Dysregulation of amyloid-β precursor protein processing by treatment with Hcy, Hcy-thiolactone, N-Hcy-proteins and deficiency of bleomycin hydrolase in mouse neuroblastoma N2a-APPswe cells

Introduction: Homocysteine (Hcy), homocysteine thiolactone (HTL), N-Hcy-proteins and dysfunction of bleomycin hydrolase (BLMH) are associated with Alzheimer's disease (AD). AD is characterized by the accumulation of amyloid-β (Aβ) plaques. Aβ is formed from amyloid-β precursor protein (APP) via the amyloidogenic pathway, involving β-secretase BACE1 and γ-secretase complex (Nicastrin, PSEN1, PEN2, APH-1). The impact of Hcy, its metabolites and BLMH dysfunction on the development and progression of AD is not fully understood.

Objective: We tested the hypothesis that Hcy, its metabolites and Blmh silencing affect APP processing pathways in mouse neuroblastoma N2a-APPswe cells.

Methods: Neuroblastoma N2a-APPswe cells harboring a human transgene with a mutation in the APP gene were treated with different concentrations of Hcy, HTL or N-Hcy-proteins. The expression of the Blmh gene was silenced using a Blmh-specific siRNA. Scrambled siRNA was used as a negative control. Proteins involved in APP metabolism pathway, i.e., APP, phospho-APP, BACE1, PSEN1, PEN2 and Nicastrin, were quantified by Western blotting. The expression of APP, Bace1 Psen1, Psenen and Ncstn mRNAs was analyzed by RT-qPCR.

Results: Hcy increased APP, phospho-APP, BACE1 and PSEN1. At mRNA level, Hcy downregulated the expression of the Psen1 gene. HTL increased protein levels of APP, BACE1, PSEN1 and Nicastrin. This treatment also decreased the expression of the Psenen gene (encoding PEN2). Treatments with N-Hcy-proteins increased level of PSEN1 protein and downregulated the expression of the Psenen gene. There was no significant impact of treatments on PEN2 protein level. Silencing of the Blmh gene resulted in upregulation of APP, Nicastrin, PEN2 and downregulation of BACE1 protein. Silencing significantly upregulated the expression of APP and Psenen mRNAs.

Conclusions: Hcy, HTL, N-Hcy-protein and Blmh gene silencing resulted in dysregulated APP processing in N2a-APPswe cells, suggesting their association with AD progression.

Acknowledgement: Supported by NCN grant 2021/43/B/NZ4/00339.