Local calcium signalling in the postsynaptic density

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.

Long-term changes in synaptic strength underlie memory formation. Calcium entry through NMDA-type glutamate receptors in the postsynaptic density can trigger either long-term potentiation (LTP) or depression (LTD) in the leading model of synaptic plasticity. The phosphatase calcineurin drives LTD following small influxes of calcium, whereas calmodulin-dependent protein kinase II (CaMKII) triggers LTP following large influxes of calcium. Since the two enzymes respond similarly to calcium in the test tube, it is not clear why they respond differently to calcium influx in the postsynaptic density (PSD). The proposed research will address this problem in two broad ways: by testing whether the two calcium enzymes are positioned at different distances from calcium entry points in the PSD; and by determining if the anchoring protein AKAP79 can operate as an override switch for calcineurin at high calcium concentrations.

In pursuit of our first major aim, we will combine crosslinking coupled to mass spectrometry to build accurate models of the PSD. The models will enable us to compare the positions of calcineurin and CaMKII relative to calcium entry points. We will also compare local calcium concentration variations within the PSD by constructing fusions with a genetically-encoded calcium sensor and applying the fusions in nerve endings. In support of our second major aim, we will solve the crystal structure of part of the anchoring protein AKAP79 and perform phosphatase assays using a novel method. This will enable us to examine whether the anchoring protein can act in concert with the cell membrane to restrict the activity of the phosphatase to low concentrations of calcium. The research will advance knowledge of a fundamental aspect of learning, and technologies developed during the project will be applicable by other researchers. The research is highly relevant to human brain disease as the research focuses on proteins that are prominent drug targets.

The award of this grant can make positive impacts in the following areas:

Developing skills in research staff & fostering research partnerships: At UCL, the appointed postdoctoral researcher will be trained to perform experimental techniques including protein and PSD purification using an AKTA FPLC, fluorescence and Alpha-Screen measurements with the plate reader, and the steps involved in protein crystallography. The researcher will develop project management skills and interpersonal skills that are advantageous in multiple employment sectors. The award will strengthen our international partnership with the laboratory of Dr. Florian Stengel at the University of Konstanz. The University of Konstanz was ranked No. 1 in the German Research Council Funding Atlas 2015 in per capita funding. Dr. Gold and the postdoctoral researcher will visit Dr. Stengel during the first year to optimize experimental design and discuss future avenues for collaboration. The requested equipment will be accessible to other resarchers at UCL enabling other researchers to incorporate Alpha-Screen measurements and dynamic high-throughput recordings using genetically encoded sensors into their research. Gold lab members including the postdoctoral researcher will also take part in hosting the annual Collyer's College student visit, and supervising summer project students including pre-university EPQ project students.

Influencing the scientific community: We anticipate that our research can make constructive impacts across the biological sciences. The proposed research provides a new perspective for the field of synaptic plasticity. Many of our experimental aims have the potential to benefit fields including the control of heart contractility, immunosuppression and blood glucose handling. Furthermore, our innovative technical approaches, and novel applications of dynamic crosslinking coupled to mass spectrometry are likely to be adopted by researchers irrespective of traditional research field boundaries.

Uncovering novel avenues for pharmaceutical intervention: The proposed research can also positively impact pharmaceutical development. Although our immediate aim is to uncover fundamental principles in the molecular basis of synaptic plasticity, in the longer-term we anticipate that the study can be of translational benefit. The synapse is a locus for mutations that cause neurological disease and psychiatric disorders. More specifically, the research will determine how the important drug targets calcineurin and CaMKII are anchored at the molecular level. Targeting the anchoring sites for signalling enzymes is a promising avenue for developing drugs with improved specificity.

Engaging the public: We will take opportunities to present our research to the wider national media assisted by UCL's Public Engagement Unit. In addition to the annual 'Crystallography Day' with Collyer's College A-level students, we will continue to supervise both pre-university and undergraduate summer project students. Overall, our research has the potential to deliver many positive social and economic impacts.