Calpain activation and disturbance of autophagy are induced in cortical neurons in vitro by exposure to HA/ β-Ga2O3:Cr3+ nanoparticles

Abstract

The toxicity of engineered nanoparticles remains a concern. The knowledge of biohazards associated with particular nanoparticles is crucial to make this cutting-edge technology more beneficial and safe. Here, we evaluated the toxicity of Ga₂O₃ nanoparticles (NPs), which are frequently used to enhance the performance of metal catalysts in a variety of catalytic reactions. The potential inflammatory signaling associated with the toxicity of HA/β-Ga₂O₃:Cr³⁺ NPs in primary cortical neurons was examined. We observed a dose-dependent decrease in cell viability and an increase in apoptosis in neurons following various concentrations (0, 1, 5, 25, 50, 100 µg/ml) of HA/β-Ga₂O₃:Cr³⁺ NPs treatment. Consistently, constitutively active forms of calcineurin (48 kDa) were significantly elevated in cultured primary cortical neurons, which was consistent with calpain activation indicated by the breakdown products of spectrin. Moreover, HA/β-Ga₂O₃:Cr³⁺ NPs result in the elevation of LC3-II formation, SQSTM/p62, and Cathepsin B, whereas phosphorylation of CaMKII (Thr286) and Synapsin I (Ser603) were downregulated in the same context. Taken together, these results demonstrate for the first time that calpain activation and a disturbance of autophagy signaling are evoked by exposure to HA/β-Ga₂O₃:Cr³⁺ NPs, which may contribute to neuronal injury in vitro.

Keywords: Autophagy; Calpain; HA/β-Ga2O3:Cr3+; Inflammation; Nanoparticles; Neurotoxicity.

Figure 1. The changes of cell morphology and viability after exposure to HA/β-Ga₂O₃:Cr³⁺ NPs in SH-SY5Y cell line.

(A) The changes of cell morphology after exposure to various doses of HA/β-Ga₂O₃:Cr³⁺ NPs for 12 h, scale bar = 50 µm. (B) MTT results of SH-SY5Y cells treated with HA/β-Ga₂O₃:Cr³⁺ NPs, **P < 0.01, ***P < 0.001 versus control group. (C) The results of TUNEL in SH-SY5Y cells after incubation with HA/β-Ga₂O₃:Cr³⁺ NPs, scale bar = 40 µm.

Figure 2. The expression level of proteins associated with synaptic structures and functions in mouse primary cortical neurons.

The primary cortical neurons were cultured for 10 days and treated with HA/β-Ga₂O₃:Cr³⁺ NPs for 12 h. (A) The representative Western blot images of P-CaMKII, P-Synapsin I, Synapsin I and P-GluR 1 in primary cortical neurons after incubation with various doses of HA/β-Ga₂O₃:Cr³⁺ NPs. (B) The representative data of spectrin and calcineurin in primary cortical neurons after treated with 10 µg/mL HA/β-Ga₂O₃:Cr³⁺ NPs for 12 h, β-actin served as the loading control. Quantification of data on the breakdown products (BDPs) of spectrin (C) and calcineurin (D) from (B). The data are expressed as the mean ± SEM for three independent experiments. **P < 0.01 versus control group.

Figure 3. The effects of HA/β-Ga₂O₃:Cr³⁺ NPs on the autophagy signaling pathway.

(A) The cultured primary cortical neurons were incubated with 10 µg/mL HA/β-Ga₂O₃:Cr³⁺ NPs for 12 h, and intracellular Cathepsin B was measured by Western blot analysis. Immunodetection of β-actin was used as a loading control. (B) Quantification of data on Cathepsin B from (A). The data are expressed as the mean ± SEM for three independent experiments, **P < 0.01 versus the control group. (C) The representative Western blot images of LC3 and P62 in primary cortical neurons after incubation with 10 µg/mL HA/β-Ga₂O₃:Cr³⁺ NPs. Quantification of data on P62 (D) and type II LC3 (16 kDa) (E) from (C), β-actin served as the loading control. The data are expressed as the mean ± SEM for three independent experiments. *P < 0.05, **P < 0.01 versus the control group.

Figure 4. Immunocytochemical analysis shown the uptaken of HA/β-Ga₂O₃:Cr³⁺ NPs and the activation of Cathepsin B in SH-SY5Y cells.

The excitation wavelength of DAPI, Cathepsin B, and HA/β-Ga₂O₃:Cr³⁺ NPs is 405 nm, 488 nm, and 641 nm, respectively. Scale bar = 10 µm.