Role of TPEN in Amyloid-β25–35-Induced Neuronal Damage Correlating with Recovery of Intracellular Zn2+ and Intracellular Ca2+ Overloading.

The overproduction of neurotoxic amyloid-β (Aβ) peptides in the brain is a hallmark of Alzheimer's disease (AD). To determine the role of intracellular zinc ion (_iZn²⁺) dysregulation in mediating Aβ-related neurotoxicity, this study aimed to investigate whether N, N, N′, N′‑tetrakis (2‑pyridylmethyl) ethylenediamine (TPEN), a Zn²⁺‑specific chelator, could attenuate Aβ_25–35‑induced neurotoxicity and the underlying mechanism. We used the 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay to measure the viability of primary hippocampal neurons. We also determined intracellular Zn²⁺ and Ca²⁺ concentrations, mitochondrial and lysosomal functions, and intracellular reactive oxygen species (ROS) content in hippocampal neurons using live-cell confocal imaging. We detected L-type voltage-gated calcium channel currents (L-I_Ca) in hippocampal neurons using the whole‑cell patch‑clamp technique. Furthermore, we measured the mRNA expression levels of proteins related to the _iZn²⁺ buffer system (ZnT-3, MT-3) and voltage-gated calcium channels (Cav1.2, Cav1.3) in hippocampal neurons using RT-PCR. The results showed that TPEN attenuated Aβ_25–35‑induced neuronal death, relieved the Aβ_25–35‑induced increase in intracellular Zn²⁺ and Ca²⁺ concentrations; reversed the Aβ_25–35‑induced increase in ROS content, the Aβ_25–35‑induced increase in the L-I_Ca peak amplitude at different membrane potentials, the Aβ_25–35‑induced the dysfunction of the mitochondria and lysosomes, and the Aβ_25–35‑induced decrease in ZnT-3 and MT-3 mRNA expressions; and increased the Cav1.2 mRNA expression in the hippocampal neurons. These results suggest that TPEN, the Zn²⁺-specific chelator, attenuated Aβ_25–35‑induced neuronal damage, correlating with the recovery of intracellular Zn²⁺ and modulation of abnormal Ca²⁺-related signaling pathways. [ABSTRACT FROM AUTHOR]