HC. CREB-activity and nmnat2 transcription are down-regulated prior to neurodegeneration, while NMNAT2 over-expression is neuroprotective, in a mouse model of human tauopathy. Hum Mol Genet. 2012 Jan 15;21(2):251-67. PubMed.

Further Reading

Papers

  1. Zhai RG, Cao Y, Hiesinger PR, Zhou Y, Mehta SQ, Schulze KL, Verstreken P, Bellen HJ. Drosophila NMNAT maintains neural integrity independent of its NAD synthesis activity. PLoS Biol. 2006 Nov;4(12):e416. PubMed.
  2. Ali YO, Ruan K, Zhai RG. NMNAT suppresses Tau-induced neurodegeneration by promoting clearance of hyperphosphorylated Tau oligomers in a Drosophila model of tauopathy. Hum Mol Genet. 2012 Jan 15;21(2):237-50. PubMed.
  3. Northington FJ, Chavez-Valdez R, Martin LJ. Neuronal cell death in neonatal hypoxia-ischemia. Ann Neurol. 2011 May;69(5):743-58. PubMed.
  4. Vohra BP, Sasaki Y, Miller BR, Chang J, DiAntonio A, Milbrandt J. Amyloid precursor protein cleavage-dependent and -independent axonal degeneration programs share a common nicotinamide mononucleotide adenylyltransferase 1-sensitive pathway. J Neurosci. 2010 Oct 13;30(41):13729-38. PubMed.
  5. 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. PubMed.
  6. Press C, Milbrandt J. Nmnat delays axonal degeneration caused by mitochondrial and oxidative stress. J Neurosci. 2008 May 7;28(19):4861-71. PubMed.

Primary Papers

  1. Verghese PB, Sasaki Y, Yang D, Stewart F, Sabar F, Finn MB, Wroge CM, Mennerick S, Neil JJ, Milbrandt J, Holtzman DM. Nicotinamide mononucleotide adenylyl transferase 1 protects against acute neurodegeneration in developing CNS by inhibiting excitotoxic-necrotic cell death. Proc Natl Acad Sci U S A. 2011 Nov 22;108(47):19054-9. PubMed.

Annotate

To make an annotation you must Login or Register.