The Role Of Rab21 In Alzheimer’s Disease

Alzheimer’s disease(AD)is one of the most common neurodegenerative disorders.The main clinical symptoms of AD include progressive memory loss,cognitive impairment,personality change,and language disabilities.Besides,the typical pathological features are senile plaques composed of extracellular accumulation and deposition of amyloid beta(Aβ),and intracellular Neurofibrillary Tangles(NFTs)formed by aggregation of hyperphosphorylated tau.Previous investigations regard AD as a multifactorial disease,in which genetic factor,environmental factor,and immune factor and as other play a role.However,the clear pathological mechanism that leads to AD is still elusive.Currently the most recognized theory is the Amyloid Hypothesis,and it supposes that,in AD progression,Aβ oligomer aggregates in brain,which is high toxic to neighboring neurons and synapses,finally leading to neuron death.Aβ is a small peptide derived from β-amyloid precursor protein(APP)through sequential cleavages by β-and y-secretase enzymatic activity.Beforeβ-secretase and y-secretase have been key targets in the therapy development of AD.Yet,there have been hundreds of inhibitors targeting β-secretase or y-secretase,and they were demonstrated to reduce the A β-amount significantly in vitro and in animal models.However,once translated to human,almost all failed due to inconspicuous effect or side effect of skin and stomach.In terms of y-secretase inhibitors,serious side effects were often observed due to inhibition of Notch signaling.As a result,the focus has been shifted to specific y-secretase modulators and interacting proteins.In this thesis,PS1,the catalytic subunit of y-secretase,was selected as the target.Starting with screening of PS1 interactors,further studies were done as follows:1.Using co-immunprecipitaion coupled with HPLC/MS/MS method to screen proteins which interact with PS1-the catalytic unit of y-secretase.After doing co-IP with PS1 antibody and analyzing by HPLC/MS/MS,Rab21 was chosen as the promising candidate from hundreds of proteins.To validate,three different PS1 antibodies were used together with different proteolysis method.Moreover,analysis of the MS data identified 340 proteins,and GO and KEGG pathway analysis found that Rab21 and another 19 proteins have the GTPase activity,among which some have been reported relevant in AD pathology.It suggests that PS1 closely relates to endocytic pathway and dysregulation of endocytic pathway plays a role in AD progression2.Verification of the interaction between PS1 and Rab21 by biological methods.We amplified RAB21 gene from HELA cDNA,and cloned it into pcDNA4-myc/his expression vector.HEK293 cells were transfected with PS1 or Rab21,and then PS1 and Rab21 antibody was used separately to do the co-IP,the result shows they co-precipitate with each other.Then the colocalization of Rab21 and PS1 in both HEK293 and SHSY5Y cells was identified through immunofluorescence experiment.The above results demonstrate that Rab21 and PS1 interact with each other.3.Regulation of Rab21 in APP processingAfter transient transfection of pcDNA4-Rab21 into 20E2 cell,We observed that the level of APP-CTF in the cell decreased,while the level of Aβ increased.These results indicate that Rab21 affects the APP cleavage.Next,APP-CTFs(C83,C89,C99)and PS1 were co-transfected with empty vector or Rab21,and it was found that the APP-CTFs level is decreased with overexpression of Rab21,which indicates that Rab21 affects the APP processing through regulation of y-secretase.Then,through co-transfection of C99 with Rab21 or shRab21,it was identified that overexpression of Rab21 promotes the formation of Aβ40 and Aβ42,while knockdown of Rab21 leads to contrary result.Fluorescent assay illustrated that overexpression of Rab21 could elevate γ-secretase activity and knockdown of Rab21 impaired y-secretase activity.Furthermore,overexpression or knockdown of Rab21 in 20E2 or 2EB2cells didn’t change the protein level of sAPP(total),sAPP和 sAPPβIt suggests that Rab21 affects APP cleavage process through y-secretase rather than α-orβ-secretase4.Mechanism study on how Rab21 affects r-secretaseFirstly,y-secretase expression level in HEK293 cells was detected,and there was no change in the protein and mRNA level of y-secretase subunits after overexpression or knockdown of Rab21,which indicates that Rab21 doesn’t affect r-secretase by regulating the expression.Secondly,the cell organelles were separated by sucrose density gradient centrifugation,and then analyzed by Western Blot.It was identified in the group transfected with Rab21,the level of PS1-NT,PS1-CT elevated significantly in 5th layer,which mainly includes late endosome and lysosome.Based on that,we speculate that Rab21 can retain PS1 in the late endosome,and therefore change its subcellular distribution.Furthermore,the results of immunofluorescent staining,in which we co-transfected Rab21-EGFP and PS1-pdsRed,showed that transfection of Rab21 promotes colocalization of PS1 with Rab7 and LAMP1,which demonstrates that Rab21 promotes PS1 migration to late endosome Meanwhile,it was identified that Rab21 promotes y-secretase endocytosis by chasing of PS1 endocytosing from cellular membrane to intra cell.These results suggest Rab21 regulate y-secretase activity through regulating y-secretase endocytosis and migration to late endosome.5.The role of Rab21 in AD pathogenesisThrough investigation of Rab21 expression in AD patient and AD transgenic mice,it was identified both mRNA and protein level of Rab21 up-regulates in AD patients compared to control.In AD transgenic mice,the expression of Rab21 varied in different brain regions,with an increase in hippocampus compared with WT mice.Moreover,expression of Rab21 increased with the aging of transgenic AD mice.All of the above suggeste a role of Rab21 in AD pathology.It is worthy to note that overexpression of Rab2l doesn’t affect the Notch cleavage by y-secretase,it further makes Rab21 a promising candidate target.In this thesis,by using co-IP coupled with HPLC/MS/MS method,hundreds of possible PS1 interactors were identified.Then Rab21 was picked out as an interesting target through GO and KEGG pathway analysis.Next,interaction of PS1 and Rab21 was proved by molecular and cellular experiments.Furthermore,to explain how Rab21 affect the APP cleavage,we investigated the possible mechanisms of Rab21.In prospect,the identification of hundreds of PS1 interactors can promote us to understand the function of PS1 and y-secretase,and the study of Rab21 function in APP processing can provide the theoretical support for AD therapy targeting Rab21.