Shank-cortactin interactions control actin dynamics to maintain flexibility of neuronal spines and synapses.

The family of Shank scaffolding molecules (comprising Shank1, 2 and 3) are core components of the postsynaptic density (PSD) in neuronal synapses. Shanks link surface receptors to other scaffolding molecules within the PSD, as well as to the actin cytoskeleton. However, determining the function of Shank proteins in neurons has been complicated because the different Shank isoforms share a very high degree of sequence and domain homology. Therefore, to control Shank content while minimizing potential compensatory effects, a miRNA-based knockdown strategy was developed to reduce the expression of all synaptically targeted Shank isoforms simultaneously in rat hippocampal neurons. Using this approach, a strong (>75%) reduction in total Shank protein levels was achieved at individual dendritic spines, prompting an approximately 40% decrease in mushroom spine density. Furthermore, Shank knockdown reduced spine actin levels and increased sensitivity to the actin depolymerizing agent Latrunculin A. A SHANK2 mutant lacking the proline-rich cortactin-binding motif (SHANK2-ΔPRO) was unable to rescue these defects. Furthermore, Shank knockdown reduced cortactin levels in spines and increased the mobility of spine cortactin as measured by single-molecule tracking photoactivated localization microscopy, suggesting that Shank proteins recruit and stabilize cortactin at the synapse. Furthermore, it was found that Shank knockdown significantly reduced spontaneous remodelling of synapse morphology that could not be rescued by the SHANK2-ΔPRO mutant. It was concluded that Shank proteins are key intermediates between the synapse and the spine interior that, via cortactin, permit the actin cytoskeleton to dynamically regulate synapse morphology and function.

Subsynaptic AMPA receptor distribution is acutely regulated by actin-driven reorganization of the postsynaptic density.

AMPA receptors (AMPARs) mediate synaptic transmission and plasticity during learning, development, and disease. Mechanisms determining subsynaptic receptor position are poorly understood but are key determinants of quantal size. We used a series of live-cell, high-resolution imaging approaches to measure protein organization within single postsynaptic densities in rat hippocampal neurons. By photobleaching receptors in synapse subdomains, we found that most AMPARs do not freely diffuse within the synapse, indicating they are embedded in a matrix that determines their subsynaptic position. However, time lapse analysis revealed that synaptic AMPARs are continuously repositioned in concert with plasticity of this scaffold matrix rather than simply by free diffusion. Using a fluorescence correlation analysis, we found that across the lateral extent of single PSDs, component proteins were differentially distributed, and this distribution was continually adjusted by actin treadmilling. The C-terminal PDZ ligand of GluA1 did not regulate its mobility or distribution in the synapse. However, glutamate receptor activation promoted subsynaptic mobility. Strikingly, subsynaptic immobility of both AMPARs and scaffold molecules remained essentially intact even after loss of actin filaments. We conclude that receptors are actively repositioned at the synapse by treadmilling of the actin cytoskeleton, an influence which is transmitted only indirectly to receptors via the pliable and surprisingly dynamic internal structure of the PSD.

Lateral organization of the postsynaptic density.

Fast excitatory synaptic transmission is mediated by AMPA-type glutamate receptors (AMPARs). It is widely accepted that the number of AMPARs in the postsynaptic density (PSD) critically determines the efficiency of synaptic transmission, but an unappreciated aspect of synapse organization is the lateral positioning of AMPARs within the PSD, that is, their distribution across the face of a single synapse. Receptor lateral positioning is important in a number of processes, most notably because alignment with presynaptic release sites heavily influences the probability of receptor activation. In this review, we summarize current understanding of the mechanisms that dynamically control the subsynaptic positioning of AMPARs. This field is still at early stages, but the recent wave of developments in super-resolution microscopy, synapse tomography, and computational modeling now enable the study of lateral protein distribution and dynamics within the nanometer-scale boundaries of the PSD. We discuss data available measuring the lateral distribution of glutamate receptors and scaffold proteins within the PSD, and discuss potential mechanisms that might give rise to these patterns. Elucidating the mechanisms that underlie the lateral organization of the PSD will be critical to improve our understanding of synaptic processes whose disruption may be unexpectedly important in neurological disorders. This article is part of a Special Issue entitled Membrane Trafficking and Cytoskeletal Dynamics in 'Neuronal Function'.

Structural plasticity with preserved topology in the postsynaptic protein network.

The size, shape, and molecular arrangement of the postsynaptic density (PSD) determine the function of excitatory synapses in the brain. Here, we directly measured the internal dynamics of scaffold proteins within single living PSDs, focusing on the principal scaffold protein PSD-95. We found that individual PSDs undergo rapid, continuous changes in morphology driven by the actin cytoskeleton and regulated by synaptic activity. This structural plasticity is accompanied by rapid fluctuations in internal scaffold density over submicron distances. Using targeted photobleaching and photoactivation of PSD subregions, we show that PSD-95 is nearly immobile within the PSD, and PSD subdomains can be maintained over long periods. We propose a flexible matrix model of the PSD based on stable molecular positioning of PSD-95 scaffolds.

Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.

Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.

A network of networks: cytoskeletal control of compartmentalized function within dendritic spines.

Almost 30 years ago, actin was identified as the major cytoskeletal component of dendritic spines. Since then, its role in the remarkable dynamics of spine morphology have been detailed with live-cell views establishing that spine shape dynamics are an important requirement for synaptogenesis and synaptic plasticity. However, the actin cytoskeleton is critical to numerous and varied processes within the spine which contribute to the maintenance and plasticity of synaptic function. Here, we argue that the spatial and temporal distribution of actin-dependent processes within spines suggests that the spine cytoskeleton should not be considered a single entity, but an interacting network of nodes or hubs that are independently regulated and balanced to maintain synapse function. Disruptions of this balance within the spine are likely to lead to psychiatric and neurological dysfunction.