Grant: Discovery Grants Program - Individual

Objective: The primary objective of my research program is to understand how gene expression is controlled, and to explore the biological functions of dynamically-regulated genes. My short-term aims are focused on the mechanisms governing the activity-dependent gene expression program, which regulates the structural plasticity of synaptic connections between neurons. Long non-coding RNAs (lncRNAs) are a newly appreciated class of gene regulatory factor that can coordinate interactions between proteins and nucleic acids. Many lncRNAs are expressed primarily in the brain, but little is known about their functions. The aim of the proposed research is to explore the roles of lncRNAs in the regulation of neuronal gene expression and activity-dependent structural plasticity. Approach: Human neurons will be generated from fibroblasts by cellular reprogramming with genetic factors. I will used molecular/biochemical approaches will to examine lncRNA splice variants and subcellular localization. lncRNAs will be manipulated using viral vector-mediated functional genetics approaches: the CRISPR/Cas9 system will be used for genome editing and to directly activate (CRISPRa) or inhibit (CRISPRi) lncRNA expression. Neurons will be stimulated by depolarization or with agonists of excitatory neurotransmitter receptors, followed by analyses of activity-dependent signaling and gene expression. Structural plasticity will be assayed using imaging approaches to assess dendrite morphology and synapse numbers. Specific Aims: As targets for functional experiments I selected two lncRNAs with potential roles in the signaling pathways that regulate synapse function: (i) PTCHD1-AS, which regulates NMDA-type excitatory neurotransmitter receptors via an unknown mechanism and (ii) the primate-specific LNC473, which is a putative regulator of cyclic AMP signaling that is robustly induced by neuronal activity. Aim 1: Examine neuronal splice variants and subcellular localization of PTCHD1-AS and LNC473. Aim 2: Evaluate the roles of PTCHD1-AS and its chromosomal neighbor DDX53 in NDMA receptor signaling. Aim 3: Determine the roles of LNC473 in regulating cyclic AMP-dependent gene expression and structural plasticity of neurons. Impact: Many lncRNAs are primarily expressed in the brain, evolve swiftly, and are not widely conserved. Innovative cellular reprogramming approaches provide a unique opportunity for functional analyses of human lncRNAs. By testing the roles of lncRNAs in regulating activity-dependent structural plasticity the proposed research will shed light on the dynamic processes that control information processing and storage in the brain. Furthermore, studies of human neuronal lncRNAs may provide insights into the regulatory and functional complexity of the human brain.