Acrylamide is a neurological and reproductive toxicant in humans and laboratory animals; however, the neuron developmental toxicity of acrylamide remains unclear. The aims of this study are to investigate the cytotoxicity and neurite outgrowth inhibition of acrylamide in nerve growth factor (NGF)- or fibroblast growth factor 1 (FGF1)-mediated neural development of PC12 cells. MTS assay showed that acrylamide treatment suppresses NGF- or FGF1-induced PC12 cell proliferation in a time- and dose-dependent manner. Quantification of neurite outgrowth demonstrated that 0.5 mM acrylamide treatment resulted in significant decrease in differentiation of NGF- or FGF1-stimulated PC12 cells. This decrease is accompanied with the reduced expression of growth-associated protein-43, a neuronal marker. Moreover, relative levels of pERK, pAKT, pSTAT3 and pCREB were increased within 5-10 min when PC12 cells were treated with NGF or FGF1. Acrylamide (0.5 mM) decreases the NGF-induced activation of AKT-CREB but not ERK-STAT3 within 20 min. Similarly, acrylamide (0.5 mM) decreases the FGF1-induced activation of AKT-CREB within 20 min. In contrast to the NGF treatment, the ERK-STAT3 activation that was induced by FGF1 was slightly reduced by 0.5 mM acrylamide. We further showed that PI3K inhibitor (LY294002), but not MEK inhibitor (U0126), could synergize with acrylamide (0.5 mM) to reduce the cell viability and neurite outgrowth in NGF- or FGF1-stimulated PC12 cells. Moreover, acrylamide (0.5 mM) increased reactive oxygen species (ROS) activities in NGF- or FGF1-stimulated PC12 cells. This increase was reversed by Trolox (an ROS scavenging agent) co-treatment. Together, our findings reveal that NGF- or FGF1-stimulation of the neuronal differentiation of PC12 cells is attenuated by acrylamide through the inhibition of PI3K-AKT-CREB signaling, along with the production of ROS.
Nerve injury of the central nervous system and the peripheral nervous system still poses a major challenge in modern clinics. Understanding the roles of neurotrophic factors and their molecular mechanisms on neuro-regeneration will not only benefit patients with neural damage but could potentially treat neurodegenerative disorders, such as amyotrophic lateral sclerosis. In this study, we showed that human IL12 p40-p40 homodimer (hIL12p80) within PLA and PLGA conduits improved sciatic nerve regeneration in mice. As such, the group of conduits with NSCs and hIL12p80 (CNI) showed the best recovery among the groups in the sciatic functional index (SFI), compound muscle action potential (CMAP), and Rotarod performance analyses. In addition, the CNI group had a faster recovery and outperformed the other groups in SFI and Rotarod performance tests beginning in the fourth week post-surgery. Immunohistochemistry showed that the CNI group increased the diameter of the newly regenerated nerve by two-fold (p < 0.01). In vitro studies showed that hIL12p80 stimulated differentiation of mouse NSCs to oligodendrocyte lineages through phosphorylation of Stat3 at Y705 and S727. Furthermore, implantation using PLGA conduits (C2.0 and C2.1) showed better recovery in the Rotarod test and CMAP than using PLA conduits in FVB mice. In B6 mice, the group with C2.1+NSCs+hIL12p80 (C2.1NI) not only promoted sciatic functional recovery but also reduced the rate of experimental autotomy. These results suggested that hIL12p80, combined with NSCs, enhanced the functional recovery and accelerated the regeneration of damaged nerves in the sciatic nerve injury mice. Our findings could further shed light on IL12′s application not only in damaged nerves but also in rectifying the oligodendrocytes’ defects in neurodegenerative diseases, such as amyotrophic lateral sclerosis and multiple sclerosis.
Previous studies showed that transplantation of cultured neural stem/progenitor cells (NSPCs) could improve functional recovery for various neurological diseases. This study aims to develop a stem cell-based model for predictive toxicology of development in the neurological system after acrylamide exposure. Treatment of mouse (KT98/F1B-GFP) and human (U-1240 MG/F1B-GFP) NSPCs with 0.5 mM acrylamide resulted in the inhibition of neurosphere formation (definition of self-renewal ability in NSPCs), but not inhibition of cell proliferation. Apoptosis and differentiation of KT98 (a precursor of KT98/F1B-GFP) and KT98/F1B-GFP are not observed in acrylamide-treated neurospheres. Analysis of secondary neurosphere formation and differentiation of neurons and glia illustrated that acrylamide-treated KT98 and KT98/F1B-GFP neurospheres retain the NSPC properties, such as self-renewal and differentiation capacity. Correlation of acrylamide-inhibited neurosphere formation with cell-cell adhesion was observed in mouse NSPCs by live cell image analysis and the presence of acrylamide. Protein expression levels of cell adhesion molecules [neural cell adhesion molecule (NCAM) and N-cadherin] and extracellular signal-regulated kinases (ERK) in acrylamide-treated KT98/F1B-GFP and U-1240 MG/F1B-GFP neurospheres demonstrated that NCAM decreased and phospho-ERK (pERK) increased, whereas expression of N-cadherin remained unchanged. Analysis of AKT (protein kinase B, PKB)/b-catenin pathway showed decrease in phospho-AKT (p-AKT) and cyclin D1 expression in acrylamide-treated neurospheres of KT98/F1B-GFP. Furthermore, PD98059, an ERK phosphorylation inhibitor, attenuated acrylamide-induced ERK phosphorylation, indicating that pERK contributed to the cell proliferation, but not in neurosphere formation in mouse NSPCs. Coimmunoprecipitation results of KT98/F1B-GFP cell lysates showed that the complex of NCAM and fibroblast growth factor receptor 1 (FGFR1) is present in the neurosphere, and the amount of this complex decreases after acrylamide treatment. Our results reveal that acrylamide inhibits neurosphere formation through the disruption of the neurosphere architecture in NSPCs. The downregulation of cell-cell adhesion resulted from decreasing the levels of NCAM as well as the formation of NCAM/ FGFR complex.
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