RESUMO
BACKGROUND: Kinase D-interacting substrate of 220 kDa (KIDINS220) is a multifunctional scaffolding protein essential for neuronal development. It has been implicated in neurological diseases with either autosomal dominant (AD) or autosomal recessive (AR) inheritance patterns. The molecular mechanisms underlying the AR/AD dual nature of KIDINS220 remain elusive, posing challenges to genetic interpretation and clinical interventions. Moreover, increased KIDINS220 exhibited neurotoxicity, but its role in neurodevelopment remains unclear. OBJECTIVE: The aim was to investigate the genotype-phenotype correlations of KIDINS220 and elucidate its pathophysiological role in neuronal development. METHODS: Whole-exome sequencing was performed in a four-generation family with cerebral palsy. CRISPR/Cas9 was used to generate KIDINS220 mutant cell lines. In utero electroporation was employed to investigate the effect of KIDINS220 variants on neurogenesis in vivo. RESULTS: We identified in KIDINS220 a pathogenic nonsense variant (c.4177C > T, p.Q1393*) that associated with AD cerebral palsy. We demonstrated that the nonsense variants located in the terminal exon of KIDINS220 are gain-of-function (GoF) variants, which enable the mRNA to escape nonsense-mediated decay and produce a truncated yet functional KIDINS220 protein. The truncated protein exhibited significant resistance to calpain and consequently accumulated within cells, resulting in the hyperactivation of Rac1 and defects in neuronal development. CONCLUSIONS: Our findings demonstrate that the location of variants within KIDINS220 plays a crucial role in determining inheritance patterns and corresponding clinical outcomes. The proposed interaction between Rac1 and KIDINS220 provides new insights into the pathogenesis of cerebral palsy, implying potential therapeutic perspectives. © 2023 International Parkinson and Movement Disorder Society.
Assuntos
Paralisia Cerebral , Neurônios , Humanos , Neurônios/metabolismo , Transdução de Sinais , Paralisia Cerebral/genética , Mutação com Ganho de Função , Neurogênese/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genéticaRESUMO
BACKGROUND: Accumulated evidence suggests that brain regions that promote wakefulness also facilitate emergence from general anesthesia (GA). Glutamatergic neurons in the substantia innominata (SI) regulate motivation-related aversive, depressive, and aggressive behaviors relying on heightened arousal. Here, we hypothesize that glutamatergic neurons in the SI are also involved in the regulation of the effects of sevoflurane anesthesia. METHODS: With a combination of fiber photometry, chemogenetic and optogenetic tools, behavioral tests, and cortical electroencephalogram recordings, we investigated whether and how SI glutamatergic neurons and their projections to the lateral hypothalamus (LH) regulate sevoflurane anesthesia in adult male mice. RESULTS: Population activity of glutamatergic neurons in the SI gradually decreased upon sevoflurane-induced loss of consciousness (LOC) and slowly returned as soon as inhalation of sevoflurane discontinued before recovery of consciousness (ROC). Chemogenetic activation of SI glutamatergic neurons dampened the animals' sensitivity to sevoflurane exposure, prolonged induction time (mean ± standard deviation [SD]; 389 ± 67 seconds vs 458 ± 53 seconds; P = .047), and shortened emergence time (305 seconds, 95% confidence interval [CI], 242-369 seconds vs 207 seconds, 95% CI, 135-279 seconds; P = .004), whereas chemogenetic inhibition of these neurons facilitated sevoflurane anesthesia. Furthermore, optogenetic activation of SI glutamatergic neurons and their terminals in LH induced cortical activation and behavioral emergence from different depths of sevoflurane anesthesia. CONCLUSIONS: Our study shows that SI glutamatergic neuronal activity facilitates emergence from sevoflurane anesthesia and provides evidence for the involvement of the SI-LH glutamatergic pathway in the regulation of consciousness during GA.
RESUMO
BACKGROUND: Exposure to general anesthesia influences neuronal functions during brain development. Recently, interneurons were found to be involved in developmental neurotoxicity by anesthetic exposure. But the underlying mechanism and long-term consequences remain elusive. METHODS: Pregnant mice received 2.5% sevoflurane for 6-h on gestational day 14.5. Pentylenetetrazole (PTZ)-induced seizure, anxiety- and depression-like behavior tests were performed in 30- and 60-day-old male offspring. Cortical interneurons were labeled using Rosa26-EYFP/-; Nkx2.1-Cre mice. Immunofluorescence and electrophysiology were performed to determine the cortical interneuron properties. Q-PCR and in situ hybridization (ISH) were performed for the potential mechanism, and the finding was further validated by in utero electroporation (IUE). RESULTS: In this study, we found that maternal sevoflurane exposure increased epilepsy susceptibility by using pentylenetetrazole (PTZ) induced-kindling models and enhanced anxiety- and depression-like behaviors in adolescent offspring. After sevoflurane exposure, the highly ordered cortical interneuron migration was disrupted in the fetal cortex. In addition, the resting membrane potentials of fast-spiking interneurons in the sevoflurane-treated group were more hyperpolarized in adolescence accompanied by an increase in inhibitory synapses. Both q-PCR and ISH indicated that CXCL12/CXCR4 signaling pathway downregulation might be a potential mechanism under sevoflurane developmental neurotoxicity which was further confirmed by IUE and behavioral tests. Although the above effects were obvious in adolescence, they did not persist into adulthood. CONCLUSIONS: Our findings demonstrate that maternal anesthesia impairs interneuron migration through the CXCL12/CXCR4 signaling pathway, and influences the interneuron properties, leading to the increased epilepsy susceptibility in adolescent offspring. Our study provides a novel perspective on the developmental neurotoxicity of the mechanistic link between maternal use of general anesthesia and increased susceptibility to epilepsy.
Assuntos
Epilepsia , Pentilenotetrazol , Humanos , Gravidez , Feminino , Camundongos , Animais , Masculino , Sevoflurano/metabolismo , Sevoflurano/farmacologia , Pentilenotetrazol/toxicidade , Pentilenotetrazol/metabolismo , Exposição Materna/efeitos adversos , Interneurônios/metabolismo , Epilepsia/induzido quimicamenteRESUMO
The establishment of neural circuits depends on precise neuronal positioning in the cortex, which occurs via a tightly coordinated process of neuronal differentiation, migration, and terminal localization. Deficits in this process have been implicated in several psychiatric disorders. Here, we show that the transcription factor Tcf4 controls neuronal positioning during brain development. Tcf4-deficient neurons become mispositioned in clusters when their migration to the cortical plate is complete. We reveal that Tcf4 regulates the expression of cell adhesion molecules to control neuronal positioning. Furthermore, through in vivo extracellular electrophysiology, we show that neuronal functions are disrupted after the loss of Tcf4. TCF4 mutations are strongly associated with schizophrenia and cause Pitt-Hopkins syndrome, which is characterized by severe intellectual disability. Thus, our results not only reveal the importance of neuronal positioning in brain development but also provide new insights into the potential mechanisms underlying neurological defects linked to TCF4 mutations.
Assuntos
Hiperventilação , Deficiência Intelectual , Adesão Celular , Fácies , Humanos , Hiperventilação/genética , Hiperventilação/metabolismo , Deficiência Intelectual/genética , Neurônios/metabolismo , Fator de Transcrição 4/genética , Fator de Transcrição 4/metabolismoRESUMO
The dentate gyrus (DG) of the hippocampal formation plays essential roles in learning and memory. Defective DG development is associated with neurological disorders. Here, we show that transcription factor 4 (Tcf4) is essential for DG development. Tcf4 expression is elevated in neural progenitors of the dentate neuroepithelium in the developing mouse brain. We demonstrate that conditional disruption of Tcf4 in the dentate neuroepithelium leads to abnormal neural progenitor migration guided by disorganized radial glial fibers, which further leads to hypoplasia in the DG. Moreover, we reveal that Wnt7b is a key downstream effector of Tcf4 in regulating neural progenitor migration. Behavioral analysis shows that disruption of integrity of the DG impairs the social memory highlighting the importance of proper development of the DG. These results reveal a critical role for Tcf4 in regulating DG development. As mutations in TCF4 cause Pitt-Hopkins syndrome (PTHS) characterized by severe intellectual disability, our data also potentially provide insights into the basis of neurological defects linked to TCF4 mutations.
Assuntos
Movimento Celular/fisiologia , Giro Denteado/crescimento & desenvolvimento , Giro Denteado/metabolismo , Células-Tronco Neurais/metabolismo , Fator de Transcrição 4/biossíntese , Animais , Giro Denteado/embriologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Fator de Transcrição 4/genéticaRESUMO
The Nedd4 family E3 ligases are key regulators of cell growth and proliferation and are often misregulated in human cancers and other diseases. The ligase activities of Nedd4 E3s are tightly controlled via auto-inhibition. However, the molecular mechanism underlying Nedd4 E3 auto-inhibition and activation is poorly understood. Here, we show that the WW domains proceeding the catalytic HECT domain play an inhibitory role by binding directly to HECT in the Nedd4 E3 family member Itch. Our structural and biochemical analyses of Itch reveal that the WW2 domain and a following linker allosterically lock HECT in an inactive state inhibiting E2-E3 transthiolation. Binding of the Ndfip1 adaptor or JNK1-mediated phosphorylation relieves the auto-inhibition of Itch in a WW2-dependent manner. Aberrant activation of Itch leads to migration defects of cortical neurons during development. Our study provides a new mechanism governing the regulation of Itch.
Assuntos
Ubiquitina-Proteína Ligases Nedd4/química , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Regulação Alostérica , Animais , Cristalografia por Raios X , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Camundongos , Ubiquitina-Proteína Ligases Nedd4/genética , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteólise , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Domínios WWRESUMO
Polycomb group (PcG) proteins form transcriptional repressor complexes with well-established functions during cell-fate determination. Yet, the mechanisms underlying their regulation remain poorly understood. Here, we extend the role of Polycomb complexes in the temporal control of neural progenitor cell (NPC) commitment by demonstrating that the PcG protein Ezh2 is necessary to prevent the premature onset of gliogenesis. In addition, we identify the chromodomain helicase DNA-binding protein 4 (Chd4) as a critical interaction partner of Ezh2 required specifically for PcG-mediated suppression of the key astrogenic marker gene GFAP. Accordingly, in vivo depletion of Chd4 in the developing neocortex promotes astrogenesis. Collectively, these results demonstrate that PcG proteins operate in a highly dynamic, developmental stage-dependent fashion during neural differentiation and suggest that target gene-specific mechanisms regulate Polycomb function during sequential cell-fate decisions.
Assuntos
Astrócitos/citologia , Diferenciação Celular , DNA Helicases/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Animais , Astrócitos/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , DNA Helicases/genética , Embrião de Mamíferos , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteína Glial Fibrilar Ácida , Histonas/química , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 2/genética , Proteínas do Grupo Polycomb/genética , Gravidez , Regiões Promotoras GenéticasRESUMO
The histone deacetylases HDAC1 and HDAC2 are crucial regulators of chromatin structure and gene expression, thereby controlling important developmental processes. In the mouse brain, HDAC1 and HDAC2 exhibit different developmental stage- and lineage-specific expression patterns. To examine the individual contribution of these deacetylases during brain development, we deleted different combinations of Hdac1 and Hdac2 alleles in neural cells. Ablation of Hdac1 or Hdac2 by Nestin-Cre had no obvious consequences on brain development and architecture owing to compensation by the paralog. By contrast, combined deletion of Hdac1 and Hdac2 resulted in impaired chromatin structure, DNA damage, apoptosis and embryonic lethality. To dissect the individual roles of HDAC1 and HDAC2, we expressed single alleles of either Hdac1 or Hdac2 in the absence of the respective paralog in neural cells. The DNA-damage phenotype observed in double knockout brains was prevented by expression of a single allele of either Hdac1 or Hdac2. Strikingly, Hdac1(-/-)Hdac2(+/-) brains showed normal development and no obvious phenotype, whereas Hdac1(+/-)Hdac2(-/-) mice displayed impaired brain development and perinatal lethality. Hdac1(+/-)Hdac2(-/-) neural precursor cells showed reduced proliferation and premature differentiation mediated by overexpression of protein kinase C, delta, which is a direct target of HDAC2. Importantly, chemical inhibition or knockdown of protein kinase C delta was sufficient to rescue the phenotype of neural progenitor cells in vitro. Our data indicate that HDAC1 and HDAC2 have a common function in maintaining proper chromatin structures and show that HDAC2 has a unique role by controlling the fate of neural progenitors during normal brain development.
Assuntos
Alelos , Encéfalo/embriologia , Encéfalo/enzimologia , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Homologia de Sequência de Aminoácidos , Acetofenonas/farmacologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzopiranos/farmacologia , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas Correpressoras/metabolismo , Dano ao DNA/genética , Perda do Embrião/enzimologia , Perda do Embrião/patologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/genética , Histona Desacetilase 2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The orientation of the mitotic spindle determines the relative size and position of the daughter cells and influences the asymmetric inheritance of localized cell fate determinants. The onset of mammalian neurogenesis, for example, coincides with changes in spindle orientation. To address the functional implications of this and related phenomena, precise methods for determining the orientation of the mitotic spindle in complex tissues are needed. Here, we present methodology for the analysis of spindle orientation in 3D. Our method allows statistical analysis and modeling of spindle orientation and involves two parameters for horizontal and vertical bias that can unambiguously describe the distribution of spindle orientations in an experimental sample. We find that 3D analysis leads to systematically different results from 2D analysis and, surprisingly, truly random spindle orientations do not result in equal numbers of horizontal and vertical orientations. We show that our method can describe the distribution of spindle orientation angles under different biological conditions. As an example of biological application we demonstrate that the adapter protein Inscuteable (mInsc) can actively promote vertical spindle orientation in apical progenitors during mouse neurogenesis.
Assuntos
Fuso Acromático , Algoritmos , Animais , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Divisão Celular , Linhagem da Célula , Polaridade Celular/genética , Simulação por Computador , Imageamento Tridimensional , Camundongos , Neurogênese/fisiologia , Neurônios/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Probabilidade , Processos EstocásticosRESUMO
The heavy metal cadmium (Cd) is known to modulate immunity and cause osteoporosis. However, how Cd influences on hematopoiesis remain largely unknown. Herein, we show that wild-type C57BL/6 (B6) mice exposed to Cd for 3months had expanded bone marrow (BM) populations of long-term hematopoietic stem cells (LT-HSCs), common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs), while having reduced populations of multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). A competitive mixed BM transplantation assay indicates that BM from Cd-treated mice had impaired LT-HSC ability to differentiate into mature cells. In accordance with increased myeloid progenitors and decreased lymphoid progenitors, the BM and spleens of Cd-treated mice had more monocytes and/or neutrophils and fewer B cells and T cells. Cd impaired the ability of the non-hematopoietic system to support LT-HSCs, in that lethally irradiated Cd-treated recipients transplanted with normal BM cells had reduced LT-HSCs after the hematopoietic system was fully reconstituted. This is consistent with reduced osteoblasts, a known critical component for HSC niche, observed in Cd-treated mice. Conversely, lethally irradiated control recipients transplanted with BM cells from Cd-treated mice had normal LT-HSC reconstitution. Furthermore, both control mice and Cd-treated mice that received Alendronate, a clinical drug used for treating osteoporosis, had BM increases of LT-HSCs. Thus, the results suggest Cd increase of LT-HSCs is due to effects on HSCs and not on osteoblasts, although, Cd causes osteoblast reduction and impaired niche function for maintaining HSCs. Furthermore, Cd skews HSCs toward myelopoiesis.
Assuntos
Cádmio/toxicidade , Células-Tronco Hematopoéticas/efeitos dos fármacos , Mielopoese/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Alendronato/farmacologia , Animais , Carga Corporal (Radioterapia) , Osso e Ossos/citologia , Osso e Ossos/efeitos dos fármacos , Cádmio/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/efeitos dos fármacosRESUMO
Polarity proteins regulate the proliferation and differentiation of neural progenitors to generate neurons during brain development through multiple signaling pathways. However, how cell polarity couples the signaling pathways remains unclear. Here, we show that coiled-coil domain-containing protein 85c (Ccdc85c) interacts with the polarity protein Par3 to regulate the proliferation of radial glial cells (RGCs) via phase separation coupled to percolation (PSCP). We find that the interaction with Ccdc85c relieves the intramolecular auto-inhibition of Par3, which leads to PSCP of Par3. Downregulation of Ccdc85c causes RGC differentiation. Importantly, the open conformation of Par3 facilitates the recruitment of the Notch regulator Numb to the Par3 condensates, which might prevent the attenuation of Notch activity to maintain RGC proliferation. Furthermore, ectopic activation of Notch signaling rescues RGC proliferation defects caused by the downregulation of Ccdc85c. These results suggest that Ccdc85c-mediated PSCP of Par3 regulates Notch signaling to control RGC proliferation during brain development.
Assuntos
Polaridade Celular , Transdução de Sinais , Polaridade Celular/fisiologia , Transdução de Sinais/fisiologia , Neurônios/metabolismo , Proliferação de Células , Receptores Notch/metabolismoRESUMO
Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of beta-actin mRNA and fewer dendritic beta-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.
Assuntos
Encéfalo/metabolismo , Dendritos , Neurônios/citologia , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Dendritos/metabolismo , Dendritos/ultraestrutura , Proteína 4 Homóloga a Disks-Large , Regulação para Baixo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Células HeLa , Hipocampo/citologia , Humanos , Hibridização In Situ , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp , Isoformas de Proteínas/genética , Interferência de RNA , Proteínas de Ligação a RNA/genética , Ratos , Sinapses/metabolismoRESUMO
A proper number of oligodendrocytes in the nerve system is essential for neuronal functions. In the olfactory bulb (OB), enriched oligodendrocytes are crucial for olfactory information processing. However, how the precise number of oligodendrocytes in the OB is regulated remains elusive. Here we identified that the transcription factor 4 (Tcf4)-mediated cell death is essential for generating an appropriate number of oligodendrocyte progenitor cells (OPCs) and thereby oligodendrocytes in the OB. We showed that Nkx2.1-positive progenitors in the medial ganglionic eminence (MGE) and anterior entopeduncular area (AEP) provide the first source of OPCs in the OB. Conditional depletion of Tcf4 leads to an increase of OPCs in the OB, which is mediated by the suppression of programmed cell death. Furthermore, we showed that Tcf4 mediated OPC survival is cell-autonomous by transplantation assay. Mechanistically, we identified Bax/Bak as a potential key pathway to promote OPC elimination during OB development. Depletion of Bax/Bak in Nkx2.1 lineage results in an increase of OPCs in the OB. Mutations in TCF4 causes Pitt-Hopkins syndrome, a severe neurodevelopmental disorder. Thus, our findings reveal an important intrinsic mechanism underlying the survival control of OPCs in the OB and provide new insights into the pathogenesis of Pitt-Hopkins syndrome.
Assuntos
Bulbo Olfatório/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Oligodendroglia/metabolismo , Fator de Transcrição 4/metabolismo , Animais , Sobrevivência Celular/fisiologia , Humanos , Camundongos , Camundongos Knockout , Neurogênese , Bulbo Olfatório/citologia , Bulbo Olfatório/crescimento & desenvolvimento , Células Precursoras de Oligodendrócitos/citologia , Oligodendroglia/citologiaRESUMO
OBJECTIVES: The effects of general anaesthetics on fetal brain development remain elusive. Radial glial progenitors (RGPs) generate the majority of neurons in developing brains. Here, we evaluated the acute alterations in RGPs after maternal sevoflurane exposure. METHODS: Pregnant mice were exposed to 2.5% sevoflurane for 6 hours on gestational day 14.5. Interkinetic nuclear migration (INM) of RGPs in the ventricular zone (VZ) of the fetal brain was evaluated by thymidine analogues labelling. Cell fate of RGP progeny was determined by immunostaining using various neural markers. The Morris water maze (MWM) was used to assess the neurocognitive behaviours of the offspring. RNA sequencing (RNA-Seq) was performed for the potential mechanism, and the potential mechanism validated by quantitative real-time PCR (qPCR), Western blot and rescue experiments. Furthermore, INM was examined in human embryonic stem cell (hESC)-derived 3D cerebral organoids. RESULTS: Maternal sevoflurane exposure induced temporary abnormities in INM, and disturbed the cell cycle progression of RGPs in both rodents and cerebral organoids without cell fate alternation. RNA-Seq analysis, qPCR and Western blot showed that the Notch signalling pathway was a potential downstream target. Reactivation of Notch by Jag1 and NICD overexpression rescued the defects in INM. Young adult offspring showed no obvious cognitive impairments in MWM. CONCLUSIONS: Maternal sevoflurane exposure during neurogenic period temporarily induced abnormal INM of RGPs by targeting the Notch signalling pathway without inducing long-term effects on RGP progeny cell fate or offspring cognitive behaviours. More importantly, the defects of INM in hESC-derived cerebral organoids provide a novel insight into the effects of general anaesthesia on human brain development.
Assuntos
Anestésicos Inalatórios/efeitos adversos , Córtex Cerebral/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Receptores Notch/metabolismo , Sevoflurano/efeitos adversos , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Córtex Cerebral/patologia , Feminino , Feto/efeitos dos fármacos , Feto/metabolismo , Feto/patologia , Humanos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/patologia , Neurogênese/efeitos dos fármacos , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Intellectual disability is closely related to impaired GABA neurotransmission. Brpf1 was specifically expressed in medial ganglionic eminence (MGE), a developmental niche of GABAergic interneurons, and patients with BRPF1 mutations showed intellectual disability. To test its role in the development and function of MGE-derived GABAergic interneurons, we performed immunofluorescence staining, whole-cell patch-clamp, MGE transplantation, and mRNA-Seq to understand its effect on neuronal differentiation, dendritic morphology, electrophysiology, migration, and gene regulation, using mouse MGE-derived GABAergic interneurons infected with AAV-shBrpf1. The results showed that Brpf1 knockdown had a decreasing trend, although not significant, on the differentiation of GABAergic interneurons into parvalbumin+ interneurons. Moreover, increased firing threshold, decreased number of evoked action potentials, and a reduced amplitude of miniature inhibitory postsynaptic currents were observed before any significant change of MAP2+ dendritic morphology and in vivo migration ability appeared. Finally, mRNA-Seq analysis revealed that genes related to neurodevelopment and synaptic transmission such as Map2k7 were dysregulated. Our results demonstrated a key role of Brpf1 in inhibitory neurotransmission and related gene expression of GABAergic interneurons.
Assuntos
Deficiência Intelectual , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação a DNA , Humanos , Deficiência Intelectual/genética , Interneurônios , Eminência Mediana , Camundongos , Transmissão SinápticaRESUMO
Septins, a highly conserved family of GTP-binding proteins, were originally identified in a genetic screen for S. cerevisiae mutants defective in cytokinesis [1, 2]. In yeast, septins maintain the compartmentalization of the yeast plasma membrane during cell division by forming rings at the cortex of the bud neck, and these rings establish a lateral diffusion barrier. In contrast, very little is known about the functions of septins in mammalian cells [3, 4] including postmitotic neurons [5-7]. Here, we show that Septin 7 (Sept7) localizes at the bases of filopodia and at branch points in developing hippocampal neurons. Upon downregulation of Sept7, dendritic branching is impaired. In mature neurons, Sept7 is found at the bases of dendritic spines where it associates with the plasma membrane. Mature Sept7-deficient neurons display elongated spines. Furthermore, Sept5 and Sept11 colocalize with and coimmunoprecipitate with Sept7, thereby arguing for the existence of a Septin5/7/11 complex. Taken together, our findings show an important role for Sept7 in regulating dendritic branching and dendritic-spine morphology. Our observations concur with data from yeast, in which downregulation of septins yields elongated buds, suggesting a conserved function for septins from yeast to mammals.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Dendritos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Dendritos/ultraestrutura , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , Hipocampo/citologia , Hipocampo/metabolismo , Complexos Multiproteicos , Mutação , Neurônios/metabolismo , Neurônios/ultraestrutura , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Septinas , Frações Subcelulares/metabolismo , TransfecçãoRESUMO
During neocortical development, there are two important events, including expansion of the neural progenitor pool through symmetric divisions, and generation of neurons via asymmetrical divisions that lead to a serial process of neuronal polarization, migration, and layer-type specific phenotype acquisition. The mechanisms underlying these processes remain poorly elucidated. Here, we show that the transcription factor Zeb1 regulates the orientation of the cleavage plane of dividing neural progenitors, neuronal polarity, and migration. Upon Zeb1 removal, the cleavage plane of mitotic neural progenitors fails to orientate vertically, resulting in random orientation and premature neuronal differentiation. Consequently, these extra number of precociously produced neurons migrate aberrantly to the upper layer. Mechanistically, we show that Zeb1 suppresses Pak3, a p21-activated serine/threonine protein kinase, through formation of a functional repressing complex together with methyltransferase PRMT5 and Pak3. Our results reveal that Zeb1 plays an essential role in neocortical development and may provide insights into the mechanisms responsible for cortical developmental diseases.
Assuntos
Células Ependimogliais/citologia , Neocórtex/embriologia , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Neurônios/citologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Quinases Ativadas por p21/metabolismoRESUMO
Neural progenitors with distinct potential to generate progeny are associated with a spatially distinct microenvironment. Neocortical intermediate progenitors (IPs) in the subventricular zone (SVZ) of the developing brain generate neurons for all cortical layers and are essential for cortical expansion. Here, we show that spatial control of IP positioning is essential for neocortical development. We demonstrate that HDAC1 and HDAC2 regulate the spatial positioning of IPs to form the SVZ. Developmental stage-specific depletion of both HDAC1 and HDAC2 in radial glial progenitors results in mispositioning of IPs at the ventricular surface, where they divide and differentiate into neurons, thereby leading to the cortical malformation. We further identified the proneural gene Neurogenin2 as a key target of HDAC1 and HDAC2 for regulating IP positioning. Our results demonstrate the importance of the spatial positioning of neural progenitors in cortical development and reveal a mechanism underlying the establishment of the SVZ microenvironment.
Assuntos
Células Ependimogliais/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Ventrículos Laterais/embriologia , Malformações do Desenvolvimento Cortical/genética , Neocórtex/embriologia , Células-Tronco Neurais/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Malformações do Desenvolvimento Cortical/embriologia , Camundongos , Proteínas do Tecido Nervoso/metabolismo , NeurogêneseRESUMO
Pumilio (Pum) protein acts as a translational inhibitor in several organisms including yeast, Drosophila, Xenopus, and mammals. Two Pumilio genes, Pum1 and Pum2, have been identified in mammals, but their function in neurons has not been identified. In this study, we found that Pum2 mRNA is expressed during neuronal development and that the protein is found in discrete particles in both the cell body and the dendritic compartment of fully polarized neurons. This finding indicates that Pum2 is a novel candidate of dendritically localized ribonucleoparticles (RNPs). During metabolic stress, Pum2 is present in stress granules (SGs), which are subsequently detected in the somatodendritic domain. It remains excluded from processing bodies under all conditions. When overexpressed in neurons and fibroblasts, Pum2 induces the formation of SGs that also contain T-cell intracellular antigen 1 (TIA-1)-related protein, eukaryotic initiation factor 4E, poly(A)-binding protein, TIA-1, and other RNA-binding proteins including Staufen1 and Barentsz. This induction of SGs is dependent on the RNA-binding domain and a glutamine-rich region in the N terminus of Pum2. This glutamine-rich region behaves in a similar manner as TIA-1 and prion protein, two molecules with known roles in protein aggregation. Pum2 downregulation in neurons via RNA interference (RNAi) interferes with the formation of SGs during metabolic stress. Cotransfection with an RNAi-resistant portion of the Pum2 mRNA restores SG formation. These results suggest a role for Pum2 in dendritic RNPs and SG formation in mammalian neurons.
Assuntos
Dendritos/metabolismo , Neurônios/citologia , Proteínas de Ligação a RNA/metabolismo , Animais , Northern Blotting/métodos , Western Blotting/métodos , Células Cultivadas , Chlorocebus aethiops , Dendritos/efeitos dos fármacos , Proteína 4 Homóloga a Disks-Large , Embrião de Mamíferos , Endorribonucleases/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Regulação da Expressão Gênica/fisiologia , Guanilato Quinases , Hipocampo/citologia , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Indóis , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Frações Subcelulares/metabolismo , Sinaptofisina/metabolismo , Transativadores/metabolismo , Transfecção/métodos , Tubulina (Proteína)/metabolismoRESUMO
Lead (Pb) is a toxic heavy metal affecting human health; it is known to be harmful to various organs or systems, yet the mechanisms by which Pb influences immune cell development remain to be defined. In this study, we show that Pb exposure (1250 ppm via drinking water) selectively impacted the development of myeloid cells (myelopoiesis). After Pb treatment of adult C57BL/6 mice, the numbers of granulocyte-macrophage progenitors (GMP) were consistently reduced, whereas the numbers of myeloid cells were increased at week (wk) 1 and decreased at wk8 after initiating the Pb exposure. Functional assays indicate that Pb accelerated GMP differentiation in a reactive oxygen species-dependent manner after treatment for 1 week and inhibited common myeloid progenitor differentiation by upregulating interferon regulatory factor 8 (IRF8) expression after treatment for 8 weeks. Consistent with the distinct Pb influences on myeloid cells observed at wk1 and wk8, Pb caused an inflammatory environment in vivo at wk8, but not at wk1. Furthermore, like the observations in mice during the Pb exposure, bloods from humans occupationally exposed to Pb had their numbers of monocytes, neutrophils and GMP negatively associated with the Pb concentration, whereas IRF8 expression in common myeloid progenitor, but not GMP, was positively correlated with the Pb concentration. These data suggest an occupationally relevant level of Pb exposure preferentially influences myelopoiesis involving reactive oxygen species and IRF8, which may contribute to the current understanding of the hematopoietic toxicology of Pb.