Abstract
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 Hdh(Q111/Q111) striatum and cultured mutant STHdh(Q111/Q111) striatal cells, compared with their wild type counterparts. Akt activation in mutant striatal cells is associated with increased Akt signaling via phosphorylation of GSK3beta 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 Ca(2+)-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.
Highlights
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]
Given the evidence in support of subacute “toxicity,” we have 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
We have investigated whether serine/threonine protein kinase B, known as Akt, which has been implicated in neuronal cell survival [16], may be activated in mutant HdhQ111/Q111 striatal cells
Summary
Chemicals and Reagents—IGF-1, LY294002, (ϩ)-MK-801, EGTA, and cyclohexamide were obtained from Sigma. Phospho-Akt (Ser473), phospho-PDK1 (Ser241), phospho-PTEN (Ser380), phospho-GSK3 (Ser9), phospho-FKHR (Ser256), phospho--catenin (Ser33/37/Thr41), total. Anti-ubiquitin (PD41) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Total GSK3 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. Total anti--catenin antibody was from Zymed Laboratories Inc. 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]. 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 bo-
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