Abstract

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The resultant mutant Htt protein (mHtt) forms aggregates in the brain and several peripheral tissues (e.g. the liver) and causes devastating neuronal degeneration. Metabolic defects resulting from Htt aggregates in peripheral tissues also contribute to HD pathogenesis. Simultaneous improvement of defects in both the CNS and peripheral tissues is thus the most effective therapeutic strategy and is highly desirable. We earlier showed that an agonist of the A2A adenosine receptor (A2A receptor), CGS21680 (CGS), attenuates neuronal symptoms of HD. We found herein that the A2A receptor also exists in the liver, and that CGS ameliorated the urea cycle deficiency by reducing mHtt aggregates in the liver. By suppressing aggregate formation, CGS slowed the hijacking of a crucial transcription factor (HSF1) and two protein chaperons (Hsp27 and Hsp70) into hepatic Htt aggregates. Moreover, the abnormally high levels of high-molecular-mass ubiquitin conjugates in the liver of an HD mouse model (R6/2) were also ameliorated by CGS. The protective effect of CGS against mHtt-induced aggregate formation was reproduced in two cells lines and was prevented by an antagonist of the A2A receptor and a protein kinase A (PKA) inhibitor. Most importantly, the mHtt-induced suppression of proteasome activity was also normalized by CGS through PKA. Our findings reveal a novel therapeutic pathway of A2A receptors in HD and further strengthen the concept that the A2A receptor can be a drug target in treating HD.

INTRODUCTION

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterized by chorea, dementia and psychiatric symptoms. The major hallmark of HD is region-specific neuronal degeneration in the striatum and cortex, which subsequently leads to movement disorders and dementia (1,2). The causative mutation is a CAG trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. The normal Htt gene has 35 or fewer CAG repeats in the N-terminal region, whereas the appearance of neurological symptoms is associated with 36 or more CAG repeats in the Htt gene (3). The expanded CAG repeat encodes a long polyglutamine (polyQ) tract of Htt which forms aggregates in both the nucleus and/or cytoplasm of affected neurons in human patients, transgenic animals and cell lines (4). Earlier studies suggested that formation of Htt aggregates was likely a result of insufficient protein degradation (5). In addition, several components of protein-folding and proteolytic mechanisms (ubiquitin, HSP40/70 and 20S proteasome) have been shown to co-localize with mutant Htt (6–10). It is generally believed that activities and regulation of proteasome and heat shock proteins (HSPs) play a critical role in HD's pathogenesis. In addition, transcriptional dysregulation is perhaps the most serious damage caused by mutant Htt (11). Htt aggregates have been shown to cause abnormal protein–protein interactions with a number of transcription factors or co-activators (e.g. p53, CREB, CBP, SP1, TAFII130 and 14-3-3 zeta) (12–18). Marked changes in the transcriptional profiles of cells expressing mHtt therefore were expected and well documented (19–24).

Marked peripheral abnormalities, which parallel the degeneration in the brain, in mouse models and patients with HD, have been clearly documented in recent years. For example, the most obvious clinical features of HD are progressive weight loss, atrophy of skeletal muscle and dysfunction of adipose tissue (25–28). Expression of mHtt by myocytes has been demonstrated to impair cardiac functions, which might lead to early-onset cardiac vascular diseases in HD patients (29). Pathogenic alterations in immune reactions were also found in both the brain and plasma of HD patients (30), suggesting a contribution of an abnormal immune response to HD pathogenesis. Deficiencies in several metabolic pathways (e.g. cholesterol biosynthesis and urea cycle metabolism) which affect the function of the entire body have also been well documented (31,32). In particular, we demonstrated that the urea cycle deficiency caused by the mHtt-evoked suppression of C/EBPα in the liver is an important pathogenic pathway of HD (32). These recent findings suggest that HD treatments which simultaneously ameliorate defects in both the central nervous system (CNS) and peripheral tissues are likely to be more effective than those that only normalize dysfunctions in the CNS (33).

The ubiquitin–proteasome system (UPS) plays an essential role in degrading misfolded or damaged proteins that are polyubiquitinated by ubiquitin ligases and targeted to proteasomes for degradation (34,35). A complete proteasome (26S) is composed of one 20S and two 19S complexes. Activities of the UPS affect many important cellular processes, including transcription, translation, DNA repair, the cell cycle, chromatin remodeling, cell survival, signal transduction, protein trafficking and synaptic plasticity (36–42). Formation of protein aggregates, which are often seen in neurodegeneration, was shown to inhibit the functions of UPS (39,43–45). Indeed, recent studies revealed that mHtt dysregulates the UPS in brains of HD animals and patients (5,46–54). In the present study, we report that the UPS activity in the liver of HD mice was also greatly reduced. In addition, treatment with a beneficial compound (CGS21680—CGS) improved the damaged UPS activity in HD livers.

The A2A adenosine receptor (A2A receptor) is a G protein-coupled receptor that is expressed in many tissues including the brain and liver (55,56). The main signal transduction activated by the A2A receptor is the cAMP/protein kinase A (PKA)-mediated pathway (57). Studies from several laboratories including our own have previously demonstrated that activation of A2A receptor protects cells against various types of stresses. Stimulation of the A2A receptor was found to protect cells from apoptosis in a cAMP/PKA-dependent manner (58,59). In addition, the A2A receptor protects the hippocampus from excitotoxicity, and a few peripheral tissues from ischemic injury (60,61). Since the A2A receptor is highly enriched in enkephalin-containing striatal neurons (62–64), the most susceptible neurons to mHtt-evoked damage in HD, the role of the A2A receptor in HD has attracted much attention in the past few years (65–71). In a well-characterized transgenic mouse model of HD [R6/2; (72)], we earlier showed that chronic treatment with an agonist (CGS) of the A2A receptor ameliorated several major symptoms (including brain atrophy, aggregation formation in the striatum and progressive reduction in rotarod performance) of HD (70). In the present study, we found that the A2A receptor also exists in the liver, and its expression in the liver was not affected by HD. In addition to the beneficial effects observed in the brain (70), chronic treatment of R6/2 mice with CGS also effectively rescued the urea cycle deficiency in the liver by enhancing the activity of the UPS through a cAMP/PKA-dependent pathway. Our findings further strengthen the concept that the A2A receptor can be a drug target in treating HD.

RESULTS

We previously demonstrated that chronic treatment with an agonist of the A2A receptor (CGS) provides beneficial effects on several major symptoms (e.g. deteriorated motor coordination, brain atrophy and formation of neuronal intranuclear inclusions) in a well-characterized transgenic mouse model of HD (70). In the brain, the A2A receptor is located in striatal GABAergic neurons, which are markedly degenerated during disease progression in several mouse models of HD and in HD patients (22,73–75). In line with the above studies and our previous findings (70), we show herein that the transcript level of the A2A receptor in the striatum was markedly lower than that in wildtype (WT) mice (Fig. 1A). In contrast to the suppression of the striatal A2A receptor, the level of the hepatic A2A receptor was not affected by mutant Htt in the liver (Fig. 1B). It is therefore of great interest to determine whether chronic CGS treatment in R6/2 mice ameliorates the defective hepatic functions in HD.

Figure 1.

Expression of the A2A receptor in the striatum and liver of R6/2 mice. RNAs were collected from the striatum (A,n = 4) and liver (B,n = 4) of 12-week-old WT or R6/2 mice and reverse-transcribed into cDNA. A real-time quantitative PCR was performed. Expression levels were normalized to that of the GAPDH. Data are presented as the mean ± SEM values from three independent experiments. ***Specific comparison to HD mice (P < 0.001; Student's t-test).

Figure 1.

Expression of the A2A receptor in the striatum and liver of R6/2 mice. RNAs were collected from the striatum (A,n = 4) and liver (B,n = 4) of 12-week-old WT or R6/2 mice and reverse-transcribed into cDNA. A real-time quantitative PCR was performed. Expression levels were normalized to that of the GAPDH. Data are presented as the mean ± SEM values from three independent experiments. ***Specific comparison to HD mice (P < 0.001; Student's t-test).

We previously demonstrated that mHtt impairs the function of the urea cycle in the liver. The urea cycle deficiency of HD was characterized by hyperammonemia, high blood citrulline and suppression of urea cycle enzymes in two mouse models of HD as well as in HD patients (32). We herein tested whether chronic CGS treatment improves this urea cycle deficiency in the liver of R6/2 mice. As shown in Figure 2, elevated blood levels of ammonia and citrulline were ameliorated by chronic CGS treatment. The beneficial effects of CGS on the urea cycle appeared to be mediated by normalizing the expressions of urea cycle enzymes. The downregulated transcript levels of three enzymes [argininosuccinase acid lyase (AL), argininosuccinic acid synthetase (AS) and arginase (AG)] and the abnormal upregulated transcript of ornithine transcarbamylase (OTC) in the HD liver returned toward more-normal values with chronic CGS treatment (Table 1). Moreover, CGS also effectively reversed the impaired activities of two affected enzymes (AS and AL) in R6/2 mice (Table 2), which subsequently rescued the elevated blood ammonia and citrulline levels.

Figure 2.

Chronic CGS treatment reduced the aberrantly elevated blood ammonia and citrulline levels in R6/2 mice. R6/2 mice were daily injected with CGS (5 µg/g body weight) for 5 weeks from the age of 7 weeks (70). The levels of blood ammonia (A,n = 6–14) and blood citrulline (B,n = 7–22) at the age of 12 weeks were determined. Data are presented as the mean ± SEM. ‘*’ and ‘***’ indicate specific comparison to the non-treated R6/2 mice (P < 0.05, P < 0.001, respectively; one-way ANOVA).

Figure 2.

Chronic CGS treatment reduced the aberrantly elevated blood ammonia and citrulline levels in R6/2 mice. R6/2 mice were daily injected with CGS (5 µg/g body weight) for 5 weeks from the age of 7 weeks (70). The levels of blood ammonia (A,n = 6–14) and blood citrulline (B,n = 7–22) at the age of 12 weeks were determined. Data are presented as the mean ± SEM. ‘*’ and ‘***’ indicate specific comparison to the non-treated R6/2 mice (P < 0.05, P < 0.001, respectively; one-way ANOVA).

Table 1.

Chronic CGS treatment rescued the gene expression of urea cycle enzymes (AS, AL, and OTC) altered by mutant Htt in the liver of R6/2 mice

Mouse Treatment % Relative transcript level
 
AS AL AG OTC 
WT (n = 3) CON 100.0*** 100.0*** 100.0* 100.0*** 
R6/2 (n = 3) CON 37.1 ± 1.8 40.8 ± 3.6 80.2 ± 2.4 182.1 ± 5.8 
R6/2 (n = 3) CGS 86.4 ± 2.8*** 84.7 ± 2.7*** 90.7 ± 1.7* 143.3 ± 5.8* 
Mouse Treatment % Relative transcript level
 
AS AL AG OTC 
WT (n = 3) CON 100.0*** 100.0*** 100.0* 100.0*** 
R6/2 (n = 3) CON 37.1 ± 1.8 40.8 ± 3.6 80.2 ± 2.4 182.1 ± 5.8 
R6/2 (n = 3) CGS 86.4 ± 2.8*** 84.7 ± 2.7*** 90.7 ± 1.7* 143.3 ± 5.8* 

The AS, AL, AG and OTC transcripts in the liver of the indicated mice were analyzed using the Q-PCR technique. R6/2 mice were injected daily for 5 weeks with CGS (5 µg/g body weight) from the age of 7 weeks. Liver RNA of the mice at the age of 12 weeks was collected and reverse-transcribed into cDNA. Real-time quantitative PCR of the indicated gene was performed and normalized to that of GAPDH. Values are expressed as percentages of the indicated transcript in WT mice and are presented as the mean ± SEM values from three independent experiments. *P < 0.05 and ***P < 0.001, by specific comparison to the vehicle (1% DMSO)-treated R6/2 mice as indicated (paired Student's t-test). AS, argininosuccinic acid synthatase; AL, argininosuccinase acid lyase; AG, arginase; OTC, ornithine transcarbamylase.

Table 2.

Improvement in the activities of AS and AL in livers of HD mice with chronic CGS treatment

Mouse Treatment Age AS activity (nmol·mg−1·min−1AL activity (nmol·mg−1·min−1
WT (n = 6) CON 12 weeks 86.2 ± 1.8*** 28.5 ± 1.4*** 
R6/2 (n = 6) CON 12 weeks 51.7 ± 1.1 8.9 ± 0.8 
R6/2 (n = 6) CGS 12 weeks 64.0 ± 2.6*** 20.8 ± 1.4*** 
Mouse Treatment Age AS activity (nmol·mg−1·min−1AL activity (nmol·mg−1·min−1
WT (n = 6) CON 12 weeks 86.2 ± 1.8*** 28.5 ± 1.4*** 
R6/2 (n = 6) CON 12 weeks 51.7 ± 1.1 8.9 ± 0.8 
R6/2 (n = 6) CGS 12 weeks 64.0 ± 2.6*** 20.8 ± 1.4*** 

R6/2 mice were injected with CGS (5 µg/g body weight) daily for 5 weeks from the age of 7 weeks (70). Liver lysates were collected from the indicated animals to determine the AS and AL activities as described earlier (32). Data are presented as the mean ± SEM of at least three mice under each condition. ***Specific comparison to the vehicle (1% DMSO)-treated R6/2 mice as indicated (P < 0.001; one-way ANOVA).

Besides the urea cycle deficiency, we found that expression of mHtt caused aberrant expressions of several HSPs. These HSPs are responsible for maintaining homeostasis in hepatocytes. Consistent with the beneficial effects of CGS on HD (70), CGS treatment markedly enhanced the reduced protein chaperones (HSP27 and 70) in the liver of R6/2 mice (Fig. 3A). In addition, abnormal elevation of HSP90 was downregulated by CGS (Fig. 3B). Moreover, the expression of heat shock transcription factor 1 (HSF1), which mediates the transcription of a subset of HSPs, was also markedly reduced in the liver of HD mice. Chronic CGS treatment rescued the decreased HSF1 in the liver of R6/2 mice (Fig. 3C). This finding is functionally important because active HSF1 has been shown to exert a strong inhibitory effect on the formation of polyQ aggregates (76). Results of the filter-retardation assays further revealed that significant amounts of HSP27, HSP70 and HSF1 were retained on filters where Htt aggregates were found (Fig. 3D). At least part of the mechanisms underlying the aberrant regulation of HSPs and HSF1 is therefore mediated through chelation by Htt aggregates. Reduction in the available chaperones and HSF1 by recruitment into Htt aggregates is likely to further impair the molecular chaperone system in the liver.

Figure 3.

Chronic CGS treatment normalized the dysregulation of protein chaperons in the liver of R6/2 mice. R6/2 mice were daily injected with CGS (5 µg/g body weight) for 5 weeks from the age of 7 weeks (70). Cytosolic (50 µg per lane; A,n = 3; B,n = 3) and nuclear fractions (50 µg per lane; C,n = 3) were collected from the indicated 12-week-old mice and subjected to western blot analysis. Levels of the indicated protein were normalized with the corresponding internal control (actin or lamin), compared with those in WT mice, and then shown in the bottom of the corresponding lane. Data are presented as the mean ± SEM values from three independent experiments. *Specific comparison to untreated R6/2 mice (P < 0.05, Student's t-test). For the filter retardation assays (D,n = 3), liver lysates (100 µg per slot) collected from the indicated 12-week-old mice were analyzed. The insoluble aggregates retained on the filters were detected using an anti-HSP27, anti-HSP70, anti-HSP90, anti-HSF1, anti-ubiquitin (Ub) or anti-Htt antibody. The level of actin in the liver lysate was assessed using western blot analyses and was used as an internal control. A representative image of three independent experiments is shown.

Figure 3.

Chronic CGS treatment normalized the dysregulation of protein chaperons in the liver of R6/2 mice. R6/2 mice were daily injected with CGS (5 µg/g body weight) for 5 weeks from the age of 7 weeks (70). Cytosolic (50 µg per lane; A,n = 3; B,n = 3) and nuclear fractions (50 µg per lane; C,n = 3) were collected from the indicated 12-week-old mice and subjected to western blot analysis. Levels of the indicated protein were normalized with the corresponding internal control (actin or lamin), compared with those in WT mice, and then shown in the bottom of the corresponding lane. Data are presented as the mean ± SEM values from three independent experiments. *Specific comparison to untreated R6/2 mice (P < 0.05, Student's t-test). For the filter retardation assays (D,n = 3), liver lysates (100 µg per slot) collected from the indicated 12-week-old mice were analyzed. The insoluble aggregates retained on the filters were detected using an anti-HSP27, anti-HSP70, anti-HSP90, anti-HSF1, anti-ubiquitin (Ub) or anti-Htt antibody. The level of actin in the liver lysate was assessed using western blot analyses and was used as an internal control. A representative image of three independent experiments is shown.

Similar to what was found earlier in the brain and neuronal cells (17,70), activation of A2A receptors using CGS markedly reduced Htt aggregates in the liver of R6/2 mice as assessed using filter-retardation assays, immunohistochemistry and western blot analyses (Figs 3D and 4A, Supplementary Material, Fig. S1). The level of Htt aggregates in the filter-retardation assays was assessed using both anti-Htt and anti-ubiquitin (Ub) antibodies (Fig. 3D). Because of the lower detection sensitivity of the filter-retardation assays (Fig. 3D) compared with immunocytochemical staining (Fig. 4A), mutant Htt aggregates were almost undetected in the CGS-treated group using the filter retardation assays (Fig. 3D). It is interesting to note that CGS appeared more effective in reducing Htt aggregates in the liver than in the brain. Unlike in the liver, CGS treatment did not affect the percentage of Htt aggregate-containing cells in the striatum, but only reduced the size of striatal Htt aggregates (70). This apparent superior effect of CGS on Htt aggregates in the liver as apposed to that in the striatum might be because a lower concentration of CGS was achievable in the brain compared with that in peripheral tissues (e.g. the liver). Importantly, by reducing Htt aggregates, CGS treatment also reduced the hijacking of HSPs and HSF1 by Htt aggregates, and thus elevated their cytosolic availabilities. No HSP90 was detected on the filters from the filter-retardation assays (Fig. 3D). Together, CGS protected the liver from mHtt-evoked toxicity by restoring normal levels of HSPs and HSF1 and therefore might improve the global protein folding, transportation and defense systems of the liver.

Figure 4.

Chronic CGS treatment reduced Huntingtin (Htt) aggregates and enhanced UPS activity in the liver of R6/2 mice. R6/2 mice were daily injected with CGS (5 µg/g body weight) for 5 weeks from the age of 7 weeks (70). (A) Htt aggregates were visualized with an antibody against ubiquitin. Representative images of the liver of 12-week-old R6/2 mice are shown. Scale bar: 20 µm. Percentages of liver cells expressing NII were quantified (>1200 cells from each liver section were quantified). ***Specific comparison to untreated R6/2 mice (P < 0.001; one-way ANOVA). (B) Liver lysates (100 µg per lane) collected from the indicated 12-week-old mice (n = 3) were subjected to western blot analysis using an anti-ubiquitin antibody (upper panel) or an anti-actin antibody (lower panel). (C) The chymotrypsin-like activity of proteasomes in liver lysates of the indicated 12-week-old mice (n = 5) was assessed as described in Materials and Methods. ‘***’ and ‘*’ indicate specific comparison to R6/2 mice on the control diet (P < 0.001, P < 0.05, respectively; one-way ANOVA).

Figure 4.

Chronic CGS treatment reduced Huntingtin (Htt) aggregates and enhanced UPS activity in the liver of R6/2 mice. R6/2 mice were daily injected with CGS (5 µg/g body weight) for 5 weeks from the age of 7 weeks (70). (A) Htt aggregates were visualized with an antibody against ubiquitin. Representative images of the liver of 12-week-old R6/2 mice are shown. Scale bar: 20 µm. Percentages of liver cells expressing NII were quantified (>1200 cells from each liver section were quantified). ***Specific comparison to untreated R6/2 mice (P < 0.001; one-way ANOVA). (B) Liver lysates (100 µg per lane) collected from the indicated 12-week-old mice (n = 3) were subjected to western blot analysis using an anti-ubiquitin antibody (upper panel) or an anti-actin antibody (lower panel). (C) The chymotrypsin-like activity of proteasomes in liver lysates of the indicated 12-week-old mice (n = 5) was assessed as described in Materials and Methods. ‘***’ and ‘*’ indicate specific comparison to R6/2 mice on the control diet (P < 0.001, P < 0.05, respectively; one-way ANOVA).

We next set out to determine the mechanism underlying the action of the A2A receptor in reducing Htt aggregates. Recent studies suggested that expression of mutant Htt markedly jeopardizes the UPS in neurons of HD mice and might contribute to the pathogenesis of HD (52–54,77–82). To assess whether UPS activity is also abnormally regulated in the liver of HD mice, total liver lysates of indicated mice were first analyzed using western blotting and an anti-ubiquitin antibody. As shown in Figure 4B, levels of high-molecular-mass ubiquitin conjugates were much higher in HD livers compared with those observed in WT livers, suggesting impaired UPS activity in the HD liver. Indeed, cymotrypsin-like activities in liver lysates of HD mice were much lower than those in WT mice (Fig. 4C). Chronic treatment with CGS significantly reduced the overall high-molecular-mass ubiquitin conjugates (Fig. 4B), as well as cymotrypsin-like activities in the HD liver (Fig. 4C).

To assess the signal transduction mediating the action of the A2A receptor, two cell lines were employed for the following experiments. We first demonstrated that HepG2, a hepatoma cell line, expressed a functional A2A receptor by showing that treating HepG2 cells with CGS elevated the cellular cAMP level. In addition, an A2A-R-selective antagonist [8-(3-chlorostyryl) caffeine (CSC)] blocked CGS-induced cAMP elevation (Fig. 5A), demonstrating the involvement of the A2A receptor. To determine whether stimulation of the A2A receptor had a direct effect on reducing aggregate formation in HepG2 cells, cells were transfected with an expression construct encoding exon 1 of Htt containing a normal number (25) or an expanded stretch (109) of CAG repeats fused to hrGFP (designated Htt-(Q)25-hrGFP and Htt-(Q)109-hrGFP, respectively). Fluorescent aggregates (≥1 µm) were observed under a fluorescent microscope and quantified. As shown in Figure 5B, treating HepG2 cells with CGS markedly reduced the percentage of cells expressing fluorescent aggregates. This effect of CGS was reversed by CSC, demonstrating that the reduction in Htt aggregates by CGS in HepG2 cells was mediated by activation of the A2A receptor. A similar suppressive effect of CGS on aggregate formation was also observed using filter retardation assays (Fig. 5C). Besides being blocked by CSC, the effect of CGS was also prevented by an inhibitor (H89) of PKA, suggesting that PKA might mediate the suppression of Htt aggregates by the A2A receptor. Interestingly, a proteasome inhibitor (MG132) completely removed the reducing effect of CGS on Htt aggregates (Fig. 5C). Moreover, expression of mutant Htt greatly enhanced the levels of high-molecular-mass ubiquitin conjugates which were reversed by CGS in HepG2. H89 also effectively blocked the effect of CGS (Fig. 5D). Collectively, A2A receptors might modulate the formation of Htt aggregates by stimulating the proteasome system via a cAMP/PKA-dependent pathway.

Figure 5.

Activation of the A2A receptor reduced mutant Huntingtin (Htt) aggregates in HepG2 cells via the cAMP/PKA pathway. (A) Cells were treated with or without the indicated reagent(s) for 20 min at RT. Data points represent the mean ± SEM of three independent experiments. (B) HePG2 cells were transfected with pcDNA3-(Htt-(Q)25-hrGFP) or pcDNA3-(Htt-(Q)109-hrGFP) and cultured in the presence of the indicated reagent(s) for 3 days. Expression of hrGFP is shown in green. Nuclei were visualized using DAPI (blue). Representative pictures of the vehicle-, CGS- (10 µm) and CGS (10 µm)/CSC (20 µm)-treated cells are shown. Percentages of cells containing fluorescent Htt aggregates were quantified using a fluorescence microscope (the bottom panel). Data points represent the mean ± SEM of three independent experiments. For each experiment, at least 130 transfected cells were scored. ***Specific comparison to cells treated with CGS (P < 0.001; one-way ANOVA). (C) HePG2 cells were transfected with pcDNA3-(Htt-(Q)25-hrGFP) or pcDNA3-(Htt-(Q)109-hrGFP) and treated with the desired reagent(s) for 3 days. Lysates (50 µg per lane) collected from the indicated condition were subjected to a filter retardation assay. The insoluble Htt aggregates retained on the filter were detected using an anti-Htt antibody. The level of actin in the lysate was assessed using a western blot analysis and was used as an internal control. A representative image of three independent experiments is shown. (D) Western blot analysis using an anti-ubiquitin antibody. Representative images of three independent experiments are shown.

Figure 5.

Activation of the A2A receptor reduced mutant Huntingtin (Htt) aggregates in HepG2 cells via the cAMP/PKA pathway. (A) Cells were treated with or without the indicated reagent(s) for 20 min at RT. Data points represent the mean ± SEM of three independent experiments. (B) HePG2 cells were transfected with pcDNA3-(Htt-(Q)25-hrGFP) or pcDNA3-(Htt-(Q)109-hrGFP) and cultured in the presence of the indicated reagent(s) for 3 days. Expression of hrGFP is shown in green. Nuclei were visualized using DAPI (blue). Representative pictures of the vehicle-, CGS- (10 µm) and CGS (10 µm)/CSC (20 µm)-treated cells are shown. Percentages of cells containing fluorescent Htt aggregates were quantified using a fluorescence microscope (the bottom panel). Data points represent the mean ± SEM of three independent experiments. For each experiment, at least 130 transfected cells were scored. ***Specific comparison to cells treated with CGS (P < 0.001; one-way ANOVA). (C) HePG2 cells were transfected with pcDNA3-(Htt-(Q)25-hrGFP) or pcDNA3-(Htt-(Q)109-hrGFP) and treated with the desired reagent(s) for 3 days. Lysates (50 µg per lane) collected from the indicated condition were subjected to a filter retardation assay. The insoluble Htt aggregates retained on the filter were detected using an anti-Htt antibody. The level of actin in the lysate was assessed using a western blot analysis and was used as an internal control. A representative image of three independent experiments is shown. (D) Western blot analysis using an anti-ubiquitin antibody. Representative images of three independent experiments are shown.

The suppressive effect of the A2A receptor on the formation of Htt aggregates was also observed in another cell line (HEK293) which also expressed endogenous A2A receptors. Treating HEK293 cells with CGS elevated the cellular cAMP content which was blocked by CSC (Fig. 6A). Cells were transfected with an expression construct encoding exon 1 of Htt containing a normal number (25) or an expanded stretch (158) of CAG repeats (designated Htt-(Q)25 and Htt-(Q)158, respectively) (17). As expected, expression of Htt-(Q)158 caused formation of Htt aggregates as assessed by filter retardation assays (Fig. 6B). Similar to what was found in HepG2 cells, CGS reduced aggregate formation, which was prevented by CSC. Moreover, a PKA inhibitor (H89) suppressed the ability of the A2A receptor to modulate aggregate formation. To evaluate the activity of the UPS, the overall ubiquitin profiles of HEK293 cells expressing either pcDNA3.1-Htt-(Q)25 or pcDNA3.1-Htt-(Q)158 were first assessed by western blot analyses using an anti-ubiquitin antibody. As shown in Figure 6C, expression of mutant Htt with polyQ expansion greatly enhanced levels of high-molecular-mass ubiquitin conjugates which were reversed by CGS. The effect of CGS was blocked by CSC and H89.

Figure 6.

Activation of the A2A receptor reduced Huntingtin (Htt) aggregates and enhanced UPS activity in HEK293 cells via the cAMP/PKA pathway. (A) Cells were treated with or without the indicated reagent(s) for 20 min at RT. Data points represent the mean ± SEM of three independent experiments. (B and C) Cells were transfected with pcDNA3-(Htt-(Q)25) or pcDNA3-(Htt-(Q)158) and treated with the desired reagent(s) for 3 days. Lysates (50 µg) harvested from the indicated condition were subjected to a filter retardation assay (B) or western blot analysis (C) using an anti-Htt and anti-ubiquitin antibody, respectively. Representative images of three independent experiments are shown. (D) HEK293 cells were co-transfected with pcDNA3-(Htt-(Q)25) or pcDNA3-(Htt-(Q)158), pZsProSensor-1 and dsRFP DNA at a molar ratio of 5:2:1. Cells were then incubated in the presence of the indicated reagents (10 µm CGS, 20 µm CSC, 10 µm H89 or 10 µm MG132) for 3 days. Representative pictures are shown (D). Transfected cells were identified by the expression of red fluorescent protein (red). Percentages of cells expressing detectable GFP were quantified using a fluorescence microscope (E). Data points represent the mean ± SEM of three independent experiments. At least 90 transfected cells were scored in each experiment. ***Specific comparison between cells transfected with the indicated construct and cells transfected with pcDNA3.1-(Q)158-Htt (P < 0.05; one-way ANOVA).

Figure 6.

Activation of the A2A receptor reduced Huntingtin (Htt) aggregates and enhanced UPS activity in HEK293 cells via the cAMP/PKA pathway. (A) Cells were treated with or without the indicated reagent(s) for 20 min at RT. Data points represent the mean ± SEM of three independent experiments. (B and C) Cells were transfected with pcDNA3-(Htt-(Q)25) or pcDNA3-(Htt-(Q)158) and treated with the desired reagent(s) for 3 days. Lysates (50 µg) harvested from the indicated condition were subjected to a filter retardation assay (B) or western blot analysis (C) using an anti-Htt and anti-ubiquitin antibody, respectively. Representative images of three independent experiments are shown. (D) HEK293 cells were co-transfected with pcDNA3-(Htt-(Q)25) or pcDNA3-(Htt-(Q)158), pZsProSensor-1 and dsRFP DNA at a molar ratio of 5:2:1. Cells were then incubated in the presence of the indicated reagents (10 µm CGS, 20 µm CSC, 10 µm H89 or 10 µm MG132) for 3 days. Representative pictures are shown (D). Transfected cells were identified by the expression of red fluorescent protein (red). Percentages of cells expressing detectable GFP were quantified using a fluorescence microscope (E). Data points represent the mean ± SEM of three independent experiments. At least 90 transfected cells were scored in each experiment. ***Specific comparison between cells transfected with the indicated construct and cells transfected with pcDNA3.1-(Q)158-Htt (P < 0.05; one-way ANOVA).

The proteasome activity of HEK293 cells was monitored using a proteasome sensor vector (pZsProSensor-1) which encodes a green fluorescent protein (GFP) fused to a degradation domain of ornithine decarboxylase. The proteasome activity was inversely correlated with the level of GFP. Cells were transfected with pZsProSensor-1 along with pcDNA3.1-Htt-(Q)25 or pcDNA3.1-Htt-(Q)158 and then treated with the indicated reagent(s). Under controlled conditions, only a very limited amount of GFP was detected in HEK293 using a fluorescent microscope. Expression of Htt-(Q)158 markedly enhanced the level of GFP, indicating impairment of proteasome activity by mutant Htt (Fig. 6D). Blockage of proteasome activity using MG132 greatly enhanced the GFP level, which supports that the level of GFP indeed reflects changes in UPS activity (Fig. 6E). In line with our hypothesis that activation of the A2A receptor might regulate proteasome activity, CGS treatment reduced the number of cells expressing GFP. The effect of CGS was reversed by a PKA-selective inhibitor (H89), demonstrating that the A2A receptor modulates proteasome activity via cAMP/PKA signaling.

DISCUSSION

In the past decade, A2A-specific drugs have been actively implicated in HD because of the expression of the A2A receptor in enkephalin-containing striatal neurons as well as in glutamatergic terminals in the corticostriatal pathway of the brain. Owing to its ability to evoke glutamate release upon activation, the presynaptic A2A receptor is suggested to be deleterious, whereas that on the postsynaptic GABAergic terminals is considered protective (67,68). In addition, the A2A receptor was recently shown to transactivate receptors of two important neurotrophic factors (brain-derived neurotrophic factor and fibroblast growth factor) and enhance their trophic functions (84–86). The role of the A2A receptor in neurodegeneration diseases is therefore of particular interest. In genetic mouse models of HD, both agonists and antagonists of the A2A receptor were shown to exhibit interesting effects to some extent. In R6/2 mice, an A2A antagonist (SCH58261) given systematically for a short period (1 week) was found to reduce NMDA-induced toxicity, although worsening the motor coordination (87). In the same mouse model, we earlier reported that chronic treatment (5 weeks) with an A2A agonist (CGS) ameliorated several major symptoms (e.g. brain atrophy, striatal aggregates and deteriorated motor coordination) (70). The disease stage, the drug administration protocol and the clinical manifestations might play critical roles in evaluating the therapeutic potential of A2A drugs (69).

Although the existence of liver Htt aggregates and their reduction by beneficial treatments have been documented (27,32,88), the contribution of hepatic deficiencies to HD pathogenesis is underappreciated. We previously reported that a urea cycle deficiency is an important factor in the pathogenesis of HD and contributes to the disease progression (32). In the present study, we show that CGS, which activates the A2A receptor, not only ameliorated several CNS symptoms of HD (70), but also improved a major function of the liver. Specifically, we found that chronic CGS treatment improved the urea cycle deficiency in R6/2 mice (Fig. 2; Tables 1 and 2). Levels of four dysregulated urea cycle enzymes (AS, AL, AG and OTC) all returned toward more-normal levels by chronic CGS treatment (Table 1). Elevated blood ammonia levels, which possibly contribute to Htt aggregates, were also reduced (Fig. 2) (32). In addition, CGS also normalized the aberrantly regulated expression of several HSPs in the liver (Fig. 3), reduced Htt aggregates (Figs 3D and 4A, and Supplementary Material, Fig. S1) and modulated UPS activity (Fig. 4). Since formation of Htt aggregates has been shown to damage several important mechanisms (including transcription and the molecular chaperone system), the beneficial effects of CGS on urea cycle deficiency and an inferior chaperone system might result from a reduction in Htt aggregates by CGS through elevating the impaired UPS activity in HD livers (Figs 4–6). We also found that the beneficial effect of the A2A receptor on the UPS was mediated by cAMP/PKA signaling (Fig. 6). Our findings are consistent with recent reports demonstrating that PKA directly phosphorylates several components and in turn promotes the activity of the UPS (89,90). To the best of our knowledge, the present study is the first report demonstrating that UPS activity is also impaired in a major peripheral tissue in an HD model. Amelioration of this defect by activation of hepatic A2A receptors appeared to contribute to an improvement in HD pathogenesis. By the same token, it is tempting to speculate that chronic CGS treatment might also ameliorate additional damage (e.g. cardiac dysfunction and insulin deficiency) induced by Htt aggregates in other peripheral tissues (29,91). Since treating the whole body is considered a more effective strategy than treating the CNS alone (33), and because the A2A receptor exists in multiple tissues where mHtt attacks, the A2A receptor appears to be a worthwhile drug target for HD that requires further exploration. Nevertheless, it should be pointed out that certain adverse effects of A2A drugs on the cardiovascular system and CNS have been reported and caution should be used when clinical application is considered (92,93). To treat neurodegeneration diseases, the ability to penetrate the brain is certainly an important factor to be considered. In addition, in order to avoid complications due to unfavorable side effects, it would be interesting to investigate whether partial A2A agonists are advantageous over full agonists as is recommended for other classes of adenosine drugs (94).

Dysregulation of molecular chaperones has long been implicated in HD's pathogenesis. Another new feature revealed by the present study is that when compared with those of WT mice, livers of HD mice possessed a weakened defense mechanism against stresses owing to altered expressions of protein chaperones in the liver (Fig. 3). Our findings agree with an earlier report demonstrating that a progressive decrease in levels of molecular chaperones was caused by sequestration of mHtt aggregates in the brain during HD progression (95). In neuronal cell lines, overexpression of several chaperone proteins (e.g. HSP27, HSP40, HSP70 and HSP104) was shown to reduce the aggregates of polyQ-expanded mutant proteins and the associated cytotoxicity (96–100). We found that at least two HSPs (HSP27 and HSP70) were downregulated in the liver of R6/2 mice. Expression of another critical transcription factor (HSF1) which is responsible for the transcription of many HSPs was also reduced in the liver of R6/2 mice. HSF1 appears to play a critical role in HD since expression of active HSF1 markedly reduced polyQ aggregate formation in both cell and mouse models (76). In contrast, the level of HSP90 in the liver of R6/2 mice was upregulated (Fig. 3). Since HSP90 has been shown to be associated with HSF1 and thereby suppresses the transcription activity of HSF1 (101), the enhancement of HSP90 in the HD liver might also contribute to the suppression of HSP27 and HSP70 via inhibiting the function of HSF1. It is interesting to note that the low level of HSP70 in the liver of R6/2 mice might also weaken the defense system against oxidative stress (102,103). Moreover, HSP90 has been shown to inhibit proteasome activity (104). Elevated HSP90 in the liver of R6/2 mice might therefore contribute to lower proteasome activity (Figs 3 and 4). Normalization of the expression profiles of HSPs and HSF1 by CGS not only amended the formation of Htt aggregates and thus ameliorated the urea cycle deficiency, but it might also improve the antioxidative mechanism and UPS activity.

Dysfunction of the 26S UPS has been shown to cause neuronal degenerative diseases and brain injuries (105,106). In HD, proteasomal subcomplexes were shown to co-localize with Htt aggregates (5,52,54). In striatal cell lines and brains of HD patients and some HD mice (HdhCAG150 and YAC72), decreased UPS activity was found to be closely associated with Htt aggregates in age- and disease-dependent manners (46,50,81,107). In some other HD mouse models (e.g. HD94 and R6/2), no reduction in the UPS activity of the brain was found compared with those of WT mice (82,108). This discrepancy might be due to compensation (52). Alternatively, dysregulation of UPS activity might occur in specific cellular compartments and would be difficult to detect when UPS is assessed in total brain lysates. Indeed, Wang et al. (47) reported that mutant Htt selectively impaired UPS activity at the synapses of striatal neurons and brains of both R6/2 and HdhCAG150 mice. In summary, UPS dysregulation in the brain is an important feature of HD and is believed to contribute to neuronal toxicity. Our results presented herein suggest that reduced UPS activity in the liver also plays a critical role in HD's pathogenesis and is a potential target for beneficial interventions. Importantly, reduced levels and/or activities of proteasomes in distinct brain areas have also been implicated in several other neuronal diseases besides HD. For example, aggregates of protein kinase Cγ, due to missense mutations in spinocerebellar ataxia type 14, were found to impair the UPS system and cause cell death (83). Proteasome-activating (or -modulating) compounds might therefore be considered for treating neurodegeneration diseases, where aggregate formations and/or UPS dysfunction are major pathogenic mechanisms (109). Our findings presented herein suggest that the A2A receptor might be considered a novel drug target for a wide variety of diseases and traumas for its ability to stimulate the activity of UPS upon encountering stress.

MATERIALS AND METHODS

Reagents

All reagents were purchased from Sigma (St Louis, MO, USA), except where otherwise specified. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA).

Animals and CGS administration

Male R6/2 mice (72) and littermate controls were originally obtained from Jackson Laboratories (Bar Harbor, ME, USA) and mated to female control mice (B6CBAFI/J). Offspring were identified by the polymerase chain reaction (PCR) genotyping and sequencing technique of genomic DNA extracted from tail tissues using primers located in the transgene (5′-CCGCTCAGGTTCTGCTTTTA-3′ and 5′-GGCTGAGGAAGCTGAGGAG-3′). The CAG repeats of the R6/2 mice used in this study was 154 ± 3 (mean ± SEM). In total, 29 R6/2 transgenic mice and 18 WT littermate controls were used in this study. Animals were housed at the Institute of Biomedical Sciences Animal Care Facility under a 12 h light/dark cycle. Animal experiments were performed under protocols approved by the Academia Sinica Institutional Animal Care and Utilization Committee, Taiwan, Republic of China. Daily injections of CGS (5 µg/g body weight) or the control vehicle (1% DMSO in saline) were given via an intraperitoneal injection between 13:00 and 18:00 as described earlier (70). As shown in our previous study, daily injections of CGS for 5 weeks did not affect the bodyweight of either R6/2 or WT mice.

RNA isolation and quantitative real-time PCR

Total RNA was isolated using the TriReagent kit (Molecular Research Center, Cincinnati, OH, USA), treated with RNase-free DNase (RQ1; Promega) to remove potential contamination by genomic DNA and transcribed into cDNA using Superscript* II reverse transcriptase. A real-time quantitative PCR was performed using a TaqMan kit (PE Applied Biosystems, Foster City, CA, USA) on a TaqMan ABI 7700 Sequence Detection System (PE Applied Biosystems) using heat-activated TaqDNA polymerase (Amplitaq Gold; PE Applied Biosystems). The PCR mixtures were incubated at 50°C for 2 min and 95°C for 10 min, and then 40 PCR cycles were conducted (95°C for 15 s and 65°C for 1 min). Primer sequences are listed in Supplementary Material, Table S1. Independent reverse-transcription PCRs were performed using the same complementary (c)DNA for both the indicated target gene and reference gene (GAPDH). A melting curve was created at the end of the PCR cycle to confirm that a single product had been amplified. Data were analyzed using ABI 7700 operating software to determine the threshold cycle above the background for each reaction. The relative transcript amount of the target gene, which was calculated using standard curves of serial RNA dilutions, was normalized to that of GAPDH of the same RNA.

Tandem mass spectrometry screening of blood citrulline

Samples were collected by impregnating filter paper (S&S 903; Schleicher & Shuell, Dassel, Germany) with blood (25 µl) from the tail vein. Before analyses, blood was eluted from the filter paper, and levels of amino acids and acylcarnitines were determined using a tandem mass spectrometer (Quattro Micro; Micromass, Beverly, MA, USA) as described elsewhere (32).

Measurements of blood ammonia level

Mice were decapitated, and blood samples (1–1.5 ml) were collected from each mouse into purple-top (EDTA) tubes. Concentrations of blood ammonia were measured using an ammonia detection kit following the manufacturer's protocol (Instruchemie, Delfzijl, The Netherlands).

AS and AL enzyme activities

Liver tissues were removed from the indicated animals, resuspended in ice-cold lysis buffer [10 mm Hepes (pH 8), 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 0.5 µg/ml aprotinin, 0.1 mm leupeptin, 4 µm pepstatin and 0.085 g sucrose per milliliter] and homogenized (Dounce, 15 strokes). Lysates were then centrifuged at 206g for 5 min to remove insoluble materials. Protein concentrations of the supernatants were determined by the Bradford analysis (110). AS and AL enzyme activities were determined spectrophotometrically as described elsewhere (32).

Immunohistochemistry and quantitation

Liver sections (5 µm) were used in the immunohistochemical analyses as described earlier (32). Cells harboring aggregates of mHtt were quantified in a blinded fashion. Single-antigen immunostaining was carried out using the avidin–biotin-peroxidase complex method as described previously (111). In general, we used a 1:2000 dilution for the polyclonal anti-ubiquitin antiserum (DakoCytomation Denmark A/S, Copenhagen, Denmark). Different sections labeled with the anti-ubiquitin antiserum and counterstained with methyl green (Vector) from one tissue sample were quantified. The number of aggregate-containing cells was normalized with the number of total cells in each section and designated as the percent of Htt aggregate-containing cells. At least 1200 cells from each liver section were quantified.

Western blot assays

Equal amounts of protein were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE) using 10% polyacrylamide gels according to the method of Laemmli (112). The resolved proteins were electroblotted onto Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% skim milk in phosphate-buffered saline (PBS) and incubated with an anti-Htt antibody (1:500; Chemicon International, Temecula, CA, USA), an anti-HSP27 antibody (1:1000; Stressgen Biotechnology, San Diego, CA, USA), an anti-HSP70 antibody (1:1000; Stressgen Biotechnology), an anti-HSP90 antibody (1:1000; Stressgen Biotechnology), an anti-HSF1 antibody (1:1000; Stressgen Biotechnology), an anti-ubiquitin antibody (1:2000; DakoCytomation Denmark A/S), an anti-actin antibody (1:2500; Chemicon International) or an anti-lamin antibody (1:2000; Santa Cruz Biotechnology) at 4°C overnight followed by the corresponding secondary antibody for 1 h at room temperature (RT). Immunoreactive bands were detected by enhanced chemiluminescence (Pierce) and recorded using Kodak XAR-5 film.

Filter retardation assay

SDS-insoluble mutant Htt aggregates were detected and quantified as described (27). A filter retardation assay was performed as described previously (32). Blots were blocked with 5% skim milk in PBS and incubated with an anti-Htt antibody (1:500; Chemicon International), an anti-ubiquitin antibody (1:2000; DakoCytomation), an anti-HSP27 antibody (1:1000; Stressgen Biotechnology), an anti-HSP70 antibody (1:1000; Stressgen Biotechnology), an anti-HSP90 antibody (1:1000; Stressgen Biotechnology) or an anti-HSF1 antibody (1:1000; Stressgen Biotechnology) at 4°C overnight followed by the corresponding secondary antibody for 1 h at RT. Immunoreactive bands were detected by enhanced chemiluminescence (Pierce) and recorded using Kodak XAR-5 film.

Nuclear extract preparation

Liver tissues were suspended, homogenized, centrifuged and collected. The nuclear extract was prepared as described previously (18).

Preparation of total and cytosolic fractions

The total lysate was prepared by resuspending liver tissues or cells in ice-cold buffer A [10 mm Hepes (pH 8), 1 mm Na3VO4 and a protease inhibitor cocktail (Roche, Basel, Switzerland)] and homogenizing samples by 15 Dounce strokes. After centrifugation at 112g for 1 min at 4°C, the supernatant was collected by centrifugation at 700g for 10 min at 4°C. The supernatant was then collected into new tubes and centrifuged at 6300g for 10 min at 4°C, followed by a final centrifugation at 97 468g for 120 min at 4°C. The protein concentration was measured using the Bio-Rad protein assay reagent.

Constructs

The human Htt exon 1 fragment of a normal allele containing the 25 CAG repeat ((Q)25) and that of a mutant Htt allele harboring the 158 CAG repeat ((Q)158) were amplified from genomic DNAs, and fragments were subcloned into the pcDNA3.1/V5-His vector (Invitrogen) (17). The pcDNA3.1-Htt-(Q)25-hrGFP and pcDNA3.1-Htt-(Q)109-hrGFP constructs encoding an N-terminal fragment of Htt with the indicated number of polyQ residues fused to hrGFP were created as described earlier (17).

Cell culture and measurement of mutant Htt aggregates

HepG2 cells were originally obtained from American Type Culture Collection (Manassas, VA, USA) and were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen) plus 1% penicillin/streptomycin (Invitrogen GibcoBRL, Carlsbad, CA, USA) in an incubation chamber gassed with 5% CO2 and 95% air at 37°C. HEK-293T cells [human embryonic kidney 293 cells expressing the large T-antigen of SV40 (simian virus 40)] were maintained in DMEM plus 10% FBS, 2 mm of l-glutamine and 1% penicillin/streptomycin at 37°C under 5% CO2. The day before transfection, cells were seeded onto a 35 mm dish at a density of 2 × 105 cells per well. To assess aggregates of mHtt, cells were transfected with pcDNA3.1-Htt-(Q)25-hrGFP or pcDNA3.1-Htt-(Q)109-hrGFP using Lipofectamine 2000 (LF2000, Invitrogen) and cultured for another 72 h in the presence or absence of the indicated reagent(s). To ensure that all reagents remained active during the incubation period, fresh reagent(s) were added to the culture medium every 24 h. Seventy-two hours after transfection, cells grown on glass coverslips were fixed with 4% paraformaldehyde in PBS for 30 min at RT and examined under a fluorescent microscope. Cells with green fluorescence were identified and scored. Percentages of cells containing visible fluorescent aggregates larger than 1 µm were quantified. At least 130 transfected cells were counted for each condition. Data are presented as the mean ± SEM of at least three independent experiments.

cAMP assay

The intracellular cAMP content was determined as described before (57) with slight modifications. Cells were washed twice with Ca2+-free Locke's solution (150 mm NaCl, 5.6 mm KCl, 5 mm glucose, 1 mm MgCl2 and 10 mm Hepes; pH 7.4) containing 0.5 mm isobutylmethylxanthine (Sigma) and were then treated with the indicated reagent(s) for 20 min at RT (25°C). The cAMP content was assayed using the 125I-cAMP assay system (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK).

Proteasome activity assay

Total liver lysate was prepared by resuspending liver tissues in ice-cold buffer A [10 mm Hepes (pH 8) and 1 mm Na3VO4] and homogenizing samples with 15 Dounce strokes. After centrifugation at 112g for 1 min at 4°C, the supernatant was collected by centrifugation at 700g for 10 min at 4°C. The protein concentration was measured using the Bio-Rad protein assay reagent. The chymotrypsin-like activity of the proteasome was determined using a specific proteasome substrate [succinyl-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin (AMC) (Sigma-Aldrich)]. Total lysates (10 µg) were incubated with the substrate (40 µm) in 100 µl of proteasome assay buffer [0.05 M Tris–HCl (pH 8.0), 0.5 mm EDTA, 1 mm ATP and 1 mm DTT] at 37°C for 60 min, where the relationship between the incubation time and product formation remained linear. The reactions were terminated by placing the reaction mixture on ice for at least 10 min. The fluorescence of the released AMC was detected by using a Fluorescence Microplate Reader System (Device, Sunnyvale, CA, USA) at 380 nm excitation and 460 nm emission wavelengths.

In living cells, proteasome activity was analyzed by transfecting a proteasome sensor vector (pZsProSensor-1; Clontech, Mountain View, CA, USA) which encoded a GFP fused to an ornithine decarboxylase degradation domain, along with DNAs of pcDNA-(CAG)25-Htt or pcDNA-(CAG)158-Htt, and deRFP at a ratio of 5:2:1. One day before transfection, cells were seeded onto a 35 mm dish at a density of 5 × 105 cells per well. Transfection was conducted using LF2000 for 5 h. Cells were then cultured for another 72 h in the presence or absence of the indicated reagent(s). To ensure that all reagents remained active during the incubation period, fresh reagent(s) were added to the culture medium every 24 h. Seventy-two hours after transfection, cells were fixed with 4% paraformaldehyde in PBS for 30 min at RT and examined under a fluorescent microscope. Transfected cells were marked by expressing deRFP (red). The accumulation of green fluorescence indicated loss of proteasome activity.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

FUNDING

This work was supported by grants from the National Science Council (NSC96-2321-B-001-015 and NSC 97-2321-B-001-012 to Y.C., and NSC96-2321-B-320-003 and NSC97-2320-B-320-008-MY2 to M.-C.C.) and Academia Sinica (AS-94-TP-B17 and AS-97-TP-B02 to Y.C.), Taiwan, Republic of China.

ACKNOWLEDGEMENTS

We thank Dr Yuh-Shan Jou for providing the HepG2 cell line, Dr Cathy S.J. Fann for consultation on statistical analyses and Drs Jer-Yuarn Wu and Yuan-Tsong Chen for their help and advice on analyzing the urea cycle deficiency. We are grateful to Mr Hao-Hung Chang for technical supports on cell cultures, Mr Wei-De Lin for the measurement of blood citrulline, Mr Chih-Ming Chang for graphic design and Mr Dan Chamberlin for reading and editing the manuscript.

Conflict of Interest statement. None declared.

REFERENCES

1
Martin
J.B.
Gusella
J.F.
Huntington's disease. Pathogenesis and management
N. Engl. J. Med.
 , 
1986
, vol. 
315
 (pg. 
1267
-
1276
)
2
Vonsattel
J.P.
Myers
R.H.
Stevens
T.J.
Ferrante
R.J.
Bird
E.D.
Richardson
E.P.
Jr
Neuropathological classification of Huntington's disease
J. Neuropathol. Exp. Neurol.
 , 
1985
, vol. 
44
 (pg. 
559
-
577
)
3
The Huntington's Disease Collaborative Research Group
A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes
Cell
 , 
1993
, vol. 
72
 (pg. 
971
-
983
)
4
Reddy
P.H.
Williams
M.
Tagle
D.A.
Recent advances in understanding the pathogenesis of Huntington's disease
Trends Neurosci.
 , 
1999
, vol. 
22
 pg. 
248
 
5
Waelter
S.
Boeddrich
A.
Lurz
R.
Scherzinger
E.
Lueder
G.
Lehrach
H.
Wanker
E.E.
Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation
Mol. Biol. Cell
 , 
2001
, vol. 
12
 (pg. 
1393
-
1407
)
6
Jana
N.R.
Tanaka
M.
Wang
G.-h.
Nukina
N.
Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated N-terminal huntingtin: their role in suppression of aggregation and cellular toxicity
Hum. Mol. Genet.
 , 
2000
, vol. 
9
 (pg. 
2009
-
2018
)
7
Wyttenbach
A.
Carmichael
J.
Swartz
J.
Furlong
R.A.
Narain
Y.
Rankin
J.
Rubinsztein
D.C.
Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease
Proc. Natl Acad. Sci. USA
 , 
2000
, vol. 
97
 (pg. 
2898
-
2903
)
8
Wyttenbach
A.
Swartz
J.
Kita
H.
Thykjaer
T.
Carmichael
J.
Bradley
J.
Brown
R.
Maxwell
M.
Schapira
A.
Orntoft
T.F.
, et al.  . 
Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington's disease
Hum. Mol. Genet.
 , 
2001
, vol. 
10
 (pg. 
1829
-
1845
)
9
Suhr
S.T.
Senut
M.-C.
Whitelegge
J.P.
Faull
K.F.
Cuizon
D.B.
Gage
F.H.
Identities of sequestered proteins in aggregates from cells with induced polyglutamine expression
J. Cell Biol.
 , 
2001
, vol. 
153
 (pg. 
283
-
294
)
10
Wanderer
J.
Morton
A.J.
Differential morphology and composition of inclusions in the R6/2 mouse and PC12 cell models of Huntington's disease
Histochem. Cell Biol.
 , 
2007
, vol. 
127
 (pg. 
473
-
484
)
11
Busch
A.
Engemann
S.
Lurz
R.
Okazawa
H.
Lehrach
H.
Wanker
E.E.
Mutant huntingtin promotes the fibrillogenesis of wild-type huntingtin: a potential mechanism for loss of huntingtin function in Huntington's disease
J. Biol. Chem.
 , 
2003
, vol. 
278
 (pg. 
41452
-
41461
)
12
Steffan
J.S.
Kazantsev
A.
Spasic-Boskovic
O.
Greenwald
M.
Zhu
Y.-Z.
Gohler
H.
Wanker
E.E.
Bates
G.P.
Housman
D.E.
Thompson
L.M.
The Huntington's disease protein interacts with p53 and CREB-binding protein and represses transcription
Proc. Natl Acad. Sci. USA
 , 
2000
, vol. 
97
 (pg. 
6763
-
6768
)
13
Nucifora
F.C.
Jr
Sasaki
M.
Peters
M.F.
Huang
H.
Cooper
J.K.
Yamada
M.
Takahashi
H.
Tsuji
S.
Troncoso
J.
Dawson
V.L.
, et al.  . 
Interference by huntingtin and atrophin-1 with CBP-mediated transcription leading to cellular toxicity
Science
 , 
2001
, vol. 
291
 (pg. 
2423
-
2428
)
14
Kegel
K.B.
Meloni
A.R.
Yi
Y.
Kim
Y.J.
Doyle
E.
Cuiffo
B.G.
Sapp
E.
Wang
Y.
Qin
Z.H.
Chen
J.D.
, et al.  . 
Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription
J. Biol. Chem.
 , 
2002
, vol. 
277
 (pg. 
7466
-
7476
)
15
Dunah
A.W.
Jeong
H.
Griffin
A.
Kim
Y.M.
Standaert
D.G.
Hersch
S.M.
Mouradian
M.M.
Young
A.B.
Tanese
N.
Krainc
D.
Sp1 and TAFII130 transcriptional activity disrupted in early Huntington's disease
Science
 , 
2002
, vol. 
296
 (pg. 
2238
-
2243
)
16
Li
S.H.
Cheng
A.L.
Zhou
H.
Lam
S.
Rao
M.
Li
H.
Li
X.J.
Interaction of Huntington disease protein with transcriptional activator Sp1
Mol. Cell. Biol.
 , 
2002
, vol. 
22
 (pg. 
1277
-
1287
)
17
Chiang
M.-C.
Lee
Y.-C.
Huang
C.-L.
Chern
Y.
cAMP-response element-binding protein contributes to suppression of the A2A adenosine receptor promoter by mutant huntingtin with expanded polyglutamine residues
J. Biol. Chem.
 , 
2005
, vol. 
280
 (pg. 
14331
-
14340
)
18
Chiang
M.-C.
Juo
C.-G.
Chang
H.-H.
Chen
H.-M.
Yi
E.C.
Chern
Y.
Systematic uncovering of multiple pathways underlying the pathology of Huntington disease by an acid-cleavable isotope-coded affinity tag approach
Mol. Cell. Proteomics
 , 
2007
, vol. 
6
 (pg. 
781
-
797
)
19
Ravikumar
B.
Stewart
A.
Kita
H.
Kato
K.
Duden
R.
Rubinsztein
D.C.
Raised intracellular glucose concentrations reduce aggregation and cell death caused by mutant huntingtin exon 1 by decreasing mTOR phosphorylation and inducing autophagy
Hum. Mol. Genet.
 , 
2003
, vol. 
12
 (pg. 
985
-
994
)
20
Ferrante
R.J.
Kubilus
J.K.
Lee
J.
Ryu
H.
Beesen
A.
Zucker
B.
Smith
K.
Kowall
N.W.
Ratan
R.R.
Luthi-Carter
R.
, et al.  . 
Histone deacetylase inhibition by sodium butyrate chemotherapy ameliorates the neurodegenerative phenotype in Huntington's disease mice
J. Neurosci.
 , 
2003
, vol. 
23
 (pg. 
9418
-
9427
)
21
Sipione
S.
Rigamonti
D.
Valenza
M.
Zuccato
C.
Conti
L.
Pritchard
J.
Kooperberg
C.
Olson
J.M.
Cattaneo
E.
Early transcriptional profiles in huntingtin-inducible striatal cells by microarray analyses
Hum. Mol. Genet.
 , 
2002
, vol. 
11
 (pg. 
1953
-
1965
)
22
Chan
E.Y.
Luthi-Carter
R.
Strand
A.
Solano
S.M.
Hanson
S.A.
DeJohn
M.M.
Kooperberg
C.
Chase
K.O.
DiFiglia
M.
Young
A.B.
, et al.  . 
Increased huntingtin protein length reduces the number of polyglutamine-induced gene expression changes in mouse models of Huntington's disease
Hum. Mol. Genet.
 , 
2002
, vol. 
11
 (pg. 
1939
-
1951
)
23
Luthi-Carter
R.
Strand
A.D.
Hanson
S.A.
Kooperberg
C.
Schilling
G.
La Spada
A.R.
Merry
D.E.
Young
A.B.
Ross
C.A.
Borchelt
D.R.
, et al.  . 
Polyglutamine and transcription: gene expression changes shared by DRPLA and Huntington's disease mouse models reveal context-independent effects
Hum. Mol. Genet.
 , 
2002
, vol. 
11
 (pg. 
1927
-
1937
)
24
Zuccato
C.
Tartari
M.
Crotti
A.
Goffredo
D.
Valenza
M.
Conti
L.
Cataudella
T.
Leavitt
B.R.
Hayden
M.R.
Timmusk
T.
, et al.  . 
Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes
Nat. Genet.
 , 
2003
, vol. 
35
 pg. 
76
 
25
Djousse
L.
Knowlton
B.
Cupples
L.A.
Marder
K.
Shoulson
I.
Myers
R.H.
Weight loss in early stage of Huntington's disease
Neurology
 , 
2002
, vol. 
59
 (pg. 
1325
-
1330
)
26
Ribchester
R.R.
Thomson
D.
Wood
N.I.
Hinks
T.
Gillingwater
T.H.
Wishart
T.M.
Court
F.A.
Morton
A.J.
Progressive abnormalities in skeletal muscle and neuromuscular junctions of transgenic mice expressing the Huntington's disease mutation
Eur. J. Neurosci.
 , 
2004
, vol. 
20
 (pg. 
3092
-
3114
)
27
Sathasivam
K.
Hobbs
C.
Turmaine
M.
Mangiarini
L.
Mahal
A.
Bertaux
F.
Wanker
E.E.
Doherty
P.
Davies
S.W.
Bates
G.P.
Formation of polyglutamine inclusions in non-CNS tissue
Hum. Mol. Genet.
 , 
1999
, vol. 
8
 (pg. 
813
-
822
)
28
Phan
J.
Hickey
M.A.
Zhang
P.
Chesselet
M.-F.
Reue
K.
Adipose tissue dysfunction tracks disease progression in two Huntington's disease mouse models
Hum. Mol. Genet.
 , 
2009
, vol. 
18
 (pg. 
1006
-
1016
)
29
Mihm
M.J.
Amann
D.M.
Schanbacher
B.L.
Altschuld
R.A.
Bauer
J.A.
Hoyt
K.R.
Cardiac dysfunction in the R6/2 mouse model of Huntington's disease
Neurobiol. Dis.
 , 
2007
, vol. 
25
 (pg. 
297
-
308
)
30
Bjorkqvist
M.
Wild
E.J.
Thiele
J.
Silvestroni
A.
Andre
R.
Lahiri
N.
Raibon
E.
Lee
R.V.
Benn
C.L.
Soulet
D.
, et al.  . 
A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington's disease
J. Exp. Med.
 , 
2008
, vol. 
205
 (pg. 
1869
-
1877
)
31
Valenza
M.
Carroll
J.B.
Leoni
V.
Bertram
L.N.
Bjorkhem
I.
Singaraja
R.R.
Di Donato
S.
Lutjohann
D.
Hayden
M.R.
Cattaneo
E.
Cholesterol biosynthesis pathway is disturbed in YAC128 mice and is modulated by huntingtin mutation
Hum. Mol. Genet.
 , 
2007
, vol. 
16
 (pg. 
2187
-
2198
)
32
Chiang
M.-C.
Chen
H.-M.
Lee
Y.-H.
Chang
H.-H.
Wu
Y.-C.
Soong
B.-W.
Chen
C.-M.
Wu
Y.-R.
Liu
C.-S.
Niu
D.-M.
, et al.  . 
Dysregulation of C/EBP{alpha} by mutant Huntingtin causes the urea cycle deficiency in Huntington's disease
Hum. Mol. Genet.
 , 
2007
, vol. 
16
 (pg. 
483
-
498
)
33
Martin
B.
Golden
E.
Keselman
A.
Stone
M.
Mattson
M.P.
Egan
J.M.
Maudsley
S.
Therapeutic perspectives for the treatment of Huntington's disease: treating the whole body
Histol. Histopathol.
 , 
2008
, vol. 
23
 (pg. 
237
-
250
)
34
DeMartino
G.N.
Gillette
T.G.
Proteasomes: machines for all reasons
Cell
 , 
2007
, vol. 
129
 (pg. 
659
-
662
)
35
Hershko
A.
Ciechanover
A.
The ubiquitin system
Ann. Rev. Biochem.
 , 
1998
, vol. 
67
 (pg. 
425
-
479
)
36
von Mikecz
A.
The nuclear ubiquitin–proteasome system
J. Cell Sci.
 , 
2006
, vol. 
119
 (pg. 
1977
-
1984
)
37
McBride
W.H.
Iwamoto
K.S.
Syljuasen
R.
Pervan
M.
Pajonk
F.
The role of the ubiquitin//proteasome system in cellular responses to radiation
Oncogene
 , vol. 
22
 (pg. 
5755
-
5773
)
38
Muratani
M.
Tansey
W.P.
How the ubiquitin–proteasome system controls transcription
Nat. Rev. Mol. Cell. Biol.
 , 
2003
, vol. 
4
 (pg. 
192
-
201
)
39
Hegde
A.N.
Upadhya
S.C.
The ubiquitin–proteasome pathway in health and disease of the nervous system
Trends Neurosci.
 , 
2007
, vol. 
30
 (pg. 
587
-
595
)
40
Ding
Q.
Cecarini
V.
Keller
J.N.
Interplay between protein synthesis and degradation in the CNS: physiological and pathological implications
Trends Neurosci.
 , 
2007
, vol. 
30
 (pg. 
31
-
36
)
41
Nandi
D.
Tahiliani
P.
Kumar
A.
Chandu
D.
The ubiquitin–proteasome system
J. Biosci.
 , 
2006
, vol. 
31
 (pg. 
137
-
155
)
42
McBride
W.H.
Iwamoto
K.S.
Syljuasen
R.
Pervan
M.
Pajonk
F.
The role of the ubiquitin/proteasome system in cellular responses to radiation
Oncogene
 , 
2003
, vol. 
22
 (pg. 
5755
-
5773
)
43
Bence
N.F.
Sampat
R.M.
Kopito
R.R.
Impairment of the ubiquitin–proteasome system by protein aggregation
Science
 , 
2001
, vol. 
292
 (pg. 
1552
-
1555
)
44
Ross
C.A.
Pickart
C.M.
The ubiquitin–proteasome pathway in Parkinson's disease and other neurodegenerative diseases
Trends Cell Biol.
 , 
2004
, vol. 
14
 (pg. 
703
-
711
)
45
van Tijn
P.
Hol
E.M.
van Leeuwen
F.W.
Fischer
D.F.
The neuronal ubiquitin–proteasome system: murine models and their neurological phenotype
Prog. Neurobiol.
 , 
2008
, vol. 
85
 (pg. 
176
-
193
)
46
Bennett
E.J.
Shaler
T.A.
Woodman
B.
Ryu
K.-Y.
Zaitseva
T.S.
Becker
C.H.
Bates
G.P.
Schulman
H.
Kopito
R.R.
Global changes to the ubiquitin system in Huntington's disease
Nature
 , 
2007
, vol. 
448
 (pg. 
704
-
708
)
47
Wang
J.
Wang
C.-E.
Orr
A.
Tydlacka
S.
Li
S.-H.
Li
J.
Impaired ubiquitin–proteasome system activity in the synapses of Huntington's disease mice
J. Cell Biol.
 , 
2008
, vol. 
180
 (pg. 
1177
-
1189
)
48
Seo
H.
Sonntag
K.C.
Isacson
O.
Generalized brain and skin proteasome inhibition in Huntington's disease
Ann. Neurol.
 , 
2004
, vol. 
56
 (pg. 
319
-
328
)
49
Doi
H.
Mitsui
K.
Kurosawa
M.
Machida
Y.
Kuroiwa
Y.
Nukina
N.
Identification of ubiquitin-interacting proteins in purified polyglutamine aggregates
FEBS Lett.
 , 
2004
, vol. 
571
 (pg. 
171
-
176
)
50
Zhou
H.
Cao
F.
Wang
Z.
Yu
Z.-X.
Nguyen
H.-P.
Evans
J.
Li
S.-H.
Li
J.
Huntingtin forms toxic NH2-terminal fragment complexes that are promoted by the age-dependent decrease in proteasome activity
J. Cell Biol.
 , 
2003
, vol. 
163
 (pg. 
109
-
118
)
51
Hunter
J.M.
Lesort
M.
Johnson
G.V.W.
Ubiquitin–proteasome system alterations in a striatal cell model of Huntington's disease
J. Neurosci. Res.
 , 
2007
, vol. 
85
 (pg. 
1774
-
1788
)
52
Seo
H.
Kim
W.
Isacson
O.
Compensatory changes in the ubiquitin–proteasome system, brain-derived neurotrophic factor and mitochondrial complex II/III in YAC72 and R6/2 transgenic mice partially model Huntington's disease patients
Hum. Mol. Genet.
 , 
2008
, vol. 
17
 (pg. 
3144
-
3153
)
53
Wang
J.
Wang
C.E.
Orr
A.
Tydlacka
S.
Li
S.H.
Li
X.J.
Impaired ubiquitin–proteasome system activity in the synapses of Huntington's disease mice
J. Cell Biol.
 , 
2008
, vol. 
180
 (pg. 
1177
-
1189
)
54
Valera
A.G.
Diaz-Hernandez
M.
Hernandez
F.
Lucas
J.J.
Testing the possible inhibition of proteasome by direct interaction with ubiquitylated and aggregated huntingtin
Brain Res. Bull.
 , 
2007
, vol. 
72
 (pg. 
121
-
123
)
55
Chern
Y.
King
K.
Lai
H.L.
Lai
H.T.
Molecular cloning of a novel adenosine receptor gene from rat brain
Biochem. Biophy. Res. Commun.
 , 
1992
, vol. 
185
 (pg. 
304
-
309
)
56
Lee
Y.-C.
Chien
C.-L.
Sun
C.-N.
Huang
C.-L.
Huang
N.-K.
Chiang
M.-C.
Lai
H.-L.
Lin
Y.-S.
Chou
S.-Y.
Wang
C.-K.L.
, et al.  . 
Characterization of the rat A2A adenosine receptor gene: a 4.8-kb promoter-proximal DNA fragment confers selective expression in the central nervous system
Eur. J. Neurosci.
 , 
2003
, vol. 
18
 (pg. 
1786
-
1796
)
57
Chern
Y.
Lai
H.L.
Fong
J.C.
Liang
Y.
Multiple mechanisms for desensitization of A2a adenosine receptor-mediated cAMP elevation in rat pheochromocytoma PC12 cells
Mol. Pharmacol.
 , 
1993
, vol. 
44
 (pg. 
950
-
958
)
58
Huang
N.-K.
Lin
Y.-W.
Huang
C.-L.
Messing
R.O.
Chern
Y.
Activation of protein kinase A and atypical protein kinase C by A2A adenosine receptors antagonizes apoptosis due to serum deprivation in PC12 cells
J. Biol. Chem.
 , 
2001
, vol. 
276
 (pg. 
13838
-
13846
)
59
Walker
B.A.
Rocchini
C.
Boone
R.H.
Ip
S.
Jacobson
M.A.
Adenosine A2a receptor activation delays apoptosis in human neutrophils
J. Immunol.
 , 
1997
, vol. 
158
 (pg. 
2926
-
2931
)
60
Day
Y.J.
Huang
L.
McDuffie
M.J.
Rosin
D.L.
Ye
H.
Chen
J.F.
Schwarzschild
M.A.
Fink
J.S.
Linden
J.
Okusa
M.D.
Renal protection from ischemia mediated by A2A adenosine receptors on bone marrow-derived cells
J. Clin. Invest.
 , 
2003
, vol. 
112
 (pg. 
883
-
891
)
61
Day
Y.J.
Marshall
M.A.
Huang
L.
McDuffie
M.J.
Okusa
M.D.
Linden
J.
Protection from ischemic liver injury by activation of A2A adenosine receptors during reperfusion: inhibition of chemokine induction
Am. J. Physiol. Gastrointest. Liver Physiol.
 , 
2004
, vol. 
286
 (pg. 
G285
-
G293
)
62
Ferre
S.
O'Connor
W.T.
Fuxe
K.
Ungerstedt
U.
The striopallidal neuron: a main locus for adenosine–dopamine interactions in the brain
J. Neurosci.
 , 
1993
, vol. 
13
 (pg. 
5402
-
5406
)
63
Weaver
D.R.
A2a adenosine receptor gene expression in developing rat brain
Brain Res. Mol. Brain Res.
 , 
1993
, vol. 
20
 (pg. 
313
-
327
)
64
Glass
M.
Dragunow
M.
Faull
R.L.
The pattern of neurodegeneration in Huntington's disease: a comparative study of cannabinoid, dopamine, adenosine and GABA(A) receptor alterations in the human basal ganglia in Huntington's disease
Neuroscience
 , 
2000
, vol. 
97
 (pg. 
505
-
519
)
65
Popoli
P.
Pezzola
A.
Reggio
R.
Caporali
M.G.
Scotti de Carolis
A.
CGS 21680 antagonizes motor hyperactivity in a rat model of Huntington's disease
Eur. J. Pharmacol.
 , 
1994
, vol. 
257
 (pg. 
R5
-
R6
)
66
Popoli
P.
Pintor
A.
Domenici
M.R.
Frank
C.
Tebano
M.T.
Pezzola
A.
Scarchilli
L.
Quarta
D.
Reggio
R.
Malchiodi-Albedi
F.
, et al.  . 
Blockade of striatal adenosine A2A receptor reduces, through a presynaptic mechanism, quinolinic acid-induced excitotoxicity: possible relevance to neuroprotective interventions in neurodegenerative diseases of the striatum
J. Neurosci.
 , 
2002
, vol. 
22
 (pg. 
1967
-
1975
)
67
Blum
D.
Galas
M.C.
Pintor
A.
Brouillet
E.
Ledent
C.
Muller
C.E.
Bantubungi
K.
Galluzzo
M.
Gall
D.
Cuvelier
L.
, et al.  . 
A dual role of adenosine A2A receptors in 3-nitropropionic acid-induced striatal lesions: implications for the neuroprotective potential of A2A antagonists
J. Neurosci.
 , 
2003
, vol. 
23
 (pg. 
5361
-
5369
)
68
Fink
J.S.
Kalda
A.
Ryu
H.
Stack
E.C.
Schwarzschild
M.A.
Chen
J.F.
Ferrante
R.J.
Genetic and pharmacological inactivation of the adenosine A2A receptor attenuates 3-nitropropionic acid-induced striatal damage
J. Neurochem.
 , 
2004
, vol. 
88
 (pg. 
538
-
544
)
69
Popoli
P.
Blum
D.
Domenici
M.R.
Burnouf
S.
Chern
Y.
A critical evaluation of adenosine A2A receptors as potentially ‘druggable’ targets in Huntington's disease
Curr. Pharm. Design
 , 
2008
, vol. 
14
 (pg. 
1500
-
1511
)
70
Chou
S.Y.
Lee
Y.C.
Chen
H.M.
Chiang
M.C.
Lai
H.L.
Chang
H.H.
Wu
Y.C.
Sun
C.N.
Chien
C.L.
Lin
Y.S.
, et al.  . 
CGS21680 attenuates symptoms of Huntington's disease in a transgenic mouse model
J. Neurochem.
 , 
2005
, vol. 
93
 (pg. 
310
-
320
)
71
Martire
A.
Calamandrei
G.
Felici
F.
Scattoni
M.L.
Lastoria
G.
Domenici
M.R.
Tebano
M.T.
Popoli
P.
Opposite effects of the A2A receptor agonist CGS21680 in the striatum of Huntington's disease versus wild-type mice
Neurosci. Lett.
 , 
2007
, vol. 
417
 (pg. 
78
-
83
)
72
Mangiarini
L.
Sathasivam
K.
Seller
M.
Cozens
B.
Harper
A.
Hetherington
C.
Lawton
M.
Trottier
Y.
Lehrach
H.
Davies
S.W.
, et al.  . 
Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice
Cell
 , 
1996
, vol. 
87
 (pg. 
493
-
506
)
73
Tarditi
A.
Camurri
A.
Varani
K.
Borea
P.A.
Woodman
B.
Bates
G.
Cattaneo
E.
Abbracchio
M.P.
Early and transient alteration of adenosine A2A receptor signaling in a mouse model of Huntington disease
Neurobiol. Dis.
 , 
2006
, vol. 
23
 (pg. 
44
-
53
)
74
Glass
M.
Dragunow
M.
Faull
R.L.M.
The pattern of neurodegeneration in Huntington's disease: a comparative study of cannabinoid, dopamine, adenosine and GABAA receptor alterations in the human basal ganglia in Huntington's disease
Neuroscience
 , 
2000
, vol. 
97
 (pg. 
505
-
519
)
75
Luthi-Carter
R.
Strand
A.
Peters
N.L.
Solano
S.M.
Hollingsworth
Z.R.
Menon
A.S.
Frey
A.S.
Spektor
B.S.
Penney
E.B.
Schilling
G.
, et al.  . 
Decreased expression of striatal signaling genes in a mouse model of Huntington's disease
Hum. Mol. Genet.
 , 
2000
, vol. 
9
 (pg. 
1259
-
1271
)
76
Fujimoto
M.
Takaki
E.
Hayashi
T.
Kitaura
Y.
Tanaka
Y.
Inouye
S.
Nakai
A.
Active HSF1 significantly suppresses polyglutamine aggregate formation in cellular and mouse models
J. Biol. Chem.
 , 
2005
, vol. 
280
 (pg. 
34908
-
34916
)
77
Mitra
S.
Tsvetkov
A.S.
Finkbeiner
S.
Single-neuron ubiquitin–proteasome dynamics accompanying inclusion body formation in Huntington's disease
J. Biol. Chem.
 , 
2009
, vol. 
284
 (pg. 
4398
-
4403
)
78
Tydlacka
S.
Wang
C.E.
Wang
X.
Li
S.
Li
X.J.
Differential activities of the ubiquitin–proteasome system in neurons versus glia may account for the preferential accumulation of misfolded proteins in neurons
J. Neurosci.
 , 
2008
, vol. 
28
 (pg. 
13285
-
13295
)
79
Bennett
E.J.
Shaler
T.A.
Woodman
B.
Ryu
K.Y.
Zaitseva
T.S.
Becker
C.H.
Bates
G.P.
Schulman
H.
Kopito
R.R.
Global changes to the ubiquitin system in Huntington's disease
Nature
 , 
2007
, vol. 
448
 (pg. 
704
-
708
)
80
Ortega
Z.
Diaz-Hernandez
M.
Lucas
J.J.
Is the ubiquitin–proteasome system impaired in Huntington's disease?
Cell. Mol. Life Sci.
 , 
2007
, vol. 
64
 (pg. 
2245
-
2257
)
81
Hunter
J.M.
Lesort
M.
Johnson
G.V.
Ubiquitin–proteasome system alterations in a striatal cell model of Huntington's disease
J. Neurosci. Res.
 , 
2007
, vol. 
85
 (pg. 
1774
-
1788
)
82
Bett
J.S.
Goellner
G.M.
Woodman
B.
Pratt
G.
Rechsteiner
M.
Bates
G.P.
Proteasome impairment does not contribute to pathogenesis in R6/2 Huntington's disease mice: exclusion of proteasome activator REGgamma as a therapeutic target
Hum. Mol. Genet.
 , 
2006
, vol. 
15
 (pg. 
33
-
44
)
83
Seki
T.
Takahashi
H.
Adachi
N.
Abe
N.
Shimahara
T.
Saito
N.
Sakai
N.
Aggregate formation of mutant protein kinase C gamma found in spinocerebellar ataxia type 14 impairs ubiquitin–proteasome system and induces endoplasmic reticulum stress
Eur. J. Neurosci.
 , 
2007
, vol. 
26
 (pg. 
3126
-
3140
)
84
Flajolet
M.
Wang
Z.
Futter
M.
Shen
W.
Nuangchamnong
N.
Bendor
J.
Wallach
I.
Nairn
A.C.
Surmeier
D.J.
Greengard
P.
FGF acts as a co-transmitter through adenosine A(2A) receptor to regulate synaptic plasticity
Nat. Neurosci.
 , 
2008
, vol. 
11
 (pg. 
1402
-
1409
)
85
Lee
F.S.
Chao
M.V.
Activation of Trk neurotrophin receptors in the absence of neurotrophins
Proc. Natl Acad. Sci. USA
 , 
2001
, vol. 
98
 (pg. 
3555
-
3560
)
86
Wiese
S.
Jablonka
S.
Holtmann
B.
Orel
N.
Rajagopal
R.
Chao
M.V.
Sendtner
M.
Adenosine receptor A2A-R contributes to motoneuron survival by transactivating the tyrosine kinase receptor TrkB
Proc. Natl Acad. Sci. USA
 , 
2007
, vol. 
104
 (pg. 
17210
-
17215
)
87
Domenici
M.R.
Scattoni
M.L.
Martire
A.
Lastoria
G.
Potenza
R.L.
Borioni
A.
Venerosi
A.
Calamandrei
G.
Popoli
P.
Behavioral and electrophysiological effects of the adenosine A2A receptor antagonist SCH 58261 in R6/2 Huntington's disease mice
Neurobiol. Dis.
 , 
2007
, vol. 
28
 (pg. 
197
-
205
)
88
Tanaka
M.
Machida
Y.
Niu
S.
Ikeda
T.
Jana
N.R.
Doi
H.
Kurosawa
M.
Nekooki
M.
Nukina
N.
Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease
Nat. Med.
 , 
2004
, vol. 
10
 (pg. 
148
-
154
)
89
Lu
H.
Zong
C.
Wang
Y.
Young
G.W.
Deng
N.
Souda
P.
Li
X.
Whitelegge
J.
Drews
O.
Yang
P.Y.
, et al.  . 
Revealing the dynamics of 20S proteasome phosphoproteome: a combined CID and ETD approach
Mol. Cell. Proteomics
 , 
2008
, vol. 
7
 (pg. 
2073
-
2089
)
90
Zhang
F.
Hu
Y.
Huang
P.
Toleman
C.A.
Paterson
A.J.
Kudlow
J.E.
Proteasome function is regulated by cyclic AMP-dependent protein kinase through phosphorylation of Rpt6
J. Biol. Chem.
 , 
2007
, vol. 
282
 (pg. 
22460
-
22471
)
91
Andreassen
O.A.
Dedeoglu
A.
Stanojevic
V.
Hughes
D.B.
Browne
S.E.
Leech
C.A.
Ferrante
R.J.
Habener
J.F.
Beal
M.F.
Thomas
M.K.
Huntington's disease of the endocrine pancreas: insulin deficiency and diabetes mellitus due to impaired insulin gene expression
Neurobiol. Dis.
 , 
2002
, vol. 
11
 (pg. 
410
-
424
)
92
Mingote
S.
Pereira
M.
Farrar
A.M.
McLaughlin
P.J.
Salamone
J.D.
Systemic administration of the adenosine A(2A) agonist CGS 21680 induces sedation at doses that suppress lever pressing and food intake
Pharmacol. Biochem. Behav.
 , 
2008
, vol. 
89
 (pg. 
345
-
351
)
93
Gordi
T.
Frohna
P.
Sun
H.L.
Wolff
A.
Belardinelli
L.
Lieu
H.
A population pharmacokinetic/pharmacodynamic analysis of regadenoson, an adenosine A2A-receptor agonist, in healthy male volunteers
Clin. Pharmacokinetics
 , 
2006
, vol. 
45
 (pg. 
1201
-
1212
)
94
Gao
Z.G.
Jacobson
K.A.
Partial agonists for A(3) adenosine receptors
Curr. Top. Med. Chem.
 , 
2004
, vol. 
4
 (pg. 
855
-
862
)
95
Hay
D.G.
Sathasivam
K.
Tobaben
S.
Stahl
B.
Marber
M.
Mestril
R.
Mahal
A.
Smith
D.L.
Woodman
B.
Bates
G.P.
Progressive decrease in chaperone protein levels in a mouse model of Huntington's disease and induction of stress proteins as a therapeutic approach
Hum. Mol. Genet.
 , 
2004
, vol. 
13
 (pg. 
1389
-
1405
)
96
Kobayashi
Y.
Kume
A.
Li
M.
Doyu
M.
Hata
M.
Ohtsuka
K.
Sobue
G.
Chaperones Hsp70 and Hsp40 suppress aggregate formation and apoptosis in cultured neuronal cells expressing truncated androgen receptor protein with expanded polyglutamine tract
J. Biol. Chem.
 , 
2000
, vol. 
275
 (pg. 
8772
-
8778
)
97
Muchowski
P.J.
Schaffar
G.
Sittler
A.
Wanker
E.E.
Hayer-Hartl
M.K.
Hartl
F.U.
Hsp70 and Hsp40 chaperones can inhibit self-assembly of polyglutamine proteins into amyloid-like fibrils
Proc. Natl Acad. Sci. USA
 , 
2000
, vol. 
97
 (pg. 
7841
-
7846
)
98
Perrin
V.
Regulier
E.
Abbas-Terki
T.
Hassig
R.
Brouillet
E.
Aebischer
P.
Luthi-Carter
R.
Deglon
N.
Neuroprotection by Hsp104 and Hsp27 in lentiviral-based rat models of Huntington's disease
Mol. Ther.
 , 
2007
, vol. 
15
 (pg. 
903
-
911
)
99
Wyttenbach
A.
Sauvageot
O.
Carmichael
J.
Diaz-Latoud
C.
Arrigo
A.P.
Rubinsztein
D.C.
Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin
Hum. Mol. Genet.
 , 
2002
, vol. 
11
 (pg. 
1137
-
1151
)
100
Sittler
A.
Lurz
R.
Lueder
G.
Priller
J.
Lehrach
H.
Hayer-Hartl
M.K.
Hartl
F.U.
Wanker
E.E.
Geldanamycin activates a heat shock response and inhibits huntingtin aggregation in a cell culture model of Huntington's disease
Hum. Mol. Genet.
 , 
2001
, vol. 
10
 (pg. 
1307
-
1315
)
101
Zou
J.
Guo
Y.
Guettouche
T.
Smith
D.F.
Voellmy
R.
Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1
Cell
 , 
1998
, vol. 
94
 (pg. 
471
-
480
)
102
Calabrese
V.
Renis
M.
Calderone
A.
Russo
A.
Barcellona
M.L.
Rizza
V.
Stress proteins and SH-groups in oxidant-induced cell damage after acute ethanol administration in rat
Free Rad. Biol. Med.
 , 
1996
, vol. 
20
 (pg. 
391
-
397
)
103
Calabrese
V.
Renis
M.
Calderone
A.
Russo
A.
Reale
S.
Barcellona
M.L.
Rizza
V.
Stress proteins and SH-groups in oxidant-induced cellular injury after chronic ethanol administration in rat
Free Rad. Biol. Med.
 , 
1998
, vol. 
24
 (pg. 
1159
-
1167
)
104
Conconi
M.
Petropoulos
I.
Emod
I.
Turlin
E.
Biville
F.
Friguet
B.
Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90
Biochem. J.
 , 
1998
, vol. 
333
 (pg. 
407
-
415
)
105
Bedford
L.
Hay
D.
Devoy
A.
Paine
S.
Powe
D.G.
Seth
R.
Gray
T.
Topham
I.
Fone
K.
Rezvani
N.
, et al.  . 
Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies
J. Neurosci.
 , 
2008
, vol. 
28
 (pg. 
8189
-
8198
)
106
Liao
C.W.
Fan
C.K.
Kao
T.C.
Ji
D.D.
Su
K.E.
Lin
Y.H.
Cho
W.L.
Brain injury-associated biomarkers of TGF-beta1, S100B, GFAP, NF-L, tTG, AbetaPP, and tau were concomitantly enhanced and the UPS was impaired during acute brain injury caused by Toxocara canis in mice
BMC Infect. Dis.
 , 
2008
, vol. 
8
 pg. 
84
 
107
Seo
H.
Sonntag
K.C.
Isacson
O.
Generalized brain and skin proteasome inhibition in Huntington's disease
Ann. Neurol.
 , 
2004
, vol. 
56
 (pg. 
319
-
328
)
108
Diaz-Hernandez
M.
Hernandez
F.
Martin-Aparicio
E.
Gomez-Ramos
P.
Moran
M.A.
Castano
J.G.
Ferrer
I.
Avila
J.
Lucas
J.J.
Neuronal induction of the immunoproteasome in Huntington's disease
J. Neurosci.
 , 
2003
, vol. 
23
 (pg. 
11653
-
11661
)
109
Dahlmann
B.
Role of proteasomes in disease
BMC Biochem.
 , 
2007
, vol. 
8
 
Suppl. 1
pg. 
S3
 
110
Bradford
M.M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
 , 
1976
, vol. 
72
 (pg. 
248
-
254
)
111
Liu
F.-C.
Wu
G.-C.
Hsieh
S.-T.
Lai
H.-L.
Wang
H.-F.
Wang
T.-W.
Chern
Y.
Expression of type VI adenylyl cyclase in the central nervous system: implication for a potential regulator of multiple signals in different neurotransmitter systems
FEBS Lett.
 , 
1998
, vol. 
436
 pg. 
92
 
112
Laemmli
U.K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4
Nature
 , 
1970
, vol. 
227
 (pg. 
680
-
685
)