RESUMEN
Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy comprise a group of neurodegenerative diseases termed synucleinopathies. Synucleinopathie are, characterized by presence of inclusion bodies in degenerating brain cells which contain aggregated α-synuclein phosphorylated on Ser129. Although the inflammation-associated serine-threonine kinase, PKR (EIF2AK2), promotes cellular protection against infection, we demonstrate a pro-degenerative role of activated PKR in an α-synuclein-dependent cell model of multiple system atrophy, where inhibition and silencing of PKR decrease cellular degeneration. In vitro phosphorylation demonstrates that PKR can directly bind and phosphorylate monomeric and filamenteous α-synuclein on Ser129. Inhibition and knockdown of PKR reduce Ser129 phosphorylation in different models (SH-SY5Y ASYN cells, OLN-AS7 cells, primary mouse hippocampal neurons, and acute brain slices), while overexpression of constitutively active PKR increases Ser129 α-syn phosphorylation. Treatment with pre-formed α-synuclein fibrils, proteostatic stress-promoting MG-132 and known PKR activators, herpes simplex virus-1-∆ICP34.5 and LPS, as well as PKR inducer, IFN-ß-1b, lead to increased levels of phosphorylated Ser129 α-synuclein that is completely blocked by simultaneous PKR inhibition. These results reveal a direct link between PKR and the phosphorylation and toxicity of α-synuclein, and they support that neuroinflammatory processes play a role in modulating the pathogenicity of α-synuclein.
Asunto(s)
Hipocampo/metabolismo , alfa-Sinucleína/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Animales Recién Nacidos , Muerte Celular/fisiología , Línea Celular Transformada , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Fosforilación/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , eIF-2 Quinasa/antagonistas & inhibidoresRESUMEN
Correct function of glutamate receptors in the postsynaptic density is crucial to synaptic function and plasticity. SorCS3 (sortilin-related receptor CNS expressed 3) is a sorting receptor which previously has been shown to interact with the key postsynaptic proteins; PSD-95 and PICK1. In this study, we employed electrophysiological analyses of acute brain slices combined with immunohistochemistry to define the role of SorCS3 in hippocampal synapses in CA1 and the dentate gyrus. We analyzed a juvenile (P17-21) and a young adult (P55-65) group of animals from a Sorcs3 knockout mouse model. We show that the basal synaptic transmission is severely affected in SorCS3-deficient neurons in CA1, while only slightly reduced in the dentate gyrus. Specifically, input/output curves of CA1 synapses revealed a 20% reduction of fEPSP (field excitatory postsynaptic potential) slopes at the highest stimulation intensity in knockouts of the juvenile group, which developed to a 33% decrease in young adult animals. These impairments may be a result of changes in the postsynaptic AMPA receptors. Interestingly, repetitive afferent stimulation demonstrated that SorCS3-deficient slices respond with an enhanced synaptic facilitation and reduced synaptic depression. These changes also developed with age. A molecular mechanism underlying this relative increase during repetitive stimulations is compatible with enhanced mobility of postsynaptic AMPA receptors resulting in faster exchange of desensitized receptors in the postsynaptic density. The altered response during repetitive stimulation was characteristic for CA1 but not the dentate gyrus. Immunohistochemical analyses of parvalbumin positive neurons combined with paired-pulse tests of network inhibition and patch-clamp recordings only showed minute inhibitory changes in SorCS3-deficient slices. Our results suggest that SorCS3 serves an important role in the postsynaptic protein network, which is more pronounced in CA1 compared to the dentate gyrus. These data support a role for SorCS3 in controlling proper positioning and mobility of glutamate receptors in the postsynaptic density. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Región CA1 Hipocampal/metabolismo , Giro Dentado/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Glutamato/metabolismo , Sinapsis/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Región CA1 Hipocampal/crecimiento & desarrollo , Región CA1 Hipocampal/patología , Recuento de Células , Giro Dentado/crecimiento & desarrollo , Giro Dentado/patología , Potenciales Postsinápticos Excitadores/fisiología , Inmunohistoquímica , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Microelectrodos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Parvalbúminas/metabolismo , Técnicas de Placa-Clamp , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Sinapsis/patologíaRESUMEN
Four 2-substituted Tet-AMPA [Tet = tetrazolyl, AMPA = 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid] analogues were characterized functionally at the homomeric AMPA receptors GluR1i, GluR2Qi, GluR3i, and GluR4i in a Fluo-4/Ca2+ assay. Whereas 2-Et-Tet-AMPA, 2-Pr-Tet-AMPA, and 2-iPr-Tet-AMPA were nonselective GluR agonists, 2-Bn-Tet-AMPA exhibited a 40-fold higher potency at GluR4i than at GluR1i. Examination of homology models of the S1-S2 domains of GluR1 and GluR4 containing 2-Bn-Tet-AMPA suggested four nonconserved residues in a region adjacent to the orthosteric site as possible determinants of the GluR4i/GluR1i selectivity. In a mutagenesis study, doubly mutating M686V/I687A in GluR1i in combination with either D399S or E683A increased both the potency and the maximal response of 2-Bn-Tet-AMPA at this receptor to levels similar to those elicited by the agonist at GluR4i. The dependence of the novel selectivity profile of 2-Bn-Tet-AMPA upon residues located outside of the orthosteric site underlines the potential for developing GluR subtype selective ligands by designing compounds with substituents that protrude beyond the (S)-Glu binding pocket.
Asunto(s)
Isoxazoles/química , Propionatos/química , Receptores AMPA/agonistas , Tetrazoles/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/análogos & derivados , Compuestos de Anilina , Animales , Sitios de Unión , Línea Celular , Femenino , Colorantes Fluorescentes , Humanos , Técnicas In Vitro , Isoxazoles/farmacología , Modelos Moleculares , Mutación , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Propionatos/farmacología , Ratas , Receptores AMPA/genética , Receptores AMPA/fisiología , Homología de Secuencia de Aminoácido , Estereoisomerismo , Relación Estructura-Actividad , Tetrazoles/farmacología , Termodinámica , Xantenos , Xenopus , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Replacement of the methyl group of the AMPA receptor agonist 2-amino-3-[3-hydroxy-5-(2-methyl-2H-5-tetrazolyl)-4-isoxazolyl]propionic acid (2-Me-Tet-AMPA) with a benzyl group provided the first AMPA receptor agonist, compound 7, capable of discriminating GluR2-4 from GluR1 by its more than 10-fold preference for the former receptor subtypes. An X-ray crystallographic analysis of this new analogue in complex with the GluR2-S1S2J construct shows that accommodation of the benzyl group creates a previously unobserved pocket in the receptor, which may explain the remarkable pharmacological profile of compound 7.
Asunto(s)
Receptores AMPA/agonistas , Tetrazoles/síntesis química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/análogos & derivados , Secuencia de Aminoácidos , Animales , Sitios de Unión , Encéfalo/metabolismo , Línea Celular , Cristalografía por Rayos X , Técnicas In Vitro , Insectos , Modelos Moleculares , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Ensayo de Unión Radioligante , Ratas , Receptores AMPA/química , Receptores AMPA/fisiología , Estereoisomerismo , Relación Estructura-Actividad , Sinaptosomas/metabolismo , Tetrazoles/química , Tetrazoles/farmacología , Xenopus laevis , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/síntesis química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/química , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Philanthotoxins are uncompetitive antagonists of Ca2+-permeable AMPA receptors presumed to bind to the pore-forming region, but a detailed molecular mechanism for this interaction is missing. Here a small library of novel philanthotoxins was designed and synthesized using a solid-phase strategy. The biological activities were investigated at cloned and "native" AMPA receptors using electrophysiological techniques. A distinct relationship between length of the polyamine moiety and the location of a secondary amino group was observed. Fitting the data to the Woodhull equation allowed the first experimental demonstration of the relative location and orientation of the philanthotoxin molecule in the receptor. These results were corroborated by in silico studies using a homology model of the AMPA receptor ion channel. Together these studies provide strong evidence for a molecular mechanism by which polyamine toxins antagonize the AMPA receptor ion channel and provide the basis for rational development of uncompetitive antagonists of AMPA receptors.
Asunto(s)
Poliaminas/síntesis química , Receptores AMPA/antagonistas & inhibidores , Toxinas Biológicas/química , Animales , Proteínas Bacterianas/química , Sitios de Unión , Calcio/fisiología , Técnicas In Vitro , Modelos Moleculares , Estructura Molecular , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Poliaminas/química , Poliaminas/farmacología , Canales de Potasio/química , Ratas , Receptores AMPA/genética , Receptores AMPA/fisiología , Estereoisomerismo , Relación Estructura-Actividad , Tirosina/análogos & derivados , Tirosina/química , Venenos de Avispas/química , Xenopus laevisRESUMEN
Polyamine toxins, such as philanthotoxins, are low-molecular-weight compounds isolated from spiders and wasps, which modulate ligand-gated ion channels in the nervous system. Philanthotoxins bind to the pore-forming region of AMPA receptors, a subtype of glutamate receptors which are important for memory formation and are involved in neurodegenerative diseases. Previous studies have demonstrated that modification of the polyamine moiety of philanthotoxins can lead to very potent and highly selective ligands for the AMPA receptor, as exemplified with philanthotoxin-56. Much less attention has been paid to the importance of the aromatic head group of philanthotoxins, but herein we demonstrate that modification of this moiety leads to a significant improvement in potency relative to philanthotoxin-56 at cloned AMPA receptors. Interestingly, the incorporation of an adamantane moiety is particularly favorable, and the most potent compound has a Ki value of 2 nM, making it the most potent uncompetitive antagonist of AMPA receptors described to date. Such compounds are potentially useful as neuroprotective agents.