Copper Overload Promotes β-amyloid Induced NLRP3/Caspase-1/GSDMD-Mediated Pyroptosis in Alzheimer's Disease

Purpose: Alzheimer's disease (AD) is characterized by cognitive decline and abnormal protein accumulation. Copper imbalance and Pyroptosis play significant roles in the pathogenesis of AD. Recent studies have suggested that dysregulated copper homeostasis contributed to β-amyloid accumulation, which may activate the NOD-like Receptor protein 3 (NLRP3)-related Pyroptosis pathway, promoting neuronal damages and AD progression. Therefore, the present study aims to investigates whether copper facilitates AD through exacerbating β-amyloid (Aβ) induced activation of NLRP3/Caspase-1/Gasdermin D (GSDMD)-mediated neuronal cell Pyroptosis.

Methods: Mouse hippocampal HT-22 cells were cultured with Aβ1-42 oligomer for 24 h as AD Model group. CuCl₂ treatment was administered to the AD cell model, and cell survivability levels were detected by Cell Counting Kit-8 (CCK-8), TdT-mediated dUTP nick end labeling (TUNEL), and Other relevant kits. Mitochondrial function was evaluated using Mitochondrial membrane potential dye JC-1 and transmission electron microscopy (TEM). After intervention with the NLRP3 Inhibitor MCC950, activation of the NLRP3/Caspase-1/GSDMD pathway by copper ions (Cu²⁺) was confirmed via Western Blot. Thioredoxin T (ThT) fluorescence assay was performed to observe the aggregation effect of Aβ induced by Cu²⁺ overload.

Results: CuCl₂ treatment of the AD cell model resulted in up-regulation of the levels of Lactate Dehydrogenase (LDH), Interleukin-1β (IL-1β), and IL-18 expression, which indicated activation of Pyroptosis. We observed a significant decrease in mitochondrial membrane potential, mitochondrial swelling, and loss of mitochondrial cristae by fluorescence microscopy and TEM. ThT fluorescence imaging showed that Cu²⁺ promoted Aβ aggregation and up-regulated NLRP3, apoptosis-associated speck-like protein containing a CARD (ACS), Caspase-1, Cleaved Caspase-1, GSDMD, and Gasdermin D N-terminal (GSDMD-NT). The NLRP3 Inhibitor MCC950 partially reversed Cu²⁺-mediated Pyroptosis in HT-22 cells.

Conclusions: Exposure to copper ions disrupt mitochondrial copper homeostasis, promotes Aβ aggregation, and activates NLRP3 inflammasomes, further promoting the Aβ aggregation activated Pyroptosis in AD cell models.

Aβ; NLRP3; copper overload; neuron; pyroptosis.