Morphological correlates of synaptic protein turnover in the mouse brain.

Synaptic proteins need to be replaced regularly, to maintain function and to prevent damage. It is unclear whether this process, known as protein turnover, relates to synaptic morphology. To test this, we relied on nanoscale secondary ion mass spectrometry, to detect newly synthesized synaptic components in the brains of young adult (6 mo old) and aged mice (24 mo old), and on transmission electron microscopy, to reveal synapse morphology. Several parameters correlated to turnover, including pre- and postsynaptic size, the number of synaptic vesicles and the presence of a postsynaptic nascent zone. In aged mice, the turnover of all brain compartments was reduced by ∼20%. The turnover rates of the pre- and postsynapses correlated well in aged mice, suggesting that they are subject to common regulatory mechanisms. This correlation was poorer in young adult mice, in line with their higher synaptic dynamics. We conclude that synapse turnover is reflected by synaptic morphology.

Trafficking proteins show limited differences in mobility across different postsynaptic spines.

The function of the postsynaptic compartment is based on the presence and activity of postsynaptic receptors, whose dynamics are controlled by numerous scaffolding, signaling and trafficking proteins. Although the receptors and the scaffolding proteins have received substantial attention, the trafficking proteins have not been investigated extensively. Their mobility rates are unknown, and it is unclear how the postsynaptic environment affects their dynamics. To address this, we analyzed several trafficking proteins (α-synuclein, amphiphysin, calmodulin, doc2a, dynamin, and endophilin), estimating their movement rates in the dendritic shaft, as well as in morphologically distinct "mushroom" and "stubby" postsynapse types. The diffusion parameters were surprisingly similar across dendritic compartments, and a few differences between proteins became evident only in the presence of a synapse neck. We conclude that the movement of trafficking proteins is not strongly affected by the postsynaptic compartment, in stark contrast to the presynapse, which regulates strongly the movement of such proteins.

The vesicle cluster as a major organizer of synaptic composition in the short-term and long-term.

For decades, the synaptic vesicle cluster has been thought of as a storage space for synaptic vesicles, whose obvious function is to provide vesicles for the depolarization-induced release of neurotransmitters; however, reports over the last few years indicate that the synaptic vesicle cluster probably plays a much broader and more fundamental role in synaptic biology. Various experiments suggest that the cluster is able to regulate protein distribution and mobility in the synapse; moreover, it probably regulates cytoskeleton architecture, mediates the selective removal of synaptic components from the bouton, and controls the responses of the presynapse to plasticity. Here we discuss these features of the vesicle cluster and conclude that it serves as a key organizer of synaptic composition and dynamics.

A minimalist model to measure interactions between proteins and synaptic vesicles.

Protein dynamics in the synaptic bouton are still not well understood, despite many quantitative studies of synaptic structure and function. The complexity of the synaptic environment makes investigations of presynaptic protein mobility challenging. Here, we present an in vitro approach to create a minimalist model of the synaptic environment by patterning synaptic vesicles (SVs) on glass coverslips. We employed fluorescence correlation spectroscopy (FCS) to measure the mobility of monomeric enhanced green fluorescent protein (mEGFP)-tagged proteins in the presence of the vesicle patterns. We observed that the mobility of all eleven measured proteins is strongly reduced in the presence of the SVs, suggesting that they all bind to the SVs. The mobility observed in these conditions is within the range of corresponding measurements in synapses of living cells. Overall, our simple, but robust, approach should enable numerous future studies of organelle-protein interactions in general.

Quantitative Synaptic Biology: A Perspective on Techniques, Numbers and Expectations.

Synapses play a central role for the processing of information in the brain and have been analyzed in countless biochemical, electrophysiological, imaging, and computational studies. The functionality and plasticity of synapses are nevertheless still difficult to predict, and conflicting hypotheses have been proposed for many synaptic processes. In this review, we argue that the cause of these problems is a lack of understanding of the spatiotemporal dynamics of key synaptic components. Fortunately, a number of emerging imaging approaches, going beyond super-resolution, should be able to provide required protein positions in space at different points in time. Mathematical models can then integrate the resulting information to allow the prediction of the spatiotemporal dynamics. We argue that these models, to deal with the complexity of synaptic processes, need to be designed in a sufficiently abstract way. Taken together, we suggest that a well-designed combination of imaging and modelling approaches will result in a far more complete understanding of synaptic function than currently possible.

A comparative analysis of the mobility of 45 proteins in the synaptic bouton.

Many proteins involved in synaptic transmission are well known, and their features, as their abundance or spatial distribution, have been analyzed in systematic studies. This has not been the case, however, for their mobility. To solve this, we analyzed the motion of 45 GFP-tagged synaptic proteins expressed in cultured hippocampal neurons, using fluorescence recovery after photobleaching, particle tracking, and modeling. We compared synaptic vesicle proteins, endo- and exocytosis cofactors, cytoskeleton components, and trafficking proteins. We found that movement was influenced by the protein association with synaptic vesicles, especially for membrane proteins. Surprisingly, protein mobility also correlated significantly with parameters as the protein lifetimes, or the nucleotide composition of their mRNAs. We then analyzed protein movement thoroughly, taking into account the spatial characteristics of the system. This resulted in a first visualization of overall protein motion in the synapse, which should enable future modeling studies of synaptic physiology.

Interrogating Synaptic Architecture: Approaches for Labeling Organelles and Cytoskeleton Components.

Synaptic transmission has been studied for decades, as a fundamental step in brain function. The structure of the synapse, and its changes during activity, turned out to be key aspects not only in the transfer of information between neurons, but also in cognitive processes such as learning and memory. The overall synaptic morphology has traditionally been studied by electron microscopy, which enables the visualization of synaptic structure in great detail. The changes in the organization of easily identified structures, such as the presynaptic active zone, or the postsynaptic density, are optimally studied via electron microscopy. However, few reliable methods are available for labeling individual organelles or protein complexes in electron microscopy. For such targets one typically relies either on combination of electron and fluorescence microscopy, or on super-resolution fluorescence microscopy. This review focuses on approaches and techniques used to specifically reveal synaptic organelles and protein complexes, such as cytoskeletal assemblies. We place the strongest emphasis on methods detecting the targets of interest by affinity binding, and we discuss the advantages and limitations of each method.