RESUMEN
Medulloblastoma is a malignant childhood brain tumor arising from the developing cerebellum. In Sonic Hedgehog (SHH) subgroup medulloblastoma, aberrant activation of SHH signaling causes increased proliferation of granule neuron progenitors (GNPs), and predisposes these cells to tumorigenesis. A second, cooperating genetic hit is often required to push these hyperplastic cells to malignancy and confer mutation-specific characteristics associated with oncogenic signaling. Somatic loss-of-function mutations of the transcriptional corepressor BCOR are recurrent and enriched in SHH medulloblastoma. To investigate BCOR as a putative tumor suppressor, we used a genetically engineered mouse model to delete exons 9/10 of Bcor (BcorΔE9-10 ) in GNPs during development. This mutation leads to reduced expression of C-terminally truncated BCOR (BCORΔE9-10). While BcorΔE9-10 alone did not promote tumorigenesis or affect GNP differentiation, BcorΔE9-10 combined with loss of the SHH receptor gene Ptch1 resulted in fully penetrant medulloblastomas. In Ptch1+/- ;BcorΔE9-10 tumors, the growth factor gene Igf2 was aberrantly up-regulated, and ectopic Igf2 overexpression was sufficient to drive tumorigenesis in Ptch1+/- GNPs. BCOR directly regulates Igf2, likely through the PRC1.1 complex; the repressive histone mark H2AK119Ub is decreased at the Igf2 promoter in Ptch1+/- ;BcorΔE9-10 tumors. Overall, our data suggests that BCOR-PRC1.1 disruption leads to Igf2 overexpression, which transforms preneoplastic cells to malignant tumors.
Asunto(s)
Neoplasias Cerebelosas/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Proteínas del Grupo Polycomb/metabolismo , Proteínas Represoras/genética , Animales , Carcinogénesis/genética , Modelos Animales de Enfermedad , Proteínas Hedgehog/genética , Humanos , Ratones , Mutación , Receptor Patched-1/genética , Proteínas del Grupo Polycomb/genética , Proteínas Represoras/metabolismo , Eliminación de SecuenciaRESUMEN
During development, neural progenitors are in proliferative and immature states; however, the molecular machinery that cooperatively controls both states remains elusive. Here, we report that cyclin D1 (CCND1) directly regulates both proliferative and immature states of cerebellar granule cell progenitors (GCPs). CCND1 not only accelerates cell cycle but also upregulates ATOH1 protein, an essential transcription factor that maintains GCPs in an immature state. In cooperation with CDK4, CCND1 directly phosphorylates S309 of ATOH1, which inhibits additional phosphorylation at S328 and consequently prevents S328 phosphorylation-dependent ATOH1 degradation. Additionally, PROX1 downregulates Ccnd1 expression by histone deacetylation of Ccnd1 promoter in GCPs, leading to cell cycle exit and differentiation. Moreover, WNT signaling upregulates PROX1 expression in GCPs. These findings suggest that WNT-PROX1-CCND1-ATOH1 signaling cascade cooperatively controls proliferative and immature states of GCPs. We revealed that the expression and phosphorylation levels of these molecules dynamically change during cerebellar development, which are suggested to determine appropriate differentiation rates from GCPs to GCs at distinct developmental stages. This study contributes to understanding the regulatory mechanism of GCPs as well as neural progenitors.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Ciclina D1/metabolismo , Gránulos Citoplasmáticos/metabolismo , Fosforilación/fisiología , Células Madre/metabolismo , Animales , Ciclo Celular/genética , Diferenciación Celular/fisiología , División Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Proteínas Hedgehog/metabolismo , Ratones , Neurogénesis/fisiología , Transducción de Señal/fisiología , Factores de TranscripciónRESUMEN
Low-grade neuroepithelial tumors are major causes of drug-resistant focal epilepsy. Clinically, these tumors are defined as low-grade epilepsy-associated neuroepithelial tumors (LEATs). The BRAF V600E mutation is frequently observed in LEAT and linked to poor seizure outcomes. However, its molecular role in epileptogenicity remains elusive. To understand the molecular mechanism underlying the epileptogenicity in LEAT with the BRAF V600E genetic mutation (BRAF V600E-LEAT), we conducted RNA sequencing (RNA-seq) analysis using surgical specimens of BRAF V600E-LEAT obtained and stored at a single institute. We obtained 21 BRAF V600E-LEAT specimens and 4 control specimens, including 24 from Japanese patients and 1 from a patient of Central Asian origin, along with comprehensive clinical data. We submitted the transcriptome dataset of 21 BRAF V600E-LEAT plus 4 controls, as well as detailed clinical information, to a public database. Preliminary bioinformatics analysis using this dataset identified 2134 differentially expressed genes between BRAF V600E-LEAT and control. Additionally, gene set enrichment analysis provided novel insights into the association between estrogen response-related pathways and the epileptogenicity of BRAF V600E-LEAT patients. Our datasets and findings will contribute toward the understanding of the pathology of epilepsy caused by LEAT and the identification of new therapeutic targets.
Asunto(s)
Neoplasias Encefálicas , Epilepsia , Neoplasias Neuroepiteliales , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/cirugía , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Epilepsia/genética , Epilepsia/complicaciones , Neoplasias Neuroepiteliales/genética , Neoplasias Neuroepiteliales/metabolismo , Neoplasias Neuroepiteliales/patología , Transcriptoma , MutaciónRESUMEN
Neural development is accomplished by differentiation events leading to metabolic reprogramming. Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo- to ganglio-series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process. Nevertheless, both the molecular mechanisms that control the glycosphingolipid switch and its function in neurodevelopment are poorly understood. Here, we describe a self-contained circuit that controls glycosphingolipid reprogramming and neural differentiation. We find that globo-series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn binds and activates the promoter of the first and rate-limiting ganglioside-producing enzyme GM3 synthase, thus fostering the synthesis of gangliosides. By this mechanism, the globo-AUTS2 axis controls glycosphingolipid reprogramming and neural gene expression during neural differentiation, which involves this circuit in neurodevelopment and its defects in neuropathology.
Asunto(s)
Diferenciación Celular/fisiología , Reprogramación Celular/fisiología , Glicoesfingolípidos/metabolismo , Neurogénesis/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Reprogramación Celular/efectos de los fármacos , Proteínas del Citoesqueleto , Epigenómica , Gangliósidos/metabolismo , Expresión Génica , Silenciador del Gen , Glicoesfingolípidos/farmacología , Células HeLa , Histonas/metabolismo , Humanos , Trastornos del Neurodesarrollo , Neurogénesis/efectos de los fármacos , Neurogénesis/genética , Neuronas/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas/genética , Proteínas/metabolismo , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Factores de TranscripciónRESUMEN
The molecular mechanisms underlying neurodevelopmental disorders (NDDs) remain unclear. We previously identified Down syndrome cell adhesion molecule like 1 (Dscaml1) as a responsible gene for Ihara epileptic rat (IER), a rat model for human NDDs with epilepsy. However, the relationship between NDDs and DSCAML1 in humans is still elusive. In this study, we screened databases of autism spectrum disorders (ASD), intellectual disability (ID)/developmental disorders (DD) and schizophrenia for genomic mutations in human DSCAML1. We then performed in silico analyses to estimate the potential damage to the mutated DSCAML1 proteins and chose three representative mutations (DSCAML1C729R , DSCAML1R1685* and DSCAML1K2108Nfs*37 ), which lacked a cysteine residue in the seventh Ig domain, the intracellular region and the C-terminal PDZ-binding motif, respectively. In overexpression experiments in a cell line, DSCAML1C729R lost its mature N-glycosylation, whereas DSCAML1K2108Nfs*37 was abnormally degraded via proteasome-dependent protein degradation. Furthermore, in primary hippocampal neurons, the ability of the wild-type DSCAML1 to regulate the number of synapses was lost with all mutant proteins. These results provide insight into understanding the roles of the domains in the DSCAML1 protein and further suggest that these mutations cause functional changes, albeit through different mechanisms, that likely affect the pathophysiology of NDDs.
Asunto(s)
Moléculas de Adhesión Celular/genética , Mutación/genética , Trastornos del Neurodesarrollo/genética , Animales , Trastorno del Espectro Autista/genética , Adhesión Celular , Membrana Celular/metabolismo , Espinas Dendríticas/metabolismo , Femenino , Glicosilación , Hipocampo/patología , Humanos , Células L , Masculino , Ratones , Anotación de Secuencia Molecular , Proteínas Mutantes/metabolismo , Proteolisis , Ratas Wistar , Esquizofrenia/genética , Sinapsis/metabolismoRESUMEN
Lewy body-related α-synucleinopathy (Lewy pathology) has been reported in patients with myotonic dystrophy (DM) type 1 (DM1), but no detailed report has described the prevalence and extent of its occurrence. We studied consecutive full autopsy cases of DM1 at the National Center of Neurology and Psychiatry (NCNP) Brain Bank for intractable psychiatric and neurological disorders. Thirty-two cases, genetically determined to be DM1 (59.0 ± 8.7 years), obtained from the NCNP Brain Bank, were compared with control cases obtained from the Brain Bank for Aging Research (BBAR) in Japan. The investigated anatomical sites followed the Dementia with Lewy Bodies Consensus Guideline, expanding to the peripheral autonomic nervous system, temporal pole, and occipital cortex, in addition to the olfactory epithelium and spinal cord. Of the 32 patients, 11 (34.4%) had Lewy pathology, with a significantly higher prevalence than that in the control cases from the BBAR (20.1%). Lewy pathology detected in DM1 was widespread, but no macroscopic depigmentation of the substantia nigra was observed in any DM1 case; this was commensurate with the microscopic paucity of Lewy pathology in the substantia nigra and amygdala. Lewy pathology in DM1 does not appear to follow either Braak's ascending paradigm or the olfactory-amygdala extension. Lewy neurites and dots in DM1 were very sparse in the cerebral cortex and distinct from those observed in BBAR control cases. This study was the first demonstration of unique Lewy pathology in DM1 and may contribute to the understanding of the protein propagation hypothesis of Lewy pathology.
Asunto(s)
Enfermedad por Cuerpos de Lewy , Distrofia Miotónica , Enfermedad de Parkinson , Encéfalo/patología , Humanos , Cuerpos de Lewy/patología , Enfermedad por Cuerpos de Lewy/patología , Distrofia Miotónica/patología , Enfermedad de Parkinson/patología , alfa-Sinucleína/metabolismoRESUMEN
A proper balance between proliferation and differentiation of cerebellar granule cell precursors (GCPs) is required for appropriate cerebellar morphogenesis. The Skp1-Cullin1-F-box (SCF) complex, an E3 ubiquitin ligase complex, is involved in polyubiquitination and subsequent degradation of various cell cycle regulators and transcription factors. However, it remains unknown how the SCF complex affects proliferation and differentiation of GCPs. In this study, we found that the scaffold protein Cullin1, and F-box proteins Skp2, ß-TrCP1 and ß-TrCP2 are expressed in the external granule layer (EGL). Knockdown of these molecules in the EGL showed that Cullin1, Skp2 and ß-TrCP2 enhanced differentiation of GCPs. We also observed accumulation of cyclin-dependent kinase inhibitor p27 in GCPs when treated with a Cullin1 inhibitor or proteasome inhibitor. Furthermore, knockdown of p27 rescued enhancement of differentiation by Cullin1 knockdown. These results suggest that the SCF complex is involved in the maintenance of the proliferative state of GCPs through p27 degradation. In addition, inhibition of Cullin1 activity also prevented cell proliferation and enhanced accumulation of p27 in Daoy cells, a cell line derived from the sonic hedgehog subtype of medulloblastoma. This suggested that excess degradation of p27 through the SCF complex causes overproliferation of medulloblastoma cells.
Asunto(s)
Cerebelo/citología , Células-Madre Neurales/metabolismo , Neurogénesis , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteínas con Repetición de beta-Transducina/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Cerebelo/metabolismo , Proteínas Cullin/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos ICR , Células-Madre Neurales/citología , Proteínas Quinasas Asociadas a Fase-S/genética , UbiquitinaciónRESUMEN
The layer structure has been intensively characterized in the developing neocortex and cerebellum based on the various molecular markers. However, as to the developing dorsal midbrain, comprehensive analyses have not been intensely carried out, and thus, the name as well as the definition of each layer is not commonly shared. Here, we redefined the three layers, such as the ventricular zone, intermediate zone and marginal zone, based on various markers for proliferation and differentiation in embryonic dorsal midbrain. Biphasic Ki67 expression defines the classical VZ, in which there is clear separation of the mitotic and interphase zones. Next, we mapped the distribution of immature neurons to the defined layers, based on markers for glutamatergic and GABAergic lineage. Interestingly, Tbr2 and Neurog2 were expressed in the postmitotic neurons. We also report that active (phosphorylated) JNK is a useful marker to demarcate layers during the embryonic stage. Finally, we validated the final arrival layers of the migratory glutamatergic and GABAergic neurons. These results form a foundation for analyses of brain development, especially in the proliferation and migration of excitatory and inhibitory neurons in the dorsal midbrain.
Asunto(s)
Desarrollo Embrionario , Mesencéfalo/citología , Mesencéfalo/embriología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Linaje de la Célula , Movimiento Celular , Proliferación Celular , Femenino , Neuronas GABAérgicas/citología , Neuronas GABAérgicas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones Endogámicos ICR , Mitosis , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis , Neuronas/citología , Neuronas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Cerebellar granule cell precursors (GCPs) and granule cells (GCs) represent good models to study neuronal development. Here, we report that the transcription factor myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse GC development. We found that Meis1 is expressed in GC lineage cells and astrocytes in the cerebellum during development. Targeted disruption of the Meis1 gene specifically in the GC lineage resulted in smaller cerebella with disorganized lobules. Knock-down/knock-out (KO) experiments for Meis1 and in vitro assays showed that Meis1 binds to an upstream sequence of Pax6 to enhance its transcription in GCPs/GCs and also suggested that the Meis1-Pax6 cascade regulates morphology of GCPs/GCs during development. In the conditional KO (cKO) cerebella, many Atoh1-positive GCPs were observed ectopically in the inner external granule layer (EGL) and a similar phenomenon was observed in cultured cerebellar slices treated with a bone morphogenic protein (BMP) inhibitor. Furthermore, expression of Smad proteins and Smad phosphorylation were severely reduced in the cKO cerebella and Meis1-knock-down GCPs cerebella. Reduction of phosphorylated Smad was also observed in cerebellar slices electroporated with a Pax6 knock-down vector. Because it is known that BMP signaling induces Atoh1 degradation in GCPs, these findings suggest that the Meis1-Pax6 pathway increases the expression of Smad proteins to upregulate BMP signaling, leading to degradation of Atoh1 in the inner EGL, which contributes to differentiation from GCPs to GCs. Therefore, this work reveals crucial functions of Meis1 in GC development and gives insights into the general understanding of the molecular machinery underlying neural differentiation from neural progenitors.SIGNIFICANCE STATEMENT We report that myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse granule cell (GC) development. Here, we show Meis1 is expressed in GC precursors (GCPs) and GCs during development. Our knock-down and conditional knock-out (cKO) experiments and in vitro assays revealed that Meis1 is required for proper cerebellar structure formation and for Pax6 transcription in GCPs and GCs. The Meis1-Pax6 cascade regulates the morphology of GCs. In the cKO cerebella, Smad proteins and bone morphogenic protein (BMP) signaling are severely reduced and Atoh1-expressing GCPs are ectopically detected in the inner external granule layer. These findings suggest that Meis1 regulates degradation of Atoh1 via BMP signaling, contributing to GC differentiation in the inner EGL, and should provide understanding into GC development.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteínas Morfogenéticas Óseas/biosíntesis , Proteínas Morfogenéticas Óseas/genética , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/fisiología , Factor de Transcripción PAX6/biosíntesis , Factor de Transcripción PAX6/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Animales , Astrocitos/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Gránulos Citoplasmáticos , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Fosforilación , Embarazo , Proteínas Smad/metabolismoRESUMEN
Synaptic AMPAR expression controls the strength of excitatory synaptic transmission and plasticity. An excess of synaptic AMPARs leads to epilepsy in response to seizure-inducible stimulation. The appropriate regulation of AMPARs plays a crucial role in the maintenance of the excitatory/inhibitory synaptic balance; however, the detailed mechanisms underlying epilepsy remain unclear. Our previous studies have revealed that a key modification of AMPAR trafficking to and from postsynaptic membranes is the reversible, posttranslational S-palmitoylation at the C-termini of receptors. To clarify the role of palmitoylation-dependent regulation of AMPARs in vivo, we generated GluA1 palmitoylation-deficient (Cys811 to Ser substitution) knock-in mice. These mutant male mice showed elevated seizure susceptibility and seizure-induced neuronal activity without impairments in synaptic transmission, gross brain structure, or behavior at the basal level. Disruption of the palmitoylation site was accompanied by upregulated GluA1 phosphorylation at Ser831, but not at Ser845, in the hippocampus and increased GluA1 protein expression in the cortex. Furthermore, GluA1 palmitoylation suppressed excessive spine enlargement above a certain size after LTP. Our findings indicate that an abnormality in GluA1 palmitoylation can lead to hyperexcitability in the cerebrum, which negatively affects the maintenance of network stability, resulting in epileptic seizures.SIGNIFICANCE STATEMENT AMPARs predominantly mediate excitatory synaptic transmission. AMPARs are regulated in a posttranslational, palmitoylation-dependent manner in excitatory synapses of the mammalian brain. Reversible palmitoylation dynamically controls synaptic expression and intracellular trafficking of the receptors. Here, we generated GluA1 palmitoylation-deficient knock-in mice to clarify the role of AMPAR palmitoylation in vivo We showed that an abnormality in GluA1 palmitoylation led to hyperexcitability, resulting in epileptic seizure. This is the first identification of a specific palmitoylated protein critical for the seizure-suppressing process. Our data also provide insight into how predicted receptors such as AMPARs can effectively preserve network stability in the brain. Furthermore, these findings help to define novel key targets for developing anti-epileptic drugs.
Asunto(s)
Hipocampo/metabolismo , Hipocampo/fisiopatología , Palmitatos/metabolismo , Receptores AMPA/deficiencia , Convulsiones/metabolismo , Convulsiones/fisiopatología , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Receptores AMPA/genética , Convulsiones/genéticaRESUMEN
The activation of phosphatidylinositol 3-kinase-AKTs-mammalian target of rapamycin cell signaling pathway leads to cell overgrowth and abnormal migration and results in various types of cortical malformations, such as hemimegalencephaly (HME), focal cortical dysplasia, and tuberous sclerosis complex. However, the pathomechanism underlying abnormal cell migration remains unknown. With the use of fetal mouse brain, we performed causative gene analysis of the resected brain tissues from a patient with HME and investigated the pathogenesis. We obtained a novel somatic mutation of the MTOR gene, having approximately 11% and 7% mutation frequency in the resected brain tissues. Moreover, we revealed that the MTOR mutation resulted in hyperphosphorylation of its downstream molecules, S6 and 4E-binding protein 1, and delayed cell migration on the radial glial fiber and did not affect other cells. We suspect cell-autonomous migration arrest on the radial glial foot by the active MTOR mutation and offer potential explanations for why this may lead to cortical malformations such as HME.
Asunto(s)
Epilepsia Refractaria/genética , Hemimegalencefalia/genética , Malformaciones del Desarrollo Cortical del Grupo II/genética , Serina-Treonina Quinasas TOR/genética , Animales , Células Cultivadas , Epilepsia Refractaria/cirugía , Electroencefalografía , Femenino , Hemimegalencefalia/cirugía , Humanos , Lactante , Malformaciones del Desarrollo Cortical del Grupo II/cirugía , Ratones , Tomografía Computarizada por Tomografía de Emisión de Positrones , Serina-Treonina Quinasas TOR/metabolismo , Transfección , Regulación hacia ArribaRESUMEN
Neuronal migration is crucial for development of the mammalian-specific six-layered cerebral cortex. Migrating neurons are known to exhibit distinct features; they form a cytoplasmic dilation, a structure specific to migrating neurons, at the proximal region of the leading process, followed by nuclear elongation and forward movement. However, the molecular mechanisms of dilation formation and nuclear elongation remain unclear. Using ex vivo chemical inhibitor experiments, we show here that rottlerin, which is widely used as a specific inhibitor for PKCδ, suppresses the formation of a cytoplasmic dilation and nuclear elongation in cortical migrating neurons. Although our previous study showed that cortical neuronal migration depends on Jnk, another downstream target of rottlerin, Jnk inhibition disturbs only the nuclear elongation and forward movement, but not the dilation formation. We found that an unconventional cyclin-dependent kinase, Cdk5, is a novel downstream target of rottlerin, and that pharmacological or knockdown-mediated inhibition of Cdk5 suppresses both the dilation formation and nuclear elongation. We also show that Cdk5 inhibition perturbs endocytic trafficking as well as microtubule organization, both of which have been shown to be required for dilation formation. Furthermore, knockdown of Dcx, a Cdk5 substrate involved in microtubule organization and membrane trafficking, or p27(kip1), another Cdk5 substrate involved in actin and microtubule organization, disturbs the dilation formation and nuclear elongation. These data suggest that Cdk5 and its substrates, Dcx and p27(kip1), characterize migrating neuron-specific features, cytoplasmic dilation formation and nuclear elongation in the mouse cerebral cortex, possibly through the regulation of microtubule organization and an endocytic pathway.
Asunto(s)
Movimiento Celular/fisiología , Núcleo Celular/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Animales , Citoplasma/metabolismo , Citoesqueleto/fisiología , Cartilla de ADN/genética , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Electroporación , Inmunohistoquímica , Ratones , Plásmidos/genéticaRESUMEN
Mammalian plexins constitute a family of transmembrane receptors for semaphorins and represent critical regulators of various processes during development of the nervous, cardiovascular, skeletal, and renal system. In vitro studies have shown that plexins exert their effects via an intracellular R-Ras/M-Ras GTPase-activating protein (GAP) domain or by activation of RhoA through interaction with Rho guanine nucleotide exchange factor proteins. However, which of these signaling pathways are relevant for plexin functions in vivo is largely unknown. Using an allelic series of transgenic mice, we show that the GAP domain of plexins constitutes their key signaling module during development. Mice in which endogenous Plexin-B2 or Plexin-D1 is replaced by transgenic versions harboring mutations in the GAP domain recapitulate the phenotypes of the respective null mutants in the developing nervous, vascular, and skeletal system. We further provide genetic evidence that, unexpectedly, the GAP domain-mediated developmental functions of plexins are not brought about via R-Ras and M-Ras inactivation. In contrast to the GAP domain mutants, Plexin-B2 transgenic mice defective in Rho guanine nucleotide exchange factor binding are viable and fertile but exhibit abnormal development of the liver vasculature. Our genetic analyses uncover the in vivo context-dependence and functional specificity of individual plexin-mediated signaling pathways during development.
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Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/genética , Animales , Ratones , Ratones TransgénicosRESUMEN
The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.
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Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Animales , Cerebelo/citología , Cerebelo/fisiopatología , Consenso , Humanos , Neurogénesis/fisiología , Neuronas/citología , Neuronas/fisiologíaRESUMEN
In the cerebellum, the bHLH transcription factors Ptf1a and Atoh1 are expressed in distinct neuroepithelial regions, the ventricular zone (VZ) and the rhombic lip (RL), and are required for producing GABAergic and glutamatergic neurons, respectively. However, it is unclear whether Ptf1a or Atoh1 is sufficient for specifying GABAergic or glutamatergic neuronal fates. To test this, we generated two novel knock-in mouse lines, Ptf1a(Atoh1) and Atoh1(Ptf1a), that are designed to express Atoh1 and Ptf1a ectopically in the VZ and RL, respectively. In Ptf1a(Atoh1) embryos, ectopically Atoh1-expressing VZ cells produced glutamatergic neurons, including granule cells and deep cerebellar nuclei neurons. Correspondingly, in Atoh1(Ptf1a) animals, ectopically Ptf1a-expressing RL cells produced GABAergic populations, such as Purkinje cells and GABAergic interneurons. Consistent results were also obtained from in utero electroporation of Ptf1a or Atoh1 into embryonic cerebella, suggesting that Ptf1a and Atoh1 are essential and sufficient for GABAergic versus glutamatergic specification in the neuroepithelium. Furthermore, birthdating analyses with BrdU in the knock-in mice or with electroporation studies showed that ectopically produced fate-changed neuronal types were generated at temporal schedules closely simulating those of the wild-type RL and VZ, suggesting that the VZ and RL share common temporal information. Observations of knock-in brains as well as electroporated brains revealed that Ptf1a and Atoh1 mutually negatively regulate their expression, probably contributing to formation of non-overlapping neuroepithelial domains. These findings suggest that Ptf1a and Atoh1 specify spatial identities of cerebellar neuron progenitors in the neuroepithelium, leading to appropriate production of GABAergic and glutamatergic neurons, respectively.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cerebelo/citología , Ácido Glutámico/metabolismo , Células-Madre Neurales/fisiología , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Cerebelo/embriología , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Antígeno Ki-67/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Factores de Transcripción/genéticaRESUMEN
Atoh1 function is required for the earliest stages of inner ear hair cell development, which begins during the second week of gestation. Atoh1 expression in developing hair cells continues until early postnatal ages, but the function of this late expression is unknown. To test the role of continued Atoh1 expression in hair cell maturation we conditionally deleted the gene in the inner ear at various embryonic and postnatal ages. In the organ of Corti, deletion of Atoh1 at E15.5 led to the death of all hair cells. In contrast, deletion at E16.5 caused death only in apical regions, but abnormalities of stereocilia formation were present throughout the cochlea. In the utricle, deletion at E14.5 or E16.5 did not cause cell death but led to decreased expression of myosin VIIa and failure of stereocilia formation. Furthermore, we show that maintained expression of Barhl1 and Gfi1, two transcription factors implicated in cochlear hair cell survival, depends upon continued Atoh1 expression. However, maintained expression of Pou4f3 and several hair cell-specific markers is independent of Atoh1 expression. These data reveal novel late roles for Atoh1 that are separable from its initial role in hair cell development.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Sáculo y Utrículo/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Biomarcadores/metabolismo , Muerte Celular , Supervivencia Celular , Cóclea/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Femenino , Eliminación de Gen , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Embarazo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Sáculo y Utrículo/embriología , Sáculo y Utrículo/metabolismo , Estereocilios/metabolismo , Tamoxifeno , Factor de Transcripción Brn-3C/genética , Factor de Transcripción Brn-3C/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The mammalian cerebral cortex can be tangentially subdivided into tens of functional areas with distinct cyto-architectures and neural circuitries; however, it remains elusive how these areal borders are genetically elaborated during development. Here we establish original bacterial artificial chromosome transgenic mouse lines that specifically recapitulate cadherin-6 (Cdh6) mRNA expression profiles in the layer IV of the somatosensory cortex and by detailing their cortical development, we show that a sharp Cdh6 gene expression boundary is formed at a mediolateral coordinate along the cortical layer IV as early as the postnatal day 5 (P5). By further applying mouse genetics that allows rigid cell fate tracing with CreERT2 expression, it is demonstrated that the Cdh6 gene expression boundary set at around P4 eventually demarcates the areal border between the somatosensory barrel and limb field at P20. In the P6 cortical cell pellet culture system, neurons with Cdh6 expression preferentially form aggregates in a manner dependent on Ca(2+) and electroporation-based Cdh6 overexpression limited to the postnatal stages perturbs area-specific cell organization in the barrel field. These results suggest that Cdh6 expression in the nascent cortical plate may serve solidification of the protomap for cortical functional areas.
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Cadherinas/metabolismo , Corteza Somatosensorial/crecimiento & desarrollo , Animales , Cadherinas/genética , Expresión Génica , Ratones , Ratones Transgénicos , Corteza Somatosensorial/metabolismoRESUMEN
Alterations in the white matter have been implicated in schizophrenia. Myelin basic protein (MBP), a component of the myelin sheath, in the cerebrospinal fluid (CSF) has been suggested as a biomarker for white matter damage in demyelinating diseases. This prompted us to examine the CSF-MBP levels in patients with schizophrenia. We analyzed the CSF-MBP levels in 152 patients with schizophrenia and 117 age- and sex-matched controls. A significant positive correlation between age and CSF-MBP levels was observed both in the patients (p < 0.001) and controls (p = 0.014). No significant difference was observed in the CSF-MBP levels between the two groups. However, among a subsample of the patients (N = 32), a significantly negative correlation was observed between CSF-MBP and age-adjusted motor speed score of the brief assessment of cognition in schizophrenia (ρ = -0.59, p < 0.001). Further, among patients who underwent diffusional magnetic resonance imaging of the brain (N = 27), the CSF-MBP levels showed a significantly negative correlation with the mean kurtosis value in the right temporo-parietal region (p < 0.001). Our results suggest that the CSF-MBP level has limited utility as a diagnostic marker; however, higher CSF-MBP levels are associated with poorer motor speed, which may be associated with regional white matter damage in the brain in patients with schizophrenia.
RESUMEN
Schizophrenia is a syndrome with multiple etiologies, one of which is the potential for an autoimmune disease of the brain such as N-methyl-d-aspartate receptor (NMDAR) encephalitis, which can induce psychosis resembling schizophrenia. Here, we examined anti-neuronal autoantibodies related to psychosis using both cell- (CBA) and tissue-based assays (TBA) in the cerebrospinal fluid (CSF) of patients with chronic schizophrenia and control participants. First, we screened for the antibodies against leucine-rich glioma-inactivated 1 (LGI1), γ-aminobutyric acid B receptor (GABABR), dipeptidyl aminopeptidase-like protein 6 (DPPX), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR1/R2), and contactin-associated protein-like 2 (CASPR2) in 148 patients with schizophrenia. No antibodies were detected. Next, we performed CBA for NMDAR antibodies in 148 patients with schizophrenia and 151 age- and sex-matched controls. Although we detected relatively weak immunoreactivity for NMDAR in the CSFs of two patients with schizophrenia and three controls, no samples were positive when strict criteria were applied. For TBA in the rat hippocampus and cerebellum, we detected positive signals in the CSFs of 13 patients with schizophrenia and eight controls. Positive samples were analyzed for paraneoplastic syndrome and antinuclear antibodies using immunoblotting. The CSFs of nine patients and six controls were positive for dense fine speckle 70 (DFS70) antibodies. Additionally, antibodies against centromere protein (CENP)-A and CENP-B were detected in patients with schizophrenia. Our results suggest that autoantibodies against NMDAR, LG1, GABABR, DPPX, AMPAR1/R2, and CASPR2 are not associated with the pathogenesis of chronic schizophrenia. Moreover, we emphasize the importance of considering the effect of anti-DFS70 antibodies when analyzing autoantibodies in CSF samples. Conclusively, we obtained no evidence suggesting that the most frequent neuronal autoantibodies in the CSF play a role in the pathogenesis of schizophrenia, even in our sample.