Cysteine oxidation in the remodelling of dendritic spines

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Overview: Synaptic plasticity in response to neurotransmission is a key component of learning and memory, requiring communication between the extracellular environment and the cytoskeletal proteins that dictate the structure of a neuron. Most excitatory postsynaptic terminals reside in dendritic spines, which are thought to serve as basic units of memory storage. Synaptic transmission through the ionotropic glutamate receptor, N-methyl-D-aspartate receptor (NMDAR), is a strong modulator of spine morphology. While activation of post-synaptic NMDARs can strengthen synaptic connections, over-activation of NMDARs results in cytoskeletal modifications that promote synaptic pruning. The equilibrium between these events governs synaptic plasticity in brain regions rich in spiny neurons such as the hippocampus, the center for learning and memory. Activation of NMDARs leads to rapid Ca+ influx and nitric oxide (NO) synthesis. Collectively, the existing evidence supports a model in which second messenger signaling by Ca+ and NO following neurotransmission confers on neurons the plasticity required for neuronal communication. While these second messengers have been extensively studied, mechanisms underlying the morphological changes to spine structure that accompany synaptic transmission are still poorly understood. By contrast to Ca+, it is estimated that NO can directly modulate up to 50% of proteins in the brain, through a process called s-nitrosylation (SNO). While much evidence supports a role for SNO in synaptic plasticity, a comprehensive analysis of the SNO-proteome that supports and regulates neurotransmission has not been performed. Approach: This proposal aims to explore the fundamental cell biology of synaptic plasticity by identifying a the role of NO in direct modulation of proteins in dendritic spines. To this end, we will capitalize on the physical properties of the NR3A subunit of NMDAR to modulate NO and Ca+ signaling. While ablation of NMDAR signaling is embryonic lethal, overexpression of NR3A dampens NMDAR signaling by decreasing the Ca+ permeability of the channel, thus reducing glutamate mediated NO synthesis. Using transgenic animals over-expressing NR3A in a tet dependant manner or NR3A KO animals, we have a means of either promoting NMDAR-mediated NO synthesis or of turning it on and off through doxycycline administration. By employing a mass spectrometry method for identification of protein S-nitrothiols, we will establish the SNO-proteome of hippocampal neurons in response to synaptic activation. This will establish a critical set of effectors of NO signal transduction, necessary for synaptic plasticity. We will subsequently determine how modulating NMDAR activity influences Ca+ influx and subsequent NO synthesis using high resolution live cell imaging. We use Crispr-generate, non-nitrosylatable variants of synaptic proteins to assess mechanism of how SNO-proteins govern synaptic plasticity.