RESUMEN
Redox-mediated posttranslational modifications represent a molecular switch that controls major mechanisms of cell function. Nitric oxide (NO) can mediate redox reactions via S-nitrosylation, representing transfer of an NO group to a critical protein thiol. NO is known to modulate neurogenesis and neuronal survival in various brain regions in disparate neurodegenerative conditions. However, a unifying molecular mechanism linking these phenomena remains unknown. Here, we report that S-nitrosylation of myocyte enhancer factor 2 (MEF2) transcription factors acts as a redox switch to inhibit both neurogenesis and neuronal survival. Structure-based analysis reveals that MEF2 dimerization creates a pocket, facilitating S-nitrosylation at an evolutionally conserved cysteine residue in the DNA binding domain. S-Nitrosylation disrupts MEF2-DNA binding and transcriptional activity, leading to impaired neurogenesis and survival in vitro and in vivo. Our data define a molecular switch whereby redox-mediated posttranslational modification controls both neurogenesis and neurodegeneration via a single transcriptional signaling cascade.
Asunto(s)
Apoptosis , Factores de Transcripción MEF2/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis , Óxido Nítrico/metabolismo , Procesamiento Proteico-Postraduccional , Activación Transcripcional , Animales , Sitios de Unión , Células Cultivadas , ADN/metabolismo , Células HEK293 , Humanos , Factores de Transcripción MEF2/química , Factores de Transcripción MEF2/genética , Ratones , Células-Madre Neurales/citología , Oxidación-Reducción , Unión ProteicaRESUMEN
HYPOTHESIS: To investigate whether OTO-104, a poloxamer-based hydrogel containing micronized dexamethasone for intratympanic delivery, can provide long-lasting inner ear exposure and be well tolerated. METHODS: OTO-104 was administered intratympanically to guinea pigs and sheep, and its pharmacokinetic and toxicity profiles were examined. RESULTS: After a single intratympanic injection of OTO-104 (from 0.6% to 20%, w/w), significant and prolonged exposure to dexamethasone in the inner ear was observed. Increasing the concentration of OTO-104 resulted in higher perilymph drug levels as well as a more prolonged duration of exposure. At the highest dose, therapeutic perilymph levels of dexamethasone could be sustained over 3 months in guinea pigs and more than 1 month in sheep. A toxicologic evaluation was conducted, including assessments of middle and inner ear function and physiology, as well as appraisal of local and systemic toxicity. A small and transient shift in hearing threshold was observed, most probably conductive in nature. No significant histologic changes in middle or inner ear tissues were noted. Although macroscopically mild erythema/inflammation was documented in a subset of guinea pigs treated with 20% OTO-104, the nature and the severity of these changes were not different between the poloxamer vehicle, saline, and 20% OTO-104 groups. No evidence of acute dermal toxicity, delayed hypersensitivity, or systemic adverse effects was found. CONCLUSION: OTO-104 is a novel proprietary therapeutic delivery system that can achieve prolonged, sustained release of dexamethasone within the inner ear fluids. The administration of this clinical candidate formulation via intratympanic injection is expected to be well tolerated both locally and systemically.
Asunto(s)
Dexametasona/administración & dosificación , Oído Interno/química , Hidrogeles/administración & dosificación , Perilinfa/química , Animales , Preparaciones de Acción Retardada , Dexametasona/análisis , Dexametasona/farmacocinética , Cobayas , Hidrogeles/análisis , Hidrogeles/farmacocinética , Inyecciones , OvinosRESUMEN
Information on inner ear pharmacokinetics is limited in the literature, especially in large animals and in humans. A preliminary study was designed to explore the differences in inner ear exposure between guinea pigs and sheep following a single intratympanic injection of a 2% dexamethasone sodium phosphate solution. In both species, significant levels of dexamethasone were observed in the perilymph within 1 h, and decreasing by 50- to 100-fold within 12 h. Overall, the exposure to dexamethasone in the inner ear was significantly lower in sheep by 17- to 27-fold than in guinea pigs. Systemic and CNS exposure were minimal in both species as indicated by the low drug levels observed in plasma and CSF. Altogether, the preliminary evidence presented herein suggests the sheep as a practical and acceptable animal model to study the inner ear pharmacokinetics of drug candidates in large mammals and its potential towards extrapolation to human exposure.
Asunto(s)
Dexametasona/farmacocinética , Membrana Timpánica , Animales , Femenino , Cobayas , Inyecciones , Perilinfa , OvinosRESUMEN
The thermo-reversible triblock copolymer poloxamer 407 was investigated as a drug delivery vehicle for micronized dexamethasone into the middle and inner ears of guinea pigs. The study characterized the gelation and in vitro release kinetics of poloxamer formulations. In vivo, the pharmacokinetic profile of formulations containing varying concentrations of poloxamer and dexamethasone was examined following intratympanic administration. Significant drug levels within the perilymph were observed for at least 10 days, while systemic exposure was minimal. The sustained-release kinetics profile could be significantly modulated by varying the concentrations of both poloxamer and dexamethasone. Assessment of auditory function revealed a small transient shift in hearing threshold, most probably of conductive nature, which resolved itself within a week. No significant histological changes of the round window membrane or cochlea could be noted. Poloxamer 407 thus represents an effective and safe delivery system to achieve sustained release of dexamethasone to the inner ear.
Asunto(s)
Dexametasona/administración & dosificación , Dexametasona/farmacocinética , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/farmacocinética , Perilinfa/efectos de los fármacos , Membrana Timpánica/efectos de los fármacos , Análisis de Varianza , Animales , Cóclea/efectos de los fármacos , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/farmacocinética , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Electrofisiología , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Femenino , Cobayas , Audición/efectos de los fármacos , Pruebas Auditivas , Poloxámero/administración & dosificación , Poloxámero/farmacocinéticaRESUMEN
Impaired adult neurogenesis has been observed in several neurodegenerative diseases, including human immunodeficiency virus (HIV-1)-associated dementia (HAD). Here we report that the HIV-envelope glycoprotein gp120, which is associated with HAD pathogenesis, inhibits proliferation of adult neural progenitor cells (aNPCs) in vitro and in vivo in the dentate gyrus of the hippocampus of HIV/gp120-transgenic mice. We demonstrate that HIV/gp120 arrests cell-cycle progression of aNPCs at the G1 phase via a cascade consisting of p38 mitogen-activated protein kinase (MAPK) --> MAPK-activated protein kinase 2 (a cell-cycle checkpoint kinase) --> Cdc25B/C. Our findings define a molecular mechanism that compromises adult neurogenesis in this neurodegenerative disorder.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/fisiología , Infecciones por VIH/patología , VIH-1/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neuronas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Células Madre/fisiología , Complejo SIDA Demencia/metabolismo , Complejo SIDA Demencia/virología , Animales , Procesos de Crecimiento Celular/fisiología , Femenino , Fase G1/genética , Fase G1/fisiología , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/enzimología , Infecciones por VIH/virología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Transgénicos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/virología , Neuronas/citología , Neuronas/enzimología , Ratas , Fosfatasas cdc25/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Nitric oxide (NO) and zinc (Zn2+) are implicated in the pathogenesis of cerebral ischemia and neurodegenerative diseases. However, their relationship and the molecular mechanism of their neurotoxic effects remain unclear. Here we show that addition of exogenous NO or NMDA (to increase endogenous NO) leads to peroxynitrite (ONOO-) formation and consequent Zn2+ release from intracellular stores in cerebrocortical neurons. Free Zn2+ in turn induces respiratory block, mitochondrial permeability transition (mPT), cytochrome c release, generation of reactive oxygen species (ROS), and p38 MAP kinase activation. This pathway leads to caspase-independent K+ efflux with cell volume loss and apoptotic-like death. Moreover, Zn2+ chelators, ROS scavengers, Bcl-xL, dominant-interfering p38, or K+ channel blockers all attenuate NO-induced K+ efflux, cell volume loss, and neuronal apoptosis. Thus, these data establish a new form of crosstalk between NO and Zn2+ apoptotic signal transduction pathways that may contribute to neurodegeneration.