RESUMO
KIF2A is a kinesin motor protein with essential roles in neural progenitor division and axonal pruning during brain development. However, how different KIF2A alternative isoforms function during development of the cerebral cortex is not known. Here, we focus on three Kif2a isoforms expressed in the developing cortex. We show that Kif2a is essential for dendritic arborization in mice and that the functions of all three isoforms are sufficient for this process. Interestingly, only two of the isoforms can sustain radial migration of cortical neurons; a third isoform, lacking a key N-terminal region, is ineffective. By proximity-based interactome mapping for individual isoforms, we identify previously known KIF2A interactors, proteins localized to the mitotic spindle poles and, unexpectedly, also translation factors, ribonucleoproteins and proteins that are targeted to organelles, prominently to the mitochondria. In addition, we show that a KIF2A mutation, which causes brain malformations in humans, has extensive changes to its proximity-based interactome, with depletion of mitochondrial proteins identified in the wild-type KIF2A interactome. Our data raises new insights about the importance of alternative splice variants during brain development.
Assuntos
Diferenciação Celular/genética , Movimento Celular/genética , Regulação da Expressão Gênica , Cinesinas/genética , Neurônios/citologia , Neurônios/metabolismo , Proteínas Repressoras/genética , Processamento Alternativo , Animais , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Perfilação da Expressão Gênica , Cinesinas/metabolismo , Camundongos , Mutação , Neurogênese/genética , Proteômica/métodos , Isoformas de RNA , Proteínas Repressoras/metabolismoRESUMO
BACKGROUND: Cellular differentiation programs are controlled, to a large extent, by the combinatorial functioning of specific transcription factors. Cortical projection neurons constitute the major excitatory neuron population within the cortex and mediate long distance communication between the cortex and other brain regions. Our understanding of effector transcription factors and their downstream transcriptional programs that direct the differentiation process of cortical projection neurons is far from complete. RESULTS: In this study, we carried out a ChIP-Seq (chromatin-immunoprecipitation and sequencing) analysis of NEUROD2, an effector transcription factor expressed in lineages of cortical projection neurons during the peak of cortical excitatory neurogenesis. Our results suggest that during cortical development NEUROD2 targets key genes that are required for Reelin signaling, a major pathway that regulates the migration of neurons from germinal zones to their final layers of residence within the cortex. We also find that NEUROD2 binds to a large set of genes with functions in layer-specific differentiation and in axonal pathfinding of cortical projection neurons. CONCLUSIONS: Our analysis of in vivo NEUROD2 target genes offers mechanistic insight into signaling pathways that regulate neuronal migration and axon guidance and identifies genes that are likely to be required for proper cortical development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Movimento Celular/genética , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Estudo de Associação Genômica Ampla , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Linhagem da Célula/genética , Córtex Cerebral/embriologia , Imunoprecipitação da Cromatina , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Modelos Biológicos , Neuropeptídeos/metabolismo , Ligação Proteica , Proteína ReelinaRESUMO
Astrocytes, often considered as secondary responders to neurodegeneration, are emerging as primary drivers of brain disease. Here we show that mitochondrial DNA depletion in astrocytes affects their primary cilium, the signaling organelle of a cell. The progressive oxidative phosphorylation deficiency in astrocytes induces FOXJ1 and RFX transcription factors, known as master regulators of motile ciliogenesis. Consequently, a robust gene expression program involving motile cilia components and multiciliated cell differentiation factors are induced. While the affected astrocytes still retain a single cilium, these organelles elongate and become remarkably distorted. The data suggest that chronic activation of the mitochondrial integrated stress response (ISRmt) in astrocytes drives anabolic metabolism and promotes ciliary elongation. Collectively, our evidence indicates that an active signaling axis involving mitochondria and primary cilia exists and that ciliary signaling is part of ISRmt in astrocytes. We propose that metabolic ciliopathy is a novel pathomechanism for mitochondria-related neurodegenerative diseases.
Assuntos
Astrócitos , Cílios , Mitocôndrias , Astrócitos/metabolismo , Cílios/metabolismo , Cílios/patologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Homeostase , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Camundongos , Animais , Fatores de Transcrição de Fator Regulador X/genética , Fatores de Transcrição de Fator Regulador X/metabolismo , DNA MitocondrialRESUMO
During cortical development, both activity-dependent and genetically determined mechanisms are required to establish proper neuronal connectivity. While activity-dependent transcription may link the two processes, specific transcription factors that mediate such a process have not been identified. We identified the basic helix-loop-helix (bHLH) transcription factor Neurogenic Differentiation 2 (NeuroD2) in a screen for calcium-regulated transcription factors and report that it is required for the proper development of thalamocortical connections. In neuroD2 null mice, thalamocortical axon terminals fail to segregate in the somatosensory cortex, and the postsynaptic barrel organization is disrupted. Additionally, synaptic transmission is defective at thalamocortical synapses in neuroD2 null mice. Total excitatory synaptic currents are reduced in layer IV in the knockouts, and the relative contribution of AMPA and NMDA receptor-mediated currents to evoked responses is decreased. These observations indicate that NeuroD2 plays a critical role in regulating synaptic maturation and the patterning of thalamocortical connections.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Vias Neurais/crescimento & desenvolvimento , Neuropeptídeos/fisiologia , Córtex Somatossensorial/crescimento & desenvolvimento , Sinapses/fisiologia , Tálamo/crescimento & desenvolvimento , 2-Amino-5-fosfonovalerato/farmacologia , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Western Blotting/métodos , Proteína de Ligação a CREB/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Quelantes/farmacologia , Cloranfenicol O-Acetiltransferase/metabolismo , Interações Medicamentosas , Ácido Egtázico/farmacologia , Estimulação Elétrica/métodos , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica/métodos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Knockout , Modelos Biológicos , Fatores de Crescimento Neural/metabolismo , Neurônios/efeitos dos fármacos , Neuropeptídeos/deficiência , Nimodipina/farmacologia , Técnicas de Patch-Clamp/métodos , Fosfopiruvato Hidratase/metabolismo , Cloreto de Potássio/farmacologia , Piridazinas/farmacologia , Compostos de Piridínio/metabolismo , Quinoxalinas/farmacologia , Receptores de AMPA/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Córtex Somatossensorial/citologia , Ativação Transcricional/genética , Transfecção/métodos , Vibrissas/crescimento & desenvolvimento , Vibrissas/inervaçãoRESUMO
High-throughput technologies for genomics, transcriptomics, proteomics, and metabolomics, and integrative analysis of these data, enable new, systems-level insights into disease pathogenesis. Mitochondrial diseases are an excellent target for hypothesis-generating omics approaches, as the disease group is mechanistically exceptionally complex. Although the genetic background in mitochondrial diseases is in either the nuclear or the mitochondrial genome, the typical downstream effect is dysfunction of the mitochondrial respiratory chain. However, the clinical manifestations show unprecedented variability, including either systemic or tissue-specific effects across multiple organ systems, with mild to severe symptoms, and occurring at any age. So far, the omics approaches have provided mechanistic understanding of tissue-specificity and potential treatment options for mitochondrial diseases, such as metabolome remodeling. However, no curative treatments exist, suggesting that novel approaches are needed. In this Review, we discuss omics approaches and discoveries with the potential to elucidate mechanisms of and therapies for mitochondrial diseases.
Assuntos
Metabolômica , Doenças Mitocondriais/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Doenças Mitocondriais/metabolismo , Especificidade de Órgãos , Locos de Características QuantitativasRESUMO
Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a group of severe, tissue-specific diseases of childhood with unknown pathogenesis. Brain-specific MDS manifests as devastating spongiotic encephalopathy with no curative therapy. Here, we report cell type-specific stress responses and effects of rapamycin treatment and ketogenic diet (KD) in mice with spongiotic encephalopathy mimicking human MDS, as these interventions were reported to improve some mitochondrial disease signs or symptoms. These mice with astrocyte-specific knockout of Twnk gene encoding replicative mtDNA helicase Twinkle (TwKOastro) show wide-spread cell-autonomous astrocyte activation and mitochondrial integrated stress response (ISRmt) induction with major metabolic remodeling of the brain. Mice with neuronal-specific TwKO show no ISRmt Both KD and rapamycin lead to rapid deterioration and weight loss of TwKOastro and premature trial termination. Although rapamycin had no robust effects on TwKOastro brain pathology, KD exacerbated spongiosis, gliosis, and ISRmt Our evidence emphasizes that mitochondrial disease treatments and stress responses are tissue- and disease specific. Furthermore, rapamycin and KD are deleterious in MDS-linked spongiotic encephalopathy, pointing to a crucial role of diet and metabolism for mitochondrial disease progression.
Assuntos
Encefalopatias/fisiopatologia , Doenças Mitocondriais/fisiopatologia , Estresse Fisiológico/fisiologia , Animais , Astrócitos/metabolismo , Encefalopatias/metabolismo , DNA Helicases/genética , Replicação do DNA/genética , DNA Mitocondrial/genética , Dieta Cetogênica/métodos , Modelos Animais de Doenças , Feminino , Cetose/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação , Neurônios/metabolismo , Sirolimo/farmacologiaRESUMO
Excitatory neurons of the mammalian cerebral cortex are organized into six functional layers characterized by unique patterns of connectivity, as well as distinctive physiological and morphological properties. Cortical layers appear after a highly regulated migration process in which cells move from the deeper, proliferative zone toward the superficial layers. Importantly, defects in this radial migration process have been implicated in neurodevelopmental and psychiatric diseases. Here we report that during the final stages of migration, transcription factor Neurogenic Differentiation 2 (Neurod2) contributes to terminal cellular localization within the cortical plate. In mice, in utero knockdown of Neurod2 resulted in reduced numbers of neurons localized to the uppermost region of the developing cortex, also termed the primitive cortical zone. Our ChIP-Seq and RNA-Seq analyses of genes regulated by NEUROD2 in the developing cortex identified a number of key target genes with known roles in Reelin signaling, a critical regulator of neuronal migration. Our focused analysis of regulation of the Reln gene, encoding the extracellular ligand REELIN, uncovered NEUROD2 binding to conserved E-box elements in multiple introns. Furthermore, we demonstrate that knockdown of NEUROD2 in primary cortical neurons resulted in a strong increase in Reln gene expression at the mRNA level, as well as a slight upregulation at the protein level. These data reveal a new role for NEUROD2 during the late stages of neuronal migration, and our analysis of its genomic targets offers new genes with potential roles in cortical lamination.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/metabolismo , Serina Endopeptidases/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação/genética , Diferenciação Celular , Movimento Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Dendritos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA-Seq , Proteína ReelinaRESUMO
Calcium signaling controls many key processes in neurons, including gene expression, axon guidance, and synaptic plasticity. In contrast to calcium influx through voltage- or neurotransmitter-gated channels, regulatory pathways that control store-operated calcium entry (SOCE) in neurons are poorly understood. Here, we report a transcriptional control of Stim1 (stromal interaction molecule 1) gene, which is a major sensor of endoplasmic reticulum (ER) calcium levels and a regulator of SOCE. By using a genome-wide chromatin immunoprecipitation and sequencing approach in mice, we find that NEUROD2, a neurogenic transcription factor, binds to an intronic element within the Stim1 gene. We show that NEUROD2 limits Stim1 expression in cortical neurons and consequently fine-tunes the SOCE response upon depletion of ER calcium. Our findings reveal a novel mechanism that regulates neuronal calcium homeostasis during cortical development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Sequência Conservada , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Íntrons , Camundongos Endogâmicos BALB C , Proteínas Nucleares , Ligação Proteica , Fatores de TranscriçãoRESUMO
The paraneoplastic neurologic disorders target several families of neuron-specific RNA binding proteins (RNABPs), revealing that there are unique aspects of gene expression regulation in the mammalian brain. Here, we used HITS-CLIP to determine robust binding sites targeted by the neuronal Elav-like (nElavl) RNABPs. Surprisingly, nElav protein binds preferentially to GU-rich sequences in vivo and in vitro, with secondary binding to AU-rich sequences. nElavl null mice were used to validate the consequence of these binding events in the brain, demonstrating that they bind intronic sequences in a position dependent manner to regulate alternative splicing and to 3'UTR sequences to regulate mRNA levels. These controls converge on the glutamate synthesis pathway in neurons; nElavl proteins are required to maintain neurotransmitter glutamate levels, and the lack of nElavl leads to spontaneous epileptic seizure activity. The genome-wide analysis of nElavl targets reveals that one function of neuron-specific RNABPs is to control excitation-inhibition balance in the brain.
Assuntos
Encéfalo/citologia , Proteínas ELAV/metabolismo , Regulação da Expressão Gênica/fisiologia , Ácido Glutâmico/metabolismo , Neurônios/fisiologia , Splicing de RNA/genética , Regiões 3' não Traduzidas/fisiologia , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Biologia Computacional , Modelos Animais de Doenças , Proteínas ELAV/deficiência , Eletroencefalografia , Epilepsia/genética , Epilepsia/fisiopatologia , Regulação da Expressão Gênica/genética , Glutaminase/genética , Glutaminase/metabolismo , Camundongos , Camundongos Knockout , Análise em Microsséries , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The establishment of functional neural connections requires the growth of axons to specific target areas and the formation of synapses with appropriate synaptic partners. Several molecules that regulate axon guidance and synapse formation have been identified in the past decade, but it is unclear how a relatively limited number of factors can specify a large number of connections. Recent evidence indicates that transcription factors make a crucial contribution to the specification of connections in the nervous system by coordinating the response of neurons to guidance molecules and neurotransmitters.