2. Universidad Cardenal Herrera-CEU

Autophagy is a highly conserved process that mediates the targeting and degradation of intracellular components to lysosomes, contributing to the maintenance of cellular homeostasis and to obtaining energy, which ensures viability under stress conditions. Therefore, autophagy defects are common to different neurodegenerative disorders. Rnd3 belongs to the family of Rho GTPases, involved in the regulation of actin cytoskeleton dynamics and important in the modulation of cellular processes such as migration and proliferation. Murine models have shown that Rnd3 is relevant for the correct development and function of the Central Nervous System and lack of its expression produces several motor alterations and neural development impairment. However, little is known about the molecular events through which Rnd3 produces these phenotypes. Interestingly we have observed that Rnd3 deficiency correlates with the appearance of autophagy impairment profiles and irregular mitochondria. In this work, we have explored the impact of Rnd3 loss of expression in mitochondrial function and autophagy, using a Rnd3 KO CRISPR cell model. Rnd3 deficient cells show no alterations in autophagy and mitochondria turnover is not impaired. However, Rnd3 KO cells have an altered mitochondria oxidative metabolism, resembling the effect caused by oxidative stress. In fact, lack of Rnd3 expression makes these cells strictly dependent on glycolysis to obtain energy. Altogether, our results demonstrate that Rnd3 is relevant to maintain mitochondria function, suggesting a possible relationship with neurodegenerative diseases.

Las GTPasas de la familia Rho están adquiriendo un gran interés en los últimos años por su papel en el desarrollo y función del sistema nervioso central (SNC). RhoE es un miembro de esta gran familia cuyas funciones in vivo no están aún bien definidas. En este trabajo estudiamos la expresión de RhoE en el desarrollo embrionario y postnatal y su papel el SNC analizando las características de ratones modificados genéticamente, que no expresan el gen RhoE. Nuestros resultados muestran que RhoE se expresa en diferentes órganos embrionarios, entre los que se encuentra el SNC, desde edades tempranas. En el SNC postnatal se expresa con altos niveles en las primeras etapas y se localiza en numerosos sitios, destacando su presencia en las células de la corriente migratoria rostral, la capa granular externa del cerebelo o en las motoneuronas La eliminación de la expresión de RhoE se traduce en la aparición de graves alteraciones en el sistema nervioso. Los ratones que no expresan RhoE presentan un acúmulo anormal en la zona subventricular de células que hemos caracterizado como neuroblastos postmitóticos, que migran en una tasa mucho menor a través de la corriente migratoria rostral. En consecuencia, a los bulbos olfatorios llegan menos células, que, además, se disponen de forma alterada, de manera que las células calbindina positivas están en menor proporción y muy desorganizadas. Además, en los ratones que carecen de RhoE la rama anterior de la comisura anterior, que conecta ambos bulbos olfatorios, está ausente, y hay en todo su cerebro una reducción en la mielinización. Por otra parte, estos ratones tienen alterado el sistema motor, muestran un retraso en la maduración de las sinapsis neuromusculares y, sorprendentemente, no forman nervio peroneo común, que está ausente en todos los ratones analizados. Estos resultados muestran que RhoE es una proteína esencial en el desarrollo del sistema nervioso, que controla eventos tan diferentes como la migración neuronal y la formación de nervios periféricos. / Rnd proteins are a subfamily of Rho GTPases involved in the control of actin cytoskeleton dynamics, which differently to the other Rho members are constitutively active. In this work we describe Rnd3/RhoE expression and we show the in vivo effect of RhoE gene ablation in the central nervous system (CNS). RhoE is widely expressed during the embryonic development, including the CNS. RhoE is found at high levels in the postnatal CNS and is extensively localized, including the cells of the rostral migratory stream, cerebral cortex and olfactory bulb and those of the external granule cell layer of the cerebellum and the motoneurons. Mice lacking RhoE expression (RhoE gt/gt) accumulate cells in the subventricular zone, which show a delayed migration through the rostral migratory stream to the olfactory bulb. As a consequence, the calbindin positive cells of this region are disorganized. Moreover, RhoE null mice have reduced central myelination, lack of the anterior branch of the anterior commissure and are occasionally hydrocephalic. Finally, these mice have an altered motor system, with a delayed maturation of the neuromuscular junctions and, strikingly, they do not form the common peroneal nerve. These results show that RhoE is an essential protein for the nervous system development controlling important event such as neuronal migration and peripheral nerve formation.

Rho small GTPases are proteins with key roles in the development of the central nervous system. Rnd proteins are a subfamily of Rho GTPases characterized by their constitutive activity. Rnd3/RhoE is a member of this subfamily ubiquitously expressed in the CNS, whose specific functions during brain development are still not well defined. Since other Rho proteins have been linked to the myelination process, we study here the expression and function of Rnd3 in oligodendrocyte development. We have found that Rnd3 is expressed in a subset of oligodendrocyte precursor cells and of mature oligodendrocytes both in vivo and in vitro. We have analyzed the role of Rnd3 in myelination using mice lacking Rnd3 expression (Rnd3gt/gt mice), showing that these mice exhibit hypomyelination in the brain and a reduction in the number of mature and total oligodendrocytes in the corpus callosum and striatum. The mutants display a decreased expression of several myelin proteins and a reduction in the number of myelinated axons. In addition, myelinated axons exhibit thinner myelin sheaths. In vitro experiments using Rnd3gt/gt mutant mice showed that the differentiation of the precursor cells is altered in the absence of Rnd3 expression, suggesting that Rnd3 is directly required for the differentiation of oligodendrocytes and, in consequence, for the correct myelination of the CNS. This work shows Rnd3 as a new protein involved in oligodendrocyte maturation, opening new avenues to further study the function of Rnd3 in the development of the central nervous system and its possible involvement in demyelinating diseases.