A nanocolumn spans the synaptic cleft between neurons, connecting regions of neurotransmitter molecule release and capture. This discovery informs on mechanisms of synaptic organization and regulation. See Letter p.210
The sophisticated human brain forms the foundation of all the cognitive processes that define us as self-conscious and social individuals. These processes are fundamentally based on the operation of a single functional unit — the synapse, which enables rapid signal transmission between neurons. Synapses are composed of two small, highly specialized compartments, one on the presynaptic (transmitting) side and one on the postsynaptic (receiving) side of the small gap that separates the two neurons. Structures spanning this synaptic cleft to coordinate these compartments have been suggested1, but direct evidence for their existence remains scarce. On page 210, Tang et al.2 use a combination of elaborate super-resolution light microscopy and mathematical modelling to provide evidence for the existence of discrete, protein-based nanocolumns that connect the pre- and postsynaptic compartments.
During neuronal signalling, electrical impulses called action potentials trigger the release of neurotransmitter molecules from the presynaptic neuron. Release involves fusion of neurotransmitter-containing synaptic vesicles with a region of the cell membrane called the active zone, which faces the synaptic cleft. Vesicle docking and fusion does not occur in isolation, but within an extended protein scaffold made up of several large multi-domain proteins3 that provides sites for synaptic-vesicle fusion.
Dissecting the organizational principles of these scaffolds was, for many years, achievable only by electron microscopy, which is not compatible with live imaging. However, this limitation has been overcome, thanks to the development of super-resolution light-microscopy techniques4, which allow the efficient visualization of distinct protein architectures. One such study5 has revealed that presynaptic scaffolds physically contact synaptic vesicles, perhaps promoting their docking and priming for neurotransmitter release at defined fusion sites. Other studies have shown that one scaffold protein, RIM, has a prominent role in synaptic-vesicle docking — RIM interacts with proteins of the MUNC-13 family6,7 to promote clustering of calcium-channel proteins8, which in turn trigger fusion processes.
In a quest to further decipher the nanoarchitecture of presynaptic active zones, Tang et al. turned to a high-resolution form of light microscopy called stochastic optical reconstruction microscopy (STORM)9. The authors used 3D STORM to study synapses between in-vitro cultured mouse neurons derived from the brain's hippocampus region, which is involved in learning and memory. The synapses under observation release the neurotransmitter glutamate. This analysis revealed that RIM is confined to protein nanoclusters of around 80 nanometres in diameter that lie close to the active zone. By contrast, other scaffold proteins and fusion factors, such as MUNC-13 and Bassoon, showed a more uniform distribution.
Is the position of these RIM-rich nanoclusters related to vesicle-fusion sites? The researchers monitored fusion events at the presynaptic membrane using a protein-based sensor that fluoresces following vesicle–membrane fusion. Mathematical modelling of the fluorescence patterns revealed that fusion sites are restricted to particular regions of the membrane. Moreover, a different form of super-resolution light microscopy called photoactivated localization microscopy that allows live imaging, confirmed that RIM density increased within 40 nm of these fusion sites.
The authors next investigated whether the RIM-rich fusion sites might be coordinated with the position of the postsynaptic apparatus dedicated to receiving the neurotransmitter signal. A sophisticated scaffold resides close to the postsynaptic membrane — a part of which is the multi-domain protein PSD-95, which is involved in the clustering of AMPA- and NMDA-type glutamate receptor proteins10,11. Precise measurement of RIM and PSD-95 densities revealed a clear spatial correlation between the components. Tang et al. therefore concluded that a nanoscale columnar structure spans the synaptic cleft, bringing RIM-enriched sites of synaptic-vesicle fusion face-to-face with postsynaptic PSD-95 nanodomains (Fig. 1).
Finally, Tang and colleagues asked if the nanocolumns could be a stable architectural motif or whether they are involved in the regulatory changes in synaptic strength that are crucial for cognitive functions. The authors pharmacologically activated NMDA receptors to depress synaptic strength. Although there was no immediate change in the architecture of the nanocolumn, after 25 minutes a subset of RIM nanoclusters suddenly grew larger — notably only those lying opposite PSD-95 nanodomains and residing in nanocolumns. Thus, retrograde signals that mediate the upregulation of presynaptic release in response to postsynaptic changes might specifically target the scaffold proteins and release machinery located opposite the postsynaptic glutamate receptors to modulate synaptic strengthening. As such, the nanocolumn could provide an important regulatory platform.
This study generates pressing questions. For instance, to understand the physical nature of the nanocolumns, it would be interesting to determine what regulates their formation. Trans-synaptic pairs of cell-adhesion membrane proteins are obvious candidates for mediating nanocolumn formation. Perhaps such adhesion molecules ultimately control the positioning and recruitment of RIM.
Alternatively, diffusible signals might cross the cleft and specifically trigger assembly of nanocolumns on the scale of a few tens of nanometres. In addition, RIM itself could be involved in nanocolumn formation — RIM contains a central domain that binds to the intracellular part of calcium channels12, which ultimately trigger synaptic-vesicle fusion.
In the future, the nanocolumn concept should be validated and extended by investigating more proteins, including synaptic cell-adhesion proteins and other cytoplasmic scaffold proteins, and by combining imaging with genetic manipulation. Although the details of trans-synaptic coordination and the proteins involved might turn out to vary between synapse types and organisms, the nanocolumnar architectural motif could be a fundamental and generic building principle for synapses.
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Chemical Reviews (2017)