RESUMO
Background: Rett syndrome (RTT) is now widely recognized as a profound neurological disorder that predominantly affects females and is closely associated with mutations in the methylated CpG binding protein 2 (MECP2) gene located on the X chromosome. The Characteristic symptoms of RTT include the loss of acquired language and motor skills, repetitive hand movements, irregular breathing, and seizures. Additionally, RTT patients may experience sporadic episodes of gastrointestinal problems, hypoplasia, early-onset osteoporosis, bruxism, and screaming episodes. It is worth noting that males exhibit a unique and variable phenotype, though rare in RTT cases, often accompanied by severe manifestations. Case Presentation: In this report, we present the case of a young male child with a de novo c.806delG hemizygous mutation, leading to an atypical presentation of RTT that remarkably mirrors the clinical features of Bartter syndrome, a genetic metabolic disorder. The clinical manifestations in this case included the loss of previously acquired language and motor skills, repetitive hand movements, breathing irregularities, seizures, sporadic episodes of gastrointestinal distress, hypoplasia, early-onset osteoporosis, bruxism, and episodes of screaming. This distinctive presentation underscores the complex diagnostic landscape of RTT, particularly in males, and highlights the need for vigilant clinical evaluation. Conclusions: This case report sheds light on an unusual and atypical presentation of RTT in a young male child with a de novo c.806delG hemizygous mutation. The resemblance of clinical features to Bartter syndrome underscores the diagnostic challenges posed by RTT and highlights the importance of comprehensive clinical assessment and genetic testing, especially in cases deviating from the typical RTT phenotype. Our findings contribute valuable insights into the early diagnosis and management of atypical RTT presentations.
Assuntos
Alcalose , Síndrome de Bartter , Bruxismo , Osteoporose , Síndrome de Rett , Criança , Feminino , Humanos , Masculino , Síndrome de Rett/complicações , Síndrome de Rett/diagnóstico , Síndrome de Rett/genética , Proteína 2 de Ligação a Metil-CpG/genética , Hipóxia , ConvulsõesRESUMO
Rett syndrome (RTT) is an X-linked neurogenetic disorder caused by mutations of the MECP2 (methyl-CpG-binding protein 2) gene. Over two decades of work established MeCP2 as a protein with pivotal roles in the regulation of the epigenome, neuronal physiology, synaptic maintenance, and behaviour. Given the genetic aetiology of RTT and the proof of concept of its reversal in a mouse model, considerable efforts have been made to design therapeutic approaches to re-express MeCP2. By being at the forefront of the development of innovative gene therapies, research on RTT is of paramount importance for the treatment of monogenic neurological diseases. Here we discuss the recent advances and challenges of promising genetic strategies for the treatment of RTT including gene replacement therapies, gene/RNA editing strategies, and reactivation of the silenced X chromosome. WHAT THIS PAPER ADDS: Recent advances shed light on the promises of gene replacement therapy with new vectors designed to control the levels of MeCP2 expression. New developments in DNA/RNA editing approaches or reactivation of the silenced X chromosome open the possibility to re-express the native MeCP2 locus at endogenous levels. Current strategies still face limitations in transduction efficiency and future work is needed to improve brain delivery.
Assuntos
Arteterapia , Síndrome de Rett , Camundongos , Animais , Humanos , Síndrome de Rett/terapia , Síndrome de Rett/tratamento farmacológico , Proteína 2 de Ligação a Metil-CpG/genética , Encéfalo/metabolismo , Mutação , NeurôniosRESUMO
Rett syndrome (RTT) is an X-linked neurological disorder caused by mutations in the transcriptional regulator MECP2. Mecp2 loss-of-function leads to the disruption of many cellular pathways, including aberrant activation of the NF-κB pathway. Genetically attenuating the NF-κB pathway in Mecp2-null mice ameliorates hallmark phenotypes of RTT, including reduced dendritic complexity, raising the question of whether NF-κB pathway inhibitors could provide a therapeutic avenue for RTT. Vitamin D is a known inhibitor of NF-κB signaling; further, vitamin D deficiency is prevalent in RTT patients and male Mecp2-null mice. We previously demonstrated that vitamin D rescues the aberrant NF-κB activity and reduced neurite outgrowth of Mecp2-knockdown cortical neurons in vitro, and that dietary vitamin D supplementation rescues decreased dendritic complexity and soma size of neocortical projection neurons in both male hemizygous Mecp2-null and female heterozygous mice in vivo. Here, we have identified over 200 genes whose dysregulated expression in the Mecp2+/- cortex is modulated by dietary vitamin D. Genes normalized with vitamin D supplementation are involved in dendritic complexity, synapses, and neuronal projections, suggesting that the rescue of their expression could underpin the rescue of neuronal morphology. Further, there is a disruption in the homeostasis of the vitamin D synthesis pathway in Mecp2+/- mice, and motor and anxiety-like behavioral phenotypes in Mecp2+/- mice correlate with circulating vitamin D levels. Thus, our data indicate that vitamin D modulates RTT pathology and its supplementation could provide a simple and cost-effective partial therapeutic for RTT.
Assuntos
Síndrome de Rett , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Transcriptoma , Vitamina D/farmacologia , Vitamina D/uso terapêuticoRESUMO
Brain organoids represent a powerful tool for studying human neurological diseases, particularly those that affect brain growth and structure. However, many diseases manifest with clear evidence of physiological and network abnormality in the absence of anatomical changes, raising the question of whether organoids possess sufficient neural network complexity to model these conditions. Here, we explore the network-level functions of brain organoids using calcium sensor imaging and extracellular recording approaches that together reveal the existence of complex network dynamics reminiscent of intact brain preparations. We demonstrate highly abnormal and epileptiform-like activity in organoids derived from induced pluripotent stem cells from individuals with Rett syndrome, accompanied by transcriptomic differences revealed by single-cell analyses. We also rescue key physiological activities with an unconventional neuroregulatory drug, pifithrin-α. Together, these findings provide an essential foundation for the utilization of brain organoids to study intact and disordered human brain network formation and illustrate their utility in therapeutic discovery.
Assuntos
Encéfalo/fisiopatologia , Epilepsia/fisiopatologia , Neurônios , Adulto , Benzotiazóis/farmacologia , Encéfalo/crescimento & desenvolvimento , Sinalização do Cálcio , Pré-Escolar , Epilepsia/diagnóstico por imagem , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas , Proteína 2 de Ligação a Metil-CpG/genética , Rede Nervosa/fisiopatologia , Neurogênese/genética , Neuroimagem , Síndrome de Rett/diagnóstico por imagem , Síndrome de Rett/fisiopatologia , Análise de Célula Única , Sinapses , Tolueno/análogos & derivados , Tolueno/farmacologia , TranscriptomaRESUMO
Mutations in the methyl-CpG binding protein 2 (MECP2) gene cause Rett syndrome (RTT), an X-linked neurodevelopmental disorder predominantly impacting females. MECP2 is an epigenetic transcriptional regulator acting mainly to repress gene expression, though it plays multiple gene regulatory roles and has distinct molecular targets across different cell types and specific developmental stages. In this review, we summarize MECP2 loss-of-function associated transcriptome and proteome disruptions, delving deeper into the latter which have been comparatively severely understudied. These disruptions converge on multiple biochemical and cellular pathways, including those involved in synaptic function and neurodevelopment, NF-κB signaling and inflammation, and the vitamin D pathway. RTT is a complex neurological disorder characterized by myriad physiological disruptions, in both the central nervous system and peripheral systems. Thus, treating RTT will likely require a combinatorial approach, targeting multiple nodes within the interactomes of these cellular pathways. To this end, we discuss the use of dietary supplements and factors, namely, vitamin D and polyunsaturated fatty acids (PUFAs), as possible partial therapeutic agents given their demonstrated benefit in RTT and their ability to restore homeostasis to multiple disrupted cellular pathways simultaneously. Further unravelling the complex molecular alterations induced by MECP2 loss-of-function, and contextualizing them at the level of proteome homeostasis, will identify new therapeutic avenues for this complex disorder.
Assuntos
Proteína 2 de Ligação a Metil-CpG/genética , Proteômica , Síndrome de Rett/genética , Síndrome de Rett/terapia , Transcrição Gênica/genética , Animais , HumanosRESUMO
Rett syndrome (RTT) is a severe, progressive X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MECP2 We previously identified aberrant NF-κB pathway upregulation in brains of Mecp2-null mice and demonstrated that genetically attenuating NF-κB rescues some characteristic neuronal RTT phenotypes. These results raised the intriguing question of whether NF-κB pathway inhibitors might provide a therapeutic avenue in RTT. Here, we investigate whether the known NF-κB pathway inhibitor vitamin D ameliorates neuronal phenotypes in Mecp2-mutant mice. Vitamin D deficiency is prevalent among RTT patients, and we find that Mecp2-null mice similarly have significantly reduced 25(OH)D serum levels compared with wild-type littermates. We identify that vitamin D rescues aberrant NF-κB pathway activation and reduced neurite outgrowth of Mecp2 knock-down cortical neurons in vitro Further, dietary supplementation with vitamin D in early symptomatic male Mecp2 hemizygous null and female Mecp2 heterozygous mice ameliorates reduced neocortical dendritic morphology and soma size phenotypes and modestly improves reduced lifespan of Mecp2-nulls. These results elucidate fundamental neurobiology of RTT and provide foundation that NF-κB pathway inhibition might be a therapeutic target for RTT.
Assuntos
Síndrome de Rett , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B , Fenótipo , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/genética , Vitamina DRESUMO
MECP2 (Methyl-CpG-binding protein 2) has been shown to have a critical role in regulating DNA methylation against smoke exposed lung injury. However, the biological function of MECP2 and the underlying molecular mechanism remains elusive. Human bronchial epithelial (16HBE) and alveolar type II epithelial cells (AECII) were exposed to increasing concentrations of cigarette smoke extracts (CSE) solution to establish CSE-induced lung epithelial cell injury models. Our findings revealed that MECP2 was down-regulated, while CYP1B1 was up-regulated in CSE-induced lung epithelial cell injury models by quantitative real time PCR, western blotting and immunoï¬uorescence staining. Down-regulated CYP1B1 was ascribed to the demethylation of its promoter by methylation-specific PCR (MSP). The in vitro experiments further showed that MECP2 overexpression significantly attenuated CSE-triggered cell growth attenuation, cell cycle arrest, apoptosis and ROS generation in lung epithelial cells by CCK-8 and flow cytometry assays. In molecular level, we further demonstrated that MECP2 overexpression obviously suppressed the expression of CYP1B1 through enhancing DNA methylation. Therefore, our data suggest that MECP2 protects against CSE-induced lung epithelial cell injury possibly through down-regulating CYP1B1 expression via elevating its methylation status.
Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Fumar Cigarros/efeitos adversos , Citocromo P-450 CYP1B1/metabolismo , Metilação de DNA , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Expressão Gênica , Pulmão/citologia , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Nicotiana/química , Extratos Vegetais/efeitos adversos , Lesão Pulmonar Aguda/patologia , Células Cultivadas , Citocromo P-450 CYP1B1/genética , Humanos , Extratos Vegetais/isolamento & purificaçãoRESUMO
Dendritic atrophy, defined as the reduction in complexity of the neuronal arborization, is a hallmark of several neurodevelopmental disorders, including Rett Syndrome (RTT). RTT, affecting 1:10,000 girls worldwide, is mainly caused by mutations in the MECP2 gene and has no cure. We describe here an in vitro model of dendritic atrophy in Mecp2-/y mouse hippocampal primary cultures, suitable for phenotypic drug-screening. Using High-Content Imaging techniques, we systematically investigated the impact of culturing determinants on several parameters such as neuronal survival, total dendritic length, dendritic endpoints, soma size, cell clusterization, spontaneous activity. Determinants included cell-seeding density, glass or polystyrene substrates, coating with poly-Ornithine with/without Matrigel and miniaturization from 24 to 96-half surface multiwell plates. We show that in all plate-sizes at densities below 320 cells/mm2, morphological parameters remained constant while spontaneous network activity decreased according to the cell-density. Mecp2-/y neurons cultured at 160 cells/mm2 density in 96 multiwell plates, displayed significant dendritic atrophy and showed a marked increase in dendritic length following treatment with Brain-derived neurotrophic factor (BDNF) or Mirtazapine. In conclusion, we have established a phenotypic assay suitable for fast screening of hundreds of compounds, which may be extended to other neurodevelopmental diseases with dendritic atrophy.
Assuntos
Dendritos/patologia , Avaliação Pré-Clínica de Medicamentos/métodos , Fármacos Neuroprotetores/farmacologia , Fenótipo , Síndrome de Rett/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células Cultivadas , Dendritos/efeitos dos fármacos , Hipocampo/citologia , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Endogâmicos C57BL , Mirtazapina/farmacologia , Síndrome de Rett/patologiaRESUMO
Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the Methyl CpG binding protein 2 (MeCP2) gene. This Science & Society article focuses on pharmacological strategies that attack RTT treatment from multiple angles, including drug repurposing and de novo discovery efforts, and discusses the impacts of preclinical study design and translationally relevant outcome measures.
Assuntos
Descoberta de Drogas/métodos , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/tratamento farmacológico , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos , Feminino , Humanos , Mutação , Projetos de Pesquisa , Síndrome de Rett/genética , Síndrome de Rett/fisiopatologiaRESUMO
How genomic DNA methylation and methyl CpG-binding protein 2 (MeCP2) gene expression affect the pathogenesis of systemic lupus erythematosus (SLE) remains poorly understood. Traditional Chinese medicine has a unique effect in the treatment of SLE patients. This study aimed to investigate the effect of Jieduquyuziyin prescription (JP)-treated rat serum on the gene expression of MeCP2 in Jurkat T cells and its role in the pathogenesis of SLE. Jurkat T cells were harvested, and drug-containing serum was prepared. The ferulic acid and paeoniflorin content in the drug-containing serum were determined by liquid chromatography-mass spectrometry (LC-MS/MS). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays were used to screen the optimal concentration of drug-containing serum. The DNA methylation level in Jurkat T cells was detected with a Methylamp™ Total DNA Methylation Kit. The methylation status of the MeCP2 promoter region was detected using bisulfite modification and methylation-specific PCR (MSP). Real-time PCR was used to measure MeCP2 mRNA expression. Western blotting and flow cytometry were done to detect MeCP2 protein expression in Jurkat cell nuclei. Paeoniflorin and ferulic acid were detected in the drug-containing serum of JP-treated rats. The results showed that cell growth was affected in the high serum-containing drug group. The experimental results showed that JP and prednisone acetate increased the level of genomic DNA methylation and MeCP2 gene promoter region methylation in Jurkat cells. MeCP2 mRNA and protein levels were also increased in the JP and prednisone acetate groups. Furthermore, flow cytometry revealed that the expression of MeCP2 protein in Jurkat T cell nuclei was higher in the drug group than the blank control group, and these results were consistent with the western blot analysis results. Our study found that there is a negative correlation between drug-containing serum and cell survival rate. JP upregulated the levels of DNA methylation, MeCP2 mRNA and protein as effectively as prednisone acetate and thus may activate the MeCP2 gene by increasing the methylation level, thereby inhibiting the pathogenesis of SLE. Therefore, JP may potentially be used to treat SLE patients. The Jurkat T lymphocyte in vitro experiments provided a foundation to study the effects of JP on the lupus mouse CD4+ T cell methylation mechanism and to further explore the pathogenesis of SLE.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Proteína 2 de Ligação a Metil-CpG/genética , Soro/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/genética , Humanos , Células Jurkat , Proteína 2 de Ligação a Metil-CpG/metabolismo , Prednisona/farmacologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-DawleyRESUMO
Rett syndrome (RTT) is a rare neurodevelopmental disorder, characterized by severe behavioural and physiological symptoms. RTT is caused by mutations in the MECP2 gene in about 95% of cases and to date no cure is available. The endocannabinoid system modulates several physiological processes and behavioural responses that are impaired in RTT and its deregulation has been associated with neuropsychiatric disorders which have symptoms in common with RTT. The present study evaluated the potential therapeutic efficacy for RTT of cannabidivarin (CBDV), a non-psychotropic phytocannabinoid from Cannabis sativa that presents antagonistic properties on the G protein-coupled receptor 55 (GPR55), the most recently identified cannabinoid receptor. Present results demonstrate that systemic treatment with CBDV (2, 20, 100 mg/Kg ip for 14 days) rescues behavioural and brain alterations in MeCP2-308 male mice, a validated RTT model. The CBDV treatment restored the compromised general health status, the sociability and the brain weight in RTT mice. A partial restoration of motor coordination was also observed. Moreover, increased levels of GPR55 were found in RTT mouse hippocampus, suggesting this G protein-coupled receptor as new potential target for the treatment of this disorder. Present findings highlight for the first time for RTT the translational relevance of CBDV, an innovative therapeutic agent that is under active investigation in the clinical setting.
Assuntos
Encéfalo/patologia , Canabinoides/administração & dosagem , Canabinoides/uso terapêutico , Fitoterapia/métodos , Síndrome de Rett/tratamento farmacológico , Animais , Ataxia/tratamento farmacológico , Atrofia/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Canabinoides/farmacologia , Relação Dose-Resposta a Droga , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Transgênicos , Tamanho do Órgão/efeitos dos fármacos , Receptores de Canabinoides/metabolismo , Síndrome de Rett/patologia , Síndrome de Rett/psicologia , Comportamento SocialRESUMO
Fine-tuning of the body's water balance is regulated by vasopressin (AVP), which induces the expression and apical membrane insertion of aquaporin-2 water channels and subsequent water reabsorption in the kidney. Here we demonstrate that silencing of microRNA-132 (miR-132) in mice causes severe weight loss due to acute diuresis coinciding with increased plasma osmolality, reduced renal total and plasma membrane expression of aquaporin-2, and abrogated increase in AVP levels. Infusion with synthetic AVP fully reversed the antagomir-132-induced diuresis, and low-dose intracerebroventricular administration of antagomir-132 similarly caused acute diuresis. Central and intracerebroventricular antagomir-132 injection both decreased hypothalamic AVP mRNA levels. At the molecular level, antagomir-132 increased the in vivo and in vitro mRNA expression of methyl-CpG-binding protein-2 (MECP2), which is a miR-132 target and which blocks AVP gene expression by binding its enhancer region. In line with this, treatment of hypothalamic N6 cells with a high-salt solution increased its miR-132 levels, whereas it attenuated endogenous Mecp2 mRNA levels. In conclusion, we identified miR-132 as a first miRNA regulating the osmotic balance by regulating the hypothalamic AVP gene mRNA expression.
Assuntos
Arginina Vasopressina/metabolismo , Homeostase/fisiologia , Proteína 2 de Ligação a Metil-CpG/genética , MicroRNAs/genética , Vasopressinas/metabolismo , Animais , Aquaporina 2/metabolismo , Expressão Gênica/genética , Hipotálamo/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Concentração Osmolar , Receptores de Vasopressinas/metabolismo , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
BACKGROUND: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MeCP2. Much of our understanding of MeCP2 function is derived from transcriptomic studies with the general assumption that alterations in the transcriptome correlate with proteomic changes. Advances in mass spectrometry-based proteomics have facilitated recent interest in the examination of global protein expression to better understand the biology between transcriptional and translational regulation. METHODS: We therefore performed the first comprehensive transcriptome-proteome comparison in a RTT mouse model to elucidate RTT pathophysiology, identify potential therapeutic targets, and further our understanding of MeCP2 function. The whole cortex of wild-type and symptomatic RTT male littermates (n = 4 per genotype) were analyzed using RNA-sequencing and data-independent acquisition liquid chromatography tandem mass spectrometry. Ingenuity® Pathway Analysis was used to identify significantly affected pathways in the transcriptomic and proteomic data sets. RESULTS: Our results indicate these two "omics" data sets supplement one another. In addition to confirming previous works regarding mRNA expression in Mecp2-deficient animals, the current study identified hundreds of novel protein targets. Several selected protein targets were validated by Western blot analysis. These data indicate RNA metabolism, proteostasis, monoamine metabolism, and cholesterol synthesis are disrupted in the RTT proteome. Hits common to both data sets indicate disrupted cellular metabolism, calcium signaling, protein stability, DNA binding, and cytoskeletal cell structure. Finally, in addition to confirming disrupted pathways and identifying novel hits in neuronal structure and synaptic transmission, our data indicate aberrant myelination, inflammation, and vascular disruption. Intriguingly, there is no evidence of reactive gliosis, but instead, gene, protein, and pathway analysis suggest astrocytic maturation and morphological deficits. CONCLUSIONS: This comparative omics analysis supports previous works indicating widespread CNS dysfunction and may serve as a valuable resource for those interested in cellular dysfunction in RTT.
Assuntos
Córtex Cerebral/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Proteoma/metabolismo , Proteômica , RNA/metabolismo , Síndrome de Rett/genética , Animais , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Feminino , Genótipo , Masculino , Proteína 2 de Ligação a Metil-CpG/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Fenótipo , Proteoma/análise , RNA/química , RNA/isolamento & purificação , Síndrome de Rett/patologia , Análise de Sequência de RNA , Espectrometria de Massas em Tandem , TranscriptomaRESUMO
Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). RTT is the second most prevalent cause of intellectual disability in girls and there is currently no cure for the disease. The finding that the deficits caused by the loss of Mecp2 are reversible in the mouse has bolstered interest in gene therapy as a cure for RTT. In order to assess the feasibility of gene therapy in a RTT mouse model, and in keeping with translational goals, we investigated the efficacy of a self-complementary AAV9 vector expressing a codon-optimized version of Mecp2 (AAV9-MCO) delivered via a systemic approach in early symptomatic Mecp2-deficient (KO) mice. Our results show that AAV9-MCO administered at a dose of 2×1011 viral genome (vg)/mouse was able to significantly increase survival and weight gain, and delay the occurrence of behavioral deficits. Apneas, which are one of the core RTT breathing deficits, were significantly decreased to WT levels in Mecp2 KO mice after AAV9-MCO administration. Semi-quantitative analysis showed that AAV9-MCO administration in Mecp2 KO mice resulted in 10 to 20% Mecp2 immunopositive cells compared to WT animals, with the highest Mecp2 expression found in midbrain regions known to regulate cardio-respiratory functions. In addition, we also found a cell autonomous increase in tyrosine hydroxylase levels in the A1C1 and A2C2 catecholaminergic Mecp2+ neurons in treated Mecp2 KO mice, which may partly explain the beneficial effect of AAV9-MCO administration on apneas occurrence.
Assuntos
Terapia Genética , Proteína 2 de Ligação a Metil-CpG/administração & dosagem , Síndrome de Rett/terapia , Aminas , Animais , Apneia/metabolismo , Apneia/patologia , Apneia/prevenção & controle , Códon , Ácidos Cicloexanocarboxílicos , Dependovirus , Modelos Animais de Doenças , Progressão da Doença , Gabapentina , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Respiração , Síndrome de Rett/metabolismo , Síndrome de Rett/patologia , Análise de Sobrevida , Tirosina 3-Mono-Oxigenase/metabolismo , Aumento de Peso , Ácido gama-AminobutíricoRESUMO
BACKGROUND: Mutations in the cyclin-dependent kinase-like 5 gene cause a clinical variant of Rett syndrome (CDKL5-RTT). A role for the acute-phase response (APR) is emerging in typical RTT caused by methyl-CpG-binding protein 2 gene mutations (MECP2-RTT). No information is, to date, available on the inflammatory protein response in CDKL5-RTT. We evaluated, for the first time, the APR protein response in CDKL5-RTT. METHODS: Protein patterns in albumin- and IgG-depleted plasma proteome from CDKL5-RTT patients were evaluated by two-dimensional gel electrophoresis/mass spectrometry. The resulting data were related to circulating cytokines and compared to healthy controls or MECP2-RTT patients. The effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) were evaluated. RESULTS: CDKL5-RTT mutations resulted in a subclinical attenuated inflammation, specifically characterized by an overexpression of the complement component C3 and CD5 antigen-like, both strictly related to the inflammatory response. Cytokine dysregulation featuring a bulk increase of anti-inflammatory cytokines, predominantly IL-10, could explain the unchanged erythrocyte sedimentation rate and atypical features of inflammation in CDKL5-RTT. Omega-3 PUFAs were able to counterbalance the pro-inflammatory status. CONCLUSION: For the first time, we revealed a subclinical smouldering inflammation pattern in CDKL5-RTT consisting in the coexistence of an atypical APR coupled with a dysregulated cytokine response.
Assuntos
Reação de Fase Aguda/imunologia , Citocinas/imunologia , Síndrome de Rett/imunologia , Espasmos Infantis/imunologia , Reação de Fase Aguda/genética , Reação de Fase Aguda/metabolismo , Adolescente , Proteínas Sanguíneas/imunologia , Proteínas Sanguíneas/metabolismo , Criança , Pré-Escolar , Citocinas/sangue , Suplementos Nutricionais , Síndromes Epilépticas , Ácidos Graxos Ômega-3/farmacologia , Feminino , Humanos , Lactente , Proteína 2 de Ligação a Metil-CpG/genética , Proteínas Serina-Treonina Quinases/genética , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Espasmos Infantis/genética , Espasmos Infantis/metabolismoRESUMO
Rett syndrome (RTT) is a postnatal neurodevelopmental disorder that primarily affects girls. Mutations in the methyl-CpG-binding protein 2 (MECP2) gene account for approximately 95 % of all RTT cases. To model RTT in vitro, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of two RTT patients with different mutations (MECP2 (R306C) and MECP2 (1155Δ32)) in their MECP2 gene. We found that these iPSCs were capable of differentiating into functional neurons. Compared to control neurons, the RTT iPSC-derived cells had reduced soma size and a decreased amount of synaptic input, evident both as fewer Synapsin 1-positive puncta and a lower frequency of spontaneous excitatory postsynaptic currents. Supplementation of the culture media with choline rescued all of these defects. Choline supplementation may act through changes in the expression of choline acetyltransferase, an important enzyme in cholinergic signaling, and also through alterations in the lipid metabolite profiles of the RTT neurons. Our study elucidates the possible mechanistic pathways for the effect of choline on human RTT cell models, thereby illustrating the potential for using choline as a nutraceutical to treat RTT.
Assuntos
Colina/farmacologia , Suplementos Nutricionais , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Síndrome de Rett/terapia , Feminino , Humanos , Técnicas In Vitro , Proteína 2 de Ligação a Metil-CpG/genética , MutaçãoRESUMO
Rett syndrome (RTT) is a neurodevelopmental disorder that results from mutations in the X-linked gene for methyl-CpG-binding protein 2 (MECP2). The underlying cellular mechanism for the sensory deficits in patients with RTT is largely unknown. This study used the Bird mouse model of RTT to investigate sensory thalamocortical synaptic transmission in the barrel cortex of Mecp2-null mice. Electrophysiological results showed an excitation/inhibition imbalance, biased toward inhibition, due to an increase in efficacy of postsynaptic GABAA receptors rather than alterations in inhibitory network and presynaptic release properties. Enhanced inhibition impaired the transmission of tonic sensory signals from the thalamus to the somatosensory cortex. Previous morphological studies showed an upregulation of NMDA receptors in the neocortex of both RTT patients and Mecp2-null mice at early ages [Blue ME, Naidu S, Johnston MV. Ann Neurol 45: 541-545, 1999; Blue ME, Kaufmann WE, Bressler J, Eyring C, O'Driscoll C, Naidu S, Johnston MV. Anat Rec (Hoboken) 294: 1624-1634, 2011]. Although AMPA and NMDA receptor-mediated excitatory synaptic transmission was not altered in the barrel cortex of Mecp2-null mice, extrasynaptic NMDA receptor-mediated responses increased markedly. These responses were blocked by memantine, suggesting that extrasynaptic NMDA receptors play an important role in the pathogenesis of RTT. The results suggest that enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses may underlie impaired somatosensation and that pharmacological blockade of extrasynaptic NMDA receptors may have therapeutic value for RTT.
Assuntos
Proteína 2 de Ligação a Metil-CpG/deficiência , Receptores de N-Metil-D-Aspartato/metabolismo , Síndrome de Rett/metabolismo , Córtex Somatossensorial/metabolismo , Animais , Feminino , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores de GABA-A/metabolismo , Síndrome de Rett/genética , Córtex Somatossensorial/fisiologia , Sinapses/metabolismo , Sinapses/fisiologia , Potenciais Sinápticos , Tálamo/metabolismo , Tálamo/fisiologiaRESUMO
Geniposide (GP), an iridoid glucoside extracted from Gardenia jasminoides Ellis fruits, has been used as a herbal medicine to treat liver and gall bladder disorders for many years. However the mechanism of anti-inflammatory is largely unknown. In this study, GP significantly attenuated inflammation in acute liver injury (ALI) mice model and in lipopolysaccharide (LPS)-induced THP-1 cells. It was demonstrated that GP obviously decreased the expression of Methyl-CpG binding protein 2 (MeCP2) in vivo and in vitro. Knockdown of MeCP2 with siRNA suppressed the expressions of IL-6 and TNF-α, while over-expression of MeCP2 had a proinflammatory effect on the expression of IL-6 and TNF-α in LPS-induced THP-1 cells. Mechanistically, it was indicated that GP had anti-inflammatory effects at least in part, through suppressing MeCP2. Interestingly, GP could attenuate expressions of Sonic hedgehog (Shh) and GLIS family zinc finger 1 (GLIS1) but increase Ptched1 (PTCH1) expression. Similar findings were also demonstrated at the protein level by siRNA MeCP2. Furthermore, over-expression of MeCP2 obviously increased Shh and GLIS1 expressions but reduced PTCH1 expression. Taken together, GP may serve as an effective modulator of MeCP2-hedgehog pathway (Hh)-axis during the pathogenesis of inflammation. Our findings shed light on the potential therapeutic feature of GP in recovering inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Intoxicação por Tetracloreto de Carbono/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Inflamação/tratamento farmacológico , Iridoides/farmacologia , Proteína 2 de Ligação a Metil-CpG/genética , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Intoxicação por Tetracloreto de Carbono/patologia , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/patologia , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteínas Hedgehog/biossíntese , Proteínas Hedgehog/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Iridoides/uso terapêutico , Fígado/patologia , Proteína 2 de Ligação a Metil-CpG/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
An involvement of the immune system has been suggested in Rett syndrome (RTT), a devastating neurodevelopmental disorder related to oxidative stress, and caused by a mutation in the methyl-CpG binding protein 2 gene (MECP2) or, more rarely, cyclin-dependent kinase-like 5 (CDKL5). To date, it is unclear whether both mutations may have an impact on the circulating cytokine patterns. In the present study, cytokines involved in the Th1-, Th2-, and T regulatory (T-reg) response, as well as chemokines, were investigated in MECP2- (MECP2-RTT) (n = 16) and CDKL5-Rett syndrome (CDKL5-RTT) (n = 8), before and after ω-3 polyunsaturated fatty acids (PUFAs) supplementation. A major cytokine dysregulation was evidenced in untreated RTT patients. In MECP2-RTT, a Th2-shifted balance was evidenced, whereas in CDKL5-RTT both Th1- and Th2-related cytokines (except for IL-4) were upregulated. In MECP2-RTT, decreased levels of IL-22 were observed, whereas increased IL-22 and T-reg cytokine levels were evidenced in CDKL5-RTT. Chemokines were unchanged. The cytokine dysregulation was proportional to clinical severity, inflammatory status, and redox imbalance. Omega-3 PUFAs partially counterbalanced cytokine changes, as well as aberrant redox homeostasis and the inflammatory status. RTT is associated with a subclinical immune dysregulation as the likely consequence of a defective inflammation regulatory signaling system.
Assuntos
Citocinas/análise , Ácidos Graxos Ômega-3/farmacologia , Inflamação , Proteína 2 de Ligação a Metil-CpG/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Síndrome de Rett/patologia , Adolescente , Adulto , Sedimentação Sanguínea/efeitos dos fármacos , Criança , Pré-Escolar , Citocinas/sangue , Suplementos Nutricionais , Ensaio de Imunoadsorção Enzimática , Eritrócitos/citologia , Ácidos Graxos Ômega-3/uso terapêutico , Feminino , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Polimorfismo de Nucleotídeo Único , Proteínas Serina-Treonina Quinases/genética , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/metabolismo , Índice de Gravidade de Doença , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Células Th1/citologia , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th2/citologia , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Adulto JovemRESUMO
Methyl CpG binding protein 2 (MeCP2) is a multifunctional protein which binds to methylated CpG, mutation of which cause a neurodevelopmental disorder, Rett syndrome. MeCP2 can function as both transcriptional activator and repressor of target gene. MeCP2 regulate gene expression in both neuron and glial cells in central nervous system (CNS). Oligodendrocytes, the myelinating cells of CNS, are required for normal functioning of neurons and are regulated by several transcription factors during their differentiation. In current study, we focused on the role of MeCP2 as transcription regulator of myelin genes in cultured rat oligodendrocytes. We have observed expression of MeCP2 at all stages of oligodendrocyte development. MeCP2 knockdown in cultured oligodendrocytes by small interference RNA (siRNA) has shown increase in myelin genes (myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), and myelin-associated oligodendrocyte basic protein (MOBP)), neurotrophin (brain-derived neurotrophic factor (BDNF)), and transcriptional regulator (YY1) transcripts level, which are involved in regulation of oligodendrocyte differentiation and myelination. Further, we also found that protein levels of MBP, PLP, DM-20, and BDNF also significantly upregulated in MeCP2 knockdown oligodendrocytes. Our study suggests that the MeCP2 acts as a negative regulator of myelin protein expression.