New mechanism of nerve injury in Alzheimer's disease: β-amyloid-induced neuronal pyroptosis

Abstract

The present study was designed to investigate the role of β-amyloid (Aβ_1-42 ) in inducing neuronal pyroptosis and its mechanism. Mice cortical neurons (MCNs) were used in this study, LPS + Nigericin was used to induce pyroptosis in MCNs (positive control group), and Aβ_1-42 was used to interfere with MCNs. In addition, propidium iodide (PI) staining was used to examine cell permeability, lactate dehydrogenase (LDH) release assay was employed to detect cytotoxicity, immunofluorescence (IF) staining was used to investigate the expression level of the key protein GSDMD, Western blot was performed to detect the expression levels of key proteins, and enzyme-linked immunosorbent assay (ELISA) was utilized to determine the expression levels of inflammatory factors in culture medium, including IL-1β, IL-18 and TNF-α. Small interfering RNA (siRNA) was used to silence the mRNA expression of caspase-1 and GSDMD, and Aβ_1-42 was used to induce pyroptosis, followed by investigation of the role of caspase-1-mediated GSDMD cleavage in pyroptosis. In addition, necrosulfonamide (NSA), an inhibitor of GSDMD oligomerization, was used for pre-treatment, and Aβ_1-42 was subsequently used to observe the pyroptosis in MCNs. Finally, AAV9-siRNA-caspase-1 was injected into the tail vein of APP/PS1 double transgenic mice (Alzheimer's disease mice) for caspase-1 mRNA inhibition, followed by observation of behavioural changes in mice and measurement of the expression of inflammatory factors and pyroptosis-related protein. As results, Aβ_1-42 could induce pyroptosis in MCNs, increase cell permeability and enhance LDH release, which were similar to the LPS + Nigericin-induced pyroptosis. Meanwhile, the expression levels of cellular GSDMD and p30-GSDMD were up-regulated, the levels of NLRP3 inflammasome and GSDMD-cleaved protein caspase-1 were up-regulated, and the levels of inflammatory factors in the medium were also up-regulated. siRNA intervention in caspase-1 or GSDMD inhibited Aβ_1-42 -induced pyroptosis, and NSA pre-treatment also caused the similar inhibitory effects. The behavioural ability of Alzheimer's disease (AD) mice was relieved after the injection of AAV9-siRNA-caspase-1, and the expression of pyroptosis-related protein in the cortex and hippocampus was down-regulated. In conclusion, Aβ_1-42 could induce pyroptosis by GSDMD protein, and NLRP3-caspase-1 signalling was an important signal to mediate GSDMD cleavage, which plays an important role in Aβ_1-42 -induced pyroptosis in neurons. Therefore, GSDMD is expected to be a novel therapeutic target for AD.

Keywords: Alzheimer's disease; GSDMD; pyroptosis; β-amyloid.