We used cell cycle pathway-specific RT² profiler PCR arrays (see Materials and methods [below]) to quantify and compare the expression of cell cycle-related genes with high sensitivity. Initially, we used RNA extracted from the cerebellum of wild-type and Wld^s mice because this tissue has proven ideal for comparative genomic experiments 22. Wld^s cerebellar granule cells are also known to express Wld^s protein at high levels and exhibit a strong neuroprotective phenotype 22. We compared expression levels of 84 genes that regulate the cell cycle, including transitions between each of the phases, DNA replication, checkpoints, and arrest. Seventeen out of the 84 genes examined (around 20%) had expression levels increased by more than twofold in Wld^s cerebellum (Figure 1 and Table 1). The array identified changes in genes associated with many different stages of the cell cycle rather than one specific stage (Table 1). Interestingly, no cell cycle related genes appeared to be suppressed greater than twofold by Wld^s (Figure 1 and Table 1).

To confirm that RNA changes led to corresponding changes in protein levels, we quantified protein expression levels in the cerebellum of Wld^s and wild-type mice in vivo. We chose to focus on one of the genes with a large RNA change and one with a smaller change, just above the twofold threshold, where good antibodies were available (cABL and Brca2, respectively; Table 1). The protein product for both of these genes exhibited corresponding increased expression levels, of a similar ratio to that seen for RNA (Figure 2). In addition, we examined protein levels of other known cell cycle regulators to show that the changes observed on the PCR arrays were not exclusive. Three of the four additional proteins examined (histone H2B, BRCA1, and phosphohistone H2Ax) exhibited significantly increased expression levels in Wld^s cerebellum, which is in keeping with the general trend observed on the PCR arrays (Figure 2).

Next, we established that protein levels of two other cell cycle regulators, not included on the PCR array chip but previously shown to be modified in Wld^s neurons, were similarly altered. Previous studies have demonstrated that protein levels of Ube1 (a protein with known cell cycle involvement 33343536) are increased in Wld^s synapses 31, and we were able to confirm this finding by showing increased total Ube1 protein levels in Wld^s cerebellum (Figure 2). In addition, immunocytochemical staining for Ube1 confirmed increased nuclear expression levels in Wld^s-expressing neurons in vivo (Figure 3). We also found that Pttg1 protein levels (another protein that regulates cell cycle pathways 32) were significantly increased in Wld^s cerebellum (Figure 2), which is in keeping with changes in all other cell cycle regulators modified by Wld^s. This result was surprising because although Pttg1 protein levels had not previously been examined in Wld^s-expressing cells, several previous reports have identified reduced mRNA levels for Pttg1 2230.

To verify that the alterations in cell cycle gene expression were occurring as a direct result of the presence of Wld^s, and to further confirm that RNA changes observed in Wld^s mouse cerebellum led to corresponding changes in protein levels, we examined the effects of Wld^s on cell cycle in human embryonic kidney (HEK293) cells after transfection with enhanced green fluorescent protein (eGFP)-tagged Wld^s constructs 22. We selected HEK293 cells for our experiments for two main reasons. First, we wanted to consider whether the expression changes observed in mouse neurons in vivo could be replicated in a human cell line, as has previously been demonstrated for other Wld^s-mediated changes in gene expression 22. Second, HEK293 cells are an experimentally amenable, homogenous cell line that is routinely used to study transcriptional effects 2250 and to model degenerative mechanisms in the human nervous system 5152.

As for the cerebellar experiments, we again chose initially to focus on one gene with a large RNA change (Abl1) and one with a change just above the twofold threshold (Brca2). The protein product for both of these genes exhibited corresponding increased expression levels, of a similar ratio to that seen for RNA (Figure 4). In addition, we once again examined protein levels of other known cell cycle regulators to show that the changes observed on the PCR arrays were not exclusive. All four additional proteins examined (HDAC2, histone H2B, acetyl histone H3, and phosphohistone H2Ax) showed increased expression levels in Wld^s-transfected cells, which is in keeping with the general trend observed on the PCR arrays (Figure 1). These experiments also provided further confirmation that both Ube1 and Pttg1 protein levels are increased by Wld^s expression (Figure 4; compare with Figures 2 and 3).

Because the Wld^s protein is known to have a predominantly nuclear distribution 2021, and most cell cycle proteins modulate cell cycle via interactions in the nucleus, we next tested whether Wld^s expression altered the nuclear expression of cell cycle proteins. We chose to investigate the nuclear distribution of phosphohistone H2Ax in Wld^s-transfected HEK293 cells because this protein has a well-established role in the cell cycle 5354 and was among the largest protein changes identified in HEK293 cells (Figure 5; see Figures 2 and 4 for phosphohistone H2Ax protein levels in vivo and in vitro). Not all cells express Wld^s using our transfection protocol, as identified by the presence of an eGFP signal (Figure 5b,e). We were therefore able to compare directly experimental cells expressing Wld^s or eGFP-only controls with neighbouring nontransfected cells. Anti-phosphohistone H2Ax antibodies revealed intense nuclear spots of phosphohistone H2Ax in all cells expressing Wld^s (Figure 5a-f). However, neighbouring cells not expressing Wld^s did not show any phosphohistone H2Ax nuclear puncta. No phosphohistone H2Ax staining was observed in control cells transfected with eGFP, indicating that the response was not simply the result of a large accumulation of foreign protein in the nucleus (Figure 5g-i).

Because we had found that a broad spectrum of cell cycle genes and proteins were modified by Wld^s (Table 1), we next tested whether Wld^s can influence neurons to pass through the complete cell cycle by quantifying proliferation rates in a human neuronal cell line (NT2 cells) using an MTT (3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) assay. Introduction of a Wld^s construct into NT2 cells did not modify cell proliferation rates compared with vector-only transfected cells, either at 48 or 72 hours after transfection, or at low, medium, or high doses (Figure 6a-b). These findings were confirmed using tritiated thymidine uptake assays where values were normalized to low dose treatment (mean count: 14,770 ± 1,259 disintegrations per minute [DPM]; Figure 6c). Tritiated thymidine uptake assays were performed at 48 hours post-transfection in order to corroborate data from MTT assays generated at the same experimental time point and because this was the time point anticipated to give the maximum chance of detecting a proliferative change in these cells. These data suggest that Wld^s upregulates the expression of a broad range of cell cycle regulators, pushing cells toward cell cycle re-entry, but that pathways influencing later stages of the cycle, such as mitotic cell division, remain inhibited.

In order to confirm that Wld^s-mediated changes in cell cycle genes/proteins were pushing terminally differentiated neurons toward cell cycle re-entry rather than inhibiting cell cycle activation, we compared the profile of Wld^s-mediated protein changes with changes induced by a well known pharmacologic inhibitor of the cell cycle: the cyclin-dependent kinase inhibitor flavopiridol. Treatment of HEK293 cells with flavopiridol at an established active concentration (10 μmol/l 48) resulted in suppression of six out of eight cell cycle proteins that were increased in Wld^s-transfected HEK293 cells (Figure 7). Thus, pharmacologic inhibition of the cell cycle also induced changes in cell cycle proteins known to be altered by Wld^s, but importantly these changes in expression levels occurred in the opposite direction. These data confirmed that Wld^s reactivates dormant cell cycle pathways, pushing cells toward cell cycle re-entry rather than inhibiting it.

After demonstrating that the Wld^s gene robustly modifies cell cycle status in a variety of cell types in vivo and in vitro, we next investigated whether any of the previously identified downstream modifications induced by Wld^s play a role in mediating cell cycle changes. First we investigated whether Pttg1 alone, as a known regulator of G₁ to S phase cell cycle transition 32 with increased protein levels in Wld^s-expressing cells (see Figures 2 and 4), was capable of mediating Wld^s-induced effects on cell cycle proteins. We compared expression levels of four previously highlighted cell cycle proteins following transfection of HEK293 cells with either a Wld^s construct 22 or a Pttg1 over-expression construct 55 (Figure 8a). Three of the four proteins examined were not modified by Pttg1 expression alone (Figure 8a), suggesting that other pathways are also required to induce the full range of cell cycle related changes (see below). However, Ube1 upregulation was induced by Pttg1 over-expression to a similar extent as seen with Wld^s. This suggests that elevated Ube1 protein levels previously reported in Wld^s synapses 31 are occurring downstream from increases in Pttg1 protein levels.

Pttg1 is currently the only known physiological substrate for the E4 ubiquitination factor Ube4b 56, which is one of the constituent parts of the chimeric Wld^s gene 13. In order to establish whether the ability of Pttg1 to be ubiquitinated is important for the regulation of Ube1, we repeated the experiment using an over-expression construct containing a non-ubiquitinatable form of Pttg1 57. The inability to be ubiquitinated completely abolished the ability of Pttg1 to increase Ube1 protein levels (Figure 8b), showing that ubiquitination of Pttg1 by Ube4b (and/or other proteins in the ubiquitin pathway) is likely to be important for Wld^s-mediated cell cycle changes.

Next, we investigated whether NAD-dependent pathways play a role in mediating cell cycle changes, because several recent studies have suggested that the Nmnat1 portion of the chimeric Wld^s gene plays a significant role in conferring a neuroprotective phenotype by elevating NAD levels and increasing sirtuin activity 232425. To examine whether NAD pathways influence cell cycle changes observed in Wld^s-expressing cells, we performed cell cycle pathway specific RT² profiler PCR arrays (using human rather than mouse arrays; see Materials and methods [below]) on HEK293 cells treated with 1 mmol/l NAD applied exogenously to the culture medium. This NAD treatment has previously been shown to confer axonal protection in vitro 23 and to mediate selected Wld^s-induced transcriptional changes 22. Forty-eight out of the 84 genes examined exhibited greater than twofold changes in expression after NAD treatment. In a similar result to that obtained in the Wld^s mouse cerebellar array experiments, the vast majority (47 out of the 84) of modified genes had increased expression levels in the NAD treated cells (Figure 9 and Table 2). Only one cell cycle related gene appeared to be suppressed greater than twofold by NAD (Figure 9). A direct comparison of SuperArray data from Wld^s cerebellum and NAD-treated HEK293 cells showed changes of a similar magnitude for eight out of the nine genes examined (Figure 10a; only nine candidate genes could be directly compared due to their presence/alteration on both arrays). Increases in protein expression levels of Pttg1, BRCA2, BRCA1, and H2Ax in NSC34 cells treated with 1 mmol/l NAD for 4 days confirmed that these NAD-induced changes extend beyond those included on the SuperArray, extend to the protein level, and can occur in neuronal cells (Figure 10b). These data suggest that elevated exogenous NAD levels can mimic many, but importantly not all, Wld^s-induced cell cycle changes.

Alongside identified changes in Pttg1/Ube1 expression and NAD pathways, previous studies have implicated VCP-mediated pathways (also known as p97 and CDC48) in Wld^s-mediated neuroprotection, via its interaction with the Ube4b component of the Wld^s chimeric protein 28. Moreover, VCP is known to be important in early stages of cell cycle progression; VCP is normally localised in the endoplasmic reticulum during nonproliferative states (for example, terminally differentiated neurons), but relocates to the nucleus during late G₁ phase in a cell cycle dependent manner 37. Thus, VCP localisation would not normally be observed in the nucleus of terminally differentiated neurons unless cell cycle had been reactivated and they are progressing toward S phase. To examine whether VCP redistribution associated with modified cell cycle status is modified by Wld^s, we examined VCP localization in the cerebellum of Wld^s and wild-type mice. These experiments revealed an expected cytoplasmic, non-nuclear localization in wild-type neurons, but distinct, strong nuclear puncta in most cerebellar granule cells in Wld^s mice (Figure 11). As predicted from the finding that VCP binds Wld^s 28, VCP localization in the nucleus was consistently found in the same nuclear puncta as Wld^s protein (Figure 11). These data provide further evidence that Wld^s-expressing cells are being pushed toward the early phases of cell cycle re-entry and suggest that VCP binding may play a role in this process.

Thus, Pttg1/Ube1, NAD, and VCP pathways are all likely to be involved in mediating Wld^s-induced modifications in cell cycle status. Taken together, these findings suggest that previous observations of apparently unrelated changes in gene and protein expression/activity downstream of Wld^s can in fact be unified by their ability to modify the cell cycle.

Changes in cell cycle status in terminally differentiated neurons are often associated with corresponding changes in cell stress pathways 585960. To examine whether cell stress pathways were also altered in Wld^s-expressing cells, we used cell stress pathway-specific RT² profiler PCR arrays (see Materials and methods [below]) to compare mRNA levels in the cerebellum of wild-type and Wld^s mice (Figure 12). Fourteen out of the 84 genes contained on the array were modified greater than twofold by Wld^s, showing that a subset of cell stress pathways are also modified in Wld^s (Figure 12 and Table 3). In contrast to the results from cell cycle arrays, however, Wld^s neurons revealed both increases and decreases across a range of different cell stress proteins.

Finally, in order to confirm that Wld^s altered nuclear localization, as well as expression, of cell stress proteins (as for cell cycle proteins shown in Figures 3 and 5), we investigated the expression and distribution of stress-induced phosphoprotein 1 (STI1) in Wld^s-transfected HEK293 cells (Figure 13). We chose to use STI1 as a marker of cell stress in vitro in order to expand our coverage of cell stress modifications beyond those genes/proteins incorporated on the array chip and also because STI1 protein levels are known to be modified in Wld^s synapses in vivo 31. Anti-STI1 antibodies revealed nuclear spots of STI1 in most cells expressing Wld^s (Figure 13a-f). However, neighbouring cells not expressing Wld^s (because of less than 100% transfection efficiency) did not show any STI1 nuclear puncta. No STI1 staining was seen in control cells transfected with eGFP, indicating that stress responses were not simply occurring due to the presence of a large amount of foreign protein in the nucleus (Figure 13g-i). These findings were supported by data from quantitative Western blotting of STI1 protein levels in whole Wld^s cerebellum in vivo, where STI1 levels were increased by 71.6 ± 6.8% (mean ± standard error of the mean; data not shown). Interestingly, we previously showed that STI1 protein levels are decreased in synapses protected by the Wld^s gene in vivo 31. The finding that nuclear STI1 immunoreactivity increases in Wld^s transfected HEK293 cells suggests that some stress proteins may exhibit differential compartmental expression via redistribution within Wld^s-expressing neurons, rather than simply having altered expression levels.

Several studies have shown, using a range of experimental approaches and platforms, that Wld^s robustly downregulates expression of Pttg1 mRNA 2230. Pttg1 plays a well established role in sister chromatid separation during mitosis, but recent data have identified an important additional role as a regulator of G₁ to S phase cell cycle transition 32. In the present study we showed that Pttg1 protein levels are significantly increased in Wld^s-expressing cells. The most parsimonious explanation for the differences between protein and mRNA levels is that decreases in mRNA are generated by a compensatory, self-regulating feedback loop responding to elevated levels of Pttg1 protein. Because Pttg1 is the only known substrate for the Ube4b component of the Wld^s gene 56, it is tempting to speculate that elevated Pttg1 protein levels result from abnormal ubiquitination and targeting for degradation, caused by Wld^s-mediated alterations in the ubiquitin-proteasome pathway 2829. This finding also has implications for previous attempts to directly link Pttg1 to neuroprotection, because earlier studies examined neurodegenerative responses in Pttg1 null mice 22. The current data suggest that repeating these experiments in Pttg1 over-expressing mice might reveal a neuroprotective phenotype, although the undoubted contribution made by other pathways (see below) suggests that Pttg1 over-expression alone would be unlikely to confer the full levels of Wld^s-mediated neuroprotection.

We have also shown that increased Pttg1 protein levels induced by Wld^s are responsible for mediating corresponding increases in expression of another cell cycle-related protein, namely Ube1. Thus, Pttg1 is likely to be a partial mediator of other cell cycle changes induced by Wld^s. We previously identified increased protein levels of Ube1 in a population of striatal synapses from Wld^s mice known to be protected from degeneration 31, suggesting that Ube1 may also play a role in directly modulating degenerative pathways in synaptic compartments of neurons. Importantly, we also found that the ability of Pttg1 to increase Ube1 protein levels was abolished if Pttg1 was expressed in a non-ubiquitinatable form. Because Pttg1 is the only known substrate for the Ube4b component of the Wld^s gene 56, these data suggest that modified ubiquitination of Pttg1 by Ube4b (either in its native form or as part of the Wld^s protein) is likely to be required to mediate downstream changes in proteins such as Ube1. However, at this stage we cannot rule out the possibility that other proteins in the ubiquitin pathway alongside Ube4b are also responsible for mediating this response.

The most convincing evidence to date for the involvement of a single pathway in downstream mediation of the Wld^s phenotype has come from studies showing that the Nmnat1 portion of the chimeric Wld^s gene confers a neuroprotective phenotype via modulation of NAD levels 232425. Here, we have shown that experimental manipulation of NAD levels mimics many (but by no means all) of the cell cycle changes induced by Wld^s. This finding raises the possibility that at least part of the NAD-mediated neuroprotective phenotype is generated by modulating cell cycle status. This suggestion is supported by several studies demonstrating that sirtuin-dependent pathways (which mediate the NAD neuroprotective phenotype 23) play important roles in regulating cell cycle 383940. However, as with the Pttg1 findings discussed above, it is unlikely that NAD-mediated changes alone are sufficient to induce the full range of Wld^s-mediated cell cycle changes, because NAD over-expression alone is not thought to be sufficient to confer the full neuroprotective phenotype in vivo or in vitro 2627.

Alongside Pttg1 and NAD, pathways mediated by VCP (also known as p97 and CDC48) have also been shown to influence cell cycle 61. For example, although VCP is predominantly a cytoplasmic protein, it is known to enter the nucleus during late G₁ phase 37. Here, we have shown that VCP, which is currently the only known binding partner for the Ube4b portion of Wld^s 2862, is localized to the nucleus in the majority of Wld^s-expressing neurons. This is further corroborating evidence that cell cycle has been reactivated in terminally differentiated Wld^s neurons and that they are progressing toward (or beyond) S phase. Although binding to VCP has not yet been demonstrated to be required for the Wld^s phenotype, the present study suggests that if this is an important event, VCP may be acting via regulation of the cell cycle in a similar manner to Pttg1. Cell cycle events potentially attributable to VCP pathways detected in the current experiments include changes in expression levels of BRCA proteins that are known to interact with VCP in the nucleus 63.

The hypothesis that Wld^s may be modifying neurodegenerative pathways in axons and synapses via modulation of the cell cycle is in keeping with other literature on somatic neurodegeneration, in which cell cycle is known to influence vulnerability significantly. For example, it is now known that postmitotic, terminally differentiated neurons in the adult nervous system are not 'permanently postmitotic', but rather depend upon the constant suppression of cell cycle pathways to maintain their arrested status 41. The ability to control cell cycle pathways is therefore a critical factor in stopping neurons entering a vulnerable state, where the risk for neurodegenerative mechanisms being instigated increases significantly 41424344. Numerous examples of cell cycle regulation gone awry, modifying neuronal vulnerability, can be found in neurodegenerative conditions such as motor neuron disease, Alzheimer's disease, and stroke 4546. Furthermore, pharmacologic manipulation of cell cycle progression has been used to confer somatic neuroprotection in animal models of traumatic brain injury and stroke 4748. The current data suggest that the influence of cell cycle status on neuronal vulnerability is likely to extend beyond neurodegenerative mechanisms resident in cell soma to incorporate independent degenerative pathways in axonal and synaptic compartments. The Wld^s gene may therefore provide an important experimental tool for future investigations into pathways through which cell cycle status modulates neuronal vulnerability.

The current data are also likely to be important for interpreting previous and future studies concerning Wld^s-mediated neuroprotection both in vivo and in vitro. Because endogenous cell cycle and cell stress pathways are robustly modified by Wld^s expression, it is difficult to imagine that Wld^s-expressing cells do not have any other covert cellular phenotypes alongside neuroprotection. These may introduce additional variables that could conceivably alter experimental outcomes (for example, comparing Nmnat over-expressing cells in vivo with cells exposed to exogenous NAD in vitro 2324252627).

Current opinion suggests that cell cycle re-entry is damaging to neurons, whereas blocking cell cycle decreases vulnerability 41424344. The finding that the neuroprotective Wld^s gene pushes neurons toward cell cycle re-entry therefore appears at odds with this hypothesis. There are two possible explanations for this discrepancy. First, it is possible that the basic principle of re-entry is bad/suppression is good may not hold for all neurodegenerative pathways. Second, and perhaps more plausibly, it is possible that Wld^s acts to 'prime' the cell against future neurodegenerative insults by inducing early-stage cell cycle changes - and cell stress modifications - without going as far as affecting proliferation and growth stages. This potential mechanism of action is in keeping with a known role for preconditioning, sublethal 'priming' events in conferring neuroprotection by modifying endogenous stress pathways 64656667.

Our finding that cell stress pathways are also modified in Wld^s mice, suggesting a possible 'primed state' of Wld^s neurons, is in keeping with the findings of other recent studies. For example, it was recently demonstrated that the NMNAT1 component of the chimeric Wld^s gene has functions alongside those involving NAD, acting as a chaperone for stress-response proteins such as heat shock protein-70 68 (Wishart TM, Gillingwater TH, unpublished observations). In addition, we have recently shown that the mitochondrial proteome is modified at a basal level in protected Wld^s synapses 31, suggesting intrinsic differences in the ability to respond to cell stress stimuli. This hypothesis has received experimental support from another recent study showing that NMNAT1 can protect against mitochondrial and oxidative stress 69.

Our study has identified possible individual stress-related proteins that may play an important role in the cell stress response in Wld^s neurons. Interestingly, several of these proteins have already been implicated in other neuroprotective situations. For example, STI1 - shown in previous work 31 and the present study to have altered levels and subcellular localization in Wld^s-expressing cells - appears to play an important role in neuroprotection and neuritogenesis 70 as well as in cell proliferation 7172. Further investigations into the in vivo role played by individual cell stress proteins modified by Wld^s, such as STI1, in modulating neuronal phenotypes may therefore provide important insights into mechanisms underlying axonal and synaptic vulnerability.

Natural mutant C57Bl6/Wld^S (Wld^S) mice and C57Bl/6 (wild type) mice (all aged 6 to 8 weeks) were obtained from Harlan Olac Laboratories (Bicester, UK) and housed within the animal care facilities in Edinburgh. Mice (minimum three mice per experimental group) were killed by cervical dislocation and the cerebellum was rapidly removed.

HEK293 and NSC34 (a mouse motor neuron-like cell line 73) cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in 5% carbon dioxide. For transfection with eGFP-Wld^s 22, Pttg1 55, and non-ubiquitinatable Pttg1 57, 5 mg of the DNA was mixed with 10% (vol/vol) 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 22. For some experiments, 1 mmol/l NAD (Sigma-Aldrich, Gillingham, UK) or 10 μmol/l Flavopiridol (obtained through the National Cancer Institute, Rockville, MD, USA) was added to the medium. All cells were incubated for 4 to 5 days and were checked on a phase contrast microscope before proceeding to either immunocytochemistry/microscopy or extraction of protein and/or RNA (at least three cultures for all transfections and treatments).

RNA was extracted from cerebella of age-matched and sex-matched mice, or HEK293 cells, in tri-reagent (Sigma) in accordance with the manufacturer's instructions, as previously described 22. Protein was extracted from cerebella of age-matched and sex-matched mice, or HEK293 cells, in RIPA buffer with 10% protease inhibitor cocktail (Sigma) 31.

Mouse cell cycle (PAMM-020A), cell stress (PAM-003A), and human cell cycle (PAHS-020A) focused pathway arrays (Tebubio Superarrays, Peterborough, UK) in 96-well plate format, compatible with an ABI 7000 real-time PCR machine, were used to assay gene expression changes (three comparisons for each array type). Samples were added to the reaction plates and signal amplification by PCR was carried out using a Sybr-Green '1 step qRT-PCR kit' (Invitrogen). Analysis was carried out using the Analysis Suite spreadsheet provided by Tebubio Superarrays. The absence of DNA contamination and efficiency of amplification was confirmed using the analysis software provided. Gene functions listed in Tables 1 to 3 were obtained from the SuperArray product specification sheets. Raw data for all of these array experiments can be found online 74.

Cerebellar/cultured cell protein was separated by SDS-PAGE on 4% to 20% pre-cast NuPage 4% to 12% Bis Tris gradient gels (Invitrogen) and then transferred to PVDF membrane overnight. The membranes were then blocked using Odyssey blocking buffer (Li-COR Biosciences, Lincoln, Nebraska, USA) and incubated with primary antibodies as per manufacturers instructions (BRCA2, cAbl, CCL3 and E1 ubiquitin activating enzyme [Abcam, Cambridge, MA, USA]; anti-HDAC2 clone 3F3, anti-histone H2B and anti acetyl histone H3 [Lake Placid Biologicals, Lake Placid, NY, USA]; Pds1 Ab1 clone DCS280/Anti-Pttg1 [LabVision Corporation, Freemont, CA, USA]; and antiphosphohistone H2Ax [Upstate, Billerica, MA, USA]). Anti-Wld^s antibodies were a kind gift from Dr Michael Coleman and were used as previously described 2021. Odyssey secondary antibodies were added in accordance with the manufacturer's instructions (Goat anti rabbit IRDye 680 and Goat anti mouse IRDye 800). Blots were imaged using an Odyssey Infrared Imaging System (Li-COR Biosciences). The scan resolution of the instrument ranges from 21 to 339 μm, and in this study blots were imaged at 169 μm. Quantification was performed on single channels with the analysis software provided. Bands were identified according to their relative molecular weight, as detailed in the manufacturer's antibody notes. Bands were delineated using Odyssey software and the arbitrary fluorescence intensity calculated by the software. For each membrane, scans were carried out at three different intensities in order to minimize possible user error in determining correct scan intensities or over-saturation of the membrane. The average of these three separate scans (giving an n of 1 per membrane) was used for further analysis.

MTT assays were carried out in 96-well plates. One hundred micrograms of MTT was added to 6,000 cells per well of NT2 cells transfected with control (vector only) or Wld^s constructs (200 ng [low dose], 600 ng [medium dose], and 1,200 ng [high dose]), and incubated for 3 hours. Media were then removed and formazan crystals dissolved in 50 μl dimethyl sulfoxide. The absorbance of the dimethyl sulfoxide was read at 545 nm to estimate cell number. For 3H-thymidine incorporation assays, 3 × 10⁵ NT2 cells per well of a 24-well plate were transfected with control (vector only) or Wld^s constructs, as detailed above, and proliferation assayed at 48 hours. Mitogenesis was estimated from the measurement of nuclear 3H-thymidine incorporation. Cells were incubated with 0.2 Ci 3H-thymidine (specific activity 70-95 Ci/mmol; Amersham Biosciences, Little Chalfont, UK) for the last 5 hours of culture incubation. Cells were then washed twice in ice-cold phosphate-buffered saline (PBS), followed by 1 ml cold 5% trichloroaceteic acid, and left on ice for 20 minutes. Then, 0.1 mol/l sodium hydroxide was added to the cells before transfer to 4 ml of scintillant. Radioactive counts were determined by scintillation counting. Experiments comprised four replicates each and were performed on at least two separate occasions.

Immunofluorescence staining was performed on either freshly cut (20 μm or 100 μm) cerebellar slices or HEK293 cells, fixed in 4% paraformaldehyde (Fisher Scientific, Loughborough, UK). Slices or cultured cells were incubated overnight in serum blocker consisting of 4% bovine serum albumin (Sigma) and 0.5% Triton X-100 (Sigma) in PBS. In cerebellar slices, anti-Wld^s antibodies (1:500 dilution in serum blocking solution 8) were applied overnight and, after washing with PBS saline, a TRITC-conjugated anti-rabbit secondary antibody (DAKO, Glostrup, Denmark) was applied overnight. Primary antibodies, including Ube1 (Abcam), VCP (Abcam), Sti1 (BD Transduction Laboratories, San Jose, CA, USA), and phosphohistone H2Ax (Upstate), were also used at concentrations specified by the manufacturers. Anti-rabbit/mouse FITC-conjugated secondary antibodies (DAKO) were used on cerebellar slices and anti-rabbit/mouse TRITC-conjugated secondary antibodies (DAKO) were used on HEK293 cells. Secondary antibody only controls were also carried out and confirmed the specificity of antibodies used (data not shown). Finally, cerebellar slices and cultured cells were then washed in PBS and incubated in TOPRO 3 (Molecular Probes, Carlsbad, CA, USA) for 10 minutes before mounting in Mowoil.

Staining was visualised on a laser scanning confocal microscope (BioRad Radiance 2000; BioRad, Hemel Hempsted, UK) and Z-series were merged using Lasersharp (BioRad) software.

All non-SuperArray data were collected in Microsoft Excel and all statistical analyses and graphs were produced using GraphPad Prism. Quantification of cytoplasmic and nuclear Ube1/TOPRO3 fluorescence was undertaken on confocal micrographs captured using identical microscope settings between images and specimens. No image manipulation was undertaken before quantification using standard fluorescence intensity tools in Image J software. Images were prepared for presentation in Adobe Photoshop.