Green William N.

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Last Name
Green
First Name
William N.
ORCID
0000-0003-2167-1391

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Now showing 1 - 8 of 8
  • Article
    Selective and regulated trapping of nicotinic receptor weak base ligands and relevance to smoking cessation
    (eLife, 2017-07-18) Govind, Anitha P. ; Vallejo, Yolanda F. ; Stolz, Jacob R. ; Yan, Jing-Zhi ; Swanson, Geoffrey T. ; Green, William N.
    To better understand smoking cessation, we examined the actions of varenicline (Chantix) during long-term nicotine exposure. Varenicline reduced nicotine upregulation of α4β2-type nicotinic receptors (α4β2Rs) in live cells and neurons, but not for membrane preparations. Effects on upregulation depended on intracellular pH homeostasis and were not observed if acidic pH in intracellular compartments was neutralized. Varenicline was trapped as a weak base in acidic compartments and slowly released, blocking 125I-epibatidine binding and desensitizing α4β2Rs. Epibatidine itself was trapped; 125I-epibatidine slow release from acidic vesicles was directly measured and required the presence of α4β2Rs. Nicotine exposure increased epibatidine trapping by increasing the numbers of acidic vesicles containing α4β2Rs. We conclude that varenicline as a smoking cessation agent differs from nicotine through trapping in α4β2R-containing acidic vesicles that is selective and nicotine-regulated. Our results provide a new paradigm for how smoking cessation occurs and suggest how more effective smoking cessation reagents can be designed.
  • Article
    Synaptic activity regulates AMPA receptor trafficking through different recycling pathways
    (eLife Sciences Publications, 2015-05-13) Zheng, Ning ; Jeyifous, Okunola ; Munro, Charlotte ; Montgomery, Johanna M. ; Green, William N.
    Changes in glutamatergic synaptic strength in brain are dependent on AMPA-type glutamate receptor (AMPAR) recycling, which is assumed to occur through a single local pathway. In this study, we present evidence that AMPAR recycling occurs through different pathways regulated by synaptic activity. Without synaptic stimulation, most AMPARs recycled in dynamin-independent endosomes containing the GTPase, Arf6. Few AMPARs recycled in dynamin-dependent endosomes labeled by transferrin receptors (TfRs). AMPAR recycling was blocked by alterations in the GTPase, TC10, which co-localized with Arf6 endosomes. TC10 mutants that reduced AMPAR recycling had no effect on increased AMPAR levels with long-term potentiation (LTP) and little effect on decreased AMPAR levels with long-term depression. However, internalized AMPAR levels in TfR-containing recycling endosomes increased after LTP, indicating increased AMPAR recycling through the dynamin-dependent pathway with synaptic plasticity. LTP-induced AMPAR endocytosis is inconsistent with local recycling as a source of increased surface receptors, suggesting AMPARs are trafficked from other sites.
  • Article
    Editorial: role of protein palmitoylation in synaptic plasticity and neuronal differentiation
    (Frontiers Media, 2020-07-09) Yoshii, Akira ; Green, William N.
    Protein palmitoylation, the reversible addition of palmitate to proteins, is a dynamic post-translational modification. Both membrane (e.g., channels, transporters, and receptors) and cytoplasmic proteins (e.g., cell adhesion, scaffolding, cytoskeletal, and signaling molecules) are substrates. In mammals, palmitoylation is mediated by 23-24 palmitoyl acyltransferases (PATs), also called ZDHHCs for their catalytic aspartate-histidine-histidine-cysteine (DHCC) domain. PATs are integral membrane proteins found in cellular membranes. In the palmitoylation cycle, palmitate is removed by the depalmitoylation enzymes, acyl palmitoyl transferases (APT1 and 2), and α/β Hydrolase domain-containing protein 17 (ABHD17A-C). These are cytoplasmic proteins that are targeted to membranes where they are substrates for PATs. The second class of depalmitoylating enzymes are palmitoyl thioesterases, PPT1 and 2, discovered through their association with infantile neuronal ceroid lipofuscinosis. These are secreted proteins found in the lumen of intracellular organelles, primarily lysosomes, where their function as depalmitoylating enzymes is unclear.
  • Article
    Activity-dependent Golgi satellite formation in dendrites reshapes the neuronal surface glycoproteome
    (eLife Sciences Publications, 2021-09-21) Govind, Anitha P. ; Jeyifous, Okunola ; Russell, Theron A. ; Yi, Zola ; Weigel, Aubrey V. ; Ramaprasad, Abhijit ; Newell, Luke ; Ramos, William ; Valbuena, Fernando M. ; Casler, Jason C. ; Yan, Jing-Zhi ; Glick, Benjamin S. ; Swanson, Geoffrey T. ; Lippincott-Schwartz, Jennifer ; Green, William N.
    Activity-driven changes in the neuronal surface glycoproteome are known to occur with synapse formation, plasticity, and related diseases, but their mechanistic basis and significance are unclear. Here, we observed that N-glycans on surface glycoproteins of dendrites shift from immature to mature forms containing sialic acid in response to increased neuronal activation. In exploring the basis of these N-glycosylation alterations, we discovered that they result from the growth and proliferation of Golgi satellites scattered throughout the dendrite. Golgi satellites that formed during neuronal excitation were in close association with endoplasmic reticulum (ER) exit sites and early endosomes and contained glycosylation machinery without the Golgi structural protein, GM130. They functioned as distal glycosylation stations in dendrites, terminally modifying sugars either on newly synthesized glycoproteins passing through the secretory pathway or on surface glycoproteins taken up from the endocytic pathway. These activities led to major changes in the dendritic surface of excited neurons, impacting binding and uptake of lectins, as well as causing functional changes in neurotransmitter receptors such as nicotinic acetylcholine receptors. Neural activity thus boosts the activity of the dendrite’s satellite micro-secretory system by redistributing Golgi enzymes involved in glycan modifications into peripheral Golgi satellites. This remodeling of the neuronal surface has potential significance for synaptic plasticity, addiction, and disease.
  • Article
    ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms
    (American Society for Cell Biology, 2020-10-28) Casler, Jason C. ; Zajac, Allison L. ; Valbuena, Fernando M. ; Sparvoli, Daniela ; Jeyifous, Okunola ; Turkewitz, Aaron ; Horne-Badovinac, Sally ; Green, William N. ; Glick, Benjamin S.
    Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block export of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescentsecretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory proteinESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used withmany model organisms.
  • Article
    CASK regulates SAP97 conformation and its interactions with AMPA and NMDA receptors
    (Society for Neuroscience, 2013-07-17) Lin, Eric I. ; Jeyifous, Okunola ; Green, William N.
    SAP97 interacts with AMPA receptors (AMPARs) and NMDA receptors (NMDARs) during sorting and trafficking to synapses. Here we addressed how SAP97 distinguishes between AMPARs and NMDARs and what role the adaptor/scaffold protein, CASK, plays in the process. Using intramolecular SAP97 Förster resonance energy transfer sensors, we demonstrated that SAP97 is in “extended” or “compact” conformations in vivo. SAP97 conformation was regulated by a direct interaction between SAP97 and CASK through L27 protein-interaction domains on each protein. Unbound SAP97 was mostly in the compact conformation, while CASK binding stabilized it in an extended conformation. In HEK cells and rat hippocampal neurons, SAP97 in the compact conformation preferentially associated and colocalized with GluA1-containing AMPARs, and in the extended conformation colocalized with GluN2B-containing NMDARs. Altogether, our findings suggest a molecular mechanism by which CASK binding regulates SAP97 conformation and its subsequent sorting and synaptic targeting of AMPARs and NMDARs during trafficking to synapses.
  • Preprint
    Palmitoylation regulates glutamate receptor distributions in postsynaptic densities through control of PSD95 conformation and orientation
    ( 2016-08) Jeyifous, Okunola ; Lin, Eric I. ; Chen, Xiaobing ; Antinone, Sarah E. ; Mastro, Ryan ; Drisdel, Renaldo ; Reese, Thomas S. ; Green, William N.
    PSD95 and SAP97 are homologous scaffold proteins with different N-terminal domains, possessing either a palmitoylation site (PSD95) or an L27 domain (SAP97). Here, we measured PSD95 and SAP97 conformation in vitro and in postsynaptic densities (PSDs) using FRET and electron microscopy, and examined how conformation regulated interactions with AMPA-type and NMDAtype glutamate receptors (AMPARs/NMDARs). Palmitoylation of PSD95 changed its conformation from a compact to an extended configuration. PSD95 associated with AMPARs (via TARP subunits) or NMDARs (via GluN2B subunits) only in its palmitoylated and extended conformation. In contrast, SAP97 in its extended conformation associates with NMDARs but not with AMPARs. Within PSDs, PSD95 and SAP97 were largely in the extended conformation, but had different orientations. PSD95 oriented perpendicular to the PSD membrane, with its palmitoylated, N-terminal domain at the membrane. SAP97 oriented parallel to the PSD membrane, likely as a dimer through interactions of its N-terminal, L27 domain. Changing PSD95 palmitoylation in PSDs altered PSD95 and AMPAR levels but did not affect NMDAR levels. These results indicate that in PSDs, PSD95 palmitoylation, conformation and its interactions are dynamic when associated with AMPARs, and more stable when associated with NMDARs. Altogether, our results are consistent with differential regulation of PSD95 palmitoylation in PSDs resulting from the clustering of palmitoylating and depalmitoylating enzymes into AMPAR nanodomains segregated away from NMDAR nanodomains.
  • Article
    Trapping of nicotinic acetylcholine receptor ligands assayed by in vitro cellular studies and in vivo PET imaging
    (Society for Neuroscience, 2023-01-04) Zhang, Hannah J. ; Zammit, Matthew ; Kao, Chien-Min ; Govind, Anitha P. ; Mitchell, Samuel J. ; Holderman, Nathanial ; Bhuiyan, Mohammed ; Freifelder, Richard ; Kucharski, Anna ; Zhuang, Xiaoxi ; Mukherjee, Jogeshwar ; Chen, Chin-Tu ; Green, William N.
    A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membrane-permeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pKa and high affinity for α4β2-type nicotinic receptors (α4β2Rs) are trapped in intracellular acidic vesicles containing α4β2Rs in vitro. Nicotine, with lower pKa and α4β2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligands in vivo in male and female mouse brain and identifying the trapping brain organelle in vitro as Golgi satellites (GSats). Two PET 18F-labeled imaging ligands were chosen: [18F]2-FA85380 (2-FA) with varenicline-like pKa and affinity and [18F]Nifene with nicotine-like pKa and affinity. [18F]2-FA PET-imaging kinetics were very slow consistent with 2-FA trapping in α4β2R-containing GSats. In contrast, [18F]Nifene kinetics were rapid, consistent with its binding to α4β2Rs but no trapping. Specific [18F]2-FA and [18F]Nifene signals were eliminated in β2 subunit knock-out (KO) mice or by acute nicotine (AN) injections demonstrating binding to sites on β2-containing receptors. Chloroquine (CQ), which dissipates GSat pH gradients, reduced [18F]2-FA distributions while having little effect on [18F]Nifene distributions in vivo consistent with only [18F]2-FA trapping in GSats. These results are further supported by in vitro findings where dissipation of GSat pH gradients blocks 2-FA trapping in GSats without affecting Nifene. By combining in vitro and in vivo imaging, we mapped both the brain-wide and subcellular distributions of weak-base nicotinic receptor ligands. We conclude that ligands, such as varenicline, are trapped in neurons in α4β2R-containing GSats, which results in very slow release long after nicotine is gone after smoking.