Experimental Studies On The Brain-derived β Amyloid _1-40 Transportation Across The Blood-brain Barrier

A central feature of Alzheimer's disease (AD) is a chronic neurodegenerative process according to the prevailing amyloid cascade hypothesis, which is initiated by the amyloid β -peptide (A β) accumulation in brain and consequent neuronal toxicity. The studies of these pathological traits of AD have dominated the field for the last decade. Indeed, much of the therapeutic armamentatium for AD are directed at developing strategies to control the A β related pathology in hopes of improving cognitive decline associated with AD. It has been suggested that decreased clearance of A β from brain and CSF is a main cause of Aβ accumulation in sporadic AD. The systemic clearance mechanisms include: a) A β degradated by action of enzymes in systemic and brain interstitial fluid (ISF) pool; b) A β removed by endocytosis of different cells, including neurons and astrocytes; c) A β transported by rapid regulation of soluble A β cross BBB. Low-density lipoprotein receptor-related protein (LRP) is linked to AD by genetic and biochemical evidence. The exact pathogenic mechanism(s) by which LRP contributes to neurotoxic A β accumulation is unclear. An earlier report suggested that A β complexed to apolipoprotein J in the circulation may utilize the lipoprotein receptor (LRP-2) at the BBB for its transport into brain ISF. Whether LRP-1 on brain endothelium may clear aggregated Aβ via an apolipoprotein E-dependent mechanism remains unknown. The recent studies reported that reduced levels of LRP-1 in AD have been associated with A β accumulations. It was suggested that brain-derived A β was transported across BBB via LRP-1. The elevated levels of A β in the plasma of transgenic mice after passive immunization with anti-A β antibodies and in PS/APP mice after active A β immunization suggested that alternation of peripheral/brain A β dynamics may be a possible therapeutic target. One of the proposed mechanisms for anti-A β antibody reducing brain A β is that the antibody significantly influence A β transfer between the brain and plasma. The periphery/brain A β dynamics may play a crucial role in the pathogenesis of AD so that the therapeutic "peripheral aggradation " strategy was put forward. Because of several problems associated with immunotherapy and current clinical trials of actively administered A β peptides suspended after adverseresponse in patients, we try to find whether some compounds unrelated to antibodies, but have the ability of binding Aβ in the periphery, might be effective in altering the periphery/brain dynamics leading to a reduction of A β in brain ISF pool.In the present study, we investigated the expression of LRP-1 on the brain microvascular endothelial cell line bEnd.3 and observed the A β transportation across a model of BBB which was established by coculture of human umbilical vein endothelial cells(HUVEC) and mouse astrocytes(As). In addition, the effects of anti-LRP-1 antibodies and RAP and gelsolin on A β in brain ISF pool clearance across BBB were also evaluated.Part I Establishment and evaluation of an in vitro blood-brain barriermodelObjective To establish a convenient and reliable model of blood-brain barrier (BBB) in vitro, and to evaluate its biological functions. Methods Astrocytes and human umbilical vein endothelial cells (HUVEC) were seeded respectively on the two opposite surface of transwell polyethylene terephtalate membrane which coated with rat tail collagen. And this coculture model was further evaluated by analyzing the morphological characteristics (electron microscopy) and biological functions, including transendothelial electrical resistance, enzyme activity of γ -glutamyl transpeptidase ( γ -GT) and amount of the paracellular transport marker ¹⁴C-sucrose and ¹²⁵I-BSA. Results The tight junction between HUVEC could be visualized by transmission electron microscope, and the transendothelial electrical resistance of the coculture model reached up to (267 ± 24) Ω/cm². There was a significant difference between the specific activity of γ -GT levels (12.41 ± 1.71U/mg protein) after coculture and the monolayer HUVEC (2.76 ± 1.00 U/mg protein). The permeability coefficient of ¹⁴C-sucrose is (0.93 ± 0.32) ×10^-3cm/min, and ¹²⁵I-BSA across the BBB model is restricted.Part II Study on the brain-derived A β clearance across an in vitro BBBmodelObjective To observe the expression of LRP-1 on brain microvascularendothelial cells, and highlight the relationship between LRP-1 and A β in brain ISF pool clearance across BBB. Methods The expression of LRP-1 mRNA and protein levels in brain microvascular endothelial cell line bEnd.3, HUVEC and As were detected by the methods of RT-PCR and Western blotting. A β_1-40 (50nM) solution was added in the lower compartment, i.e. the abluminal side. At time 10, 20, 30, 60 and 120min, an aliquot of 20ul from each lower compartment were taken, and the concentration of A β _1-40 was detected by using ELISA. Finally, we observed the effects of anti-LRP-1 antibodies and RAP and gelsolin on brain-derived A β clearance across the vitro model of BBB. Results Satisfied quantity of LRP-1 mRNA and/or protein were detected in both bEnd.3 and HUVEC, while none were observed in astrocytes. The concentration of A β_1-40 in lower compartment reached downward with time, from (200.12±15.01) pg/ml at time 0 min to (99.07 ±11.92) pg/mlat time 20 min. At time 2h, it reached the lowest(44.92±7.40)pg/ml. The concentration of Aβ_1-40 was significantly different after RAP or anti-LRP-1 pretreatment (P<0.05) on the five time points, compared with the control. There was no significant difference between RAP and anti-LRP-1 antibodies pretreatment on the effect of brain-derived A 3 clearance across the in vitro model of BBB(P>0.05). There was a significant difference after gelsolin pretreatment (P<0.05) on the five time points, compared with the control. The A β_1-40 clearance did not impact on the BBB tight junction in vitro.Part â…¢ Study on the relationship between LRP-1 and brain-derived A βclearance across mouse BBBObjective To study the relationship between LRP-1 and the brain-derived A β clearance across BBB, and to investigate the effect of peripheral gelsolin on brain-derived A β clearance across BBB. Methods RAP (5uM) or anti-LRP-1 antibodies (60ug/ml) or normal saline was administered intracranially at a volume of 0.3ul per time, and was injected every 2d for 2 weeks? Blood was collected at predrug(d0), mid-drug(d8) and postdrug treatment(d15) times into preweighed tubes. The proteins of different brain tissues were extracted by two steps extraction. Gelsolin or normal saline was administered intraperitoneally at a dose of 0.6mg/kg body weight, every 2d for 3 weeks. Blood was collected at predrug(d0), mid-drug(d12) and postdrug treatment(d22) times into preweighed tubes. The proteins of different braintissues were extracted by two steps extraction. Levels of mice A β_1-40 in plasma and brain extracts were qualified by ELISA. Results After anti-LRP-1 antibodies administration, the levels of A β_1-40 in brain extracts were (47.29±6.80) pg/g (d0),(69.03±9.06) pg/g (d8) and (86.63±7.78) pg/g (d15), respectively. There were significant difference on levels of A β_1-40 in brain extracts between anti-LRP-1 antibodies administration and normal saline administration or the control (P<0.05) . After anti-LRP-1 antibodies administration, the levels of A β_1-40 in plasma were(14.67 ±3.37)pg/ml (d0), (8.59±1.32)pg/ml(d8) and (4.56 ± 0.89)pg/ml(d15), respectively. There were significant difference on levels of A β_1-40 in plasma between anti-LRP-1 antibodies administration and normal saline administration or the control(P<0.05) . After RAP administration, the levels of Aβ_1-40 in brain extracts were(40.38 ±7.93) pg/g (d0), (57.86 ± 8.85) pg/g (d8) and (74.73±8.54) pg/g (d15), respectively. There were significant difference on levels of A 3 1.40 in brain extracts between RAP administration and normal saline administration or the control (P<0.05), while no significant difference existed between RAP administration and anti-LRP-1 antibodies administration. After RAP administration, the levels of A 3 1-40 in plasma were (16.21±2.71)pg/ml (d0), (7.56±0.85) pg/ml(d8) and (5.26±1.65 )pg/ml (d15), respectively. There were significant difference on levels of A 3 1-40 in plasma between RAP administration and normal saline administration or the control(P<0.05), while no significant difference existed between RAP administration and anti-LRP-1 antibodies administration(P>0.05). After gelsolin administration, the levels of A 3 1-40 in brain extracts were (45.33±7.63)pg/g (d0) ,(32.28±4.24)pg/g (d12) and (24.17 ± 3.73) pg/g (d22), respectively. There were significant difference on levels of A 3 _1-40 in brain extracts between gelsolin administration and normal saline administration intraperitonially or the control (P<0.05). After gelsolin administration, the levels of A 3 1.40 in plasma were (13.18 ± 2.03) pg/ ml (d0), (23.60±3.35) pg/ml (d12) and(28.93±3.57) pg/ml (d22), respectively. There were significant difference on levels of A β_1-40 in plasma between gelsolin administration and normal saline administration intraperitonially or the control (P<0.05) .Conclusions1. This coculture system sustained the morphological, enzymatic characteristics and restricted paracellular diffusion of BBB in vivo.2. There is LRP-1 expression on brain microvascular endothelial cell line bEnd.3and HUVEC, while none were observed on normal human astrocytes. The A β_1-40 at a concentration of 50 nM in lower compartment diminished half in 20 minutes, which suggested a rapid clearance across the BBB. There was significant inhibition after RAP or anti-LRP-1 pretreatment on A β_1-40 clearance across BBB, while acceleration after gelsolin administration on brain-derived A 3 clearance across BBB.3. The vivo experiment also suggested that both anti-LRP-1 antibodies and RAP administration intracranially could increase the levels of A 3 1-40 in brain interstitial fluid pool, while the levels of A 3 1-40 in plasma were decreased correspondingly. On contrary, after gelsolin administration intraperitoneally, the levels of A β_1-40 in brain extracts were decreased, while the levels of A β_1-40 in plasma were increased.