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Originally published In Press as doi:10.1074/jbc.M309348200 on October 1, 2003

J. Biol. Chem., Vol. 278, Issue 50, 50514-50522, December 12, 2003
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Enhanced Akt Signaling Is an Early Pro-survival Response That Reflects N-Methyl-D-aspartate Receptor Activation in Huntington's Disease Knock-in Striatal Cells*

Silvia Gines{ddagger}§, Elena Ivanova{ddagger}, Ihn-Sik Seong{ddagger}, Carlos A. Saura¶, and Marcy E. MacDonald{ddagger}

From the {ddagger}Molecular Neurogenetics Unit, Massachusetts General Hospital, Charlestown Massachusetts 02129 and the Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, Massachusetts 02115

Received for publication, August 22, 2003 , and in revised form, September 22, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Huntington's disease features the loss of striatal neurons that stems from a disease process that is initiated by mutant huntingtin. Early events in the disease cascade, which predate overt pathology in Hdh CAG knock-in mouse striatum, implicate enhanced N-methyl-D-aspartate (NMDA) receptor activation, with excitotoxity caused by aberrant Ca2+ influx. Here we demonstrate in precise genetic Huntington's disease mouse and striatal cell models that these early phenotypes are associated with activation of the Akt pro-survival signaling pathway. Elevated levels of activated Ser(P)473-Akt are detected in extracts of HdhQ111/Q111 striatum and cultured mutant STHdhQ111/Q111 striatal cells, compared with their wild type counterparts. Akt activation in mutant striatal cells is associated with increased Akt signaling via phosphorylation of GSK3{beta} at Ser9. Consequent decreased turnover of transcription co-factor {beta}-catenin leads to increased levels of {beta}-catenin target gene cyclin D1. Akt activation is phosphatidylinositol 3-kinase dependent, as demonstrated by increased levels of Ser(P)241-PDK1 kinase and decreased Ser(P)380-PTEN phosphatase. Moreover, Akt activation can be normally stimulated by treatment with insulin growth factor-1 and blocked by treatment with the phosphatidylinositol 3-kinase inhibitor LY294002. However, in contrast to wild type cells, Akt activation in mutant striatal cells can be blocked by the addition of the NMDA receptor antagonist MK-801. Akt activation in mutant striatal cells is Ca2+-dependent, because treatment with EGTA reduces levels of Ser(P)473-Akt. Thus, consistent with excitotoxicity early in the disease process, activation of the Akt pro-survival pathway in mutant knock-in striatal cells predates overt pathology and reflects mitochondrial dysfunction and enhanced NMDA receptor signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Huntington's disease (HD)1 is a dominantly inherited disorder that is characterized by choreiform movements, psychiatric and cognitive decline, and the graded loss of medium spiny projection neurons in the striatum (1). HD is initiated by an unstable CAG repeat that lengthens a polyglutamine tract in huntingtin above ~37 residues, such that disease severity is increased with increased polyglutamine length (2). Disease symptoms typically manifest in mid-life caused by mutant huntingtin with about 50 glutamines or in juvenile years caused by mutant huntingtin with more than ~55 glutamines. However, although clinical symptoms manifest after decades, genotype-phenotype studies with neuropathologically graded HD postmortem brain samples strongly suggest that the disease process is likely to begin at birth (3).

Evidence from precise genetic HD mouse and striatal cell models and HD patient cells implicates a chronic disease cascade, in which mutant cells may be progressively sensitized to further insults. Dominantly inherited abnormalities manifest in the striatum of HdhCAG150, HdhCAG94, and HdhQ111 knock-in mice, at only a few months of age, predating signs of neuropathology, such as intranuclear inclusions, by nearly a year (4–11). These early molecular phenotypes support excitotoxicity, via Ca2+ influx, stemming from decreased mitochondrial ATP synthesis (8) and enhanced NMDA receptor activation (7). They also suggest a role for loss of neuroprotective factors, such as brain-derived neurotrophic factor (12) and proenkephalin (5), caused by reduced levels of cAMP and diminished cAMP signaling via cAMP-dependent protein kinase and CREB-binding protein/cAMP-responsive element-binding protein (CBP/CREB) (8). The dominant phenotypes detected in vivo are also manifest in a genetically precise HD cell model: immortalized STHdhQ111/Q111 striatal neuronal cells, derived from HdhQ111/Q111 embryos (13). STHdhQ111/Q111striatal cells do not exhibit markers of pathology but instead display enhanced sensitivity to metabolic stressors, including 3-nitropropionic acid (8). Furthermore, lymphoblastoid cells from HD patients display polyglutamine length-dependent deficits in mitochondrial calcium handling (14, 15) and ATP synthesis (8),2 although these peripheral cells are not overtly affected in HD patients.

Given the evidence in support of subacute "toxicity," we have now examined the hypothesis that mutant striatal neuronal cells may activate compensatory pro-survival pathways, countering potentially toxic metabolic changes that emanate from the expression of mutant huntingtin. Specifically, we have investigated whether serine/threonine protein kinase B, also known as Akt, which has been implicated in neuronal cell survival (16), may be activated in mutant HdhQ111/Q111 striatal cells. Akt can be activated through the interaction of a trophic factor, such as insulin growth factor-1 (IGF-1), with its cognate receptor. However, in neuronal cells Akt can also be activated via N-methyl-D-aspartate (NMDA) receptor signaling, because rapid glutamate-induced Ca2+- and phosphatidylinositol (PI) 3-kinase-dependent Akt phosphorylation at Ser473 can be blocked by the NMDA receptor antagonist MK-801 (17). Furthermore, Akt signaling has been implicated in HD by findings in acute cell models that achieve pathology by overexpression of mutant huntingtin fragment. In these systems, IGF-1 treatment ameliorates mutant fragment-induced toxicity, and Akt phosphorylation of mutant fragment decreases intranuclear inclusions (18).

Now our investigations using genetically accurate HD mouse and striatal cell models reveals enhanced Ca2+- and PI 3-kinase-dependent Akt signaling that appears to be due to enhanced NMDA receptor activation. Thus, modulation of the Akt pathway may provide a means to counter the early excitotoxic effects of mutant huntingtin that predate overt neuronal cell pathology.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents—IGF-1, LY294002, (+)-MK-801, EGTA, and cyclohexamide were obtained from Sigma. Phospho-Akt (Ser473), phospho-PDK1 (Ser241), phospho-PTEN (Ser380), phospho-GSK3{beta} (Ser9), phospho-FKHR (Ser256), phospho-{beta}-catenin (Ser33/37/Thr41), total Akt, and anti-ubiquitin (PD41) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Total GSK3{beta} antibody and NMDAR1 antibody were purchased from BD Transduction Laboratories (San Diego, CA). NMDAR2A and NMDAR2B affinity purified rabbit polyclonal antibodies were purchased from Chemicon International, Inc. (Temecula, CA). Total anti-{beta}-catenin antibody was from Zymed Laboratories Inc. (San Francisco, CA). c-Myc (9E10) antibody and LEF-1 (N-17) antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cyclin D1 (Ab-3) monoclonal antibody was from Oncogene Research Products (Boston, MA). Anti-{alpha}-tubulin was from Sigma-Aldrich. pcDNA3c-MycGSK3{beta} mammalian cell expression vector was a kind gift of Dr. Miguel Medina (Brigham's and Women's Hospital).

Genetic HD Mouse and Striatal Cell Culture Models—Mice carrying the HdhQ111 knock-in allele, expressing mutant huntingtin with 111 glutamine residues, have been described previously (10, 11). Striatum was dissected from genotyped 8-month-old homozygous mutant HdhQ111/Q111 and wild type HdhQ7/Q7 littermate offspring of matings between male and female HdhQ111/Q7 heterozygotes. Conditionally immortalized wild type STHdhQ7/Q7 striatal neuronal progenitor cells expressing endogenous normal huntingtin and homozygous mutant STHdhQ111/Q111 striatal neuronal progenitor cell lines expressing endogenous mutant huntingtin with 111-glutamines generated from HdhQ111/Q111 and wild type HdhQ7/Q7 littermate embryos have been described previously (13). The striatal cell lines were grown at 33 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 2 mM L-glutamine, and 400 µg/ml G418 (Geneticin; Invitrogen). For overexpression of Myc-GSK3{beta}, striatal cells were transfected with pcDNA3c-MycGSK3{beta}, or, in the control with pcDNA3 vector alone, using LipofectAMINETM 2000, according to the protocol of the supplier (Invitrogen). The protein extracts were prepared 24 h post-transfection.

Immunoblot Analysis—In general, the protein extracts were prepared from striatal tissue and from cultured striatal cells by lysis on ice for 30 min in a buffer containing 50 mM Tris base (pH 7.4), 150 mM NaCl, 2 mM EDTA, protease inhibitor mixture, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin and supplemented with phosphatase inhibitors (1 mM Na3VO4 and 50 mM NaF). The total cell lysates were then cleared by centrifugation at 10,000 x g, and the supernatants were collected.

To analyze Akt activation, striatal cells were placed in Dulbecco's modified Eagle's serum-free medium 3 h before protein extracts were prepared. For treatment with drugs, striatal cells were first placed in Dulbecco's modified Eagle's serum-free medium for 3 h and then exposed to IGF-1 (50 ng/ml for 30 min), LY294002 (25 µM for 30 min), MK-801 (2 µM for 10 min), or EGTA (400 µM for 5 or 20 min) and analyzed by immunoblot for levels of phospho-Akt (Ser473). To analyze the expression of NMDA receptor subtypes in membrane fraction, striatal cells grown in complete Dulbecco's modified Eagle's medium were lysed in buffer A for 10 min with 40 strokes in a Dounce homogenizer, and the cell lysates were centrifuged at 2,000 x g for 15 min. The supernatant was further centrifuged at 100,000 x g. The membrane fraction was obtained by resuspending the high speed pellet in buffer A containing 1% Nonidet P-40.

Following determination of protein concentration, by Bio-Rad (detergent compatible) protein assay, 30 µg of total protein extract prepared as detailed above was mixed with 4x SDS sample buffer, boiled for 5 min, and resolved on 8% SDS-PAGE. For immunoblot analysis the proteins were transferred to nitrocellulose membranes (Schleicher & Schuell) and incubated for 30 min in blocking buffer containing 10% nonfat powdered milk in TBS-T (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.05% Tween 20). The blots were then probed overnight at 4 °C with primary antibodies: phospho-Akt (Ser473) (1:1,000), phospho-PDK1 (Ser241) (1:1,000), phospho-PTEN (Ser380) (1:1,000), phospho-GSK3{beta} (Ser9) (1:1,000), phospho-FKHR (Ser256) (1:1,000), total Akt (1:1,000), total GSK3{beta} (1:1,000), cyclin D1 (1:100), NMDAR1 (1:1,000), NMDAR2A (1:500), or NMDAR2B (1:500). Immunoblots were rinsed three times for 10 min in TBS-T and incubated1hat room temperature with horseradish peroxidase-conjugated goat anti-mouse (1:10,000) or anti-rabbit (1:10,000) antibodies. After being washed extensively for 30 min, the membranes were processed using an ECL chemiluminiscence substrate kit (New England Biolabs, Beverly, MA) and exposed to autoradiographic film (Hyperfilm ECL; Amersham Biosciences). Quantification of the immunoreactive bands was performed by scanning and analysis using the Scion densitometry program (Scion Images for Windows-Release Beta 4.0.2).

Turnover of Cytosolic {beta}-Catenin—Wild type STHdhQ7/Q7 and mutant STHdhQ111/Q111 cells plated on 10-mm tissue culture dishes were incubated in growth medium containing cyclohexamide at a final concentration of 30 µg/ml and chased at 0, 0.5, 1, and 2 h after the addition of cyclohexamide. At each time point the cells were washed once with ice-cold phosphate-buffered saline and mechanically lysed by incubation for 10 min in ice-cold buffer A (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, protease inhibitor mixture, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin) followed by 40 strokes in a Dounce homogenizer. The cell lysates were then centrifuged at 2,000 x g for 15 min, and the supernatant was further centrifuged at 100,000 x g for 30 min to provide the cytoplasmic fraction. The protein concentration of the cytoplasmic extracts was quantified by Bio-Rad (detergent compatible) protein assay, and equal amounts of protein from each lysate were resolved by 8% SDS-PAGE. The proteins were then transferred to nitrocellulose membranes, blocked in 10% nonfat milk TBS-T, and incubated overnight at 4 °C with a monoclonal anti-{beta}-catenin antibody. The immunoblot was then probed with horseradish peroxidase-conjugated secondary antibody and visualized by ECL reagents (New England Biolabs).

Immunoprecipitation—To isolate cytosolic and nuclear fractions, wild type STHdhQ7/Q7 and mutant STHdhQ111/Q111 cells were incubated in ice-cold hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, protease inhibitor mixture, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin) for 10 min and mechanically lysed by 40 strokes in a Dounce homogenizer. The homogenate was centrifuged at 2,000 x g for 15 min, and the resulting supernatant was further centrifuged at 100,000 x g for 30 min to isolate the cytoplasmic fraction. The washed pellet from the 2,000 x g spin (nuclear 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 mixture, and 1 mM phenylmethylsulfonyl fluoride) with gentle rotation for up to 1 h at 4 °C and centrifuged at 12,000 x g for 10 min, and the supernatant was collected to provide the nuclear fraction.

Immunoprecipitation was performed by incubation of cytosolic or nuclear fractions (500 µg of protein) with 3 µg of anti-{beta}-catenin antibody overnight at 4 °C followed by a 2-h incubation with 50 µl of protein A-Sepharose Cl-4B (Sigma). The beads were washed by centrifugation three times, then resuspended in ice-cold phosphate-buffered saline, and then boiled for 5 min in reducing SDS loading buffer. The immunocomplexes were resolved by SDS-PAGE on 12% polyacrylamide gel and transferred to nitrocellulose membranes. Immunoblot analysis was carried out as described above. Briefly, the blots were incubated with anti-{beta}-catenin, anti-phospho-{beta}-catenin, anti-ubiquitin, or anti-LEF-1 antibody and detected using ECL chemiluminescent reagents.

Statistical Analysis—The statistical significance of observations from independent experiments was determined by one-way analysis of variance and Scheffe's S post-test for comparisons between multiple groups.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
STHdhQ111/Q111 Striatal Cells Exhibit Enhanced Akt Activation—Akt can be activated as a direct downstream target of PI 3-kinase. Upon stimulation of PI 3-kinase, PDK1 can activate Akt by phosphorylation of Thr308 and Ser473. Phosphorylation of Akt kinase at Ser473 is required for its full activation (19). Therefore, we evaluated Akt activation in Hdh CAG knock-in mice by using an antibody that specifically recognizes Akt phosphorylated at Ser473. Extracts of striatum dissected from three homozygous mutant HdhQ111/Q111 and three wild type HdhQ7/Q7 littermate mice were individually analyzed by immunoblotting with antibodies that detect Ser(P)473-Akt or total Akt, as shown in Fig. 1A. Quantification of band intensity revealed that the average levels of Ser(P)473-Akt in the striata of mutant mice was significantly increased by 1.8-fold (p = 0.0045), compared with levels in wild type striata. Similar results were obtained at 2 and 18 months of age (data not shown). These data indicate that enhanced Akt activation is a consequence of the disease process in vivo, in Hdh knock-in striatum.



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  		FIG. 1.
Enhanced Akt activation in mutant HdhQ111/Q111 striatum and STHdhQ111/Q111 striatal cells. A, immunoblot of striatal extracts from three homozygous HdhQ7/Q7wild type (7/7) and three homozygous HdhQ111/Q111mutant (111/111) littermates, and in B, homozygous wild type STHdhQ7/Q7 (ST7/7) and homozygous STHdhQ111/Q111 mutant (ST111/111) striatal cells probed for Ser(P)473-Akt (p-Akt) or total Akt (Akt). The corresponding histograms (right panels) plot the relative Ser(P)473-Akt to total Akt ratio (Relative p-Akt/Akt ratio), determined by band intensities that were analyzed by Scion software, with the ratio in mutant extracts (111/111) normalized to the ratio determined in wild type extracts (7/7). The relative Ser(P)473-Akt/totalAkt ratio is significantly increased in mutant HdhQ111/Q111striatum (n = 3 experiments; *, p = 0.0045) and in mutant STHdhQ111/Q111 striatal cells (n = 8 experiments; *, p = 0.0006).

 
Next, Akt kinase activation was measured in extracts prepared from immortalized wild type STHdhQ7/Q7 and mutant STHdhQ111/Q111striatal cells, a manipulable genetic HD neuronal striatal cell model. As shown in Fig. 1B, a significant ~4-fold increase in the ratio of Ser(P)473-Akt-signal versus the total Akt-signal (p = 0.0006) was apparent in mutant cell extracts compared with wild type cell extracts. This finding, which is consistent with the in vivo results, demonstrates an increase in Akt activation in mutant striatal neuronal cells.

Increased Akt Activation Is Associated with GSK3{beta} Inactivation—To test whether increased Akt activation might be associated with altered Akt signaling in STHdhQ111/Q111 striatal cells, we assessed Ser(P)9-GSK3{beta}, which is a well known downstream target of the Akt pathway that is implicated in neuronal cell survival (16). Phosphorylation of GSK3{beta} at Ser9 by Akt inhibits GSK3{beta} kinase activity. Immunoblot analyses of Ser(P)9-GSK3{beta} and total GSK3{beta} in extracts prepared from wild type STHdhQ7/Q7 and mutant STHdhQ111/Q111 cells are shown in Fig. 2A. Consistent with increased Akt activation, the ratio of Ser(P)9-GSK3{beta} versus total GSK3{beta} was increased 3-fold (p = 0.0028) in mutant cell extracts compared with wild type cell extracts.



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  		FIG. 2.
GSK3{beta} is specifically inactivated in STHdhQ111/Q111 striatal cells. A, immunoblot of STHdhQ7/Q7 (ST7/7) and STHdhQ111/Q111 (ST111/111) striatal cell extracts detected with an antibody specific for Ser(P)9-GSK3{beta} (p-GSK3{beta}) or an antibody that recognizes total GSK3{beta} (GSK3{beta}). Analysis of the band intensities using Scion software indicates that the Ser(P)9-GSK3{beta}/total GSK3{beta} ratio is significantly increased in mutant cell extracts by ~3-fold (n = 3 experiments; p = 0.0028), relative to the ratio detected in wild type striatal cell extracts. B, immunoblot of STHdhQ7/Q7 (ST7/7) and STHdhQ111/Q111 mutant (ST111/111) striatal cell extracts detected with an antibody that is specific for Ser(P)256-FKHR (p-FKHR) or an antibody specific for {alpha}-tubulin. The relative ratios of the band intensities, from three independent experiments, of Ser(P)256-FKHR/{alpha}-tubulin in each lane revealed no difference in the levels of Ser(P)256-FKHR between wild type and mutant striatal cell extracts (p = 0.01).

 
To determine whether increased levels of Ser9-GSK3{beta} phosphorylation might be a specific consequence of enhanced Akt activation in STHdhQ111/Q111 cells, we examined the Akt-regulated phosphorylation of pro-apoptotic transcription factor FKHR at Ser256 by immunoblot analysis. In contrast to GSK3{beta}, levels of Ser(P)256-FKHR, normalized to {alpha}-tubulin levels, were similar in mutant compared with wild type striatal extracts (Fig. 2B). Thus, increased Akt activation in STHdhQ111/Q111 cells does not appear to have consequences for a downstream target that is involved in apoptosis but instead is associated with neuronal cell pro-survival signaling via GSK3{beta} inactivation.

Increased {beta}-Catenin Stabilization in STHdhQ111/Q111 Striatal Cells—To further study the functional significance of Akt phosphorylation in mutant striatal cells, we determined whether elevated Ser(P)9 GSK3{beta} in STHdhQ111/Q111 might alter the levels of {beta}-catenin, an essential transcriptional co-activator downstream of GSK3{beta} whose turnover and translocation to the nucleus is regulated by GSK3{beta} phosphorylation (20). We first tested whether the turnover of cytosolic {beta}-catenin differs in STHdhQ111/Q111 and wild type STHdhQ7/Q7 cells. Cyclohexamide, an inhibitor of protein synthesis, was added to the growth medium, and cells were then harvested after 0, 0.5 1, and 2 h and processed by differential centrifugation to isolate cytosolic and nuclear protein fractions. {beta}-Catenin levels in equal amounts of cytosolic protein fractions were analyzed by immunoblot analysis. Fig. 3A shows that although the half-life of the 92-kDa {beta}-catenin-reactive band in wild type cytosolic extracts was about 30 min, the {beta}-catenin band in STHdhQ111/Q111 cytosolic extracts remained stable over the 2-h chase period, implying decreased turnover.



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  		FIG. 3.
Increased stability of cytosolic {beta}-catenin in STHdhQ111/Q111 striatal cells. A, immunoblot of cytosolic extracts prepared from STHdhQ7/Q7 (ST7/7) and STHdhQ111/Q111 (ST111/111) striatal cells at various times after cyclohexamide was added to the growth medium (0, 0.5, 1, and 2 h), probed for {beta}-catenin or {alpha}-tubulin, indicating the relative stability of cytosolic {beta}-catenin in mutant striatal cells. B, immunoblot of proteins immunoprecipitated with anti-{beta}-catenin antibody from cytosolic extracts prepared from STHdhQ7/Q7 (ST7/7) and STHdhQ111/Q111 (ST111/111) striatal cells, probed for {beta}-catenin, ubiquitin, or {beta}-Ser(P)33, or Ser37 or Thr41 {beta}-catenin (p-{beta}-catenin), showing decreased levels of ubiquitinated and phosphorylated cytosolic {beta}-catenin in mutant cell extracts (n = 3 experiments). C, immunoblot of cytosolic extracts prepared from STHdhQ7/Q7 (ST7/7) and STHdhQ111/Q111 (ST111/111) cells transfected with pcDNA3 vector (pcDNA3) or pcDNAc-Myc-tagged GSK3{beta} (pcDNA3-c-myc-GSK3{beta}), probed for {beta}-catenin, indicating decreased levels of cytosolic {beta}-catenin (n = 3 experiments) caused by c-Myc-GSK3{beta} expression in wild type and in mutant striatal cells. The membrane was then reprobed with anti-c-Myc antibody to confirm c-Myc-GSK3{beta} expression and with {alpha}-tubulin antibody to indicate equal loading of cytosolic extracts.

 
Because {beta}-catenin turnover is regulated by GSK3{beta}-mediated phosphorylation, which targets {beta}-catenin for degradation by the ubiquitin/proteosome pathway, we next assessed the levels of ubiquitinated and phosphorylated {beta}-catenin, immunoprecipitated by anti-{beta}-catenin antibody from wild type and mutant cytosolic extracts. Detection of immunoblots with anti-ubiquitin antibody (Fig. 3B) revealed that multiple bands of ubiquitinated {beta}-catenin are immunoprecipitated from wild type striatal cell cytosolic extracts, whereas the {beta}-catenin band precipitated from mutant striatal cell extracts is not detected by anti-ubiquitin reagent. Reprobing immunoblots for phospho-{beta}-catenin using a phospho-specific anti-{beta}-catenin antibody that detects Ser33-, Ser37-, or Thr41-{beta}-catenin revealed phosphorylated {beta}-catenin bands in wild type immunoprecipitates. By contrast, phospho-{beta}-catenin was almost undetectable in anti-{beta}-catenin immunocomplexes from mutant cytosolic extracts.

To determine whether the increased {beta}-catenin levels that are detected in STHdhQ111/Q111 cell extracts might be a direct consequence of GSK3{beta} inactivation, mutant and wild type striatal cells were transfected with a cDNA plasmid that drives overexpression of c-Myc-tagged GSK3{beta} (c-Myc-GSK3{beta}). Immunoblot analysis of cytosolic protein extracts immunoprecipitated by anti-{beta}-catenin antibody (Fig. 3C) revealed that exogenous c-Myc-GSK3{beta}, detected using a specific anti-Myc antibody, was associated with decreased levels of cytosolic {beta}-catenin in both wild type and mutant cell extracts, eliminating the discrepancy between the two genotypes. Thus, because c-Myc-GSK3{beta} can appropriately regulate the turnover of {beta}-catenin in transfected STHdhQ111/Q111 cells, increased levels of cytosolic {beta}-catenin detected in untransfected mutant striatal cells seem likely to reflect decreased GSK3{beta} activity.

Increased Levels of Cyclin D1 in STHdhQ111/Q111 Striatal Cells—Cytosolic {beta}-catenin can bind to the T-cell factor/LEF-1 family of transcription factors, translocating to the nucleus and regulating the expression of specific target genes. Consequently, we assessed {beta}-catenin-LEF complexes immunoprecipitated with an anti-{beta}-catenin antibody from nuclear extracts of wild type and mutant striatal cells. The immunocomplexes were analyzed by immunoblot using anti-{beta}-catenin and anti-LEF-1 antibodies. The results in Fig. 4A demonstrate that as expected {beta}-catenin was immunoprecipitated from the nuclear extracts from wild type and mutant striatal cells. By contrast, a 55-kDa band of LEF-1 was immunoprecipitated from mutant nuclear extracts but was barely apparent in the immunoprecipitates from wild type nuclear extracts. Because {beta}-catenin-LEF complexes activate cyclin D1 gene transcription, cyclin D1 protein levels were measured in striatal extracts from three wild type and three homozygous mutant mice, by immunoblot analysis with anti-cyclin D1 antibody (Fig. 4B). In equal amounts of protein extract (anti-{alpha}-tubulin load control), the intensity of the 36-kDa cyclin D1 band is, on average, increased in mutant compared with wild type extracts by ~1.8-fold. The increase in cyclin D1 levels in mutant striatal cells was confirmed by immunoblot analysis of STHdhQ111/Q111 and STHdhQ7/Q7 cell extracts (Fig. 4C). In equal amounts of protein extract (anti-{alpha}-tubulin load control), cyclin D1 is increased by ~1.9-fold in mutant, compared with wild type extracts. Thus, stabilization of {beta}-catenin in mutant striatal cells is accompanied by increased levels of cyclin D1, indicating elevated {beta}-catenin signaling in association with enhanced Akt activation.



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  		FIG. 4.
Increased cyclin D1 levels in STHdhQ111/Q111 striatal cells. A, immunoblot of proteins immunoprecipitated (IP) with a monoclonal {beta}-catenin antibody, or control IgG, from nuclear STHdhQ7/Q7 (ST7/7) and STHdhQ111/Q111 (ST111/111) striatal cell extracts, probed for {beta}-catenin and transcription factor LEF-1. The IgG band is indicated. B, immunoblot of proteins extracted from striata of three wild type (7/7) and three homozygous mutant HdhQ111/Q111 mice (111/111) at 8 months of age, probed for cyclin D1. Analysis of the ratio of cyclin D1/{alpha}-tubulin band intensities revealed an average ~1.8-fold increase (ratios of 0.14, 0.19, and 0.18 for wild type mice and 0.3, 0.38, and 0.27 for mutant mice). C, immunoblot of total protein extracts prepared from STHdhQ7/Q7 (ST7/7) and STHdhQ111/Q111 (ST111/111) striatal cells, probed with specific antibodies to detect cyclin D1 or {alpha}-tubulin, as a loading control. Analysis of the ratio of cyclin D1/{alpha}-tubulin band intensities indicated an ~2-fold increase (n = 2 experiments; p = 0.03) in cyclin D1 levels in extracts from mutant striatal cells, compared with their wild type counterparts.

 
PI 3-Kinase Mediates Akt Activation in STHdhQ111/Q111 Striatal Cells—To explore the basis of enhanced Akt signaling in STHdhQ111/Q111 striatal cells, we analyzed the upstream signaling molecules that regulate Akt activation. PDK1, the Ser/Thr kinase that transduces the PI 3-kinase signal and regulates Akt phosphorylation, is activated by phosphorylation at Ser241. Phosphatase-PTEN, which blocks PI 3-kinase signaling, thereby reducing Akt activation, is activated by phosphorylation at Ser380. Ser(P)241-PDK1 and Ser(P)380-PTEN levels were assessed by immunoblot analyses of proteins extracted from wild type STHdhQ7/Q7 and mutant STHdhQ111/Q111 cells (Fig. 5A). Consistent with enhanced Akt activation, detection of immunoblots with anti-phospho-specific antibodies revealed that, compared with wild type cell extracts, the level of Ser(P)241-PDK1 is increased by 2.4-fold (p = 0.003), and the level of Ser(P)380-PTEN is decreased by 1.7-fold (p = 0.03) in mutant cell extracts.



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  		FIG. 5.
Enhanced Akt activation in STHdhQ111/Q111 striatal cells is PI 3-kinase-dependent. A, immunoblot of STHdhQ7/Q7 (ST7/7) and STHdhQ111/Q111 (ST111/111) striatal cell extracts, probed to specifically detect Ser(P)241-PDK1 (p-PDK1) (left panel) or Ser(P)380-PTEN (p-PTEN) (right panel) and as load control {alpha}-tubulin. The corresponding histograms (middle panels) plot the relative ratios of Ser(P)241-PDK1/{alpha}-tubulin or Ser(P)380-PTEN/{alpha}-tubulin band intensities detected in mutant striatal cell extracts, normalized to the ratio detected in wild type extracts, indicating a significant increase in Ser(P)241-PDK1 (n = 3 experiments; p = 0.003) and a decrease in Ser(P)380-PTEN (n = 2 experiments; p = 0.027). B, immunoblot of protein extracts prepared from STHdhQ7/Q7 (ST7/7) and STHdhQ111/Q111 (ST111/111) striatal cells, after treatment with IGF-1 or LY-294002, probed for Ser(P)473-Akt (p-Akt) or total Akt (Akt). The corresponding histogram plots the relative ratio of Ser(P)473-Akt/total Akt band intensities, in mutant relative to untreated wild type cell extracts. IGF treatment increased Akt Ser437 phosphorylation in wild type extracts by 3.9-fold (n = 4 experiments; *, p = 0.0016), whereas Akt activation was further increased in mutant cell extracts by only by 1.3-fold (n = 4 experiments; #, p = 0.0054). LY-294002 dramatically reduced Akt activation in both wild type (n = 4 experiments; **, p = 0.0001) and mutant (n = 4 experiments; ##, p = 0.0001) cell extracts, indicating that enhanced Akt activation in mutant cells is also PI 3-kinase-dependent.

 
We next determined whether the PI 3-kinase/Akt pathway is appropriately regulated in mutant and wild type striatal cells by supplementing the growth medium with IGF-1 and then evaluating the levels of Ser(P)473-Akt in total protein extracts by probing immunoblots using the specific Ser473-Akt antibody (Fig. 5B). IGF-1 treatment increased the levels of Akt phosphorylation at Ser473 in both wild type and mutant cell extracts, although there was an ~4.7-fold response in wild type cells (p = 0.005), whereas in mutant striatal cells the already high levels of Ser(P)473-Akt were only augmented by ~1.3-fold (p = 0.0016). These results indicate that although Akt is already activated in mutant striatal cells, the pathway can be appropriately induced by IGF-1.

To confirm PI 3-kinase regulation of Akt activation, wild type and mutant striatal cells were treated for 30 min with LY294002, a selective inhibitor of PI 3-kinases. The immunoblot results presented in Fig. 5B demonstrate that pretreatment of cells with LY294002 completely prevented Akt phosphorylation at Ser473 in wild type and mutant cell extracts, whereas the total levels of Akt were not altered. Thus, as it is in wild type striatal cells, Akt activation in mutant striatal cells is appropriately regulated by PI 3-kinase. This finding implies that increased Akt activation in mutant cells may reflect enhanced upstream receptor signaling.

MK-801 Blocks Akt Phosphorylation in STHdhQ111/Q111 Striatal Cells—Consequently, given evidence of excitotoxicity in HD patient cells (14, 15) and genetic knock-in mouse models (5–8), we tested whether increased activation of Akt in STHdhQ111/Q111 striatal cells might be mediated through NMDA receptor signaling. Wild type STHdhQ7/Q7 and mutant STHdhQ111/Q111 striatal cells express NMDA receptor subunits, NMDAR1, NMDAR2A, and NMDAR2B, as demonstrated by immunoblot analyses of proteins extracted from cell membrane fractions (Fig. 6A). To test whether Akt activation is associated with NMDA receptor activity, wild type and mutant striatal cells were treated with MK-801, a specific NMDA receptor antagonist that blocks Ca2+ influx associated with NMDA receptor signaling. Akt activation was judged by monitoring the levels of Ser(P)473-Akt in total cell extracts as assayed by immunoblot analyses. The results presented in Fig. 6B reveal that phosphorylation of Akt at Ser473 in STHdhQ111/Q111cell extracts was dramatically decreased (~10-fold, p = 0.0002) by treatment with MK-801. By contrast, the same MK-801 pretreatment had no significant effect (~1.2-fold, p = 0.1) on the levels of Ser(P)473 Akt in wild type cell extracts. These data indicate that Akt activation in mutant but not wild type cells is directly associated with stimulation of NMDA receptors.



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  		FIG. 6.
Enhanced Akt activation is mediated by NMDA receptors in STHdhQ111/Q111 striatal cells. A, immunoblot of proteins solubilized from membrane fractions prepared from STHdhQ7/Q7 (ST7/7) and STHdhQ111/Q111 (ST111/111) striatal cells probed with specific antibodies to detect different NMDA receptor subunits (NMDAR1, NMDAR2B, or NMDAR2A). B, immunoblot of extracts prepared from STHdhQ7/Q7 (ST7/7) and STHdhQ111/Q111 (ST111/111) striatal cells that were untreated or treated with 2 µM MK-801, probed for Ser(P)473-Akt (p-Akt) or total Akt (Akt). The corresponding histogram plots the ratio of band intensities of Ser(P)473-Akt/total Akt, compared with the ratio in untreated wild type cell extracts. MK-801 did not alter the levels of Ser(P)473-Akt in wild type striatal cells (n = 3 experiments; p = 0.1) but dramatically reduced the level of activated Akt in mutant striatal cell extracts (n = 3 experiments; p = 0.0002). C, immunoblot of extracts prepared from STHdhQ111/Q111 (ST111/111) striatal cells that were untreated or treated with 2 µM MK-801 for 10 min or 400 µM EGTA for 5 or 20 min, probed for Ser(P)473-Akt (p-Akt) or total Akt (Akt). The corresponding histogram plots the ratio of band intensities of Ser(P)473-Akt/total Akt, relative to the ratio in untreated mutant cell extracts, revealing that, consistent with the reduction produced by the NMDA receptor inhibitor MK-801 in these experiments (n = 3 experiments; **, p = 0.0001), both treatments with EGTA significantly reduced Akt activation (n = 3 experiments; *, p = 0.001).

 
Therefore, to address whether Ca2+ influx through the NMDA receptor pore might be involved in Akt phosphorylation in mutant cells, EGTA (400 µM) was added to the growth medium to chelate extracellular Ca2+. Immunoblot analyses of protein extracts prepared from untreated and treated mutant cells (Fig. 6C) revealed that EGTA treatment reduced the levels of phospho-Akt by ~3-fold (p = 0.001). These data strongly implicate Ca2+ influx via NMDA receptors in the PI 3-kinase-dependent enhanced activation of Akt signaling in STHdhQ111/Q111 cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
HD is a chronic neurodegenerative disorder that is initiated by a novel property that is conferred on mutant huntingtin by a lengthened amino-terminal polyglutamine segment. Evidence from HD patient samples and precise genetic Hdh CAG knock-in mouse and STHdhQ111/Q111 striatal neuronal cell models has revealed abnormal biochemical phenotypes that suggest excitotoxicity and loss of neuroprotective factors that must precede later pathologic markers and even later neuronal cell death.

Now our data demonstrate that mutant huntingtin leads to enhanced activation of protein kinase B/Akt signaling via GSK3{beta} inhibition and {beta}-catenin stabilization. These data are consistent with a pro-survival response. For example, constitutive {beta}-catenin signaling via LEF-1 has been shown to protect against neuronal cell death induced by the toxic {beta}-amyloid protein (21). Therefore, enhanced {beta}-catenin signaling via LEF-1 may be expected to protect mutant STHdhQ111/Q111 striatal cells from the effects of mutant huntingtin. Notably, the precise downstream {beta}-catenin target genes that mediate neuronal cell survival remain to be elucidated.

In STHdhQ111/Q111 mutant striatal cells, the activation of Akt is appropriately dependent on PI 3-kinase and Ca2+. However, in contrast to Akt activation in wild type striatal cells, the enhanced Akt activation that is observed in mutant striatal cells can be abrogated by MK-801 and therefore is largely determined by Ca2+ influx via the NMDA receptor. Thus, as depicted in Fig. 7, our results link the PI 3-kinase-dependent activation of the Akt pathway with the enhanced NMDA receptor activation and mitochondrial deficits that support excitotoxicity in mutant striatal cells (7, 8, 14, 22). In the latter long standing hypothesis, decrements in mitochondrial calcium handling and ATP synthesis lead to lowered resting membrane potential alleviating the voltage-dependent Mg2+ blockade on the NMDA receptor, permitting Ca2+ influx via the NMDA receptor at physiologic glutamate concentrations (22–25). Therefore, the early disease cascade that is initiated by mutant huntingtin comprises a mixture of events. Some of these can lead to detrimental consequences, whereas other events represent the recruitment of signaling pathways that are activated in response to these deficits. Thus, the prosurvival Akt pathway is activated as an early disease event in mutant striatal neuronal cells in conjunction with NMDA receptor-mediated excitotoxicity.



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  		FIG. 7.
Activation of the Akt pro-survival in response to enhanced NMDA receptor activity and mitochondrial dysfunction in mutant striatal cells. Depicted are components of a hypothetical model to illustrate the links between activation of the pro-survival Akt signaling pathway and excitotoxicity, which is evident from enhanced NMDA receptor signaling and mitochondrial deficits, in mutant striatal cells (6–8). In this scheme, mutant huntingtin, perhaps by direct interaction with the mitochondrion (14, 15) or via as yet unknown processes, leads to decrements in mitochondrial calcium handling and ATP synthesis (7, 8, 14, 15). Energy deficit in turn leads to decreased resting plasma membrane potential that relieves (+ in circle) the voltage-dependent Mg2+ block on the NMDA receptor, permitting Ca2+. influx at physiologic glutamate concentrations. Enhanced activation of NMDA receptors may also reflect an altered interaction of mutant huntingtin with PSD95 (25). In turn, increased intracellular Ca2+.may further exacerbate mitochondrial dysfunction perhaps by further depolarizing the mitochondrial membrane ({Delta}{Psi}m in box) (21–24). As our data demonstrate, enhanced NMDA receptor signaling can lead to Ca2+ and PI 3-kinase-dependent activation of the Akt pathway (through inactivation of PTEN and activation of PDK1) in manner that can be blocked by the NMDA receptor antagonist MK-801. Enhanced activation of the Akt pro-survival response leads to the stabilization of cytosolic {beta}-catenin, via the inactivation of GSK3{beta}, with a consequent increase in the expression of {beta}-catenin-LEF-1-dependent downstream target genes such as cyclin D1.

 
Importantly, these consequences of mutant huntingtin are manifest before mutant striatal neuronal cells become more severely compromised and start to exhibit markers of overt pathology (6–8). Therefore, in genetically accurate HD models, in vivo and in cell culture, the end products of cellular dysfunction and toxicity, such as intranuclear inclusions, are not involved in determining the altered signaling state of mutant striatal cells early in the disease process. Instead, enhanced Akt signaling appears to reflect the consequences of mutant huntingtin on processes that lead to abnormally enhanced NMDA receptor activation. This may involve the decreased interaction of mutant huntingtin with the NMDA receptor-associated protein PSD95, which has been shown to increase NMDA receptor-mediated glutamate toxicity (26). Alternatively, mutant huntingtin may interact directly with the mitochondrion, resulting in aberrant calcium handling (7, 14, 15) and impaired ATP synthesis, which is hypothesized to lead to abnormal NMDA receptor activation. Moreover, Akt signaling may be enhanced because of an increased association of mutant huntingtin with the type 1 inositol 1,4,5-triphosphate receptor, which elevates intracellular calcium (27). However, because our data demonstrate that Akt activation in mutant cells can be fully blocked by MK-801, a specific NMDA receptor antagonist, participation of the type 1 inositol 1,4,5-triphosphate receptor pathway would be expected to involve an effect on NMDA receptor activity.

Activation of Akt, without changes in the total levels of the protein, early in the disease cascade appears to contrast with data suggesting that the Akt protein is diminished in HD postmortem brain (18). However, in contrast to this tissue, with extensive loss of neuronal cells, the Hdh CAG knock-in models do not feature overt neuronal cell death (11, 13). Therefore, it is possible that in severely compromised neurons at end stage disease, Akt is decreased, and the earlier pro-survival Akt response is lost. Our data therefore suggest that strategies to boost Akt signaling early in the disease process may not be beneficial, because the pathway is already induced, although stimulating the Akt pathway late in the disease cascade may be ameliorative. Importantly, our data demonstrate that strategies designed to circumvent or alleviate the effects of enhanced NMDA receptor activation and other consequences of mitochondrial dysfunction in mutant cells merit particular investigation.


   FOOTNOTES
 
* This work was supported by NINDS, National Institutes of Health Grants NS32765 and NS16367 (Huntington's Disease Center Without Walls), an anonymous donor, and the Huntington's Disease Society of America (Coalition for the Cure). 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

§ To whom correspondence should be addressed: Molecular Neurogenetics Unit, Massachusetts General Hospital, Bldg. 149, 13th St., Charlestown, MA 02129. Tel.: 617-726-5726; Fax: 617-726-5735; E-mail: gines{at}helix.mgh.harvard.edu.

1 The abbreviations used are: HD, Huntington's disease; IGF-1, insulin growth factor-1; NMDA, N-methyl-D-aspartate; PI, phosphatidylinositol. Back

2 I.-S. Seong, E. Ivanova, S. Gines, and M. E. MacDonald, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Elisa Fossale, Vanessa Wheeler, and Vladimir Vrbanac for providing HdhQ111 striatal tissue and Miguel Medina for the kind gift of pcDNA3c-Myc-GSK3{beta} plasmid.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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