Changes in miR-134-3p expression and zDHHC3-AMPARs axis in association with aluminum neurotoxicity.

Aluminum (Al) is a neurotoxic substance associated with cognitive dysfunction and neurodegenerative diseases, such as Alzheimer's disease, but the mechanisms for aluminum neurotoxicity remain to be identified. In this work, we try to investigate a novel potential biomarker of cognitive dysfunction following aluminum exposure and the mechanism involved. Recently, miR-134-3p was reported as a novel regulator of cognitive function. To address this, we investigate the expression level of miR-134-3p in plasma from 280 aluminum factory workers and analyzed the correlation between miRNA-134-3p, blood Al concentration, and Montreal Cognitive Assessment Scale (MoCA scale) score. The results implied that occupational aluminum exposure elevated miR-134-3p expression in the plasma of workers accompanied by cognitive impairment. Our experiment studies using both animal models and PC12 cells validated the upregulation of miR-134-3p caused by aluminum. In addition, we identified that palmitoylation enzyme zDHHC3 was the target of miR-134-3p, and the decreasing AMPAR receptor (AMPAR) trafficking was related to the learning and memory impairment induced by aluminum. More importantly, using transfection and interference approaches in PC12 cells, inhibition of miR-134-3p resulted in a recovery of zDHHC3-AMPARs axis to a certain extent in response to aluminum. In summary, miR-134-3p was found to be involved in aluminum neurotoxicity by targeting zDHHC3-AMPARs axis and could serve as a potential biomarker or helpful target.

The Palmitoylation/Depalmitoylation Cycle is Involved in the Inhibition of AMPA Receptor Trafficking Induced by Aluminum In Vitro.

To study the effect of the palmitoylation/depalmitoylation cycle on the inhibition of ɑ-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA) receptor trafficking induced by aluminum (Al) in vitro. Five different doses of aluminum-maltolate complex (Al(mal)3) were administered to rat adrenal pheochromocytoma cells (PC12 cells) for three exposure time durations, and the cell activity was measured by the CCK-8 method to obtain the optimal doses and time of Al(mal)3 exposure. Following Al(mal)3 exposure, membrane protein (M) and total protein (T) were extracted. The expression levels of GluR1 and GluR2, which are AMPA receptor subunits, were determined by Western blot analysis, and the levels with respect to membrane and total protein were calculated. The ratio of membrane protein to total protein (M/T) was used to measure the rate of AMPA receptor transport. The palmitoylation levels of GluR1 and GluR2 were detected by immunoprecipitation-acyl-biotin exchange (IP-ABE) assay. Western blotting was performed to detect the protein expression of acyltransferase (zDHHC3) and palmitoyl protein thioesterase 1 (PPT1). Following depalmitoylation inhibitor (palmostatin B) treatment of PC12 cells, the effect of aluminum on AMPA receptor trafficking was detected through the aforementioned methods. With increasing Al(mal)3 doses administered to PC12 cells, a gradual decrease in the trafficking of AMPA receptor subunits GluR1 and GluR2 and in the palmitoylation levels of GluR1 and GluR2 was found; the expression of zDHHC3 was decreased; and the expression of PPT1 was increased. In addition, palmostatin B reduced the effects of Al(mal)3 on AMPA receptor palmitoylation and trafficking. Al can inhibit the trafficking of the AMPA receptor in vitro, and a decrease in the palmitoylation level of the AMPA receptor may be a mechanism of Al action. The palmitoylation/depalmitoylation cycle of the AMPA receptor is influenced by Al through the actions of zDHHC3 and PPT1.

APP palmitoylation is involved in the increase in Aß1-42 induced by aluminum.

The increase in Aß1-42 is a neurotoxic effect induced by aluminum which can lead to impairment of learning and memory, but its mechanism has yet to be fully elucidated. Studies have shown that APP palmitoylation is appears to be involved in the production process of Aß1-42. Here, we investigated whether APP palmitoylation is related to the increase in Aß caused by aluminum and its specific mechanism of action. In this study, APP palmitoylation was studied in the setting of aluminum-induced increases in Aß1-42 from two perspectives: whole animal experiments and in vitro cell experiments. First, the learning and memory of rats were impaired and the number of rat cortical neurons was decreased after staining with aluminum. Second, the expression of palmitoyl APP, APP in lipid rafts and palmitoyl acyltransferase zDHHC7 both in rat cerebral cortex and PC12 cells increased with the production of Aß1-42 induced by aluminum in a dose-dependent manner. Finally, the intervention with the palmitoylation inhibitors 2-BP and siRNA zDHHC7 in PC12 cells reduced levels of palmitoyl APP, the expression of APP in lipid rafts and the content of Aß1-42 induced by aluminum to a certain extent. Our results indicate that increased APP palmitoylation levels may be related to the increase in Aß1-42 caused by aluminum, and the mechanism may involve APP palmitoylation promoting the accumulation of APP protein on lipid rafts and the cleavage of APP by BACE1 in amyloidogenic pathway. The increase in expression of zDHHC7 may be one of the reasons for the increase in levels of APP palmitoylation caused by aluminum.

Regulation of mGluR1 on the Expression of PKC and NMDAR in Aluminum-Exposed PC12 Cells.

Aluminum demonstrates clear neurotoxicity and can cause Alzheimer's disease (AD)-like symptoms, including cognitive impairment. One toxic effect of aluminum is a decrease in synaptic plasticity, but the specific mechanism remains unclear. In this study, PC12 cells were treated with Al(mal)3 to construct a toxic cell model. (S)-3,5-Dihydroxyphenylglycine (DHPG), α-methyl-4-carboxyphenylglycine (MCPG), and mGluR1-siRNA were used to interfere with the expression of metabotropic glutamate receptor subtype 1 (mGluR1). Polymerase chain reaction and western blotting were used to investigate the expression of mGluR1, protein kinase C (PKC), and N-methyl-D-aspartate receptor (NMDAR) subunits. ELISA was used to detect PKC enzyme activity. In PC12 cells, mRNA and protein expressions of PKC and NMDAR subunits were inhibited by Al(mal)3. Aluminum may further regulate the expression of NMDAR1 and NMDAR2B through mGluR1 to regulate PKC enzyme activity, thereby affecting learning and memory functions. Furthermore, the results implied that the mGluR1-PKC-NMDAR signaling pathway may predominately involve positive regulation. These findings provide new targets for studying the neurotoxic mechanism of aluminum.