| Background and objectiveAlzheimer’s disease(AD) is the most common form of neurodegenerative disease affecting the elderly, with the etiology and pathogenesis largely unknown. Currently, therapies for this disease are mostly symptomatic treatment and no strategy is available to reverse or stop the disease progression. Amyloid-beta(Aβ) is the core pathological agent and senile plaque containing Aβ is the major pathological hallmark of AD. Immunotherapies, including active(Aβ vaccine) and passive ones(antibodies), are suggested to be effective in clearance of Aβ in animal models of AD. However, clinical trials of immunotherapies for AD are not successful due to adverse effects. Naturally occurring antibodies of Aβ(NAbs-Aβ)ubiquitously exist in human body. Previous study indicates that NAbs-Aβ antagonize the neurotoxicity of Aβ, thus is protective against AD. Invtrovenous immunoglobulin(IVIg) contains NAbs-Aβ and were proved to be effective in improving AD-like pathologies in animals. But the clinical trials also failed to improve the cognition of AD patients. The site for aggregation and neurotoxicity of Aβ both locate at the middle domain of this peptide. Conformational analysis indicates that the N-terminus is exposed outside of Aβ fibrils but the middle domain and C-terminus are embedded inside the core. Our previous study found out that monoclonal antibodies targeting the N-terminus of Aβ increase its production. Currently used antibodies in immunotherapies are mostly targeting the N-terminus of Aβ. In the present study, we aim to investigate the epitope-related functional heterogeneity of NAbs-Aβ. Besides, as the adverse effects associated with immunotherapies are mostly caused by the entry of antibodies into the brain. We proposed that clearance of Aβ in periphery might be safer. We also investigated the function of kidney in peripheral clearance of Aβ in patients with chronic kidney disease(CKD). Furthermore, due to the adverse effects of novel therapeutic agents for AD, we investigated the therapeutic potential of Edaravone, clinically used for the treatment of acute ischemic stroke, in transgenic mouse model of AD. Matierals and methodsPart 1: We compared the serum and cerebral spinal fluid(CSF) levels of NAbs-Aβ between AD patients and healthy control(HC). We tested the total levels of NAbs-Aβ utilizting Elisa and Western Blot. We mapped the epitopes of NAbs-Aβ in AD patients and HC. We analyzed the correlations of serum and CSF levels of NAbs-Aβ with Aβ levels, MMSE and CDR scores. We purified NAbs-Aβ targeting different epitopes of Aβ from IVIg. In APPswePS1wt/CHO cell lines, we investigated the effects of NAbs-Aβ targeting different epitopes on Aβ precursor protein(APP) processing and Aβ production. We investigated the antagonisitic effects of NAbs-Aβ on Aβ aggregation using ThT and TEM assays. In SYH-SY5 Y cell lines, we investigated the antagonisitic effects of NAbs-Aβ against the neurotoxicity of Aβ using MTT and dendrite outgrowth assays. After intracranial injections of purified NAbs-Aβ, we investigated the effects of NAbs-Aβ on neuronal apoptosis in 3 months old APP-Swe PSEN1dE9 transgenic mice.Part 2: Anti-Aβ antibodies, including 6E10(which targets the N-terminus of Aβ), 4G8(which targets the middle domain of Aβ) and 8G7(which targets the C-terminus of Aβ) were tested for the capacity for binding and disaggregating pre-formed Aβ fibrils. The co-incubates of Aβ antibodies and pre-formed fibrils were tested for the neurotoxicities in vitro and in vivo.Part 3: We recruited 47 CKD patients(including 31 patients who had peritoneal dialysis and 16 patients who never had dialysis) and 43 healthy controls(HC). Blood was sampled and tested for serum Aβ40 and Aβ42 levels. The correlations between Aβ levels and renal functions were analyzed.ResultsPart 1: The levels of NAbs-Aβ to different epitopes of Aβ were decreased in AD serum and CSF. However, the ratio of N-terminal NAbs-Aβ was increased while that of the middle domain NAbs-Aβ was decreased in AD serum and CSF. NAbs-Aβ to the middle domain of Aβ had a better antagonistic capacity against Aβ neurotoxicity and aggregation in comparisom with NAbs-Aβ targeting N-terminus and C-terminus. NAbs-Aβ to the N-terminus of Aβ could bind to human hippocampal neurons and neuronal cells overexpressing APP. In addition they promoted the amyloidogenic processing of APP in vitro and induced neuronal apoptosis in vivo. These results suggest that in AD patients, N-NAbs-Aβ are neurotoxic and relatively increased, while M-NAbs-Aβ are protective and relatively decreased.Part 2: After incubation of preformed Aβ fibrils with N-terminal antibody 6E10, but the fibrils were decreased while oligomers, mostly dimers and trimmers, were significantly increased. However, we observed no such effects of antibodies targeting middle domain(4G8) and C-terminus of Aβ(8G7). The co-incubates of preformed Aβ fibrils with 6E10 are more neurotoxic, both in vitro and in vivo, than that with 4G8 and 8G7.Part 3: We found that serum Aβ levels were significantly higher in CKD patients. Moreover, dialysis CKD patients had significantly lower serum Aβ levels than patients who never had dialysis, being comparable to NC. Furthermore, we found correlations of serum Aβ levels with estimated glomerular filtration rate(eGFR) according to serum creatinine levels and residual GFR(rGFR) estimated by serum cystatin C levels.Conclusions1. Functions of NAbs-Aβ are closely associated with epitopes. A disrupted profile of NAbs-Aβ might contribute to the pathogenesis of AD, and immunotherapies should avoid using antibodies to the N-terminus of Aβ. 2. N-terminal antibodies promote the transformation of Aβ from fibrils into oligomers and increase the neurotoxicity. Immunotherapies should take into consideration the enhanced neurotoxicity associated with solubilization of Aβ deposits by antibodies against N-terminal of Aβ. 3. Renal functions may be essential for peripheral clearance of Aβ and insufficient Aβ clearance in CKD patients might predispose towards AD. |
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