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*

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 GSK3β at Ser9. Consequent decreased turnover of transcription co-factor β-catenin leads to increased levels of β-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.

unstable CAG repeat that lengthens a polyglutamine tract in huntingtin above ϳ37 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 ϳ55 glutamines. However, although clinical symptoms manifest after decades, genotypephenotype 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 Hdh CAG150 , HdhCAG94, and Hdh Q111 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 Ca 2ϩ 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 STHdh Q111/Q111 striatal neuronal cells, derived from Hdh Q111/Q111 embryos (13). STHdh Q111/Q111 striatal 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 Hdh Q111/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 Ca 2ϩand phosphatidylinositol (PI) 3-kinase-dependent Akt phosphorylation at Ser 473 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 Ca 2ϩ -and PI 3-kinasedependent 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.
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 g/ml aprotinin, 10 g/ml leupeptin, 10 g/ml pepstatin and supplemented with phosphatase inhibitors (1 mM Na 3 VO 4 and 50 mM NaF). The total cell lysates were then cleared by centrifugation at 10,000 ϫ 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 M for 30 min), MK-801 (2 M for 10 min), or EGTA (400 M for 5 or 20 min) and analyzed by immunoblot for levels of phospho-Akt (Ser 473 ). 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 ϫ g for 15 min. The supernatant was further centrifuged at 100,000 ϫ g. The membrane fraction was obtained by resuspending the high speed pellet in buffer A containing 1% Nonidet P-40.
Following . Immunoblots were rinsed three times for 10 min in TBS-T and incubated 1 h at 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). Quan-
Turnover of Cytosolic ␤-Catenin-Wild type STHdh Q7/Q7 and mutant STHdh Q111/Q111 cells plated on 10-mm tissue culture dishes were incubated in growth medium containing cyclohexamide at a final concentration of 30 g/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 MgCl 2 , 10 mM KCl, protease inhibitor mixture, 10 g/ml aprotinin, 10 g/ml leupeptin, and 10 g/ml pepstatin) followed by 40 strokes in a Dounce homogenizer. The cell lysates were then centrifuged at 2,000 ϫ g for 15 min, and the supernatant was further centrifuged at 100,000 ϫ 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-␤-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 STHdh Q7/Q7 and mutant STHdh Q111/Q111 cells were incubated in ice-cold hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, protease inhibitor mixture, 10 g/ml aprotinin, 10 g/ml leupeptin, and 10 g/ml pepstatin) for 10 min and mechanically lysed by 40 strokes in a Dounce homogenizer. The homogenate was centrifuged at 2,000 ϫ g for 15 min, and the resulting supernatant was further centrifuged at 100,000 ϫ g for 30 min to isolate the cytoplasmic fraction. The washed pellet from the 2,000 ϫ 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 MgCl 2 , 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 ϫ 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 g of protein) with 3 g of anti-␤-catenin antibody overnight at 4°C followed by a 2-h incubation with 50 l 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-␤-catenin, anti-phospho-␤-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.

STHdh Q111/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 Thr 308 and Ser 473 . Phosphorylation of Akt kinase at Ser 473 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 Ser 473 . Extracts of striatum dissected from three homozygous mutant Hdh Q111/Q111 and three wild type Hdh Q7/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 ϭ 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.
Next, Akt kinase activation was measured in extracts pre-pared from immortalized wild type STHdh Q7/Q7 and mutant STHdh Q111/Q111 striatal cells, a manipulable genetic HD neuronal striatal cell model. As shown in Fig. 1B, a significant ϳ4-fold increase in the ratio of Ser(P) 473 -Akt-signal versus the total Akt-signal (p ϭ 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.
shown in Fig. 2A. Consistent with increased Akt activation, the ratio of Ser(P) 9 -GSK3␤ versus total GSK3␤ was increased 3-fold (p ϭ 0.0028) in mutant cell extracts compared with wild type cell extracts.
To determine whether increased levels of Ser 9 -GSK3␤ phosphorylation might be a specific consequence of enhanced Akt activation in STHdh Q111/Q111 cells, we examined the Akt-regulated phosphorylation of pro-apoptotic transcription factor FKHR at Ser 256 by immunoblot analysis. In contrast to GSK3␤, levels of Ser(P) 256 -FKHR, normalized to ␣-tubulin levels, were similar in mutant compared with wild type striatal extracts (Fig. 2B). Thus, increased Akt activation in STHdh Q111/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␤ inactivation.
Increased ␤-Catenin Stabilization in STHdh Q111/Q111 Striatal Cells-To further study the functional significance of Akt phosphorylation in mutant striatal cells, we determined whether elevated Ser(P) 9 GSK3␤ in STHdh Q111/Q111 might alter the levels of ␤-catenin, an essential transcriptional coactivator downstream of GSK3␤ whose turnover and translocation to the nucleus is regulated by GSK3␤ phosphorylation (20). We first tested whether the turnover of cytosolic ␤-catenin differs in STHdh Q111/Q111 and wild type STHdh Q7/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. ␤-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 ␤-catenin-reactive band in wild type cytosolic extracts was about 30 min, the ␤-catenin band in STHdh Q111/Q111 cytosolic extracts remained stable over the 2-h chase period, implying decreased turnover.
To determine whether the increased ␤-catenin levels that are

FIG. 6. Enhanced Akt activation is mediated by NMDA receptors in STHdh Q111/Q111 striatal cells.
A, immunoblot of proteins solubilized from membrane fractions prepared from STHdh Q7/Q7 (ST7/7) and STHdh Q111/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 STHdh Q7/Q7 (ST7/7) and STHdh Q111/Q111 (ST111/111) striatal cells that were untreated or treated with 2 M 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 ϭ 3 experiments; p ϭ 0.1) but dramatically reduced the level of activated Akt in mutant striatal cell extracts (n ϭ 3 experiments; p ϭ 0.0002). C, immunoblot of extracts prepared from STHdh Q111/Q111 (ST111/111) striatal cells that were untreated or treated with 2 M MK-801 for 10 min or 400 M 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 ϭ 3 experiments; **, p ϭ 0.0001), both treatments with EGTA significantly reduced Akt activation (n ϭ 3 experiments; *, p ϭ 0.001). detected in STHdh Q111/Q111 cell extracts might be a direct consequence of GSK3␤ inactivation, mutant and wild type striatal cells were transfected with a cDNA plasmid that drives overexpression of c-Myc-tagged GSK3␤ (c-Myc-GSK3␤). Immunoblot analysis of cytosolic protein extracts immunoprecipitated by anti-␤-catenin antibody (Fig. 3C) revealed that exogenous c-Myc-GSK3␤, detected using a specific anti-Myc antibody, was associated with decreased levels of cytosolic ␤-catenin in both wild type and mutant cell extracts, eliminating the discrepancy between the two genotypes. Thus, because c-Myc-GSK3␤ can appropriately regulate the turnover of ␤-catenin in transfected STHdh Q111/Q111 cells, increased levels of cytosolic ␤-catenin detected in untransfected mutant striatal cells seem likely to reflect decreased GSK3␤ activity.
Increased Levels of Cyclin D1 in STHdh Q111/Q111 Striatal Cells-Cytosolic ␤-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 ␤-catenin-LEF complexes immunoprecipitated with an anti-␤-catenin antibody from nuclear extracts of wild type and mutant striatal cells. The immunocomplexes were analyzed by immunoblot using anti-␤-catenin and anti-LEF-1 antibodies. The results in Fig. 4A demonstrate that as expected ␤-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 ␤-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-␣-tubulin load control), the intensity of the 36-kDa cyclin D1 band is, on average, increased in mutant compared with wild type extracts by ϳ1.8-fold. The increase in cyclin D1 levels in mutant striatal cells was confirmed by immunoblot analysis of STHdh Q111/Q111 and STHdh Q7/Q7 cell extracts (Fig. 4C). In equal amounts of protein extract (anti-␣-tubulin load control), cyclin D1 is increased by ϳ1.9-fold in mutant, compared with wild type extracts. Thus, stabilization of ␤-catenin in mutant striatal cells is accompanied by increased levels of cyclin D1, indicating elevated ␤-catenin signaling in association with enhanced Akt activation.
PI 3-Kinase Mediates Akt Activation in STHdh Q111/Q111 Striatal Cells-To explore the basis of enhanced Akt signaling in STHdh Q111/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 Ser 241 . Phosphatase-PTEN, which blocks PI 3-kinase signaling, thereby reducing Akt activation, is activated by phosphorylation at Ser 380 . Ser(P) 241 -PDK1 and Ser(P) 380 -PTEN levels were assessed by immunoblot analyses of proteins extracted from wild type STHdh Q7/Q7 and mutant STHdh Q111/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 ϭ 0.003), and the level of Ser(P) 380 -PTEN is decreased by 1.7-fold (p ϭ 0.03) in mutant cell extracts.
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 Ser 473 -Akt antibody (Fig. 5B). IGF-1 treatment increased the levels of Akt phospho-rylation at Ser 473 in both wild type and mutant cell extracts, although there was an ϳ4.7-fold response in wild type cells (p ϭ 0.005), whereas in mutant striatal cells the already high levels of Ser(P) 473 -Akt were only augmented by ϳ1.3-fold (p ϭ 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 Ser 473 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 STHdh Q111/Q111 Striatal Cells-Consequently, given evidence of excitotoxicity in HD patient cells (14,15) and genetic knock-in mouse models (5)(6)(7)(8), we tested whether increased activation of Akt in STHdh Q111/Q111 striatal cells might be mediated through NMDA receptor signaling. Wild type STHdh Q7/Q7 and mutant STHdh Q111/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 Ca 2ϩ 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 im- 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 (ϩ in circle) the voltage-dependent Mg2ϩ block on the NMDA receptor, permitting Ca 2ϩ. 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 Ca 2ϩ .may further exacerbate mitochondrial dysfunction perhaps by further depolarizing the mitochondrial membrane (⌬⌿ m in box) (21)(22)(23)(24). As our data demonstrate, enhanced NMDA receptor signaling can lead to Ca 2ϩ 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 ␤-catenin, via the inactivation of GSK3␤, with a consequent increase in the expression of ␤-catenin-LEF-1-dependent downstream target genes such as cyclin D1. munoblot analyses. The results presented in Fig. 6B reveal that phosphorylation of Akt at Ser 473 in STHdh Q111/Q111 cell extracts was dramatically decreased (ϳ10-fold, p ϭ 0.0002) by treatment with MK-801. By contrast, the same MK-801 pretreatment had no significant effect (ϳ1.2-fold, p ϭ 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.
Therefore, to address whether Ca 2ϩ influx through the NMDA receptor pore might be involved in Akt phosphorylation in mutant cells, EGTA (400 M) was added to the growth medium to chelate extracellular Ca 2ϩ . 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 ϳ3-fold (p ϭ 0.001). These data strongly implicate Ca 2ϩ influx via NMDA receptors in the PI 3-kinasedependent enhanced activation of Akt signaling in STHdh Q111/Q111 cells. DISCUSSION 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 STHdh Q111/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␤ inhibition and ␤-catenin stabilization. These data are consistent with a pro-survival response. For example, constitutive ␤-catenin signaling via LEF-1 has been shown to protect against neuronal cell death induced by the toxic ␤-amyloid protein (21). Therefore, enhanced ␤-catenin signaling via LEF-1 may be expected to protect mutant STHdh Q111/Q111 striatal cells from the effects of mutant huntingtin. Notably, the precise downstream ␤-catenin target genes that mediate neuronal cell survival remain to be elucidated.
In STHdh Q111/Q111 mutant striatal cells, the activation of Akt is appropriately dependent on PI 3-kinase and Ca 2ϩ . 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 Ca 2ϩ 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 voltagedependent Mg2ϩ blockade on the NMDA receptor, permitting Ca 2ϩ influx via the NMDA receptor at physiologic glutamate concentrations (22)(23)(24)(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.
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 dysfunc-tion 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 receptorassociated 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.