Dissertations / Theses on the topic 'Neurogenetic syndrome'

Le neurotransmetteur sérotonine contrôle une grande variété de mécanismes physiologiques et comportementaux. Chez les humains, les mutations qui affectent la monoamine oxydase (MAO), l’enzyme qui dégrade la sérotonine, sont hautement délétères. Pourtant, le morphotype cavernicole aveugle de l’espèce Astyanax mexicanus (téléostéens) porte une mutation (P106L) induisant une perte de fonction partielle de cette enzyme, et semble prospérer dans son habitat souterrain naturel. Cette thèse décrit les effets de cette mutation, depuis l’échelle moléculaire à l’échelle des populations, afin de mieux cerner sa contribution à l’évolution des traits neuro-comportementaux du poisson cavernicole lors de son adaptation au milieu souterrain.Dans une première publication, nous avons établi 4 lignées de poissons, correspondant aux deux morphotypes de l’espèce Astyanax mexicanus (poisson cavernicole et poisson vivant en surface), chacun porteur ou non de la mutation P106L. La mutation P106L affecte le stress en augmentant l’amplitude de la réponse aigue au stress, tout en diminuant l’anxiété. Nous avons aussi étudié la distribution des allèles mutés dans les populations naturelles au Mexique et trouvé que l’allèle mutant est présent dans plusieurs grottes. L’évolution de l’allèle muté sous sélection ou dérive génétique est discutée.Dans une seconde publication, nous avons étudié les conséquences structurelles et biochimiques de la mutation P106L. La réduction de l’activité enzymatique de la MAO mutée est probablement due à une réduction de la flexibilité de l’une des boucles formant l’entrée au site actif, limitant l’accès des substrats. Des mesures d’HPLC ont montré des perturbations majeures de taux de sérotonine, dopamine et noradrénaline (et métabolites) dans le cerveau des poissons mutants. La mutation P106L mao est donc entièrement responsable du déséquilibre monoaminergique observé dans le cerveau des poissons cavernicoles. Enfin, l’effet de la mutation est partiellement compensé par une réduction de l’activité de synthèse de la sérotonine par la TPH. Nos résultats en révèlent plus sur les spécificités des systèmes monoaminergiques des poissons.Enfin, pour aggraver l’inhibition de la MAO, nous avons généré le premier poisson mutant knockout pour la MAO. Les mutants KO homozygotes sont chétifs et meurent durant les premières semaines de développement. Contrairement au système dopaminergique, le système sérotoninergique des poissons KO homozygotes est très fortement altéré : aucun neurone positif à la sérotonine n’est détecté dans l’hypothalamus<br>The neurotransmitter serotonin controls a great variety of physiological and behavioral processes. In humans, mutations affecting the monoamine oxidase or MAO, the serotonin-degrading enzyme, are highly deleterious. Yet, blind cavefish of the species A. mexicanus carry a partial loss-of-function mutation in MAO (P106L) and seem to thrive in their subterranean environ-ment. This thesis describes the effects of this mutation, from the molecular level to the population level, in order to decipher the exact contribution of mao P106L in the evolution of cavefish neuro-behavioral traits, during their adaptation to the cave environment.In a first paper, we established 4 fish lines, corresponding to the blind cave-dwelling and the sighted river-dwelling morphs of this species, with or without the mutation. We found that mao P106L strongly affected anxiety-like behaviors. Cortisol measurements showed lower basal levels and an increased amplitude of stress response after a change of environment in fish carrying the mutation. Finally, we studied the distribution of the P106L mao allele in wild populations of cave and river A. mexicanus, and discovered that the mutant allele was present – and sometimes fixed - in all populations inhabiting caves of the Sierra de El Abra. The possibility that this partial loss-of-function mao allele evolves under a selective or a genetic drift regime in the particular cave environment is discussed.In a second paper, we assessed the structural and biochemical consequences of the mutation. We found that the reduction of enzymatic activity of mutant MAO is probably caused by a decrease of flexibility in one of the three loops forming the entrance of the active site, thus reducing the access of substrates. HPLC measurements in brains of mutant and non-mutant larvae and adult fish showed major disturbances in serotonin, dopamine and noradrenalin (and metabolites) contents, demonstrating that the P106L mao mutation is fully responsible for monoaminergic disequilibrium in the cavefish brain. We also discovered that the effects of the mutation were partially compensated by a decrease in activity of the TPH, the serotonin biosynthesis rate-limiting enzyme. Our results shade light on the specificities of fish monoaminergic systems.Finally in order to aggravate the MAO inhibition, we generated the first fish knockout MAO mutant. The KO homozygous mutants were stunted and died during the first weeks of development. Contrary to the dopaminergic system which seemed normal in the KO homozygous mutants, the serotonergic system was strongly impaired; no serotonin positive neuron was detected in the hypothalamus

Synaptic connections in the brain respond throughout their lives to the activity of incoming neurons, adjusting their biological properties to increment activity-dependent changes. Hippocampal neuronal plasticity disruptions have been suggested as mechanisms underlying cognitive impairments in Down syndrome (DS). However, it remains unknown whether specific candidate genes are implicated in these phenotypes in the multifactorial context of trisomy 21. DYRK1A is a serine/threonine kinase, which overexpression is sufficient to recapitulate hippocampal learning and memory deficits characteristic of DS individuals and trisomic mouse models. In this Thesis we have studied the effects of DYRK1A overexpression on activity-dependent plasticity in the hippocampus. We found that transgenic mice overexpressing Dyrk1A (TgDyrk1A) present hippocampal morphological alterations in CA1 and CA3 that may constrain network connectivity, and therefore are relevant to the structure-function relationship. We also found reduced LTP that may derive from the changes in connectivity and in dendritic occupancy. Dendritic excitability and neuronal morphology are determinants of synaptic efficacy and thus may contribute to the hippocampal learning and memory deficits detected. In addition, we demonstrated important defects in adult neurogenesis in the dentate gyrus including reduced cell proliferation rate, altered cell cycle progression and reduced cell cycle exit leading to premature migration, altered differentiation and reduced survival of newly born cells. Moreover, less proportion of newborn hippocampal TgDyrk1A neurons are activated upon learning, suggesting reduced integration in learning circuits. Some of these alterations were rescued by normalizing DYRK1A kinase using a DYRK1A inhibitor, epigallocatechin-3-gallate. Interestingly, environmental stimulation also normalized DYRK1A kinase overdosage in the hippocampus, and rescued hippocampal morphology, synaptic plasticity and adult neurogenesis alterations in TgDyrk1A mice. We conclude that Dyrk1A is a good candidate gene to explain neuronal plasticity deficits in DS and that targeting DYRK1A kinase activity excess either pharmacologically or using environmental stimulation in the adult could correct these defects in DS.<br>Les connexions sinàptiques tenen la capacitat de respondre a l’activitat de neurones ajustant les seves propietats biològiques per incrementar els canvis activitat-depenents. Alteracions en la plasticitat neuronal de l’hipocamp s'han suggerit com a mecanismes subjacents als deterioraments cognitius característics en la síndrome de Down (SD). No obstant, es desconeix quins gens específics estan implicats en aquests fenotips en el context de la trisomia del cromosoma 21. DYRK1A és una serina / treonina quinasa, que quan es troba sobreexpressada recapitula el dèficit d'aprenentatge i de memòria depenent de l'hipocamp característic de la SD. En aquesta tesi, s’han estudiat els efectes de la sobreexpressió de DYRK1A en la plasticitat activitat-dependent de l’hipocamp. Hem descobert que ratolins transgènics amb sobreexpressió de Dyrk1A (TgDyrk1A) presenten alteracions morfològiques en les regions CA1 i CA3 de l'hipocamp, una limitació estructural en les connexions neuronals que és rellevant per entendre la relació entre estructura i funció. A més, hem trobat una reducció en la LTP possiblement deguda als canvis en la connectivitat i ocupació dendrítica. L’excitabilitat de les dendrites i la morfologia neuronal són factors determinants de l'eficàcia sinàptica i per tant poden contribuir als dèficits d'aprenentatge i la memòria de l'hipocamp detectats. Hem demostrat defectes importants en la neurogènesi adulta en el gir dentat incloent una reduïda taxa de proliferació cel·lular, alteracions en el cicle cel·lular i reducció de cèl·lules que surten del cicle cel·lular que condueix a una migració precoç de les noves cèl·lules generades i una reducció de la supervivència. D'altra banda, en ratolins TgDyrk1A hi ha menys proporció de neurones generades de novo que s'activen amb l'aprenentatge, indicant una menor integració d’aquestes en els circuits implicats en l'aprenentatge. Algunes d'aquestes alteracions han estat rescatades per la normalització de DYRK1A quinasa utilitzant un inhibidor de DYRK1A, epigallocatechin-3-gallate. L'estimulació del medi ambient també normalitza la sobreexpressió de DYRK1A quinasa en l'hipocamp, i rescata la morfologia, la plasticitat sinàptica i les alteracions en la neurogènesi adulta en ratolins TgDyrk1A. Arribem a la conclusió que Dyrk1A és un bon gen candidat per explicar els dèficits de plasticitat neuronal en la SD i que tractant l’excés d'activitat de la quinasa DYRK1A farmacològicament o mitjançant l'estimulació ambiental en l'adult podria corregir aquests defectes en la SD.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Brain and Cognitive Sciences, 2009.<br>This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.<br>Cataloged from student submitted PDF version of thesis.<br>Includes bibliographical references.<br>The elucidation of the biological bases of a complex trait like human language proceeds from identification of precise behavioral phenotypes to investigation of the underlying genes. The human behavioral parts of this dissertation focus on understanding the reasons for children's overuse of definite article 'the', to refer to one of several objects in a context set, as opposed to the unique established referent. Competing theories argue the deficit is either in children's semantic computational knowledge (of uniqueness/maximality), or in their pragmatic/social awareness/theory-of-mind development. Experiments in this dissertation focused on children's comprehension and interpretation of the indefinite and definite determiners, as well as 'that', anaphors 'another' and 'same', and free relative clauses. The results in this thesis suggest that in typically developing (TD) children the late acquisition of determiner 'the' is due to the late maturation of the semantic principle of maximality. Children with autism spectrum disorders (ASD) and with Williams syndrome (WS) either manifested an adult-like competence, an absence of manifestation of knowledge, or a pattern found in TD younger children (where 'that' is understood better than 'the' as referring to the salient unique referent) -- indicating delay of development of the language faculty, but no deviance. This suggests that the observed deficits in ASD and WS pattern with those in TD, and hence are also semantic in nature. The mouse neurogenetic part of this dissertation investigates whether the GTF2I family of genes, causal to WS behavioral phenotype, also contributes to WS cortical development.<br>(cont.) By overexpression of Gtf2i and Gtf2ird1 in the mouse neocortex via in utero electroporation, their effects on laminar patterning and cell morphogenesis during brain development are characterized. The present results suggest that these genes can synergistically contribute to the abnormal neocortical development in WS, and thereby could contribute to language deficits in WS. Beyond posing an explanatory challenge to linguistic theories, the research comparing typical and atypical development sheds light on the mechanisms of language development and impairment, and provides endophenotypic descriptions of ASD and WS, which are crucial for elucidating not only genetics of neurodevelopmental disorders, but also the genetic basis of the human language faculty.<br>by Nadezhda N. Modyanova.<br>Ph.D.

Le syndrome de Coffin Lowry est une forme syndromique rare de déficience intellectuelle liée au chromosome X. Ce syndrome est dû à des mutations du gène Rsk2 codant la protéine kinase RSK2 dans la voie de signalisation des MAPK/ERK. La caractérisation phénotypique du modèle murin Rsk2-KO a principalement mis en évidence un retard d’acquisition ainsi qu’un déficit de mémoire spatiale à long terme, suggérant une altération des fonctions hippocampiques. Nous avons montré que les souris Rsk2-KO présentent également des déficits dans une forme d’apprentissage et de mémoire mettant en jeu la fonction de séparation de patterns dépendante du gyrus denté. Plusieurs études montrent que la genèse de nouveaux neurones dans le gyrus denté chez l’adulte constitue une forme de plasticité jouant un rôle important dans l’apprentissage et la mémoire dépendante de l’hippocampe, en particulier dans les tâches spatiales et de séparation de patterns. En raison des déficits observés chez les souris Rsk2-KO, nous nous sommes intéressés à la neurogenèse adulte chez ce modèle murin. Aucune différence de prolifération, de survie ou de maturation n’a été observée dans le gyrus denté des souris Rsk2-KO à l’état basal, ni après une tâche de séparation de patterns. Cependant, nous avons observé un déficit de survie des nouvelles cellules chez les souris Rsk2-KO après apprentissage dans la piscine de Morris. La littérature montre que l’enrichissement environnemental a des effets bénéfiques sur les performances cognitives des rongeurs et est notamment capable d’augmenter la neurogenèse adulte hippocampique. Nous avons donc analysé les effets de l’enrichissement sur les performances comportementales et la neurogenèse adulte des souris Rsk2-KO. Nos résultats montrent qu’un protocole d’enrichissement environnemental de 3 heures par jours durant 24 jours est capable de compenser ou d’améliorer les performances des souris Rsk2-KO dans les tâches de mémoire spatiale et de séparation de patterns et aussi d’augmenter la neurogenèse hippocampique adulte<br>The Coffin-Lowry Syndrome is a rare syndromic form of X-linked intellectual disability. This syndrome is caused by mutations of the Rsk2 gene that encodes a protein kinase, RSK2, in the MAPK/ERK signaling pathway. Characterization of the behavioural phenotype of Rsk2-KO mice mainly showed that they display delayed acquisition and long-term deficits in a spatial reference memory task, suggesting an alteration in hippocampal function. Here, we show that Rsk2-KO mice are also deficient in a learning and memory task that involves dentate gyrus-dependent pattern separation function. Several studies showed the formation of new neurons in the adult dentate gyrus by neurogenesis is a form of plasticity that plays a significant role in hippocampal-dependent learning and memory, in particular for spatial learning and memory and pattern separation. As these functions are altered in Rsk2-KO mice, we studied hippocampal adult neurogenesis in these mice. No difference in proliferation, survival and maturation of newborn neurons was found in the dentate gyrus of the mutant mice in basal conditions, nor after a pattern separation task. However, we found a deficit in the survival of newborn cells in Rsk2-KO mice submitted to spatial learning and memory in the Morris water maze task. According to several studies, environmental enrichment in rodents has beneficial effects on cognitive performance and is associated with an enhancement of adult hippocampal neurogenesis. Thus, we assessed the potential effect of environmental enrichment on spatial learning and memory performance and adult hippocampal neurogenesis in Rsk2-KO mice. Our results show that an environmental enrichment protocol of 3h per day during 24 days can rescue or ameliorate spatial learning and memory performance and pattern separation function in Rsk2-KO mice and increase adult hippocampal neurogenesis

In this work we have assessed the possible contribution of the human chromosome-21 gene DYRK1A in the developmental cortical alterations associated with Down Syndrome using the mBACTgDyrk1a mouse, which carries 3 copies of Dyrk1a, and a trisomic model of the syndrome, the Ts65Dn mouse. We show that trisomy of Dyrk1a changes the cell cycle parameters of dorsal telencephalic radial glial (RG) progenitors and the division mode of these progenitors leading to a deficit in glutamatergic neurons that persist until the adulthood. We demonstrate that Dyrk1a is the triplicated gene that causes the deficit in early-born cortical glutamatergic neurons in Ts65Dn mice. Moreover, we provide evidences indicating that DYRK1A-mediated degradation of Cyclin D1 is the underlying mechanism of the cell cycle defects in both, mBACTgDyrk1a and Ts65Dn dorsal RG progenitors. Finally, we show that early neurogenesis is enhanced in the medial ganglionic eminence of mBACTgDyrk1a embryos resulting in an altered proportion of particular cortical GABAergic neuron types. These results indicate that the overexpression of DYRK1A contributes significantly to the formation of the cortical circuitry in Down syndrome.<br>En aquest treball s'ha avaluat la possible contribució del gen DYRK1A, localitzat en el cromosoma humà 21, en les alteracions del desenvolupament de l’escorça cerebral associades a la Síndrome de down (SD) mitjançant l’estudi de dos models murins: el ratolí mBACTgDyrk1a, el qual conté 3 còpies de Dyrk1a, i el ratolí Ts65Dn, un dels models trisòmics de la SD més ben caracteritzats. Els nostres resultats mostren que la trisomia de Dyrk1A altera alguns paràmetres del cicle cel•lular i el tipus de divisió dels progenitors neurals del telencèfal dorsal, donant lloc a un dèficit de neurones glutamatèrgiques que persisteix fins l’edat adulta. Hem demostrat que Dyrk1a és el gen triplicat responsable del dèficit inicial en la generació de neurones glutamatèrgiques corticals observat en el ratolí Ts65Dn. A més a més, hem proporcionat evidències de que la degradació de Ciclina D1 induïda per DYRK1A és el mecanisme molecular subjacent a les alteracions de cicle cel•lular observades en els progenitors neuronals dels embrions mBACTgDyrk1a i Ts65Dn. Per altra banda, hem demostrat que la neurogènesis inicial està incrementada en l’eminència ganglionar medial dels embrions mBACTgDyrk1a, fet que altera la proporció de subtipus específics d’interneurones GABAèrgiques en l’escorça cerebral adulta. En conclusió, els nostres resultats indiquen que la sobreexpressió de DYRK1A contribueix significativament a la formació dels circuits cortical en la SD.

L’autisme est un trouble envahissant du développement défini uniquement sur des critères comportementaux et l’âge d’apparition. Le X fragile est une pathologie d’origine monogénique dont 15-25% des patients présente le diagnostique complet de l’autisme et dont de nombreux symptômes chevauchent avec l’autisme. Une souris Fmr1 KO a été créée et validée comme modèle pour le X fragile. A l’instar de la variabilité des phénotypes du X fragile chez l’humain, les données préliminaires montrent que la mutation nulle Fmr1 chez la souris interagit avec l'arrière fond génétique. Les travaux présentés visaient à déterminer les caractéristiques autistiques exprimées par les souris Fmr1 KO, ainsi que l’interaction de la mutation nulle avec le fond génétique (souches C57BL/6J, FVB.129P2tm1Cgr /J et leurs hybrides). Les résultats de ces travaux montrent notamment que les souris Fmr1 KO présentent un évitement initial d’approche sociale, des altérations principalement qualitatives des vocalisations, de l’hyperactivité et une augmentation de l’activité diurne. La mutation interagit avec le fond génétique et les résultats actuels indiquent que les KO de fond FVB.129P2tm1Cgr /J ont le phénotype le plus marqué<br>Autism is a pervasive developmental disorder defined by behavioural criteria and age of onset. Fragile X is a disorder due to the silencing of the Fmr1 gene. About 15-25% of Fragile X patients are diagnosed as autistic and many symptoms overlap between the two disorders. A mouse Fmr1 KO was created and validated as a model for Fragile X Syndrome. Preliminary data also show that the null mutation interacts with the genetic background. The work presented in this thesis aimed to determine the autistic features expressed in Fmr1 KO mice, as well as the influence of the genetic background (C57BL/6J and FVB.129P2tm1Cgr/J strains, and their reciprocal hybrids) on the expression of the Fmr1 mutation. Our results show an initial inhibition of social approach in Fmr1 KO mice and a qualitative alteration of ultrasonic vocalizations in isolated pups, as well as an increase in activity, especially during the diurnal period. The Fmr1 mutation interacts with the genetic background and the results indicate that KO on the FVB.129P2tm1Cgr/J background show the most marked phenotype

Les malformations du cortex cérébral (MDC) représentent une cause importante de handicap et d'épilepsie pharmaco-résistante. Le séquençage à haut débit a permis une amélioration considérable de l'identification des bases moléculaires des MDC non syndromiques. Toutefois, certaines formes, notamment les MDC complexes, demeurent inexpliquées. Mon projet de thèse a pour objectif de progresser dans la compréhension des MDC complexes en utilisant deux modèles : les microlissencéphalies (MLIS) et le syndrome d'Aicardi (AIC), une forme syndromique particulière associant des malformations de l'oeil et du cerveau uniquement rapporté chez les filles. L'étude par séquençage d'exome en trios de 16 familles MLIS m'a permis d'identifier et de caractériser un nouveau gène, WDR81, impliqué dans le cycle cellulaire. Par la même stratégie, j'ai pu identifier un variant homozygote pathogène dans TLE1, un partenaire majeur de FOXG1 dans la balance prolifération/différenciation de progéniteurs neuronaux, dans une famille consanguine de microcéphalie postnatale dont le phénotype est proche du syndrome FOXG1. En parallèle, mes travaux ont permis de préciser les spectres phénotypiques associés à RTTN, EPG5, COL4A1, COL4A2, TBR1, KIF5C, KIF2A et FOXG1. La deuxième partie de mon projet avait pour objet l'identification des bases moléculaires du syndrome d'Aicardi à partir d'une cohorte internationale de 19 patientes. Après avoir exclu un biais d'inactivation du chromosome X et la présence de microremaniements chromosomiques, j'ai réalisé un séquençage d'exome en trio. Aucun variant récurrent n'a été retrouvé dans les séquences codantes. Dans un second temps, j'ai testé une approche combinant les données du séquençage de génome et l'analyse du transcriptome (RNA-Seq) sur fibroblastes, me permettant d'identifier des transcrits dérégulés qui étaient impliqués dans le développement du cerveau et de l'oeil. J'ai comparé les résultats de cette analyse avec ceux de l'analyse du génome dans le but d'identifier des variants dans ces gènes candidats. En conclusion, mon travail de thèse a permis d'améliorer la connaissance des bases moléculaires des MDC complexes et d'ouvrir des perspectives de nouveaux mécanismes tels que ceux engageant les gènes WDR81 et EPG5, et le rôle des endosomes et de l'autophagie dans les MDC, et aussi TLE1 comme nouvelle cause de microcéphalies postnatales. Mes travaux ont également permis de générer une collection de données de séquençage haut débit (WES, WGS et RNA-Seq) qui seront mises en commun dans le cadre d'un consortium international afin de développer des nouvelles stratégies d'analyse en particulier pour les séquences non codantes. Cette approche permettra également d'ouvrir la voie vers la compréhension des mécanismes cellulaires impliqués dans la formation du cerveau et de l' œil<br>Malformations of cortical development (MCD) are a major cause of intellectual disability and drug-resistant epilepsy. Next Generation Sequencing (NGS) has considerably improved the identification of the molecular basis of non-syndromic MCD. However, certain forms, including complex MCD, remain unexplained. My PhD project aimed to improve the understanding of complex MCD using two disorders: Microlissencephaly (MLIS) and Aicardi Syndrome (AIC), the latter associating brain and eye malformations and only reported in girls. Trio Whole Exome Sequencing (WES) performed in 16 MLIS families allowed me to identify and functionally characterize a new MLIS gene, WDR81, in which mutations lead to cell cycle alteration. Moreover, using the same strategy, I was able to identify a pathogenic homozygous variant in TLE1 in a patient from consanguineous family with a postnatal microcephaly, suggestive of a FOXG1-like presentation. Interestingly, TLE1 is a major partner of FOXG1, a gene involved in maintaining the balance between progenitor proliferation and differentiation. In parallel, my work allowed me to redefine the phenotypic spectrum associated with RTTN, EPG5, COL4A1 and COL4A2, TBR1, KIF5C, KIF2A and FOXG1. The second part of my PhD program was aimed at identifying the genetic basis of AIC in an international cohort of 19 patients. After excluding a skewed X chromosome inactivation and the presence of chromosomal rearrangements, I performed WES in trios. The analysis of the data from WES did not allow me to identify any recurrent variants. I therefore tested a new approach combining Whole Genome Sequencing (WGS) and RNA-Sequencing (RNA-Seq) on fibroblast cells. I identified a number of deregulated transcripts implicated in brain and eye development. I compared the results of this analysis with the WGS analysis in order to find variants in these candidate genes. In conclusion, these studies have improved the knowledge of the molecular basis of complex MCD, such as TLE1 in postnatal microcephaly, and revealed the pathogenic mechanisms such as WDR81 in cell cycle progression and EPG5 in endosomes and autophagy. My work has also generated a collection of NGS data (WES, WGS and RNA-Seq) that will be shared in an international consortium to develop new analytical strategies, in particular for the non-coding DNA regions. This novel strategy provides opportunities to improve understanding of the cellular mechanisms involved in brain and eye development

O sistema da procineticina desempenha um papel importante na migração dos neurônios secretores de GnRH e na neurogênese do bulbo olfatório. Camundongos com ablação dos genes que codificam a procineticina 2 (PROK2) e seu receptor (PROKR2) apresentaram fenótipos semelhantes ao da síndrome de Kallmann descrita em humanos. Mutações inativadoras nos genes PROK2 e PROKR2 foram identificadas em pacientes com hipogonadismo hipogonadotrófico isolado. Com base nestes achados, investigamos a presença de alterações estruturais nos genes PROK2 e PROKR2 em 107 pacientes brasileiros (63 com síndrome de Kallmann e 47 com hipogonadismo hipogonadotrófico isolado normósmico). Cem indivíduos brasileiros que relataram desenvolvimento puberal normal foram utilizados como grupo controle. As regiões codificadoras dos genes PROK2 e PROKR2 foram amplificadas utilizando-se oligonucleotídeos intrônicos específicos, seguida de purificação enzimática e sequenciamento automático. Duas mutações no gene PROK2 foram identificadas: a mutação p.G100fsX121 em homozigose presente em dois irmãos com síndrome de Kallmann; e a mutação p.I55fsX56 em heterozigose identiificada em um paciente com HHIn. Quatro mutações foram identificadas no gene PROKR2 (p.R80C, p.Y140X, p.L173R e p.R268C) em cinco pacientes com síndrome de Kallmann e um paciente com HHIn. Essas mutações não foram encontradas no grupo controle. As mutações do tipo missense, p.R80C, p.L173R e p.R268C foram identificadas em heterozigose. Mutações nos genes FGFR1, GnRHR, KiSS-1 e GPR54 foram excluídas nesses pacientes. O paciente portador da mutação p.R268C do PROKR2 apresentou deleção dos exons 1 e 2 do gene KAL1. Adicionalmente, as mutações p.R80C e p.R268C foram identificadas em heterozigose em parentes de primeiro grau assintomáticos dos casos índices. A nova mutação p.Y140X do PROKR2, única alteração em homozigose, foi identificada em um paciente com micropênis, criptorquidia bilateral, anosmia e palato ogival. Os pais deste paciente eram portadores da mutação p.Y140X em heterozigose e relataram desenvolvimento puberal normal e ausência de anormalidades olfatórias. Estudos in vitro da nova mutação p.R80C localizada na primeira alça intracelular demonstraram que o acúmulo de fofatidil-inositol (IP), assim como a ativação da via da MAPK foram significativamente afetadas em células transfectadas com o receptor mutado em relação ao receptor selvagem, indicando que a mutação p.R80C determina uma menor atividade do receptor. Avaliação da expressão por Western blot mostrou uma diminuição na expressão do receptor mutado R80C e uma maior expressão de receptores imaturos. Esses achados sugeriram o papel crítico da arginina localizada na posição 80 na atividade normal do receptor. Em conclusão, expandimos o repertório de mutações deletérias nos genes PROK2 e PROKR2 em pacientes com hipogonadismo hipogonadotrófico isolado. A haploinsuficiência do PROKR2 não foi suficiente para causar síndrome de Kallmann ou HHIn, entretanto mutações inativadoras em homozigose nos genes PROK2 e PROKR2 foram responsáveis pelo fenótipo reprodutivo e olfatório anormal, em concordância com os estudos prévios de ablação gênica em modelos animais. Arginina localizada na posição 80 do PROKR2 desempenha um papel crucial na adequada maturação do receptor<br>Physiological activation of the prokineticin pathway has a critical role in olfactory bulb morphogenesis and GnRH secretion. Knock-out mice for genes that encode prokineticin 2 (PROK2) and the prokineticin receptor 2 (PROKR2) exhibited a phenotype similar to the Kallmann syndrome (KS). Inactivating mutations in PROK2 and PROKR2 have been identified in patients with isolated hypogonadotropic hypogonadism. Based on these findings, we investigated the presence of inactivating mutations of the genes PROK2 and PROKR2 in Brazilian patients with isolated hypogonadotropic hypogonadism associated or not with olfactory abnormalities and performed in vitro studies of the new identified mutations. We studied 107 patients with HH (63 with Kallmann syndrome and 44 with normosmic HH) and 100 control individuals. The coding regions of PROK2 and PROKR2 were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In PROK2, two known frameshift mutations were identified. Two brothers with Kallmann syndrome harbored the homozygous p.G100fsX121 mutation, whereas one male with normosmic HH harbored the heterozygous p.I55fsX56 mutation. In PROKR2, four distinct mutations (p.R80C, p.Y140X, p.L173R and p.R268C) were identified in five patients with Kallmann syndrome and in one patient with normosmic HH. These mutations were not found in the control group. The p.R80C and p.R268C missense mutations were identified in heterozygous state in the HH patients and in their asymptomatic first-degree relatives. The p.L173R was also identified in heterozygous state. In addition, no mutations of FGFR1, GnRHR, KiSS-1 or GPR54 were identified in these patients. The patient with the PROKR2 mutation p.R268C also has a deletion of the exon 1 and 2 in the gene KAL1. Notably, the new nonsense mutation (p.Y140X) was identified in homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism and high-arched palate. His asymptomatic parents were heterozygous for this severe defect. In vitro studies of the new mutation, p.R80C, were performed in order to access the mechanism by which this mutation could affect the activity of the PROKR2. In vitro studies showed that the amount of fofatidil-inositol (PI) and the activation of MAPK were significantly lower in cells transfected with the R80C mutant receptor than in cells transfected with the wild receptor, indicating that this variant is a loss-of-function mutation. Binding studies and Western blot showed a reduction in the expression levels of the receptor in the plasma membrane and in whole cell, respectively. Additionally, Western blot analysis of R80C PROKR2 revealed an additional smaller molecular weight band that represents the presence of immature unglycosylated receptors. The arginine 80 in ICL1 is important for post-translational processing of PROKR2. In conclusion, we expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH and showed that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROK2 or PROKR2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models. In vitro studies suggested that the arginine located at position 80 of the receptor seems to play an important role in the receptor function

Le Syndrome d’Impatiences Musculaires de l’Éveil (SIME) est une maladie neurologique caractérisée par un besoin urgent de bouger les jambes. C’est également l’une des causes les plus fréquentes d’insomnie. C’est une maladie très répandue, avec une prévalence de presque 15 % dans la population générale. Les maladies multifactorielles comme le SIME sont souvent le résultat de l’évolution d’une composante génétique et d’une composante environnementale. Dans le cadre du SIME, les études d’association génomique ont permis l’identification de 4 variants à effet modéré ou faible. Cependant, ces quatre variants n’expliquent qu’une faible partie de la composante génétique de la maladie, ce qui confirme que plusieurs nouveaux variants sont encore à identifier. Le rôle des déséquilibres génomiques (Copy Number Variations ou CNVs) dans le mécanisme génétique du SIME est à ce jour inconnu. Cependant, les CNVs se sont récemment positionnés comme une source d’intérêt majeur de variation génétique potentiellement responsable des phénotypes. En collaboration avec une équipe de Munich, nous avons réalisé deux études CNVs à échelle génomique (biopuces à SNP et hybridation génomique comparée (CGH)) sur des patients SIME d’ascendance germanique. À l’aide d’une étude cas-contrôle, nous avons pu identifier des régions avec une occurrence de CNVs différentes pour les patients SIME, comparés à différents groupes contrôles. L’une de ces régions est particulièrement intéressante, car elle est concordante à la fois avec des précédentes études familiales ainsi qu’avec les récentes études d’associations génomiques.<br>Restless Legs syndrome (RLS) is a neurological disorder characterized by the urge to move one’s limbs. It is also one of the most frequent causes of insomnia. The prevalence of RLS is estimated to be around 15% in the general population. Complexes disorders like RLS are often the result of the evolution of genetic and environmental components. For RLS, recent Genome Wide Association Study (GWAS) have identified four variants with mild to moderate effects. However, those four variants explain only a small part of the disease heritability and thus, we expect that many new variants are still to be found. The impact of Copy-Number Variation (CNV) in the genetic mechanism of RLS is still unknown. However, many studies have recently position the CNVs as a significant source of genetic variation potentially responsible of phenotypes. In collaboration with a team from Munich, we conducted two genome-wide CNVs studies (Genome Wide SNP chips and Comparative Genomic Hybridization (CGH)) on RLS patients from Germany. Using cases-controls studies, we identified regions with a different occurrence of CNVs for RLS patients, compared to different groups of controls. One of these regions is particularly interesting, as it has already been identified by both linkage and association studies.

碩士<br>國立臺灣大學<br>藥理學研究所<br>103<br>Dravet syndrome is a refractory seizure characterized by severe infant-onset myoclonic epilepsy, delayed psychomotor development and autism-spectrum behaviours. Loss-of-function mutations in Scn1a gene which encodes the type Ⅰvoltage-gated sodium channel (Nav1.1) is the predominant molecular cause in most patients. Children with Dravet syndrome are significantly impaired in visuo-perceptual skill performance, and the spatial memory have been reported close relative to hippocampus neurogenesis. However, the underlying dysfunction of the Scn1a gene might confer to the brain neurogenesis is largely unknown. Here, we constructed a transgenic mice with Scn1a1099X knock-in (KI) allele which presented an animal model of Dravet syndrome, exhibited spontaneous epileptic discharges and susceptible to hyperthermia-induced seizures. Our results show that Nav1.1 deficiency lead to significant increase in neural stem cell-derived neurospheres number and accompanied with increasing neural stem cell (NSC) marker, GFAP. Quantification of cell division with the proliferation indicator, Ki67 or PCNA, showed that cell proliferation was significant increase result from Nav1.1 dysfunction. Measurement of [Ca2+]i by fura-2 AM Ca2+ imaging demonstrated that [Ca2+]i was markedly elevated in neurospheres derived from Scn1a mutation mice. NSC are characterized as cells with potential to produce a large amount of progeny that grow and differentiate into neuron, astrocyte and oligodendrocyte. Immunocytochemistry result indicated that loss of Nav1.1 in NSC result in significant increase the neuroblast and immature neuron but oligodendrocyte in neurosphere-derived cells which isolated from postnatal day 1 mice. In contrast, NSC obtained from postnatal day 8 Scn1a-deficit animal showed an upregulation of immature neuron and oligodendrocyte lineage but neuroblast. Moreover, prolonged differentiation for 2 weeks of neurosphere-derived cell culture isolated from postnatal day 1 Scn1a-mutant mice revealed that the ratio of mature neuron and GABAergic neuron were dramatically increased. GABA has been known to regulate neurogenesis, thus we further to investigate the effects of GABA on Scn1a deficiency neurosphere. Our results show that GABA promote neurosphere formation in PD1 neurosphere, but inhibit neurosphere formation in PD8 neurosphere. When neurosphere were induced to differentiation for 5 days, we found abnormal neuronal differentiation were reverse by GABA. These results revealed that loss of Scn1a gene might contribute to promote NSCs differentiate into neuronal series. Our results support that Scn1a gene play an important role in postnatal neuronal development.

Motor deficits are a hallmark of Down syndrome (DS), yet little is known about their exact cause. Despite the rich understanding of the neurobiology of DS, there is still a lack of targetable mechanisms for early intervention aimed at alleviating motor changes in people with DS. Therefore, we utilized a mouse model of DS known as Ts65Dn to characterize for the first time the effects of trisomy 21 on spinal cord (SC) development. A central molecular player in SC patterning and cell-type specification, Oligodendrocyte transcription factor 2 (Olig2), is located on human chromosome 21 (Hsa21) and is triplicated in both people with DS and in Ts65Dn mice. To observe the effects of the supernumerary Olig2, we used immunohistochemistry to visualize the OLIG2-derived cellular populations (i.e., motor neurons (MNs) and oligodendrocytes (OLs)), as well as adjacent and interacting cell populations (i.e., ventral spinal interneurons (INs)). We limited our analyses to two embryonic ages—embryonic days (E) 12.5 and 14.5. Our results indicate that there is no overall change in the numbers of OLs at either E12.5 or E14.5. However, there tend to be more OL-fated cells within the pMN domain, where they originate, and migrating cells tend to be clustered closer to the pMN domain at E12.5. IN populations show some changes in Ts65Dn mice at E12.5, with both total and abventricular PAX6+ cell numbers and abventricular NKX2.2+ cell numbers increased in Ts65Dn embryos compared to euploid mice. However, at E14.5 the number of NKX2.2+ cells is unchanged. No difference in the NKX6.1+ population was seen at either time-point. In contrast, there are significant changes in the MN population at both E12.5 and E14.5. Specifically, at E12.5, the total ISL1+ MN population is significantly increased and shows altered regional distribution in the ventral horn of Ts65Dn SCs. Conversely, the Ts65Dn spinal MN population is normalized to euploid levels at E14.5. Overall, our results suggest that neurogenesis, gliogenesis, and cell-type specification of OLIG2-lineage cells are altered in the developing SC of Ts65Dn mice. Thus, this work identifies a novel target for future therapeutic interventions aimed at ameliorating motor changes in DS.

Morfin is a clinically used analgesic drug but also an abusive drug. It has an impact on a wide range of CNS regions (nucleus accumbens, ventral tegmentum, hippocampus, etc.) and affects their functions, e.g. cognitive functions or anxiety. Although the results of so far published studies are often contradictory, the effects on cell death and proliferation in the CNS have been demonstrated. In this work, we focused on how chronic administration of morphine and subsequent withdrawal of this drug affects neurogenesis and neurodegeneration in the rat brain and how it affects some markers involved in the addiction and post-drug-induced condition. We have succeeded in introducing immunohistochemical markers for monitoring neurogenesis (bromodeoxyuridine and doublecortin) and neurodegeneration (Fluoro-Jade C) and for detection of selected neuromodulatory peptides (cholecystokinin and neuropeptide Y). We have found that morphine may influence the process of neurogenesis and neurodegeneration, but its effects differ in different CNS structures (nucleus accumbens, hippocampus, and amygdala). Key words: Morphine, brain, rat, withdrawal syndrom, neurogenesis, neurodegeneration

Alcohol consumption during pregnancy is detrimental to the developing nervous system of the unborn offspring. The hippocampus, one of the two brain regions where neurogenesis persists into adulthood, is particularly sensitive to the teratogenic effects of alcohol. The present study examined the effects of alcohol exposure throughout all three trimester equivalents on the stages of adult neurogenesis. Prenatal and early postnatal alcohol exposure (PPAE) altered cell proliferation in adult but not adolescent animals and increased early neuronal differentiation without affecting cell survival in both age groups. The levels of brain-derived neurotrophic factor (BDNF) were not affected by PPAE in the dentate gyrus but were significantly decreased in the Cornu ammonis region of the hippocampus. These results might explain the functional deficits seen in this hippocampal sub-region. This study identified that voluntary wheel running increased cell proliferation, differentiation and survival as well as BDNF expression in both PPAE and control animals.<br>Graduate

Non-syndromic intellectual disability (NS-ID) is a widespread neurodevelopmental disorder in which the major phenotypic manifestation is low IQ. Given the known genetic overlaps between the two conditions, it was hypothesize that autosomal recessive NS-ID (NS-ARID) genes may also play a role in autism. In this thesis, autism probands with CNVs overlapping NS-ARID genes were screened for additional mutations by sequencing. In addition, TRAPPC9 was identified as a novel cause of NS-ARID in two unrelated consanguineous families. TRAPPC9 (NIBP) is believed to function in the NF-kB pathway and the TRAPP complex. Multiple probands with developmental delays and CNVs overlapping TRAPPC9 were also identified. A potential mechanism for the CNV-related phenotype is that TRAPPC9 may be partially paternally imprinted in brain, and overlapping CNVs may cause loss of regulation. Identification of genes for autism and ID will translate into earlier diagnosis and better clinical care for this population in the future.

Down syndrome (DS) is a developmental disorder caused by a triplication of human chromosome 21, which contains approximately 550 genes. DS is the most common autosomal aneuploidy occurring with an incidence of 1 in 793 live births. Hallmarks of DS include abnormal central nervous system (CNS) development and function resulting in intellectual disability (ID), motor dysfunction, and early onset Alzheimer’s neuropathology. Studies have elucidated widespread neurohistological abnormalities in brains of fetuses with DS as early as 20 weeks of gestation, suggesting that early dysfunction in neural development may set the stage for exacerbated CNS abnormalities throughout life. Additionally, the complex constellation of symptoms associated with DS changes over the lifespan, particularly in adolescence and in middle to old age. Thus, these periods may represent opportune windows for age-specific therapeutic interventions. Due to ethical and practical constraints, use of human samples is alone insufficient to characterize the etiological underpinnings of DS phenotypes across the lifespan. Furthermore, while human data are instructive for drug development, preclinical trials are necessary for target validation, to establish dosage, and to prove safety and efficacy of any proposed therapeutic. With the advent of mouse models of DS, informative studies on the neurobiology of DS as well as preclinical testing of proposed therapies are possible. Here, we use a multi-pronged approach to assess molecular, neuroanatomical, and behavioral phenotypes indicative of brain and SC function in three distinct mouse models of DS: Ts1Cje, Ts65Dn, and Dp16. We identify neurodevelopment phenotypes, cytoarchitectural aberrations, bioenergetic abnormalities, myelination deficits, and motor/cognitive dysfunction at multiple ages spanning the period between embryonic day 12.5 and 6-7 months in trisomic mice. Additionally, we show that while Ts65Dn mice recapitulate all known phases of histological, functional, and behavioral phenotypes typical of DS starting from prenatal development and into middle age, this is not true for the Ts1Cje or Dp16 models. Lastly, we present promising outcomes of two possible therapies for cognitive and motor dysfunction in Ts65Dn mice. Altogether our findings provide insights into the underlying neurobiology of ID and motor dysfunction in DS and elucidate molecular changes that can be targeted for future therapeutic intervention.<br>2018-07-09T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI)<br>Down syndrome (DS) is caused by three copies of human chromosome 21 (Hsa21) and results in an array of phenotypes including intellectual disability. Ts65Dn mice, the most extensively studied DS model, have three copies of ~50% of the genes on Hsa21 and display many phenotypes associated with DS, including cognitive deficits. DYRK1A is found in three copies in humans with Trisomy 21 and in Ts65Dn mice, and is involved in a number of critical pathways including CNS development and osteoclastogenesis. Epigallocatechin-3-gallate (EGCG), the main polyphenol in green tea, inhibits Dyrk1a activity. We have shown that a three-week EGCG treatment (~10mg/kg/day) during adolescence normalizes skeletal abnormalities in Ts65Dn mice, yet the same dose did not rescue deficits in the Morris water maze spatial learning task (MWM) or novel object recognition (NOR). Others have reported that An EGCG dose of 2-3 mg per day (90mg/ml) improved hippocampal-dependent task deficits in Ts65Dn mice. The current study investigated deficits in a radial arm maze pattern separation task in Ts65Dn mice. Pattern separation requires differentiation between similar memories acquired during learning episodes; distinguishing between these similar memories is thought to depend on distinctive encoding in the hippocampus. Pattern separation has been linked to functional activity of newly generated granule cells in the dentate gyrus. Recent studies in Ts65Dn mice have reported significant reductions in adult hippocampal neurogenesis, and after EGCG treatment, enhanced hippocampal neurogenesis. Thus, it was hypothesized that Ts65Dn mice would be impaired in the pattern separation task, and that EGCG would alleviate the pattern separation deficits seen in trisomic mice, in association with increased adult hippocampal neurogenesis. At weaning, Ts65Dn mice and euploid littermates were randomly assigned to the water control, or EGCG [0.4 mg/mL], with both treatments yielding average daily intakes of ~50 mg/kg/day. Beginning on postnatal day 75, all mice were trained on a radial arm maze-delayed non-matching-to-place pattern separation task. Euploid mice performed significantly better over training than Ts65Dn mice, including better performance at each of the three separations. EGCG did not significantly alleviate the pattern separation deficits in Ts65Dn mice. After the behavioral testing commenced, animals were given ad libitum food access for five days, received a 100mg/kg injection of BrdU, and were perfused two hours later. Coronal sections through the dorsal hippocampus were processed for BrdU labeling, and cells were manually counted throughout the subgranular zone of the dentate gyrus. The euploid controls had significantly more BrdU labeled cells than Ts65Dn mice, however, EGCG does not appear to increase proliferation of the hippocampal neuroprogenitor cells. This is the first report of deficits in Ts65Dn mice on a pattern separation task. To the extent that pattern separation depends on the functional involvement of newly generated neurons in an adult dentate gyrus, this approach in Ts65Dn mice may help identify more targeted pharmacotherapies for cognitive deficits in individuals with DS.

Reduction in dopamine receptor D2, has been associated with insufficient brain reward, food addiction, obesity, and type 2 diabetes (T2D). Our aim was to assess whether the genetic variability responsible for this reduction is associated with poor dietary compliance and life style habits in T2D patients. Genetic-analysis was done for 109 T2D individuals who completed a 24-week randomized clinical trial and were assigned to follow either a low-GI or a high-fibre diet. Polymorphisms of TaqIA and C957T were compared with physical and biochemical measures. Regardless of dietary treatments, individuals with the C957T-T allele and the TaqIA-A2 allele were significantly associated with blood pressure reduction. Carriers of the T allele significantly lowered their body mass index (BMI) over the 24-week trial. Our findings suggest that the presence of the TaqIA-A2 allele is associated with a decrease in blood pressure. The C957T-T allele was associated with decrease in pressure and body weight.