Publications by authors named "Jonathan G Hanley"

36 Publications

c-Abl regulates a synaptic plasticity-related transcriptional program involved in memory and learning.

Prog Neurobiol 2021 Oct 18;205:102122. Epub 2021 Jul 18.

Department of Cell & Molecular Biology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago de Chile, Chile; Centre for Aging and Regeneration (CARE-UC), Chile. Electronic address:

Memory consolidation requires activation of a gene expression program that allows de novo protein synthesis. But the molecular mechanisms that favour or restrict that program are poorly understood. The kinase c-Abl can modulate gene expression through transcription factors and chromatin modifiers. Here, we show that c-Abl ablation in the brain improves learning acquisition and memory consolidation in mice. Its absence also affects gene expression profiles in the mouse hippocampus. We found that genes involved in synaptic plasticity and actin cytoskeleton dynamics, such as Arp2 and Thorase, are up-regulated at the mRNA and protein levels in trained c-Abl KO mice and by a chemical-LTP stimulus. Trained c-Abl KO mice also show that dendritic spines are larger than in wild-type mice and present at a higher density. These results indicate that c-Abl kinase is an important part of the mechanism that limits or restricts signalling of relevant gene programs involved in morphological and functional spine changes upon neuronal stimulation.
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http://dx.doi.org/10.1016/j.pneurobio.2021.102122DOI Listing
October 2021

Regulation of AMPAR expression by microRNAs.

Neuropharmacology 2021 Jul 15;197:108723. Epub 2021 Jul 15.

School of Biochemistry, Biomedical Sciences Building, University of Bristol, University Walk, Bristol, BS8 1TD, UK. Electronic address:

AMPA receptors (AMPARs) are the major excitatory neurotransmitter receptor in the brain, and their expression at synapses is a critical determinant of synaptic transmission and therefore brain function. Synaptic plasticity involves increases or decreases in synaptic strength, caused by changes in the number or subunit-specific subtype of AMPARs expressed at synapses, and resulting in modifications of functional connectivity of neuronal circuits, a process which is thought to underpin learning and the formation or loss of memories. Furthermore, numerous neurological disorders involve dysregulation of excitatory synaptic transmission or aberrant recruitment of plasticity processes. MicroRNAs (miRNAs) repress the translation of target genes by partial complementary base pairing with mRNAs, and are the core component of a mechanism widely used in a range of cell processes for regulating protein translation. MiRNA-dependent translational repression can occur locally in neuronal dendrites, close to synapses, and can also result in relatively rapid changes in protein expression. MiRNAs are therefore well-placed to regulate synaptic plasticity via the local control of AMPAR subunit synthesis, and can also result in synaptic dysfunction in the event of dysregulation in disease. Here, I will review the miRNAs that have been identified as playing a role in physiological or pathological changes in AMPAR subunit expression at synapses, focussing on miRNAs that target mRNAs encoding AMPAR subunits, and on miRNAs that target AMPAR accessory proteins involved in AMPAR trafficking and hence the regulation of AMPAR synaptic localisation.
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http://dx.doi.org/10.1016/j.neuropharm.2021.108723DOI Listing
July 2021

Mechanisms of AMPA Receptor Endosomal Sorting.

Front Mol Neurosci 2018 5;11:440. Epub 2018 Dec 5.

Centre for Synaptic Plasticity and School of Biochemistry, University of Bristol, Bristol, United Kingdom.

The regulation of synaptic AMPA receptors (AMPARs) is critical for excitatory synaptic transmission, synaptic plasticity and the consequent formation of neural circuits during brain development and their modification during learning and memory processes. The number of synaptic AMPARs is regulated through endocytosis, exocytosis and endosomal sorting that results in recycling back to the plasma membrane or degradation in the lysosome. Hence, endo-lysosomal sorting is vitally important in maintaining AMPAR expression at the synapse, and the dynamic regulation of these trafficking events is a key component of synaptic plasticity. A reduction in synaptic strength such as in long-term depression (LTD) involves AMPAR sorting to lysosomes to reduce synaptic AMPAR number, whereas long-term potentiation (LTP) involves an increase in AMPAR recycling to increase the number of AMPARs at synapses. Here, we review our current understanding of the endosomal trafficking routes taken by AMPARs, and the mechanisms involved in AMPAR endosomal sorting, focussing on the numerous AMPAR associated proteins that have been implicated in this complex process. We also discuss how these events are dysregulated in brain disorders.
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http://dx.doi.org/10.3389/fnmol.2018.00440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289981PMC
December 2018

The Regulation of AMPA Receptor Endocytosis by Dynamic Protein-Protein Interactions.

Front Cell Neurosci 2018 11;12:362. Epub 2018 Oct 11.

Centre for Synaptic Plasticity and School of Biochemistry, University of Bristol, Bristol, United Kingdom.

The precise regulation of AMPA receptor (AMPAR) trafficking in neurons is crucial for excitatory neurotransmission, synaptic plasticity and the consequent formation and modification of neural circuits during brain development and learning. Clathrin-mediated endocytosis (CME) is an essential trafficking event for the activity-dependent removal of AMPARs from the neuronal plasma membrane, resulting in a reduction in synaptic strength known as long-term depression (LTD). The regulated AMPAR endocytosis that underlies LTD is caused by specific modes of synaptic activity, most notably stimulation of NMDA receptors (NMDARs) and metabotropic glutamate receptors (mGluRs). Numerous proteins associate with AMPAR subunits, directly or indirectly, to control their trafficking, and therefore the regulation of these protein-protein interactions in response to NMDAR or mGluR signaling is a critical feature of synaptic plasticity. This article reviews the protein-protein interactions that are dynamically regulated during synaptic plasticity to modulate AMPAR endocytosis, focussing on AMPAR binding proteins and proteins that bind the core endocytic machinery. In addition, the mechanisms for the regulation of protein-protein interactions are considered, as well as the functional consequences of these dynamic interactions on AMPAR endocytosis.
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http://dx.doi.org/10.3389/fncel.2018.00362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193100PMC
October 2018

NMDAR-dependent Argonaute 2 phosphorylation regulates miRNA activity and dendritic spine plasticity.

EMBO J 2018 06 30;37(11). Epub 2018 Apr 30.

Centre for Synaptic Plasticity and School of Biochemistry, University of Bristol, Bristol, UK

MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins to form the RNA-induced silencing complex (RISC), underpinning a powerful mechanism for fine-tuning protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)-dependent synaptic plasticity by modulating the translation of proteins involved in dendritic spine morphogenesis or synaptic transmission. However, it is unknown how NMDAR stimulation stimulates RISC activity to rapidly repress translation of synaptic proteins. We show that NMDAR stimulation transiently increases Akt-dependent phosphorylation of Ago2 at S387, which causes an increase in binding to GW182 and a rapid increase in translational repression of via miR-134. Furthermore, NMDAR-dependent down-regulation of endogenous LIMK1 translation in dendrites and dendritic spine shrinkage requires phospho-regulation of Ago2 at S387. AMPAR trafficking and hippocampal LTD do not involve S387 phosphorylation, defining this mechanism as a specific pathway for structural plasticity. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA-mediated translational repression to control dendritic spine morphology.
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http://dx.doi.org/10.15252/embj.201797943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983126PMC
June 2018

Cortactin regulates endo-lysosomal sorting of AMPARs via direct interaction with GluA2 subunit.

Sci Rep 2018 03 7;8(1):4155. Epub 2018 Mar 7.

Centre for Synaptic Plasticity and School of Biochemistry, Biomedical Sciences Building, University of Bristol, University Walk, Bristol, BS8, 1TD, UK.

AMPA receptor (AMPAR) trafficking is a key determinant of synaptic strength and synaptic plasticity. Under basal conditions, constitutive trafficking maintains surface AMPARs by internalization into the endosomal system, where the majority are sorted and targeted for recycling back to the plasma membrane. NMDA receptor (NMDAR)-dependent Long-Term Depression (LTD) is characterised by a reduction in synaptic strength, and involves endosomal sorting of AMPARs away from recycling pathways to lysosomes. The mechanisms that determine whether AMPARs are trafficked to lysosomes or to recycling endosomes, especially in response to NMDAR stimulation, are unclear. Here, we define a role for the actin-regulatory protein cortactin as a mediator of AMPAR endosomal sorting by direct interaction with the GluA2 subunit. Disrupting GluA2-cortactin binding in neurons causes the targeting of GluA2/A3-containing receptors to lysosomes and their consequent degradation, resulting in a loss of surface and synaptic GluA2 under basal conditions and an occlusion of subsequent LTD expression. Furthermore, we show that NMDAR stimulation causes a dissociation of endogenous cortactin from GluA2 via tyrosine phosphorylation of cortactin. These results demonstrate that cortactin maintains GluA2/A3 levels by directing receptors away from lysosomes, and that disrupting GluA2-cortactin interactions to target GluA2/A3 to lysosomes is an essential component of LTD expression.
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http://dx.doi.org/10.1038/s41598-018-22542-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841360PMC
March 2018

Differential regulation of the Rac1 GTPase-activating protein (GAP) BCR during oxygen/glucose deprivation in hippocampal and cortical neurons.

J Biol Chem 2017 12 18;292(49):20173-20183. Epub 2017 Oct 18.

Centre for Synaptic Plasticity and School of Biochemistry, Biomedical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.

Brain ischemia causes oxygen and glucose deprivation (OGD) in neurons, triggering a cascade of events leading to synaptic accumulation of glutamate. Excessive activation of glutamate receptors causes excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts, but the mechanisms that underlie this difference are unclear. Signaling via Rho-family small GTPases, their upstream guanine nucleotide exchange factors, and GTPase-activating proteins (GAPs) is differentially dysregulated in response to OGD/ischemia in hippocampal and cortical neurons. Increased Rac1 activity caused by OGD/ischemia contributes to neuronal death in hippocampal neurons via diverse effects on NADPH oxidase activity and dendritic spine morphology. The Rac1 guanine nucleotide exchange factor Tiam1 mediates an OGD-induced increase in Rac1 activity in hippocampal neurons; however, the identity of an antagonistic GAP remains elusive. Here we show that the Rac1 GAP breakpoint cluster region (BCR) associates with NMDA receptors (NMDARs) along with Tiam1 and that this protein complex is more abundant in hippocampal compared with cortical neurons. Although total BCR is similar in the two neuronal types, BCR is more active in hippocampal compared with cortical neurons. OGD causes an NMDAR- and Ca-permeable AMPAR-dependent deactivation of BCR in hippocampal but not cortical neurons. BCR knockdown occludes OGD-induced Rac1 activation in hippocampal neurons. Furthermore, disrupting the Tiam1-NMDAR interaction with a fragment of Tiam1 blocks OGD-induced Tiam1 activation but has no effect on the deactivation of BCR. This work identifies BCR as a critical player in Rac1 regulation during OGD in hippocampal neurons.
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http://dx.doi.org/10.1074/jbc.M117.796292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5724004PMC
December 2017

Cadherin-10 Maintains Excitatory/Inhibitory Ratio through Interactions with Synaptic Proteins.

J Neurosci 2017 11 13;37(46):11127-11139. Epub 2017 Oct 13.

Departments of Physiology,

Appropriate excitatory/inhibitory (E/I) balance is essential for normal cortical function and is altered in some psychiatric disorders, including autism spectrum disorders (ASDs). Cell-autonomous molecular mechanisms that control the balance of excitatory and inhibitory synapse function remain poorly understood; no proteins that regulate excitatory and inhibitory synapse strength in a coordinated reciprocal manner have been identified. Using super-resolution imaging, electrophysiology, and molecular manipulations, we show that cadherin-10, encoded by within the ASD risk locus 5p14.1, maintains both excitatory and inhibitory synaptic scaffold structure in cultured cortical neurons from rats of both sexes. Cadherin-10 localizes to both excitatory and inhibitory synapses in neocortex, where it is organized into nanoscale puncta that influence the size of their associated PSDs. Knockdown of cadherin-10 reduces excitatory but increases inhibitory synapse size and strength, altering the E/I ratio in cortical neurons. Furthermore, cadherin-10 exhibits differential participation in complexes with PSD-95 and gephyrin, which may underlie its role in maintaining the E/I ratio. Our data provide a new mechanism whereby a protein encoded by a common ASD risk factor controls E/I ratios by regulating excitatory and inhibitory synapses in opposing directions. The correct balance between excitatory/inhibitory (E/I) is crucial for normal brain function and is altered in psychiatric disorders such as autism. However, the molecular mechanisms that underlie this balance remain elusive. To address this, we studied cadherin-10, an adhesion protein that is genetically linked to autism and understudied at the cellular level. Using a combination of advanced microscopy techniques and electrophysiology, we show that cadherin-10 forms nanoscale puncta at excitatory and inhibitory synapses, maintains excitatory and inhibitory synaptic structure, and is essential for maintaining the correct balance between excitation and inhibition in neuronal dendrites. These findings reveal a new mechanism by which E/I balance is controlled in neurons and may bear relevance to synaptic dysfunction in autism.
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http://dx.doi.org/10.1523/JNEUROSCI.1153-17.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5688522PMC
November 2017

Endocytosis and lysosomal degradation of GluA2/3 AMPARs in response to oxygen/glucose deprivation in hippocampal but not cortical neurons.

Sci Rep 2017 09 26;7(1):12318. Epub 2017 Sep 26.

Centre for Synaptic Plasticity and School of Biochemistry, Biomedical Sciences Building, University of Bristol, University Walk, Bristol, BS8 1TD, UK.

Global cerebral ischemia results in oxygen and glucose deprivation (OGD) and consequent delayed cell death of vulnerable neurons, with hippocampal CA1 neurons more vulnerable than cortical neurons. Most AMPA receptors (AMPARs) are heteromeric complexes of subunits GluA1/GluA2 or GluA2/GluA3, and the presence of GluA2 renders AMPARs Ca-impermeable. In hippocampal CA1 neurons, OGD causes the synaptic expression of GluA2-lacking Ca-permeable AMPARs, contributing to toxic Ca influx. The loss of synaptic GluA2 is caused by rapid trafficking of GluA2-containing AMPARs from the cell surface, followed by a delayed reduction in GluA2 mRNA expression. We show here that OGD causes endocytosis, lysosomal targeting and consequent degradation of GluA2- and GluA3-containing AMPARs, and that PICK1 is required for both OGD-induced GluA2 endocytosis and lysosomal sorting. Our results further suggest that GluA1-containing AMPARs resist OGD-induced endocytosis. OGD does not cause GluA2 endocytosis in cortical neurons, and we show that PICK1 binding to the endocytic adaptor AP2 is enhanced by OGD in hippocampal, but not cortical neurons. We propose that endocytosis of GluA2/3, caused by a hippocampal-specific increase in PICK1-AP2 interactions, followed by PICK1-dependent lysosomal targeting, are critical events in determining changes in AMPAR subunit composition in the response to ischaemia.
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http://dx.doi.org/10.1038/s41598-017-12534-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614987PMC
September 2017

PICK1 regulates AMPA receptor endocytosis via direct interactions with AP2 α-appendage and dynamin.

J Cell Biol 2017 10 30;216(10):3323-3338. Epub 2017 Aug 30.

Centre for Synaptic Plasticity and School of Biochemistry, University of Bristol, Bristol, England, UK

Clathrin-mediated endocytosis (CME) is used to internalize a diverse range of cargo proteins from the cell surface, often in response to specific signals. In neurons, the rapid endocytosis of GluA2-containing AMPA receptors (AMPARs) in response to NMDA receptor (NMDAR) stimulation causes a reduction in synaptic strength and is the central mechanism for long-term depression, which underlies certain forms of learning. The mechanisms that link NMDAR activation to CME of AMPARs remain elusive. PICK1 is a BAR domain protein required for NMDAR-dependent reductions in surface GluA2; however, the molecular mechanisms involved are unclear. In this study, we show that PICK1 makes direct, NMDAR-dependent interactions with the core endocytic proteins AP2 and dynamin. PICK1-AP2 interactions are required for clustering AMPARs at endocytic zones in dendrites in response to NMDAR stimulation and for consequent AMPAR internalization. We further show that PICK1 stimulates dynamin polymerization. We propose that PICK1 is a cargo-specific endocytic accessory protein required for efficient, activity-dependent AMPAR endocytosis.
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http://dx.doi.org/10.1083/jcb.201701034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626541PMC
October 2017

The PICK1 Ca sensor modulates -methyl-d-aspartate (NMDA) receptor-dependent microRNA-mediated translational repression in neurons.

J Biol Chem 2017 06 12;292(23):9774-9786. Epub 2017 Apr 12.

From the School of Biochemistry and the Centre for Synaptic Plasticity, University of Bristol, Bristol BS8 1TD, United Kingdom

MicroRNAs (miRNAs) are important regulators of localized mRNA translation in neuronal dendrites. The presence of RNA-induced silencing complex proteins in these compartments and the dynamic miRNA expression changes that occur in response to neuronal stimulation highlight their importance in synaptic plasticity. Previously, we demonstrated a novel interaction between the major RNA-induced silencing complex component Argounaute-2 (Ago2) and the BAR (bin/amphiphysin/rvs) domain protein PICK1. PICK1 recruits Ago2 to recycling endosomes in dendrites, where it inhibits miRNA-mediated translational repression. Chemical induction of long-term depression via NMDA receptor activation causes the dissociation of Ago2 from PICK1 and a consequent increase in dendritic miRNA-mediated gene silencing. The mechanism that underlies the regulation of PICK1-Ago2 binding is unknown. In this study, we demonstrate that the PICK1-Ago2 interaction is directly sensitive to Ca ions so that high [Ca] reduces PICK1 binding to Ago2. Mutating a stretch of C-terminal Ca-binding residues in PICK1 results in a complete block of NMDA-induced PICK1-Ago2 disassociation in cortical neurons. Furthermore, the same mutant also blocks NMDA-stimulated miRNA-mediated gene silencing. This study defines a novel mechanism whereby elevated [Ca] induced by NMDA receptor activation modulates Ago2 and miRNA activity via PICK1. Our work suggests a Ca-dependent process to regulate miRNA activity in neurons in response to the induction of long-term depression.
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http://dx.doi.org/10.1074/jbc.M117.776302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465499PMC
June 2017

Identification of novel nesprin-1 binding partners and cytoplasmic matrin-3 in processing bodies.

Mol Biol Cell 2016 12 12;27(24):3894-3902. Epub 2016 Oct 12.

Cardiovascular Division, King's College London, London SE5 9NU, United Kingdom.

Nesprins are highly conserved spectrin repeat-containing scaffold proteins predominantly known to function at the nuclear envelope (NE). However, nesprin isoforms are emerging with localizations and scaffolding functions at sites away from the NE, suggesting their functions are more diverse than originally thought. In this study, we combined nesprin-1 coimmunoprecipitations with mass spectrometry to identify novel nesprin-1 binding partners for isoforms that localize to subcellular compartments beyond the NE. We show that one of these interactors, matrin-3 (matr3), localizes to mRNA processing bodies (PBs), where we have previously shown a nesprin-1 isoform to localize. Furthermore, we show that Matr3 is part of PB mRNP complexes, is a regulator of miRNA-mediated gene silencing, and possibly shuttles to stress granules in stressed cells. More importantly, we identify a new C-terminally truncated Matr3 isoform that is likely to be involved in these functions and PB localization. This study highlights several novel nesprin-1 binding partners and a new function and localization for Matr3 in cytoplasmic RNA granules.
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http://dx.doi.org/10.1091/mbc.E16-06-0346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5170611PMC
December 2016

Chitin and carbon nanotube composites as biocompatible scaffolds for neuron growth.

Nanoscale 2016 Apr;8(15):8288-99

Advanced Composites Centre for Innovation and Science, Department of Aerospace Engineering, University of Bristol, Bristol BS8 1TR, UK.

The design of biocompatible implants for neuron repair/regeneration ideally requires high cell adhesion as well as good electrical conductivity. Here, we have shown that plasma-treated chitin carbon nanotube composite scaffolds show very good neuron adhesion as well as support of synaptic function of neurons. The addition of carbon nanotubes to a chitin biopolymer improved the electrical conductivity and the assisted oxygen plasma treatment introduced more oxygen species onto the chitin nanotube scaffold surface. Neuron viability experiments showed excellent neuron attachment onto plasma-treated chitin nanotube composite scaffolds. The support of synaptic function was evident on chitin/nanotube composites, as confirmed by PSD-95 staining. The biocompatible and electrically-conducting chitin nanotube composite scaffold prepared in this study can be used for in vitro tissue engineering of neurons and, potentially, as an implantable electrode for stimulation and repair of neurons.
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http://dx.doi.org/10.1039/c5nr06595jDOI Listing
April 2016

The Ins and Outs of miRNA-Mediated Gene Silencing during Neuronal Synaptic Plasticity.

Noncoding RNA 2016 Jan 11;2(1). Epub 2016 Jan 11.

Department of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK.

Neuronal connections through specialized junctions, known as synapses, create circuits that underlie brain function. Synaptic plasticity, , structural and functional changes to synapses, occurs in response to neuronal activity and is a critical regulator of various nervous system functions, including long-term memory formation. The discovery of mRNAs, miRNAs, ncRNAs, ribosomes, translational repressors, and other RNA binding proteins in dendritic spines allows individual synapses to alter their synaptic strength rapidly through regulation of local protein synthesis in response to different physiological stimuli. In this review, we discuss our understanding of a number of miRNAs, ncRNAs, and RNA binding proteins that are emerging as important regulators of synaptic plasticity, which play a critical role in memory, learning, and diseases that arise when neuronal circuits are impaired.
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http://dx.doi.org/10.3390/ncrna2010001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831896PMC
January 2016

Protein interacting with C kinase 1 suppresses invasion and anchorage-independent growth of astrocytic tumor cells.

Mol Biol Cell 2015 Dec 14;26(25):4552-61. Epub 2015 Oct 14.

School of Biochemistry, University of Bristol, Bristol BS8 1TD, United Kingdom

Astrocytic tumors are the most common form of primary brain tumor. Astrocytic tumor cells infiltrate the surrounding CNS tissue, allowing them to evade removal upon surgical resection of the primary tumor. Dynamic changes to the actin cytoskeleton are crucial to cancer cell invasion, but the specific mechanisms that underlie the particularly invasive phenotype of astrocytic tumor cells are unclear. Protein interacting with C kinase 1 (PICK1) is a PDZ and BAR domain-containing protein that inhibits actin-related protein 2/3 (Arp2/3)-dependent actin polymerization and is involved in regulating the trafficking of a number of cell-surface receptors. Here we report that, in contrast to other cancers, PICK1 expression is down-regulated in grade IV astrocytic tumor cell lines and also in clinical cases of the disease in which grade IV tumors have progressed from lower-grade tumors. Exogenous expression of PICK1 in the grade IV astrocytic cell line U251 reduces their capacity for anchorage-independent growth, two-dimensional migration, and invasion through a three-dimensional matrix, strongly suggesting that low PICK1 expression plays an important role in astrocytic tumorigenesis. We propose that PICK1 negatively regulates neoplastic infiltration of astrocytic tumors and that manipulation of PICK1 is an attractive possibility for therapeutic intervention.
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http://dx.doi.org/10.1091/mbc.E15-05-0270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678014PMC
December 2015

PICK1 links AMPA receptor stimulation to Cdc42.

Neurosci Lett 2015 Jan 2;585:155-9. Epub 2014 Dec 2.

School of Biochemistry, Medical Sciences Building,University of Bristol, University Walk, Bristol BS8 1TD, UK. Electronic address:

Rho-family GTPases control numerous cell biological processes via effects on actin dynamics, such as cell migration, cell adhesion, morphogenesis and vesicle traffic. In neurons, they are involved in dendritic spine morphogenesis and other aspects of neuronal morphology via regulation of the actin cytoskeleton. The Rho-family member Cdc42 regulates dendritic spine morphology via its effector N-WASP, which activates the actin-nucleating Arp2/3 complex. Excitatory synaptic transmission is known to regulate actin dynamics in dendritic spines to bring about changes in spine morphology or motility, however, the details of the signalling pathways that transduce glutamate receptor activation to Rho GTPase function are unclear. PICK1 is a PDZ and BAR domain protein that interacts with the Arp2/3 complex and the GTPase Arf1 to regulate actin polymerisation in dendritic spines. PICK1 also binds AMPA receptor subunits GluA2/3 and is involved in GluA2-dependent AMPAR trafficking. Here, we show that PICK1 binds Rac1 and Cdc42, via distinct but overlapping binding sites. Furthermore, AMPAR stimulation deactivates Cdc42 and alters its detergent solubility in neurons via a PICK1-dependent process. This work suggests a novel role for PICK1 in transducing AMPAR stimulation to Cdc42 function in neurons.
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http://dx.doi.org/10.1016/j.neulet.2014.11.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4300402PMC
January 2015

Actin-dependent mechanisms in AMPA receptor trafficking.

Front Cell Neurosci 2014 12;8:381. Epub 2014 Nov 12.

School of Biochemistry, University of Bristol Bristol, UK.

The precise regulation of AMPA receptor (AMPAR) number and subtype at the synapse is crucial for the regulation of excitatory neurotransmission, synaptic plasticity and the consequent formation of appropriate neural circuits for learning and memory. AMPAR trafficking involves the dynamic processes of exocytosis, endocytosis and endosomal recycling, all of which involve the actin cytoskeleton. The actin cytoskeleton is highly dynamic and highly regulated by an abundance of actin-binding proteins and upstream signaling pathways that modulate actin polymerization and depolymerization. Actin dynamics generate forces that manipulate membranes in the process of vesicle biogenesis, and also for propelling vesicles through the cytoplasm to reach their destination. In addition, trafficking mechanisms exploit more stable aspects of the actin cytoskeleton by using actin-based motor proteins to traffic vesicular cargo along actin filaments. Numerous studies have shown that actin dynamics are critical for AMPAR localization and function. The identification of actin-binding proteins that physically interact with AMPAR subunits, and research into their mode of action is starting to shed light on the mechanisms involved. Such proteins either regulate actin dynamics to modulate mechanical forces exerted on AMPAR-containing membranes, or associate with actin filaments to target or transport AMPAR-containing vesicles to specific subcellular regions. In addition, actin-regulatory proteins that do not physically interact with AMPARs may influence AMPAR trafficking by regulating the local actin environment in the dendritic spine.
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http://dx.doi.org/10.3389/fncel.2014.00381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4228833PMC
November 2014

Differential Tiam1/Rac1 activation in hippocampal and cortical neurons mediates differential spine shrinkage in response to oxygen/glucose deprivation.

J Cereb Blood Flow Metab 2014 Dec 24;34(12):1898-906. Epub 2014 Sep 24.

School of Biochemistry, University of Bristol, University Walk, Bristol, UK.

Distinct neuronal populations show differential sensitivity to global ischemia, with hippocampal CA1 neurons showing greater vulnerability compared to cortical neurons. The mechanisms that underlie differential vulnerability are unclear, and we hypothesize that intrinsic differences in neuronal cell biology are involved. Dendritic spine morphology changes in response to ischemic insults in vivo, but cell type-specific differences and the molecular mechanisms leading to such morphologic changes are unexplored. To directly compare changes in spine size in response to oxygen/glucose deprivation (OGD) in cortical and hippocampal neurons, we used separate and equivalent cultures of each cell type. We show that cortical neurons exhibit significantly greater spine shrinkage compared to hippocampal neurons. Rac1 is a Rho-family GTPase that regulates the actin cytoskeleton and is involved in spine dynamics. We show that Rac1 and the Rac guanine nucleotide exchange factor (GEF) Tiam1 are differentially activated by OGD in hippocampal and cortical neurons. Hippocampal neurons express more Tiam1 than cortical neurons, and reducing Tiam1 expression in hippocampal neurons by shRNA enhances OGD-induced spine shrinkage. Tiam1 knockdown also reduces hippocampal neuronal vulnerability to OGD. This work defines fundamental differences in signalling pathways that regulate spine morphology in distinct neuronal populations that may have a role in the differential vulnerability to ischemia.
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http://dx.doi.org/10.1038/jcbfm.2014.158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4269742PMC
December 2014

PICK1 links Argonaute 2 to endosomes in neuronal dendrites and regulates miRNA activity.

EMBO Rep 2014 May 10;15(5):548-56. Epub 2014 Apr 10.

School of Biochemistry, University of Bristol, Bristol, UK.

MicroRNAs fine-tune gene expression by inhibiting the translation of mRNA targets. Argonaute (Ago) proteins are critical mediators of microRNA-induced post-transcriptional silencing and have been shown to associate with endosomal compartments, but the molecular mechanisms that underlie this process are unclear, especially in neurons. Here, we report a novel interaction between Ago2 and the BAR-domain protein, PICK1. We show that PICK1 promotes Ago2 localization at endosomal compartments in neuronal dendrites and inhibits Ago2 function in translational repression following neuronal stimulation. We propose that PICK1 provides a link between activity-dependent endosomal trafficking and local regulation of translation in neurons.
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http://dx.doi.org/10.1002/embr.201337631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210090PMC
May 2014

Distinct subunit-specific α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking mechanisms in cultured cortical and hippocampal neurons in response to oxygen and glucose deprivation.

J Biol Chem 2014 Feb 8;289(8):4644-51. Epub 2014 Jan 8.

From the School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.

Brain ischemia occurs when the blood supply to the brain is interrupted, leading to oxygen and glucose deprivation (OGD). This triggers a cascade of events causing a synaptic accumulation of glutamate. Excessive activation of glutamate receptors results in excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts. The mechanisms that underlie this difference are unclear. Cultured hippocampal neurons respond to OGD with a rapid internalization of AMPA receptor (AMPAR) subunit GluA2, resulting in a switch from GluA2-containing Ca(2+)-impermeable receptors to GluA2-lacking Ca(2+)-permeable subtypes (CP-AMPARs). GluA2 internalization is a critical component of OGD-induced cell death in hippocampal neurons. It is unknown how AMPAR trafficking is affected in cortical neurons following OGD. Here, we show that cultured cortical neurons are resistant to an OGD insult that causes cell death in hippocampal neurons. GluA1 is inserted at the plasma membrane in both cortical and hippocampal neurons in response to OGD. In contrast, OGD causes a rapid endocytosis of GluA2 in hippocampal neurons, which is absent in cortical neurons. These data demonstrate that populations of neurons with different vulnerabilities to OGD recruit distinct cell biological mechanisms in response to insult, and that a crucial aspect of the mechanism leading to OGD-induced cell death is absent in cortical neurons. This strongly suggests that the absence of OGD-induced GluA2 trafficking contributes to the relatively low vulnerability of cortical neurons to ischemia.
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http://dx.doi.org/10.1074/jbc.M113.533182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3931026PMC
February 2014

Subunit-specific trafficking mechanisms regulating the synaptic expression of Ca(2+)-permeable AMPA receptors.

Semin Cell Dev Biol 2014 Mar 15;27:14-22. Epub 2013 Dec 15.

School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom. Electronic address:

AMPA receptors are the main excitatory neurotransmitter receptor in the brain, and hence regulating the number or properties of synaptic AMPA receptors brings about critical changes in synaptic transmission. Synaptic plasticity is thought to underlie learning and memory, and can be brought about by decreasing or increasing the number of AMPA receptors localised to synaptic sites by precisely regulating AMPA receptor trafficking. AMPA receptors are tetrameric assemblies of subunits GluA1-4, and the vast majority are GluA1/2 and GluA2/3 heteromers. The inclusion of GluA2 subunit is critical because it renders the AMPA receptor channel impermeable to Ca(2+) ions. The vast majority of synaptic AMPA receptors in the brain contain GluA2, but relatively recent discoveries indicate that an increasing number of specific forms of synaptic plasticity involve not only an alteration of the number of synaptic AMPA receptors, but also changes to their GluA2 content. The resulting change in AMPA receptor Ca(2+) permeability clearly has profound consequences for synaptic transmission and intracellular signalling events. The subunit-specific trafficking mechanisms that cause such changes represent an emerging field of research with implications for an increasing number of physiological or pathological situations, and are the topic of this review.
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http://dx.doi.org/10.1016/j.semcdb.2013.12.002DOI Listing
March 2014

The small GTPase Arf1 modulates Arp2/3-mediated actin polymerization via PICK1 to regulate synaptic plasticity.

Neuron 2013 Jul;79(2):293-307

School of Biochemistry, Centre for Synaptic Plasticity, Medical Sciences Building and Dorothy Hodgkin Building, University of Bristol, University Walk, Bristol BS8 1TD, UK.

Inhibition of Arp2/3-mediated actin polymerization by PICK1 is a central mechanism to AMPA receptor (AMPAR) internalization and long-term depression (LTD), although the signaling pathways that modulate this process in response to NMDA receptor (NMDAR) activation are unknown. Here, we define a function for the GTPase Arf1 in this process. We show that Arf1-GTP binds PICK1 to limit PICK1-mediated inhibition of Arp2/3 activity. Expression of mutant Arf1 that does not bind PICK1 leads to reduced surface levels of GluA2-containing AMPARs and smaller spines in hippocampal neurons, which occludes subsequent NMDA-induced AMPAR internalization and spine shrinkage. In organotypic slices, NMDAR-dependent LTD of AMPAR excitatory postsynaptic currents is abolished in neurons expressing mutant Arf1. Furthermore, NMDAR stimulation downregulates Arf1 activation and binding to PICK1 via the Arf-GAP GIT1. This study defines Arf1 as a critical regulator of actin dynamics and synaptic function via modulation of PICK1.
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http://dx.doi.org/10.1016/j.neuron.2013.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725416PMC
July 2013

The antagonistic modulation of Arp2/3 activity by N-WASP, WAVE2 and PICK1 defines dynamic changes in astrocyte morphology.

J Cell Sci 2013 Sep 10;126(Pt 17):3873-83. Epub 2013 Jul 10.

School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.

Astrocytes exhibit a complex, branched morphology, allowing them to functionally interact with numerous blood vessels, neighboring glial processes and neuronal elements, including synapses. They also respond to central nervous system (CNS) injury by a process known as astrogliosis, which involves morphological changes, including cell body hypertrophy and thickening of major processes. Following severe injury, astrocytes exhibit drastically reduced morphological complexity and collectively form a glial scar. The mechanistic details behind these morphological changes are unknown. Here, we investigate the regulation of the actin-nucleating Arp2/3 complex in controlling dynamic changes in astrocyte morphology. In contrast to other cell types, Arp2/3 inhibition drives the rapid expansion of astrocyte cell bodies and major processes. This intervention results in a reduced morphological complexity of astrocytes in both dissociated culture and in brain slices. We show that this expansion requires functional myosin II downstream of ROCK and RhoA. Knockdown of the Arp2/3 subunit Arp3 or the Arp2/3 activator N-WASP by siRNA also results in cell body expansion and reduced morphological complexity, whereas depleting WAVE2 specifically reduces the branching complexity of astrocyte processes. By contrast, knockdown of the Arp2/3 inhibitor PICK1 increases astrocyte branching complexity. Furthermore, astrocyte expansion induced by ischemic conditions is delayed by PICK1 knockdown or N-WASP overexpression. Our findings identify a new morphological outcome for Arp2/3 activation in restricting rather than promoting outwards movement of the plasma membrane in astrocytes. The Arp2/3 regulators PICK1, and N-WASP and WAVE2 function antagonistically to control the complexity of astrocyte branched morphology, and this mechanism underlies the morphological changes seen in astrocytes during their response to pathological insult.
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http://dx.doi.org/10.1242/jcs.125146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757329PMC
September 2013

PICK1 mediates transient synaptic expression of GluA2-lacking AMPA receptors during glycine-induced AMPA receptor trafficking.

J Neurosci 2012 Aug;32(34):11618-30

Medical Research Council Centre for Synaptic Plasticity and School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol, BS8 1TD, United Kingdom.

The number and subunit composition of postsynaptic AMPA receptors (AMPARs) is a key determinant of synaptic transmission. The vast majority of AMPARs contain GluA2 subunit, which renders the channel impermeable to calcium. However, a small proportion are GluA2 lacking and therefore calcium permeable (CP-AMPARs). It has been proposed recently that long-term potentiation (LTP) involves not only an increase in the total number of AMPARs at the synapse but also a transient switch to CP-AMPARs in the first few minutes after LTP induction. The molecular mechanisms that underlie this switch to CP-AMPARs and the subsequent switch back to calcium-impermeable AMPARs are unknown. Here, we show that endogenous GluA1 is rapidly inserted at the synaptic plasma membrane of rat hippocampal neurons immediately after stimulation with elevated glycine, a treatment known to induce LTP. In contrast, GluA2 is restricted from trafficking to the cell surface by a glycine-induced increase in PICK1-GluA2 binding on endosomal compartments. Between 5 and 20 min after stimulus, activation of CP-AMPARs triggers a release of GluA2 from PICK1, allowing GluA2-containing AMPARs to traffic to the synaptic plasma membrane. These results define a PICK1-dependent mechanism that underlies transient alterations in the subunit composition and calcium permeability of synaptic AMPARs that is important during the early phase after stimulation with glycine and therefore is likely to be important during the expression of LTP.
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http://dx.doi.org/10.1523/JNEUROSCI.5068-11.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6703756PMC
August 2012

Oxygen/glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors.

J Neurosci 2011 Aug;31(33):11941-52

Medical Research Council Centre for Synaptic Plasticity, School of Physiology and Pharmacology, University of Bristol, Bristol BS8 1TD, UK.

Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca(2+), resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A(3) receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca(2+) also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A(3) receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection.
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http://dx.doi.org/10.1523/JNEUROSCI.1183-11.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3160741PMC
August 2011

PICK1 inhibition of the Arp2/3 complex controls dendritic spine size and synaptic plasticity.

EMBO J 2011 Feb 21;30(4):719-30. Epub 2011 Jan 21.

MRC Centre for Synaptic Plasticity, School of Biochemistry, University of Bristol, University Walk, Bristol, UK.

Activity-dependent remodelling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the precise molecular mechanisms that regulate this process are unclear. Activators of Arp2/3-mediated actin polymerisation are required for spine enlargement; however, during long-term depression (LTD), spines shrink via actin depolymerisation and Arp2/3 inhibitors in this process have not yet been identified. Here, we show that PICK1 regulates spine size in hippocampal neurons via inhibition of the Arp2/3 complex. PICK1 knockdown increases spine size, whereas PICK1 overexpression reduces spine size. NMDA receptor activation results in spine shrinkage, which is blocked by PICK1 knockdown or overexpression of a PICK1 mutant that cannot bind Arp2/3. Furthermore, we show that PICK1-Arp2/3 interactions are required for functional hippocampal LTD. This work demonstrates that PICK1 is a novel regulator of spine dynamics. Via Arp2/3 inhibition, PICK1 has complementary yet distinct roles during LTD to regulate AMPA receptor trafficking and spine size, and therefore functions as a crucial factor in both structural and functional plasticity.
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http://dx.doi.org/10.1038/emboj.2010.357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3041953PMC
February 2011

Endosomal sorting of AMPA receptors in hippocampal neurons.

Biochem Soc Trans 2010 Apr;38(2):460-5

MRC Centre for Synaptic Plasticity and Department of Anatomy, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.

An important mechanism for the regulation of excitatory synaptic transmission in the hippocampus involves tight control of AMPAR [AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor] trafficking to alter the number or subtype of synaptic receptors. This is achieved via the multiple stages of the endosomal system. AMPARs constitutively cycle through early endosomes and recycling endosomes to maintain synaptic receptor numbers. However, on induction of synaptic plasticity, subtle alterations are made to this cycle by the action of specific AMPAR-interacting proteins and also via a number of additional proteins that regulate endosomal sorting more generally. During long-term depression, receptors are diverted to late endosomes and lysosomes rather than recycling back to the plasma membrane, hence reducing the number of receptors at the synapse. The increased number of synaptic AMPARs after induction of LTP (long-term potentiation) originates from the recycling compartment. In addition, transient changes in subunit composition may arise as a result of retention of AMPAR subtypes within the endosome during LTP. Aberrant trafficking after pathological insults such as oxygen/glucose deprivation or mechanical trauma also involves alterations in synaptic AMPAR subunit composition, leading to calcium influx that ultimately results in cell death.
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http://dx.doi.org/10.1042/BST0380460DOI Listing
April 2010

PICK1-mediated glutamate receptor subunit 2 (GluR2) trafficking contributes to cell death in oxygen/glucose-deprived hippocampal neurons.

J Biol Chem 2009 May 25;284(21):14230-5. Epub 2009 Mar 25.

Medical Research Council (MRC) Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.

Oxygen and glucose deprivation (OGD) induces delayed cell death in hippocampal CA1 neurons via Ca(2+)/Zn(2+)-permeable, GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents in hippocampal neurons show marked inward rectification and increased sensitivity to channel blockers selective for GluR2-lacking AMPARs. This occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression and a rapid internalization of GluR2-containing AMPARs during the OGD insult, which are replaced by GluR2-lacking receptors. The mechanisms that underlie this rapid change in subunit composition are unknown. Here, we demonstrate that this trafficking event shares features in common with events that mediate long term depression and long term potentiation and is initiated by the activation of N-methyl-d-aspartic acid receptors. Using biochemical and electrophysiological approaches, we show that peptides that interfere with PICK1 PDZ domain interactions block the OGD-induced switch in subunit composition, implicating PICK1 in restricting GluR2 from synapses during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at synapses during OGD as a result of PICK1 PDZ interactions are involved in OGD-induced delayed cell death. This work demonstrates that PICK1 plays a crucial role in the response to OGD that results in altered synaptic transmission and neuronal death and has implications for our understanding of the molecular mechanisms that underlie cell death during stroke.
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http://dx.doi.org/10.1074/jbc.M901203200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682871PMC
May 2009

AMPA receptor trafficking pathways and links to dendritic spine morphogenesis.

Cell Adh Migr 2008 Oct-Dec;2(4):276-82. Epub 2008 Oct 27.

MRC Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, Bristol, UK.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633691PMC
http://dx.doi.org/10.4161/cam.2.4.6510DOI Listing
May 2009
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