Study On The Protective Action Of Anti-Aβ_31-35 Antibody On The Rat Hippocampal Long-Term Potentiation, Learning And Memory Behavior And Cultured Cortical Neurons

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive impairment of learning, memory and unrelenting cognitive decline. AD not only seriously affects the health, life expectancy and the quality of life in the aged, but also brings a huge burden to the family wotj AD patients and the society. The typical pathological feature of AD is the presence of high density amyloid plaques in the brain. A primary component of the amyloid plaques is amyloid-β(Aβ) protein, a short natively unfolded protein with 39⁴2 amino acids, which aggregates into a number of soluble oligomers, protofibrils, and insoluble fibrils. All of these various forms of Aβseem to be neurotoxic. Although the toxicity of Aβhas been widely reported, the underlying mechanism and the shortest active fragment of Aβis not well known. Especially, it is still seriously lack of the effective measures for relieving Aβtoxicity and the symptoms of AD.The amyloid hypothesis of AD has promoted studies on a variety of therapeutic strategies aiming to clear Aβin the brain. Although the booming immunotherapy for AD in recent years has achieved some exciting results, active immunization with Aβor its different peptides has a typical disadvantage including the generation of very robust immune response, particularly cellular immune response that may not be desirable in patients suffering from AD. Reasonable passive immunization with anti-Aβantibody is safer than active immunization, because it avoids cellular immune response. Although conventional administration of anti-Aβantibody risks the adverse inflammatory reactions and cerebral hemorrhaging, there are some researches showed that using intracerebroventricular (ICV) passive immunization with anti-Aβantibody reduced cerebral plaques and did not trigger inflammation and microhemorrhage in the brain.According to our recent studies, the sequence 31-35 in full length of Aβmight be the active center of Aβtoxicity, which can induce apoptosis in cultured cortical neurons; suppress delayed redtifying potassium channels and large conductance Ca2+-activated potassium channels on hippocampal membrane patches and depress the hippocampal long term potentiation (LTP), a very useful electrophysiological model of learning and memory. Therefore, the present study, on the basis of making a specific antibody against the sequence 31-35 of Aβ, investigated the effects of Aβ_1-42 and anti-Aβ_31-35 antibody on HFS-induced hippocampal LTP, the spatial learning and memory behaviour and cultured cortical neurons of rats. Especially, we further investigated whether anti-Aβ_31-35 antibody can inhibit Aβ_1-42-induced impairment in hippocampal LTP, learning and memory as well as cultured cortical neurons of rats.Partâ… : Anti-Aβ_31-35 Antibody Protects against Aβ_1-42-Induced Suppression of Long-Term Potentiation in Rat Hippocampal CA1 Region in vivoThis electrophysiological study was designed to observe the effects of Aβ_1-42 and anti-Aβ_31-35 antibody on baseline fEPSPs and HFS-induced LTP in hippocampal CA1 region, and further to investigate the effects of co-application of anti-Aβ_31-35 antibody and Aβ_1-42 on hippocampal LTP by acute i.c.v. injection drugs into urethane anesthetized rats.The results showed that: (1) Aβ_1-42 significantly suppressed HFS-induced LTP, but did not affect the basal synaptic transmission. In control rats (only normal saline), the amplitude of the fEPSPs was increased to 213.7±6.0%, 175.9±6.3 and 166.3±7.1% at 1, 30 and 60 min post-HFS, respectively, indicating a successful LTP induction. After intracerebralventricular (i.c.v) administration of Aβ_1-42, the average amplitude of the fEPSPs decreased to 162±7.6%, 126.5±6.7% and 114±5.4%, respectively, being significantly lower than those in control group (p<0.01). (2) In anti-Aβ_31-35 antibody alone group, both the baseline fEPSPs and the HFS-induced LTP did not change. After i.c.v. injection of 5 nmol anti-Aβ_31-35 antibody, the average amplitude of fEPSPs after HFS increased to 220.6±7.4%, 177±5.9% and 166.1±7.0%, at 1, 30 and 60 min post-HFS, respectively, without any significant difference compared to control rats (p>0.05). (3) Importantly, pretreatment with anti-Aβ_31-35 antibody avoided the LTP suppression induced by Aβ_1-42 in a dose-dependent manner. co-injection of 0.05 nmol anti-Aβ_31-35 antibody and Aβ_1-42 almost did not produce any effect on the impairment of LTP induced by Aβ_1-42, and the average amplitude of fEPSPs were 167±6.3%, 126.6±4.9% and 112±6.7% at 1, 30 and 60 min following HFS, respectively, without any significant difference when compared with those of Aβ_1-42 only group (p>0.05); after administration of 0.5 nmol anti-Aβ_31-35 antibody produced a moderate reversal of the Aβ_1-42-induced suppression of LTP, with an average fEPSP amplitude of 190.3±7.5%, 155.8±7.5% and 142.6±5.9% at 1, 30 and 60 min post-HFS, respectively, significantly higher than those in Aβ_1-42 group (p<0.05), but still lower than those of 5 nmol anti-Aβ_31-35 antibody group (p<0.05); further,i.c.v. injection of 5 nmol of anti-Aβ_31-35 antibody produced a complete protection against the suppression of LTP induced by Aβ_1-42, and the LTP values were 225±9.3%, 180.6±7.7% and 163.3±6.5% at 1, 30 and 60 min following HFS, respectively, being significantly higher than the values (162±7.6%, 126.5±6.7% and 114±5.4%) in Aβ_1-42 alone group (p<0.01). Morover, there is a significant statistical difference between these co-application groups (p<0.01 between 0.1 nmol and 5 nmol; p<0.05 between 0.1 nmol and 0.5 nmol). (4) Both Aβ_1-42 and anti-Aβ_31-35 antibody did not affect PPF. The quantified data of PPF ratio indicated that there was no significant difference (p>0.05) between the PPF values calculated before and after i.c.v. injection of vehicle, Aβ_1-42, anti-Aβ_31-35 antibody and co-application of anti-Aβ_31-35 antibody and Aβ_1-42.These results demonstrate that 31-35 sequence may be a shorter active fragment of Aβ; anti-Aβ_31-35 antibody can inhibit the Aβ_1-42-induced suppression of LTP, probably through specifically combining to and shielding the active center of Aβ. The present study also suggests that 31-35 sequence may be a new therapy target for AD, and anti-Aβ_31-35 antibody will be probably a more effective and safer strategy in the immunotherapy of AD.Partâ…¡: The Effects of Anti-Aβ_31-35 Antibody on the Aβ_1-42-Induced Impairment of Spatial Learning and Memory in RatsThe present study, by using Morris water maze test, investigated the effects of Aβ_1-42 and anti-Aβ_31-35 antibody as well as co-application of these two drugs on the spatial learning and memory of rats. The escape latencies, swim distance in hidden platform tests, and target quadrant preference in probe trials were monitored by a CCD camera connected to computer and analiezd by Ethervision software system.The results showed that: (1) Aβ_1-42 impaired the spatial learning and memory of rats. i.c.v. injection of 5 nmol Aβ_1-42 resulted in a significant decline in spatial learning, with longer latencies and distances for searching for the platform under water, being 93.1±6.1 s, 79.6±5.8 s, 60.3±6.0 s and 45.5±6.5 s as well as 2185.1±143.2 cm, 1868.2±136.1 cm, 1415.2±140.8 cm and 1067.9±152.6 cm, at 1, 2, 3 and 4 training day, respectively, significantly longer (p<0.01) than 62.5±5.9 s, 41.7±6.5 s, 22.4±6.0 s and 9.8±5.5 s as well as 1609.0±161.9 cm, 1073.8±167.4 cm, 576.8±154.5 cm and 252.4±141.6 cm in control; Aβ_1-42 also impaired the spatial reference memory as indicated by the reduced total time and distance swam in the previous target quadrant. The percentage of total time elapsed and distance swam in right quadrant were 37.6±2.3% and 35.6±4.0%, respectively after injection of Aβ_1-42, significantly lower (p<0.01) than 50.2±3.1% and 49.8±2.9% in control group. (2) Anti-Aβ_31-35 antibody itself had no effects on the spatial learning and memory of rats. After i.c.v. injection of 5 nmol anti-Aβ_31-35 antibody, the escape latencies were 67.4±5.6 s, 46.7±6.0 s, 24.6±6.3 s and 13.9±5.5 s, the distances swam were 1760.5±146.3 cm, 1219.8±156.7 cm, 642.6±164.6 cm and 363.1±143.7 cm at 1, 2, 3 and 4 training day, respectively, similar to the values in control (p>0.05). The percentage of total time elapsed and distance swam in the target quadrant were 48.5±2.8% and 50.0±3.3%, also equivalent (p>0.05) with those in control group. (3) Larger dosage of anti-Aβ_31-35 antibody inhibited the spatial learning and memory impairment induced by Aβ_1-42. After co-application of 0.05 nmol anti-Aβ_31-35 antibody and Aβ_1-42, the escape latencies and distances swam were 98.3±5.9 s, 75.8±6.4 s, 59.3±6.6 s and 40.4±6.0 s, 2325.0±148.6 cm, 1958.6±161.2 cm, 1257.2±166.2 cm and 1017.3±151.1 cm at 1, 2, 3 and 4 training day, respectively; the percentages of total time elapsed and distance swam in the target quadrant were 38.4±2.0% (time) and 36.0±3.0% (distance), respectively, without significant differences (p>0.05) compared with those of Aβ_1-42 group. However, 0.5 nmol or 5 nmol anti-Aβ_31-35 antibody inhibited the spatial learning and memory impairment induced by Aβ_1-42. The escape latencies and distances swam were 64.0±6.3 s, 43.2±5.9 s, 27.1±6.5 s and 11.9±5.4 s as well as 1663.0±173.6 cm, 1190.6±162.6 cm, 746.9±179.1 cm and 328.0±148.8 cm at 1, 2, 3 and 4 training day, respectively in 0.5 nmol anti-Aβ_31-35 antibody group, and the corresponding values were 59.4±6.5 s, 40.8±5.7 s, 28.7±7.0 s and 13.0±6.0 s as well as 1543.4±216.0 cm, 1091.44±162.6 cm, 697.4±170.1 cm and 315.9±145.8 cm respectively, in 5 nmol anti-Aβ_31-35 antibody group, significantly shorter than 0.05 nmol group (p<0.01) and Aβ_1-42 only group (p<0.01). The percentage of total time elapsed and distance swam in the target quadrant were 51.3±1.4% (time) and 51.6±2.5% (distance) in 0.5 nmol anti-Aβ_31-35 antibody group, 49.0±3.0% (time) and 48.7±1.8% (distance) in 5 nmol anti-Aβ_31-35 antibody group, the values in both groups significantly larger than that in 0.05 nmol group (p<0.01) and in Aβ_1-42 only group (p<0.01), and nearly equivalent to that in control group. (4) All drugs in the present experiment did not affect both the vision and the swimming speeds of rats.These data indicated that Aβ_1-42 but not anti-Aβ_31-35 antibody resulted in a significant decline in spatial learning and memory, and the larger dosage of anti-Aβ_31-35 antibody protected the spatial learning and memory against Aβ_1-42-induced impairment, suggesting that 31-35 sequence may be the shorter active fragment of Aβneurotoxicity and a new therapy target. Anti-Aβ_31-35 antibody will be probably a new more specifical and more effective anti-Aβantibody in the treatment of AD.Partâ…¢: Anti-Aβ_31-35 Antibody Protects Cultured Cortial Neurons against Aβ_1-42-Induced CytotoxicityThe present experiment examined the effects of Aβ_1-42 and anti-Aβ_31-35 antibody on cultured cortical neurons, and further investigated anti-Aβ_31-35 antibody whether can inhibit Aβ_1-42-induced cytotoxicity as indicated by the decline of cell viability (CCK-8 reduction), the increase of LDH release and the decrease of living cells.The results showed that: (1) anti-Aβ_31-35 antibody itself had not effects on cultured cortical neurons but significantly inhibited Aβ_1-42-induced neurotoxicity on cultured cortical neurons in a dose-dependent manner. 20μM Aβ_1-42 but not anti-Aβ_31-35 antibody exhibited obvious cytotoxicity, with only 42.6±4.7% (p<0.01) of cell viability (%control) in Aβ_1-42 group compared to 100% in control group and 97.3±3.9% in anti-Aβ_31-35 antibody alone group. However, after co-incubation of different concentrations (5, 10 and 20μM) of anti-Aβ_31-35 antibody and Aβ_1-42 for 24 h, 5μM anti-Aβ_31-35 antibody showed a little protective action, the cell viability being 48.7±5.2%, but without no statistical differences (p>0.05) compared with Aβ_1-42 group; 10μM anti-Aβ_31-35 antibody showed significant neuroprotection against Aβ_1-42-induced insults on cultured cortical neurons, the cell viability being 66.8±3.5%, which is higher than the values in Aβ_1-42 alone group (p<0.05); 20μM anti-Aβ_31-35 antibody showed nearly complete protection, with a 98.5±4.1% of cell viability, significantly higher than Aβ_1-42 alone (p<0.01) and very similar to that in control (p>0.05). (2) anti-Aβ_31-35 antibody inhibited Aβ_1-42-induced increase of LDH release in a dose-dependent manner. Cells treated with 20μM Aβ_1-42 in absence of anti-Aβ_31-35 antibody for 24 h showed a high LDH release, the LDH value (% control) being 157.1±3.6% (p<0.01), while in anti-Aβ_31-35 antibody alone group, it was 103.5±4.2% (p>0.05). But, after treatment with 5, 10 and 20μM anti-Aβ_31-35 antibody, the values of LDH release decreased to 150.2±4.5% (p>0.05), 123.9±4.0% (p<0.05) , and 101.9±5.1% (p<0.01), respectively, (3) anti-Aβ_31-35 antibody inhibited Aβ_1-42-induced decrease of living cells in a dose-dependent manner. After treatment with 20μM Aβ_1-42 for 24 h, the cells survival rate (%) significantly decreased to 41.0±4.0% (p<0.01) from 99.1±0.8% of control. Administration of 20μM anti-Aβ_31-35 antibody for 24 h did not alter the cells survival rate, being of 98.2±1.5% (p>0.05). However, anti-Aβ_31-35 antibody inhibited Aβ_1-42-induced decrease of living cells. In the presence of 5, 10 and 20μM anti-Aβ_31-35 antibody, Aβ_1-42-induced cells survival rates were 45.3±4.2%, 63.4±2.9% and 98.8±1.0%, respectively. All of three concentrations of anti-Aβ_31-35 antibody, except to 5μM group (p>0.05), significantly increased the cell survival rate (p<0.05 in 10μM; p<0.01 in 20μM) compared with Aβ_1-42 alone.These data indicate that anti-Aβ_31-35 antibody itself does not exhibit cytotoxicity on cultured cortical neurons, but can inhibite Aβ_1-42-induced cytotoxicity in a dose-dependent manner, implying 31-35 sequence may be an effective therapeutic target in the prevention and treatment of AD by using immunotherapy.In conclusion, the present study, by using field potential recording, Morris water maze test, cell viability assays and confocal imaging technique, observed the effects of Aβ_1-42, anti-Aβ_31-35 antibody and co-application of these two drugs on hippocampal LTP in vivo, the spatial learning and memory behavior and cultured cortical neurons of rats. The results reveal that anti-Aβ_31-35 antibody itself has no effects on the physiological activities observed in the present study, but can inhibit Aβ_1-42-induced impairment on hippocampal LTP, learning and memory, as well as cultured cortical neuron survival, indicatting that 31-35 sequence may be the shortest active center of Aβresponsible for all neurotoxicity of Aβ, and suggesting that anti-Aβ_31-35 antibody will be probably a more specific, more effective, and more safer antibody alternative for the AD treatment in the future.