Human Molecular Genetics, 2003, Vol. 12, No. 5 497-508
© 2003 Oxford University Press
Specific progressive cAMP reduction implicates energy deficit in presymptomatic Huntington's disease knock-in mice
Molecular Neurogenetics Unit, Massachusetts General Hospital, Building 149, 13th St, Charlestown, MA 02129, USA
Received October 23, 2002; Accepted December 20, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Defects in gene transcription and mitochondrial function have been implicated in the dominant disease process that leads to the loss of striatal neurons in Huntington's disease (HD). Here we have used precise genetic HD mouse and striatal cell models to investigate the hypothesis that decreased cAMP responsive element (CRE)-mediated gene transcription may reflect impaired energy metabolism. We found that reduced CRE-signaling in HdhQ111 striatum, monitored by brain derived neurotrophic factor and phospho-CRE binding protein (CREB), predated inclusion formation. Furthermore, cAMP levels in HdhQ111 striatum declined from an early age (10 weeks), and cAMP was significantly decreased in HD postmortem brain and lymphoblastoid cells, attesting to a chronic deficit in man. Reduced CRE-signaling in cultured STHdhQ111 striatal cells was associated with cytosolic CREB binding protein that mirrored diminished cAMP synthesis. Moreover, mutant cells exhibited mitochondrial respiratory chain impairment, evidenced by decreased ATP and ATP/ADP ratio, impaired MTT conversion and heightened sensitivity to 3-nitropropionic acid. Thus, our findings strongly suggest that impaired ATP synthesis and diminished cAMP levels amplify the early HD disease cascade by decreasing CRE-regulated gene transcription and altering energy dependent processes essential to neuronal cell survival.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In Huntington's disease (HD), an elongated glutamine tract at the amino terminus of huntingtin leads to the loss of vulnerable neurons, primarily in the striatum, and to chorea, cognitive and psychiatric decline (1). Huntingtin is a large nuclear and cytoplasmic HEAT domain protein that has been implicated in mRNA production (2–5) and vesicle trafficking (2,6–10). The polyglutamine expansion appears to confer a novel structural property on mutant huntingtin that does not impair the protein's essential developmental activities (11), but which instead triggers a dominant disease process (12).
HD-like neuropathology in animals treated with mitochondrial toxins and loss of mitochondrial respiratory chain activities in end-stage HD patient brain (13) have suggested that energy deficits might contribute to the disease process (reviewed in 14). However, studies with HD transgenic systems have implicated decreased transcription of genes regulated by cyclic adenosine 3',5'-monophosphate (cAMP) responsive element (CRE) binding protein (CREB) (15–19). These genes include brain derived neurotrophic factor (BDNF) (20) and a host of others involved in diverse processes ranging from neurotransmission (15,21) to cholesterol metabolism (22).
Reduced gene transcription has been ascribed to sequestration of coactivators that bind huntingtin amino terminus, such as CREB binding protein (CBP) or TAFI1130, into inclusions formed from mutant amino terminal product (16–19,23). However, CBP is localized to inclusions in distinct polyglutamine neurodegenerative disorders, DRPLA (18) and SBMA (24), implying that sequestration may be a downstream consequence of the disease process. Moreover, CBP sequestration seems unlikely to explain the dominant reduction in BDNF transcription observed in immortalized STHdhQ111 striatal cells (20), which accurately express normal and mutant huntingtin and do not exhibit amino terminal product inclusions (5).
An alternative possibility for diminished CRE-mediated gene transcription, which is linked to energy impairment, is a deficiency in cAMP, the second messenger in CRE-mediated signaling. This scenario is supported by evidence for low cAMP in the cerebral spinal fluid of symptomatic HD patients (25), and by the capacity of forskolin, which stimulates adenylyl cyclases to produce cAMP from ATP, to ameliorate mutant huntingtin-fragment induced phenotypes in PC12 cells (19). Moreover, recent data suggests a direct impact of mutant huntingtin on mitochondrial function. Ectopic mutant huntingtin amino terminal fragment was able to induce deficits in calcium handling found in mitochondria isolated from HD patient lymphoblastoid cells (26,27) and HD transgenic mouse brain (27).
Consequently, we have used precise genetic HD mouse and striatal cell models to test the hypothesis that reduced cAMP may link aberrant CRE signaling and potential energy deficits early in the disease process. HdhQ111 mice accurately express 111-glutamine mutant huntingtin and exhibit early dominant abnormalities selective for medium spiny striatal neurons, including nuclear retention of full-length mutant huntingtin (28). These changes predate late onset mutant amino terminal product (as inclusions and insoluble-aggregate), at 
12 months of age, and overt neurodegeneration, evident at 24 months (28,29). The cognate immortalized STHdhQ111 striatal neuronal cell lines accurately express endogenous normal and mutant huntingtin and display dominant mutant phenotypes (5), including decreased BDNF transcription (20), consistent with aberrant PKA-CREB signaling in vivo.
Our results show for the first time that the dominant disease process triggered by mutant huntingtin features early, unremitting energy deficits, which are likely to amplify the disease cascade by affecting a host of homeostatic processes involved in cell survival.
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RESULTS |
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Dominantly reduced PKA–CREB signaling and cAMP in HdhQ111 brain
We first investigated whether, as suggested by decreased BDNF transcription in STHdhQ111 striatal cells (20), altered cAMP PKA-CREB signaling might be an early consequence of the HD mutation in vivo. BDNF and phospho-CREB levels in extracts of dissected HdhQ111 brain were assessed by immunoblot analyses at 5 months of age. The results, shown in Figure 1A, revealed that, when normalized to ß-actin in the same lane, BDNF levels in cortical and striatal extracts from HdhQ111 homozygotes were decreased from wild-type levels by 2.2- and 1.4-fold, respectively. Moreover, although total CREB levels were similar (data not shown), the level of the transcriptionally active form of CREB, phospho-CREB, in cortical extracts from HdhQ111 homozygotes and heterozygotes was reduced, by 4.5- and 2.2-fold, respectively (Fig. 1B).
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As these data supported dominantly diminished PKA/CREB signaling, we tested cAMP levels in cortical tissue extracts from HdhQ111 homozygote and heterozygote mice. The results of the competitive enzyme immunoassay (EIA) (Fig. 1B histogram) revealed significantly reduced cAMP levels in HdhQ111 homozygote (1.5-fold; P<0.001) and HdhQ111 heterozygote (1.2-fold; P<0.05) extracts, compared with wild-type littermate extracts. These findings, therefore, demonstrated that the HD mutation led to a dominant reduction in cAMP by 5 months of age.
cAMP is selectively reduced early in the disease process
As the early HdhQ111 disease process is progressive and selective for striatum, we next determined the timing and regional selectivity of decreased cAMP. EIA experiments were performed with striatum, cortex and cerebellum dissected from HdhQ111 homozygote and wild-type mice at various ages ranging from 3 weeks to 16 months. The results, summarized in Figure 2, revealed reduced cAMP concentrations in striatal 1.5-fold (P<0.001) and cortical extracts 1.2-fold (P<0.001), compared with wild-type littermate extracts, that manifest between 6 and 10 weeks of age, worsening thereafter. By contrast, cAMP levels in mutant and wild-type cerebellar extracts were similar even at 16 months. Therefore, the decline in cAMP was progressive and selectively manifest in HdhQ111 striatum, and cerebral cortex, as expected for a consequence of mutant huntingtin that may be involved in the disease process in man.
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Reduced cAMP in HD lymphoblasts and postmortem HD brain
To test this possibility directly, we used EIA to determine cAMP concentrations in HD patient and non-HD control postmortem brain and lymphoblastoid cell extracts. The results are summarized in the scatter plot shown in Figure 3. For parietal cortex extracts from the three control postmortem brains, the group mean cAMP level was 0.84±0.21 pmol/ml (range mean±SD 0.64±0.11 to 0.97±0.31 pmol/ml). By contrast, the extracts from five age-matched HD postmortem brains (Vonsattel neuropathologic grade 3) exhibited a group mean cAMP level of 0.29±0.07 pmol/ml (range mean±SD 0.22±0.06 to 0.39±0.11 pmol/ml), disclosing a striking 2.8-fold reduction (P<0.001). Similarly, HD and control lymphoblastoid cell extracts exhibited group mean cAMP levels of 0.30±0.09 pmol/ml (range mean±SD 0.28±0.01 to 0.31±0.16 pmol/ml) and 0.61±0.15 pmol/ml (range mean±SD 0.55±0.07 to 0.67±0.22 pmol/ml), respectively, revealing a significant
2.1-fold decrease (P<0.005). Thus, decreased cAMP reflects a dominant disease process that is manifest in non-neuronal as well as brain cells from HD patients.
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STHdhQ111 striatal cells exhibit decreased PKA–CREB signaling
We then investigated the basis of decreased cAMP and CRE signaling, using striatal neuronal cell lines generated from homozygous and heterozygous HdhQ111 and wild-type HdhQ7 littermate embryos (5). First, immunoblot analyses confirmed decreased CRE signaling in mutant STHdhQ111 cells, compared with wild-type striatal cells. BDNF signal was decreased (data not shown) (20) and, as shown in Figure 4A, phospho-CREB signal was reduced by
1.3-fold. To determine whether decreased cAMP–PKA signaling might involve altered CBP, CBP complexes were immunoprecipitated from cytosolic and nuclear fractions of wild-type STHdhQ7 and homozygous STHdhQ111 cells. As shown in the immunoblot in Figure 4B, the CBP reagent detected a nuclear CBP band in precipitates from wild-type and mutant cells, although in the latter cytoplasmic CBP was also detected. Confocal immunostaining of wild-type and heterozygous and homozygous mutant striatal cells, in Figure 4C, confirmed a nuclear pattern of CBP-stain in wild-type cells. By contrast, the majority of heterozygous and homozygous mutant cells exhibited both perinuclear and nuclear CBP stain, indicating that perinuclear CBP was a dominant effect of mutant huntingtin. Quantification (Fig. 4D) revealed CBP signal confined to the nucleus in 100% of wild-type cells but only in 18% of homozygous mutant cells.
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Since mutant striatal cells do not display accumulated mutant amino terminal huntingtin inclusions (5), CBP sequestration seemed unlikely to explain perinuclear CBP in mutant cells. Therefore, to determine whether this distinct distribution might instead be dynamic and reflect altered cAMP–PKA signaling, wild-type and homozygous mutant cells were treated with forskolin. As shown in Figure 4D, forskolin restored a wild-type nuclear CBP pattern in the majority (88%) of mutant striatal cells. Thus, perinuclear CBP in mutant striatal cells appears to be due to decreased cAMP–PKA signaling, implying reduced cAMP as a consequence of mutant huntingtin.
STHdhQ111 striatal cells exhibit decreased cAMP synthesis
Therefore, we measured cAMP in extracts of wild-type STHdhQ7 and mutant STHdhQ111 cells. The results of EIA cAMP assays, in Figure 5A, disclosed significantly decreased cAMP levels in homozygous and heterozygous STHdhQ111 cell extracts, of 1.7-fold (P<0.001) and 1.4-fold (P<0.005), respectively, compared with wild-type levels. Moreover, when adenylyl cyclase activity was stimulated by addition of forskolin to the growth medium, cAMP concentration was increased for all genotypes, but levels in extracts from homozygous and heterozygous cells remained lower than wild-type by 1.5-fold (P<0.001) and 1.2-fold (P<0.05), respectively (Fig. 5A). As these data suggested dominantly altered cAMP synthesis or degradation, we determined cAMP levels in extracts of homozygous STHdhQ111 cells and STHdhQ7 cells as a function of time after forskolin treatment. The results, in Figure 5B, revealed a shallower slope and cAMP maximum for mutant cells that was approximately two-thirds that achieved in wild-type striatal cells. This discrepancy was not eliminated by the addition of the phosphodiesterase inhibitor Ro-201724, although basal and forskolin-induced cAMP levels were elevated (data not shown), demonstrating that differential degradation was unlikely to explain restricted cAMP levels observed in mutant striatal cell extracts.
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STHdhQ111 striatal cells display reduced ATP and ATP/ADP ratio
We measured ATP, the substrate for adenylyl cyclases, using an assay based on firefly luciferase activity, for which ATP is an essential and specific co-factor. As shown in Figure 6A, compared with wild-type extract, homozygous and heterozygous STHdhQ111 cell extracts exhibited a significant reduction in ATP, of 2.2-fold (P<0.001) and 1.4-fold (P<0.001), respectively, disclosing decreased ATP as a dominant consequence of mutant huntingtin.
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To determine whether reduced ATP might reflect a deficit in energy metabolism, the relative levels of adenosine nucleotides were measured by HPLC in equivalent protein extracts prepared from STHdhQ7 and homozygous STHdhQ111 cells. Typical HPLC profiles, in Figure 6B, and calculated summary data, in Figure 6C, disclosed a relative reduction in cAMP, as well as ATP, but an increase in ADP in mutant cell extracts. The ratio of ATP/ADP, a measure of mitochondrial energy function, is plotted in Figure 6D, indicating a significantly reduced ratio in mutant striatal cells (P<0.001), suggesting a respiratory chain defect.
STHdhQ111 striatal cells exhibit mitochondrial respiratory chain deficits
We therefore assessed mitochondrial energy metabolism in naive wild-type and homozygous mutant STHdhQ111 cells, and in cells treated with the oxidative stressor H2O2, using the conversion of MTT to formazan to assess the mitochondrial electron transport chain. The results, summarized in Figure 7A, revealed that compared with untreated wild-type cells formazan production was significantly reduced by 30% in untreated mutant cells (P<0.001). Furthermore, H2O2 treatment caused a more dramatic reduction in formazan production in mutant cells compared with wild-type cells (41 and 18%, respectively), consistent with a defect of mitochondrial respiratory chain activity.
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To directly test respiratory chain function, we assessed the sensitivity of wild-type STHdhQ7 and homozygous mutant striatal cells to 3-nitropropionic acid (3-NP), a succinate dehydrogenase inhibitor, by measuring cell survival. DAPI staining revealed nuclear blebbing that was more prominent in treated mutant STHdhQ111 cells (data not shown). Quantification of all DAPI stained nuclei in treated and parallel mock treated cultures (Fig. 7B) revealed a significant
2.5-fold decrease (P<0.001) in the survival of 3-NP challenged mutant cells, compared to their wild-type counterparts, consistent with a mitochondrial respiratory chain deficit that reduces cell survival. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In HD, the graded loss of vulnerable neurons in the striatum underlies the distinctive clinical symptoms of the disorder. We have tested the hypothesis that the dominant pathogenic process triggered by mutant huntingtin might lead to an early metabolic deficit that amplifies the disease cascade by altering cAMP-dependent processes, including CRE-mediated gene transcription.
Our findings in genetically accurate HD mouse and striatal cell models provide the first evidence that a striatal specific energy deficit, manifest as reduced cAMP and ATP/ADP ratio, is an early, unremitting consequence of mutant huntingtin. Indeed, decreased cAMP is evident in HdhQ111 striatum by 10 weeks of age, preceding amino terminal aggregate and intranuclear inclusions by almost a year and predating reactive gliosis and overt neurodegenerative changes by 2 years. These data, therefore, strongly suggest that selective metabolic impairment precedes neuropathology and clinical symptoms in HD patients. Moreover, consistent with low cAMP in cerebral spinal fluid (25), dramatically reduced cAMP in HD postmortem brain and lymphoblastoid cells indicates a chronic deficit in HD patients that ultimately involves many cell types.
The precise steps by which mutant huntingtin leads to impaired cAMP and energy metabolism are not known, but may involve a direct interaction of the mutant protein with the mitochondrion (27). Our results in STHdhQ111 cells demonstrate a reduced ATP/ADP ratio that directly implicates a mitochondrial respiratory chain deficit, supported by reduced respiratory chain activity in the MTT assay and the hypersensitivity of mutant striatal cells to mitochondrial toxins. This deficit would be consistent with respiratory complex deficiencies reported in cells from symptomatic HD patients (13).
ATP deficit, which was associated with low cAMP in mutant striatal cells, may underlie declining HdhQ111 striatal cAMP levels in vivo. Limiting ATP substrate and/or free radicals produced during oxidative stress would be expected to decrease adenylyl cyclase activity (30), restricting cAMP synthesis. However, it is also possible that reduced ATP may be a consequence of decreased cAMP. The disease process may first lead to a reduction in cAMP, for example via a reduction in the levels of specific adenylyl cyclases (15), with consequent impact on mitochondrial energy function.
Decreased CRE-mediated signaling in HdhQ111 striatal cells appears to be explained by reduced cAMP, which occurs early in the disease cascade in the absence of amino terminal huntingtin inclusions, rather than by transcription factor sequestration or loss of huntingtin pro-survival activity. This finding suggests that low cAMP may give rise to altered CRE-regulated gene transcription observed in HD postmortem brain (20,23) and in transgenic HD mouse and cell culture models (15,19–22).
Energy deficit and decreased CRE-mediated signaling might amplify the disease cascade in multiple ways. Our findings, together with defective NMDA receptor-mediated neuronal cell swelling found in HdhCAG94 knock-in brain (31) and decreased aconitase in R6/2 brain (32), support NMDA receptor mediated excitotoxicity (reviewed in 14) early in the disease process. Moreover, CREB function, which is required to sustain adult CNS neurons (33), mediates nuclear calcium regulated gene transcription following neuronal cell membrane depolarization (34). Furthermore, survival factors, such as BDNF, and a host of other CRE-regulated gene products altered in HD model systems, including DARPP-32, enkephalin and somatostatin, are essential for normal neuronal cell function.
Notably, cAMP-mediated signaling is integral to mitochondrial function. CREB regulates cytochrome c transcription (35) and cAMP directly modulates mitochondrial membrane potential, respiration and formation of reactive oxygen species (ROS) (36). Thus, our findings support an early disease process in which mutant huntingtin may lead to mitochondrial respiratory chain impairment and reduced cAMP. Consequently decreased CRE-regulated gene transcription may ‘feedback’, further depressing energy metabolism. This proposal is consistent with the hypothesis that energy impairment in HD neuronal cells may lead to catastrophic ATP deficiency (13). Our data also provide strong evidence that ATP/ADP ratio and cAMP merit investigation as early progressive biochemical markers in HD and suggest that pharmacological manipulation of energy metabolism at an early stage may slow the pathogenic process in HD brain.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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HdhQ111 knock-in mice
HdhQ111 knock-in mice, with targeted insertion of 109 CAG repeats that extends the glutamine segment in murine huntingtin to 111 residues (37), were maintained on a CD1 outbred genetic background (Charles River Laboratories, Wilmington, MA, USA). HdhQ111 heterozygote males and females were intercrossed to produce HdhQ111 heterozygotes and homozygotes and wild-type littermates. Genotyping for CAG repeat size was performed as described (37).
Immortalized STHdhQ111 striatal neuronal cell lines
Conditionally immortalized wild-type STHdhQ7 striatal neuronal progenitor cells expressing endogenous normal huntingtin, and homozygous and heterozygous mutant STHdhQ111 striatal neuronal progenitor cell lines, expressing endogenous mutant huntingtin with 111-glutamines, have been described previously (5). The striatal cell lines were grown at 33°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids, 2 mM L-glutamine and 400 µg/ml G418 (Geneticin; GIBCO-BRL, Life Technologies, Gaithersburg, MD, USA). Cells were treated by addition of agents (Sigma-Aldrich, St Louis, MO, USA) to growth medium: forskolin (10 µM) for 30 min, with or without Ro 20-1724 [4-(3-butoxy-4-methoxy-benzyl)-2-imidazolidinone; 0.5 mM], or with 3-nitropropionic acid. Cell survival was determined with striatal cells plated on tissue culture ‘welled’ glass slides (Becton Dickinson, Franklin Lakes, NJ, USA) at density of 1.5x105/well (triplicate wells). Twenty-four hours later fresh growth medium without (control) or with 3-nitropropionic acid (1, 2.5 or 5 mM) was added. After 3 days the cells were rinsed, fixed for 15 min in 4% paraformaldehide, permeabilized with 0.1% Triton-X100 for 5 min and incubated with DAPI (0.5 µg/ml, Molecular Probes, Eugene, OR, USA) for 5 min. DAPI-stained nuclei were counted (n=3 independent experiments) using confocal microscope (MRC-1024 laser confocal microscope, BioRad, Hercules, CA, USA). Survival represents the proportion of nuclei counted in treated wells relative to nuclei counted in parallel untreated wells (100%).
Postmortem brain tissue and lymphoblastoid cell lines
The Harvard Brain Tissue Resource Center, McLean Hospital (Belmont, MA, USA) supplied the postmortem brain tissue. Three control (non-HD) cases, which had postmortem intervals of 2–32 h were from two males and one female (mean age 76 years). Cause of death was listed as myocardial infarct. Five HD cases (HD neuropathology Vonsattel grade 3) had postmortem intervals of 8–30 h and were from five males (mean age 70 years), with death at end-stage disease. The HD CAG allele sizes were determined previously: control brain 25/17, 21/21, 20/18; HD brain 41/18, 41/37, 42/23, 43/17. Tissue was used pursuant to the guidelines of the MGH IRB for discarded human tissue. Previously genotyped lymphoblastoid cell lines from two control non-HD individuals (HD CAG repeats; 17/18, 17/15 units) and three HD patients (39/17; 56/21; 52/18 units) have been described (1,38).
Subcellular fractionation and immunoprecipitation
Wild-type STHdhQ7/HdhQ7 and homozygous mutant STHdhQ111/HdhQ111 cells were harvested and cell pellets used to prepare cytosolic and nuclear fractions. The cell pellet was suspended in buffer A [10 mM Hepes (pH 7.9) containing 1.5 mM MgCl2, 10 mM KCl, protease inhibitor cocktail and 1 mM PMSF] left on ice for 10 min, subjected to homogenization by 40 strokes in a Douncer homogenizer and centrifuged at 2000g for 15 min. The supernatant was collected for provide the cytosolic fraction. The pelleted fraction was resuspended in buffer C [20 mM Hepes (pH 7.9) containing 25% glycerol, 0.42 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, protease inhibitor cocktail and 1 mM PMSF] with gentle rotation (30 min at 4°C). The mixture was centrifuged at 12 000g for 10 min to yield the nuclear fraction The protein content of each fraction was determined using Bio-Rad DC (Detergent Compatible) protein assay (Bio-Rad, Hercules, CA, USA).
Immunoprecipitation was done by incubation (overnight at 4°C) of nuclear and cytosolic cell fractions (500 µg) with 3 µg of anti-CBP-A22 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The complexes were then incubated for further 2 h with protein A Sepharose CL-4B (Sigma) with gentle rotation. The immunocomplexes were then washed by centrifugation and resuspension two times in 1% NP-40 buffer and once in PBS. After boiling for 5 min in reducing SDS sample buffer the immunoprecipitated proteins were resolved by SDS–PAGE on 6% polyacrylamide gel and transferred to nitrocellulose membranes. Immunoprecipitate complexes were detected by incubation with anti-CBP antibody followed by HRP-conjugated secondary antibody and enhanced chemiluminescence as described above.
Immunoblot analyses
Protein extracts of cerebral cortex and striatum dissected from HdhQ111 and wild-type mice (5 months age) were produced by lysis of minced tissue in ice-cold buffer (50 mM Tris–HCl pH 7.4–150 mM NaCl–2 mM EDTA–1% NP-40–1 mM PMSF). The supernatant was recovered after centrifugation at 10 000g. Protein extracts (30 µg) were resolved on 10 and 15% SDS–PAGE, proteins were transferred to nitrocellulose membranes (Schleicher & Schuell), blocked with 10% non-fat milk and incubated with primary antibodies overnight at 4°C and detected by horseradish peroxidase-conjugated secondary antibody-enhanced chemiluminescence (ECL) reagents (New England Biolabs, Beverly, MA, USA). Phosphoimager analyses were used to quantify the intensity of BDNF and phospho-CREB relative to the intensity of the ß-actin band in the same lane. Antibody reagents were anti-BDNF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-CREB Ser133 (Cell Signaling, Beverly, MA, USA) and anti ß-actin (Sigma-Aldrich, St Louis, MO, USA).
Immunofluorescence and confocal analysis
Wild-type STHdhQ7/HdhQ7 and homozygous mutant STHdhQ111/HdhQ111 cells were grown on four chambers glass slides (Becton Dickinson, Franklin Lakes, NJ, USA) at a density of 6x105/well (duplicate wells). The cells were fixed in 4% paraformaldehide, permeabilized for 5 min in 0.1% Triton X-100 in PBS treated for 30 min in blocking buffer (1% bovine serum albumin in PBS) and incubated for 2 h in blocking solution containing anti-CBP-A22. After several washes in PBS (3x5 min), cells were then incubated for 1 h in blocking solution containing goat anti-rabbit Cy3 secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). Cells were imaged with a laser confocal microscope (Bio Rad, Hercules, CA, USA) using the 20x objective.
cAMP competitive enzyme immunoassay
cAMP measurements were performed using the Direct cAMP enzyme immunoassay (EIA) kit CA-200 (Sigma-Aldrich, St Louis, MO, USA), according to the protocol supplied by the manufacturer. Frozen dissected mouse or human brain tissue was lysed directly in 0.1 M HCl (20 min) and supernatant recovered after centrifugation at 9000g. Dissected HdhQ7 and HdhQ111 knock-in mouse tissue was pooled: 6 weeks (n=2) 10 weeks (n=3); 5 months (n=4); and 16 months of age (n=3). For cultured cells, PBS washed pellets were lysed in 0.1 M HCl (10 min) and supernatant recovered after centrifugation at 2000g. An aliquot of cleared lysate was used to determine protein concentration using Bio-Rad DC (Detergent Compatible) protein assay (Bio-Rad, Hercules, CA, USA). EIA was performed with equal protein. Each EIA determination represents the mean of values obtained with quadruplicate (brain) or triplicate (cells) protein aliquots. cAMP concentration (pmol/ml) was determined by interpolation to a curve produced in the same assay with known concentrations of cAMP (standard curve) supplied by the manufacturer.
Measurement of ATP
ATP was determined luminometrically (MicroLumat LB96P) using the CytoLux L001-100 kit (PerkinElmer Life Sciences Inc., Boston, MA, USA) to measure light-emitting luciferase-catalyzed oxidation of luciferin, according to the manufacturer. PBS-washed cell pellets were lysed in non-ionic detergent buffer (Perkin-Elmer) for 5 min, lysates were cleared by centrifugation at 2000g and an aliquot of the supernatant was used for protein determination using Bio-Rad DC (Detergent Compatible) protein assay (Bio-Rad, Hercules, CA, USA). ATP determinations were performed with equal protein. Each ATP determination represents the mean of values obtained with triplicate aliquots. ATP concentration (nmol of ATP per mg of total protein) was calculated by interpolation to values (triplicates) determined with known concentrations of ATP (standard curve) in the same assay. The ATP standard was provided by the manufacturer.
Nucleotide extraction and HPLC analysis
HPLC analysis was performed on wild-type STHdhQ7/HdhQ7 and homozygous mutant STHdhQ111/HdhQ111 cells to analyze quantitative differences on cellular nucleotide pools. One million cells were pelleted at 800g and the pellets washed twice in ice-cold PBS (pH 7.4). Nucleotide extraction was done with 50 µl of ice-cold acetonitrile followed by 150 µl of cold water (39). The soluble and precipitated fractions were centrifuged at 14 000g for 10 min at -20°C and the supernatant fraction, kept on ice, was then gassed with N2 for 15 min to evaporate acetonitrile. The pellet was solubilized with 1 N NaOH, and the protein content was analyzed by Bio-Rad DC protein assay. The column used to perform the HPLC analysis was a 4 µm Nova-Pack C18 cartridge (100x8 mm inner diameter) equipped with a radial compression chamber (Waters, Milford, MA, USA). The buffer consisted of 20% acetonitrile, 10 mM ammonium phosphate, and 2 mM PIC-A ion-pairing reagent (Waters) and was run isocratically at 1 min/ml (40). Waters HPLC model 510 was used (Waters, Milford, MA, USA), and the ultraviolet (UV) detector model 440 (Waters) was set at 254 nm. HPLC-grade nucleotide standards were used to calibrate the signals. Internal standards were occasionally added to the samples to test recovery, which exceeded 90% for all nucleotides. All data were quantified by Millenium32 Chromatography Manager (Waters) software.
MTT assay
The metabolic activity of mitochondria was estimated by using MTT assay. This method is based on the reduction of the soluble yellow MTT tetrazolium salt to blue insoluble MTT formazan product by mitochondrial succinic dehydrogenase. After treatment with H2O2 (0.8 or 2 mM, 2 h) the cells were washed twice with PBS and the MTT (0.5 mg/ml in PBS) was added to each well and incubated for 2 h at 33°C. The medium was carefully removed and 200 µl of acidified isopropyl alcohol was added to solubilize the colored formazan product. Absorbance was read at 550 nm on a multiwell spectophotometer (Molecular Devices).
Statistical analysis
All values are expressed as mean±SD. Statitical significance was determined by one-way analysis of variance (ANOVA) and Scheffe's S post-test for comparisons between multiple groups.
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ACKNOWLEDGEMENTS |
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We thank Drs R. Shapiro and J. Jenkins for assistance with HPLC and M. Anderson, P. Crawford and F. Puglisi in the MGH Genomics Core Facility for tissue culture. This work was supported by NINDS grants NS32765 and NS16367 (HD Center Without Walls), an Anonymous Donor, and the Huntington's Disease Society of America (Coalition for the Cure). S.G. is a recipient of a fellowship from the Huntington's Disease Society of America.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 6177265089; Fax: +1 6177265735; Email: macdonam{at}helix.mgh.harvard.edu
Present address: Department of Pharmacology, University of Rome La Sapienza, P. le A Moro 5, I00185 Rome, Italy.
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