Effects Of Sub-chronic Exposure To Aluminum Of Abeta Production And Its Role In Aluminum-induced Neurobehavioral Change

Part I Study on the effects of sub-chronic exposure to aluminum on neurobehavior and its mechanism in ratsAlinninum(Al), the most abundant metal and the third largest chemical element in the earth crust,is widely used in our day-to-day life and in the course of industrial production and gains an easy access to our body through using of drinking water, foods, cooking utensils, some antacids, Al-containing dusts and fumes. Al is a potent neurotoxicant and has been proposed as one of the critical environmental factors in several neurodegenerative diseases such as Alzheimer's disease (AD). The role of Al in AD remains controversial, which may be related with the chemical species of Al and the chemical form of the metal ion at neutral pH. Aluminum-mallolate complex [Al(mal)3] is a electroneutral complex of Al. which forms a metastable solution and can deliver a significant amount of free aqueous Al at physiological pH. Behavioural changes may be an monitoring index of neurotoxicity and may be observed early during the exposure to Al. Long term potentiation(LTP) is widely used as a well-known model to investigate the cellular mechanisms underlying some kinds of learning and memory. Al accumulation in both the brain and serum is assumed to impair learning and memory, lead to dementia-like symptoms. Changes in the structure of the nervous system is the basis of Al neurotoxicity. In the section, neurological damage model was established by sub-chronic intraperitoneal injections of Al(mal)3in rats. The neurotoxicity of Al was evaluated by neuroethology, electrophysiology. histopathology and the level of Al in both the brain and serum.Chapter I Effect of sub-chronic exposure to aluminum on neurobehavior in ratsObjective To expose the neurotoxicity of Al(mal)3. Methods Behavior screening on two months old Sprague Dawley(SD) male rats was carried out by Morris water maze(MWM) and open-field test(OFT). Thirty two male SD rats were divided randomly into four subgroups according to the body weight:saline group,10,20. and40μM/kg Al(mal)3treated groups. Over two months, rats in the saline group received daily intraperitoneal (i.p.) injections0.9%saline, rats in the10,20, and40μM/kg Al(mal)3groups received i.p. administrations of these three doses, respectively. After exposure, the MWM, step-down test(SDT), step-through test(STT) were performed to examine the learning and memory abilities of rats, the OFT was performed to exmine the general activities of rats. Subsequently, LTP in hippocampus was measured. Al concentration in both cerebral cortex and blood were determined by graphite furnace atomic absorption spectrometry. The morphological changes of hippocampus were observed by optical microscope and electron microscopy. Results In the same day, escape latencies in the learning acquisition test of Al(mal)3groups were relatively prolonged when compared to those of the saline group, but there was no statistically significant difference (P>0.05). In the probe trail, the time of finding platform site of20,40μM/kg Al(mal)3groups increased progressively compared to those of saline group (P <0.05), the average number of crossings of the platform site was significantly reduced in the Al(mal)3group compared to those of saline group (P<0.05). There were significantly correlationship between the dose of Al exposure and the time of finding the platform site (r,=0.664, P<0.05), Al exposure dose and the average number of crossings of the platform site (rs=-0.64l. P<0.05). In the SDT. the significant decrease was found in the mean latency time of all Al(mal)3groups when compared with those of saline group (P<0.05). while errorcountland2were increased significantly in Al(mal)3groups, there were obvious significance in all Al(malb groups, except errorcount2in10uM/kg Al(mal)3groups, compared to the saline group (P<0).05). There were correlationship between the dose of Al exposure and errorcountl of SDT (rs=0.663. P<0.01). Al exposure dose and errorcount2of SDT (rs=0.943. P <0.01). The latency time of SIT in Al(mal)3exposed rats decreased gradually, while the errorcount1and2was increased significantly along with the increase of Al dosage. There were obvious significance in all Al(mal)3groups when compared to the saline group (P<0.05). Statistical analysis confirms that a correlationships between the dose of Al exposure and errorcount1(rs=0.789. P<0.01), the dose of Al exposure and latent period of STT (rs=-0.708. P<0.01), furthermore, the dose of Al exposure and errorcount2(rs=0.466, P<0.05). The retention time in the centre of the arena was significantly longer, while The total number of squares crossed and the total numbers of rearings in OFT were decreased significantly along with the increase of Al dosage, except the total numbers of rearings in10μM/kg Al(mal)3groups (P <0.05). Conclution Sub-chronic exposure to Al(mal)3has a significant injury of the nervous system in rats.Chapter Ⅱ Effect of sub-exposure to aluminum on LTP in hippocampal CA1area in ratsObjective To investigate the effects of Al(mal)3on the hippoeampal Long-term potentiation (LTP) in vivo. Methods Animals and treatments were same with chapter I. After the behavioral investigations, four rats were randomly elected from every group, LTP of the Schaffer collateral-CA1pathway was recorded. Results Compared with saline group, a significant suppression of LTP by Al(mal)3was found. Although the average standardized fEPSP amplitude at the beginning did not obviously change, the values gradually decayed towards the baseline during1h of recording. At10min,20min,30min,40min,50min and60min after HFS, the average standardized fEPSP amplitude in all of Al(mal)3groups, except10μM/kg Al(mal)3group at10min after HFS, were significantly decreased compared to those in saline group (P<0.05). Conclution The result indicates that the Al(mal)3induced impairment of LTP in hippocamcal CA1of rats.Chapter III Effect of sub-exposure to aluminum on morphological structure of hippocampus in ratsObjective To investigate the effects of Al(mal)3on morphological structure of hippocampus in rats. Methods Animals and treatments were same with chapter Ⅰ. Morphological structure of hippocampus was observed with light microscope and electron microscope. Results The observation by optical microscope showed:arrangement of the neurocytes in rats'hippocampus was orderly in control group, but with the increasing of the doses of Al exposure, the neurocytes displayed disorderly, the connections among the neurocytes were loosened, the karyon shrinked, some rings around the neurocyte were observed. Picture by electron microscope displayed:the karyotin distributed symmetrically, and its form was regular, the karyon was unbroken, and with the increasing of the doses of Al exposure, the karyon shrinked, the karyotin assembled in the edge, karyotheca was broken. Conclution The result indicates that the Al(mal)3can cause obviously serious damage of hippocampal formation.Chapter Ⅳ Effect of sub-exposure to aluminum on Al contents in both cerebral cortex and blood in ratsObjective To investigate the effects of Al(mal)3on Al contents in both cerebral cortex and blood. Methods Animals and treatments were same with chapter Ⅰ. After the LTP were recorded. Al contents in both cerebral cortex and blood were assayed by graphite furnace atomic spectrophotometry. Results Al contents in both cerebral cortex and blood in Al(mal)3exposed groups were increased significantly along with the increased of Al(mal)3exposure dosage, which was significantly higher than those in saline group(P<0.05). Conclution The result indicates that the Al(mal)3can cause obviously Al accumulation.Part II Effect of exposure to aluminum on the production of amyloid protein in vitro and in vivo AD, a devastating neurodegenerative disease, which is clinically characterized by the progressive memory loss, cognitive impairment, personality changes, and is pathologically characterized by senile plaques (SPs), intraneuronal neurofibrillary tangles (NFTs), and severe neuronal loss. Aβ, a39to43amino acid peptide, is not only a major component of senile plaque but also the biochemical basis of NFTs and vascular amyloidosis. A(3, generated by proteolytic cleavage of the amyloid precursor protein(APP), plays a pivotal role in the pathogenesis of Alzheimer's disease (AD). the excessive producation and accumulation of Aβ may be the common way to AD induced by various reasons. Walton et al. founded that the gene expression of APP in the hippocampal and cortical tissue was elevated, when rats were chronically ingested Al in amounts equivalent to total dietary Al levels that Americans routinely ingest. Al may thus launch the cascade that results in the formation of amyloid plaques. It was reported that long-term exposure to sublethal levels of aluminium chloride (50μM) for more than3weeks caused depositions immunopositive to Aβ in cultured neurons. Al can promote the structural changes of Aβ from a-helix to β-sheet, accelerate Aβ generation and aggregation, increase the stability of Aβ oligomers. Some studies suggested that Al may cause Aβ increased in experimental animals by promoting directly the synthesis of Aβ. but its exact mechanism is unclear. A disintegrin and metalloproteinase10(ADAM10) is the constitutive α-sccretase.β-site AβPP cleaving enzyme (BACE1) is thought to be the major β-secretase involved in Aβ production in the brain.The BACE1enzyme is essential for the generation of Aβ. BACE1cleavage of APP is a prerequisite for Aβ formation and is putatively the rate-limiting step in Aβ genesis. Presenilin1(PSI) are the crucial catalytic components of y-secretase.γ-cleavage can yield both AP40and AP42. AP42is more hydrophobic and more prone to fibril formation than others, and is also the predominant isoform found in cerebral plaques in AD. In this section, we will test the protein and gene expression of APP. ADAM10, ADAM17, ADAM9, BACE1and PS1in brain in rats and in PC12cell, respectively. At the same time, BACE1activity and Aβ40and Aβ42content was determined to explore the effects of Al on the metabolism of APP.Chapter I Effect of sub-chronic exposure to aluminum on the production of amyloid protein in ratsObjective To expose the effect of Al(mal)3on the catabolism of amyloid precursor protein in rats. Methods Animal and treatment were same with those of part Ⅰ. After exposure, the protein and mRNA expression of ADAM10. ADAM17, ADAM9. BACE1, PS1were detected by quantitative PCR and ELISA in cerebral cortex. The BACE1activity were detected by β-secretase activity assay kit. The protein expression of Aβ40and Aβ42in cerebral cortex were detected by ELISA. Results The protein and mRNA expression of APP were increased gradually with increasing Al(mal)3dosage. The protein and mRNA expression of APP in40μM/kg AI(mal)3-exposed group were significantly higher than those of the saline group (P<0.05). The protein and mRNA expression of ADAM10, ADAM17, ADAM9in40μM/kg Al(mal)3-exposed group were significantly higher than those of the saline group (P<0.05). The protein expression of BACE1in20,40μM/kg Al(mal)3-exposed groups were significantly increased when compared to those of the saline group (P<0.05). The mRNA expression of BACE1in20,40μM/kg Al(mal)3-exposed group was significantly higher than those of the saline group (P<0.05). When compared with the saline group, the enzyme activity of BACE1it20,40μM/kg Al(mal)3-exposed groups were significantly increased(P<0.05). The protein expression of PS1were increased gradually with increasing Al(mal)3dosage. The protein expression of PS1in all Al(mal)3-exposed group were increased significantly than those in saline group(P<0.05). The mRNA expression of PS1in40μM/kg Al(mal)3-exposed group was significantly higher than those of the saline group (P<0.05). The protein expression of Aβ40were decreased gradually and the protein expression of Aβ42were increased gradually with increasing Al(mal)3dosage. The protein expression of Aβ40in20,40μM/kg Al(mal)3-exposed groups were significantly decreased when compared to those of the saline group (P<0.05). The protein expression of Aβ42in all Al(mal)3-exposed group were significantly increased when compared to those of the saline group (P<0.05). Conclution Aluminum can lead to an increase in the protein expression of Aβ42by enhancing the amyloid hydrolysis of APP and inhibiting the non-amyloid hydrolysis of APP in rats.Chapter II Effect of exposure to aluminum on the production of amyloid protein in PC12cellsObjective To expose the effect of Al(mal)3on the catabolism of amyloid precursor protein in PC12cells. Methods Rat pheochromocytoma cell line(PCl2cells) were incubated in culture media with Al(mal)3at final concentration of0,50,100,200,400μM for12h.24h. and48h, respectively. The protein expression of APP, ADAM10, ADAM17, ADAM9, BACE1, PS1, A(340, and A(342were determined by ELISA. The mRNA expression of APP. ADAM10, ADAM17. ADAM9, BACEI, PS1were detected by quantitative PCR. The enzymatic activity of BACE1were determined by ELISA. Results Treatment of PC12cells with200μM.400μM Al(mal)3for12h,24h and48h did show a significant increase in the protein and mRNA expression of APP except the protein expression of APP at200μM/kg AI(mal)3group for24h (P<0.05). Al(mal)3can increase the protein and mRNA expression of BACE I in PC12cells. When the PC12cells were treated with Al(mal)3for12h.24h and48h. the protein and mRNA expression of ADAM10in50,100,200,400μM Al(mal)3groups were significantly decreased when compared to those of control groups(P<0.05). The protein and mRNA expression of ADAM17, ADAM9in PC12cells were treated with400μM Al(mal)3for24h and48h were significantly decreased when compared to those of control groups(P<0.05). The protein and mRNA expression of BACE1in PC12cells were treated with400μM Al(mal)3for12h,24h and48h were significantly increased when compared to those of control groups(P<0.05). The protein expression of Aβ40in PC12cells were decreased gradually and the protein expression of Aβ42in PC12cells were increased significantly along with the increase of Al(mal)3dosage and exposed time(P<0.05). Conclution Results in vitro experiment indicated that Al(mal)3can accelerate the amyloidogenic processing of APP, at the same time, inhibit the non-amyloidogenic processing of APP.Part III Effect of exposure to aluminum on Reelin-ApoER2/VLDLR-Dabl in RatsThe excess expression of Aβ in brain is a major factor in the pathogenesis of Alzheimer's disease (AD). A(3is produced through a sequential cleavage of APP by (3-secretase and y-secretase. Some studies suggested that APP and β-secretase, both present on cell surface, are endocytosed into endosomes to produce Aβ. The molecular mechanism by which neurons trigger the production of Aβ is poorly understood.Reelin is an extracellular glycoprotein that binds to the transmembrane receptors apolipoprotein-E receptor type2(ApoER2) and very low density lipoprotein receptor (VLDLR). which transduce the Reelin signal through the intracellular adapter Disabled-1(Dabl). Reelin signaling triggers a Dab-1-dependent signaling cascade involving several kinases, which ultimately controls proper neuronal migration and positioning during CNS development. Furthermore, the involvement of the Reelin signaling pathway in neurodegeneration has also been proposed. In recent years, increasing evidence supports a link between Reelin and components of its signaling pathway with the main hallmarks of AD pathology. Recent study indicate that Reelin is present in β-amyloid plaques in a transgenic mouse model of AD. Dabl increases cell surface expression of APP. increases α-cleavage of APP, and decreases Ap in transfected cells and in primary neurons, and this effect is influenced by Reelin. Reelin treatment significantly increases co-immunoprccipitation of Dabl with APP. Moreover, Reelin decreases production of the Aβ. Therefore. Reelin and components of its signaling pathway may participate in APP trafficking and processing. The certain mechanism is largely unknown. In this part, the expression of Reelin and components of its signaling pathway were detected by qRT-PCR. when APP abnormal metabolic process were induced by Al (mal)3in vivo and in vitro. Chapter Ⅰ Effect of sub-chronic aluminum exposure on the expression of Reelin and components of its signaling pathway in ratsObjectsive To expose the effect of sub-chronic exposure to Al(mal)3on the gene expression of Reelin and components of its signaling pathway in rats. Methods Animal and treatment were same with Part Ⅰ. Results The mRNA and protein expression of Reelin decreased gradually with increasing of Al(mal)3doseage. The mRNA and protein expression of RELN in10,20,40μM Al(mal)3groups had statistically significant when compared with those of control groups(P<0.05). The mRNA expression of VLDLR increased significantly but the protein expression of VLDLR decreased significantly in20,40μM Al(mal)3groups when compared with those of control groups(P<0.05). The mRNA expression of ApoER2in all Al(mal)3groups decreased significantly when compared with those of saline groups(P<0.05). The protein expression of ApoER2in40μM Al(mal)3groups decreased significantly when compared with those of saline groups(P<0.05). The mRNA expression of Dabl in all Al(mal)3groups decreased significantly when compared with those of control groups(P<0.05). the protein expression of Dabl decreased significantly in20.40μM Al(mal)3groups when compared with those of control groups (P<0.05). Conclution Sub-chronic exposure to Al(mal)3had a significant effect on the protein and mRNA expression of Reelin and components of its signaling pathway in rats, which may be related to the abnormal composition of Aβ induced by Sub-chronic exposure to Al(mal)3.Chapter Ⅱ Effect of aluminum exposure on the expression of Reelin and components of its signaling pathway in PC12cellsObjectsive To expose the effect of Al(mal)3exposure on the protein and gene expression of Reelin and components of its signaling pathway in PC12cells. Methods PC12cells were incubated in culture media with Al(mal)3at final concentration of0,50,100,200,400μM for12h,24h, and48h, respectively. The protein and gene expression of Reelin, VLDLR, ApoER2, and Dab1were detected by ELISA and qRT-PCR, respectively. Results The protein and gene expression of Reelin decreased gradually with increasing Al(mal)3dosage. The protein and gene expression of Reelin in PC12cells which exposured to200,400μM Al(mal)3for12h,24h. and48h increased significantly when compared with those of control groups(P<0.05). The protein and gene expression of VLDLR in PC12cells treated with200,400μM Al(mal)3for12h,24h, and48h were decreased obviously(P<0.05). Treatment of PC12cells with50,100.200.400μM Al(mal)3for12h,24h and48h did show a significant decrease in the protein and mRNA expression of ApoER2(P<0.05). except the protein expression of ApoER2at50μM/kg AI(mal)3for24h (P>0.05). The mRNA and protein expression of Dabl in PC12cells treated with50,100,200,400μM Al(mal)3for24h,48h were decreased significantly when compared with those of control groups(P<0.05). Conclution The exposure to Al(mal)3had a significant effect on the protein and gene expression of Reelin and components of its signaling pathway in PC12cells.Conclusions:1. The excessive production of Aβ induced by sub-chronic exposure to aluminum may be play a major role in aluminum-induced learning and memory disorder, and several neurodegenerative diseases, particularly AD. Its main mechanism is Al accelerates amyloidiosis pathway of APP through promoting the expression of BACE1and PS1, at same time, Al inhibits nonamyloidosis pathway through reducing the expression of ADAM10, ADAM17, and ADAM9.2. Reelin signal transduction pathways may be involved in the adjustment of the abnomal hydrolysis of APP and the excessive production of Aβ induced by Al (mal)3.