RESUMEN
Here we report on a novel fluorescent analog of phenytoin as a potential inhibitor of neuropathic pain with potential use as an imaging agent. Compound 2 incorporated a heptyl side chain and dansyl moiety onto the parent compound phenytoin and produced greater displacement of BTX from sodium channels and greater functional blockade with greatly reduced toxicity. Compound 2 reduced mechano-allodynia in a rat model of neuropathic pain and was visualized ex vivo in sensory neuron axons with two-photon microscopy. These results suggest a promising strategy for developing novel sodium channel inhibitors with imaging capabilities.
Asunto(s)
Fluorescencia , Colorantes Fluorescentes/farmacología , Neuralgia/tratamiento farmacológico , Fenitoína/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Animales , Modelos Animales de Enfermedad , Diseño de Fármacos , Femenino , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Modelos Moleculares , Estructura Molecular , Fenitoína/síntesis química , Fenitoína/química , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/síntesis química , Bloqueadores de los Canales de Sodio/química , Relación Estructura-ActividadRESUMEN
Synucleinopathies, such as Parkinson's disease and diffuse Lewy body disease, are progressive neurodegenerative disorders characterized by selective neuronal death, abnormal accumulation of misfolded α-synuclein, and sustained microglial activation. In addition to inducing neuronal toxicity, higher-ordered oligomeric α-synuclein causes proinflammatory responses in the brain parenchyma by triggering microglial activation, which may exacerbate pathogenic processes by establishing a chronic neuroinflammatory milieu. We found that higher-ordered oligomeric α-synuclein induced a proinflammatory microglial phenotype by directly engaging the heterodimer TLR1/2 (Toll-like receptor 1 and 2) at the cell membrane, leading to the nuclear translocation of NF-κB (nuclear factor κB) and the increased production of the proinflammatory cytokines TNF-α (tumor necrosis factor-α) and IL-1ß (interleukin-1ß) in a MyD88-dependent manner. Blocking signaling through the TLR1/2 heterodimer with the small-molecule inhibitor CU-CPT22 reduced the nuclear translocation of NF-κB and secretion of TNF-α from cultured primary mouse microglia. Candesartan cilexetil, a drug approved for treating hypertension and that inhibits the expression of TLR2, reversed the activated proinflammatory phenotype of primary microglia exposed to oligomeric α-synuclein, supporting the possibility of repurposing this drug for synucleinopathies.
Asunto(s)
Factor 88 de Diferenciación Mieloide/metabolismo , Pliegue de Proteína , Transducción de Señal , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 2/metabolismo , alfa-Sinucleína/metabolismo , Animales , Células HEK293 , Humanos , Enfermedad por Cuerpos de Lewy/genética , Enfermedad por Cuerpos de Lewy/metabolismo , Ratones , Factor 88 de Diferenciación Mieloide/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/metabolismo , Receptor Toll-Like 1/genética , Receptor Toll-Like 2/genética , alfa-Sinucleína/genéticaRESUMEN
Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder typified by tremor, rigidity, akinesia and postural instability due in part to the loss of dopamine within the nigrostriatal system. The pathologic features of this disorder include the loss of substantia nigra dopamine neurons and attendant striatal terminals, the presence of large protein-rich neuronal inclusions containing fibrillar α-synuclein and increased numbers of activated microglia. Evidence suggests that both misfolded α-synuclein and oxidative stress play an important role in the pathogenesis of sporadic PD. Here we review evidence that α-synuclein activates glia inducing inflammation and that Nrf2-directed phase-II antioxidant enzymes play an important role in PD. We also provide new evidence that the expression of antioxidant enzymes regulated in part by Nrf2 is increased in a mouse model of α-synuclein overexpression. We show that misfolded α-synuclein directly activates microglia inducing the production and release of the proinflammatory cytokine, TNF-α, and increasing antioxidant enzyme expression. Importantly, we demonstrate that the precise structure of α-synuclein is important for induction of this proinflammatory pathway. This complex α-synuclein-directed glial response highlights the importance of protein misfolding, oxidative stress and inflammation in PD and represents a potential locus for the development of novel therapeutics focused on induction of the Nrf2-directed antioxidant pathway and inhibition of protein misfolding.
Asunto(s)
Antioxidantes/farmacología , Activación de Macrófagos/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/inmunología , Enfermedad de Parkinson/inmunología , alfa-Sinucleína/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Western Blotting , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Microscopía de Fuerza Atómica , Factor 2 Relacionado con NF-E2/fisiología , Estrés Oxidativo/efectos de los fármacos , Placenta/enzimología , Embarazo , Conformación Proteica , ARN/biosíntesis , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Necrosis Tumoral alfa/metabolismo , alfa-Sinucleína/químicaRESUMEN
Parkinson's disease (PD) is typified by the loss of midbrain dopamine neurons, the presence of large proteinaceous α-synuclein-positive intracellular inclusions, oxidatively modified molecules and activated microglia. The etiology of sporadic PD is not fully understood but several lines of evidence suggest that genetic vulnerability and environmental toxicants converge to incite pathology--the multiple hit hypothesis. One gene linked to both familial and sporadic PD is SNCA, which encodes for the protein α-synuclein that has a propensity to misfold into toxic moieties. Here we show that α-synuclein directly activates microglia inciting the production of proinflammatory molecules and altering the expression of Toll-like receptors (TLRs). We discuss the role for α-synuclein-directed TLR expression changes in PD and the therapeutic potential of modifying this response.
Asunto(s)
Sistemas de Liberación de Medicamentos , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/terapia , Pliegue de Proteína , Receptores Toll-Like/metabolismo , alfa-Sinucleína/fisiología , Animales , Antiparkinsonianos/metabolismo , Antiparkinsonianos/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Humanos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Pliegue de Proteína/efectos de los fármacos , Receptores Toll-Like/biosíntesis , Receptores Toll-Like/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Parkinson's disease, an age-related neurodegenerative disorder, is characterized by the loss of dopamine neurons in the substantia nigra, the accumulation of α-synuclein in Lewy bodies and neurites, and neuroinflammation. While the exact etiology of sporadic Parkinson's disease remains elusive, a growing body of evidence suggests that misfolded α-synuclein promotes inflammation and oxidative stress resulting in neurodegeneration. α-Synuclein has been directly linked to microglial activation in vitro and increased numbers of activated microglia have been reported in an α-synuclein overexpressing mouse model prior to neuronal loss. However, the mechanism by which α-synuclein incites microglial activation has not been fully described. Microglial activation is governed in part, by pattern recognition receptors that detect foreign material and additionally recognize changes in homeostatic cellular conditions. Upon proinflammatory pathway initiation, activated microglia contribute to oxidative stress through release of cytokines, nitric oxide, and other reactive oxygen species, which may adversely impact adjacent neurons. Here we show that microglia are directly activated by α-synuclein in a classical activation pathway that includes alterations in the expression of toll-like receptors. These data suggest that α-synuclein can act as a danger-associated molecular pattern.