Effects of the Stargazin Mutation on the Scaffolding Proteins GRIP and PICK1 in the Stargazer Mutant Mouse

Abstract

AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid receptors) are glutamatergic receptors required for the fast component of excitatory neurotransmission. There are numerous AMPAR interacting proteins involved in the regulation of AMPAR function and changes in these AMPAR interacting proteins can result in neurological dysfunction. Glutamate receptor interacting proteins (GRIP1&2) and protein interacting with C kinase 1 (PICK1) are scaffolding proteins involved in GluA2/3-AMPAR anchorage, stability and recycling at synapses. AMPAR phosphorylation leads to their internalisation as a result of decreased GRIP affinity, but not PICK1 affinity for AMPARs. Gain-of-function GRIP1 mutations have been shown to accelerate the rate of AMPAR recycling and influence social behaviour in autism. Transmembrane AMPAR regulatory proteins (TARPs) play a key role in AMPAR trafficking to the synapse. Stargazer mice have a TARP-γ2 (stargazin) mutation leading to impaired AMPAR trafficking to synapses in the cerebellum and thalamus, resulting in ataxia and absence epilepsy respectively. The aim of this study was to investigate the effects of the stargazin mutation on cerebellar and thalamic GRIP and PICK1 levels in stargazers and control littermates. It was hypothesised that the deficits in AMPAR trafficking in stargazers would lead to a compensatory change in synaptic GRIP and PICK1 levels in these brain regions.

This study used a combination of Western blot analysis, immunofluorescence confocal microscopy and immunogoldcytochemistry electron microscopy to investigate GRIP and PICK1 expression in the cerebellum and thalamus of stargazers and matched controls. The global levels of GRIP1&2, but not PICK1, were significantly increased in stargazer cerebella. Further analysis showed GRIP expression was significantly elevated in the soma of inhibitory Purkinje cells (PCs) and Golgi cells (GoCs). However, synaptic GRIP levels were unchanged at the excitatory synapses on to PCs namely, parallel fibres-PC and climbing fibres-PC synapses, which have impaired GluA2/3-AMPAR trafficking in stargazers. Previous research has shown that GRIP levels are also unchanged at excitatory mossy fibre-granule cell synapses, which are devoid of GluA2/3-AMPARs in stargazers. Thus, the elevated cerebellar GRIP levels in stargazers is not due to compensatory changes in synaptic GRIP at excitatory synapses with impaired AMPAR trafficking, as a result of the stargazin mutation. GRIP and PICK1 expression was also examined in the thalamus for the first time using double immunofluorescence labelling, as AMPAR expression is also altered in the thalamus of stargazers. Western blot analysis showed a trend towards an increase in reticular thalamic nucleus GRIP levels in stargazers, however this did not reach significance. PICK1 levels also showed no significant difference between stargazers and controls in thalamus.

Overall, data presented indicates the stargazin mutation is associated with a selective increase in cerebellar GRIP levels. This may reflect compensatory changes in GRIP in inhibitory PC and GoC neurons or at inhibitory synapses. GRIP has been identified at inhibitory synapses and is proposed to facilitate the stabilisation and recycling of GABAA receptors similar to AMPARs. Further research is required to understand the cell-specific changes in stargazer GRIP expression, which may help identify common molecular mechanisms present in comorbid disorders such as ataxia, epilepsy and autism.