Publications by authors named "David S Bredt"

86 Publications

Targeting receptor complexes: a new dimension in drug discovery.

Nat Rev Drug Discov 2020 12 11;19(12):884-901. Epub 2020 Nov 11.

Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.

Targeting receptor proteins, such as ligand-gated ion channels and G protein-coupled receptors, has directly enabled the discovery of most drugs developed to modulate receptor signalling. However, as the search for novel and improved drugs continues, an innovative approach - targeting receptor complexes - is emerging. Receptor complexes are composed of core receptor proteins and receptor-associated proteins, which have profound effects on the overall receptor structure, function and localization. Hence, targeting key protein-protein interactions within receptor complexes provides an opportunity to develop more selective drugs with fewer side effects. In this Review, we discuss our current understanding of ligand-gated ion channel and G protein-coupled receptor complexes and discuss strategies for their pharmacological modulation. Although such strategies are still in preclinical development for most receptor complexes, they exemplify how receptor complexes can be drugged, and lay the groundwork for this nascent area of research.
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http://dx.doi.org/10.1038/s41573-020-0086-4DOI Listing
December 2020

Functional α6β4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties.

J Clin Invest 2020 11;130(11):6158-6170

The α6β4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6β4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6β4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6β4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6β4 receptor assembly. Effects on α6β4 involve BARP's N-terminal region and IRE1α's splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6β4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6β4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6β4 receptor.
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http://dx.doi.org/10.1172/JCI140311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598046PMC
November 2020

Hair cell α9α10 nicotinic acetylcholine receptor functional expression regulated by ligand binding and deafness gene products.

Proc Natl Acad Sci U S A 2020 09 14;117(39):24534-24544. Epub 2020 Sep 14.

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, San Diego, CA 92121

Auditory hair cells receive olivocochlear efferent innervation, which refines tonotopic mapping, improves sound discrimination, and mitigates acoustic trauma. The olivocochlear synapse involves α9α10 nicotinic acetylcholine receptors (nAChRs), which assemble in hair cells only coincident with cholinergic innervation and do not express in recombinant mammalian cell lines. Here, genome-wide screening determined that assembly and surface expression of α9α10 require ligand binding. Ion channel function additionally demands an auxiliary subunit, which can be transmembrane inner ear (TMIE) or TMEM132e. Both of these single-pass transmembrane proteins are enriched in hair cells and underlie nonsyndromic human deafness. Inner hair cells from TMIE mutant mice show altered postsynaptic α9α10 function and retain α9α10-mediated transmission beyond the second postnatal week associated with abnormally persistent cholinergic innervation. Collectively, this study provides a mechanism to link cholinergic input with α9α10 assembly, identifies unexpected functions for human deafness genes TMIE/TMEM132e, and enables drug discovery for this elusive nAChR implicated in prevalent auditory disorders.
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http://dx.doi.org/10.1073/pnas.2013762117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533656PMC
September 2020

NACHO Engages N-Glycosylation ER Chaperone Pathways for α7 Nicotinic Receptor Assembly.

Cell Rep 2020 08;32(6):108025

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors.
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http://dx.doi.org/10.1016/j.celrep.2020.108025DOI Listing
August 2020

NACHO Engages N-Glycosylation ER Chaperone Pathways for α7 Nicotinic Receptor Assembly.

Cell Rep 2020 08;32(6):108025

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors.
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http://dx.doi.org/10.1016/j.celrep.2020.108025DOI Listing
August 2020

α7 nicotinic acetylcholine receptor upregulation by anti-apoptotic Bcl-2 proteins.

Nat Commun 2019 06 21;10(1):2746. Epub 2019 Jun 21.

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA, 92121, USA.

Nicotinic acetylcholine receptors (nAChRs) mediate and modulate synaptic transmission throughout the brain, and contribute to learning, memory, and behavior. Dysregulation of α7-type nAChRs in neuropsychiatric as well as immunological and oncological diseases makes them attractive targets for pharmaceutical development. Recently, we identified NACHO as an essential chaperone for α7 nAChRs. Leveraging the robust recombinant expression of α7 nAChRs with NACHO, we utilized genome-wide cDNA library screening and discovered that several anti-apoptotic Bcl-2 family proteins further upregulate receptor assembly and cell surface expression. These effects are mediated by an intracellular motif on α7 that resembles the BH3 binding domain of pro-apoptotic Bcl-2 proteins, and can be blocked by BH3 mimetic Bcl-2 inhibitors. Overexpression of Bcl-2 member Mcl-1 in neurons enhanced surface expression of endogenous α7 nAChRs, while a combination of chemotherapeutic Bcl2-inhibitors suppressed neuronal α7 receptor assembly. These results demonstrate that Bcl-2 proteins link α7 nAChR assembly to cell survival pathways.
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http://dx.doi.org/10.1038/s41467-019-10723-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588605PMC
June 2019

Modulation of TARP 8-Containing AMPA Receptors as a Novel Therapeutic Approach for Chronic Pain.

J Pharmacol Exp Ther 2019 06 25;369(3):345-363. Epub 2019 Mar 25.

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (K.L.K., R.M.A.S., W.G., B.L.A., K.M.G., D.L.G., P.L.O., W.P., J.R., C.D., H.W., Y.Q., K.D.B., A.N., V.B., S.S., J.C., J.M.W., K.-C.C., T.M.W., D.S., C.C.F., A.S.K., D.S.B., E.S.N.) and Lilly Research Centre, Eli Lilly and Company Ltd., Erl Wood Manor, Windlesham, Surrey, United Kingdom (R.Z., E.S.).

Nonselective glutamate -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists are efficacious in chronic pain but have significant tolerability issues, likely arising from the ubiquitous expression of AMPA receptors in the central nervous system (CNS). Recently, LY3130481 has been shown to selectively block AMPA receptors coassembled with the auxiliary protein, transmembrane AMPA receptor regulatory protein (TARP) 8, which is highly expressed in the hippocampus but also in pain pathways, including anterior cingulate (ACC) and somatosensory cortices and the spinal cord, suggesting that selective blockade of 8/AMPA receptors may suppress nociceptive signaling with fewer CNS side effects. The potency of LY3130481 on recombinant 8-containing AMPA receptors was modulated by coexpression with other TARPs; 2 subunits affected activity more than 3 subunits. Consistent with these findings, LY3130481 had decreasing potency on receptors from rat hippocampal, cortical, spinal cord, and cerebellar neurons that was replicated in tissue from human brain. LY3130481 partially suppressed, whereas the nonselective AMPA antagonist GYKI53784 completely blocked, AMPA receptor-dependent excitatory postsynaptic potentials in ACC and spinal neurons in vitro. Similarly, LY3130481 attenuated short-term synaptic plasticity in spinal sensory neurons in vivo in response to stimulation of peripheral afferents. LY3130481 also significantly reduced nocifensive behaviors after intraplantar formalin that was correlated with occupancy of CNS 8-containing AMPA receptors. In addition, LY3130481 dose-dependently attenuated established gait impairment after joint damage and tactile allodynia after spinal nerve ligation, all in the absence of motor side effects. Collectively, these data demonstrate that LY3130481 can suppress excitatory synaptic transmission and plasticity in pain pathways containing 8/AMPA receptors and significantly reduce nocifensive behaviors, suggesting a novel, effective, and safer therapy for chronic pain conditions.
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http://dx.doi.org/10.1124/jpet.118.250126DOI Listing
June 2019

α6-Containing Nicotinic Acetylcholine Receptor Reconstitution Involves Mechanistically Distinct Accessory Components.

Cell Rep 2019 01;26(4):866-874.e3

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

Acetylcholine gates a large family of nicotinic receptor cation channels that control neuronal excitation and neurotransmitter release. These receptors are key targets for neuropsychiatric disorders; however, difficulties in expressing nicotinic acetylcholine (nACh) receptors hamper elaboration of their pharmacology and obscure elucidation of their biological functions. Particularly intriguing are α6-containing nACh receptors, which mediate nicotine-induced dopamine release in striatum-nucleus accumbens. Using genome-wide cDNA screening, we identify three accessory proteins, β-anchoring and -regulatory protein (BARP), lysosomal-associated membrane protein 5 (LAMP5), and SULT2B1, that complement the nACh receptor chaperone NACHO to reconstitute α6β2β3 channel function. Whereas NACHO mediates α6β2β3 assembly, BARP primarily enhances channel gating and LAMP5 and SULT2B1 promote receptor surface trafficking. BARP knockout mice show perturbations in presynaptic striatal nACh receptors that are consistent with BARP modulation of receptor desensitization. These studies unravel the molecular complexity of α6β2β3 biogenesis and enable physiological studies of this crucial neuropharmacological target.
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http://dx.doi.org/10.1016/j.celrep.2018.12.103DOI Listing
January 2019

Getting a Handle on Neuropharmacology by Targeting Receptor-Associated Proteins.

Neuron 2017 Dec;96(5):989-1001

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

Targeted therapy for neuropsychiatric disorders requires selective modulation of dysfunctional neuronal pathways. Receptors relevant to CNS disorders typically have associated proteins discretely expressed in specific neuronal pathways; these accessory proteins provide a new dimension for drug discovery. Recent studies show that targeting a TARP auxiliary subunit of AMPA receptors selectively modulates neuronal excitability in specific forebrain pathways relevant to epilepsy. Other medicinally important ion channels, gated by glutamate, γ-aminobutyric acid (GABA), and acetylcholine, also have associated proteins, which may be druggable. This emerging pharmacology of receptor-associated proteins provides a new approach for improving drug efficacy while mitigating side effects.
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http://dx.doi.org/10.1016/j.neuron.2017.10.001DOI Listing
December 2017

NACHO Mediates Nicotinic Acetylcholine Receptor Function throughout the Brain.

Cell Rep 2017 04;19(4):688-696

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine. Neuronal nAChRs derive from combinations of α and β subunits, whose assembly is tightly regulated. NACHO was recently identified as a chaperone for α7-type nAChRs. Here, we find NACHO mediates assembly of all major classes of presynaptic and postsynaptic nAChR tested. NACHO acts at early intracellular stages of nAChR subunit assembly and then synergizes with RIC-3 for receptor surface expression. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin, epibatidine, and conotoxin MII, illustrating essential roles for NACHO in proper assembly of α7-, α4β2-, and α6-containing nAChRs, respectively. By contrast, GABA receptors are unaffected consistent with NACHO specifically modulating nAChRs. NACHO knockout mice show abnormalities in locomotor and cognitive behaviors compatible with nAChR deficiency and underscore the importance of this chaperone for physiology and disease associated with nAChRs.
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http://dx.doi.org/10.1016/j.celrep.2017.04.008DOI Listing
April 2017

Forebrain-selective AMPA-receptor antagonism guided by TARP γ-8 as an antiepileptic mechanism.

Nat Med 2016 12 7;22(12):1496-1501. Epub 2016 Nov 7.

Lilly Research Laboratory, Eli Lilly and Company, Indianapolis, Indiana, USA.

Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.
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http://dx.doi.org/10.1038/nm.4221DOI Listing
December 2016

Brain α7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO.

Neuron 2016 Mar 11;89(5):948-55. Epub 2016 Feb 11.

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

Nicotine exerts its behavioral and additive actions through a family of brain nicotinic acetylcholine receptors (nAChRs). Enhancing α7-type nAChR signaling improves symptoms in Alzheimer's disease and schizophrenia. The pharmaceutical study of α7 receptors is hampered because these receptors do not form their functional pentameric structure in cell lines, and mechanisms that underlie α7 receptor assembly in neurons are not understood. Here, a genomic screening strategy solves this long-standing puzzle and identifies NACHO, a transmembrane protein of neuronal endoplasmic reticulum that mediates assembly of α7 receptors. NACHO promotes α7 protein folding, maturation through the Golgi complex, and expression at the cell surface. Knockdown of NACHO in cultured hippocampal neurons or knockout of NACHO in mice selectively and completely disrupts α7 receptor assembly and abolishes α7 channel function. This work identifies NACHO as an essential, client-specific chaperone for nAChRs and has implications for physiology and disease associated with these widely distributed neurotransmitter receptors.
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http://dx.doi.org/10.1016/j.neuron.2016.01.018DOI Listing
March 2016

Porcupine Controls Hippocampal AMPAR Levels, Composition, and Synaptic Transmission.

Cell Rep 2016 Feb 14;14(4):782-794. Epub 2016 Jan 14.

Neuroscience Discovery, Janssen Pharmaceutical Companies of Johnson & Johnson, 3210 Merryfield Row, San Diego, CA 92121, USA. Electronic address:

AMPA receptor (AMPAR) complexes contain auxiliary subunits that modulate receptor trafficking and gating. In addition to the transmembrane AMPAR regulatory proteins (TARPs) and cornichons (CNIH-2/3), recent proteomic studies identified a diverse array of additional AMPAR-associated transmembrane and secreted partners. We systematically surveyed these and found that PORCN and ABHD6 increase GluA1 levels in transfected cells. Knockdown of PORCN in rat hippocampal neurons, which express it in high amounts, selectively reduces levels of all tested AMPAR complex components. Regulation of AMPARs is independent of PORCN's membrane-associated O-acyl transferase activity. PORCN knockdown in hippocampal neurons decreases AMPAR currents and accelerates desensitization and leads to depletion of TARP γ-8 from AMPAR complexes. Conditional PORCN knockout mice also exhibit specific changes in AMPAR expression and gating that reduce basal synaptic transmission but leave long-term potentiation intact. These studies define additional roles for PORCN in controlling synaptic transmission by regulating the level and composition of hippocampal AMPAR complexes.
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http://dx.doi.org/10.1016/j.celrep.2015.12.078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4955627PMC
February 2016

Translating depression biomarkers for improved targeted therapies.

Neurosci Biobehav Rev 2015 Dec 1;59:1-15. Epub 2015 Oct 1.

Neuroscience at Janssen Pharmaceutical Companies of Johnson & Johnson, 1125 Trenton Harbourton Rd, Titusville, NJ 08560, United States.

Mood disorders are among the most common medical conditions and cause amongst the greatest disease burden. Currently approved antidepressants target monoamine pathways; these medicines take many weeks to relieve symptoms, and most patients do not have sustained responses. This review will highlight recent advances in translational science identifying dysfunctional biochemical processes and neuronal circuits associated with mood disorders. We will also summarize strategies for targeting these pathways and for enhancing synaptic plasticity to develop most effective and rapidly acting antidepressant therapies.
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http://dx.doi.org/10.1016/j.neubiorev.2015.09.013DOI Listing
December 2015

Acute inactivation of PSD-95 destabilizes AMPA receptors at hippocampal synapses.

PLoS One 2013 16;8(1):e53965. Epub 2013 Jan 16.

Department of Anatomy & Neurobiology, School of Medicine, University of Puerto Rico, San Juan, Puerto Rico.

Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053965PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3546964PMC
July 2013

Glutamate receptor δ2 associates with metabotropic glutamate receptor 1 (mGluR1), protein kinase Cγ, and canonical transient receptor potential 3 and regulates mGluR1-mediated synaptic transmission in cerebellar Purkinje neurons.

J Neurosci 2012 Oct;32(44):15296-308

Lilly Research Laboratory, Indianapolis, Indiana 46285, USA.

Cerebellar motor coordination and cerebellar Purkinje cell synaptic function require metabotropic glutamate receptor 1 (mGluR1, Grm1). We used an unbiased proteomic approach to identify protein partners for mGluR1 in cerebellum and discovered glutamate receptor δ2 (GluRδ2, Grid2, GluΔ2) and protein kinase Cγ (PKCγ) as major interactors. We also found canonical transient receptor potential 3 (TRPC3), which is also needed for mGluR1-dependent slow EPSCs and motor coordination and associates with mGluR1, GluRδ2, and PKCγ. Mutation of GluRδ2 changes subcellular fractionation of mGluR1 and TRPC3 to increase their surface expression. Fitting with this, mGluR1-evoked inward currents are increased in GluRδ2 mutant mice. Moreover, loss of GluRδ2 disrupts the time course of mGluR1-dependent synaptic transmission at parallel fiber-Purkinje cells synapses. Thus, GluRδ2 is part of the mGluR1 signaling complex needed for cerebellar synaptic function and motor coordination, explaining the shared cerebellar motor phenotype that manifests in mutants of the mGluR1 and GluRδ2 signaling pathways.
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http://dx.doi.org/10.1523/JNEUROSCI.0705-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6621574PMC
October 2012

AMPA receptor modulation by cornichon-2 dictated by transmembrane AMPA receptor regulatory protein isoform.

Eur J Neurosci 2012 Jan 30;35(2):182-94. Epub 2011 Dec 30.

Neuroscience Discovery Research and Clinical Investigation, Eli Lilly and Company, Indianapolis, IN 46285, USA.

Transmembrane AMPA receptor regulatory proteins (TARPs) are auxiliary subunits that modulate AMPA receptor trafficking, gating and pharmacology throughout the brain. Why cornichon-2 (CNIH-2), another AMPA receptor-associated protein, modulates AMPA receptor gating and pharmacology in hippocampal neurons but not cerebellar granule neurons remains unresolved. Here, we report that CNIH-2 differentially impacts Type-Ia (γ-2 or γ-3) vs. Type-Ib (γ-4 or γ-8) TARP-containing AMPA receptors. Specifically, with AMPA receptors comprising γ-2, the cerebellar-enriched TARP isoform, CNIH-2 decreases I(KA) /I(Glu) ratio and decreases cyclothiazide efficacy while having minimal impact on recovery from desensitization and deactivation kinetics. By contrast, with AMPA receptors comprising γ-8, the hippocampal-enriched TARP isoform, we find that CNIH-2 slows deactivation kinetics, increases cyclothiazide potency and occludes a novel AMPA receptor kinetic phenomenon, namely resensitization. Additionally, we find that CNIH-2 differentially modulates the glutamate off-kinetics of γ-8-containing, but not γ-2-containing, AMPA receptors in a manner dependent upon the duration of agonist application. Together, these data demonstrate that the modulation of AMPA receptors by CNIH-2 depends upon the TARP isoform composition within the receptor complex.
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http://dx.doi.org/10.1111/j.1460-9568.2011.07948.xDOI Listing
January 2012

PDZ binding of TARPγ-8 controls synaptic transmission but not synaptic plasticity.

Nat Neurosci 2011 Oct 16;14(11):1410-2. Epub 2011 Oct 16.

Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut, USA.

The reduction in synaptic transmission and plasticity in mice lacking the hippocampus-enriched AMPA receptor (AMPAR) auxiliary subunit TARPγ-8 could be a result of a reduction in AMPAR expression or of the direct action of γ-8. We generated TARPγ-8Δ4 knock-in mice lacking the C-terminal PDZ ligand. We found that synaptic transmission and AMPARs were reduced in the mutant mice, but extrasynaptic AMPAR expression and long-term potentiation (LTP) were unaltered. Our findings suggest that there are distinct TARP-dependent mechanisms for synaptic transmission and LTP.
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http://dx.doi.org/10.1038/nn.2952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3206644PMC
October 2011

Cornichon-2 modulates AMPA receptor-transmembrane AMPA receptor regulatory protein assembly to dictate gating and pharmacology.

J Neurosci 2011 May;31(18):6928-38

Neuroscience Discovery Research, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.

Neuronal AMPA receptor complexes comprise a tetramer of GluA pore-forming subunits as well as accessory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon-2/3 (CNIH-2/3). The mechanisms that control AMPA receptor complex assembly remain unclear. AMPA receptor responses in neurons differ from those in cell lines transfected with GluA plus TARPs γ-8 or γ-7, which show unusual resensitization kinetics and non-native AMPA receptor pharmacologies. Using tandem GluA/TARP constructs to constrain stoichiometry, we show here that these peculiar kinetic and pharmacological signatures occur in channels with four TARP subunits per complex. Reducing the number of TARPs per complex produces AMPA receptors with neuron-like kinetics and pharmacologies, suggesting a neuronal mechanism controls GluA/TARP assembly. Importantly, we find that coexpression of CNIH-2 with GluA/TARP complexes reduces TARP stoichiometry within AMPA receptors. In both rat and mouse hippocampal neurons, CNIH-2 also associates with AMPA receptors on the neuronal surface in a γ-8-dependent manner to dictate receptor pharmacology. In the cerebellum, however, CNIH-2 expressed in Purkinje neurons does not reach the neuronal surface. In concordance, stargazer Purkinje neurons, which express CNIH-2 and γ-7, display AMPA receptor kinetics/pharmacologies that can only be recapitulated recombinantly by a low γ-7/GluA stoichiometry. Together, these data suggest that CNIH-2 modulates neuronal AMPA receptor auxiliary subunit assembly by regulating the number of TARPs within an AMPA receptor complex to modulate receptor gating and pharmacology.
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http://dx.doi.org/10.1523/JNEUROSCI.6271-10.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562416PMC
May 2011

Transmembrane AMPA receptor regulatory proteins and cornichon-2 allosterically regulate AMPA receptor antagonists and potentiators.

J Biol Chem 2011 Apr 22;286(15):13134-42. Epub 2011 Feb 22.

Discovery Neuroscience Research, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, USA.

AMPA receptors mediate fast excitatory transmission in the brain. Neuronal AMPA receptors comprise GluA pore-forming principal subunits and can associate with multiple modulatory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and CNIHs (cornichons). AMPA receptor potentiators and non-competitive antagonists represent potential targets for a variety of neuropsychiatric disorders. Previous studies showed that the AMPA receptor antagonist GYKI-53655 displaces binding of a potentiator from brain receptors but not from recombinant GluA subunits. Here, we asked whether AMPA receptor modulatory subunits might resolve this discrepancy. We find that the cerebellar TARP, stargazin (γ-2), enhances the binding affinity of the AMPA receptor potentiator [(3)H]-LY450295 and confers sensitivity to displacement by non-competitive antagonists. In cerebellar membranes from stargazer mice, [(3)H]-LY450295 binding is reduced and relatively resistant to displacement by non-competitive antagonists. Coexpression of AMPA receptors with CNIH-2, which is expressed in the hippocampus and at low levels in the cerebellar Purkinje neurons, confers partial sensitivity of [(3)H]-LY450295 potentiator binding to displacement by non-competitive antagonists. Autoradiography of [(3)H]-LY450295 binding to stargazer and γ-8-deficient mouse brain sections, demonstrates that TARPs regulate the pharmacology of allosteric AMPA potentiators and antagonists in the cerebellum and hippocampus, respectively. These studies demonstrate that accessory proteins define AMPA receptor pharmacology by functionally linking allosteric AMPA receptor potentiator and antagonist sites.
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http://dx.doi.org/10.1074/jbc.M110.212522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075660PMC
April 2011

Hippocampal AMPA receptor gating controlled by both TARP and cornichon proteins.

Neuron 2010 Dec;68(6):1082-96

Department of Neuroscience, Eli Lilly and Company, Indianapolis, IN 46285, USA.

Transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon proteins (CNIH-2/3) independently modulate AMPA receptor trafficking and gating. However, the potential for interactions of these subunits within an AMPA receptor complex is unknown. Here, we find that TARPs γ-4, γ-7, and γ-8, but not γ-2, γ-3, or γ-5, cause AMPA receptors to "resensitize" upon continued glutamate application. With γ-8, resensitization occurs with all GluA subunit combinations; however, γ-8-containing hippocampal neurons do not display resensitization. In recombinant systems, CNIH-2 abrogates γ-8-mediated resensitization and modifies AMPA receptor pharmacology and gating to match that of hippocampal neurons. In hippocampus, γ-8 and CNIH-2 associate in postsynaptic densities and CNIH-2 protein levels are markedly diminished in γ-8 knockout mice. Manipulating neuronal CNIH-2 levels modulates the electrophysiological properties of extrasynaptic and synaptic γ-8-containing AMPA receptors. Thus, γ-8 and CNIH-2 functionally interact with common hippocampal AMPA receptor complexes to modulate synergistically kinetics and pharmacology.
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http://dx.doi.org/10.1016/j.neuron.2010.11.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3034222PMC
December 2010

An emerging role for TARPs in neuropsychiatric disorders.

Neuropsychopharmacology 2011 Jan;36(1):362-3

Neuroscience Discovery Research, Eli Lilly and Company, Indianapolis, IN, USA.

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http://dx.doi.org/10.1038/npp.2010.149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3055496PMC
January 2011

TARPs differentially decorate AMPA receptors to specify neuropharmacology.

Trends Neurosci 2010 May 8;33(5):241-8. Epub 2010 Mar 8.

Department of Neuroscience, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285-0510, USA.

Transmembrane AMPA receptor regulatory proteins (TARPs) are the first identified auxiliary subunits for a neurotransmitter-gated ion channel. Although initial studies found that stargazin, the prototypical TARP, principally chaperones AMPA receptors, subsequent research demonstrated that it also regulates AMPA receptor kinetics and synaptic waveforms. Recent studies have identified a diverse collection of TARP isoforms--types Ia, Ib II--that distinctly regulate AMPA receptor trafficking, gating and neuropharmacology. These TARP isoforms are heterogeneously expressed in specific neuronal populations and can differentially sculpt synaptic transmission and plasticity. Whole-genome analyses also link multiple TARP loci to childhood epilepsy, schizophrenia and bipolar disorder. TARPs emerge as vital components of excitatory synapses that participate both in signal transduction and in neuropsychiatric disorders.
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http://dx.doi.org/10.1016/j.tins.2010.02.004DOI Listing
May 2010

Mobile DHHC palmitoylating enzyme mediates activity-sensitive synaptic targeting of PSD-95.

J Cell Biol 2009 Jul;186(1):147-60

Division of Membrane Physiology, Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki, Aichi, Japan.

Protein palmitoylation is the most common posttranslational lipid modification; its reversibility mediates protein shuttling between intracellular compartments. A large family of DHHC (Asp-His-His-Cys) proteins has emerged as protein palmitoyl acyltransferases (PATs). However, mechanisms that regulate these PATs in a physiological context remain unknown. In this study, we efficiently monitored the dynamic palmitate cycling on synaptic scaffold PSD-95. We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors. A dendritically localized DHHC2 but not the Golgi-resident DHHC3 mediates this activity-sensitive palmitoylation. Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect. These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.
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http://dx.doi.org/10.1083/jcb.200903101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2712995PMC
July 2009

AMPA receptor subunit-specific regulation by a distinct family of type II TARPs.

Neuron 2008 Sep;59(6):986-96

Department of Neuroscience, Eli Lilly and Company, Indianapolis, IN 46285, USA.

AMPA-type glutamate receptors (GluRs) play major roles in excitatory synaptic transmission. Neuronal AMPA receptors comprise GluR subunits and transmembrane AMPA receptor regulatory proteins (TARPs). Previous studies identified five mammalian TARPs, gamma-2 (or stargazin), gamma-3, gamma-4, gamma-7, and gamma-8, that enhance AMPA receptor function. Here, we classify gamma-5 as a distinct class of TARP that modulates specific GluR2-containing AMPA receptors and displays properties entirely dissimilar from canonical TARPs. Gamma-5 increases peak currents and decreases the steady-state currents selectively from GluR2-containing AMPA receptors. Furthermore, gamma-5 increases rates of GluR2 deactivation and desensitization and decreases glutamate potency. Remarkably, all effects of gamma-5 require editing of GluR2 mRNA. Unlike other TARPs, gamma-5 modulates GluR2 without promoting receptor trafficking. We also find that gamma-7 regulation of GluR2 is dictated by mRNA editing. These data establish gamma-5 and gamma-7 as a separate family of "type II TARPs" that impart distinct physiological features to specific AMPA receptors.
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http://dx.doi.org/10.1016/j.neuron.2008.07.034DOI Listing
September 2008

Fibroblast growth factor-regulated palmitoylation of the neural cell adhesion molecule determines neuronal morphogenesis.

J Neurosci 2008 Sep;28(36):8897-907

Abteilung Neuro- und Sinnesphysiologie, Physiologisches Institut, Universität Göttingen, 37073 Göttingen, Germany.

During development of the nervous system, short- and long-range signals cooperate to promote axonal growth, guidance, and target innervation. Particularly, a short-range signal transducer, the neural cell adhesion molecule (NCAM), stimulates neurite outgrowth via mechanisms that require posttranslational modification of NCAM and signaling via receptors to a long-range messenger, the fibroblast growth factor (FGF). In the present study we further characterized a mechanism which regulates the functional interplay between NCAM and FGF receptor(s). We show that activation of FGF receptor(s) by FGF2 leads to palmitoylation of the two major transmembrane NCAM isoforms, NCAM140 and NCAM180, translocation of NCAM to GM1 ganglioside-containing lipid rafts, and stimulation of neurite outgrowth of hippocampal neurons. Ablation of NCAM, mutation of NCAM140 or NCAM180 palmitoylation sites, or pharmacological suppression of NCAM signaling inhibited FGF2-stimulated neurite outgrowth. Of the 23 members of the aspartate-histidine-histidine-cysteine (DHHC) domain containing proteins, DHHC-7 most strongly stimulated palmitoylation of NCAM, and enzyme activity was enhanced by FGF2. Thus, our study uncovers a molecular mechanism by which a growth factor regulates neuronal morphogenesis via activation of palmitoylation, which in turn modifies subcellular location and thus signaling via an adhesion molecule.
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http://dx.doi.org/10.1523/JNEUROSCI.2171-08.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301590PMC
September 2008

TARP redundancy is critical for maintaining AMPA receptor function.

J Neurosci 2008 Aug;28(35):8740-6

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143, USA.

Transmembrane AMPA receptor regulatory proteins (TARPs) are AMPA receptor auxiliary subunits that influence diverse aspects of receptor function. However, the full complement of physiological roles for TARPs in vivo remains poorly understood. Here we find that double knock-out mice lacking TARPs gamma-2 and gamma-3 are profoundly ataxic and fail to thrive. We demonstrate that these TARPs are critical for the synaptic targeting and kinetics of AMPA receptors in cerebellar Golgi cells, but that either alone is sufficient to fully preserve function. By analyzing the few remaining synaptic AMPA receptors in the gamma-2, gamma-3 double knock-out mice, we unexpectedly find that these TARPs specify AMPA receptor subunit composition. This study establishes a new role for TARPs in regulating AMPA receptor assembly and suggests that TARPs are necessary for proper AMPA receptor localization and function in most, if not all, neurons of the CNS.
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http://dx.doi.org/10.1523/JNEUROSCI.1319-08.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159041PMC
August 2008

Postsynaptic density-93 clusters Kv1 channels at axon initial segments independently of Caspr2.

J Neurosci 2008 May;28(22):5731-9

Department of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA.

Postsynaptic density-93 (PSD-93)/Chapsyn-110 is a PDZ (PSD-95/Discs large/zona occludens-1) domain-containing membrane-associated guanylate kinase (MAGUK) that functions as a scaffold to assemble channels, receptors, and other signaling proteins at cell membranes. PSD-93 is highly enriched at synapses, but mice lacking this protein have no synaptic structural abnormalities, probably because of overlapping expression and redundancy with other MAGUKs. Consequently, the function of PSD-93 is not well understood. Here, we show that PSD-93, but not other MAGUKs, is enriched at the axon initial segment (AIS), where it colocalizes with Kv1.1, Kv1.2, Kv1.4, and Kvbeta2 subunit-containing K(+) channels, Caspr2, and TAG-1 (transient axonal glycoprotein-1). When coexpressed with Kv1 channels in heterologous cells, PSD-93 induces formation of large cell-surface clusters. Knockdown of PSD-93 in cultured hippocampal neurons by RNA interference disrupted Kv1 channel localization at the AIS. Similarly, PSD-93-/- mice failed to cluster Kv1 channels at the AIS of cortical and hippocampal neurons. In contrast, Caspr2, which mediates Kv1 channel clustering at the juxtaparanode, is not required for localization of Kv1 channels at the AIS. These results show PSD-93 mediates AIS accumulation of Kv1 channels independently of Caspr2.
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http://dx.doi.org/10.1523/JNEUROSCI.4431-07.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2729199PMC
May 2008

MALS-3 regulates polarity and early neurogenesis in the developing cerebral cortex.

Development 2008 May 9;135(10):1781-90. Epub 2008 Apr 9.

Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.

Apicobasal polarity plays an important role in regulating asymmetric cell divisions by neural progenitor cells (NPCs) in invertebrates, but the role of polarity in mammalian NPCs is poorly understood. Here, we characterize the function of the PDZ domain protein MALS-3 in the developing cerebral cortex. We find that MALS-3 is localized to the apical domain of NPCs. Mice lacking all three MALS genes fail to localize the polarity proteins PATJ and PALS1 apically in NPCs, whereas the formation and maintenance of adherens junctions appears normal. In the absence of MALS proteins, early NPCs progressed more slowly through the cell cycle, and their daughter cells were more likely to exit the cell cycle and differentiate into neurons. Interestingly, these effects were transient; NPCs recovered normal cell cycle properties during late neurogenesis. Experiments in which MALS-3 was targeted to the entire membrane resulted in a breakdown of apicobasal polarity, loss of adherens junctions, and a slowing of the cell cycle. Our results suggest that MALS-3 plays a role in maintaining apicobasal polarity and is required for normal neurogenesis in the developing cortex.
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http://dx.doi.org/10.1242/dev.013847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3016226PMC
May 2008