Bioinformática de circRNAS : análise em modelos animais do Transtorno do Espectro Autista e desenvolvimento de um método de avaliação da usabilidade dos softwares utilizados para identificação

O Transtorno do Espectro Autista (TEA) é um transtorno do neurodesenvolvimento caracterizado por prejuízos na comunicação e interação social, comportamentos repetitivos e interesses restritos. Estima-se que, mundialmente, 1% da população é afetada pela condição. A etiologia do TEA é desconhecida, porém sabe-se que existe uma contribuição complexa de fatores ambientais e genéticos para o desenvolvimento do transtorno. Alterações transcritômicas são consistentemente observadas no sistema nervoso central de indivíduos com TEA e não estão restritas a expressão de genes codificantes. Uma classe de RNAs não-codificantes de interesse especialmente recente é a dos RNAs circulares (circRNAs). Os circRNAs são abundantes no encéfalo e podem desempenhar funções variadas como competir pela ligação de miRNAs, estimular a transcrição do gene parental e ser traduzido em proteínas funcionais. O envolvimento dos circRNAs no TEA ainda é pouco explorado. Recentemente foram feitas as primeiras observações de circRNAs diferencialmente expressos (CDE) no córtex cerebral de indivíduos com TEA. Adicionalmente, CDE também foram observados em dois modelos animais de TEA. Modelos animais são essenciais para o estudo de transtornos do neurodesenvolvimento e podem auxiliar os pesquisadores a investigar a etiologia e os mecanismos biológicos subjacentes. A detecção em massa e a descoberta de novos circRNAs depende quase exclusivamente de softwares de identificação de circRNA. Entretanto, possíveis problemas na usabilidade dessas ferramentas são relatados na literatura e podem prejudicar os usuários ao consumir tempo e dificultar a reprodutibilidade das análises. Considerando o potencial envolvimento de circRNAs no TEA e a dependência metodológica de softwares para identificação, a presente tese analisa por bioinformática a expressão gênica de circRNAs no encéfalo de diferentes modelos animais de TEA, assim como desenvolver um método de avaliação e melhoramento da usabilidade das ferramentas de bioinformática utilizadas para a identificação dos circRNAs. No Capítulo I, identificamos milhares de circRNAs em diferentes modelos animais de TEA em camundongos e demonstramos que as alterações na expressão estão relacionadas principalmente às funcionalidades sinápticas. No Capítulo II, identificamos parcialmente a sequência do circRNA ciRS-7 no modelo animal de ratos induzidos por ácido valproico. Demonstramos que a expressão de ciRS-7 está elevada, assim como encontrada nos modelos Ash1L, BTBR e Kmt5b no Capítulo I, e que funções sinápticas estão entre os possíveis grupos funcionais afetados. Por fim, no Capítulo III, identificamos os problemas de usabilidade das ferramentas de identificação de circRNAs utilizadas nos Capítulos I e II. Também criamos um conjunto de regras para auxiliar os desenvolvedores desse tipo de software para criarem ferramentas mais utilizáveis para os usuários finais. Em conjunto, os resultados apresentam um panorama do envolvimento de circRNAs no TEA a partir de modelos animais. Além disso, apresentamos um novo método de avaliação de usabilidade de software que deve possibilitar que futuras análises estejam alinhadas com as necessidades e expectativas de uso de ferramentas de identificação de circRNAs.Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by impairments in communication and social interaction, repetitive behaviors, and restricted interests. It is estimated that the condition affects 1% of the population worldwide. The etiology of ASD is unknown, but it is known that there is a complex contribution of environmental and genetic factors to the development of the disorder. Transcriptomic alterations are consistently observed in the central nervous system of individuals with ASD and are not restricted to the expression of coding genes. Circular RNAs (circRNAs) are a class of noncoding RNAs of recent interest. CircRNAs are abundant in the brain and can perform varied functions, such as competing for binding miRNAs, stimulating parental gene transcription, and being translated into functional proteins. The involvement of circRNAs in ASD is still poorly explored. Recently, the first observations of differentially expressed circRNAs (DECs) were made in the cerebral cortex of individuals with ASD. Additionally, DECs were also observed in two animal models of ASD. Animal models are essential for studying neurodevelopmental disorders and can help researchers investigate the etiology and underlying biological mechanisms. The mass detection and discovery of novel circRNAs rely almost exclusively on circRNA identification software. However, potential problems in the usability of these tools are reported in the literature and may harm users by consuming time and hindering the reproducibility of analyses. Considering the potential involvement of circRNAs in ASD and the methodological dependence on software for identification, this thesis analyzes by bioinformatics the gene expression of circRNAs in the brain of different animal models of ASD, as well as develops a method to evaluate and improve the usability of bioinformatics tools used for circRNA identification. In Chapter I, we identify thousands of circRNAs in different animal models of ASD in mice and demonstrate that the changes in expression are mainly related to synaptic functionalities. In Chapter II, we partially identify the sequence of the circRNA ciRS-7 in the valproic acid-induced rat model. We demonstrate that ciRS- 7 expression is elevated, as found in the Ash1L, BTBR, and Kmt5b models in Chapter I, and that synaptic functions are among the possible functional groups affected. Finally, in Chapter III, we identify the usability problems of the circRNAs identification tools used in Chapters I and II. We also create a set of rules to assist developers of such software in creating more usable tools for end users. Taken together, the results present an overview of the involvement of circRNAs in ASD from the perspective of animal models. In addition, we present a new software usability evaluation method that should enable future analyses to be aligned with the needs and expectations of using circRNAs identification tools

Abnormal empathy-like pro-social behaviour in the valproic acid model of autism spectrum disorder

Impairments in social behaviour are a defining feature of autism spectrum disorder (ASD). Individuals with ASD also usually present some difficulty to recognise or understand another person's feelings. Therefore, it is possible that altered empathy processing could hinder typical social interaction in ASD. Recently, robust paradigms confirmed that rodents show primordial forms of empathy-like behaviour. Therefore, in this work, we used one of these new protocols to test pro-social behaviour in the rat model of autism induced by Valproic Acid (VPA). We also evaluated possible beneficial effects of Resveratrol, since it can prevent social deficits in the VPA model. Rats were tested on their ability to open a restrainer to release a trapped conspecific. Exposure to VPA precludes the timely manifestation of this empathy-like behaviour, but does not affect its continuation after its first expression. We also found a significant correlation between average speed during the first day of test and becoming an Opener. Similarly, rats able to open the restrainer on the first day had an increased likelihood of repeating this behaviour in the later days of the testing programme. We did not find any protective effects of Resveratrol. Further investigation of empathy-like behaviour in the VPA model and in other models of autism could help to clarify the behavioural and neural processes underpinning the basic aspects of empathy alterations in autistic individuals. [Abstract copyright: Copyright © 2019. Published by Elsevier B.V.

Data on social transmission of food preference in a model of autism induced by valproic acid and translational analysis of circulating microRNA

This article contains data of Social Transmission of Food Preference in an animal model of autism and the evaluation of a set of microRNA analyzed in autistic patients and animal model of autism. The analyses of the absolute consumption of two flavored food by male rats prenatally exposed to valproic acid (VPA) and treated with resveratrol (RSV), showed that VPA animals show a trend to eat less of the flavored food presented by a demonstrator rat. We also identified 13 microRNA with similar levels among rodents' experimental groups, as well as 11 microRNA with no alterations between autistic and control subjects. Further evaluation of mechanisms of VPA and RSV actions on behavioral and molecular alterations can shed light in important biomarkers and etiological triggers of autistic spectrum disorders

Resveratrol prevents cellular and behavioral sensory alterations in the animal model of autism induced by valproic acid

Autism spectrum disorder (ASD) is characterized by impairments in both social communication and interaction and repetitive or stereotyped behaviors. Although its etiology remains unknown, genetic and environmental risk factors have been associated with this disorder, including the exposure to valproic acid (VPA) during pregnancy. Resveratrol (RSV) is an anti-inflammatory and antioxidant molecule known to prevent social impairments in the VPA animal model of autism. This study aimed to analyze the effects of prenatal exposure to VPA, as well as possible preventive effects of RSV, on sensory behavior, the localization of GABAergic parvalbumin (PV+) neurons in sensory brain regions and the expression of proteins of excitatory and inhibitory synapses. Pregnant rats were treated daily with RSV (3.6 mg/kg) from E6.5 to E18.5 and injected with VPA (600 mg/kg) in the E12.5. Male pups were analyzed in nest seeking behavior and in whisker nuisance task. At P30, the tissues were removed and analyzed by immunofluorescence and western blotting. Our data showed for the first time an altered localization of PV+-neurons in primary sensory cortex and amygdala. We also showed a reduced level of gephyrin in the primary somatosensory area of VPA animals. The treatment with RSV prevented all the aforementioned alterations triggered by VPA. Our data shed light on the relevance of sensory component in ASD and highlights the interplay between RSV and VPA animal model as an important tool to investigate the pathophysiology of ASD

Resveratrol prevents cytoarchitectural and interneuronal alterations in the valproic acid rat model of autism

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder characterized by several alterations, including disorganized brain cytoarchitecture and excitatory/inhibitory (E/I) imbalance. We aimed to analyze aspects associated with the inhibitory components in ASD, using bioinformatics to develop notions about embryonic life and tissue analysis for postnatal life. We analyzed microarray and RNAseq datasets of embryos from different ASD models, demonstrating that regions involved in neuronal development are affected. We evaluated the effect of prenatal treatment with resveratrol (RSV) on the neuronal organization and quantity of parvalbumin-positive (PV+), somatostatin-positive (SOM+), and calbindin-positive (CB+) GABAergic interneurons, besides the levels of synaptic proteins and GABA receptors in the medial prefrontal cortex (mPFC) and hippocampus (HC) of the ASD model induced by valproic acid (VPA). VPA increased the total number of neurons in the mPFC, while it reduced the number of SOM+ neurons, as well as the proportion of SOM+, PV+, and CB+ neurons (subregion-specific manner), with preventive effects of RSV. In summary, metabolic alterations or gene expression impairments could be induced by VPA, leading to extensive damage in the late developmental stages. By contrast, due to its antioxidant, neuroprotective, and opposite action on histone properties, RSV may avoid damages induced by VPA

Reduced CD4 T Lymphocytes in Lymph Nodes of the Mouse Model of Autism Induced by Valproic Acid

Considering the potential role of lymphocytes in the pathophysiology of autism spectrum disorder (ASD), we aimed to evaluate possible alterations of T cell pools in the lymphoid organs of an animal model of autism induced by valproic acid (VPA). Pregnant Swiss mice received a single intraperitoneal injection of 600 mg/kg of VPA (VPA group) or saline (control group) on day 11 of gestation. Male offspring were euthanized on postnatal day 60 for removal of thy-muses, spleens, and a pool of inguinal, axillary and brachial lymph nodes. Cellularity was evaluated, and flow cytometry analysis was performed on cell suspensions incubated with the mouse antibodies anti-CD3-FITC, anti-CD4-PE, and anti-CD8-PE-Cy7. We observed that the prenatal exposure to VPA induced a reduction in the numbers of CD3+CD4+ T cells in their lymph nodes when compared to the control animals. This was specific since it was not seen in the thymus or spleen. The consistent decrease in the number of CD4+ T cells in subcutaneous lymph nodes of mice from the animal model of autism may be related to the allergic symptoms frequently observed in ASD. Further research is necessary to characterize the immunological patterns in ASD and the connection with the pathophysiology of this disorder. [Abstract copyright: © 2018 S. Karger AG, Basel.

Behavioral alterations in autism model induced by valproic acid and translational analysis of circulating microRNA

Autism spectrum disorder (ASD) is characterized by difficulties in social interaction, communication and language, and restricted repertoire of activities and interests. The etiology of ASD remains unknown and no clinical markers for diagnosis were identified. Environmental factors, including prenatal exposure to valproic acid (VPA), may contribute to increased risk of developing ASD. MicroRNA (miRNA) are small noncoding RNA that regulate gene expression and are frequently linked to biological processes affected in neurodevelopmental disorders. In this work, we analyzed the effects of resveratrol (an antioxidant and anti-inflammatory molecule) on behavioral alterations of the VPA model of autism, as well as the levels of circulating miRNA. We also evaluated the same set of miRNA in autistic patients. Rats of the VPA model of autism showed reduced total reciprocal social interaction, prevented by prenatal treatment with resveratrol (RSV). The levels of miR134-5p and miR138-5p increased in autistic patients. Interestingly, miR134-5p is also upregulated in animals of the VPA model, which is prevented by RSV. In conclusion, our findings revealed important preventive actions of RSV in the VPA model, ranging from behavior to molecular alterations. Further evaluation of preventive mechanisms of RSV can shed light in important biomarkers and etiological triggers of ASD

Effects of single-dose antipurinergic therapy on behavioral and molecular alterations in the valproic acid-induced animal model of autism

Autism spectrum disorder (ASD) is characterized by deficits in communication and social interaction, restricted interests, and stereotyped behavior. Environmental factors, such as prenatal exposure to valproic acid (VPA), may contribute to the increased risk of ASD. Since disturbed functioning of the purinergic system has been associated with the onset of ASD and used as a potential therapeutic target for ASD in both clinical and preclinical studies, we analyzed the effects of suramin, a non-selective purinergic antagonist, on behavioral, molecular and immunological in an animal model of autism induced by prenatal exposure to VPA. Treatment with suramin (20 mg/kg, intraperitoneal) restored sociability in the three-chamber apparatus and decreased anxiety measured by elevated plus maze apparatus, but had no impact on decreased reciprocal social interactions or higher nociceptive threshold in VPA rats. Suramin treatment had no impact on VPA-induced upregulation of P2X4 and P2Y2 in hippocampus, and P2X4 in medial prefrontal cortex, but normalized an increased level of interleukin 6 (IL-6). Our results suggest an important role of purinergic modulation in behavioral, molecular, and immunological aberrations described in VPA model, and suggest that purinergic system might be a potential target for pharmacotherapy in preclinical studies of ASD