Accumulation of ubiquitin conjugates in a polyglutamine disease model occurs without global ubiquitin/proteasome system impairment

Abstract

Aggregation-prone proteins have been suggested to overwhelm and impair the ubiquitin/proteasome system (UPS) in polyglutamine (polyQ) disorders, such as Huntington's disease (HD). Overexpression of an N-terminal fragment of mutant huntingtin (N-mutHtt), an aggregation-prone polyQ protein responsible for HD, obstructs the UPS in cellular models. Furthermore, based on the accumulation of polyubiquitin conjugates in brains of R6/2 mice, which express human N-mutHtt and are one of the most severe polyQ disorder models, it has been proposed that UPS dysfunction is a consistent feature of this pathology, occurring in both in vitro and in vivo models. Here, we have exploited transgenic mice that ubiquitously express a ubiquitin fusion degradation proteasome substrate to directly assess the functionality of the UPS in R6/2 mice or the slower onset R6/1 mice. Although expression of N-mutHtt caused a general inhibition of the UPS in PC12 cells, we did not observe an increase in the levels of proteasome reporter substrate in the brains of R6/2 and R6/1 mice. We show that the increase in ubiquitin conjugates in R6/2 mice can be primarily attributed to an accumulation of large ubiquitin conjugates that are different from the conjugates observed upon UPS inhibition. Together our data show that polyubiquitylated proteins accumulate in R6/2 brain despite a largely operative UPS, and suggest that neurons are able to avoid or compensate for the inhibitory effects of N-mutHtt.

The primary proteolytic machinery responsible for the turnover of proteins in the cytosol and nuclei of cells, including the destruction of misfolded or otherwise abnormal proteins, is the ubiquitin/proteasome system (UPS) (1). The UPS is in essence a two-step process: the targeting of proteins through the covalent linkage of polyubiquitin chains (2), and the destruction of ubiquitylated proteins by the proteasome (3). A number of studies have implicated UPS dysfunction in a range of polyglutamine (polyQ) neurodegenerative diseases (4). The aggregation-prone polyQ proteins are postulated to impair the UPS in these diseases, either by overloading the capacity of the cell's UPS machinery (5), by sequestration of essential components of the UPS into inclusions (6), or by obstruction of the proteasome (7). If polyQ proteins themselves inhibit the system crucial to their own degradation, this could elicit a self-perpetuating pathogenic cascade of events, both accelerating the accumulation of the toxic protein, and impairing essential regulatory functions of the UPS (4). With the help of specifically designed reporter substrates, it has been shown that polyQ proteins can cause UPS impairment in cell lines (5, 8, 9). However, since these experiments rely on acute overexpression of the polyQ protein in cells with limited physiological relevance, it is difficult to extrapolate these findings to the status of the UPS during the progression of the disease in patients.

In contrast to the observation with in vitro models, no signs of UPS impairment were found in mouse models for spinocerebellar ataxia 7 (SCA7) (10), or spinal and bulbar muscular atrophy (11), two diseases that are caused by polyQ proteins (12). Huntington's disease (HD), which is caused by a polyQ repeat expansion in the protein huntingtin (13), has appeared to be an exceptional case, as several lines of data have implicated UPS impairment in HD pathology (14). A number of studies have provided evidence in favor of UPS impairment in cellular models, as a consequence of the expression of the N-terminal fragment of mutant huntingtin (N-mutHtt) (5, 8, 9, 15, 16). Moreover, a recent study suggested that global UPS impairment is a consistent feature of HD pathology based on a striking increase in the levels of various types of polyubiquitin conjugates in HD postmortem brain and in R6/2 mice, a commonly used disease model expressing human N-mutHtt (17). The N-mutHtt fragment of the human huntingtin protein contains the polyQ repeat, and is more toxic than the full-length protein, resulting in a rapid and aggressive pathogenesis in animal models (18). In the present study, we evaluated ubiquitin-dependent proteasomal degradation in R6/2 and the milder R6/1 mouse models (18) using transgenic UPS reporter mice (19). These UPS reporter mice express a green fluorescent protein (GFP) fusion that is constitutively targeted for ubiquitin-dependent proteasomal degradation through the presence of a ubiquitin fusion degradation (UFD) signal. Global impairment of the UPS is expected to give a general accumulation of proteasome substrates, including UFD substrates, which can be readily assessed in these mice by the levels of the GFP reporter. Our data demonstrate that global impairment of the UPS does not occur in R6 mice and suggest that the accumulation of polyubiquitin conjugates in HD is not a direct reflection of UPS functionality.

Results

N-mutHtt Impairs Degradation of Two Unrelated Reporter Substrates in Cells.

We generated a PC12 cell line that inducibly expresses a cyan fluorescent protein (CFP)-tagged N-mutHtt, harboring a pathogenic polyQ repeat of 94 residues (N-mutHtt-CFP). To this cell line, we subsequently introduced the UFD reporter substrate ubiquitin^G76V-yellow fluorescent protein (Ub^G76V-YFP) or the YFP-CL1 reporter substrate, which carries the CL1 degradation signal (20), and for which it has been shown that expression of N-mutHtt impairs its degradation (5, 8). The functionality of the reporters in the PC12 cell lines was confirmed by treatment with a specific proteasome inhibitor (Fig. S1). Induction of N-mutHtt-CFP expression caused accumulation of both the Ub^G76V-YFP (Fig. 1 A and C) and YFP-CL1 substrates. (Fig. 1 B and C). Thus, N-mutHtt causes a general inhibition of the UPS in cell lines.

Fig. 1.

N-mutHtt impairs degradation of two unrelated reporter substrates in cells. PC12 cells expressing Ub^G76V-YFP (A) and YFP-CL1 (B) in which expression of N-mutHtt-CFP had not been induced (Left) or had been induced for 4 days by omitting doxycyclin from the medium (Right). (Scale bar, 20 μm.) (C) Quantitation of YFP fluorescence in Ub^G76V-YFP and YFP-CL1 PC12 cells in the absence or presence of expression of N-mutHtt-CFP. The fluorescence intensity in the absence of N-mutHtt-CFP expression has been standardized to 1.0. (SEM, n > 75 cells counted per group, t test ***, P < 0.001).

Functional Analysis of UFD Reporter Substrate in UbGFP Mouse Brain.

We recently developed two reporter mouse lines, UbGFP/1 and UbGFP/2, that ubiquitously express the UFD substrate ubiquitin^G76V-green fluorescent protein (Ub^G76V-GFP) (19). In situ hybridization showed that the reporter mRNA transcript is present throughout the brain including the cortex and striatum, regions that are particularly affected in HD and in R6 mice (Fig. 2A). Quantification revealed that UbGFP/1 mice have higher expression levels than UbGFP/2 mice in all regions examined (Fig. S2). The GFP protein levels in the brains of both lines are too low to be detected by native GFP fluorescence (19). Immunostaining with a GFP specific antibody revealed a weak but specific staining in neurons of UbGFP/1 mice (Fig. 2B and Fig. S3), whereas the reporter remained undetectable in UbGFP/2 mice (Fig. 2B).

Fig. 2.

Functional analysis of UFD reporter substrate in the UbGFP mouse brain. (A) In situ hybridization of Ub^G76V-GFP reporter mRNA expression in 30-to 40-week-old non-transgenic (NTg), UbGFP/1, and UbGFP/2 brain. Sagittal sections are shown. [Labels, cortex (Ctx), striatum (Str), hippocampus (Hc), and cerebellum (Cb)]. (B) Anti-GFP immunostaining of Ub^G76V-GFP reporter protein expression in 12-week NTg, UbGFP/1, and UbGFP/2 cortex. Nuclei are stained with Hoechst. (C) Native GFP fluorescence in striatum of UbGFP/1 (Upper) and UbGFP/2 mice (Lower) 24 h after stereotactic injection of vehicle only (Left) or 50 nmol of the proteasome inhibitor lactacystin (Right). (Scale bar, 50 μm.) (D) Quantitation of number of GFP-positive cells/mm² in striata from lactacystin treated UbGFP/1 and UbGFP/2 mice. Sections from lactacystin treated UbGFP/1 (E), and UbGFP/2 (F) mice were probed with antibodies specific for neurons (NeuN), microglia (Iba1), astrocytes (GFAP), and oligodendrocytes (CNPase). The percentage of GFP-positive cells double stained for the respective markers were quantitated. Average counts of 2 sections from each of 2 mice ± SD. (G) Western blot analysis of GFP products in homogenates of forebrain, cerebellum and pancreas of UbGFP/1 and UbGFP/2 mice. Blots were probed with a monoclonal GFP antibody. Astrices mark non-specific bands. (H) GFP products were immunoprecipitated (IP) from forebrain, cerebellum, and pancreas homogenates with a polyclonal GFP antibody before detection of the products by Western blotting with a monocolonal GFP antibody.

Whilst no native GFP fluorescence was detected in untreated mice, direct administration of proteasome inhibitor into the brains of UbGFP/1 and UbGFP/2 mice, which caused an approximate 80% and 50% reduction in the chymotrypsin-like and caspase-like activities, respectively (Fig. S4A and B), resulted in the appearance of cells with detectable fluorescence (Fig. 2 C and D). Subsequent analysis with markers for different cell types demonstrated that neurons, microglia, astrocytes, and oligodendrocytes accumulated the reporter substrate in inhibitor-treated UbGFP/1 (Fig. 2E and Fig. S4C) and UbGFP/2 mice (Fig. 2F and Fig. S4D).

We next analyzed the levels of the reporter substrate in the forebrain and cerebellum of UbGFP/1 and UbGFP/2 mice by Western blotting. As a reference tissue, we included the pancreas since it exhibits particularly high reporter levels in UbGFP/1 mice. In tissue homogenates, the reporter protein could only be detected in UbGFP/1 mice (Fig. 2G), whereas concentrating the reporter by immunoprecipitation before Western blotting was required to detect the reporter in tissues from UbGFP/2 mice (Fig. 2H). Notably, two specific GFP bands were detected in all three analyzed tissues of both mouse lines. The upper band corresponded in molecular weight with the full-length Ub^G76V-GFP fusion and the lower band corresponded in size with unmodified GFP (Fig. S5A). We reported earlier that expression in cell lines gave rise to small amounts of a similar truncated product (21). In primary fibroblast cultures from UbGFP/1 mice, the full length Ub^G76V-GFP had a very short half-life whereas the truncated product was long-lived and remained stable during an 8-h chase (Fig. S5B). Western blot analysis of Ub^G76V-GFP/1 striatal tissue after injection with proteasome inhibitor showed that the full length UbGFP but not the smaller GFP fragment accumulated (Fig. S4E).

Together our data show that the mouse lines express the UPS reporter throughout the brain and that both neuronal and non-neuronal cell types accumulate the reporter substrate in response to UPS dysfunction. Moreover, in addition to the short-lived Ub^G76V-GFP fusion, which accumulates in response to UPS inhibition, the tissues also contain a long-lived fragment corresponding in size with GFP.

Absence of Global UPS Impairment in R6/2 and R6/1 Mice.

Next, we crossed the UPS reporter mice with the R6/2 and R6/1 mouse models, which express human N-mutHtt (22). Due to differences in expression levels and repeat length, R6/2 and R6/1 mice exhibit different rates of disease onset and pathogenesis, developing late-stage symptoms at 12 and 40 weeks of age, respectively (22).

We first analyzed the effect of N-mutHtt on expression of the reporter in UbGFP mice. Although we observed no gross changes in the expression pattern of the reporter transcript in brains of late stage R6/2 and R6/1 mice (Fig. S6A), levels of the transcripts were slightly elevated in the striatum and cortex (Fig. S6B). To investigate the effect of N-mutHtt on the levels of the reporter protein substrate, we first used the UbGFP/2 reporter mouse line, because the absence of a constitutively detectable GFP signal enables clear detection of impairment of the UPS. Detailed immunocytochemical analysis of the brains of UbGFP/2:R6/2 (6–14 weeks) and UbGFP/2:R6/1 (6–40 weeks) mice, revealed however, no accumulation of the GFP reporter early or late in the disease process (Fig. 3A and Fig. S7A).

Fig. 3.

Absence of global UPS impairment in R6/2 mice. (A) Representative cryosections from brains of non-transgenic (NTg), UbGFP/2 and UbGFP/2:R6/2 mice co-immunostained for Htt and GFP. Nuclei were counterstained with Hoechst. Micrographs show 12-week non-transgenic (NTg), UbGFP/2 and UbGFP/2:R6/2 Cortex (Ctx) and striatum (Str). (Scale bar, 20 μm.) (B) Activity labeling of proteolytic active proteasome subunits in brain homogenates of four 14-week-old NTg controls and R6/2 mice using the fluorescent activity probe MV151. Image shows in-gel fluorescence readout. Labeled β-1, β-2, and β-5 proteasome subunits are indicated. (C) Densitometric analysis of band intensities from B (n = 4, SEM).

Consolidating these findings, we also investigated proteasome activity using the activity probe MV151 that covalently labels the proteolytically active subunits of the proteasome (23). Earlier studies based on cleavage of fluorogenic substrates reported an increase in proteasome activity in N-mutHtt mice including R6/2 (24, 25). Using the activity probe, we observed however no differences in the activities of the constitutive subunits of the proteasome in brain lysates from late-stage R6/2 mice compared with age-matched controls (Fig. 3 B and C). We cannot exclude the possibility that the levels of inducible proteasome subunits are elevated, as reported previously for a similar N-mutHtt mouse model (25), since they remained below the detection level in our assay.

An advantage of the UbGFP/1 reporter mouse line is that it can be used for detection of more subtle differences in the levels of the reporter, since the higher transgene expression levels result in detectable basal levels of the reporter. However, consistent with our data obtained using UbGFP/2 mice, we observed no increase in fluorescence intensity in late-stage UbGFP/1:R6/2 (Fig. 4A) or UbGFP/1:R6/1 mice (Fig. S7B). Instead, we noticed that the neuronal GFP staining was reduced compared to age-matched UbGFP/1 control mice (Fig. 4A and Fig. S7B). Western blot analysis confirmed this finding and revealed that despite there being no significant increase in the levels of full-length Ub^G76V-GFP, consistent with the absence of UPS impairment, the levels of the truncated product were significantly reduced in UbGFP/1:R6/2 mice (Fig. 4 B and C). Our data therefore suggest that N-mutHtt does not affect the clearance of the short-lived reporter but reduces the levels of the long-lived GFP fragment. We conclude that global UPS impairment does not occur in the R6 mouse models.

Fig. 4.

No changes in Ub^G76V-GFP and decrease in long-lived GFP product in R6/2 mice. (A) Cortex (Ctx) and striatum (Str) of 12-week-old NTg, UbGFP/1, and UbGFP/1:R6/2 mice co-immunostained for N-mutHtt and GFP. Nuclei counterstained with Hoechst (Lower). (Scale bar, 20 μm.) (B) Western blot anaysis of UbGFP and GFP protein products in forebrain homogenates of 14-week UbGFP/1 and UbGFP/1:R6/2 mice using α-GFP antibody. (C) Quantification of band intensities in B (n = 6, SEM; t test, ***, P < 0.001).

Polyubiquitylated Proteins Accumulating in R6/2 Mice Differ from Those Observed upon Proteasome Inhibition.

It has been recently shown that the levels of ubiquitin conjugates are significantly increased in R6/2 mice, which has been interpreted as evidence for the occurrence of UPS dysfunction in R6/2 mice (17). We indeed found that the levels of ubiquitin conjugates were dramatically increased in late stage R6/2 mice (Fig. 5A). Ubiquitin conjugates and the GFP reporter accumulated in primary UbGFP/1 fibroblasts at a similar level of proteasome inhibition excluding the possibility that ubiquitin conjugates are a more sensitive readout for UPS impairment (Fig. 5B and Fig. S8A and B). A more detailed analysis of the ubiquitin conjugates in the soluble fraction of R6/2 brain lysates showed that the increase was primarily due to an elevation in the levels of large ubiquitin complexes (Fig. 5 C and D). This was even more striking when the brain lysates were separated using a stacking gel consisting of a low-percentage of agarose to enable separation of larger protein complexes (Fig. 5E). Notably, the N-mutHtt also migrated as large complexes (Fig. 5F). In contrast, administration of proteasome inhibitor to the brain (Fig. 5G) or cells (Fig. S8C) caused an increase in high molecular weight polyubiquitin conjugates whereas no increase was detected in large complexes remaining in the stacking gel. We conclude that the polyubiquitin conjugates that accumulate in brains of R6/2 mice do not correspond with those observed upon proteasome inhibition but are primarily attributed to a pool of large polyubiquitylated complexes. These findings suggest that the elevation in ubiquitin conjugates in R6/2 mice is not a consequence of global UPS impairment, consistent with the absence of accumulation of UPS reporter substrates.

Fig. 5.

Polyubiquitylated proteins accumulating in R6/2 mice differ from those observed upon proteasome inhibition. (A) A slot blot of 14-week-old UbGFP/1 and UbGFP/1:R6/2 brain homogenates using a monoclonal antibody specific for conjugated polyubiquitin (FK1). (B) Accumulation of Ub^G76V-GFP reporter and polyubiquitin conjugates in UbGFP/1 primary fibroblast cultures treated with increasing concentrations of epoxomycin. Standardised signals from densitometric analysis of the Western blot in Fig. S8A. Representative of 3 independent experiments. (C) Separation of high and low MW polyubiquitinylated material from the same samples as in A by SDS/PAGE Western blotting. Ubiquitin conjugates in the stacking gel (a); upper resolving gel >250kDa (b); resolving gel 20–150 kDa (c) are indicated. (i) shorter exposure, (ii) longer exposure. (D) Densitomentric analysis of the ubiquitin conjugates in the stacking gel, upper resolving gel and resolving gel shown in C (n = 6, SEM; t test ***, P < 0.001). (E) Polyacrylamide stacking gel was replaced by a 1% agarose stacking gel to allow entry of larger protein aggregates. Blot was probed with the FK1 antibody. (F) Immunostaining of the same samples as shown in E with α-Htt antibody. (G) Western blot analysis of polyubiquitin conjugates in striatal tissue of UbGFP/1 mice 24 h after lactacystin injection or vehicle control. Far right lane, 14-week R6/2 brain homogenate.

Discussion

Since the UPS is important for protein quality control (26), but unable to degrade aggregated proteins (27), it is feasible that futile attempts to degrade protein aggregates hinders the proteasome from fulfilling other tasks, which in turn, may cause cellular dysfunction or death. We indeed found, in line with earlier studies (5, 8, 9), that N-mutHtt caused functional impairment of ubiquitin-dependent proteasomal degradation in cell lines but did not observe global UPS impairment in brains of mice expressing N-mutHtt. Although we cannot exclude the occurrence of spatially or temporally confined impairment of the UPS (28) or more subtle changes affecting a selective subpopulation of substrates, our data unequivocally shows that the UPS is largely operative and not globally impaired. This conclusion is strengthened by a recent study demonstrating that the degradation of a CL1-based UPS reporter is also unconpromised in R6/2 mice (29). An important difference between the in vitro and in vivo models is that in the cellular model, the cells are acutely challenged with high levels of the N-mutHtt, whereas the R6 mice are chronically exposed to more moderate expression levels of this protein (22). As global impairment of the UPS is lethal for cells (30), it is not unlikely that the chronic presence of N-mutHtt may trigger adaptive responses that allow the cells to cope with this toxic protein. It has indeed been shown that chronic exposure of cell cultures to a high dose of proteasome inhibitor results in the appearance of adapted cells that have compensated for the curtailed proteasome activity by up-regulating other proteases (31), such as the tripeptidyl peptidase II (32). In respect to polyQ proteins, the puromycin-sensitive aminopeptidase is also of interest since it has been shown that this protease is able to degrade expanded polyQ repeats (33). However, we did not detect any striking differences in the levels of these proteases in R6/2 mice (Fig. S9 A and B).

Our data suggest that polyubiquitylated proteins accumulate not as a consequence of UPS impairment but despite the presence of a largely functional UPS. If ubiquitylated substrates are sequestered into larger protein aggregates before reaching the proteasome for degradation, this could account for the observed elevation in high molecular weight polyubiquitin conjugates. Furthermore, since polyubiquitin modifications are involved in a large number of cellular processes other than proteasomal degradation (34), it is feasible that the increase in polyubiquitylated material reflects changes in ubiquitin-dependent processes unrelated to UPS function, either as a consequence of impairment of these processes, or alternatively, as part of an adaptive response. An interesting possibility can be found in the fact that ubiquitylation plays pivotal roles in the formation of aggresomes (35) and the clearance of aggregated proteins by macroautophagy (36, 37). Both events are stimulated by polyQ proteins (38) and are believed to be largely protective responses that defend the cell against the toxic insults of aggregation-prone proteins (38, 39) and can restore UPS function (16, 40). Since macroautophagy is primarily responsible for the turnover of long-lived proteins (41), and induction of this pathway does not affect the levels of UFD substrates (16), this may also provide an explanation for the selective decrease in the long-lived GFP product in the brains of R6/1 and R6/2 mice.

The same reporter mice that were applied in our study have confirmed UPS dysfunction in prion-infected mice (42) and in transgenic mice overexpressing a mutant SOD1 responsible for familial amyotrophic lateral sclerosis (43) showing that these mice are suited for in vivo assessment of the UPS. In contrast, in three polyQ disease models, namely SCA7 (10), spinal and bulbar muscular atrophy (11) and mice expressing N-mutHtt, the UPS remains operative. Although it may be premature to generalize these observations as pertaining to all polyQ pathologies, these observations together suggest that the UPS manages to escape global impairment even in the face of a pathological polyQ challenge. We conclude that global UPS impairment is not a unanimous casualty amongst protein misfolding diseases. The fact that the UPS appears to be functional, even at late stages of the pathology in R6 mice when many cellular pathways are disturbed, underscores the robustness of this proteolytic machinery and suggests that the UPS may be a reliable partner to exploit for reducing the load of the toxic proteins in these pathologies.

Materials and Methods

Cell Lines.

Exon 1 of human Htt containing 94 polyQ repeats was ligated to the N terminus of CFP (N-mutHtt-CFP), cloned into the pTRE-tight vector (Clontech) and introduced into PC12 cells carrying the tet-transactivator (Clontech). Subsequently, the Ub^G76V-YFP or YFP-CL1 reporter (44) was integrated to generate double stable cell lines. Cells were analyzed with a confocal laser scanning microscope (Zeiss LSM510 META). Images were quantified using Volocity software (Improvision).

Transgenic Mice.

All animal experiments were approved by local ethical committees and conformed to international animal welfare guidelines. The UbGFP mouse lines were maintained in a heterozygous state on a C57/BL6 background, and R6/1 and R6/2 mice on a BL6/CBA background. Double transgenic UbGFP:R6 mice were obtained by crossing R6/1 or R6/2 males with UbGFP females. Mice were decapitated or perfused transcardially with PBS. For immunohistochemical analyses, mice were perfused transcardially with PBS followed by 4% paraformaldehyde. Tissues were postfixed in 4% paraformaldehyde, cryopreserved in 30% sucrose/PBS, and mounted in Tissue-Tec O.C.T. compound (Sakura).

In Situ Hybridization.

In situ hybridization was performed as described in SI Text using the specific antisense oligonucleotide to green fluorescent protein (GFP) (5′ CAC CTA CGG CAA GCT GAC CCT GAA GTT CAT CTG CAC CAC CGG C 3′) and the corresponding sense sequence used as a negative control. Quantitation was performed using Image J software (National Institutes of Health). For each brain region analyzed, the signal intensity was taken as the average of at least three sections for each mouse analyzed, and corrected for the background signal from NTg controls.

Immunostaining.

Immunostaining was performed on cryotome-cut brain sections using rabbit polyclonal anti-GFP (Invitrogen); mouse monoclonal anti-human Htt antibody, MAB5374 (Millipore); mouse monoclonal anti-NeuN, MAB377 (Chemicon), in PBS and 0.2% Triton X-100, pH 7.4. Alexafluor conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were applied in PBS and 0.2% Triton X-100. Immunofluorescence staining combined with native GFP fluorescence detection was performed by incubating free-floating slices at room temperature in PBS, 1% BSA, and 0.1% Triton X-100 with: anti-NeuN antibody; anti-GFAP polyclonal antibody (Promega); anti-CNPase monoclonal antibody (11–5B, AbCam); or anti-Iba1 polyclonal antibody (Wako). The reaction was visualized with TexasRed-conjugated anti-mouse or anti-rabbit IgG (Molecular Probes). Sections were examined with a confocal laser scanning microscope (Zeiss LSM510 META).

Western Blotting.

Frozen tissues were homogenized in 5–10× wt/vol ice cold homogenization buffer [20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM NaF, 1% Triton X-100, 1 mM orthovanadate, 10 mM EDTA, 20 mM N-ethylmaleimide (Sigma-Aldrich), and complete miniprotease inhibitor mixture (Roche)]. Homogenization was carried out by sonication on a Bandelin Sonopuls ultrasonic homogenizer. Lysates were cleared by centrifugation at 14,000 × g. Equal amounts of protein were separated on Tris-glycine polyacrylamide gels or NuPAGE (Bio-Rad) gels, and electroblotted onto 0.45-μM nitrocellulose membranes. Agarose stacking gel (1% agarose and 0.05% SDS in 200 mM Tris, pH 8.8) preparation was adapted from Warren et al. (45). Briefly, stacking gels were poured at 65 °C, gels were run at 15 mA and transferred at 40 mV for 4 h on ice. GFP protein, ubiquitin conjugates, and GAPDH were detected using a mixed-mouse monoclonal anti-GFP antibody (Roche), the monoclonal antibody FK1 (Chemicon), and GAPDH antibody (Fitzgerald Industries International), respectively, followed by horse radish peroxidase-conjugated goat-anti-mouse secondary antibody (GE Healthcare). Enhanced chemiluminescence (ECL) and Fuji Super RX film were used for detection. Band intensities were measured using Image J software (NIH). Slot-blots were performed on an SDS minifold I (Bio-Rad) using 0.2-μm nitrocellulose and 0.1% SDS in PBS for equilibration and washing.

Immunoprecipitation.

Brain homogenates were prepared as for Western blotting, and immunoprecipitation was performed on 2 mg total protein in a 1 mL final volume. Samples were precleared with protein A Sepharose (GE Healthcare), followed by incubation with 1 μL rabbit α-GFP antibody (A6455, Molecular Probes) overnight 4 °C, and immunoprecipitation with protein A Sepharose. GFP immunoreactivity was detected after immunoblotting using a monoclonal α-GFP antibody as for western blotting.

Proteasome Activity Measurements.

For proteasome subunit activity labeling, brain samples were homogenized in buffer containing 50 mM Tris (pH 7.5), 5 mM MgCl₂, 250 mM sucrose, 1 mM DTT, and 2 mM ATP, centrifuged at 14,000 × g and the supernatant used for analysis. For labeling reactions, 50 μg protein was incubated for 1 h at 37 °C with MV151 (500 nM). The reaction was ceased by boiling in Laemmli's buffer and 5 μg total protein was resolved by 12.5% SDS/PAGE. In-gel visualization of labeled proteasome subunits was performed as described previously (23). Proteasome activities in brain tissue and cell lysates were measured with fluorogenic substrates as described previously (46). Non-proteasomal activity was determined by performing the assay after addition of high concentrations of proteasome inhibitor (20 μM epoxomycin for cell lysates; 50 μM lactacystin or 10 μM MG-132 for striatal homogenates) to the in vitro reaction.