RESUMO
MNX1 encodes a homeobox transcription factor with conserved embryonic requirements in spinal motor neuron formation and pancreatic beta-cell differentiation. Mutations in MNX1 are associated with dominantly inherited Currarino syndrome and neonatal diabetes. To better understand embryonic MNX1 functions we generated an hiPSC-1 knock-in line heterozygously expressing MNX1 C-terminally tagged with 2xTY1 together with a T2A-separated red fluorescent reporter mScarlet. The TY1 epitope tag was introduced to enable immunoprecipitation based analyses on molecular MNX1 interactions and mScarlet was included for enrichment of MNX1 expressing cell populations. This cell line shows normal karyotype, pluripotency marker expression and differentiation potential in vitro.
Assuntos
Células-Tronco Pluripotentes Induzidas , Siringomielia , Anormalidades do Sistema Digestório , Proteínas de Homeodomínio/genética , Humanos , Proteínas Luminescentes , Fatores de Transcrição/genética , Proteína Vermelha FluorescenteRESUMO
Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase - enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.
Assuntos
Sistemas CRISPR-Cas , Neurônios Dopaminérgicos/metabolismo , Edição de Genes , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/biossíntese , Células-Tronco Pluripotentes Induzidas/metabolismo , Reação em Cadeia da Polimerase , Transgenes , Linhagem Celular , Neurônios Dopaminérgicos/citologia , Proteínas de Fluorescência Verde/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Microscopia de FluorescênciaRESUMO
The Ca2+-dependent activator protein for secretion 2 (CADPS2) is a member of the CAPS/CADPS protein family that plays crucial roles in synaptic vesicle dynamics. Genomic variability in the CADPS2 gene has been associated to autism spectrum disorders and Alzheimer's disease, both characterized by altered neurotransmission. Biological evidence also linked CADPS2 to Parkinson's disease (PD), as a disease-causing mutation in leucine-rich repeat kinase 2 (LRRK2) was reported to increase CADPS2 gene and protein expression. Furthermore, restoration of CADPS2 physiologic levels was able to provide neuroprotection in patient-derived neurons, consistent with the synaptic dysfunction postulated to underlie PD. However, little is known about the influence of PD-related proteins on transcriptional regulation of critical synaptic genes such as CADPS2. Here we aimed at investigating the transcriptional effects of LRRK2 and alpha-synuclein (aSyn) on CADPS2 gene expression, using a combination of in silico analyses and cell biology techniques. First, we identified a predicted promoter in the human CADPS2 genomic sequence, which we then utilized in a luciferase-based gene reporter assay. This approach enabled us to disclose a differential effect of high levels of LRRK2 and aSyn on CADPS2 promoter activity. Specifically, CADPS2 transcriptional activity was enhanced by high cellular levels of LRRK2 and reduced by overexpression of aSyn. Consistently, CADPS2 mRNA levels were diminished in aSyn overexpressing cells. Our results indicate that LRRK2 and aSyn participate in the dysregulation of CADPS2 by altering transcription and support the hypothesis that synaptic dysfunctions, through different mechanisms, might contribute to the neuronal defects of diseases such as PD.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Regulação da Expressão Gênica , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Proteínas de Transporte Vesicular/genética , alfa-Sinucleína/genética , Sequência de Bases , Linhagem Celular , Expressão Gênica , Humanos , Neurônios/metabolismo , Doença de Parkinson/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Ativação Transcricional , Regulação para CimaRESUMO
Alpha-synuclein is central to Parkinson's disease pathogenesis and pathology, however its precise functions are still unclear. It has been shown to bind both PLCß1 and MAPKs, but how this property influences the downstream signaling of Gq protein-coupled receptors has not been elucidated. Here we show that recombinant expression of alpha-synuclein in human neuroblastoma cells enhances cellular levels of PLCß1 but blunts its signaling pathway, preventing the agonist-dependent rise of cytoplasmic Ca2+. In addition, overexpressing alpha-synuclein abolishes the activation of ERK1/2 upon agonist stimulation, indicating an upstream action in the signal transduction pathway. This data demonstrates that alpha-synuclein, when recombinantly expressed, interferes with the normal signaling of Gq-protein coupled receptors, which are then dysfunctional. Since many neurotransmitter systems utilize these receptor signaling pathways to mediate different abilities affected in Parkinson's disease, we argue this novel perspective might be helpful in designing treatment strategies for some of the non-motor symptoms in Parkinson's disease and synucleinopathies.
