Title:
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Lentiviral Delivery of RNAi to Neurons to Study Kainate Receptor Trafficking
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Kainate receptors (KAR) are a subtype of ionotropic glutamate receptors implicated in
excitatory transmission and neuronal excitability in the CNS. A number of KAR
interacting proteins have been identified including, GRIP1, PICK1 and syntenin (Garcia
et aI., 1998; Hirbec et aI., 2003) all of which have been shown to directly interact with
KAR GluR5/6 subunits via a C-terminus PDZ domain protein-protein interaction. These
proteins are thought to playa role in KAR trafficking. In this study I have investigated the
effects on the surface expression of KAR subunits when over expressing the PDZ
domain containing KAR interacting proteins. Over expressing GRIP1 or syntenin
resulted in an increased surface expression of GluR6 and KA2 subunits. To investigate
this mechanism further, lentiviral RNA interference (RNAi) was developed to enable
short hairpin RNA (shRNA)-mediated specific knockdown of KAR interacting proteins.
RNAi a natural innate mechanism in cells which can be mimicked experimentally to
specifically reduce target protein levels via post transcriptional gene silencing. Lentiviral
delivery of shRNA constructs has been successfully validated in both cortical cultured
cells in vitro, in long term hippocampal slices and in the perirhinal cortex in vivo. In proof
of concept experiments significant PICK1 knockdown caused marked morphological
changes in neurons and PICK1 is thought to be involved in Arp2/3 mediated inhibition of
actin polymerisation (Rocca et aI., 2008). These initial experiments demonstrate lentiviral
RNAi is a powerful approach that can be used to study the specific roles of targeted
proteins in KAR trafficking which, in turn, is a key mechanism for regulating synaptic
strength and synaptic plasticity.
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