Paul M. Jenkins, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a better understanding of the mechanisms underlying protein transport into olfactory sensory neuron (OSN) cilia. Cilia are complex, microtubule-rich, organelles uniquely adapted for diverse cellular functions. Defects in cilia structure and function have been shown to underlie a number of human diseases. Sensory cilia on dendritic endings of OSNs compartmentalize signaling molecules, including cyclic nucleotide-gated (CNG) channels that are the primary targets of odorant-induced signaling. Olfactory CNG channels are responsible for the translation of the chemical signal from the odorant receptor into action potentials that are processed in the brain. Despite a wealth of knowledge about CNG channel structure and function, little is known about the mechanisms of their subcellular targeting. Movement of proteins within the cilia is governed by intraflagellar transport (IFT), an evolutionary conserved process that facilitates movement of cargo along cilia and flagella. In mammalian systems, the heterotrimeric Kinesin II plays a clear role in IFT; however, a role in ciliary transport has not yet been established for the homodirrieric KIF17. Newly published experiments I performed in Dr. Martens' laboratory have shown that KIF17 is required for ciliary enrichment of the olfactory CNG channel in a heterologous system; however, the role for KIF17 in native olfactory epithelium remains unknown. Recently it has been shown that the proper targeting of another ciliary protein, nephrocystin 1, in nasal respiratory epithelial cells requires the binding of phosphofurin acidic cluster sorting protein 1 (PACS-1). PACS-1 is an intracellular sorting protein that binds to phosphorylated serine residues contained in a cluster of acidic residues on the cargo protein. PACS-1 has been shown to interact with several other acidic cluster-containing ion channel, however no role has been defined for PACS-1 in olfactory cilia transport. It is my hypothesis that the ciliary targeting of olfactory CNG channels is dependent on both KIF17 and PACS-1 in native olfactory epithelium. The following specific aims are designed to test this hypothesis: Specific Aim 1: To investigate the role of KIF17 in the ciliary transport of the olfactory CNG channel in native olfactory sensory neurons. Specific Aim 2: To determine the effects of PACS-1 on CNG channel ciliary targeting. I will address these aims using confocal imaging, assays of olfactory function, and viral expression systems. My proposal provides a plan to further elucidate the mechanisms of ciliary enrichment of the olfactory CNG channel. Characterizing the general mechanisms of channel targeting will provide a better understanding of ciliary transport and give us further insight into the underlying etiologies of human ciliary disorders. [unreadable] [unreadable] [unreadable] [unreadable]

Developmental and epileptic encephalopathies (DEEs) are severe epilepsy syndromes that manifest in infancy or early childhood and are characterized by intractable seizures, neurological and behavioral deficits, and a high risk of Sudden Unexpected Death in Epilepsy (SUDEP). While most DEEs are linked to variants in genes encoding ion channels, especially that of voltage-gated sodium (NaV) and potassium (KV) channels, DEE-linked variants in non-ion-channel genes may provide important insights into the etiology of disease. Recently, whole exome sequencing of DEE patients by our Peking University colleagues in China identified variants of uncertain significance (VUS) in ANK3, suggesting that deficits in ion channel localization may contribute to disease mechanisms. A large body of literature has shown that ankyrin-G, encoded by the ANK3 gene, plays a fundamental role in the localization of voltage-gated ion channels to critical neuronal plasma membrane subdomains, including the axon initial segment (AIS) and nodes of Ranvier, which are the sites of action potential (AP) initiation and propagation, respectively. Recently, we have discovered novel functions for ankyrin-G in the regulation of inhibitory synapses and control of neuronal excitability. However, the mechanisms underlying the link between ANK3 and epilepsy are incompletely understood. The long-term goal of our work is to understand how ankyrin-G dysfunction contributes to the etiology of neurological disorders, like DEE. The objective of this application is to use a knockout and rescue strategy to understand the cellular and electrophysiological effects of variants identified in the Peking University DEE cohort. Our central hypothesis is that DEE-associated ANK3 variants affect ankyrin-G function in controlling localization and function of voltage- and ligand-gated ion channels resulting in pyramidal cell dysfunction, contributing to the pathophysiology of DEE. We will test our hypothesis by pursuing two Specific Aims: 1) To understand the effects of human DEE-associated ANK3 variants on ankyrin- G-mediated ion channel localization. 2) To determine the electrophysiological consequences of ANK3 DEE- associated variants. The mechanisms underlying the link between ANK3 and epilepsy are incompletely understood, yet the recently discovered VUS from the DEE patient cohort in China suggest a novel DEE mechanism. The results of this work will have an important positive impact on the understanding of how ankyrin- G regulates neuronal excitability and how ankyrin-G loss-of-function contributes to complex neurological disorders, such as DEE. Most of the published work investigating DEE mechanisms has focused on patient variants within ion channel genes. However, treatment strategies targeted against perturbations in ion channel function have proven ineffective for many DEEs. Our approach may yield novel targets, which could serve as a guide to develop innovative therapeutic strategies to treat DEE and possibly other idiopathic forms of epilepsy. In addition, the results of these studies may uncover previously unappreciated functions of AnkG in neurotransmission.

Thanks to the Simons Simplex Collection, the scientific community possesses dozens of highly reliable risk genes through the identification of rare de novo variants in patients with autism spectrum disorder (ASD). What is currently missing is a mechanism linking these genes into a convergent pathway that gives insight into disease etiology. In this proposal, we will test the hypothesis that ANK2 and SCN2A, two of the top genes implicated in ASD, are linked at the molecular level to control dendritic excitability. NaV1.2, product of the SCN2A gene, is a voltage-gated sodium channel found primarily in pyramidal neurons where it localizes to the axon initial segment (AIS) early in development. Later in development, NaV1.2 relocalizes to dendrites where it plays critical roles in synaptic plasticity and stability. The timing of the subcellular relocalization of NaV1.2 away from the AIS (~ 1 year in humans) also correlates with the onset of symptoms in ASD patients, suggesting that understanding the new role for NaV1.2 in dendrites may be critical for determining the etiology of SCN2A-associated ASD. While our group and others have shown that the intracellular scaffolding protein, ankyrin-G (ANK3), is necessary for NaV1.2 localization to the AIS, very little is known about the mechanisms underlying the dendritic localization of NaV1.2. Ankyrin-B, product of the ANK2 gene, is a member of the ankyrin gene family that shares significant homology with ankyrin-G, yet it is localized at high levels to dendrites where it may play a key role in NaV1.2 dendritic localization. Testing the hypothesis that ANK2 and SCN2A are linked at the molecular level to control dendritic excitability will have a positive impact by increasing our understanding of the mechanisms underlying synaptic alterations in ASD, which may provide novel targets for therapeutic intervention.