Análisis de la neurogénesis cortical en el desarrollo cerebral de ratones carentes del receptor de ácido lisofosfatídico LPA1

El ácido lisofosfatídico (LPA) es un fosfolípido sencillo con propiedades de señalización extracelular mediadas por receptores de membrana específicos acoplados a proteínas G. Actualmente se conocen hasta 6 tipos de receptores diferentes para el LPA. El receptor LPA1 se expresa en la zona neurogénica del cerebro en desarrollo, en la zona ventricular (VZ), lo que sugiere su implicación en la neurogénesis. A pesar de los numerosos estudios farmacológicos que han aportado datos de los efectos del LPA en el sistema nervioso central (SNC) utilizando modelos in vitro, no es sino hasta que se dispuso de animales carentes del receptor, cuando se avanzó en el estudio de la función específica del receptor. Los primeros ratones obtenidos que permitían el estudio de pérdida de función del receptor LPA1 mostraron una alta mortalidad perinatal pero abrían una puerta excelente a nuevos estudios de caracterización del SNC en ausencia de vías específicas de señalización por LPA. En el presente trabajo se muestran resultados que demuestran una función destacada del receptor LPA1 en los precursores neuronales corticales durante el desarrollo cerebral, resultantes del análisis de la neurogénesis en una variante, que hemos venido a denominar Málaga, de un ratón nulo para-LPA1. Esta variante surge de forma espontánea durante la expansión de la colonia original y porta un fenotipo con defectos observables en el SNC, a la vez que muestra una viabilidad perinatal casi completa, lo que ha permitido su caracterización. Nuestros resultados muestran alteraciones significativas en la neurogénesis cortical embrionaria, en el patrón proliferativo de la zona ventricular, afectando al tipo de división y la posterior diferenciación, con expresión de marcadores neuronales de forma prematura en la capa cortical y alteración de la expresión de factores de transcripción. Estos defectos de la neurogénesis en ausencia de la vía de señalización por LPA1 se asocian con defectos en el patrón migratorio neuronal, indicativos de alteraciones de tipo estructural y funcional, y que generan, en última instancia, una reducción del grosor de la pared cortical y del número de neuronas en diferentes capas corticales, especialmente las profundas donde se detecta, además, un nivel inusualmente mayor de apoptosis. Los resultados que mostramos en esta memoria reflejan, con ello, la necesidad del receptor LPA1 para el desarrollo normal cerebral y acentúan el importante papel que el modelo de animal nulo para LPA1 de la variedad Málaga ha representado para el estudio de la señalización mediada por este receptor. A la fecha actual, el uso de este ratón ha permitido un avance muy significativo en el campo y sigue siendo objeto de estudio por nuestro grupo de investigación y por diferentes colaboradores a nivel nacional e internacional

Correlation between IL1β expression level and morphological parameters proves the usefulness of morphology measures to predict the degree of activation of microglial cells

It is well known that microglial cells undergo an important change in morphology upon activation, so that form and function are intimately related. Upon activation, microglia cell body enlarges, its ramifications shortens and become thicker. In parallel, a variety of cytokines and inflammatory mediators such as IL1β are released. However the activation process is not all-or-nothing. Rather, cells in subtle activation states or in a deactivation process can occur, so intermediate not obvious phenotypes may appear. Thus, we aimed to correlate the expression level of a well-defined marker of activation, IL1β, with different morphological parameters. To do so, we used an intracerebroventricular injection of neuraminidase to produce an acute inflammation in rats. Brain sections were double-stained with IBA1 to have an image of the whole cell and its ramifications, and with IL1β to assess the level of activation. Images were captured from septofimbria (close to the injection site) and from the hypothalamus. A ratio of IL1β-positive pixels to IBA1-positive pixels was used to estimate the level of IL1β expression for each cell. Single microglial cell images were processed with ImageJ software to obtain outlined and filled shapes, which were used to obtain (by means of FracLac plug in) the following morphological parameters: fractal dimension, lacunarity, area, density and perimeter. All parameters showed a significant correlation with the level of expression of IL1β. This occurred in cells sampled from the two brain areas studied. Density, lacunarity and perimeter resulted as the best predictor parameters of activation, that is, those with a better correlation with the level of expression of IL1β. Area, an extensively used parameter to assess microglial activation, presented the least significant correlation. Thus, objectively measured morphological parameters correlate with the level of expression of IL1β, and could therefore be used as predictors of the activation level of microglial cells.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

FGF2, but not EGF, Induces multiciliated ependymal cells to dedifferentiate and adopt radial glial features in vitro

Multiciliated ependymal cells form an epithelium lining most of the ventricular cavities of vertebrates brain. Although considered postmitotic and completely differentiated, ependymal cells maintain some phenotypic characteristics of neural stem cells. Thereby, under specific conditions they behave as neural stem cells, developing radial glia characteristics, and undergoing asymmetric division. Our group is searching for factors that promote dedifferentiation of ependymal cells in vitro. We developed a simple method to obtain pure cultures of non-adherent multiciliated ependymal cells from adult rats. These cultures were used to investigate the effect of FGF2 on the differentiation state and the aggregation of ependymal cells. Thus, FGF2 treated ependymal cells lose cilia and hence mobility, and after 7 days they aggregate to form irregular spheres (diameter ≥ 20-30 μm). Such changes were not observed when EFG was used instead of FGF2. To assess the specificity of FGF2 action on cell aggregation, the FGF receptor inhibitor PD166866 and an anti-FGF2 neutralizing antibody were used. In both conditions the aggregative effect of FGF2 was abolished. No cell proliferation was observed during sphere formation, at least in such experimental conditions. Spheres were analyzed by immunocytochemistry using radial glia markers. They were positive for GFAP, vimentin, BLBP and GLAST. These data suggest that FGF2 promotes the identity loss in multiciliated ependymal cells in vitro, which are transformed into cells with radial glia features.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Neuraminidase-activated microglia compromise the viability of ependymocytes

Neuraminidase (NA) is a sialidase present in the envelope/wall of some virus/bacteria responsible for brain infections, such as flu, mumps or meningitis. The intracerebroventricular injection of NA in the rat brain provokes ependymal detachment and death, and an acute inflammatory process. Although inflammation reverses, ependymal lining is not regenerated. Complement system activation within the CSF contributes to ependymal damage, but is not the only cause (Granados-Duran et al, 2016). Here we aimed to investigate if microglial activation might also play a role. For this purpose we used pure isolated ependymocytes (Grondona et al, 2013) and ventricular wall explants, which were co-cultured with microglial cells, both in basal conditions and with agents that induce microglial activation: NA, LPS, or Pam3CSK4 (synthetic lipopeptide). The viability of the ependymal cells was assessed by trypan blue exclusion. The viability of isolated ependymocytes was reduced when NA or LPS were added to the culture, compared to controls without additives. In the absence of microglia, NA or LPS did not compromise viability significantly, indicating that microglia was involved in ependymocytes death. The addition of NA to cultured explants reduced ependymocytes viability only when microglial cells were present in the culture; a similar reduction was observed when LPS or Pam3CSK4 were added. Conversely, explants cultured in the absence of microglia did not suffer a significant decrease in ependymocytes viability upon NA addition to the medium. We hypothesized that cytokines released by activated microglia, such as IL1β or TNFα, could mediate ependymocytes death. RT-PCR performed in RNA obtained from pure ependymocytes confirmed the presence of IL1β and TNFα receptors in ependymal cells. Nevertheless further experiments are required to confirm this hypothesis. We conclude that microglia activated by NA mediates, at least in part, ependymal cell death, what might be relevant for neuroinflammatory diseases mediated by NA bearing virus/bacteria.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Neuraminidase-induced neuroinflammation is largely dependent on microglial TLR4 receptor

The sialidase neuraminidase (NA) cleaves terminal sialic acid from glycoproteins and glycolipids. Among its various locations, it is present in the envelope/membrane of some bacteria/viruses (e.g. influenza virus), where it is involved in infectiveness and dispersion. The injection of NA within the brain lateral ventricle represents a model of acute sterile inflammation. The relevance of the toll-like receptors TLR2 and TLR4 (particularly those in microglial cells) in such process was investigated using mouse strains deficient in these receptors. In septofimbria and hypothalamus, IBA1-positive and IL-1β-positive cell counts increased after NA injection in wild type (WT) mice. In TLR4-/- mice such increases were largely abolished, while only slightly affected in TLR2-/- mice. Similarly, the NA-induced expression of IL-1β, TNFα and IL-6 (evaluated by qPCR) was completely blocked in TLR4-/- mice, and only partially reduced in TLR2-/- mice. Microglia was isolated from the three mouse strains and exposed to NA or to specific TLR2 and TLR4 agonists (Pam3CSK4 and LPS respectively) in vitro. NA induced a cytokine response (IL-1β, TNFα and IL-6) in WT microglia, but was unable to do so in TLR4-/- microglia; TLR2 deficiency partially affected the NA-induced microglia response. To investigate if such response of microglial cells to NA was dependent on the sialidase activity of the enzyme, WT microglia was exposed in vitro to NA previously inactivated with heat, or inhibited with two different sialidase inhibitors (oseltamivir phosphate and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid). In all cases, NA- induced microglia activation was dependent on the intact sialidase activity of NA. Therefore, we conclude that NA is able to directly activate microglial cells, mostly through TLR4 receptor and due to its sialidase activity. Accordingly, the inflammatory reaction induced by NA in vivo is partially dependent on TLR2, while TLR4 plays a crucial role.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Morphological traits of surveillant/activated microglia during an experimentally induced acute neuroinflammatory process

Poster en CongresoSeveral studies show that morphological changes of microglia over the course of inflammation are tightly coupled to function. However the progressive transformation into activated microglia is poorly characterized. AIMS: This study aimed to establish a spatiotemporal correlation between quantifiable morphological parameters of microglia and the spread of an acute ventricular inflammatory process. METHODS: Inflammation was induced by a single injection of the enzyme neuraminidase within the lateral ventricle of rats. Animals were sacrificed 2, 4 and 12 hours after injection. Coronal slices were immunostained with Iba1 to label microglia and with IL1β to delimit the spread of inflammation. Digital images were obtained by scanning the labelled sections. Single microglia images were randomly selected from periventricular areas of caudate putamen, hippocampus and hypothalamus. FracLac for ImageJ software was used to measure the following morphological parameters: fractal dimension, lacunarity, area, perimeter and density. RESULTS: Significant differences were found in fractal dimension, lacunarity, perimeter and density of microglia cells of neuraminidase injected rats compared to sham animals. However no differences were found in the parameter “area”. In hipoccampus there was a delay in the significant change of the measured parameters. These morphological changes correlated with IL1β-expression in the same areas. CONCLUSIONS: Ventricular inflammation induced by neuraminidase provokes quantifiable morphological changes in microglia restricted to areas labelled with IL1β. Morphological parameters of microglia such as fractal dimension, lacunarity, perimeter and density are sensitive and valuable tools to quantify activation. However, the extensively used parameter “area” did not change upon microglia activation.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Grant: Junta de Andalucía (Spain) P11-CVI-07637 Ibima, Andalucía Tech, UM

A model of neuroinflammation and demyelination by intracerebroventricular injection of microbial neuraminidase

Comunicación en forma de poster al congresoNeuraminidase from Clostridium perfringens, which cleaves terminal sialic acid from carbohydrate chains, was injected in the lateral ventricle of rats. It diffused in the ipsilateral ventricle, the third ventricle, and also towards the periventricular brain parenchyma. Soon after, the complement system activated, and some ependymal cells detached and died. In the affected zones, there was an increased expression of GFAP in astrocytes, IBA1 in microglia, and ICAM1 in the endothelial cells of blood vessels. Cytokines, such as IL1β secreted by activated macrophages and microglia, provoked the extravasation of leucocytes from about 4 h post-injection. The main sources of cells were large venules located in the choroid plexus, the meninges and the subependyma around the foramen interventricularis. Invading cells arrived orderly: first neutrophils, then macrophage-monocytes, and last lymphocytes (mainly CD8α-positive T-lymphocytes). Leucocytes invaded the ventricle and the meninges, and also penetrated the brain parenchyma, sometimes passing through the ependyma and the glia limitans. As a result, some myelinated tracts suffered vacuolar degeneration, being the stria medullaris consistently affected. Oligodendrocytes in the damaged tracts were not affected. Vacuolated myelin recovered with time. Thus, the intracerebroventricular injection of neuraminidase may represent a novel reversible animal model to study experimental neuroinflammation and myelin vacuolization.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Ministerio de Ciencia e Innovación, SAF2010-19087; Junta de Andalucía, Consejería de Sanidad, SAS 08-0029; Junta de Andalucía, Proyecto de excelencia P-11-CVI-07637

Complement system activation contributes to the ependymal damage induced by microbial neuraminidase

Background In the rat brain, a single intracerebroventricular injection of neuraminidase from Clostridium perfringens induces ependymal detachment and death. This injury occurs before the infiltration of inflammatory blood cells; some reports implicate the complement system as a cause of these injuries. Here, we set out to test the role of complement. Methods The assembly of the complement membrane attack complex on the ependymal epithelium of rats injected with neuraminidase was analyzed by immunohistochemistry. Complement activation, triggered by neuraminidase, and the participation of different activation pathways were analyzed by Western blot. In vitro studies used primary cultures of ependymal cells and explants of the septal ventricular wall. In these models, ependymal cells were exposed to neuraminidase in the presence or absence of complement, and their viability was assessed by observing beating of cilia or by trypan blue staining. The role of complement in ependymal damage induced by neuraminidase was analyzed in vivo in two rat models of complement blockade: systemic inhibition of C5 by using a function blocking antibody and testing in C6-deficient rats. Results The complement membrane attack complex immunolocalized on the ependymal surface in rats injected intracerebroventricularly with neuraminidase. C3 activation fragments were found in serum and cerebrospinal fluid of rats treated with neuraminidase, suggesting that neuraminidase itself activates complement. In ventricular wall explants and isolated ependymal cells, treatment with neuraminidase alone induced ependymal cell death; however, the addition of complement caused increased cell death and disorganization of the ependymal epithelium. In rats treated with anti-C5 and in C6-deficient rats, intracerebroventricular injection of neuraminidase provoked reduced ependymal alterations compared to non-treated or control rats. Immunohistochemistry confirmed the absence of membrane attack complex on the ependymal surfaces of neuraminidase-exposed rats treated with anti-C5 or deficient in C6. Conclusions These results demonstrate that the complement system contributes to ependymal damage and death caused by neuraminidase. However, neuraminidase alone can induce moderate ependymal damage without the aid of complement

Microglial Morphometric Parameters Correlate With the Expression Level of IL-1β, and Allow Identifying Different Activated Morphotypes.

Microglia are the resident macrophages in the brain. Traditionally, two forms of microglia have been described: one considered as a resting/surveillant state in which cells have a highly branched morphology, and another considered as an activated state in which they acquire a de-ramified or amoeboid form. However, many studies describe intermediate microglial morphologies which emerge during pathological processes. Since microglial form and function are closely related, it is of interest to correlate microglial morphology with the extent of its activation. To address this issue, we used a rat model of neuroinflammation consisting in a single injection of the enzyme neuraminidase (NA) within the lateral ventricle. Sections from NA-injected animals were co-immunolabeled with the microglial marker IBA1 and the cytokine IL-1β, which highlight features of the cell's shape and inflammatory activation, respectively. Activated (IL-1β positive) microglial cells were sampled from the dorsal hypothalamus nearby the third ventricle. Images of single microglial cells were processed in two different ways to obtain (1) an accurate measure of the level of expression of IL-1β (indicating the degree of activation), and (2) a set of 15 morphological parameters to quantitatively and objectively describe the cell's shape. A simple regression analysis revealed a dependence of most of the morphometric parameters on IL-1β expression, demonstrating that the morphology of microglial cells changes progressively with the degree of activation. Moreover, a hierarchical cluster analysis pointed out four different morphotypes of activated microglia, which are characterized not only by morphological parameters values, but also by specific IL-1β expression levels. Thus, these results demonstrate in an objective manner that the activation of microglial cells is a gradual process, and correlates with their morphological change. Even so, it is still possible to categorize activated cells according to their morphometric parameters, each category presenting a different activation degree. The physiological relevance of those activated morphotypes is an issue worth to be assessed in the future