Neurotoxicity In Neuroblastoma SH-SY5Y Cells Research Articles

KMUP-1 attenuates serum deprivation-induced neurotoxicity in SH-SY5Y cells: Roles of PKG, PI3K/Akt and Bcl-2/Bax pathways

Aging populations with neurodegenerative disorders will gradually become a greater problem for society. Serum deprivation-induced cell death is recognized as one of the standard models for the study of neurotoxicity. Increasing evidence indicates that cGMP/PKG pathway may play a rescue role in serum deprivation-induced toxicity. The aim of this study was to investigate protective effects of KMUP-1, an enhancer of cGMP/PKG signaling on serum deprivation-induced neurotoxicity in SH-SY5Y neuroblastoma cells. Under normal serum condition, KMUP-1 enhanced protein expression of nNOS, PKG and sGCα1, increased intracellular cyclic GMP level, and attenuated PDE5 expression. KMUP-1 also increased expression of BDNF and Bcl-2, but it did not affect Bax expression. The phosphorylation of Akt and CREB induced by KMUP-1 was inhibited by tyrosine kinase (TrK) inhibitor K252a and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, respectively. Under serum deprivation condition, flow cytometric analysis using Annexin V showed KMUP-1 increased cell viability, but lacked protective effects in the presence of nitric oxide synthase inhibitor l-NAME, PKG inhibitor Rp-8-pCPT-cGMPS or LY294002. KMUP-1 not only enhanced expression of nNOS, sGCα1, PKG, p-CREB, p-Akt and Bcl-2, but also attenuated Bax expression in serum deprivation-treated cultures. In conclusion, cGMP/PKG, PI3K/Akt/CREB and Bcl-2/Bax signals play critical roles in the neuroprotective effects of KMUP-1 on serum deprivation-induced toxicity.

Indirubin-3′-monoxime inhibits β-amyloid-induced neurotoxicity in neuroblastoma SH-SY5Y cells

Increasing evidence suggests that the inappropriate activation of cyclin-dependent kinases (CDKs) could induce neuronal apoptosis in Alzheimer's disease (AD), which means that the pharmacological inhibitors of cell-cycle progression may effectively impede the development or progression of AD. Indirubin-3′-monoxime (IMX), a known effective inhibitor of CDKs, has been shown to have therapeutic effects on learning and memory deficits induced by beta-amyloid (Aβ) intracerebroventricular infusion in rats. In the present study, we investigated the neuroprotective effects of IMX on Aβ25–35-induced neuronal apoptosis and its potential mechanisms in human neuroblastoma SH-SY5Y cells. Aβ25–35-induced apoptosis, characterized by decreased cell viability, neuronal DNA condensation, and fragmentation, was associated with an increase in tau protein hyperphosphorylation. IMX, however, attenuated Aβ25–35-induced cell death in a dose-dependent manner. Furthermore, expression of hyperphosphorylation tau protein was significantly decreased with IMX treatment. Our study suggests that IMX may usefully prevent or delay the neuronal loss of AD.

A rapid method to measure beta-amyloid induced neurotoxicity in vitro

β-Amyloid (Aβ) is the primary protein component of senile plaques in Alzheimer's disease (AD) and is believed to be associated with neurotoxicity in the disease. Aβ-induced neurotoxicity is strongly dependent on its structure. The aggregation of the peptide from monomeric form to fibrils is a function of many variables including time. It is because of this dynamic nature of Aβ structure that there is a necessity for an in vitro toxicity assay that is rapid enough to eliminate or at least lower the possibility of Aβ structural changes during the time required for the assay. Here we describe a fast and sensitive method with which to assess Aβ-induced neurotoxicity in SH-SY5Y neuroblastoma cells. The method employs two-color flow cytometry using annexin-phycoerythrin, a marker for cell surface phosphatidylserine that typically indicates early stages of apoptosis, and 7-amino-actinomycin D, a membrane impermeant nucleic acid dye. We compare results using the two-color assay to those obtained using the propidium iodide toxicity assay and demonstrate comparability of results, but in 2 h or less with the two-color assay as opposed to 24–48 h with the propidium iodide assay. The assay described could be a useful tool in evaluating the role of Aβ structure in biological activity of the peptide.