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In vitro analysis of the biochemical pathways activated by cholesteryl glucoside in a motor neuron hybrid model of amyotrophic lateral sclerosis-parkinsonism dementia complex Ly, Philip Tuan Thanh

Abstract

Steryl glycosides are a family of compounds commonly found in the environment with unclear biological roles. Cycad seeds, a dietary link to the etiology of the Guamanian amyotrophic lateral sclerosis-Parkinsonism dementia complex (ALS-PDC) have an abundant amount of steryl glycosides. Previously, our laboratory demonstrated that several members of the cycad-derived steryl glycosides have toxic properties. Cholesteryl glucosides (CG), a variant form of the cycad-derived steryl glycosides have been found to induce cell death in primary rat cortical neurons. However, other groups have demonstrated that CG can protect fibroblast cells from heat shock. In the present study, we showed that the motor neuronderived cell line, NSC34 cells, exposed to CG resulted in a concentration- and timedependent reduction in cell viability. However, a brief exposure of CG for one hour to NSC34 cells induced cytoprotection against serum deprivation stress. The Kinetworks™ KPSS-1.3 phosphosite screen was used to examine phosphorylation changes during CG preconditioning and indicated elevated AktSer⁻⁴⁷³ phosphorylation. Suppression of CG- stimulated Akt phosphorylation with pharmacological inhibitors abolished CG preconditioning. Furthermore, the results indicated AktSer⁻⁴⁷³ phosphorylation via a PI3K-dependent and independent way. Erk1, but not Erk2 phosphorylation at its activation site was inhibited with CG treatment. The role of Erk1 inhibition in CG toxicity remains unclear. JNK and p38 MAPK were activation were temporally delayed, but were found to not involved in mediating cell death. A sub-population of NSC34 cells treated with CG for at least 4 days displayed a differentiated phenotype with enlarged soma and extended neurites. Moreover, focal neurite swellings with accumulated cytoskeletal proteins and disease-associated phospho-Tau were observed. CG-treatment did not induce abnormal cytoplasmic accumulation of TDP43, as seen in sporadic ALS and Guamanian ALS and PDC cases. Trypan blue staining indicated that the treated-cells with abnormal morphology were viable. Taken together, these results demonstrated that CG is toxic to NSC34 cells. As a cellular response to stress, these cells upregulated survival signals and/or undergo differentiation to resist CG toxicity. A better understanding of the biochemical pathways triggered by CG in NSC34 cells may provide insights to a common aberrant mechanism underlying the cause of neuronal death in ALS-PDC.

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