RESUMEN
In this study, we describe a simple and reliable method to study neuroprotective effects in living and organized neural tissue. This method, which was based on retinal explants for in vivo focal lesions, was conceived as a collection of modular procedures, which can be customized for particular demands. With this model, it is possible to combine immunohistochemistry with image data analysis to track the two- or three-dimensional redistribution of proteins as a time/space function of primary cell loss. At the same time, it is possible to finely control the exposure of the tissue to specific drugs and molecules. In order to illustrate the use of the proposed method, we tested the effects of two different nanotube compounds on retinal explant viability. Transcriptome analyses can be separately performed in the lesion focus and penumbra with laser capture microdissection followed by polymerase chain reaction analyses. In addition, other common experimental drawbacks, such as high individual variance, are eliminated. With intraocular injections, treatments can be verified in vivo, with one eye serving as the experimental tissue and the other serving as the control tissue. In summary, we describe a flexible and easy method, which can be useful in combination with a broad variety of recently developed neuroprotective strategies, to study neurodegeneration.
Asunto(s)
Proteínas del Ojo/genética , Fármacos Neuroprotectores/farmacología , Retina/citología , Neuronas Retinianas/citología , Técnicas de Cultivo de Tejidos , Animales , Aptámeros de Nucleótidos/farmacología , Aptámeros de Péptidos/farmacología , Pollos , Proteínas del Ojo/antagonistas & inhibidores , Proteínas del Ojo/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Inyecciones Intraoculares , Masculino , Imagen Molecular , Nanotubos , ARN Interferente Pequeño/genética , Ratas , Retina/efectos de los fármacos , Retina/lesiones , Retina/metabolismo , Neuronas Retinianas/efectos de los fármacos , Neuronas Retinianas/metabolismo , Análisis de la Célula IndividualRESUMEN
To test for a role for the cellular prion protein (PrP(c)) in cell death, we used a PrP(c)-binding peptide. Retinal explants from neonatal rats or mice were kept in vitro for 24 h, and anisomycin (ANI) was used to induce apoptosis. The peptide activated both cAMP/protein kinase A (PKA) and Erk pathways, and partially prevented cell death induced by ANI in explants from wild-type rodents, but not from PrP(c)-null mice. Neuroprotection was abolished by treatment with phosphatidylinositol-specific phospholipase C, with human peptide 106-126, with certain antibodies to PrP(c) or with a PKA inhibitor, but not with a MEK/Erk inhibitor. In contrast, antibodies to PrP(c) that increased cAMP also induced neuroprotection. Thus, engagement of PrP(c) transduces neuroprotective signals through a cAMP/PKA-dependent pathway. PrP(c) may function as a trophic receptor, the activation of which leads to a neuroprotective state.
Asunto(s)
Anisomicina/farmacología , Apoptosis/efectos de los fármacos , AMP Cíclico/análogos & derivados , AMP Cíclico/fisiología , Proteínas del Ojo/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas PrPC/metabolismo , Retina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Animales Recién Nacidos , Anisomicina/antagonistas & inhibidores , Anticuerpos Monoclonales/farmacología , Apoptosis/fisiología , Colforsina/farmacología , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas del Ojo/antagonistas & inhibidores , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/inmunología , Flavonoides/farmacología , Regulación del Desarrollo de la Expresión Génica , Sueros Inmunes , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Fosforilación , Proteínas PrPC/química , Procesamiento Proteico-Postraduccional , Ratas , Ratas Endogámicas , Retina/metabolismo , Tionucleótidos/farmacología , Fosfolipasas de Tipo C/farmacologíaRESUMEN
Fasciculin 2 (FAS), an acetylcholinesterase (AChE) peripheral site ligand that inhibits mammalian AChE in the picomolar range and chicken AChE only at micromolar concentrations, was used in chick retinal cell cultures to evaluate the influence of AChE on neuronal development. The effects of other AChE inhibitors that bind the active and/or the peripheral site of the enzyme [paraoxon, eserine, or 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51)] were also studied. Morphological changes of cultured neurons were observed with the drugs used and in the different cell culture systems studied. Cell aggregates size decreased by more than 35% in diameter after 9 days of FAS treatment, mainly due to reduction in the presumptive plexiform area of the aggregates. Eserine showed no effect on the morphology of the aggregates, although it fully inhibited the activity of AChE. In dense stationary cell culture, cluster formation increased after 3 days and 6 days of FAS treatment. However, FAS, at concentrations in which changes of morphological parameters were observed, did not inhibit the AChE activity as measured histochemically. In contrast, paraoxon treatment produced a slight morphological alteration of the cultures, while a strong inhibition of enzyme activity caused by this agent was observed. BW284c51 showed a harmful, probably toxic effect, also causing a slight AChE inhibition. It is suggested that the effect of an anticholinesterase agent on the morphological modifications of cultured neurons is not necessarily associated with the intensity of the AChE inhibition, especially in the case of FAS. Moreover, most of the effects of AChE on culture morphology appear to be independent of the cholinolytic activity of the enzyme. The results obtained demonstrate that FAS is not toxic for the cells and suggest that regions of the AChE molecule related to the enzyme peripheral site are likely to be involved with the nonclassical role of AChE.