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.