Abstract
The slow Wallerian degeneration protein (WldS), a fusion protein incorporating full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by injury, toxins and genetic mutation. Nmnat1 overexpression is reported to protect axons in vitro, but its effect in vivo and its potency remain unclear. We generated Nmnat1-overexpressing transgenic mice whose Nmnat activities closely match that of WldS mice. Nmnat1 overexpression in five lines of transgenic mice failed to delay Wallerian degeneration in transected sciatic nerves in contrast to WldS mice where nearly all axons were protected. Transected neurites in Nmnat1 transgenic dorsal root ganglion explant cultures also degenerated rapidly. The delay in vincristine-induced neurite degeneration following lentiviral overexpression of Nmnat1 was significantly less potent than for WldS, and lentiviral overexpressed enzyme-dead WldS still displayed residual neurite protection. Thus, Nmnat1 is significantly weaker than WldS at protecting axons against traumatic or toxic injury in vitro, and has no detectable effect in vivo. The full protective effect of WldS requires more N-terminal sequences of the protein.
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Main
Axon degeneration is an early, often contributory, event in many neurodegenerative disorders including amyotrophic lateral sclerosis, multiple sclerosis and Alzheimer's disease.1, 2, 3, 4, 5 Wallerian degeneration distal to an axonal injury is an important and relevant experimental model of axon degeneration in disease. A dominant mutation that delays Wallerian degeneration 10-fold in slow Wallerian degeneration (WldS) mice and rats,6, 7 also delays axon degeneration in genetic and toxic ‘dying-back’ disorders,2, 8, 9, 10, 11 indicating similarities in the underlying mechanisms. Thus, understanding the Wallerian degeneration mechanism should help to understand how axons degenerate in disease. Moreover, the influence of genetics on axon degeneration suggests a regulated, proactive pathway that could be manipulated both as an experimental tool and as a novel protective treatment for axonal degeneration. A clear understanding of the mechanism of axonal protection by WldS is thus essential.
The mechanism for axonal protection by WldS has been in question since the discovery of the WldS mouse.6 Our groups showed that the gene identified by positional cloning12, 13, 14 delays Wallerian degeneration in transgenic mice and virally transduced dorsal root ganglion (DRG) explant cultures.15, 16 WldS encodes a fusion protein containing 70 N-terminal amino acids of ubiquitination factor Ube4b, full-length nicotinamide adenine dinucleotide (NAD+)-synthesising enzyme nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), and a unique 18-amino-acid joining sequence.15 Surprisingly, WldS is abundant in neuronal nuclei8, 11, 15, 18 and so far undetectable in axons in vivo. However, it does enter neurites in virally transduced primary cultures.16, 19 Thus, while a direct protective effect in axons remains possible,19 WldS appears likely to act within nuclei. Thus, it is important to identify downstream factors mediating WldS action, to understand and perhaps mimic the protective mechanism.
An important first step is to determine the parts of the WldS fusion protein required to delay axon degeneration. Two recent reports19, 20 suggest a primary role for Nmnat1 and its synthetic product NAD+, but provide conflicting data on how NAD+ mediates axonal protection. One report concludes that raised NAD+ levels increase activity of the Sir2 deacetylase Sirt1.20 Sirtinol, which reduces Sir2 activity, was reported to block NAD+-dependent axonal protection, whereas resveratrol, a Sir2 enhancer, had the opposite effect. Sirt1 siRNA negated the protective effects of NAD+ in transected DRG neurites. The other study could not reproduce these results in Sirt1-null mice, and instead concluded that NAD+ works directly within axons independently of Sir2.19 Both papers also reported that exogenous NAD+ mimics the protection of neurites by WldS but disagreed on the amount of NAD+ required (0.01–1 and 5–20 mM, respectively).
The implications of these studies are profound. Not only is it important to understand how Wallerian degeneration is controlled, but protection by exogenous agents such as NAD+ and resveratrol suggests possible leads into pharmacological intervention in axonal disease. Moreover, a role for Sirt1 would link Wallerian degeneration with other forms of ageing-related cell death.21 However, both studies were carried out exclusively in vitro and the potencies of Nmnat1 and WldS were not compared. Thus, we studied Wallerian degeneration using lentiviral vectors and transgenic mice to overexpress Nmnat1. Despite matching the Nmnat enzyme activity of WldS heterozygotes, which have a robust slow Wallerian degeneration phenotype, Nmnat1 transgenic mice consistently showed a wild-type rate of Wallerian degeneration. Furthermore, we find that Nmnat1 has significantly lower potency to protect neurites from traumatic or toxic injury in vitro. Although we cannot rule out the possibility that Nmnat1 may delay Wallerian degeneration when expressed at very high levels, our data show conclusively that the ability of WldS to protect axons is far greater than that of Nmnat1 alone.
Results
Generation of transgenic mice overexpressing Nmnat1
Twenty-three lines of transgenic mice were generated and six high-copy number lines (Supplementary Figure 1) were selected to test for overexpression of enzymatically active Nmnat1. All lines were healthy, fertile and overtly normal.
We tested for transgenic lines expressing Nmnat1 at levels similar to or greater than WldS heterozygotes to use for phenotypic analysis. C57BL/WldS heterozygotes express increased Nmnat1 as part of the WldS protein and show a strong axon protection phenotype.13, 15, 22 Western blots of sagittally divided half-brain homogenates, probed with anti-Nmnat1 antibody 183, showed that lines 881, 891, 2460, 7103 and 7104 overexpressed Nmnat1 (Figure 1a). Nonexpressing line 884 (data not shown) was not studied further. Nmnat1 levels in lines 881 and 7104 matched or exceeded that of WldS protein in WldS heterozygotes, suggesting that these lines were particularly useful to test whether Nmnat1 and WldS are functionally interchangeable in their ability to preserve transected axons in vivo.
We then tested whether transgenically expressed Nmnat1 possessed NAD+ synthesis activity. Total Nmnat activity in sagittally divided half-brains of Nmnat1 transgenic line 881 and 7104 hemizygotes was very highly significantly greater than in wild-type mice (P=0.0073 and P<0.0001, respectively; n=8–9). Mean values were increased 3.2–3.5 times, slightly more (although not significantly) than WldS heterozygotes (Figure 1b) and only a little less than the four-fold increase reported in WldS homozygotes.15
Wallerian degeneration was tested in sciatic nerve, so we first confirmed transgene expression in neurons projecting axons to this nerve. RT-PCR confirmed the presence of a transgene-specific Nmnat1 mRNA in lumbar DRG (Figure 1c). The same mRNA was detected in DRG explant cultures almost devoid of non-neuronal cells, strongly suggesting the transgene is expressed in sensory neurons (Figure 6n). In motor neurons, we found more definitive evidence that Nmnat1 was overexpressed and targeted to neuronal nuclei like WldS. Immunocytochemistry of Nmnat1 transgenic, WldS and wild-type lumbar spinal cords showed clearly stronger signal intensity in nuclei of all transgenic motor neurons compared to wild-type motor neurons (Figure 2), data which were reproducible also with a different anti-Nmnat1 antibody (Supplementary Figure 2). WldS mice showed an intermediate level of nuclear staining (Figure 2b). We cannot rule out targeting of some additional overexpressed Nmnat1 to the cytoplasm or axons, but these data confirm that overexpressed Nmnat1 is targeted to neuronal nuclei at levels exceeding that of WldS in WldS mice.
Thus, line 881 and 7104 hemizygotes express enzymatically active Nmnat1 at levels similar to, if not greater than WldS heterozygotes, mice where most axons survive 5 days after nerve lesion.9, 15, 22 We therefore chose these lines for most of our subsequent investigations, but used also the other expressing lines, 891, 2460 and 7103, in some assays.
Wallerian degeneration is not delayed in Nmnat1 transgenic mice
We transected sciatic nerves in transgenic, wild-type and WldS heterozygote mice and tested axon survival in the distal stump, studying ultrastructure, axon continuity and protein integrity. To detect even a small delay in Wallerian degeneration, axon ultrastructure and continuity were analysed up to 72 h after nerve lesion, when over 80% of axons remain intact in WldS heterozygotes, but only a negligible proportion survive in wild-type mice.15 Intact neurofilament protein was assessed at 5 days. To put this low stringency in perspective, most axons in WldS homozygotes survive structurally even 14 days after lesioning.6, 15, 23, 24
First, we studied 3-day lesioned distal nerve stumps using electron microscopy (Figure 3 and Supplementary Figure 3). In WldS heterozygotes, most axons displayed normal cytoskeleton, unswollen mitochondria and a regular myelin sheath of normal thickness. In contrast, not one preserved axon was found in an extensive search of nerves from homozygotes or hemizygotes of Nmnat1 transgenic lines 881, 7104 and 891 (n=5), despite the low stringency of the assay (Figure 3c–f and Supplementary Figure 3). A very low rate of preservation (<1%) was observed in line 7103, a rate similar to that previously reported in wild-type nerves.15 Extensive analysis of semithin sections by light microscopy also revealed no evidence of axon preservation in Nmnat1 transgenics (data not shown). Thus, Nmnat1 expressed at levels similar to, or higher than WldS protein, does not preserve axon ultrastructure in vivo.
We then studied the longitudinal continuity of lesioned, fluorescently labelled distal axons in Nmnat1 transgenic mice crossed with the YFP-H strain.25 We previously showed that in the absence of WldS all labelled axons fragment within 52 h of nerve transection,26 whereas WldS/YFP-H axons remain continuous after 20 days, showing only localised atrophy.23 Homozygotes of highly expressing Nmnat1 transgenic line 7104 (Figure 4f) were indistinguishable from wild type (Figure 4b), with every labelled axon fragmented 72 h after nerve transection. Similar results were seen in each line where we obtained Nmnat1/YFP-H double transgenics: lines 7103 (Figure 4e) and 2460 (data not shown). WldS heterozygous nerves showed no loss of continuity at 72 h (Figure 4c), consistent with our previous report that heterozygous WldS is sufficient to maintain axon continuity for 5 days.9 Thus, Nmnat1 expressed at levels similar to, or higher than WldS protein, does not preserve axon continuity in vivo.
We then looked for biochemical preservation of heavy neurofilament protein (NF-H) in distal stumps of lesioned Nmnat1 transgenic nerves. As reported previously,9 heterozygous WldS partially preserved NF-H 5 days after nerve lesion. However, transected nerves from transgenic lines 881 and 7104 were indistinguishable from wild-type nerves with no intact NF-H remaining (Figure 5). Thus, Nmnat1 expression at these levels does not preserve the integrity of cytoskeletal proteins in lesioned axons in vivo. Together with its inability to preserve axon ultrastructure and continuity, this leads us to conclude that Nmnat1 is not functionally equivalent to WldS. The ability of WldS to preserve lesioned axons in vivo requires more N-terminal sequences.
Lesioned DRG neurites in Nmnat1 transgenic primary cultures degenerate rapidly
The above data contrast sharply with reports that viral overexpression of Nmnat1 constructs in primary DRG cultures delays degeneration of transected neurites for at least 3 days.19, 20 Thus, we tested the effect of Nmnat1 in vitro by transecting neurites of DRG explant cultures from highly expressing lines 881 and 7104. Homozygous, hemizygous and wild-type embryos were produced by intercrossing hemizygous transgenic parents, and genotypes were determined only after degeneration had been scored independently by two experienced investigators.
In all 30 heterozygous WldS explants studied, transected neurites survived for more than 3 days (Figure 6a–d). In contrast, cut neurites of all 15 wild-type explants beaded and fragmented within 12 h, and detached or lost all semblance of neurite morphology by 24 h (Figure 6e–g). In every explant from Nmnat1 transgenic embryos (24 homozygous, 60 hemizygous; 39 line 881; 45 line 7104), neurite degeneration was indistinguishable from wild-type cultures. Interestingly, neither WldS nor Nmnat1 could be detected at the protein level in WldS and transgenic cultures, respectively (although for WldS there is clearly enough protein to confer the phenotype). However, transgene expression was confirmed at the mRNA level using RT-PCR (Figure 6n). We could find only occasional non-neuronal cells migrating out from aphidicolin-treated explants (Supplementary Figure 4). Thus, it is unlikely that non-neuronal cells made more than a negligible contribution to these strong RT-PCR products, although we cannot completely rule out this possibility. This suggests that Nmnat1 does not prolong the survival of injured axons in vitro or in vivo when expressed at a similar level to that in WldS heterozygotes.
Lentiviral expression of WldS shows significantly more axonal protection than Nmnat1
The lack of axonal protection in Nmnat1 transgenic mice led us to re-examine the reported protection provided by in vitro expression of Nmnat1 in cultured DRG.19, 20 Lentiviral expression of WldS and Nmnat1 was confirmed by Western blotting and endogenous fluorescence, respectively, and the signal in neurites in vitro confirmed neuronal expression (Figure 7). Expression of GFP alone showed no protection against vincristine toxicity when compared with uninfected DRG cultures (Figure 8). Expression of WldS showed a remarkable level of protection at both 7 and 13 days, similar to that reported previously using adenoviral delivery.16 Expression of Nmnat1 provided an intermediate level of protection that was significantly less than WldS at 7 days (P<0.01). Nmnat1 overexpression showed no protection compared to controls after 13 days of vincristine exposure (P>0.05).
We also constructed a lentivirus that expressed only the Ube4b portion of WldS and tested it in the same experimental paradigm. As with the expression of Nmnat1, there was intermediate protection at day 7 but no protection at day 13 of vincristine exposure. Infection with both the Ube4b and the Nmnat1 showed no additive protective effect (Figure 8b).
The W258A mutation reduces but does not abolish protection by WldS
In order to test whether WldS confers neuroprotection through its Nmnat1 enzymatic activity, we introduced a specific point mutation in WldS that abolishes Nmnat1 activity and thus the additional NAD+ production.20 Lentiviral expression of W258A mutant in DRG was comparable to the expression of wild-type WldS (Figure 7). However, W258A provided a reduced level of protection at both 7 and 13 days of vincristine exposure (Figure 8). Direct comparison of expression of WldS Nmnat1 and W258A showed that the protection provided by W258A was indistinguishable from that provided by Nmnat1. These results suggest that at least a portion of the protective activity of WldS relates to the enzymatic activity of Nmnat1. However, the protection provided by W258A was not reduced compared to wild-type Nmnat1. These seemingly conflicting data could reflect a balance between the weakening effect of W258A and the strengthening effect of the N-terminal region of WldS, which is not included in the Nmnat1 construct.
Exogenous NAD+ and resveratrol do not protect against axonal degeneration
To further address the question of whether increased levels NAD+ is the mediator of axonal protection by WldS,20 we added NAD+ (up to 1 mM) in DRG cultures 24 h prior to the addition of vincristine. NAD+ provided no significant protection against vincristine-induced axonal degeneration (P>0.05, Figure 9). Treatment of DRG cultures with resveratrol, a compound that increases Sir2 activity, also showed no protective effect (P>0.05). Furthermore, neither NAD+ nor resveratrol were protective against transection injury (Figure 9). These results do not support the hypothesis that the direct pathway for axonal protection by WldS is through the production of NAD+ and interaction with Sir2.
Discussion
The data we report led our groups to conclude independently that full-length WldS protein delays axon degeneration more effectively than Nmnat1. Transgenically overexpressed Nmnat1 was unable to delay Wallerian degeneration in vivo or in vitro. Nmnat1 overexpressed using a lentivirus vector in vitro, protected far more weakly than WldS and, at later time points, made no significant difference from wild-type neurons. Moreover, expression of the Ube4b component of WldS provided similar levels of protection to Nmnat1, demonstrating that this limited protection is not restricted to the Nmnat1 component. Neither of these constructs showed any significant protection at the 13 days. The fact that lentiviral expression of both the Ube4b sequence and Nmnat1, either separately or together, did not match protection by full-length WldS argues that the functional mechanism of WldS is more than the sum of its parts. Thus, the proposal that the direct action of WldS is mediated solely by Nmnat119, 20 must be revised to incorporate a role for more N-terminal WldS sequence.
Hemizygotes of Nmnat1 transgenic lines 881 and 7104 matched or exceeded the increase in Nmnat1 protein and enzyme activity in WldS heterozygotes. The 3.2- to 3.5-fold increase in total Nmnat enzyme activity probably underestimates the increase in Nmnat1, as the mitochondrial and cytosolic isoforms of Nmnat also contribute to the baseline figure.27, 28, 29 However, by ultrastructural, topological and biochemical criteria, Wallerian degeneration was not delayed in either hemi- or homozygous Nmnat1 transgenic mice in vivo. We previously generated transgenic mice expressing full-length WldS protein using the same promoter as here, enabling us to identify the WldS gene.15 Full-length WldS was targeted to motor neuron nuclei, the same location that we now observe for overexpressed Nmnat1 (Figure 2 and Supplementary Figure 2). Full-length WldS significantly delayed Wallerian degeneration for 5 days, even at lower expression levels than in WldS heterozygotes (e.g., hemizygotes of WldS transgenic line 4830). Many axons in transgenic lines expressing higher levels of WldS survived for 2 weeks. In contrast, Nmnat1 targeted to the same subcellular compartment fails to protect any axons for 3 days, even if the level exceeds that of WldS in WldS heterozygotes.
Also in vitro, overexpressed Nmnat1 could not robustly delay axon degeneration like full-length WldS protein. DRG cultures established from homozygous embryos of our strongest lines were indistinguishable from wild-type following neurite transection. In contrast, heterozygous WldS routinely delayed degeneration of transected neurites for 3 days. Although both WldS and Nmnat1 proteins were not detected in these cultures, the lentiviral experiments did provide clear evidence of Nmnat1 expression in nuclei (Figure 7b). Lentiviral expression of WldS protected DRG neurites from vincristine-induced axonal degeneration, consistent with our previous report of protection by WldS expression using adenoviral vectors.16 However, expression of Nmnat1 at similar levels to WldS did not protect effectively. If Nmnat1 were the sole functional component of WldS, it should substitute fully in this system. While the possibility remains that very strong expression of Nmnat1 might protect axons, our data show conclusively that the potency of Nmnat1 does not match that of WldS. Thus, another part of the WldS protein is necessary to delay Wallerian degeneration.
Araki et al. (2004) also reported a protective effect of 0.01–1.0 mM exogenous NAD+ on transected neurites.20 However, like Wang et al.,19 neither of our groups found significant neurite protection at the upper end of this concentration range in either transection or toxic models (Figure 9). Exogenous NAD+ has also been ineffective at neuroprotection in other laboratories (Pierluigi Nicotera, personal communication). The lack of a robust and fully reproducible effect with exogenous NAD+ raises questions about the role of Sirt1 in delaying Wallerian degeneration, as Sirt1 was implicated in RNAi experiments using the exogenous NAD+ method.20 Resveratrol was also reported to protect axons from degeneration by increasing the activity of Sir2 proteins, analogous to the proposed mechanism of NAD+,20 but resveratrol also could not reproduce the WldS phenotype in our laboratories. Our data, together with the recent report that WldS delays degeneration of transected neurites even in Sirt1−/− mice,19 support a different functional mechanism for WldS.
Exactly how the N-terminal Ube4b-derived sequence contributes to the WldS phenotype remains unclear, but recent data suggest two possibilities. First, valosin-containing protein (VCP/p97) binds to the N-terminal 16 amino acids of WldS and Ube4b.30 Consequently, WldS partially redistributes VCP into punctate intranuclear foci in some neuronal subtypes. Removing the N-terminal 16 amino acids of WldS abolishes the targeting of VCP to intranuclear foci, and we confirm here that overexpressed Nmnat1 also fails to target VCP to intranuclear foci (Supplementary Figure 5). Second, expression of full-length WldS protein alters the expression of a small subset of genes, but these changes are not fully reproduced when N-terminal sequences are deleted.31 It is not yet known whether either of these effects is required for axon protection, or whether they are linked with one another. Importantly, however, both involve nuclear events, and the nucleus remains the only intracellular location in vivo where WldS has been detected. Thus, one can hypothesise that binding of WldS to VCP alters ubiquitin-proteasome-mediated turnover of a transcription factor, which in turn alters the expression of an axonal regulator of Wallerian degeneration. Our demonstration here that the N-terminal region (including the VCP binding site) is required for strong axon protection supports this model.
The existence of WldS is an extraordinary experiment of nature that will no doubt provide valuable clues to the causes of axonal degeneration in human diseases. WldS is truly neuroprotective and has been shown to modify disease course in animal models of toxic neuropathy,10 Charcot–Marie–Tooth neuropathy,8 motor neuronopathy,2 amyotrophic lateral sclerosis32 and Parkinson's disease.11 Defining the precise mechanism of axonal protection in WldS mice is thus an important step for developing novel therapeutics for peripheral neuropathies and other disorders of axonal degeneration. Our data are inconsistent with the premise that overexpression of Nmnat1 is the sole mechanism underlying the WldS phenotype, and we believe that the question needs further examination. We have no conclusive explanation for why our findings differ from those reported previously,19, 20 but suggest differences in expression level as one possibility. However, the data we report, repeated many times in both our laboratories, conclusively show that Nmnat1 cannot substitute for a similar level of WldS.
In summary, we conclude that Nmnat1 and WldS protein are not equivalent in their ability to delay Wallerian degeneration in injured axons. Specifically, robust protection of axons by WldS is not reproduced when Nmnat1 is expressed at a similar level, as demonstrated by ultrastructural, morphological and biochemical assays in vivo, and using both traumatic and toxic lesions in vitro. We do not rule out possible involvement of Nmnat1 in the WldS phenotype, but the significantly reduced potency compared to WldS and the lack of effectiveness in providing axonal protection in vivo suggest that more N-terminal sequences of the WldS protein should now be investigated.
Materials and Methods
Generation of transgenic mice
A transgene construct designed to express Nmnat1 from a β-actin promoter was generated as previously described in our successful generation of WldS transgenic mice,15 except for the use of a different forward PCR primer as follows (underlined bases are 5' HindIII cloning tag and start codon of Nmnat1): 5′-TAGATCCCAAGCTTAACTTCTCCCCATGGACTCAT-3′.
Pronuclear injection of the ca. 5.7 kbp EcoRI/NdeI into an F1 C57BL/CBA strain was performed by the in-house Gene Targeting Facility of the Babraham Institute. All animal work in the UK was carried out in accordance with the Animals (Scientific Procedures) Act, 1986, under Project Licence PPL 80/1778.
Genotyping
Founder mice, and their transgene-positive offspring, were identified by Southern blotting of BamHI plus HindIII double-digested genomic tail DNA hybridised with a 32P-labelled WldS cDNA probe (Supplementary Figure 1). Twenty-three positive founders with varying transgene copy number were identified from a total of 152 mice. Founders with high-copy number integrations were selected for further study. YFP-H mice25 were obtained from the Jackson Laboratories and genotyped as described previously.26
Western blotting
Sagittally-divided half brains were homogenised in five volumes of RIPA buffer (phosphate-buffered saline (PBS) containing 1% NP40, 0.5% deoxycholate, 0.1% sodium dodecylsulphate). High-speed supernatant was diluted to approximately 0.5 mg/ml total protein according to Bradford Assay (BioRad) and fractionated by standard SDS-PAGE. After semidry blotting (BioRad), nitrocellulose membranes (BioRad) were blocked in PBS plus 0.02% Tween-20 and 5% low-fat milk powder, before incubation with primary antibody and then horseradish peroxidase (HRP)-conjugated secondary antibody (1 : 3000; Amersham Biosciences). Proteins were visualised using the ECL detection kit (Amersham Biosciences) according the manufacturer's instructions. Nmnat1 and WldS protein were detected on Western blots using rabbit polyclonal antiserum 183 as described previously,14 and mouse monoclonal anti-β-actin (Abcam) was used as a loading control.
The distal stumps of transected sciatic nerves (ca. 0.5 cm) were homogenised in 25 μL RIPA buffer. High-speed supernatant was fractionated by SDS-PAGE and gels blotted onto nitrocellulose membranes and blocked as above. Blots were incubated in mouse anti-neurofilament 200 antibody (N52; 1 : 2000; Sigma) and then HRP-conjugated secondary antibody (1:3000; Amersham Biosciences). Proteins were visualised using the ECL detection kit (Amersham Biosciences).
RT-PCR
Lumbar DRG were removed from freshly killed mice, or approximately 20 DRG explants from a primary culture dish and extracted in Trizol (Invitrogen) according to the manufacturer's instructions. First-strand cDNA synthesis and 30 cycles of PCR was then performed as described previously14 using the following primers:
to detect transgene-specific Nmnat1 mRNA:
5′-GAGCCTCGCCTTTGCCGAT-3′ and
5′-TTCCCACGTATCCACTTCCA-3′
to detect WldS mRNA:
5′-CTTGCTGGTGGACAGACCT-3′ and
5′-TTCCCACGTATCCACTTCCA-3′
Assay of Nmnat enzyme activity
Sagittally divided half mouse brains were flash-frozen immediately post mortem and stored at −80°C until use. Tissue was suspended in six volumes of 50 mM Hepes, pH 7.4, 0.5 mM EDTA, 1 mM MgCl2, 1 mM phenylmethylsulphonyl fluoride and 0.02 mg/ml each of leupeptin, antipain, chymostatin and pepstatin, and homogenised on ice (3 × 4 s with 10 s intervals at medium speed). Nmnat activity assay was performed at 37°C in a 0.1 ml reaction mixture containing 30 mM Tris-HCl, pH 7.5, 2 mM nicotinamide mononucleotide (NMN), 2 mM ATP, 20 mM MgCl2, 10 mM NaF and an appropriate aliquot of brain homogenate. The reaction was started by adding 4 μl of 50 mM NMN and stopped by the addition of a half-volume of ice-cold 1.2 M HClO4. After 10 min at 0°C, the mixture was centrifuged and 135 μl of supernatant was neutralised by the addition of 36 μl of 0.8 M K2CO3. Nmnat activity was calculated after HPLC identification and quantification of the product (NAD+).33 One unit of enzyme was defined as the amount capable of producing 1 μmol of NAD+ per minute at 37°C. Pairwise statistical analyses (unpaired Student's t-test) were performed using Prism.
Immunocytochemistry
Cryostat sections (20 μm) were prepared from lumbar spinal cord of TgNmnat1 line 7104 homozygous, WldS homozygous or C57BL/6 control mice that had been perfusion-fixed with 4% paraformaldehyde. Sections were pretreated by incubating overnight at 45°C in 1 mM sodium citrate, pH 6.0, followed by 10 min in 0.1% Trypsin in PBS. They were then immunostained for Nmnat1 using two different affinity-purified rabbit polyclonal antibodies (each diluted 1 : 50) that detect only Nmnat1 on Western blots: one kindly supplied by Professor Mathias Ziegler (Figure 2),34 and the other (D-20) obtained from Santa Cruz Biotechnology Inc. (Supplementary Figure 2). For each, we used Alexa 568-conjugated anti-rabbit secondary antibody (1 : 200; Molecular Probes). Dispersed primary cultures of DRG neurons were fixed with 4% paraformaldehyde and immunostained for NF-H using monoclonal antibody N52 (1 : 200; Sigma) and Alexa 568 anti-mouse secondary (1 : 200 Molecular Probes).
Nerve lesion
Mice were anaesthetised with a mixture of ketamine (100 mg/kg; Fort Dodge Animal Health, Southampton) and xylazine (5 mg/kg; Parke Davis/Pfizer, Karlsruhe, Germany). Right sciatic nerves were transected at the upper thigh and wounds closed with sutures. After 60 to 72 h, mice were killed by cervical dislocation and the swollen first 2 mm of distal nerve discarded. The remaining distal sciatic nerve stump was used for light and electron microscopy (see below) or Western blotting with antibody to NF-H (see above). For mice carrying a YFP-H transgene, tibial nerve was also removed for confocal microscopy. Except where otherwise indicated, the mutant mice used were hemizygous for Nmnat1 or WldS heterozygotes, produced by crossing C57BL/WldS homozygous males (Harlan UK) to C57BL/6 females.
Light and electron microscopy
Nerves were fixed for at least 24 h in 0.1 M phosphate buffer containing 4% paraformaldehyde and 2.5% glutardialdehyde, embedded in Durcupan resin (Fluka) and processed for light and electron microscopy as described previously.26
Analysis of YFP-labelled nerves
Tibial nerves were quickly removed from humanely killed mice and processed as described previously26 for analysis on a Zeiss LSM 510 Meta Confocal system (LSM software release 3.2) coupled to a Zeiss Axiovert 200 microscope. Only lines 2460, 7103 and 7104 were studied in this assay, as initial crosses of other lines produced no double mutants.
Primary culture for transgenic experiments (Coleman group)
DRGs were dissected from E15.5 mouse embryos using sterile technique, initially into L15 medium (Invitrogen) for removal of other tissue, and then plated ca. 20 per dish in 3.5 cm dishes precoated with poly-L-lysine (20 μg/ml for 1 h; Sigma) and laminin (20 μg/ml for 1 h; Invitrogen). Explants were then cultured in Neurobasal medium (Invitrogen) containing 4 μM aphidicolin (Sigma) (which we find reduces non-neuronal cells ca. 250-fold), 2% B27-supplement, 2 mM glutamine, 1% penicillin, 1% streptomycin and 100 ng/ml NGF (all from Invitrogen). Scratches were made on dishes prior to plating the explants to aid subsequent orientation of photographs (e.g., Figure 6e–g). After 5–7 days of neurite outgrowth in standard culture conditions, neurites were transected using a scalpel, immediately photographed with phase contrast using an Olympus IX81 inverted fluorescent microscope, coupled to a PC running SIS imaging software. Further photographs were taken at 3, 6, 9, 12 and 24 h, and after approximately each subsequent 24 h period if neurites had survived (on WldS neurites survived this long). Homozygous and hemizygous transgenic and wild-type embryos were produced by intercrossing hemizygous parents of lines 881 and 7104. Heterozygous WldS embryos were produced by crossing WldS homozygotes with C57BL/6 mice. The liver and tail of each embryo were retained for DNA extraction using the Nucleon II kit (Amersham) and genotyping.
Primary culture for lentiviral vector experiments (Glass group)
DRG explant cultures were generated from E15 rats as described previously.16, 35 After allowing for neurite extension for 6 days in culture, lentiviruses titered at 1 × 109 were added to the culture media at a dose of 10 μl in 1 ml (final viral titer 1 × 107). The cultures were maintained under standard conditions for an additional 7 days to allow for the expression of exogenous proteins. For vectors expressing GFP, transduction was monitored in real time by fluorescence microscopy. After 7 days (total 13 days in culture), vincristine sulphate was added to a final concentration 0.02 μM and the cultures monitored by phase-contrast microscopy for an additional 13 days. Quantitative analysis of vincristine neurotoxicity was performed as described previously.35 Data were compared using a one-way analysis of variance with post-test correction for multiple comparisons (Tukey–Kramer). The expression of exogenous proteins was assessed by immunocytochemistry on cultures prepared at the same sitting as those exposed to vincristine, but fixed and stained after 7 days in culture. Two antibodies raised against the WldS protein, 183 and Wld-18, were used to demonstrate expression of Ube4b/Nmnat1 and W258A, as described previously.8, 14 Expression of Nmnat1 was demonstrated with a polyclonal antibody to Nmnat1 (D20 from Santa Cruz Biotech, 1 : 100 concentration).
Addition of resveratrol and NAD+ to primary cultures
Resveratrol (100 μM) and NAD+ (1 mM, both from Sigma) were tested for their potential neuroprotective effects against vincristine-induced axonal degeneration in the Glass culture system (above) and against transection-induced degeneration in the Coleman culture system (above). These agents were added 24 h before vincristine (0.02 μM) to 6-day-old cultures and the degree of axonal degeneration was assessed after 6 and 9 days of exposure. For transection experiments, these agents were added for 24 h before neurite transection to 5-day-old cultures, and replaced with new medium immediately before transection containing NAD+ or resveratrol as appropriate.
Generation of lentiviral vectors
Lentivirus vectors were created on the FUGW backbone using a CMV promotor36 (Figure 7). The following cDNAs were cloned into the vector: Ube4b/Nmnat1 (WldS), mutant Ube4b/Nmnat1 (W258A), Nmnat1 fused to GFP (Nmnat1) and the 70-amino-acid N-terminal portion of the WldS protein fused to GFP (Ube4b). The WldS cDNA was isolated by RT-PCR from the brain of a WldS mouse.16 The W258A point mutation was generated by PCR-based site-directed mutagenesis.
Vector particles were produced in HEK293 T cells by transient cotransfection with the transfer vector, the HIV-1 packaging vector R8.9 and the VSVG envelope glycoprotein. Approximately 72 h after transfection, virus-containing supernatant was removed, filtered through a 0.45 μM filter unit and concentrated by centrifugation, aliquoted and frozen at −80°C. Viral titres were calculated using a serial dilution method. Prior to use in DRG cultures, lentiviral expression of the inserted protein was confirmed by Western blot analysis of infected HEK293 cells (Figure 7).
Abbreviations
- DRG:
-
dorsal root ganglion
- NAD:
-
nicotinamide adenine dinucleotide
- Nmnat:
-
nicotinamide mononucleotide adenylyltransferase
- Sir2, Sirt1:
-
silent information regulator 2, and its mammalian homologue
- WldS, WldS:
-
slow Wallerian degeneration gene and protein
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Acknowledgements
We thank the Gene Targeting Facility of the Babraham Institute for assistance with production of transgenic mice, and Dr. Simon Walker for expert advice on confocal microscopy. We thank Dr. Giacomo Morreale, Ms. Anna Wilbrey and Ms. Elisabetta Babetto for practical assistance, Professor Richard Ribchester (University of Edinburgh) and Lindsey Fischer for constructive comments on the manuscript, and Professor Mathias Ziegler (Bergen) for Nmnat1 antibody. This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), NS40405 from the NINDS (JDG) and the Robert Packard Center for ALS Research (JDG).
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Conforti, L., Fang, G., Beirowski, B. et al. NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration. Cell Death Differ 14, 116–127 (2007). https://doi.org/10.1038/sj.cdd.4401944
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DOI: https://doi.org/10.1038/sj.cdd.4401944
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