Abstract
Investigating the roles of synaptogenic adhesion molecules during synapse formation has proven challenging, often due to compensatory functions between additional family members. The synaptic cell adhesion molecules 1–3 (SynCAM1–3) are expressed both pre- and postsynaptically, share highly homologous domains and are synaptogenic when ectopically presented to neurons; yet their endogenous functions during synaptogenesis are unclear. Here we report that SynCAM1–3 are functionally redundant and collectively necessary for synapse formation in cultured hippocampal neurons. Only triple knockdown (KD) of SynCAM1–3 using highly efficient, chained artificial microRNAs (amiRNAs) reduced synapse density and increased synapse area. Electrophysiological recordings of quantal release events supported an increase in synapse size caused by SynCAM1–3 depletion. Furthermore, a combinatorial, mosaic lentiviral approach comparing wild type (WT) and SynCAM1–3 KD neurons in the same culture demonstrate that SynCAM1–3 set synapse number and size through postsynaptic mechanisms. The results demonstrate that the redundancy between SynCAM1–3 has concealed their synaptogenic function at the postsynaptic terminal.
Highlights
Synapse formation is initiated by physical contact of adhesion molecules between axons and dendrites which triggers recruitment of molecular complexes to the presynaptic active zone or the postsynaptic density (PSD) (Washbourne et al, 2004)
We generated a separate lentiviral vector driving expression of a plasma membrane-localized GFP reporter used to label dendrites and spines (Figure 1B). We previously characterized these chained artificial microRNAs (amiRNAs) targeting SynCAM1–3, which are encoded by the cell adhesion molecule 1–3 genes, as highly efficient for KD of SynCAM1–3 in rat primary hippocampal cultures at 13–15 days in vitro (DIV) by western blot (Fowler et al, 2016b)
This report describes the first investigation into the functional redundancy of SynCAM1–3 during excitatory synapse development
Summary
Synapse formation is initiated by physical contact of adhesion molecules between axons and dendrites which triggers recruitment of molecular complexes to the presynaptic active zone or the postsynaptic density (PSD) (Washbourne et al, 2004). Several observations suggest that the nectin-like synaptic cell adhesion molecule (SynCAM) family may share redundant functions during synapse formation. Knockdown (KD) of any single, or two SynCAMs does not affect excitatory synapse formation; rather triple KD of SynCAM1–3 shows they are necessary for and compensate to set synapse density and limit synapse size We further investigate these phenotypes using a novel method that generates a traceable mosaic of KD and wild type (WT) cells on the same coverslip. Electrophysiological recordings confirm postsynaptic effects of SynCAM1–3 KD with broadened event peaks of miniature excitatory postsynaptic currents (mEPSCs) consistent with an increase in synapse size These results suggest that a postsynaptic mechanism for SynCAM1–3 in synaptogenesis has been heretofore concealed by overlapping functions of the gene family members
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