Publications by authors named "Robert T Fremeau"

26 Publications

  • Page 1 of 1

1,2,4-Triazolsulfone: A novel isosteric replacement of acylsulfonamides in the context of Na1.7 inhibition.

Bioorg Med Chem Lett 2018 06 17;28(11):2103-2108. Epub 2018 Apr 17.

Amgen, Inc., 360 Binney Street, Cambridge, MA 02142, United States.

Recently, the identification of several classes of aryl sulfonamides and acyl sulfonamides that potently inhibit Na1.7 and demonstrate high levels of selectivity over other Na isoforms have been reported. The fully ionizable nature of these inhibitors has been shown to be an important part of the pharmacophore for the observed potency and isoform selectivity. The requirement of this functionality, however, has presented challenges associated with optimization toward inhibitors with drug-like properties and minimal off-target activity. In an effort to obviate these challenges, we set out to develop an orally bioavailable, selective Na1.7 inhibitor, lacking these acidic functional groups. Herein, we report the discovery of a novel series of inhibitors wherein a triazolesulfone has been designed to serve as a bioisostere for the acyl sulfonamide. This work culminated in the delivery of a potent series of inhibitors which demonstrated good levels of selectivity over Na1.5 and favorable pharmacokinetics in rodents.
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http://dx.doi.org/10.1016/j.bmcl.2018.04.035DOI Listing
June 2018

Pharmacologic Characterization of AMG8379, a Potent and Selective Small Molecule Sulfonamide Antagonist of the Voltage-Gated Sodium Channel Na1.7.

J Pharmacol Exp Ther 2017 07 4;362(1):146-160. Epub 2017 May 4.

Department of Neuroscience (T.J.K., R.Y., S.A, C.P.I., M.J., D.J., J.H.L., S.G.L., J.Li., D.L., J.Lu., D.M., D.O., K.T., J.W., V.Y., D.X.D.Z., R.T.F., B.D.M.), Department of Medicinal Chemistry (M.M.W.), and Department of Pharmacokinetics and Drug Metabolism (X.B., V.B., J.R.), Amgen Inc., Cambridge, Massachusetts and Thousand Oaks, California

Potent and selective antagonists of the voltage-gated sodium channel Na1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human Na1.7 channels with an IC of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other Na family members, including Na1.4 expressed in muscle and Na1.5 expressed in the heart, as well as TTX-resistant Na channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses. In vivo target engagement of AMG8379 in mice was evaluated in multiple Na1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels. AMG8379 is a potent and selective Na1.7 inhibitor that blocks sodium current in heterologous cells as well as DRG neurons, inhibits action potential firing in peripheral nerve fibers, and exhibits pharmacodynamic effects in translatable models of both itch and pain.
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http://dx.doi.org/10.1124/jpet.116.239590DOI Listing
July 2017

Sulfonamides as Selective Na1.7 Inhibitors: Optimizing Potency, Pharmacokinetics, and Metabolic Properties to Obtain Atropisomeric Quinolinone (AM-0466) that Affords Robust in Vivo Activity.

J Med Chem 2017 07 20;60(14):5990-6017. Epub 2017 Apr 20.

Department of Neuroscience, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.

Because of its strong genetic validation, Na1.7 has attracted significant interest as a target for the treatment of pain. We have previously reported on a number of structurally distinct bicyclic heteroarylsulfonamides as Na1.7 inhibitors that demonstrate high levels of selectivity over other Na isoforms. Herein, we report the discovery and optimization of a series of atropisomeric quinolinone sulfonamide inhibitors [ Bicyclic sulfonamide compounds as sodium channel inhibitors and their preparation . WO 2014201206, 2014 ] of Na1.7, which demonstrate nanomolar inhibition of Na1.7 and exhibit high levels of selectivity over other sodium channel isoforms. After optimization of metabolic and pharmacokinetic properties, including PXR activation, CYP2C9 inhibition, and CYP3A4 TDI, several compounds were advanced into in vivo target engagement and efficacy models. When tested in mice, compound 39 (AM-0466) demonstrated robust pharmacodynamic activity in a Na1.7-dependent model of histamine-induced pruritus (itch) and additionally in a capsaicin-induced nociception model of pain without any confounding effect in open-field activity.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01850DOI Listing
July 2017

Sulfonamides as Selective Na1.7 Inhibitors: Optimizing Potency and Pharmacokinetics While Mitigating Metabolic Liabilities.

J Med Chem 2017 07 20;60(14):5969-5989. Epub 2017 Apr 20.

Department of Neuroscience, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.

Several reports have recently emerged regarding the identification of heteroarylsulfonamides as Na1.7 inhibitors that demonstrate high levels of selectivity over other Na isoforms. The optimization of a series of internal Na1.7 leads that address a number of metabolic liabilities including bioactivation, PXR activation, as well as CYP3A4 induction and inhibition led to the identification of potent and selective inhibitors that demonstrated favorable pharmacokinetic profiles and were devoid of the aforementioned liabilities. The key to achieving this within a series prone to transporter-mediated clearance was the identification of a small range of optimal cLogD values and the discovery of subtle PXR SAR that was not lipophilicity dependent. This enabled the identification of compound 20, which was advanced into a target engagement pharmacodynamic model where it exhibited robust reversal of histamine-induced scratching bouts in mice.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01851DOI Listing
July 2017

Sulfonamides as Selective Na1.7 Inhibitors: Optimizing Potency and Pharmacokinetics to Enable in Vivo Target Engagement.

ACS Med Chem Lett 2016 Dec 21;7(12):1062-1067. Epub 2016 Sep 21.

Department of Medicinal Chemistry, Department of Molecular Engineering, Department of Pharmacokinetics and Drug Metabolism, Department of Neuroscience, and Department of Biologics, Amgen, Inc. , 360 Binney Street, Cambridge, Massachusetts 02142, and One Amgen Center Drive, Thousand Oaks, California 91320, United States.

Human genetic evidence has identified the voltage-gated sodium channel Na1.7 as an attractive target for the treatment of pain. We initially identified naphthalene sulfonamide as a potent and selective inhibitor of Na1.7. Optimization to reduce biliary clearance by balancing hydrophilicity and hydrophobicity (Log ) while maintaining Na1.7 potency led to the identification of quinazoline (AM-2099). Compound demonstrated a favorable pharmacokinetic profile in rat and dog and demonstrated dose-dependent reduction of histamine-induced scratching bouts in a mouse behavioral model following oral dosing.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5150675PMC
December 2016

An Orally Available BACE1 Inhibitor That Affords Robust CNS Aβ Reduction without Cardiovascular Liabilities.

ACS Med Chem Lett 2015 Feb 29;6(2):210-5. Epub 2014 Dec 29.

Department of Medicinal Chemistry, Department of Molecular Structure, Department of Neuroscience, Department of HTS and Molecular Pharmacology, and Department of Pharmacokinetics and Drug Metabolism, Comparative Biology and Safety Sciences, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.

BACE1 inhibition to prevent Aβ peptide formation is considered to be a potential route to a disease-modifying treatment for Alzheimer's disease. Previous efforts in our laboratory using a combined structure- and property-based approach have resulted in the identification of aminooxazoline xanthenes as potent BACE1 inhibitors. Herein, we report further optimization leading to the discovery of inhibitor 15 as an orally available and highly efficacious BACE1 inhibitor that robustly reduces CSF and brain Aβ levels in both rats and nonhuman primates. In addition, compound 15 exhibited low activity on the hERG ion channel and was well tolerated in an integrated cardiovascular safety model.
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http://dx.doi.org/10.1021/ml500458tDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329588PMC
February 2015

Development of 2-aminooxazoline 3-azaxanthenes as orally efficacious β-secretase inhibitors for the potential treatment of Alzheimer's disease.

Bioorg Med Chem Lett 2015 Feb 8;25(4):767-74. Epub 2015 Jan 8.

Department of Medicinal Chemistry, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA.

The β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is one of the most hotly pursued targets for the treatment of Alzheimer's disease. We used a structure- and property-based drug design approach to identify 2-aminooxazoline 3-azaxanthenes as potent BACE1 inhibitors which significantly reduced CSF and brain Aβ levels in a rat pharmacodynamic model. Compared to the initial lead 2, compound 28 exhibited reduced potential for QTc prolongation in a non-human primate cardiovascular safety model.
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http://dx.doi.org/10.1016/j.bmcl.2014.12.092DOI Listing
February 2015

Lead optimization and modulation of hERG activity in a series of aminooxazoline xanthene β-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors.

J Med Chem 2014 Dec 12;57(23):9796-810. Epub 2014 Nov 12.

Departments of Therapeutic Discovery, ‡Neuroscience, §Molecular Structure and Characterization, ∥Pharmacokinetics and Drug Metabolism, and ⊥Comparative Biology and Safety Sciences, Amgen Inc. , 360 Binney Street, Cambridge, Massachusetts 02142, One Amgen Center Drive, Thousand Oaks, California 91320, and 1120 Veterans Boulevard, South San Francisco, California 94080, United States.

The optimization of a series of aminooxazoline xanthene inhibitors of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is described. An early lead compound showed robust Aβ lowering activity in a rat pharmacodynamic model, but advancement was precluded by a low therapeutic window to QTc prolongation in cardiovascular models consistent with in vitro activity on the hERG ion channel. While the introduction of polar groups was effective in reducing hERG binding affinity, this came at the expense of higher than desired Pgp-mediated efflux. A balance of low Pgp efflux and hERG activity was achieved by lowering the polar surface area of the P3 substituent while retaining polarity in the P2' side chain. The introduction of a fluorine in position 4 of the xanthene ring improved BACE1 potency (5-10-fold). The combination of these optimized fragments resulted in identification of compound 40, which showed robust Aβ reduction in a rat pharmacodynamic model (78% Aβ reduction in CSF at 10 mg/kg po) and also showed acceptable cardiovascular safety in vivo.
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http://dx.doi.org/10.1021/jm501266wDOI Listing
December 2014

Inhibitors of β-site amyloid precursor protein cleaving enzyme (BACE1): identification of (S)-7-(2-fluoropyridin-3-yl)-3-((3-methyloxetan-3-yl)ethynyl)-5'H-spiro[chromeno[2,3-b]pyridine-5,4'-oxazol]-2'-amine (AMG-8718).

J Med Chem 2014 Dec 14;57(23):9811-31. Epub 2014 Nov 14.

Departments of Therapeutic Discovery, ‡Neuroscience, §Molecular Structure and Characterization, ∥Pharmacokinetics and Drug Metabolism, and ⊥Comparative Biology and Safety Sciences, Amgen, Inc. , 360 Binney Street, Cambridge, Massachusetts 02142, and One Amgen Center Drive, Thousand Oaks, California 91320, United States.

We have previously shown that the aminooxazoline xanthene scaffold can generate potent and orally efficacious BACE1 inhibitors although certain of these compounds exhibited potential hERG liabilities. In this article, we describe 4-aza substitution on the xanthene core as a means to increase BACE1 potency while reducing hERG binding affinity. Further optimization of the P3 and P2' side chains resulted in the identification of 42 (AMG-8718), a compound with a balanced profile of BACE1 potency, hERG binding affinity, and Pgp recognition. This compound produced robust and sustained reductions of CSF and brain Aβ levels in a rat pharmacodynamic model and exhibited significantly reduced potential for QTc elongation in a cardiovascular safety model.
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http://dx.doi.org/10.1021/jm5012676DOI Listing
December 2014

Structure- and property-based design of aminooxazoline xanthenes as selective, orally efficacious, and CNS penetrable BACE inhibitors for the treatment of Alzheimer's disease.

J Med Chem 2012 Nov 11;55(21):9156-69. Epub 2012 Sep 11.

Department of Medicinal Chemistry, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States.

A structure- and property-based drug design approach was employed to identify aminooxazoline xanthenes as potent and selective human β-secretase inhibitors. These compounds exhibited good isolated enzyme, cell potency, and selectivity against the structurally related aspartyl protease cathepsin D. Our efforts resulted in the identification of a potent, orally bioavailable CNS penetrant compound that exhibited in vivo efficacy. A single oral dose of compound 11a resulted in a significant reduction of CNS Aβ40 in naive rats.
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http://dx.doi.org/10.1021/jm300598eDOI Listing
November 2012

Chromenones as potent bradykinin B1 antagonists.

Bioorg Med Chem Lett 2012 Jan 28;22(1):619-22. Epub 2011 Oct 28.

Department of Chemistry Research and Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.

A series of fused 6,6-bicyclic chromenones was investigated for activity against the bradykinin B1 receptor. SAR studies based on a pharmacophore model revealed compounds with high affinity for both human and rabbit B1. These compounds demonstrated favorable pharmacokinetic properties and 5-chlorochromenone 15 was efficacious in a carrageenan-induced mechanical hyperalgesia model for chronic pain.
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http://dx.doi.org/10.1016/j.bmcl.2011.10.068DOI Listing
January 2012

Discovery of potent, orally bioavailable phthalazinone bradykinin B1 receptor antagonists.

J Med Chem 2011 Oct 26;54(20):7232-46. Epub 2011 Sep 26.

Department of Chemistry Research and Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States.

The bradykinin B1 receptor is rapidly induced upon tissue injury and inflammation, stimulating the production of inflammatory mediators resulting in plasma extravasation, leukocyte trafficking, edema, and pain. We have previously reported on sulfonamide and sulfone-based B1 antagonists containing a privileged bicyclic amine moiety leading to potent series of 2-oxopiperazines. The suboptimal pharmacokinetics and physicochemical properties of the oxopiperazine sulfonamides led us to seek B1 antagonists with improved druglike properties. Using a pharmacophore model containing a bicyclic amine as anchor, we designed a series of amide antagonists with targeted physicochemical properties. This approach led to a novel series of potent phthalazinone B1 antagonists, where we successfully replaced a sulfonamide acceptor with a cyclic carbonyl unit. SAR studies revealed compounds with subnanomolar B1 binding affinity. These compounds demonstrate excellent cross-species PK properties with high oral bioavailability and potent activity in a rabbit biochemical challenge pharmacodynamic study.
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http://dx.doi.org/10.1021/jm200808vDOI Listing
October 2011

From fragment screening to in vivo efficacy: optimization of a series of 2-aminoquinolines as potent inhibitors of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1).

J Med Chem 2011 Aug 29;54(16):5836-57. Epub 2011 Jul 29.

Chemistry Research and Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, USA.

Using fragment-based screening of a focused fragment library, 2-aminoquinoline 1 was identified as an initial hit for BACE1. Further SAR development was supported by X-ray structures of BACE1 cocrystallized with various ligands and molecular modeling studies to expedite the discovery of potent compounds. These strategies enabled us to integrate the C-3 side chain on 2-aminoquinoline 1 extending deep into the P2' binding pocket of BACE1 and enhancing the ligand's potency. We were able to improve the BACE1 potency to subnanomolar range, over 10(6)-fold more potent than the initial hit (900 μM). Further elaboration of the physical properties of the lead compounds to those more consistent with good blood-brain barrier permeability led to inhibitors with greatly improved cellular activity and permeability. Compound 59 showed an IC(50) value of 11 nM on BACE1 and cellular activity of 80 nM. This compound was advanced into rat pharmacokinetic and pharmacodynamic studies and demonstrated significant reduction of Aβ levels in cerebrospinal fluid (CSF).
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http://dx.doi.org/10.1021/jm200544qDOI Listing
August 2011

Discovery of alpha-amidosulfones as potent and selective agonists of CB2: synthesis, SAR, and pharmacokinetic properties.

Bioorg Med Chem Lett 2009 Jan 13;19(1):31-5. Epub 2008 Nov 13.

Department of Medicinal Chemistry, Amgen Inc, One Kendall Square, Cambridge, MA 02139, USA.

A series of alpha-amidosulfones were found to be potent and selective agonists of CB(2). The discovery, synthesis, and structure-activity relationships of this series of agonists are reported. In addition, the pharmacokinetic properties of the most promising compounds are profiled.
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http://dx.doi.org/10.1016/j.bmcl.2008.11.026DOI Listing
January 2009

Discovery and optimization of a novel series of N-arylamide oxadiazoles as potent, highly selective and orally bioavailable cannabinoid receptor 2 (CB2) agonists.

J Med Chem 2008 Aug 5;51(16):5019-34. Epub 2008 Aug 5.

Chemistry Research and Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, USA.

The CB2 receptor is an attractive therapeutic target for analgesic and anti-inflammatory agents. Herein we describe the discovery of a novel class of oxadiazole derivatives from which potent and selective CB2 agonist leads were developed. Initial hit 7 was identified from a cannabinoid target-biased library generated by virtual screening of sample collections using a pharmacophore model in combination with a series of physicochemical filters. 7 was demonstrated to be a selective CB2 agonist (CB2 EC50 = 93 nM, Emax = 98%, CB1 EC50 > 10 microM). However, this compound exhibited poor solubility and relatively high clearance in rat, resulting in low oral bioavailability. In this paper, we report detailed SAR studies on 7 en route toward improving potency, physicochemical properties, and solubility. This effort resulted in identification of 63 that is a potent and selective agonist at CB2 (EC50 = 2 nM, Emax = 110%) with excellent pharmacokinetic properties.
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http://dx.doi.org/10.1021/jm800463fDOI Listing
August 2008

Structural modifications of N-arylamide oxadiazoles: Identification of N-arylpiperidine oxadiazoles as potent and selective agonists of CB2.

Bioorg Med Chem Lett 2008 Aug 3;18(15):4267-74. Epub 2008 Jul 3.

Department of Medicinal Chemistry, Amgen Inc., One Kendall Square, Building 1000, Cambridge, MA 02139, USA.

Structural modifications to the central portion of the N-arylamide oxadiazole scaffold led to the identification of N-arylpiperidine oxadiazoles as conformationally constrained analogs that offered improved stability and comparable potency and selectivity. The simple, modular scaffold allowed for the use of expeditious and divergent synthetic routes, which provided two-directional SAR in parallel. Several potent and selective agonists from this novel ligand class are described.
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http://dx.doi.org/10.1016/j.bmcl.2008.06.096DOI Listing
August 2008

Vesicular glutamate transporter 1 is required for photoreceptor synaptic signaling but not for intrinsic visual functions.

J Neurosci 2007 Jul;27(27):7245-55

Department of Ophthalmology, University of California School of Medicine, San Francisco, California 94143, USA.

Glutamatergic neurotransmission requires vesicular glutamate transporters (VGLUTs) to sequester glutamate into synaptic vesicles. Generally, VGLUT1 and VGLUT2 isoforms show complementary expression in the CNS and retina. However, little is known about whether isoform-specific expression serves distinct pathways and physiological functions. Here, by examining visual functions in VGLUT1-null mice, we demonstrate that visual signaling from photoreceptors to retinal output neurons requires VGLUT1. However, photoentrainment and pupillary light responses are preserved. We provide evidence that melanopsin-containing, intrinsically photosensitive retinal ganglion cells (RGCs), signaling via VGLUT2 pathways, support these non-image-forming functions. We conclude that VGLUT1 is essential for transmitting visual signals from photoreceptors to second- and third-order neurons, but VGLUT1 is not necessary for intrinsic visual functions. Furthermore, melanopsin and VGLUT2 expression in a subset of RGCs immediately after birth strongly supports the idea that intrinsic vision can function well before rod- and cone-mediated signaling has matured.
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http://dx.doi.org/10.1523/JNEUROSCI.0815-07.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2443709PMC
July 2007

Distribution of vesicular glutamate transporters 1 and 2 in the rat spinal cord, with a note on the spinocervical tract.

J Comp Neurol 2006 Aug;497(5):683-701

Department of Experimental Medical Science, Division for Neuroscience, and Lund University Pain Research Center, Lund University, S-221 84 Lund, Sweden.

To evaluate whether the organization of glutamatergic fibers systems in the lumbar cord is also evident at other spinal levels, we examined the immunocytochemical distribution of vesicle glutamate transporters 1 and 2 (VGLUT1, VGLUT2) at several different levels of the rat spinal cord. We also examined the expression of VGLUTs in an ascending sensory pathway, the spinocervical tract, and colocalization of VGLUT1 and VGLUT2. Mainly small VGLUT2-immunoreactive varicosities occurred at relatively high densities in most areas, with the highest density in laminae I-II. VGLUT1 immunolabeling, including small and medium-sized to large varicosities, was more differentiated, with the highest density in the deep dorsal horn and in certain nuclei such as the internal basilar nucleus, the central cervical nucleus, and the column of Clarke. Lamina I and IIo displayed a moderate density of small VGLUT1 varicosities at all spinal levels, although in the spinal enlargements a uniform density of such varicosities was evident throughout laminae I-II in the medial half of the dorsal horn. Corticospinal tract axons displayed VGLUT1, indicating that the corticospinal tract is an important source of small VGLUT1 varicosities. VGLUT1 and VGLUT2 were cocontained in small numbers of varicosities in laminae III-IV and IX. Anterogradely labeled spinocervical tract terminals in the lateral cervical nucleus were VGLUT2 immunoreactive. In conclusion, the principal distribution patterns of VGLUT1 and VGLUT2 are essentially similar throughout the rostrocaudal extension of the spinal cord. The mediolateral differences in VGLUT1 distribution in laminae I-II suggest dual origins of VGLUT1-immunoreactive varicosities in this region.
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http://dx.doi.org/10.1002/cne.20987DOI Listing
August 2006

Expression of the vesicular glutamate transporters during development indicates the widespread corelease of multiple neurotransmitters.

J Comp Neurol 2004 Dec;480(3):264-80

Institute of Basic Medical Sciences and the Centre for Molecular Biology and Neuroscience, University of Oslo, N-0317 Oslo, Norway.

Three closely related proteins transport glutamate into synaptic vesicles for release by exocytosis. Complementary patterns of expression in glutamatergic terminals have been reported for VGLUT1 and VGLUT2. VGLUT3 shows expression by many cells not considered to be glutamatergic. Here we describe the changes in VGLUT expression that occur during development. VGLUT1 expression increases gradually after birth and eventually predominates over the other isoforms in telencephalic regions. Expressed at high levels shortly after birth, VGLUT2 declines with age in multiple regions, in the cerebellum by 14-fold. In contrast, Coexpression of the two isoforms occurs transiently during development as well as permanently in a restricted subset of glutamatergic terminals in the adult. VGLUT3 is transiently expressed at high levels by select neuronal populations, including terminals in the cerebellar nuclei, scattered neurons in the cortex, and progenitor-like cells, implicating exocytotic glutamate release in morphogenesis and development. VGLUT3 also colocalizes extensively during development with the neuronal vesicular monoamine transporter VMAT2, with the vesicular acetylcholine transporter VAChT, and with the vesicular gamma-aminobutyric acid transporter VGAT. Such coexpression occurs particularly at some specific developmental stages and is restricted to certain sets of cells. In skeletal muscle, VGLUT3 localizes to granular organelles in the axon terminal as well as in the muscle sarcoplasm. The results suggest novel mechanisms and roles for regulated transmitter release.
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http://dx.doi.org/10.1002/cne.20354DOI Listing
December 2004

Vesicular glutamate transporter 3 expression identifies glutamatergic amacrine cells in the rodent retina.

J Comp Neurol 2004 Sep;477(4):386-98

Department of Ophthalmology, University of California School of Medicine, San Francisco, San Francisco, California 94143, USA.

Synaptic transmission from glutamatergic neurons requires vesicular glutamate transporters (VGLUTs) to concentrate cytosolic glutamate in synaptic vesicles. In retina, glutamatergic photoreceptors and bipolar cells exclusively express the VGLUT1 isoform, whereas ganglion cells express VGLUT2. Surprisingly, the recently identified VGLUT3 isoform was found in presumed amacrine cells, generally considered to be inhibitory interneurons. To investigate the synaptic machinery and conceivable secondary neurotransmitter composition of VGLUT3 cells, and to determine a potential functional role, we further investigated these putative glutamatergic amacrine cells in adult and developing rodent retina. Reverse transcriptase-PCR substantiated VGLUT3 expression in mouse retina. VGLUT3 cells did not immunostain for ganglion or bipolar cell markers, providing evidence that they are amacrine cells. VGLUT3 colocalized with synaptic vesicle markers, and electron microscopy showed that VGLUT3 immunostained synaptic vesicles. VGLUT3 cells were not immunoreactive for amacrine cell markers gamma-aminobutyric acid, choline acetyltransferase, calretinin, or tyrosine hydroxylase, although they immunostain for glycine. VGLUT3 processes made synaptic contact with ganglion cell dendrites, suggesting input onto these cells. VGLUT3 immunostaining was closely associated with the metabotropic glutamate receptor 4, which is consistent with glutamatergic synaptic exocytosis by these cells. In the maturing mouse retina, Western blots showed VGLUT3 expression at postnatal day 7/8 (P7/8). VGLUT3 immunostaining in retinal sections was first observed at P8, achieving an adult pattern at P12. Thus, VGLUT3 function commences around the same time as VGLUT1-mediated glutamatergic transmission from bipolar cells. Furthermore, a subset of VGLUT3 cells expressed the circadian clock gene period 1, implicating VGLUT3 cells as part of the light-entrainable retina-based circadian system.
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http://dx.doi.org/10.1002/cne.20250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2586940PMC
September 2004

Endocannabinoid-independent retrograde signaling at inhibitory synapses in layer 2/3 of neocortex: involvement of vesicular glutamate transporter 3.

J Neurosci 2004 May;24(21):4978-88

Unit of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden.

Recent studies implicate dendritic endocannabinoid release from subsynaptic dendrites and subsequent inhibition of neurotransmitter release from nerve terminals as a means of retrograde signaling in multiple brain regions. Here we show that type 1 cannabinoid receptor-mediated endocannabinoid signaling is not involved in the retrograde control of synaptic efficacy at inhibitory synapses between fast-spiking interneurons and pyramidal cells in layer 2/3 of the neocortex. Vesicular neurotransmitter transporters, such as vesicular glutamate transporters (VGLUTs) 1 and 2, are localized to presynaptic terminals and accumulate neurotransmitters into synaptic vesicles. A third subtype of VGLUTs (VGLUT3) was recently identified and found localized to dendrites of various cell types. We demonstrate, using multiple immunofluorescence labeling and confocal laser-scanning microscopy, that VGLUT3-like immunoreactivity is present in dendrites of layer 2/3 pyramidal neurons in the rat neocortex. Electron microscopy analysis confirmed that VGLUT3-like labeling is localized to vesicular structures, which show a tendency to accumulate in close proximity to postsynaptic specializations in dendritic shafts of pyramidal cells. Dual whole-cell recordings revealed that retrograde signaling between fast-spiking interneurons and pyramidal cells was enhanced under conditions of maximal efficacy of VGLUT3-mediated glutamate uptake, whereas it was reduced when glutamate uptake was inhibited by incrementing concentrations of the nonselective VGLUT inhibitor Evans blue (0.5-5.0 microm) or intracellular Cl- concentrations (4-145 mm). Our results present further evidence that dendritic vesicular glutamate release, controlled by novel VGLUT isoforms, provides fast negative feedback at inhibitory neocortical synapses, and demonstrate that glutamate can act as a retrograde messenger in the CNS.
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http://dx.doi.org/10.1523/JNEUROSCI.4884-03.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6729377PMC
May 2004

Vesicular glutamate transporters 1 and 2 target to functionally distinct synaptic release sites.

Science 2004 Jun 29;304(5678):1815-9. Epub 2004 Apr 29.

Department of Neurology, Graduate Programs in Neuroscience and Cell Biology, University of California San Francisco School of Medicine, CA 94143, USA.

Vesicular glutamate transporters (VGLUTs) 1 and 2 show a mutually exclusive distribution in the adult brain that suggests specialization for synapses with different properties of release. Consistent with this distribution, inactivation of the VGLUT1 gene silenced a subset of excitatory neurons in the adult. However, the same cell populations exhibited VGLUT1-independent transmission early in life. Developing hippocampal neurons transiently coexpressed VGLUT2 and VGLUT1 at distinct synaptic sites with different short-term plasticity. The loss of VGLUT1 also reduced the reserve pool of synaptic vesicles. Thus, VGLUT1 plays an unanticipated role in membrane trafficking at the nerve terminal.
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http://dx.doi.org/10.1126/science.1097468DOI Listing
June 2004

VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate.

Trends Neurosci 2004 Feb;27(2):98-103

Department of Neurology, Graduate Program in Neuroscience, UCSF School of Medicine, 600 16th Street, San Francisco, CA 94143-2140, USA.

Exocytotic release of the excitatory neurotransmitter glutamate depends on transport of this amino acid into synaptic vesicles. Recent work has identified a distinct family of proteins responsible for vesicular glutamate transport (VGLUTs) that show no sequence similarity to the other two families of vesicular neurotransmitter transporters. The distribution of VGLUT1 and VGLUT2 accounts for the ability of most established excitatory neurons to release glutamate by exocytosis. Surprisingly, they show a striking complementary pattern of expression in adult brain that might reflect differences in membrane trafficking. By contrast, VGLUT3 is expressed by many cells traditionally considered to release a different classical transmitter, suggesting novel roles for glutamate as an extracellular signal. VGLUT3 also differs from VGLUT1 and VGLUT2 in its subcellular location, with somatodendritic as well as axonal expression.
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http://dx.doi.org/10.1016/j.tins.2003.11.005DOI Listing
February 2004

Complementary distribution of vesicular glutamate transporters 1 and 2 in the nucleus accumbens of rat: Relationship to calretinin-containing extrinsic innervation and calbindin-immunoreactive neurons.

J Comp Neurol 2003 Oct;465(1):1-10

Paul Flechsig Institute for Brain Research, University of Leipzig, D-04109 Leipzig, Germany.

The caudomedial shell of the rat nucleus accumbens exhibits inhomogeneous distribution patterns of the vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2). This paper focuses on the question of whether patterns of VGLUT1 and VGLUT2 correspond to cytoarchitectonically and cytochemically defined subterritories of the caudomedial shell region. VGLUT2 was shown to be coexpressed with calretinin in the dense axonal plexus known to emanate from the paraventricular thalamic nucleus. In regions termed corridors, which are spared by this paraventricular thalamic innervation, axonal terminals were found to be clustered and VGLUT1-immunoreactive. It is assumed that these fibers originate in the prelimbic cortex and/or in the parvicellular basal amygdaloid nucleus known to project to accumbal shell components. Our findings confirm the existence of two well-separated neuronal circuits in the caudomedial shell that are dominated by two different excitatory input systems originating from either thalamic, cortical, or cortex-like amygdaloid sources. The large lateral corridors-which resemble the accumbal core not only in respect to their VGLUT1 immunolabeling but also concerning their content of calbindin-positive cells-may represent a component of the anatomically weakly defined accumbal shore region.
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http://dx.doi.org/10.1002/cne.10789DOI Listing
October 2003

The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate.

Proc Natl Acad Sci U S A 2002 Oct 18;99(22):14488-93. Epub 2002 Oct 18.

Department of Neurology, University of California School of Medicine, San Francisco, CA 94143, USA.

Quantal release of the principal excitatory neurotransmitter glutamate requires a mechanism for its transport into secretory vesicles. Within the brain, the complementary expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the release of glutamate by all known excitatory neurons. We now report the identification of VGLUT3 and its expression by many cells generally considered to release a classical transmitter with properties very different from glutamate. Remarkably, subpopulations of inhibitory neurons as well as cholinergic interneurons, monoamine neurons, and glia express VGLUT3. The dendritic expression of VGLUT3 by particular neurons also indicates the potential for retrograde synaptic signaling. The distribution and subcellular location of VGLUT3 thus suggest novel modes of signaling by glutamate.
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http://dx.doi.org/10.1073/pnas.222546799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137910PMC
October 2002