Autophagy dysfunction plays a significant role in the neurotoxicity of cadmium. This study aims to screen autophagy-related hub genes in cadmium-exposed HT22 cells and evaluate the role of the hub gene-related regulatory networks in cadmium neurotoxicity.

CCK-8 assay was used to detect the survival rate of HT22 cells exposed to CdCl₂ at concentrations of 0, 5, 10, 15, 20 and 25 μmol/L for 24 h, and the half-maximal inhibitory concentration (IC₅₀) was calculated. The control group (n=4) was not treated with cadmium, while the cadmium exposure group (n=4) was treated with an IC₅₀ of CdCl₂ for 24 h. RNA samples from the control group and the cadmium exposure group were extracted for transcriptome sequencing. Autophagy-related differentially expressed genes (DEGs) were screened based on transcriptomic results of cadmium-exposed HT22 cells and public databases, and GO and KEGG analyses were performed to identify the main enriched pathways. Further screening of autophagy-related hub genes was carried out by protein-protein interaction (PPI) network, and the relative expression levels of these hub genes were validated by qPCR. Multiple regulatory networks related to hub genes (miRNAs, drugs, transcription factors, RNA binding proteins) were further constructed.

Cadmium exposure caused to a dose-dependent decrease in the survival rate of HT22 cells (P<0.01), with an IC₅₀ value of about 10 μmol/L. Compared with the control group, the transcriptomic expression profile of the cadmium exposure group underwent significant global changes. A total of 36 autophagy-related DEGs and 13 hub genes (Atf3, Csf2, Cdkn1a, Cd68, Cryab, Hbegf, Adam8, Areg, Gadd45a, Plk3, Aldh3a1, Adm, Dusp4) were identified, mainly enriched in oxidative stress and signal transduction-related pathways. Among them, the expression of 11 hub genes (Atf3, Csf2, Cdkn1a, Cd68, Cryab, Hbegf, Adam8, Areg, Gadd45a, Plk3, Dusp4) was upregulated significantly (P<0.05). The hub genes were involved in the multi-regulatory networks of 271 miRNAs, 86 drugs, 20 transcription factors, and 17 RNA-binding proteins.

Cadmium exposure induces alterations in the expression of 11 autophagy-related hub genes in HT22 cells, and the multiple regulatory networks involving autophagy-related hub genes mediate cadmium-induced neurotoxicity in neuronal cells.

To investigate the effect of kinesin family member 5A (KIF5A) mediated lysosomal functions in cadmium (Cd) exposure-induced neurotoxicity.

Primary cortical neurons isolated and primarily cultured from specific pathogen free (SPF) C57BL/6J mice were used and divided into control and Cd-exposed groups. The 50% inhibitory concentration (IC₅₀) of cortical neurons after 24 h of Cd exposure was measured. Furthermore, the neurons were treated with 1, 2 and 3 μmol/L cadmium chloride (CdCl₂) for 24 h, and then cell viability, neuronal differentiation and lysosomal function were measured. RT-qPCR and Western blotting were used to detect the changes of KIF5A expression at mRNA and protein levels. The effects of overexpression of KIF5A by adenoviral vector on cell viability, neuronal differentiation and lysosomal function were also investigated in the Cd-exposed cortical neurons.

The IC₅₀ value in neurons exposure to CdCl₂ for 24 h was determined to be 2.9 μmol/L. Compared with the control group, the cell viability was inhibited (P<0.01) and the total length of protrusion growth and the number of branching points were obviously reduced (P<0.01) after 2 and 3 μmol/L CdCl₂ exposure for 24 h. In addition, 2 and 3 μmol/L CdCl₂ treatment also resulted in dramatically decreased lysosomal activity of cathepsin B (CTSB) and altered acidic environment when compared with the control group (P<0.01). However, the activity cathepsin D(CTSD) was not significantly changed in CdCl₂-exposed neuronal cells. More importantly, the mRNA and protein levels of KIF5A were remarkably decreased after Cd exposure (P<0.01). Overexpression of KIF5A effectively increased the CTSB activity, stabilized the lysosomal pH value, and antagonized the inhibitory effect on cell viability and protrusion growth induced by Cd exposure (P<0.01).

Cd exposure significantly reduces KIF5A protein expression in neurons, and thereby impairs lysosomal function and inhibits neuronal cell viability and protrusion growth.