Tesis sobre el tema "Syndrome de Rett – Génétique"

Le syndrome de Rett (RTT) est une encéphalopathie neurodéveloppementale sévère d'origine génétique affectant presque exclusivement les filles avec une incidence de une naissance de filles sur 15 000. Dans plus de 95% des cas de forme classique, le syndrome de Rett est associé à une mutation dans le gène MECP2 (methyl-CpG-binding protein 2) codant pour une protéine modulatrice de l'expression des gènes. Dans ce travail, une altération de la stabilité du réseau de microtubules a été identifiée dans les astrocytes et les fibroblastes cutanés en tant que phénotype cellulaire associé à la déficience en Mecp2/MECP2. Cette perturbation de la dynamique des microtubules induit des dysfonctionnements cellulaires dans les astrocytes déficients en Mecp2/MECP2 comme une désorganisation du transport des vésicules dépendant des microtubules. Les perturbations de la dynamique des microtubules et du transport vésiculaire ont été corrigées in vitro dans les astrocytes murins et humains déficients en Mecp2/MECP2 par un traitement par une faible concentration d'épothilone D, molécule stabilisatrice des microtubules. De plus, un premier essai de traitement in vivo par l'épothilone D a été réalisé et suggère un effet bénéfique de cette molécule sur le comportement exploratoire des souris déficientes en Mecp2
Rett syndrome (Ri I) is a severe neurodevelopmental encephalopathy affecting almost exclusively girls with an incidence of 1/15000 female birth. In more than 95% of the typical cases, Rett syndrome is associated with a mutation in the MECP2 gene (methyl-CpG binding protein 2) coding for a modulator of gene expression. Here we show that alteration of microtubule network stability in astrocytes and skin fibroblasts is a cellular phenotype associated with Mecp2/MECP2 deficiency. This alteration of microtubule dynamics triggers cellular dysfunctions in Mecp2-deficient astrocytes such as disorganization of microtubule dependent vesicular transport. Disturbances of microtubule dynamics and vesicular transport have been corrected in vitro in human and mouse astrocytes by a treatment with low level of epothilone D, a microtubule stabilizer. Moreover, a first try of in vivo epothilone D treatment was realized and indicated a beneficial effet of this molecule on Mecp2-deficient mite exploratory behavior

Le syndrome de Rett (RTT) est une encéphalopathie neurodéveloppante sévère touchant presque exclusivement les filles. Sur le plan clinique, la pathologie est caractérisée par un développement apparemment normal jusqu'à 6-18 mois, suivi d'une période de régression suggérant un arrêt de développement du cerveau, et durant laquelle une perte des acquisitions motrices et psychiques est observée. Sur le plan neuro-anatomique, les malades présentent une microcéphalie associée à des anomalies qualitatives des cellules neuronales, tant fonctionnelles que morphologiques. En 1999, des mutations du gène MECP2, codant pour une protéine de liaison à l'ADN, ont été identifiées chez la grande majorité des patientes présentant la forme typique du RTT, ainsi que chez la moitié des patientes présentant une forme atypique. En 2004, le gène CDKL5 codant pour une protéine kinase, a été impliqué dans les formes atypiques avec épilepsie précoce. Malgré dix années de recherches intensives tentant de lier les mutations de ces gènes aux désordres neurologiques observés, nous ignorons toujours quel est le rôle précis de MeCP2, sur quels gènes agit cette protéine, et quelles sont les conséquences moléculaires du déficit de MeCP2. De plus, la similarité des phénotypes et la mise en évidence d'une interaction in vitro entre les protéines MeCP2 et CDKL5 suggèrent que ces deux protéines pourraient intervenir dans une même voie de signalisation. Mes travaux de thèse se sont articulés autour de trois axes : (i) l'identification des cibles moléculaires de la protéine MeCP2, (ii) l'identification des gènes dérégulés lors d'un déficit en CDKL5, et l'étude de la potentialité d'une voie commune de signalisation entre MeCP2 et CDKL5, et (iii) l'étude des conséquences fonctionnelles de la dérégulation des cibles de MeCP2. Grâce à l'utilisation d'un modèle de cultures clonales de fibroblastes humains issus de patientes atteintes du syndrome de Rett, nous avons pu mettre en évidence des cibles directes et indirectes de l'action de MeCP2. Certaines de ces cibles sont notamment impliquées dans la régulation de l'arborisation dendritique, la modulation de la synaptogénèse et la dynamique du cytosquelette et tendent à confirmer les anomalies de maturation et de maintien du réseau neuronal à l'origine des perturbations neuropathologiques observées chez les patientes. Nous avons également pu identifier les dérégulations transcriptionnelles consécutives au déficit en CDKL5 et montrer que MeCP2 et CDKL5 ne partagaient probablement pas la même voie de signalisation. De façon intéressante, les anomalies d'expression transcriptionnelles mises en évidence lors de la perte de fonction de CDKL5 pourraient également être impliquées dans l'altération de la morphogénèse dendritique observée chez les patientes, suggérant que, même si les protéines MeCP2 et CDKL5 n'empruntent pas les mêmes voies, un défaut de l'une ou l'autre de ces protéines aboutirait à des conséquences similaires qur la morphologie neuronale. Dans l'ensemble, nos travaux ont permis une progression de la compréhension des mécanismes physiopathologiques à l'origine du syndrome de Rett, mettant en lumière l'importance du rôle de MeCP2 dans la régulation de la stabilité des microtubules, et soulignant le rôle majeur des astrocytes dans cette pathologie
Pas de résumé en anglais

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

Le syndrome de Rett est une maladie génétique rare conduisant à un désordre neurologique grave, causée par des mutations de la protéine MeCP2. MeCP2 est une protéine de liaison à l’ADN dont le domaine MBD lie spécifiquement les cytosines hydroxyméthylées au sein de répétitions de dinucléotides CA. Dans cette thèse, je présente une étude structurale par cristallographie aux rayons-X et une caractérisation biophysique par ITC et NanoDSF des cinq mutations faux-sens les plus fréquentes du MBD de MeCP2 (T158M, R133C, R106, P152R et S134C) qui sont naturellement présentes chez les patients atteints du syndrome de Rett. Des structures comprenant les MBD P152R et S134C en complexe avec un ADN contenant une cytosine hydroxyméthylées au sein d’une répétition de dinucléotides CA ont pu être déterminées. Ces structures révèlent que les mutations causent une altération de l’interaction spécifique et critique de MeCP2, qui permet normalement la reconnaissance des cytosines hydroxyméthylées. De plus, la caractérisation biophysique des mutations du domaine MBD de MeCP2 révèle une baisse de l’affinité de liaison à l’égard des répétitions de dinucléotides CA hydroxyméthylées et une baisse de la stabilité du repliement du domaine pour certaines mutations
Rett syndrome is a rare genetic disorder leading to severe neurological impairments. It is caused by mutations of the MeCP2 protein. MeCP2 is a DNA binding protein whose MBD domain specifically binds to hydroxymethylated cytosines in the context of CA dinucleotide repeats. This thesis presents a structural study by X-ray crystallography and a biophysical characterization by ITC and NanoDSF of the five most frequent mutations that are naturally affecting the MBD domain of MeCP2 in patients with Rett syndrome.The structures of the MBD P152R and S134C in complex with DNA containing a hydroxymethylated cytosine within a CA dinucleotide repeat have been determined. These structures reveal an alteration caused by the mutation of the specific and critical interaction of MeCP2 that usually enables the recognition of hydroxymethylated cytosines. In addition, the biophysical characterization of the mutations reveals a decrease of the binding affinity of the MBD toward hydroxymethylated CA dinucleotide repeats along with a decrease in the MBD folding stability for some mutations

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. .

Rett syndrome is a profoundly handicapping neurological disorder with an incidence of approximately 1 in 10,000 live female births. The recently acknowledged syndrome has neither known cause nor cure. The nature of the syndrome is particularly distressing for parents, since regression occurs in infants after apparently normal early development. Rapid therapeutic intervention after diagnosis is considered vital in order to ameliorate detrimental stereotypical behaviours and to limit painful physical deterioration. However, there is little published research dealing with the effects of therapies or the manner which they are delivered in schools. The National Curriculum in Britain aims to ensure that pupils have access to a broad and balanced education, regardless of ability. The rights of children are recognised in legislation, and it is accepted that some pupils will need specialist support. Recent trends are towards greater involvement of parents in educational issues and towards encouraging the development of partnerships in education. This research examines therapy provision for girls with Rett syndrome aged between 7 and 12 years, investigates parental opinions regarding effectiveness of therapies in key skill areas of communication, hand function, motor ability and learning ability, and touches upon the broader issues of how essential needs can be considered alongside the rights of children in the classroom environment. The study shows that the roles of teachers and therapists are complementary, and suggests that enhanced professional interaction may be a future objective. Music therapy, physiotherapy, hydrotherapy and speech therapy are the most common forms of intervention, and the perceived values of each are reported. Inconsistency in provision is apparent, and whilst all therapies are considered beneficial, no single therapy is identified as significantly more beneficial than others.

Rett syndrome is a debilitating autistic spectrum disorder affecting one in ten thousand girls. Patients develop normally for up to eighteen months before a period of regression involving stagnation in head growth, loss of speech, hand use and mobility. It is almost exclusively caused by mutation in Methyl CpG binding Protein 2 (MeCP2). MeCP2 has traditionally been thought of as a transcriptional repressor, although its exact function remains unknown and it has recently been shown that the protein can also bind to hydroxymethylation and non-CpG methylation, which occurs predominantly at CAC sites in the mature nervous system. Genotype-phenotype studies of the most common Rett-causing mutations in affected patients revealed that a missense mutation, R133C results in a milder form of Rett syndrome. The reasons for this are unclear, as the mutation lies right in the heart of the methylated DNA binding domain. Previous in vitro studies of R133C showed a severe deficit in binding to methylated cytosine. A subsequent study found that R133C binding to hydroxymethylated cytosine was specifically impaired, whereas binding to methylated cytosine was indistinguishable from wildtype. Defining the DNA binding impairment of MeCP2R133C would yield important insights into Rett disease pathophysiology and provide an explanation for the phenotypic spectrum seen in patients. To shed light on these matters, a novel mouse model of the R133C mutation was created. The R133C mouse had a phenotype that was less severe than other missense mutant mice, in terms of survival, growth, Rett-like phenotypic score and some behavioural paradigms thus recapitulating the patient data. At the molecular level in adult mouse brain, MeCP2R133C protein abundance was reduced. Immunohistochemistry showed that MeCP2R133C had an abnormal pattern of localisation in the nucleus of neurons. In vitro electrophoretic mobility shift assays suggested that MeCP2R133C binding to (hydroxy)methyl-CAC may be reduced to a greater extent than binding to mCpG. Chromatin immunoprecipitation experiments confirmed the deficit in binding to methylated sites and supported a disproportionate reduction in binding to methylation in a CAC sequence context. Analysis of adult mouse cerebellar gene expression revealed a subtle upregulation of long genes and downregulation of short genes. Based on these data, it is proposed that Rett syndrome caused by the R133C mutation results from a combination of protein instability and defective binding to methylated DNA. Methyl-CAC binding is potentially abolished. The downstream biological consequence of this is a length-dependent deregulation of gene expression in the brain.

Rett syndrome (RTT) is a rare paediatric disorder of females that leads to lifelong cognitive, motor, and respiratory impairment . In the vast majority of cases the disorder is caused by de novo mutations in the X-linked gene MECP2. There are currently no treatments, but genetic studies in mice have shown that the disease is reversible, even after the onset of symptoms. Since there remains a fundamental lack of knowledge about the downstream pathways involved in gene function, current therapeutic efforts are focused on targeting the disease at the gene level, mainly using viral based delivery of Mecp2 gene products. Recent work in mouse models has shown that exogenous delivery of a wild-type (WT) copy of the Mecp2 gene can lead to significant improvements in RTT-like symptoms, but significant challenges remain, both in the delivery of gene constructs to target cells, and in maintaining gene transcription within physiological tolerance. The work in this thesis explores an alternative therapeutic approach, using newly developed genome editing technology. There were two major aims of this thesis. The first aim was to examine the role of the peripheral tissues in the development of the RTT phenotype. In order to develop correctly targeted therapies it is crucial to know what tissues are most relevant to disease development. It has been widely assumed that the major RTT symptoms can be explained solely by an absence of MeCP2 from cells in the nervous system. However, this was based on mouse studies in which only a few gross aspects of the disorder were examined. In this thesis a newly created peripheral knock-out (KO) mouse model, in which Mecp2 transcription is silenced in peripheral tissues but selectively reactivated in the cells of the nervous system, was comprehensively phenotyped in order to determine the role of peripheral MeCP2 in RTT. The second major aim of this thesis was to develop a novel strategy for Mecp2 mutation repair, using recently developed genome editing tools. Based on the results from the peripheral KO phenotyping, this strategy was designed to overcome the particular challenges associated with genome editing in the nervous system, and involved the insertion of a therapeutic construct directly into a non-coding region of the Mecp2 gene using TALEN and CRISPR. This construct was designed to splice to upstream Mecp2 exons in order to replace downstream mutated exons in the final mRNA transcript. To generate the peripheral KO model (stop-cre), mice in which Mecp2 transcription was globally silenced by a cre-excisable stop cassette (stop-y) were crossed with a nestin-cre mouse line to selectively reactivate gene transcription in the nervous system. Southern blot analysis of tissues showed reactivation in a large number of cells (91.9%) in whole brain samples. Reactivation was particularly high in the cerebellum which showed 96.4% efficiency. Robust silencing was shown in peripheral tissues with only very small levels of reactivation in liver (0.9%), spleen (0.5%), skeletal muscle (1.2%) and heart (7.4%). Higher levels were seen in lung (14.3%) and kidney (24.4%) tissue. Peripheral KO mice did not show the early death phenotype seen in global KO mice and showed only very subtle RTT symptoms when examined using a well-established RTT scoring system. These mice also did not display any of the gait, balance, or respiratory dysfunction typical of RTT mouse models. However, peripheral KO mice did show a reduction in activity levels and exercise capacity across a number of tests. In the open-field, spontaneous activity levels were significantly reduced compared with WT (total distance moved = 3523 cm ± 215 SEM vs 4242 cm ± 167 in WT), on the accelerating rotarod, latency to fall was significantly reduced (168 s ± 14.9 vs 243.5 s ± 11.5 in WT) and on an inclined accelerating treadmill, the time lasted before exhaustion was markedly reduced (8.7 min ± 1.6 vs 16.5 min ± 1.3 in WT). In addition, peripheral KO mice also displayed the biomechanical abnormalities of bone seen in global KO mice, including reduced cortical stiffness and hardness. The genome editing mutation repair strategy developed in this thesis required a non-coding target region free of repetitive sequence to be identified upstream of exons 3 and 4, where most of the disease causing mutations occur. A suitable 900 bp region of unique sequence was identified 1.6 – 0.7 kb upstream of the beginning of exon 3. To design TALEN pairs targeting this region, the Cornell University TALEN design tool was used to identify 100s of possible TALEN pairs, which were then filtered based on best practice-TALEN design and for the presence of unique restriction sites at the break site. Four pairs remained after filtering and these were assembled using a two stage cloning process based on the Golden Gate method. The efficiency of each pair was assessed using a restriction digest based assay and an online tool (TIDE) which relied on the decomposition of Sanger sequencing traces. The results showed a range of cutting efficiencies from 2.1% of cells (TALEN # 63) to 42.9% of cells (TALEN # 333). A CRISPR design tool was used to generate CRISPR guide target sequences. The four guides with the lowest predicted off-target effects were selected, synthesised as complementary oligonucleotides, and cloned upstream of a guide RNA scaffold in a CRISPR guide expression plasmid. Cutting efficiency was assessed using TIDE, and the results again showed a range of efficiencies from 60.4% (B52) to 22% (A65). To assess if the best performing TALEN and CRISPR-Cas9 constructs could successfully target exogenous DNA into intron 2 of the Mecp2 gene, a repair construct was designed. This contained WT sequence for exons 3 and 4 of Mecp2 in a minigene format as well as appropriate splice elements and an mCherry fluorescent tag for easy detection. The repair construct was synthesised and cloned into a mammalian expression plasmid, with flanking regions, containing either TALEN or CRISPR target sites, inserted at either end of the construct. For unknown reasons the repair construct was toxic to bacterial cells when flanked by the CRISPR target sites and this version could therefore not be used for transfection experiments. The TALEN repair construct was transfected into cells along with TALEN pair # 333 and successful insertion was assessed using a PCR-based assay. Results showed that the repair construct was successfully inserted into non-coding genomic DNA in the correct location, and that a mutant version of the TALEN construct, designed to increase specificity, led to an increase in the levels of insertion in the correct orientation. To assess if this led to the production of corrected protein, cells were examined for mCherry protein expression using flow cytometry. Results showed that there was no significant increase in mCherry expression in transfected cells, suggesting the repair construct did not successfully splice to upstream Mecp2 exons. In summary, the results from this thesis show that RTT is primarily a disorder of the nervous system, and that this should therefore be the main target of new therapies. However, they also show that an absence of MeCP2 from the peripheral tissues leads to a markedly reduced exercise capacity, and is also likely to be the primary cause for the bone dysfunction seen in RTT patients and mouse models. In this thesis, a novel therapeutic strategy for RTT was developed using genome editing tools. A number of TALEN and CRISPR constructs were designed that could successfully target specific non-coding regions of the Mecp2 gene, and were shown to enable the insertion of an exogenous DNA repair construct into the genome. However, further flow cytometry analysis showed that this did not lead to the expected protein repair suggesting further work is required on the design of the repair construct to enable splicing to endogenous Mecp2 exons. Overall, the results show that genome editing has a potential role in the treatment of genetic disorders like RTT, but that further work is required to enable successful repair of disease causing mutations.

Rett syndrome is a severe neurodevelopmental disorder. The condition affects approximately one in every 10.000 females and is only rarely seen in males. Causative mutations in the transcriptional regulator MeCP2 have been identified in more than 95% of classic Rett patients; mutations in CDKL5 are responsible for the early onset seizures Rett variant and mutations in FOXG1 gene lead to the congenital Rett variant. To shed light on molecular mechanisms underlying Rett syndrome onset and progression in disease-relevant cells, we took advantage of the breakthrough genetic reprogramming technology and we investigated changes in iPSC-derived neurons from patients with different MECP2 and FOXG1 mutations and in the brain of Foxg1+/- mice. In total brains from Foxg1+/ − mutants we noticed a statistically significant overexpression of a group of neuropeptides expressed in the basal ganglia, cortex, hippocampus and hypothalamus: Oxytocin (Oxt), Arginine vasopressin (Avp) and Neuronatin (Nnat).Moreover, in iPSC-derived neuronal precursors and neurons mutated in FOXG1 and in Foxg1+/− mouse embryonic brain (E11.5) compared to wild type controls we found an increase in the expression of GluD1 and inhibitory synaptic markers, such as GAD67 and GABA AR-α1 and a decreased expression of excitatory synaptic markers, such as VGLUT1, GluA1, GluN1 and PSD-95, suggesting an excitation/inhibition imbalance in the developing brain of the congenital RTT variant. Furthermore, we investigated transcriptome changes in neurons differentiated from MECP2 mutated iPSC-derived neurons and we noticed a prominent GABAergic circuit disruption and a perturbation of cytoskeleton dynamics. In particular, in MECP2-mutated neurons we identified a significant decrease of acetylated α-tubulin which can be reverted by treatment with a selective inhibitor of HDAC6, the main α-tubulin deacetylase. Taken togheter, these findings contribute to shed light on Rett pathogenic mechanisms and provide hints for the definition of new therapeutic strategies for Rett syndrome.

This thesis was submitted in May 2018 for the partial fulfilment of the award of Doctor in Clinical Psychology (DClinPsy) at Cardiff University. The thesis investigated a review of non-medical interventions in Rett Syndrome and the prevalence of attenuated behaviours in Rett Syndrome. Rett Syndrome is a rare neurodevelopmental genetic disorder which is characterised by period of outwardly typical development followed by a period of regression around 12-18 months. The regression is causes progressive disabilities in speech, motor and hand use. Physical comorbidities are often present including breathing difficulties, the requirement of feeding tubes, anxiety, gastrointestinal difficulties and orthopaedic issues. The condition is almost exclusive to females. Despite the inability to use their body to communicate, research has shown that individuals with Rett Syndrome are more intellectually capable than their body allows them to present. Paper 1 describes a systematic review of non-medical interventions researched into Rett Syndrome. The electronic databases were searched (Embase, PsychINFO and MEDLINE). Thirteen papers which met the quality rating threshold were reviewed and the methodology was assessed. Communication interventions were the most researched intervention. Alternative interventions included fitness and brainstem activation. Eleven of the studies described positive results. Recommendations for clinical practice and future research are made. Paper 2 describes an empirical study investigating the prevalence of attenuated behaviours [Autistic Catatonia] in Rett Syndrome and its presence during ‘Rett Episodes’. Parents of 28 individuals with Rett Syndrome completed questionnaires relating to attenuated behaviours and Rett Episodes. The findings revealed the presence of attenuated behaviour in individuals with Rett Episodes but this was not specific to Rett Episodes. The severity of attenuated behaviour was negatively correlated with age. Recommendations for clinical implications and further research are made. Paper 3 discusses an evaluation of the research process. The paper critically appraises the research across both papers including the strengths and limitations. The paper includes key reflections and process information that was not permitted within the constraints of the author guidelines for submission.

Rett syndrome (RTT), a rare but severe neurological disorder is associated with a mutation in the methyl CpG binding protein 2 (MECP2) gene on the X chromosome occurring in 1:9000 live female births. Apparently normal development is followed by a regression in hand and communication skills, and subsequent development of hand stereotypies and abnormal gait. These characteristics are usually accompanied with comorbidities such as sleep problems and scoliosis. Over 80% of RTT individuals have specific sleep problems including night waking and difficulty falling asleep. Using a cross-sectional survey design, this study explored types of sleeping problems observed and relationships with variables such as age and genotype; and finally investigated specific management strategies. Participants mainly comprised families registered in the International Rett Syndrome Phenotype Database (InterRett) with a child of any age who has a confirmed RTT diagnosis and/or a MECP2 mutation. New families were invited via InterRett Facebook page and parent Listserve RettNet. A web-based questionnaire using freely available software, “REDCap” was developed to collect data. The independent variables tested were age group, mutation type, epilepsy, scoliosis, mobility and uses of sleep hygiene strategies while dependent variables tested include the presence, nature and frequency of sleep problems. Descriptive statistics were used to examine each variables and regression models including simple logistic regression and multinomial logistic regression models were employed to investigate the relationships between phenotype, treatments and sleep problems. Findings from this study showed sleep problems were more pronounced in RTT cases than in the general population. The prevalence of most sleep problems were higher in younger children and those with a p.Arg294* mutation. Other covariates including severe seizure activity was associated with poor sleep by taking into account the effect of age, mutation type, scoliosis and mobility. Non-pharmacological interventions were more perceived by families as effective in comparison to pharmacological medications.

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

Rett syndrome is the manifestation of an X linked, mainly female, genetic, neurodevelopmental disorder that usually produces profound intellectual and physical disabilities including abnormal muscle tone, with a tendency to develop limb contractures, scoliosis, epilepsy and irregular respiration. There is characteristic hand stereotypy with· poor voluntary hand use, locomotion is compromised and speech is rare. Although the disorder is not progressive many sequele shorten life especially in the most severely affected. Subtle abnormalities, present from birth, are frequently overlooked because there is some developmental progress until a period of regression at around one year of age when speech and hand use diminish. This thesis gives an account of clinical, physiological and genetic studies carried out between 1982 and 2005 with the aim of recording the natural history of the disorder and understanding its clinical manifestations. The subjects of these studies have been people of all ages, mainly from the British Isles, reported to have Rett syndrome by their physicians and families or carers (British Isles Survey, n=l228). Most have been examined and recorded on video by myself, many repeatedly. Fully informed parental consent and appropriate ethical approval has been given for all procedures. The early manifestations of the disorder were investigated from developmental histories and donated videos (78) taken by families before they were aware of the problem. The abnormal respiratory rhythms were investigated and characterised, using non-invasive measures of respiratory rhythm, carbon dioxide, oxygen, heart rate and blood pressure. The poor control of voluntary movement was investigated using electromagnetic stimulation of the cortex to record conduction in the motor pathways. Stereotyped hand movements were analysed from three-dimensional live recording and informal two-dimensional video. The prevalence of a toe anomaly was estimated, visual evoked potentials were recorded and a reported increase in urinary neopterin was investigated. The health of people in the British Survey was monitored longitudinally from family and physician reports and direct clinical examinations, data being stored on computer. Simple scores were generated to indicate separately the severity of the condition and health of the individual. The survey data has been used to estimate the prevalence of the disorder (I in 10,000 females), natural history from birth to death, the predictive value of the earliest signs, survival at different levels of severity, the impact of scoliosis surgery on health and has provided a foundation for studies relating clinical manifestations to specific mutations on the affected gene MECP 2 (Xq28). The studies have indicated the nature of the Rett disorder to be developmental and non-progressive, with primary impact on the processing functions of the brain, probably beginning in the brain stem before birth.

Rett Syndrome (RTT) is an X-linked genetic disorder and a major cause of intellectual disability in girls. Mutations in the methyl-CpG binding protein 2 (MECP2) gene, are the primary cause of the disorder. Despite the dominant neurological phenotypes that characterise RTT, MECP2 is expressed ubiquitously throughout the body and a number of peripheral phenotypes such as growth retardation (reduced height and weight), skeletal deformities (scoliosis/kyphosis), reduced bone mass and low energy fractures are also common yet under-reported clinical features of the disorder. In order to explore whether MeCP2 protein deficiency results in altered structural and functional properties of bone and to test the potential reversibility of any such defects, I have conducted series of histological, imaging and biomechanical tests of bone using an accurate genetic (functional knockout) mouse model of RTT. Initial experiments using a GFP reporter mouse line demonstrated the presence of MeCP2 in bone cells and the effective silencing on the gene in functional knockout mice. Different aspects of the study were conducted in different types of bone tissues that were especially suited for individual assays. For instance, biomechanical three point bending tests were conducted in long bone (femur) whilst trabecular geometry measures were measured in spinal vertebrae. Both hemizygous Mecp2stop/y male mice in which Mecp2 is silenced in all cells and female Mecp2stop/+ mice in which Mecp2 is silenced in ~50% of cells as a consequence of random X-chromosome inactivation (XCI), revealed, lighter and smaller long bones and significant reductions in cortical bone mechanical properties (~ 39.5% reduction in stiffness, 31% reduction in ultimate load and 37% reduction in Young’s modulus respectively in Mecp2stop/y male mice; %) and material properties (microhardess reduced 12.3% in Mecp2stop/y male mice and 14% inMecp2stop/+ female mice) as compared to age wild type control mice. Micro structural analysis conducted using µCT also revealed a significant reduction in cortical (54% reduction in cortical thickness, 30% in bone volume, 20% in total area, and 38% in marrow area) and trabecular (~30% in trabecular thickness) bone parameters as compared to age matched wild-type controls MeCP2-deficent mice. Histological analysis using Sirius red staining as a marker of collagen revealed a ~25% reduction in collagen content in MeCP2 deficient mice as compared to age matched wild type controls. In experiments designed to establish the potential for reversal of MeCP2-related deficits, unsilencing of Mecp2 in adult mice by tamoxifen-induced and cre-mediated excision of a stop cassette located at the endogenous Mecp2 locus (male; Mecp2stop/y, CreER and female; Mecp2+/stop, CreER), resulted in a restoration of biomechanical properties towards the wild-type levels. Specifically, Male Mecp2stop/y, CreER mice displayed improvement in mechanical properties (stiffness 40%, ultimate load 10%, young’s modulus 61% and micro hardness 12%) and structural bone parameter (trabecular thickness 80%) as compared to Mecp2stop/y male mice. Female Mecp2+/stop, CreER, displayed a significant improvement (19%) in microhardess measures as compared to Mecp2 deficient mice. Overall, the results of my studies show that MeCP2-deficiency results in overt, but potentially reversible, alterations in the biomechanical integrity of bone and highlights the importance of targeting skeletal phenotypes in considering the development of pharmacological and gene-based therapies for Rett Syndrome.

Irrespective of the great individual variation, people diagnosed with RTT largely rely on support from others to be able to do and participate in activities throughout their lives. This thesis focuses on which activities are done and liked/disliked by girls and women with RTT in Sweden. The overall aim was to describe the everyday lives of female individuals with Rett syndrome. Two studies are included in this thesis. The first is a descriptive study, using secondary data from three earlier questionnaires, encompassing data from 175 participants (girls/women) described by 365 informants (parents/staff). Content analysis was used to analyse the openended questions. In the second study a Time-geographic diary method and the software VISUAL-TimePAcTs computer program, DAILY LIFE 2011 were used. Ten participants (teenagers/young female adults) with RTT and their 63 informants participated in the diary study. The main findings in the first study (I) were that the girls and women with RTT enjoyed activities that included aspects of ‘contact’, ‘sensory impression’ and ‘motion’. The activities most enjoyed over the years were bathing/swimming, listening to music or being outdoors/walking. The parents and staff also liked to do the same activities that the girls or women enjoyed doing, described as sharing their joy. Of the few activities that were reported as being unenjoyable, most were daily care activities. The diary study (II) showed that the most frequently reported activities were hygiene/toilet, moving around indoors, eating and getting dressed. Most time was spent in sleeping, daily care, medical and health care activities and also for travel/transportation. Little time remained for other kinds of activities especially for the young adults. Most time was spent with staff, thereafter with families, and the least time was spent with friends. The participant response that was reported most often during activities was ‘interested’, while ‘opposed’ was the least reported. Responses of ‘opposition’ were primarily seen during caring activities such as toileting, using the breathing mask, stretching, brushing teeth, being woken up, dressing and putting on orthoses. Responses of ‘engagement’ were noted in contexts of socialising, playing and communicating activities with friends or staff. Engagement responses were also reported during activities of ‘motion’ such as changing body position, moving in the water or gymnastics, eating food and snacks, and even when watching/listening to films, books or music. Thus, increased knowledge concerning the importance of activities for girls and women with RTT is essential for their well-being, participation and continued development. Increased knowledge could facilitate the choice of activities and a more varied use of activities. Regardless of age, severity of symptoms or developed skills, it is important that not only basic needs such as sleep, daily care and medical health care activities are fulfilled for individuals with Rett syndrome. It is also essential for them to spend time with friends, family and staff doing enjoyable activities both at home and in other places.

Funding:

Linnéa and Josef Carlsson’s Foundation, Helsingborg, Sweden and the Folke Bernadotte Foundation, Stockholm, Sweden.

The Forkhead box G1 (FoxG1) is a transcription factor essential for the forebrain development and involved in pathogenesis of Rett syndrome. Notwithstanding the importance of this protein, little is known about the modalities by which it exerts its cellular functions. In this thesis I investigated, in cell culture and in animal model, the molecular mechanisms of Foxg1 action and the pathophysiological consequences of Foxg1 haploinsufficiency. Using fluorescence recovery after photobleaching strategy, I investigated the chromatin binding dynamics of Foxg1 in its wild-type form or carrying some Rett syndrome-causing mutations. The experiments show that GFP-Foxg1 mobile molecules have a chromatin affinity that decreases in truncated mutants with the extension of the protein deletion. Conversely, the immobile fraction does not decrease gradually with the extension of protein deletion but is significantly higher for a truncation associated with a severe phenotype, suggesting a possible dominant negative function of some RTT mutants. As already present in literature, GFP-Foxg1 localizes almost exclusively in the nucleus. However, no one reported data on Foxg1-GFP C-terminal fusion protein. Surprisingly, Foxg1 wt-GFP, in addition to the preserved nuclear localization, shows a clear mitochondrial localization. Further investigation led me to discover that Foxg1 undergoes a proteolytic cleavage and the resulting C-terminal peptide localizes to mitochondria, as evidenced by CFP-Foxg1 wt-YFP fusion protein as well as by other biochemical and immunohistochemical assays. Tripan Blue and proteinase K protection experiments in living cells show that nearly 80% of the protein reside in the mitochondrial matrix. Considering that Foxg1 does not have a classical N terminal mitochondrial targeting signal, I looked for Foxg1 amino acids able to drive a fused fluorescent tag into mitochondria and identified amino acids 277-302 as critical. A pull down experiment showed that a "mitochondrial" Foxg1 peptide interacts with some proteins involved in mitochondrial trafficking and function such as voltage dependent anion channel 1 and 2. Functionally, a C-terminal "mitochondrial" peptide of Foxg1 (aa 272-481) is able to enhance ATP production and mitochondrial membrane potential. Full length Foxg1 strikingly enhances mitochondrial potential and promotes cellular proliferation and mitochondrial fission; on the contrary, "mitochondrial" Foxg1 promotes mitochondrial fusion and an initial stage of cellular differentiation. In an animal model, I investigated the functional result of Foxg1 haploinsufficiency in mouse visual system. We chose visual system since it is known that Foxg1 is expressed in the developing retina and chiasm and the knockout mouse embryos show defects in retinal ganglion cells axonal navigation. It is also known that Foxg1 haploinsufficiency in mice results in subtle cortical defects. My data show that Foxg1+/Cre heterozygous mice have a profound impairment in visual acuity, despite a preserved retinal organization. Defects in visual cortical circuitry suggest that acuity loss may be due to altered cortical mechanisms. Taken together, these results suggest the following hypothesis: Foxg1 is a nuclear and mitochondrial protein that upon its internal processing localizes in the mitochondrial compartment and becomes a key coordination part of the machinery that brings about cell differentiation, replication and bioenergetics.

Rett syndrome is a rare genetic neurodevelopmental disorder. Cross sectional and longitudinal densitometry assessment in Rett syndrome found decreased bone density with age but muscle mass and mensus afforded some protection. Consensus-based clinical management guidelines provided recommendations for clinical assessment and pharmacological and non-pharmacological interventions. These data have the potential to reduce the frequency of fractures in Rett syndrome and stimulate further research that aims to ameliorate the impacts of poor bone health.

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.

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.

Rett syndrome (RTT) is a progressive non degenerative neurodevelopmental disease affecting mainly females, with an incidence of 1 out 10.000 newborn girls. Its symptomatology is very variable and may include: motor deficits, intellectual disability, cardiorespiratory alterations and epilepsy, among other alterations. RTT diagnosis is complex and mainly based on clinical evaluations. All patients apparently develop normally until approximately the first year of life. Then, a developmental stagnation occurs, followed by a regression period in which patients lose acquired skills. In most cases, RTT is due to loss-of-function mutations in the MECP2 gene, which encodes the methyl-CpG-binding protein 2 (MeCP2), a widespread chromatin organizer that controls the transcription of hundreds of genes. Even if experiments performed in a mouse model showed a reversion of RTT-like phenotypes after the re-expression of murine Mecp2, at present there is no a cure for RTT. Pharmacological and physical therapies are the only available techniques to alleviate RTT symptomatology. Regarding drugs, all treatments used on RTT patients are used only for specific symptoms. Since monoaminergic systems are downregulated in both RTT patients and mouse models, antidepressants have been proposed as candidate drugs to treat this disease. Among them, mirtazapine (MTZ) has showed an excellent safety profile through years and the ability to rescue several behavioural, physiological and neuroanatomical phenotypes in a male mouse model of RTT. The present project aimed to complete and to extend these results, as well as to find some mechanisms of action of MTZ. To do this, we used Mecp2tm1.1Bird female mice, a RTT model with a verified face validity, and treated them at different ages and for different durations. First, we verified the safety of MTZ, as we did not observe any adverse effects, even when we treated young female mice with a high dosage of MTZ for one month. Second, we found several improvements in motor, somatosensory and cognitive domains. In one case, we were able to propose a mechanism for the behavioural rescue, as MTZ normalized parvalbumin expression in a related brain area. Results in female mice were completed by a retrospective analysis of a heterogeneous cohort of adult RTT patients that had been treated with MTZ for long periods. Only 2 out 11 patients showed adverse effects and were discontinued, while others showed an improvement of several clinical features in the motor domain, as well as in multiple behavioural alterations. In summary, results obtained in this thesis strongly supports MTZ as a promising treatment for RTT. They represent a robust proof-of-concept and will constitute the base of a dossier aimed to obtain the ethical authorization to initiate a randomized clinical trial. In this way, it will be possible to verify whether MTZ can effectively alleviate RTT symptomatology and, thus, improve quality of life of affected people and their families.

Rett syndrome (RTT) is a neurodevelopmental disorder affecting the central nervous system and is one of the most common causes of intellectual disability in girls, resulting in severe cognitive and physical disabilities. Mutations in MECP2 and FOXG1 genes cause the classic form and the congenital variant of Rett syndrome, respectively. Both genes are transcriptional regulators and both under- and over-expression of these gene cause disease in humans. To characterize the biological mechanisms implicated in disease pathogenesis, we established and characterized a human neuronal model based on genetic reprogramming of patient fibroblasts into induced Pluripotent Stem Cells (iPSCs). Functional analyses performed in MECP2 iPSC-derived neurons demonstrated that these cells closely mimic the impairment of molecular pathway characterizing the disease revealing defects in GABAergic system and cytoskeleton dynamics. Furthermore, we explored the possibility to use iPSC-derived neurons to develop and study a new treatment for RTT patients. Effective therapies are not currently available and the need for tight regulation of MeCP2 and FOXG1 expression for proper brain functioning makes gene replacement therapy risky. Therefore, gene editing would be much more effective. Gene editing based on CRISPR/Cas9 technology and Homology Directed Repair appears an appealing option for the development of new therapeutic approaches. We have engineered a two-plasmid system to correct FOXG1 (c.688C>T (p(Arg230Cys)); C.765G>A (p.Trp255Ter)) and MECP2 (c.473C>T-p.Thr158Met) variants.. Mutation-specific sgRNAs and donor DNAs have been selected and cloned together with an mCherry/GFP reporter system. Cas9 flanked by sgRNA recognition sequences for auto-cleaving has been cloned in a second plasmid. The system has been designed to be ready for in vivo delivery via Adeno-Associated Viral (AAV) vectors. NGS analysis of corrected cells from MECP2 and FOXG1 patients demonstrated an high editing efficiency, ranging from 20 to 80 % of HDR and confirmed that this correction strategy is feasible in neurons. Functional analyses in edited cells confirm the correction of molecular defects due to the mutation. Based on the use of AAV viruses and their capacity to cross the Blood Brain Barrier (BBB) following intravenous injection these experiments will allow us to demonstrate the full potential of gene editing as a therapeutic option for RTT and for other neurodevelopmental disorders currently lacking an effective treatment.

Le sommeil est essentiel pour maintenir une santé optimale. Le sommeil problématique se retrouve avec une plus grande fréquence et sévérité chez les enfants atteints de troubles neurodéveloppementaux et psychiatriques. De plus, le sommeil problématique est associé à un fonctionnement psychosocial plus faible pendant la journée. Le syndrome de Rett (RTT), en tant que l'un des handicaps multiples génétiques les plus courants et les plus graves chez la femme, est fortement lié au gène mutant de la protéine de liaison méthyl-CpG 2 (MECP2) sur le chromosome X. Les formes phénotypiques variantes de l'RTT présentent un spectre de symptomatologie similaire à celui de l'RTT classique, mais présentent des différences subtiles dans certaines caractéristiques cliniques, variante d’épilepsie précoce (variante de Hanefeld, liée au gène mutant X-linked cyclin-dependent kinase-like 5, CDKL5), variante congénitale (variante de Rolando, liée au gène forkhead box G1, FOXG1) et variante de la parole préservée (variante de Zappella, également liée à MECP2). Le syndrome de Rett concerne 1 naissance sur 10 000 à 15 000, ce qui représente 40 à 50 nouveaux enfants malades chaque année en France. Le RTT se caractérise par un arrêt du développement environ 6 à 18 mois après la naissance, la présence de mouvements stéréotypés de la main et des anomalies de la démarche coïncidant avec la perte des compétences acquises de la main intentionnelle et du langage parlé. L’enfant se retire socialement. D'autres signes également décrits dans les profils cliniques du RTT comprennent les crises d'épilepsie, les difficultés respiratoires, le tonus musculaire anormal, la scoliose, ainsi que les troubles du sommeil. En général, les résultats physiopathologiques du RTT suggèrent des activités corticales anormales et une dysmaturité de la fonction du tronc cérébral, ce qui est essentiel pour maintenir un état adéquat pendant le sommeil ou l'éveil. Cependant, il n'existe pas d'étude scientifique sur la relation entre les anomalies du sommeil et les troubles sociaux dans le RTT. Ainsi, ce travail de doctorat s'est orienté vers ce sujet pour lier le jour et la nuit en RTT. Premièrement, nous avons entrepris au total cinq revues systématique de toutes les études précédentes sur les performances sociales non verbales et le sommeil réalisé sur des personnes atteintes de RTT. Deuxièmement, nous avons analysé les enregistrements polysomnographiques dans un échantillon clinique d'individus atteints de RTT présentant les mutations MECP2. Nous avons étudié leur macrostructure du sommeil et leur respiration pendant le sommeil. En outre, nous avons examiné les traits phénotypiques possibles via une approche analytique stratifiée par caractéristiques cliniques et génétiques. Pour examiner les profils sociaux chez les personnes atteintes de RTT, nous avons extrait 25 items liés au comportement social du questionnaire ‘Rett Syndrome Behavior Questionnaire’, qui étaient corrélés à leur sommeil.De manière générale, nous pouvons conclure que le sommeil dans le phénotype social des individus atteints de RTT est lié à des déficiences sensorimotrices progressives. Par conséquent, à l'avenir, la physiopathologie du système sensorimoteur devrait faire l'objet d'une plus grande attention dans l'étude du sommeil et de la vie sociale des personnes atteintes de RTT. En outre, nous attendons avec impatience de nouvelles recherches sur la démonstration des effets des thérapies sensorimotrices sur les troubles du sommeil et les déficiences sociales
Sleep is essential for maintaining optimal health. In children with neurodevelopmental and psychiatric disorders, problematic sleep is found with greater frequency and severity. Furthermore, problematic sleep is associated with poorer psychosocial functioning during the daytime. Rett Syndrome (RTT), one of the most common and severe genetic multi-disabilities in females, is strongly linked to the mutant methyl-CpG binding protein 2 gene (MECP2) on the X chromosome. Variant phenotypic forms of RTT present a spectrum of symptomatology similar to that of classical RTT but show subtle differences in some clinical features, including the Early Seizure Variant (ESV, Hanefeld variant, linked to mutant gene X-linked cyclin-dependent kinase-like 5, CDKL5), congenital variant (CV, Rolando variant, linked to the forkhead box G1 gene, FOXG1) and preserved speech variant (PSV, Zappella variant, also linked to MECP2). RTT affects 1 in 10,000 to 15,000 births, which represents 40 to 50 new cases each year in France. RTT is characterized by developmental arrest around 6-18 months after birth, the presence of stereotypical hand movements, and gait abnormalities coinciding with the loss of acquired purposeful hand skills and spoken language. The child withdraws socially. Other signs also described in RTT clinical profiles include epileptic seizure, breathing difficulties, abnormal muscle tone, scoliosis/kyphosis, as well as disturbed sleep. Accumulating pathophysiological findings in RTT suggest abnormal cortical activities and dysmaturity of the brainstem function, which is key in maintaining proper status during sleep or wakefulness. However, there is no scientific study investigating the relationship between sleep abnormalities and social impairments in RTT. Therefore, this doctoral work is subjected to this topic to link the day and night together in RTT. First, we undertook five systematic reviews of all previous studies on non-verbal social performance and sleep in RTT. Then, we analyzed polysomnographic recordings in a clinical sample of RTT individuals with MECP2 mutations. We studied their sleep macrostructure and respiration during sleep. In addition, we examined possible phenotypic traits via a stratified analytical approach to clinical and genetic characteristics. Lastly, to examine social profiles in RTT individuals, we extracted 25 social behavior items from the Rett Syndrome Behavior Questionnaire, and correlated them to their sleep. Overall, we can conclude that sleep in the social phenotype of individuals with RTT is related to progressive sensorimotor impairments. Therefore, in the future, the pathophysiology of the sensorimotor system should receive more attention in the study of sleep and the social life of individuals with RTT. In addition, we look forward to furthering research demonstrating the effects of sensorimotor therapies on sleep and social impairments

Rett syndrome (FlTT) is a severe neurological disorder that primarily affects females, with an incidence of approximately 1:10 000. RTT shows marked clinical variability in its presentation. Mutations in the X-linked Methyl-CpG-Binding Protein 2 (MECPZ) have been found in approximately 65% of cases. The MeCP2 protein binds methylated DNA via a Methyl Binding Domain (MBD), and recruits a silencing complex via a Transcription Repression Domain (TRD). The aim of the present study was to investigate the causes for phenotypic variability in R'lT, in the hope of understanding its aetiology and prognosis. A large, clinically well characterised cohort of RTT patients was utilised to investigate the incidence of MECP2 mutations and phenotypic correlations with different mutations and skewing of X-inactivation. Truncation mutations were associated with a more severe phenotype in general, as were early mutations affecting the Methyl-Binding Domain (MBD). The incidence of skewing of X-inactivation was significantly increased in RTT patients compared to normal female populations. Varying degrees of skewing in individual mutant cell lines were associated with differences in protein function. Different mutations also altered protein function in different ways in these cell lines. A proportion of RTT cases may have mutations in genes other than MECP2. A family has been described with no pathogenic MECP2 mutation. The ARX gene was screened in this family, but no mutations were found. Future work will examine other genes that may be responsible for RTT in a minority of cases. In conclusion, the phenotypic variability observed in RTT appears to arise from several interacting factors. Protein function and phenotypic outcome may vary depending on the mutation and the importance of particular residues for protein structure or function. Varying degrees of skewing of X-inactivation may then modify these effects. In addition, a small proportion of RTT cases may be phenocopies resulting from mutations in other genes.

STUDY I HYPOTHESIS Genetic alterations have already been recognized as major etiological factors for ASD and ID, of which RTT is an example. In addition to the contribution of polymorphic variants that confer low or moderate risk of appearance of these neurodevelopmental defects, de novo mutations affecting genes in a number of cellular pathways have been reported to be a cause of ASD, ID and associated NDDs (Vissers et al., 2010; Ronemus et al., 2014; Gilissen et al., 2014; Deciphering Developmental Disorders Study, 2015). Although the strong correlation between NDDs and genetic factors has been long established, the exact genetic background of ASD and ID remains unclear because of the strong heterogeneity of these disabilities. Genetic research has focused on the use of unbiased genome-wide approaches, including genomic microarrays and, more recently, NGS technology with the use of extensive gene panels, the exome or the whole genome. Consequently, new ASD and ID genes are now being identified in rapid succession. Among these new candidate genes, supportive role for the Jumonji Domain Containing 1C (JMJD1C) histone demethylase in ASD and ID has been fostered by both positional cloning strategies (Castermans et al., 2007) and exome-sequencing studies (Neale et al., 2012; Iossifov et al., 2014). Thus, based on the strong genetic architecture underlying NDDs and the emerging evidences for a role of JMJD1C as potential candidate genes, in the present PhD thesis we proposed to study the involvement of JMJD1C alterations in ASD, ID and RTT and their effects on the protein function. OBJECTIVES The specific objectives of study I are as follows: 1. To investigate the occurrence of JMJD1C mutations in the mentioned disabilities, a comprehensive mutational analysis was performed in samples from ASD, ID and RTT, searching for SNVs and indels, as well as larger genetic defects. 2. To address the functional consequences of the identified de novo JMJD1C- Pro163Leu mutation, the intracellular localization of the wildtype and mutated JMJD1C protein, as well as the efficiency in demethylating a non-histone target of JMJD1C, MDC1 (mediator of DNA-damage checkpoint 1), was studied in immunofluorescence, fractionation and immunoprecipitation experiments in HEK293 cells. 3. To direct a molecular explanation for the implication of JMJD1C in Rett Syndrome, the interaction between MeCP2 and JMJD1C, in the wildtype and mutated form, was assessed in an immunoprecipitation assay. 4. To study the cellular effects of the disruption of wildtype JMJD1C on a neuronal system, we analyzed the existence of changes in dendritic branching of primary neuron cultures from neonatal mouse hippocampus. STUDY II HYPOTHESIS Mutations in MECP2 cause most of the classical or typical forms of RTT (Chahrour and Zoghbi, 2007). Approximately 8 % of classic RTT and 42 % of variant RTT patients are MECP2 mutation-negative (Monrós et al. 2001; Percy, 2008). Some of the latter group have mutations in other genes, such as that of CDKL5, which is described in individuals with an early seizure onset variant of RTT (Kalscheuer et al., 2003) or FOXG1, which is responsible for the congenital variant of RTT (Ariani et al., 2008). However, there remains a subset of patients with a clinical diagnosis of RTT who are mutation-negative for all the aforementioned genes. In the present PhD thesis, we proposed to identify new candidate genes that could explain the RTT-like phenotype of several clinical cases without mutations in MECP2, CDKL5 and FOXG1, using NGS, with the purpose of expanding the knowledge of the impaired biological pathways in RTT. OBJECTIVES The specific objectives of study II are as follows: 1. To identify previously undescribed variants potentially implicated in RTT-like phenotype, WES was performed on a cohort of 19 Spanish parent–child trios and one family with two affected daughters presenting features associated with RTT. A bioinformatics process of WES data was realized to filter and select putative pathogenic de novo variants absent or present with very low frequency in the control population, and putatively dangerous for protein function. 2. To realize a differential diagnosis among diverse neurodevelopmental disorders with overlapping phenotype, a gene-association analysis was carried out to define a list of variants previously associated with neurodevelopmental disorders and another one with undescribed new variants not previously associated with. 3. To demonstrate a neurological implication for a loss of function of detected candidate genes not previously associated with neurodevelopmental disorders, the model organism C. elegans was used to confirm a genotype-phenotype correlation by performing locomotion assays in worm mutants that carry deleterious mutations in the orthologous genes to those human genes with potentially pathogenic mutations in the patients.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.
Includes bibliographical references.
(cont.) the RTT phenotype.
Deletion of the Xist gene results in skewed X-inactivation. To distinguish primary non-random choice from post-choice selection, we analyzed X-inactivation in early embryonic development in the presence of two different Xist deletions. We found that Xist is an important choice element, and that in the absence of an intact Xist gene, the X chromosome will never be chosen as the active X. To understand the molecular mechanisms that affect choice we analyzed the role of replication timing prior to X-inactivation. The X chromosomes replicated asynchronously before X-inactivation but analysis of cell-lines with skewed X-inactivation showed no preference for one of the two Xist alleles to replicate early, indicating that asynchronous replication timing prior to X-inactivation does not play a role in skewing of X-inactivation. Expression of the Xist is negatively regulated by its antisense gene, Tsix. In order to determine the role of transcription in Tsix function, we modulated Tsix transcription with minimal disturbance of the genomic sequence. Loss of Tsix transcription lead to non-random inactivation of the targeted chromosome, whereas induction of Tsix expression caused the targeted chromosome always to be chosen as the active X. These results for the first time establish a function for antisense transcription in the regulation of Xist expression. The X-linked disease Rett syndrome (RTT), a neurodevelopmental disorder, is caused by mutations in the MECP2 gene. We used a mouse model to test the hypothesis that RTT is exclusively caused by neuronal MeCP2 deficiency. Expression of an Mecp2 transgene in postmitotic neurons resulted in symptoms of severe motor dysfunction. Transgene expression in Mecp2 mutant mice, however, rescued
by Sandra Luikenhuis.
Ph.D.

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.