AMPAR and spine dysfunction or dysregulation underlies many CNS diseases including depression, autism, PTSD, epilepsy, and stroke-induced neuronal damage. Precise postsynaptic localization of AMPARs is critical for fast synaptic transmission. It depends on PSD-95 and its interaction with auxiliary AMPAR subunits called TARPs. Despite its central role in targeting AMPARs, it is unknown how PSD-95 itself is anchored at the postsynapse. Our preliminary data suggest that A) ?-actinin binds to the first 13 residues of the N-terminus of PSD-95;B) knock-down (KD) of ?-actinin reduces postsynaptic PSD-95 content and mEPSCs, the latter phenocopying KD of PSD-95;C) mutating either Lys10 or Lys11 to Glu (K10E, K11E) specifically impairs PSD- 95 binding to ?-actinin and postsynaptic targeting of PSD-95 and of AMPARs;D) peptide PSD95(1-13) displaces PSD-95 from ?-actinin;E) injection of PSD95(1-13) decreases mEPSC amplitude. We hypothesize that ?-actinin is critical for postsynaptic anchoring of PSD-95 and thereby AMPAR-TARP complexes. Proving this hypothesis will fundamentally advance our understanding of postsynaptic AMPAR localization. A), B), and D) are final data.
Aims 1 and 2 will further scrutinize C) and E), i.e., whether mutating K10E and K11E or injecting PSD95(1-13) affect synaptic PSD-95 and AMPAR taregting using fluorescence microscopy and mEPSC. NMR structural analysis will identify residues in ?-actinin that are important for PSD-95 binding. KD of endogenous ?-actinin and replacement with either WT or mutant ?-actinin will show whether mutant ?-actinin is not able to rescue the KD effect on PSD-95, in contrast to our rescue with WT ?-actinin.
Aim 3 is to test whether NMDA-induced Ca2+ influx displaces PSD-95 from ?-actinin and thereby from postsynaptic sites along with AMPARs via calmodulin (CaM). We found that Ca2+/CaM binds to the N-terminus of PSD-95 to dislodge ?-actinin. Our structural NMR analysis of a complex between Ca2+/CaM and the first 71 aa of PSD-95 identified Y12 in PSD-95 as critical for CaM binding. Mutating Y12 to Glu (Y12E) prevents Ca2+/CaM binding without affecting ?-actinin binding or postsynaptic localization of PSD-95. NMDA-induced Ca2+ influx displaces a portion of WT but not Y12E PSD-95 from spines. In fact, Y12E exhibits a large increase rather than decrease in spines upon Ca2+ influx. We will test whether this mutation and other manipulations unmask a mechanism that leads to postsynaptic accumulation of PSD-95 and AMPARs rather than their decrease. Such a decrease is usually seen following 5 min NMDA treatment and is referred to as chemical LTD. This exciting new direction will elucidate how Ca2+ influx can cause LTD rather than LTP.

Public Health Relevance

This project is to investigate the role of a-actinin in anchoring glutamate receptors at postsynaptic sites. Aberrant functioning and localization of glutamate receptors are implicated in mental and neurological diseases such as schizophrenia, autism, depression, posttraumatic stress disorder, epipelsy, stroke-induced neuronal damage, and Alzheimer's disease. This work will define fundamental molecular mechanisms of regulating glutamate receptor localization, thereby providing a foundation for advancing our understanding of this important process under physiological as well as pathological conditions.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
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Synapses, Cytoskeleton and Trafficking Study Section (SYN)
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Asanuma, Chiiko
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University of California Davis
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Matt, Lucas; Kirk, Lyndsey M; Chenaux, George et al. (2018) SynDIG4/Prrt1 Is Required for Excitatory Synapse Development and Plasticity Underlying Cognitive Function. Cell Rep 22:2246-2253
Chowdhury, Dhrubajyoti; Turner, Matthew; Patriarchi, Tommaso et al. (2018) Ca2+/calmodulin binding to PSD-95 mediates homeostatic synaptic scaling down. EMBO J 37:122-138
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Matt, Lucas; Kim, Karam; Hergarden, Anne C et al. (2018) ?-Actinin Anchors PSD-95 at Postsynaptic Sites. Neuron 97:1094-1109.e9
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Tseng, Pang-Yen; Henderson, Peter B; Hergarden, Anne C et al. (2017) ?-Actinin Promotes Surface Localization and Current Density of the Ca2+ Channel CaV1.2 by Binding to the IQ Region of the ?1 Subunit. Biochemistry 56:3669-3681

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