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1.
EMBO Rep ; 19(10)2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30177554

RESUMO

MacroH2A histone variants suppress tumor progression and act as epigenetic barriers to induced pluripotency. How they impart their influence on chromatin plasticity is not well understood. Here, we analyze how the different domains of macroH2A proteins contribute to chromatin structure and dynamics. By solving the crystal structure of the macrodomain of human macroH2A2 at 1.7 Å, we find that its putative binding pocket exhibits marked structural differences compared with the macroH2A1.1 isoform, rendering macroH2A2 unable to bind ADP-ribose. Quantitative binding assays show that this specificity is conserved among vertebrate macroH2A isoforms. We further find that macroH2A histones reduce the transient, PARP1-dependent chromatin relaxation that occurs in living cells upon DNA damage through two distinct mechanisms. First, macroH2A1.1 mediates an isoform-specific effect through its ability to suppress PARP1 activity. Second, the unstructured linker region exerts an additional repressive effect that is common to all macroH2A proteins. In the absence of DNA damage, the macroH2A linker is also sufficient for rescuing heterochromatin architecture in cells deficient for macroH2A.


Assuntos
Cromatina/genética , Epigênese Genética/genética , Histonas/química , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/genética , Cromatina/química , Cristalografia por Raios X , Dano ao DNA/genética , Heterocromatina/química , Heterocromatina/genética , Histonas/genética , Humanos , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/genética , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética
2.
Nature ; 503(7475): 267-71, 2013 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-24132240

RESUMO

Phelan-McDermid syndrome (PMDS) is a complex neurodevelopmental disorder characterized by global developmental delay, severely impaired speech, intellectual disability, and an increased risk of autism spectrum disorders (ASDs). PMDS is caused by heterozygous deletions of chromosome 22q13.3. Among the genes in the deleted region is SHANK3, which encodes a protein in the postsynaptic density (PSD). Rare mutations in SHANK3 have been associated with idiopathic ASDs, non-syndromic intellectual disability, and schizophrenia. Although SHANK3 is considered to be the most likely candidate gene for the neurological abnormalities in PMDS patients, the cellular and molecular phenotypes associated with this syndrome in human neurons are unknown. We generated induced pluripotent stem (iPS) cells from individuals with PMDS and autism and used them to produce functional neurons. We show that PMDS neurons have reduced SHANK3 expression and major defects in excitatory, but not inhibitory, synaptic transmission. Excitatory synaptic transmission in PMDS neurons can be corrected by restoring SHANK3 expression or by treating neurons with insulin-like growth factor 1 (IGF1). IGF1 treatment promotes formation of mature excitatory synapses that lack SHANK3 but contain PSD95 and N-methyl-D-aspartate (NMDA) receptors with fast deactivation kinetics. Our findings provide direct evidence for a disruption in the ratio of cellular excitation and inhibition in PMDS neurons, and point to a molecular pathway that can be recruited to restore it.


Assuntos
Transtornos Cromossômicos/fisiopatologia , Fator de Crescimento Insulin-Like I/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Linhagem Celular , Criança , Deleção Cromossômica , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 22/genética , Feminino , GABAérgicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lentivirus/genética , Masculino , Neurônios/citologia , Neurônios/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Receptores de Glutamato/genética , Deleção de Sequência , Sinapses/genética , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética
3.
Nature ; 476(7359): 228-31, 2011 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-21753754

RESUMO

Neurogenic transcription factors and evolutionarily conserved signalling pathways have been found to be instrumental in the formation of neurons. However, the instructive role of microRNAs (miRNAs) in neurogenesis remains unexplored. We recently discovered that miR-9* and miR-124 instruct compositional changes of SWI/SNF-like BAF chromatin-remodelling complexes, a process important for neuronal differentiation and function. Nearing mitotic exit of neural progenitors, miR-9* and miR-124 repress the BAF53a subunit of the neural-progenitor (np)BAF chromatin-remodelling complex. After mitotic exit, BAF53a is replaced by BAF53b, and BAF45a by BAF45b and BAF45c, which are then incorporated into neuron-specific (n)BAF complexes essential for post-mitotic functions. Because miR-9/9* and miR-124 also control multiple genes regulating neuronal differentiation and function, we proposed that these miRNAs might contribute to neuronal fates. Here we show that expression of miR-9/9* and miR-124 (miR-9/9*-124) in human fibroblasts induces their conversion into neurons, a process facilitated by NEUROD2. Further addition of neurogenic transcription factors ASCL1 and MYT1L enhances the rate of conversion and the maturation of the converted neurons, whereas expression of these transcription factors alone without miR-9/9*-124 was ineffective. These studies indicate that the genetic circuitry involving miR-9/9*-124 can have an instructive role in neural fate determination.


Assuntos
Diferenciação Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , MicroRNAs/genética , Neurônios/citologia , Neurônios/metabolismo , Adulto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Linhagem Celular , Linhagem da Célula/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Humanos , Recém-Nascido , MicroRNAs/metabolismo , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/análise , Tubulina (Proteína)/metabolismo
4.
Brain Stimul ; 15(3): 586-597, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35395424

RESUMO

BACKGROUND: Modulation of pathological neural circuit activity in the brain with a minimum of complications is an area of intense interest. OBJECTIVE: The goal of the study was to alter neurons' physiological states without apparent damage of cellular integrity using stereotactic radiosurgery (SRS). METHODS: We treated a 7.5 mm-diameter target on the visual cortex of Göttingen minipigs with doses of 40, 60, 80, and 100 Gy. Six months post-irradiation, the pigs were implanted with a 9 mm-wide, eight-shank multi-electrode probe, which spanned the radiation focus as well as the low-exposure neighboring areas. RESULTS: Doses of 40 Gy led to an increase of spontaneous firing rate, six months post-irradiation, while doses of 60 Gy and greater were associated with a decrease. Subjecting the animals to visual stimuli resulted in typical visual evoked potentials (VEP). At 40 Gy, a significant reduction of the P1 peak time, indicative of higher network excitability was observed. At 80 Gy, P1 peak time was not affected, while a minor reduction at 60 Gy was seen. No distance-dependent effects on spontaneous firing rate, or on VEP were observed. Post-mortem histology revealed no evidence of necrosis at doses below 60 Gy. In an in vitro assay comprising of iPS-derived human neuron-astrocyte co-cultures, we found a higher vulnerability of inhibitory neurons than excitatory neurons with respect to radiation, which might provide the cellular mechanism of the disinhibitory effect observed in vivo. CONCLUSION: We provide initial evidence for a rather circuit-wide, long-lasting disinhibitory effect of low sub-ablative doses of SRS.


Assuntos
Potenciais Evocados Visuais , Radiocirurgia , Animais , Encéfalo , Radiação Ionizante , Radiocirurgia/métodos , Suínos , Porco Miniatura
5.
Elife ; 82019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31868578

RESUMO

The syndromic autism spectrum disorder (ASD) Timothy syndrome (TS) is caused by a point mutation in the alternatively spliced exon 8A of the calcium channel Cav1.2. Using mouse brain and human induced pluripotent stem cells (iPSCs), we provide evidence that the TS mutation prevents a normal developmental switch in Cav1.2 exon utilization, resulting in persistent expression of gain-of-function mutant channels during neuronal differentiation. In iPSC models, the TS mutation reduces the abundance of SATB2-expressing cortical projection neurons, leading to excess CTIP2+ neurons. We show that expression of TS-Cav1.2 channels in the embryonic mouse cortex recapitulates these differentiation defects in a calcium-dependent manner and that in utero Cav1.2 gain-and-loss of function reciprocally regulates the abundance of these neuronal populations. Our findings support the idea that disruption of developmentally regulated calcium channel splicing patterns instructively alters differentiation in the developing cortex, providing important in vivo insights into the pathophysiology of a syndromic ASD.


Assuntos
Processamento Alternativo/fisiologia , Transtorno do Espectro Autista/metabolismo , Canais de Cálcio/metabolismo , Diferenciação Celular/fisiologia , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Transtorno Autístico , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Cálcio , Canais de Cálcio/genética , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do QT Longo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Modelos Animais , Mutação , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Splicing de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sindactilia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
6.
PLoS Biol ; 3(5): e159, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15836427

RESUMO

Two ETS transcription factors of the Pea3 subfamily are induced in subpopulations of dorsal root ganglion (DRG) sensory and spinal motor neurons by target-derived factors. Their expression controls late aspects of neuronal differentiation such as target invasion and branching. Here, we show that the late onset of ETS gene expression is an essential requirement for normal sensory neuron differentiation. We provide genetic evidence in the mouse that precocious ETS expression in DRG sensory neurons perturbs axonal projections, the acquisition of terminal differentiation markers, and their dependence on neurotrophic support. Together, our findings indicate that DRG sensory neurons exhibit a temporal developmental switch that can be revealed by distinct responses to ETS transcription factor signaling at sequential steps of neuronal maturation.


Assuntos
Gânglios Espinais/fisiologia , Neurônios/fisiologia , Proteínas Proto-Oncogênicas c-ets/fisiologia , Transdução de Sinais/fisiologia , Animais , Sequência de Bases , Diferenciação Celular , Primers do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Éxons , Gânglios Espinais/crescimento & desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/fisiologia , Músculo Esquelético/inervação , Neurônios/citologia , Neurônios Aferentes/fisiologia , Proteínas Recombinantes/metabolismo , Medula Espinal/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Front Cell Neurosci ; 12: 159, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29970990

RESUMO

Recent advances in single-cell technologies are paving the way to a comprehensive understanding of the cellular complexity in the brain. Protocols for single-cell transcriptomics combine a variety of sophisticated methods for the purpose of isolating the heavily interconnected and heterogeneous neuronal cell types in a relatively intact and healthy state. The emphasis of single-cell transcriptome studies has thus far been on comparing library generation and sequencing techniques that enable measurement of the minute amounts of starting material from a single cell. However, in order for data to be comparable, standardized cell isolation techniques are essential. Here, we analyzed and simplified methods for the different steps critically involved in single-cell isolation from brain. These include enzymatic digestion, tissue trituration, improved methods for efficient fluorescence-activated cell sorting in samples containing high degree of debris from the neuropil, and finally, highly region-specific cellular labeling compatible with use of stereotaxic coordinates. The methods are exemplified using medium spiny neurons (MSN) from dorsomedial striatum, a cell type that is clinically relevant for disorders of the basal ganglia, including psychiatric and neurodegenerative diseases. We present single-cell RNA sequencing (scRNA-Seq) data from D1 and D2 dopamine receptor expressing MSN subtypes. We illustrate the need for single-cell resolution by comparing to available population-based gene expression data of striatal MSN subtypes. Our findings contribute toward standardizing important steps of single-cell isolation from adult brain tissue to increase comparability of data. Furthermore, our data redefine the transcriptome of MSNs at unprecedented resolution by confirming established marker genes, resolving inconsistencies from previous gene expression studies, and identifying novel subtype-specific marker genes in this important cell type.

8.
Elife ; 52016 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-27458797

RESUMO

Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing, which were rescued by a Nav1.1 transgene, whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons.


Assuntos
Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/patologia , Mutação , Canal de Sódio Disparado por Voltagem NAV1.1/deficiência , Neurônios/fisiologia , Telencéfalo/fisiologia , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia
9.
Autism Res ; 8(5): 507-21, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25663600

RESUMO

Recurrent deletions and duplications at chromosomal region 16p11.2 are variably associated with speech delay, autism spectrum disorder, developmental delay, schizophrenia, and cognitive impairments. Social communication deficits are a primary diagnostic symptom of autism. Here we investigated ultrasonic vocalizations (USVs) in young adult male 16p11.2 deletion mice during a novel three-phase male-female social interaction test that detects vocalizations emitted by a male in the presence of an estrous female, how the male changes its calling when the female is suddenly absent, and the extent to which calls resume when the female returns. Strikingly fewer vocalizations were detected in two independent cohorts of 16p11.2 heterozygous deletion males (+/-) during the first exposure to an unfamiliar estrous female, as compared to wildtype littermates (+/+). When the female was removed, +/+ emitted calls, but at a much lower level, whereas +/- males called minimally. Sensory and motor abnormalities were detected in +/-, including higher nociceptive thresholds, a complete absence of acoustic startle responses, and hearing loss in all +/- as confirmed by lack of auditory brainstem responses to frequencies between 8 and 100 kHz. Stereotyped circling and backflipping appeared in a small percentage of individuals, as previously reported. However, these sensory and motor phenotypes could not directly explain the low vocalizations in 16p11.2 deletion mice, since (a) +/- males displayed normal abilities to emit vocalizations when the female was subsequently reintroduced, and (b) +/- vocalized less than +/+ to social odor cues delivered on an inanimate cotton swab. Our findings support the concept that mouse USVs in social settings represent a response to social cues, and that 16p11.2 deletion mice are deficient in their initial USVs responses to novel social cues.


Assuntos
Transtorno Autístico/fisiopatologia , Comportamento Animal/fisiologia , Transtornos Cromossômicos/fisiopatologia , Deficiência Intelectual/fisiopatologia , Comportamento Social , Vocalização Animal/fisiologia , Animais , Deleção Cromossômica , Cromossomos Humanos Par 16 , Modelos Animais de Doenças , Masculino , Camundongos
10.
Cell Rep ; 7(4): 1077-1092, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24794428

RESUMO

A deletion on human chromosome 16p11.2 is associated with autism spectrum disorders. We deleted the syntenic region on mouse chromosome 7F3. MRI and high-throughput single-cell transcriptomics revealed anatomical and cellular abnormalities, particularly in cortex and striatum of juvenile mutant mice (16p11(+/-)). We found elevated numbers of striatal medium spiny neurons (MSNs) expressing the dopamine D2 receptor (Drd2(+)) and fewer dopamine-sensitive (Drd1(+)) neurons in deep layers of cortex. Electrophysiological recordings of Drd2(+) MSN revealed synaptic defects, suggesting abnormal basal ganglia circuitry function in 16p11(+/-) mice. This is further supported by behavioral experiments showing hyperactivity, circling, and deficits in movement control. Strikingly, 16p11(+/-) mice showed a complete lack of habituation reminiscent of what is observed in some autistic individuals. Our findings unveil a fundamental role of genes affected by the 16p11.2 deletion in establishing the basal ganglia circuitry and provide insights in the pathophysiology of autism.


Assuntos
Transtorno Autístico/genética , Gânglios da Base/anormalidades , Deleção Cromossômica , Transtornos Cromossômicos/genética , Modelos Animais de Doenças , Deficiência Intelectual/genética , Transtornos Mentais/genética , Animais , Gânglios da Base/patologia , Cromossomos Humanos Par 16/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
11.
PLoS One ; 8(4): e60526, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23613729

RESUMO

The C-terminus of the voltage-gated calcium channel Cav1.2 encodes a transcription factor, the calcium channel associated transcriptional regulator (CCAT), that regulates neurite extension and inhibits Cav1.2 expression. The mechanisms by which CCAT is generated in neurons and myocytes are poorly understood. Here we show that CCAT is produced by activation of a cryptic promoter in exon 46 of CACNA1C, the gene that encodes CaV1.2. Expression of CCAT is independent of Cav1.2 expression in neuroblastoma cells, in mice, and in human neurons derived from induced pluripotent stem cells (iPSCs), providing strong evidence that CCAT is not generated by cleavage of CaV1.2. Analysis of the transcriptional start sites in CACNA1C and immune-blotting for channel proteins indicate that multiple proteins are generated from the 3' end of the CACNA1C gene. This study provides new insights into the regulation of CACNA1C, and provides an example of how exonic promoters contribute to the complexity of mammalian genomes.


Assuntos
Canais de Cálcio Tipo L/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Animais , Northern Blotting , Western Blotting , Encéfalo/embriologia , Encéfalo/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Éxons/genética , Humanos , Imunoprecipitação , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Ratos , Fatores de Transcrição/genética , Sítio de Iniciação de Transcrição
12.
Nat Med ; 17(12): 1657-62, 2011 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-22120178

RESUMO

Monogenic neurodevelopmental disorders provide key insights into the pathogenesis of disease and help us understand how specific genes control the development of the human brain. Timothy syndrome is caused by a missense mutation in the L-type calcium channel Ca(v)1.2 that is associated with developmental delay and autism. We generated cortical neuronal precursor cells and neurons from induced pluripotent stem cells derived from individuals with Timothy syndrome. Cells from these individuals have defects in calcium (Ca(2+)) signaling and activity-dependent gene expression. They also show abnormalities in differentiation, including decreased expression of genes that are expressed in lower cortical layers and in callosal projection neurons. In addition, neurons derived from individuals with Timothy syndrome show abnormal expression of tyrosine hydroxylase and increased production of norepinephrine and dopamine. This phenotype can be reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type-channel blocker. These findings provide strong evidence that Ca(v)1.2 regulates the differentiation of cortical neurons in humans and offer new insights into the causes of autism in individuals with Timothy syndrome.


Assuntos
Sinalização do Cálcio , Células-Tronco Pluripotentes Induzidas/citologia , Síndrome do QT Longo/fisiopatologia , Neurônios/citologia , Sindactilia/fisiopatologia , Tirosina 3-Mono-Oxigenase/genética , Transtorno Autístico/genética , Transtorno Autístico/fisiopatologia , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular , Dopamina/metabolismo , Regulação da Expressão Gênica , Humanos , Síndrome do QT Longo/enzimologia , Análise em Microsséries , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Norepinefrina/metabolismo , Fenótipo , Purinas/farmacologia , Roscovitina , Sindactilia/enzimologia , Tirosina 3-Mono-Oxigenase/metabolismo
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