RESUMEN
The intracellular mechanisms driving postmitotic development of cortical γ-aminobutyric acid (GABA)ergic interneurons are poorly understood. We have addressed the function of Rac GTPases in cortical and hippocampal interneuron development. Developing neurons express both Rac1 and Rac3. Previous work has shown that Rac1 ablation does not affect the development of migrating cortical interneurons. Analysis of mice with double deletion of Rac1 and Rac3 shows that these GTPases are required during postmitotic interneuron development. The number of parvalbumin-positive cells was affected in the hippocampus and cortex of double knockout mice. Rac depletion also influences the maturation of interneurons that reach their destination, with reduction of inhibitory synapses in both hippocampal CA1 and cortical pyramidal cells. The decreased number of cortical migrating interneurons and their altered morphology indicate a role of Rac1 and Rac3 in regulating the motility of cortical interneurons, thus interfering with their final localization. While electrophysiological passive and active properties of pyramidal neurons including membrane capacity, resting potential, and spike amplitude and duration were normal, these cells showed reduced spontaneous inhibitory currents and increased excitability. Our results show that Rac1 and Rac3 contribute synergistically to postmitotic development of specific populations of GABAergic cells, suggesting that these proteins regulate their migration and differentiation.
Asunto(s)
Corteza Cerebral/citología , Neuronas GABAérgicas/fisiología , Hipocampo/citología , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/metabolismo , 4-Aminopiridina/farmacología , Animales , Animales Recién Nacidos , Bicuculina/farmacología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Neuronas GABAérgicas/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Potenciales Postsinápticos Inhibidores/genética , Interneuronas/efectos de los fármacos , Interneuronas/fisiología , Ratones , Ratones Noqueados , Piperazinas/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismo , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1/genéticaRESUMEN
The nervous system influences organ development by direct innervation and the action of hormones. We recently showed that the specific absence of Rac1 in neurons (Rac1(N) ) in a Rac3-deficient (Rac3(KO) ) background causes motor behavioural defects, epilepsy, and premature mouse death around postnatal day 13. We report here that Rac1(N) /Rac3(KO) mice display a progressive loss of immune-competence. Comparative longitudinal analysis of lymphoid organs from control, single Rac1(N) or Rac3(KO) , and double Rac1(N) /Rac3(KO) mutant animals showed that thymus development is preserved up to postnatal day 9 in all animals, but is impaired in Rac1(N) /Rac3(KO) mice at later times. This is evidenced by a drastic reduction in thymic cell numbers. Cell numbers were also reduced in the spleen, leading to splenic tissue disarray. Organ involution occurs in spite of unaltered thymocyte and lymphocyte subset composition, and proper mature T-cell responses to polyclonal stimuli in vitro. Suboptimal thymus innervation by tau-positive neuronal terminals possibly explains the suboptimal thymic output and arrested thymic development, which is accompanied by higher apoptotic rates. Our results support a role for neuronal Rac1 and Rac3 in dictating proper lymphoid organ development, and suggest the existence of lymphoid-extrinsic mechanisms linking neural defects to the loss of immune-competence.
Asunto(s)
Inmunocompetencia , Neuronas/fisiología , Bazo/inmunología , Timo/inmunología , Proteínas de Unión al GTP rac/fisiología , Proteína de Unión al GTP rac1/fisiología , Animales , Apoptosis , Diferenciación Celular , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Citometría de Flujo , Ratones , Ratones Noqueados , Bazo/citología , Bazo/crecimiento & desarrollo , Timo/citología , Timo/crecimiento & desarrollo , Timo/inervación , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1/deficiencia , Proteína de Unión al GTP rac1/genéticaRESUMEN
Rac GTPases are members of the Rho family regulating the actin cytoskeleton and implicated in neuronal development. Ubiquitous Rac1 and neuron-specific Rac3 GTPases are coexpressed in the developing mammalian brain. We used Cre-mediated conditional deletion of Rac1 in neurons combined with knockout of neuron-specific Rac3 to study the role of these GTPases in neural development. We found that lack of both genes causes motor behavioral defects, epilepsy, and premature death of mice. Deletion of either GTPase does not produce evident phenotypes. Double-knockout mice show specific defects in the development of the hippocampus. Selective impairment of the dorsal hilus of double-knockout animals is associated with alteration in the formation of the hippocampal circuitry. Axonal pathways to and from the dorsal hilus are affected because of the deficit of hilar mossy cells. Moreover, analysis of Rac function in hippocampal cultures shows that spine formation is strongly hampered only in neurons lacking both Rac proteins. These findings show for the first time that both Rac1 and Rac3 are important for the development of the nervous system, wherein they play complementary roles during late stages of neuronal and brain development.
Asunto(s)
Neurogénesis/fisiología , Proteínas de Unión al GTP rac/fisiología , Proteína de Unión al GTP rac1/fisiología , Animales , Apoptosis/fisiología , Espinas Dendríticas/fisiología , Giro Dentado/fisiología , Giro Dentado/ultraestructura , Hipocampo/citología , Hipocampo/embriología , Ratones , Ratones Noqueados , Neuronas/citología , Transgenes/fisiologíaRESUMEN
Rho family GTPases have been implicated as important regulators of neuronal development. Rac3 is a member of this family specifically expressed in vertebrate developing neurons, where it is coexpressed with the ubiquitous Rac1 GTPase. We have previously shown that Rac3 knockout mice are viable and fertile. The Rac3 protein shows highest expression around postnatal day 7 in brain regions relevant for cognitive behaviors. In this study we find that Rac3 knockout mice do not show defects in spatial reference memory assessed with water maze task, but they show a reduced behavioral flexibility to novel situations. Analysis of explorative behavior revealed hyperactive behavior and hyperreactivity to the presentation of new stimuli, as assessed by dark/light box, emergence and novel object tests. These defects were not due to reduced visual abilities, since visual acuity and contrast sensitivity were comparable in Rac3 knockout and wildtype littermates. Our data reinforce the notion that Rho family GTPases are important for normal cognitive development, and highlight specific functions of Rac3 that cannot be compensated by the coexpressed homologous Rac1.
Asunto(s)
Conducta Animal/fisiología , Conducta Exploratoria/fisiología , Hipercinesia/genética , Hipercinesia/fisiopatología , Proteínas de Unión al GTP rac/deficiencia , Análisis de Varianza , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Sensibilidad de Contraste/genética , Regulación del Desarrollo de la Expresión Génica , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Noqueados , Estimulación Luminosa/métodos , Factores de Tiempo , Grabación en Video/métodos , Agudeza Visual/genética , Proteínas de Unión al GTP rac/inmunologíaRESUMEN
BACKGROUND INFORMATION: Rho family GTPases are required for cytoskeletal reorganization and are considered important for the maturation of neurons. Among these proteins, Rac1 is known to play a crucial role in the regulation of actin dynamics, and a number of studies indicate the involvement of this protein in different steps of vertebrate neuronal maturation. There are two distinct Rac proteins expressed in neurons, namely the ubiquitous Rac1 and the neuron-specific Rac3. The specific functions of each of these GTPases during early neuronal development are largely unknown. RESULTS: The combination of the knockout of Rac3 with Rac1 down-regulation by siRNA (small interfering RNA) has been used to show that down-regulation of Rac1 affects dendritic development in mouse hippocampal neurons, without affecting axons. F-actin levels are strongly decreased in neuronal growth cones following down-regulation of Rac1, and time-lapse analysis indicated that the reduction of Rac1 levels decreases growth-cone dynamics. CONCLUSIONS: These results show that normal levels of endogenous Rac1 activity are critical for early dendritic development, whereas dendritic outgrowth is not affected in hippocampal neurons from Rac3-null mice. On the other hand, early axonal development appears normal after Rac1 down-regulation. Our findings also suggest that the initial establishment of neuronal polarity is not affected by Rac1 down-regulation.
Asunto(s)
Axones/enzimología , Dendritas/enzimología , Hipocampo/citología , Hipocampo/enzimología , Proteína de Unión al GTP rac1/metabolismo , Actinas/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Conos de Crecimiento/enzimología , Ratones , Ratones Noqueados , ARN Interferente Pequeño/metabolismo , Sinapsis/metabolismo , Proteínas de Unión al GTP rac/deficiencia , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Rac proteins are members of the Rho family of GTPases involved in the regulation of actin dynamics. The three highly homologous Rac proteins in mammals are the ubiquitous Rac1, the hematopoiesis-specific Rac2, and the least-characterized Rac3. We show here that Rac3 mRNA is widely and specifically expressed in the developing nervous system, with highest concentration at embryonic day 13 in the dorsal root ganglia and ventral spinal cord. At postnatal day 7 Rac3 appears particularly abundant in populations of projection neurons in several regions of the brain, including the fifth layer of the cortex and the CA1-CA3 region of the hippocampus. We generated mice deleted for the Rac3 gene with the aim of analyzing the function of this GTPase in vivo. Rac3 knockout animals survive embryogenesis and show no obvious developmental defects. Interestingly, specific behavioral differences were detected in the Rac3-deficient animals, since motor coordination and motor learning on the rotarod was superior to that of their wild-type littermates. No obvious histological or immunohistological differences were observed at major sites of Rac3 expression. Our results indicate that, in vivo, Rac3 activity is not strictly required for normal development in utero but may be relevant to later events in the development of a functional nervous system.
Asunto(s)
Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Conducta Animal , Química Encefálica , ADN Complementario , Electroporación , Embrión de Mamíferos , Expresión Génica , Marcación de Gen , Vectores Genéticos , Genoma , Inmunohistoquímica , Hibridación in Situ , Aprendizaje , Ratones , Ratones Noqueados , Actividad Motora/genética , Actividad Motora/fisiología , Pruebas de Precipitina , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , beta-Galactosidasa/genética , Proteínas de Unión al GTP rac/deficienciaRESUMEN
Rac3 is one of the three known Rac GTPases in vertebrates. Rac3 shows high sequence homology to Rac1, and its transcript is specifically expressed in the developing nervous system, where its localization and function are unknown. By using Rac3-specific antibodies, we show that the endogenous Rac3 protein is differentially expressed during mouse brain development, with a peak of expression at times of neuronal maturation and synaptogenesis. Comparison with Rac1 shows clear-cut differences in the overall distribution of the two GTPases in the developing brain, and in their subcellular distribution in regions of the brain where both proteins are expressed. At P7, Rac3 staining is particularly marked in the deep cerebellar nuclei and in the pons, where it shows a discontinuous distribution around the neuronal cell bodies, in contrast with the diffuse staining of Rac1. Rac3 does not evidently co-localize with pre- and post-synaptic markers, nor with GFAP-positive astrocytes, but it clearly co-localizes with actin filaments, and with the terminal portions of calbindin-positive Purkinje cell axons in the deep cerebellar nuclei. Our data implicate Rac3 in neuronal differentiation, and support a specific role of this GTPase in actin-mediated remodelling of Purkinje cell neuritic terminals at time of synaptogenesis.