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Snyder, Joshua Clair

Overview:

My research objective is to translate basic science discoveries into treatments and cures for cancer. My work primarily focuses on G protein-coupled receptors (GPCR)s as a primary target in cancer. GPCRs are the largest family of receptors encoded by the genome, tightly control cell signaling, and regulate physiology in a diversity of tissues. As such, they are historically among the best targets for small molecule therapy in the clinic. The leucine-rich G protein-coupled receptor-5 (Lgr5) is particularly interesting since it is expressed in stem and cancer stem cells in a myriad of tissues. However, the function of Lgr5 is still largely unknown. Currently, my work utilizes cutting-edge multidisciplinary approaches to tackle this important challenge. This includes genetic engineering of fluorescently labelled mice, high-content confocal microscopy and cell behavior modeling, organoid culturing and genome editing, and fluorescent based approaches for high-throughput screening of receptor trafficking.


 


Using these approaches, we have made several important discoveries regarding Lgr5 that are facilitating future studies. We found that https://www.youtube.com/watch?v=7Hm-DOA_SO0&list=PLzVgBTaUqsfVDUvjVM... drives the formation of very long cellular protrusions that serve as scaffolds for cell signaling. We are continuing to investigate the https://www.youtube.com/watch?v=sTh9XzIX0xI">mechanistic importance of this finding using mouse models and intestinal organoid cultures to view this process in living mice. Another key observation was our discovery that Lgr5 internalization and trafficking are critical for regulating its function. Current work is now working toward a more mechanistic characterization of Lgr5 trafficking using fluorescent sensors that are capable of quantitatively assessing this dynamic process. We are also actively screening small molecule libraries in an effort to discover potential agonists/antagonists of Lgr5 that may be useful clinically in cancer treatment or in tissue regeneration. Lastly, we are continuing to develop additional technologies for directing gene expression in vivo in order to study the structure/function of tumor driver genes with greater sensitivity and more cellular resolution. Our strategy enables the https://www.youtube.com/watch?v=J6RjTv-QeyY">simultaneous expression of multiple driver genes in vivo along with the ability to monitor their effects on cell fate and behavior. Importantly, many of the tools that we have developed are broadly applicable to other receptors and candidate tumor driver genes for which we are open for collaboration.


 


 

Positions:

Assistant Professor of Surgery

Surgery, Surgical Sciences
School of Medicine

Assistant Research Professor of Cell Biology

Cell Biology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 2009

Ph.D. — University of Pittsburgh School of Medicine

Grants:

Akt/GSK-3 Signaling Cascade and the Actions of Dopamine

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Senior Research Scientist
Start Date
May 17, 2005
End Date
December 31, 2020

Establishing the molecular and cellular mechanisms of Lgr5 signaling for controlling cancer stem cell behavior

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 01, 2017
End Date
August 31, 2020

Drug Abuse: Discovering Ligands for Pertinent GPCRs

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Assistant Research Professor
Start Date
July 01, 2010
End Date
April 30, 2019

A Cancer Rainbow Mouse for Simultaneous Assessment of Multiple Oncogenes

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Assistant Research Professor
Start Date
February 09, 2015
End Date
January 31, 2018

Only the strong survive: Microenvironmental and genetic determinants of organotropism

Administered By
Surgery, Surgical Sciences
AwardedBy
Sage Bionetworks
Role
Principal Investigator
Start Date
July 05, 2017
End Date
July 31, 2017

Beta-catenin modulates dopamine dependent signal transduction and behavior.

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
PI-Fellow
Start Date
August 08, 2011
End Date
September 30, 2013
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Publications:

Inhibiting clathrin-mediated endocytosis of the leucine-rich G protein-coupled receptor-5 diminishes cell fitness.

The leucine-rich G protein-coupled receptor-5 (LGR5) is expressed in adult tissue stem cells of many epithelia, and its overexpression is negatively correlated with cancer prognosis. LGR5 potentiates WNT/β-catenin signaling through its unique constitutive internalization property that clears negative regulators of the WNT-receptor complex from the membrane. However, both the mechanism and physiological relevance of LGR5 internalization are unclear. Therefore, a natural product library was screened to discover LGR5 internalization inhibitors and gain mechanistic insight into LGR5 internalization. The plant lignan justicidin B blocked the constitutive internalization of LGR5. Justicidin B is structurally similar to more potent vacuolar-type H+-ATPase inhibitors, which all inhibited LGR5 internalization by blocking clathrin-mediated endocytosis. We then tested the physiological relevance of LGR5 internalization blockade in vivo A LGR5-rainbow (LBOW) mouse line was engineered to express three different LGR5 isoforms along with unique fluorescent protein lineage reporters in the same mouse. In this manner, the effects of each isoform on cell fate can be simultaneously assessed through simple fluorescent imaging for each lineage reporter. LBOW mice express three different forms of LGR5, a wild-type form that constitutively internalizes and two mutant forms whose internalization properties have been compromised by genetic perturbations within the carboxyl-terminal tail. LBOW was activated in the intestinal epithelium, and a year-long lineage-tracing course revealed that genetic blockade of LGR5 internalization diminished cell fitness. Together these data provide proof-of-concept genetic evidence that blocking the clathrin-mediated endocytosis of LGR5 could be used to pharmacologically control cell behavior.

Authors
Snyder, JC; Rochelle, LK; Ray, C; Pack, TF; Bock, CB; Lubkov, V; Lyerly, HK; Waggoner, AS; Barak, LS; Caron, MG
MLA Citation
Snyder, JC, Rochelle, LK, Ray, C, Pack, TF, Bock, CB, Lubkov, V, Lyerly, HK, Waggoner, AS, Barak, LS, and Caron, MG. "Inhibiting clathrin-mediated endocytosis of the leucine-rich G protein-coupled receptor-5 diminishes cell fitness." The Journal of biological chemistry 292.17 (April 2017): 7208-7222.
PMID
28275053
Source
epmc
Published In
The Journal of biological chemistry
Volume
292
Issue
17
Publish Date
2017
Start Page
7208
End Page
7222
DOI
10.1074/jbc.m116.756635

Distinct cortical and striatal actions of a β-arrestin-biased dopamine D2 receptor ligand reveal unique antipsychotic-like properties.

The current dopamine (DA) hypothesis of schizophrenia postulates striatal hyperdopaminergia and cortical hypodopaminergia. Although partial agonists at DA D2 receptors (D2Rs), like aripiprazole, were developed to simultaneously target both phenomena, they do not effectively improve cortical dysfunction. In this study, we investigate the potential for newly developed β-arrestin2 (βarr2)-biased D2R partial agonists to simultaneously target hyper- and hypodopaminergia. Using neuron-specific βarr2-KO mice, we show that the antipsychotic-like effects of a βarr2-biased D2R ligand are driven through both striatal antagonism and cortical agonism of D2R-βarr2 signaling. Furthermore, βarr2-biased D2R agonism enhances firing of cortical fast-spiking interneurons. This enhanced cortical agonism of the biased ligand can be attributed to a lack of G-protein signaling and elevated expression of βarr2 and G protein-coupled receptor (GPCR) kinase 2 in the cortex versus the striatum. Therefore, we propose that βarr2-biased D2R ligands that exert region-selective actions could provide a path to develop more effective antipsychotic therapies.

Authors
Urs, NM; Gee, SM; Pack, TF; McCorvy, JD; Evron, T; Snyder, JC; Yang, X; Rodriguiz, RM; Borrelli, E; Wetsel, WC; Jin, J; Roth, BL; O'Donnell, P; Caron, MG
MLA Citation
Urs, NM, Gee, SM, Pack, TF, McCorvy, JD, Evron, T, Snyder, JC, Yang, X, Rodriguiz, RM, Borrelli, E, Wetsel, WC, Jin, J, Roth, BL, O'Donnell, P, and Caron, MG. "Distinct cortical and striatal actions of a β-arrestin-biased dopamine D2 receptor ligand reveal unique antipsychotic-like properties." Proceedings of the National Academy of Sciences of the United States of America 113.50 (December 2016): E8178-E8186.
PMID
27911814
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
113
Issue
50
Publish Date
2016
Start Page
E8178
End Page
E8186
DOI
10.1073/pnas.1614347113

Chapter One - Ubiquitination and Deubiquitination of G Protein-Coupled Receptors.

The seven-transmembrane containing G protein-coupled receptors (GPCRs) constitute the largest family of cell-surface receptors. Transmembrane signaling by GPCRs is fundamental to many aspects of physiology including vision, olfaction, cardiovascular, and reproductive functions as well as pain, behavior and psychomotor responses. The duration and magnitude of signal transduction is tightly controlled by a series of coordinated trafficking events that regulate the cell-surface expression of GPCRs at the plasma membrane. Moreover, the intracellular trafficking profiles of GPCRs can correlate with the signaling efficacy and efficiency triggered by the extracellular stimuli that activate GPCRs. Of the various molecular mechanisms that impart selectivity, sensitivity and strength of transmembrane signaling, ubiquitination of the receptor protein plays an important role because it defines both trafficking and signaling properties of the activated GPCR. Ubiquitination of proteins was originally discovered in the context of lysosome-independent degradation of cytosolic proteins by the 26S proteasome; however a large body of work suggests that ubiquitination also orchestrates the downregulation of membrane proteins in the lysosomes. In the case of GPCRs, such ubiquitin-mediated lysosomal degradation engenders long-term desensitization of transmembrane signaling. To date about 40 GPCRs are known to be ubiquitinated. For many GPCRs, ubiquitination plays a major role in postendocytic trafficking and sorting to the lysosomes. This chapter will focus on the patterns and functional roles of GPCR ubiquitination, and will describe various molecular mechanisms involved in GPCR ubiquitination.

Authors
Jean-Charles, P-Y; Snyder, JC; Shenoy, SK
MLA Citation
Jean-Charles, P-Y, Snyder, JC, and Shenoy, SK. "Chapter One - Ubiquitination and Deubiquitination of G Protein-Coupled Receptors." Progress in molecular biology and translational science 141 (January 2016): 1-55. (Review)
PMID
27378754
Source
epmc
Published In
Progress in Molecular Biology and Translational Science
Volume
141
Publish Date
2016
Start Page
1
End Page
55
DOI
10.1016/bs.pmbts.2016.05.001

A rapid and affordable screening platform for membrane protein trafficking.

Membrane proteins regulate a diversity of physiological processes and are the most successful class of targets in drug discovery. However, the number of targets adequately explored in chemical space and the limited resources available for screening are significant problems shared by drug-discovery centers and small laboratories. Therefore, a low-cost and universally applicable screen for membrane protein trafficking was developed.This high-throughput screen (HTS), termed IRFAP-HTS, utilizes the recently described MarsCy1-fluorogen activating protein and the near-infrared and membrane impermeant fluorogen SCi1. The cell surface expression of MarsCy1 epitope-tagged receptors can be visualized by simple addition of SCi1. User-friendly, rapid, and quantitative detection occurs on a standard infrared western-blotting scanner. The reliability and robustness of IRFAP-HTS was validated by confirming human vasopressin-2 receptor and dopamine receptor-2 trafficking in response to agonist or antagonist. The IRFAP-HTS screen was deployed against the leucine-rich G protein-coupled receptor-5 (Lgr5). Lgr5 is expressed in stem cells, modulates Wnt/ß-catenin signaling, and is therefore a promising drug target. However, small molecule modulators have yet to be reported. The constitutive internalization of Lgr5 appears to be one primary mode through which its function is regulated. Therefore, IRFAP-HTS was utilized to screen 11,258 FDA-approved and drug-like small molecules for those that antagonize Lgr5 internalization. Glucocorticoids were found to potently increase Lgr5 expression at the plasma membrane.The IRFAP-HTS platform provides a versatile solution for screening more targets with fewer resources. Using only a standard western-blotting scanner, we were able to screen 5,000 compounds per hour in a robust and quantitative assay. Multi-purposing standardly available laboratory equipment eliminates the need for idiosyncratic and more expensive high-content imaging systems. The modular and user-friendly IRFAP-HTS is a significant departure from current screening platforms. Small laboratories will have unprecedented access to a robust and reliable screening platform and will no longer be limited by the esoteric nature of assay development, data acquisition, and post-screening analysis. The discovery of glucocorticoids as modulators for Lgr5 trafficking confirms that IRFAP-HTS can accelerate drug-discovery and drug-repurposing for even the most obscure targets.

Authors
Snyder, JC; Pack, TF; Rochelle, LK; Chakraborty, SK; Zhang, M; Eaton, AW; Bai, Y; Ernst, LA; Barak, LS; Waggoner, AS; Caron, MG
MLA Citation
Snyder, JC, Pack, TF, Rochelle, LK, Chakraborty, SK, Zhang, M, Eaton, AW, Bai, Y, Ernst, LA, Barak, LS, Waggoner, AS, and Caron, MG. "A rapid and affordable screening platform for membrane protein trafficking." BMC biology 13 (December 17, 2015): 107-.
PMID
26678094
Source
epmc
Published In
BMC Biology
Volume
13
Publish Date
2015
Start Page
107
DOI
10.1186/s12915-015-0216-3

Lgr4 and Lgr5 drive the formation of long actin-rich cytoneme-like membrane protrusions.

Embryonic development and adult tissue homeostasis require precise information exchange between cells and their microenvironment to coordinate cell behavior. A specialized class of ultra-long actin-rich filopodia, termed cytonemes, provides one mechanism for this spatiotemporal regulation of extracellular cues. We provide here a mechanism whereby the stem-cell marker Lgr5, and its family member Lgr4, promote the formation of cytonemes. Lgr4- and Lgr5-induced cytonemes exceed lengths of 80 µm, are generated through stabilization of nascent filopodia from an underlying lamellipodial-like network and functionally provide a pipeline for the transit of signaling effectors. As proof-of-principle, we demonstrate that Lgr5-induced cytonemes act as conduits for cell signaling by demonstrating that the actin motor and filopodial cargo carrier protein myosin X (Myo10) and the G-protein-coupled receptor (GPCR) signaling effector β-arrestin-2 (Arrb2) transit into cytonemes. This work delineates a biological function for Lgr4 and Lgr5 and provides the rationale to fully investigate Lgr4 and Lgr5 function and cytonemes in mammalian stem cell and cancer stem cell behavior.

Authors
Snyder, JC; Rochelle, LK; Marion, S; Lyerly, HK; Barak, LS; Caron, MG
MLA Citation
Snyder, JC, Rochelle, LK, Marion, S, Lyerly, HK, Barak, LS, and Caron, MG. "Lgr4 and Lgr5 drive the formation of long actin-rich cytoneme-like membrane protrusions." Journal of cell science 128.6 (March 2015): 1230-1240.
PMID
25653388
Source
epmc
Published In
Journal of cell science
Volume
128
Issue
6
Publish Date
2015
Start Page
1230
End Page
1240
DOI
10.1242/jcs.166322

The complete mitochondrial genome sequence of the Canada goose (Branta canadensis).

The Canada goose (Branta canadensis) entire mitochondrial genome of a bird from Western Pennsylvania has 16,760 bp (GenBank accession number NC 007011) and has been analyzed for gene locations, length, start codon and stop codons. This genome from a bird harvested during the non-migratory season is the REFSEQ and the haplotype is designated GCC-A. There are two rRNAs, 22 tRNAs, 13 protein-coding regions, and 1 displacement loop region. The base composition of mtDNA was A (30.2%), G (15.1%), C (32.1%), and T (22.6%), so the percentage of A and T (52.8%) was slightly higher than G and C. All genes except ND6 and eight tRNA genes (Gln, Ala, Asn, Cys, Tyr, Ser, Pro and Glu) are encoded on the heavy strand. The gene arrangement is the same as most birds and differs from mammals by an inversion of the mtDNA at the connection between the D-loop and the ND5 junctions.

Authors
Snyder, JC; Mackaness, CA; Sopher, MR; Huber, JP; Disantis, EJ; Senecal, AJ; Vaughn, BP; Desantis, RS; Tobelmann, PE; Balauff, NMH; Barry, PM; Show, MD; Speering, LH; Genareo, CA; Brenner, FJ; Ray, DB
MLA Citation
Snyder, JC, Mackaness, CA, Sopher, MR, Huber, JP, Disantis, EJ, Senecal, AJ, Vaughn, BP, Desantis, RS, Tobelmann, PE, Balauff, NMH, Barry, PM, Show, MD, Speering, LH, Genareo, CA, Brenner, FJ, and Ray, DB. "The complete mitochondrial genome sequence of the Canada goose (Branta canadensis)." Mitochondrial DNA 26.5 (January 2015): 672-673.
PMID
24148019
Source
epmc
Published In
Mitochondrial DNA
Volume
26
Issue
5
Publish Date
2015
Start Page
672
End Page
673
DOI
10.3109/19401736.2013.840601

Correction: The Stem Cell-Expressed Receptor Lgr5 Possesses Canonical and Functionally Active Molecular Determinants Critical to β-arrestin-2 Recruitment.

[This corrects the article on p. e84476 in vol. 8.].

Authors
Snyder, JC; Rochelle, LK; Barak, LS; Caron, MG
MLA Citation
Snyder, JC, Rochelle, LK, Barak, LS, and Caron, MG. "Correction: The Stem Cell-Expressed Receptor Lgr5 Possesses Canonical and Functionally Active Molecular Determinants Critical to β-arrestin-2 Recruitment." PloS one 9.1 (2014).
PMID
24427241
Source
epmc
Published In
PloS one
Volume
9
Issue
1
Publish Date
2014
DOI
10.1371/annotation/7f83735d-53d4-42f3-b3b2-b05dd5db059c

Integrated Approaches to Understand the Actions of GPCRs: The b-Arrestin-Dependent D2R Mediated Signaling Through Akt/GSK3

Authors
Caron, MG; Urs, NM; Snyder, JC; Peterson, SM
MLA Citation
Caron, MG, Urs, NM, Snyder, JC, and Peterson, SM. "Integrated Approaches to Understand the Actions of GPCRs: The b-Arrestin-Dependent D2R Mediated Signaling Through Akt/GSK3." (December 1, 2013): 99-100. (Chapter)
Source
scopus
Publish Date
2013
Start Page
99
End Page
100
DOI
10.1016/B978-0-12-800044-1.00086-6

Triphenylmethane dye activation of beta-arrestin.

β-Arrestins regulate G protein-coupled receptor signaling as competitive inhibitors and protein adaptors. Low molecular weight biased ligands that bind receptors and discriminate between the G protein dependent arm and β-arrestin, clathrin-associated arm of receptor signaling are considered therapeutically valuable as a result of this distinctive pharmacological behavior. Other than receptor agonists, compounds that activate β-arrestins are not available. We show that within minutes of exposure to the cationic triphenylmethane dyes malachite green and brilliant green, tissue culture cells recruit β-arrestins to clathrin scaffolds in a receptor-activation independent manner. In the presence of these compounds, G protein signaling is inhibited, ERK and GSK3β signaling are preserved, and the recruitment of the beta2-adaptin, AP2 adaptor complex to clathrin as well as transferrin internalization is reduced. Moreover, malachite green binds β-arrestin2-GFP coated immunotrap beads relative to GFP only coated beads. Triphenylmethane dyes are FDA approved for topical use on newborns as components of triple-dye preparations and are not approved but used effectively as aqueous antibiotics in fish husbandry. As possible carcinogens, their chronic ingestion in food preparations, particularly through farmed fish, is discouraged in the U.S. and Europe. Our results indicate triphenylmethane dyes as a result of novel pharmacology may have additional roles as β-arrestin/clathrin pathway signaling modulators in both pharmacology research and clinical therapy.

Authors
Barak, LS; Bai, Y; Snyder, JC; Wang, J; Chen, W; Caron, MG
MLA Citation
Barak, LS, Bai, Y, Snyder, JC, Wang, J, Chen, W, and Caron, MG. "Triphenylmethane dye activation of beta-arrestin." Biochemistry 52.32 (August 13, 2013): 5403-5414.
PMID
23865508
Source
pubmed
Published In
Biochemistry
Volume
52
Issue
32
Publish Date
2013
Start Page
5403
End Page
5414
DOI
10.1021/bi400217r

Constitutive internalization of the leucine-rich G protein-coupled receptor-5 (LGR5) to the trans-Golgi network.

LGR5 is a Wnt pathway associated G protein-coupled receptor (GPCR) that serves as a molecular determinant of stem cells in numerous tissues including the intestine, stomach, hair follicle, eye, and mammary gland. Despite its importance as a marker for this critical niche, little is known about LGR5 signaling nor the biochemical mechanisms and receptor determinants that regulate LGR5 membrane expression and intracellular trafficking. Most importantly, in cells LGR5 is predominantly intracellular, yet the mechanisms underlying this behavior have not been determined. In this work we elucidate a precise trafficking program for LGR5 and identify the motif at its C terminus that is responsible for the observed constitutive internalization. We show that this process is dependent upon dynamin GTPase activity and find that wild-type full-length LGR5 rapidly internalizes into EEA1- and Rab5-positive endosomes. However, LGR5 fails to rapidly recycle to the plasmid membrane through Rab4-positive vesicles, as is common for other GPCRs. Rather, internalized LGR5 transits through Rab7- and Rab9-positive vesicles, co-localizes in vesicles with Vps26, a retromer complex component that regulates retrograde trafficking to the trans-Golgi network (TGN) and reaches a steady-state distribution in the TGN within 2 h. Using mutagenesis, particularly of putative phosphorylation sites, we show that the amino acid pair, serine 861 and 864, is the principal C-tail determinant that mediates LGR5 constitutive internalization. The constitutive internalization of LGR5 to the TGN suggests the existence of novel biochemical roles for its Wnt pathway related, but ill defined signaling program.

Authors
Snyder, JC; Rochelle, LK; Lyerly, HK; Caron, MG; Barak, LS
MLA Citation
Snyder, JC, Rochelle, LK, Lyerly, HK, Caron, MG, and Barak, LS. "Constitutive internalization of the leucine-rich G protein-coupled receptor-5 (LGR5) to the trans-Golgi network." J Biol Chem 288.15 (April 12, 2013): 10286-10297.
PMID
23439653
Source
pubmed
Published In
The Journal of biological chemistry
Volume
288
Issue
15
Publish Date
2013
Start Page
10286
End Page
10297
DOI
10.1074/jbc.M112.447540

The stem cell-expressed receptor Lgr5 possesses canonical and functionally active molecular determinants critical to β-arrestin-2 recruitment.

Lgr5 is a membrane protein related to G protein-coupled receptors (GPCR)s whose expression identifies stem cells in multiple tissues and is strongly correlated with cancer. Despite the recent identification of endogenous ligands for Lgr5, its mode of signaling remains enigmatic. The ability to couple to G proteins and βarrestins are classical molecular behaviors of GPCRs that have yet to be observed for Lgr5. Therefore, the goal of this study was to determine if Lgr5 can engage a classical GPCR behavior and elucidate the molecular determinants of this process. Structural analysis of Lgr5 revealed several motifs consistent with its ability to recruit βarr2. Among them, a "SSS" serine cluster located at amino acid position 873-875 within the C-terminal tail (C-tail), is in a region consistent with other GPCRs that bind βarr2 with high-affinity. To test its functionality, a ligand-independent βarr2 translocation assay was implemented. We show that Lgr5 recruits βarr2 and that the "SSS" amino acids (873-875) are absolutely critical to this process. We also demonstrate that for full efficacy, this cluster requires other Lgr5 C-tail serines that were previously shown to be important for constitutive and βarr2 independent internalization of Lgr5. These data are proof of principle that a classical GPCR behavior can be manifested by Lgr5. The existence of alternative ligands or missing effectors of Lgr5 that scaffold this classical GPCR behavior and the downstream signaling pathways engaged should be considered. Characterizing Lgr5 signaling will be invaluable for assessing its role in tissue maintenance, repair, and disease.

Authors
Snyder, JC; Rochelle, LK; Barak, LS; Caron, MG
MLA Citation
Snyder, JC, Rochelle, LK, Barak, LS, and Caron, MG. "The stem cell-expressed receptor Lgr5 possesses canonical and functionally active molecular determinants critical to β-arrestin-2 recruitment. (Published online)" PLoS One 8.12 (2013): e84476-.
PMID
24386388
Source
pubmed
Published In
PloS one
Volume
8
Issue
12
Publish Date
2013
Start Page
e84476
DOI
10.1371/journal.pone.0084476

Deletion of GSK3β in D2R-expressing neurons reveals distinct roles for β-arrestin signaling in antipsychotic and lithium action.

Several studies in rodent models have shown that glycogen synthase kinase 3 β (GSK3β) plays an important role in the actions of antispychotics and mood stabilizers. Recently it was demonstrated that GSK3β through a β-arrestin2/protein kinase B (PKB or Akt)/protein phosphatase 2A (PP2A) signaling complex regulates dopamine (DA)- and lithium-sensitive behaviors and is required to mediate endophenotypes of mania and depression in rodents. We have previously shown that atypical antipsychotics antagonize DA D2 receptor (D2R)/β-arrestin2 interactions more efficaciously than G-protein-dependent signaling, whereas typical antipsychotics inhibit both pathways with similar efficacy. To elucidate the site of action of GSK3β in regulating DA- or lithium-sensitive behaviors, we generated conditional knockouts of GSK3β, where GSK3β was deleted in either DA D1- or D2-receptor-expressing neurons. We analyzed these mice for behaviors commonly used to test antipsychotic efficacy or behaviors that are sensitive to lithium treatment. Mice with deletion of GSK3β in D2 (D2GSK3β(-/-)) but not D1 (D1GSK3β(-/-)) neurons mimic antipsychotic action. However, haloperidol (HAL)-induced catalepsy was unchanged in either D2GSK3β(-/-) or D1GSK3β(-/-) mice compared with control mice. Interestingly, genetic stabilization of β-catenin, a downstream target of GSK3β, in D2 neurons did not affect any of the behaviors tested. Moreover, D2GSK3β(-/-) or D1GSK3β(-/-) mice showed similar responses to controls in the tail suspension test (TST) and dark-light emergence test, behaviors which were previously shown to be β-arrestin2- and GSK3β-dependent and sensitive to lithium treatment. Taken together these studies suggest that selective deletion of GSK3β but not stabilization of β-catenin in D2 neurons mimics antipsychotic action without affecting signaling pathways involved in catalepsy or certain mood-related behaviors.

Authors
Urs, NM; Snyder, JC; Jacobsen, JPR; Peterson, SM; Caron, MG
MLA Citation
Urs, NM, Snyder, JC, Jacobsen, JPR, Peterson, SM, and Caron, MG. "Deletion of GSK3β in D2R-expressing neurons reveals distinct roles for β-arrestin signaling in antipsychotic and lithium action." Proceedings of the National Academy of Sciences of the United States of America 109.50 (December 2012): 20732-20737.
PMID
23188793
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
109
Issue
50
Publish Date
2012
Start Page
20732
End Page
20737
DOI
10.1073/pnas.1215489109

β-Catenin-SOX2 signaling regulates the fate of developing airway epithelium.

Wnt-β-catenin signaling regulates cell fate during organ development and postnatal tissue maintenance, but its contribution to specification of distinct lung epithelial lineages is still unclear. To address this question, we used a Cre recombinase (Cre)-LoxP approach to activate canonical Wnt signaling ectopically in developing lung endoderm. We found that persistent activation of canonical Wnt signaling within distal lung endoderm was permissive for normal development of alveolar epithelium, yet led to the loss of developing bronchiolar epithelium and ectasis of distal conducting airways. Activation of canonical Wnt led to ectopic expression of a lymphoid-enhancing factor and a T-cell factor (LEF and TCF, respectively) and absence of SRY (sex-determining region Y)-box 2 (SOX2) and tumor protein p63 (p63) expression in proximal derivatives. Conditional loss of SOX2 in airways phenocopied epithelial differentiation defects observed with ectopic activation of canonical Wnt. Our data suggest that Wnt negatively regulates a SOX2-dependent signaling program required for developmental progression of the bronchiolar lineage.

Authors
Hashimoto, S; Chen, H; Que, J; Brockway, BL; Drake, JA; Snyder, JC; Randell, SH; Stripp, BR
MLA Citation
Hashimoto, S, Chen, H, Que, J, Brockway, BL, Drake, JA, Snyder, JC, Randell, SH, and Stripp, BR. "β-Catenin-SOX2 signaling regulates the fate of developing airway epithelium." J Cell Sci 125.Pt 4 (February 15, 2012): 932-942.
PMID
22421361
Source
pubmed
Published In
Journal of cell science
Volume
125
Issue
Pt 4
Publish Date
2012
Start Page
932
End Page
942
DOI
10.1242/jcs.092734

β-catenin-SOX2 signaling regulates the fate of developing airway epithelium

Wnt-β-catenin signaling regulates cell fate during organ development and postnatal tissue maintenance, but its contribution to specification of distinct lung epithelial lineages is still unclear. To address this question, we used a Cre recombinase (Cre)-LoxP approach to activate canonical Wnt signaling ectopically in developing lung endoderm. We found that persistent activation of canonical Wnt signaling within distal lung endoderm was permissive for normal development of alveolar epithelium, yet led to the loss of developing bronchiolar epithelium and ectasis of distal conducting airways. Activation of canonical Wnt led to ectopic expression of a lymphoid-enhancing factor and a T-cell factor (LEF and TCF, respectively) and absence of SRY (sex-determining region Y)-box 2 (SOX2) and tumor protein p63 (p63) expression in proximal derivatives. Conditional loss of SOX2 in airways phenocopied epithelial differentiation defects observed with ectopic activation of canonical Wnt. Our data suggest that Wnt negatively regulates a SOX2- dependent signaling program required for developmental progression of the bronchiolar lineage. © 2012. Published by The Company of Biologists Ltd.

Authors
Hashimoto, S; Chen, H; Que, J; Brockway, BL; Drake, JA; Snyder, JC; Randell, SH; Stripp, BR
MLA Citation
Hashimoto, S, Chen, H, Que, J, Brockway, BL, Drake, JA, Snyder, JC, Randell, SH, and Stripp, BR. "β-catenin-SOX2 signaling regulates the fate of developing airway epithelium." Development 139.9 (2012): 1-11.
Source
scival
Published In
Development (Cambridge)
Volume
139
Issue
9
Publish Date
2012
Start Page
1
End Page
11
DOI
10.1242/jcs.092734

Clara cells attenuate the inflammatory response through regulation of macrophage behavior.

Chronic lung diseases are marked by excessive inflammation and epithelial remodeling. Reduced Clara cell secretory function and corresponding decreases in the abundance of the major Clara cell secretory protein (CCSP) are characteristically seen in these disease states. We sought to define the impact of Clara cell and CCSP depletion on regulation of the lung inflammatory response. We used chemical and genetic mouse models of Clara cell and CCSP deficiency (CCSP(-/-)) coupled with Pseudomonas aeruginosa LPS elicited inflammation. Exposure of Clara cell-depleted or CCSP(-/-) mice to LPS resulted in augmented inflammation as assessed by polymorphonuclear leukocyte recruitment to the airspace. Gene expression analysis and pathway modeling of the CCSP(-/-) inflammatory response implicated increased TNF-alpha signaling. Consistent with this model was the demonstration of significantly elevated TNF-alpha in airway fluid of LPS-stimulated CCSP(-/-) mice compared with similarly exposed wild-type mice. Increased LPS-elicited TNF-alpha production was also observed in cultured lung macrophages from CCSP(-/-) mice compared with wild-type mice. We demonstrate that macrophages from Clara cell-depleted and CCSP(-/-) mice displayed increased Toll-like receptor 4 surface expression. Our results provide evidence that Clara cells can attenuate inflammation through regulation of macrophage behavior, and suggest that epithelial remodeling leading to reduced Clara cell secretory function is an important factor that increases the intensity of lung inflammation in chronic lung disease.

Authors
Snyder, JC; Reynolds, SD; Hollingsworth, JW; Li, Z; Kaminski, N; Stripp, BR
MLA Citation
Snyder, JC, Reynolds, SD, Hollingsworth, JW, Li, Z, Kaminski, N, and Stripp, BR. "Clara cells attenuate the inflammatory response through regulation of macrophage behavior." Am J Respir Cell Mol Biol 42.2 (February 2010): 161-171.
PMID
19423773
Source
pubmed
Published In
American journal of respiratory cell and molecular biology
Volume
42
Issue
2
Publish Date
2010
Start Page
161
End Page
171
DOI
10.1165/rcmb.2008-0353OC

Stem cells are dispensable for lung homeostasis but restore airways after injury.

Local tissue stem cells have been described in airways of the lung but their contribution to normal epithelial maintenance is currently unknown. We therefore developed aggregation chimera mice and a whole-lung imaging method to determine the relative contributions of progenitor (Clara) and bronchiolar stem cells to epithelial maintenance and repair. In normal and moderately injured airways chimeric patches were small in size and not associated with previously described stem cell niches. This finding suggested that single, randomly distributed progenitor cells maintain normal epithelial homeostasis. In contrast we found that repair following severe lung injury resulted in the generation of rare, large clonal cell patches that were associated with stem cell niches. This study provides evidence that epithelial stem cells are dispensable for normal airway homeostasis. We also demonstrate that stem cell activation and robust clonal cellular expansion occur only during repair from severe lung injury.

Authors
Giangreco, A; Arwert, EN; Rosewell, IR; Snyder, J; Watt, FM; Stripp, BR
MLA Citation
Giangreco, A, Arwert, EN, Rosewell, IR, Snyder, J, Watt, FM, and Stripp, BR. "Stem cells are dispensable for lung homeostasis but restore airways after injury." Proc Natl Acad Sci U S A 106.23 (June 9, 2009): 9286-9291.
PMID
19478060
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
106
Issue
23
Publish Date
2009
Start Page
9286
End Page
9291
DOI
10.1073/pnas.0900668106

Reparative capacity of airway epithelium impacts deposition and remodeling of extracellular matrix.

Defective epithelial repair in the setting of chronic lung disease has been suggested to contribute to uncontrolled extracellular matrix (ECM) deposition and development of fibrosis. We sought to directly test this hypothesis through gene expression profiling of total lung RNA isolated from mouse models of selective epithelial cell injury that are associated with either productive or abortive repair. Analysis of gene expression in repairing lungs of naphthalene-exposed mice revealed prominent clusters of up-regulated genes with putative roles in regulation of the extracellular matrix and cellular proliferation. Further analysis of tenascin C (Tnc), a representative matrix protein, in total lung RNA revealed a transient 4.5-fold increase in mRNA abundance 1 day after injury and a return to steady-state levels by Recovery Day 3. Tnc was deposited by the peribronchiolar mesenchyme immediately after injury and was remodeled to basement membrane subtending the bronchiolar epithelium during epithelial repair. Epithelial restitution was accompanied by a decrease in Tnc mRNA and protein expression to steady-state levels. In contrast, abortive repair using a transgenic model allowing ablation of all reparative cells led to a progressive increase in Tnc mRNA within lung tissue and accumulation of its gene product within the subepithelial mesenchyme of both conducting airways and alveoli. These data demonstrate that the ECM is dynamically remodeled in response to selective epithelial cell injury and that this process is activated without resolution in the setting of defective airway epithelial repair.

Authors
Snyder, JC; Zemke, AC; Stripp, BR
MLA Citation
Snyder, JC, Zemke, AC, and Stripp, BR. "Reparative capacity of airway epithelium impacts deposition and remodeling of extracellular matrix." Am J Respir Cell Mol Biol 40.6 (June 2009): 633-642.
PMID
18978301
Source
pubmed
Published In
American journal of respiratory cell and molecular biology
Volume
40
Issue
6
Publish Date
2009
Start Page
633
End Page
642
DOI
10.1165/rcmb.2008-0334OC

Molecular staging of epithelial maturation using secretory cell-specific genes as markers.

Bronchiolar Clara cells undergo phenotypic changes during development and in disease. These changes are poorly described due to a paucity of molecular markers. We used chemical and transgenic approaches to ablate Clara cells, allowing identification of their unique gene expression profile. Flavin monooxygenase 3 (Fmo3), paraoxonase 1 (Pon1), aldehyde oxidase 3 (Aox3), and claudin 10 (Cldn10) were identified as novel Clara cell markers. New and existing Clara cell marker genes were categorized into three classes based on their unique developmental expression pattern. Cldn10 was uniformly expressed in the epithelium at Embryonic Day (E)14.5 and became restricted to secretory cells at E18.5. This transition was defined by induction of CCSP. Maturation of secretory cells was associated with progressive increases in the expression of Fmo3, Pon1, Aox3, and Cyp2f2 between late embryonic and postnatal periods. Messenger RNA abundance of all categories of genes was dramatically decreased after naphthalene-induced airway injury, and displayed a sequence of temporal induction during repair that suggested sequential secretory cell maturation. We have defined a broader repertoire of Clara cell-specific genes that allows staging of epithelial maturation during development and repair.

Authors
Zemke, AC; Snyder, JC; Brockway, BL; Drake, JA; Reynolds, SD; Kaminski, N; Stripp, BR
MLA Citation
Zemke, AC, Snyder, JC, Brockway, BL, Drake, JA, Reynolds, SD, Kaminski, N, and Stripp, BR. "Molecular staging of epithelial maturation using secretory cell-specific genes as markers." Am J Respir Cell Mol Biol 40.3 (March 2009): 340-348.
PMID
18757308
Source
pubmed
Published In
American journal of respiratory cell and molecular biology
Volume
40
Issue
3
Publish Date
2009
Start Page
340
End Page
348
DOI
10.1165/rcmb.2007-0380OC

Endogenous lung stem cells and contribution to disease.

Epithelial branching during the process of lung development results in the establishment of distinct functional zones, each of which is characterized by a unique cellular composition and repertoire of local progenitor cells. Significant new insights into cellular and molecular mechanisms of epithelial maintenance that provide insights into the pathophysiology of lung disease have been made in recent years. This review focuses on the complex structure-function relationship in the airway epithelium, how this epithelium is maintained in the normal state and repaired following injury, and how deregulation may contribute to airway disease and cancer.

Authors
Snyder, JC; Teisanu, RM; Stripp, BR
MLA Citation
Snyder, JC, Teisanu, RM, and Stripp, BR. "Endogenous lung stem cells and contribution to disease." J Pathol 217.2 (January 2009): 254-264. (Review)
PMID
19039828
Source
pubmed
Published In
The Journal of Pathology
Volume
217
Issue
2
Publish Date
2009
Start Page
254
End Page
264
DOI
10.1002/path.2473

CCSP regulates cross talk between secretory cells and both ciliated cells and macrophages of the conducting airway.

Pulmonary host defense employs a combination of biochemical and biophysical activities to recognize, inactivate, and mediate clearance of environmental agents as well as modulate the overall response to such challenge. Dysregulation of the inflammatory arm of this response is associated with chronic lung diseases (CLD) including cystic fibrosis and chronic obstructive lung disease. Although mechanisms mediating immunoregulation are incompletely characterized, decrements in levels of the nonciliated secretory cell product Clara cell secretory protein (CCSP) in numerous CLD and identification of proinflammatory state in mice homozygous for a null allele of the CCSP gene (CCSP-/-) suggest a central role for the nonciliated secretory cell in this process. In an effort to determine the molecular basis for immunoregulatory defects associated with CCSP deficiency, we utilized difference gel electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight to compare the proteomes of wild-type and CCSP-/- mice. We demonstrate a shift in the isoelectric point of the immunomodulatory protein annexin A1 (ANXA1) to more acidic isoforms in CCSP-/- mice. Similar ANXA1 mRNA and protein abundance in wild-type and CCSP-/- tissue and identical localization of ANXA1 protein to alveolar macrophages and the ciliary bed of ciliated cells demonstrated that CCSP deficiency was associated exclusively with altered posttranslational modification of ANXA1. These results suggest that both long- and short-range paracrine signaling between nonciliated secretory cells and cells of the immune system and epithelium impact modification of cell type-specific proteins and implicate nonciliated secretory cells in a regulatory axis that might integrate critical aspects of host defense.

Authors
Reynolds, SD; Reynolds, PR; Snyder, JC; Whyte, F; Paavola, KJ; Stripp, BR
MLA Citation
Reynolds, SD, Reynolds, PR, Snyder, JC, Whyte, F, Paavola, KJ, and Stripp, BR. "CCSP regulates cross talk between secretory cells and both ciliated cells and macrophages of the conducting airway." Am J Physiol Lung Cell Mol Physiol 293.1 (July 2007): L114-L123.
PMID
17384087
Source
pubmed
Published In
American journal of physiology. Lung cellular and molecular physiology
Volume
293
Issue
1
Publish Date
2007
Start Page
L114
End Page
L123
DOI
10.1152/ajplung.00014.2007
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