Effect of aluminum-maltolate on primary hippocampal neuron neurite damage by regulating CRMP2 through GSK-3β in mice

Background  Aluminum can induce irreversible structural and synaptic functional damage, and the associated mechanism may be related to the neurite damage regulated by glycogen synthase kinase-3β (GSK-3β)/collapsin response mediator protein 2 (CRMP2).

Objective  This experiment is conducted to investigate the effect of aluminum-maltolate Al(mal)₃ on primary hippocampal neuron neurites in mice, and reveal the role of GSK-3β-CRMP2 in this process.

Methods  The hippocampus of newborn ICR mice (≤ 24 h old) was used for primary neuronal cultures. On the 5th day in vitro (DIV5), neuron purity detection were performed by confocal laser scanning microscopy. On DIV7, the neurons were transfected with lentiviral vector-mediated mNeonGreen. On DIV10, the neurons with mNeonGreen fluorescence in good growth state were treated with Al(mal)₃. The stage I experimental groups were blank control group, maltol group, 10 µmol·L⁻¹ Al group, 20 µmol·L⁻¹ Al group, and 40 µmol·L⁻¹ Al group. Then 20 µmol·L⁻¹ Al was used to establish a model of neurite injury and for the intervention. The stage II experimental groups were blank control group, dimethyl sulfoxide (DMSO) group, Al (20 µmol·L⁻¹) group, SB (GSK-3β inhibitor, 1 µmol·L⁻¹), and SB (1 µmol·L⁻¹)+Al (20 µmol·L⁻¹) group. CCK-8 method was used to detect the viability of neurons. The primary hippocampal neurons of mice were scanned with high content analysis system at 0 h and 48 h after Al or SB treatment, and the density and length of neurites were analyzed. Western blotting was used to detect the expression and phosphorylation levels of CRMP2 and GSK-3β in primary hippocampal neurons of mice.

Results  The immunofluorescence results showed that the purity of primary neurons was more than 90%. Compared with the blank control group in stage I, the cell viability rates of the 10, 20, and 40 µmol·L⁻¹ Al groups were decreased after 48h of Al(mal)₃ treatment (P<0.05), while the cell viability rate of the maltol group had no significant change. There was no significant difference in cell viability rate among the DMSO group, the SB group, and the control group after 48h of SB treatment, and the viability rate of neurons in the SB+Al group was higher than that in the Al group (P<0.05) in stage II. The 48 h/0 h ratios of average number and length of neurites in the control group were 90.13%±11.70% and 113.24%±8.34%, respectively. The 48 h/0 h ratios in the Al group were 56.47%±16.36% and 62.06%±6.75%, respectively, which were lower than those in the control group (P<0.05). The 48 h/0 h ratios of average number of neurites in the SB group (99.03%±21.83%) was not significantly different from that in the control group, but the 48 h/0 h ratio of average length of neurites in the SB group (128.72%±15.39%) was higher than that in the control group (P<0.05). The 48 h/0 h ratios of average number (72.59%±10.89%) and length of neurites (93.84%±14.65%) in the SB+Al group were significantly increased compared with those in the Al group (P<0.05). Western blotting results showed that: There was no significant difference in GSK-3β protein level among all groups; compared with the control group (1.00±0.18), the protein level of p-GSK-3β in the Al group (0.45±0.05) was significantly decreased, and that in the SB group (1.32±0.23) was significantly increased; the protein level of p-GSK-3β in the SB+Al group (0.80±0.05) was significantly higher than that in the Al group (P<0.05). Compared with the control group (1.00±0.07), the CRMP2 protein level in the Al group (0.66±0.11) was significantly decreased (P<0.05), while that in the SB group (1.01±0.02) was not significantly changed. Compared with the control group (1.00±0.13), the p-CRMP2 protein level in the Al group (1.50±2.18) was significantly increased, and that in the SB group (0.62±0.09) was significantly decreased (P<0.05); the protein level of p-CRMP2 in the SB+Al group (1.28±0.24) was lower than that in the Al group (P<0.05).

Conclusion  Aluminum may activate GSK-3β, increase CRMP2 phosphorylation level, and damage neurite growth.