Profiling activity-dependent synaptic translation

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ABSTRACT Synaptic plasticity allows for dynamic changes in the strength of neuronal connections, a hallmark of neural circuits that is thought to underlie learning and memory. Activity-dependent plasticity can strengthen or weaken synapses in response to calcium influx and other signaling pathways triggered by neuronal activity. These signals drive short- and long-term changes in synaptic strength through a variety of mechanisms. Local translation at active synapses is required for many long-term changes, likely through the synthesis of synaptic proteins. Subtle dysregulation of synaptic protein synthesis is thought to explain the neurodevelopmental features of fragile X syndrome, and is linked more broadly with autism spectrum disorders. These findings highlight the importance of regulated synaptic translation. Synaptic activity is transient, however, and active synapses are interspersed amongst many other synapses and cell bodies, posing serious technical challenges to profiling synaptic translation. Here, we propose to overcome these limitations and achieve global translational profiling at active synapses. We demonstrate a system for "tagging" ribosomes at active synapses using an engineered, calcium-dependent biotin ligase. Purification of these tagged ribosomes enriches for synaptic transcripts and confirms a key role for FMRP as a regulator of synaptic translation. In order to gain tighter spatiotemporal control needed for in vivo experiments, we have further engineered an optogenetic light-dependent biotin ligase substrate. Based on these promising preliminary data, we propose to develop and apply our calcium and light-dependent ribosome biotinylation to investigate activity-dependent translation. Results from this work will provide new insights into synaptic translation and produce widely applicable tools for neurobiology.