NMDA receptor function and NMDA receptor-dependent phosphorylation of huntingtin is altered by the endocytic protein HIP1

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.