isolation-propagation-prion-protein-expression-during-neuronal

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very ...

Sapienza University of Rome

Since stressing conditions induce a relocalization of endogenous human neuroglobin (NGB) to mitochondria, this research is aimed to evaluate the protective role of NGB overexpression against neurotoxic stimuli, through mitochondrial lipid raft-associated complexes. To this purpose, we built a neuronal model of oxidative stress by the use of human dopaminergic neuroblastoma cells, SK-N-BE2, stably overexpressing NGB by transfection and treated with 1-methyl-4-phenylpyridinium ion (MPP+). We preliminary observed the redistribution of NGB to mitochondria following MPP+ treatment. The analysis of mitochondrial raft-like microdomains revealed that, following MPP+ treatment, NGB translocated to raft fractions (Triton X-100-insoluble), where it interacts with ganglioside GD3. Interestingly, the administration of agents capable of perturbating microdomain before MPP+ treatment, significantly affected viability in SK-N-BE2-NGB cells. The overexpression of NGB was able to abrogate the mitochondrial injuries on complex IV activity or mitochondrial morphology induced by MPP+ administration. The protective action of NGB on mitochondria only takes place if the mitochondrial lipid(s) rafts-like microdomains are intact, indeed NGB fails to protect complex IV activity when purified mitochondria were treated with the lipid rafts disruptor methyl-β-cyclodextrin. Thus, our unique in vitro model of stably transfected cells overexpressing endogenous NGB allowed us to suggest that the role in neuroprotection played by NGB is reliable only through interaction with mitochondrial lipid raft-associated complexes.

Neuroglobin overexpression plays a pivotal role in neuroprotection through mitochondrial raft-like microdomains in neuroblastoma SK-N-BE2 cells.

Several studies demonstrated that cannabinoids reduce tumor growth, inhibit angiogenesis, and decrease cancer cell migration. As these molecules are well tolerated, it would be interesting to investigate the potential benefit of newly synthesized compounds, binding cannabinoid receptors (CBRs). In this study, we describe the synthesis and biological effect of 2-oxo-1,8-naphthyridine-3-carboxamide derivative LV50, a new compound with high CB2 receptor (CB2R) affinity. We demonstrated that it decreases viability of Jurkat leukemia cells, evaluated by Trypan Blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), but mainly induces a proapoptotic effect. We observed an increase of a hypodiploid peak by propidium iodide staining and changes in nuclear morphology by Hoechst 33258. These data were confirmed by a significant increase of Annexin V staining, cleavage of the nuclear enzyme poly(ADP-ribose)-polymerase (PARP), and caspases activation. In addition, in order to exclude that LV50 non-specifically triggers death of all normal leukocytes, we tested the new compound on normal peripheral blood lymphocytes, excluding the idea of general cytotoxicity. To characterize the involvement of CB2R in the anti-proliferative and proapoptotic effect of LV50, cells were pretreated with a specific CB2R antagonist and the obtained data showed reverse results. Thus, we suggest a link between inhibition of cell survival and proapoptotic activity of the new compound that elicits this effect as selective CB2R agonist.

Anti-Proliferative Properties and Proapoptotic Function of New CB2 Selective Cannabinoid Receptor Agonist in Jurkat Leukemia Cells.

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) in the neuronal differentiation process, and in the signal pathway activation of ERK 1/2 and Akt. We demonstrated that recPrP 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) was necessary to mediate the signaling induced by recPrP, we silenced PrP, demonstrating that the presence of endogenous PrP was essential for ERK 1/2 and Akt phosphorylation. Since endogenous PrP is a well-known lipid rafts component, we evaluated the role of these structures in the signal pathway induced by recPrP. Our results suggest that lipid rafts integrity play a key role in recPrP activity. In fact, lipid rafts inhibitors, such as fumonisin B1 and MβCD, significantly prevented ERK 1/2 and Akt phosphorylation induced by recPrP. In addition, we investigated the capacity of recPrP 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, we observed the inhibition of neuronal differentiation induced by recPrP. The combined data suggest that recPrP plays a key role in the neuronal differentiation process and in the activation of specific intracellular signal pathways in hDPSCs.

Article	Year
Neuroglobin overexpression plays a pivotal role in neuroprotection through mitochondrial raft-like microdomains in neuroblastoma SK-N-BE2 cells. Molecular and cellular neurosciences\| PubMed ID: 29378245	2018
Anti-Proliferative Properties and Proapoptotic Function of New CB2 Selective Cannabinoid Receptor Agonist in Jurkat Leukemia Cells. International journal of molecular sciences\| PubMed ID: 29973514	2018
Cellular and Molecular Mechanisms Mediated by recPrP Involved in the Neuronal Differentiation Process of Mesenchymal Stem Cells. International journal of molecular sciences\| PubMed ID: 30654447	2019

Roberta Misasi

Biography

JoVE Journal Publications

Other Publications