Figure 4.5. Recruitment of Clusters of Presynaptic and Postsynaptic Proteins at Early Sites of Contact Between Axons and Dendrites. (A) Shows an accumulation of a synaptophysin cluster at a contact site between dendritic filopodia of a cell transfected with a membrane targeted GFP and an axon from a neuron transfected with synaptophysin tagged with DsRed (SYN DsRed). (B) Time-lapse images showing accumulation of SYN DsRed at a site apposed to an existing PSD-95 GFP cluster, occurred over a time (t) period of 20 min. See Colorplate 5.

However, a recent study by our group in young hippocampal neurons has shown that clusters of PSD-95 can also be found associated with neuroligin-1 in the absence of an active presynaptic terminal. In these young neurons, protein complexes exist in two distinct subpopulations which differ in their content, mobility, and involvement in synapse formation. (One subpopulation of clusters is mobile and rely on actin transport to nascent and existing synapses. Further, the majority of mobile clusters, containing the scaffolding proteins PSD-95, GKAP, and Shank, lack neuroligin-1. The second subpopulation consist of stationary nonsynaptic scaffold complexes. These complexes contain neuroligin-1 and recruit synaptophysin-containing vesicles to the opposing axonal site of contact. Importantly, these sites were shown to become functional presynaptic contacts through FM 4-64 dye loading. In this case, the clustering of postsynaptic proteins such as neuroligin-1 may facilitate recruitment of presynaptic proteins important for vesicular release. Another study looking at new contacts made by axon filopodia demonstrated the rapid recruitment of NMDA-type glutamate receptor clusters independent of PSD-95, and before the establishment of an active presynaptic terminal (see Chapter 14)47. Although critical for synapse maturation, it remains unknown how clustering of these proteins is developmentally and spatially regulated, and what adhesion systems are involved.

Recent in vitro studies showed that several of the identified cell adhesion molecules modulate synaptic contact number, morphology, and function. However, strong evidence that any of these molecules is indispensable for synapse formation in vivo is lacking, suggesting a redundancy in their function. Thus, synapse formation and maturation may rely on assembly of several adhesion systems. Whether the numerous adhesion families act in parallel or in a hierarchical manner is unknown, and future studies will be required to tease apart the nuances of how these adhesion systems work together in the establishment and function of the synapse. To study the ability of cell adhesion molecules to cluster presynaptic proteins at contact sites, a clever assay has been developed using co-culture of transfected heterologous cells with neurons. In a series of experiments by Scheiffele's group, HEK cells were transfected with DNA encoding the adhesion molecule neuroligin-1 and co-cultured with developing neurons52. Remarkably, the expression of this postsynaptic adhesion molecule in heterologous cells resulted in the differentiation of presynaptic terminals at sites of axon--HEK cell contact. Furthermore, these contacts were found to have not only morphological but functional characteristics of actual presynaptic contacts, with the accumulation of synaptic vesicles52. In hippocampal neurons, overexpression of neuroligins increased the number of both excitatory and inhibitory presynaptic terminals53-57. Similar assays have been used to show that SYNCAM can also induce clustering of presynaptic proteins at contact sites. Conversely P-neurexin, a binding partner of neuroligins, presented to dendrites via heterologous cells or beads has been demonstrated to cluster postsynaptic proteins (see Chapter 19)58,59. These studies suggest that initial interaction between adhesion molecules initiates53,55 clustering of postsynaptic proteins and recruitment of neurotransmitter receptors to newly formed neuronal contacts52,58,59. Thus, early events that involve clustering and assembly of protein complexes appear to regulate not only initial contact formation and stabilization but also drive synapse maturation.

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