Bleomycin hydrolase deficiency and Homocysteine metabolism promote ER Stress, UPR dysregulation and apoptosis in mouse neuroblastoma N2a-APPSwe cells

Background: Alzheimer's disease (AD) is a neurodegenerative disorder characterised by endoplasmic reticulum (ER) stress, unfolded protein response (UPR) and apoptosis. Elevated plasma total homocysteine (tHcy), known as Hyperhomocysteinemia (HHcy), has been linked to an increased risk of AD. HHcy and related metabolites such as Hcy thiolactone (HTL), N-homocysteinylated (N-Hcy)-protein and bleomycin hydrolase (BLMH), an enzyme that detoxifies HTL, are also associated with AD pathology. However, it is not fully understood how Blmh gene deficiency, Hcy and its metabolites promote ER stress, UPR dysregulation and apoptosis in the development of AD.

Aim: The aim of this research was to study the effects of Hcy, HTL, and N-Hcy-protein treatments and Blmh gene silencing on the expression of genes involved in ER stress, UPR and the apoptosis pathway in mouse N2a-APPswe cells.

Method: N2a-APPswe mouse neuroblastoma cells, which contain a human APP transgene with Swedish mutations, were cultured in complete DMEM/F12 medium. Cells were treated with different concentrations of Hcy, HTL, and N-Hcy-protein in methionine-free medium for 24 h. Blmh gene expression was silenced using Blmh-specific siRNA with Lipofectamine in Opti-MEM medium for 48 h. Proteins involved in ER stress, UPR, and apoptosis were quantified by Western blotting, and the corresponding mRNAs were analysed by RT-qPCR.

Results: Blmh gene silencing, Hcy, HTL, and N-Hcy-protein increased levels of ER chaperone GRP78, indicating the induction of ER stress, which further activated the UPR pathway by increased levels of ATF3 and CHOP. The proapoptotic proteins BAX and CASPASE 3 were increased, whereas the anti-apoptotic protein BCL-2 was reduced, suggesting a shift towards cell death. RT-qPCR analysis confirmed that mRNA expression changes were similar to the protein-level changes.

Conclusion: Blmh gene silencing, Hcy, HTL and N-Hcy-protein may contribute to the development and progression of AD by inducing ER stress, dysregulation of the UPR pathway and apoptosis.