RESUMO
Increased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup), carrying different duplication sizes, to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition, using multi-electrodes arrays, we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level, we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor, NCH-51, was validated as a potential clinical candidate. Interestingly, this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders.
Assuntos
Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/fisiologia , Rede Nervosa/metabolismo , Diferenciação Celular , Dendritos/metabolismo , Dosagem de Genes/fisiologia , Duplicação Gênica/genética , Estudos de Associação Genética , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Neurogênese , NeurôniosRESUMO
An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs), and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here, we report a de novo balanced translocation disruption of TRPC6, a cation channel, in a non-syndromic autistic individual. Using multiple models, such as dental pulp cells, induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models, we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development, morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin, a TRPC6-specific agonist, suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome, revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population, and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together, these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.
Assuntos
Transtorno Autístico/patologia , Neurônios/patologia , Canais de Cátion TRPC/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Transtorno Autístico/genética , Transtorno Autístico/fisiopatologia , Carboplatina/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Células Cultivadas , Criança , Modelos Animais de Doenças , Embrião de Mamíferos , Etoposídeo/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/fisiologia , Potenciais Pós-Sinápticos Inibidores/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitoxantrona/metabolismo , Mutação/genética , Neurônios/metabolismo , Prednisolona/metabolismo , Transdução de Sinais/genética , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6RESUMO
Human pluripotent stem cells bring promise in regenerative medicine due to their self-renewing ability and the potential to become any cell type in the body. Moreover, pluripotent stem cells can produce specialized cell types that are affected in certain diseases, generating a new way to study cellular and molecular mechanisms involved in the disease pathology under the controlled conditions of a scientific laboratory. Thus, induced pluripotent stem cells (iPSC) are already being used to gain insights into the biological mechanisms of several human disorders. Here we review the use of iPSC as a novel tool for disease modeling in the lab.
Assuntos
Modelos Biológicos , Doenças do Sistema Nervoso/patologia , Células-Tronco Pluripotentes/patologia , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/patologia , Humanos , FenótipoRESUMO
A deeper understanding of early disease mechanisms occurring in Parkinson's disease (PD) is needed to reveal restorative targets. Here we report that human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons (DAn) obtained from healthy individuals or patients harboring LRRK2 PD-causing mutation can create highly complex networks with evident signs of functional maturation over time. Compared to control neuronal networks, LRRK2 PD patients' networks displayed an elevated bursting behavior, in the absence of neurodegeneration. By combining functional calcium imaging, biophysical modeling, and DAn-lineage tracing, we found a decrease in DAn neurite density that triggered overall functional alterations in PD neuronal networks. Our data implicate early dysfunction as a prime focus that may contribute to the initiation of downstream degenerative pathways preceding DAn loss in PD, highlighting a potential window of opportunity for pre-symptomatic assessment of chronic degenerative diseases.
RESUMO
Anorexia nervosa (AN) is a complex and multifactorial disorder occurring predominantly in women. Despite having the highest mortality among psychiatric conditions, it still lacks robust and effective treatment. Disorders such as AN are most likely syndromes with multiple genetic contributions, however, genome-wide studies have been underpowered to reveal associations with this uncommon illness. Here, we generated induced pluripotent stem cells (iPSCs) from adolescent females with AN and unaffected controls. These iPSCs were differentiated into neural cultures and subjected to extensive transcriptome analysis. Within a small cohort of patients who presented for treatment, we identified a novel gene that appears to contribute to AN pathophysiology, TACR1 (tachykinin 1 receptor). The participation of tachykinins in a variety of biological processes and their interactions with other neurotransmitters suggest novel mechanisms for how a disrupted tachykinin system might contribute to AN symptoms. Although TACR1 has been associated with psychiatric conditions, especially anxiety disorders, we believe this report is its first association with AN. Moreover, our human iPSC approach is a proof-of-concept that AN can be modeled in vitro with a full human genetic complement, and represents a new tool for understanding the elusive molecular and cellular mechanisms underlying the disease.
Assuntos
Anorexia Nervosa/genética , Neurônios/metabolismo , Receptores da Neurocinina-1/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Células-Tronco Pluripotentes Induzidas , Modelos NeurológicosRESUMO
Mutations in MECP2 cause Rett syndrome, a severe neurological disorder with autism-like features. Duplication of MECP2 also causes severe neuropathology. Both diseases display immunological abnormalities that suggest a role for MECP2 in controlling immune and inflammatory responses. Here, we used mecp2-null zebrafish to study the potential function of Mecp2 as an immunological regulator. Mecp2 deficiency resulted in an increase in neutrophil infiltration and upregulated expression of the pro- and anti-inflammatory cytokines Il1b and Il10 as a secondary response to disturbances in tissue homeostasis. By contrast, expression of the proinflammatory cytokine tumor necrosis factor alpha (Tnfa) was consistently downregulated in mecp2-null animals during development, representing the earliest developmental phenotype described for MECP2 deficiency to date. Expression of tnfa was unresponsive to inflammatory stimulation, and was partially restored by re-expression of functional mecp2 Thus, Mecp2 is required for tnfa expression during zebrafish development and inflammation. Finally, RNA sequencing of mecp2-null embryos revealed dysregulated processes predictive for Rett syndrome phenotypes.
Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Inflamação/embriologia , Inflamação/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Fator de Necrose Tumoral alfa/genética , Peixe-Zebra/embriologia , Animais , Trato Gastrointestinal/patologia , Perfilação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Larva/crescimento & desenvolvimento , Contagem de Leucócitos , Proteína 2 de Ligação a Metil-CpG/deficiência , Neutrófilos/patologia , Fenótipo , Síndrome de Rett/genética , Síndrome de Rett/patologia , Análise de Sequência de RNA , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Electronic excited molecular oxygen (singlet oxygen, (1)O(2)) is known to damage DNA, yielding mutations. In this work, the mutagenicity induced by (1)O(2) in a defined sequence of DNA was investigated after replication in Escherichia coli mutants deficient for nucleotide and base excision DNA repair pathways. For this purpose a plasmid containing a (1)O(2)-damaged 14 base oligonucleotide was introduced into E.coli by transfection and mutations were screened by hybridization with an oligonucleotide with the original sequence. Mutagenesis was observed in all strains tested, but it was especially high in the BH20 (fpg), AYM57 (fpg mutY) and AYM84 (fpg mutY uvrC) strains. The frequency of mutants in the fpg mutY strain was higher than in the triple mutant fpg mutY uvrC, suggesting that activity of the UvrABC excinuclease can favor the mutagenesis of these lesions. Additionally, most of the mutations were G-->T and G-->C transversions, but this was dependent on the position of the guanine in the sequence and on repair deficiency in the host bacteria. Thus, the kind of repair and the mutagenesis associated with (1)O(2)-induced DNA damage are linked to the context of the damaged sequence.
Assuntos
DNA Glicosilases , Reparo do DNA/genética , Endodesoxirribonucleases , Proteínas de Escherichia coli , Escherichia coli/genética , Mutagênese/efeitos dos fármacos , Mutação/genética , Oxigênio/farmacologia , Proteínas de Bactérias/genética , Sequência de Bases , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Replicação do DNA , DNA-Formamidopirimidina Glicosilase , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Vetores Genéticos/efeitos dos fármacos , Vetores Genéticos/genética , Mutagênese/genética , Mutação/efeitos dos fármacos , N-Glicosil Hidrolases/genética , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Oxigênio Singlete , Transformação BacterianaRESUMO
Photolyase absorbs blue light and employs the energy to remove UV-induced DNA damage, cyclobutane pyrimidine dimers, or pyrimidine pyrimidone (6-4) lesions. These enzymes have been found in many living organisms ranging from bacteria to aplacental mammals, but their photoreactivation effect, such as survival increase of UV-irradiated cells by light-illumination, has not been identified in placental mammals, including humans. Therefore, we introduced a photolyase gene derived from the marsupial rat kangaroo, Potorous tridactylus, into HeLa cells and established the first human cell line capable of photorepairing UV-induced pyrimidine dimers. Several clones were found to increase cell survival after UV irradiation when illuminated by fluorescent light. The induction of apoptosis by UV irradiation was investigated in these photoreactivation-proficient cells. Several typical features of the programmed cell death, such as internucleosomal DNA degradation, presence of subdiploid cells, loss of membrane integrity, and chromosomal condensation, were found to be induced by UV in the HeLa cells, but they can be reduced by photorepair. This implicates that cyclobutane pyrimidine dimers cause UV-induced apoptosis in human cells.
Assuntos
Apoptose , Reparo do DNA , Desoxirribodipirimidina Fotoliase/genética , Macropodidae/genética , Raios Ultravioleta , Animais , Sobrevivência Celular/efeitos da radiação , DNA/efeitos da radiação , Fragmentação do DNA , Relação Dose-Resposta à Radiação , Citometria de Fluxo , Células HeLa , Humanos , Microscopia de Fluorescência , Dímeros de Pirimidina , Fatores de Tempo , TransfecçãoRESUMO
Fibroblasts are cells widely used in cell culture, both for transient primary cell culture or permanent as transformed cell lines. Lately, fibroblasts become cell sources for use in disease modeling after cell reprogramming because it is easily accessible in the body. Fibroblasts in patients will maintain all genetic background during reprogramming into induced pluripotent stem cells. In spite of their large use, fibroblasts are obtained after an invasive procedure, a superficial punch skin biopsy, collected under patient's local anesthesia. Taking into consideration the minimum patient's discomfort during and after the biopsy procedure, as well as the aesthetics aspect, it is essential to reflect on the best site of the body for the biopsy procedure combined with the success of getting robust fibroblast cultures in the lab. For this purpose, we compared the efficiency of four biopsy sites of the body (skin from eyelid, back of the ear, abdominal cesarean scar and groin). Cell proliferation assays and viability after cryopreservation were measured. Our results revealed that scar tissue provided fibroblasts with higher proliferative rates. Also, fibroblasts from scar tissues presented a higher viability after the thawing process.
RESUMO
Monge's disease, also known as chronic mountain sickness (CMS), is a disease that potentially threatens more than 140 million highlanders during extended time living at high altitudes (over 2500m). The prevalence of CMS in Andeans is about 15-20%, suggesting that the majority of highlanders (non-CMS) are rather healthy at high altitudes; however, CMS subjects experience severe hypoxemia, erythrocytosis and many neurologic manifestations including migraine, headache, mental fatigue, confusion, and memory loss. The underlying mechanisms of CMS neuropathology are not well understood and no ideal treatment is available to prevent or cure CMS, except for phlebotomy. In the current study, we reprogrammed fibroblast cells from both CMS and non-CMS subjects' skin biopsies into the induced pluripotent stem cells (iPSCs), then differentiated into neurons and compared their neuronal properties. We discovered that CMS neurons were much less excitable (higher rheobase) than non-CMS neurons. This decreased excitability was not caused by differences in passive neuronal properties, but instead by a significantly lowered Na(+) channel current density and by a shift of the voltage-conductance curve in the depolarization direction. Our findings provide, for the first time, evidence of a neuronal abnormality in CMS subjects as compared to non-CMS subjects, hoping that such studies can pave the way to a better understanding of the neuropathology in CMS.
Assuntos
Doença da Altitude/fisiopatologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Neurônios/fisiologia , Canais de Sódio/metabolismo , Potenciais de Ação/fisiologia , Adulto , Técnicas de Cultura de Células , Células Cultivadas , Doença Crônica , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Neurônios/citologia , Técnicas de Patch-Clamp , Peru , Adulto JovemRESUMO
Ribozymes are RNA molecules that possess the dual properties of RNA sequence-specific recognition and site-specific cleavage of other RNA molecules. These properties provide powerful tools for studies requiring gene inhibition, when the DNA sequence is known. The use of these molecules goes beyond basic research, with a potential impact in therapeutical practice in medicine in the near future. In this review, we briefly describe the progress towards developing this class of molecules and its applications for the control of gene expression.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , RNA Catalítico/uso terapêutico , Animais , Sequência de Bases , HumanosRESUMO
Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK) gene, followed by administration of the antiviral drug ganciclovir (GCV). The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK) driven by three strong promoters (P CMV IE, SV40 and EN1) and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system.
Assuntos
Adenoviridae/genética , Antivirais/farmacologia , Ganciclovir/farmacologia , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Timidina Quinase/genética , Animais , Genes Virais , Vetores Genéticos/genética , Células HeLa , Humanos , Camundongos , Regiões Promotoras Genéticas , Timidina Quinase/uso terapêutico , Células Tumorais CultivadasRESUMO
Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK) gene, followed by administration of the antiviral drug ganciclovir (GCV). The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK) driven by three strong promoters (P CMV IE, SV40 and EN1) and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system