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.

Methyl-CpG binding protein 2 (MeCP2) is an epigenetic regulator in brain that binds to both methylated and hyroxymethylated DNA with similar affinities. MeCP2 has two isoforms, MeCP2E1 and MeCP2E2 with distinct N-terminus. These isoforms are generated by alternative splicing of the MECP2/Mecp2 gene. Despite the wealth of knowledge about the molecular properties of MeCP2 as a whole protein, little is known regarding its isoform components. Importantly, basic fundamental questions such as MeCP2 isoform-specific expression profile and the underlying regulatory mechanisms are yet to be fully addressed. To further explore the role(s) of MeCP2 isoforms, we have characterized MeCP2 isoform-specific expression profiles. In this study, we show that MeCP2 isoforms are differentially distributed in the adult murine brain, and are temporally expressed during mouse brain development. Our study allows us to gain further insight into the function(s) of MeCP2 isoforms in the central nervous system.

Methyl-CpG binding protein 2 (MeCP2) is an epigenetic regulator in brain that binds to both methylated and hyroxymethylated DNA with similar affinities. MeCP2 has two isoforms, MeCP2E1 and MeCP2E2 with distinct N-terminus. These isoforms are generated by alternative splicing of the MECP2/Mecp2 gene. Despite the wealth of knowledge about the molecular properties of MeCP2 as a whole protein, little is known regarding its isoform components. Importantly, basic fundamental questions such as MeCP2 isoform-specific expression profile and the underlying regulatory mechanisms are yet to be fully addressed. To further explore the role(s) of MeCP2 isoforms, we have characterized MeCP2 isoform-specific expression profiles. In this study, we show that MeCP2 isoforms are differentially distributed in the adult murine brain, and are temporally expressed during mouse brain development. Our study allows us to gain further insight into the function(s) of MeCP2 isoforms in the central nervous system.

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.

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.

Methyl-CpG binding protein 2 (MeCP2) and histone H1 are important chromatin associated proteins. Both exhibit their own extent of complexity as MeCP2 is an intrinsically disordered protein (IDP) that interacts with many different partners involved in several cellular processes and histone H1 consists of 11 different subtypes each of them associated with different posttranslational modifications (PTMs). An interesting avenue for the study of these proteins is in neurons where MeCP2 is very abundant and histone H1 level is half that observed in other somatic tissues. Several reports in the past have proposed that this lower level of histone H1 is due to the abundance of MeCP2 which displaces histone H1. However, this hypothesis has been debated and there is no clear consensus. In an attempt to study this controversy, a cell model system ReNCell WT and MeCP2-KO was used that can be induced to differentiate into neurons. The protein levels, transcript levels and localization of histone H1 subtypes in these cells were analyzed using HPLC, RT-qPCR and immunofluorescence, respectively. The results show that ReNCell WT and MeCP2-KO do not exhibit significant differences in their relative amount of histone H1 protein and transcript level neither at the proliferative nor at the later differentiated stages. However, HPLC analyses show that the histone H1 subtypes of these two cell types exhibit significant elution differences probably resulting from differences in their PTM content. Immunofluorescence analyses show that WT ReNCell differentiation as determined by extension of dendritic or axonic processes can be seen to occur over the course of one week and there is a significant difference in the nuclear area of these two cells at 8 DIV. This study provides important preliminary data for future research in MeCP2 and histone H1 using this cell model system and show that MeCP2 may have a bearing on histone H1 PTMs. / Graduate

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.

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.

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.