S100A6 Inhibits Amyloid-? Aggregation In The Brain Of Alzheimer's Disease

Objective:Alzheimer's disease?AD?,a heterogeneous,progressive neurodegenerative disorder,is the main cause of dementia in the elderly,with no disease-modifying therapy available until now.The typical symptoms of the disease are memory impairment and cognitive decline.?-amyloid?A??fibrils associate into amyloid plaque structures to form one of hallmarks of the Alzheimer's disease pathology.A?is a cleavage product of?-amyloid precursor protein?APP?.It is generated by the combined actions of the proteolytic enzymes?-and?-secretases and secreted out of cells.A?is the key factor in the pathologic process of AD.Latest research showed that metal ions play an important role in the assemble of A?and formation of plaque.Sevaral study reported that metal chelating agents?clioquinol,PBT2 and DP-109,both membrane permeable chelators,?can reduce the cortical and hippocampal amyloid burden and improve the cognitive performance by decreacing the brain zinc level.Therefore,it suggested that modulation of zinc homeostasis and keep the dynamic equilibrium of generation of degradation of A?have became one of the therapic methods in AD.S100A6 is a small EF-hand acidic Ca²⁺/Zn²⁺binding proteins of 10kDa belonging to the large S100 protein family.Monomer of S100A6 binds two Ca²⁺that induces conformational changes which are sufficient to induce interaction with target proteins and transduction of Ca²⁺signals.Under physiological condition in neural system,S100A6 is mainly expressed in neurons in restricted areas of the brain?amygdala and entorhinal cortex?and found in a few astrocytes.Interestingly,overexpression of S100A6in astrocytes has been observed arround amyloid plaques deposits in AD patients and AD transgenic animals models.It suggested that S100A6 maybe involved in AD process.But the function of overexpression of S100A6 in astrocytes in AD is unclear.It has been researched that S100A6 has two zinc binding-sites and has an even higher affinity for zinc ions than for calcium ions.Binding of Zn²⁺also induces conformational changes in the S100A6 molecule but they are different from those observed after binding of Ca²⁺.Because zinc is enriched in the neocortical areas and even further concentrated in the amyloid deposits in AD,and S100A6 around the amyloid plaques has two zinc binding-sites,we supposed that overexpression of S100A6 around the amyloid plaques in AD maybe play an important role to inhibit the aggregation of A?and the information of plaques through competition for zinc.In the present study,first,we probed correlation of zinc and overexpression of S100A6 in astrocytes in APP/PS1 transgenic mouse brain.Then we investigated the potential of S100A6 to degrade A?deposited in APP/PS1 mouse brain section in situ.To further identify the function of S100A6 overpressed arround amyloid plaques in APP/PS1 mouse brain,we constructed S100A6/APP/PS1 three-transgenic-mouse which overpressed the S100A6 in APP/PS1 mouse astrocyte,and examined the A?burden and cognitive performance in elder S100A6/APP/PS1 mouse compared with APP/PS1 mouse.Based on the results of the current study,it is reasonable to emply that expression of S100A6 in AD will play an important role in the neuropathophysiology of AD.Methods:1.Adopt double transgenic mice?APP/PS1 mice?with APP695swe gene and the mutant presenilin,PS-1 and APP/PS1 mice with S100A6 special overexpression in astrocyte as the research object,detected the correlation of high zinc feeding and the overexpression of S100A6 in APP/PS1 transgenic mouse by Confocal Laser Scanning Microscopy?CLSM?,detected the expression changes of S100A6 in APP/PS1 transgenic mouse feded with high zinc by RT-PCR and Western blot,applied primary cell culture techniques to cultrue cerebral cortex astrocytes from new born mice,and detected the expression changed of S100A6 in primary astrocytes sitimulated with zinc.2.Established the Cos-7 cell overexpressed S100A6 using transfection method,detected the changes of zinc ions and senile plaques in the APP/PS1 mice brain slice co-cultured with Cos-7 cells transfected hS100A6 by cell culture techniques,TSQ technology,immunohistochemical technique and ELISA technique.3.Established the APP/PS1transgenic mice overexpressed S100A6 in astrocyte specially with transgenic technology,and detected the DNA and protein expression of S100A6 to identify the transgenic mice,and the detected the learning memory ability using water maze test and detected the senile plaques in the cerebral cortex and hippocampus of S100A6/APP/PS1 mice.Results:1.Study on relationship between high-Zn and the high level expression of S100A6 protein.Results of confocal scanning immunofluorescence showed that,in APP/PS1 mice fed with high zinc diet group,A?positive plaque was significantly higher than that in normal feeding group,zinc chelator feeding group and chelating agent feeding group.The positive expression of S100A6 protein was mainly distributed in the senile plaques.S100A6 in APP/PS1 mice fed with high zinc diet group was significantly higher than that in normal group in feeding,zinc chelator feeding group and chelating agent feeding group.Immunofluorescence results showed,primary astrocyte of neonatal mouse displayed GFAP positive.It was suggested that the culture of astrocyte were successful.RT-PCR results showed that mRNA of S100A6 have no significantly difference in high zinc diet group,in normal feeding group,zinc chelator feeding group and chelating agent feeding group.Western blot results showed that S100A6 was significantly higher in APP/PS1 mice than those other groups.2.S100A6 can bind the zinc ion to promote the depolymerization of senile plaques.Western results showed that,S100A6 could promote the expression of hS100A6 in Cos-7 cell.TSQ results showed that,zinc ion specific positive staining decreased in APP/PS1 transgenic mouse brain sliceswithfreshstabletransfectionofculturedhS100A6Cos-7cells.Immunohistochemistry results showed,senile plaque A?positive staining was shallowed and A?polymer visible in senile plaques depolymerization when APP/PS1 transgenic mouse brain slices were cultured with fresh S100A6 protein or transfection of cultured hS100A6 Cos-7 cells.3.Expression of astrocyte specific S100A6 effect on learning and memory ability of APP/PS1 mice in vivo.PCR results showed that 252kb bands is the positive for S100A6 transgenic mice.RT-PCR results showed that expression of S100A6in mouse cerebral cortex,hippocampus,cerebellum,spinal cord,adrenal and testis were exists in transfected human S100A6.Other tissue does not detected the expression of S100A6.Western blot results showed that the expression of S100A6 in transgenic mice brain were higher than wild type mice.Immunofluorescence results showed S100A6expression was exits in S100A6 transgenic mice astrcyte.The expression of S100A6 in wild-type mouse is negative.PCR results showed that the location of the 252kb both the S100A6 band and APP/PS1 band is S100A6/APP/PS1 three transgenic mice.Water maze results showed that,the path and the time of S100A6/APP/PS1 three transgenic mice are less than APP/PS1 transgenic mice.“Shuttle back and forth”test showed that three transgenic mice through the platform were more than APP/PS1 transgenic mice,there was significant difference.Water maze experiment confirmed the ability of learning and memory in three transgenic mice were improved compared to APP/PS1 transgenic mice.A?immunohistochemistry results showed,quantity and load of senile plaques in S100A6/APP/PS1 three transgenic mice are less than APP/PS1 transgenic mice.There were significant differences.Conclusion:1.High zinc could increase the expression of S100A6 in brain astrocyte.2.S100A6 can combine with the zinc competitively to make A?-zinc compound depolymerization.3.Astrocyte overexpressed S100A6 can reduce the formation of senile plaques and improve the ability of learning and memory of the APP/PS1 transgenic mouse.