Araki T, Sasaki Y, Milbrandt J.
Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration.
Science. 2004 Aug 13;305(5686):1010-3.
PubMed.
This is a powerful and noteworthy article. The authors show with strong and well-considered data that there is a link between NAD metabolism and axonal degeneration rates. Through the use of an identified mouse mutant, Wlds, which has long-lived axons, the authors used the knowledge that a protein produced in this mouse is a fusion of parts of a ubiquitin ligase and an enzyme involved in NAD synthesis. Careful experiments dissected the roles of these disparate arms of neuronal metabolism. A noted article for contrast is that of Zhai et al., implicating the proteasome in axonal degeneration.
The authors begin by examining the N-terminus of the Wld fusion, which comprises the first 70 amino acids of the ubiquitin ligase Ufd2a. In yeast, this protein ortholog is known to mediate cell survival under conditions of duress, so it was reasonable to hypothesize that Ufd2a activity might mediate axonal survival when toxins or neuronal transection were applied. It is arguable that different Ufd2a fusions provide different binding sites on their folded surfaces, and the authors furthermore make no attempt to discuss how a small domain of only 70 residues could possess such activity when even ubiquitin itself is larger. Nonetheless, this criticism is irrelevant, and the best available experiment as performed by the authors of inserting a GFP-Ufd2 construct into the mouse shows no effect. Furthermore, though not mentioned, the U-Box is present at the C-terminus of Ufd2, and not in the domain fused to the NAD relevant portion. It is, therefore, accepted from this evidence that the Ub ligase fusion is random and spurious, and not physiologically related to the function of the NAD-enzyme in alleviating disease phenotype. This is strongly correlated by confirmation of the lack of effect in the neurons with transfection of a dominant negative Ufd2a mutant (molecular lesion not described), and siRNA for the same. Ufd2a is not responsible for the physiological effect of the Wld fusion.
The carboxyl terminus of the Wld fusion is Nmnat1, a nicotinamide adenylyl transferase expressed highly in neuron. Introduction of the GFP-Nmnat1 fusion isolated away from the Ub ligase produces a similar cellular phenotype as does the neuron control. Additionally, carefully constructed point mutants in the adenylase which did not synthesize NAD were unable to rescue axons that were dying back, or suppress their shrinkage post-trauma by two mechanisms. This suggested that the increased NAD synthesis was responsible for the action of the fusion in the disease process. Support for this idea came in the form of addition of NAD to cultures, which again suppressed axonal retraction.
The report continues with a somewhat disjunct discussion of Sir2 and PARP action, two selected NAD utilizing enzymes. Inhibitors of PARP had no effect on the experimentation, and the negative results are accepted. However, inhibitors of Sir2, a neuronal histone deacetylase, blocked axonal regression. Equally, stimulators of Sir2 slowed the axonal degeneration. This is a confusing result, and resolution depends heavily on the actual biochemical activities of the sirtrinol and resveratrol used, as well as their specificity towards other NAD utilizing enzymes (not at all discussed). These results are not as strong as the first set, but imply that Sir2 may be involved in generating new transcripts or signaling which results in axonal decay.
Next, the authors take the implied Sir2 connection further and begin a screen of bioinformatically identified proteins with a Sir2 domain. Only one of those Sir2 domain-containing proteins inhibited by siRNA methods blocked axon degeneration as well as sirtrinol. This protein is SIRT1. The report ends with the suggestion that SIRT1 may regulate the neuronal breakdown by unknown processes.
In total, the report adequately shows that the Ubox ligase Ufd2 is not involved in the (presumably) proteasome-mediated axonal degeneration. This is not surprising, as there are many ligases in the cell, perhaps 500, and statistically the result is not unacceptable. Furthermore, the idea that NAD levels in the nucleus mediate the processes is backed by solid evidence, and in this light the paper presents its strongest foot forward. More murky are the suggestions that Sir1 is the actual mediator of these processes, and the pharmacology of the drugs used are not investigated at a deep enough level to adequately dissect from this report what is going on metabolically. Nonetheless, it is an interesting implication, and the SIRT1 data is not weak. We may assume from all the results that an unknown NAD-requiring enzyme, perhaps SIRT1 but not necessarily, is proceeding faster, as a result of higher substrate concentration beyond normal nuclear physiological levels.
It remains to be seen if other NAD utilizing enzymes are involved in the process, which transcriptions may be affected, by deacetylation of which proteins, and how the time course of NAD addition can shed light on the specific transcriptional or post-translational events that lead to axonal protection. The paper is strong, unique, and welcome.
Comments
Cognia Bioinformatics
This is a powerful and noteworthy article. The authors show with strong and well-considered data that there is a link between NAD metabolism and axonal degeneration rates. Through the use of an identified mouse mutant, Wlds, which has long-lived axons, the authors used the knowledge that a protein produced in this mouse is a fusion of parts of a ubiquitin ligase and an enzyme involved in NAD synthesis. Careful experiments dissected the roles of these disparate arms of neuronal metabolism. A noted article for contrast is that of Zhai et al., implicating the proteasome in axonal degeneration.
The authors begin by examining the N-terminus of the Wld fusion, which comprises the first 70 amino acids of the ubiquitin ligase Ufd2a. In yeast, this protein ortholog is known to mediate cell survival under conditions of duress, so it was reasonable to hypothesize that Ufd2a activity might mediate axonal survival when toxins or neuronal transection were applied. It is arguable that different Ufd2a fusions provide different binding sites on their folded surfaces, and the authors furthermore make no attempt to discuss how a small domain of only 70 residues could possess such activity when even ubiquitin itself is larger. Nonetheless, this criticism is irrelevant, and the best available experiment as performed by the authors of inserting a GFP-Ufd2 construct into the mouse shows no effect. Furthermore, though not mentioned, the U-Box is present at the C-terminus of Ufd2, and not in the domain fused to the NAD relevant portion. It is, therefore, accepted from this evidence that the Ub ligase fusion is random and spurious, and not physiologically related to the function of the NAD-enzyme in alleviating disease phenotype. This is strongly correlated by confirmation of the lack of effect in the neurons with transfection of a dominant negative Ufd2a mutant (molecular lesion not described), and siRNA for the same. Ufd2a is not responsible for the physiological effect of the Wld fusion.
The carboxyl terminus of the Wld fusion is Nmnat1, a nicotinamide adenylyl transferase expressed highly in neuron. Introduction of the GFP-Nmnat1 fusion isolated away from the Ub ligase produces a similar cellular phenotype as does the neuron control. Additionally, carefully constructed point mutants in the adenylase which did not synthesize NAD were unable to rescue axons that were dying back, or suppress their shrinkage post-trauma by two mechanisms. This suggested that the increased NAD synthesis was responsible for the action of the fusion in the disease process. Support for this idea came in the form of addition of NAD to cultures, which again suppressed axonal retraction.
The report continues with a somewhat disjunct discussion of Sir2 and PARP action, two selected NAD utilizing enzymes. Inhibitors of PARP had no effect on the experimentation, and the negative results are accepted. However, inhibitors of Sir2, a neuronal histone deacetylase, blocked axonal regression. Equally, stimulators of Sir2 slowed the axonal degeneration. This is a confusing result, and resolution depends heavily on the actual biochemical activities of the sirtrinol and resveratrol used, as well as their specificity towards other NAD utilizing enzymes (not at all discussed). These results are not as strong as the first set, but imply that Sir2 may be involved in generating new transcripts or signaling which results in axonal decay.
Next, the authors take the implied Sir2 connection further and begin a screen of bioinformatically identified proteins with a Sir2 domain. Only one of those Sir2 domain-containing proteins inhibited by siRNA methods blocked axon degeneration as well as sirtrinol. This protein is SIRT1. The report ends with the suggestion that SIRT1 may regulate the neuronal breakdown by unknown processes.
In total, the report adequately shows that the Ubox ligase Ufd2 is not involved in the (presumably) proteasome-mediated axonal degeneration. This is not surprising, as there are many ligases in the cell, perhaps 500, and statistically the result is not unacceptable. Furthermore, the idea that NAD levels in the nucleus mediate the processes is backed by solid evidence, and in this light the paper presents its strongest foot forward. More murky are the suggestions that Sir1 is the actual mediator of these processes, and the pharmacology of the drugs used are not investigated at a deep enough level to adequately dissect from this report what is going on metabolically. Nonetheless, it is an interesting implication, and the SIRT1 data is not weak. We may assume from all the results that an unknown NAD-requiring enzyme, perhaps SIRT1 but not necessarily, is proceeding faster, as a result of higher substrate concentration beyond normal nuclear physiological levels.
It remains to be seen if other NAD utilizing enzymes are involved in the process, which transcriptions may be affected, by deacetylation of which proteins, and how the time course of NAD addition can shed light on the specific transcriptional or post-translational events that lead to axonal protection. The paper is strong, unique, and welcome.
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