Navegando por Autor "Farias, Ana Raquel Melo de"
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Artigo Differential transcript usage unravels gene expression alterations in Alzheimer’s disease human brains(Springer Science and Business Media LLC, 2021-01-04) Coelho, Diego Marques; Iohan, Lukas da Cruz Carvalho; Farias, Ana Raquel Melo de; Flaig, Amandine; Lambert, Jean-Charles; Costa, Marcos RomualdoAlzheimer’s disease (AD) is the leading cause of dementia in aging individuals. Yet, the pathophysiological processes involved in AD onset and progression are still poorly understood. Among numerous strategies, a comprehensive overview of gene expression alterations in the diseased brain could contribute for a better understanding of the AD pathology. In this work, we probed the differential expression of genes in different brain regions of healthy and AD adult subjects using data from three large transcriptomic studies: Mayo Clinic, Mount Sinai Brain Bank (MSBB), and ROSMAP. Using a combination of differential expression of gene and isoform switch analyses, we provide a detailed landscape of gene expression alterations in the temporal and frontal lobes, harboring brain areas affected at early and late stages of the AD pathology, respectively. Next, we took advantage of an indirect approach to assign the complex gene expression changes revealed in bulk RNAseq to individual cell types/subtypes of the adult brain. This strategy allowed us to identify previously overlooked gene expression changes in the brain of AD patients. Among these alterations, we show isoform switches in the AD causal gene amyloid-beta precursor protein (APP) and the risk gene bridging integrator 1 (BIN1), which could have important functional consequences in neuronal cells. Altogether, our work proposes a novel integrative strategy to analyze RNAseq data in AD and other neurodegenerative diseases based on both gene/transcript expression and regional/cell-type specificitiesTese Probing the Alzheimer’s disease risk gene PTK2B using human-derived induced neurons(Universidade Federal do Rio Grande do Norte, 2023-10-20) Farias, Ana Raquel Melo de; Costa, Marcos Romualdo; http://lattes.cnpq.br/6118493598074445; http://lattes.cnpq.br/9402603894122604; Girault, Jean-Antoine; Crozet, Carole; Lambert, Jean-Charles; Hedin-Pereira, Cecília; Leão, Emelie Katarina SvahnAlzheimer's disease (AD) is the main type of dementia and poses a significant global public health challenge. It is characterized by a progressive decline in cognition, memory, and behavioral functions and affects more than 55 million people worldwide. At the molecular level, AD is defined by the presence of aggregated neurofibrillary tangles (NFTs) within neurons and the accumulation of amyloid-β (Aβ) plaques in the brain. These pathological features are associated with alterations in neuronal activity, synapse loss, gliosis, and neuroinflammation, leading to irreversible neurodegeneration. AD etiology and pathophysiology involves a complex interplay between genetic and environmental factors. Genome-Wide Association Studies (GWAS) have successfully identified more than 75 genetic loci carrying single nucleotide polymorphisms (SNPs) associated with AD risk. Among these loci, the one harboring the Protein Tyrosine Kinase 2β (PTK2B) is highlighted in the present work. This gene encodes a protein tyrosine kinase that is involved in calciuminduced regulation of ion channels and activation of numerous signaling pathways, such as MAP kinase. Non-synonymous genetic variations in the PTK2B locus have been associated with an increased risk of AD and are thought to regulate PTK2B expression. However, both the physiological and pathophysiological roles of PTK2B are not fully understood. In the human brain, PTK2B expression is mainly observed in glutamatergic neurons and this expression declines during AD progression. This reduced PTK2B expression in the brain of patients with AD may contribute to neuronal dysfunctions observed in the disease, such as increased electrical excitability and synaptic alterations. Therefore, understanding the role of PTK2B in human neurons may contribute to reveal the mechanisms of neuronal dysfunctions in AD. Considering that, the aims of this thesis are to uncover the cellular processes and molecular pathways regulated by PTK2B in human neurons. To that, we took advantage of isogenic human induced-pluripotent stem cells (hiPSCs) to generate neurons expressing different levels of PTK2B. Next, we employed functional and molecular assays to probe the consequences of altered PTK2B expression both in a physiological and in an AD-like context. We show that reduced PTK2B expression leads to increased TAU phosphorylation at various epitopes associated with AD pathology, suggesting a central role of PTK2B in regulating TAU aggregation. Using single-cell transcriptomics, we also show that reduced PTK2B expression leads to specific transcriptional alterations related to neuronal electrical activity and synaptic transmission mainly in glutamatergic neurons. Calcium imaging experiments indicate that PTK2B downregulation contributes to increased calcium spikes frequency without affecting synchronization, indicating an elevated neuronal electrical activity. Additionally, results from electrophysiological recordings from multi-electrode array (MEA) show increased electrical activity and disrupted bursting patterns in PTK2B mutant neurons. Overall, this work sheds light on the involvement of PTK2B in AD-related cellular processes, providing insights into the molecular mechanisms and functional alterations associated with PTK2B dysregulation in human iPSC-derived neural cells.TCC Reprogramação de astrócitos corticais em neurônios utilizando coquetel de pequenas moléculas(Universidade Federal do Rio Grande do Norte, 2016-11-02) Farias, Ana Raquel Melo de; Costa, Marcos Romualdo; Velho, Tarciso André Ferreira; Golbert, Daiane Cristina FerreiraRecentemente, diversas técnicas têm sido descritas visando à geração de neurônios que podem ser usados em terapias celulares objetivando o tratamento de doenças neurodegenerativas ou lesões agudas no sistema nervoso central. Uma dessas técnicas inclui a reprogramação de células já diferenciadas em neurônios, de forma direta ou indireta. Entretanto, a maioria dos protocolos de reprogramação celular depende da expressão de genes ectópicos, o que pode ocasionar outras alterações transitórias não esperadas nas células reprogramadas. A fim de contornar esses efeitos colaterais, investigou-se neste trabalho a possibilidade de reprogramação de astrócitos corticais de camundongos pós-natais em neurônios, através da exposição transitória das células a um coquetel de pequenas moléculas adicionado ao meio de cultivo celular. O referido coquetel inclui moléculas que atuam em diferentes vias celulares, dentre as quais a regulação da expressão gênica, modulação da neurogênese e controle do ciclo celular, tendo sido previamente utilizado na reprogramação de fibroblastos em neurônios. Para demonstrar o fenótipo neuronal após o tratamento dos astrócitos cultivados na presença das pequenas moléculas, foi avaliada a expressão de proteínas tipicamente neuronais, a morfologia celular e a expressão gênica, através dos métodos de imunocitoquímica e RT-qPCR. A partir dos dados preliminares observou-se que os astrócitos tratados adquirem morfologia e expressão gênica compatíveis com um fenótipo neuronal, indicando que o coquetel de pequenas moléculas pode ser utilizado na reprogramação de astrócitos em neurônios induzidos.Dissertação Reprogramação de células astrogliais em neurônios utilizando pequenas moléculas orgânicas(2018-11-28) Farias, Ana Raquel Melo de; Costa, Marcos Romualdo; ; ; Pereira, Cecilia Hedin; ; Leão, Emelie Katarina Svahn;A reprogramação de diferentes tipos celulares especializados em outros tem sido um campo amplamente estudado nos últimos anos. Mais especificamente, a geração de neurônios induzidos (iNs) a partir de outras células já diferenciadas é aplicada para estudar os mecanismos moleculares da diferenciação neuronal, gerar modelos humanizados de doenças neurológicas e psiquiátricas, assim como obter iNs que podem ser utilizados em terapias celulares para doenças neurodegenerativas ou lesões agudas no sistema nervoso central. O primeiro tipo de célula não-progenitora convertido em iNs através da expressão de um gene exógeno foi a astroglia. Em seguida, células nãoneurais, tais como fibroblastos e hepatócitos, também foram reprogramados em iNs através de manipulação gênica. Mais recentemente, o uso de pequenas moléculas orgânicas - capazes de interferir em cascatas de sinalização intracelular específicas, modulando processos biológicos - tem sido proposto como alternativa para a reprogramação de células diferenciadas em iNs sem manipulação gênica direta. No entanto, ainda não está claro qual seria a melhor combinação de pequenas moléculas para reprogramar células astrogliais isoladas do cérebro pós-natal. Neste trabalho, avaliamos a possibilidade de reprogramação de astrócitos em iNs, utilizando uma combinação de pequenas moléculas previamente utilizada para reprogramar fibroblastos embrionários. Para isto, astrócitos isolados do neocórtex e cerebelo de camundongos pós-natais foram cultivados e expostos às pequenas moléculas durante 8-20 dias. Findo este período, o fenótipo das células em cultura foi avaliado através das técnicas de imunocitoquímica, RT-qPCR e vídeo-microscopia de tempo intervalado. Foram avaliados aspectos como a expressão de RNAm e proteínas específicas de neurônios e astrócitos, morfologia, sobrevivência e proliferação celular. Nossos resultados indicam que apenas uma fração dos astrócitos em cultura adquirem características tipicamente neuronais quando expostos às pequenas moléculas. Além disso, algumas destas células mantém propriedades astrocitárias, indicando um estado de reprogramação incompleto. Análises de vídeo-microscopia de tempo intervalado também indicam que o tratamento com pequenas moléculas propicia um aumento da mortalidade celular, o que poderia contribuir para a baixa taxa de conversão em iNs observada. Alternativamente, a combinação de moléculas utilizadas pode não ser a mais adequada para reprogramar astrócitos em iNs, indicando a necessidade de diferentes combinações para este fim.Artigo The Alzheimer susceptibility gene BIN1 induces isoform-dependent neurotoxicity through early endosome defects(2022-01-08) Lambert, Erwan; Saha, Orthis; Landeira, Bruna Soares; Farias, Ana Raquel Melo de; Hermant, Xavier; Carrier, Arnaud; Pelletier, Alexandre; Gadaut, Johanna; Davoine, Lindsay; Dupont, Cloé; Amouyel, Philippe; Bonnefond, Amélie; Lafont, Frank; Abdelfettah, Farida; Verstreken, Patrik; Chapuis, Julien; Barois, Nicolas; Delahaye, Fabien; Dermaut, Bart; Lambert, Jean‑Charles; Costa, Marcos Romualdo; Dourlen, PierreThe Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer’s disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to earlyendosome size deregulation, which is an early pathophysiological hallmark of AD pathologyArtigo The Alzheimer’s disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons(Springer Science and Business Media LLC, 2024-03) Saha, Orthis; Farias, Ana Raquel Melo de; Pelletier, Alexandre; Siedlecki-Wullich, Dolores; Landeira, Bruna Soares; Gadaut, Johanna; Carrier, Arnaud; Vreulx, Anaïs-Camille; Guyot, Karine; Shen, Yun; Bonnefond, Amelie; Amouyel, Philippe; Tcw, Julia; Kilinc, Devrim; Queiroz, Claudio Marcos Teixeira de; Delahaye, Fabien; Lambert, Jean-Charles; Costa, Marcos RomualdoBridging Integrator 1 (BIN1) is the second most important Alzheimer’s disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. Using single-cell RNA-sequencing on cerebral organoids generated from isogenic BIN1 wild type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs), we show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons, like in the human brain. Both BIN1 HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of hiPSC-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological blocking of this channel with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs that affect biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. Together, these findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to slow disease-onset and progression