Antonio Sanz-clemente - US grants

DESCRIPTION (provided by applicant): N-methyl-D-aspartate receptors (NMDARs) play a central role in development, learning, memory, and in many neurological disorders. In cerebral cortex NMDARs are mainly composed of two GluN1 and two GluN2A or GluN2B subunits. Many functional properties of NMDARs are determined by GluN2 subunits, so they are subjected to strict control mechanisms. My long-term research objective is to understand the role that glutamate receptors dysregulation (in particular, NMDARs) plays in the development of Alzheimer's disease (AD) and other age-related neurodegenerative diseases. Therefore, the goal of this K99/R00 award proposal is to define the precise molecular mechanisms that regulate synaptic GluN2 composition during the switch from GluN2B to GluN2A that occurs during synaptic maturation and to determine if they are involved in synaptic dysfunction in AD. Specifically, the role of casein kinase 2 (CK2) in these processes will be analyzed, since I have previously demonstrated that CK2 regulates GluN2 synaptic composition by phosphorylating the PDZ binding domain of GluN2B (S1480) and that CK2 activity is required for the GluN2 subunit switch. Although synaptic CK2 has been shown to be important, how synaptic activity regulates this kinase remains obscure, since CK2 is considered a constitutively active kinase and it is not regulated by calcium. Specific Aim 1 will test the hypothesis that synaptic recruitment of CK2 by CaMKII is a key step for GluN2B S1480 phosphorylation, with CaMKII acting as a scaffolding protein to link GluN2B and CK2 after NMDAR activation. Therefore, GluN2B S1480 phosphorylation will be determined after disruption of the GluN2B/CaMKII/CK2 complex, using biochemistry and immunofluorescence microscopy. My central hypothesis is that the GluN2 subunit switch is a process with two sequential and coupled steps, in which the synaptic removal of GluN2B by CK2 phosphorylation is required to allow synaptic incorporation of GluN2A. This will be tested using biochemical and electrophysiological approaches in the Specific Aim 2, analyzing the synaptic GluN2 composition after the replacement of endogenous GluN2B by mutated GluN2B with defective S1480 phosphorylation (GluN2B E1479Q). Several molecular genetic approaches will be used for this replacement including lentivirus infection and the generation of a genetically-altered mouse line expressing GluN2B E1479Q. Recent reports support a role for extrasynaptic NMDARs overactivation in AD. Therefore, using the data and tools generated in my two previous Aims I will analyze if Abeta oligomers, main neurotoxins in AD, leads to a redistribution in GluN2B subunit (from synaptic to extrasynaptic sites) via aberrant CK2 overactivation (Specific Aim 3). The successful completion of this proposal will have a significant positive impact by elucidating the mechanisms regulating GluN2 subunit composition during development and identifying a potential new pharmacological target in AD. )

Excitotoxicity is defined as the deterioration of neuronal function/structure caused by excessive glutamatergic stimulation. It is a shared major pathological hallmark in many neurodegenerative diseases (ND), including Alzheimer?s disease (AD), Huntington?s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). Excitotoxicity is mostly mediated by the activation of the NMDA-type of glutamate receptors (NMDARs). However, the NMDAR function is indispensable for normal neuronal function. This conundrum is explained by the fact that NMDARs are segregated in two populations: synaptic (sNMDARs) and extrasynaptic (exNMDARs). While sNMDARs are linked to pro-survival signaling, over-activation of exNMDARs triggers excitotoxicity. Therefore, exNMDAR are obvious pharmacological targets in a broad range of ND and, in fact, blocking NMDAR activity strongly ameliorates cognitive defects in AD and HD mouse models. However, selective inhibition of exNMDARs is challenging, and the vast majority of NMDAR antagonists have failed in clinic due to side effects mediated by sNMDAR blockade. We propose to test a novel therapeutic strategy based on the fact that s- and exNMDARs are not independent populations. On the contrary, s- and exNMDARs pools are physiologically connected via lateral diffusion. We hypothesize that shifting the s/exNMDAR balance towards synaptic expression would be beneficial two-folds (i) promoting survival cascades (sNMDAR-mediated) and (ii) decreasing pro-death signaling (exNMDAR-mediated). We are ideally suited to test this strategy because we have previously identified several of the mechanisms controlling s/exNMDAR balance. Those include different protein interactions with the GluN2B-subunit of NMDARs and a particular phosphorylation on GluN2B (at S1480) that promotes sNMDAR clearance and receptor stabilization at extrasynaptic sites. The goal of this proposal is to validate the proof-of-principle that reducing excitotoxicity by preventing sNMDAR clearance and/or promoting exNMDAR reinsertion into synaptic sites is an effective therapeutic strategy in ND. In Aim 1, we will evaluate novel molecular tools to modulate s/exNMDAR balance in culture and in vivo, including (i) small interfering peptides (sIPs) and (ii) pharmacology to modulate GluN2B phosphorylation. Our study includes the repurposing of an anti-tumoral drug currently in phase 1/2 of clinical trial. Also, we will use proteomics to compare the posttranslational modification profile of s- vs. exNMDARs, aiming to identify novel mechanisms regulating the balance. In Aim 2, we will evaluate the suitability of this strategy as a common therapeutic strategy in ND. First, we will test the efficacy of our tools in ameliorating excitotoxicity-mediated pathological outcomes in several models of AD, both in culture and in vivo. Finally, we will use our strategy in primary cultures from models of HD (associated by excitotoxicity) and Parkinson?s disease (excitotoxicity is not a primary pathomechanism). If successful, this proposal, based on reducing excitotoxicity by regulating NMDAR trafficking but not by inhibiting NMDAR function, will have a groundbreaking translational impact on the identification of innovative therapeutics for a wide range of ND.