Methyl-CpG binding protein 2 deregulation: from Rett syndrome to MeCP2 duplication disorder

[eng] NTRODUCTION To answer many complex and fascinating biological phenomena, we must go over or above (epi-) genetics because the DNA blueprint is identical in each of the abovementioned somatic cells. The mechanisms by which epigenetics affects so deeply the cell physiology are mediated by a large number of actors, most of them represented by covalently modified nucleotides and amino acids, non-coding RNAs and proteins. MeCP2 is an epigenetic reader able to bind to methylated and 5- hydroxymethylated cytosines. Despite all the initial evidences proposing a chromatin-repression role for MeCP2, many other functions have been demonstrated, including transcriptional activation, mRNA splicing regulation and protein synthesis modulation. Importantly, MeCP2 impairments are the primary responsible for RTT syndrome and have also been shown to be involved in several other disorders, albeit in very few patients, as Prader-Willi syndrome, Angelman syndrome, nonsyndromic mental retardation, and autism. AIMS To date, no MeCP2-regulated gene has been successfully targeted in order to improve the severe symptoms of RTT. In the present Doctoral Thesis we sought to identify new MeCP2 targets through different approaches with the purpose of expanding the knowledge of the impaired biological pathways in RTT. * In the first study we focused on a class of transcriptional regulators called long non-coding RNAs (lncRNAs). * In the second study we took advantage of RNA sequencing, a powerful high-throughput technique with the ability to detect very low amounts of transcript. Then, we proposed to investigate also the consequence of MeCP2 over-expression in a well-known developmental model such as the chicken embryo. RESULTS STUDY I DYSREGULATION OF THE LONG NON-CODING RNA TRANSCRIPTOME IN A RETT SYNDROME MOUSE MODEL * We found 701 lncRNAs that had a different expression pattern in wild-type and Mecp2-null brain with a score of 1.5-fold expression change. Among the altered lncRNAs, downregulation of transcripts was predominant (520 of 701, 74%), whereas upregulation occurred in the minority of differentially expressed genes (181 of 701, 26%). * Following a selection of lncRNAs with a fold-change >2 that were associated with an annotated protein-coding gene involved in neuronal or glial functions, we validated two up-regulated lncRNAs, AK081227 and AK087060, in the Mecp2-null brain using qRT-PCR on independent samples. * We showed that AK081227 and AK087060 promoters were occupied by the MeCP2 protein in wild-type mouse brains. * We reported that the up-regulation of AK081227 in Mecp2-null mice was associated with a down-regulation of its host gene Gabrr2 in four brain regions (frontal cortex, hypothalamus, thalamus and cerebellum) (Pearson's correlation test = 0.44, p = 0.06). * In the case of AK087060, we found that the up-regulation of this 1ncRNA was correlated with an increase in the expression of its host gene Arhgef26 in the four studied brain regions (Pearson's correlation test = 0.41, p = 0.08). STUDY II RNA-SEQUENCING OF A RETT SYNDROME MOUSE MODEL REVEALS GLOBAL IMPAIRMENT OF IMMEDIATE-EARLY GENES EXPRESSION * We sequenced the transcriptome of Mecp2-null and control mice and we detected 1049 and 1154 differentially expressed genes in HIP and PFC, respectively. The ratio of up- and down-regulated genes was different between the two regions. In the HIP the ratio was favorable to the less expressed genes, being 388 (37%) and 661 (63%) the up- and down-regulated genes, respectively. On the other hand, in the PFC there were slightly more up-regulated genes, 630 (55%), compared to the down-regulated ones, 523 (45%). In addition we reported that only a small fraction of genes, 76 and 109, were up- and down-regulated, respectively, in both brain areas. * Gene Ontology (GO) analysis of differentially expressed transcripts revealed that both HIP and PFC up-regulated genes were enriched in neuronal function terms and, to a lesser extent, signal transduction ones. The scenario was similar for the down-regulated genes but in this case we found many inflammatory, apoptosis, oxidative stress and immune system-related terms. * We found several members of the immediate-early genes (IEGs) family to be up-regulated both in the PFC and HIP of Mecp2-null mouse. Consistent with the findings from the RNA-sequencing analysis in the HIP, qRT-PCR showed significant alterations in the expression of Fos, Junb, Egr2, Nr4a1, Npas4, Fosb and Egr1. Furthermore, Fos, Junb, Npas4 and Fosb were validated also in the PFC. * We demonstrated the binding of MeCP2 upon the regulatory regions of IEGs. In both PFC and HIP wild-type brain, we observed a reduction of MeCP2 occupancy upon the regions associated with high CpG content of Fos, Junb, Nr4a1, Npas4, Fosb and Egr1 promoters. We also found that the HIP chromatin was more accessible to MNase digestion in the Mecp2-null brain. * Then, we showed that four IEGs (Fos, Junb, Egr2, Npas4) displayed altered expression in Mecp2-null cultured neurons treated with forskolin. Precisely, this four IEG exhibited an aberrant kinetic of recovery to the basal state. One hour after forskolin withdrawal, Fos, Junb, Egr2 and Npas4 expression levels in the Mecp2-null hippocampal neurons continue to increase, while in wild-type they did not change or even decrease. The situation is the opposite in cortical neurons, where Fos, Junb, Egr2 and Npas4 are less expressed after forskolin withdrawal in Mecp2-null samples. * Finally, we evaluated whether the IEGs response was impaired in vivo as well. Indeed, we observed a significant increase of Junb expression in the hippocampus of Mecp2-null animals treated with kainic acid, when compared to treated wild type mice. STUDY III AN INCREASE IN MECP2 DOSAGE IMPAIRS NEURAL TUBE FORMATION * We detected the expression of both chicken MeCP2 (cMECP2) transcript and protein in a wide window of developmental stages. In addition, we showed that nuclear localization and the sequence of the region encompassing the methyl-CpG binding domain are conserved between human and chicken. * We found that the overexpression of MeCP2 in the neural tube of chicken embryos provokes an overall decrease in the number of proliferating BrdUpositive cells, with the most affected part being the ventricular zone. In addition, normal H3S1Op pattern along the lumen is disrupted upon MeCP2 overexpression. * Also, MeCP2 increase in dosage cause a clear decrease in the amounts of differentiated neuronal population located at the mantle zone, as it was demonstrated through immunostaining of neural tubes with TUJ1 and HUC/D, two neuronal-lineage restricted markers. Moreover, MeCP2 overexpression leads to a decrease of a neuroepithelial polarity marker such as N-cadherin. * Finally, we showed that one of the possible explanations of our phenotype is the increased cell death occurring upon MeCP2 increase in dosage. We reported an increment of apoptotic cells in MeCP2-overexpressing neural tubes immunostained with Caspase-3 and -8. Furthermore, we described also an increase of pyknotic cells number in MeCP2 electroporated neural tubes.[spa] Introducción: El síndrome de Rett (RTT, OMIM#312750) fue por primera vez descrito en 1966 por el pediatra austriaco Andreas Rett. El síndrome de Rett causa retraso mental en 1 de cada 10000 niñas, lo que hace que sea la segunda causa de retraso mental en niñas. En 1999 en el laboratorio de Huda Zoghbi descubrieron las bases genéticas de la enfermedad. El 95% de los casos de Rett clásico se produce por mutaciones en MeCP2. Es interesante el hecho de que mutaciones que provocan el incremento de copias del gen MECP2 también llevan a enfermedades neurológicas, como es el caso del trastorno provocado por la duplicación de MeCP2. MeCP2 es una proteína nuclear, que se expresa en diferentes tejidos, pero es especialmente abundante en neuronas del sistema nervioso maduro. MeCP2 es una proteína con capacidad para unirse a dinucleótidos CpG. Entre las varias funciones biológicas propuesta para MeCP2 se encuentran: 1) Silenciamento transcripcional; 2) activador transcripcional; 3) regulador de splicing; 4) Regulador de la cromatina. Objetivos del estudio: El principal objetivo de esta tesis es evaluar el impacto del incremento o disminución de expresión de MeCP2 , tanto a nivel transcripcional como de desarrollo, al fin de caracterizar las vías moleculares desreguladas en las manifestaciones clínicas relacionadas con MeCP2. En los primeros dos estudios se buscarán nuevos targets de MeCP2 a través de dos diferentes tecnologías, secuenciación del ARN y microarray. En ambos estudios utilizaremos un modelo murino bien establecido (MeCP2-null), obtenido mediante supresión del gen MeCP2, que simula el síndrome de Rett. Las diferencias entre los primeros dos estudios es que mientras en el primero se buscarán solo "long non-coding RNA" relacionados con MeCP2, el segundo será enfocado en todos los ARN codificantes. En el tercer estudio evaluaremos el efecto de la sobreexpresión de MeCP2 en un bien establecido modelo de desarrollo embrionario como es el embrión de pollo. Resultados y conclusiones: Parte 1 * Se han encontrado 701 lncRNAs diferencialmente expresados entre el cerebro del ratón Mecp2-null y el control (salvaje). * MeCP2 está unido a los promotores de los lncRNAs AK081227 y AK087060. * El incremento de expresión de AK081227 en ratones Mecp2-null está asociado con la bajada de expresión de su gen huésped Gabrr2 en cuatro regiones del cerebro. * La sobre regulación de AK087060 se correlaciona con un aumento en la expresión de su gen huésped Arhgef26 en las cuatro regiones cerebrales estudiadas. Parte 2 * Hemos encontrados 1049 y 1154 transcritos diferencialmente expresado en el hipocampo (HIP) y la corteza pre-frontal (PFC), respectivamente, del ratón Mecp2- null. * Los genes "immediate early genes" (IEGs) Fos, JunB, EGR2, NR4A1, Npas4, FosB y Egrl están sobreexpresados en el HIP de Mecp2-null. Además, Fos, JunB, Npas4 y FosB están sobreexpresados también en el PFC. * En tanto la PFC como en el HIP del ratón wild-type, la unión de MeCP2 se reduce en las regiones asociadas con alto contenido de CpG de los genes Fos, JunB, NR4A1, Npas4, FosB y Egr1. Además, los promotores de Fos, JunB y Npas4 son más accesibles a la digestión con nucleasas micrococales (MNase) en el HIP de ratones Mecp2-null. * Cuatro IEGs (Fos, JunB, Egr2, Npas4) muestran un patrón de expresión alterado en neuronas derivadas de animales Mecp2-null y tratadas con forskolina. * La expresión de JunB es incrementada significativamente en el hipocampo de los animales Mecp2-null tratados con ácido kaínico, en comparación con ratones controles tratados. Parte 3 * El transcrito y la proteína de MeCP2 de pollo se expresan en varios estadio del desarrollo embrionario y especialmente en el tubo neural * La sobreexpresión de MeCP2 en el tubo neural de embriones de pollo provoca una disminución general en el número de células proliferantes. Además, el patrón de localización del marcador mitótico H3S1Op es aberrante en tubos neurales que sobreexpresan MeCP2. * Una dosis elevada de MeCP2 provoca una clara disminución de las neuronas diferenciadas localizadas en la zona del mantel. Por otra parte, la sobreexpresión de MeCP2 conduce a una disminución del marcador de polaridad neuroepitelia Ncadherin. * La sobreexpresión de MeCP2 en tubos neurales provoca un aumento de apoptosis

Sull’incontro di Kandinskij e Schönberg, sotto il segno della “necessità interiore”

Il 2 gennaio 1911 Kandinskij ascoltò a Monaco un concerto con alcuni Lieder, i Quartetti op. 7 e op. 10 e i Klavierstücke di Schönberg, e pochi giorni dopo sentì il bisogno di scrivere al compositore (che non era presente al concerto): le lettere che si scambiarono negli anni 1911-14 formano il nucleo essenziale di un dialogo epistolare intorno alle affinità e alle convergenze che Kandinskij e Schönberg immediatamente avvertirono nella loro ricerca sotto il segno della “necessità interiore”, nella fase del primo astrattismo per l’uno e della sospensione della tonalità per l’altro. Senza indulgere ad approssimativi giochi di corrispondenze tra linguaggi e personalità differenti, è possibile tentare un confronto tra la sconvolta visione dello spazio pittorico del primo astrattismo di Kandinskij (uno spazio privato di un “centro” di riferimento) e il vertiginoso stravolgimento dello spazio sonoro che negli stessi anni produce in Schönberg l’abolizione di un centro tonale. Tra gli aspetti più interessanti dell’epistolario tra Schönberg e Kandinskij, che costituisce un contrappunto alle loro riflessioni teoriche e alle loro creazioni degli anni 1911-14, ci sono le ricerche sinestesiche che caratterizzano Il suono giallo e La mano felice, due esperienze teatrali diversissime e concepite in modo del tutto indipendente. E va ricordata la collaborazione del compositore all’almanacco Der Blaue Reiter con Herzgewächse su testo di Maeterlinck.On the Meeting of Kandinskij and Schönberg, under the Sign of “Inner Need”On January 2, 1911 Kandinskij attended concert in Münich with some Lieder, the Quartets op. 7 and op. 10 and the Klavierstücke by Schönberg – a few days later he felt the need to write to the composer (who didn’t attend the concert). The letters exchanged in the years 1911-14 form the essential nucleus of an epistolary dialogue about the affinities and convergences that Kandinsky and Schönberg immediately felt in their research under the sign of “inner need”, in the phase of the first abstractionism for one and the suspension of the tonality for the other. Without indulging in approximate games of correspondence between different languages and personalities, it is possible to attempt a comparison between the upset vision of the pictorial space of Kandinsky’s first abstractionism (a space deprived of a “centre” of reference) and the dizzying distortion of the sound space that in the same years produced in Schönberg the abolition of a tonal centre. Among the most interesting aspects of the correspondence between Schönberg and Kandinsky, which is a counterpoint to their theoretical reflections and their creations of the years 1911-14, there are the synaesthetic researches that characterize Der gelbe Klang and Die glückliche Hand, two very different theatrical experiences and conceived in a completely independent way. Schönberg also composed Herzgewächse on a text by Maeterlinck for the almanac Der Blaue Reiter

Generation of Cholinergic and Dopaminergic Interneurons from Human Pluripotent Stem Cells as a Relevant Tool for In Vitro Modeling of Neurological Disorders Pathology and Therapy

The cellular and molecular bases of neurological diseases have been studied for decades; however, the underlying mechanisms are not yet fully elucidated. Compared with other disorders, diseases of the nervous system have been very difficult to study mainly due to the inaccessibility of the human brain and live neurons in vivo or in vitro and difficulties in examination of human postmortem brain tissue. Despite the availability of various genetically engineered animal models, these systems are still not adequate enough due to species variation and differences in genetic background. Human induced pluripotent stem cells (hiPSCs) reprogrammed from patient somatic cells possess the potential to differentiate into any cell type, including neural progenitor cells and postmitotic neurons; thus, they open a new area to in vitro modeling of neurological diseases and their potential treatment. Currently, many protocols for generation of various neuronal subtypes are being developed; however, most of them still require further optimization. Here, we highlight accomplishments made in the generation of dopaminergic and cholinergic neurons, the two subtypes most affected in Alzheimer's and Parkinson's diseases and indirectly affected in Huntington's disease. Furthermore, we discuss the potential role of hiPSC-derived neurons in the modeling and treatment of neurological diseases related to dopaminergic and cholinergic system dysfunction

Circadian cycle-dependent MeCP2 and brain chromatin changes

Abstract Methyl CpG binding protein 2 (MeCP2) is a chromosomal protein of the brain, very abundant especially in neurons, where it plays an important role in the regulation of gene expression. Hence it has the potential to be affected by the mammalian circadian cycle. We performed expression analyses of mice brain frontal cortices obtained at different time points and we found that the levels of MeCP2 are altered circadianly, affecting overall organization of brain chromatin and resulting in a circadian-dependent regulation of well-stablished MeCP2 target genes. Furthermore, this data suggests that alterations of MeCP2 can be responsible for the sleeping disorders arising from pathological stages, such as in autism and Rett syndrome

Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies

Methyl CpG binding protein 2 (MeCP2) binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome, a neurological disease, 80% of patients carry a mutation in the MECP2 gene. Since the MECP2 gene lies on the X chromosome and is subjected to X chromosome inactivation, affected patients are usually chimeric for wild type and mutant MeCP2. Here, we present the generation and characterization of the first rat monoclonal MeCP2 specific antibodies as well as mouse monoclonal antibodies and a rabbit polyclonal antibody. We demonstrate that our antibodies are suitable for immunoblotting, (chromatin) immunoprecipitation and immunofluorescence of endogenous and ectopically expressed MeCP2. Epitope mapping revealed that most of the MeCP2 monoclonal antibodies recognize the C-terminal domain and one the N-terminal domain of MeCP2. Using slot blot analysis, we determined a high sensitivity of all antibodies, detecting amounts as low as 1 ng of MeCP2 protein. Moreover, the antibodies recognize MeCP2 from different species, including human, mouse, rat and pig. Lastly, we have validated their use by analyzing and quantifying X chromosome inactivation skewing using brain tissue of MeCP2 heterozygous null female mice. The new MeCP2 specific monoclonal antibodies described here perform well in a large variety of immunological applications making them a very valuable set of tools for studies of MeCP2 pathophysiology in situ and in vitro

The hypomethylation of imprinted genes in IVF/ICSI placenta samples is associated with concomitant changes in histone modifications

Although more and more children are born by Assisted Reproductive Technologies (ART), ART safety has not fully been demonstrated. Notably, ART could disturb the delicate step of implantation, and trigger placenta-related adverse outcomes with potential long-term effects, through disrupted epigenetic regulation. We have previously demonstrated that placental DNA methylation was significantly lower after IVF/ICSI than following natural conception at two differentially methylated regions (DMRs) associated with imprinted genes (IGs): H19/IGF2 and KCNQ1OT1. As histone modifications are critical for placental physiology, the aim of this study was to profile permissive and repressive histone marks in placenta biopsies to reveal a better understanding of the epigenetic changes in the context of ART. Utilizing chromatin immunoprecipitation (ChIP) coupled with quantitative PCR, permissive (H3K4me3, H3K4me2, and H3K9ac) and repressive (H3K9me3 and H3K9me2) post-translational histone modifications were quantified. The analyses revealed a significantly higher quantity of H3K4me2 precipitation in the IVF/ICSI group than in the natural conception group for H19/IGF2 and KCNQ1OT1 DMRs (P = 0.016 and 0.003, respectively). Conversely, the quantity of both repressive marks at H19/IGF2 and SNURF DMRs was significantly lower in the IVF/ICSI group than in the natural conception group (P = 0.011 and 0.027 for H19/IGF2; and P = 0.010 and 0.035 for SNURF). These novel findings highlight that DNA hypomethylation at imprinted DMRs following ART is linked with increased permissive/decreased repressive histone marks, altogether promoting a more permissive chromatin conformation. This concomitant change in epigenetic state at IGs at birth might be an important developmental event because of ART manipulations

Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope

Altres ajuts: Funding This work was supported by the Obra Social La Caixa (LCF/PR/HR19/52160011), the Spanish Cancer Association and Leo Messi Foundation to PM.Background There are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19 - either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22 + CD19 - B-ALL relapses and CD19 - preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied. Methods Here, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays. Results Conformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy. Conclusions We report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22 high and CD22 low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22-CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL

Robustness of dead Cas9 activators in human pluripotent and mesenchymal stem cells

Human pluripotent stem cells (hPSCs) and mesenchymal stromal/stem cells (hMSCs) are clinically relevant sources for cellular therapies and for modeling human development and disease. Many stem cell-based applications rely on the ability to activate several endogenous genes simultaneously to modify cell fate. However, genetic intervention of these cells remains challenging. Several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains can modulate gene expression when directed to their regulatory regions by a specific single-guide RNA (sgRNA). In this study, we have compared the ability of the first-generation dCas9-VP64 activator and the second-generation systems, dCas9-SAM and dCas9-SunTag, to induce gene expression in hPSCs and hMSCs. Several stem cell lines were tested for single and multiplexed gene activation. When the activation of several genes was compared, all three systems induced specific and potent gene expression in both single and multiplexed settings, but the dCas9-SAM and dCas9-SunTag systems resulted in the highest and most consistent level of gene expression. Simultaneous targeting of the same gene with multiple sgRNAs did not result in additive levels of gene expression in hPSCs nor hMSCs. We demonstrate the robustness and specificity of second-generation dCas9 activators as tools to simultaneously activate several endogenous genes in clinically relevant human stem cells.We thank CERCA/Generalitat de Catalunya and Fundació Josep Carreras-Obra Social la Caixa for their institutional support. We thank Jose Luis Sardina (IJC, Barcelona) for technical assistance with the teratoma assays. Financial support for this work was obtained from the Catalunya Goverment (SGR330 and PERIS 2017-2019), the Spanish Ministry of Economy and Competitiveness (SAF2016-80481-R), the European Research Council (CoG-2014-646903), and the Fundación Leo Messi to P.M.; the Spanish Association against Cancer (AECC-CI-2015) and the Health Institute Carlos III (ISCIII/FEDER, PI17/01028) to C.B.; the Biotechnology and Biological Sciences Research Council (BBRSC) to L.M.F. and A.F.; and the Spanish National Research and Development Plan (ISCIII/FEDER, PI17/02303) and the AEI/MICIU EXPLORA Project (BIO2017-91272-EXP) to S.R.-P. P.M. is an investigator of the Spanish Cell Therapy Cooperative Network (TERCEL). R.T.-R. is supported by a postdoctoral fellowship from the Asociación Española Contra el Cáncer (AECC).S

In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells

This work was supported by CaixaImpulse (CI18-00017;FuGe) to S.R-P. RT-R. is supported by a postdoctoral fellowship from the Asociación Española Contra el Cáncer (AECC). J.C.S. is supported by the Spanish Cell Therapy cooperative research network (TERCEL)(RD16/0011/0011). P.M. also acknowledges the financial support from the Obra Social La Caixa-Fundaciò Josep Carreras. P.M. is an investigator of the Spanish Cell Therapy cooperative research network (TERCEL). A.M.C. acknowledges funding fromXarxa de Bancs de Tumors de Catalunya (XBTC; sponsored by Pla Director d'Oncologia de Catalunya).Fusion oncogenes (FOs) are common in many cancer types and are powerful drivers of tumor development. Because their expression is exclusive to cancer cells and their elimination induces cell apoptosis in FO-driven cancers, FOs are attractive therapeutic targets. However, specifically targeting the resulting chimeric products is challenging. Based on CRISPR/Cas9 technology, here we devise a simple, efficient and non-patient-specific gene-editing strategy through targeting of two introns of the genes involved in the rearrangement, allowing for robust disruption of the FO specifically in cancer cells. As a proof-of-concept of its potential, we demonstrate the efficacy of intron-based targeting of transcription factors or tyrosine kinase FOs in reducing tumor burden/mortality in in vivo models. The FO targeting approach presented here might open new horizons for the selective elimination of cancer cells

Inhibition of Gsk3b Reduces Nfkb1 Signaling and Rescues Synaptic Activity to Improve the Rett Syndrome Phenotype in Mecp2-Knockout Mice

Rett syndrome (RTT) is the second leading cause of mental impairment in girls and is currently untreatable. RTT is caused, in more than 95% of cases, by loss-of-function mutations in the methyl CpG- binding protein 2 gene (MeCP2). We propose here a molecular target involved in RTT: the glycogen synthase kinase-3b (Gsk3b) pathway. Gsk3b activity is deregulated in Mecp2-knockout (KO) mice models, and SB216763, a specific inhibitor, is able to alleviate the clinical symptoms with consequences at the molecular and cellular levels. In vivo, inhibition of Gsk3b prolongs the lifespan of Mecp2-KO mice and reduces motor deficits. At the molecular level, SB216763 rescues dendritic networks and spine density, while inducing changes in the properties of excitatory synapses. Gsk3b inhibition can also decrease the nuclear activity of the Nfkb1 pathway and neuroinflammation. Altogether, our findings indicate that Mecp2 deficiency in the RTT mouse model is partially rescued following treatment with SB216763