Palmitoylation-dependent regulation of the actin cytoskeleton in dendritic spines

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DESCRIPTION (provided by applicant): This project will address whether direct palmitoylation, the covalent attachment of the lipid palmitate, accounts for the unique ability of LIM Kinase-1 (LIMK1) to regulate the morphology and stability of dendritic spines. Dendritic spines are small, actin-rich protrusions that are the sites of most excitatory synapses. The importance of spatially precise regulation of dendritic spine actin for neuronal development and for neuronal plasticity is well established. Conversely, impaired spine structure is a hallmark of conditions such as Intellectual Disability (ID) and Autism Spectrum Disorders (ASDs). However, how neurons spatially restrict actin regulation to ensure that only appropriate spines are modified is unclear. We have found that LIMK1, a key actin regulator linked to higher brain function, is directly palmitoylated and that palmitoylation targets LIMK1 to spines. Palmitoylation is known to target non-enzymatic 'scaffold' proteins to spines and synapses, but roles for palmitoylation in the control of actin polymerization, and in the regulation of neuronal kinase signaling are completely undescribed. We have established an shRNA-mediated knockdown/rescue system to replace endogenous LIMK1 in neurons with a form that cannot be palmitoylated. We now propose a series of experiments using this system to determine whether palmitoyl-LIMK1 is functionally required for normal spine actin polymerization, for spine- specific morphological plasticity, and for long-term spine stability. Complementary experiments will determine whether palmitoylation is necessary and sufficient to explain differences in localization and function between LIMK1 and its closest homolog LIMK2 and will identify the enzyme that controls LIMK1 palmitoylation. These experiments will not only shed light on new mechanisms of spine regulation but may reveal new targets for therapy to ameliorate conditions such as ID and ASDs.