Final Report Summary - AMPAZETA (Regulation of AMPA type of glutamate receptor surface diffusion by Protein Kinase ζ) 1.1.1 A summary description of project context and objectives:SummaryIncreases in the number of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate type of glutamate receptor (AMPAR) at the synapse, such as following the induction of long-term potentiation (LTP), is the leading cellular mechanism thought to underlay information storage in brain circuits. Synaptic strengthening or LTP is triggered following strong activation of the N-methyl D-aspartate receptors which leads to an increase in postsynaptic Ca2+ and the activation of a whole array of signaling mechanisms1. In general terms, these signaling mechanisms regulate synaptic accumulation of AMPAR via the regulation of local exocytosis/endocytosis mechanisms and through the recruitment of new receptors via lateral diffusion along the plane of the plasma membrane2,3.In recent years significant progress has been made in understanding the induction mechanisms responsible of synaptic strengthening. Contrarily, very few studies address the question of what molecular mechanisms are responsible for the maintenance of LTP. Recently two proteins have been implicated in the maintenance of long-term potentiation in hippocampal CA1 synapse; the protein kinase M zeta (PKMζ, an atypical protein kinase C)4,5 and the Interacting with Never in Mitosis 1 (Pin1) a phosphorylation dependent peptidyl-prolyl cis/trans isomerase6. Although PKMζ and Pin1 activity are crucial for the persistence of LTP, very little is known about their precise mode of action at synapses. The aim of the project is to combine single-molecule imaging techniques, molecular biology, protein biochemistry and single cell electrophysiology to study the precise molecular mechanism by which Pin1 regulate hippocampal excitatory synapses. Specifically, the project focuses in understanding if Pin1 regulate surface AMPA receptors lateral diffusion properties7, identifying novel protein targets, discovering novel sites of posttranslational modification or changes in protein conformation, determining changes in spine morphology and excitatory synaptic strength.In the work of Dr. Jary Delgado in the laboratory of Dr. Daniel Choquet, he has identified the Postsynaptic Density Protein 95 (PSD-95) protein as a novel Pin1 binding partner. Pin1 preferentially binds to phosphorylated threonine 19 (T19) and serine 25 (S25), two sites known to regulate PSD-95 multimerization, ubiquitination and endocytosis of AMPA receptors, via its N-terminus WW domain. Furthermore, PSD-95 phosphorylation increases cis N-terminus phosphorylated PSD-95 of which Pin1 isomerises the prolyl bonds from cis to trans via its C-terminal isomerase domain. Binding of Pin1 to PSD-95, both in vitro and in vivo, prevents PP2A binding and dephosphorylation of T19. Given that T19 and S25 phosphorylation regulates PSD-95 multimerization, we tested whether Pin1 binding interferes with this process. As expected, downregulation of Pin1 increases the amount of multimeric PSD-95 as measured from CO-IP assays but paradoxically reduced the frequency of PSD-95 containing synapses along dendritic spines and increase the amount of overexpressed extrasynaptic PSD-95. This requires the constitutive activity of the ubiquitin-proteosome pathway which is also responsible for an observed increase in the mobile pool of PSD-95::EGFP, as measured by FRAP. Finally, we are currently testing the role of Pin1 on AMPAR mediated excitatory synaptic transmission and have found that Pin1 inhibition reduces the frequency but not the amplitude of mEPSCs while overexpression of the WW domain of Pin1 or an isomerase deficient mutant decrease the amplitude of the mEPSCs but increases the frequency of mEPSCs. Our results are the first to demonstrate the precise series of signaling events following T19 and S25 phosphorylation which in turn regulate PSD-95 life-time at excitatory synapses.1.1.2 A description of the work performed since the beginning of the project and the main results achieved so far.During the past year I have tested the role of two different proteins, PKMzeta and Pin1, in the regulation of excitatory synapses. I have assayed whether they regulate AMPAR surface diffusion, spine morphology, and the abundance of the postsynaptic density protein 95 (PSD-95). I have made significant progress on two of the three originally proposed specific aims and have several final figures which will be the main framework for an upcoming manuscript. I expect to be done with experiments on this first part of the project by the middle of next year. For all intents of the discussion, this proposal is mainly focused on the role of Pin1 in the regulation of excitatory synapses and not on the originally proposed idea of studying PKMzeta. To briefly summarize my results, I had tested whether overexpression of PKMzeta regulate AMPAR surface diffusion using antibody-coupled quantum dots experiments and found that it do not regulate the diffusional properties of GluR1 and GluR2 containing AMPA receptors. In contrast, inhibition of Pin1 strongly regulated the diffusional properties of GluR1 and GluR2 containing AMPA receptors, the shape of dendritic spines, AMPAR-mediated miniature excitatory postsynaptic potentials and the abundance of PSD-220.127.116.11 The expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far).We expect these findings will lead to better understanding of the molecular and signaling mechanisms regulating excitatory synapse formation, maintenance and synaptic plasticity. More specifically by regulating the conformation of serine/threonine phosphorylated proteins embedded in the postsynaptic density such as the Postsynaptic Density Protein 95 (PSD-95). Because Pin1 plays a key role in age dependent neurodegeneration8 these findings will help understand the precise ways by which Pin1 maintain healthy synapses. Specifically, Alzheimer’s disease a neurodegenerative disorder which triggers a time dependent loss of synaptic connections. Given that Pin1 regulates Amyloid Precursor Protein processing9, which is concentrated in the Alzheimer’s disease brain, understanding the precise molecular mechanisms by which Pin1 regulates excitatory synapses will undoubtedly lead to the development of better pharmacological targets which may ameliorate the devastating effects of this neurological disease.