Physiological Amyloid-beta Clearance In The Periphery And Its Therapeutic Potential For Alzheimer’s Disease

BackgroundAlzheimer’s disease is an aging closely related neurodegeneration with the clinical features including progressive cognitive decline and social dysfunction. However, the current clinical medicines are only symptomatic treatments due to AD pathogenesis which is not fully elucidated. Amyloid β (Aβ) plays a pivotal role in AD pathogenesis, of which the disruption of the balance between the production and clearance has been considered to be the main cause of the sporadic AD which accounts for approximately 99% of AD. The clearance of Aβ includes central clearance and peripheral clearance. The central clearance has been investigated well, nevertheless, a serial of clinical trials for the anti-Aβ antibodies based on the central clearance theory have been failed because of the adverse effects including encephalitis, cerebral vascular damage and so on. All of the adverse effects are associated with entering in the brain of the antibodies. Thus, the peripheral clearance is getting increasing expecting as a new target for AD prevention and treatment. Yet the peripheral clearance of Aβ has not been clarified as far. Firstly, it is still unknown whether the entire periphery system can clear the brain derived Aβ physiologically though many studies have indicated the potential of the tissues and molecules in periphery on Aβ clearance. Secondly, the previous researches have the controversial results about brain Aβ clearance by administration of antibodies or medicines in periphery. Therefore, determining the capacity and related mechanism of peripheral clearance of Aβ as well as the role of Aβ peripheral clearance in AD pathogenesis will be helpful to the prevention and treatment of AD.Materials and methods1. The capacity of clearance of Aβ by the organs and tissues in periphery. We collected the blood samples from superior vena cava (SVC), inferior vena cava (IVC) (proximal to the hepatic vein), femoral vein (FV) and femoral artery in 30 patients underwent the Atrioventricular reentrant tachycardia in radiofrequency ablation. Meanwhile, we collected the blood samples from the jugular vein, abdominal aorta and posterior vena cava (PVC) in 10 APP/PS1 transgenic AD mice. We measured the Aβ concentrations in different locations in the circulation system of humans and APP/PS1 transgenic AD mice by ELISA assay and compared the venous/arterial ratios of Aβ concentrations.2. The capacity of metabolism of Aβ by the organs and tissues in periphery. We investigated the Aβ biodistribution by measuring the radioactive levels in different organs and tissues in the wild type C57BL/6J mice aged of 3 months after the administration of 125I labeled Aβ.3. Establishment of the parabiosis models between APP/PS1 mice and wild type C57BL/6J mice. We established the parabiosis animal models by surgically connecting the APP/PS1 mice and the gender/age matched littermates. Based on the production and deposition of Aβ in different ages, we established the prevention group using APP/PS1 mice aged of 3 months and collected the tissues at 9 months of age. Besides, we established the treatment group using APP/PS1 mice aged of 9 months and collected the tissues at 12 months of age.4. Measurement of the Aβ levels in the brain of AD. We investigated the influence of parabiosis on the senile plaques, brain Aβ deposition and Aβ concentrations in brain extracts by means of the Congo red staining, immunohistochemical staining and ELISA assay. We also observed the effects of parabiosis on the cerebral amyloid angiopathy (CAA) and brain microbleeds using the Congo red staining, immunofluorescence and Prussian blue staining. Besides, we determined the effects of parabiosis on the amyloid precursor protein and its metabolites as well as the Aβ production and central clearance related enzymes and transport receptors by Western blotting.5. Detection of the AD like pathologies in the brain of AD. We determined the effects of parabiosis on the microgliosis, astrocytosis, cytokines levels and hyperphosphorylation of Tau protein using immunohistochemical staining, immunofluorescence, ELISA assay and Western blotting. Furthermore, we explored the effects of parabiosis on the loss and apoptosis of neurons and synaptic degeneration by means of Western blotting.Results1. The capacity of peripheral clearance of Aβ exists in vivo. We measured and compared the venous/arterial ratios of Aβ concentrations where the arteriaβ concentrations were considered as the references. The Aβ40 and total Aβ levels in blood from SVC are significantly higher than that from FV, whose blood Aβ40 and total Aβ levels are significantly higher than that from IVC in humans. Besides, the Aβ40 and total Aβ levels in blood from jugular vein (JV) are significantly higher than that from PVC in APP/PS 1 mice. At 2 hours after administration of 125Ⅰ labeled Aβ, the ratios of radioactivity levels in the skin, gastrointestinal tract and liver are higher than the heart, spleen, lung and brain in the wild type mice. Further, the radioactivity levels per gram in liver, kidney, gastrointestinal tract and skin are higher than the others organs or tissues in the wild type mice.2. Parabiosis significantly prevented Aβ deposition in the AD brain. Compared with the control group of APP/PS 1 mice, the area fraction% and No./mm of Congo red staining positive plaques in the 9 months aged parabiotic APP/PS 1 mice are significantly lower. Specifically, the area fraction% reduce by 77.43% (0.3164±0.0259 vs 1.4016±0.0497, P<0.01) in neocortex and 71.22%(0.02636±0.0236 vs 0.9159±0.0631, P<0.01) in hippocampus, as well as the plaques No./mm2 decrease by 80.10%(13.7758±1.5062 vs 69.2223±3.4455, P<0.01) in neocortex and 76.58%(10.8991±1.3055 vs 46.5435±3.1947, P<0.01) in hippocampus. Meanwhile, Compared with the control group of APP/PS 1 mice, the area fraction% and No./mm of 6E10 antibody immunohistochemical staining positive plaques in the 9 months aged parabiotic APP/PS 1 mice are significantly lower. Specifically, the area fraction% reduce by 85.97% (5.8096±0.9244 vs 41.4155±2.0209, P<0.01) in neocortex and 82.62%(3.3584±0.6359 vs 19.3194±1.4791, P<0.01) in hippocampus, as well as the plaques No./mm2 decrease by 80.49%(20.3321±2.4963 vs 104.2200±4.4021, P<0.01) in neocortex and 83.56%(9.0656±1.0702 vs 55.1584±2.1844, P<0.01) in hippocampus. Besides, the Aβ40 concentrations in the brain abstracts in TBS,2% SDS and 70% formic acid of 9 months aged parabiotic APP/PS 1 mice are significantly lower than the control APP/PS 1 mice (TBS:0.1148±0.0100 vs 0.0436±0.0056, P<0.01; 2% SDS: 0.7417±0.0835 vs 0.1333±0.0236, P<0.01; 70% formic acid:3.0781±0.3233 vs 1.0512±0.1440, P<0.01). The Aβ42 concentrations in the three brain abstracts have the similar differences (TBS:0.5219±0.0519 vs 0.2251±0.0328, P<0.01; 2% SDS: 0.8295±0.1211 vs 0.3249±0.0521, P<0.01; 70% formic acid:3.5536±0.2662 vs 1.0570±0.1129, P<9.01). Further, the total Ap (Aβ40+Aβ42) concentrations in those brain abstracts of 9 months aged parabiotic APP/PS 1 mice are significantly lower (TBS: 0.6367±0.0577 vs 0.2687±0.0338, P<0.01; 2% SDS:1.5712±0.0901 vs 0.4582±0.0639, P<0.01; 70% formic acid:6.6317±0.3913 vs 2.1083±0.2173, P<0.01; in all:8.8396±0.4034 vs 2.8352±0.2520, P<0.01). In addition, the No./section and fraction% of CAA as well as the No./section of brain microbleeds of 9 months aged parabiotic APP/PS 1 mice are significantly lower than the control APP/PS 1 mice. Further, there are no significant differences in the expression levels of APP, CTF-a, CTF-β, BACE1, IDE, NEP, LRP-1 and RAGE between the 9 months aged parabiotic and control APP/PS 1 mice.3. Parabiosis restrained the growth of Aβ deposition in the brain of AD. Compared with the control group of APP/PS 1 mice, the area fraction% reduces by 17.10% (1.4134±0.0888 vs 1.7049±0.0396, P<0.05) and the plaques No./mm2 decreases by 16.94% (76.0826±3.7654 vs 87.9899±4.6522, P<0.05) in neocortex of the 12 months aged parabiotic APP/PS 1 mice. Besides, compared with the control group of APP/PS 1 mice, the area fraction% reduces by 36.02%(48.6341±2.2949 vs 76.0197±3.7302, P<0.01) in neocortex and 26.72%(23.2100±1.6363 vs 36.6794±2.4699, P<0.01) in hippocampus, as well as the plaques No./mm2 decreases by 28.60% 115.1200±4.1091 vs 161.2300±5.9683, P<0.01) in neocortex of the 12 months aged parabiotic APP/PS 1 mice.4. Parabiosis significantly attenuated the AD like pathologies. Compared with the control group of APP/PS 1 mice, the area fraction % and No./mm2 of CD45 antibody, GFAP antibody and pS396 antibody immunohistochemical staining positive cells, the expression levels of pS199 and pS396, and the levels of cytokines including IL-1β, IL-6 and TNF-a of 9 months aged parabiotic APP/PS 1 mice are significantly lower. Meanwhile, the area fraction% of immunofluorescence labeled positive cells for NeuN antibody in CA1 and CA3 regions of hippocampus, as well as MAP-2 antibody in CA1 region of hippocampus in 9 months aged parabiotic APP/PS 1 mice are significantly higher than the control APP/PS 1 mice, while the area fraction% of Caspase-3 antibody immuno fluorescence labeled positive cells in the CA3 region of hippocampus is lower. In addition, the expression levels of the synaptic markers including PSD-93, PSD-95, Synapsin-1 and Synaptophysin of 9 months aged parabiotic APP/PS 1 mice are significantly higher than the control APP/PS 1 mice.Conclusions1. The periphery organs and tissues have the capacity to clear brain derived Aβ physiologically. Particularly, the liver, kidney, gastrointestinal tract and skin play pivotal roles in Aβ peripheral clearance.2. Increasing the capacity of Aβ peripheral clearance is able to reduce Aβ senile plaques, Aβ deposition and Aβ concentrations in different brain extracts, as well as the CAA and related cerebral microbleeds.3. Increasing the capacity of Aβ peripheral clearance is able to attenuate the microgliosis and astrocytosis, reduce the cytokines levels and decrease the hyperphosphorylation of Tau protein in AD. In addition, the capacity of Aβ peripheral clearance can ameliorate the brain neurons loss and apoptosis as well as the synaptic degeneration.4. The peripheral clearance of Aβ is a potential way to prevent and treat AD, which is worthy of further exploration.5. The peripheral organs and tissues play a substantial role in brain derived Aβ clearance reflected as around 40% of brain derived Aβ is cleared in periphery by calculating, suggesting that the defects of the capacity of Aβ peripheral clearance participate the pathogenesis of AD.