Biology subjects

Dharmasri, P. A.

Publications and source records attributed to Dharmasri, P. A..

5 recordsLinked to original sources

bioRxiv2024

Loss of postsynaptic NMDARs drives nanoscale reorganization of Munc13-1 and PSD-95

Nanoscale protein organization within the active zone (AZ) and post-synaptic density (PSD) influences synaptic transmission. Nanoclusters of presynaptic Munc13-1 are associated with readily releasable pool size and neurotransmitter vesicle priming, while postsynaptic PSD-95 nanoclusters coordinate glutamate receptors across from release sites to control their opening probability. Nanocluster number, size, and protein density vary between synapse types and with development and plasticity, supporting a wide range of functional states at the synapse. Whether or how the receptors themselves control this critical architecture remains unclear. One prominent PSD molecular complex is the NMDA receptor (NMDAR). NMDARs coordinate several modes of signaling within synapses, giving them the potential to influence synaptic organization through direct protein interactions or through signaling. We found that loss of NMDARs results in larger synapses that contain smaller, denser, and more numerous PSD-95 nanoclusters. Intriguingly, NMDAR loss also generates retrograde reorganization of the active zone, resulting in denser, more numerous Munc13-1 nanoclusters, more of which are aligned with PSD-95 nanoclusters. Together, these changes to synaptic nanostructure predict stronger AMPA receptor-mediated transmission in the absence of NMDARs. Notably, while prolonged antagonism of NMDAR activity increases Munc13-1 density within nanoclusters, it does not fully recapitulate these trans-synaptic effects. Thus, our results confirm that NMDARs play an important role in maintaining pre- and postsynaptic nanostructure and suggest that both decreased NMDAR expression and suppressed NMDAR activity may exert distinct effects on synaptic function, yet through unique architectural mechanisms. Significance StatementSynaptic transmission is shaped by the trans-synaptic coordination of molecular ensembles required for neurotransmitter release and receptor retention, but how receptors themselves influence this critical architecture remains unclear. Using state-of-the-art super-resolution microscopy, we report that loss of NMDA receptors from excitatory synapses alters both pre- and postsynaptic nano-organizational features. Notably, pharmacological antagonism of NMDA receptors also alters presynaptic features, but without fully mimicking effects of the knockout. This suggests that both NMDA receptor activity and presence at the synapse exert retrograde influence on active zone organization. Because numerous disease and activity states decrease expression or function of NMDA receptors, our results suggest that distinct nanostructural states contribute to the unique functional status of synapses in these disorders.

neuroscience↗

bioRxiv2023

Trans-synaptic molecular context of NMDA receptor nanodomains

Tight coordination of the spatial relationships between protein complexes is required for cellular function. In neuronal synapses, many proteins responsible for neurotransmission organize into subsynaptic nanoclusters whose trans-cellular alignment modulates synaptic signal propagation. However, the spatial relationships between these proteins and NMDA receptors (NMDARs), which are required for learning and memory, remain undefined. Here, we mapped the relationship of key NMDAR subunits to reference proteins in the active zone and postsynaptic density using multiplexed super-resolution DNA-PAINT microscopy. GluN2A and GluN2B subunits formed nanoclusters with diverse configurations that, surprisingly, were not localized near presynaptic vesicle release sites marked by Munc13-1. Despite this, we found a subset of release sites was enriched with NMDARs, and modeling of glutamate release and receptor activation in measured synapses indicated this nanotopography promotes NMDAR activation. This subset of release sites was internally denser with Munc13-1, aligned with abundant PSD-95, and associated closely with specific NMDAR nanodomains. Further, NMDAR activation drove rapid reorganization of this release site/receptor relationship, suggesting a structural mechanism for tuning NMDAR-mediated synaptic transmission. This work reveals a new principle regulating NMDAR signaling and suggests that synaptic functional architecture depends on the assembly of and trans-cellular spatial relationships between multiprotein nanodomains.

neuroscience↗

bioRxiv2023

Molecular definition of distinct active zone protein machineries for Ca2+ channel clustering and synaptic vesicle priming

Action potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with CaV2 Ca2+ channels. The presumed model is that scaffolding proteins directly tether primed vesicles to CaV2s. We find that CaV2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, CaV2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins, but distinct interaction motifs independently execute these functions. In heterologous cells, Liprin- and RIM from co- assemblies that are separate from CaV2-organizing complexes upon co-transfection. At synapses, Liprin-1-4 knockout impairs vesicle priming, but not CaV2 clustering. The cell adhesion protein PTP{sigma} recruits Liprin-, RIM and Munc13 into priming complexes without co- clustering of CaV2s. We conclude that active zones consist of distinct complexes to organize CaV2s and vesicle priming, and Liprin- and PTP{sigma} specifically support priming site assembly.

neuroscience↗

bioRxiv2023

Distinct SAP102 and PSD-95 nano-organization defines multiple types of synaptic scaffold protein domains at single synapses

The MAGUK family of scaffold proteins plays a central role in maintaining and modulating synaptic signaling, providing a framework to retain and position receptors, signaling molecules, and other synaptic components. Of these scaffold proteins, SAP102 and PSD-95 are essential for synaptic function at distinct developmental timepoints and perform overlapping as well as unique roles. While their similar structures allow for common binding partners, SAP102 is expressed earlier in synapse development and is required for synaptogenesis, whereas PSD-95 expression peaks later in development and is associated with synapse maturation. PSD-95 and other key synaptic proteins organize into subsynaptic nanodomains that have a significant impact on synaptic transmission, but the nanoscale organization of SAP102 is unknown. How SAP102 is organized within the synapse, and how it relates spatially to PSD-95 on a nanometer scale, could impact how SAP102 clusters synaptic proteins and underlie its ability to perform its unique functions. Here we used DNA-PAINT super-resolution microscopy to measure SAP102 nano-organization and its spatial relationship to PSD-95 at individual synapses. We found that like PSD-95, SAP102 accumulates in high-density subsynaptic nanoclusters. However, SAP102 nanoclusters were smaller and denser than PSD-95 nanoclusters across development. Additionally, only a subset of SAP102 nanoclusters co-organized with PSD-95, revealing that within individual synapses there are nanodomains that contain either one or both proteins. This organization into both shared and distinct subsynaptic nanodomains may underlie the ability of SAP102 and PSD-95 to perform both common and unique synaptic functions. Significance statementSAP102 and PSD-95 are two key members of the MAGUK family of synaptic scaffold proteins that are critical for synapse development, maintenance, and modification during plasticity. Because PSD-95 has a highly complex subsynaptic nanostructure that impacts synaptic function, we asked if SAP102 is similarly organized into nanoclusters at individual synapses and how it relates to PSD-95 within synapses. We found that SAP102 forms subsynaptic nanoclusters with unique properties, and that within individual synapses proteins both concentrate into overlapping nanodomains, as well as form MAGUK-specific nanodomains. This demonstrates that organization of synaptic proteins into nanoclusters is likely to be maintained within the family of MAGUK proteins and reveals potential mechanism for specializing functions within individual synapses based on scaffold protein nanodomains.

neuroscience↗

bioRxiv2023

Differential nanoscale organization of excitatory synapses onto excitatory vs inhibitory neurons

A key feature of excitatory synapses is the existence of subsynaptic protein nanoclusters whose precise alignment across the cleft in a trans-synaptic nanocolumn influences the strength of synaptic transmission. However, whether nanocolumn properties vary between excitatory synapses functioning in different cellular contexts is unknown. We used a combination of confocal and DNA-PAINT super-resolution microscopy to directly compare the organization of shared scaffold proteins at two important excitatory synapses - those forming onto excitatory principal neurons (Ex[->]Ex synapses) and those forming onto parvalbumin-expressing interneurons (Ex[->]PV synapses). As in Ex[->]Ex synapses, we find that in Ex[->]PV synapses presynaptic Munc13-1 and postsynaptic PSD-95 both form nanoclusters that demonstrate alignment, underscoring synaptic nanostructure and the trans-synaptic nanocolumn as conserved organizational principles of excitatory synapses. Despite the general conservation of these features, we observed specific differences in the characteristics of pre-and postsynaptic Ex[->]PV nanostructure. Ex[->]PV synapses contained larger PSDs with fewer PSD-95 NCs when accounting for size than Ex[->]Ex synapses. Furthermore, the PSD-95 NCs were larger and denser. The identity of the postsynaptic cell also had a retrograde impact on Munc13-1 organization, as Ex[->]PV synapses hosted larger Munc13-1 puncta that contained less dense but larger and more numerous Munc13-1 NCs. Moreover, we measured the spatial variability of trans-synaptic alignment in these synapse types, revealing protein alignment in Ex[->]PV synapses over a distinct range of distances compared to Ex[->]Ex synapses. We conclude that while general principles of nanostructure and alignment are shared, cell-specific elements of nanodomain organization likely contribute to functional diversity of excitatory synapses. Understanding the rules of synapse nanodomain assembly, which themselves are cell-type specific, will be essential for illuminating brain network dynamics.

neuroscience↗