The neuroprotective Wld gene regulates ... - Semantic Scholar

Human Molecular Genetics, 2006, Vol. 15, No. 4 doi:10.1093/hmg/ddi478 Advance Access published on January 10, 2006

625–635

The neuroprotective Wld S gene regulates expression of PTTG1 and erythroid differentiation regulator 1-like gene in mice and human cells Thomas H. Gillingwater1,2,*,{, Thomas M. Wishart1,{, Philip E. Chen1, Jane E. Haley1, Kevin Robertson3, Stephen H.-F. MacDonald2, Susan Middleton1, Kolja Wawrowski4, Michael J. Shipston2, Shlomo Melmed4, David J.A. Wyllie1, Paul A. Skehel1, Michael P. Coleman5 and Richard R. Ribchester1 1

Centre for Neuroscience Research, University of Edinburgh, 1 George Square, Edinburgh EH8 9JZ, UK, 2Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, UK, 3Scottish Centre for Genomic Technology and Informatics, University of Edinburgh, Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK, 4Burns and Allen Research Institute, Cedars-Sinai Medical Centre, Los Angeles, CA, USA and 5The Babraham Institute, Babraham, Cambridge CB2 4AT, UK

Received October 7, 2005; Revised November 23, 2005; Accepted January 4, 2006

Wallerian degeneration of injured neuronal axons and synapses is blocked in Wld S mutant mice by expression of an nicotinamide mononucleotide adenylyl transferase 1 (Nmnat-1)/truncated-Ube4b chimeric gene. The protein product of the Wld S gene localizes to neuronal nuclei. Here we show that WldS protein expression selectively alters mRNA levels of other genes in Wld S mouse cerebellum in vivo and following transfection of human embryonic kidney (HEK293) cells in vitro. The largest changes, identified by microarray analysis and quantitative real-time polymerase chain reaction of cerebellar mRNA, were an approximate 10-fold down-regulation of pituitary tumour-transforming gene-1 ( pttg1) and an approximate 5-fold upregulation of a structural homologue of erythroid differentiation regulator-1 (edr1l-EST ). Transfection of HEK293 cells with a Wld S-eGFP construct produced similar changes in mRNA levels for these and seven other genes, suggesting that regulation of gene expression by Wld S is conserved across different species, including humans. Similar modifications in mRNA levels were mimicked for some of the genes (including pttg1) by 1 mM nicotinamide adenine dinucleotide (NAD). However, expression levels of most other genes (including edr1l-EST ) were insensitive to NAD. Pttg1 2/2 mutant mice showed no neuroprotective phenotype. Transfection of HEK293 cells with constructs comprising either full-length Nmnat-1 or the truncated Ube4b fragment (N70-Ube4b) demonstrated selective effects of Nmnat-1 (down-regulated pttg1) and N70-Ube4b (up-regulated edr1l-EST ) on mRNA levels. Similar changes in pttg1 and edr1l-EST were observed in the mouse NSC34 motor neuron-like cell line following stable transfection with Wld S. Together, the data suggest that the WldS protein co-regulates expression of a consistent subset of genes in both mouse neurons and human cells. Targeting Wld S-induced gene expression may lead to novel therapies for neurodegeneration induced by trauma or by disease in humans.

INTRODUCTION Traumatic physical or chemical injury in the nervous system normally triggers Wallerian degeneration (WD), a breakdown

of axons and their associated neuroglial cells (1 –3). In rodents, distal axons and their sensory or motor terminals degenerate within 18 –36 h (4). However, in Wld S mutant mice (Wallerian degeneration slow; 5,6) axotomy-induced

*To whom correspondence should be addressed. Tel: þ44 1316503724; Fax: þ44 1316506545; Email: [email protected] { The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

# The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

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degeneration is delayed about 10-fold (5). Many severed axons in these mice survive and retain the ability to conduct action potentials for more than 3 weeks, whereas most motor nerve terminals continue to release neurotransmitter for more than 4 days (7 – 9). Moreover, the characteristic morphological signs accompanying the ultimate demise of the severed axons and synaptic terminals are quite different from those of axotomized wild-type axons (3,10). The protection from WD appears to be an intrinsic property of axons and is also slow in vitro (11 – 13). Expression of the Wld S gene in several mouse models of neurodegenerative disease delays the onset and retards progression of neurological signs and axon pathology (14 – 18; but see 19). Thus, a deeper understanding of the cellular functions and molecular mechanisms of the Wld S gene could lead to the identification of novel therapeutic targets for the treatment of neurodegeneration, ultimately in humans. The Wld S locus comprises an 85 kb region embracing the complete coding sequences of two genes, Rbp7 and nicotinamide mononucleotide adenylyl transferase 1 (Nmnat-1), plus a partial sequence of a third, encoding the N-terminal 70 amino acids of Ube4b (N70-Ube4b; Fig. 1A). Spontaneous triplication of this region generated in-frame fusion of the Nmnat-1 and N70-Ube4b components in Wld S mutant mice. Over-expression of Rbp7 appears to play no role in generation of the axon-protection phenotype (20), but transgenic expression of the chimeric Nmnat1/N70-Ube4b protein in mice and rats was sufficient to reproduce it completely in vivo (9,10,21). The chimeric WldS protein also contains a unique 18 amino acid peptide (‘Wld-18’), linking the Nmnat-1 and Ube4b components (Fig. 1A). This arises from a 50 UTR, not normally translated during expression of the wild-type Nmnat-1 gene, but which forms part of the open-reading frame in Wld S. A highly specific antibody raised against the Wld-18 peptide uniquely recognizes the full-length Wld S protein (14,17). Remarkably, the WldS protein is strongly localized to neuronal nuclei in vivo (9,17). Thus, WldS protein itself is in the wrong place to exert a direct neuroprotective effect on isolated distal axons and synapses. Its neuroprotective functions and mechanisms must, therefore, be exerted indirectly and on the constitutive, basal state of the neuron (i.e. prior to an isolating axonal injury), either by altering downstream structural proteins or by modulating intracellular signalling pathways. Here we tested the hypothesis that WldS protein selectively alters transcription of other specific genes. The data suggest that WldS protein expression brings about regulation of several genes and that these effects are conserved across cell types and species, including humans. The largest changes were down-regulation of pituitary tumour transforming gene1 ( pttg1), an oncogene involved in the regulation of sister chromatid separation during cell division (22,23), and upregulation of an erythroid differentiation regulator 1-like gene (edr1l-EST; 24). We also show that mRNAs for pttg1 and edr1l-EST in HEK293 cells are affected independently by the separate Nmnat-1 and N70-Ube4b components of the Wld S gene.

RESULTS Wld S-associated gene expression in mouse cerebellum in vivo Cellular heterogeneity in most regions of the brain is problematical for accurate gene expression profiling (25). We, therefore, searched for regions where Wld S might be uniformly expressed in a defined neuronal cell population. The cerebellum is composed predominantly of cerebellar granule cells (CGC), a virtually homogenous cell population (26). We found by immunostaining with Wld-18 antibody that almost all CGCs (.95%) strongly express WldS protein (Fig. 1B and C). CGCs from Wld S mice also show a strong neuroprotective phenotype in vitro (Supplementary Material, Fig. S1A and B; 13). Comparative gene expression profiling was performed in a pilot study of three independently prepared and analysed preparations of mRNA extracted from Wld S and wild-type mouse cerebellum. The global mRNA expression profile was determined using Mouse genome MOE-430A arrays (Affymetrix, USA), containing the majority (23 000) of known mouse genes. The expression array data revealed seven genes that showed a difference in relative expression levels in all three experiments of more than 2-fold compared to wild-type (Fig. 1D; Table 1). Four other genes showed an average and consistent difference of .1.5-fold, with at least one of the three microarrays showing a greater than 2-fold difference. All 10 genes were selected for further investigation and validation (Table 1). The largest difference from wild-type was for pituitary tumour-transforming gene-1 ( pttg1) which showed approximately 7– 8 times lower expression on all three microarrays. Importantly, two independent probe sets representing the expression of pttg1 were contained on the array chip and both demonstrated clear down-regulation. The identities of the other selected genes are known, with the exception of those represented by the probe sets 1427055_at and 1427820_at. A BLAST search for Affymetrix EST 1427055_at yielded no overt homology with genes of known function. We, therefore, refer to this gene here simply as ‘EST ’. There is also currently no evidence that the putative transcript 1427820_at has any functional protein associated with it. However, a BLAST sequence comparison showed a 91% homology to a GenBank sequence submitted by the MGC Program Team (27). The nearest homologous known protein to this transcript, as determined by sequence alignment, is Mus musculus edr1. However, our novel transcript is unlikely to be an exact match for this gene as levels of edr1 transcripts were below the detectable threshold in all experiments and were not altered on any of our microarray chips (Affymetrix 1439200_x_at; data not shown). Biochemical analysis of our novel transcript and its possible products is beyond the scope of the current study, so we have therefore named this transcript edr1l-EST for the purposes of this paper. This gene consistently showed the greatest up-regulation on the Wld S cerebellar microarray, about 3.4 times higher than in wild-type mice. Preliminary assessment of any functional linkages between individual altered genes (see Materials and Methods) revealed no specific pathway(s) in which two or more of the candidate


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Figure 1. Bidirectional changes in mRNA expression in Wld S mouse cerebellum. (A) The 85 kDa region of chromosome 4 triplicated in the Wld S mutation (upper) and the resulting WldS chimeric protein (lower). (B, C) Confocal images of a cerebellar folium immunocytochemically labelled for WldS protein (red), with strong expression in the granule cell layer (Topro-3; blue). Scale bars: 700 mm (B); 5 mm (C). (D) Wld S mRNA expression versus wild-type from a single microarray experiment. Diagonals bracket 2-fold up- and down-regulation. The blue box contains genes whose expression levels fell below the reliable detection threshold. Rbp-7, Nmnat-1 and edr1l-EST are clearly up-regulated. Two independent probe sets, both representing the expression of pttg1, are clearly down-regulated. (E, F) Validation of expression levels indicated from microarray analysis of cerebellar mRNA, using RT–PCR (E) and qRT–PCR (F), showing consistent down-regulation of pttg1 and up-regulation of edr1l-EST, together with the expected up-regulation of rbp7 (see A). qRT–PCR data for pttg1 and edr1l-EST (Edr1) are expressed as fold changes (versus background beta-tubulin levels; mean + SEM; N ¼ 3 mice). Both pttg1 and edr1l-EST levels were significantly different compared with beta-tubulin (P , 0.01, one-way ANOVA, Dunnett’s post hoc test).

genes constitute significant components (data not shown). As internal controls, we noted that Rbp7 was up-regulated in Wld S mice by approximately a factor of 8 and Nmnat-1 mRNA expression levels were also consistently elevated by

a mean factor of 8. Both were to be expected, as both genes are contained within the triplicated 85 kb Wld gene locus and their protein expression levels are at least 3-fold higher than in wild-type mice (9,20).

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Table 1. Genes highlighted by microarray analysis selected for further investigation Affymetrix ID

Gene title

Gene symbol

Up-regulated 1427820_at 1418551_at 1417764_at 1450779_at 1427689_a_at 1420819_at

Erythroid differentiation regulator 1 like-EST Myosin-binding protein C, cardiac Signal sequence receptor, alpha Fatty-acid-binding protein 7, brain TNFAIP3-interacting protein 1 Homologous to human/rat Src-like adaptor

Edr1l-EST Mybpc3 Ssr1 Fabp7 Tnip1 Src-LA

Death-associated protein 3 RIKEN cDNA 4921507I02 gene DNA segment, Chr 11, ERATO Doi 730, expressed Pituitary tumor transforming 1 Pituitary tumor transforming 1

Dap3 EST D11Ertd730e

21.7 22.0 22.5

Pttg1 Pttg1

27.1 28.6

Down-regulated 1450848_at 1427055_at 1447146_s_at 1438390_s_at 1424105_a_at

Re-analysis of the cerebellar mRNAs using gene-specific primers for reverse transcriptase – polymerase chain reaction (RT – PCR) confirmed each provisional modification in gene expression highlighted by the microarray analysis. For example, expression of pttg1 was consistently down-regulated in separate mRNA preparations and edr1l-EST was consistently up-regulated. As expected (see above), rbp7 was also consistently up-regulated, by an amount that appeared qualitatively similar to that of edr1l-EST (Fig. 1E). In addition, we used quantitative real-time PCR analysis (qRT-PCR) to measure the expression levels of the two candidate genes that were altered the most but in opposite directions, namely pttg1 and edr1l-EST (Fig. 1F). This analysis showed that pttg1 mRNA levels were even more strongly suppressed in Wld S mouse cerebellum than indicated by the microarray data: 14.1 + 2.2-fold down-regulated compared with wildtype tissue (N ¼ 3 mice). Likewise, edr1l-EST mRNA was 8.6 + 1.4-fold up-regulated in Wld S cerebellum (N ¼ 3), also greater than indicated by the microarray analysis. All the other genes were respectively reduced or increased in expression in RT –PCR, as indicated by the microarray data (Supplementary Material, Fig. S2). Thus, our validated microarray data show unequivocally that Wld S mouse cerebellar neurons have altered mRNA levels for several unrelated genes in vivo and that this association is bidirectional: specifically, pttg1 is about 10-fold down-regulated and edr1l-EST is about 5-fold up-regulated by Wld S. Wld S alters pttg1 and edr1l-EST mRNA expression in HEK293 cells To verify that the alteration in mRNA expression levels for pttg1, edr1l-EST and other genes was occurring as a direct result of the presence of Wld S, we measured RNA levels in human embryonic kidney (HEK293) cells after transfecting them with a Wld S-eGFP construct (Fig. 2A – C; Supplementary Material, Fig. S3). We selected HEK293 cells for two main reasons. First, we wanted to ask whether the expression changes observed in mouse neurons in vivo could be replicated in human cells. Second, HEK293 cells are an experimentally amenable, homogenous cell line that have repeatedly been used to study transcriptional effects (28) and to model disorders

Approx fold change 3.4 2.1 2.0 1.7 1.5 1.5

of the human nervous system (29,30). Transfection efficiency, as measured by counting cells that showed both eGFP fluorescence and Wld-18 immunostaining, was 18 + 9% of total cells (mean + SD; N ¼ 4). Transfected cells localized the fluorescent Wld S gene product to their nuclei, forming either discrete puncta or diffuse nuclear fluorescence (Fig. 2A –C). We observed similar diversity of nuclear localization patterns in Wld S mouse brains and dorsal root ganglia (J.E. Haley, T.M. Wishart and R.R. Ribchester, unpublished data). RT – PCR analysis confirmed that both pttg1 and edr1l-EST mRNA levels were altered when HEK293 cells were transfected with WldS-eGFP (Fig. 2D). We ruled out the possibility that transfection with large amounts of Wld S-eGFP cDNA might have altered gene expression in a non-specific fashion. HEK293 cell cultures were transfected with 5 mg of eGFP (Clontech) and Wld S-eGFP cDNA, but with varying proportions of Wld S-eGFP. The data show that changes in expression of both pttg1 and edr1l-EST were proportional to the amount of Wld S-eGFP in the transfection mixture rather than the total amount of construct (eGFP plus Wld SeGFP) added to the cells. These findings are also consistent with the gene dose dependence of Wld S protein expression, as measured in different transgenic lines in vivo (9). Analysis of mRNA levels using qRT – PCR, normalized for transfection efficiency (see Materials and Methods), showed significant changes in expression in nearly all (eight out of nine; Fig. 2E) of the candidate genes we examined, compared with actin controls (P , 0.05; ANOVA with Tukey’s post hoc test; N ¼ 6 for actin, pttg1 and edr1l-EST; N ¼ 3 for all other genes). The only exception was Tnip1. qRT – PCR analysis of these cells showed pttg1 down-regulated by 77.6 + 10.0% and edr1l-EST up-regulated by 103.0 + 10.8%. Nicotinamide adenine dinucleotide only partially mimicked Wld S-induced changes in gene transcription Araki et al. (31) and Wang et al. (32) recently reported that the Wld S phenotype could be reproduced in vitro by increasing nicotinamide adenine dinucleotide (NAD) biosynthesis or by adding NAD to cultures of dorsal root ganglion explants. These effects were blocked by sirtinol (100 mM ), a specific inhibitor of the nuclear transcriptional regulator SIRT-1,


Figure 2. Wld S produces gene dose-dependent regulation of gene expression in a transfected human HEK293 cell line in vitro. Confocal micrographs of HEK293 cells 4 days after transfection with eGFP-WldS. (A) eGFP-Wld S signal; (B) WldS immunostaining. (C) Topro-3 staining showing nuclei of all the cells present. Only about 18% of the total cell population was transfected (N ¼ 4 preparations). (D) HEK293 cells were transfected with 5 mg of total construct, and the proportion of eGFP-Wld S was varied between 1 and 5 mg. RT–PCR bands showing gene dose-dependent effects of Wld S transfection on pttg1 and edr1l-EST expressions. (E) Percentage changes in gene expression for nine of the candidate genes listed in Table 1 (and actin controls) following transfection with eGFP-Wld S and measured by qRT–PCR (P , 0.05 for all genes except Tnip1; ANOVA with Tukey’s post hoc test; N ¼ 6 for actin, pttg1 and edr1l-EST; N ¼ 3 for all other genes). Scale bar: 15 mm (A–C).

suggesting that SIRT-1 regulates a molecular program leading to axon protection. We, therefore, tested whether any of the candidate genes we identified were also subject to NAD-dependent regulation via the SIRT-1 pathway. Only three of the candidate genes ( pttg1, fabp7 and src-LA) showed significantly altered expression when compared with actin, 4 days after incubation in medium containing 1 mM NAD (Fig. 3A; Supplementary Material, Table S1). Moreover,

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Figure 3. NAD/Nmnat-1 and Ube4b components selectively regulate pttg1 and edr1l-EST in HEK293 cells. (A) qRT– PCR measurements of pttg1 and edr1l-EST mRNA expression, 4 days after adding NAD (1 mM ; N ¼ 6 cultures) or 1 mM NAD and 100 mM sirtinol (N ¼ 3 cultures). Only pttg1 showed a significant response to NAD ( P , 0.001; ANOVA with Tukey’s post hoc test). This response was blocked by 100 mm sirtinol. (B) Confocal micrographs of GFP signal in cells transfected with either eGFP-Nmnat-1 (left) or eGFP-N70-Ube4b (right). Ube4b became distributed throughout the cell, whereas Nmnat-1 aggregated in the nucleus, consistent with the nuclear localization motif in Nmnat-1. Scale bars: 6 and 3 mm, respectively. (C) qRT–PCR of pttg1 and edr1l-EST mRNA after transfection with full-length eGFP-Wld S construct (N ¼ 3 cultures), eGFP-N70-Ube4b construct alone (N ¼ 6 cultures) or eGFP-Nmnat-1 construct alone (N ¼ 6 cultures). Nmnat-1 down-regulated pttg1 expression but had no significant effect on edr1l-EST expression, whereas N70-Ube4b up-regulated edr1l-EST but had no significant effect on pttg1 expression. Neither individual construct elicited a response as great as that observed following transfection with the full-length Wld S construct.

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the effects were modest compared with the changes in expression following eGFP-WldS transfection. The largest change was observed for pttg1 (214.9 + 2.7%; P , 0.001; ANOVA with Tukey’s post hoc test; N ¼ 6), about five times less than that observed following Wld S transfection (Fig. 2). Thus, exogenous administration of 1 mM NAD is less effective in suppressing pttg1 mRNA levels than transfection of HEK293 cells with eGFP-WldS. Nevertheless, downregulation of pttg1 by exogenous NAD was blocked by 100 mM sirtinol (Fig. 3A; P , 0.02; Mann – Whitney test; N ¼ 3). Transfection of HEK293 cells with Wld S also produced up-regulation of fabp7 (Fig. 2), a gene encoding a cytosolic protein involved in fatty acid uptake, transport and targeting (33). A small, but statistically significant, 10% change in expression of this gene was also produced by exogenous NAD and this response was also blocked by sirtinol (Supplementary Material, Table S1). The only other gene to show significant NAD sensitivity was Src-LA. This changed by only 6% (compared with about 25% following Wld S transfection; P , 0.01; Supplementary Material, Table S1). This effect was also antagonized by sirtinol (P , 0.04; Supplementary Material, Table S1). Edr1l-EST, D11Ertd730e, EST, Mybpc3, Ssr1 and Tnip1 all showed no response to either exogenous NAD or sirtinol (Supplementary Material, Table S1; Fig. 3A). That is, adding 1 mM NAD to the culture medium did not significantly change the expression levels of these genes when compared with control actin levels. Curiously, Ssr1 expression appeared to be potentiated, rather than blocked, by the addition of sirtinol (P , 0.04; Supplementary Material, Table S1). This suggests that SIRT-1 may act as repressor of Ssr1, at least in HEK293 cells, or that sirtinol affects other pathways as well. Taken together, most of the Wld S-mediated changes in mRNA levels, including edr1l-EST, appear not to be mimicked by NAD, the product of Nmnat-1 enzymic activity, but those three that were, including pttg1, appear to be mediated by SIRT-1-dependent signalling pathways. Different components of the Wld S gene regulate bidirectional gene transcription Araki et al. (31) also showed that over-expression of Nmnat-1 was sufficient to reproduce the Wld S phenotype in vitro, but over-expression of Ube4b was not. We, therefore, asked whether transfection of HEK293 cells with either component of the Wld S gene would produce changes in expression of the two most strongly regulated genes we identified in the present study, namely pttg1 and edr1l-EST. We used qRT – PCR to measure mRNA levels following transfection of HEK293 cells with either eGFP-Nmnat-1 or eGFP-N70Ube4b constructs (Supplementary Material, Fig. S1). Expression of eGFP-Nmnat-1 in transfected HEK293 cells was localized almost exclusively to the nucleus, whereas expression of eGFP-N70-Ube4b was found in both the nucleus and cytoplasm (Fig. 3B). As expected from the effects of NAD on cultured HEK293 cells, Nmnat-1 transfection caused a significant downregulation of pttg1 compared with actin controls (Fig. 3C; P , 0.001, ANOVA with Tukey’s post hoc test, N ¼ 6). Also as expected (from the lack of effect of NAD on edr1l-EST ),

transfection with Nmnat-1 construct had no effect on the level of edr1l-EST mRNA. Conversely, the Ube4b N-terminal construct had no effect on the expression level of pttg1 mRNA but caused a highly significant up-regulation of edr1l-EST mRNA levels (Fig. 3C; P , 0.01). Interestingly, transfection with full-length Wld S construct produced a significantly greater suppression of pttg1 (P , 0.05; Mann –Whitney test, two-tailed) compared with transfection of Nmnat-1 construct alone (Fig. 3C). The changes in edr1l-EST mRNA following transfection with the Ube4b construct alone also appeared to be smaller than with transfection of the full-length Wld S construct. However, the difference was not quite statistically significant. But overall, the data suggest that the Ube4b N-terminal sequence and Nmnat-1 components, respectively, of the Wld S gene have independent, selective capacities for regulating expression levels of the pttg1 and edr1l-EST genes. Mice with a null mutation in pttg1 do not show the Wld S phenotype The Wld S mutation delays WD by about a factor of 10. As pttg1 was the most strongly down-regulated of the candidate genes we tested (also about 10-fold), and this down-regulation was mimicked by NAD in a sirtinol-sensitive fashion, we hypothesized that down-regulation of pttg1 alone would be sufficient to delay WD. We tested this in vivo using pttg1 2/2 mice (34). We compared the amount of distal axon degeneration up to 6 days after unilateral sciatic nerve section, in seven pttg1 2/2 mice, compared with four littermate (þ/þ) mice. Pre-synaptic motor nerve terminals at neuromuscular junctions in lumbrical muscles degenerated completely within 48 h of sciatic nerve injury in the pttg1 2/2 mice, as in wild-type. Clear evidence of axon degeneration was also observed in neurofilament-immunostained teased nerve fibre preparations. Axon counts made in toluidine-blue-stained sections of the distal nerve, 1– 6 days after nerve lesions, also suggested that axon degeneration occurs the same rate in both 2/2 and þ/þ mice (Fig. 4). These data indicate that pttg1 down-regulation alone is not sufficient to reproduce the Wld S phenotype. It was not possible to test the effects of edr1l-EST overexpression on WD in vivo, because transgenic mice overexpressing edr1l-EST or edr1 are not available. In any case, we showed (above) that edr1l-EST up-regulation occurred in response to over-expression of N70-Ube4b but not Nmnat-1. Previous studies have shown that over-expression of N70Ube4b alone, either in vivo or in vitro, is itself insufficient for axon protection (31). Therefore, we would not expect selective up-regulation of edr1l-EST alone to produce a discernible neuroprotective phenotype in vivo. Wld S also alters pttg1 and edr1l-EST mRNA expression levels in an NSC34 neuronal cell line Finally, in order to examine whether the changes in gene expression detailed earlier could also be replicated in a neuronal cell line in vitro, we made a stably transfected Wld S-expressing cell line, based on the mouse motor neuron-like cell line (NSC34; 35,36) (Fig. 5). As in Wld Sexpressing neurones in vivo and transfected HEK293 cells


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expression manifested itself in the variability of mRNA expression changes that we measured following WldS-eGFP transfection (see error bars in Fig. 5C; N¼3 separate culture preparations). Nevertheless, both pttg1 and edr1l-EST expression levels were strongly down- and up-regulated, respectively, by more than 5-fold compared with background beta-tubulin levels. Thus, the ability of Wld S to regulate expression levels of other genes was conserved in all cell types we examined, including mouse neurons in vivo and in vitro, as well as human cells in vitro.

DISCUSSION

Figure 4. pttg1 2/2 mutant mice show no neuroprotective phenotype in vivo. (A) pttg1 2/2 mouse tibial nerve immunostained for 165–kDa neurofilaments, showing fragmentation of axon profiles 3 days after sciatic nerve lesion. (B) pttg1 2/2 mouse deep lumbrical muscle 2 days after sciatic nerve lesion stained with FM1-43 (green) and TRITC-conjugated alpha-bungarotoxin (red). No FM1-43-positive motor nerve terminals were found in any of the muscles examined (N ¼ 6). All terminals in contralateral unoperated muscles stained with FM1-43 (data not shown). (C) Unlesioned pttg1 2/2 mouse tibial nerve stained with toluidine blue. (D) pttg1 2/2 mouse tibial nerve 6 days after sciatic nerve lesion. Almost all axon profiles were undergoing degeneration. (E) Quantification of axon profile preservation in the tibial nerve of pttg1 2/2 (ko) and pttg1 þ/þ (wt) mice at 2, 3 and 6 days post-sciatic nerve lesion (mean + SD; N ¼ 2 for pttg1 2/2 and 1 for pttg1 þ/þ per time-point). There was no difference in the onset or rate of degeneration between the two experimental groups. Scale bars: 50 mm (A, B); 10 mm (C, D).

in vitro, confocal analysis of the eGFP signal in the stably transfected NCS34 cells showed that Wld S was localized to the nucleus (see also 9,14,17; Fig. 5A and B). However, there was a high level of variability in the nuclear expression patterns of Wld S, as well as heterogeneous cellular morphology in our NSC34 cultures (data not shown). This suggests that the NSC34 cell line is not as homogeneous as HEK293 cells (cf. Fig. 2) and that there may be different sites of integration and transgene copy number among the cell population. This heterogeneity of cell type and Wld S

Our study has generated four significant findings. First, the data show that the neuroprotective WldS protein regulates mRNA levels of other genes. Second, we identified several genes with expression levels altered by more than a factor of 2. Of these, two genes in particular, Pttg1 and Edr1l, showed mRNA expression levels altered more than 5-fold. All these genes, either singly or in combination, should presently be considered candidates for downstream mediation of the Wld S neuroprotective phenotype, although the data do not rule out alternative possibilities such as post-translational effects on mRNA turnover. Third, we show that human cells respond to Wld S, in a similar fashion to Wld S mouse neurones, at least with respect to regulation of specific mRNAs. Together, these findings may represent a significant step towards understanding the mechanisms of neuroprotection conferred by the Wld S gene. Finally, the unexpected nature of the genes that are regulated by Wld S implies that the chimeric protein may constitute a useful investigative tool that could open up new avenues of cell biological and molecular genetic research, in areas other than neurodegeneration. Before the discovery of the Wld S mutant mouse (5), it was presumed that axotomy either starves the isolated distal axon of essential maintenance factors normally synthesized in neuronal cell bodies and propagated through the neuron by axonal transport or that injury generates a signal at the site of injury triggering rapid necrosis in the distal stump (4). The absence of WD in Wld S mice suggests that WD is an active, genetically regulated process (1,2,37 – 39). Three plausible hypotheses, none of them mutually exclusive, could account for the mechanism of axon protection by the chimeric WldS protein. First, WD may be blocked by dominant-negative inhibition of the ubiquitin-proteasome system, an effect of the truncated N70Ube4b component integral to the WldS protein structure (ubiquitination hypothesis; see 2,40,41). Second, axon protection may be conferred by over-expression of the enzyme Nmnat-1—the largest component of the Wld S gene—and its consequential effects on levels of NAD in the nucleus (NAD hypothesis; 31). A third plausible hypothesis, that excludes neither of the above, is that WldS protein exerts its protective effects by modulating expression levels of genes and proteins that normally execute a program of axon degeneration (gene regulation hypothesis; Fig. 6; see 31). The present data satisfy one requirement of the gene regulation hypothesis, namely that changes in specific and unrelated mRNAs should occur when WldS protein is expressed. We demonstrated this in Wld S mutant mouse cerebellar tissue, following transfection of non-neuronal human cells, and

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Figure 5. Bidirectional changes in mRNA expression can also be replicated in eGFP-Wld S-transfected NSC34 cells in vitro. (A, B) Confocal micrographs of eGFP signal (A) and Topro-3 signal (B) in an NSC34 cell transfected with WldS-eGFP. WldS-eGFP became aggregated in the nucleus, consistent with the nuclear localization motif in Nmnat-1. Scale bar: 4 mm. (C) qRT– PCR data for pttg1 and edr1l-EST mRNA levels in transfected NSC34 cells, expressed as fold changes (versus background beta-tubulin levels; mean + SEM; N ¼ 3 cultures).

Figure 6. Working hypothesis linking WldS phenotype to bidirectional gene transcription. (A) The data suggest that Nmnat-1 over-expression in HEK293 cells may selectively down-regulate pttg1 transcription, probably via transcription factors (TFs) linked to the SIRT-1 signalling pathway. However, suppression of pttg1 confers no axosynaptic protective phenotype on mice in vivo (see Fig. 4). N70-Ube4b over-expression in HEK293 cells up-regulates edr1l-EST in an SIRT-1 independent fashion, possibly via transcriptional regulation mediated by factors such as VCP. Other studies show that N70-Ube4b confers no phenotype in vitro (31,39). (B) Transfection of HEK293 cells with full-length Wld S strongly co-regulates pttg1 and edr1l-EST expression, possibly through synergistic, synchronous and bidirectional regulation of expression of pttg1, edr1l-EST and/or other genes. Future studies should establish whether bidirectional changes in the levels of these genes, and/or others in combination, are responsible for the Wld S phenotype in vivo.

following transfection of neuronal mouse cells. The largest and most consistent changes induced by WldS protein expression were in levels of pttg1 and edr1l-EST. The present data do not resolve whether these changes are caused by modifications in transcriptional pathways, or whether changes in the specificity or rate of mRNA degradation are responsible. None the less, neither pttg1 nor edr1l-EST has previously been implicated in mechanisms regulating axonal or neuronal degeneration. Our findings, therefore, add to a growing list of surprising cellular and molecular features associated with the remarkable, covert neuroprotective phenotype displayed by the Wld S mutant mouse. This study, by linking Wld S expression to genes involved both in cell biology of cancer (via pttg1 down-regulation) or development (via edr1l up-regulation), indicates that the functional genetic significance of Wld S may be much more wide ranging than that appreciated hitherto, possibly opening up several new avenues for cell biological research. Despite highly significant changes in specific mRNA levels, it remains an open question whether any of the genes we identified (or others yet to be identified) cause the neuroprotective Wld S phenotype in vivo. Neither of the two genes we focussed on here, namely pttg1 and the edr1l-EST, is likely to play an exclusive role (Fig. 6A), for the following reasons. First, pttg1 2/2 mice did not show axonal or synaptic protection after sciatic nerve injury (Fig. 4). These findings indicate the need for more caution when basing conclusions on in vitro models of neuroprotection (31,32). Underscoring this point, recent studies of mice transgenically over-expressing Nmnat-1 also do not show protection from WD


(M.P. Coleman et al., submitted for publication), which is in contrast to some published in vitro studies (31,32). Second, edr1l-EST was up-regulated by transfection of HEK293 cells either with a full-length Wld S construct or by N70-Ube4b, but not by Nmnat-1. But as N70-Ube4b over-expression also fails to induce phenotype (9,31), it follows that edr1l-EST up-regulation alone will also be insufficient (Fig. 6). One of the several alternative possibilities is that simultaneous coregulation of pttg1 and edr1l-EST genes by both the Nmnat1 and N70-Ube4b components of the WldS protein confers resistance to WD, by signalling pathways that are also yet to be identified (Fig. 6B). Equally, it is possible that one or more of the other eight genes we identified might directly mediate the Wld S phenotype. Further biochemical and bioinformatic analyses of the Wld S microarray data set provided here could perhaps add additional plausible candidates to the short list we have generated thus far. How might WldS protein regulate expression levels of pttg1, edr1l-EST or any of the other genes we have highlighted? The primary structure of the chimeric protein gives few indications that the regulation of transcription occurs directly by selective DNA binding. For example, the WldS protein contains neither zinc fingers nor any other motifs common to known transcription factors. Perhaps Wld S provides an activation signal for signaldependent transcription factors that specifically regulate downstream genes (Fig. 6). Binding of WldS protein to transcription factors has not yet been documented in detail. However, Wld S binds strongly to valosin-containing protein (VCP; H. Laser, M.P. Coleman et al., in preparation). This protein may regulate transcription directly (42). Therefore, binding of Wld S to VCP, or to other nuclear proteins, could be sufficient for the transcriptional regulation of some of the genes reported here. Preliminary data suggest that Wld S co-localizes with other histone deactylases (HDACs) (J.E. Haley, T.M. Wishart, G. Hardingham and R.R. Ribchester, unpublished observations). Disruption of the nuclear organization of HDACs and their binding partners has been implicated in other forms of degeneration, including spinal muscular atrophy (43) and activity-dependent rescue of compromised neurons from apoptosis (44). In summary, the findings and measurements contained in this study provide direct evidence that WldS protein regulates steady-state mRNA levels of other specific genes, both in murine brain tissue in vivo and in human cells in vitro. Transferring the Wld S gene to other neurological mouse mutants has been shown to confer significant levels of neuroprotection and to alleviate clinical signs, including mouse models of motor neuron disease (15), de-myelinating neuropathies (14), Parkinson’s disease (16), global cerebral ischaemia (17) and gracile axonal dystrophy (18). The fact that the effects of Wld S on gene expression are conserved between cells of different species encourages optimism for further translation of the neuroprotective effects of Wld S, either pharmacologically or genetically, ultimately to humans.

MATERIALS AND METHODS Immunocytochemistry Wld-18 immunostaining was performed and visualized as described previously (1:500 dilution; 10,14,21).

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RNA extraction Cerebellums from age- and sex-matched mice, or HEK293 cells, were frozen in liquid nitrogen for 1 min, then placed in 1 ml of tri-reagent (Sigma) on dry ice. mRNA was extracted using standard techniques. Microarray analyses RNA quality was initially assessed using formaldehyde gels and by measurement of absorbance at 260/280 nm following standard procedures. A further assessment of RNA quality was undertaken using an Agilent Bioanalyser (Agilent Technologies, GmBH). Samples (10 mg) were labelled, hybridized to Affymetrix MOE-430A GeneChip arrays and scanned according to standard Affymetrix protocols. To ensure quality and consistency of the sample labelling process and array hybridizations, control information from all six arrays was collated and reviewed prior to the data analysis (data not shown). Experimental data and links to supplementary data can be obtained from the Scottish Centre for Genomic Technology and Informatics GPX database (http://www.gti. ed.ac.uk/ GPX; experiment accession number GPX-000048). All analyses were undertaken in Affymetrix Microarray Suite 5.1 software as follows: arrays were scaled (normalized) to an overall target intensity of 100 prior to comparison and notable differences in gene expression were identified by pair-wise comparison of appropriate samples. Differentially expressed genes were annotated using the Affymetrix online analysis resource ‘NetAffx’ (http://www.affymetrix.com/ analysis). We assessed all candidate genes for potential functional linkages by using Pathway Assist software (Ariadne Genomics). Polymerase chain reaction RT – PCR was carried out in ‘ready-to-go’ RT – PCR tubes (Amersham Biosciences) using a T-Personal PCR machine (Biometra). Beta-tubulin was used as a loading control for all RT –PCR experiments. qRT – PCR was carried out using a Sybr-Green ‘1 step qRT – PCR kit’ (Invitrogen) on a Model 7700 instrument (Applied Biosystems). The control gene was actin. Primers were designed to span an intron eliminating the possibility of genomic DNA contamination. PCR bands were removed and the product extracted using a Q-gel extraction kit (Qiagen). Bands were sequenced to confirm product identity. Primers used are shown in Supplementary Material, Table S2. We were unable to obtain human Dap3 primers. Transfection of HEK293 cultures The eGFP-Wld S construct we used encodes the N-terminal 70 amino acids of Ube4b, the full 285 amino acids of Nmnat-1, plus the Wld-18 region located normally in the 50 UTR region of Nmnat-1 in wild-type mice (9). cDNA sequence encoding full-length WldS protein, Nmnat-1 or Ube4b amino acids 1– 70 (N70) was PCR amplified from the Wld S transgene template (9) using the high-fidelity enzyme Pfu (Stratagene) and appropriate combinations of the following primers

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(1 þ 2, 4þ2 and 1 þ 3, respectively). 50 restriction enzyme tags, added for cloning purposes, are shown in bold and the first or last three bases of sequence derived from the Wld S gene are underlined. A single base change to repress the stop codon of Wld S Rev and allow read-through of the C-terminal EGFP is double underlined: (1) Wld S For: 50 -TAGATCCCAAGCTTAACCTTTCACC ATTAAGAGGAAAGCGATG-30 ; Rev: 50 -GCGGGATCCCGTCCCAGAGTGG (2) Wld S AATGGTTGTG-30 ; (3) Ube4b Rev: 50 -TCCTCCCCGCGGGTCTGCTGCACCT ATGGGGGA-30 ; (4) Nmnat-1 For: 50 -GACTAGCTAGCATGGACTCATCC AAGAAGACAG-30 . After cloning of pEGFP-N1 (Clontech), the sequences of individual clones were verified and plasmid DNA isolated using the endonuclease-free plasmid kit (QIAGEN). HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS and 1% penicillin/ streptomycin (Invitrogen) at 378C in 5% CO2. For transfection with eGFP-Wld S, 5 mg of the DNA was mixed with 10% (v/v) CaCl2. An equal volume of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was added and the solution gently dropped onto the HEK293 cell culture. Transfection efficiency was 18 + 9%. qRT –PCR data from transfected HEK293 cells were, therefore, normalized, by multiplying the mRNA levels relative to actin expression by a factor of 100/18 (i.e. 5.56), in order to compare changes in the subpopulation of transfected cells with the changes measured in Wld S mouse cerebellum. In some experiments, 1 mM NAD (Sigma) was added to the medium +100 mM sirtinol, and cells were incubated for 4 days (31). mRNA levels measured by qRT –PCR were not corrected in these experiments, as NAD was uniformly applied to all cells. Transfection of NSC34 cultures The mouse neural hybrid cell line NSC34 was developed and kindly provided by Cashman et al. (35). Cells were cultured at 378C under 5%CO2 – 95% air in DMEM containing 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin sulphate. For transfections, 106 cells were electroporated using an Amaxa Nucleofector following the manufacturer’s recommendations. Cells were transfected with 2 mg of eGFP-Wld S DNA (see earlier) that had been linearized by restriction endonuclease digestion with MluI. Two days following transfection, G418 was added to the culture media to 50 mg/ml. Cells were cultured under G418 selection for three further passages and subsequently maintained as a mixed population of stable transfectants. Quantification of axon and synapse degeneration Unilateral sciatic nerve lesions were performed on pttg1 null (2/2) and wild-type litter mate (þ/þ) mice (34) under general anaesthesia (100 mg kg21 ketamine; 5 mg kg21 xylazine; IP injection), under the licence authority of the Los Angeles Cedars-Sinai animal facility. Qualitative and quantitative assessments of axon and synapse degeneration using

immunocytochemistry and FM1-43 staining were performed as described previously (10).

SUPPLEMENTARY MATERIAL Supplementary Material is available at HMG Online.

ACKNOWLEDGEMENTS We thank Prof P. Dormer, Prof A. Grinnell, Dr S.-G. Ren, D. Thomson, A. Thomson and J. Gilley for advice and assistance; Dr N. Cashman for providing us with NSC34 cells; and Prof A.J. Harmar, M.C. Raff, C.R. Slater and Drs G. Blanco and G. Hardingham for helpful comments on the manuscript. This work was supported by the BBSRC, Wellcome Trust, MRC and NIH Grant CA 75979. Conflict of Interest statement. The authors have no conflicts of interest.

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