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Human Molecular Genetics Advance Access originally published online on January 9, 2007
Human Molecular Genetics 2007 16(5):483-498; doi:10.1093/hmg/ddl481
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Dysregulation of C/EBP{alpha} by mutant Huntingtin causes the urea cycle deficiency in Huntington's disease

Ming-Chang Chiang1,2, Hui-Mei Chen1, Yi-Hsin Lee1, Hao-Hung Chang1, Yi-Chih Wu1, Bing-Wen Soong3, Chiung-Mei Chen4, Yih-Ru Wu4, Chin-San Liu5, Dau-Ming Niu6, Jer-Yuarn Wu1, Yuan-Tsong Chen1 and Yijuang Chern1,2,*

1 Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei, Taiwan, 2 Institute of Neuroscience, National Yang-Ming University, Taipei, Taiwan, 3 Department of Neurology, Yang-Ming University School of Medicine and Neurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan, 4 Department of Neurology, Chang Gung Memorial Hospital, Taipei, Taiwan, 5 Department of Neurology, Changhua Christian Hospital, Changhua, Taiwan and 6 Department of Pediatrics, Taipei Veterans General Hospital, Taipei, Taiwan

* To whom correspondence should be addressed. Tel: +886 226523913; Fax: +886 227829143; Email: bmychern{at}ibms.sinica.edu.tw

Received August 10, 2006; Revised December 26, 2006; Accepted December 28, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. Using two mouse models of HD, we demonstrate that the urea cycle deficiency characterized by hyperammonemia, high blood citrulline and suppression of urea cycle enzymes is a prominent feature of HD. The resultant ammonia toxicity might exacerbate the neurological deficits of HD. Suppression of C/EBP{alpha}, a crucial transcription factor for the transcription of urea cycle enzymes, appears to mediate the urea cycle deficiency in HD. We found that in the presence of mutant Htt, C/EBP{alpha} loses its ability to interact with an important cofactor (CREB-binding protein). Moreover, mutant Htt recruited C/EBP{alpha} into aggregates, as well as suppressed expression of the C/EBP{alpha} gene. Consumption of protein-restricted diets not only led to the restoration of C/EBP{alpha}'s activity, and repair of the urea cycle deficiency and hyperammonemia, but also ameliorated the formation of Htt aggregates, the motor deterioration, the suppression of striatal brain-derived neurotrophic factor and the normalization of three protein chaperones (Hsp27, Hsp70 and Hsp90). Treatments aimed at repairing the urea cycle deficiency may provide a new strategy for dealing with HD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Huntington's disease (HD) is a neurodegenerative disease characterized by intranuclear and cytoplasmic aggregates and cell death in the brain. The causative mutation is a CAG trinucleotide expansion in exon 1 of the Huntingtin (Htt) gene. Normal chromosomes have 35 or fewer repeats in the N-terminal region, whereas HD is associated with 36 or more repeats (1). The major characteristic of HD is a selective loss of neurons in the striatum and the cortex, which leads to movement disorders, dementia and eventual death (2). The time of disease onset is correlated with the length of polyglutamine (polyQ) expansion, but the mechanism of toxicity remains largely controversial (3). Interestingly, polyQ expansion may alter transcription by squelching transcription factors such as CREB (4) and the CREB-binding protein (CBP) (5).

Besides the well-characterized neurological deficits, metabolic abnormalities have also been reported in HD (6–11). For example, levels of several metabolic markers (including glycerol, malonate, aliphatic amino acids, neopterin and lipid peroxidation products) were found to be altered in the blood of HD patients (10,11). The role of metabolic dysfunction in the progression of HD is therefore of great interest. In addition to the brain, formation of Htt aggregates has also been documented in peripheral tissues including the liver (12,13). The urea cycle is a major function of the liver and is responsible for transforming toxic nitrogenous compounds into urea, which is then disposed of in the urine. Several enzymes including argininosuccinic acid synthetase (AS), argininosuccinase acid lyase (AL), arginase (AG), ornithine transcarbamylase (OTC), N-acetylglutamate synthetase (NAGS) and carbamyl phosphate synthetase (CPS) are known to play key roles in the urea cycle (14). Hyperammonemia can result from genetic defects of urea cycle enzymes and transporters of amino acids related to the urea cycle, or other metabolic abnormalities and liver failure (15). Since Htt aggregation has been found in the liver (12,13,16), it is plausible that mutant Htt aggregation in the liver may cause liver dysfunction and trigger hyperammonemia. The possibility of liver dysfunction in HD and the underlying mechanisms have never been explored before.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Urea cycle deficiency contributes to the progression of HD
In addition to the elevated blood glucose (17), we found that the blood ammonia levels of R6/2 mice were also higher than those of wild-type (WT) mice (Fig. 1A). This is of great interest because ammonia toxicity has long been associated with psychosis (18), and therefore might contribute to the psychotic symptoms of HD (19). Since hyperammonemia might result from several different causes including liver diseases, organic acid disorders, defects in fatty acid oxidation and urea cycle disorders, we performed the following experiments to identify the causative defect. We first measured blood levels of alanine transaminase (ALT) and aspartate transaminase (AST) and found no differences between WT and R6/2 mice. The mean ALT levels of R6/2 and WT mice were 37.1 ± 8.02 and 43.3 ± 12.0 U/l (mean ± SD, n = 7–9 mice/condition), respectively. The mean AST levels of R6/2 and WT mice were 264.5 ± 51.0 and 269.4 ± 77.8 U/l (mean ± SD, n = 7–9 mice/condition), respectively. There was no hepatomegaly, and furthermore, hematoxylin and eosin (H&E) staining revealed no apparent liver pathology in R6/2 mice (data not shown). Urine organic acid profiles also revealed no differences between 10.5-week-old R6/2 and WT mice (n = 3 mice/condition, data not shown), and no abnormal metabolites were noted. The observed hyperammonemia in HD mice therefore was unlikely to have resulted from defects in organic acid metabolism or fatty acid oxidation.


Figure 1
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  		Figure 1. The urea cycle deficiency is a prominent feature of HD. (A–F) Mice were fed with indicated diets from the age of 4 weeks. Protein contents of the indicated diets are listed in Supplementary Material, Table S3. The blood ammonia levels of 10.5-week-old R6/2 (A, C, n = 5–15) and 5-month-old Hdh mice (E, n = 7–8) were determined. The blood citrulline levels of R6/2 (B, D, n = 15–54) and Hdh mice (F, n = 13–48) at the indicated age were determined. Data are presented as the mean ± SD. Specific comparison between the indicated group and WT mice on the control or K1 diet is denoted as ‘a’ (P < 0.01; Student's t-test). Specific comparison between the indicated group and HD mice on the diet with 22% of the energy provided by protein denoted as ‘b’ (control or K1 as indicated; P < 0.01; Student's t-test). CON, the control Diet 20; LPD, the EUROdent diet which contains a lower protein content than the CON diet.

 
In contrast, the level of blood citrulline, a marker of the urea cycle function, was elevated. As shown in Figure 1B, the amount of blood citrulline was ~2-fold than that of WT mice. A significant difference was found at an age as early as 6 weeks, when deterioration of motor coordination had not yet been observed in R6/2 mice. We next determined the levels of blood ammonia and citrulline in a knock-in HD mouse model [Hdh(CAG)150; (20)] which exhibits late onset symptoms. These mice harbor the Htt homolog gene (Hdh) containing 150 copies of polyQ and show neurological abnormalities at ~15 months of age when only one copy of the poly-Q-expanded Hdh gene exists. As shown in Figure 1E, at 5-months old (an early presymptomatic age), heterozygous Hdh(CAG)150 mice already exhibited elevated blood ammonia levels when compared with their WT littermates. Consistently, blood citrulline levels of Hdh(CAG)150 mice were also elevated (Fig. 1F). The enhanced blood citrulline levels could be detected at an age as early as 1 month. Collectively, these observations led to the hypothesis that a urea cycle deficiency might occur and contribute to the progression of HD. Note that this increase in blood citrulline was selective because among the amino acids and acylcarnitines examined, citrulline was the only component consistently regulated in both R6/2 and Hdh mice (Supplementary Material, Tables S1 and S2).

Because protein-restricted diets have been shown to ameliorate the symptoms of urea cycle deficiency (21), we first examined the disease progression in R6/2 mice on a regular control diet (Diet 20) in which 22% of the energy is provided by protein and on a commercially available low-protein diet (LPD; EUROdent diet; Supplementary Material, Table S3) in which only 17% of the energy is from protein. As expected, blood ammonia and citrulline levels of R6/2 mice on the LPD diet were reduced (Fig. 1A and B). We then designed two isocaloric diets (K1 and K3, Supplementary Material, Table S3), in which proteins provided 22 and 16% of the energy, respectively. Note that K1 contained the same amounts of protein, fat and carbohydrate as the CON diet (Diet 20) and was used as a control diet for the K3 diet in the following experiments. No difference in any analyses of disease progression employed in the present study was found between mice fed with the CON and the K1 diets. As predicted, R6/2 mice that fed with K3 diet also had lower blood ammonia and citrulline levels than those that fed with K1 diet (Fig. 1C and D). Most importantly, R6/2 mice on both diets with lower protein contents (LPD and K3) exhibited improved motor coordination (Fig. 2). Nevertheless, neither the LPD nor K3 diet ameliorated the body weight loss (data not shown), nor did they affect the lifespan of R6/2 mice. The mean respective survival times of R6/2 mice fed with control and LPD diets were 103.9 ± 14.4 and 95.3 ± 11.1 days (mean ± SD, n = 20–32 mice/condition). The mean respective survival times of R6/2 mice fed with K1 and K3 diets were 101.8 ± 12.7 and 102.6 ± 12.6 days (mean ± SD, n = 8 mice/condition).


Figure 2
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  		Figure 2. Diets composed of lower protein contents (LPD and K3) improved movement coordination in R6/2 mice. Mice were fed with indicated diet from the age of 4 weeks. Rotarod performance was assessed. Latency to falling was automatically recorded. Data are presented as mean ± SD (n > 8 for each condition). Specific comparison between the indicated group and WT mice on the control or K1 diet is denoted as ‘a’ (P < 0.01; Student's t-test). Specific comparison between the indicated group and R6/2 mice on the diet with 22% of the energy provided by protein is denoted as ‘b’ (control or K1 as indicated; P < 0.01; Student's t-test).

 
An important hallmark of HD is the formation of Htt aggregates in the brain. As shown in Figure 3A–F, mice fed with LPD and K3 diets exhibited reduced numbers of cells containing Htt aggregates in the striatum. Previous studies also showed that the levels of an important neurotrophic factor (brain-derived neurotrophic factor; BDNF) are decreased in the brains of HD mice (22). Using the quantitative PCR technique, we confirmed the previous results and found that 12-week-old R6/2 mice fed with LPD and K3 diets contained more striatal BDNF than those fed with control and K1 diets as indicated (Fig. 3G). Collectively, lower protein contents (with the LPD and K3 diets) resulted in marked improvement in several major deficits of HD including hyperammonemia, rotarod performance, neurotrophic deficiency and aggregate formation in the striatum.


Figure 3
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  		Figure 3. The LPD and K3 diets decreased the number of mutant huntingtin (Htt) aggregates and enhanced the reduced brain-derived neurotrophic factor (BDNF) expression in the striatum of R6/2 mice. Mice were fed with LPD or K3 diet from the age of 4 weeks. Htt aggregates were visualized with an antibody against ubiquitin. Representative images of the striatum (A, B, D, E) of 12-week-old R6/2 mice are shown. Scale bar, 20 µm. (C, F) The percentages of striatal cells expressing NII were quantified at the age of 12 weeks. At the age of 12 weeks, striatal tissues of R6/2 mice were collected to determine the gene expression of BDNF using the Q-PCR technique (G). The expression levels of BDNF were normalized to that of GAPDH. Data are presented as mean ± SD values from three independent experiments. Specific comparison between the indicated and WT mice is denoted as ‘a’ (P < 0.001; one-way ANOVA). Specific comparison between R6/2 mice fed with control or K1 diet in each group is denoted as ‘b’ (P < 0.05; one-way ANOVA).

 
In addition to the brain, the liver also appears to be an important target tissue of mutant Htt. We first examined whether Htt aggregates also exist in the liver where the urea cycle occurs. Although at a much lower frequency than in the striatum, Htt aggregates were detected in the liver of R6/2 mice. Htt-aggregate formation in the livers of R6/2 mice on the LPD and K3 diets was also markedly reduced (Fig. 4A–F). Moreover, R6/2 mice fed with LPD and K3 diets exhibited markedly increased reductions in protein chaperones in the liver (Hsp70 and Hsp27; Fig. 4G and H). This is of great interest because both Hsp27 and Hsp70 have been found to reduce the aggregation and toxicity of mutant Htt (23,24). In addition, the elevated expression of Hsp90 was normalized in the livers of R6/2 mice fed with either of the LPDs (Fig. 4I and J). Inhibition of Hsp90 using specific inhibitors has been shown to induce the expression of several molecular chaperons (including Hsp70) and to exert a protective effect in HD (25–27). Down-regulation of Hsp90 in R6/2 mice consuming the LPD or K3 diets might therefore be responsible for the up-regulation of molecular chaperons (e.g. Hsp70) and, in turn, for reducing the formation of Htt aggregates in the liver. We also measured the levels of Hsp27, Hsp70 and Hsp90 in the striatum. However, the amount of Hsp27 was too low to be detected, and those of Hsp70 and Hsp90 were not altered in the striatum of R6/2 mice when compared with those of WT mice (Supplementary Material, Fig. S2). We therefore did not further examine the effect of the LPDs on these three chaperons in the striatum.


Figure 4
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  		Figure 4. The LPD and K3 diets decreased the number of mutant huntingtin (Htt) aggregates and normalized the expression of protein chaperones in the liver of R6/2 mice. R6/2 mice were fed with LPD or K3 diet from the age of 4 weeks. Representative images of the liver (A, B, D, E) of 12-week-old R6/2 mice are shown. (C, F) Htt aggregates were visualized with an antibody against ubiquitin, and the percentages of liver cells expressing NII were quantified at the age of 12 weeks. Scale bar, 20 µm. (G–J) The cytosolic fractions from the liver were analyzed for the expressions of HSP27, HSP70 and HSP90 as indicated, and normalized to actin. Data are presented as mean ± SD values from three independent experiments. Specific comparison with WT mice fed with control or K1 diet as indicated is denoted as ‘a’ (P < 0.05; one-way ANOVA). Specific comparison between R6/2 mice fed with control or K1 diet in each group is denoted as ‘b’ (P < 0.05; one-way ANOVA).

 
Suppression of C/EBP{alpha} by mutant Htt leads to urea cycle deficiency in HD
We set out to identify the target enzyme(s) of the urea cycle that might be altered by mutant Htt and were responsible for the elevated blood levels of ammonia and citrulline. Expressions of four major enzymes (AS, AL, AG and OTC; Supplementary Material, Fig. S1) of the urea cycle were determined in the livers of R6/2 mice. Quantitative RT-PCR analyses revealed that the levels of AL, AS and AG were reduced, whereas that of OTC was elevated in the livers of R6/2 mice (Fig. 5A, Table 1). Most importantly, R6/2 mice on the lower-protein diets (LPD and K3) exhibited effective reversal of the suppressed gene expression of the two affected enzymes (AS and AL), which might contribute to normalization of blood citrulline and ammonia levels (Fig. 5A, Table 1).


Figure 5
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  		Figure 5. Suppression of the expression of the AL gene at the transcriptional level by mutant huntingtin (Htt) leads to urea cycle deficiency in HD. Mice were fed with indicated diet from the age of 4 weeks. (A) The AL transcripts in the liver of the indicated mice of 10.5 weeks were analyzed using the Q-PCR technique. (B) A typical CCAAT site (underlined) is located in the promoter region of the mouse AL gene (28) and mutated residues are shown in bold. (C) The indicated Htt construct with 109 copies of polyQ was cotransfected with the WT or the C/EBP-null AL gene [pGL2-(–235/–1)] into HepG2 cells, and luciferase activity was normalized to the protein content. Values are expressed as percentages of the WT promoter activity in the presence of pcDNA3.1-(Q)25-Htt-hrGFP, and are presented as mean ± SD of at least three independent experiments. Total lysates collected from HepG2 cells transfected with pcDNA3.1-(Q)25-Htt-hrGFP or pcDNA3.1-(Q)109-Htt-hrGFP as indicated were subjected to western blot analyses (D, 75 µg per lane) to detect the transgene expression and a filter retardation assay (E; 30 µg per lane) to monitor aggregate formation. Specific comparison with R6/2 mice fed with control or K1 diet in each group is denoted as ‘a’ (P < 0.001; one-way ANOVA). Specific comparison between cells transfected with pcDNA3.1-(Q)109-Htt-hrGFP and cells transfected with pcDNA3.1-(Q)25-Htt-hrGFP is denoted by double asterisk (P < 0.001; one-way ANOVA).

 


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  		Table 1. Mutant Htt altered the gene expression of urea cycle enzymes (AS, AG and OTC) in the liver of R6/2 mice

 
We next examined the activities of AL and AS in the livers of HD mice fed with various diets. In agreement with the gene expression study, the AS and AL activities of R6/2 mice fed with control or K1 diet were much lower than those of WT mice. Similar to those in R6/2 mice, the liver AL and AS activities of heterozygous Hdh(CAG)150 mice were markedly lower than those of their WT littermates (Tables 2 and 3). Moreover, consistent with the beneficial effects observed, liver AL and AS activities of R6/2 mice fed with diets having lower protein contents (LPD and K3) were also normalized (Tables 2 and 3). Consistent with the early onset of elevated blood citrulline, reduced activities of AL and AS in HD mice were evident when major symptoms (e.g. deterioration of motor coordination) had not yet been observed in either of the HD mouse models examined. To understand the molecular mechanism underlying the dysregulation of urea cycle enzymes in HD, we chose to further examine AL because its gene structure and regulation have been well characterized (28).


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  		Table 2. AL activities in the livers of HD mice under various treatments

 


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  		Table 3. AS activities in the livers of HD mice under various treatments

 
Transcriptional dysfunction is a major mechanism proposed for HD (4,29). To determine whether the suppression of AL occurs at the transcriptional level, we created a mouse AL promoter construct (pGL2-AL(–235/–1);+1 as the transcriptional start site) based on a previously characterized rat AL gene (28). In the promoter region of the AL gene, a CCAAT site which binds to C/EBP{alpha} was previously shown to play an important role in its basal promoter activity (Fig. 5B) (28). Expression of the mutant Htt with 109 copies of CAG [Htt-(Q)109-hrGFP] markedly reduced the AL promoter activity (Fig. 5C) in HepG2 cells. Because C/EBP{alpha} has been shown to play a critical role in ammonia detoxification (30), we mutated the CCAAT box of the AL promoter (Fig. 5B). The resultant AL promoter mutant exhibited lower promoter activity and could not be suppressed by mutant Htt (Fig. 5C). These results support the hypothesis that a functional CCAAT site is located in the promoter region of the AL gene and that C/EBP{alpha} is involved in mutant Htt-evoked suppression of the AL gene. Using electrophoretic gel mobility shift assay (EMSA) analyses, we also demonstrated that C/EBP{alpha} and its cofactor [CBP (31)] bind to a 25 bp region of the AL promoter harboring the CCAAT site (Fig. 6A). Selectivity of the above binding was demonstrated by competition using the original AL probe or an irrelevant 25 bp probe in a 50-fold molar excess (Supplementary Material, Fig. S3). Mutations of the CCAAT site (28) completely eliminated the binding. Adding antibodies against C/EBP{alpha} or CBP (Fig. 6A, Supplementary Material, Fig. S4a), but not an irrelevant antibody (AC6D, Fig. 6B, Supplementary Material, Fig. S4b), created supershifts in the EMSA of WT mice, suggesting that the C/EBP{alpha} and CBP complex binds the CCAAT box in WT mice. For HD mice fed with K1 and control diets, the anti-C/EBP{alpha} antibody caused a supershift, while the anti-CBP antibody did not. Thus, under the influence of mutant Htt, CBP was no longer bound to the AL promoter while C/EBP{alpha} remained on the CCAAT site. Since the binding of CBP is critical for the activity of C/EBP family members (31), the loss of CBP binding at the CCAAT site of the AL promoter in R6/2 mice fed with K1 and control diets likely caused reduced AL expression. The binding of C/EBP{alpha} and CBP to the AL promoter was further verified using a DNA-mediated pull-down assay using a double-stranded oligo (AL–90/–66), which contains the C/EBP{alpha} binding site from the AL promoter to isolate the DNA/protein complex from the liver of WT and R6/2 mice. The complexes were first resolved and isolated from non-denaturing gels and then subjected to separation by SDS–PAGE, followed by western blot analyses. As shown in Figure 7, both C/EBP{alpha} and CBP could be detected in the DNA/protein complex of WT mice. In contrast, although expression of both CBP and C/EBP{alpha} could be detected in the livers of R6/2 mice (Fig. 8A), only C/EBP{alpha} existed in the DNA/protein complex of R6/2 mice fed with control diets (CON and K1). No binding of C/EBP{alpha} or CBP was found when the CCAAT site was mutated, demonstrating the selectivity of this assay. Most importantly, both EMSA (Fig. 6, Supplementary Material, Fig. S4) and the DNA-pull down assays (Fig. 7A and B) showed that the lower-protein diets (LPD and K3) restored the complex formation of C/EBP{alpha} and CBP in R6/2 mice, indicating that the LPDs rescued the binding of CBP and C/EBP{alpha} at the CCAAT box of the mouse AL promoter.


Figure 6
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  		Figure 6. Mutant huntingtin (Htt) suppressed the expression of the AL gene by altering the binding ability of C/EBP{alpha} to the AL promoter. R6/2 mice were fed with K1 or K3 diet from the age of 4 weeks. Liver nuclear extract (NE, 5 µg) prepared from the liver in the indicated 10.5-week-old mice was pre-incubated for 1 h with anti-C/EBP{alpha}, anti-CBP (A) or an irrelevant anti-AC6D antibody (B), followed by incubation with the 32P-labeled double-stranded oligonucleotide probe (25 bp; AL – 90/–66) for EMSA. The NE (5 µg) was reacted with a 25 bp 32P-labeled AL – 90/–66 oligonucleotide probe containing the WT or mutated CCAAT site as indicated. Reaction products were separated in 6% polyacrylamide gels by electrophoresis. This is one representative result of the three independent experiments performed.

 


Figure 7
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  		Figure 7. The functional CCAAT site was critical for AL promoter activity and contributed to suppression of the AL gene by huntingtin (Htt) with expanded poly(Q). (A, B) A combination of EMSA and western blot analyses was performed as follows: 10 µg of the unlabeled AL (–90/–66) probe containing the WT or mutated CCAAT site was incubated with liver nuclear fractions (0.25 mg) collected from the indicated 10.5-week-old mice, and then subjected to EMSA electrophoresis. Gels were stained with Coomassie blue to identify the location of the protein/DNA complex. The complex was then excised and separated by SDS–PAGE, followed by western blot analyses using an anti-C/EBP{alpha} and an anti-CBP antibody. Representative images of mice fed with indicated diets are shown. Data are presented as mean ± SD values from three independent experiments. Specific comparison with the R6/2 mice fed with control or K1 diet in each group denoted as ‘a’ and ‘b’ (P < 0.001, P < 0.05, respectively; one-way ANOVA). (C) Immunostaining of the liver of R6/2 mice at the age of 12 weeks using anti-Htt and anti-C/EBP{alpha} antiserum. Htt and C/EBP{alpha} were visualized by the Avidin-Alexa Fluor® 488- (green) and 568 (red)-conjugated secondary antibodies, respectively. The nucleus was visualized by Hoechst 33342 (blue). Animals were fed with LPD (D) or K3 (E) diet from the age of 4 weeks. Liver lysate (250 µg per lane) collected from the indicated 10.5-week-old mice were subjected to a filter retardation assay. The insoluble Htt aggregates retained on the filter were detected using an anti-C/EBP{alpha} or anti-Htt antibody. Representative images of three independent experiments are shown.

 


Figure 8
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  		Figure 8. Mutant Htt reduced the expression of C/EBP{alpha} in the liver of R6/2 mice. Mice were fed with indicated diet from the age of 4 weeks. (A) Liver and striatum nuclear fractions (50 µg per lane) collected from the indicated 10.5-week-old mice were subjected to western blot analysis. Levels of the indicated protein normalized with an internal control (lamin) were compared with that in WT mice fed with control or K1 diet, and are shown in the bottom of the corresponding lane. Data are presented as mean ± SD from three independent experiments. (B) Analysis of the C/EBP{alpha} transcripts in the liver of the indicated mice was performed using the Q-PCR technique. Liver RNA of the indicated 10.5-week-old mice was collected, and reverse-transcribed into cDNA. Real-time quantitative PCR of C/EBP{alpha} was performed and normalized to that of the GAPDH. Data are presented as mean ± SD from three independent experiments. (C) The indicated Htt construct with 109 copies of polyQ was cotransfected with the C/EBP{alpha} promoter [pGL2-C/EBP{alpha}(–365/+50)] into HepG2 cells, and luciferase activity was normalized to the protein content. Values are expressed as percentages of the WT promoter activity in the presence of pcDNA3.1-(Q)25-Htt-hrGFP, and are presented as mean ± SD of three independent experiments. Data are presented as mean ± SD from three independent experiments. Specific comparison with WT mice on the control or K1 diet is denoted as ‘a’ (P < 0.001; one-way ANOVA). Specific comparison between cells transfected with pcDNA3.1-(Q)109-Htt-hrGFP and cells transfected with pcDNA3.1-(Q)25-Htt-hrGFP is denoted as 'double asterisk' (P < 0.001; one-way ANOVA).

 
Importantly, the amount of C/EBP{alpha} bound to the CCAAT site of the AL promoter appeared to be reduced in the liver lysate of R6/2 mice (Fig. 7). Filter retardation assays (Fig. 7D and E) revealed that C/EBP{alpha} was recruited into aggregates, which was further demonstrated by immunocytochemical analyses (Fig. 7C). Expression of polyQ-expanded mutant Htt therefore reduced the availability of C/EBP{alpha} for gene transcription. Consistent with Figure 4, the lower-protein diets (K3 and LPD) reduced Htt aggregates and therefore ameliorated the hijacking of C/EBP{alpha} (Fig. 7D and E). Moreover, we found that the protein and transcript levels of C/EBP{alpha} in the livers of R6/2 mice were significantly lower than those in WT mice. Neither of these decreases in C/EBP{alpha} expression could be rescued by the lower-protein diets (K3 and LPD) (Fig. 8). We next created a mouse C/EBP{alpha} promoter construct (pGL2-C/EBP{alpha}(–365/+50); with + 1 as the transcriptional start site) based on a previously characterized mouse C/EBP{alpha} gene (32). As shown in Figure 8C, marked repression of the C/EBP{alpha} promoter was observed in the presence of Htt-(Q)109-hrGFP in HepG2 cells, indicating that suppression of C/EBP{alpha} by mutant Htt occurred at the transcriptional level. Note that the defects resulting from reduced activity and expression of C/EBP{alpha} by mutant Htt primarily occurred in the liver, not in the striatum because expression of C/EBP{alpha} was only detected in the liver, not in the striatum (Fig. 8A).

HD patients exhibited higher blood citrulline levels
To determine whether elevated blood citrulline was also observed in human patients, we carried out a pilot study to assess the fasting blood citrulline levels of 21 HD patients and 23 healthy volunteers (age- and gender-matched; see Materials and Methods). As in the R6/2 and Hdh mice, HD patients exhibited statistically higher blood citrulline levels when compared with the healthy controls (Fig. 9; Supplementary Material, Table S4).


Figure 9
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  		Figure 9. HD patients exhibited higher blood citrulline levels. Blood samples were collected from 23 control subjects (CON) and 21 HD patients (HD) for citrulline measurement by tandem mass spectrometry. Each solid circle represents the mean of five determinations for each subject. The median value of blood citrulline of each group is shown as a solid line. Specific comparison with control subjects (CON) is denoted as ‘a’ (P < 0.001; the non-parametric Mann–Whitney–Wilcoxon test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The existence of liver Htt aggregates and their reduction by beneficial treatments were previously documented (12,13). However, the contribution of such a deficit to the progression of HD has never been explored. In the present study, we demonstrated elevated blood ammonia, enhanced blood citrulline and reduced activity of two urea cycle enzymes (AL and AS) in two different HD mouse models (R6/2 and Hdh(CAG)150; Fig. 1; Tables 2 and 3). The existence of a urea cycle deficiency in two HD mouse models of different designs demonstrated that the deficient urea cycle existed in HD mice, and was not due to the position effect of the transgene or any other artifact of R6/2 mice. Hyperammonemia can also be caused by liver dysfunction or secondary to other metabolic abnormalities such as organic acidemia. However, liver function, acylcarnitine profiles and urine organic acids have been normal in HD mice. Most importantly, we showed that protein-restricted diets ameliorated the urea cycle deficiency in R6/2 mice, the formation of Htt aggregates, the deterioration of motor coordination, the suppression of BDNF expression in the brain and the decreased expressions of HSP27 and HSP70 (Figs 2–4). Such a close association between the urea cycle function and neurological symptoms suggests that the urea cycle deficiency might contribute to the pathogenesis of HD.

Consistent with a previous study (13), we found that mutant Htt aggregates also appeared in the livers of R6/2 mice. The altered liver expressions of protein chaperones in R6/2 mice fed with control or K1 diet suggest a weakened defense mechanism against stress, which might further enhance the ammonia toxicity (Fig. 4). This finding agrees with an early report which suggested that a progressive decrease in the levels of molecular chaperones caused by sequestration of mutant Htt aggregates occurs during HD progression (25). Note that hyperammonemia has been shown to cause elevated oxidative stress (33). In addition, elevated oxidative stress promotes aggregate formation and cell death evoked by mutant Htt (34). Most importantly, over-expression of Hsp70 effectively rescues the mutant Htt-induced cytotoxicity described earlier (34). Consumption of the lower-protein diets (LPD and K3) normalized the elevated blood ammonia, and thus would be expected to decrease oxidative stress and reduce Htt aggregates. The reduction in Htt aggregates resulted in more chaperones (Hsp27 and Hsp70) being available, and this in turn suppressed the aggregation of mutant Htt and restored the proper physiological functions (e.g. transcription) of the liver. Alternatively, diet interventions (e.g. nutrition deprivation or caloric restriction), which cause deficiency in the levels of amino acids, have been shown to activate autophagy (35,36). This is of great interest because autophagy is a major clearance pathway for the removal of both aggregated and soluble forms of mutant Htt. Induction of autophagy thus exerts a protective effect against the cytotoxicity caused by mutant Htt (37–39). Nevertheless, besides normalizing the elevated citrulline level, chronic consumption of LPDs did not decrease blood amino acids in R6/2 mice to the abnormally low levels (Supplementary Material, Table S1). Induction of autophagy therefore was unlikely to mediate the protective effect of LPDs in the present study.

Inhibition of urea cycle enzymes appears to occur, at least in part, at the transcriptional level (Fig. 5, Table 1). Similar to what was observed in the central nervous system (29), transcriptional dysfunction evoked by mutant Htt plays a key role. Suppression of the AL and AS genes and enhancement of the OTC gene in R6/2 mice resulted in elevated blood citrulline levels (Figs 1 and 5; Table 1). In contrast, expression of NO synthases, another family of enzymes involving free citrulline metabolism (40), was not affected in the livers of R6/2 mice (data not shown). Thus, modification of urea cycle genes is very specific. Our study revealed that suppression of C/EBP{alpha}, a crucial transcription factor for the urea cycle, might mediate the urea cycle deficit in R6/2 mice. C/EBP{alpha} binds and activates genes at the CCAAT box which can be found in ~67% of human promoters (41). We tested the involvement of C/EBP{alpha} because it has been implicated in ammonia detoxification. Disruption of the C/EBP{alpha} gene led to decreased expressions of urea cycle enzymes (including AL) and several-fold elevation of blood ammonia (30). In line with the above report, we found that mutation of the CCAAT box in the AL gene markedly suppressed the activity of the AL promoter and eliminated its sensitivity to mutant Htt-evoked suppression (Fig. 5). We demonstrated herein that multiple pathways are involved in the inhibition of C/EBP{alpha} by mutant Htt. First, although mutant Htt did not eliminate the binding of C/EBP{alpha} to the CCAAT box of the AL gene, it prevented the association of C/EBP{alpha} with its cofactor (CBP) and therefore suppressed transcription of the AL gene (Figs 6 and 7; Supplementary Material, Fig. S4). Secondly, mutant Htt formed aggregates in the liver and recruited C/EBP{alpha} (Fig. 7D and E), which reduced the availability of C/EBP{alpha} for active transcription (Fig. 7D and E). Finally, mutant Htt suppressed the gene and protein expression levels of C/EBP{alpha} (Fig. 8). Consumption of the K3 and LPD diets resulted in the former two being blocked, but not the last interfering action of mutant Htt. The inability of the K3 and LPD diets to restore proper expression of C/EBP{alpha} (Fig. 8) might be related to its inability to improve tissue wasting and prolong the lifespan, because C/EBP{alpha} is important for fat cell differentiation and maturation (42). Results in C/EBP{alpha}-deficient mice also indicated that C/EBP{alpha} is critical for energy homeostasis and that it affects energy-related genes (30,43). Reduction of C/EBP{alpha} therefore is likely to contribute to body weight loss, a major hallmark of HD. In addition to the urea cycle and energy homeostasis, C/EBP{alpha} has also been implicated in the transcription of genes regulating hepatocyte differentiation, cell cycle progression, the membrane/extracellular matrix structure and other metabolic functions (44). Inhibition of C/EBP{alpha} by mutant Htt thus might lead to disorders with a wide spectrum of responses.

Excitotoxicity is one of the leading hypotheses for HD (45), and has also been shown to play a major role in the neurological injury caused by hyperammonemia (46). Elevated ammonia levels alter glutamate receptor-mediated synaptic transmission cause NMDA receptor-mediated excitotoxicity, and result in mitochondrial dysfunction (46–48). In brains of OTC-deficient mice which contain elevated ammonia, the density of medium spiny striatal neurons is greatly reduced (48) as in the brains of HD mice. Although the elevated ammonia levels in R6/2 or Hdh(CAG)150 mice are less than 1-fold higher when compared with WT littermates (Figs. 1A, C and E), the hyperammonemia is chronic and might begin at very early ages when significant damage can be done. In addition, chronic ammonia toxicity might enhance the sensitivity of HD mice to excitotoxicity. It is therefore not surprising to find that antagonists of NMDA receptors have been used to treat hyperammonemia and to confer beneficial effects on HD (49–51). Both chronic treatment with an NMDA antagonist (52) and LPDs (the present study) ameliorated the formation of Htt aggregates, suggesting a potential overlap of the underlying mechanism. These observations are consistent with our hypothesis that ammonia toxicity might contribute to the progression of HD.

In a pilot study of 21 HD patients and 23 healthy volunteers, we found that the blood citrulline levels of HD patients were statistically higher than those of healthy controls (Fig. 9), suggesting that a urea cycle deficiency might also occur in humans with HD. The blood ammonia levels were not measured because an accurate ammonia determination requires arterial punctures, which were not accessible to most of our outpatients and controls. In addition, the average human protein consumption represents ~15% of total daily calories (53), which is relatively low when compared with a regular mouse diet (22% protein, Supplementary Material, Table S3). A smaller difference in human subjects than in the mouse models fed with regular rodent diets would therefore be expected. For patients on a regular diet with a 15% protein content, the contribution of the urea cycle to HD pathology might be less significant than that found in the mouse models we used. Nevertheless, our findings suggest that chronic high-protein intake might worsen HD pathogenesis. This is of particular importance because although the length of the CAG repeat is the key factor for the onset HD, environmental factors markedly affect the onset and severity of HD (54,55). A closely monitored diet with proper protein content therefore may be critical for HD patients.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Animals and diet administration
Male R6/2 mice (56) and littermate controls were originally obtained from Jackson Laboratories (Bar Harbor, ME, USA) and were mated to female control mice (B6CBAFI/J). Offspring were identified by genotyping of tail DNA as previously described (17). In total, 128 R6/2 transgenic mice and 59 WT littermate control mice were used in this study. The mice used for the knock-in HD mouse model (Hdh, B6.129P2-Hdhtm2Detl/J) harboring a mutant mouse Htt gene with 150 copies of CAG (20) were also purchased from Jackson Laboratories, and these were mated to C57BL/6J mice. PCR genotyping was performed using the following primers: 5'-CCCATTCATTGCCTTGCTG-3' and 5'-GCGGCTGAGGGGGTTGA-3'. In total, 36 heterozygote Hdh mice and 29 WT littermates were used in this study. Animals were housed at the Institute of Biomedical Sciences Animal Care Facility under a 12-h light/dark cycle. Diets used in the present study were all purchased from LabDiet® (Richmond, VA, USA). Body weights of mice were recorded once daily. Animal experiments were performed under protocols approved by the Academia Sinica Institutional Animal Care and Utilization Committee, Taiwan.

Tandem mass spectrometry (MS) screening of blood amino acids and acylcarnitines
Samples were collected by impregnating filter paper (S&S 903; Schleicher & Shuell) with blood (25 µl) from the tail vein, and then levels of amino acids and acylcarnitines were determined by multiple reaction monitoring using an electrospray tandem mass spectrometry (ESI/MS/MS; Quattro Micro, Waters Corporation, Milford, MA, USA) as described elsewhere (57).

Study subjects for blood citrulline
Twenty-one patients with HD (11 women and 10 men, 47.14 ± 2.8 years old, with 44.29 ± 1.1 CAG repeats) and 23 age- and gender-matched controls with no known neurological disease (11 women and 12 men, 42.26 ± 2.6 years old) were studied. Diagnosis of HD was established by a neurological examination and genetic assessment of CAG expansion in the Htt gene. A blood sample was drawn from the vein (venipuncture) of each overnight-fasting subject after obtaining informed consent. The protocol was in compliance with the guidelines of the Institutional Review Boards of Chang Gung Memorial Hospital, Taipei Veterans General Hospital and Academia Sinica (Taipei, Taiwan). Normal values and higher cutoff values (mean + 4SD) established from 2100 newborns (58) and a pilot study of 48 healthy adults are listed in Supplementary Material, Table S5. It should be pointed out that these normal values were based on samples collected from individuals without fasting and are bound to have varied more than fasting samples. Thus, although the blood citrulline levels of HD patients were lower than the higher cutoff of individuals without fasting, the statistically higher blood citrulline levels of HD patients than those of the age- or gender-matched controls under the same fasting condition in the present study are important and imply a deficient urea cycle in HD patients.

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).

Rotarod performance
Motor coordination was assessed using a rotarod apparatus (UGO BASILE, Comerio, VA, Italy) at a constant speed (12 rpm) over a period of 2 min. We performed the rotarod test as described earlier (17). Briefly, the animals were pre-trained for 2 days at the age of 6 weeks to allow them to become acquainted with the rotarod apparatus. Animals were then tested three times per week from the age of 7 weeks. Each mouse was given three trials for a maximum of 2 min for each trial. Latency to falling was automatically recorded. The best performance (i.e. the longest time spent on the rod) out of three trials for each animal was used for the analysis. All WT animals tested remained on the rotarod at 12 rpm for the full 120 s.

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, Madison, WI, USA) to remove the potential contamination of genomic DNA, and then transcribed into cDNA using Superscript* II reverse transcriptase. 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 sequences of primers are listed in Supplementary Material, Table S6. Independent reverse-transcription PCRs were performed as described elsewhere (4). 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. Consistent with an earlier study using R6/2 mice (59), expression of GAPDH was not altered in the striatum or liver of R6/2 mice when compared with WT animals using the hypoxanthine guanine phosphoribosyl transferase (HPRT) gene as the reference gene (60) (data not shown).

Immunohistochemistry and quantitation
Striatum and liver sections (20 µm) were used in the immunohistochemical analyses as described earlier (17). Cells harboring aggregates of mutant Htt were quantitated in a blinded fashion. Single-antigen immunostaining was carried out using the avidin–biotin–peroxidase complex (ABC) method as previously described (61). In general, we used a 1:2000 dilution for the polyclonal anti-ubiquitin antiserum (DakoCytomation Denmark A/S, Glostrup, Denmark). In total, three to five mice for each treatment at the age of 12 weeks were analyzed. Three 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 percentage of Htt aggregate-containing cells. At least 800 cells from each brain or liver section were quantified. We observed no changes in the number of cells among all of the groups tested.

Double immunostaining of liver sections (20 µm) using anti-Htt and anti-C/EBP{alpha} antibodies was conducted as described earlier (62). Nuclei were stained with Hoechst 33342. Patterns of immunostaining were analyzed with the aid of a laser confocal microscope (LSM510, Carl Zeiss, Germany).

Constructs
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 poly(Q) residues fused to hrGFP were created as described elsewhere (4). The pGL2-AL-promoter constructs, which contain 235 bp of the mouse promoter were prepared by PCR using the following primers: 5'-GCAGGTACCACAAACCTGTCGTCTGTCTT-3' and 5'-GGTGAGCTCTGGCTTTTTCTGGTCCGGAT-3'. The resultant fragments were subcloned into the pGL2-basic luciferase reporter construct (Promega). To create the mutant AL-promoter with a defective C/EBP{alpha} site [–84 to – 77; AACATGTT (28)], two complementary 47 bp oligonucleotides (–103 to – 57; 5'-TGAGTCACACCCACCTCTCAACATGTTCTCTACTCTTCCAGGAGGCG-3' and 5'-CGCCT CCTGGAAGAGTAGAGAACATGTTGAGAGGTGGGTGTGACTCA-3’) bracketing mutations at the CCAAT site were annealed to produce a double-stranded 47 bp fragment harboring the Tsp45I and BslI sites at its 5' and 3' ends, respectively. The pGL2-AL-promoter construct was then cleaved with Tsp45I and BslI and ligated with the 47 bp fragment containing a null C/EBP{alpha} site. Mutations were confirmed by DNA sequencing. The pGL2-C/EBP{alpha}-promoter constructs, which contain 416 bp of the mouse promoter, were prepared by PCR using the following primers: 5'-TCGCTCGGCCTCTATATGCTCCCGG-3' and 5'-CCCACCCAGTGCCCCAACTGGCTC-3' (32). The resultant fragments were subcloned into the pGL2-basic construct which contains the luciferase gene as the reporter (Promega).

Cell culture and transfection
HepG2 cells were originally obtained from the American Type Culture Collection (Manassas, VA, USA) and were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, San Diego, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) plus penicillin (10 000 U/ml; Invitrogen), and streptomycin (10 mg/ml; Invitrogen) in an incubation chamber gassed with 5% CO2 and 95% air at 37°C. The day before transfection, cells were seeded onto a 35-mm dish at a density of 2 x 105 cells per well. Transfection was measured using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol.

AL enzyme activity
AL enzyme activity was determined spectrophotometrically as described elsewhere (63). In brief, liver lysate (5 µg) was added to 300 µl of reaction buffer containing sodium argininosuccinate (11.7 mM, Sigma–Aldrich, St Louis, MO, USA) and potassium phosphate (100 mM, pH 7.5) and incubated for 90 min at 37°C. The formation of fumarate ({lambda}max=240 nm; {epsilon} = 244 mM–1 cm–1) was determined by UV absorption at 240 nm using an Ultrospec 1100 pro UV/Visible Spectrophotometer (Amersham Biosciences, Piscataway, NJ, USA). Assays were performed in the linear range and in triplicate.

AS enzyme activity
Liver tissues were prepared from indicated animals to determine enzyme activity as described elsewhere (64). AS enzyme activity was determined spectrophotometrically as described elsewhere (64). In brief, liver lysate (5 µg) was added to 300 µl of reaction buffer containing 20 mM Tris–HCl (pH 7.8), 2 mM ATP, 2 mM citrulline (Sigma–Aldrich), 2 mM aspartate (Sigma–Aldrich), 6 mM MgCl2, 20 mM KCl and 0.2 units of pyrophosphatase and incubated for 20 min at 37°C. The reactions were then terminated by adding 300 µl of Taussky-Shorr Reagent (10% ammonium molybdate and 5% ferrous sulfate). The UV absorption at 650 nm was measured at the end of the reaction using an Ultrospec 1100 pro UV/Visible spectrophotometer (BioChrom Ltd, Cambridge, UK). Assays were performed in the linear range, and triplicate analyses were run.

Luciferase assays
Luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega) as described elsewhere (4). The protein concentrations of lysates were determined by the Bradford analysis, and the luciferase activities were normalized to the amount of proteins in the lysate. At least three independent transfections were performed for each experiment.

Western and dot blot assays
Equal amounts of protein were separated by SDS–PAGE using 10% polyacrylamide gels as described earlier (4). The resolved proteins were electroblotted onto Immobilon polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk in PBS and incubated with an anti-CBP antibody (1:1000; Santa Cruz Biotech), an anti-C/EBP{alpha} antibody (1:1000; Santa Cruz Biotech), 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 (1:300; Stressgen Biotechnology), an anti-actin antibody (1:2500; Chemicon International) or an anti-lamin antibody (1:2000; Santa Cruz Biotech) at 4°C overnight followed by the corresponding secondary antibody for 1 h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence (Pierce Chemical, Woburn, MA, USA) and recorded using Kodak XAR-5 film.

Nuclear extract preparation
Striatum and liver tissues were suspended, homogenized, centrifuged and then collected. The nuclear extract was prepared as previously described (4).

Cytosolic fraction preparation
Liver tissues were suspended in 1 ml of buffer A [10 mM Hepes (pH 8), 1 mM Na3VO4 and protease inhibitor cocktail tablets (Roche, Basel, Switzerland)], and homogenized by 15 Dounce strokes in buffer A. After centrifugation at 112g for 1 min at 4°C, the supernatant was collected and then centrifuged at 700g for 10 min at 4°C. The supernatant was 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 of the cytosolic extracts in the supernatant was measured using the Bio-Rad protein assay reagent.

Electrophoretic gel mobility shift assay
The double-stranded DNA fragment comprising the core promoter region of the AL gene [–90 to – 66 (28)] of 25 bp containing the WT or mutated CCAAT site was prepared by annealing two complementary oligonucleotides (for the WT, 5'-CCTCTCCCAATTGGCTCTACTCTTC-3' and 3'-GGAGAGGGTTAACCGAGATGAGAAG-5'; and for the mutant, 5'-CCTCTCAACATGTTCTCTACTCTTC-3' and 3'-GGAGAGTTGTACAAGAGATGAGAAG-5'; and for the control probe 5'-ATGCTTCGTTAGTAGTGCTGTGTTG-3'and 3'-TACGAAGCAATCATCACGACACAAC-5’) as previously described (4). For the supershift analyses, 1 µg of an anti-CBP antibody, an anti-C/EBP{alpha} antibody or an unrelated AC6D antibody (65) was incubated with 5 µg of nuclear extract (for 1 h at 4°C) prior to the addition of the binding buffer and a radiolabelled probe.

DNA pull-down assay
To determine whether CBP and C/EBP{alpha} exist in the protein–AL promoter complexes, a combination of EMSA and western blot analyses was performed as described (4). In brief, unlabeled AL–90/–66 probes (10 or 20 µg) harboring the WT or the mutated C/EBP{alpha} site were incubated with nuclear extract (0.5 mg) of the liver from WT or R6/2 mice under the same conditions described above for EMSA. The resultant gels were first stained with Coomassie blue to identify the location of the protein/DNA complex. The complex was excised, mixed with 1 x SDS sample treatment buffer, boiled for 5 min and separated by 10% SDS–PAGE. Following electrophoresis, gels were transferred to polyvinylidene difluoride membranes for western blot analysis using an anti-C/EBP{alpha} antibody or an anti-CBP antibody as described above.

Filter retardation assay
Detection and quantification of SDS-insoluble mutant Htt aggregates were carried out as described (66). Briefly, livers were suspended in ice-cold lysis buffer (5 mM Tris–HCl (pH 8.8), 1 mM EDTA, 100 mM NaCl, 5 mM MgCl2, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 0.5 µg/ml aprotinin, 0.1 mM leupeptin and 4 µM pepstatin), and homogenized (by 15 Dounce strokes). After centrifugation at 573g for 1 min at 4°C, the supernatant was collected into new tubes and then centrifuged at 18 000g for 20 min at 4°C. The pellet was resuspended in 100 µl of sample buffer (10 mM Tris–HCl (pH 8), 40 mM EDTA, 4% SDS, 100 mM DTT, 5 mM MgCl2 and 500 µM CaCl2), mixed with 100 µl of 4% SDS, and boiled for 5 min. For slot blotting, proteins were applied to OE66 membrane filters (0.2 µm pore size, Whatman Schleicher and Schuell, Middlesex, UK) in triplicate through a slot-blot manifold (Bio-Rad, Irvine, CA, USA). Blots were blocked with 5% skim milk in PBS and incubated with an anti-C/EBP{alpha} antibody (1:1000; Santa Cruz) or an anti-Htt antibody (1:500; Chemicon) at 4°C overnight followed by the corresponding secondary antibody for 1 h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence (Pierce) and recorded using Kodak XAR-5 film.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We are grateful to Drs Wuh-Liang Hwu, Yu-May Lee, Cathy S.-J. Fann, Hwei-Jen Lee, Cheng-Ming Chiang, Shwu-Yuon Wu, Hsu-Ming Shih, Ying-Hue Lee and Pei Chen for valuable suggestions; to Mr Dan Chamberlin and Ms Christine Hsieh for reading and editing the manuscript; to Ms Hsing-Lin Lai for the measurement of blood ammonia; to Mr Michael J.B. Lin for statistic analyses; and to Dr Fuu-Jen Tsai, Mr Wei-De Lin and Mr Gau-Chyi Young for the measurement of blood amino acids. This work was supported by grants from the National Science Council (NSC93–2321-B-001–012) and the Institute of Biomedical Sciences (CRC projects), Academia Sinica, Taipei, Taiwan.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

  1. Group T.H.s.D.C.R. (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell 72:971–983.[CrossRef][ISI][Medline]

  2. Reiner A., Albin R.L., Anderson K.D., D'Amato C.J., Penney J.B., Young A.B. (1988) Differential loss of striatal projection neurons in Huntington disease. PNAS 85:5733–5737.[Abstract/Free Full Text]

  3. Martindale D., Hackam A., Wieczorek A., Ellerby L., Wellington C., McCutcheon K., Singaraja R., Kazemi-Esfarjani P., Devon R., Kim S.U., et al. (1998) Length of huntingtin and its polyglutamine tract influences localization and frequency of intracellular aggregates. Nat. Genet. 18:150.[CrossRef][ISI][Medline]

  4. Chiang M.-C., Lee Y.-C., Huang C.-L., Chern Y. (2005) cAMP-response element-binding protein contributes to suppression of the A2A adenosine receptor promoter by mutant Huntingtin with expanded polyglutamine residues. J. Biol. Chem. 280:14331–14340.[Abstract/Free Full Text]

  5. 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. (2000) The Huntington's disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc. Natl Acad. Sci. USA 97:6763–6768.[Abstract/Free Full Text]

  6. Duan W., Guo Z., Jiang H., Ware M., Li X.-J., Mattson M.P. (2003) Dietary restriction normalizes glucose metabolism and BDNF levels, slows disease progression, and increases survival in huntingtin mutant mice. PNAS 100:2911–2916.[Abstract/Free Full Text]

  7. Podolsky S., Leopold N.A., Sax D.S. (1972) Increased frequency of diabetes mellitus in patients with Huntington's chorea. Lancet 1:1356–1358.[CrossRef][ISI][Medline]

  8. Garseth M., Sonnewald U., White L.R., Rod M., Zwart J.A., Nygaard O., Aasly J. (2000) Proton magnetic resonance spectroscopy of cerebrospinal fluid in neurodegenerative disease: indication of glial energy impairment in Huntington chorea, but not Parkinson disease. J. Neurosci. Res. 60:779–782.[CrossRef][ISI][Medline]

  9. Hurlbert M.S., Zhou W., Wasmeier C., Kaddis F.G., Hutton J.C., Freed C.R. (1999) Mice transgenic for an expanded CAG repeat in the Huntington's disease gene develop diabetes. Diabetes 48:649–651.[Abstract]

  10. Stoy N., Mackay G.M., Forrest C.M., Christofides J., Egerton M., Stone T.W., Darlington L.G. (2005) Tryptophan metabolism and oxidative stress in patients with Huntington's disease. J. Neurochem. 93:611–623.[CrossRef][ISI][Medline]

  11. Underwood B.R., Broadhurst D., Dunn W.B., Ellis D.I., Michell A.W., Vacher C., Mosedale D.E., Kell D.B., Barker R.A., Grainger D.J., et al. (2006) Huntington disease patients and transgenic mice have similar pro-catabolic serum metabolite profiles. Brain 129:877–886.[Abstract/Free Full Text]

  12. Tanaka M., Machida Y., Niu S., Ikeda T., Jana N.R., Doi H., Kurosawa M., Nekooki M., Nukina N. (2004) Trehalose alleviates polyglutamine-mediated pathology in a mouse model of Huntington disease. Nat. Med. 10:148.[CrossRef][ISI][Medline]

  13. Sathasivam K., Hobbs C., Turmaine M., Mangiarini L., Mahal A., Bertaux F., Wanker E.E., Doherty P., Davies S.W., Bates G.P. (1999) Formation of polyglutamine inclusions in non-CNS tissue. Hum. Mol. Genet. 8:813–822.[Abstract/Free Full Text]

  14. Jackson M.J., Beaudet A.L., O'Brien W.E. (1986) Mammalian urea cycle enzymes. Annu. Rev. Genet. 20:431–464.[CrossRef][ISI][Medline]

  15. Endo F., Matsuura T., Yanagita K., Matsuda I. (2004) Clinical manifestations of inborn errors of the urea cycle and related metabolic disorders during childhood. J. Nutr. 134:1605S–1609S discussion 1630S–1632S, 1667S–1672S.[Abstract/Free Full&n