Cycle 5 project 1

AMPAR auxiliary subunits Stargazin and Shisa6: role in AMPAR trafficking and physiology


P01-ZamriPhD student: Azra Elia Zamri, Malaysia
Home Institute: Bordeaux Neuroscampus; Principle Investigator: Daniel Choquet
Host Institute: Amsterdam Neuroscience; Principle Investigator: Guus Smit

Executive Summary

Regulation of AMPAR stabilization at synapses is a hallmark of synaptic plasticity. Despite over a decade of research, we still do not understand the molecular basis of activity-dependent AMPAR stabilization, likely originating from the incomplete view we had of the AMPAR complex composition that contains many auxiliary subunits in addition to the core GluA subunits. Indeed, AMPARs form complexes with several auxiliary proteins that play key roles in their trafficking, stability, gating properties and pharmacology.

Research rationale and aims
An essential AMPAR auxiliary subunit is Stargazin that facilitates synaptic targeting and controls AMPAR synaptic stability. Since the identification of Stargazin and related TARPs, the list of AMPAR interactors has grown. We have discovered a new AMPAR auxiliary protein (collaboration with A.B. Smit, Amsterdam). Our preliminary data shows that GluA2 mobility at the plasma membrane is highly regulated by interaction between this protein and PSD95. It’s overexpression immobilizes GluA2 at the synapse, while disrupting the interaction with PSD95 by deleting the PDZ binding domain restores the mobility of GluA2. Interestingly, this protein shares some striking properties with Stargazin, such as the ability to stabilize AMPAR at synapses.

Aim 1: We will determine the respective contributions of the various auxiliary subunits to AMPAR synaptic stability using high resolution microscopy, at rest and during activity-dependent plasticity

In addition, we postulate, based on preliminary proteomics data from the Smit lab that composition of individual AMPAR complexes is heterogeneous and that it determines the trafficking and functional properties of the receptor. Post translational modifications are very important for the trafficking of the receptors, likely playing an important role during plasticity.

Aim 2: Using state-of-the-art proteomics technology we will identify the respective intracellular interactors of the two auxiliary proteins. In addition, we will test how phosphorylation contributes to their role in AMPAR function.

A true collaborative effort:
This project can only be achieved through a collaborative effort as it crucially needs integration of high-resolution proteomics (Amsterdam), with high-resolution imaging and physiology (Bordeaux), in order to:

  1. determine the respective roles of the auxiliary proteins for the stabilization of AMPAR at synapse (imaging expertise Bordeaux),
  2. identify the respective interactors that bind to the c-terminus of both auxiliary proteins (MS expertise Amsterdam),
  3. analyze the role of post-translational modifications of auxiliary proteins intracellular domains in the binding to interactors and synaptic stabilization (various expertise, microscopy, affinity analysis, etc., unique to each lab).

Originality and relevance
The originality of this project lies in the novelty of the characterization of AMPAR auxiliary subunit and the unique integration of complementary approaches that we propose to determine its role. A gene-knockout has been made and is only available in our labs. Shedding light on new mechanisms of AMPAR modulation is of utmost importance to understanding synaptic plasticity, and mechanisms of learning and memory.

Nair, D., Hosy, E., Petersen, J.D., Constals, A., Giannone, G., Choquet, D., and Sibarita, J.B. (2013). Super-Resolution Imaging Reveals That AMPA Receptors Inside Synapses Are Dynamically Organized in Nanodomains Regulated by PSD95. J Neurosci 33, 13204-13224.
Von Engelhardt J, M.V., Sprengel R, Kavenstock N, Li KW, Stern-Bach Y, Smit AB, Seeburg PH, Monyer H. (2010). CKAMP44: A Brain-Specific Protein Attenuating Short-Term Synaptic Plasticity in the Dentate Gyrus. Science 327, 1518-1522.
Guillem K, Bloem B, Poorthuis RB, Loos M, Smit AB, Maskos U, Spijker S, Mansvelder HD. Nicotinic acetylcholine receptor β2-subunits in the medial prefrontal cortex are necessary and sufficient for attention. Science, 2011 333(6044):888-91.
Rao-Ruiz P, Rotaru DC, van der Loo RJ, Mansvelder HD, Stiedl O, Smit AB, Spijker S. Retrieval-specific endocytosis of GluA2-AMPARs underlies adaptive reconsolidation of contextual fear. Nat. Neurosci. 2011 14(10):1302-8.

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