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GluD1 is a common altered player in neuronal differentiation from both MECP2-mutated and CDKL5-mutated iPS cells.

ABSTRACT: Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.

SUBMITTER: Livide G

PROVIDER: S-EPMC4172451 | biostudies-literature |

REPOSITORIES: biostudies-literature

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iPS cells to model CDKL5-related disorders.

Project description:Rett syndrome (RTT) is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in three genes have been associated with this condition. Classic RTT is caused by mutations in the MECP2 gene, whereas variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of RTT and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons, but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types, including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation, affected by early onset seizure variant and X-linked epileptic encephalopathy, respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore, the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro.

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Altered neuronal network and rescue in a human MECP2 duplication model.

Project description: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.

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Expression profiling in WT vs CDKL5 mutated IPSCs

Project description:Mutations in the human CDKL5 gene have been associated with early onset seizure variant of Rett Syndrome. In order to investigate the potential involvement of CDKL5 in the regulation of gene expression, we compared expression profiles in WT vs CDKL5-mutated IPS clones from two patients (one male and one female) with different mutations using two-colour microarray experiments. For the female patient (CDKL5 mutation p.Gln347X) we compared one clone expressing the mutant CDKL5 allele to another clone from the same patient that expresses the wild-type allele. Maintenance of X chromosome inactivation and the resulting mono-allelic expression of CDKL5 were confirmed by androgen receptor assay and direct sequencing of CDKL5 mRNA. The clone derived from the male patient (CDKL5 mutation p.Thr288Ile) was compared to a clone derived from a normal newborn male. For each mutant/control pair four technical replicates were performed for a total of eight chip hybridizations.

2014-07-02 | E-MTAB-2223 | biostudies-arrayexpress

CDKL5/STK9 is mutated in Rett syndrome variant with infantile spasms.

Project description:Rett syndrome is a severe neurodevelopmental disorder, almost exclusively affecting females and characterised by a wide spectrum of clinical manifestations. Both the classic form and preserved speech variant of Rett syndrome are due to mutations in the MECP2 gene. Several other variants of Rett syndrome have been described. In 1985, Hanefeld described a variant with the early appearance of convulsions. In this variant, the normal perinatal period is soon followed by the appearance of seizures, usually infantile spasms. We have observed two patients with signs of Rett syndrome showing acquired microcephaly and stereotypic midline hand movements. The disease started with generalised convulsions and myoclonic fits at 1.5 months in the first patient and with spasms at 10 days in the other, suggesting a diagnosis of the Hanefeld variant. In these patients, MECP2 point mutations and gross rearrangements were excluded by denaturing high performance liquid chromatography and real time quantitative PCR. The ARX and CDKL5 genes have been associated with West syndrome (infantile spasms, hypsarrhythmia, and mental retardation).Based on the clinical overlap between the Hanefeld variant and West syndrome, we analysed ARX and CDKL5 in the two girls.We found frameshift deletions in CDKL5 in both patients; one in exon 5 (c.163_166delGAAA) and the other in exon 18 (c.2635_2636delCT). CDKL5 was then analysed in 19 classic Rett and 15 preserved speech variant patients, all MECP2 negative, but no mutations were found.Our results show that CDKL5 is responsible for a rare variant of Rett syndrome characterised by early development of convulsions, usually of the spasm type.

| S-EPMC1735977 | biostudies-other

Transgenic expression of Glud1 (glutamate dehydrogenase 1) in neurons: in vivo model of enhanced glutamate release, altered synaptic plasticity, and selective neuronal vulnerability.

Project description:The effects of lifelong, moderate excess release of glutamate (Glu) in the CNS have not been previously characterized. We created a transgenic (Tg) mouse model of lifelong excess synaptic Glu release in the CNS by introducing the gene for glutamate dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. Glud1 is, potentially, an important enzyme in the pathway of Glu synthesis in nerve terminals. Increased levels of GLUD protein and activity in CNS neurons of hemizygous Tg mice were associated with increases in the in vivo release of Glu after neuronal depolarization in striatum and in the frequency and amplitude of miniature EPSCs in the CA1 region of the hippocampus. Despite overexpression of Glud1 in all neurons of the CNS, the Tg mice suffered neuronal losses in select brain regions (e.g., the CA1 but not the CA3 region). In vulnerable regions, Tg mice had decreases in MAP2A labeling of dendrites and in synaptophysin labeling of presynaptic terminals; the decreases in neuronal numbers and dendrite and presynaptic terminal labeling increased with advancing age. In addition, the Tg mice exhibited decreases in long-term potentiation of synaptic activity and in spine density in dendrites of CA1 neurons. Behaviorally, the Tg mice were significantly more resistant than wild-type mice to induction and duration of anesthesia produced by anesthetics that suppress Glu neurotransmission. The Glud1 mouse might be a useful model for the effects of lifelong excess synaptic Glu release on CNS neurons and for age-associated neurodegenerative processes.

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Transcriptomic Analysis of Mecp2 Mutant Mice Reveals Differentially Expressed Genes and Altered Mechanisms in Both Blood and Brain.

Project description:Rett syndrome is a rare neuropsychiatric disorder with a wide symptomatology including impaired communication and movement, cardio-respiratory abnormalities, and seizures. The clinical presentation is typically associated to mutations in the gene coding for the methyl-CpG-binding protein 2 (MECP2), which is a transcription factor. The gene is ubiquitously present in all the cells of the organism with a peak of expression in neurons. For this reason, most of the studies in Rett models have been performed in brain. However, some of the symptoms of Rett are linked to the peripheral expression of MECP2, suggesting that the effects of the mutations affect gene expression levels in tissues other than the brain. We used RNA sequencing in Mecp2 mutant mice and matched controls, to identify common genes and pathways differentially regulated across different tissues. We performed our study in brain and peripheral blood, and we identified differentially expressed genes (DEGs) and pathways in each tissue. Then, we compared the genes and mechanisms identified in each preparation. We found that some genes and molecular pathways that are differentially expressed in brain are also differentially expressed in blood of Mecp2 mutant mice at a symptomatic-but not presymptomatic-stage. This is the case for the gene Ube2v1, linked to ubiquitination system, and Serpin1, involved in complement and coagulation cascades. Analysis of biological functions in the brain shows the enrichment of mechanisms correlated to circadian rhythms, while in the blood are enriched the mechanisms of response to stimulus-including immune response. Some mechanisms are enriched in both preparations, such as lipid metabolism and response to stress. These results suggest that analysis of peripheral blood can reveal ubiquitous altered molecular mechanisms of Rett and have applications in diagnosis and treatments' assessments.

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Potentiation of neuronal activity by tonic GluD1 current in brain slices

Project description:Ion channel function of native delta glutamate receptors (GluDRs) is incompletely understood. Previously, we and others have shown that activation of Gαq protein-coupled receptors (GqPCR) produces a slow inward current carried by GluD1R. GluD1R also carry a tonic cation current of unknown cause. Here, using voltage-clamp electrophysiological recordings from adult mouse brain slices containing the dorsal raphe nucleus, we find no role of on-going G protein-coupled receptor activity in generating or sustaining tonic GluD1R currents. Neither augmentation nor disruption of G protein activity affects tonic GluD1R currents, suggesting that on-going G protein-coupled receptor activity does not give rise to tonic GluD1R currents. Further, the tonic GluD1R current is unaffected by the addition of external glycine or D-serine, which influence GluD2R current at millimolar concentrations. Instead, GqPCR-stimulated and tonic GluD1R currents are regulated by physiological levels of external calcium. In current-clamp recordings, block of GluD1R channels hyperpolarizes the membrane by ~7 mV at subthreshold potentials, reducing excitability. Thus, GluD1R carry a G protein-independent tonic current that contributes to subthreshold neuronal excitation in the dorsal raphe nucleus.

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CDKL5 and Shootin1 Interact and Concur in Regulating Neuronal Polarization.

Project description:In the last years, the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene has been associated with epileptic encephalopathies characterized by the early onset of intractable epilepsy, severe developmental delay, autistic features, and often the development of Rett syndrome-like features. Still, the role of CDKL5 in neuronal functions is not fully understood. By way of a yeast two hybrid screening we identified the interaction of CDKL5 with shootin1, a brain specific protein acting as a determinant of axon formation during neuronal polarization. We found evidence that CDKL5 is involved, at least in part, in regulating neuronal polarization through its interaction with shootin1. Indeed, the two proteins interact in vivo and both are localized in the distal tip of outgrowing axons. By using primary hippocampal neurons as model system we find that adequate CDKL5 levels are required for axon specification. In fact, a significant number of neurons overexpressing CDKL5 is characterized by supernumerary axons, while the silencing of CDKL5 disrupts neuronal polarization. Interestingly, shootin1 phosphorylation is reduced in neurons silenced for CDKL5 suggesting that the kinase affects, directly or indirectly, the post-translational modification of shootin1. Finally, we find that the capacity of CDKL5 to generate surplus axons is attenuated in neurons with reduced shootin1 levels, in agreement with the notion that two proteins act in a common pathway. Altogether, these results point to a role of CDKL5 in the early steps of neuronal differentiation that can be explained, at least in part, by its association with shootin1.

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Human iPSC-Derived Neuronal Cells From CTBP1-Mutated Patients Reveal Altered Expression of Neurodevelopmental Gene Networks.

Project description:A recurrent de novo mutation in the transcriptional corepressor CTBP1 is associated with neurodevelopmental disabilities in children (Beck et al., 2016, 2019; Sommerville et al., 2017). All reported patients harbor a single recurrent de novo heterozygous missense mutation (p.R342W) within the cofactor recruitment domain of CtBP1. To investigate the transcriptional activity of the pathogenic CTBP1 mutant allele in physiologically relevant human cell models, we generated induced pluripotent stem cells (iPSC) from the dermal fibroblasts derived from patients and normal donors. The transcriptional profiles of the iPSC-derived "early" neurons were determined by RNA-sequencing. Comparison of the RNA-seq data of the neurons from patients and normal donors revealed down regulation of gene networks involved in neurodevelopment, synaptic adhesion and anti-viral (interferon) response. Consistent with the altered gene expression patterns, the patient-derived neurons exhibited morphological and electrophysiological abnormalities, and susceptibility to viral infection. Taken together, our studies using iPSC-derived neuron models provide novel insights into the pathological activities of the CTBP1 p.R342W allele.

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MECP2 impairs neuronal structure by regulating KIBRA.

Project description:Using a Drosophila model of MECP2 gain-of-function, we identified memory associated KIBRA as a target of MECP2 in regulating dendritic growth. We found that expression of human MECP2 increased kibra expression in Drosophila, and targeted RNAi knockdown of kibra in identified neurons fully rescued dendritic defects as induced by MECP2 gain-of-function. Validation in mouse confirmed that Kibra is similarly regulated by Mecp2 in a mammalian system. We found that Mecp2 gain-of-function in cultured mouse cortical neurons caused dendritic impairments and increased Kibra levels. Accordingly, Mecp2 loss-of-function in vivo led to decreased Kibra levels in hippocampus, cortex, and cerebellum. Together, our results functionally link two neuronal genes of high interest in human health and disease and highlight the translational utility of the Drosophila model for understanding MECP2 function.

| S-EPMC5609186 | biostudies-literature

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