Probing the interaction between huntingtin and NMDA receptors

• Main Author: Icton, Carolyn D.
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/11765

Huntington's Disease (HD) is caused by an expansion (>35) of a polyglutamine (polyQ) tract, in the N-terminus of the protein, huntingtin (htt). Evidence suggests that the selective degeneration of striatal neurons seen in HD is, in part, caused by overactivation of N-methyl-D-aspartate (NMDA) type glutamate receptors (NMDARs). Previously, our lab has shown that co-expression of NMDARs and full-length htt (138Q), in HEK 293 cells, results in a significant increase in glutamate-evoked current amplitude for NR1/NR2B but not NRl/NR2A-type NMDARs. Since channel function and/or receptor distribution of NMDARs may be modulated by interactions with cytoskeletal proteins, we postulate that the polyQ expansion in mutant htt permits the indirect interaction of htt with NMDARs through cytoskeletal proteins, contributing to changes in NMDAR properties and resulting in overactivation of the receptors. We began by characterizing protein expression of htt, htt interacting proteins (i.e. HIP-1), PSD-95 family members and components of the actin cytoskeleton (actin and a-actinin) in HEK 293 cells and striatal, hippocampal and cortical tissue from wildtype and transgenic mice (18Q and 46Q). HEK 293 cells were shown to endogenously express HIP-1, a-actinin and actin. The wildtype and transgenic mice expressed all proteins of interest with relatively high expression of PSD-95 and SAP-102. In addition, NR2A vs. NR2B expression in the mouse striatal, hippocampal and cortical tissues was compared. Densitometric analysis revealed expression levels of NR2B in the striatum, relative to the cortex, is higher as compared to NR2A. The expression of a-actinin was further examined in both HEK cells and mouse tissues using antibodies specific for two isoforms of a-actinin, a-actinin-2 and a-actinin-4. Alpha-actinin-4 was present in HEK 293 cells and striatal, hippocampal and cortical tissue from both wildtype and transgenic mice. In contrast to a-actinin-4, no cc-actinin-2 was detected. Following completion of protein characterization we began to address our hypothesis by investigating a potential interaction between HIP-1 and a-actinin-4 in transfected HEK 293 cells. We have established that there is an interaction between HIP-1 and a-actinin-4. This interaction could prove to be a mechanism by which htt indirectly binds to NMDARs.