RESUMEN

BACKGROUND: Mainly known as a transcription factor patterning the rostral brain and governing its histogenesis, FOXG1 has been also detected outside the nucleus; however, biological meaning of that has been only partially clarified. RESULTS: Prompted by FOXG1 expression in cytoplasm of pallial neurons, we investigated its implication in translational control. We documented the impact of FOXG1 on ribosomal recruitment of Grin1-mRNA, encoding for the main subunit of NMDA receptor. Next, we showed that FOXG1 increases GRIN1 protein level by enhancing the translation of its mRNA, while not increasing its stability. Molecular mechanisms underlying this activity included FOXG1 interaction with EIF4E and, possibly, Grin1-mRNA. Besides, we found that, within murine neocortical cultures, de novo synthesis of GRIN1 undergoes a prominent and reversible, homeostatic regulation and FOXG1 is instrumental to that. Finally, by integrated analysis of multiple omic data, we inferred that FOXG1 is implicated in translational control of hundreds of neuronal genes, modulating ribosome engagement and progression. In a few selected cases, we experimentally verified such inference. CONCLUSIONS: These findings point to FOXG1 as a key effector, potentially crucial to multi-scale temporal tuning of neocortical pyramid activity, an issue with profound physiological and neuropathological implications.

Asunto(s)

Factores de Transcripción Forkhead , Neocórtex , Proteínas del Tejido Nervioso , Neuronas , Receptores de N-Metil-D-Aspartato , Animales , Femenino , Masculino , Ratones , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Neocórtex/metabolismo , Neocórtex/embriología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Biosíntesis de Proteínas/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo

RESUMEN

Foxg1 masters telencephalic development via a pleiotropic control over its progression. Expressed within the central nervous system (CNS), L1 retrotransposons are implicated in progression of its histogenesis and tuning of its genomic plasticity. Foxg1 represses gene transcription, and L1 elements share putative Foxg1-binding motifs, suggesting the former might limit telencephalic expression (and activity) of the latter. We tested such a prediction, in vivo as well as in engineered primary neural cultures, using loss- and gain-of-function approaches. We found that Foxg1-dependent, transcriptional L1 repression specifically occurs in neopallial neuronogenic progenitors and post-mitotic neurons, where it is supported by specific changes in the L1 epigenetic landscape. Unexpectedly, we discovered that Foxg1 physically interacts with L1-mRNA and positively regulates neonatal neopallium L1-DNA content, antagonizing the retrotranscription-suppressing activity exerted by Mov10 and Ddx39a helicases. To the best of our knowledge, Foxg1 represents the first CNS patterning gene acting as a bimodal retrotransposon modulator, limiting transcription of L1 elements and promoting their amplification, within a specific domain of the developing mouse brain.

Asunto(s)

Factores de Transcripción Forkhead , Regulación del Desarrollo de la Expresión Génica , Neocórtex , Proteínas del Tejido Nervioso , ARN Mensajero , Animales , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Ratones , Neocórtex/metabolismo , Neocórtex/embriología , Neocórtex/crecimiento & desarrollo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Retroelementos/genética , ADN/metabolismo , ADN/genética , Neuronas/metabolismo

RESUMEN

Sizes of neuronal, astroglial and oligodendroglial complements forming the neonatal cerebral cortex largely depend on rates at which pallial stem cells give rise to lineage-committed progenitors and the latter ones progress to mature cell types. Here, we investigated the spatial articulation of pallial stem cells' (SCs) commitment to astrogenesis as well as the progression of committed astroglial progenitors (APs) to differentiated astrocytes, by clonal and kinetic profiling of pallial precursors. We found that caudal-medial (CM) SCs are more prone to astrogenesis than rostro-lateral (RL) ones, while RL-committed APs are more keen to proliferate than CM ones. Next, we assessed the control of these phenomena by 2 key transcription factor genes mastering regionalization of the early cortical primordium, Emx2 and Foxg1, via lentiviral somatic transgenesis, epistasis assays, and ad hoc rescue assays. We demonstrated that preferential CM SCs progression to astrogenesis is promoted by Emx2, mainly via Couptf1, Nfia, and Sox9 upregulation, while Foxg1 antagonizes such progression to some extent, likely via repression of Zbtb20. Finally, we showed that Foxg1 and Emx2 may be implicated-asymmetrically and antithetically-in shaping distinctive proliferative/differentiative behaviors displayed by APs in hippocampus and neocortex.

Asunto(s)

Neocórtex , Neurogénesis , Humanos , Recién Nacido , Astrocitos/metabolismo , Astrocitos/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Factor de Transcripción COUP I/genética , Regulación del Desarrollo de la Expresión Génica/genética , Hipocampo/metabolismo , Hipocampo/fisiología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Neocórtex/metabolismo , Neurogénesis/genética , Neurogénesis/fisiología , Neuronas/metabolismo , Neuronas/fisiología , Oligodendroglía/metabolismo , Oligodendroglía/fisiología

RESUMEN

FOXG1 is an ancient transcription factor gene mastering telencephalic development. A number of distinct structural FOXG1 mutations lead to the "FOXG1 syndrome", a complex and heterogeneous neuropathological entity, for which no cure is presently available. Reconstruction of primary neurodevelopmental/physiological anomalies evoked by these mutations is an obvious pre-requisite for future, precision therapy of such syndrome. Here, as a proof-of-principle, we functionally scored three FOXG1 neuropathogenic alleles, FOXG1G224S, FOXG1W308X, and FOXG1N232S, against their healthy counterpart. Specifically, we delivered transgenes encoding for them to dedicated preparations of murine pallial precursors and quantified their impact on selected neurodevelopmental and physiological processes mastered by Foxg1: pallial stem cell fate choice, proliferation of neural committed progenitors, neuronal architecture, neuronal activity, and their molecular correlates. Briefly, we found that FOXG1G224S and FOXG1W308X generally performed as a gain- and a loss-of-function-allele, respectively, while FOXG1N232S acted as a mild loss-of-function-allele or phenocopied FOXG1WT. These results provide valuable hints about processes misregulated in patients heterozygous for these mutations, to be re-addressed more stringently in patient iPSC-derivative neuro-organoids. Moreover, they suggest that murine pallial cultures may be employed for fast multidimensional profiling of novel, human neuropathogenic FOXG1 alleles, namely a step propedeutic to timely delivery of therapeutic precision treatments.

Asunto(s)

Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas del Tejido Nervioso/genética , Trastornos del Neurodesarrollo/genética , Alelos , Animales , Encéfalo/metabolismo , Corteza Cerebral/metabolismo , Factores de Transcripción Forkhead/metabolismo , Expresión Génica/genética , Frecuencia de los Genes/genética , Humanos , Ratones , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis , Neuronas/metabolismo , Cultivo Primario de Células , Prueba de Estudio Conceptual

RESUMEN

Chromatin organization plays a crucial role in tissue homeostasis. Heterochromatin relaxation and consequent unscheduled mobilization of transposable elements (TEs) are emerging as key contributors of aging and aging-related pathologies, including Alzheimer's disease (AD) and cancer. However, the mechanisms governing heterochromatin maintenance or its relaxation in pathological conditions remain poorly understood. Here we show that PIN1, the only phosphorylation-specific cis/trans prolyl isomerase, whose loss is associated with premature aging and AD, is essential to preserve heterochromatin. We demonstrate that this PIN1 function is conserved from Drosophila to humans and prevents TE mobilization-dependent neurodegeneration and cognitive defects. Mechanistically, PIN1 maintains nuclear type-B Lamin structure and anchoring function for heterochromatin protein 1α (HP1α). This mechanism prevents nuclear envelope alterations and heterochromatin relaxation under mechanical stress, which is a key contributor to aging-related pathologies.

Asunto(s)

Proteínas de Drosophila/metabolismo , Heterocromatina/metabolismo , Lamina Tipo B/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Estrés Mecánico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Células Cultivadas , Homólogo de la Proteína Chromobox 5/genética , Homólogo de la Proteína Chromobox 5/metabolismo , Elementos Transponibles de ADN/genética , Drosophila/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Humanos , Lamina Tipo B/química , Ratones , Ratones Endogámicos C57BL , Peptidilprolil Isomerasa de Interacción con NIMA/antagonistas & inhibidores , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Neocórtex/citología , Neocórtex/metabolismo , Neuronas/citología , Neuronas/metabolismo , Membrana Nuclear/química , Isomerasa de Peptidilprolil/antagonistas & inhibidores , Isomerasa de Peptidilprolil/genética , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo

RESUMEN

Foxg1 is an ancient transcription factor gene orchestrating a number of neurodevelopmental processes taking place in the rostral brain. In this study, we investigated its impact on neocortical activity. We found that mice overexpressing Foxg1 in neocortical pyramidal cells displayed an electroencephalography (EEG) with increased spike frequency and were more prone to kainic acid (KA)-induced seizures. Consistently, primary cultures of neocortical neurons gain-of-function for Foxg1 were hyperactive and hypersynchronized. That reflected an unbalanced expression of key genes encoding for ion channels, gamma aminobutyric acid and glutamate receptors, and was likely exacerbated by a pronounced interneuron depletion. We also detected a transient Foxg1 upregulation ignited in turn by neuronal activity and mediated by immediate early genes. Based on this, we propose that even small changes of Foxg1 levels may result in a profound impact on pyramidal cell activity, an issue relevant to neuronal physiology and neurological aberrancies associated to FOXG1 copy number variations.

Asunto(s)

Factores de Transcripción Forkhead/metabolismo , Neocórtex/fisiología , Proteínas del Tejido Nervioso/metabolismo , Células Piramidales/metabolismo , Animales , Variaciones en el Número de Copia de ADN , Electroencefalografía , Factores de Transcripción Forkhead/genética , Ratones , Proteínas del Tejido Nervioso/genética , Convulsiones/genética , Convulsiones/metabolismo , Regulación hacia Arriba

RESUMEN

Friedreich's ataxia (FRDA) is an untreatable disorder with neuro- and cardio-degenerative progression. This monogenic disease is caused by the hyper-expansion of naturally occurring GAA repeats in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Thus, current therapeutic strategies are mostly aimed to restore physiological FXN expression. We have previously described SINEUPs, natural and synthetic antisense long non-coding RNAs, which promote translation of partially overlapping mRNAs through the activity of an embedded SINEB2 domain. Here, by in vitro screening, we have identified a number of SINEUPs targeting human FXN mRNA and capable to up-regulate frataxin protein to physiological amounts acting at the post-transcriptional level. Furthermore, FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial aconitase defects in FRDA-derived cells. In summary, we provide evidence that SINEUPs may be the first gene-specific therapeutic approach to activate FXN translation in FRDA and, more broadly, a novel scalable platform to develop new RNA-based therapies for haploinsufficient diseases.

Asunto(s)

Ataxia de Friedreich/genética , Regulación de la Expresión Génica , Proteínas de Unión a Hierro/genética , Modelos Biológicos , ARN no Traducido/metabolismo , Aconitato Hidratasa/metabolismo , Línea Celular , Fibroblastos/metabolismo , Humanos , Linfocitos/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/genética , Frataxina

RESUMEN

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Asunto(s)

Ventrículos Cerebrales/citología , Células-Madre Neurales/trasplante , Animales , Diferenciación Celular/genética , Movimiento Celular , Femenino , Masculino , Ratones , Neuritas , Neuronas/fisiología , Ingeniería de Tejidos

RESUMEN

Neocortical astrogenesis follows neuronogenesis and precedes oligogenesis. Among key factors dictating its temporal articulation, there are progression rates of pallial stem cells (SCs) towards astroglial lineages as well as activation rates of astrocyte differentiation programs in response to extrinsic gliogenic cues. In this study, we showed that high Foxg1 SC expression antagonizes astrocyte generation, while stimulating SC self-renewal and committing SCs to neuronogenesis. We found that mechanisms underlying this activity are mainly cell autonomous and highly pleiotropic. They include a concerted downregulation of 4 key effectors channeling neural SCs to astroglial fates, as well as defective activation of core molecular machineries implementing astroglial differentiation programs. Next, we found that SC Foxg1 levels specifically decline during the neuronogenic-to-gliogenic transition, pointing to a pivotal Foxg1 role in temporal modulation of astrogenesis. Finally, we showed that Foxg1 inhibits astrogenesis from human neocortical precursors, suggesting that this is an evolutionarily ancient trait.

Asunto(s)

Astrocitos/citología , Factores de Transcripción Forkhead/metabolismo , Neocórtex/embriología , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/citología , Neurogénesis/fisiología , Animales , Astrocitos/metabolismo , Diferenciación Celular/fisiología , Humanos , Ratones , Neocórtex/metabolismo , Células-Madre Neurales/metabolismo

RESUMEN

The architecture of neocortical projection neurons is subject of a complex gene control. Here we demonstrated that Foxg1, a transcription factor gene which patterns the early rostral brain and sets the pace of telencephalic neuronogenesis, specifically stimulates dendrite elongation. This phenomenon occurs in vivo like in vitro, and it is detectable even upon moderate changes of Foxg1 expression levels. We found that Foxg1 acts by stimulating Hes1, which in turn upregulates pCreb1, a well-known pro-dendritogenic effector, and downregulates Syt and Ndr1, namely two established antagonizers of dendrite elongation. Moreover, Foxg1-driven pCreb1 upregulation requires PKA and AKT, and correlates with reduced PP1 and PP2A phosphatase activity. These findings contribute to clarify normal neurodevelopmental and activity-related regulation of neuritogenesis. They further suggest that an abnormal sizing of the dendritic tree of neocortical projection neurons may occur in West and Rett syndrome patients with anomalous FOXG1 allele dosages and contribute to their neurolopathological profiles.

Asunto(s)

Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dendritas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células Piramidales/metabolismo , Factor de Transcripción HES-1/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo , Femenino , Regulación de la Expresión Génica , Masculino , Ratones , Fosforilación , Transducción de Señal , Regulación hacia Arriba

RESUMEN

Specification of dorsoventral regional identity in progenitors of the developing telencephalon is a first pivotal step in the development of the cerebral cortex and basal ganglia. Previously, we demonstrated that the two zinc finger doublesex and mab-3 related (Dmrt) genes, Dmrt5 (Dmrta2) and Dmrt3, which are coexpressed in high caudomedial to low rostrolateral gradients in the cerebral cortical primordium, are separately needed for normal formation of the cortical hem, hippocampus, and caudomedial neocortex. We have now addressed the role of Dmrt3 and Dmrt5 in controlling dorsoventral division of the telencephalon in mice of either sex by comparing the phenotypes of single knock-out (KO) with double KO embryos and by misexpressing Dmrt5 in the ventral telencephalon. We find that DMRT3 and DMRT5 act as critical regulators of progenitor cell dorsoventral identity by repressing ventralizing regulators. Early ventral fate transcriptional regulators expressed in the dorsal lateral ganglionic eminence, such as Gsx2, are upregulated in the dorsal telencephalon of Dmrt3;Dmrt5 double KO embryos and downregulated when ventral telencephalic progenitors express ectopic Dmrt5 Conditional overexpression of Dmrt5 throughout the telencephalon produces gene expression and structural defects that are highly consistent with reduced GSX2 activity. Further, Emx2;Dmrt5 double KO embryos show a phenotype similar to Dmrt3;Dmrt5 double KO embryos, and both DMRT3, DMRT5 and the homeobox transcription factor EMX2 bind to a ventral telencephalon-specific enhancer in the Gsx2 locus. Together, our findings uncover cooperative functions of DMRT3, DMRT5, and EMX2 in dividing dorsal from ventral in the telencephalon.SIGNIFICANCE STATEMENT We identified the DMRT3 and DMRT5 zinc finger transcription factors as novel regulators of dorsoventral patterning in the telencephalon. Our data indicate that they have overlapping functions and compensate for one another. The double, but not the single, knock-out produces a dorsal telencephalon that is ventralized, and olfactory bulb tissue takes over most remaining cortex. Conversely, overexpressing Dmrt5 throughout the telencephalon causes expanded expression of dorsal gene determinants and smaller olfactory bulbs. Furthermore, we show that the homeobox transcription factor EMX2 that is coexpressed with DMRT3 and DMRT5 in cortical progenitors cooperates with them to maintain dorsoventral patterning in the telencephalon. Our study suggests that DMRT3/5 function with EMX2 in positioning the pallial-subpallial boundary by antagonizing the ventral homeobox transcription factor GSX2.

Asunto(s)

Proteínas de Homeodominio/fisiología , Células-Madre Neurales/fisiología , Neuronas/fisiología , Telencéfalo/embriología , Factores de Transcripción/fisiología , Animales , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Telencéfalo/metabolismo , Factores de Transcripción/genética

RESUMEN

Small activating RNAs (saRNAs), targeting endogenous genes and stimulating their transcription, are a promising tool for implementing a variety of neurotherapeutic strategies. Among these there is the stimulation of select histogenetic subroutines for purposes of cell-based brain repair, as well as the therapeutic treatment of gene expression deficits underlying severe neurological disorders.We employed RNA activation (RNAa) to transactivate the Emx2 transcription factor gene in embryonic cortico-cerebral precursor cells. This led to enhanced self-renewal, delayed differentiation, and reduced death of neuronally committed precursors, resulting in a remarkable expansion of the neuronogenic precursors pool. These results are of paramount interest for purposes of gene-promoted brain repair. As such, RNAa makes therapeutic stimulation of neuronogenesis via Emx2 overexpression a feasible goal, preventing the drawbacks of exogenous gene copies introduction.Moreover, we employed RNAa to achieve a gentle transactivation of the Foxg1 transcription factor gene, specifically in cortico-cerebral cells. This manipulation led to an appreciable biological outcome, while complying with endogenous gene tuning linked to early central nervous system regionalization and late activity of neocortical projection neurons. Foxg1-activating miRNAs stimulated RNApolII recruitment, possibly via Ago1. One of them worked promisingly in vivo. As such, RNAa can be a valuable approach for therapeutic treatment of the FOXG1-haploinsufficiency-linked variant of the Rett syndrome. Remarkably, hemizygosity for specific genes and polygenic chromosomal segments underlies a huge number of neuropathological entities for which no cure is presently available. Based on the results reported above, RNAa might be a simple and scalable approach for fixing this class of problems.

Asunto(s)

Haploinsuficiencia , Neurogénesis , ARN Pequeño no Traducido/genética , Activación Transcripcional , Diferenciación Celular , Factores de Transcripción Forkhead/genética , Humanos , Proteínas del Tejido Nervioso/genética , Síndrome de Rett

RESUMEN

The post-genomic era has unveiled the existence of a large repertory of non-coding RNAs and repetitive elements that play a fundamental role in cellular homeostasis and dysfunction. These may represent unprecedented opportunities to modify gene expression at the right time in the correct space in vivo, providing an almost unlimited reservoir of new potential pharmacological agents. Hijacking their mode of actions, the druggable genome can be extended to regulatory RNAs and DNA elements in a scalable fashion. Here, we discuss the state-of-the-art of nucleic acid-based drugs to treat neurodegenerative diseases. Beneficial effects can be obtained by inhibiting (Yin) and increasing (Yang) gene expression, depending on the disease and the drug target. Together with the description of the current use of inhibitory RNAs (small inhibitory RNAs and antisense oligonucleotides) in animal models and clinical trials, we discuss the molecular basis and applications of new classes of activatory RNAs at transcriptional (RNAa) and translational (SINEUP) levels.

Asunto(s)

Encéfalo/efectos de los fármacos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Ácidos Nucleicos/farmacología , Animales , Humanos

RESUMEN

More than one hundred distinct gene hemizygosities are specifically linked to epilepsy, mental retardation, autism, schizophrenia and neuro-degeneration. Radical repair of these gene deficits via genome engineering is hardly feasible. The same applies to therapeutic stimulation of the spared allele by artificial transactivators. Small activating RNAs (saRNAs) offer an alternative, appealing approach. As a proof-of-principle, here we tested this approach on the Rett syndrome-linked, haploinsufficient, Foxg1 brain patterning gene. We selected a set of artificial small activating RNAs (saRNAs) upregulating it in neocortical precursors and their derivatives. Expression of these effectors achieved a robust biological outcome. saRNA-driven activation (RNAa) was limited to neural cells which normally express Foxg1 and did not hide endogenous gene tuning. saRNAs recognized target chromatin through a ncRNA stemming from it. Gene upregulation required Ago1 and was associated to RNApolII enrichment throughout the Foxg1 locus. Finally, saRNA delivery to murine neonatal brain replicated Foxg1-RNAa in vivo.

Asunto(s)

Proteínas Argonautas/genética , Factores Eucarióticos de Iniciación/genética , Factores de Transcripción Forkhead/genética , Haploinsuficiencia/genética , MicroARNs/genética , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/genética , ARN Polimerasa III/genética , Activación Transcripcional/genética , Células 3T3 , Animales , Línea Celular , Células HEK293 , Humanos , Ratones , Tejido Nervioso/crecimiento & desarrollo , Interferencia de ARN , ARN Interferente Pequeño/genética , Síndrome de Rett/genética , Síndrome de Rett/terapia , Regulación hacia Arriba/genética

RESUMEN

Glioblastoma is a devastating CNS tumour for which no cure is presently available. We wondered if manipulation of Emx2, which normally antagonizes cortico-cerebral astrogenesis by inhibiting proliferation of astrocyte progenitors, may be employed to counteract it. We found that Emx2 overexpression induced the collapse of seven out of seven in vitro tested glioblastoma cell lines. Moreover, it suppressed four out of four of these lines in vivo. As proven by dedicated rescue assays, the antioncogenic activity of Emx2 originated from its impact on at least six metabolic nodes, which accounts for the robustness of its effect. Finally, in two out of two tested lines, the tumor culture collapse was also achieved when Emx2 was driven by a neural stem cell-specific promoter, likely active within tumor-initiating cells. All that points to Emx2 as a novel, promising tool for therapy of glioblastoma and prevention of its recurrencies.

Asunto(s)

Glioblastoma/terapia , Proteínas de Homeodominio/fisiología , Factores de Transcripción/fisiología , Adulto , Animales , Diferenciación Celular/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Terapia Genética , Glioblastoma/genética , Glioblastoma/patología , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/prevención & control , Células-Madre Neurales/patología , Células-Madre Neurales/fisiología , Factores de Transcripción/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

To promote expression of endogenous genes ad libitum, we developed a novel, programmable transcription factor prototype. Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Gene upregulation was small, however sufficient to robustly inhibit neuronal differentiation. The transactivator interacted with target gene chromatin via its RNA cofactor. Its activity was restricted to cells in which the target gene is normally transcribed. Our device might be useful for specific applications. However for this purpose, it will require an improvement of its transactivation power as well as a better characterization of its target specificity and mechanism of action.

Asunto(s)

ARN/metabolismo , Transactivadores/química , Activación Transcripcional , Animales , Encéfalo/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Ratones , Células-Madre Neurales/metabolismo , Estructura Terciaria de Proteína , ARN/química , Transactivadores/metabolismo , Regulación hacia Arriba

RESUMEN

Generation of astrocytes within the developing cerebral cortex is a tightly regulated process, initiating at low level in the middle of neuronogenesis and peaking up after its completion. Astrocytic outputs depend on two primary factors: progression of multipotent precursors toward the astroglial lineage and sizing of the astrogenic proliferating pool. The aim of this study was to investigate the role of the Emx2 homeobox gene in the latter process. We addressed this issue by combined gain- and loss-of-function methods, in vivo as well as in primary cultures of cortico-cerebral precursors. We found that Emx2 overexpression in cortico-cerebral stem cells shrinked the proliferating astrogenic pool, resulting in a severe reduction of the astroglial outcome. We showed that this was caused by EgfR and Fgf9 downregulation and that both phenomena originated from exaggerated Bmp signaling and Sox2 repression. Finally, we provided evidence that in vivo temporal progression of Emx2 levels in cortico-cerebral multipotent precursors contributes to confine the bulk of astrogenesis to postnatal life. Emx2 regulation of astrogenesis adds to a number of earlier developmental processes mastered by this gene. It points to Emx2 as a new promising tool for controlling reactive astrogliosis and optimizing cell-based designs for brain repair.

Asunto(s)

Astrocitos/fisiología , Diferenciación Celular/fisiología , Receptores ErbB/metabolismo , Factor 9 de Crecimiento de Fibroblastos/metabolismo , Proteínas de Homeodominio/metabolismo , Células-Madre Neurales/fisiología , Factores de Transcripción/metabolismo , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Células Cultivadas , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/fisiología , Proteínas de Homeodominio/genética , Ratones , Ratones Transgénicos , ARN Mensajero/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Factores de Transcripción/genética

RESUMEN

Granule cells (GCs) in the dentate gyrus are generated mainly postnatally. Between embryonic day 10 and 14, neural precursors migrate from the primary dentate matrix to the dentate gyrus where they differentiate into neurons. Neurogenesis reaches a peak at the end of the first postnatal week and it is completed at the end of the first postnatal month. This process continues at a reduced rate throughout life. Interestingly, immediately after birth, GCs exhibit a clear GABAergic phenotype. Only later they integrate the classical glutamatergic trisynaptic hippocampal circuit. Here, whole cell patch clamp recordings, in current clamp mode, were performed from immature GCs, intracellularly loaded with biocytin (in hippocampal slices from P0 to P3 old rats) in order to compare their morphological characteristics with their electrophysiological properties. The vast majority of GCs were very immature with small somata, few dendritic branches terminating with small varicosities and growth cones. In spite of their immaturity their axons reached often the cornu ammonis 3 area. Immature GCs generated, upon membrane depolarization, either rudimentary sodium spikes or more clear overshooting action potentials that fired repetitively. They exhibited also low threshold calcium spikes. In addition, most spiking neurons showed spontaneous synchronized network activity, reminiscent of giant depolarizing potentials (GDPs) generated in the hippocampus by the synergistic action of glutamate and GABA, both depolarizing and excitatory. This early synchronized activity, absent during adult neurogenesis, may play a crucial role in the refinement of local neuronal circuits within the developing dentate gyrus.

RESUMEN

A remarkable body of research over the last 15 years has been aimed at disentangling the cellular and molecular mechanisms which regulate murine cortico-cerebral astrogenesis. This research effort has allowed the reconstruction of the actual sizing of this process, as well as a better definition of its temporal, spatial and clonal articulation. Moreover, these investigations have shed substantial light on the cardinal molecular mechanisms governing the transition from pallial neuronogenesis to astrogenesis, as well as subsequent progress of the latter. It has turned out that proper temporal articulation of astrogenesis relies on a plethora of tightly interlaced mechanisms, which synergistically dampen astrogenesis prior to birth and promote it during peri- and postnatal life. The aim of this review is to provide a comprehensive and organic synthesis of these mechanisms, as well as a critical evaluation of their specific relevance to proper articulation of cerebral cortex astrogenesis in time and space.

Asunto(s)

Astrocitos/citología , Corteza Cerebral/embriología , Células-Madre Neurales/citología , Animales , Astrocitos/metabolismo , Proliferación Celular , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Cromatina , Epigénesis Genética , Ratones , Células-Madre Neurales/metabolismo , Neurogénesis , Neuronas/fisiología