Natriuretic peptides are neuroprotective on in vitro models of PD and promote dopaminergic differentiation of hiPSCs-derived neurons via the Wnt/β-catenin signaling

Over the last 20 years, the efforts to develop new therapies for Parkinson’s disease (PD) have focused not only on the improvement of symptomatic therapy for motor and non-motor symptoms but also on the discovering of the potential causes of PD, in order to develop disease-modifying treatments. The emerging role of dysregulation of the Wnt/β-catenin signaling in the onset and progression of PD, as well as of other neurodegenerative diseases (NDs), renders the targeting of this signaling an attractive therapeutic opportunity for curing this brain disorder. The natriuretic peptides (NPs) atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), are cardiac and vascular-derived hormones also widely expressed in mammalian CNS, where they seem to participate in numerous brain functions including neural development/differentiation and neuroprotection. We recently demonstrated that ANP affects the Wnt/β-catenin pathway possibly through a Frizzled receptor-mediated mechanism and that it acts as a neuroprotective agent in in vitro models of PD by upregulating this signaling. Here we provide further evidence supporting the therapeutic potential of this class of natriuretic hormones. Specifically, we demonstrate that all the three natriuretic peptides are neuroprotective for SHSY5Y cells and primary cultures of DA neurons from mouse brain, subjected to neurotoxin insult with 6-hydroxydopamine (6-OHDA) for mimicking the neurodegeneration of PD, and these effects are associated with the activation of the Wnt/β-catenin pathway. Moreover, ANP, BNP, CNP are able to improve and accelerate the dopaminergic differentiation and maturation of hiPSCs-derived neural population obtained from two differed healthy donors, concomitantly affecting the canonical Wnt signaling. Our results support the relevance of exogenous ANP, BNP, and CNP as attractive molecules for both neuroprotection and neurorepair in PD, and more in general, in NDs for which aberrant Wnt signaling seems to be the leading pathogenetic mechanism.

Dynamic landscape of chromatin accessibility and transcriptomic changes during differentiation of human embryonic stem cells into dopaminergic neurons

Chromatin architecture influences transcription by modulating the physical access of regulatory factors to DNA, playing fundamental roles in cell identity. Studies on dopaminergic differentiation have identified coding genes, but the relationship with non-coding genes or chromatin accessibility remains elusive. Using RNA-Seq and ATAC-Seq we profiled differentially expressed transcripts and open chromatin regions during early dopaminergic neuron differentiation. Hierarchical clustering of differentially expressed genes, resulted in 6 groups with unique characteristics. Surprisingly, the abundance of long non-coding RNAs (lncRNAs) was high in the most downregulated transcripts, and depicted positive correlations with target mRNAs. We observed that open chromatin regions decrease upon differentiation. Enrichment analyses of accessibility depict an association between open chromatin regions and specific functional pathways and gene-sets. A bioinformatic search for motifs allowed us to identify transcription factors and structural nuclear proteins that potentially regulate dopaminergic differentiation. Interestingly, we also found changes in protein and mRNA abundance of the CCCTC-binding factor, CTCF, which participates in genome organization and gene expression. Furthermore, assays demonstrated co-localization of CTCF with Polycomb-repressed chromatin marked by H3K27me3 in pluripotent cells, progressively decreasing in neural precursor cells and differentiated neurons. Our work provides a unique resource of transcription factors and regulatory elements, potentially involved in the acquisition of human dopaminergic neuron cell identity.

The natural functional biopolymer matrix demonstrates to be a promise for safe cell therapy in Parkinson’s disease

Background Mesenchymal stem cells (MSCs) are undifferentiated cells and can be isolated from many tissues, including adipose tissue. MSCs neuronal differentiation ability has arisen interest in research with these cells in neurodegenerative diseases, such as Parkinson’s disease (PD). To differentiate MSCs in cells that produce dopamine that posteriorly potential to be a safe cell therapy for PD. Methods MSCs were isolated from adipose tissue, characterized by flow cytometry and trilineage differentiation, and cultivated seeded on natural functional biopolymer matrix (NFBX) to differentiate in neuronal precursors. Finally, a neural precursor was cultivated in the dopaminergic differentiation medium. The immunocytochemistry was performed with antibody anti-Nestin for precursor neural and antibodies anti-ß III-tubulin and hydroxylase tyrosine. Then, quantification of dopamine was made by the ELISA kit in the culture medium. Results The cytometric analysis of MSCs and the trilineage test to chondrocyte, osteocyte, and adipocyte demonstrated their pluripotency. Cells seeded and cultivated over NFBX have developed neurospheres, and their mechanical dissociated cells were Nestin positive. Dopaminergic differentiation was confirmed with positivity for ß-III tubulin and hydroxylase tyrosine. The dopamine concentration was very high in one sample (74.91 ng/mL). Without this sample, the media was 2.34 ± 2.13 ng/mL. The difference between dopamine concentrations was probably due to donors' metabolic characteristics. Conclusions The MSCs differentiated in neural precursor cells without genetic modification or growth factors, using only this NFBX. When these neural precursors were induced to differentiate, they were available to produce dopamine, demonstrating a functional neuronal differentiation.

Reprogramming By Cytosolic Extract of Human Embryonic Stem Cells Improves Dopaminergic Differentiation Potential of Human Adipose Tissue-Derived Stem Cells

The extract of pluripotent stem cells induces dedifferentiation of somatic cells with restricted plasticity. In this study, we used the extract of human embryonic stem cells (hESC) to dedifferentiate adipose tissue-derived stem cells (ADSCs) and examined the impact of this reprogramming event on dopaminergic differentiation of the cells. For this purpose, cytoplasmic extract of ESCs was prepared by repeated cycles of freezing and thawing. The plasma membrane of hADSCs was reversibly permeabilized by Streptolysin O (SLO), exposed to hESC extract and resealed by CaCl2-containing medium. As revealed by qPCR analysis, expression of OCT4, SOX2, NANOG, LIN28A and KLF4 mRNAs were downregulated in the ADSCs one week after extract incubation, while all except for KLF4 were upregulated at the end of second week. For dopaminergic differentiation, control and reprogrammed ADSCs were induced by serum-free neurobasal medium containing B27 and a cocktail of SHH, FGF8, bFGF and BDNF for 12 days. After differentiation, expression levels of some neuronal and dopaminergic-related genes, including PAX6, NESTIN, NEFL, GLI1, LMXB1, EN1, NURR1 and TH, were significantly increase in the reprogrammed ADSCs compared to the control group. As a whole, two weeks after reprogramming by ESC extract, ADSCs showed an improved dopaminergic differentiation potential. These findings suggest that the cytoplasmic extract of hESCs is containing some regulatory factors which induce the expression of pluripotency-associated markers in somatic cells and that the exposure to ESC extract may serve as a simple and rapid strategy to enhance plasticity of somaticstem cells for cell replacement therapy purposes.