Dynamic Structural Properties of Synapses

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications, trials & patents

Abstract

The postsynaptic density (PSD) at excitatory glutamatergic synapses is a large molecular machine that is known to be a key site of memory, information processing, and storage. To map the molecular organization of the PSD, we freeze-substitute hippocampal cultures and examine them in plastic embedded sections by EM tomography. This reveals individual protein complexes within the PSD. Our early tomography work revealed that the core of the PSD is an array of membrane-associated, vertically oriented filaments like PSD-95. This finding provided insight into the overall organization of the PSD. For instance, scaffolding proteins like PSD-95 have multiple common binding sites arrayed along their length, such that, regular arrays of vertically oriented PSD-95 filaments impose order on other PSD proteins, including the glutamate receptors, and provide an overall plan for the core structure of the PSD. The projects outlined below use and refine our fundamental insight into the organization of synaptic structures to explore the dynamics of molecular changes in the PSD and understand how these molecules contribute to synaptic function. We have several lines of ongoing research and collaboration to investigate specific synaptic proteins. Recently, we collaborated with Rumbaugh Lab to characterize the structural role of SynGAP, which negatively regulates the glutamate AMPA receptor binding to the PDZ domain of PSD-95 at the PSD. We have used immunoEM to map the location, orientation, and conformation of SynGAP at the PSD to arrive at a structural model of how SynGAP might regulate and control synaptic excitability. In collaboration with the Roger Nicoll Lab, we are studying the effects of overexpressing constitutively activated CaMKII on synaptic structure and function. To ameliorate potential artifacts due to overexpressing of CaMKII, we are also using a newly developed CaMKII CRISPR knock-in construct which allows expression and localization of endogenous CaMKIIs in neurons. Electrophysiology measurements show that activated CaMKII expression enhances synaptic transmission, and we plan to analyze changes in spine sizes and PSD structure, using serial section EM or thick section STEM tomography. We are finishing up the work directly identifying NMDARs in the PSD in intact hippocampal synapses by using CRISPR-Cas9 construct developed in the Nicoll Lab. The knockout eliminates the required GluN1 subunit of NMDARs. We made 3Dreconstructions of the resulting PSDs with dark field scanning EM tomography. As result, we now have evidence that individual NMDARs and AMPARs can be identified by EM tomography, and their organization and connections with other molecules can be delineated. We are in the process of finishing up this work and preparing it for publication. Also, we just published a major study with M. DellAcqua's lab at the University of Colorado on the conformations and distribution of Anchoring Proteins (AKAPs) in the hippocampal synapse. In this work, we used immunoEM on thick sections and visualized them using STEM tomography. The results showed palmitoylation effects on AKAP150/79 membrane organization, trafficking, and mobility. Membrane-associated AKAPs are known to interact with PSD-95 MAGUKs and anchor several classes of kinases (PKA and PKC) important for synaptic plasticity (LTP and LTD). This work demonstrates that there is a conformational change in AKAPs in the PSD, different than that at the extrasynaptic membrane, and this distinction may have important functional implications in understanding the role of AKAPs in regulating AMPARs at the PSDs. We also found extensive AKAP association with recycling endosomes and that depalmitolylation appeared to diminish such association. This project opened an exciting new front in imaging AMPAR trafficking. EM tomography has allowed the creation of 3D reconstructions to delineate the organization of subsynaptic organelles, key synaptic proteins, and macromolecular complexes at synapses. Reconstructions provide the size, shape, and location of structures at 2-4 nm resolution but cannot guarantee unambiguous molecular identification of the individual structures. While we had success using immunogold to label endogenous and overexpressed GFP-tagged PSD-95, the large antibody complexes that also manifest as filamentous structures in tomograms confound the identification of the target PSD proteins. Now we have a major technical breakthrough with APEX2 and nanobody labeling to solve this problem. In one localization project, we are using the genetic tag APEX2 to localize CaMKII. The CaMKII-APEX2 construct in the presence of DAB and peroxide has revealed individual CaMKIIs. We are studying CaMKII in the spine and membrane in basal and high K stimulated conditions by EM tomography, and we are in the process of finishing up the work and preparing a publication. In the meantime, we are expanding the APEX2 work to Shank and Homer. Furthermore, another localization project uses an advance in nanobody labeling. In this parallel method, we developed a method to use nanobodies with EM tomography to directly identify PSD proteins in spines. This is a swift method, and we are expecting exciting new results in the coming years on a slew of synaptic molecules. Recently, a major issue has cropped up in the field regarding PSD-95. Several super-resolution light microscopy studies have suggested that PSD-95 forms 100 nm subsynaptic nanoclusters at the PSD. This is significantly smaller than the average size of a PSD, yet we considered PSD-95 to be uniformly distributed. Currently, we are re-examining the endogenous PSD-95 distribution at the PSD with thick section tomography to further study PSD-95 distribution and clustering at the PSD. We are also further analyzing the distribution of all vertical filaments in tomograms using the methods outlined above to see if any clustering of the vertical filaments ever exists within the PSD and if we can reconcile the super-resolution findings with immuno-labeling and tomographic EM. Outside our work in mammalian neurons, an ongoing collaboration with Carolyn Smith in the NINDS Light Microscopy Facility and Adriano Senatore (University of Toronto Mississauga) sheds light on the evolution of cell types and pre-neural regulations in a primitive animal, Trichoplax. Although lacking muscles, nerves, and synapses, Trichoplax demonstrates different types of behaviors indicative of neural function. We identified a cell that senses gravity and described the consequence of stages, which occurs during the differentiation of this cell. We also described a population of cells functioning as macrophages. Our collaborative effort provided evidence that Trichoplax can sense the pH of ambient water and demonstrates avoidance of low pH (acidic) regions. However, the regulatory mechanisms as well as the ways Trichoplax cells communicate with each other are not yet clear. Our previous results and elsewhere obtained data showed that this organism utilizes neuropeptide signaling pathways dependent on many of the same proteins found at synapses in higher animals. In our next steps, we will characterize different types of secretory cells in Trichoplax, which presumably regulate and integrate functional activities of the cells in the nerveless animal. Knowing exactly how these unconventional, nonsynaptic systems function to control behaviors is expected to provide previously overlooked information on non-synaptic signaling mechanisms in mammalian brains.