Study On The Transport Pathway Of Tetherin,a New Interacting Protein For Amyloid β Peptide

Alzheimer’s disease（AD）is the most common dementia in the elderly.Typical neuropathologies of AD include extracellular deposition of senile plaques composed of amyloidβpeptides,intraneuronal fibrillary tangles formed principally by hyperphosphorylated tau,and progressive loss of neurons in vulnerable brain areas.The pathogenic mechanism of AD is unclear.Our current understandings about AD pathogenesis stem mainly from the Aβcascade hypothesis,which posits the deposition of Aβto be a central event in the pathogenesis of AD.Aβis produced through sequential cleavage of amyloid precursor protein（APP）byβ-andγ-secretases.The equilibrium between the production and removal of Aβpeptides in AD brains is disrupted,thereby leading to gradual buildup of Aβplaques and subsequently triggering a cascade of pathological changes including the formation of NFT in surviving neurons.However,therapeutic strategies that target the generation or clearance of Aβfail in stage-III clinical trials one after another,substantially challenging the Aβcascade hypothesis.Mounting evidence links microbial infection to the etiology of AD;on one hand,viral infection promotes the deposition of Aβ,on the other hand,Aβexhibits activities against various microbes including herpes simplex virus1,which is frequently linked to AD.Yet how Aβpolymerization in AD brains shifts from protection against microbial infection to pathological deposition of Aβfibrils remains to be clarified.Tetherin has an unusual topology shared by a pathological isoform of Aβreceptor,cellular prion protein;association with membranes is mediated by a transmembrane domain near the NH₂ terminus and glycosylphosphatidylinositol moity at the COOH terminus.Tetherin is well known for its function in preventing the spreading of a wide range of encapsulated viruses,but its function in healthy cells is largely unknown.We took the advantage of the tripartite GFP fluorescence complementation（Tri FC）assay to examine the potential interaction between tetherin and Aβ₄₂.Fluorescent signals were detected in cells transiently transfected with Tri FC expressing the ectodomain of tetherin and Aβ₄₂,which were fused with the 11^th and 10^thβ-sheet of GFP,respective,indicating that there is bona fide interaction between tetherin and Aβ₄₂.Taken with the finding of tetherin to be a signal molecule in the NF-k B pathway,we reasoned that tetherin might play a role in the development of AD.We constructed GFP fused tetherin or h GREG and red fluorescent protein Ds Red fused vesicle regulators including arf6,rab4,rab5,rab11 and vps35 to track the trafficking pathways of tetherin in cells.Confocol microscopy and quantitative analysis revealed that the majority（aproximately 90%）of tetherin was colocalized with vps35,with minor portion localized to rab4 or rab5 positive early endosomes,indicating that tetherin undergoes vps35 positive retromer dependent recycling through early endosomes.Although there was only 30%of tetherin was colocalized with rab11 in transiently transfected cells,about 80%of rab11 positive structures were found to contain tetherin-GFP.Our previous studies show that the hamster ortholog of tetherin,GREG,contains three tandem EQ repeats（EQEAQIK）that entrap tetherin in the Golgi apparatus.While tetherin in human has no such EQ repeats,bioinformatics analysis retrieved EQ-like repeats in other proteins of human genome.We generated constructs expressing chimeric h GREG-GFP（h GREG-GFPc）with or without EQ-like repeats.Confocal microscopy and quantitative analysis showed that although EQ-like repeats were not able to trap h GREG-GFPc in the Golgi,more accurately cis-Golgi,signals of h GREG-GFPc with EQ-like repeats were more concentrated at areas close to cis-Golgi than those of h GREG-GFPc without EQ-like repeats.In addition,EQ-like repeats altered levels of h GREG-GFPc on the cell surface.In summary,this thesis identifies tetherin as a new interaction partner for Aβ₄₂.Following endocytosis,tetherin recycles through both rab11 and vps35 dependent pathways,specifically,recycling endosomes and retromer-dependent endosome-to-Golgi pathways.Unlike GREG in CHO cells,human GREG（tetherin engineered with EQ-like repeats）does not reside at the Golgi.However,human EQ-like repeats alter the distribution of tetherin.