Functional significance of the metabotropic glutamate receptor 1 (mGlu1) splice variants

The metabotropic glutamate receptor 1 (mGlu1) is probably the most extensively studied mGlu receptor and it has been shown to be involved in many neural functions including the regulation of neuronal excitability, synaptic plasticity and synapse elimination. Four translated alternatively spliced variants of the mGlu1 receptor mRNA have been identified, which are characterized by a common N-terminal extracellular domain and different C-termini of variable length. Very little is known about the functional significance of the distinct mGlu1 receptor isoforms, although differences in their cellular distribution and efficiency in G-protein coupling have been reported. However, most of the data concerning mGlu1 receptors relate to the longest variant (mGlu1a), despite the predominant expression of the short isoforms (mGlu1ß and -1) in most areas of the brain. So far, the intrinsic complexity of the brain has largely hindered a detailed analysis of the cellular responses elicited by individual mGlu1 splice variants in neurons. Our primary aim is to elucidate the functional significance of mGlu1 receptor splice variants in neurons addressing their interaction with scaffolding proteins, specific cellular and subcellular localization and coupling to intracellular transduction pathways. For some of our studies, we will take advantage of the availability of different genetically modified mouse lines that we have previously generated, such as Purkinje cell-specific mGlu1a and mGlu1ß receptor-rescued mice. Using cell cultures we will investigate the interaction between mGlu1 receptors and a novel interactor partner that we have recently identified, namely KCTD12.. Furthermore, using the detergent- digested-freeze fracture replica labelling (SDS-FRL) technique, we will establish whether individual short mGlu1 variants, when expressed independently from other isoforms, can be targeted to the plasma membrane and maintain a similar subcellular distribution in neurons. We also propose to develop new critical tools to further study the role of individual mGlu1 splice variants such as selective antibodies and a new conditional KO/KI mouse line in which the mGlu1 and mGlu1a receptors are tagged and can be selectively knocked out in discrete brain areas. Given the considerable attention that in the last decade inhibition of mGlu1 receptors has received as a potential novel therapeutic approach for anxiety disorders and schizophrenia, a better understanding of the role of distinct mGlu1 spice variants is thus critical for the interpretation of the therapeutic potential of new selective compounds at these receptors.

Metabotropic glutamate receptors (mGluRs) upon activation by the neurotransmitter glutamate modulate synaptic transmission via intracellular second messengers. The first cloned mGluR, mGluR1, regulates motor coordination, synaptic plasticity and synapse elimination. Severe ataxia has been observed in mice lacking this receptor and in patients with Hodgkins disease who produce autoantibodies to the extracellular domain of mGluR1. The mGluR1 undergoes alternative splicing giving rise to four translated variants that differ in their intracellular C-terminal domains. Our current knowledge about mGluR1 relates almost entirely to the long mGluR1 isoform, whereas little is known about the other short variants. To study the expression of the mGluR1 variant, we have generated, using a novel gene- targeting technology, mice in which the C-terminus of this variant carries two short tags. With this approach, we could establish that mGluR1 is either untranslated or in amounts that are undetectable in the mouse cerebellum. Therefore, only the mGluR1 and mGluR1 variants are present and active at cerebellar synapses. The trafficking and function of mGluR1 appear strongly influenced by adaptor proteins that bind to its intracellular C-terminus. To investigate the mechanisms that regulate the subcellular localization of mGluR1 at cerebellar synapses, we generated a second transgenic mouse line carrying a single amino acid mutation disrupting the binding between mGluR1 and the scaffolding protein Homer. Disruption of this interaction did not result in an overt behavioural phenotype, e.g. these mice showed a normal motor coordination. Despite the typical mGluR1 perisynaptic distribution was not significantly changed in these transgenic animals, we observed a higher probability of intrasynaptic diffusion suggesting that long Homer proteins regulate the lateral mobility of mGluR1. We extended our ultrastructural analysis of mGluR1 perisynaptic clustering using a high-resolution technique to other mouse lines in which only one mGluR1 variant was reintroduced specifically in Purkinje cells only of mGluR1-knock out mice. This work revealed that distinct mGluR1 splice variants have a different targeting with respect to the edge of the postsynaptic density which could influence their activity in terms of regulation of synaptic activity, synaptic elimination and motor coordination.