RESUMEN
Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.
Asunto(s)
Conducta Animal/fisiología , Distrofia Miotónica/genética , Distrofia Miotónica/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Adulto , Anciano , Animales , Western Blotting , Electroforesis en Gel Bidimensional , Electrofisiología , Humanos , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Distrofia Miotónica/complicaciones , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Expansión de Repetición de TrinucleótidoRESUMEN
INTRODUCTION: Failures in drug development often result from the emergence of unexpected adverse drug reactions. It is clear that adverse drug reactions, including seizure liability, should be assessed earlier. The goal of the present work was to develop a new platform of in vitro assays, NS-PC set (for Neuroservice proconvulsive set), to determine the proconvulsive potential of compounds earlier in preclinical development. METHODS: Assays were based on electrophysiological recordings in acute hippocampal slices performed with multielectrode arrays. 4 reference proconvulsive/seizurogenic compounds (4-aminopyridine, bicuculline, kainate and carbachol) and 4 anti-epileptic drugs (AEDs; phenobarbital, carbamazepine, clonazepam and valproic acid) were evaluated on electrophysiological endpoints involved in seizure risk (neuronal excitability, balance of excitatory/inhibitory synaptic transmission, occurrence of neuronal synchronization mechanisms materialized by epileptiform discharges). RESULTS: The reference compounds increased the number and area under the curve of population spikes, triggered epileptiform discharges and enhanced the firing rate of CA1 neurons. The effects of the 4 antiepileptic drugs were assessed on these 3 parameters. They were able to partially of completely reverse the effects of proconvulsive compounds. DISCUSSION: The use of reference proconvulsive compounds and AEDs validated the electrophysiological parameters to detect proconvulsive risk. Systematic evaluation of compounds with the 3 complementary endpoints increase the probability to detect seizure liability in vitro. Depending on the compound mechanism of action, only one or two of the identified parameters might be modified.
RESUMEN
Histaminergic H3 inverse agonists, by stimulating central histamine release, represent attractive drug candidates to treat cognitive disorders. The present studies aimed to describe the mechanistic profile of S 38093 a novel H3 receptors inverse agonist. S 38093 displays a moderate affinity for rat, mouse and human H3 receptors (Ki=8.8, 1.44 and 1.2µM, respectively) with no affinity for other histaminergic receptors. In cellular models, the compound was able to antagonize mice H3 receptors (KB=0.65µM) and to suppress cAMP decrease induced by an H3 agonist via human H3 receptors (KB=0.11µM). The antagonism properties of the compound were confirmed by electrophysiological studies on rat hippocampal slices (from 0.1µM). In cells expressing a high H3 density, S 38093 behaved as a moderate inverse agonist at rat and human H3 receptors (EC50=9 and 1.7µM, respectively). S 38093 was rapidly absorbed in mouse and rat (Tmax=0.25-0.5h), slowly in monkey (2h), with a bioavailability ranging from 20% to 60% and t1/2 ranging from 1.5 to 7.4h. The compound was widely distributed with a moderate volume of distribution and low protein binding. The brain distribution of S 38093 was rapid and high. In mice, S 38093 significantly increased ex vivo N-tele-Methylhistamine cerebral levels from 3mg/kg p.o. and antagonized R-α-Methylhistamine-induced dipsogenia from 10mg/kg i.p. Taken together, these data suggest that S 38093, a novel H3 inverse agonist, is a good candidate for further in vivo evaluations, in particular in animal models of cognition.
Asunto(s)
Compuestos de Azabiciclo/farmacología , Benzamidas/farmacología , Agonismo Inverso de Drogas , Agonistas de los Receptores Histamínicos/farmacocinética , Antagonistas de los Receptores Histamínicos H3/farmacocinética , Receptores Histamínicos H3/metabolismo , Animales , Ácido Araquidónico/metabolismo , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Histamina/metabolismo , Agonistas de los Receptores Histamínicos/metabolismo , Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos H3/metabolismo , Antagonistas de los Receptores Histamínicos H3/farmacología , Humanos , Masculino , Ratones , RatasRESUMEN
The multi-electrode arrays (MEA) technology for the recording of brain slices is available for more than 10 years. However, despite its relative simplicity, this recording technique is not widely used in academic or pharmaceutical research laboratories. We illustrate here that MEA provide multiple possibilities to investigate some network physiological properties as well as to evaluate the pharmacological effects of compounds. We first document that MEA allow to trigger and to record conventional FP which are inhibited by the block of action potential propagation (with 500 nM TTX). FP recorded with MEA are sensitive to ionic substitutions, to ionotropic glutamate receptor antagonists (CNQX or NBQX) and to energetic failure. Second, we illustrate that different "classical" protocols (paired-pulse, LTP, chemical LTD), revealing synaptic plasticity mechanisms, could be performed. Third, we document that MEA allow spatial and temporal discriminations for the effects of known pharmacological compounds such as competitive antagonist (gabazine, bicuculline) and allosteric modulators (steroids) of GABA(A) receptors. In conclusion, we illustrate that MEA recordings of adult rat hippocampal slices constitute a powerful and sensitive system to evaluate the effect of molecules on basic synaptic propagation/transmission and on synaptic plasticity processes.
Asunto(s)
Electrofisiología/instrumentación , Hipocampo/fisiología , Animales , Interpretación Estadística de Datos , Estimulación Eléctrica , Electrodos , Antagonistas del GABA/farmacología , Técnicas In Vitro , Canales Iónicos/antagonistas & inhibidores , Potenciación a Largo Plazo/efectos de los fármacos , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Neurotransmisores/farmacología , Piridazinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/agonistas , Receptores Presinapticos/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)pyrazine-2-carboxamide) and MPX-007 (5-(((3-fluoro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)methylpyrazine-2-carboxamide). MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC50s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.
Asunto(s)
Subunidades de Proteína/metabolismo , Pirazinas/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Sulfonamidas/farmacología , Animales , Región CA1 Hipocampal/citología , Calcio/metabolismo , Células Cultivadas , Perros , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Ácido Glutámico/metabolismo , Glicina/metabolismo , Humanos , Activación del Canal Iónico/efectos de los fármacos , Células de Riñón Canino Madin Darby , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oocitos/metabolismo , Pirazinas/química , Ratas Sprague-Dawley , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Relación Estructura-Actividad , Sulfonamidas/química , XenopusRESUMEN
Dysregulation of the kynurenine (Kyn) pathway has been associated with the progression of Huntington's disease (HD). In particular, elevated levels of the kynurenine metabolites 3-hydroxy kynurenine (3-OH-Kyn) and quinolinic acid (Quin), have been reported in the brains of HD patients as well as in rodent models of HD. The production of these metabolites is controlled by the activity of kynurenine mono-oxygenase (KMO), an enzyme which catalyzes the synthesis of 3-OH-Kyn from Kyn. In order to determine the role of KMO in the phenotype of mouse models of HD, we have developed a potent and selective KMO inhibitor termed CHDI-340246. We show that this compound, when administered orally to transgenic mouse models of HD, potently and dose-dependently modulates the Kyn pathway in peripheral tissues and in the central nervous system. The administration of CHDI-340246 leads to an inhibition of the formation of 3-OH-Kyn and Quin, and to an elevation of Kyn and Kynurenic acid (KynA) levels in brain tissues. We show that administration of CHDI-340246 or of Kyn and of KynA can restore several electrophysiological alterations in mouse models of HD, both acutely and after chronic administration. However, using a comprehensive panel of behavioral tests, we demonstrate that the chronic dosing of a selective KMO inhibitor does not significantly modify behavioral phenotypes or natural progression in mouse models of HD.
Asunto(s)
Fenómenos Electrofisiológicos/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/fisiopatología , Quinurenina 3-Monooxigenasa/antagonistas & inhibidores , Pirimidinas/uso terapéutico , Análisis de Varianza , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Fenómenos Electrofisiológicos/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Hipocampo/efectos de los fármacos , Humanos , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Técnicas In Vitro , Ácido Quinurénico/metabolismo , Quinurenina 3-Monooxigenasa/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microdiálisis , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacología , Ácido Quinolínico/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transfección , Repeticiones de Trinucleótidos/genética , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
Huntington's disease (HD) symptoms are driven to a large extent by dysfunction of the basal ganglia circuitry. HD patients exhibit reduced striatal phoshodiesterase 10 (PDE10) levels. Using HD mouse models that exhibit reduced PDE10, we demonstrate the benefit of pharmacologic PDE10 inhibition to acutely correct basal ganglia circuitry deficits. PDE10 inhibition restored corticostriatal input and boosted cortically driven indirect pathway activity. Cyclic nucleotide signaling is impaired in HD models, and PDE10 loss may represent a homeostatic adaptation to maintain signaling. Elevation of both cAMP and cGMP by PDE10 inhibition was required for rescue. Phosphoproteomic profiling of striatum in response to PDE10 inhibition highlighted plausible neural substrates responsible for the improvement. Early chronic PDE10 inhibition in Q175 mice showed improvements beyond those seen with acute administration after symptom onset, including partial reversal of striatal deregulated transcripts and the prevention of the emergence of HD neurophysiological deficits. VIDEO ABSTRACT.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Enfermedad de Huntington/fisiopatología , Neostriado/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Pirazoles/farmacología , Quinolinas/farmacología , Animales , Ganglios Basales/diagnóstico por imagen , Ganglios Basales/efectos de los fármacos , Ganglios Basales/metabolismo , Ganglios Basales/fisiopatología , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Enfermedad de Huntington/metabolismo , Ratones , Neostriado/diagnóstico por imagen , Neostriado/metabolismo , Neostriado/fisiopatología , Hidrolasas Diéster Fosfóricas , Tomografía de Emisión de Positrones , Núcleo Subtalámico/diagnóstico por imagen , Núcleo Subtalámico/efectos de los fármacos , Núcleo Subtalámico/metabolismo , Núcleo Subtalámico/fisiopatología , TritioRESUMEN
Huntington's disease is a neurodegenerative disorder caused by mutations in the CAG tract of huntingtin. Several studies in HD cellular and rodent systems have identified disturbances in cyclic nucleotide signaling, which might be relevant to pathogenesis and therapeutic intervention. To investigate whether selective phosphodiesterase (PDE) inhibitors can improve some aspects of disease pathogenesis in HD models, we have systematically evaluated the effects of a variety of cAMP and cGMP selective PDE inhibitors in various HD models. Here we present the lack of effect in a variety of endpoints of the PDE subtype selective inhibitor SCH-51866, a PDE1/5 inhibitor, in the R6/2 mouse model of HD, after chronic oral dosing.
RESUMEN
A simple and versatile fabrication process is used to define conducting polymer microelectrode arrays (MEAs), patterning at the same time the recording electrodes as well as the insulating layer. Thanks to the low impedance of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) electrodes, these MEAs allow in vitro recording of action potentials from rat hippocampus slices.
RESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive death of cortical and spinal motor neurons, for which there is no effective treatment. Using a cell-based assay for compounds capable of preventing motor neuron cell death in vitro, a collection of approximately 40,000 low-molecular-weight compounds was screened to identify potential small-molecule therapeutics. We report the identification of cholest-4-en-3-one, oxime (TRO19622) as a potential drug candidate for the treatment of ALS. In vitro, TRO19622 promoted motor neuron survival in the absence of trophic support in a dose-dependent manner. In vivo, TRO19622 rescued motor neurons from axotomy-induced cell death in neonatal rats and promoted nerve regeneration following sciatic nerve crush in mice. In SOD1(G93A) transgenic mice, a model of familial ALS, TRO19622 treatment improved motor performance, delayed the onset of the clinical disease, and extended survival. TRO19622 bound directly to two components of the mitochondrial permeability transition pore: the voltage-dependent anion channel and the translocator protein 18 kDa (or peripheral benzodiazepine receptor), suggesting a potential mechanism for its neuroprotective activity. TRO19622 may have therapeutic potential for ALS and other motor neuron and neurodegenerative diseases.