Organizing Synapses via Trans-synaptic Adhesion
Synapses are specialized contact sites between neurons that allow for neuronal transmission. Synapses therefore wire neurons into networks. The precise spatial alignment of pre- and postsynaptic sites and their temporally coordinated assembly are strong evidence for critical roles of local cell-cell interactions in synapse development. Several such trans-synaptic complexes are now known. We have identified and characterized SynCAM proteins as a class of synaptic cell adhesion molecules. We use SynCAMs and other synaptic adhesion proteins as entry points to delineate how interactions across the synaptic cleft organize synapses. Our progress has established that SynCAMs are unique among synapse-organizing proteins as they control consecutive steps of synaptic development, from axo-dendritic contact to synapse induction (Fig. 1) and maintenance in vivo.
Figure 1. SynCAMs are synaptic plasma membrane proteins that induce synaptic specializations. A. Synaptic plasma membrane fractionation of SynCAM proteins. The indicated subcellular fractions were prepared from rat forebrain at postnatal day 9 and equal protein amounts were analyzed by immunoblotting for the four SynCAM family members. B. Synapse induction in a reconstituted cell culture system. SynCAM 2 (green) heterologous expressed in a HEK 293 cell binds and retains SynCAM 1 (red) from a co-cultured hippocampal neuron. This induces the neuron to form clusters of the presynaptic protein synapsin (blue) atop the HEK cell surface. C. Expression of SynCAM 1 or SynCAM 2 in HEK 293 cells co-cultured with hippocampal neurons causes a significant increase of synapsin-positive puncta covering the cell surface area compared to control HEK 293 cells expressing CFP alone. The synaptogenic activity of both SynCAMs is similar.
Synaptogenic Signalling Pathways
Understanding the mechanisms mediating synaptogenic signaling can provide points of therapeutic intervention in human brain disorders. We have demonstrated that SynCAM protein have signaling roles and are elucidating the pathways involved. In an unbiased proteomic analysis, we identified proteins that undergo SynCAM 1-dependent recruitment and stabilization at synaptic membranes. The analysis of our first hit, the protein Farp1, showed that it is a novel synapse-organizing molecule that binds to the cytosolic tail of SynCAM 1. Notably, SynCAM 1 requires Farp1 to promote synapse formation, and Farp1 specifically binds the GTPase Rac1 to activate it at postsynaptic sites. Moreover, SynCAM 1 and postsynaptic Farp1 signal retrogradely across the synaptic cleft to modulate the composition of presynaptic active zones (Fig. 2). As we elucidate synaptogenic signaling by the proteins identified in our proteomic screen, we combine biochemical assays with live imaging of optical probes. In order to understand the functions of synapse-organizing proteins, we additionally address where they localize while synapses assemble, and use live and super-resolution imaging.
Figure 2. Trans-synaptic organization by the SynCAM 1/Farp1 pathway. A, Confocal images of dendritic segments from hippocampal neurons in which Farp1 was knocked-down (shFarp1) or overexpressed (GFP-Farp1) and controls. Circles mark presynaptic terminals positive for bassoon (red) apposed to postsynaptic protrusions termed spines. B, The intensity of the presynaptic active zone marker bassoon is regulated by Farp1 across the synaptic cleft. Bassoon intensity was imaged as in A and was normalized to staining atop control dendrites. C, Model of trans-synaptic organization by the synaptic SynCAM 1/Farp1 complex. SynCAM 1 recruits and stabilizes Farp1 at synaptic membranes to increase excitatory synapse number, with Farp1 activating postsynaptic Rac1 and promoting F-actin assembly in postsynaptic spines heads. In addition, postsynaptic Farp1 organizes presynaptic active zones via SynCAM 1.
Remodeling Synapses in Health and Disease
Synapse-organizing proteins impact dynamic changes of networks as supported for example by the SynCAM-mediated regulation of synaptic connectivity in the adult brain (Fig. 3). Building on our progress, our group analyzes to what extent synaptic adhesion contributes to the activity-dependent remodeling of synapses. We additionally develop transgenic mouse models that express synapse-organizing proteins in subsets of neurons to investigate how dynamic changes in synapse formation contribute to memory processes.
Moreover, our advances enable us to address trans-synaptic interactions in human disorders. Among the translational research areas we pursue is the link of synapse-organizing mechanisms to synaptic and behavioral responses to drugs of abuse.
Figure 3. Synapses are organized by SynCAM adhesion molecules in vivo as determined by comparative studies of mouse models lacking SynCAM 1 and inducible overexpressor mice.
Impact of our Research Program
Once the mechanisms of synapse development have been determined, our knowledge of how neuronal circuits are wired will be advanced profoundly. Further, our evidence already supports that the mechanisms organizing synapses in the developing brain can also contribute to the remodeling of mature synapses, and impact spatial reference memory or cue-dependent conditioning. We therefore expect that our progress will advance the molecular understanding of memory processes. Our work can also lead to the identification of targets for therapeutic intervention to ameliorate synaptic deficits associated with neurodevelopmental and synaptopathic brain disorders, addiction, and brain injury.
Together, our research gains comprehensive multidisciplinary insights into the mechanisms that instruct synaptogenesis and remodel synapses and neuronal networks in health and disease.