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1.
Annu Rev Neurosci ; 41: 1-23, 2018 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-29490194

RESUMO

The mechanistic target of rapamycin (mTOR) is an important signaling hub that integrates environmental information regarding energy availability and stimulates anabolic molecular processes and cell growth. Abnormalities in this pathway have been identified in several syndromes in which autism spectrum disorder (ASD) is highly prevalent. Several studies have investigated mTOR signaling in developmental and neuronal processes that, when dysregulated, could contribute to the development of ASD. Although many potential mechanisms still remain to be fully understood, these associations are of great interest because of the clinical availability of mTOR inhibitors. Clinical trials evaluating the efficacy of mTOR inhibitors to improve neurodevelopmental outcomes have been initiated.


Assuntos
Transtorno Autístico/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Transtorno Autístico/genética , Transtorno Autístico/patologia , Transtorno Autístico/fisiopatologia , Humanos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética
2.
Cell ; 147(1): 235-46, 2011 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-21962519

RESUMO

Although many genes predisposing to autism spectrum disorders (ASD) have been identified, the biological mechanism(s) remain unclear. Mouse models based on human disease-causing mutations provide the potential for understanding gene function and novel treatment development. Here, we characterize a mouse knockout of the Cntnap2 gene, which is strongly associated with ASD and allied neurodevelopmental disorders. Cntnap2(-/-) mice show deficits in the three core ASD behavioral domains, as well as hyperactivity and epileptic seizures, as have been reported in humans with CNTNAP2 mutations. Neuropathological and physiological analyses of these mice before the onset of seizures reveal neuronal migration abnormalities, reduced number of interneurons, and abnormal neuronal network activity. In addition, treatment with the FDA-approved drug risperidone ameliorates the targeted repetitive behaviors in the mutant mice. These data demonstrate a functional role for CNTNAP2 in brain development and provide a new tool for mechanistic and therapeutic research in ASD.


Assuntos
Transtorno Autístico/genética , Encéfalo/crescimento & desenvolvimento , Modelos Animais de Doenças , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Animais , Transtorno Autístico/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Movimento Celular , Epilepsia/genética , Humanos , Interneurônios/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos Knockout , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/patologia
3.
Neurobiol Dis ; 190: 106386, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38110041

RESUMO

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. SSADHD leads to impaired GABA metabolism and results in accumulation of GABA and γ-hydroxybutyrate (GHB), which alter neurotransmission and are thought to lead to neurobehavioral symptoms. However, why increased inhibitory neurotransmitters lead to seizures remains unclear. We used induced pluripotent stem cells from SSADHD patients (one female and two male) and differentiated them into GABAergic and glutamatergic neurons. SSADHD iGABA neurons show altered GABA metabolism and concomitant changes in expression of genes associated with inhibitory neurotransmission. In contrast, glutamatergic neurons display increased spontaneous activity and upregulation of mitochondrial genes. CRISPR correction of the pathogenic variants or SSADHD mRNA expression rescue various metabolic and functional abnormalities in human neurons. Our findings uncover a previously unknown role for SSADHD in excitatory human neurons and provide unique insights into the cellular and molecular basis of SSADHD and potential therapeutic interventions.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Células-Tronco Pluripotentes Induzidas , Humanos , Masculino , Feminino , Células-Tronco Pluripotentes Induzidas/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Neurônios/metabolismo , Ácido gama-Aminobutírico/metabolismo , Succinato-Semialdeído Desidrogenase/genética
4.
Mod Pathol ; 34(2): 264-279, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33051600

RESUMO

Subependymal giant-cell astrocytomas (SEGAs) are slow-growing brain tumors that are a hallmark feature seen in 5-10% of patients with Tuberous Sclerosis Complex (TSC). Though histologically benign, they can cause serious neurologic symptoms, leading to death if untreated. SEGAs consistently show biallelic loss of TSC1 or TSC2. Herein, we aimed to define other somatic events beyond TSC1/TSC2 loss and identify potential transcriptional drivers that contribute to SEGA formation. Paired tumor-normal whole-exome sequencing was performed on 21 resected SEGAs from 20 TSC patients. Pathogenic variants in TSC1/TSC2 were identified in 19/21 (90%) SEGAs. Copy neutral loss of heterozygosity (size range: 2.2-46 Mb) was seen in 76% (16/21) of SEGAs (44% chr9q and 56% chr16p). An average of 1.4 other somatic variants (range 0-7) per tumor were identified, unlikely of pathogenic significance. Whole transcriptome RNA-sequencing analyses revealed 190 common differentially expressed genes in SEGA (n = 16, 13 from a prior study) in pairwise comparison to each of: low grade diffuse gliomas (n = 530) and glioblastoma (n = 171) from The Cancer Genome Atlas (TCGA) consortium, ganglioglioma (n = 10), TSC cortical tubers (n = 15), and multiple normal tissues. Among these, homeobox transcription factors (TFs) HMX3, HMX2, VAX1, SIX3; and TFs IRF6 and EOMES were all expressed >12-fold higher in SEGAs (FDR/q-value < 0.05). Immunohistochemistry supported the specificity of IRF6, VAX1, SIX3 for SEGAs in comparison to other tumor entities and normal brain. We conclude that SEGAs have an extremely low somatic mutation rate, suggesting that TSC1/TSC2 loss is sufficient to drive tumor growth. The unique and highly expressed SEGA-specific TFs likely reflect the neuroepithelial cell of origin, and may also contribute to the transcriptional and epigenetic state that enables SEGA growth following two-hit loss of TSC1 or TSC2 and mTORC1 activation.


Assuntos
Astrocitoma/genética , Neoplasias Encefálicas/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Adolescente , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Transcriptoma , Adulto Jovem
5.
J Neurosci ; 39(47): 9294-9305, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31591157

RESUMO

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in TSC1 or TSC2 Patients frequently have epilepsy, autism spectrum disorder, and/or intellectual disability, as well as other systemic manifestations. In this study, we differentiated human induced pluripotent stem cells (iPSCs) from a female patient with TSC with one or two mutations in TSC2 into neurons using induced expression of NGN2 to examine neuronal dysregulation associated with the neurological symptoms in TSC. Using this method, neuronal differentiation was comparable between the three genotypes of iPSCs. We observed that TSC2+/- neurons show mTOR complex 1 (mTORC1) hyperactivation and associated increased cell body size and process outgrowth, as well as exacerbation of the abnormalities by loss of the second allele of TSC2 in TSC2-/- neurons. Interestingly, iPSC-derived neurons with either a single or biallelic mutation in TSC2 demonstrated hypersynchrony and downregulation of FMRP targets. However, only neurons with biallelic mutations of TSC2 demonstrated hyperactivity and transcriptional dysregulation observed in cortical tubers. These data demonstrate that loss of one allele of TSC2 is sufficient to cause some morphological and physiological changes in human neurons but that biallelic mutations in TSC2 are necessary to induce gene expression dysregulation present in cortical tubers. Finally, we found that treatment of iPSC-derived neurons with rapamycin reduced neuronal activity and partially reversed gene expression abnormalities, demonstrating that mTOR dysregulation contributes to both phenotypes. Therefore, biallelic mutations in TSC2 and associated molecular dysfunction, including mTOR hyperactivation, may play a role in the development of cortical tubers.SIGNIFICANCE STATEMENT In this study, we examined neurons derived from induced pluripotent stem cells with two, one, or no functional TSC2 (tuberous sclerosis complex 2) alleles and found that loss of one or both alleles of TSC2 results in mTORC1 hyperactivation and specific neuronal abnormalities. However, only biallelic mutations in TSC2 resulted in elevated neuronal activity and upregulation of cell adhesion genes that is also observed in cortical tubers. These data suggest that loss of heterozygosity of TSC1 or TSC2 may play an important role in the development of cortical tubers, and potentially epilepsy, in patients with TSC.


Assuntos
Alelos , Células-Tronco Pluripotentes Induzidas/fisiologia , Mutação/genética , Neurônios/fisiologia , Proteína 2 do Complexo Esclerose Tuberosa/genética , Esclerose Tuberosa/genética , Células Cultivadas , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Neurônios/patologia , Esclerose Tuberosa/patologia
6.
Mol Psychiatry ; 23(11): 2167-2183, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29449635

RESUMO

Accumulating evidence suggests that cerebellar dysfunction early in life is associated with autism spectrum disorder (ASD), but the molecular mechanisms underlying the cerebellar deficits at the cellular level are unclear. Tuberous sclerosis complex (TSC) is a neurocutaneous disorder that often presents with ASD. Here, we developed a cerebellar Purkinje cell (PC) model of TSC with patient-derived human induced pluripotent stem cells (hiPSCs) to characterize the molecular mechanisms underlying cerebellar abnormalities in ASD and TSC. Our results show that hiPSC-derived PCs from patients with pathogenic TSC2 mutations displayed mTORC1 pathway hyperactivation, defects in neuronal differentiation and RNA regulation, hypoexcitability and reduced synaptic activity when compared with those derived from controls. Our gene expression analyses revealed downregulation of several components of fragile X mental retardation protein (FMRP) targets in TSC2-deficient hiPSC-PCs. We detected decreased expression of FMRP, glutamate receptor δ2 (GRID2), and pre- and post-synaptic markers such as synaptophysin and PSD95 in the TSC2-deficient hiPSC-PCs. The mTOR inhibitor rapamycin rescued the deficits in differentiation, synaptic dysfunction, and hypoexcitability of TSC2 mutant hiPSC-PCs in vitro. Our findings suggest that these gene expression changes and cellular abnormalities contribute to aberrant PC function during development in TSC affected individuals.


Assuntos
Células de Purkinje/metabolismo , Esclerose Tuberosa/metabolismo , Adulto , Transtorno do Espectro Autista/complicações , Transtorno do Espectro Autista/metabolismo , Doenças Cerebelares/metabolismo , Cerebelo/metabolismo , Criança , Pré-Escolar , Feminino , Proteína do X Frágil da Deficiência Intelectual/efeitos dos fármacos , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Modelos Biológicos , Células de Purkinje/patologia , Sirolimo/farmacologia , Sinapses/metabolismo , Sinapses/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Esclerose Tuberosa/fisiopatologia , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética
7.
Hum Mol Genet ; 25(11): 2168-2181, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27005422

RESUMO

Spinal muscular atrophy (SMA) is an autosomal-recessive pediatric neurodegenerative disease characterized by selective loss of spinal motor neurons. It is caused by mutation in the survival of motor neuron 1, SMN1, gene and leads to loss of function of the full-length SMN protein. microRNAs (miRNAs) are small RNAs that are involved in post-transcriptional regulation of gene expression. Prior studies have implicated miRNAs in the pathogenesis of motor neuron disease. We hypothesized that motor neuron-specific miRNA expression changes are involved in their selective vulnerability in SMA. Therefore, we sought to determine the effect of SMN loss on miRNAs and their target mRNAs in spinal motor neurons. We used microarray and RNAseq to profile both miRNA and mRNA expression in primary spinal motor neuron cultures after acute SMN knockdown. By integrating the miRNA:mRNA profiles, a number of dysregulated miRNAs were identified with enrichment in differentially expressed putative mRNA targets. miR-431 expression was highly increased, and a number of its putative mRNA targets were significantly downregulated in motor neurons after SMN loss. Further, we found that miR-431 regulates motor neuron neurite length by targeting several molecules previously identified to play a role in motor neuron axon outgrowth, including chondrolectin. Together, our findings indicate that cell-type-specific dysregulation of miR-431 plays a role in the SMA motor neuron phenotype.


Assuntos
MicroRNAs/genética , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/biossíntese , Análise em Microsséries , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/fisiopatologia , Neuritos/metabolismo , Neuritos/patologia
8.
Neurobiol Dis ; 78: 35-44, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25818007

RESUMO

The molecular basis of epileptogenesis is poorly characterized. Studies in humans and animal models have identified an electrophysiological signature that precedes the onset of epilepsy, which has been termed fast ripples (FRs) based on its frequency. Multiple lines of evidence implicate regions generating FRs in epileptogenesis, and FRs appear to demarcate the seizure onset zone, suggesting a role in ictogenesis as well. We performed gene expression analysis comparing areas of the dentate gyrus that generate FRs to those that do not generate FRs in a well-characterized rat model of epilepsy. We identified a small cohort of genes that are differentially expressed in FR versus non-FR brain tissue and used quantitative PCR to validate some of those that modulate neuronal excitability. Gene expression network analysis demonstrated conservation of gene co-expression between non-FR and FR samples, but examination of gene connectivity revealed changes that were most pronounced in the cm-40 module, which contains several genes associated with synaptic function and the differentially expressed genes Kcna4, Kcnv1, and Npy1r that are down-regulated in FRs. We then demonstrate that the genes within the cm-40 module are regulated by seizure activity and enriched for the targets of the RNA binding protein Elavl4. Our data suggest that seizure activity induces co-expression of genes associated with synaptic transmission and that this pattern is attenuated in areas displaying FRs, implicating the failure of this mechanism in the generation of FRs.


Assuntos
Giro Denteado/fisiopatologia , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/fisiopatologia , Animais , Modelos Animais de Doenças , Expressão Gênica , Redes Reguladoras de Genes , Ratos
9.
Semin Neurol ; 35(3): 277-87, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26060907

RESUMO

Megalencephaly is a developmental disorder characterized by brain overgrowth secondary to increased size and/or numbers of neurons and glia. These disorders can be divided into metabolic and developmental categories based on their molecular etiologies. Metabolic megalencephalies are mostly caused by genetic defects in cellular metabolism, whereas developmental megalencephalies have recently been shown to be caused by alterations in signaling pathways that regulate neuronal replication, growth, and migration. These disorders often lead to epilepsy, developmental disabilities, and behavioral problems; specific disorders have associations with overgrowth or abnormalities in other tissues. The molecular underpinnings of many of these disorders are now understood, providing insight into how dysregulation of critical pathways leads to disease. The advances in molecular understanding are leading to improved diagnosis of these conditions, as well as providing new avenues for therapeutic interventions.


Assuntos
Epilepsia/etiologia , Megalencefalia/complicações , Deficiências do Desenvolvimento/etiologia , Humanos , Transtornos Mentais/etiologia
10.
Nature ; 462(7270): 213-7, 2009 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-19907493

RESUMO

The signalling pathways controlling both the evolution and development of language in the human brain remain unknown. So far, the transcription factor FOXP2 (forkhead box P2) is the only gene implicated in Mendelian forms of human speech and language dysfunction. It has been proposed that the amino acid composition in the human variant of FOXP2 has undergone accelerated evolution, and this two-amino-acid change occurred around the time of language emergence in humans. However, this remains controversial, and whether the acquisition of these amino acids in human FOXP2 has any functional consequence in human neurons remains untested. Here we demonstrate that these two human-specific amino acids alter FOXP2 function by conferring differential transcriptional regulation in vitro. We extend these observations in vivo to human and chimpanzee brain, and use network analysis to identify novel relationships among the differentially expressed genes. These data provide experimental support for the functional relevance of changes in FOXP2 that occur on the human lineage, highlighting specific pathways with direct consequences for human brain development and disease in the central nervous system (CNS). Because FOXP2 has an important role in speech and language in humans, the identified targets may have a critical function in the development and evolution of language circuitry in humans.


Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transcrição Gênica , Animais , Encéfalo/citologia , Linhagem Celular , Evolução Molecular , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Humanos , Idioma , Pan troglodytes/embriologia , Pan troglodytes/genética , Pan troglodytes/metabolismo , Regiões Promotoras Genéticas/genética , Especificidade da Espécie , Fala/fisiologia , Ativação Transcricional
11.
Nat Commun ; 15(1): 584, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38233389

RESUMO

Unbiased phenotypic screens in patient-relevant disease models offer the potential to detect therapeutic targets for rare diseases. In this study, we developed a high-throughput screening assay to identify molecules that correct aberrant protein trafficking in adapter protein complex 4 (AP-4) deficiency, a rare but prototypical form of childhood-onset hereditary spastic paraplegia characterized by mislocalization of the autophagy protein ATG9A. Using high-content microscopy and an automated image analysis pipeline, we screened a diversity library of 28,864 small molecules and identified a lead compound, BCH-HSP-C01, that restored ATG9A pathology in multiple disease models, including patient-derived fibroblasts and induced pluripotent stem cell-derived neurons. We used multiparametric orthogonal strategies and integrated transcriptomic and proteomic approaches to delineate potential mechanisms of action of BCH-HSP-C01. Our results define molecular regulators of intracellular ATG9A trafficking and characterize a lead compound for the treatment of AP-4 deficiency, providing important proof-of-concept data for future studies.


Assuntos
Paraplegia Espástica Hereditária , Humanos , Paraplegia Espástica Hereditária/tratamento farmacológico , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Proteômica , Neurônios/metabolismo , Transporte Proteico , Proteínas/metabolismo , Mutação
12.
Cell Rep ; 42(8): 112838, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37494191

RESUMO

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder, but new therapies have been impeded by a lack of understanding of the pathological mechanisms. Tuberous sclerosis complex (TSC) and fragile X syndrome are associated with alterations in the mechanistic target of rapamycin (mTOR) and fragile X messenger ribonucleoprotein 1 (FMRP), which have been implicated in the development of ASD. Previously, we observed that transcripts associated with FMRP were down-regulated in TSC2-deficient neurons. In this study, we find that FMRP turnover is dysregulated in TSC2-deficient rodent primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons and is dependent on the E3 ubiquitin ligase anaphase-promoting complex. We also demonstrate that overexpression of FMRP can partially rescue hyperexcitability in TSC2-deficient iPSC-derived neurons. These data indicate that FMRP dysregulation represents an important pathological mechanism in the development of abnormal neuronal activity in TSC and illustrate a molecular convergence between these two neurogenetic disorders.


Assuntos
Transtorno do Espectro Autista , Células-Tronco Pluripotentes Induzidas , Esclerose Tuberosa , Humanos , Transtorno do Espectro Autista/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
13.
Res Sq ; 2023 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-37398196

RESUMO

Unbiased phenotypic screens in patient-relevant disease models offer the potential to detect novel therapeutic targets for rare diseases. In this study, we developed a high-throughput screening assay to identify molecules that correct aberrant protein trafficking in adaptor protein complex 4 (AP-4) deficiency, a rare but prototypical form of childhood-onset hereditary spastic paraplegia, characterized by mislocalization of the autophagy protein ATG9A. Using high-content microscopy and an automated image analysis pipeline, we screened a diversity library of 28,864 small molecules and identified a lead compound, C-01, that restored ATG9A pathology in multiple disease models, including patient-derived fibroblasts and induced pluripotent stem cell-derived neurons. We used multiparametric orthogonal strategies and integrated transcriptomic and proteomic approaches to delineate putative molecular targets of C-01 and potential mechanisms of action. Our results define molecular regulators of intracellular ATG9A trafficking and characterize a lead compound for the treatment of AP-4 deficiency, providing important proof-of-concept data for future Investigational New Drug (IND)-enabling studies.

14.
J Biol Chem ; 286(46): 39794-803, 2011 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-21868380

RESUMO

A subset of non-steroidal anti-inflammatory drugs modulates the γ cleavage site in the amyloid precursor protein (APP) to selectively reduce production of Aß42. It is unclear precisely how these γ-secretase modulators (GSMs) act to preferentially spare Aß40 production as well as Notch processing and signaling. In an effort to determine the substrate requirements in NSAID/GSM activity, we determined the effects of sulindac sulfide and flurbiprofen on γ-cleavage of artificial constructs containing several γ-secretase substrates. Using FLAG-tagged constructs that expressed extracellularly truncated APP, Notch-1, or CD44, we found that these substrates have different sensitivities to sulindac sulfide. γ-Secretase cleavage of APP was altered by sulindac sulfide, but CD44 and Notch-1 were either insensitive or only minimally altered by this compound. Using chimeric APP constructs, we observed that the transmembrane domain (TMD) of APP played a pivotal role in determining drug sensitivity. Substituting the APP TMD with that of APLP2 retained the sensitivity to γ-cleavage modulation, but replacing TMDs from Notch-1 or ErbB4 rendered the resultant molecules insensitive to drug treatment. Specifically, the GXXXG motif within APP appeared to be critical to GSM activity. Consequently, the modulatory effects on γ-cleavage appears to be substrate-dependent. We hypothesize that the substrate present in the γ-secretase complex influences the conformation of the complex so that the binding site of GSMs is either stabilized or less favorable to influence the cleavage of the respective substrates.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Receptores de Hialuronatos/metabolismo , Receptor Notch1/metabolismo , Motivos de Aminoácidos , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Anti-Inflamatórios não Esteroides/farmacologia , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Estrutura Terciária de Proteína , Receptor Notch1/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sulindaco/análogos & derivados , Sulindaco/farmacologia
15.
Bio Protoc ; 12(9): e4407, 2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35800463

RESUMO

Mammalian tissues are highly heterogenous and complex, posing a challenge in understanding the molecular mechanisms regulating protein expression within various tissues. Recent studies have shown that translation at the level of the ribosome is highly regulated, and can vary independently of gene expression observed at a transcriptome level, as well as between cell populations, contributing to the diversity of mammalian tissues. Earlier methods that analyzed gene expression at the level of translation, such as polysomal- or ribosomal-profiling, required large amounts of starting material to isolate enough RNA for analysis by microarray or RNA-sequencing. Thus, rare or less abundant cell types within tissues were not able to be properly studied with these methods. Translating ribosome affinity purification (TRAP) utilizes the incorporation of an eGFP-affinity tag on the large ribosome subunit, driven by expression of cell-type specific Cre-lox promoters, to allow for identification and capture of transcripts from actively translating ribosomes in a cell-specific manner. As a result, TRAP offers a unique opportunity to evaluate the entire mRNA translation profile within a specific cell type, and increase our understanding regarding the cellular complexity of mammalian tissues. Graphical abstract: Schematic demonstrating TRAP protocol for identifying ribosome-bound transcripts specifically within cerebellar Purkinje cells.

16.
Front Psychiatry ; 13: 924956, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36405918

RESUMO

16p13.11 copy number variants (CNVs) have been associated with autism, schizophrenia, psychosis, intellectual disability, and epilepsy. The majority of 16p13.11 deletions or duplications occur within three well-defined intervals, and despite growing knowledge of the functions of individual genes within these intervals, the molecular mechanisms that underlie commonly observed clinical phenotypes remain largely unknown. Patient-derived, induced pluripotent stem cells (iPSCs) provide a platform for investigating the morphological, electrophysiological, and gene-expression changes that result from 16p13.11 CNVs in human-derived neurons. Patient derived iPSCs with varying sizes of 16p13.11 deletions and familial controls were differentiated into cortical neurons for phenotypic analysis. High-content imaging and morphological analysis of patient-derived neurons demonstrated an increase in neurite branching in patients compared with controls. Whole-transcriptome sequencing revealed expression level changes in neuron development and synaptic-related gene families, suggesting a defect in synapse formation. Subsequent quantification of synapse number demonstrated increased numbers of synapses on neurons derived from early-onset patients compared to controls. The identification of common phenotypes among neurons derived from patients with overlapping 16p13.11 deletions will further assist in ascertaining common pathways and targets that could be utilized for screening drug candidates. These studies can help to improve future treatment options and clinical outcomes for 16p13.11 deletion patients.

17.
Nat Commun ; 12(1): 2897, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006844

RESUMO

Reciprocal copy number variations (CNVs) of 16p11.2 are associated with a wide spectrum of neuropsychiatric and neurodevelopmental disorders. Here, we use human induced pluripotent stem cells (iPSCs)-derived dopaminergic (DA) neurons carrying CNVs of 16p11.2 duplication (16pdup) and 16p11.2 deletion (16pdel), engineered using CRISPR-Cas9. We show that 16pdel iPSC-derived DA neurons have increased soma size and synaptic marker expression compared to isogenic control lines, while 16pdup iPSC-derived DA neurons show deficits in neuronal differentiation and reduced synaptic marker expression. The 16pdel iPSC-derived DA neurons have impaired neurophysiological properties. The 16pdel iPSC-derived DA neuronal networks are hyperactive and have increased bursting in culture compared to controls. We also show that the expression of RHOA is increased in the 16pdel iPSC-derived DA neurons and that treatment with a specific RHOA-inhibitor, Rhosin, rescues the network activity of the 16pdel iPSC-derived DA neurons. Our data suggest that 16p11.2 deletion-associated iPSC-derived DA neuron hyperactivation can be rescued by RHOA inhibition.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 16/genética , Neurônios Dopaminérgicos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Rede Nervosa/metabolismo , Transmissão Sináptica/genética , Proteína rhoA de Ligação ao GTP/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Variações do Número de Cópias de DNA , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Rede Nervosa/efeitos dos fármacos , Compostos Orgânicos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transmissão Sináptica/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismo
18.
Elife ; 102021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34259631

RESUMO

Tuberous sclerosis complex (TSC) is a genetic disorder that is associated with multiple neurological manifestations. Previously, we demonstrated that Tsc1 loss in cerebellar Purkinje cells (PCs) can cause altered social behavior in mice. Here, we performed detailed transcriptional and translational analyses of Tsc1-deficient PCs to understand the molecular alterations in these cells. We found that target transcripts of the Fragile X Mental Retardation Protein (FMRP) are reduced in mutant PCs with evidence of increased degradation. Surprisingly, we observed unchanged ribosomal binding for many of these genes using translating ribosome affinity purification. Finally, we found that multiple FMRP targets, including SHANK2, were reduced, suggesting that compensatory increases in ribosomal binding efficiency may be unable to overcome reduced transcript levels. These data further implicate dysfunction of FMRP and its targets in TSC and suggest that treatments aimed at restoring the function of these pathways may be beneficial.


Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Células de Purkinje/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Animais , Modelos Animais de Doenças , Expressão Gênica , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Ribossomos/metabolismo , Esclerose Tuberosa/genética , Esclerose Tuberosa/metabolismo
19.
Cell Rep ; 37(4): 109902, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34706228

RESUMO

Rheumatoid arthritis is a systemic autoimmune disease, but disease flares typically affect only a subset of joints, distributed in a distinctive pattern for each patient. Pursuing this intriguing pattern, we show that arthritis recurrence is mediated by long-lived synovial resident memory T cells (TRM). In three murine models, CD8+ cells bearing TRM markers remain in previously inflamed joints during remission. These cells are bona fide TRM, exhibiting a failure to migrate between joints, preferential uptake of fatty acids, and long-term residency. Disease flares result from TRM activation by antigen, leading to CCL5-mediated recruitment of circulating effector cells. Correspondingly, TRM depletion ameliorates recurrence in a site-specific manner. Human rheumatoid arthritis joint tissues contain a comparable CD8+-predominant TRM population, which is most evident in late-stage leukocyte-poor synovium, exhibiting limited T cell receptor diversity and a pro-inflammatory transcriptomic signature. Together, these findings establish synovial TRM as a targetable mediator of disease chronicity in autoimmune arthritis.


Assuntos
Artrite Reumatoide/imunologia , Linfócitos T CD8-Positivos/imunologia , Células T de Memória/imunologia , Membrana Sinovial/imunologia , Transcriptoma/imunologia , Animais , Artrite Reumatoide/patologia , Linfócitos T CD8-Positivos/patologia , Humanos , Células T de Memória/patologia , Camundongos , Camundongos Knockout , Membrana Sinovial/patologia
20.
Mol Syst Biol ; 5: 291, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19638972

RESUMO

Genome-wide expression profiling has aided the understanding of the molecular basis of neuronal diversity, but achieving broad functional insight remains a considerable challenge. Here, we perform the first systems-level analysis of microarray data from single neuronal populations using weighted gene co-expression network analysis to examine how neuronal transcriptome organization relates to neuronal function and diversity. We systematically validate network predictions using published proteomic and genomic data. Several network modules of co-expressed genes correspond to interneuron development programs, in which the hub genes are known to be critical for interneuron specification. Other co-expression modules relate to fundamental cellular functions, such as energy production, firing rate, trafficking, and synapses, suggesting that fundamental aspects of neuronal diversity are produced by quantitative variation in basic metabolic processes. We identify two transcriptionally distinct mitochondrial modules and demonstrate that one corresponds to mitochondria enriched in neuronal processes and synapses, whereas the other represents a population restricted to the soma. Finally, we show that galectin-1 is a new interneuron marker, and we validate network predictions in vivo using Rgs4 and Dlx1/2 knockout mice. These analyses provide a basis for understanding how specific aspects of neuronal phenotypic diversity are organized at the transcriptional level.


Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Neurônios , Animais , Galectina 1 , Camundongos , Mitocôndrias/genética , Neurônios/ultraestrutura , Sinapses
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