title: NMDA receptor function and NMDA receptor-dependent phosphorylation of huntingtin is altered by the endocytic protein HIP1.
abstract: Huntingtin-interacting protein 1 (HIP1) is an endocytic adaptor protein that plays a role in clathrin-mediated endocytosis and the ligand-induced internalization of AMPA receptors (AMPARs) (Metzler et al., 2003). In the present study, we investigated the role of HIP1 in NMDA receptor (NMDAR) function by analyzing NMDA-dependent transport and NMDA-induced excitotoxicity in neurons from HIP1-/- mice. HIP1 colocalizes with NMDARs in hippocampal and cortical neurons and affinity purifies with NMDARs by GST (glutathione S-transferase) pull down and coimmunoprecipitation. A profound decrease in NMDA-induced AMPAR internalization of 75% occurs in neurons from HIP1-/- mice compared with wild type, using a quantitative single-cell-based internalization assay. This defect in NMDA-dependent removal of surface AMPARs is in agreement with the observed defect in long-term depression induction in hippocampal brain slices of HIP1-/- mice and supports a role of HIP1 in AMPAR internalization in vivo. HIP1-/- neurons are partially protected from NMDA-induced excitotoxicity as assessed by LDH (lactate dehydrogenase) release, TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling) and caspase-3 activation assays, which points to a role of HIP1 in NMDA-induced cell death. Interestingly, phosphorylation of Akt and its substrate huntingtin (htt) decreases during NMDA-induced excitotoxicity by 48 and 31%, respectively. This decrease is significantly modulated by HIP1, resulting in 94 and 48% changes in P-Akt and P-htt levels in HIP1-/- neurons, respectively. In summary, we have shown that HIP1 influences important NMDAR functions and that both HIP1 and htt participate in NMDA-induced cell death. These findings may provide novel insights into the cellular mechanisms underlying enhanced NMDA-induced excitotoxicity in Huntington's disease.
authors.affiliation: Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4.
authors.forename: Martina
authors.initials: M
authors.lastname: Metzler
authors.forename: Lu
authors.initials: L
authors.lastname: Gan
authors.forename: Tak Pan
authors.initials: TP
authors.lastname: Wong
authors.forename: Lidong
authors.initials: L
authors.lastname: Liu
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Helm
authors.forename: Lili
authors.initials: L
authors.lastname: Liu
authors.forename: John
authors.initials: J
authors.lastname: Georgiou
authors.forename: Yushan
authors.initials: Y
authors.lastname: Wang
authors.forename: Nagat
authors.initials: N
authors.lastname: Bissada
authors.forename: Kevin
authors.initials: K
authors.lastname: Cheng
authors.forename: John C
authors.initials: JC
authors.lastname: Roder
authors.forename: Yu Tian
authors.initials: YT
authors.lastname: Wang
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hayden
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D016923:Cell Death; D002540:Cerebral Cortex / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism*; D000071058:Huntingtin Protein; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*
chemical_list: D004268:DNA-Binding Proteins; C489536:Hip1 protein, mouse; C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D016194:Receptors, N-Methyl-D-Aspartate; D005982:Glutathione Transferase