Saturday, November 23, 2024
11/23/2024
PROJECT SUMMARY/ABSTRACT Synaptic plasticity is a fundamental mechanism through which synapses can modify their communication efficacy in response to neural activity patterns. Long-term potentiation (LTP) is a well-studied form of synaptic plasticity that creates a long-lasting strengthening of synaptic connections. LTP requires NMDA receptors, which activate calcium-dependent signaling pathways that promote phosphorylation and insertion of synaptic AMPA receptors. Weak stimulation induces a temporary potentiation known as early phase LTP, whereas a strong stimulus can elicit a persistent strengthening termed late phase LTP. The late phase of LTP requires new protein translation and well as new gene transcription. One of the most extensively studied activity-dependent transcription factors is the cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). A major mode of CREB activation occurs through phosphorylation of serine 133 (Ser133), which can be mediated by a variety of synapse-to-nucleus signaling pathways and kinases. Upon phosphorylation, CREB binds to DNA sequences known as cAMP response elements (CREs) and recruits co-activators to initiate gene transcription of numerous target genes. CREB is canonically considered to be permanently residing in the nucleus within the brain, and there is little recognition in the field of a potential extra-nuclear CREB function in the CNS. However, our novel preliminary data suggest that CREB is found in both pre -and postsynaptic sites at excitatory synapses in hippocampal neurons. To further investigate this surprising observation, Aim 1 of the proposal will conduct a comprehensive CREB localization, epitope mapping, and phosphomapping study at pre- and postsynaptic sites, using knockdown and knockout cells and tissues as specificity controls; test different pharmacological stimulation paradigms using cultured neurons as well as electrophysiological LTP stimulation of hippocampal acute slices; employ pharmacological approaches to inhibit various channels and kinases to determine mechanisms of activation, and use STED superresolution microscopy to obtain a more precise view of CREB colocalization with pre- and postsynaptic markers. To understand the function of CREB at synapses, Aim 2 will determine which regions of CREB are required for targeting to synapses through deletion analysis, and use synapse-deficient CREB mutants to determine the importance of synaptic CREB for gene expression as well as synaptic morphology and transmission. We will also use unbiased mass spectrometry to determine additional CREB post- translational modifications, isoforms, and protein-protein associations specifically in synaptosome fractions, which will give insights into the functional pathways that CREB may be involved in synapses. These studies will revise our view of CREB and expand the roles of transcription factors at synapses. This research has significance for basic understanding of synaptic plasticity as well as promoting long-lasting plasticity in neurodegenerative disorders such as Alzheimer’s disease.
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