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Originally published In Press as doi:10.1074/jbc.M708192200 on December 13, 2007

J. Biol. Chem., Vol. 283, Issue 12, 7390-7400, March 21, 2008
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Histone Deacetylase Inhibition Modulates Kynurenine Pathway Activation in Yeast, Microglia, and Mice Expressing a Mutant Huntingtin Fragment*

Flaviano Giorgini{ddagger}1, Thomas Möller§, Wanda Kwan, Daniel Zwilling, Jennifer L. Wacker{ddagger}, Soyon Hong§, Li-Chun L. Tsai{ddagger}, Christine S. Cheah{ddagger}, Robert Schwarcz||, Paolo Guidetti||, and Paul J. Muchowski{ddagger}**2

From the {ddagger}Departments of Pharmacology and §Neurology, University of Washington, Seattle, Washington 98195, Gladstone Institute of Neurological Disease, University of California, San Francisco, California 94158, ||Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland 21228, and the **Departments of Biochemistry and Biophysics and of Neurology, University of California, San Francisco, California 94158

Received for publication, October 2, 2007 , and in revised form, November 30, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The kynurenine pathway of tryptophan degradation is hypothesized to play an important role in Huntington disease, a neurodegenerative disorder caused by a polyglutamine expansion in the protein huntingtin. Neurotoxic metabolites of the kynurenine pathway, generated in microglia and macrophages, are present at increased levels in the brains of patients and mouse models during early stages of disease, but the mechanism by which kynurenine pathway up-regulation occurs in Huntington disease is unknown. Here we report that expression of a mutant huntingtin fragment was sufficient to induce transcription of the kynurenine pathway in yeast and that this induction was abrogated by impairing the activity of the histone deacetylase Rpd3. Moreover, numerous genetic suppressors of mutant huntingtin toxicity that are functionally unrelated converged unexpectedly on the kynurenine pathway, supporting a critical role for the kynurenine pathway in mediating mutant huntingtin toxicity in yeast. Histone deacetylase-dependent regulation of the kynurenine pathway was also observed in a mouse model of Huntington disease, in which treatment with a neuroprotective histone deacetylase inhibitor blocked activation of the kynurenine pathway in microglia expressing a mutant huntingtin fragment in vitro and in vivo. These findings suggest that a mutant huntingtin fragment can perturb transcriptional programs in microglia, and thus implicate these cells as potential modulators of neurodegeneration in Huntington disease that are worthy of further investigation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Huntington disease (HD)3 is a fatal neurodegenerative disorder characterized by abnormal motor movements, personality changes, and dementia (1). This autosomal dominant disease results from an expansion of a CAG repeat tract in the gene IT-15, which encodes a polyglutamine (poly(Q)) tract in the protein Huntingtin (htt) (2). Medium spiny projection neurons are strikingly and selectively lost from the striatum in HD (3, 4), despite the fact that mutant htt is expressed ubiquitously (5–7). However, it is still unclear whether nerve cells succumb in a cell-autonomous fashion, through pathogenic cell-cell interactions or through a combination of these two processes.

Inflammatory responses involving reactive astrocytes and microglia have been documented in affected regions of HD brain tissue (8–12). Reactive gliosis was also observed in early stages of neuronal dysfunction in mouse models of HD (12–15), and ferritin immunostaining in dystrophic microglia was increased in the striatum of pre-symptomatic HD mice and in early grade HD brain tissue (12). Consistent with these results, microglia in late stage HD mice have condensed nuclei and fragmented processes (16). Recent positron emission tomography studies using a radiotracer specific to activated microglia have found evidence of microglial activation in both presymptomatic and symptomatic HD patients (17, 18). In total, this evidence raises the possibility that microglial activation and/or dysfunction may be an early and important contributor to HD, as has been suggested for Alzheimer disease and other neurodegenerative disorders involving neuroinflammation (19).

Accumulating evidence supports the hypothesis that the kynurenine pathway (KP) of tryptophan degradation, which in the brain is expressed predominantly in microglia and astrocytes, may play an important role in HD (20). Many of the distinct neuropathological features in HD neostriatum can be duplicated in experimental animals by an intrastriatal injection of the excitotoxin quinolinic acid (QUIN) (21–23). These findings led to the suggestion that QUIN, a selective N-methyl-D-aspartate receptor agonist endogenous to the mammalian brain, might be causally involved in HD. QUIN is a metabolite of the KP (Fig. 1). This pathway contains two additional neuroactive products, the neurotoxic QUIN precursor 3-hydroxykynurenine (3-HK) and the neuroinhibitory and neuroprotective agent kynurenic acid (KYNA), which is formed in a side arm of the metabolic cascade (Fig. 1). We recently reported that neostriatal and neocortical levels of 3-HK and QUIN are significantly increased in early grade HD brains (24) and in three mouse models of HD (25). These results suggest that enhanced flux through the KP to generate 3-HK and QUIN in microglia might contribute to neuronal degeneration in the early phases of HD. The signals and mechanism of KP up-regulation in the microglia of HD brains are unknown.

There is widespread evidence that mutant htt induces transcriptional dysregulation in HD (26, 27). DNA microarray studies in HD mice indicate that transcriptional abnormalities precede overt behavioral phenotypes (28–30). Mutant htt may repress transcription in neurons by directly interacting and interfering with proteins that mediate transcription. Histone deacetylase (HDAC) inhibitors, which in general are thought to increase gene transcription, cause significant improvements in behavior and survival in fly and mouse models of HD (31–34).

In this study, we undertook a series of genetic and biochemical experiments in yeast, microglia, and mice expressing a mutant htt fragment to test the hypothesis that transcriptional dysregulation underlies activation of the KP. Using gene expression profiling, we showed that a mutant htt fragment caused specific repression of targets regulated by the HDAC Rpd3 in yeast, and that reduced 3-HK and QUIN levels in a suppressor mutant encoding a subunit of the Rpd3 HDAC complex were mediated by transcriptional repression of the KP. We then demonstrated that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) completely blocks increases in KP metabolites in microglia of HD mice in vitro and in vivo, indicating that HDAC activity also regulates transcription of the KP in mammalian cells. These studies begin to elucidate the molecular basis of mutant htt-induced activation of the KP, and raise the possibility that abnormal transcription in microglia may contribute to neurodegeneration in HD.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and DNA Constructs—The strains used were from the yeast gene deletion set in the MATa (BY4741) strain background (Research Genetics (Huntsville, AL)). The constructs pYES2-Htt25Q-GFP and pYES2-Htt103Q-GFP (35) were used for all yeast studies. Htt103Q is a galactose-inducible, FLAG- and green fluorescent protein-tagged construct encoding the first 17 amino acids of Htt fused to a poly(Q) tract of 103 glutamines.

Biochemical Analysis of Yeast Extracts—Yeast cultures were inoculated in triplicate at a starting A600 of 0.2 in 8 ml of SC-Ura medium supplemented with 2% galactose and grown in a shaking incubator at 30 °C for 14 h. Yeast extracts were prepared using glass bead lysis as described (36). 3-HK and QUIN levels were determined from the same yeast extracts in triplicate by HPLC and gas chromatography-mass spectrometry analyses (24).

Yeast Total RNA Preparation—SC-Ura galactose (2%) cultures (12 ml) were inoculated at A600 0.2 and incubated with shaking at 30 °C until reaching an A600 of ~1.0. Cells were harvested and lysed with acid-washed glass beads. Total RNA was isolated with Qiagen RNeasy midi kits, following standard protocols.

Gene Expression Analysis by DNA Oligonucleotide Arrays—Double-stranded cDNA was synthesized from total RNA, amplified as cRNA, labeled with biotin, and hybridized to Affymetrix Yeast Genome S98 Array GeneChips, which were washed and scanned at the University of Washington Center for Expression Arrays according to manufacturer protocols. Images were processed with Affymetrix Microarray Suite 5.0 (MAS-5). The quality of hybridization and overall chip performance were determined from the MAS-5 generated report file. Microarray data were analyzed as described (37). Gene ontology searches were performed with DAVID 2.0 and GOTERM_BP_ALL annotations (david.niaid.nih.gov).

Preparation of Primary Mouse Microglia Cells—Microglial cells were prepared from the cortex of newborn (P2–P4) mice, as described previously (38). Cortical tissue from a single mouse pup was freed from blood vessels and meninges. A tail sample of the same mouse was collected for genotyping. Brain tissue was minced, trypsinized for 10 min, triturated with a fire-polished pipette, and washed twice in Hanks' Buffer. The resulting cell suspension of a single mouse was cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a 25-cm2 flask. Cultures were kept at 37 °C in a 5% CO2, 95% air incubator, and medium was changed every 3–4 days. After astrocytes reached confluence (5–7 days), the culture medium was supplemented with 20% L-929 conditioned medium. 9–21 days after the initiation of cultures, microglial cells were separated from the underlying astrocytic monolayer by gentle agitation, using their differential adhesive properties. The resulting cell suspension was spun down at 200 x g for 10 min. The cell pellet was resuspended in Dulbecco's modified Eagle's medium, and 10% fetal bovine serum and cells were plated in a Primaria® 24-well plate at a density of 1 x 105 cells per well. Cells were allowed to settle for 20 min, and nonadhesive cells were removed by washing in Ca2+- and Mg2+-free phosphate-buffered saline (PBS). Cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 20% L-929 for 24 h and then changed to macrophage serum-free medium supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin for 24 h before being used for experiments. Cultures routinely consisted of >98% microglial cells as determined by staining with CD11b. 15 mice (7 R6/2 and 8 wild type) from three litters were used for isolation of primary microglia. Microglia from the same litter and genotype were pooled for the experiments. Extracts were generated by lysing the cells with a cell scraper. 3-HK and QUIN levels were determined by HPLC and gas chromatography-mass spectrometry analyses as described (24). The data in Fig. 4, A and B, were calculated in microglia isolated from n ≥ 4 per treatment group.

RT-PCR and Quantitative RT-PCR of Microglial mRNAs—Kmo Total mouse RNA was isolated from primary microglia, cortical neurons, or astrocytes with TRIzol reagent and used as a template for RT-PCR. mRNA levels were determined by quantitative real time RT-PCR using a primer set for a murine Kmo mRNA amplicon. Quantitative analysis was conducted by a Stratagene Mx3000P real time detection system using SYBR Green reagent (Applied Biosystems, Foster City, CA). Primer verification was performed by melting curve analysis. Expression of Kmo mRNA in microglia was determined relative to that detected in astrocytes, as calculated from the cycle threshold (Ct, the number of cycles required to cross an arbitrary threshold fluorescence value). Kmo mRNA was not detected in neurons after 40 cycles of PCR amplification, whereas the Ct for microglia and astrocytes was 30 and 36, respectively. All experiments were performed in triplicate, and values were normalized to mRNA levels of the housekeeping enzyme acidic ribosomal phosphoprotein.

For analysis of mutant htt mRNA expression in primary murine microglia, RNA was reverse-transcribed using 1.25 units/µl Multiscribe RT (Applied Biosystems), 5.5 mM MgCl2, 500 µM dNTPs, and 2.5 µM random hexamers with 0.4 units/µl RNase inhibitor with incubations of 10 min at 25 °C, 30 min at 48 °C, 4 min at 95 °C, 5 min at 4 °C. Specific sense and antisense primers for detection of the mutant htt transgene were employed. The PCR was performed in TAE buffer, 10% Me2SO, 200 µM dNTPs, and 10 ng/µl primer with 0.5 units/µl Taq polymerase (Invitrogen). Cycling conditions were an initial incubation of 5 min at 94 °C, and 34 cycles of 30 min at 94 °C, 30 min at 55 °C, 30 min at 72 °C, followed by a final incubation of 5 min at 72 °C. GAPDH-specific primers were used to detect GAPDH transcripts as a positive control.

Immunoprecipitation Experiments—Brain tissue and primary microglia were homogenized in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% IGEPAL) with "complete" protease inhibitors (Roche Applied Science), 1 mM phenylmethylsulfonyl fluoride, and 1 mM β-mercaptoethanol. Brain and cell homogenates diluted in 200 µl of buffer were precleared by incubation with mixture of protein A-Sepharose and protein G-agarose (Invitrogen) for 1 h at 4 °C. The cleared lysate was subjected to immunoprecipitation with the addition of 1:200 of anti-S830 in a total volume of 200 µl overnight at 4 °C. After incubation, protein A-Sepharose/protein G-agarose beads were added for 2 h before being washed four times with RIPA buffer. The final protein-agarose pellets were resuspended in 1x sample buffer, and the immunoprecipitated complexes were eluted by boiling in water. Protein samples were fractionated on 12% SDS-polyacrylamide gels and blotted onto nitrocellulose membrane (Schleicher & Schuell) by submerged transfer apparatus (Bio-Rad) in transfer buffer (25 mM Tris, 192 mM glycine, and 20% v/v methanol). Membranes were blocked for 1 h in 4% nonfat dried milk in PBS, 0.1% Tween 20 (PBST) and incubated with gentle agitation for 1 h at room temperature with anti-EM48 (1:500, Chemicon) PBST with 0.5% nonfat dried milk. Blots were washed three times in PBST, probed with horseradish peroxidase-linked anti-mouse secondary antibody (1:10,000, Jackson ImmunoResearch) in PBST with 0.5% nonfat dried milk for 1 h at room temperature, and washed three times in PBST. Protein was detected by chemiluminescence (ECL kit, Amersham Biosciences) according to the manufacturer's instructions.

Analysis of Microarray Data—Experiments were performed in triplicate, and the microarray data were analyzed with MAS-5 by making all nine possible pairwise comparisons between RNA samples and their respective base-line controls (WT Htt103Q versus WT Htt25Q or ume1{Delta} Htt103Q versus WT Htt103Q). The difference calls calculated by this software were converted to numbers (increase, moderate increase = 1, no call = 0, decrease, moderate decrease = –1), and the sum of the difference calls was used to identify genes most consistently up-regulated or down-regulated. Genes were considered significant if 7/9 (78%) of the pairwise comparisons were called changed. MAS version 5.0 signal log ratio values were converted to fold-change values and averaged across all pairwise comparisons, and the genes were ordered by these values.

SAHA Treatment of R6/2 Mice—Three R6/2 and WT mice received either a solution of SAHA (1.00 g/kg) dissolved in hydroxypropyl β-cyclodextrin (MP Biomedicals, CA; 27.75 g/liter) in distilled water or vehicle. Three R6/2 and WT mice received a lower dose of SAHA (0.67g/liter), in a lower concentration of hydroxypropyl β-cyclodextrin (18 g/liter) in distilled water or vehicle. SAHA was added to the hydroxypropyl β-cyclodextrin solution and then carefully heated until fully dissolved. Part of this solution was then further dissolved to obtain the lower dose solution. The solutions were given to the mice in their drinking water and replaced by freshly prepared solutions twice weekly. SAHA levels were measured in total brain homogenates by HPLC after 4 weeks of treatment.

Sodium Butyrate Treatment of WT Mice—FVB/N mice (2 months old) were used for this experiment. Ten mice were treated for 14 days with sodium butyrate (1 g/day in sterile PBS intraperitoneal) as described (32). Ten controls received vehicle only. At day 13, five treated mice and five controls received LPS (50 µg of intraperitoneally). After 24 h, the mice were sacrificed, and the brains were rapidly removed, frozen, and KYN, KYNA, 3-HK, and QUIN were measured as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Decreased KP Metabolite Levels in Yeast Suppressors of Mutant Htt Toxicity—We recently identified 28 yeast gene deletion strains that suppress the toxicity of a mutant htt fragment (Htt103Q) (36). Among the most potent suppressors was BNA4, which encodes the yeast homolog of kynurenine 3-monooxygenase (KMO), an enzyme in the KP that catalyzes the hydroxylation of kynurenine (KYN). As Bna4 is the only suppressor from our genetic screen known to function specifically in the KP, we asked if suppressors that function outside of the KP affect levels of the KP metabolites 3-HK and QUIN, as these KP metabolites have been shown to have neurotoxic properties. We measured 3-HK and QUIN levels in all of the suppressors expressing Htt103Q (Table 1). Compared with WT cells expressing Htt103Q, 17 of the 28 suppressors (61%) had significant (p < 0.05) reductions in 3-HK alone, 21 (75%) in QUIN alone, 23 (82%) in 3-HK or QUIN, and 15 (54%) in 3-HK and QUIN (Table 1). Thus, suppression of Htt103Q toxicity in yeast correlates strongly with decreased levels of 3-HK or QUIN in the majority of suppressor strains. Because 25% of the suppressors function in transcription and/or maintenance of chromatin architecture (Table 1), we speculated that decreased levels of 3-HK and QUIN in these suppressors are because of transcriptional repression of the KP.


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  		TABLE 1
Kynurenine levels in toxicity suppressor yeast strains expressing Htt103Q

Numbers listed refer to relative percentage of 3-HK and QUIN levels in deletion strains expressing Htt103Q as compared with the parental strain (BY4741) expressing Htt103Q.

 
The Rpd3 HDAC Complex Regulates Transcription of the KP in Yeast—The Rpd3 HDAC complex was initially characterized as a transcriptional repressor, but global studies of its function by a modified chromatin immunoprecipitation method showed that it also increases transcription at many promoters (39, 40). Two components of the Rpd3 complex in yeast, Ume1 and Rxt3, were isolated as suppressors of Htt103Q toxicity (36). Aside from bna4{Delta} cells, ume1{Delta} cells expressing Htt103Q had the largest combined reduction in 3-HK and QUIN levels (87 and 69% decrease in levels compared with WT cells expressing Htt103Q, respectively) of any of the suppressors tested. Complementing these observations, a partial loss-of-function allele of Sin3A, a conserved component of the Drosophila Rpd3 complex, reduces neurodegeneration and increases viability in a fly model of poly(Q) disease (31). In addition, Rpd3 is a class I HDAC and is sensitive to the inhibitors sodium butyrate and SAHA, which are neuroprotective in fly and mouse models of HD (31–33).

To measure global poly(Q)-dependent transcriptional perturbations in WT yeast expressing Htt103Q, we used a two-color cDNA microarray hybridization assay to compare mRNA expression profiles of an isogenic, control yeast strain expressing Htt103Q or Htt25Q. As predicted from our genetic data (41), functional groups up-regulated significantly in parental yeast cells expressing Htt103Q included many genes involved in protein folding and response to stress (Table 2). The expression of two KP genes, BNA2 (tryptophan 2,3-dioxygenase or TDO) and ARO9 (kynurenine aminotransferase), was up-regulated significantly in parental yeast cells expressing Htt103Q (not shown). As Bna2 (TDO) is the first enzyme in the KP (Fig. 1), this increase in mRNA expression (and presumably in TDO levels and activity) may mediate the increases in 3-HK and QUIN in yeast expressing Htt103Q. Functional groups down-regulated significantly in the parental strain expressing Htt103Q as compared with the parental strain expressing Htt25Q included genes involved in ribosome biogenesis and rRNA processing and metabolism (Table 3). Similar functional categories were enriched among Rpd3 gene targets identified in yeast by a modified chromatin immunoprecipitation method (40). In total, of the 295 most highly enriched Rpd3 targets (≥3-fold enrichment) and the 231 most consistently down-regulated genes in yeast expressing Htt103Q, 40 genes were present in both sets, a nearly 4-fold increase over that predicted by random chance (Fig. 2 and Table 4). These results indicate that expression of Htt103Q in yeast selectively represses genes targeted by Rpd3.


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  		TABLE 2
Gene ontology groups significantly up-regulated in isogenic yeast cells expressing Htt103Q as compared with Htt25Q

An arbitrary cut-off p value (—log10) of 1 was selected for this table. Gene count indicates the number of genes within a particular functional group. Percentage indicates what percentage of the total genes in the functional group that the gene count represents.

 


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  		TABLE 3
Gene ontology for functional groups significantly down-regulated in isogenic yeast cells expressing Htt103Q as compared with Htt25Q

An arbitrary cutoff p value (-log10) of 10 was selected for this table. Gene count indicates the number of genes within a particular functional group. Percentage indicates what percentage of the total genes in the functional group that the gene count represents.

 


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  		TABLE 4
Genes common to Rpd3 targets and Htt103Q down-regulated genes

A total of 40 genes overlap these two data sets, a 3.6-fold enhancement over random chance. Gene sets are enumerated using Venn diagram in Fig. 2. Both data sets are enriched for genes involved in ribosome biogenesis, rRNA processing, and rRNA metabolism. The selected Rpd3 gene targets were enriched 3-fold or more over background (295 genes). The 231 most consistently down-regulated genes in parental yeast expressing Htt103Q represent genes with a sum of difference calls equaling -7, -8, -9 (see "Experimental Procedures").

 


Figure 1
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  		FIGURE 1.
The kynurenine pathway (KP) of tryptophan degradation. The neurotoxic metabolites assayed in this study, 3-HK and QUIN, are indicated in the boxes.

 
We next generated and compared expression profiles of the suppressor mutant ume1{Delta} and the parental WT strain expressing Htt103Q. Of 56 genes down-regulated significantly in ume1{Delta} versus WT cells expressing Htt103Q, four members of the KP were present: BNA1 (3-hydroxyanthranilate 3,4-dioxygenase), BNA2 (TDO), BNA4 (KMO), and BNA5 (kynureninase) (Table 5). Classification of expression profile changes by gene ontology indicated that this gene cluster was the functional category most significantly enriched in this experiment (p < 0.0001). As Htt103Q induces BNA2 (TDO) and ARO9 (kynurenine aminotransferase) in WT cells, whereas KP genes are repressed in ume1{Delta} cells, these results indicate a direct correlation between Rpd3-mediated transcription of the KP and Htt103Q-mediated toxicity. Moreover, because full activity of Rpd3 is required for transcriptional induction of the KP in yeast, these results further suggest that pharmacological inhibition of this HDAC complex, or its mammalian equivalent, would lead to repression of the KP.


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  		TABLE 5
Genes down-regulated in ume1{Delta} expressing Htt103Q versus a parental yeast strain expressing Htt103Q

Four genes (shown in boldface) encoding kynurenine pathway enzymes are among the strongest and most consistently down-regulated genes.

 
KMO Is Expressed Predominantly in Microglia—Enzymes of the KP are expressed constitutively in human microglia and astrocytes but not in neurons (42–44). In human cells, KMO is expressed exclusively in microglia, whereas the enzymatic transamination of kynurenine to KYNA occurs mainly in astrocytes (44, 45). To confirm that KMO expression occurs solely in murine microglia, we performed quantitative real time RT-PCR with RNA samples to measure transcript levels of Kmo in primary microglia, astrocytes, and cortical neurons from WT mice. Kmo mRNA was not detected in primary neurons, and microglia expressed at least 50-fold more Kmo mRNA than astrocytes (relative expression levels of 57.7 and 1.0, respectively), confirming that microglia are the predominant cell type for KMO expression in mice.

Mutant Htt Exon 1 Is Expressed in Microglia—Two independent studies showed by in situ hybridization that mRNA for htt is expressed ubiquitously in human and rat tissues, including in microglia (5, 46). In addition, electron microscopic and immunohistochemical analyses of brain sections from R6/1 (47) and R6/2 transgenic mouse models of HD (12), respectively, detected expression of mutant htt in microglia. To confirm these observations, we assayed for the presence of mutant htt exon 1 mRNA transcripts using RT-PCR in RNA samples from primary microglia derived from R6/2 or WT mice (Fig. 3A). We found that mutant htt mRNA is expressed in microglia from R6/2 mice, but not in WT mice. Furthermore, immunoprecipitation experiments with the anti-htt antibody S830 followed by immunoblotting with the anti-htt antibody EM48 detected expression of mutant htt exon 1 protein in primary microglia derived from R6/2 mice and in brain homogenates from early postnatal R6/2 mice, but not in primary microglia or brain homogenates from WT mice (Fig. 3B). These analyses, not unexpectedly, confirm that mutant htt exon 1 is indeed expressed in microglia from R6/2 mice.


Figure 2
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  		FIGURE 2.
Expression of a mutant htt fragment in yeast selectively represses genes targeted by the HDAC Rpd3. Shown is a Venn diagram indicating the overlap between Rpd3 target genes and Htt103Q down-regulated genes in yeast. Of 6200 genes in the Saccharomyces cerevisiae genome, 295 (or 4.8%) appear to be Rpd3 targets (3-fold or more enrichment) (40). Therefore, by random chance 11 of the 231 Htt103Q down-regulated genes are predicted Rpd3 targets. The actual overlap of 40 genes, indicates a 3.6-fold (11/40) enrichment of Rpd3 targets among the down-regulated genes.

 
SAHA Blocks the KP in Primary Microglia from R6/2 Mice in Vitro—We recently found that 3-HK, but not QUIN, levels are elevated in the striatum and cortex of the R6/2 (48) transgenic mouse model of HD (25). Because KMO catalyzes the formation of 3-HK from KYN predominantly in microglia, we next determined whether 3-HK or QUIN levels were elevated in primary microglia isolated from day 2–4 neonatal R6/2 mice. Indeed, 3-HK levels were 52% higher in R6/2 than WT microglia (p < 0.01) (Fig. 4), but QUIN levels did not differ, consistent with previous observations in dissected brain regions (25). In R6/2 microglia treated with Ro 61-8048 (an IC50 ~ 37 nM inhibitor of KMO) (49) (Fig. 4A) or the HDAC inhibitor SAHA (Fig. 4B), 3-HK levels were significantly lower (p < 0.05) than in vehicle-treated cells. Thus, a mutant htt fragment induces microglial 3-HK at a very early stage in R6/2 mice and treatment with SAHA or a KMO inhibitor return 3-HK to levels observed in WT mice.

SAHA Blocks the KP in R6/2 Mice in Vivo—We next determined whether SAHA modulates KMO activity and 3-HK levels in R6/2 mice, under conditions in which SAHA has been shown to cross the blood-brain barrier, increase histone acetylation in the brain, and dramatically improve motor impairment (33). Three R6/2 or WT mice per treatment group received SAHA (1.0 g/liter) or vehicle in drinking water daily between 4 and 8 weeks of age. Quantification of brain levels of SAHA by HPLC demonstrated that no significant difference was detected between genotypes (data not shown). As observed previously in R6/2 mice (25), 3-HK levels were significantly increased (p < 0.05) in the cortex (1.5-fold) and striatum (1.6-fold) as compared with WT mice (Fig. 5, A and B). Interestingly, SAHA treatment in WT mice did not significantly alter brain levels of 3-HK (Fig. 5, A and B) or QUIN (not shown). In contrast, SAHA significantly (p < 0.05) reduced 3-HK in the cortex (40%) and striatum (31%) of R6/2 mice (Fig. 5, A and B). KMO enzymatic assays, performed to determine whether fluctuations in 3-HK levels were accompanied by changes in biosynthetic activity, showed that activity was 1.4-fold higher (p < 0.05) in the brains of R6/2 mice than in WT mice (Fig. 5C). Moreover, SAHA reduced KMO activity by 20% (p < 0.05) in R6/2 mice (Fig. 5C). These results indicate that treatment of R6/2 mice with an HDAC inhibitor blocks increases in 3-HK levels and KMO activity mediated by a mutant htt fragment, and indicate that the activity of HDAC complexes targeted by SAHA regulate KP activation in microglia in vivo.

Sodium Butyrate Blocks the KP in Vivo after Microglial Activation—To confirm that the effects of SAHA on 3-HK levels and KMO activity were related to its effects on HDAC activity, we next asked if sodium butyrate, a class I/II HDAC inhibitor structurally dissimilar to SAHA, could modulate the KP in WT mice. We also assayed the effects of LPS treatment, which robustly activates microglia and the KP in the central nervous system (50), in combination with sodium butyrate (Fig. 6). Sodium butyrate was tested at a concentration (1 g daily) that was neuroprotective and increased histone acetylation in R6/2 mice (32). Ten mice were treated for 14 days with sodium butyrate or vehicle. On day 13, half of the treated mice and controls received LPS. After 24 h, the animals were euthanized, and KP metabolites were analyzed in total brain extracts. Consistent with the lack of effects of SAHA on the KP in WT mice (Fig. 6), sodium butyrate alone did not affect brain levels of KP metabolites (Fig. 6A) or KMO activity (Fig. 6B) in vivo. However, in LPS-treated mice, sodium butyrate substantially attenuated LPS-induced increases in KYN and 3-HK levels and KMO activity (Fig. 6, A and B). Together with the SAHA data, these results demonstrate that two structurally distinct HDAC inhibitors can block the KP in vivo, but apparently only when microglia are activated and/or dysfunctional.


Figure 3
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  		FIGURE 3.
Mutant htt exon 1 mRNA and protein are expressed in primary microglia from R6/2 mice. A, expression of mutant htt exon 1 mRNA was detected in primary microglia derived from R6/2 transgenic mice, and not in WT control mice. RT-PCR was used to detect the presence of human mutant htt exon1 transcripts in primary microglia from either R6/2 mice (lanes 3 and 4) or WT littermate controls (lanes 1 and 2), as indicated by arrow. The quality of the RNA samples was confirmed using primers specific for GAPDH (lanes 5 and 7), as indicated by the arrowhead. Negative controls without reverse transcriptase (RT) were also performed (lanes 2, 4, 6, and 8), to ensure samples were free of genomic DNA contamination. B, immunoprecipitation (IP) with anti-htt antibody S830 followed by immunoblotting with anti-htt antibody EM48 (1:500 dilution) was used to detect mutant htt exon 1 protein in R6/2 primary microglia (lane 9) or early postnatal and 14-week-old total brain homogenates (lanes 7 and 5, respectively). In 14-week-old brain homogenates, immunoprecipitation with S830 resulted in detection of aggregated species of mutant htt exon 1 protein by EM48 that did not enter the gel matrix (arrowhead), whereas immunoprecipitation of primary microglia or early postnatal brain homogenates from R6/2 mice resulted in detection of soluble mutant htt exon 1 protein (arrow). Mutant htt exon 1 was not detected in primary microglia or total brain homogenates from WT mice (lanes 4, 6 and 8). Preclear (lanes 1–3) controls are included. MG, microglia; PN, postnatal brain homogenate; 14 wk, 14-week-old brain homogenate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The KP of tryptophan degradation is hypothesized to play an important role in HD. Increased levels of neurotoxic metabolites in this pathway are generated in microglia at very early stages of the disease only in affected brain regions, but the molecular mechanisms responsible for up-regulation of the KP have not been investigated. In this study, we begin to elucidate the molecular basis for activation of the KP, demonstrating that its induction occurs in a manner that is dependent on HDAC activity in yeast, microglia, and mice expressing a mutant htt fragment.


Figure 4
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  		FIGURE 4.
The HDAC inhibitor SAHA decreases 3-HK levels in primary microglia from R6/2 mice in vitro. A and B, elevated 3-HK levels in primary microglia from R6/2 mice. 3-HK and QUIN levels were analyzed in primary microglia after 6 h in culture after treatment with either vehicle alone, 10 µM Ro 61-8048 (A), or 10 µM SAHA (B). The data were calculated in microglia isolated from n ≥ 4 per treatment group. 3-HK levels were significantly higher (*, p < 0.01) in R6/2 microglial cultures than in WT controls, and significantly lower in both Ro 61-8048 and SAHA-treated cells than in vehicle-treated R6/2 microglia (#, p < 0.05). Statistical comparisons were performed using two-way ANOVA and the Bonferroni post hoc test on the log transformed data (p = 0.0115, F1,43 = 6.96 for genotype, and p = 0.0197, F1,43 = 5.87 for treatment (A); p = 0.0007, F1,9 = 25.86 for genotype, and p = 0.0079, F1,9 = 11.54 for treatment (B)). Statistically significant changes in QUIN levels were not observed.

 
Biochemical and genetic analyses in yeast showed that the levels of 3-HK and QUIN were decreased in the majority of 28 gene deletion strains that suppress toxicity of a mutant htt fragment. Because most of the suppressors have distinct cellular functions not previously associated with the KP, these results indicate that multiple independent modes of suppression of mutant htt toxicity unexpectedly converge on the KP, suggesting that the KP plays a critical downstream role in mediating mutant htt toxicity in yeast. Deletion of UME1, which encodes a component of the class I HDAC Rpd3 complex, dramatically reduced 3-HK and QUIN levels as compared with a control strain that expressed Htt103Q, and these decreases were mediated by transcriptional repression of the KP as shown by DNA microarrays. Because Ume1 is required for full activity of Rpd3, the most parsimonious interpretation of these data is that Rpd3 normally activates transcription at KP promoters and that this activity is decreased in ume1{Delta} cells, resulting in repression of KP gene transcription. This interpretation is consistent with a previous microarray study showing that WT yeast cells treated with the HDAC inhibitor trichostatin A or mutant cells lacking Rpd3 strongly repress BNA1 (3-hydroxyanthranilate 3,4-dioxygenase) (51). Our interpretation is also consistent with a previous study in mammalian cells showing that treatment with trichostatin A causes histone hyperacetylation and repression of transcription of kynurenine formamidase, another member of the KP (52). Nevertheless, our current findings do not allow us to formally exclude an alternative model in which Rpd3 normally represses KP gene transcription in WT cells but is inhibited in cells expressing a mutant htt fragment, resulting in transcriptional induction of the KP. In line with this model, our DNA microarrays showed that gene targets of Rpd3 are abnormally regulated in cells expressing Htt103Q as compared with Htt25Q. Because Ume1 normally recruits Rpd3 to repress transcription (39), it is possible that in WT cells mutant htt may physically interact with Ume1 and prevent it from recruiting Rpd3 to repress transcription of KP promoters, whereas in ume1{Delta} cells Rpd3 activity is normalized, allowing repression of KP genes. Future experiments will discriminate between these and other possibilities.


Figure 5
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  		FIGURE 5.
The HDAC inhibitor SAHA decreases brain 3-HK levels and KMO activity in R6/2 mice in vivo. A and B, levels of 3-HK in the cortex and striatum, respectively, of WT and R6/2 mice (n = 3 for each genotype). In both brain regions, 3-HK levels were significantly higher in R6/2 mice than in WT mice (*, p < 0.05). Treatment with SAHA significantly reduced 3-HK levels as compared with vehicle alone (#, p < 0.05). Statistical comparisons were performed by two-way ANOVA and the Fisher LSD comparison test on the log transformed data (p < 0.0001, F1,19 = 37.49 for genotype, and p < 0.0001, F1,19 = 52.29 for treatment, p = 0.0053, F1,19 = 9.87, for interaction (A); p < 0.0001, F1,19 = 42.13 for genotype, and p = 0.0145, F1,19 = 7.24 for treatment (B)). C, KMO activity in whole brain extracts from WT and R6/2 mice treated with vehicle alone or SAHA. KMO activity was significantly higher in R6/2 mice than in WT mice (*, p < 0.05). Treatment with SAHA significantly reduced KMO activity as compared with vehicle alone (#, p = 0.05). Statistical comparisons were performed by two-way ANOVA and the Fisher LSD comparison test on the log transformed data (p < 0.0001, F1,19 = 37.97 for genotype, and p < 0.0405, F1,19 = 4.83, for treatment).

 


Figure 6
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  		FIGURE 6.
The HDAC inhibitor sodium butyrate decreases brain 3-HK levels, KYN levels, and KMO activity in LPS-stimulated WT mice in vivo. A, levels of 3-HK, QUIN, KYN, and KYNA were measured in the brains of WT mice (vehicle alone, LPS alone, sodium butyrate alone, and LPS with sodium butyrate; n = 5 for each treatment group). Administration of sodium butyrate to WT mice prevented LPS-induced increases in 3-HK and KYN levels. *, p < 0.05 versus vehicle; #, p < 0.05 versus LPS alone; two-way ANOVA and the Fisher LSD comparison test on the log transformed data (3-HK, p < 0.0001, F1,15 = 56.79 for LPS treatment, and p < 0.0006, F1,15 = 18.91 for HDAC inhibitor treatment, p = 0.0005, F1,15 = 19.19 for interaction. QUIN, p < 0.0004, F1,15 = 20.61, for LPS treatment. KYN, p < 0.0001, F1,15 = 52.93, for LPS treatment, and p = 0.0193, F1,15 = 6.87, for interaction). B, KMO activity in whole brain extracts from WT mice (vehicle alone, LPS alone, sodium butyrate alone, and LPS with sodium butyrate; n = 5 for each treatment group). Administration of sodium butyrate to WT mice prevented LPS-induced increases in KMO activity. *, p < 0.05 versus vehicle; #, p < 0.05 versus LPS alone; two-way ANOVA and the Fisher LSD comparison test on the log transformed data (p < 0.0056, F1,15 = 10.46, for LPS treatment).

 
HDAC-dependent modulation of the KP in microglia, both in vitro and in vivo, was remarkably similar to that observed in yeast. Levels of 3-HK in primary microglia cultures from R6/2 mice were >50% higher than in WT littermates, consistent with previous observations in yeast, HD mouse models, and HD patients (24, 25, 36, 53, 54). Because the microglia were cultured from neonatal mice, this result indicates not only that the KP is abnormal in microglia (as previously hypothesized) but that mutant htt-induced perturbation of the pathway occurs at a stage that precedes the onset of motor symptoms in R6/2 mice. Treatment of R6/2 microglia with Ro 61-8048 (a KMO inhibitor) or SAHA (an HDAC inhibitor) reduced 3-HK levels by equivalent amounts, demonstrating that HDAC inhibition mimics the effect of direct inhibition of KMO. Thus, mutant htt-induced perturbation of HDAC-dependent transcription in the nuclei of microglia may occur upstream of abnormal increases in KP metabolites.

As observed in vitro, SAHA treatment in R6/2 mice reduced levels of 3-HK and KMO activity in vivo. HDAC-dependent modulation of the KP was also shown in WT mice treated with sodium butyrate, an HDAC inhibitor structurally dissimilar to SAHA. However, sodium butyrate reduced levels of KYN and 3-HK only in mice treated with LPS, suggesting that microglial activation in R6/2 mice may be associated with transcriptional induction of the KP. This finding is supported by recent studies that reported compelling evidence of microglial activation in brains of HD patients and R6/2 mice (17, 18). Although it is not clear if the effects we observed of HDAC inhibitors on the KP in vivo are direct or indirect, our yeast experiments suggest that these effects could be due to a direct effect upon transcription of KP genes. This question will be dissected in future studies.

HDAC inhibitors have shown promising results in preclinical studies in HD mice (32–34) and are being tested in a clinical trial for HD. They are thought to exert beneficial effects in HD mice by increasing the transcription of multiple target genes in neurons that are repressed because of expression of mutant htt. Indeed, in this study we observed that many targets of the Rpd3 HDAC complex in yeast are repressed by mutant htt expression. However, we have also shown that the HDAC inhibitors SAHA and sodium butyrate block up-regulation of the KP in vivo in HD mice and in LPS-stimulated WT mice. The mechanism that underlies these effects is currently being investigated, but is likely mediated by HDAC-dependent transcriptional repression of KP genes and other pro-inflammatory responses in microglia, as was observed in LPS-stimulated mice after treatment with SAHA (55). These results demonstrate that HDAC inhibitors can modulate mutant htt-induced transcriptional perturbations not only by increasing transcription in neurons but also by possibly repressing transcription of the KP in microglia.

In summary, our results indicate that a mutant htt fragment perturbs transcriptional programs in microglia in vitro and in vivo. As mutant htt is ubiquitously expressed (5–7, 46), including in astrocytes (56) and microglia (12, 47), these results suggest that cell types other than neurons may contribute to neurodegeneration in HD (56). Indeed, several recent studies showed that accumulation of misfolded disease-causing proteins in glia contributes to ongoing neurodegeneration in mouse models of amyotrophic lateral sclerosis (57, 58) and spinocerebellar ataxia type 7 (SCA7) (59). It will be interesting to determine whether similar findings are obtained in HD models.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by the HighQ Foundation, Medical Research Council, and the Royal Society. Present address: Dept. of Genetics, University of Leicester, Leicester LE1 7RH, UK. Back

2 Supported by NINDS, National Institutes of Health and the HighQ Foundation. To whom correspondence should be addressed: Gladstone Institute of Neurological Disease, Depts. of Biochemistry and Biophysics and of Neurology, University of California, San Francisco, 1650 Owens St., San Francisco, CA 94158. Tel.: 415-734-2515; Fax: 415-355-0824; E-mail: pmuchowski{at}gladstone.ucsf.edu.

3 The abbreviations used are: HD, Huntington disease; HDAC, histone deacetylase; SAHA, suberoylanilide hydroxamic acid; RT, reverse transcription; ANOVA, analysis of variance; LPS, lipopolysaccharide; WT, wild type; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; 3-HK, 3-hydroxykynurenine; KMO, kynurenine 3-monooxygenase; KYN, kynurenine; QUIN, quinolinic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TDO, tryptophan 2,3-dioxygenase; KYNA, kynurenic acid. Back


   ACKNOWLEDGMENTS
 
We thank M. Sherman and A. Meriin for the pYES2-Htt25Q and pYES2-Htt103Q constructs. We are grateful to M. Grunstein and S. Kurdistani for providing the Rpd3 gene target data. SAHA-treated R6/2 mice were kindly provided by L. Menalled at Psychogenics Corp. and D. Howland at the HighQ Foundation. We thank N. Stella and E. Cudaback for providing mRNA from primary neuron, microglia, and astrocyte cultures for real time, quantitative RT-PCR analysis. We also thank the Center for Expression Arrays at the University of Washington for providing RNA quality control, target labeling, Affymetrix chip processing, and Affymetrix chip scanning services. We thank A. Strand and J. Olson for advice on data analysis of our gene expression experiments. We also thank S. Ordway and G. Howard for editorial assistance and S. Finkbeiner and L. Mucke for useful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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