Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-02-09T10:36:15Z No. of bitstreams: 2 Dissertação - Nara Lígia Leão Casa - 2015.pdf: 3128154 bytes, checksum: 290faf6f7e255eb06dcfd7b0baf48e03 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-02-09T10:36:36Z (GMT) No. of bitstreams: 2 Dissertação - Nara Lígia Leão Casa - 2015.pdf: 3128154 bytes, checksum: 290faf6f7e255eb06dcfd7b0baf48e03 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-02-09T10:36:36Z (GMT). No. of bitstreams: 2 Dissertação - Nara Lígia Leão Casa - 2015.pdf: 3128154 bytes, checksum: 290faf6f7e255eb06dcfd7b0baf48e03 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-12-02 / Introduction: Lumbar disc herniation (LDH) is the most common diagnosis among the degenerative changes in the lumbar spine and it can lead to functional limitations. Although several studies have shown a positive association between polymorphisms in the aggrecan gene and LDH, results are conflicting and vary across populations. Until now, such studies have not been carried out in Brazil. An understanding of this association can help in the prevention and intervention processes in patients with LDH. Objective: Investigate the association between polymorphisms in the aggrecan gene and LDH. Methods: This is a Case-Control study, paired by gender and age. A total of 119 male and female individuals from Goiania (Brazil) participated in the study; 39 individuals in the Case Group (CaG) and 80 in the Control Group (CtG). For each individual, sociodemographic and clinical data were collected as well as blood samples for genetic analysis. DNA was isolated and genotyped at the variable number of tandem repeats (VNTR) region of the aggrecan gene using polymerase chain reaction. Results: Both groups were homogeneous for socio-demographic, anthropometric, and life style variables. The sum of allele sizes at the VNTR region of the aggrecan gene were significantly lower in individuals with LDH, with allele A22 showing significantly higher frequency in this group. Comparison of allele size distribution indicated that shorter alleles (i.e., A13-A25) were more frequent in individuals with LDH, whereas alleles with a higher number of repeats were more frequent among healthy individuals. It should be noted, however, that differences were not significant. The most frequent alleles in both groups were A28, A27 and A29, respectively. Genotype A26/A26 was the most common among individuals in the CaG. Conclusions: An association between short alleles and LDH was observed in this study. This finding is in agreement with other reports in the literature, corroborating the hypothesis that individuals with shorter alleles have an aggrecan gene with fewer repeats. Therefore, a smaller number of chondroitin sulfate chains bind to the aggrecan in the intervertebral disc, leading to a decrease in its physiological function of hydration of the intervertebral disc, and thus to an increase in LDH susceptibility. / Introdução: A hérnia de disco lombar (HDL) é um diagnóstico frequente dentre as alterações degenerativas da coluna lombar e causa limitações funcionais importantes. Alguns estudos apontam para uma associação positiva entre polimorfismos no gene aggrecan e a HDL, porém os resultados ainda são conflitantes e variam entre as populações, não havendo pesquisas sobre o tema na população brasileira até o momento. Compreender essa associação pode auxiliar na prevenção e intervenção junto às pessoas com HDL. Objetivo: Investigar a associação entre polimorfismos no gene aggrecan com a hérnia de disco lombar. Métodos: Estudo caso-controle, pareado em quinquênio por idade e sexo. Participaram do estudo 119 pessoas de ambos os sexos, domiciliadas em Goiânia (Brasil), sendo 39 no Grupo Caso (Ca) e 80 no Controle (Ct). Foram coletados dados sociodemográficos e clínicos e amostras de sangue periférico; o DNA foi isolado para posterior genotipagem do número variável de repetições em Tandem (VNTR) do gene aggrecan (ACAN) através de PCR convencional. Resultados: Os grupos mostraram-se homogêneos quanto às variáveis sociodemográficas, antropométricas e hábitos de vida. O escore alélico do VNTR do gene aggrecan foi significativamente menor nos voluntários com HDL; o alelo A22 mostrou-se significativamente mais prevalente nesse mesmo grupo; no grupo Ca houve maior frequência dos alelos pequenos A13 - A25, enquanto no Ct maior frequência dos alelos grandes, porém, sem diferença estatisticamente significativa; em ambos os grupos os alelos mais frequentes foram A28, A27 e A29, respectivamente; o genótipo A26/A26 foi significativamente mais comum no Grupo Ca. Conclusões: Houve associação entre alelos pequenos com a HDL nos adultos pesquisados e compatibilidade com a literatura internacional, corroborando a hipótese de que indivíduos que possuem alelos pequenos possuem um aggrecan com menor número de repetições. Portanto, um número menor de cadeias de sulfato de condroitina se ligam no aggrecan do disco intervertebral, o que resulta na diminuição da sua função fisiológica de hidratação do disco e, consequentemente, aumenta a susceptibilidade individual à HDL.

Mutations in genes of importance for cartilage development may lead to skeletal malformations, chondroskeletal dysfunction and increased susceptibility to degenerative joint disease. Characterization of these mutations and identification of molecular pathways for the corresponding gene products have contributed to our understanding of mechanisms regulating skeletal patterning, endochondral ossification and joint formation. A five generation family segregating autosomal dominant osteochondritis dissecans (OCD) was identified. Affected family members presented with OCD in knees, hips and elbows, short stature, and early osteoarthritis. A genome wide scan and a multipoint linkage analysis identified aggrecan (ACAN) as a prime candidate gene. DNA sequence analysis of the ACAN-gene revealed heterozygosity for a missense mutation (c.6907G>A) in affected subjects, resulting in a p.V2303M substitution in the aggrecan G3 domain C-type lectin. This domain is important for the interaction with other proteins in the cartilage extracellular matrix. To determine the effect of the V2303M substitution on secretion and interaction, we performed binding studies with recombinant mutated and wild type G3 proteins. We found decreased affinity or complete loss of interaction between V2303M aggrecan and fibulin1, fibulin2 and tenascin-R. Analysis of articular cartilage from an affected family member confirmed that V2303M aggrecan is produced and present. In search for gene mutations associated with multiple epiphyseal dysplasia (MED) we considered the ACAN-gene a likely candidate. The ACAN-gene was analysed in 39 individuals with MED and screened negative for mutations in six previously known MED genes. Sequence analysis revealed a heterozygous missense mutation (c.1448G>T) in one adult male and compound heterozygous missense mutations (c.1366T>C and c.836G>A) in a five year old boy with healthy parents, each of them carrier for one of the mutations. A large family segregating autosomal dominant brachymesophalangia and OCD in finger joints was characterised. The clinical presentation in six affected family members was consistent with the diagnosis Brachydactyly type A1, in this family characterized by shortening of the middle phalanges, short ulnar styloid process, flattening of the metacarpal heads and mild osteoarthritis. The condition may be caused by mutations in the Indian hedgehog gene (IHH) or a yet unidentified gene on chromosome 5p13. Sequence analysis of the IHH-gene in affected individuals revealed a novel C to T transition (c.472C>T) leading to a p.158Arg>Cys substitution. Residue 158 in IHH is highly conserved throughout evolution and molecular structure modelling of IHH suggests that the R158C substitution leads to a conformational change at the site of interaction with the IHH-receptor. This supports that the substitution causes Brachydactyly type A1 in this family. In summary, we report on the clinical, radiological and molecular genetic characteristics of the three skeletal disorders OCD, MED and BDA1. Our results provide a novel molecular mechanism in the pathophysiology of familial osteochondritis dissecans confirming the importance of aggrecan C-type lectin for cartilage function. We also show that ACAN-gene mutations may be associated with MED extending the spectrum of skeletal dysplasias associated with the aggrecan gene. Finally, we report on a novel missense mutation in a conserved region of the IHH-gene associated with BDA1.

La plasticité cérébrale est régulée de façon dynamique au cours d’une vie : atteignant des sommets au cours de l’enfance, elle est réduite chez l’adulte. Toutefois, des circonstances particulières appellent à vouloir stimuler la malléabilité du cerveau adulte : pour favoriser la réhabilitation suite à un accident vasculaire cérébral ou un traumatisme crânien, pour aider l’adaptation spécifique nécessaire pour vivre avec une nouvelle prothèse ou encore pour améliorer l’efficacité de la thérapie cognitivo-comportementale suite à un traumatisme émotionnel qui a laissé un souvenir de peur qui s’est développé en syndrome de choc post-traumatique. Toutes ces situations demandent des capacités d’adaptation et une flexibilité exceptionnelles au système nerveux central. Or, pour retrouver une plasticité cérébrale telle qu’au niveau juvénile, la littérature nous apprend qu’il faut diminuer la puissance inhibitrice générée par un type d’interneurones particuliers, ceux exprimant la parvalbumine (PV+). Les fonctions des interneurones PV+ dépendent autant de leur patron de connectivité que de l’environnement extracellulaire dans lequel ils évoluent. En effet, en innervant des centaines de neurones cibles, délivrant une forte inhibition périsomatique en formant de multiples synapses autour de leur corps cellulaire et de leurs dendrites proximales, ils ont été impliqués dans l’intégration synaptique des neurones pyramidaux et dans la synchronisation des circuits corticaux. Toute manipulation ciblant cette arborescence axonale complexe pourrait s’avérer efficace à l’augmentation de la plasticité cérébrale en diminuant l’inhibition qu’elle génère. Ainsi, comprendre la signalisation moléculaire restreignant la croissance de l’arborescence axonale et la formation de boutons fonctionnels au cours de la longue phase développementale qui caractérise les interneurones PV+ aiderait à identifier des méthodes efficaces afin d’activer cette signalisation moléculaire chez l’adulte. De plus, comprendre les régulations épigénétiques liées au développement et à la maturation structurelle et fonctionnelle des interneurones PV+ offrirait une cible de choix afin de dématurer ces circuits inhibiteurs et lever un frein sur la plasticité cérébrale adulte. Nous démontrons ici que l’expression du récepteur des neurotrophines p75NTR chez les interneurones PV+ au cours de leur développement restreint la maturation de leur arborescence axonale, autant in vitro que in vivo, ainsi que l’agglomération des filets périneuronaux, autour de leur corps cellulaire. Aussi, en utilisant une version modifiée du test de ligation de proximité, nous avons résolu une controverse et démontré que le récepteur est toujours exprimé chez les interneurones PV+ du cortex adulte. Enfin, l’activation de la signalisation via p75NTR des interneurones PV+ par son ligand proBDNF est suffisante pour déstabiliser leur connectivité et restaurer la plasticité du cortex visuel suite à une privation monoculaire. Également, l’inactivation d’un régulateur épigénétique, l’histone déacétylase Hdac2, spécifiquement chez les interneurones PV+ suffit à diminuer leur connectivité efférente ainsi que l’agglomération des filets périneuronaux autour de leurs corps cellulaire tout en augmentant la rétention de l’extinction des souvenirs de peur, témoignant d’une augmentation de la plasticité cérébrale adulte. Par le séquençage d’ARNm en cellule unique, suivi de l’hybridation in situ RNAscope, nous avons identifié le gène Acan, codant pour aggrécane, une composante protéique des filets périneuronaux, comme étant exprimé de façon autonome à la cellule par les interneurones PV+ du cortex préfrontal adulte. Enfin, nous avons démontré qu’une seule injection d’un nouvel inhibiteur spécifique pour Hdac2 avant le paradigme d’extinction suffit à augmenter la rétention des souvenirs d’extinction chez l’adulte, tout en réduisant l’expression de Acan et l’agglomération des filets périneuronaux dans le cortex préfrontal. En somme, nos travaux ont montré que le remodelage des circuits des interneurones PV+ en ciblant soit le récepteur p75NTR, soit l’histone déacétylase Hdac2, peut efficacement augmenter la plasticité cérébrale chez l’adulte. / Brain plasticity is dynamically regulated during a lifespan: it reaches a peak during juvenile age and decreases in adulthood. However, exceptional circumstances can drive the need to foster adult brain plasticity: to help rehabilitation after a stroke or a head trauma, to increase the adaptability of an individual facing a new life with a prosthetic, to improve the efficiency of cognitive behavioral therapy to cope with the indelible fear memory trace created by an emotional trauma. All these situations require exceptional adaptation capabilities and cognitive flexibility. Several studies have suggested that reducing inhibitory drive, in particular of a specific GABAergic interneuron population, the parvalbumin-expressing interneurons (PV+), could be an effective approach to recover juvenile brain plasticity, thereby increasing adult brain plasticity. PV+ interneuron functions depend on their axonal connectivity pattern as well as their specific extracellular environment. Indeed, by contacting hundreds of postsynaptic neurons and delivering a strong perisomatic inhibitory drive by forming multiple synapses on their somata and proximal dendrites, PV+ interneurons strongly regulate pyramidal cell synaptic integration and cortical circuit synchronisation. PV+ interneuron maturation is a prolonged process, which reaches plateau only after the end of adolescence, and correlates with the decline of developmentally regulated- brain plasticity. We hypothesize that manipulations specifically targeting PV+ interneuron highly complex axonal arborisation, and thus reducing their inhibitory drive, could be efficient tools to foster adult brain plasticity. Understanding the molecular signalling that restricts PV+ cell axonal arborisation growth and the formation of functional presynaptic boutons during their long developmental phase may help identifying efficient methods to activate this molecular pathway, thus reducing PV+ interneuron connectivity, in adults. In addition, understanding the epigenetic regulation of structural and functional maturation of PV+ interneurons may offer a choice target to dematurate these inhibitory circuits and lift a brake on adult brain plasticity. Here, we demonstrate that the expression levels of neurotrophin receptor p75NTR during PV+ interneurons development constrain the maturation of their connectivity as well as the perineuronal net agglomeration around their cell bodies in a cell-autonomous fashion, both in vitro and in vivo. Also, by using a modified version of the proximity ligand assay, we solve a long-standing debate by demonstrating p75NTR expression in PV+ interneurons in adult cortex. Finally, we show that promoting p75NTR signalisation in PV+ cortical interneurons by its ligand proBDNF is sufficient to destabilize their connectivity and restore cortical plasticity following monocular deprivation in the adult visual cortex. We further show that the deletion of the epigenetic regulator histone deacetylase 2 (Hdac2), specifically in PV+ interneurons, is sufficient to decrease their efferent connectivity and perineuronal net agglomeration around their cell bodies, while increasing fear extinction retention, a measure of brain plasticity. By single-cell RNA sequencing, followed by RNAscope in situ hybridization, we found that the Acan gene, which encodes for aggrecan, a critical perineuronal net protein component, is expressed cell-autonomously by PV+ interneurons in adult prefrontal cortex. Finally, we showed that a single injection of a novel Hdac2 specific inhibitor before extinction training is sufficient to increase fear extinction retention in adults, while reducing Acan expression and perineuronal net agglomeration in prefrontal cortex. In summary, our work shows that increasing remodeling of PV+ interneuron circuits by targeting either p75NTR receptor or histone deacetylase Hdac2 efficiently foster adult brain plasticity.