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First published online 3 April 2007
doi: 10.1242/jcs.03438

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Dose-dependent inhibition of proteasome activity by a mutant ubiquitin associated with neurodegenerative disease

Paula van Tijn^1,*, Femke M. S. de Vrij^1,*,, Karianne G. Schuurman¹, Nico P. Dantuma², David F. Fischer^1,, Fred W. van Leeuwen^1,¶ and Elly M. Hol^1,**

¹ Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands
² Department of Cell and Molecular Biology, The Medical Nobel Institute, Karolinska Institutet, Box 285, SE-17177 Stockholm, Sweden

^** Author for correspondence (e-mail: e.hol{at}nin.knaw.nl)

Accepted 21 February 2007

	Summary

Top Summary Introduction Results Discussion Materials and Methods References

 
The ubiquitin-proteasome system is the main regulated intracellular proteolytic pathway. Increasing evidence implicates impairment of this system in the pathogenesis of diseases with ubiquitin-positive pathology. A mutant ubiquitin, UBB⁺¹, accumulates in the pathological hallmarks of tauopathies, including Alzheimer's disease, polyglutamine diseases, liver disease and muscle disease and serves as an endogenous reporter for proteasomal dysfunction in these diseases. UBB⁺¹ is a substrate for proteasomal degradation, however it can also inhibit the proteasome. Here, we show that UBB⁺¹ properties shift from substrate to inhibitor in a dose-dependent manner in cell culture using an inducible UBB⁺¹ expression system. At low expression levels, UBB⁺¹ was efficiently degraded by the proteasome. At high levels, the proteasome failed to degrade UBB⁺¹, causing its accumulation, which subsequently induced a reversible functional impairment of the ubiquitin-proteasome system. Also in brain slice cultures, UBB⁺¹ accumulation and concomitant proteasome inhibition was only induced at high expression levels. Our findings show that by varying UBB⁺¹ expression levels, the dual proteasome substrate and inhibitory properties can be optimally used to serve as a research tool to study the ubiquitin-proteasome system and to further elucidate the role of aberrations of this pathway in disease.

Key words: Ubiquitin, Ubiquitin-proteasome system, Neurodegeneration, Alzheimer's disease, Protein aggregation, Protein degradation

	Introduction

Top Summary Introduction Results Discussion Materials and Methods References

 
The main function of the ubiquitin-proteasome system (UPS) is the proteolytic degradation of target substrates, including aberrant and misfolded proteins, to maintain cellular homeostasis (Glickman and Ciechanover, 2002). UPS-mediated post-translational regulation is also involved in many other cellular pathways such as transcription, DNA repair and endocytosis (Welchman et al., 2005). Ubiquitin (Ub) tags proteins for degradation through an enzymatic cascade, consisting of Ub-activating (E1), Ub-conjugating (E2) and Ub-ligating (E3) enzymes. Via this pathway, Ub is conjugated to internal lysine residues in substrate proteins. Through the sequential addition of Ub molecules to the substrate-bound Ub, a poly-Ub tree is formed which targets the substrate protein for degradation by the 26S proteasome (Glickman and Ciechanover, 2002; Pickart and Cohen, 2004).

As the UPS is important for maintaining intracellular homeostasis, it is not surprising that impairment of the UPS has been observed to occur in the pathogenesis of numerous diseases, often demonstrated by the accumulation of Ub conjugates or other components of the UPS machinery in protein aggregates (Ciechanover and Brundin, 2003). One of the disease-specific proteins that accumulates is ubiquitin-B⁺¹ (UBB⁺¹), a mutant form of Ub formed by a di-nucleotide deletion in the mRNA of the ubiquitin B gene (van Leeuwen et al., 1998). Previous in vitro results showed that UBB⁺¹ is ubiquitylated and appears to be a protein with dual properties; it acts as a ubiquitin-fusion-degradation (UFD) substrate for the proteasome and also acts as a specific inhibitor of the proteasome (Lam et al., 2000; Lindsten et al., 2002). UBB⁺¹ accumulation eventually leads to apoptotic cell death (De Pril et al., 2004; De Vrij et al., 2001) and induces expression of heat-shock proteins (Hope et al., 2003).

In the diseased brain, UBB⁺¹ accumulates in the neuropathological hallmarks of tauopathies; e.g. in neuronal tangles in Alzheimer's disease (AD), but also in astrocytes in Progressive Supranuclear Palsy (Fischer et al., 2003; van Leeuwen et al., 2006). UBB⁺¹ is also found in intranuclear inclusions characteristic of polyglutamine diseases (De Pril et al., 2004). Outside the nervous system, UBB⁺¹ accumulates in the inclusion bodies of the muscle disease inclusion-body myositis (Fratta et al., 2004) and in Mallory bodies of chronic liver disease (McPhaul et al., 2002). We reported that UBB⁺¹ mRNA is present in equal levels in non-demented control individuals compared with AD patients (Fischer et al., 2003; Gerez et al., 2005). This suggests that the UBB⁺¹ mRNA transcript is always present. The UBB⁺¹ protein, however, seems to be efficiently degraded in healthy control subjects. Through a decline in UPS activity, for example by aging or disease-related processes, the degradation of the UBB⁺¹ protein might be affected to such an extent that accumulation of the protein commences. Therefore we have proposed that UBB⁺¹ accumulation can serve as an endogenous reporter for decreased UPS activity (Fischer et al., 2003). Once accumulated, UBB⁺¹ can contribute to disease pathogenesis by inhibiting the UPS (e.g. Hol et al., 2005). It is conceivable that this accumulation of UBB⁺¹ will only occur after exceeding a threshold level, causing a shift in the protein properties from proteasome substrate to proteasome inhibitor.

View larger version (82K): [in this window] [in a new window]   Fig. 1. UBB+1 is degraded by the UPS at low expression levels. (A,B) Western blot of cell lysates of HeLa cells transiently transfected with inducible Tet-off CMV-UBB+1 vectors treated with decreasing Dox concentrations. UBB+1 was detected with anti-UBB+1 antibody (Ub3) (De Vrij et al., 2001) in cells without proteasome inhibitor treatment (A) or in cells treated overnight with 1 µM proteasome inhibitor MG132 (B). CMV-UBB+1 pcDNA3 (UBB+1) and empty pcDNA3 vector (control) transfections served as controls. Arrows in B indicate additional UBB+1 expression after proteasome inhibitor treatment. Protein input was equal in all lanes as determined by Bradford protein measurement (not shown); this is a representative experiment of two duplicate experiments. Molecular mass in kDa is indicated on the left. *UBB+1; **Ub-UBB+1.

	Results

Top Summary Introduction Results Discussion Materials and Methods References

 
UBB⁺¹ accumulates at high expression levels
We hypothesised that the previously described opposing properties of UBB⁺¹ (Hol et al., 2005; Lindsten et al., 2002) might be explained by a dose-dependent shift from UPS substrate to inhibitor. In this study we quantified this effect in living cells using a HeLa cell line stably expressing the Ub^G76V-GFP UPS reporter (Dantuma et al., 2000). Inducible levels of UBB⁺¹ expression were achieved using the Tet-off gene-expression system. UBB⁺¹ protein expression levels were analyzed by western blot (Fig. 1) and Ub^G76V-GFP reporter accumulation was measured in the same sample using flow cytometry (Fig. 2). HeLa cells were transiently transfected with the UBB⁺¹ Tet-off constructs and after 16 hours, doxycycline (Dox) was added to the culture medium for 48 hours to regulate UBB⁺¹ expression. Western blot analysis on cell lysates showed that UBB⁺¹ protein accumulation was present only at high expression levels induced by absence of Dox (maximal expression) or low Dox concentrations ranging from 0 to 0.01 ng/ml (Fig. 1A). In addition, ubiquitylation of UBB⁺¹ (Ub-UBB⁺¹) was present in cells that showed UBB⁺¹ accumulation and in cells transiently transfected with a CMV-UBB⁺¹ pcDNA3 high expression control plasmid, as expected (Lindsten et al., 2002) (Fig. 1A). Ubiquitylation of UBB⁺¹ is essential for targeting UBB⁺¹ to the proteasome. A double lysine mutant of UBB⁺¹, UBB^+1K29,48R, cannot be ubiquitylated and is not targeted to the proteasome (Lindsten et al., 2002). Transient transfection with a CMV-UBB^+1K29,48R pcDNA3 plasmid indeed showed an increased accumulation of the non-ubiquitylated form of this protein compared with CMV-UBB⁺¹ pcDNA3 (not shown).

View larger version (60K): [in this window] [in a new window]   Fig. 2. High levels of UBB+1 inhibit the proteasome. Flow cytometric analysis of UbG76V-GFP HeLa cells for GFP fluorescence as an indication of UPS inhibition (% of cells that are GFP positive). (A) Cells were transiently transfected with empty pcDNA3 vector (control), CMV-Ub wild-type pcDNA3 (Ubwt), CMV-UBB+1K29,48R pcDNA3 (+1K29,48R) or CMV-UBB+1 pcDNA3 (UBB+1) or treated with 100 nM epoxomicin (epox) or 1 µM MG132. Significant accumulation of GFP compared with empty vector control is marked with an asterisk (*P<0.005, ANOVA Bonferroni). (B) UbG76V-GFP cells were transiently transfected with the inducible Tet-off UBB+1 expression system and treated with decreasing concentrations of Dox. Significant increase in the percentage of GFP-positive cells compared with levels in cells treated with 1000 ng/ml Dox is marked by an asterisk (*P<0.05, ANOVA Bonferroni). Results are the means ± s.e.m. of three or four independent duplicate experiments. (C) Representative flow cytometric scatter plots of UBB+1 Tet-off transfected cells treated with 1000, 0.01, 0.0001 and 0 ng/ml Dox as shown in B. GFP-positive cells were detected in the region set as R1. The mean GFP fluorescence and the percentage of cells that are GFP-positive are indicated on the right.

UBB⁺¹ induces dose-dependent UPS inhibition
Our previous work showed that UBB⁺¹ can act as a proteasome inhibitor (Lindsten et al., 2002). Thus, we next determined the expression level at which inducible UBB⁺¹ inhibited the proteasome and whether this proteasome inhibition was dose-dependent. Therefore, we transfected stable HeLa Ub^G76V-GFP cells with the inducible Tet-off UBB⁺¹ vectors as described in Fig. 1. General inhibition of the proteasome leads to accumulation of GFP in this Ub^G76V-GFP HeLa cell line, a reporter cell line for UPS activity (Dantuma et al., 2000). Indeed, flow cytometric analysis of HeLa Ub^G76V-GFP cells treated overnight with proteasome inhibitors epoxomicin (irreversible inhibitor, 100nM) or MG132 (reversible inhibitor, 1 µM) showed a large increase in the percentage of GFP-positive cells to 90% (Fig. 2A). Transient transfection with CMV-UBB⁺¹ pcDNA3 resulted in significant accumulation of the GFP reporter in 10% of living cells (Fig. 2A), reaffirming our previous observations that UBB⁺¹ acts as a proteasome inhibitor (Lindsten et al., 2002). The discrepancy between the high levels of UPS inhibition achieved with classical inhibitors (90% GFP positive) compared with UBB⁺¹ (10% GFP positive) is partially due to the transfection efficiency of UBB⁺¹, which is routinely 40% in this cell line (data not shown) and probably also due to the fact that not all UBB⁺¹-positive cells have built up enough expression to surpass the threshold level for UPS inhibition, corroborating our hypothesis that a critical level of UBB⁺¹ must be reached before the protein is stabilised. As expected, transient transfection with CMV-UBB^+1K29,48R pcDNA3, which is not directed to the UPS, did not lead to significant UPS inhibition (Fig. 2A). Also transfection of wild-type Ub (CMV-Ub pcDNA3) did not lead to significant accumulation of the GFP reporter (Fig. 2A).

We determined whether the proteasome inhibition induced by UBB⁺¹ was dose dependent by transfecting HeLa Ub^G76V-GFP cells with the inducible Tet-off UBB⁺¹ vectors. Maximal UBB⁺¹ expression (absence of Dox) was sufficient to induce UPS inhibition, shown by significant accumulation of the GFP reporter, although the percentage of GFP-positive cells (4.4%) was lower than after transient transfection with CMV-UBB⁺¹ pcDNA3 (Fig. 2B). Furthermore, increasing GFP reporter accumulation was visible in a range from 0.01 to 0.0001 ng/ml Dox, reaching significance at Dox concentrations of 0.001 and 0.0001 ng/ml (Fig. 2B). This increase in the percentage of GFP-positive cells and in the mean GFP fluorescence intensity is visualised in representative plots in Fig. 2C. These results indicate that UBB⁺¹ inhibited the proteasome in a dose-dependent manner, starting from expression levels at which UBB⁺¹ accumulation commenced.

Accumulation of UBB⁺¹ and UPS inhibition is reversible in living cells
The previous results showed that increasing UBB⁺¹ expression gave rise to dose-dependent UPS inhibition. It is conceivable that this UPS inhibition is irreversible, causing a defective degradation of previously accumulated UBB⁺¹ even after UBB⁺¹ expression is shut down. To verify this we measured the percentage of remaining UBB⁺¹-positive cells after shutting down UBB⁺¹ expression. Ub^G76V-GFP HeLa cells were transiently transfected with the Tet-off UBB⁺¹ vectors and expressed high levels of UBB⁺¹ for 64 hours, after which a baseline sample was taken (time point 0 hours). UBB⁺¹ expression was continued at high levels and additional samples were taken at 12 hours and 36 hours after the baseline measurement. Alternatively, UBB⁺¹ expression was shut down by adding 10 ng/ml Dox and samples were taken at similar time points. The percentages of UBB⁺¹-positive cells (Fig. 3A) and GFP/UBB⁺¹ double-positive cells indicative for UPS inhibition (Fig. 3B) were determined for every time point using flow cytometry (minimum of 10,000 cells counted per sample).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Accumulation of UBB+1 is reversible after shutting off expression. Flow cytometric analysis of the percentage of UBB+1 (A) or UBB+1/GFP (B) positive cells at 0 hours, 12 hours and 36 hours after shutting down maximal UBB+1 expression. UbG76V-GFP HeLa cells were not transfected (control), transfected with empty pcDNA3 vector (e) or transfected with the UBB+1 Tet-off vectors. After 64 hours of maximal expression (absence of Dox) a baseline sample was taken (0 hours). UBB+1 expression was continued at maximal levels (Dox–) or shut down by addition of 10 ng/ml Dox (Dox+) and samples were analyzed after 12 hours and 36 hours of Dox treatment. Results are the means ± s.e.m. of two or three independent duplicate experiments, *P<0.01; #p=0.099 ANOVA between groups indicated at 36 hours.

The percentage of UBB⁺¹/GFP double-positive cells remained stable after 12 hours in both the Dox-treated and untreated condition (Fig. 3B). After 36 hours, this population of cells decreased in both the continuous UBB⁺¹ expression condition (Dox–) and after shutting down expression (Dox+). However, similarly to the results for UBB⁺¹ accumulation, the UBB⁺¹/GFP-positive cell population was lower in the Dox-treated condition than in the condition with constant UBB⁺¹ expression (Dox–), although this effect was not significant (Fig. 3B). This lower amount of GFP reporter accumulation in the condition where UBB⁺¹ expression has been shut down for 36 hours indicated that the UPS inhibition induced by UBB⁺¹ was a reversible process. Moreover, possible UBB⁺¹-induced cell death should be lower when UBB⁺¹ expression is shut down, so the decrease in GFP accumulation can then only be attributed to the reversal of UPS inhibition.

To corroborate these data, we also used an experimental setup where high levels of UBB⁺¹ expression were induced with lentiviral (LV) vectors in a human neuroblastoma cell line (SH-SY5Y). By using LV transduction, the UBB⁺¹ cDNA integrated in the cellular DNA, leading to stable expression of the protein. With this high continuous LV-UBB⁺¹ expression, experiments could be done in a shorter time span in which UBB⁺¹-induced cell death did not occur yet. In this setup, LV-UBB⁺¹ transduction induced UBB⁺¹ accumulation, which increased further after overnight inhibition of the UPS with the reversible inhibitor MG132. After restoring proteasome activity by removing MG132, the percentage of UBB⁺¹-positive cells decreased significantly compared with the condition in which MG132 was not removed (data not shown), indicating that UBB⁺¹ accumulation is a reversible process. The percentage of UBB⁺¹-positive cells in the condition with maximal UBB⁺¹ expression was comparable to the percentage of positive cells after a control LV-UBB^+1K29,48R infection. It is known from previous experiments that this control construct does not induce cell death (De Pril et al., 2004), thereby ruling out the possibility that UBB⁺¹-induced cell death affected the reversibility of UBB⁺¹ accumulation in this setup.

Degradation of UPS substrates in organotypic cortex cultures
We then investigated whether these results in cell lines also hold true in primary (neuronal) cultures using mouse organotypic cortex slice cultures, so that we could extrapolate our data to neurodegenerative disease conditions. First, we verified that transduction of cells was possible in this setup using LV vectors. Cortex slice cultures of C57Bl/6 mice were infected with Ub-M-GFP – a ubiquitin fusion protein that results in a stable form of GFP (Dantuma et al., 2000). This gave rise to many GFP immunopositive cells two days after transduction (Fig. 4). The transduced cell population consisted mainly of astrocytes, although also a substantial amount of GFP-positive neurons were present in the slice, as demonstrated by double-staining with neuronal (NeuN) and glial [glial fibrillary acidic protein (GFAP)] markers (Fig. 4).

View larger version (148K): [in this window] [in a new window]   Fig. 4. Lentiviral transduction targets a heterogeneous cell population in cortex slice cultures. GFAP (red) and NeuN (blue) double staining on LV-Ub-M-GFP transduced organotypic cortex slice cultures of C57Bl/6 mice revealed mostly GFAP-labelled GFP-positive glia, but also GFP-positive neurons. Arrows indicate transduced neurons, positive for both GFP and Neu-N. Bar, 50 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 5. UBB+1 is degraded by the proteasome in cortex slice cultures. Organotypic cortex slices of C57Bl/6 mice were transduced with LV-UbG76V-GFP, LV-UBB+1 or LV-UBB+1K29,48R. Both the UPS reporter protein UbG76V-GFP (green) and UBB+1 (red) are efficiently degraded by the 26S proteasome and only accumulate after treatment with proteasome inhibitor. The lysine mutant of UBB+1, UBB+1K29,48R, is not degraded by the proteasome and accumulates without inhibitor treatment. – epox, not treated with epoxomicin; + epox, treated overnight with 1 µM epoxomicin. Bar, 100 µm.

UBB⁺¹ accumulation is not reversible in cortex cultures
We made use of the reversible proteasome inhibitor MG132 to study whether accumulation of UBB⁺¹ in cortex cultures also decreased after wash out of the proteasome inhibitor as we observed in the neuroblastoma cell line experiments. Consistent with results obtained with epoxomicin treatment shown above, applying MG132 overnight to LV-transduced cultures resulted in strong accumulation of both Ub^G76V-GFP and UBB⁺¹ (Fig. 6). When MG132-treated cultures were rinsed and allowed to recover, proteasome activity was restored, as demonstrated by the regained capacity to completely degrade the GFP reporter protein (Fig. 6). This restored proteasome activity did not seem capable of degrading accumulated UBB⁺¹ under these washout conditions, because the number of cells containing accumulated UBB⁺¹ after washout of the inhibitor was similar to the number of UBB⁺¹-positive cells after initial transduction (Fig. 6). This indicated that, in contrast to the results obtained in cell lines, UBB⁺¹ accumulation in cortex cultures remained present after washout of the proteasome inhibitor, which might be due to the recovery time needed after washout to degrade UBB⁺¹.

View larger version (67K): [in this window] [in a new window]   Fig. 6. UBB+1 remains present after washout of inhibitor in cortex slice cultures. Overnight incubation of cortex cultures transduced with LV-UbG76V-GFP or LV-UBB+1 with the reversible proteasome inhibitor MG132 (10 µM) resulted in accumulation of both proteins. Washing out the reversible inhibitor reactivated the proteasome, as shown by the degradation of the proteasome reporter substrate UbG76V-GFP. However, UBB+1 remained in a considerable number of cells after reactivation of the proteasome. Transduction with the LV-UBB+1K29,48R control construct gave rise to accumulation of the UBB+1 protein regardless of proteasome inhibitor treatment. UbG76V-GFP is depicted in green, UBB+1 in red, and the nuclear staining (TO-PRO) in blue. Bar, 500 µm.

Accumulated UBB⁺¹ inhibits the UPS in cortex cultures
In vitro, UBB⁺¹ inhibits the UPS after exceeding a threshold as shown in a Ub^G76V-GFP stable HeLa cell line. We used a Ub^G76V-GFP transgenic (tg) mouse line (Ub^G76V-GFP/2) to translate these results to our cortex culture setup. In this mouse model, the UPS reporter is ubiquitously expressed and similar to the HeLa cell line, GFP accumulates only after proteasome inhibition (Lindsten et al., 2003). To verify the GFP proteasome reporter system in organotypic cortex slices of Ub^G76V-GFP/2 tg mice, the cortex slices were cultured and treated with 1 µM epoxomicin. Indeed, the GFP reporter only accumulated in the cortex slice cultures after treatment with proteasome inhibitor (Fig. 7). Similar to cortex slices from non-tg mice, in the Ub^G76V-GFP/2 cortex slices, LV-UBB⁺¹ transduction did not lead to UBB⁺¹ accumulation (Fig. 8B) unless additional proteasome inhibitors were applied (not shown). This additional proteasome inhibitor treatment led to GFP reporter accumulation in the tg cultures regardless of UBB⁺¹ expression, making it impossible to distinguish proteasome inhibition by the inhibitor or by UBB⁺¹. Therefore, we used adenoviral (Ad) instead of LV transduction of UBB⁺¹ to induce higher expression levels, which might exceed the accumulation threshold. We confirmed the increased expression of Ad-UBB⁺¹ (right lane, Fig. 8A) compared with LV-UBB⁺¹ (left lane, Fig. 8A) in HEK293 cells, showing that Ad-UBB⁺¹ transduction resulted in four- to fivefold higher expression of UBB⁺¹ compared with LV transduction (Fig. 8A). Transduction of Ub^G76V-GFP tg cortex cultures with Ad-UBB⁺¹ indeed resulted in accumulation of UBB⁺¹ in many cells (Fig. 8C), in contrast to LV-UBB⁺¹ transduction (Fig. 8B). The majority of cells that were positive for UBB⁺¹ after Ad-UBB⁺¹ transduction clearly accumulated the GFP reporter (Fig. 8D). UBB⁺¹ accumulation was observed mainly in the cytosol, whereas the GFP reporter accumulation was present in the cytosol as well as in the nucleus. Similarly to the in vitro results, inducing a high level of UBB⁺¹ expression in organotypic cortex slice resulted in surpassing the accumulation threshold and subsequent inhibition of the proteasome.

View larger version (76K): [in this window] [in a new window]   Fig. 7. The UPS reporter system in cortex cultures of UbG76V-GFP transgenic mice. (A) UbG76V-GFP tg organotypic cortex cultures without treatment with proteasome inhibitors. (B) UbG76V-GFP tg cortex cultures treated with 1 µM epoxomicin. The GFP-reporter substrate only accumulated after proteasome inhibition. Bars, 50 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 8. High Ad-UBB+1 expression causes proteasome inhibition in cortex cultures. High levels of UBB+1 expression with Ad-UBB+1 led to accumulation of UBB+1 without inhibitor treatment. (A) Representative western blot of HEK293 cell lysates transduced with equal MOI of LV-UBB+1 (left lane) or Ad-UBB+1 (right lane). Equal amounts of protein were loaded per lane, as confirmed by Coomassie Blue staining of total protein load of the same lanes shown on the right. The blot was stained with anti-UBB+1 antibody Ub3 and quantified with Imagepro software (quantification not shown). (B,C) Organotypic cortex slice cultures of UbG76V-GFP tg mice were transduced with LV-UBB+1, which did not induce UBB+1 accumulation (B) or Ad-UBB+1, which did result in many UBB+1-immunopositive cells (C). (D):UBB+1 accumulation after adenoviral transduction led to accumulation of UbG76V-GFP (arrows). Bars, 250 µm (B); 500 µm (C); 50 µm (D).

	Discussion

Top Summary Introduction Results Discussion Materials and Methods References

 
Our previous results indicated that UBB⁺¹ behaves both as a substrate as well as an inhibitor of the UPS (Fischer et al., 2003; Lindsten et al., 2002). In this study we further explored the dual UPS substrate and UPS inhibitor properties of UBB⁺¹, using novel Tet-off inducible UBB⁺¹ expression vectors to vary levels of UBB⁺¹ expression. We show here that a concentration-dependent shift in UBB⁺¹ properties from UPS substrate to inhibitor takes place with increasing expression levels. UBB⁺¹ accumulation commenced only at high expression levels and preceded the induction of UPS inhibition. In addition, we show in this study that both UBB⁺¹ accumulation and UPS inhibition were partially reversible after ceasing UBB⁺¹ expression. We further studied UBB⁺¹ characteristics in organotypic cortex slice cultures, a system that reflects a multicellular environment in which neuronal connectivity and neuron-glia interactions are preserved (Sundstrom et al., 2005). This study is, to our knowledge, the first to use organotypic cultures to assess UBB⁺¹ properties. In these cultures, UBB⁺¹ accumulation and subsequent UPS inhibition only occurred at high levels of expression, similar to the results obtained in cell lines. In summary, the current study shows that UBB⁺¹ properties dose-dependently shift from a proteasome substrate to a partially reversible proteasome inhibitor after a critical level of accumulation is reached.

We hypothesised that UBB⁺¹ might be able to irreversibly sustain or even increase its own accumulation through a `feedback loop' of UBB⁺¹-induced UPS inhibition. Surprisingly, a clear decrease was observed in the percentage of UBB⁺¹-positive cells after shutting down expression of UBB⁺¹ (Fig. 3A) or recovery after UPS inhibition, although UBB⁺¹ levels remained elevated compared with the levels before treatment. It is conceivable that this decline of UBB⁺¹ accumulation continues over time, clearing the remaining accumulation after a longer period of recovery. This reversible accumulation was not as clear in cortex slices; many UBB⁺¹-positive cells remained present after recovery of proteasomal inhibition in a setup where full degradation of the UPS reporter Ub^G76V-GFP was observed (Fig. 6). In these slices, 16 hours of UPS recovery might not be sufficient to observe a clear UBB⁺¹ degradation. These results indicate that the UBB⁺¹ protein probably has a longer half-life than Ub^G76V-GFP in cortex slices, corresponding to previous cell line observations regarding the half-life of UBB⁺¹ compared with Ub^G76V-GFP (Lindsten et al., 2002). Also, a slight decrease in UBB⁺¹ intensity or number of UBB⁺¹-positive cells might not be detectable in cortex slices because exact quantification is not feasible owing to variation. Alternatively, primary neurons and astrocytes might respond differently to the inhibition of the UPS compared with tumour cell lines.

A point of interest is that LV vectors are known to efficiently transduce neuronal cells in culture (Ehrengruber et al., 2001). In our organotypic slices we observed that the majority of the infected population consisted of GFAP-positive glial cells, although neuronal cells were also transduced (Fig. 4). In this respect it is important to note that in the human brain, UBB⁺¹ not only accumulates in neurons in for instance AD, but also in glial cells of white matter in e.g. Progressive Supranuclear Palsy (Fischer et al., 2003). The UBB⁺¹ protein was mainly localised in the cytosol of the transfected cells in the slices. Surprisingly, UBB⁺¹-positive cells showed accumulation of the Ub^G76V-GFP reporter in both the cytosol and in the nucleus (Fig. 8D). Intranuclear localisation of the GFP reporter was also seen in Ub^G76V-GFP tg cortex cultures (Fig. 4) and in neuronal cultures from a comparable Ub^G76V-GFP tg line (Lindsten et al., 2003) treated solely with proteasome inhibitor, indicating that general UPS inhibition results in both cytosolic and nuclear accumulation of the GFP reporter.

Besides the UBB⁺¹ protein, shown in this study to be a dose-dependent reversible UPS inhibitor, many other compounds are known that inhibit the proteolytic activity of the 26S proteasome. These inhibitors can be divided into several major classes such as the synthetic peptide aldehydes, which act upon reversible binding (e.g. MG 132, MG 115 and PSI), the peptide boronates, a class of highly selective reversible inhibitors that have a slow on-off rate (e.g. MG262 and PS-341) and peptide vinyl-sulfones which bind irreversibly to the 20S core. Lactacystin and the specific 26S proteasome inhibitor epoxomicin are natural non-peptide irreversible proteasome inhibitors (reviewed by Kisselev and Goldberg, 2001; Myung et al., 2001). In this respect, UBB⁺¹ can be classified as an endogenously encoded inhibitor of the UPS, which induces dose-dependent reversible UPS inhibition (Fig. 1B). UPS inhibition by UBB⁺¹ is a specific effect of UBB⁺¹ and is not due to overloading the UPS by overexpressing a UPS substrate, because comparable expression of other UPS substrates such as ^FLAGUb^G76V-nfGFP, ^FLAGUb-R-nfGFP or ^FLAGp53 did not inhibit the UPS (Lindsten et al., 2002). Through the possibility to vary UBB⁺¹ expression levels in different models systems (e.g. cell lines, primary cultures and transgenic animal models) using inducible vectors, the dual UPS substrate and UPS inhibitory properties of this protein can be optimally employed.

In this study we further validate our hypothesis that the presence of UBB⁺¹ serves as a marker for proteasomal dysfunction in human neurodegenerative disease (Fischer et al., 2003; Hol et al., 2005), showing in human cell lines and neuronal slice cultures that inhibition of the proteasome using proteasome inhibitors can induce the accumulation of UBB⁺¹, even if at these specific expression levels UBB⁺¹ is normally degraded. Accordingly, UBB⁺¹ accumulation in human pathology can serve as an endogenous marker for UPS inhibition, which holds UPS inhibitory properties itself (Fig. 9). Recently, it was shown that a decline in proteasome activity induced by classical proteasome inhibitors impaired protein synthesis in neuronal cells (Ding et al., 2006) and disrupted multiple processes in ribosome biogenesis (Stavreva et al., 2006). After washout of the reversible proteasome inhibitor MG262, levels of protein synthesis were restored (Ding et al., 2006). As UBB⁺¹ is also an inhibitor of the proteasome, it is possible that we underestimated the extent of UBB⁺¹ accumulation in our experiments owing to a decreased UBB⁺¹ synthesis. Also the protein synthesis levels of the GFP-reporter might be decreased. Conversely, the reversal of UBB⁺¹ accumulation after shutting down UBB⁺¹ expression might be influenced by an increased level of protein synthesis, possibly slowing the process of accumulation reversal. Decreases in ribosome function and protein synthesis are also associated with aging (reviewed by Rattan, 1996) and neurodegenerative disease (Ding et al., 2005). Possibly, the accumulated UBB⁺¹ in the hallmarks of neurodegenerative disease further contributes to these processes via its UPS inhibitory properties. Future experiments could clarify this issue.

View larger version (32K): [in this window] [in a new window]   Fig. 9. UBB+1 properties shift from UPS substrate to inhibitor. (1) UBB+1 mRNA and translation levels are constant throughout life (Fischer et al., 2003; Gerez et al., 2005). In non-diseased tissue, the 26S proteasome is capable of degrading all the translated UBB+1 and accumulation of UBB+1 is not present. (2) Owing to various causes such as disease or aging, the efficiency of proteasomal degradation can decrease, leading to a diminished degradation of UBB+1. (3) The levels of translated UBB+1 exceed the degradation capacity of the proteasome and surpass the accumulation threshold. Accumulated UBB+1 now holds UPS inhibitory properties itself, which can aggravate the initial decrease in UPS activity.

The reversible inhibitory properties of UBB⁺¹ can prove to be useful in many research fields besides neurobiology. The UPS is well known is its function in protein quality control, but increasing significance is attributed to its role in development, endocytosis, DNA repair and transcriptional regulation (Welchman et al., 2005). In this respect, the role of UPS-regulated protein turnover in cell cycle progression has become of major importance in cancer research because oncogenic mutations can been found that perturb ubiquitylation of cell cycle proteins and induce the disruption of intracellular balance between the cell growth and death characteristic of cancer cells (Mani and Gelmann, 2005). Therefore it is not surprising that proteasome inhibitors have emerged as attractive drug targets for e.g. cancer therapy (Hol et al., 2006; Nalepa et al., 2006). The first clinically approved anti-cancer drug in this respect is bortezomib (Velcade®), a small-molecule UPS inhibitor used to treat malignant multiple myeloma (reviewed by Rajkumar et al., 2005), which can induce apoptosis in tumour cells (Adams, 2004). As UBB⁺¹ is an endogenously encoded UPS inhibitor that induces apoptosis, it is possible to mediate tissue-specific gene delivery by viral vectors. This unique combination makes it worthwhile to further explore the potential of UBB⁺¹ as a tissue-specific UPS inhibitor in disease.

	Materials and Methods

Top Summary Introduction Results Discussion Materials and Methods References

 
Plasmid construction and viral constructs
Ub, UBB⁺¹ and UBB^+1K29,48R open reading frames were cloned in pcDNA3 (Invitrogen) as described earlier (Lindsten et al., 2002). For the Tet-off inducible expression system the UBB⁺¹ open reading frame was cloned downstream of the TRE-minimal cytomegalovirus immediate early (CMV) sequence of a Tet-off expression vector (pRevTRE; Clontech) into pcDNA3 and co-transfected with pRev Tet-Off (Clontech). First generation recombinant adenoviral vectors Ad-UBB⁺¹ and Ad-Ub were generated, purified and titred as described elsewhere (De Vrij et al., 2001; Hermens et al., 1997). Adenoviral vectors were based on the Ad5 mutant dl309 (Jones and Shenk, 1979) and used the CMV promoter to drive transgene expression. Titration of double CsCl gradient-purified Ad-CMV-UBB⁺¹ and Ad-CMV-Ub on the permissive cell line 911 (Fallaux et al., 1996) revealed titres of 1x10⁹ plaque forming units/ml. Lentiviral vectors were generated by cloning DNA encoding Ub-M-GFP, Ub^G76V-GFP, UBB⁺¹ or UBB^+1K29,48R into the lentiviral transfer plasmid pRRLsin-ppThCMV. Lentivirus was produced according to Naldini et al. (Naldini et al., 1996) and harvested and titred as described previously using a HIV-1 p24 coat protein ELISA (NEN Research, Boston, USA) (De Pril et al., 2004). Virus titres were correlated to titres determined by counting GFP fluorescent cells of an LV-Ub-M-GFP stock. In this way titres of adenoviral and lentiviral stocks could be correlated.

Cell lines and transfections
The human cervical epithelial carcinoma cell line HeLa stably transfected with Ub^G76V-GFP (Dantuma et al., 2000) was cultured in high-glucose Dulbecco's modified Eagle medium, containing 10% FCS, supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (all Gibco). Stable cell line HeLa Ub^G76V-GFP was maintained on 60 µg/ml geneticin (G418; Gibco) selection. For western blots and flow cytometry, HeLa Ub^G76V-GFP cells were plated on 6- or 12-well plates with 1x10⁵ cells/well or 5000 cells/well respectively 1 day prior to transfection. Cells were transiently transfected with the calcium phosphate method using 1 µg/ml DNA per vector. Where mentioned doxycycline (Sigma-Aldrich) treatment was applied 16 hours after transfection. Samples were harvested 48 hours after continuous doxycycline treatment, unless stated otherwise. Where indicated cells were additionally treated with the proteasome inhibitors MG132 (1 µM; Affiniti Research) or epoxomicin (100 nM; Affiniti Research) for 16 hours before samples were taken.

Western blotting
For western blotting, cell pellets were resuspended in suspension buffer (0.1 M NaCl, 0.01 M Tris-HCl pH 7.6, 1 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin) and lysed by sonification for 2x 20 seconds. Total protein content was quantified by Bradford assay, equal protein amounts were fractionated by SDS-PAGE and blotted semi-dry to nitrocellulose filters (Schleicher and Schuell, Germany). UBB⁺¹ was detected using rabbit polyclonal anti-UBB⁺¹ antibody [Ub3 serum; 05/08/97; 1:1000 overnight (De Vrij et al., 2001)] and secondary swine anti-rabbit HRP (Dako; 1:1000) diluted in Supermix (0.05 M Tris-HCl, 0.9% NaCl, 0.25% gelatine and 0.5% Triton-X-100, pH 7.4). Blots were developed by enhanced chemoluminescence (Lumilight ECL, Perkin Elmer).

Flow cytometry
For flow cytometry, cell suspensions were fixed in 4% formalin in PBS and resuspended in PBS-0.5% bovine serum albumin (Roche). GFP could be directly visualised. For UBB⁺¹ cytometry, cells were stained with primary antibody anti-UBB⁺¹ (Ub3 serum; 1:500) and secondary antibody anti-rabbit Cy5 (Jackson ImmunoResearch; 1:400). Analysis was performed on at least 10,000 cells per sample with a flow cytometer (FACSCalibur, Becton Dickinson Biosciences); data were analyzed using CellQuest software (Becton Dickinson Biosciences).

Organotypic cortex slice cultures
C57BL/6 or C57BL/6 Ub^G76V-GFP/2 tg mice (Lindsten et al., 2003) were decapitated at post natal day 5; the brain was transferred to ice-cold Gey's Balanced Salt Solution (Sigma-Aldrich) containing 5.4 mg/ml glucose, 100 U/ml penicillin and 100 µg /ml streptomycin (all Gibco). After removal of the meninges, the fronto-parietal part was sliced into 300 µm coronal sections per hemisphere using a tissue chopper (McIlwain). The first four slices were discarded. Slices were cultured on an air-fluid interface on culture plate inserts (Millipore; 0.4 µm pore; 30 mm diameter; three cultures per insert) on medium containing 50% Minimum Essential Medium alpha, 25% HBSS, 25% horse serum, 6.5 mg/ml glucose, 2 mM glutamine (all Gibco) and penicillin/streptomycin (100 U/ml, 100 µg/ml). Viral transduction of cultures was achieved by applying 1x10⁶ transducing units of virus in a 10 µl droplet of culture medium on top of the slices. Treatment with epoxomicin (1 µM; Affiniti Research) or MG132 (10 µM; Affiniti Research) was performed in the same manner. Inhibitors were applied for 6 hours and subsequently left on or washed out overnight. Slices were stained free floating with rabbit polyclonal anti-UBB⁺¹ (Ub3 serum; 1:1000), rabbit polyclonal anti-GFAP (DAKO; 1:4000), monoclonal anti-GFP (Chemicon; 1:500) and monoclonal NeuN (Chemicon; 1:400) diluted in Supermix, followed by Cy2 and Cy3 staining (Jackson ImmunoResearch; 1:800) Nuclei were visualised with TO-PRO-3 (Molecular Probes; 1:1000). Subsequently, slices were mounted in mowiol (0.1 M Tris-HCl pH 8.5, 25% glycerol, 10% w/v Mowiol 4-88) images were acquired using confocal laser scanning microscopy (Zeiss 510) and accompanying software (Zeiss LSM Image Browser).

	Acknowledgments

 
We would like to thank L. Naldini (University of Torino, Torino, Italy) for the lentiviral constructs and K. Lindsten and V. Menéndez-Benito (Karolinska Institute, Stockholm, Sweden) for assistance with the work on the Ub^G76V-GFP transgenic mice. This research was supported by HFSPO grant RG0148/1999B, the "Hersenstichting Nederland" H00.06 and 12F04.01, ISAO (01504, 04507 and 04830), MW-NWO/Swedish MRC 910-32-401 and Stichting "De drie lichten" 00/56.

	Footnotes

 
^* These authors contributed equally to this work

Present address: Department of Clinical Genetics, Erasmus MC, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands

Present address: Galapagos Genomics, PO Box 2048, 2301 CA Leiden, The Netherlands

^¶ Present address: Department of Psychiatry and Neuropsychology, Division of Cellular Neuroscience, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands

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