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Synergistic Effects of Combined Nurr1 Overexpression and Natural Inducers on the More Efficient Production of Dopaminergic Neuron-Like Cells From Stem Cells.

ABSTRACT: The development of dopaminergic (DA) neurons is a very complex process, and a combination of extrinsic and intrinsic factors involves their differentiation. Transcription factor, Nurr1 plays an essential role in the differentiation and maintenance of midbrain DA neurons. Nurr1-based therapies may restore DA function in Parkinson's disease (PD) by replacing damaged cells with differentiated cells derived from stem cells. Providing tissue-specific microenvironments such as brain extract can effectively induce dopaminergic gene expression in stem cells. The present study aimed to investigate the combined effects of Nurr1 gene overexpression and a neonatal rat brain extract (NRBE) induction on dopaminergic differentiation of P19 stem cells. In order to neural differentiation induction, stably Nurr1-transfected cells were treated with 100 μg/ml of NRBE. The differentiation potential of the cells was then evaluated during a period of 1-3 weeks via various methods. The initial evaluation of the cells by direct observation under a light microscope and cresyl violet specific staining, confirmed neuron-like morphology in the differentiated cells. In addition, different molecular and cellular techniques, including real-time PCR, immunofluorescence, and flow cytometry, demonstrated that the treated cells expressed pan-neuronal and dopaminergic markers. In all experimental groups, neuronal phenotype with dopaminergic neuron-like cells characteristics mainly appeared in the second week of the differentiation protocol. Overall, the results of the present study revealed for the first time the synergistic effects of Nurr1 gene overexpression and possible soluble factors that existed in NRBE on the differentiation of P19 stem cells into dopaminergic neuron-like cells.

SUBMITTER: Beiki R

PROVIDER: S-EPMC8787052 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Obsessive-compulsive disorder (OCD) is a disabling condition that often begins in childhood. Genetic studies in OCD have pointed to SLC1A1, which encodes the neuronal glutamate transporter EAAT3, with evidence suggesting that increased expression contributes to risk. In mice, midbrain Slc1a1 expression supports repetitive behavior in response to dopaminergic agonists, aligning with neuroimaging and pharmacologic challenge studies that have implicated the dopaminergic system in OCD. These findings suggest that Slc1a1 may contribute to compulsive behavior through altered dopaminergic transmission; however, this theory has not been mechanistically tested. To examine the developmental impact of Slc1a1 overexpression on compulsive-like behaviors, we, therefore, generated a novel mouse model to perform targeted, reversible overexpression of Slc1a1 in dopaminergic neurons. Mice with life-long overexpression of Slc1a1 showed a significant increase in amphetamine (AMPH)-induced stereotypy and hyperlocomotion. Single-unit recordings demonstrated that Slc1a1 overexpression was associated with increased firing of dopaminergic neurons. Furthermore, dLight1.1 fiber photometry showed that these behavioral abnormalities were associated with increased dorsal striatum dopamine release. In contrast, no impact of overexpression was observed on anxiety-like behaviors or SKF-38393-induced grooming. Importantly, overexpression solely in adulthood failed to recapitulate these behavioral phenotypes, suggesting that overexpression during development is necessary to generate AMPH-induced phenotypes. However, doxycycline-induced reversal of Slc1a1/EAAT3 overexpression in adulthood normalized both the increased dopaminergic firing and AMPH-induced responses. These data indicate that the pathologic effects of Slc1a1/EAAT3 overexpression on dopaminergic neurotransmission and AMPH-induced stereotyped behavior are developmentally mediated, and support normalization of EAAT3 activity as a potential treatment target for basal ganglia-mediated repetitive behaviors.

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Synergistic Effects of Combined Nurr1 Overexpression and Natural Inducers on the More Efficient Production of Dopaminergic Neuron-Like Cells From Stem Cells.

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