Implicación de acuaporina-4 en las bases celulares y moleculares del desarrollo postnatal del sistema nervioso central murino

Abstract

El agua es un elemento básico en la fisiología de los seres vivos. A escala celular, su transporte se encuentra facilitado por las acuaporinas (AQPs), una familia de proteínas integrales de membrana ubicuamente distribuidas por los distintos sistemas del organismo. En el Sistema Nervioso Central (SNC), acuaporina-4 (AQP4) es la AQP más ampliamente expresada y juega un papel clave en la homeostasis de los distintos líquidos que fluyen en el sistema. No obstante, pocos estudios han abordado el papel de esta proteína durante el desarrollo. En este trabajo, hemos caracterizado el curso de aparición de la proteína AQP4 durante el desarrollo postnatal del SNC, destacando dos regiones que manifestaron perfiles de expresión singulares: el acueducto cerebral y el cuerpo calloso. Profundizando en el sentido fisiológico de estas observaciones, utilizamos el ratón transgénico con deleción de AQP4 (AQP4-KO) y evaluamos parámetros relacionados con el neurodesarrollo postnatal en ambas regiones. Mediante varias aproximaciones, determinamos la existencia de defectos asociados a la diferenciación y desarrollo de estructuras ciliares en las células ependimarias del acueducto cerebral del ratón AQP4-KO. Asimismo, en el cuerpo calloso de este animal, identificamos déficits en la abundancia y maduración de oligodendrocitos, así como en la formación de mielina. Interesantemente, el análisis de ambas regiones nos hizo hallar un subtipo microglial CD11c+ que, con una huella transcripcional asociada al neurodesarrollo, mostró una presencia exclusiva en el animal AQP4- KO durante el estadio postnatal estudiado. Por último, para analizar la posible influencia de esta población microglial, evaluamos el efecto que su depleción (realizada por vía farmacológica con el compuesto PLX5622) generó en las regiones de interés, indicando un papel relevante en el desarrollo del tejido ependimario. En conclusión, este trabajo apoya la importancia de la expresión de AQP4 en el desarrollo temprano del SNC, y plantea cómo su defecto podría subyacer a alteraciones estructurales y funcionales ligadas a patologías del neurodesarrollo.

Water transport facilitated by aquaporins (AQPs) is an indispensable mechanism to achieve fluid homeostasis in the different compartments of the organism. In the central nervous system (CNS), aquaporin-4 (AQP4) is the most abundantly expressed AQP and, given its localised pattern in the border regions, it is considered a determining element in the aqueous exchange between the different fluids of the system. In recent years, this property has been especially reinforced by the proposal of the existence of the glymphatic system, which highlights AQP4 as a key component in the establishment of a convective flow linked to the clearance of compounds in the brain parenchyma. This new scenario has prompted numerous studies focused on understanding how brain clearance of substances occurs and developing new clinical strategies to eliminate neurotoxic protein accumulations, a plausible therapy against neurodegenerative diseases such as Alzheimer's disease. A major role in these approaches has been played by the characterisation of the AQP4 knockout mouse (AQP4-KO), a model that, in addition to presenting a greater susceptibility to develop symptoms compatible with Alzheimer's disease, has been associated with a predisposition to suffer pathologies associated with early CNS development, such as congenital hydrocephalus. Postnatal CNS development is one of the major phases of activation of processes such as proliferation, differentiation and changes in cellular and molecular components of the parenchyma. These involve significant water movement and osmotic imbalances in which the role of AQP4 has not been studied in detail. In this work, we provide a better understanding of the distribution of AQP4 during postnatal murine CNS development and its contribution to the development of different periventricular neural tissues. To this end, we first sought to characterise when and where AQP4 expression arises in the CNS, assessing the levels and distribution of its gene expression. From this analysis, we highlighted two regions, the cerebral aqueduct and the corpus callosum, which followed specific dynamics regarding the evolution of the abundance of AQP4 during development. Thus, in the aqueductal ependyma, AQP4 expression showed an increase during the first two postnatal weeks and then stabilised in adult tissue. In contrast, in the corpus callosum, AQP4 expression gradually decreased until the adult stage, following an expression profile that results similar to that of oligodendrocytic progenitor (OPCs) markers and opposed to tissue myelination. This expression was identified in developing astrocytes, following delocalised expression patterns, which did not correspond to those identified in astrocytes of adult tissue (highly polarised toward the perivascular space). To determine the functional relevance of early expression of AQP4 in both regions, we used the AQP4-KO model and explored by microarrays the transcriptomic profiles of the tissues of interest from 11-day-old mice (P11). In the cerebral aqueduct, reductions in the expression levels of genes involved in the development of ependymal and ciliary were highlighted, which were translated into alterations, evidenced in this study by immunohistochemical and electron microscopy approaches. On the other hand, we also found increases in the expression of genes related to components of the immune system, which we attribute to the presence of a CD11c+ microglial subtype preferentially located in the subependymal space and that showed a transcriptomic signature associated with neurodevelopment. Transcriptomic analysis of the corpus callosum also showed differential overexpression in genes linked to the microglial subtype, strongly suggesting that the microglial population, CD11c+, detected in both tissues (aqueduct and corpus callosum), corresponds to the same microglial population or subtype. In the corpus callosum, we also observed gene changes in oligodendroglial development that we confirmed histologically by changes in the proportions of cell types of this lineage, as well as in the degree of myelination of the axons that make up the tissue. To identify the influence of the microglial population on tissue development, we implemented a microglial depletion system based on the administration of the drug PLX5622 during the postnatal window (P6-P11) of development. The results of this study have pointed to the existence of defects in the maturation of ependymal cells of the depleted animals, however, no relevant changes were identified in the corpus callosum. In conclusion, the results shown in the present PhD Thesis propose that AQP4 has a relevant role in the neurodevelopment of wild-type mice and that its loss in the AQP4-KO animal could contribute to the development of congenital hydrocephalus by altering the normal structural and functional development of the aqueductal ependyma. A role for AQP4 in the process of myelination has also been highlighted and remains to be tested.