| Background and objectiveAlzheimer’s disease (AD) is the most common neurodegenerative disease.Extracellular senile plaques comprising amyloid-beta (Aβ) peptide are the histopathological hallmarks of AD. It has been suggested that excessive A(3 production or deficits in Aβclearance play pivotal or causative roles in AD pathogenesis. AD comprises familial AD and sporadic AD. Familial AD afflicts approximately 1% of total AD patients and is caused by mutations in APP, PS1 or PS2 genes, leading to the overproduction of Aβ, whereas sporadic AD affects over 99% of AD patients and primarily attributes to impaired Aβ clearance in the brain. Therefore, the enhancement of brain Aβ clearance is the most promising strategy for AD prevention and treatment.Current Aβ clearing strategies mainly focus on enhancing brain A(3 clearing capacities by introducing agents into the brain. However, introducing exogenous substance into the brain likely cause adverse effects. For example, in clinical trials of immunotherapy, which target Aβ clearance, the entry of therapeutic antibodies leads to various adverse effects, such as neuroinflammation, vasogenic oedema, microhaemorrhage, neuronal hyperactivity and the increasing conversion of Aβ fibrils to more toxic Aβ oligomers. Thus, to avoid these adverse effects, using a peripheral approach to clear brain Aβ which reduces brain-derived Aβ in the blood, would be a safer strategy.In previous studies, we revealed that the physiological Aβclearance capacity in peripheral organs and tissues plays an important role in the clearance of brain Aβ by catabolizing brain-derived A(3 in the blood, suggesting that clearing blood Aβ may be a potential approach to removing brain Aβ. In the clinic, dialysis, including haemodialysis and peritoneal dialysis, is an effective method to remove metabolites and wastes from blood to maintain the homeostasis of the internal environment of the body. Dialysis can also recover the homeostasis of the microenvironment in the brain, as indicated by the effectiveness of dialysis in treating encephalopathies due to liver and kidney failures or toxicosis. Previous clinical studies have suggested that dialysis reduces blood Aβ levels. Thus, we investigated whether peritoneal dialysis can reduce brain Aβand exert therapeutic benefits to AD in the present study.Materials and methods1. A total of 30 consecutive patients newly diagnosed with chronic kidney disease(CKD) were enrolled in the study. The blood was sampled before and immediately after the first time peritoneal dialysis, and the peritoneal dialysis solution was also collected after dialysis.2. Peritoneal dialysis model. The transgenic mouse line expressing APP/PS1 transgenic mice was obtained from the Jackson Laboratory. Female wild type and APP/PS1 mice were used in the present study. Prevention or treatment experiments were conducted.Mice aged 6 months, upon the initiation of Aβ deposition in the brain, and mice aged 9 months, when abundant deposits were formed in the brain, were subjected to daily Peritoneal dialysis for 30 days. Age-matched APP/PS1 mice with sham-operation were used as controls without peritoneal dialysis.3. Microdialysis model. A separate group of 9-month-old APP/PS1 mice were used for microdialysis. Guide cannulas were stereotactically inserted into CA1 area of the hippocampus. The mice were kept awake during microdialysis.4. Brain sampling. The brains were sampled and weighed. The left hemispheres were fixed in 4 % paraformaldehyde for 24 h, followed by incubation with 30% sucrose for 24 h.Brain sections were cut coronally at a thickness of 35 μm and stored at 4℃ in phosphate-buffered saline containing 0.1% sodium azide. The right hemispheres were snap frozen in liquid nitrogen and stored at -80 ℃ for future biochemical analysis.5. AD type pathology and quantitative image analysis. A series of five equally spaced brain sections (～1.3 mm apart) were used for each type of stain. Congo red staining was used for compact Aβ plaques, and total Aβ plaques containing both compact and diffuse Aβplaques were visualized using antibody 6E10 immunohistochemistry as previously described.The apoptosis of neuronal cells was detected using NeuN and Caspase-3 double immunofluorescence staining. Neuronal loss and neurite degeneration were detected using NeuN and microtubule-associated protein (MAP)-2 double immunofluorescence staining.Immunohistochemistry of anti-CD45 antibody detecting activated microglia, and anti-glial fibrillary acidic protein antibody detecting astrocytes were used to visualize astrocytosis and microgliosis. Double immunofluorescence staining of Iba-1 and 6E10 was performed to verify the phagocytic ability of microglia.6. ELISA assays. Frozen brains were homogenized in liquid nitrogen and extracted with TBS, 2% SDS, and 70% formic acid (FA) solutions as previously described. The levels of Aβ40 and Aβ42 in brain extracts, plasma, peritoneal dialysis solution and microdialysis fluid were measured using ELISA kits. The concentrations of the inflammatory cytokines IL-6, IL-1β, IFN-γ and TNF-α were quantitatively measured using ELISA kits. All experiments were performed according to the manufacturer’s instructions.7. Western blot. Western blotting was used to analyse the levels of molecules or enzymes involving Aβ metabolism, phosphorylated Tau, and synapse-related proteins.Proteins in the animal brain homogenate were extracted using RIPA buffer. The blots were probed with the following antibodies: anti-APP C-terminal antibody to detect C-terminal fragment (CTF)-α (CTF-α) and CTF-β,anti-Aβ antibody was used to detect A(3, full-length APP (APPfl), secreted APP (sAPP)-α (sAPPα), anti-sAPP antibody to detect sAPPa and sAPPβ; anti-BACE1; anti-IDE; anti-NEP; anti-receptor for advanced glycosylation products(RAGE); anti-LRP-1; anti-phosphorylated-Tau antibodies, including anti-pS396 and anti-pS199; anti-total tau; anti-Synaptophysin; anti-Synapsin-1; anti-PSD95; anti-PSD93;and anti-β-actin8. Animals in the treatment experiments underwent Golgi staining using the manufacturer’s protocols (FD rapid Golgi Stain kit).9. Behavioural Tests and Electrophysiology. Y-maze and open-field tests were performed following as previously described.Results1. Peritoneal dialysis decreases plasma Aβ in both human and mice.2. Dynamic changes of A(3 in blood and ISF during peritoneal dialysis. We further investigated the dynamic interaction between blood Aβ levels and brain ISF Aβ levels. Aβlevels in microdialysis fluid were used to estimate ISF A(3 levels. These results suggest that the changes of Aβ levels between the brain ISF and blood are dynamically correlated in AD mice.3. Peritoneal dialysis reduces brain Aβ burden. We investigated whether long-term peritoneal dialysis reduces brain Aβ In a prevention study, compared with control mice,mice treated with peritoneal dialysis had a significantly lower area fraction of both compact plaques stained with Congo red and total plaques stained with 6E10 in neocortex, and lower area fraction of total plaques in the hippocampus. The levels of Aβ40 and Aβ42 in the brain homogenates were also significantly reduced in mice treated with peritoneal dialysis relative to control mice. We next investigated whether peritoneal dialysis is effective in reducing brain Aβ after abundant deposition of Aβ. In a treatment study, peritoneal dialysis-treated mice had significantly less Congo red and 6E 10-positive Aβ plaques in the neocortex and hippocampus than control mice.4. Peritoneal dialysis attenuates neuroinflammation and enhances Aβ phagocytosis by microglia. Compared with control mice, peritoneal dialysis-treated mice had a lower area fraction of activated astrocytosis (GFAP positive) and microgliosis (CD45 positive) in both the neocortex and hippocampus and reduced levels of proinflammatory cytokines, including TNF-α, IFN-γ and IL-6, in brain homogenates.5. There were more microglial cells containing intercellular Aβ in the CA1 of the hippocampus of peritoneal dialysis-treated mice compared with control mice. These findings suggest that peritoneal dialysis enhances Aβ phagocytosis by microglia in the brain.6. Peritoneal dialysis alleviates neurodegeneration in the brain. The levels of phosphorylated Tau (pS396) were significantly reduced in the brains of mice after peritoneal dialysis. Compared with control mice, the neuronal apoptosis was also significantly reduced in the peritoneal dialysis-treated mice compared with the control mice, as detected by caspase-3 staining in the hippocampus. The levels of synapse-associated protein expression,including PSD93, PSD95, synapsin-1, and synaptophysin, in the brain homogenates, and the number of dendritic spines detected via Golgi staining in the hippocampus were increased in the peritoneal dialysis-treated mice compared with control mice. These data suggest that neurodegeneration was attenuated in the brains of APP/PS1 mice after peritoneal dialysis.7. Peritoneal dialysis rescues LTP and cognition impairment. APP/PS1 mice treated with peritoneal dialysis and their age-matched controls were subjected to cognitive tests. In the open-field test, a higher number of rearing and a longer distance travelled were observed for peritoneal dialysis-treated mice compared with the controls. However, no difference in the number of grooming behaviours was observed between the two groups. In the Y maze test, peritoneal dialysis-treated mice had better performance than controls, reflected by a higher spontaneous alternation percentage, increased number of total entries into the three arms in alternation tests, and increased number of entries and time spent in the novel arm.These results indicate that peritoneal dialysis can prevent or halt cognitive decline in APP/PS1 mice. High-frequency stimulation (HFS) induced reliable LTP, the cellular mechanism underlying learning and memory, in wild-type mice, whereas the strength of LTP was significantly decreased in APP/PS1 mice compared with wild-type mice, suggesting that the plasticity of the synapse is impaired in the brains of APP/PS1 mice. Notably, a higher LTP was observed in peritoneal dialysis-treated mice compared with control mice, indicating that peritoneal dialysis can substantially rescue impaired hippocampal LTP induction in APP/PS1 mice.DiscussionIn the present study, peritoneal dialysis effectively decreased blood Aβ and brain ISF soluble Aβ levels. After one month of peritoneal dialysis, brain Aβ burden,neuroinflammation, neurodegeneration and cognitive deficits in AD mice were significantly alleviated, suggesting that peritoneal dialysis might enhance the Aβ efflux from brain to blood, attenuate the brain inflammatory microenvironment, and improve the phagocytosis of Aβ by microglia.Previous anti-A(3 therapeutic strategies have primarily focused on reducing A(3 production, inhibiting Aβdeposition and facilitating Aβ clearance, all of which depend on Aβ clearing agents entering the brain through the BBB. The BBB restricts the entry of peripheral blood proteins into the brain to maintain the homeostasis of the brain internal environment. Thus, the entry of therapeutic agents into the brain may disrupt the brain internal environment and induce various adverse effects. Numerous studies have shown that the Aβ antibodies used in AD clinical trials cause neuroinflammation, vasogenic oedema,microhaemorrhage and neuronal hyperactivity, likely reflecting the entry of antibodies into the brain. Therefore, clearing blood Aβ and accelerating the efflux of Aβ from the brain into the periphery would be a better strategy for clearing brain Aβ Previous studies have tested the therapeutic efficacy of clearing blood Aβ for AD. For example, brain Aβ could be significantly reduced by increasing Aβ degradation in the liver via the oral administration of Withania somnifera extracts, and the peripheral expression of the Aβ-degrading enzyme neprilysin was also effective in clearing blood and brain Aβ,despite conflicting findings suggest that the peripheral injection of neprilysin only reduces blood Aβ but not brain Aβ. In the present study, peritoneal dialysis effectively decreased both blood Aβ and blood Aβburden in CKD patients and in AD mice, indicating that enhancing the clearance of Aβ from the periphery is a promising approach for reducing brain Aβ burden.The most important prerequisite to peripheral Aβ clearance is the efflux transport of Aβacross the BBB. Previous studies have shown that BBB transporters were changed in AD in a pattern that decreases Aβ efflux and/or increases Aβ influx across the BBB and contributes to AD pathogenesis, and a contrasting view has been suggested in a recent study which indicates that there is not the lack of widespread disruption of BBB in AD mouse models. In the present study, we conducted the first investigation of the dynamic effects of decreasing Aβ levels in blood on the soluble Aβ levels in ISF. The compelling finding of the present study was that the ISF soluble Aβ levels were decreased with the reduction of the blood Aβlevels during peritoneal dialysis. Our findings suggest that the transport of Aβ across the BBB is functional in AD mice, and this lays the foundation for therapeutic development focusing on peripheral A(3 clearance approaches to reduce brain Aβ burden.Is the removal of blood Aβby peritoneal dialysis the only explanation for the reduction of Aβ from the brain? To address the question, we calculated the total A(3 removal via peritoneal dialysis. Total A(3 burden in the brain of 10-month-old AD mice is approximately 360 ng as calculated by brain Aβ concentration (1 ng/mg) × brain weight (360 mg). The total brain Aβ at the end of peritoneal dialysis is composed of the existing Aβ at the beginning of peritoneal dialysis and newly produced A(3 during the 30 days of peritoneal dialysis. The total Aβ removal in dialysis solution is 7.2 ng as calculated by the Aβ concentration in dialysis solution (200 pg/ml) × the volume of recovered dialysis solution (1.2 ml) × dialysis duration (30 day). While the total reduction of brain Aβ after peritoneal dialysis = brain Aβconcentration (1 ng/mg) × brain weight (360 mg) × the percentage decrease in brain Aβburden (20.44%) = 72 ng. Thus, Aβ removal in dialysis solution was only 10% of the reduction in brain Aβ burden, suggesting that other A(3 clearing pathways may be enhanced through peritoneal dialysis. In our present study chronic peritoneal dialysis increased the expression of LRP-1 and decreased the expression of RAGE in the brain of AD mice, these changes could favor the net efflux of A(3 across BBB from the brain into the blood. In addition to the removal of Aβthe convection between dialysate and blood produced by hypertonic dialysis solution can also promote the efflux of other metabolites into the peritoneal dialysate through the peritoneum and omentum. Thus, peritoneal dialysis could significantly improve the internal environment of the brain, as reflected by the decreased pro-inflammatory cytokines and increased anti-inflammatory cytokines in the brain,suggesting that there might be a shift of microglia polarization state from M1 to M2.Enhancement of the Aβ phagocytic ability of microglial cells reflects the improvement of microglia functions in the brain after peritoneal dialysis. Furthermore, as the level of Aβ in the omentum in the peritoneal dialysis-treated mice was much higher than the controls,entrapment of Aβ in omentum also contributes to the reduction of brain Aβ burden. Taken together, peritoneal dialysis clears brain Aβ through multiple pathways.Previous studies suggest that A(3 levels in the blood are not correlated with that in the brain and disease severity of AD, partially due to the difference in the Aβ solubility and the many confounding factors which influence the brain-derived Aβ levels in the blood such as binding of Aβ with albumin, blood cells and catabolism of Aβ in the periphery by enzymes,tissues and organs. But in the present study the changes of soluble Aβ levels in ISF were closely correlated with that in blood during peritoneal dialysis, suggesting that the soluble brain Aβ pool and blood Aβ pool are communicative. Our recent study suggests that the physiological Aβ clearance in the periphery plays a significant role in removing brain A(3,and the clearance of Aβ in the periphery is a potential route for brain Aβclearance. In addition, previous studies suggest that Aβ can also deposit in the peripheral tissues, such as intestine, skin and heart, of AD patients. In our present study, we provided the evidence that Aβ also accumulates in the omentum in AD mice. The higher levels of Aβ42 over Aβ40 in the omentum might be because that Aβ42 is more prone to deposit in tissues than A(340.Moreover, previously we found that blood Aβ levels were significantly increased in patients with peripheral disorders, such as hepatic and renal failures, chronic obstructive pulmonary disease, and systemic infection. These findings suggest that the disturbance of Aβ clearance in the periphery may contribute to AD pathogenesis, and AD may be a disorder related not only to the brain but also to the peripheral system.It is worthy to note that CKD patients are different from AD mice in the present study.The first difference is that patients were suffering from chronic renal failure while renal functions of AD mice were normal. Our previous study suggests that the A(3 clearance capacity of the kidney is impaired in CKD patients. In the present study, we found that peritoneal dialysis is potent in removing Aβ from the blood in CKD patients. While in AD mice, the blood Aβlevels were dramatically higher than that of wild type mice, suggesting that the renal function in AD mice is not sufficient to remove A(3 in blood. In this regard,enhancement of Aβ clearance from blood is a necessary therapeutic approach for AD, even though their renal functions are normal. The second difference between CKD patients and AD mice is different procedures of peritoneal dialysis. CKD patients usually received continuous ambulatory peritoneal dialysis (CAPD) which consists of three daytime exchanges (i.e. 4-6 h dwell-time) and one nightly (i.e. 8-12 h dwell-time) in clinical settings.While in our study AD mice received only two hours of dialysis per day. Even with this shorter time of dialysis, significant reduction of brain A(3 burden was also achieved in AD mouse, suggesting that CAPD would be more potent in removing brain Aβ if it is applied for AD patients. It is of importance to note that brain Aβ deposition is lower in CDK patients who received hemodialysis than those who did not, suggesting that reducing blood Aβ by dialysis would be a promising and effective therapeutic approach for AD.In conclusion, the efficacy of peritoneal dialysis for clearing brain Aβ in our present study provides proof-of-concept evidence that the restoration of the AD brain microenvironment and the clearance of brain Aβ could be realized via peripheral approaches.The findings of the present study could also provide implication for the prevention and treatment of other neurodegenerative diseases,such as Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis, through peripheral approaches.ConclusionIn the study, peritoneal dialysis could significant improve the AD-type pathology,which shows that peripheral clearance is effective and feasible for AD prevention and treatment. |
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