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Role of lateral diffusion of AMPA Receptors in LTP

Final Report Summary - TRAPAMPAR (Role of lateral diffusion of AMPA Receptors in LTP)

Executive summary

The Ca2+/Calmodulin-dependent protein kinase II (CaMKII) is critically required for the synaptic recruitment of AMPA-type glutamate receptors (AMPARs) during both development and plasticity. However, the underlying mechanism is unknown. Using single particle tracking of AMPARs, I show that CaMKII activation and postsynaptic translocation induce the synaptic trapping of AMPARs diffusing in the membrane. AMPAR immobilisation requires both phosphorylation of the auxiliary subunit Stargazin. Using fluorescence lifetime imaging microscopy (FLIM_FRET), I went on to demonstrate that phosphorylation triggered a conformational change in the cytoplasmatic tail of stargazin that i) decrease binding between the tail and the plasma membrane and ii) facilitates binding to the synaptic scaffold PSD95. Finally, CaMKII dependent AMPAR immobilisation regulates both short and long term plasticity. Thus, N-methyl-D-aspartate (NMDA)-dependent Ca2+ influx in the post-synapse triggers a CaMKII and Stargazin-dependent decrease in AMPAR diffusional exchange at synapses that controls synaptic plasticity and thus might be at the core of learning and memory .

Project context and objectives

Activity-dependent modifications of synaptic efficacy, such as long term potentiation (LTP), are thought to underlie learning and memory processes. Although discovered more than 30 years ago, the molecular mechanism underlying LTP are still unclear. The current model indicates that activation of NMDA receptors trigger the activation of protein kinases which in turn promotes the insertion of new AMPA receptors at the synapse. Among these kinases, numerous studies have shown that activation of calcium/calmodulin dependent protein kinase II (CamkII) is both necessary and sufficient for LTP induction. On one hand, pharmacological and genetic manipulations that abolish CamkII activity have consistently blocked LTP. On the other hand, intracellular perfusion of active CamkII is sufficient to induce LTP. More importantly, knock out mice lacking CamkII showed dramatic impairments in LTP and in learning and memory. Although it is clear that CamkII has a critical role in LTP and memory, the underlying mechanisms remain unclear. The first model postulated that CamkII could directly phosphorylate AMPA receptor and thus increase conductance. Although CamkII phosphorylate the GluR1 subunit (Ser831) of the AMPA receptor and this in turn increase channel conductance, mutation of this phosphorylation site (Ser831Ala) have no effect in LTP, ruling it out as a LTP mechanism. A second model proposes that CamkII could induce LTP by promoting the exocytosis of vesicle containing AMPA receptors. Although this model is the current favoured, there is surprisingly not clear data validating it. Lately, a third 'structural' model has proposed that CamkII increases synaptic transmission by organising new anchoring sites for AMPA receptors at the synapse. Although there is not direct evidence for this hypothesis, a number of findings indirectly support it: First, the abundance of CamkII at the postsynaptic density (PSD) is more consistent with a structural than an enzymatic function. Second, NMDA receptor activation causes a translocation of CamkII from the cytoplasm to the PSD. Third, CamkII bind strongly to NMDA receptors and thus can be specifically localised to active synapses. This alternative model of LTP implies that AMPA receptors diffuse freely in the neuronal surface and can be synaptically 'trapped' by new anchoring sites organised by CamKII. Studies done by the host laboratory using innovative imaging techniques to track the lateral mobility of individual receptors with quantum dots (QDs) have showed that surface AMPA receptors are highly mobile (60 % mobile fraction) and they can easily move in and out of synapses. These finding provide a conceptual and experimental framework to investigate the structural function of CamKII. The main objective of this project was to directly assess whether CamKII activation and synaptic translocation can lead to the anchoring and stabilisation of new AMPA receptors.

The specific objectives of this project were: i) to determine the role of CaMKII activation and synaptic translocation in endogenous AMPA receptors immobilisation, ii) determine the molecular mechanism of CaMKII-dependent immobilisation of AMPA receptors and iii) to determine the role of diffusional trapping of AMPAR in synaptic plasticity

Main results

Aim one. Determine the role of CaMKII activation and synaptic translocation in endogenous AMPA receptors immobilisation As proposed in Annex one, I used a number of pharmacological and electrophysiological techniques to activate NMDA Receptors and promote the consequently translocation of CamkII. I tracked the mobility of AMPA receptor before and after CamKII translocation using QDs coupled to antibodies against either the GluR1 or the GluR2 subunit. In particular, I activated NMDA receptors using two different protocols. First, I bath applied a combination of glutamate and glycine (100 uM and 10 uM, respectively) for two minutes. Second, I stimulated a small network of neurons using a field electrode. As previously shown, I found that NMDAR activation trigger the activation and synaptic translocation of CaMKII. More importantly for the objective of this project I found that CamKII translocation triggered the immobilisation of passing AMPAR. In addition I found that the synaptic translocation of a kinase-death form of CaMKII is unable to immobilise AMPAR suggesting that CaMKII immobilised AMPAR trhough phosphorylation events.

Aim two. Determine the molecular mechanism of CaMKII-dependent immobilisation of AMPA receptors Because CamkII does not interact directly with AMPA receptors, I studied the role of scaffolding proteins of the MAGUK family in mediating the effect of CamkII activation. To study the role of the PDZ binding domain of the GluR1 subunit known to interact with SAP97, I overexpressed GluR1 carrying a deletion in the PDZ binding domain (HA-GluR1DC) and determine whether CamKII activation was still able to immobilise it. I found that CaMKII still immobilised this mutant form of GluR1 suggesting a different mode of AMPA immobilisation. Since previous studies performed at the host laboratory indicate that AMPAR is indirectly stabilised at the synapse by the binding of its auxiliatoy subnit Stargazin (a CamkII substrate) to PSD95, I determined whether CamkII activation still immobilise AMPAR in the presence of Stargazin DC, a mutant lacking the PDZ-binding domain or Stargazin Ser9Ala., a mutant that can not longer be phosphorylated by CamkII. I found that both mutants form of Stargazin prevented the CaMKII-dependent immobilisation of AMPAR. These results indicated that CaMKII directly immobilised Stargazin and indirectly AMPAR. As a confirmation, I found that CaMKII strongly immobilised recombinant stargazin.

Aim three: Determine the role of diffusional trapping of AMPAR in synaptic plasticity

Since synaptic plasticity is not reliably induced in hippocampal cultured, I tried to determine the role of AMPAR surface diffusion and trapping in organotypic hippocampal slices, a more physiological preparation which preserved most of the in vivo connectivity and consequently LTP is easily induced. First, I demonstrated that CaMKII activation also trigger the immobilisation of AMPAR in organotypic slices. Using Biolistic, I expressed recombinant AMPAR (GluR1:pHGFP, extracellularly tagged) and active CaMKII RFP between one or two weeks to ensure all surface AMPAR carry the recombinant subunit. To immobilise AMPAR I will use two different approaches. In the first approach, I will then evaluate the degree of AMPAR immobilisation in organotypic slices using FRAP. I also demonstrated that a chemical LTP protocol cause the immobilisation of AMPAR in a CaMKII dependent manner.

Results highlights
1) CaMKII triggers the synaptic immobilisation of AMPAR
2) CamkII triggered the immobilisation of AMPAR via the phosphorylation of Stargazin
3) Stargazin phosphorylation facilitates binding to PSD95
4) CaMKII trigger the immobilisation of AMPAR in organotypic slices
5) CamKII immobilisation regulates both short and long term plasticit
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