Local calcium signalling in the postsynaptic density

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Within the brain, tens of billions of electrically active cells communicate with each other at junctions called synapses. Long-lasting changes in the strength of synapses are thought to underlie learning and memory. Research has focused on understanding how synaptic plasticity is brought about at the level of molecules in model organisms including sea slugs and rats. Some components of a 'core program' for synaptic plasticity have emerged. Notably, long-lasting potentiation and depression of synapses both follow from influx of calcium. Two calcium-sensitive enzymes are critical for this process: one modifies synaptic proteins to strengthen the synapse following large influxes of calcium whereas the other has the opposite effect following small influxes of calcium. However, both enzymes exhibit equal sensitivity to calcium in the test tube so it is not obvious why they respond differently to calcium within the synapse. An important consideration is that calcium is locally concentrated in cells near to its points of entry. Our research will pursue two major goals. First, we propose to determine whether the relative positions of the two enzymes from calcium entry points, within a massive protein assembly called the postsynaptic density (PSD), explain why one of the enzymes requires larger influxes of calcium for activation. Second, we aim to determine why the enzyme that depresses synaptic strength is inactive following large influxes of calcium.We will apply complementary approaches to examine the positions of the two enzymes relative to calcium entry points in the PSD. We will chemically crosslink PSD samples and map the exact sites of crosslinking between different proteins within the protein assembly. This will enable us to build accurate structural models so we can compare the relative positions of the key enzymes with calcium entry points. We will also measure how a calcium sensor responds to experimentally controlled influxes of calcium when co-localised with different proteins within the PSD. In pursuit of our second major aim, we will focus on understanding how an anchoring protein regulates the activity of the calcium-sensitive enzyme that depresses synaptic strength. We will determine the high-resolution three-dimensional structure of a fragment of the anchoring protein to understand how it is regulated by calcium within the PSD. We will also use a novel enzymatic assay to examine whether the anchoring protein is able to tune the enzyme that brings about long-term synaptic depression such that it is active at low but not high concentrations of calcium.The research will benefit from collaboration with proteomics specialists in Germany thereby cementing an international partnership. In addition to addressing a fundamental aspect of how the brain lays down memories, the novel technological approaches that we develop will be widely applicable to other areas of biology. The research is highly relevant to human brain disease as the synapse is a locus for mutations that cause neurological disease and psychiatric disorders, and aberrant synaptic plasticity may be fundamental to many brain disorders including Alzheimer's and autism. Both the calcium-sensitive enzymes and the anchoring protein that are the focus of the proposed research are drug targets. Overall, the research can provide insights at a molecular level that is informative for drug design.