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1.
Int J Mol Sci ; 20(10)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117273

RESUMO

Chromatin modifiers play a crucial role in maintaining cell identity through modulation of gene expression patterns. Their deregulation can have profound effects on cell fate and functions. Among epigenetic regulators, the MECP2 protein is particularly attractive. Mutations in the Mecp2 gene are responsible for more than 90% of cases of Rett syndrome (RTT), a progressive neurodevelopmental disorder. As a chromatin modulator, MECP2 can have a key role in the government of stem cell biology. Previously, we showed that deregulated MECP2 expression triggers senescence in mesenchymal stromal cells (MSCs) from (RTT) patients. Over the last few decades, it has emerged that senescent cells show alterations in the metabolic state. Metabolic changes related to stem cell senescence are particularly detrimental, since they contribute to the exhaustion of stem cell compartments, which in turn determine the falling in tissue renewal and functionality. Herein, we dissect the role of impaired MECP2 function in triggering senescence along with other senescence-related aspects, such as metabolism, in MSCs from a mouse model of RTT. We found that MECP2 deficiencies lead to senescence and impaired mitochondrial energy production. Our results support the idea that an alteration in mitochondria metabolic functions could play an important role in the pathogenesis of RTT.


Assuntos
Senescência Celular , Proteína 2 de Ligação a Metil-CpG/genética , Mitocôndrias/metabolismo , Mutação , Síndrome de Rett/metabolismo , Animais , Reparo do DNA , Modelos Animais de Doenças , Feminino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Síndrome de Rett/fisiopatologia
2.
PLoS Genet ; 15(4): e1008043, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30973874

RESUMO

Mounting evidence supports that LINE-1 (L1) retrotransposition can occur postzygotically in healthy and diseased human tissues, contributing to genomic mosaicism in the brain and other somatic tissues of an individual. However, the genomic distribution of somatic human-specific LINE-1 (L1Hs) insertions and their potential impact on carrier cells remain unclear. Here, using a PCR-based targeted bulk sequencing approach, we profiled 9,181 somatic insertions from 20 postmortem tissues from five Rett patients and their matched healthy controls. We identified and validated somatic L1Hs insertions in both cortical neurons and non-brain tissues. In Rett patients, somatic insertions were significantly depleted in exons-mainly contributed by long genes-than healthy controls, implying that cells carrying MECP2 mutations might be defenseless against a second exonic L1Hs insertion. We observed a significant increase of somatic L1Hs insertions in the brain compared with non-brain tissues from the same individual. Compared to germline insertions, somatic insertions were less sense-depleted to transcripts, indicating that they underwent weaker selective pressure on the orientation of insertion. Our observations demonstrate that somatic L1Hs insertions contribute to genomic diversity and MeCP2 dysfunction alters their genomic patterns in Rett patients.


Assuntos
Elementos Nucleotídeos Longos e Dispersos , Síndrome de Rett/genética , Adolescente , Adulto , Sequência de Bases , Encéfalo/metabolismo , Estudos de Casos e Controles , Córtex Cerebral/metabolismo , Feminino , Mutação em Linhagem Germinativa , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Mosaicismo , Mutação , Neurônios/metabolismo , Síndrome de Rett/metabolismo , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual , Transcrição Genética , Adulto Jovem
3.
Proc Natl Acad Sci U S A ; 116(14): 7071-7076, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30890637

RESUMO

Parvalbumin-positive (PV+) interneurons play a pivotal role in orchestrating windows of experience-dependent brain plasticity during development. Critical period closure is marked by the condensation of a perineuronal net (PNN) tightly enwrapping subsets of PV+ neurons, both acting as a molecular brake on plasticity and maintaining mature PV+ cell signaling. As much of the molecular organization of PNNs exists at length scales near or below the diffraction limit of light microscopy, we developed a superresolution imaging and analysis platform to visualize the structural organization of PNNs and the synaptic inputs perforating them in primary visual cortex. We identified a structural trajectory of PNN maturation featuring a range of net structures, which was accompanied by an increase in Synaptotagmin-2 (Syt2) signals on PV+ cells suggestive of increased inhibitory input between PV+ neurons. The same structural trajectory was followed by PNNs both during normal development and under conditions of critical period delay by total sensory deprivation or critical period acceleration by deletion of MeCP2, the causative gene for Rett syndrome, despite shifted maturation levels under these perturbations. Notably, superresolution imaging further revealed a decrease in Syt2 signals alongside an increase in vesicular glutamate transporter-2 signals on PV+ cells in MeCP2-deficient animals, suggesting weaker recurrent inhibitory input between PV+ neurons and stronger thalamocortical excitatory inputs onto PV+ cells. These results imply a latent imbalanced circuit signature that might promote cortical silencing in Rett syndrome before the functional regression of vision.


Assuntos
Proteína 2 de Ligação a Metil-CpG/metabolismo , Rede Nervosa/metabolismo , Plasticidade Neuronal , Síndrome de Rett/metabolismo , Sinapses/metabolismo , Sinaptotagmina II/metabolismo , Córtex Visual/metabolismo , Animais , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Rede Nervosa/diagnóstico por imagem , Síndrome de Rett/diagnóstico por imagem , Síndrome de Rett/genética , Sinapses/genética , Sinaptotagmina II/genética , Córtex Visual/diagnóstico por imagem
4.
Cells ; 8(2)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30781346

RESUMO

Rett syndrome (RTT) is a human neurodevelopmental disorder, whose pathogenesis has been linked to both oxidative stress and subclinical inflammatory status (OxInflammation). Methylglyoxal (MG), a glycolytic by-product with cytotoxic and pro-oxidant power, is the major precursor in vivo of advanced glycation end products (AGEs), which are known to exert their detrimental effect via receptor- (e.g., RAGE) or non-receptor-mediated mechanisms in several neurological diseases. On this basis, we aimed to compare fibroblasts from healthy subjects (CTR) with fibroblasts from RTT patients (N = 6 per group), by evaluating gene/protein expression patterns, and enzymatic activities of glyoxalases (GLOs), along with the levels of MG-dependent damage in both basal and MG-challenged conditions. Our results revealed that RTT is linked to an alteration of the GLOs system (specifically, increased GLO2 activity), that ensures unchanged MG-dependent damage levels. However, RTT cells underwent more pronounced cell death upon exogenous MG-treatment, as compared to CTR, and displayed lower RAGE levels than CTR, with no alterations following MG-treatment, thus suggesting that an adaptive response to dicarbonyl stress may occur. In conclusion, besides OxInflammation, RTT is associated with reshaping of the major defense systems against dicarbonyl stress, along with an altered cellular stress response towards pro-glycating insults.


Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Síndrome de Rett/metabolismo , Adolescente , Adulto , Sobrevivência Celular/efeitos dos fármacos , Criança , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glicosilação , Humanos , Lactoilglutationa Liase/metabolismo , Aldeído Pirúvico/farmacologia , Síndrome de Rett/patologia , Tioléster Hidrolases/metabolismo , Adulto Jovem
5.
Neurosci Biobehav Rev ; 98: 154-163, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30639673

RESUMO

Rett syndrome (RTT) is a neurodevelopmental disorder affecting mostly girls. A seemingly normal initial development is followed by developmental stagnation and regression, leading to severe mental impairment with autistic features, motor dysfunction, irregular breathing and epilepsy. Currently, a cure does not exist. Due to the close association of RTT with mitochondrial alterations, cellular redox-impairment and oxidative stress, compounds stabilizing mitochondrial function, cellular redox-homeostasis, and oxidant detoxification are increasingly considered as treatment concepts. Indeed, antioxidants and free-radical scavengers ameliorate certain aspects of the complex and severe clinical presentation of RTT. To further evaluate these strategies, reliable biosensors are needed to quantify redox-conditions in brain and peripheral organs of mouse models or in patient-derived cells. Genetically-encoded redox-sensors meet these requirements. Expressed in transgenic mouse-models such as our unique Rett-redox indicator mice, they will report for any cell type desired the severity of oxidant stress throughout the various disease stages of RTT. Furthermore, these sensors will be crucial to evaluate in vitro and in vivo the outcome of mitochondria- and redox-balance targeted treatments.


Assuntos
Encéfalo/fisiopatologia , Homeostase/fisiologia , Estresse Oxidativo/fisiologia , Síndrome de Rett/fisiopatologia , Animais , Modelos Animais de Doenças , Humanos , Mitocôndrias/metabolismo , Síndrome de Rett/metabolismo
6.
Interdiscip Sci ; 11(1): 10-20, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30673959

RESUMO

MeCP2 is a protein highly expressed in the brain that participates in the genetic expression and RNA splicing regulation. MeCP2 binds preferably to methylated DNA and other nuclear corepressors to alter chromatin. MECP2 gene mutations can cause rett syndrome (RTT), a severe neurological disorder that affects around one in ten thousand girls. In this paper, Molecular Dynamics (MD) simulations were performed to scrutinize how the MeCP2 P152R mutation influences the protein binding to DNA. Also, the Umbrella Sampling technique was used to obtain the potential mean forces (PMFs) of both wild-type and mutated MeCP2 Methyl-CpG-binding domain (MBD) binding to both non-methylated and methylated DNA. P152R is a common missense mutation in MBD associated with RTT; however, there are no studies that explain how it causes protein dysfunction. The results from this study hypothesize that P152R mutation leads to MBD binding more strongly to DNA, while selectively decreasing binding affinity to methylated DNA. These provide an explanation for previous not conclusive experimental results regarding the mechanism of how this mutation affects the binding of the protein to DNA, and subsequently its effects on RTT. Furthermore, the results of this research-in-progress can be used as the basis for further investigations into the molecular basis of RTT and to potentially reveal a target for therapy in the future.


Assuntos
DNA/genética , Proteína 2 de Ligação a Metil-CpG/genética , Mutação de Sentido Incorreto , Síndrome de Rett/genética , DNA/metabolismo , Humanos , Proteína 2 de Ligação a Metil-CpG/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Síndrome de Rett/metabolismo
7.
Brain ; 142(2): 239-248, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649225

RESUMO

With the recent 50th anniversary of the first publication on Rett syndrome, and the almost 20 years since the first report on the link between Rett syndrome and MECP2 mutations, it is important to reflect on the tremendous advances in our understanding and their implications for the diagnosis and treatment of this neurodevelopmental disorder. Rett syndrome features an interesting challenge for biologists and clinicians, as the disorder lies at the intersection of molecular mechanisms of epigenetic regulation and neurophysiological alterations in synapses and circuits that together contribute to severe pathophysiological endophenotypes. Genetic, clinical, and neurobiological evidences support the notion that Rett syndrome is primarily a synaptic disorder, and a disease model for both intellectual disability and autism spectrum disorder. This review examines major developments in both recent neurobiological and preclinical findings of Rett syndrome, and to what extent they are beginning to impact our understanding and management of the disorder. It also discusses potential applications of knowledge on synaptic plasticity abnormalities in Rett syndrome to its diagnosis and treatment.


Assuntos
Plasticidade Neuronal/fisiologia , Síndrome de Rett/diagnóstico , Síndrome de Rett/terapia , Sinapses/metabolismo , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Sinapses/patologia , Resultado do Tratamento
8.
Orphanet J Rare Dis ; 14(1): 13, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30630505

RESUMO

BACKGROUND: Conversion of human somatic cells into induced pluripotent stem cells (iPSCs) is often an inefficient, time consuming and expensive process. Also, the tendency of iPSCs to revert to their original somatic cell type over time continues to be problematic. A computational model of iPSCs identifying genes/molecules necessary for iPSC generation and maintenance could represent a crucial step forward for improved stem cell research. The combination of substantial genetic relationship data, advanced computing hardware and powerful nonlinear modeling software could make the possibility of artificially-induced pluripotent stem cells (aiPSC) a reality. We have developed an unsupervised deep machine learning technology, called DeepNEU that is based on a fully-connected recurrent neural network architecture with one network processing layer for each input. DeepNEU was used to simulate aiPSC systems using a defined set of reprogramming transcription factors. Genes/proteins that were reported to be essential in human pluripotent stem cells (hPSC) were used for system modelling. RESULTS: The Mean Squared Error (MSE) function was used to assess system learning. System convergence was defined at MSE < 0.001. The markers of human iPSC pluripotency (N = 15) were all upregulated in the aiPSC final model. These upregulated/expressed genes in the aiPSC system were entirely consistent with results obtained for iPSCs. CONCLUSION: This research introduces and validates the potential use of aiPSCs as computer models of human pluripotent stem cell systems. Disease-specific aiPSCs have the potential to improve disease modeling, prototyping of wet lab experiments, and prediction of genes relevant and necessary for aiPSC production and maintenance for both common and rare diseases in a cost-effective manner.


Assuntos
Reprogramação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Aprendizado de Máquina , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Reprogramação Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Neurônios , Doenças Raras/metabolismo , Síndrome de Rett/metabolismo
9.
Neuroscience ; 397: 107-115, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30458221

RESUMO

People with Rett Syndrome (RTT), a neurodevelopmental disorder caused by mutations in the MECP2 gene, have breathing abnormalities manifested as periodical hypoventilation with compensatory hyperventilation, which are attributable to a high incidence of sudden death. Similar breathing abnormalities have been found in animal models with Mecp2 disruptions. Although RTT-type hypoventilation is believed to be due to depressed central inspiratory activity, whether this is true remains unknown. Here we show evidence for reshaping in firing activity and patterns of medullary respiratory neurons in RTT-type hypoventilation without evident depression in inspiratory neuronal activity. Experiments were performed in decerebrate rats in vivo. In Mecp2-null rats, abnormalities in breathing patterns were apparent in both decerebrate rats and awake animals, suggesting that RTT-type breathing abnormalities take place in the brainstem without forebrain input. In comparison to their wild-type counterparts, both inspiratory and expiratory neurons in Mecp2-null rats extended their firing duration, and fired more action potentials during each burst. No changes in inspiratory or expiratory neuronal distributions were found. Most inspiratory neurons started firing in the middle of expiration and changed their firing pattern to a phase-spanning type. The proportion of post-inspiratory neurons was reduced in the Mecp2-null rats. With the increased firing activity of both inspiratory and expiratory neurons in null rats, phrenic discharges shifted to a slow and deep breathing pattern. Thus, the RTT-type hypoventilation appears to result from reshaping of firing activity of both inspiratory and expiratory neurons without evident depression in central inspiratory activity.


Assuntos
Potenciais de Ação/fisiologia , Bulbo/metabolismo , Proteína 2 de Ligação a Metil-CpG/deficiência , Neurônios/metabolismo , Respiração , Síndrome de Rett/metabolismo , Animais , Estado de Descerebração , Modelos Animais de Doenças , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Nervo Frênico/metabolismo , Ratos Sprague-Dawley , Ratos Transgênicos , Vigília
10.
Nat Neurosci ; 21(12): 1670-1679, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30455458

RESUMO

In females with X-linked genetic disorders, wild-type and mutant cells coexist within brain tissue because of X-chromosome inactivation, posing challenges for interpreting the effects of X-linked mutant alleles on gene expression. We present a single-nucleus RNA sequencing approach that resolves mosaicism by using single-nucleotide polymorphisms in genes expressed in cis with the X-linked mutation to determine which nuclei express the mutant allele even when the mutant gene is not detected. This approach enables gene expression comparisons between mutant and wild-type cells within the same individual, eliminating variability introduced by comparisons to controls with different genetic backgrounds. We apply this approach to mosaic female mouse models and humans with Rett syndrome, an X-linked neurodevelopmental disorder caused by mutations in the gene encoding the methyl-DNA-binding protein MECP2, and observe that cell-type-specific DNA methylation predicts the degree of gene upregulation in MECP2-mutant neurons. This approach can be broadly applied to study gene expression in mosaic X-linked disorders.


Assuntos
Encéfalo/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/genética , Alelos , Metilação de DNA , Feminino , Humanos , Proteína 2 de Ligação a Metil-CpG/metabolismo , Mosaicismo , Mutação , Neurônios/metabolismo , Polimorfismo de Nucleotídeo Único , Síndrome de Rett/metabolismo , Análise de Sequência de RNA
11.
PLoS One ; 13(9): e0202802, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30256804

RESUMO

Excess glutamate during intense neuronal activity is not instantly cleared and may accumulate in the extracellular space. This has various long-term consequences such as ectopic signaling, modulation of synaptic efficacy and excitotoxicity; the latter implicated in various neurodevelopmental and neurodegenerative diseases. In this study, the quantitative imaging of glutamate homeostasis of hippocampal slices from methyl-CpG binding protein 2 knock-out (Mecp2-/y) mice, a model of Rett syndrome (RTT), revealed unusual repetitive glutamate transients. They appeared in phase with bursts of action potentials in the CA1 neurons. Both glutamate transients and bursting activity were suppressed by the blockade of sodium, AMPA and voltage-gated calcium channels (T- and R-type), and enhanced after the inhibition of HCN channels. HCN and calcium channels in RTT and wild-type (WT) CA1 neurons displayed different voltage-dependencies and kinetics. Both channels modulated postsynaptic integration and modified the pattern of glutamate spikes in the RTT hippocampus. Spontaneous glutamate transients were much less abundant in the WT preparations, and, when observed, had smaller amplitude and frequency. The basal ambient glutamate levels in RTT were higher and transient glutamate increases (spontaneous and evoked by stimulation of Schaffer collaterals) decayed slower. Both features indicate less efficient glutamate uptake in RTT. To explain the generation of repetitive glutamate spikes, we designed a novel model of glutamate-induced glutamate release. The simulations correctly predicted the patterns of spontaneous glutamate spikes observed under different experimental conditions. We propose that pervasive spontaneous glutamate release is a hallmark of Mecp2-/y hippocampus, stemming from and modulating the hyperexcitability of neurons.


Assuntos
Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/metabolismo , Potenciais de Ação , Animais , Canais de Cálcio/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Masculino , Camundongos , Neurônios/fisiologia , Síndrome de Rett/genética
12.
eNeuro ; 5(5)2018.
Artigo em Inglês | MEDLINE | ID: mdl-30255129

RESUMO

Rett syndrome (RTT) is caused in most cases by loss-of-function mutations in the X-linked gene encoding methyl CpG-binding protein 2 (MECP2). Understanding the pathological processes impacting sensory-motor control represents a major challenge for clinical management of individuals affected by RTT, but the underlying molecular and neuronal modifications remain unclear. We find that symptomatic male Mecp2 knockout (KO) mice show atypically elevated parvalbumin (PV) expression in both somatosensory (S1) and motor (M1) cortices together with excessive excitatory inputs converging onto PV-expressing interneurons (INs). In accordance, high-speed voltage-sensitive dye imaging shows reduced amplitude and spatial spread of synaptically induced neuronal depolarizations in S1 of Mecp2 KO mice. Moreover, motor learning-dependent changes of PV expression and structural synaptic plasticity typically occurring on PV+ INs in M1 are impaired in symptomatic Mecp2 KO mice. Finally, we find similar abnormalities of PV networks plasticity in symptomatic female Mecp2 heterozygous mice. These results indicate that in Mecp2 mutant mice the configuration of PV+ INs network is shifted toward an atypical plasticity state in relevant cortical areas compatible with the sensory-motor dysfunctions characteristics of RTT.


Assuntos
Interneurônios/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Plasticidade Neuronal/fisiologia , Parvalbuminas/metabolismo , Síndrome de Rett/metabolismo , Animais , Modelos Animais de Doenças , Camundongos Knockout , Neurônios/metabolismo , Sinapses/metabolismo
14.
Hum Mol Genet ; 27(23): 4077-4093, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30137367

RESUMO

Mutations in the X-linked gene MECP2 cause the majority of Rett syndrome (RTT) cases. Two differentially spliced isoforms of exons 1 and 2 (MeCP2-e1 and MeCP2-e2) contribute to the diverse functions of MeCP2, but only mutations in exon 1, not exon 2, are observed in RTT. We previously described an isoform-specific MeCP2-e1-deficient male mouse model of a human RTT mutation that lacks MeCP2-e1 while preserving expression of MeCP2-e2. However, RTT patients are heterozygous females that exhibit delayed and progressive symptom onset beginning in late infancy, including neurologic as well as metabolic, immune, respiratory and gastrointestinal phenotypes. Consequently, we conducted a longitudinal assessment of symptom development in MeCP2-e1 mutant females and males. A delayed and progressive onset of motor impairments was observed in both female and male MeCP2-e1 mutant mice, including hind limb clasping and motor deficits in gait and balance. Because these motor impairments were significantly impacted by age-dependent increases in body weight, we also investigated metabolic phenotypes at an early stage of disease progression. Both male and female MeCP2-e1 mutants exhibited significantly increased body fat compared to sex-matched wild-type littermates prior to weight differences. Mecp2e1-/y males exhibited significant metabolic phenotypes of hypoactivity, decreased energy expenditure, increased respiratory exchange ratio, but decreased food intake compared to wild-type. Untargeted analysis of lipid metabolites demonstrated a distinguishable profile in MeCP2-e1 female mutant liver characterized by increased triglycerides. Together, these results demonstrate that MeCP2-e1 mutation in mice of both sexes recapitulates early and progressive metabolic and motor phenotypes of human RTT.


Assuntos
Proteína 2 de Ligação a Metil-CpG/genética , Atividade Motora/genética , Síndrome de Rett/genética , Animais , Modelos Animais de Doenças , Éxons/genética , Feminino , Regulação da Expressão Gênica/genética , Heterozigoto , Humanos , Masculino , Camundongos , Atividade Motora/fisiologia , Mutação , Fenótipo , Isoformas de Proteínas/genética , Síndrome de Rett/metabolismo , Síndrome de Rett/fisiopatologia
15.
Neural Plast ; 2018: 9726950, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29977282

RESUMO

CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five) gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous female Cdkl5 knockout (Cdkl5 +/-) mouse, has been little characterized. The lack of detailed behavioral profiling of this model remains a crucial gap that must be addressed in order to advance preclinical studies. Here, we provide a behavioral and molecular characterization of heterozygous Cdkl5 +/- mice. We found that Cdkl5 +/- mice reliably recapitulate several aspects of CDKL5 disorder, including autistic-like behaviors, defects in motor coordination and memory performance, and breathing abnormalities. These defects are associated with neuroanatomical alterations, such as reduced dendritic arborization and spine density of hippocampal neurons. Interestingly, Cdkl5 +/- mice show age-related alterations in protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling, two crucial signaling pathways involved in many neurodevelopmental processes. In conclusion, our study provides a comprehensive overview of neurobehavioral phenotypes of heterozygous female Cdkl5 +/- mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder.


Assuntos
Encéfalo/metabolismo , Modelos Animais de Doenças , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Síndrome de Rett/genética , Espasmos Infantis/genética , Animais , Comportamento Animal , Síndromes Epilépticas , Feminino , Heterozigoto , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome de Rett/metabolismo , Síndrome de Rett/psicologia , Transdução de Sinais , Espasmos Infantis/metabolismo , Espasmos Infantis/psicologia
16.
Proc Natl Acad Sci U S A ; 115(31): 7991-7996, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30012595

RESUMO

Rett syndrome (RTT) is a genetic disorder resulting from a loss-of-function mutation in one copy of the X-linked gene methyl-CpG-binding protein 2 (MECP2). Typical RTT patients are females and, due to random X chromosome inactivation (XCI), ∼50% of cells express mutant MECP2 and the other ∼50% express wild-type MECP2. Cells expressing mutant MECP2 retain a wild-type copy of MECP2 on the inactive X chromosome (Xi), the reactivation of which represents a potential therapeutic approach for RTT. Previous studies have demonstrated reactivation of Xi-linked MECP2 in cultured cells by biological or pharmacological inhibition of factors that promote XCI (called "XCI factors" or "XCIFs"). Whether XCIF inhibitors in living animals can reactivate Xi-linked MECP2 in cerebral cortical neurons, the cell type most therapeutically relevant to RTT, remains to be determined. Here, we show that pharmacological inhibitors targeting XCIFs in the PI3K/AKT and bone morphogenetic protein signaling pathways reactivate Xi-linked MECP2 in cultured mouse fibroblasts and human induced pluripotent stem cell-derived postmitotic RTT neurons. Notably, reactivation of Xi-linked MECP2 corrects characteristic defects of human RTT neurons including reduced soma size and branch points. Most importantly, we show that intracerebroventricular injection of the XCIF inhibitors reactivates Xi-linked Mecp2 in cerebral cortical neurons of adult living mice. In support of these pharmacological results, we also demonstrate genetic reactivation of Xi-linked Mecp2 in cerebral cortical neurons of living mice bearing a homozygous XCIF deletion. Collectively, our results further establish the feasibility of pharmacological reactivation of Xi-linked MECP2 as a therapeutic approach for RTT.


Assuntos
Córtex Cerebral/metabolismo , Proteína 2 de Ligação a Metil-CpG , Mutação , Neurônios/metabolismo , Síndrome de Rett/metabolismo , Animais , Linhagem Celular , Córtex Cerebral/patologia , Humanos , Proteína 2 de Ligação a Metil-CpG/biossíntese , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Camundongos Knockout , Neurônios/patologia , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/patologia
17.
Proc Natl Acad Sci U S A ; 115(23): E5363-E5372, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29769330

RESUMO

Rett syndrome (RTT) is a severe neurodevelopmental disorder that affects about 1 in 10,000 female live births. The underlying cause of RTT is mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2); however, the molecular mechanism by which these mutations mediate the RTT neuropathology remains enigmatic. Specifically, although MeCP2 is known to act as a transcriptional repressor, analyses of the RTT brain at steady-state conditions detected numerous differentially expressed genes, while the changes in transcript levels were mostly subtle. Here we reveal an aberrant global pattern of gene expression, characterized predominantly by higher levels of expression of activity-dependent genes, and anomalous alternative splicing events, specifically in response to neuronal activity in a mouse model for RTT. Notably, the specific splicing modalities of intron retention and exon skipping displayed a significant bias toward increased retained introns and skipped exons, respectively, in the RTT brain compared with the WT brain. Furthermore, these aberrations occur in conjunction with higher seizure susceptibility in response to neuronal activity in RTT mice. Our findings advance the concept that normal MeCP2 functioning is required for fine-tuning the robust and immediate changes in gene transcription and for proper regulation of alternative splicing induced in response to neuronal stimulation.


Assuntos
Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Síndrome de Rett/genética , Processamento Alternativo/genética , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Éxons/genética , Expressão Gênica/genética , Genes Ligados ao Cromossomo X , Hipocampo/metabolismo , Íntrons/genética , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Síndrome de Rett/metabolismo , Transcriptoma/genética
18.
Lipids ; 53(4): 363-373, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29770459

RESUMO

Rett syndrome (RS) is a pervasive neurodevelopmental disorder resulting from loss-of-function mutations in the X-linked gene methyl-Cpg-binding protein 2 (MECP2). Using a well-defined model for RS, the C57BL6/Mecp2tm1.1Bird mouse, we have previously found a moderate but persistently lower rate of cholesterol synthesis, measured in vivo, in the brains of Mecp2-/y mice, starting from about the third week after birth. There was no genotypic difference in the total cholesterol concentration throughout the brain at any age. This raised the question of whether the lower rate of cholesterol synthesis in the mutants was balanced by a fall in the rate at which cholesterol was converted via cholesterol 24-hydroxylase (Cyp46A1) to 24S-hydroxycholesterol (24S-OHC), the principal route through which cholesterol is ordinarily removed from the brain. Here, we show that while there were no genotypic differences in the concentrations in plasma and liver of three cholesterol precursors (lanosterol, lathosterol, and desmosterol), two plant sterols (sitosterol and campesterol), and two oxysterols (27-hydroxycholesterol [27-OHC] and 24S-OHC), the brains of the Mecp2 -/y mice had significantly lower concentrations of all three cholesterol precursors, campesterol, and both oxysterols, with the level of 24S-OHC being ~20% less than in their Mecp2 +/y controls. Together, these data suggest that coordinated regulation of cholesterol synthesis and catabolism in the central nervous system is maintained in this model for RS. Furthermore, we speculate that the adaptive changes in these two pathways conceivably resulted from a shift in the permeability of the blood-brain barrier as implied by the significantly lower campesterol and 27-OHC concentrations in the brains of the Mecp2-/y mice.


Assuntos
Encéfalo/metabolismo , Modelos Animais de Doenças , Hidroxicolesteróis/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Síndrome de Rett/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Hidroxicolesteróis/análise , Lovastatina/farmacologia , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Endogâmicos C57BL
19.
Exp Neurol ; 307: 74-81, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29782864

RESUMO

We tested the claim that the dopaminergic dysfunction of Rett Syndrome (RTT) also occurs in Mecp2-deficient mice that serve as a model of the syndrome. We used positron emission tomography (PET) to image dopamine D2 receptors (D2R) and transporters (DAT) in women with RTT and in Mecp2-deficient mice, and D1R and D2R density was measured in postmortem human tissue by autoradiography. Results showed 1) significantly reduced D2R density in the striatum of women with RTT compared to control subjects. 2) PET imaging of mouse striatum similarly demonstrated significant reductions in D2R density of 7-10 week-old hemizygous (Mecp2-null) and heterozygous (HET) mice compared to wild type (WT) mice. With age, the density of D2R declined in WT mice but not HET mice. 3) In contrast, postmortem autoradiography revealed no group differences in the density of D1R and D2R in the caudate and putamen of RTT versus normal control subjects. 4) In humans and in the mouse model, PET revealed only marginal group differences in DAT. The results confirm that dopaminergic dysfunction in RTT is also present in Mecp2-deficient mice and that reductions in D2R more likely explain the impaired ambulation and progressive rigidity observed rather than alterations in DAT.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/biossíntese , Proteína 2 de Ligação a Metil-CpG/deficiência , Receptores de Dopamina D2/biossíntese , Síndrome de Rett/diagnóstico por imagem , Síndrome de Rett/metabolismo , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Corpo Estriado/diagnóstico por imagem , Corpo Estriado/metabolismo , Feminino , Humanos , Camundongos , Camundongos Knockout , Adulto Jovem
20.
Exp Cell Res ; 368(2): 225-235, 2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29730163

RESUMO

Mutations in MECP2 gene have been identified in more than 95% of patients with classic Rett syndrome, one of the most common neurodevelopmental disorders in females. Taking advantage of the breakthrough technology of genetic reprogramming, we investigated transcriptome changes in neurons differentiated from induced Pluripotent Stem Cells (iPSCs) derived from patients with different mutations. Profiling by RNA-seq in terminally differentiated neurons revealed a prominent GABAergic circuit disruption along with a perturbation of cytoskeleton dynamics. In particular, in mutated neurons we identified a significant decrease of acetylated α-tubulin which can be reverted by treatment with selective inhibitors of HDAC6, the main α-tubulin deacetylase. These findings contribute to shed light on Rett pathogenic mechanisms and provide hints for the treatment of Rett-associated epileptic behavior as well as for the definition of new therapeutic strategies for Rett syndrome.


Assuntos
Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/fisiologia , Desacetilase 6 de Histona/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Síndrome de Rett/metabolismo , Síndrome de Rett/fisiopatologia , Tubulina (Proteína)/metabolismo , Acetilação , Diferenciação Celular/fisiologia , Feminino , Humanos , Masculino
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