Analysis of Reelin function in brain development and in adult neurogenesis = Análisis de la función de Reelina en el desarrollo del cerebro y la neurogénesis adulta

Abstract

Reelin is a large extracellular matrix glycoprotein with a crucial role both during brain development, where it is key for neuronal migration and for the formation of the layered structure of cerebral cortex and cerebellum, and in the adulthood, where it is involved in adult synaptic plasticity, including neurogenesis in the dentate gyrus and dendritogenesis amongst other processes. Reelin acts through the binding to its canonical receptors (apolipoprotein E receptor 2, ApoER2; and very low density lipoprotein receptor, VLDLR) which trigger a complex signaling cascade involving numerous kinases and the adaptor protein Dab1. At the embryonic stage, Reelin is expressed mainly by Cajal-Retzius cells on the developing brain whereas at perinatal stages its expression gradually disappears from Cajal-Retzius cells and starts to be expressed by GABAergic interneurons of the cortex and hippocampus. In the neocortex, postmitotic neurons migrate in an ordered sequence that determines the normal “inside-out” layer formation. The malpositioning of cortical neurons is a result of abnormal migration and could cause severe layering malformations with functional consequences related with neurodevelopmental diseases such as Schizophrenia, Autism and Epilepsy. In this context, one of the most studied models has been the reeler mouse which presents a characteristic phenotype caused by an autosomal mutation in the Rln gene. The reeler mouse presents several morphological defects including a failed pre-plate splitting that causes a roughly inverted neuronal layering in the cortex, mispositioning of pyramidal neurons as well as granular cells on the dentate gyrus and profound cerebellar hypoplasia. However the study of the effects of Reelin signaling in the adult brain is difficult in the reeler mouse model due to the failed migration and mispositioning during development. Thus, to unravel the function of Reelin at different developmental stages (from embryonic to adult) as well as to gain insight in the potential distinct contribution of Reelin from different cell-types, we have generated three Reelin deficient conditional transgenic lines which allow us to ubiquitously delete Reelin in a temporally-controlled manner (Cre fR/fR) or selectively remove Reelin from Cajal-Retzius cells (CR fR/fR) or GABAergic interneurons (Gad fR/fR). Analysis of the cortical organization using layer-specific markers reveals that, unlike the reeler mouse, none of our transgenic lines shows the characteristic inversion of cortical layers. Moreover, our data strongly indicates that Reelin from Cajal-Retzius cells is important for the typical inside-out laminar cortical development but seems to be dispensable for pre-plate splitting. Furthermore, our results suggest that the absence of Reelin during early postnatal and adult stages seems to impact on the well-defined laminar structure of the cortex, leading to an invasion of layer I by late-born neurons from layer II-III. Regarding the hippocampus, our results suggest, on the one hand, a differential contribution of Reelin expressed by Cajal-Retzius cells and by GABAergic interneurons in the formation of the laminar structures of the hippocampus. On the other hand, temporally-controlled removal of Reelin at postnatal stages demonstrates that it is essential for the correct formation of the hippocampus whereas in the adult seems to be key for several aspects of hippocampus neurogenesis, including neuronal positioning in the dentate gyrus and dendritic orientation at different maturation stages of adult new-born granule cells. Finally, our findings also support the importance of Reelin expression for proper Purkinje cell migration, but not for granule cell disposition in the cerebellum at early postnatal and adult stages. Taken altogether, our results suggest a causal relation between the absence of Reelin and structural alterations in the hippocampus, cortex and cerebellum, either at developments stages or adult stages.

Reelina es una glicoproteína extracelular de matriz esencial para la regulación de los procesos de migración neuronal y posicionamiento de las neuronas corticales durante el desarrollo del encéfalo. Durante la embriogénesis, Reelina es producida por las células Cajal-Retzius de la superficie de la corteza en desarrollo. En este estadío, las neuronas postmitóticas migran de forma ordenada originando una estructura laminar en seis capas, en las cuáles las neuronas más jóvenes se sitúan en las capas más externas. La pérdida de Reelina durante el desarrollo comporta fallos en la migración de las neuronas, provocando a su vez grandes alteraciones en la estructuración de la corteza que contribuyen a la patogénesis de muchos trastornos neurológicos como el autismo, la epilepsia, la esquizofrenia, o el trastorno bipolar. En este contexto, uno de los fenotipos más estudiado es el del ratón mutante de Reelina, reeler, que presenta una estructura cortical alterada con las capas invertidas. Sin embargo, dado que la expresión de Reelina durante el desarrollo ocurre a edades embrionarias muy tempranas, es difícil estudiar el efecto de su pérdida en este tipo de mutantes a edades más tardías, en los que los primeros efectos de su pérdida son tan profundos. Todo ello evidencia la necesidad de desarrollar otro tipo de modelos en los que la pérdida de Reelina sea más gradual o selectiva. En estadíos perinatales y en el cerebro adulto Reelina es expresada principalmente por interneuronas gabaérgicas, donde presumiblemente Reelina controla funciones de formación de sinapsis y mantenimiento de la plasticidad sináptica de las neuronas del córtex y del hipocampo. Nuestros resultados muestran un fenotipo diferencial para cada uno de nuestros mutantes, sugiriendo un papel diferente de Reelina expresada por cada tipo celular, o en función del estadío en el cuál la deleccionamos. En concreto, hemos visto que la función de Reelina es imprescindible para la correcta laminación del córtex, para la formación del hipocampo, y para el correcto posicionamiento de las células Purkinje del cerebelo. Además la pérdida de Reelina en estadíos adultos comporta fallos en la neurogénesis de la zona subventricular del giro dentado.