Synaptic transmission is maintained by a delicate, subsynaptic molecular architecture, and even mild alterations in synapse structure drive functional changes during experience-dependent plasticity and pathological disorder1,2. Key to this architecture is how the distribution of presynaptic vesicle fusion sites corresponds to the position of receptors in the postsynaptic density. However, despite long recognition that this spatial relationship modulates synaptic strength3, it has not been precisely described, due in part to the limited resolution of light microscopy. Using localization microscopy, we report here that key proteins mediating vesicle priming and fusion are mutually co-enriched within nanometer-scaled subregions of the presynaptic active zone. Through development of a new method to map vesicle fusion positions within single synapses, we found that action potential evoked fusion was guided by this protein gradient and occurred preferentially in confined areas with higher local density of RIM within the active zones. These presynaptic RIM nanoclusters closely aligned with concentrated postsynaptic receptors and scaffolding proteins4–6, suggesting a transsynaptic molecular “nanocolumn.” Thus, we propose that the nanoarchitecture of the active zone directs action potential evoked vesicle fusion to occur preferentially at sites directly opposing postsynaptic receptor-scaffold ensembles. Remarkably, NMDA receptor activation triggered distinct phases of plasticity in which postsynaptic reorganization was followed by transsynaptic nanoscale realignment. This architecture thus suggests a simple organizational principle of CNS synapses to maintain and modulate synaptic efficiency.
Mechanisms regulating lateral diffusion and positioning of glutamate receptors within the postsynaptic density (PSD) determine excitatory synaptic strength. Scaffold proteins in the PSD are abundant receptor binding partners, yet electron microscopy suggests that the PSD is highly crowded, potentially restricting the diffusion of receptors regardless of binding. However, the contribution of macromolecular crowding to receptor retention remains poorly understood. We combined experimental and computational approaches to test the effect of synaptic crowding on receptor movement and positioning in Sprague Dawley rat hippocampal neurons. We modeled AMPA receptor diffusion insynapseswherethedistributionofscaffoldproteinswasdeterminedfromphotoactivatedlocalizationmicroscopyexperiments,andreceptorscaffold association and dissociation rates were adjusted to fit single-molecule tracking and fluorescence recovery measurements. Simulations predicted that variation of receptor size strongly influences the fractional synaptic area the receptor may traverse, and the proportion that may exchange in and out of the synapse. To test the model experimentally, we designed a set of novel transmembrane (TM) probes. A single-pass TM protein with one PDZ binding motif concentrated in the synapse as do AMPARs yet was more mobile there than the much larger AMPAR. Furthermore, either the single binding motif or an increase in cytoplasmic bulk through addition of a single GFP slowed synaptic movement of a small TM protein. These results suggest that both crowding and binding limit escape of AMPARs from the synapse. Moreover, tight protein packing within the PSD may modulate the synaptic dwell time of many TM proteins important for synaptic function.
Osteoarthritis, a highly prevalent degenerative joint disorder, is characterized by joint pain and disability. Available treatments fail to modify osteoarthritis progression and decrease joint pain effectively. Here, we show that intermittent parathyroid hormone (iPTH) attenuates osteoarthritis pain by inhibiting subchondral sensory innervation, subchondral bone deterioration, and articular cartilage degeneration in a destabilized medial meniscus (DMM) mouse model. We found that subchondral sensory innervation for osteoarthritis pain was significantly decreased in PTH-treated DMM mice compared with vehicle-treated DMM mice. In parallel, deterioration of subchondral bone microarchitecture in DMM mice was attenuated by iPTH treatment. Increased level of prostaglandin E2 in subchondral bone of DMM mice was reduced by iPTH treatment. Furthermore, uncoupled subchondral bone remodeling caused by increased transforming growth factor β signaling was regulated by PTH-induced endocytosis of the PTH type 1 receptor–transforming growth factor β type 2 receptor complex. Notably, iPTH improved subchondral bone microarchitecture, and decreased level of prostaglandin E2 and sensory innervation of subchondral bone in DMM mice by acting specifically through PTH type 1 receptor in Nestin+ mesenchymal stromal cells. Thus, iPTH could be a potential disease-modifying therapy for osteoarthritis.
Postsynaptic transmembrane proteins are critical elements of synapses, mediating trans-cellular contact, sensitivity to neurotransmitters and other signaling molecules, and flux of Ca and other ions. Positioning and mobility of each member of this large class of proteins is critical to their individual function at the synapse. One critical example is that the position of glutamate receptors within the postsynaptic density (PSD) strongly modulates their function by aligning or misaligning them with sites of presynaptic vesicle fusion. In addition, the regulated ability of receptors to move in or out of the synapse is critical for activity-dependent plasticity. However, factors that control receptor mobility within the boundaries of the synapse are not well understood. Notably, PSD scaffold molecules accumulate in domains much smaller than the synapse. Within these nanodomains, the density of proteins is considerably higher than that of the synapse as a whole, so high that steric hindrance is expected to reduce receptor mobility substantially. However, while numerical modeling has demonstrated several features of how the varying protein density across the face of a single PSD may modulate receptor motion, there is little experimental information about the extent of this influence. To address this critical aspect of synaptic organizational dynamics, we performed single-molecule tracking of transmembrane proteins using universal point accumulation-for-imaging-in-nanoscale-topography (uPAINT) over PSDs whose internal structure was simultaneously resolved using photoactivated localization microscopy (PALM). The results provide important experimental confirmation that PSD scaffold protein density strongly influences the mobility of transmembrane proteins. A protein with a cytosolic domain that does not bind PSD-95 was still slowed in regions of high PSD-95 density, suggesting that crowding by scaffold molecules and perhaps other proteins is sufficient to stabilize receptors even in the absence of binding. Because numerous proteins thought to be involved in establishing PSD structure are linked to disorders including autism and depression, this motivates further exploration of how PSD nanostructure is created. The combined application PALM and uPAINT should be invaluable for distinguishing the interactions of mobile proteins with their nano-environment both in synapses and other cellular compartments.
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