| Aluminum(A1) is the most abundant metal in the earth crust. O f all the influence factors to the AD, Al is the only one of environmental impact factors which was widely studied and can cause histopathological changes of AD in multiple aspects. One important clinical feature of AD is progressive memory loss, the earliest and the most basic symptom of AD is learning and memory dysfunction. So the study on mechanisms of learning and memory impairment induced by Al has always been the hot spot of the aluminium neurotoxicity research. Long-term potentiation(LTP) is one the major fo rms of synaptic plasticity. Variations of hippocampal LTP have been widely used to elucidate the synaptic mechanisms involved in impairments of learning and memory. Since Al impairs learning and memory, and has been reported to affect LTP, understanding the mechanism of Al- induced LTP impairment may also reveal the molecular mechanism of Al neurotoxicity. It is now generally accepted that LTP typically results from an increased concentration of AMPARs at the neuronal membrane surface. A previous study of our group found that Al- induced LTP impairment reduces while the expression of AMPAR subunits also decreases. However, the detailed mechanisms of how Al exerts its effects on LTP in the hippocampal CA1 region are still only partially understood. Therefore, it is necessary to further explore this effect to elucidate its underlying mechanisms. The activity and trafficking of AMPAR are modulated by GSK-3β, and the inhibition or activation of GSK-3β via Akt also influences the externalization and internalization of AMPAR. A mechanism by which caspase-3 cleaves Akt to inhibit its function is involved in synaptic plasticity. Hence, whether the Caspase-3 regulated Akt/GSK-3β signaling pathway is involved in the suppressive effects of Al on LTP and AMPAR levels is still not known, and few reports of synaptic plasticity in this context have been published to date. Objective:(1) To investigate the effects of Al(mal)3 on LTP and AMPA receptor subunits delivery by subchronic intraperitoneal injection(i.p.) for 60 days and intracerebroventricular(i.c.v.) injection treatment for 10 days; To investigate the effects of Al(mal)3 on caspase-3、Akt、GSK-3β for determining whether caspase 3, Akt and GSK-3β are involved in the impairment of LTP induced by Al;(2)To investigate whether caspase-3 is involved in Al- induced impairment of LTP and AMPA receptor subunits delivery through Akt/GSK-3β pathway by using caspase-3 inhibitor N- benzyloxycarbonyl- Asp(OMe)- Glu(OMe)Val- Asp(OMe)- fluoromethyl ketone(z-DEVD- fmk) and GSK-3β inhibitor SB216763. Methods:(1)32 adult male rats were randomly divided into four groups( 8 rats per group):control group,10μM/kg, 20μM/kg, and 40μM/kg Al(mal)3 treated group. Rats were treated with saline(control group) or different doses of Al(mal)3 via i.p. injection for 8 weeks. Hippocampal LTP in CAl region were recorded in vivo. The expression of AMPAR subunit proteins(Glu Rl and Glu R2) in total and membrane extracts from the CA 1 area of rat hippocampus were detected by Western blot assay. The expression of caspase-3、 Akt、GSK-3β and p-GSK-3β(Ser9) from the CA 1 region of rat hippocampus were detected by Western blot assay.(2)Animals under chloral hydrate anesthesia were held in a stereotaxic apparatus. A stainless steel guide cannula, inserted with a stainless steel stylet, was placed through holes drilled in the skull and then the cannula was secured to the skull with dental acrylic. Ten to fourteen days after surgery each rat received an i.c.v. injection of 5 μl Al(mal)3(equating to 2, 5 or 10 m M) or saline. The administration was performed once every 2 days, for 10 days.Hippocampal LTP in CAl region were recorded in vivo. The expression of AMPAR subunit proteins(Glu Rl and Glu R2) in total and membrane extracts from the CA 1 region of rat hippocampus were detected by Western blot assay. The expression of caspase-3, Akt, GSK-3βand p-GSK-3β(Ser9) from the CA 1 region of rat hippocampus were detected by Western blot assay.(3)Rats were divided into control group,z-DEVD-fmk group,z-DEVD-fmk +A1 group and Al group randomly, then received i.c.v. injection of 5 μl Al(mal)3, saline or z-DEVD- fmk. The administration was performed once every 2 days, for 10 days.Hippocampal LTP in C Al region were recorded in vivo. The expression of AMPAR subunit proteins(Glu Rl and Glu R2) in total and membrane extracts from the C A 1 region of rat hippocampus were detected by Western blot assay. The expression of caspase-3, Akt, GSK-3β and p-GSK-3β(Ser9)from the CA 1 region of rat hippocampus were detected by Western blot assay too.(4)Rats were divided into control group, SB216763 group, SB216763+A1(50m M) group and Al(50m M) group randomly, then received i.c.v. injection of 5 μl Al(mal)3, saline or SB216763. The administration was performed once every 2 days, for 10 days.Hippocampal LTP in C Al region were recorded in vivo. The expression of AMPAR subunit proteins(Glu Rl and Glu R2) in total and membrane extracts from the C A 1 region of rat hippocampus were detected by Western blot assay. The expression of GSK-3β and p-GSK-3β(Ser9) from the CA 1 region of rat hippocampus were detected by Western blot assay too. Results:1.The damage of aluminum exposure on hippocampal LTP and AMPA receptor delivery in rats through i.p. injection subchronicallyThe results of LTP test : The f EPSP amplitudes of the control group were(176.04±6.2)%,(154.5±10.4)% and(146.8±4.2)% at 1, 30, and 60 min after HFS, respectively, and those in the 10μM/kg Al(mal)3 group were(170.3±4.8) %,(155.0±5.8)%and(145.8±5.0)% at 1, 30, and 60 min post HFS, respectively, not significantly different between the two groups(P >0.05). In the 20μM/kg Al(mal)3 group the f EPSP amplitudes were(154.7±8.0)%,(141.1 士 4.4)% and(135.7 士 7.7)% at 1, 30, and 60 min post HFS, respectively, and in the 40μM/kg Al(mal)3 group the f EPSP amplitudes were(141.0±9.8)%,(122.7±9.3)% and(121.0±9.3)% at 1, 30, and 60 min post HFS, respectively, significa ntly suppressed compared with the controls(P <0.05).The results of Western-blot assay :(1) Subchronic A1 treatment caused dose-dependent decreases in Glu Rl and Glu R2 in the total extracts and the membrane extracts(P <0.05). The protein level of Glu Rl and Glu R2 in the total extracts and the membrane extracts in the 20μM/kg Al(mal)3 group and 40μM/kg Al(mal)3 group were significantly decreased compared with the controls(P <0.05). By Analysis of the ratio of membrane protein and total protein of Glu Rl, Glu R2, we found that the ratio of Glu Rl in membrane protein and in total protein declined dose-dependently, significantly decreased by 41.1% in high dose group, compared with the control group(P <0.05). The ratio of Glu R2 in membrane protein and in total prote in also declined dose-dependently, significantly decreased by 41.2% and 73% in middle-dose group and high dose group respectively, compared with the control group(P <0.05).(2) The protein expression of active caspase-3 increased gradually with increasing of Al(mal)3 dosage, statistically significant in 20,40 μM Al(mal)3 groups compared with the control groups(P <0.05). The protein expression of Akt decreased significantly in 20,40 μM Al(mal)3 groups compared with the control group(P <0.05). The protein expressions of p-GSK-3β(Ser9) in 20,40 μM Al(mal)3 groups were statistically different compared with the control group(P <0.05). By further analysis of the ratio of p-GSK-3β(Ser9) and GSK-3β, we found that the ratio of p-GSK-3β(Ser9) and GSK-3β declined dose-dependently, significantly decreased in 40 μM Al(mal)3 group, compared with the control group(P <0.05).2.The damage of aluminum exposure on hippocampal LTP and AMPA receptor delivery in rats through i.c. v. injection.The results of LTP test : The f EPSP amplitudes of the control group were(176.2±7.0)%,(155.8±11.3)% and(148.0±3.8)% at 1, 30, and 60 min after HFS, respectively, and those in the 2 m M Al(mal)3 group were(166.5±6.0)%,(148.8±10.1)%and(143.3±4.2)% at 1, 30, and 60 min post HFS, respectively, not significantly different between the two groups(P >0.05). In the 10 m M Al(mal)3 group the f EPSP amplitudes were(141.9±8.2)%,(126.2 士 9.3)%and(124.3 士 8.5)% at 1, 30, and 60 min post HFS, respectively, while in the 50 m M Al(mal)3 group those were(128.5±4.4) %,(106.7±5.7) % and(104.0±5.1) %, at 1, 30, and 60 min post HFS, respectively, significantly suppressed compared with the controls(P <0.05).The results of Western-blot assay:(1) I.c.v. injection of A1 caused dose-dependent decreases in Glu Rl and Glu R2 in the total extracts and the membrane extracts(P <0.05). The protein level of Glu Rl and Glu R2 in the total extracts and the membrane extracts in in the 10 m M and 50 m M Al(mal)3 group were significantly decreased,compared with the controls(P <0.05). By Analysis of the ratio of membrane protein and total protein of Glu Rl and Glu R2, we found that the ratio of Glu Rl in membrane protein and in total protein declined dose-dependently, significantly decreased by 33.7% and 34.8% in 10 m M and 50 m M dose group, compared with control group(P <0.05). The ratio of Glu R2 in membrane protein and in total protein also declined dose-dependently, significantly decreased by 14.9% and 20.7% in 10 m M and 50 m M dose group, respectively, compared with the control group(P <0.05).(2) The protein expression of active caspase-3 increased gradually with increasing of Al(mal)3 doseage.The protein expression of active caspase-3, Akt and p-GSK-3β(Ser9)in 10 and 50 m M Al(mal)3 groups showed statistically significant difference compared with t he control groups(P <0.05).By further analysis of the ratio of p-GSK-3β(Ser9)and GSK-3β, we found that the ratio of p-GSK-3β(Ser9)and GSK-3β declined dose-dependently, significantly decreased in 50 m M Al(mal)3 group, compared with the control group(P <0.05).3. Effect of caspase-3 inhibitor z-DEVD-fmk on impairment of hippocampal LTP induced by aluminum.The results of LTP test : The f EPSP amplitudes of the control group were(174.6±6.9) %,(152.4±11.1) %,(146.1±3.2) % at 1, 30, and 60 min after HFS, respectively, and those in the z-DEVD- fmk group were(170.3±3.6)%、(147.8±8.6)%and(143.2±4.6)%at 1, 30, and 60 min post HFS, respectively, no significant difference between the the two groups(P >0.05). The f EPSP amplitudes in 50 m M Al(mal)3 group were(130.6±5.1)%,(108.0±5.2)% and(105.2±6.1)%at 1, 30, and 60 min post HFS, respectively, significantly suppressed compared with the controls(P <0.05). Al- induced suppression of LTP was partially, but significantly(p < 0.05), reversed by z-DEVD-fmk in the z-DEVD- fmk + 50 m M Al(mal)3 group, the average standardized f EPSP amplitudes were 149.2 % ± 2.3 %, 133.0 % ± 10.3 %, and 126.6 % ± 10.4 % at 1, 30, and 60 min post HFS, respectively.The results of Western-blot assay:(1) compared with the control group, we did not find any significant change in the protein concentrations of Glu R1 and Glu R2 in either the total or membrane-enriched extracts following z-DEVD- fmk injection(P >0.05). However, z-DEVD- fmk reversed the decrease of Glu R1 and Glu R2 in both the total and membrane-enriched extracts in the z-DEVD- fmk + 50 m M Al(mal)3 group, compared with the 50 m M Al(mal)3 group. By Analysis of the ratio of membrane protein and total protein of Glu Rl and Glu R2, we found that the ratios of Glu Rl and Glu R2 in membrane protein and in total protein significantly decreased by 20.22%, 26.25% in the Al group compared with the control group(P <0.05), but significantly increased by 26.76%, 62.71% in z-DEVD-fmk +A1 group compared with the Al group(P <0.05).(2) The protein expression of active caspase-3 increased gradually with increasing of Al(mal)3 dosage, statistically significant in 20,40 μM Al(mal)3 groups compared with the control groups(P <0.05).The protein expression of Akt decreased significantly in 20,40 μM Al(mal)3 groups compared with the control group(P <0.05).The protein expressions of p-GSK-3β(Ser9) in 20,40 μM Al(mal)3 groups were statistically different compared with the control group(P <0.05). By further analysis of the ratio of p-GSK-3β(Ser9)and GSK-3β, we found that the ratio of p-GSK-3β( Ser9) and GSK-3β declined dose-dependently, significantly decreased in 40 μM Al(mal)3 group, compared with the control group(P <0.05).4. Effect of GSK-3β inhibitor SB216763 on impairment of hippocampal LTP induced by aluminumThe results of LTP test:The f EPSP amplitudes in the SB216763 group were(187.9±17.1)%,(165.0±18.7)% and(157.6±13.7)%at 1, 30, and 60 min post HFS, respectively, higher than(174.8±6.1)%,(150.6±10.3)% and(146.0±5.9)% at 1, 30, and 60 min after HFS in the control group, but not significantly(P >0.05). The f EPSP amplitudes in 50 m M Al(mal)3 group were(130.6±5.1) %,(108.0±5.2) % and(105.2±6.1)% at 1, 30, and 60 min post HFS, respectively, significantly suppressed at 1, 30, and 60 min post HFS, compared with the controls(p <0.05). Al- induced suppression of LTP was significantly(P < 0.05) reversed by SB216763 in the SB216763 + 50 m M Al(mal)3 group, the average standardized f EPSP amplitudes were(157.7±2.1) %,(136.0±16.1) and(128.0±15.3) % at 1, 30, and 60 min post HFS, respectively.The results of Western-blot assay:(1) Compared with the control group, we did not find any significant change in the protein concentrations of Glu R1 and Glu R2 in either the total or membrane-enriched extracts following SB216763 injection(P > 0.05). However, SB216763 reversed the decrease of Glu R1 and Glu R2 in both the total and membrane-enriched extracts in the SB216763+50m M Al(mal)3 group, compared with the 50 m M Al(mal)3 group(p <0.05). By Analysis of the ratio of membrane protein and total protein of Glu Rl, Glu R2, we found that the ratios of Glu Rl, Glu R2 in membrane protein and in total protein significantly decreased by 26.76%,18.28% in Al group compared with the control group(p <0.05), and the ratios of Glu Rl, Glu R2 in membrane protein and in total protein significantly increased by 23.19%, 27.63% in SB216763+A1 group compared with Al group(p <0.05)(2) SB216763 induced significant changes in p GSK-3β protein level compared with the control group(p <0.05), the expressions of p GSK-3β in the SB216763+50m M Al(mal)3 group were significantly higher than in the 50 m M Al(mal)3 group(p < 0.05). Conclusions: ·Subchronic Al exposure and i.c.v. injection of Al obviously suppressd the LTP in hippocampal CAl region in rat in vivo in a dose-dependent manner, induced total protein level of AMPA receptor subunits decrease, reduced the expression of Glu R1 and Glu R2 subunit on the surface of neurons, and facilitated Glu R1 and Glu R2 subunit removal from the synaptic site. ·The result of i.c.v. injection model was consistent with that of intraperitoneal injection model, intraperitoneal injection model can be replaced by the way of i.c.v. injection in order to achieve precise doses and to reduce the interference caused by absorption, metabolism and other factors. ·The molecular mechanism of Al- induced LTP impairment might be related to the activation of caspase-3, cleavage of Akt, activation of GSK-3b, and inhibition of the externalization of AMPAR. |
PDF Full Text Request