While the neurotrophic factor neuritin is known to be involved in neurodevelopment, the effects of this compound on cell differentiation remain unclear. The present study demonstrated that neuritin treatment induced the differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) into neuron-like (NL) cells. For these analyses, rBM-MSCs were incubated with 0.5 µg/ml neuritin for 24 h. Following induction, 27% of the rBM-MSCs exhibited typical NL cell morphologies. Subsequently, NL cells were characterized by examining the expression of neuronal markers and by analysis of cell functions. The findings demonstrated that the NL cells produced by neuritin treatment expressed the neuronal markers neuron-specific enolase and microtubule associate protein 2, and secreted the neurotransmitter 5-hydroxytryptamine. Furthermore, the NL cells exhibited certain partial neural-electrophysiological functions. In conclusion, neuritin treatment may be an effective method for inducing the differentiation of BM-MSCs towards NL cells. This may provide an alternative, potentially complementary tool for disease modeling and the development of cell-based therapies.
Objective: To obtain the long-acting protein neuritin we fused the carboxyl-terminal peptide (CTP) to the C-terminal of neuritin and expressed it in Chinese hamster ovarian (CHO) cells. Methods:The plasmid was constructed by fusion PCR. Affinity chromatography is used for protein purification. Thermal stability and serum stability were used to evaluate protein stability. Results: The molecular weight of the neuritin-CTP was determined to be approximately 20 kDa. Subsequent functional analysis showed that the purified neuritin-CTP promoted neurite outgrowth in PC12 cells at a rate equivalent to that observed with neuritin. The stability experiments showed that the degradation rate of neuritin was 100% after incubation at 37°C for 72 h, whereas only approximately 20% of the neuritin-CTP was degraded under the same conditions. Similarly, the serum stability analysis results showed that neuritin degraded by approximately 90% and neuritin-CTP degraded by approximately 30% after incubation at 37°C for 72 h. Conclusions: Fusion with CTP can effectively increase the stability of neuritin without affecting its secretion and activity. These results provide a basis for the construction of long-acting neuritin proteins.
Neuritin plays a key role in neural development and regeneration by promoting neurite outgrowth and synapse maturation. Our previous research revealed the mechanism by which neuritin inhibits Notch signaling through interaction with neuralized-like 1 (Neurl1) to promote neurite growth. However, how neuritin regulates Notch signaling through Neurl1 has not been elucidated. Here, we first confirm that neuritin is an upstream regulator of Neurl1 and inhibits Notch signaling through Neurl1. Neurl1 is an E3 ubiquitin ligase that can promote ubiquitination and endocytosis of the Notch1 ligand Jagged1. Therefore, we observe the effect of neuritin on the ligase activity of Neurl1. The results indicate that neuritin inhibits Neurl1 activity by reducing the ubiquitination level and endocytosis of the target protein Jagged1. Moreover, we find that decreased activity of Neurl1 results in reduced expression of Notch receptor Notch intracellular domain (NICD) and downstream target gene hairy and enhancer of split-1 ( HES1 ). Furthermore, we investigate how neuritin affects Neurl1 enzyme activity. The results show that neuritin not only weakens the affinity between Neurl1 and Jagged1 but also promotes the degradation of Neurl1 by the 26S proteasome pathway. Taken together, our results suggest that neuritin negatively regulates Notch signaling by inhibiting the activity of Neurl1, promoting the degradation of Neurl1 and weakening the affinity of Neurl1 for Jagged1. Our study clarifies the molecular mechanisms of neuritin in regulating the Notch signaling pathway and provides new clues about how neuritin mediates neural regeneration and plasticity.
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