S-Palmitoylation as a Regulatory Mechanism for Localization and Function of Neuronal Substrates Involved In Action Potential Initiation and Propagation

Abstract

S-palmitoylation, the covalent addition of a 16-carbon fatty acid to cysteine residues of proteins, anchors proteins to membrane domains to regulate protein localization, trafficking, stability, and function, and plays broad roles in promoting, facilitating, and fine-tuning critical neuronal processes such as development, synaptic plasticity, axonal growth, dendritogenesis, neuronal transmission, and neuronal excitability. Understanding the role of palmitoylation for neuronal substrate function provides important insight into how normal processes may be regulated and in turn how pathophysiological processes may arise when palmitoylation goes awry. The goal of this thesis work was to explore the role of S-palmitoylation for proper localization, stability, and functions of two neuronal proteins, the voltage-gated sodium channel (VGSC) β1 subunit and ankyrin-B, both previously unknown to be palmitoylated but highly associated with neurological diseases such as Early Infantile-Developmental and Epileptic Encephalopathy (EIEE52) and autism spectrum disorder (ASD), respectively.

VGSC β1 subunits are multifunctional proteins that canonically modulate VGSC α subunit biophysical properties and cell surface localization, while also participating in cell-cell and cell-matrix adhesion, necessary for intracellular signal transduction, cell migration, and differentiation. Recently, VGSC β1 subunits were also shown to undergo proteolytic cleavage to generate a β1 intracellular C-terminal domain that translocates to the nucleus and participates in transcriptional regulation, a potentially important mechanism underlying EIEE52. However, the mechanisms that regulate proteolysis of β1 were unknown. We demonstrate in this work that β1 subunit palmitoylation regulates β1 cell surface localization and subsequent β1 proteolytic cleavage at the plasma membrane of heterologous cells. This work provides important insight into a regulatory mechanism that regulates β1 cleavage, which has important consequences for downstream cellular excitability via alterations in gene transcription.

Ankyrins act as general adaptor proteins that localize membrane, cytoskeletal, and cytoplasmic proteins at specialized membrane domains, and promote cell polarity and function in many vertebrate tissues. One ankyrin family member, ankyrin-B, has recently been found to scaffold VGSC Nav1.2 at the dendritic membrane of cortical pyramidal neurons to promote dendritic excitability and synaptic plasticity. How ankyrin-B itself targets to dendritic membranes to cluster VGSC Nav1.2 there was previously unknown. We show here that ankyrin-B is modified by S-palmitoylation, and that palmitoylation of ankyrin-B is required for ankyrin-B localization at dendritic membranes necessary for subsequent Nav1.2 clustering at dendrites. We demonstrate that ankyrin-B palmitoylation is mediated by the palmitoyl acyl transferase enzyme zDHHC17. We also demonstrate that the axonal cargo transport of ankyrin-B is unaffected by palmitoylation, thereby highlighting two pools of ankyrin-B: a dendritic palmitoylation-dependent pool and a vesicular, axonal palmitoylation-independent pool. Targeting palmitoylation could represent an opportunity to specifically target the dendritic pool of ankyrin-B without affecting the axonal pool. These findings reveal an important regulatory mechanism underlying dendritic localization and function of ankyrin-B in neurons, which may have important implications in the etiology of ankyrin-B-associated autism spectrum disorders.

Given that VGSCs, β subunits, and ankyrins form macromolecular complexes essential for proper neuronal activity, and that disruption of any of these members in complex can lead to severe neurological diseases, this work provides a glimpse into a more mechanistic understanding of the regulatory mechanisms underlying proper localization and function of these substrates individually, so that future work can investigate the role palmitoylation plays for the formation and maintenance of ankyrin-B/Nav1.2/β1 macromolecular complexes under normal and pathophysiological conditions.