Dissertations / Theses on the topic 'Rett syndrome MeCP2'

Rett syndrome (RTT) is a severe neurological disorder that affects approximately 1:10000 girls. Classical RTT is defined by a developmental regression phase and subsequent stabilisation of diagnostic criteria, which include partial or complete loss of spoken language, dyspraxic gait and stereotypic hand movements such as hand mouthing. RTT is a monogenic disorder, with the majority of cases being due to loss-of-function mutations in MeCP2 (methyl-CpG binding protein 2). Due to this clear genotype-phenotype link multiple RTT mouse models have been used to elucidate the molecular details, and consequent neuropathogenesis, of this complex neurological disease, as well as for the development of potential therapeutics for RTT. However, as the molecular details become clearer, the need for a simpler model system becomes evident. Human induced pluripotent stem cells (hiPSCs) generated from RTT patient fibroblasts are an option; however the handling of these cells is laborious, time-consuming and expensive and they often differentiate into a heterogeneous population of cells. To explore an alternative human model system I have been genetically engineering and experimenting with the human dopaminergic LUHMES cell line. LUHMES cells are an immortalised pre-neuronal cell line derived from an 8-week old, female foetus and can readily be differentiated into a homogeneous population of mature, electrically active neurons in just one week. In this thesis I have assessed the phenotypic properties of the wild-type cell line, demonstrated the ease of genetic manipulation of LUHMES cells by CRISPR/Cas9 approaches, generated seven mutant MECP2 LUHMES cell lines and explored the potential of protein therapy as a therapeutic approach for RTT. The LUHMES cell line proves to be extremely easy to handle and robust and has yielded novel molecular insights into the function of MeCP2 in human neurons. In particular, MeCP2-null cells show a striking relationship between the level of gene body methylation and the extent of transcriptional upregulation when compared to wild-type neurons. In contrast neurons that express a form of MeCP2 that can bind to DNA but cannot recruit a transcriptional corepressor complex (the R306C mutant) do not exhibit substantial gene expression alterations, yet do display a consistent decrease in total RNA amount. This decrease in total RNA is recapitulated in MeCP2-null LUHMES-derived neurons and in brain regions from MeCP2-R306C mice. The requirement for functional DNA binding for normal gene-body methylation dependent gene repression is demonstrated by assessing LUHMES cells that overexpress MeCP2-R111G, a protein that cannot bind to DNA. Furthermore, overexpression of the MeCP2-R306C protein highlights the importance of NCoR binding for normal gene repression, but also demonstrates that MeCP2-R306C protein retains some gene repression activity. Thinking more broadly, this cell line also has applications as a model system for a variety of other neurological disorders; as a simplified model system to elucidate molecular and neurological phenotypes, and as a relevant human system that can be cultured in a high-throughput manner for testing therapeutic strategies.

It is now commonly agreed that Rett Syndrome is a monogenic neurological disease caused by mutations in MECP2 gene. Rett Syndrome mainly occurs in girls and it is characterised by a period of normal development until around 6­18 months, followed by a rapid regression. After the regression, symptoms persist as severe mental retardation, reduced head size, seizures, ataxia, hyperventilation and repetitive hand wringing movements. The phenotype of mice with a deleted Mecp2 gene mimics some Rett Syndrome symptoms. The Mecp2­null mouse develops normally until about 6 weeks of age after which tremors, irregular breathing, lack of mobility and hindlimb clasping develop. To understand how the lack of MeCP2 causes Rett Syndrome, the search for MeCP2 regulated genes was initiated in Mecp2-null mouse brain. Examination of candidate genes revealed that Bdnf is down-regulated and Hes1 is up-regulated in pre, early and late symptomatic Mecp2-null mice. Further, global analysis of gene expression was examined by ADDER differential display. Some mis-regulated genes were identified, two of which are involved in mitochondrial respiration. Oxygen electrode measurements revealed defects in brain mitochondrial respiration, which commenced coincident with symptom onset in Mecp2-null mice. This finding suggests mitochondrial involvement in the pathogenesis of Rett Syndrome symptoms. In the course of these studies, the structure of the Mecp2 gene was re-investigated, leading to the identification of a new MeCP2 isoform. Data in this thesis demonstrates that the new isoform is the major form of MeCP2 in both mouse and human brain.

INTRODUCTION 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 <0.05 in the false discovery rate (FDR) test and a >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.
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.

Methyl-CpG binding protein 2 (MeCP2) was discovered as a protein binding to methylated DNA more than 20 years ago. It is very abundant in the brain and was shown to be able to repress transcription. The mutations in MeCP2 cause Rett syndrome, an autism-spectrum neurological disorder affecting girls. Yet, the exact role of MeCP2 in Rett disease, its function and mechanism of action are not fully elucidated. In order to shed some light on its role in the disease the aim of this project was to identify proteins interacting with MeCP2. Affinity purification of MeCP2 from mouse brains and mass spectrometry analysis revealed new interactions between MeCP2 and protein complexes. Detailed analysis confirmed the findings and narrowed down the top interactions to distinct regions of MeCP2. One of the domains interacts with identified NCoR/SMRT co-repressor complex and is mutated in many patients with Rett syndrome. In vitro assays proved that these mutations abolish the putative transcriptional repressor function of MeCP2. We propose a model in which Rett syndrome is caused by two types of mutations: either disrupting the interaction with DNA or affecting the interaction with the identified complex, which has an effect on the global state of chromatin. The presented findings can help to develop new therapies for Rett syndrome in the future.

MeCP2 was initially identified as an abundant protein in the brain, with an affinity for methylated DNA in vitro. Interestingly, both deficiency and excess of the protein leads to severe neurological problems, such as Rett syndrome, which is the result of mutations in the MECP2 gene. Subsequent transfection experiments showed that MeCP2 can recruit corepressor complexes and inhibit gene expression in vivo. MeCP2 was therefore thought to repress specific gene targets and the aetiology of Rett syndrome was proposed to result from aberrant gene expression in the MeCP2-deficient brain. Although gene expression is perturbed in the Mecp2-null mouse brain, few specific targets have been verified and alternative hypotheses for MeCP2 function have been put forward. Previous binding studies have also failed to clearly identify MeCP2 targets. To shed light on these matters, a novel technique was generated to isolate neuronal and glial nuclei and established that the amount of MeCP2 is unexpectedly high in neurons, with an abundance approaching that of the histone octamer. Chromatin immunoprecipitation experiments on mature mouse brain showed widespread binding of MeCP2, consistent with its high abundance, tracking the methyl-CpG density of the genome. MeCP2 deficiency results in global changes in neuronal chromatin structure, including elevated histone acetylation and a doubling of histone H1. The mutant brain also shows elevated transcription of repetitive elements, which are distributed throughout the mouse genome. Based on this data, we propose that MeCP2 binds genome wide and suppresses spurious transcription through binding in a DNA methylation dependent manner.

Rett syndrome (RTT) is an X-linked neurological disorder primarily caused by mutations in the MECP2 gene. The majority of RTT mutations disrupt the interaction of MeCP2 with DNA or TBL1X/TBL1XR1, which forms the scaffold of NCoR/SMRT co-repressor complex. Patients with RTT show no signs of neuronal death, although they have abnormal neuronal morphology, indicating that it is a neurodevelopmental rather than a neurodegenerative disease. It has been shown that reactivation of silenced MeCP2 in mice rescues the RTT phenotype, which implies that the disease is treatable. The RTT mutations in MeCP2 cluster to two regions - the methyl-CpG-binding domain (MBD) and NCoR/SMRT Interaction Domain (NID). While the interaction between MBD and DNA has been biochemically and structurally characterised, there are no structural data about the interaction between MeCP2 NID and TBL1XR1. The aim of this work was to understand how mutations in the NID cause RTT by characterising the interaction between MeCP2 and TBL1XR1. I have solved the structure of MeCP2 NID bound to TBL1XR1 WD40 domain. I show that a small region of the MeCP2 NID makes extensive contacts with TBL1XR1, and that these contacts are mediated primarily by MeCP2 residues known to be mutated in RTT. I also measured the affinities between TBL1XR1 and MeCP2-derived peptides using fluorescence anisotropy and surface plasmon resonance assays. I determined the affinity between MeCP2 NID peptide and TBL1XR1 to be around 10- 20 μM, and show that mutations in either MeCP2 or TBL1XR1 can abolish this interaction. Taken together, these data strongly suggest that the abolition of the interaction between MeCP2 NID and TBL1XR1 WD40 domain is sufficient to cause RTT. This knowledge can help with the rational design of small drug-like molecules that might be able to mediate the interaction between mutated MeCP2 and TBL1XR1, potentially helping to treat the disease.

Chez l’homme, des mutations dans le gène MECP2 sont à l’origine de maladies neurologiques dont la principale est le Syndrome de Rett (RTT). Le décours de la pathologie est caractérisé par un arrêt du développement entre 6 et 18 mois après la naissance, suivi par un ensemble de signes cliniques, dont une phase de régression importante avec des troubles moteurs, autonomes et cognitifs. Parmi les facteurs qui participent au développement de la pathologie, le BDNF joue un rôle clé. En effet l'expression du Bdnf est diminuée de moitié dans le système nerveux central en l’absence de celui-ci. C'est un acteur prépondérant dans l’apparition et dans la progression du phénotype anormal des neurones dans cette pathologie et donc une excellente cible thérapeutique. L’utilisation directe du Bdnf n’est actuellement pas envisageable car cette protéine ne traverse pas la BHE et que son temps de demi-vie est très court. Nous avons donc élaboré des stratégies alternatives afin d’agir indirectement sur le Bdnf, de façon à stimuler son transport vésiculaire et compenser les déficits en contenus, par une augmentation de sa biodisponibilité et de sa libération à la synapse. Dans cette optique j’ai utilisé deux approches visant à stimuler le transport axonal du Bdnf par la phosphorylation d'Htt afin d’augmenter la vitesse de transport antérograde de vésicules de Bdnf dans les neurones. Mes conclusions sont que la phosphorylation d'Htt corrige le déficit de transport axonal du Bdnf in vitro. Elle permet d'améliorer la survie et certains symptômes de la souris modèle de RTT. Cette phosphorylation apparait ainsi comme une piste thérapeutique intéressante
Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the MECP2 gene, located on the X chromosome. After a period of apparent normal development, females with MECP2 mutations undergo a regression of early developmental milestones, resulting in the deterioration of motor skills, eye contact, speech, and hand movements and ultimately resulting in severe breathing disturbances, as the disease progresses, and severe handicap. Bdnf, a neuronal modulator that plays a key role in neuronal survival, development, and plasticity has been found to be one of the main factors altered in the absence of Mecp2. The Bdnf pathway is one of the most appealing pathways to target in RTT. Bdnf itself is unable to cross the blood-brain barrier (BBB) and needs to be indirectly activated. Thus, we developed an indirect strategy to enhance Bdnf trafficking in neurons. Huntingtin (Htt) phosphorylation of Serine 421 enhances Bdnf transport and promoting Htt phosphorylation may restore Bdnf homeostasis in Mecp2 KO brain. We tested this possibility using two approaches to promote Htt phosphorylation of S421 in Mecp2-deficient neurons and Mecp2 KO mice. We evaluated the consequences of Htt S421 phosphorylation on BDNF axonal trafficking in projecting corticostriatal neurons in vitro, and in vivo on the behavior of Mecp2 KO mice. Our findings demonstrate that pharmacological and genetic stimulation approaches correct Bdnf trafficking in vitro and improve longevity and behavioural features in Mecp2 KO mice. Htt S421 phosphorylation appears to be a possible target for the development of treatments in RTT

La sindrome di Rett è una grave malattia del neurosviluppo, causata da mutazioni del gene MECP2 presente nel cromosoma X, che agisce principalmente come repressore trascrizionale. Nonostante la Rett si sia dimostrata reversibile nel topo, non è ancora disponibile una cura per questa devastante patologia. Negli ultimi vent’anni sono stati sviluppati numerosi modelli animali mutati in grado di riprodurre i classici segni della patologia e i difetti molecolari osservati nei pazienti, contribuendo alla scoperta che il fenotipo patologico è principalmente causato dalla mancanza di MeCP2 a livello centrale. Tuttavia, l’uso di questi modelli per testare librerie di farmaci richiederebbe dei costi elevati e lunghe tempistiche sperimentali. Pertanto, per supportare e accelerare gli screening farmacologici in vivo, sono stati sviluppati nuovi sistemi di screening in vitro, basati sulla valutazione degli effetti del farmaco sulla morfologia neuronale, che solitamente è difettiva nei pazienti affetti da sindrome di Rett. Tuttavia, il nostro laboratorio ha recentemente dimostrato che il recupero dei difetti trascrizionali tipici della malattis assicurano un maggiore miglioramento funzionale dei neuroni rispetto a quello causato dal recupero dei difetti morfologici. Sulla base di questi nostri dati preliminari, il mio progetto di dottorato prevedeva lo sviluppo di un nuovo sistema di drug screening in vitro, basato sulla valutazione del recupero trascrizionale indotto da diversi trattamenti farmacologici, utilizzando piastre high-throughput 96x96 di RT-qPCR. Per sviluppare questa nuova piattaforma trascrizionale, abbiamo eseguito un’analisi di trascrittomica longitudinale su colture in differenziamento di precursori neuronali privi di Mecp2, identificando dei difetti trascrizionali tipici dei neuroni Rett. In seguito ad un processo di prioritizzazione e selezione dei geni deregolati, abbiamo validato un gruppo di geni in diverse piastre 96x96 di RT-qPCR e abbiamo in questo modo identificato un gruppo di geni deregolati in modo consistente da usare come readout per misurare l’efficacia farmacologica. Per stabilire se questi geni potessero realmente riflettere la potenziale efficacia di un farmaco in vivo, abbiamo testato la capacità di recuperare l’espressione dei geni difettivi in seguito al trattamento con l’ampachina CX546, per la quale avevamo precedentemente dimostrato un’efficacia in vitro e nel modello murino nullo. I neuroni trattati con CX546 hanno dimostrato un miglioramento trascrizionale del 75% dei geni difettivi testati, anche se negli esperimenti di RT-qPCR abbiamo dovuto aumentare il numero di campioni necessari a riprodurre i dati trascrizionali di trascrittomica. Per questo motivo proponiamo il nostro sistema di screening farmacologico come un approccio di conferma di molecole già precedentemente selezionate da uno screening morfologico in vitro oppure per scegliere un potenziale candidato farmaco tra alcuni scelti sulla base delle loro funzioni farmacologiche. In parallelo, i dati di trascrittomica ci hanno permesso di identificare e di caratterizzare due geni consistentemente deregolati, Haus7 e Nsdhl, in colture cellulari e tessuti cerebrali privi di Mecp2, dimostrando un loro possibile coinvolgimento nella patogenesi della malattia e offrendo dei nuovi possibili target terapeutici per la sindrome di Rett.
Abstract Rett syndrome (RTT) is a devastating neurodevelopmental disorder caused by mutations in the X-linked MECP2 gene, primarily acting as transcriptional repressor. Although RTT proved to be reversible in mice, no cure is yet available. Several Mecp2-muntant mouse models have been developed and they generally reproduce behavioral and physiological phenotypes observed in RTT patients, establishing that disease phenotypes are widely due to neuronal dysfunctions. However, their use in large drug screening programs require a great number of animals, elevated costs and time-consuming experimental approaches. To support the in vivo evaluation, new drug screening systems have emerged in vitro, usually based on the analysis of neuronal defective morphology. We previously demonstrated that the amelioration of the transcriptional profile in Mecp2-null neurons appears as a better indicator of functional rescue than morphological readouts. For this reason, we aimed at developing a cell-based drug screening system for RTT therapy, based on customized high-throughput 96x96 qRT-PCR arrays. To this purpose, a longitudinal RNASeq analysis performed in differentiating Mecp2-null neuronal precursors cells identified consistent transcriptional defects of RTT neurons. By using different prioritization criteria and testing selected neuronal differentially expressed genes (DEGs) on 96x96 qRT-PCR cards, we established a group of reproducible DEGs which represent our quantitative probes to measure the transcriptional amelioration induced by the drugs tested. To assess whether the selected DEGs are able to reflect the efficacy of drugs in vivo, we analyzed the effects of the ampakine CX546, for which we previously published positive results in vitro and in vivo. The drug demonstrated to rescue 75% of our selected DEGs, though a sample size larger than expected was required to reproduce RNASeq data in qRT-PCR experiments, forcing us to reconsider its use for the screening of large drug libraries in a laboratory scale. Thus, we propose the use of our screening system as either a confirmatory approach of a previously produced selection of molecules or as a useful system to validate rationally deduced pharmacological approaches. As secondary outcome, we identified and further characterized a consistent defect in the expression of two genes, Haus7 and Nsdhl, in cultured neurons and Mecp2-defective tissues, prompting further investigations of their role and functions in RTT pathogenesis. A comprehensive analysis of their expression across different stages and models of the disorder lay the foundation for novel possible pathogenic mechanisms of RTT and hopefully will provide new potential targets for RTT therapy.

Typical Rett syndrome (RTT) is a paediatric neurological disorder caused in >95 % of cases by loss-of-function mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2). The gene product, MeCP2, is a widely expressed nuclear protein that is especially abundant in postmitotic neurons of the central nervous system (CNS). Knocking out Mecp2 function in mice recapitulates many of the overt neurological features seen in RTT patients and provides a very useful model for testing potential therapeutic applications. The absence of a curative therapy together with the monogenecity of the disorder and established reversibility of the phenotype in mice suggest that replacement of the MECP2 gene is a potential therapeutic option worthy of exploration. In this study I used several viral vectors to test the potential of gene therapy in RTT mice. First, I generated different viral vectors which can express tagged-MeCP2 under the control of ubiquitous and cell-type specific promoters. Secondly, I assessed the ability of these vectors to deliver the Mecp2 transgene into Mecp2 knockout mice at both neonatal and adult stages of development. I then aimed to investigate the effect of exogenously delivered Mecp2 on RTT-like phenotypes. The results demonstrated that lentiviral vectors were able to effectively deliver an RFP-tagged Mecp2 minigene into neurons of Mecp2-null mice both in vitro and in vivo. Exogenous Mecp2 was targeted to the nucleus and displayed heterochromatin localization with no evidence of ectopic expression. Use of the synapsin1 (syn1, neuron-specific) promoter resulted in cellular levels of Mecp2 equal to 85 ± 0.1% of endogenous protein levels, whereas the phosphoglycerate kinase (PGK) promoter produced cellular levels of exogenous Mecp2 at a relatively high levels (210 ± 0.1 % of endogenous levels). Direct brain injection of Lentiviral vector was able to deliver exogenous Mecp2 into the CA1 region of the hippocampus and to produce high transduction efficiency around the injection sites but with limited spread. The early mortality of the injected mice precluded assessment of the functional consequences of exogenous Mecp2 expression. However, assessment of the cellular morphology was possible and this analysis revealed delivery of exogenous Mecp2 to normalise neuronal nuclear volume deficits seen in the Mecp2stop/y mice from 86 ± 0.1 % of WT values to 100 ± 0.04 % of WT levels. At the molecular level, I showed that exogenous Mecp2 3 becomes phosphorylated at serine 421 under basal conditions and that the level of phosphorylation of exogenous Mecp2 is disproportionately higher (5.5 ± 0.4 times) than that seen for endogenous Mecp2. I also showed the Mecp2 overexpression in WT neurons is associated with a reduction in the cellular levels of total histone 4 (78 ± 0.01 % of the endogenous level) and a parallel reduction in cellular levels of acetylated histone 4 (79 ± 0.01 % of the endogenous levels). In second phase of experiments, I showed that the single stranded Adeno-associated virus (ssAAV)-based vector with chicken beta actin (CBA) promoter and encapsulated with capsid of AAV serotype 9, was able to efficiently deliver exogenous MECP2 into the brain of Mecp2-null as well as WT neonatal mice after intracranial (IC) injection. In contrast to lentiviral vectors, there was widespread transduction of cells throughout the nervous system with transduction efficiency varying between 6.8 ± 2.3 % and 41.5 ± 11.3% of all cells dependent on the brain region. The transgene was mostly expressed in neurons which represented 67 ± 11.8 % to 98 ± 0.8 % of all transduced cells. ssAAV9 vector expressed exogenous MeCP2 at near-physiological levels (100-125 % of endogenous levels). At the cellular level, exogenous MeCP2 was able to rescue the neuronal nuclear volume of Mecp2-null mice (69 ± 0.02 % of WT values) to WT comparable values (97 ± 0.03 % of WT values). At the organismal levels Mecp2-null mice treated with ssAAV9/MECP2 showed extended survival (median survival of 16.7 weeks compared to 9.3 weeks for the GFP-treated control) and also displayed a modest, but significant, reduction in the RTT-like phenotype severity score compared to the GFP-treated control group. The most robust improvement reported in this study was in the locomotion activity (velocity and total distance moved in the open field test and in performance on a forced motor task). Interestingly, WT mice receiving neonatal injections of ssAAV9/CBA-MECP2 did not show any significant deficits, suggesting a tolerance for modest MeCP2 overexpression. In a further experiment, I showed that the self-complementary AAV 9 (scAAV9) vector with an Mecp2-endogenous core promoter fragment was able to deliver exogenous MECP2 into the brain of neonatal mice after intravenous (IV) or (IC) injection. Brain transduction efficiency was 8 - 12 % after IV injection and 48 - 68 % after IC injection in neonatal mice. Cellular levels of exogenous MeCP2 were between 1.4-1.8 times the endogenous levels. At the organismal level, 4 scAAV9/MECP2-injected mice displayed an overt hindlimb motor dysfunction which was observed 3 and 5 weeks post-injection after IV and IC injection respectively. The stereotyped hindlimb dysfunction suggested a toxicity issues with this vector and examination of the lumbar segment of the spinal cord confirmed evidence of axonal degeneration in the dorsal columns. IV injection of scAAV9/MECP2 into RAG-/- knockout mice (immunocompromised) displayed hindlimb motor dysfunction similar to that observed with Mecp2stop/y mice suggesting that the adaptive immune response is not likely to be involved in the pathogenesis of this phenotype. MECP2stop/y mice treated with scAAV9/MECP2 displayed higher RTT-like phenotype severity score than GFP-treated controls which is probably due to the effect produced by the hindlimb motor dysfunction on regular RTT-like phenotype (gait, mobility and hindlimb clasping). In summary, I have demonstrated the successful application of lentiviral and AAV2/9 vectors to deliver exogenous MECP2 both in vitro and in vivo. I showed that lentiviral vectors are unlikely to be useful for global brain delivery of MECP2 due to limited spread of the virus. However the lentiviral vectors I developed are potentially useful where localized brain injection is desirable. The main translational finding is the first, at the proof of concept level, demonstration of therapeutic benefits (including enhanced survival) of exogenously delivered MECP2 using the ssAAV9/CBA-MECP2 vector. I also however, identify potential toxicity issues of exogenous MECP2 delivery whereby a scAAV9 vector was found to produce overt neuromotor deficits. Overall, my data supports the potential of gene therapy in RTT but also emphasises the importance of issues including careful vector design, choice of delivery methods and the timing of treatment in any future clinical translation.

Rett Syndrome is a neurological disorder that affects primarily females, it is characterised by apparent normal development for the first 6-18 months of life followed by a loss of both fine and gross motor skills. Diagnosis is based on a set of clinical criteria that includes stereotypical hand wringing and a decrease in head growth. Between 60-80% of Rett syndrome patients have pathogenic mutations in the gene methyl CpG binding protein 2 (MECP2). Following the initial reports of MECP2 mutations, the identification of a sub set of pathogenic mutations in which whole exons were deleted and the identification of a second isoform of MECP2 resulted in extended screening of otherwise MECP2 mutation negative patients. In this study extended mutation screening of a Rett syndrome cohort was undertaken to investigate the contributions of large deletions and exon 1 mutations, in addition MECP2 screening was also performed on individuals with non-syndromic mental retardation. Presented here is the complete list of mutations and polymorphisms of the cohort. Six reoccurring mutations account for almost 60% of pathogenic mutations, large deletions of whole exons were identified in 7 individuals accounting for almost 6% of identified mutations, and only one pathogenic mutation in exon 1 was identified in an individual with a poorly defined phenotype. Apart from this one individual with the exon 1 mutation pathogenic mutations were only identified in Rett syndrome patients. A mutation in a second gene, Cyclin Dependent Kinase Like 5 (CDKL5) was identified in one family with multiple affected individuals but no MECP2 mutation and subsequently a second mutation was identified in another atypical Rett syndrome patient. Investigation of CDKL5 transcripts identified an alternative isoform. The examination of the tissue distribution of both isoforms indicated that the novel isoform is expressed ubiquitously while the recognised transcript was detected in only the testis. An attempt to characterise the kinase properties of CDKL5 was made but was prevented by the inability to produce and purify recombinant CDKL5, qPCR and western analysis indicates that this is because of regulation at the translation level. MeCP2 is a transcriptional repressor that binds to methylated CpG and mediates transcription repression by modifying chromatin structure, the functional characteristics of MeCP2 were also investigated in this project. The secondary structure of the transcription repression domain was examined by NMR, however no stable secondary structure was identified. This led to the exploration of the potential novel protein binding partners of MeCP2 using GST-pull downs from rat brains. The final area of investigation was the phosphorylation of MeCP2. Phosphorylation has been shown to be an important factor in regulating MeCP2 function, 32F labelling of recombinant MeCP2 and investigation by mass spectrometry identified serine 229 as the major site of phosphorylation. Further investigation of this residue indicates that there are other residues that are phosphorylated and that serine 229 does not influence the nuclear localisation or ability of MeCP2 to bind to methylated Cst in mouse cells. The identification of those other residues as well as the kinases responsible for the phosphorylation of MeCP2 will allow the pathways of MeCP2 regulation to be explored.

La méthylation de l’ADN est une modification majeure du génome des eucaryotes permettant de moduler l’expression génique et contrôler le développement des mammifères. La protéine Mecp2 (Methyl CpG binding protein 2), dont le gène est situé sur le chromosome X, appartient à la famille des protéines de liaison à l’ADN méthylé. Sur la base de sa structure et de ses interactions Mecp2 a été décrit comme un répresseur de l’expression des gènes. A l’heure actuelle, son implication en tant qu’activateur de la transcription et organisateur de la structure chromatinienne lui confère un rôle plus global dans la régulation de l’épigénome. Des mutations de MECP2 conduisent à des troubles neurologiques dont le principal est le syndrome de Rett (RTT). Cette pathologie dominante liée à l’X affecte principalement les jeunes filles (incidence: 1/15000 naissances). Même si les causes précises du phénotype RTT ne sont pas connues, le profil d’expression de Mecp2 est en lien avec la synaptogenèse, la maturation et la maintenance des réseaux neuronaux. A mon arrivée en thèse l’équipe qui m’a accueilli venait d’identifier des déficits neuronaux, affectant notamment les groupes catécholaminergiques bulbaires et périphériques, à l’origine de troubles respiratoires chez un modèle murin de cette pathologie. Mon travail de thèse a permis de caractériser l’évolution postnatale des déficits moteurs et physiologiques affectant la souris Mecp2-déficiente. L’étude de structures catécholaminergiques d’intérêt telles que la Substantia Nigra et le Locus Coeruleus a révélé que les neurones dopaminergiques et noradrénergiques centraux ont un métabolisme affecté. Le nombre de neurones immunomarqués apparait significativement réduit dans ces groupes ce qui résulterait d’une perte progressive du phénotype « catécholaminergique », en l’absence de mort cellulaire. Nos données suggèrent que ces atteintes constituent un corrélat neuropathologique aux troubles comportementaux observés chez les souris Mecp2-déficientes. Ainsi certains troubles moteurs ont pu être améliorés, à l’aide d’un agent pharmacologique pro-dopaminergique, la L-Dopa. En relation avec les déficits en Bdnf (Brain-derived neurotrophic factor) décrits chez les patientes et les souris Mecp2-déficientes, nous avons identifié qu’une modification du dosage de Mecp2 induit une dérégulation de gènes (Htt, Hap1) codant des protéines impliquées dans le transport intracellulaire des vésicules de Bdnf. Nos travaux nous permettent de postuler que chez la souris Mecp2-déficiente, une altération de la dynamique de transport des vésicules chargées en Bdnf pourrait exacerber le déficit d’expression de cette neurotrophine. Notre traitement des souris Mecp2-déficientes par la cystéamine, une molécule capable d’agir sur les contenus, la libération et la sécrétion du Bdnf permet d’augmenter la survie des animaux et de réduire leurs troubles moteurs. Nos résultats montrent que les déficiences en Mecp2 entrainent des déficits de transport axonal du Bdnf qui s’ajoutent aux déficits de production du Bdnf. Par ailleurs, avec l’utilisation d’agents pharmacologiques agissant sur ce transport, nous offrons de nouvelles perspectives thérapeutiques
DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian development. The protein Mecp2 (Methyl CpG binding protein 2), encoded by a gene located on the X chromosome, belongs to the ‘Methyl Binding domain’ protein family. Based on its structure and its interactions Mecp2 has historically been described as a repressor of expression for many genes. Currently, its involvement as an activator of transcription and its role in chromatin architecture suggests that it could be a global regulator of the epigenome. Mutations in MECP2 lead to neurological disorders, among which Rett syndrome (RTT). This dominant X-linked pathology mainly affects girls (incidence: 1/15000 live births). Although the precise causes of the RTT phenotype are unknown, the pattern of Mecp2 expression is related to synaptogenesis, maturation and neuromaintenance. Before my integration in the ‘Human Neurogenetics’ team, this group identified neural deficits, affecting brainstem and peripheral catecholaminergic cell groups, causing respiratory disturbances in a mouse model of this disease. My thesis work enabled the characterization of the postnatal physiological and motor deficits affecting the Mecp2-deficient mice. The study of catecholaminergic structures of interest such as the substantia nigra pars compacta and the locus coeruleus has revealed that the central noradrenergic and dopaminergic neurons are affected in their metabolism. The number of immunolabelled neurons of these groups appears significantly reduced and would result in a gradual loss of the mature ‘catecholaminergic’ phenotype, in the absence of cell death. Our data suggest that these defects are a neuropathological correlate for behavioral disorders observed in Mecp2-deficient mice. Some motor deficits have been improved, with L-Dopa, a pro-dopaminergic drug. In relation with Bdnf (Brain-derived neurotrophic factor) reduction described in patients and Mecp2-deficient mice, we identified that a change in the dosage of Mecp2 deregulates genes (Htt, Hap1) encoding proteins involved in the intracellular transport of Bdnf. Our work allows to postulate that in the Mecp2-deficient neurons, an altered dynamics of Bdnf vesicles transport could exacerbate the deficit of expression of this neurotrophin. Our treatment of Mecp2-deficient mice with cysteamine, a molecule able to increase Bdnf contents and enhancing its release and secretion, increased the survival of the animals and reduced their motor defects. Our results show that the Mecp2-deficiencies lead to alteration in the axonal transport of Bdnf in addition to deficits in Bdnf production. In addition, by the use of pharmacological agents that affect this transport, we offer new therapeutic perspectives

Le syndrome de Rett est une maladie neurodéveloppementale progressive causée par des mutations du gène mecp2. Elle touche essentiellement les filles avec une fréquence d'environ 1/10000 naissances. Différentes fonctions ont été attribuées à MeCP2 : modulateur transcriptionnel, épissage alternatif de certains ARN, maintien de l'état de méthylation des gènes et modification de la structure tridimensionnelle de la chromatine. Initialement, mes travaux de thèse ont consisté à explorer l'hypothèse que MeCP2 aurait la capacité de passer d'une cellule à l'autre. Les résultats obtenus suggèrent que le transfert intercellulaire de MeCP2 ne se produise pas in vivo mais serait dû à une diffusion intercellulaire de la protéine suite à l'étape de fixation cellulaire à l'acétone durant l'expérimentation. Cependant, ces travaux ont permis de mettre au point une nouvelle méthode pour la détection des protéines dans les cellules de mammifères basée sur le système de split-GFP. Dans le cadre de mon projet de thèse, j'ai également produit et caractérisé des anticorps dirigés spécifiquement contre chacune des 2 isoformes de MeCP2. Ces anticorps originaux vont permettre d'étudier les niveaux d'expression et le rôle de chaque isoforme dans l'organisme. Cela va pouvoir améliorer notre compréhension de la pathologie du syndrome de Rett. Plus récemment, mes travaux se sont focalisés sur la relation entre MeCP2 et les mécanismes de réparation de l'ADN, et nous ont permis de mettre en évidence la capacité de MeCP2 de s'accumuler sur l'ADN endommagé. Les futurs projets de l'équipe viseront à élucider les mécanismes impliqués dans cette nouvelle fonction de MeCP2
Rett syndrome is a severe and progressive X-linked neurodevelopmental disorder that affects 1/10000 female birth. RTT is caused by mutations in the mecp2 gene, encoding the Methyl CpG binding Protein 2. MeCP2 binds to methylated DNA and has several roles in: transcription activation or repression, chromatin remodeling, alternative splicing of mRNA. . . Initially, my thesis project was to explore the hypothesis that MeCP2 may be able to transfer between cells. My results suggest that this phenomenon appears after cell fixation with acetone and doesn't occur in vivo. This work, however, allowed us to develop a new staining method to detect and localize proteins in mammalian cells using the split-GFP system. Within the frame of this project, I have also produced antibodies specific for each of the two MeCP2 isoforms. These novel antibodies should prove to be interesting tools to understand the role of each isoform in the pathology of Rett syndrome. More recently, my work was focalized on the relationship between MeCP2 and DNA damage. I was able to show that MeCP2 accumulates on DNA damage. Future work will be aimed at understanding the mechanisms involved in this newly uncovered function of MeCP2, and will hopefully improve our understanding of Rett syndrome pathogenesis

2012/2013
RIASSUNTO La syndrome di Rett (RTT) è una patologia dello sviluppo neuronale postnatale causata dalle mutazioni del gene MeCP2, situato nel cromosoma X, codificante per la Methyl CpG binding protein 2, un modulatore della trascrizione. La forma classica si manifesta in 1:10,000 bambine ed è caratterizzata da una progressiva regressione generale fisica e mentale, in seguito ad un normale sviluppo nei primi 2 anni di vita. Molti degli aspetti della patologia sono stati riprodotti in diversi modelli murini deleti per il gene MeCP2 (MeCP2-/y), inclusi la riduzione della massa cerebrale, l’atrofia neuronale e le disfunzioni cardiorespiratorie, che costituiscono i parametri più robusti e riproducibili tra i diversi modelli murini, accanto ai meno conservati parametri comportamentali, come l’ansia, la socievolezza e l’aspetto motorio. Il fenotipo Rett è caratterizzato inoltre da una riduzione dei livelli di espressione della serotonina (5HT), norepinefrina (NE) e del BDNF (Brain Derived Neurotrophic Factor). Tuttavia, è noto che i farmaci antidepressivi sono in grado di modulare i livelli di BDNF in parte regolando il sistema monoaminergico. Lo scopo di questo lavoro consiste perciò nel valutare gli effetti del trattamento cronico con antidepressivi in un modello della sindrome di Rett. Abbiamo scelto la Desipramina (DMI) come farmaco di controllo, dal momento che è già stata precedentemente utilizzata per un trial clinico della sindrome di Rett. La Desipramina è un antidepressivo che blocca il recupero di 5HT e NE a livello dello spazio sinaptico, tuttavia presenta delle complicanze cliniche a livello cardiaco. Per evitare tale effetto collaterale della DMI, abbiamo selezionato un antidepressivo altamente tollerabile, la Mirtazapina (MIR), un antagonista degli α2 autorecettori ed eterorecettori centrali e uno specifico inibitore dei recettori 5HT2 e 5HT3. Il lavoro si divide in 4 fasi: Fase 1: analisi degli effetti del trattamento con antidepressivi sul peso del corpo e del cervello ed analisi della morfologia dei neuroni piramidali della corteccia somatosensoriale in un modello murino della sindrome di Rett Fase 2: analisi degli effetti del trattamento con antidepressivi sui parametri vitali, inclusi il battito cardiaco e la frequenza respiratoria nel modello murino della sindrome di Rett Fase 3: analisi degli effetti del trattamento con antidepressivi sul comportamento nel modello murino della sindrome di Rett Fase 4: analisi degli effetti del trattamento con antidepressivi sul livello di espressione del BDNF Fase 1: analisi degli effetti del trattamento con antidepressivi sul peso del corpo e del cervello ed analisi della morfologia dei neuroni piramidali della corteccia somatosensoriale in un modello murino della sindrome di Rett Prima di tutto abbiamo valutato le caratteristiche generali del modello murino della sindrome di Rett (MeCP2-/y), osservando che il peso del corpo e del cervello dell’animale era significativamente ridotto a 42 giorni dalla nascita. Inoltre, come osservato in precedenza (Kishi and Macklis, 2004, Fukuda et al., 2005), abbiamo confermato la significativa riduzione dello spessore totale della corteccia somatosensoriale (la più compromessa in questa patologia), in particolare degli strati II-III e VI a 42 giorni dalla nascita. Abbiamo quindi trattato gli animali per due settimane a partire dal 28° giorno dalla nascita con DMI alla concentrazione 10 mg/Kg e con MIR a due differenti concentrazioni (10 o 50 mg/Kg) ed analizzato gli effetti del trattamento sul peso del corpo e del cervello. Non abbiamo riscontrato differenze per quanto riguarda il peso del corpo dopo trattamento farmacologico, tuttavia abbiamo notato un significativo aumento del peso del cervello in topi MeCP2-/y dopo 2 settimane di trattamento con MIR 50 mg/Kg, confrontato con il peso del cervello di topi MeCP2-/y della stessa età non trattati. Per meglio definire le strutture coinvolte nel recupero del peso cerebrale dopo trattamento con MIR 50 mg/Kg, abbiamo effettuato una colorazione Nissl su sezioni coronali di cervello di topo e abbiamo analizzato l’ippocampo e la corteccia somatosensoriale. Abbiamo osservato che non c’erano differenze nelle proporzioni di ogni strato ippocampale rispetto allo spessore totale dell’ippocampo lungo l’asse rostro-caudale. Tuttavia, l’analisi della corteccia somatosensoriale ha rivelato che il trattamento con DMI 10 mg/Kg e MIR 50 mg/Kg fa recuperare lo spessore totale della corteccia in topi MeCP2-/y a 42 giorni dalla nascita ed in particolare lo spessore degli strati II-III e VI che sono principalmente compromessi nel modello murino della sindrome di Rett (Kishi and Macklis, 2004, Fukuda et al., 2005). Per avere maggiori informazioni sull’effetto della MIR 50 mg/Kg a livello dei neuroni corticali, abbiamo esaminato la morfologia dei neuroni piramidali dello strato II-III della corteccia somatosensoriale in topi MeCP2-/y a 42 giorni dalla nascita utilizzando la colorazione di Golgi. Abbiamo osservato che il trattamento con MIR 50 mg/Kg induce un recupero dei deficit morfologici presenti nel modello murino (Kishi and Macklis, 2004, Fukuda et al., 2005) inclusi, la ridotta area del soma, il diametro ridotto dei dendriti apicali, l’atrofia dell’albero dendritico apicale ed in particolare quello basale, il numero delle spine “stubby” sia nei dendriti secondari apicali che basali. Infine, dal momento che è stato precedentemento osservato un deficit di rilascio del GABA in topi MeCP2-/y (Chao et al., 2010), abbiamo deciso di valutare se la MIR 50 mg/Kg era in grado di recuperare questo deficit. Abbiamo quindi dimostrato che le correnti GABA sono parzialmente recuperate dopo trattamento con MIR 50 mg/Kg nella corteccia di topi MeCP2-/y a 42 giorni dalla nascita. Fase 2: analisi degli effetti del trattamento con antidepressivi sui parametri vitali, inclusi il battito cardiaco e la frequenza respiratoria nel modello murino della sindrome di Rett I pazienti Rett e i topi MeCP2-/y presentano alterazioni cardiache e un respiro anomalo allo stato avanzato della patologia. Attraverso uno strumento non invasivo (MouseOX), abbiamo raccolto i dati relativi alla saturazione dell’ossigeno (percentuale di siti dell’emoglobina occupati dalle molecole di ossigeno), il battito cardiaco e la frequenza respiratoria (numero di battiti e respiri al minuto) e la distensione dell’arteria in base al battito cardiaco in topi Wild Type e MeCP2-/y non trattati e trattati con DMI 10 mg/Kg or MIR 50 mg/Kg. Abbiamo osservato che non ci sono alterazioni nella saturazione dell’ossigeno, tuttavia la frequenza dei battiti cardiaci e del respiro, che è ridotta nei topi MeCP2-/y non trattati, viene recuperata in seguito a trattamento con gli antidepressivi, in particolare con la MIR. Inoltre, l’effetto negativo sulla distensione dell’arteria osservato per la DMI 10 mg/Kg, non viene alterato dal trattamento con MIR 50 mg/Kg. Fase 3: analisi degli effetti del trattamento con antidepressivi sul comportamento nel modello murino della sindrome di Rett I topi MeCP2-/y sono caratterizzati dal disturbi motori e una ridotta ansia (Chahrour and Zoghbi, 2007), cosi abbiamo deciso di testare gli effetti degli antidepressivi sul comportamento del modello murino della sindrome di Rett. Attraverso il test dell’”open field”, abbiamo dimostrato che i topi MeCP2-/y trattati con i farmaci trascorrono la maggior parte del tempo del test immobili, e la loro attività in termini di capacità di alzarsi e tenersi sulle zampe posteriori e di cura personale è ridotta. Queste osservazioni sono probabilmente dovute all’effetto sedativo indotto dal trattamento con antidepressivi. Tuttavia, l’ansia che è ridotta nei topi MeCP2-/y non trattati osservata nel test dell’”elevated plus maze”, ritorna a valori normali dopo trattamento con gli antidepressivi. Fase 4: analisi degli effetti del trattamento con antidepressivi sul livello di espressione del BDNF Precedenti studi hanno dimostrato che il livello di espressione del BDNF totale è significativamente ridotto nel cervello dei topi MeCP2-/y (Chang et al., 2006, Wang et al., 2006). In questo lavoro abbiamo dapprima dimostrato come i livelli delle diverse isoforme del BDNF variano sulla base della mutazione del gene MeCP2 nei pazienti Rett. Successivamente abbiamo valutato le diverse isoforme del BDNF nel prosencefalo di topi MeCP2-/y dimostrando come esse siano significativamente ridotte a 42 giorni dalla nascita. Tuttavia, il trattamento con DMI 10 mg/Kg e MIR 50 mg/Kg non è in grado di recuperare in modo significativo il livello di mRNA. Abbiamo quindi valutato il livello proteico del BDNF, dimostrando un aumento della neurotrofina a livello corticale e una diminuzione a livello ippocampale in topi MeCP2-/y non trattati ma non statisticamente significativo. Tuttavia il trattamento con MIR 50 mg/Kg sembra recuperare il livello del BDNF, sebbene non sia significativo.
ABSTRACT Rett syndrome (RTT) is an X-linked postnatal neurodevelopmental disorder caused by the mutations on MeCP2 gene which encodes for the Methyl CpG binding protein 2, a transcriptional regulator. The classical form manifests in girls with an incidence of 1:10,000 with a progressive general physical and mental regression after a normal development during the first two years of age. Several clinical features are recapitulated in MeCP2-/y mice, including the reduced brain mass, neuronal atrophy and the cardiorespiratory abnormalities, which are considered the most robust and reproducible parameters among the Rett mouse models and the less conserved alterations on mice behavior. Rett phenotype was characterized by a reduction on serotonin, norepinephrine (5HT; NE) and BDNF (Brain Derived Neurotrophic Factor) expression level. However, it is known that the antidepressants drugs modulate BDNF expression level partly by regulation of monoamine systems. The aim of the project is to evaluate the effects of repeated antidepressant treatments in a Rett mouse model. We choose Desipramine (DMI) as control drug because it was previously used in a clinical trial of Rett syndrome. DMI blocks the reuptake of 5HT and NE, but it has some cardiac complications. To overcame the cardiac side effect of DMI, we selected the highly tolerable antidepressant Mirtazapine (MIR), which is an antagonist of central α2 autoreceptors and α2 heteroreceptors and a specific blocker of 5HT2 and 5HT3 receptors. The project comprises four phases: Phase1: Analysis of the effects of antidepressant treatments on body and brain weight, including the morphology of the somatosensory pyramidal neurons in a model of Rett syndrome (MeCP2-/y) Phase2: Analysis of the effects of antidepressant treatments on the vital signs parameters, including heart and breath rate in MeCP2-/y mice Phase3: Analysis of the effects of antidepressant treatments on the behavior of the mice (open field and plus maze test) in MeCP2-/y mice Phase4: Analysis of the effects of antidepressant treatments on brain derived neurotrophic factor (BDNF) expression level Phase1: Analysis of the effects of the drugs on body and brain weight, including the morphology of the somatosensory pyramidal neurons in a model of Rett syndrome (MeCP2-/y) First of all, we evaluated the general features of the Rett mouse model, observing that the body and the brain weight of MeCP2-/y mice were reduced at postnatal day 42 (p42). We found also that there is a significant reduction on total cortical thickness, in particular of layers II-III and VI at p42 as observed in previous studies (Kishi and Macklis, 2004, Fukuda et al., 2005). Then, we analyzed the effects of DMI 10 mg/Kg and MIR (at two different concentration: 10 or 50 mg/Kg) treatments on body and brain weight. No difference was observed for body weight, while an increase in brain weight was noticed after treatment with MIR 50 mg/Kg in p42 MeCP2-/y mice compared to MeCP2-/y untreated mice. To better define the brain structures involved in the rescue of the brain weight after MIR 50 mg/Kg treatment, we performed a Nissl staining and we analyzed the hippocampus and the somatosensory cortex. We found that among p42 MeCP2-/y treated mice, there were no differences in the proportion of each hippocampal layer to the total thickness along the rostro-caudal axis. However, the analysis of the somatosensory cortex revealed that DMI 10 mg/Kg and MIR 50 mg/Kg rescued the total cortical thickness in p42 MeCP2-/y mice and in particular the layers II-III and VI which are principally compromised in Rett mouse model (Kishi and Macklis, 2004, Fukuda et al., 2005). To gain further insight regarding the effect of Mirtazapine treatment on cortical neurons, we investigated the morphology of layer II-III pyramidal neurons of the somatosensory cortex in MeCP2-/y mice using Golgi staining. We observed that MIR 50 mg/Kg treatment was able to recover the neuronal morphology deficits of p42 MeCP2-/y mice (Kishi and Macklis, 2004, Fukuda et al., 2005), including, the small soma area, the reduced diameter of apical dendrites, the atrophy of apical and, in particular, the basal dendritic arborization, the number of secondary basal dendrites, the number of stubby spines both in secondary apical and basal dendrites. Finally, as a deficit on GABA release in MeCP2-/y mice was previously described (Chao et al., 2010), we investigatd if Mirtazapine could rescue this deficit. Indeed, we found that GABA currents were rescued by MIR 50 mg/Kg treatment in the cortex of p42 MeCP2-/y mice, although without reaching full recovery. Phase2: Analysis of the effects of the drugs on the vital signs parameters, including heart and breath rate in MeCP2-/y mice Rett patients and MeCP2-/y mice presents cardiac alterations and breathing abnormalities in a later stage of the disorder. Through a non-invasive instrument (MouseOX) we collected the data regarding the Oxygen Saturation (percentage of sites of arterial hemoglobin occupied by oxygen molecules), the Hearth and the Breath Rate (number of beats or breaths per minute) and the Pulse Distention (change in distension of the arterial blood vessels due to a cardiac pulse) on Wild Type and MeCP2-/y mice untreated or treated with DMI 10 mg/Kg or MIR 50 mg/Kg. We found that no alterations was observed for the oxygen saturation, however the frequency of heart and breath are rescued after drug treatments. A negative effect of Desipramine was observed in pulse distention which is not affected with Mirtazapine treatment. Phase3: Analysis of the effects of the drugs on the behavior of the mice (open field and plus maze test) MeCP2-/y mice are characterized by motor abnormalities and a decreased anxiety (Chahrour and Zoghbi, 2007), thus, we tested the effects of the antidepressant drugs on the behavior of MeCP2-/y mice. Through an open field test, we found that the MeCP2-/y mice treated with the drugs spent more of the time immobile, and their activity in terms of number of rearing and grooming was reduced. These observations are probably due to the sedative effect of antidepressant treatments. However, the anxiety was recover to normal levels in MeCP2-/y mice treated with the antidepressants in the elevated plus maze. Phase4: Analysis of the effects of treatments on BDNF expression level Previous studies showed that total BDNF expression level was significantly reduced in the brain of MeCP2-/y mice (Chang et al., 2006, Wang et al., 2006). First of all, we demonstrated that the levels of BDNF isoforms depend on mutations in MeCP2 gene in Rett patients. Then, we evaluated the BDNF splice variants in the forebrain of MeCP2-/y mice and we demonstrated that they were significantly reduced at p42. However, treatments with DMI 10 mg/Kg or MIR 50 mg/Kg not rescue significantly the mRNA of BDNF. Therefore, we evaluated the protein level of BDNF and we demonstrated a no statistically significant increase of the neurotrophin in the cortex and a decrease in the hippocampus in MeCP2-/y untreated mice. However, the treatment with MIR 50 mg/Kg seemed to rescue the protein level of BDNF, even if no statistically significant.
XXVI Ciclo
1985

Le syndrome de Rett est une pathologie neurodéveloppementale progressive sévère, provoquée par des mutations dominantes du gène MECP2. La protéine MeCP2, fortement exprimée dans les neurones matures, est un répresseur transcriptionnel de la famille des methyl-CpG-binding proteins (MBP) caractérisées par leur capacité de liaison aux nucléotides CpG méthylés. Bien que les mécanismes moléculaires responsables du syndrome de Rett demeurent obscurs, il semble probable que cette pathologie soit due à l'expression incontrôlée de gènes normalement réprimés par MeCP2 dans les neurones. Une étape clé dans la compréhension de cette maladie sera donc d'identifier les gènes cibles de MeCP2. Mes travaux de thèse ont consisté à évaluer l'hypothèse selon laquelle les gènes codant pour les molécules du CMH de classe I, riches en îlots CpG, pourraient être contrôlés par MeCP2 au sein du système nerveux central (SNC). En effet, ces molécules sont impliquées dans l'établissement des connexions neuronales fines au cours de la synaptogenèse et dans la plasticité cérébrale puis sont activement réprimées dans les neurones matures. Les molécules de CMH de classe I présentent donc un profil d'expression au sein du SNC complexe qui doit être finement et activement régulé. Nous avions donc envisagé qu'en absence de MeCP2, une dérégulation des gènes du CMH pourrait non seulement altérer le statut immunologique des tissus nerveux mais également perturber l'établissement et le remodelage des connexions neuronales, et ainsi contribuer à la progression de la pathologie qui se développe chez les patientes atteintes du syndrome de Rett. .
Mutations in the X-linked MECP2 gene are the cause of the Rett Syndrome (RTT), a progressive neurodevelopmental disorder leading to important neurological deficiencies, including motor, vegetative and cognitive dysfunctions. To date, despite the identification of MECP2 mutations as the central cause for RTT and the generation of several mouse models mimicking the human disease, the molecular basis for the pathogenesis of the syndrome still remains to be clearly elucidated. MeCP2 (Methyl-CpG-binding protein 2) has been shown to harbour a transcriptional repression activity by its ability to bind methylated CpG nucleotides and to recruit co-repressor complexes. Moreover, MeCP2 is involved in RNA splicing regulation of target genes, takes part in the chromatin architecture and can also associate with Dnmt1, the maintenance DNA methyl-transferase. MeCP2 is therefore a multifunctional protein that is involved at many levels of genes' regulation. MHC class I molecules, whose genes are particularly rich in CpG islands, are required in the brain for the establishment and maintenance of neuronal connections during development, in plastic remodelling in the hippocampus and in neuronal signalling in specific brain areas. .

Le syndrome de Rett (SR) est une encéphalopathie progressive sévère touchant presque exclusivement les filles avec une prévalence de 1 cas sur 15000 naissances féminines. Défini par des critères cliniques précis, le SR typique se caractérise par un développement initial apparemment normal, suivi d'une perte progressive des fonctions psychomotrices. Cinq variants cliniques de SR ont étés définis, qui diffèrent du SR typique par la chronologie et/ou la sévérité des symptôme. Des mutations du gène MECP2,localisé en Xq28, ont été identifiées chez des patients présentant un SR typique, un variant clinique du SR chez des garçons souffrant d'encéphalopathies néonatales sévères. Nous avons identifié des mutations de MECP2 chez environ 65% des patientes (30/46) présentant un syndrome de Rett typique. Le développement d'une stratégie d'amplification par PCR allèle spécifique nous a alors permis de montrer dans la majorité des cas, les mutations de novo de MECP2 se produisaient sur le chromosome d'origine paternelle, raison principale pour laquelle seules les filles étaient touchées par le syndrome de Rett. Devant l'hétérogénéité phénotypique des mutations de MECP2, nous avons recherché des mutations de ce gène chez les garçons atteints de retard mental non syndromique. Des mutations faux-sens, différentes de celles retrouvées dans les cas de SR, ont été identifiées chez des garçons présentant un retard mental isolé sporadique. Afin d'essayer de savoir si la physiopathologie de ces retards mentaux était semblable à celle du SR, nous nous sommes intéressés aux conséquences de ces mutations sur la capacité de MeCP2 à lier l'ADN méthylé in vitro. Les résultats observés montrent que la mutation R453Q augmente l'affinité de MeCP2 pour l'A2DN méthylé. Le retard mental isolé lié à cette mutation pourrait donc être la conséquence d'une répression transcriptionnelle accrue de MeCP2.

2009/2010
Rett syndrome (RTT, MIM 312750) is a debilitating neurodevelopmental disorder that manifests in early childhood and affects almost exclusively girls. It 's a genetic disorder and is present worldwide with an estimated average incidence of 1:10,000 / 15,000 newborn girls. RTT is the second leading cause of mental retardation in female often misdiagnosed as autism or an unspecified developmental delay. In its classic version, this disease is caused by mutations in the transcription factor Methyl-CpG binding protein 2 (MeCP2) located on the female X chromosome (Amir et al., 1999). MeCP2 is a transcriptional regulator of many genes, including the neurotrophic factor Brain Derived Neurotrophic Factor (BDNF). BDNF is a member of the neurotrophins family and represents a key molecule for neuronal survival and development, and it is involved in learning and memory processes. BDNF levels significantly increase during the first period of postnatal life till it reaches the threshold necessary for the maturations of neurons with the development of dendrites, axons, dendritic spines and synapses. Recent studies have shown that in transgenic mice, in which MECP2 gene has been inactivated to mimic the symptoms of RTT, the mRNA levels of BDNF are reduced and, in these animals at 6-8 weeks of age, the total content of BDNF protein is reduced by 70%. Furthermore, the overexpression of BDNF in MeCP2-/Y mice leads to an increase in lifespan, improved locomotor deficits and electrophysiological defects, while the deletion of the gene coding for BDNF in the same animals leads to an early onset of the disease symptoms. The main goal of this thesis is to identify a possible drug treatment for the treatment of RTT. The idea is based on the fact that in animal models of RTT there are residual levels of mRNA coding for BDNF and that many drugs, mostly used for the treatment of depression, are able to increase the synthesis of BDNF. In this study we analyzed the residual expression of different BDNF transcripts in human brain and in two models of RTT (the mouse and the cellular model). Using quantitative PCR, we determined the residual expression of BDNF transcripts in post mortem brain samples of RTT patients, using tissues from somatosensory and motor cortex (Broadmann areas 1-5), which are the most affected by the disease. In addition, we determined the expression of BDNF transcripts in the cortex and hippocampus of MeCP2-/Y at different of post-natal ages. Finally, in order to establish a cellular model of disease, we disrupted the expression of MeCP2 gene in human neuroblastoma cell line SHSY-5Y (via RNA interference), and then we measured the expression levels of BDNF transcripts. Using the same cell line we obtained information on the translatability of the different BDNF transcripts through a luminescence assay, based on firefly luciferase as a reporter gene. We observed that in the absence of stimuli, each exon contained the 5'UTR of the BDNF leads to a different level of translation of the reporter gene. We therefore conducted a systematic pharmacological translation of each BDNF transcript to determine what compounds may be used to stimulate the synthesis of BDNF starting from its own specific transcripts. The treatment with serotonin and norepinephrine separately, or with two antidepressants such as desipramine and mirtazapine were found to be most effective in stimulating the BDNF synthesis. These results encourage the planning of future experiments to test the efficacy of existing antidepressant drugs in restoring the compromised BDNF levels in RTT.
La sindrome di Rett (RTT, MIM 312750) è un debilitante disordine neurologico dello sviluppo, che si manifesta nella prima infanzia e colpisce quasi esclusivamente le bambine. E’ una malattia genetica ed è presente in tutto il mondo con una incidenza media stimata di 1:10.000 / 15.000 bambine nate. La RTTè la seconda causa di ritardo mentale femminile spesso erroneamente diagnosticata come autismo o un non specificato ritardo dello sviluppo. Nella sua variante classica, questa malattia è causata da mutazioni nel fattore di trascrizione Methyl-CpG binding protein 2 (MeCP2) localizzato sul cromosoma femminile X (Amir et al., 1999). In condizioni normali MeCP2 è un regolatore della trascrizione di numerosi geni, tra cui il fattore neurotrofico Brain Derived Neurotrophic Factor (BDNF). Il BDNF è un membro della famiglia delle neurotrofine e rappresenta una molecola chiave per la sopravvivenza, lo sviluppo neuronale e i processi di apprendimento e memoria. I livelli di BDNF aumentano significativamente durante il primo periodo di vita postnatale fino a raggiungere la soglia necessaria per lo sviluppo di neuroni maturi con lo sviluppo di dendriti, assoni, spine dendritiche e sinapsi. Studi recenti hanno dimostrato che in topi transgenici in cui il gene MeCP2 è stato inattivato per mimare la sintomatologia della RTT, i livelli di mRNA del BDNF risultano ridotti e, in tali animali, a 6-8 settimane di età il contenuto totale di proteina BDNF è ridotto del 70% rispetto agli animali selvatici. Inoltre, l’overespressione di BDNF in topi privi del gene funzionale per MeCP2 porta a un aumento della durata della vita, a un miglioramento dei deficit locomotori e dei difetti elettrofisiologici, mentre la delezione del gene che codifica per bdnf negli stessi animali porta a una precoce insorgenza dei sintomi della patologia. Lo scopo di questa tesi è individuare un possibile trattamento farmacologico per la cura della RTT. L’idea si basa sul fatto che in modelli animali di RTT sono presenti dei livelli residui dell’mRNA codificante per il BDNF e che sono noti numerosi farmaci, utilizzati per lo più per la terapia della depressione che aumentano la sintesi del BDNF. In questo studio abbiamo analizzato l’espressione residuale dei diversi trascritti del BDNF in cervelli umani RTT e in due modelli di RTT. Mediante PCR quantitativa, abbiamo determinato l’espressione residuale dei trascritti del BDNF in cervelli umani di soggetti normali e affetti da RTT di età comparabile usando tessuti delle aree di Broadmann 1-5 che includono le cortecce somatosensoriali e motorie e che risultano essere le più colpite dalla malattia. Inoltre, abbiamo determinato i livelli dei trascritti del BDNF nella corteccia e nell’ippocampo di topi normali e in topi transgenici con delezione del gene MeCP2 (Bird) a differenti stadi post-natali. Infine, per stabilire un modello cellulare della malattia abbiamo distrutto l’espressione di MeCP2 nella linea cellulare di neuroblastoma umano SHSY-5Y mediante la tecnica dell’RNA interference e misurato l’espressione dei trascritti di BDNF. Utilizzando la stessa linea cellulare abbiamo ottenuto informazioni sulla traducibilità dei diversi trascritti del BDNF grazie a un saggio di luminescenza basato sull’utilizzo della luciferasi come gene reporter. Abbiamo osservato che in assenza di stimoli, ogni esone contenuto al 5’UTR del BDNF determina una diverso livello di traduzione del gene reporter. Abbiamo quindi condotto un’analisi farmacologica sistematica della traduzione di ciascun trascritto di BDNF per determinare quali composti possono essere usati per stimolare la sintesi BDNF partendo da un suo specifico trascritto. La combinazione di serotonina e norepinefrina, che si può ottenere anche mediante trattamento con antidepressivi quali desipramina e mirtazapina, è risultata essere la più efficace nello stimolare la sintesi del BDNF. Questi risultati ci incoraggiano a progettare futuri esperimenti per testare farmaci già esistenti sul mercato farmaceutico usando il modello cellulare ed il modello animale RTT.
XXIII Ciclo
1982

MeCP2 est une protéine multifonctionnelle agissant à de nombreux niveaux de contrôle des programmes génétiques. Un mauvais dosage de MeCP2 cause un groupe de maladies neurologiques dont le point commun est une déficience intellectuelle sévère. Des mutations ou une délétion de MECP2 causent le syndrome de Rett chez les filles, alors que sa surexpression cause chez les garçons le syndrome de duplication de MECP2. Plusieurs modèles murins de Mecp2-pathies ont été générés qui permettent d’expliciter les mécanismes qui sous-tendent l’apparition des symptômes dans ces différentes maladies. Dans notre laboratoire, deux modèles murins sont utilisés: le modèle Mecp2tm1Bird qui présente une déficience en Mecp2 et le modèle Mecp2Tg1 présentant une surexpression de Mecp2. Ce travail de thèse a permis de caractériser l’évolution postnatale des déficits moteurs et physiologique affectant la souris Mecp2Tg1. Nos résultats montrent que la surexpression de Mecp2 conduit à l’apparition de problèmes moteurs, et des convulsions chez la souris. En parallèle, nous avons étudié les déficits neuronaux affectants la voie GABAergique et glutamatergique chez la souris déficiente en Mecp2. Nous avons montré que la déficience en Mecp2 cause une dérégulation de la transmission synaptique dépendante du ‘territoire’ et de l’âge de la maladie. Ces dérégulations sous-tendent vraisemblablement des différences neurophysiologiques importantes entre les régions du cerveau qu’il nous reste encore à découvrir. Par ailleurs, nous avons constaté que la stimulation pharmacologique du système GABAergique par la Tiagabine, permet d’augmenter la survie des animaux Mecp2-déficients
MeCP2 is a multifunctional protein acting on many levels of control of genetic programs. Thus, an abnormal dosage of MeCP2 protein causes a group of neurological disorders with a common feature of severe intellectual disability. Mutations or deletions in MECP2 gene cause Rett Syndrome in females, whereas in boys its overexpression causes the MECP2-duplication Syndrome. Several mouse models of MECP2-pathologies were generated. The use of these models is crucial for understanding the mechanisms underlying the onset of symptoms related to the pathology. In our laboratory, two mouse models are under study: The Mecp2tm1Bird model with an Mecp2 deficiency and the transgenic Mecp2Tg1 model with a double expression of Mecp2. My thesis work enabled the characterization of the postnatal physiological and motor deficits affecting Mecp2Tg1 mice. My work led to a better understanding of the gene dosage effect. Our results showed that overexpression of Mecp2 in mice, led to the occurrence of motor problems, and seizures. In parallel, we studied the neural deficits affecting the GABA and the glutamate pathway in several structures of the Mecp2 deficient brain (Mecp2tm1bird). We showed that Mecp2-deficiency causes deregulation of the synaptic transmission, which is dependent on the area, and the age of the study. These deregulations underlie significant neurophysiological differences between the different regions of the brain that we still have to uncover. Furthermore, we found that pharmacological stimulation of the GABA system with Tiagabine, a molecule capable of acting on GABA transporters to prevent its uptake, increases the survival of Mecp2-deficients animals

Rett syndrome is a neurological condition that affects primarily girls. Approximately 40% of Rett syndrome cases arise from nonsense mutations. Several studies have shown that certain aminoglycosides can suppress some types of nonsense mutations in a context dependent manner, and allow the generation of a full length protein. It remains mostly unclear whether different nonsense mutations of MECP2 will be responsive to aminoglycoside treatment. In this study I tested whether some nonsense mutations of MECP2 seen clinically in Rett syndrome girls can be partially suppressed by aminoglycoside administration. My results show that aminoglycosides allow different mutant forms of MECP2 to be overcome in transiently transfected HEK-293 cells, but with differing levels of efficiency. Furthermore, I also show that aminoglycosides increased the prevalence of full length MeCP2 protein in a lymphocyte cell line derived from a Rett girl with R255X mutation. This study establishes the “proof of principle” that some nonsense mutations causing Rett syndrome can be suppressed by drμg treatment.

碩士
國立臺灣大學
臨床醫學研究所
97
Background: Mutations in the methy-CpG-binding protein 2 (MECP2) gene are well known to cause Rett syndrome (RTT), a severe neurodevelopmental disorder, occurs almost exclusively in females, with an estimated prevalence of approximately 1 in 10,000-15,000 females. RTT is often considered an X-linked dominant condition with male lethality (Hagberg 1985) characterized by a progressive loss of intellectual function, fine gross motor skills, and communicative abilities; deceleration of head growth; and the development of stereotypic hand movements, all occurring after a period of normal development. To date, mutations have been identified in the MECP2 sequences of approximately 80-90% of all RTT; the remaining 10-20% may possess noncoding regions of this gene, or they may harbor a second RTT inducing gene or locus. Several reports have identified large gene deletions in RTT patients that escaped detection by PCR-based screening strategies (Laccone et al. 2004; Schollen et al. 2003). This study reports the result of the mutation analysis of MECP2 in 97 patients with classic or atypical RTT or RTT-like, conducted to obtain a genotypeic representation of the mutational spectrum in Taipei, Taiwan. Patients and Methods: A total of 97 clinically suspicious RTT or RTT-like patients, referred to National Taiwan University Hospital for MECP2 gene mutation analysis, were involved in this study. Patients were grouping according to the clinical diagnostic criteria for the classic or variant Rett syndrome proposed by The Rett Syndrome Diagnostic Criteria Work Group (Hagberg et al. 2002), consisting of 23 classic, 37 atypical or RTT-like, and 37 grouped into idiopathic psychomotor retardation patients.The first group composed of RTT or RTT-like patients who were fulfilled the diagnostic criteria, and this group underwent DNA direct sequencing. In the mutation-negative patient, we used multiplex ligation-dependent probe amplification (MLPA) kit P015C to screen for large deletions. The other group who weren’t fulfill the classic or atypical RTT diagnostic criteria, were considered as idiopathic psychomotor retardation, but who may be related to the MECP2-related disorders were underwent MECP2-MLPA test directly to study the possible role of MECP2 duplication in this group. Results: Mutations in MECP2 were detected in 60.1% (14/23) of the patients presenting with classic RTT, and in 8.1% (3/37) of those with atypical RTT or RTT-like features. In total, 14 different MECP2 mutations, and 1 unclassified variant or mutation were identified in 17 of the 60 diagnosed as classical or atypical RTT or RTT-like patients. Most of the variants were missense mutations, accounting for 57.1% (8/14), followed by nonsense mutations 21.4% (3/14), and frame shift mutations 21.4% (3/14). We identified five novel mutations, four of which were point mutations, and one was deletion. The first, a nonsense mutation, C>T transition at the cDNA 844 (c.844C>T), lead to CGA change to TGA and became a stop codon (p.R282X). The other three were missense mutations (p.S149Y, p.V207M, p.Q437H), and one was a 83bp deletion (c.1117_1199del83) happened in exon 4 and lead to frameshift (p.S373fs). No mutations were found in exon 1 or 2 based on our study. However, we did not pinpoint a significant relationship between genotype and phenotype in these cases. In the group of 37 patients with idiopathic psychomotor retardation, we identified one case with Xq28 duplication including MECP2 gene. Conclusion: In Taiwan, most of the screenings are PCR-based and restricted to the coding part of the gene and therefore prone to miss gene dosage changes. To our knowledge, this is the first study in Taiwan to determine the role of large deletions of the MECP2 in RTT or RTT-like patients using MLPA method. Also, it is the first study in Taiwan to determine the possible role of duplications of the MECP2 region in children with psychomotor retardation using MLPA method. Although there was no large deletion of MECP2 found in our patients with Rett or Rett-like syndrome, it remains important to screen large deletions in these patients. Because duplication of MECP2 gene remains one of the possibilities in patients with psychomotor retardation of unknown etiology, we recommend screening MECP2 gene duplications in males with moderate to severe developmental delay, especially when a history of recurrent infections has been documented. We will continue our work to screen the large deletion of MECP2 gene in RTT or RTT-like patients, and the duplication of MECP2 gene in children with psychomotor retardation of unknown etiology to clarify the role of such changes in these patients.

Intractable epilepsy remains one of the top issues affecting the quality of living in Rett children. While several MeCP2-deficient mouse models of Rett Syndrome have been established, minimal information exists on how the loss of MeCP2 affects brain network activity. To address this issue, in vivo recordings of the hippocampus and somatosensory cortex of MeCP2-deficient mice were taken during exploration, immobility, and sleep. The frequency of hippocampal theta oscillations was significantly attenuated in MeCP2-deficient mice during exploration. A subset of MeCP2-heterozygotes displayed spontaneous, cortical epileptiform-like discharges in the immobile-awake state. Similar epileptiform-like discharges were observed in one of the four Mecp2-null mice recorded. Aside from these EEG abnormalities, basal network activity was preserved. Further, convulsive seizures were not seen. Collectively, these findings indicate that a deficiency of MeCP2 in mice leads to only subtle alterations in brain wave activity, contrasting the severely abnormal EEG observed in Rett girls.

abstract: Methyl-CpG binding protein 2 (MECP2) is a widely abundant, multifunctional regulator of gene expression with highest levels of expression in mature neurons. In humans, both loss- and gain-of-function mutations of MECP2 cause mental retardation and motor dysfunction classified as either Rett Syndrome (RTT, loss-of-function) or MECP2 Duplication Syndrome (MDS, gain-of-function). At the cellular level, MECP2 mutations cause both synaptic and dendritic defects. Despite identification of MECP2 as a cause for RTT nearly 16 years ago, little progress has been made in identifying effective treatments. Investigating major cellular and molecular targets of MECP2 in model systems can help elucidate how mutation of this single gene leads to nervous system and behavioral defects, which can ultimately lead to novel therapeutic strategies for RTT and MDS. In the work presented here, I use the fruit fly, Drosophila melanogaster, as a model system to study specific cellular and molecular functions of MECP2 in neurons. First, I show that targeted expression of human MECP2 in Drosophila flight motoneurons causes impaired dendritic growth and flight behavioral performance. These effects are not caused by a general toxic effect of MECP2 overexpression in Drosophila neurons, but are critically dependent on the methyl-binding domain of MECP2. This study shows for the first time cellular consequences of MECP2 gain-of-function in Drosophila neurons. Second, I use RNA-Seq to identify KIBRA, a gene associated with learning and memory in humans, as a novel target of MECP2 involved in the dendritic growth phenotype. I confirm bidirectional regulation of Kibra by Mecp2 in mouse, highlighting the translational utility of the Drosophila model. Finally, I use this system to identify a novel role for the C-terminus in regulating the function of MECP in apoptosis and verify this finding in mammalian cell culture. In summary, this work has established Drosophila as a translational model to study the cellular effects of MECP2 gain-of-function in neurons, and provides insight into the function of MECP2 in dendritic growth and apoptosis.
Dissertation/Thesis
Doctoral Dissertation Neuroscience 2015

Das Rett-Syndrom ist eine postnatal progressiv verlaufende neurologische Entwicklungsstörung, die x-chromosomal vererbt. Das klassische Rett-Syndrom entsteht durch eine spontane Mutation des MECP2-Gens, welches für die Kodierung des Transkriptionsfaktors MeCP2 (methyl CpG binding protein 2) verantwortlich ist. Das Krankheitsbild verläuft in vier Stadien und ist vor allem von geistiger Retadierung, motorischer Dysfunktion und Unregelmäßigkeiten der Atmung geprägt. In verschiedenen Untersuchungen wurden bereits verschiedene zelluläre Dysfunktionen und Beeinträchtigungen der Mitochondrien bestätigt, weshalb wir weitere Untersuchungen anstrebten. Ziel dieser Dissertation war es, mögliche Einflüsse verschiedener Enzyme auf die ROS-Produktion zu untersuchen und somit die Erkenntnisse aus vorangegangenen Arbeiten zu erweitern. Im Fokus dieser Untersuchungen lagen zum einen die mitochondriale ROS-Produktion sowie die extramitochondriale ROS-Produktion. Durch unterschiedliche pharmakologische Modulationen wurden beide Systeme beeinflusst, um den jeweiligen Beitrag zur gesamten ROS-Produktion abzuschätzen.

Das Rett-Syndrom ist eine postnatale neurologische Entwicklungsstörung, der eine Mutation im Methyl-CPG-bindenden Protein 2 (MECP2) zugrunde liegt. Es betrifft überwiegend Mädchen und geht mit kognitiven Beeinträchtigungen, motorischen Stereotypien und Atmungsstörungen einher. Es existieren vielfältige Hinweise dafür, dass die Pathogenese des Rett-Syndroms im Zusammenhang mit einer beeinträchtigten Mitochondrienfunktion steht. Genetische Untersuchungen des Rett-Genoms zeigten, dass eine Untereinheit des Komplex III der Atmungskette dysreguliert ist und die innere Mitochondrienmembran ein Protonenleck aufweist. Weiterhin fanden sich Hinweise für erhöhten oxidativen Stress in Blut- und Liquoruntersuchungen. Um den intrazellulären Redox-Status zu quantifzieren, wurde die genetisch kodierte optische Sonde roGFP1 verwendet, die semiquantitative Messungen reaktiver Sauerstoffspezies ermöglichte. Es zeigte sich, dass Mecp2(-/y)-Hirnschnitte bereits unter Ruhebedingungen erhöhtem oxidativen Stress ausgesetzt sind. Auf der Suche nach der Ursache wurden die intrazellulären antioxidativen Schutzenzyme Superoxid-Dismutase und Katalase sowie das Glutathionsystem überprüft. Alle drei Enzymsysteme zeigten Funktionsstörungen und waren nicht in der Lage, extern applizierten oxidativen Stress im gleichen Umfang zu kompensieren wie die Enzyme der Wildtyp-Vergleichsgruppe. Um die zytosolischen Redox-Verhältnisse zu beeinflussen, wurden Untersuchungen mit den Antioxidantien Ascorbat, Trolox und Melatonin vorgenommen. Dabei zeigte sich, dass Antioxidantien eine potentielle pharmakologische Maßnahme darstellen, um die zu oxidativen Verhältnissen verschobene Redox-Homöostase in Mecp2(-/y)-Hippokampi zu senken und folglich zu normalisieren. Vor allem das Vitamin E-Derivat Trolox stellte sich als wirkungsvoller Radikalfänger heraus und bietet sich für weitere detaillierte Untersuchungen hinsichtlich einer therapeutischen Option des Rett-Syndroms an. Die externe Störung der mitochondrialen Funktion durch die Induktion einer transienten Hypoxie sowie die gezielte Inhibition verschiedener Atmungskettenkomplexe zeigte eine deutlich erhöhte Hypoxieempfindlichkeit der Mecp2(-/y)-Hippokampi und war mit einer erhöhten ROS-Produktion verbunden. In der Arbeit gelang es erstmals, die bereits mehrfach postulierte Störung der Redox-Homöostase im Rett-Syndrom direkt auf zellulärer Ebene nachzuweisen. Die erhobenen Befunde liefern mögliche mechanistische Erklärungsansätze für die Störung der synaptischen Plastizität im Rett-Syndrom, da es klare Verbindungen zwischen dem zellulären Redox-Status und dem Kalziumhaushalt gibt, der durch redoxsensitive Proteine mitreguliert wird. Somit konnte eine zentrale Dysregulation der Erkrankung identifziert werden, die unter Umständen auch neue pharmakologische Angriffspunkt aufzeigt, um die Symptomatik des Rett-Syndroms zu mildern.

Rett syndrome is a neurodevelopmental disorder that is predominately caused by mutations of the MECP2 gene. As neuronal apoptosis is not observed in RTT patients and MeCP2-deficient mice, the neurological deficits may be reversible. To address this, we reactivated MeCP2 expression ubiquitously in MeCP2-deficient mice after symptom onset. Our results showed that life span, behavioural performances, EEG activity, thermoregulation, and daily rhythmic activity were significantly improved after MeCP2 reactivation. Furthermore, the extent of improvement was dependent upon the efficiency of MeCP2 reactivation. To assess the role of the catecholaminergic system in Rett syndrome pathophysiology, we selectively preserved MeCP2 function within tyrosine hydroxylase expressing cells. We observed a significant improvement in the life span of male rescue mice and reduced sudden unexplained death rates in female rescue mice. Behavioural performances and EEG patterns were also significantly improved.

Für das Rett Syndrom, eine der häufigsten genetischen Ursachen für mentale Retardie-rung bei Frauen, gibt es bisher keine kausale Therapie, obwohl gentherapeutische Studi-en mit konditionellen knockout Mäusen gezeigt haben, dass es sich um eine therapierbare Erkrankung handelt. Um neue Therapien entwickeln zu können, werden Mausmodelle benötigt, die auf den beim Menschen am häufigsten gefundenen Mutation beruhen. In der vorliegenden Arbeit wurde ein Mausmodell mit der häufigsten humanen Nonsense-Mutation R168X im Mecp2 Gen charakterisiert. Mit Hilfe dieses Mausmodells wurden dann die Therapieansätze der „Stop-Codon Readthrough-Therapie“ und einer Knochenmarktransplantation auf ihre Wirksamkeit in vitro und in vivo untersucht. Die Charakterisierung der Mauslinie zeigte, dass männliche MeCP2R168X-Mäuse im Gegensatz zu anderen MeCP2-Mausmodellen kein verkürztes MeCP2 Protein exprimieren. Desweiteren weisen männliche MeCP2R168X-Mäuse einen Phänotyp, inklu-sive der drastisch verkürzten Lebenspanne, auf, wie er bei bereits etablierten Mausmo-dellen für das Rett Syndrom beschrieben wurde. Dagegen zeigten weibliche, heterozy-gote MeCP2R168X-Mäuse nur einen sehr mild ausgeprägten Phänotyp verglichen mit bereits etablierten MeCP2-Mauslinien. Für die „Stop-Codon Readthrough-Therapie“ wurde die Effizienz der Aminoglykoside Geniticin, Gentamicin und Neomycin, der Komponenten NB54, NB84 und NB124, sowie der niedermolekularen Substanz PTC124 auf ihre Wirksamkeit bei der Induktion eines Readthroughs mit transfizierten HeLa-Zellen und MeCP2R168X/y-Mausohrfibroblasten in vitro untersucht. Dabei zeigte sich eine deutliche Steigerung der Readthrough-Effizienz der NB-Komponenten, gemessen an der detektierbaren Menge an MeCP2, mit zunehmender Generation (NB54 --> NB84 --> NB124) und gegenüber dem klinisch angewandten Gentamicin. Während die Behandlung mit Neomycin zu einem minimalen Readthrough-Produkt führte, zeigte die Behandlung mit PTC124 kei-nen messbaren Readthrough. Anschließend wurden männliche MeCP2R168X-Mäuse mit den in vitro getesteten Sub-stanzen, mit Ausnahme von Geniticin, behandelt. Die Expression eines MeCP2-Proteins voller Länge konnte durch keine der applizierten Substanzen induziert werden. Auch bei Behandlungen über einen längeren Zeitraum mit hohen Dosierungen, im Fall von Gentamicin nahe der LD50-Dosis und nachweisbarer intrazellulärer Aufnahme, konnte in den behandelten Tieren weder ein verkürztes noch ein MeCP2 Protein nativer Länge detektiert werden. Die Ergebnisse dieser Arbeit zeigen, dass für die „Stop-Codon Readthrough-Therapie“ für das Rett Syndrom neue Komponenten entwickelt werden oder andere Applikationswege gewählt werden müssen, da mit den derzeit verfügbaren Substanzen kein therapeutischer Erfolg erzielt werden kann. Im letzten Teil dieser Arbeit wurde die Theorie einer gestörten Phagozytose MeCP2-defizienter Mikroglia, sowie die Therapie von MeCP2-defizienten Mäusen durch eine Knochenmarktransplantation überprüft. Dabei konnte weder in vitro noch in vivo eine Veränderung der Phagozytoseaktivität der MeCP2-defizienten Mikroglia nachgewiesen werden, wie sie von Derecki und Kollegen publiziert wurde. Die Transplantation von gesundem Knochenmark führte bei männlichen MeCP2R168X-Tieren zu keiner Verlängerung der Überlebensspanne oder einer allgemeinen Abmilde-rung der Symptomatik, wie sie ebenfalls von Derecki und Kollegen publiziert wurde. Bei weiblichen Tieren führte die Transplantation gesunden Knochenmarks zu einer Verschlechterung der motorischen Fähigkeiten. Diese Ergebnisse sind im Einklang mit denen Ergebnissen der Arbeitsgruppen von An-drew Pieper, Antonio Bedalov und Jeffrey Neul, die in anderen Mausmodellen die Wir-kung der Knochenmarktransplantation untersuchten. Die Ergebnisse aller beteiligten Arbeitsgruppen legen daher nahe, dass eine Knochen-marktransplantation nach einer Ganzkörperbestrahlung keine geeignete Therapie für das Rett Syndroms darstellt.

Rett Syndrome (RTT) is a developmental disorder that affects numerous neuronal systems that underlie problems with breathing, movement, cognition and sleep. RTT is caused by mutations in the methyl-CpG-binding protein 2 (Mecp2) gene. MeCP2 is a ubiquitous protein that is found in all mature neurons and binds to methylated DNA to repress transcription; thus regulating protein expression levels in neurons. The mutations in Mecp2 affect a large number of proteins that are crucial for regulating neuronal activity. Despite the abnormal expression of many of these proteins, mice with a total loss of MeCP2 can live to adulthood and some people with RTT can live to a very late age as well. It is possible that mutations in the Mecp2 gene not only cause widespread defects, but also elicit neuroadaptive processes that may limit the impact of the MeCP2 dysfunction. To test this hypothesis we performed these studies in which we focused on how synaptic and membrane currents were altered to maintain normal neuronal activity in Mecp2-null mice. We show two examples from different neurons where neuroadaptations of ion channel expression allowed the neuron to remain viable. First, the properties of the nicotinic acetylcholine receptor (nAChR) current were altered in LC neurons in Mecp2-null mice. This was caused by changes in the nicotinic receptor subunit expression. Despite the changes in the nAChR current, the cholinergic modulation of LC neuron activity in WT and Mecp2-null mice were similar. Secondly, we show that the fast Na+ voltage-gated and the hyperpolarization-activated currents were altered in mesencephalic trigeminal V (Me5) propriosensory neurons. The changes in the hyperpolarization-activated current caused a smaller sag and post-inhibitory rebound. Opposite to what we expected, these cells were hyperexcitable. The hyperexcitability was due to changes in the fast Na+ voltage-gated current causing a decreased action potential threshold. Alterations in the ionic currents in Me5 neurons seem to be due to changes in subunit expression patterns. These results indicate that despite the complications caused by defects in the Mecp2 gene, neurons respond by rearranging receptor / ion channel expression. This reorganization allows neurons to remain viable despite the MeCP2 deficiency.

Rett Syndrome (RTT) is a neurodevelopmental disorder affecting 1 out of 10,000 females worldwide. Mutations of the X-linked MECP2 gene encoding methyl CpG binding protein 2 (MeCP2) accounts for >90% of RTT cases. People with RTT and mice with Mecp2 disruption show autonomic dysfunction, especially life-threatening breathing disorders, which involves defects in brainstem neurons for breathing controls, including neurons in the locus coeruleus (LC). Accumulating evidence obtained from Mecp2−/Y mice suggests that imbalanced excitation/inhibition or the impaired synaptic communications in central neurons plays a major role. LC neurons in Mecp2−/Ymice are hyperexcited, attributable to the deficiency in GABA synaptic inhibition. Several previous studies indicate that augmenting synaptic GABA receptors (GABARs) leads to a relief of RTT-like symptoms in mice. The extrasynaptic GABARs located outside synaptic cleft, which have the capability to produce sustained inhibition, and may be a potential therapeutic target for the rebalance of excitation/inhibition in RTT. In contrast to the rich information of the synaptic GABARs in RTT research, however, whether Mecp2 gene disruption affects the extrasynaptic GABARs remains unclear. In this study, we show evidence that the extrasynaptic GABAR mediated tonic inhibition of LC neurons was enhanced in Mecp2−/Ymice, which seems attributable to the augmented δ subunit expression. Low-dose THIP exposure, an agonist specific to δ subunit containing extrasynaptic GABARs, extended the lifespan, alleviated breathing abnormalities, enhanced motor function, and improved social behaviors of Mecp2−/Ymice. Such beneficial effects were associated with stabilization of brainstem neuronal hyperexcitability, including neurons in the LC and the mesencephalic trigeminal V nucleus (Me5), and improvement of norepinephrine (NE) biosynthesis. Such phenomena were found in symptomatic Mecp2+/− (sMecp2+/−) female mice model as well, in which the THIP exposure alleviated the hyperexcitability of both LC and Me5 neurons to a similar level as their counterparts in Mecp2−/Y mice, and improved breathing function. In identified LC neurons of sMecp2+/− mice, the hyperexcitability appeared to be determined by both MeCP2 expression and their environmental cues. In conclusion, intervention to extrasynaptic GABAAR by chronic treatment with THIP might be a therapeutic approach to RTT-like symptoms in both Mecp2−/Y and Mecp2+/− mice models and perhaps in people with RTT as well.

Rett syndrome (RTT), a severe neurodevelopmental disorder, affects females resulting from loss-of-function mutations in the X-linked transcription factor methyl-CpG-binding protein 2 (MECP2). RTT patients show severe verbal, motor, respiratory, and intellectual impairments. We studied two forms of activity-dependent plasticity in Mecp2 mutant mice to better understand the loss of MECP2 function in neuronal circuit and sensory processing. Sensory deprivation was applied by trimming one whisker to 3 mm to study long-term cortical plasticity in Mecp2-/y mice. Intrinsic optical signaling (IOS) imaging showed the neuronal response to wiggling a non-trimmed was consistent from day 0 to 14 but reduced for the trimmed whisker by 49.0 ± 4.3% in wild type (WT) and 22.7 ± 4.6% (p=0.0135) in RTT mice. Primary hindlimb (HL) somatosensory cortical responses to vibratory stimulation were assessed by IOS and intracortical local field potential (LFP). Responses were assessed before, during and, after 1 hour of repeated HL vibratory stimulation (100Hz,1sec, ISI 6 sec) in symptomatic male (4-6 week), female (10-12 month) and pre-symptomatic young female (4 week) RTT model mice. After 1-hour, cortical responses to test vibrations were reduced by approximately 40% in RTT and WT mice as assessed by both methods. Recovery of the IOS responses (1 sec vibration at 100Hz) and LFP (300µm below pia, 7 stimuli, 100mse ISI) were tested at 15 min intervals for 1 hour after ceasing the repeated stimulation. Reduced responses persisted for at least 60 min in WT but recovered to 90-100% of normal within 15-30 min in RTT. Analysis of the LFP responses within the test train indicated that the reduced cortical sensitivity during and after continuous stimulation resulted primarily from an increase in adaptation during the 7-stimulus test train rather than a reduction in the response to a single vibratory stimulus in all groups. Retention of this increased STA is the primary cause of the persistently reduced tactile response in young WT female mice, while in RTT mice the rapid recovery of tactile sensitivity was due to the return of STA to lower, baseline levels. Male RTT mice exhibited a marked increased excitability to the first stimulus in the test train resulting in hypersensitivity to a single vibration by 45 minutes. Old females exhibited the same pattern of adaptation and recovery but retention of adaptation was less pronounced in both WT and RTT compared to younger animals suggesting an age-dependent reduction in neural plasticity may mask deficits specific to RTT. Recording sciatic nerve sensory afferent activity did not reveal any STA, persistent adaptation or sensitization of peripheral afferent endings in any groups. I propose persistent sensory adaptation mediated by increased short-term adaptation may reflect enhanced feedback by inhibitory elements of circuits within the sensory pathway. The rapid recovery of responsiveness in young female RTT mice may therefore reflect a deficit in the capacity for activity dependent plasticity to consolidate and thus could provide a platform to understand the causes of learning and cognitive deficits in RTT patients.
Graduate

Mutations in the X-linked gene encoding Methyl-CpG-binding protein 2 (MECP2) cause the neurodevelopmental disorder Rett syndrome, a common genetic cause of mental retardation in females. Although alterations in performance of MeCP2-deficient mice in specific behavioural tasks have been documented, it remains unclear if, and to what degree, MeCP2 dysfunction affects patterns of periodic behavioural and electroencephalographic activity. To address this, we monitored daily rhythmic patterns of core body temperature, gross motor activity, and cortical delta power from MeCP2-deficient mice and correlated it against regional MeCP2 expression levels. Our results show that normal daily rhythmic behavioural patterning of delta wave activity, body temperature and mobility are disrupted in these mice. Moreover, MeCP2-deficient mice displayed lower average core body temperature and significantly greater body temperature fluctuation than wild-type female mice. Finally, we also found that epileptiform discharge activity in MeCP2-deficient mice is more predominant during times of behavioural activity compared to inactivity.

Rett Syndrome (RTT) is a neurodevelopmental disorder primarily caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). The mosaic brain environment in heterozygous (MECP2+/-) females consists of both MeCP2-wildtype (MeCP2+) and Mecp2-mutant (MeCP2-) neurons. To separate possible cell autonomous and cell non-autonomous effects three-dimensional morphological analysis was performed on individually genotyped layer V pyramidal neurons in the primary motor cortex of heterozygous (Mecp2+/-) and wild-type (Mecp2+/+) mature female mice (>8 months old) from the Mecp2tm1.1Jae line. Mecp2+/+ neurons and Mecp2+ were found to be indistinguishable while Mecp2- neurons have significantly reduced basal dendritic length (p<0.05), predominantly in the region 70-130 μm from the cell body, culminating in a total reduction of 15%. Mecp2- neurons have three (17%) fewer total branch points, lost specifically at the second and third branch orders. Thus the reduced total dendritic length in Mecp2- neurons is a result of fewer higher-order branches. Soma and nuclear areas of 30 Mecp2+/- female mice (5-21 months) with X chromosome inactivation (XCI) ratios ranging from 12% to 56% were analyzed. On average Mecp2- somata and nuclei were 15% and 13% smaller than Mecp2+ neurons respectively. The variation observed in the soma and nuclear sizes of Mecp2- neurons was not due to age, but was found to be correlated with the XCI ratio. Animals with a balanced XCI ratio (approximately 50% Mecp2-) were found to have Mecp2- neurons with a less severe cellular phenotype (11-17% smaller than Mecp2+). Animals with a highly skewed XCI ratio favouring expression of the wild-type allele (less than 30% Mecp2-) were found to have a more severe Mecp2- cellular phenotype (17-22% smaller than Mecp2+). These data support indicate that mutations in Mecp2 exert both cell autonomous and cell non- autonomous effects on neuronal morphology.
Graduate

In this study we tested whether the prevalence or synaptic distribution of NMDA receptor subunits would be altered in the brain of the MeCP2-null mouse model of Rett syndrome. Detergent resistant membranes (DRMs) and post-synaptic densities (PSDs) were isolated from the synaptic membranes treated with TritonX-100, and resolved by sucrose density gradient centrifugation. Immunoblot analysis of the resulting density gradient fractions revealed that the relative distribution of the different NMDA receptor subunits between the DRM fractions, soluble fractions, and insoluble postsynaptic density fractions was preserved in the MeCP2-null brain. However, analysis of the overall NMDA receptor subunit prevalence within these fractions revealed a significant decrease in the expression of the NR1 and NR2A subunits, but not the NR2B subunit, in the MeCP2-null brain. The preservation of distribution of NMDAR subunits to the synaptic membranes, together with the decrease in NR1 and NR2A prevalence, suggest an imbalance in equilibrium between the mature and the immature synapses in a mouse model of Rett syndrome.