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1.
Int J Mol Sci ; 24(19)2023 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-37834379

RESUMO

Fragile X syndrome (FXS) is caused by a repression of the FMR1 gene that codes the Fragile X mental retardation protein (FMRP), an RNA binding protein involved in processes that are crucial for proper brain development. To better understand the consequences of the absence of FMRP, we analyzed gene expression profiles and activities of cortical neural progenitor cells (NPCs) and neurons obtained from FXS patients' induced pluripotent stem cells (IPSCs) and IPSC-derived cells from FMR1 knock-out engineered using CRISPR-CAS9 technology. Multielectrode array recordings revealed in FMR1 KO and FXS patient cells, decreased mean firing rates; activities blocked by tetrodotoxin application. Increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed using RNA sequencing on FMR1 KO neurons and validated using quantitative PCR in both models. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and autism-related genes found amongst downregulated genes. Our findings suggest that the absence of FMRP affects transcriptional profiles since the NPC stage, and leads to impaired activity and neuronal differentiation over time, which illustrates the critical role of FMRP protein in neuronal development.


Assuntos
Síndrome do Cromossomo X Frágil , Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Camundongos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Neurogênese/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , RNA Mensageiro/genética , Camundongos Knockout
2.
Cells ; 12(4)2023 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-36831212

RESUMO

A multitude of in vitro models based on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) have been developed to investigate the underlying causes of selective MN degeneration in motor neuron diseases (MNDs). For instance, spheroids are simple 3D models that have the potential to be generated in large numbers that can be used across different assays. In this study, we generated MN spheroids and developed a workflow to analyze them. To start, the morphological profiling of the spheroids was achieved by developing a pipeline to obtain measurements of their size and shape. Next, we confirmed the expression of different MN markers at the transcript and protein levels by qPCR and immunocytochemistry of tissue-cleared samples, respectively. Finally, we assessed the capacity of the MN spheroids to display functional activity in the form of action potentials and bursts using a microelectrode array approach. Although most of the cells displayed an MN identity, we also characterized the presence of other cell types, namely interneurons and oligodendrocytes, which share the same neural progenitor pool with MNs. In summary, we successfully developed an MN 3D model, and we optimized a workflow that can be applied to perform its morphological, gene expression, protein, and functional profiling over time.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular/genética , Fluxo de Trabalho , Neurônios Motores/metabolismo
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