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Cellular and Molecular Mechanisms Mediated by recPrP^C Involved in the Neuronal Differentiation Process of Mesenchymal Stem Cells.

ABSTRACT: Human Dental Pulp Stem Cells (hDPSCs) represent a type of adult mesenchymal stem cells that have the ability to differentiate in vitro in several lineages such as odontoblasts, osteoblasts, chondrocytes, adipocytes and neurons. In the current work, we used hDPSCs as the experimental model to study the role of recombinant prion protein 23?231 (recPrP^C) in the neuronal differentiation process, and in the signal pathway activation of ERK 1/2 and Akt. We demonstrated that recPrP^C was able to activate an intracellular signal pathway mediated by extracellular-signal-regulated kinase 1 and 2 (ERK 1/2) and protein kinase B (Akt). Moreover, in order to understand whether endogenous prion protein (PrP^C) was necessary to mediate the signaling induced by recPrP^C, we silenced PrP^C, demonstrating that the presence of endogenous PrP^C was essential for ERK 1/2 and Akt phosphorylation. Since endogenous PrP^C is a well-known lipid rafts component, we evaluated the role of these structures in the signal pathway induced by recPrP^C. Our results suggest that lipid rafts integrity play a key role in recPrP^C activity. In fact, lipid rafts inhibitors, such as fumonisin B1 and M?CD, significantly prevented ERK 1/2 and Akt phosphorylation induced by recPrP^C. In addition, we investigated the capacity of recPrP^C to induce hDPSCs neuronal differentiation process after long-term stimulation through the evaluation of typical neuronal markers expression such as B3-Tubulin, neurofilament-H (NFH) and growth associated protein 43 (GAP43). Accordingly, when we silenced endogenous PrP^C, we observed the inhibition of neuronal differentiation induced by recPrP^C. The combined data suggest that recPrP^C plays a key role in the neuronal differentiation process and in the activation of specific intracellular signal pathways in hDPSCs.

SUBMITTER: Martellucci S

PROVIDER: S-EPMC6358746 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Cellular and Molecular Mechanisms Mediated by recPrPC Involved in the Neuronal Differentiation Process of Mesenchymal Stem Cells.

Martellucci Stefano S Santacroce Costantino C Santilli Francesca F Piccoli Luca L Delle Monache Simona S Angelucci Adriano A Misasi Roberta R Sorice Maurizio M Mattei Vincenzo V

International journal of molecular sciences 20190116 2

Human Dental Pulp Stem Cells (hDPSCs) represent a type of adult mesenchymal stem cells that have the ability to differentiate in vitro in several lineages such as odontoblasts, osteoblasts, chondrocytes, adipocytes and neurons. In the current work, we used hDPSCs as the experimental model to study the role of recombinant prion protein 23⁻231 (recPrPC) in the neuronal differentiation process, and in the signal pathway activation of ERK 1/2 and Akt. We demonstrated that recPrPC</su ...[more]

PMID: 30654447

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Project description:UnlabelledLow-to-moderate levels of reactive oxygen species (ROS) govern different steps of neurogenesis via molecular pathways that have been decrypted only partially. Although it has been postulated that redox-sensitive molecules are involved in neuronal differentiation, the molecular bases for this process have not been elucidated yet. The aim of this work was therefore to study the role played by the redox-sensitive, multifunctional protein APE1/Ref-1 (APE1) in the differentiation process of human adipose tissue-derived multipotent adult stem cells (hAT-MASC) and embryonic carcinoma stem cells (EC) towards a neuronal phenotype.Methods and resultsApplying a definite protocol, hAT-MASC can adopt a neural fate. During this maturation process, differentiating cells significantly increase their intracellular Reactive Oxygen Species (ROS) levels and increase the APE1 nuclear fraction bound to chromatin. This latter event is paralleled by the increase of nuclear NF-?B, a transcription factor regulated by APE1 in a redox-dependent fashion. Importantly, the addition of the antioxidant N-acetyl cysteine (NAC) to the differentiation medium partially prevents the nuclear accumulation of APE1, increasing the neuronal differentiation of hAT-MASC. To investigate the involvement of APE1 in the differentiation process, we employed E3330, a specific inhibitor of the APE1 redox function. The addition of E3330, either to the neurogenic embryonic carcinoma cell line NT2-D1or to hAT-MASC, increases the differentiation of stem cells towards a neural phenotype, biasing the differentiation towards specific subtypes, such as dopaminergic cells. In conclusion, during the differentiation process of stem cells towards a neuroectodermic phenotype, APE1 is recruited, in a ROS-dependent manner, to the chromatin. This event is associated with an inhibitory effect of APE1 on neurogenesis that may be reversed by E3330. Therefore, E3330 may be employed both to boost neural differentiation and to bias the differentiation potential of stem cells towards specific neuronal subtypes. These findings provide a molecular basis for the redox-mediated hypothesis of neuronal differentiation program.

Project description:ObjectiveTo investigate the modulation of microRNAs (miRNAs) upon the neuronal differentiation of mesenchymal stem cells (MSCs) through targeting RE-1 Silencing Factor (REST), a mature neuronal gene suppressor in neuronal and un-neuronal cells.MethodsRat bone marrow derived-MSCs were induced into neuron-like cells (MSC-NCs) by DMSO and BHA in vitro. The expression of neuron specific enolase (NSE), microtubule-associated protein tau (Tau), REST and its target genes, including synaptosomal-associated protein 25 (SNAP25) and L1 cell adhesion molecular (L1CAM), were detected in MSCs and MSC-NCs. miRNA array analysis was conducted to screen for the upregulated miRNAs after neuronal differentiation. TargetScan was used to predict the relationship between these miRNAs and REST gene, and dual luciferase reporter assay was applied to validate it. Gain and loss of function experiments were used to study the role of miR-29a upon neuronal differentiation of MSCs. The knockdown of REST was conducted to show that miR-29a affected this process through targeting REST.ResultsMSCs were induced into neuron-like cells which presented neuronal cell shape and expressed NSE and Tau. The expression of REST declined and the expression of SNAP25 and L1CAM increased upon the neuronal differentiation of MSCs. Among 14 upregulated miRNAs, miR-29a was validated to target REST gene. During the neuronal differentiation of MSCs, miR-29a inhibition blocked the downregulation of REST, as well as the upregulation of SNAP25, L1CAM, NSE and Tau. REST knockdown rescued the effect of miR-29a inhibition on the expression of NSE and Tau. Meanwhile, miR-29a knockin significantly decreased the expression of REST and increased the expression of SNAP25 and L1CMA in MSCs, but did not significantly affect the expression of NSE and Tau.ConclusionmiR-29a regulates neurogenic markers through targeting REST in mesenchymal stem cells, which provides advances in neuronal differentiation research and stem cell therapy for neurodegenerative diseases.

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Cellular and Molecular Mechanisms Mediated by recPrP^C Involved in the Neuronal Differentiation Process of Mesenchymal Stem Cells.

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Cellular and Molecular Mechanisms Mediated by recPrP<sup>C</sup> Involved in the Neuronal Differentiation Process of Mesenchymal Stem Cells.

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