Dynamic Structural Properties of Synapses

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications & trials

Abstract

Progress Summary: The postsynaptic density (PSD) at excitatory glutamatergic synapses is crucial for memory, information processing, and storage. To map the PSD's molecular organization, we freeze-substitute hippocampal cultures and examine them in plastic-embedded sections using EM tomography. This approach reveals individual protein complexes within the PSD. Early tomography work showed that the PSD core consists of membrane-associated, vertically oriented filaments, such as PSD-95. This finding provided insights into the overall organization of the PSD. For example, scaffolding proteins like PSD-95 have multiple common binding sites along their length, which imposes order on other PSD proteins, including glutamate receptors, and provides a structural plan for the PSD core. Our ongoing research and collaborations aim to investigate the organization of specific synaptic proteins within the PSD. We collaborated with the Rumbaugh Lab to study SynGAP's structural role in the PSD. SynGAP negatively regulates the binding of the glutamate AMPA receptor to the PDZ domain of PSD-95. Using immuno-electron microscopy (immunoEM), we mapped the location, orientation, and conformation of SynGAP in the PSD, including a SynGAP mutant that abolishes PDZ domain binding to PSD-95. We are obtaining EM tomography data of immunogold-labeled SynGAP to develop a structural model of how SynGAP regulates synaptic excitability. We are identifying NMDA receptors (NMDARs) in the PSD using EM tomography on intact hippocampal synapses with a CRISPR-Cas9 construct from the Nicoll Lab. This construct eliminates the GluN1 subunit, effectively removing NMDARs. We created 3D reconstructions of the PSDs using dark-field scanning EM tomography, comparing structures in wild-type and knockout samples at different time points. This comparison allows us to identify NMDARs structurally and characterize their organization and interactions with other molecules. Additionally, we are using FIB-SEM to image AMPA receptor distributions in resting and activated synapses. Membrane-associated AKAPs interact with PSD-95 MAGUKs and anchor kinases like PKA and PKC, essential for synaptic plasticity. We are extending our study with M. Dell'Acqua's lab to investigate the conformations and distribution of AKAPs in the hippocampal synapse, focusing on their role in organizing PKA and PKC in inhibitory synapses. Using dark-field STEM tomography of rodent synapses immunogold-labeled for endogenous PSD-95, we discovered that PSD-95 is involved in presynaptic vesicle tethering, priming, and fusing and is widely distributed across the PSD. We propose a new structural model where transsynaptic assembly involving PSD-95 underlies synaptic transmission and plasticity. Additionally, EM tomography has revealed new images of vesicle fusion and trafficking events at presynaptic terminals, providing insights into vesicle exocytosis in synapses. This work was presented at the 2024 “Molecular and Cell Biology of the Neuromuscular System “, 2024 SFN meeting and 2025 Gordon conference on “Excitatory Synapses and Brain Function” is being prepared for publication. We developed two new methods to identify proteins in intact synapses: APEX2 tagging and nanobody nanogold labeling, which were used alongside EM tomography. These methods complement our previous use of immunogold labeling, which was successful for PSD-95 but problematic due to large antibody complexes. Using the APEX2 genetic tag, we visualize CaMKII holoenzymes. The CaMKII-APEX2 construct, combined with DAB and peroxide, reveals individual CaMKII holoenzymes. We are studying CaMKII distribution in the spine and membrane under basal and high potassium (high K+) stimulated conditions using EM tomography. Size analysis shows that in the basal state, CaMKII is mostly in inactivated configurations due to prevalent phosphatases. In contrast, high K+ stimulation significantly increases DAB-stained proteins at the PSD, indicating active CaMKII. These CaMKII holoenzymes cluster prominently at specific sites likely associated with NMDAR complexes. Even in the basal state, CaMKII is found within the core PSD structure, suggesting it is a critical structural element, likely complex with NMDARs and AMPARs. We are analyzing over 70 dual-axis STEM tomograms to investigate these findings further. Additionally, we are developing a method to combine immunogold labeling of glutamate receptors with APEX2-labeled CaMKII to elucidate their organizational correlation within the PSD. Initial results have been presented at two SFN nano symposiums, and we are preparing a publication. We are also expanding the APEX2 approach by creating a CRISPR knockin APEX2-tagged CaMKII construct, allowing labeling of endogenous CaMKII. This method has successfully identified endogenous APEX2-labeled CaMKII in synapses of neuronal cultures expressing the new construct. We plan to apply APEX2 tagging to CaMKII mutants to explore their role in LTP and extend this approach to other proteins like Shank and Homer. We are using nanobodies with EM tomography to swiftly and directly identify PSD proteins in spines. We analyzed nanobody-labeled tomograms in intact synapses for PSD-95, CaMKII, and Homer1b. Nanobody labeling has identified these proteins in spine synapses, providing compelling evidence for their labeling with small gold particles. We are working on completing the analysis on Homer1b and hope to develop a new structural model involving Homer in the PSD. We also plan to use this approach to label and identify Shank. In collaboration with Shahid Khan, we examine the higher-order association of CaMKII, stabilized with a β-hydroxybutyrate analog, with isolated NMDAR NR2B subunits and PSDs. While β-hydroxybutyrate analogs are known as neuroprotective agents, they have been recently established to bind specifically to the hub of the CaMKII isoform. This hub-stabilized CaMKII offers a novel opportunity to explore the formation of CaMKII-mediated condensates with NMDA NR2B subunits and PSDs. We are using two-color fluorescence and cryo-EM for this study. Additionally, Khan used the NIH Biowulf supercomputer to show that the phylogenetics of the CaMKII hub tracks the evolution of memory with high fidelity. He found that the CaMKII kinase domain, calmodulin, autophosphorylation, and substrate (e.g., NR2B) binding sites are remarkably well-conserved across species. Since T. adhaerens do not have a nervous system, this suggests that CaMKII architecture initially developed for a primitive sensory or stress response and was later repurposed for memory storage. Experimental validation will require proof of T. adhaerens CaMKII holoenzyme formation, calcium-calmodulin-triggered autophosphorylation, and NR2B binding affinity.