Abstract

AIM:

To investigate the effect of N-acetylcysteine (NAC), a potent antioxidant, on neuron differentiation of cultured mouse embryonic stem cells (ESCs) induced by retinoic acid (RA) in vitro. Superior cervical ganglion (SCG) neurons were used to study the effect of NAC on neuritogenesis.

METHODS:

Immunoblotting was performed to detect the expression of microtubule-associated protein 2 (MAP2). MTT assays were used to determine cell viability. Cell death was estimated with trypan blue exclusion and Hoechst 33342 staining. Immunocytochemical analysis was carried out to identify neurons.

RESULTS:

We obtained a high percentage of MAP2-positive neurons derived from embryoid bodies (EBs) induced by RA by administering 1 mmol/L NAC at differentiation day 0. On differentiation day 8, the expression of MAP2 protein was strongly upregulated in the presence of NAC. NAC promoted neuron differentiation of ES cells in a dose- and time-dependent manner. Notably, NAC suppressed cell death caused by RA during neuron differentiation. In addition, neurite extension of SCG neurons was greatly stimulated in the presence of NAC.

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RA-induced neuron differentiation of ESCs was enhanced in the presence of NAC. (A) EBs were induced to differentiate into neurons by RA with (right) or without (left) 1 mmol/L NAC from differentiation day 0. Phase-contrast photomicrographs were taken on day 8. * indicate the location of EBs. Scale bars, 85 μm. (B) Cells were then lysed and subjected to immunoblotting with anti-MAP2 antibody. The upregulation of MAP2 expression was detected in the presence of NAC. Actin immunoreactive bands were used for normalization. (C) EBs with the similar diameter were picked up, plated onto 96-well plates, and differentiated by RA with or without NAC. On differentiation day 0, 4, and 8, cell proliferation was determined by MTT assay.
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NAC promotes ESCs neuron differentiation in a dose- and time-dependent manner. (A) Immunocytochemical analysis of MAP2-positive neurons in differentiated ESCs. PI nuclear staining (red) was used as the marker of total number of cells. Note that the average nuclear area was similar, while MAP2-positive (green) neurons with dendritic processes were significantly increased in the presence of NAC (1 mmol/L), compared with untreated control. Scale bars, 50 μm. (B) NAC promoted neuron differentiation in a dose-dependent manner. Different concentrations of NAC were added into culture medium on differentiation day 0. Eight days later, cells were subjected to immunocytochemical analysis with anti-MAP2 antibody. The number of MAP2-positive cells was counted per area and was presented as a percentage of the total number of cells. At least 15 photomicrographs were evaluated randomly per treatment and each treatment was performed in triplicate. cP<0.01 compared with cultures without NAC treatment. (C) NAC enhanced neuron differentiation in a time-dependent manner. Cultures of different differentiation stage (differentiation day 0, 4, and 8) were exposed to 1 mmol/L NAC for two days. On day 12, neuron differentiation was estimated as described in (B). Cultures without NAC treatment were used as control (Ctrl). cP<0.01 compared with control.
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RA-induced neuron differentiation of ESCs was enhanced in the presence of NAC. (A) EBs were induced to differentiate into neurons by RA with (right) or without (left) 1 mmol/L NAC from differentiation day 0. Phase-contrast photomicrographs were taken on day 8. * indicate the location of EBs. Scale bars, 85 μm. (B) Cells were then lysed and subjected to immunoblotting with anti-MAP2 antibody. The upregulation of MAP2 expression was detected in the presence of NAC. Actin immunoreactive bands were used for normalization. (C) EBs with the similar diameter were picked up, plated onto 96-well plates, and differentiated by RA with or without NAC. On differentiation day 0, 4, and 8, cell proliferation was determined by MTT assay.
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NAC promoted neurite extension in SCG neurons. NAC 1 mmol/L was added to explant SCG culture on the day when ganglia were planted on tissue dishes. (A) Phase-contrast photomicrographs of cultures with or without NAC treatment were taken on DIV 4 after dissection. Scale bar, 300 μm. (B) Average neurite length in control or NAC-treated cultures from day 0 to day 8. cP<0.01 compared with control.

CONCLUSION:

These results show that NAC enhanced both neuron differentiation and neuritogenesis, suggesting that it may be used in the development of novel therapeutic approaches targeting neuron loss and neurite dystrophy in neurodegenerative diseases.Acta Pharmacologica Sinica (2009) 30: 907-912; doi: 10.1038/aps.2009.72.