| Alzheimer's disease(AD),one of the most prevalent neurodegenerative diseases, geatly threatens the health of the elderly and has been the fourth killing disease.AD holds about 60-70%of the totally 32 kinds of dementia.Mainly due to the uncertain aetiology of AD,the complexity of this disease,limited therapeutic drugs,it is hard to make great progress on the prevention and cure of AD in the near future.Cholinesterase inhibitors are most widely used drugs for the treatment of AD now,but their effects are only to ameliorate symptoms but do not achieve any permanent improvement.Therefore, to develop more effective AD therapeutic drugs with lower toxicity and lower side-effect remains an urgent task.The definite targets for anti-AD drugs restrict the development of new promising therapeutic drugs for AD and the breakthrough in this field is urgently calling for discovery and verification of the new targets for anti-AD drugs.However,for any of the compounds,aiming at a sole target for AD,it is indeed a difficulty to obtain the satisfied therapeutic effects since AD is with such a complex pathogenesis which is still unclear till now,and also partly due to the serious consequences of AD,which are linked to abnormal construction and anormal functions of multi-systems.This fact suggests that intervention of multiple targets for AD is more important and which may be a feasible strategy to make great progress in prevention and cure of this stubborn disease.Thus,we propose a hypothesis that "combination targets" may be of great value to design,screen and further evaluate new drugs for AD. Traditional Chinese medicine(TCM)are composed of several herbs and have many ingredients,their effects have the characteristics of multi-mechanisms, multi-pathway and multi-targets,likewise AD is a complex,multi-factorial and genetically heterogeneous syndrome.So the character of the whole traditional Chinese medicinal prescription is consistent with AD,the complicated disease,It is well known that many widely used traditional Chinese medicinal(TCM)prescriptions,including Liu-Wei-Di-Huang decoction(LW),Ba-Wei-Di-Huang decoction(BW), Huang-Lian-Jie-Du decoction(HL),Dang-Gui-Shao-Yao decoction(DSS), Tiao-Xin-Fang decoction(TXF)and Huperzine A(HupA),play important roles in the clinical therapy of AD patients,and also have beneficial effects on cognitive abilities in animal models.Senescence-accelerated mouse(SAM)prone/8(SAMP8)is a good animal model to investigate the fundamental mechanisms of age-related learning and memory deficits such as Alzheimer's disease(AD)at the gene and protein levels,and SAM resistant/1(SAMR1)is its normal control.It has been proved that all of the above-mentioned five TCM,including LW,BW,HL,DSS and TXF,can improve the functional impairments of learning and memory in SAMP8 in our previous studies. Although these five TCM are initially designed for different diseases,they do show the common therapeutic effects on AD patients and animal models.However,they may indeed have the different characters and through the different action mechanisms on the cognitive functions.Having influences on gene expression is one of the mechanisms on drug action. Cerebral cortex and hippocampus are the key regions which are closely linked to learning and memory.Since these five TCM can improve the ability of learning and memory in AD cases,gene expression pattern of SAMP8 should be changed after SAMP8 were given to these TCM.To study these different expressed genes in SAMP8, drug reaction genes could be picked out.Then,to further compare the difference and identity of these drug reaction genes,the common genes responding to all these drugs and also the special genes responding to certain drug could be discovered.The common responding genes may reflect the similar action mechanism of all these drugs while the specific responding genes may represent the distinct action mechanism of the special drug.Thus,it will be very helpful to investigate the changed genes during the degenerative process of cognitive ability in SAMP8,which will not only give cues to find new targets for anti-AD drags but also be an important way to study and search the molecular targets for AD.Consequently,the distinct anti-AD drugs such as TCM LW,BW,HL,DSS,TXF and natural product Hupzine A(HupA)were used as tools and experimental therapy had been conducted to investigate their effects on cognitive ability of SAMP8.Meantime,by Suppression subtractive hybridization(SSH)and cDNA microarray technique,our team bad successfully screened out the candidate "combination targets" for anti-AD drugs, which are composed by various molecules,such as protein degradation related genes AMFR and STUB1,neural cystoskeleton related gene NF-L,mitochondria function related gene NDUFS,signal transduction related genes CAMK2A,EphB6 and CSTN1, and neurogenesis related genes NRXN1 and NGRN.However,these results are mainly based on the cDNA technique,which has a relatively higher false positive rate(FPR).So the necessary verification procedure is needed for the further validation of these targets for anti-AD drugs.In order to preliminary verification of the candidate "combination targets" for anti-AD drugs,from which,this paper selected totally 9 representative genes belonging to five types and used SAMP8 as an animal model for AD.By the methods such as real time Quantitative fluorescence RT-PCR,Western blot,Immunohistochemistry and Immunofluorescence.the auther firstly investigated the age-related expression changes of these 9 molecules in SAMP8 and SAMR1 with aging.Then,the expression changes of these 9 molecules in 12-month SAMP8 treated with anti-AD drugs LW,BW,HL, DSS,TXF and natural product HupA were also investigated.Finally,the respective networks of these 9 molecules,the interaction of these 9 molecules and their possible roles in AD process had also been studied by bioinformatics.This paper have provided the evidences for supporting the hypothesis that "'combination targets",based on the bio-regulative network of targets for anti-AD drugs,may be of great value to design, screen and further evaluate new drugs for AD.Part one.Expression changes of APP and Tau in the cerebral cortex and hippocampus of SAMR1 and SAMP8 and their responses to anti-AD drugsThe main pathological feature of AD is neuron loss,senile plaques(SP)and neurofibrillary tangles(NFT)in the key brain regions.SP and NFT are formed by the intercellular and intracellular deposition and aggregation byβamyloid protein(Aβ)and hyperphosphorylation form of Tau(HP Tau),respectively.Thus,Aβand Tau has been considered as the key factors that lead to the onset and progress of AD.Aβis produced byβ-amyloid precursor protein(APP).Consequently,this paper firstly investigated the expression changes of APP and Tau in the key brain regions related to cognitive functions in SAM.1.Age-related expression changes of APP and its responses to anti-AD drugs With aging process,the expression of APP mRNA is maintained at certain level in the hippocampus and cerebral cortex from SAMR1,while it was increased after 2-month-old in the hippocampus and cerebral cortex from SAMP8.Compared with SAMR1,the expression of APP is increased significantly in SAMP8;The results from immunohistochemistry(IHC)showed that the number of positive cells is increased with aging in the cortex and hippocampus in SAMP8.After SAMP8 was given anti-AD drugs LW,BW,HL,DSS,TXF and HupA for a month,the abnormal high mRNA expression of APP was significantly down-regulated in cerebral cortex of 12-month-old SAMP8 treated with LW,HL,DSS,TXF and HupA. In hippocampus of SAMP8,only BW and DSS treated groups showed down-regulative effects on mRNA expression of APP.The results indicated that these anti-AD drugs could regulate the abnormal mRNA expression of APP and meantime they indicated that APP could actively respond to these anti-AD drugs.2.Age-related expression changes of Tau and its responses to anti-AD drugsWith aging process,the expression of Tau mRNA is maintained at certain level in the cerebral cortex from SAMR1,while it was significantly increased in cerebral cortex in 12-month-old SAMP8.Compared with SAMR1,the expression of Tau is increased significantly in the cerebral cortex in 12-month-old SAMP8.In the hippocampus from both SAMP8 and SAMR1,Tau mRNA was increased with aging.Compared with SAMR1,the expression of Tau is significantly increased in hippocampus in every month of SAMP8.The different expression pattern for Tau in SAMP8 indicated that the abnormal higher expression of Tau may result in the degeneration ability in learning and memory.After SAMP8 was given to anti-AD drugs LW,BW,HL,DSS,TXF and HupA for a month,the abnormal high mRNA expression of Tau was significantly down-regulated in cerebral cortex of 12-month-old SAMP8 treated with all these anti-AD drugs.In hippocampus of SAMP8,only HL-treated group didn't show down-regulative effects on mRNA expression of Tau.The results indicated that these anti-AD drugs could regulate the abnormal mRNA expression of Tau and meantime they indicated that Tau could actively respond to these anti-AD drugs.Many studies have indicated that,there were age-related defects in cognitive abilities in SAMP8.Our studies showed that in SAMP8,both APP and Tau were increased with aging process both in the cerebral cortex and hippocampus,the key brain regions closely linked to learning and memory.At the same time,they showed the apparent responses to these anti-AD drugs.These results further indicated that SAMP8 is an excellent animal model and which could be used to preliminarily verify the candidate obtained "combination targets" for anti-AD drugs.Part two.Expression changes of the 9 molecules from the candidate "combination targets" for anti-AD drugs and their responses to anti-AD drugs1.Abnormal protein degradation related molecules AMFR and STUB11.1 AMFRAMFR,a G-protein-coupled receptor,shows an ubiquitin protein ligase(E3) activity and participates in degradation of abnormal proteins from the smooth endoplasmic reticulumits-associated degradation(ERAD).Studies showed that after its binding with its endogenous ligand Autocrine motility factor(AMF),AMFR played various roles such as stimulating cell migration,facilitating the cell survival.To study whether AMFR involves in the AD process or not,this paper had studied the age-related mRNA and protein expression of AMFR and its distribution in SAM with aging.1.1.1 Age-related expression of AMFR and its ligand AMF in the cerebral cortex and hippocampus of SAM with agingIn both cerebral cortex and hippocampus,AMFR mRNA increased significantly after 2-month-old in SAMR1 while it decreased significantly in both cerebral cortex and hippocampus from SAMP8.Compared with SAMR1,the expression of AMFR mRNA decreased significantly in both the cerebral cortex and hippocampus in 12-month-old SAMP8.In the cerebral cortex of SAMR1,AMFR protein increased after 2-months of age while in SAMP8,it showed an apparent decrease tendency after 6-months of age. Compared with SAMR1,AMFR protein decreased significantly after 6-months of age in SAMP8.In the hippocampus of SAMR1,AMFR protein increased after 2-months of age while it decreased with aging in SAMP8.Compared with SAMR1,protein expression of AMFR increased significantly at the 2-month old SAMP8,but decreased significantly at 6-and 15-month old SAMP8.In the cerebral cortex of SAMR1,AMF protein increased significantly at 10-and 15-month-old SAMR1 than its expression level at 2-and 6-month-old SAMR1 while in samples from SAMP8,its expression decreased after 6-month-old with aging. Compared with SAMR1,AMF protein decreased significantly after 6-months of age in SAMP8.In the hippocampus of SAMR1,AMF protein maintained at a certain level with aging process while its expression showed an increase before 10-month-old and a decrease since 10-month-old.Compared with SAMR1.AMF protein decreased at all of age. 1.1.2 Expression of AMFR in the cerebral cortex and hippocampus of SAMP8 treated with anti-AD drugsCompared with the normal control group,the mRNA and protein expression in the cerebral cortex and hippocampus of SAMP8 was decreased significantly.After SAMP8 was treated with the six anti-AD drugs,the abnormal low expression of AMFR was up-regulated by LW and HupA for mRNA and LW,BW and HupA for protein in the cerebral cortex of SAMP8.In the hippocampus of SAMP8,the abnormal low expression of AMFR was up-regulated by BW,HL,DSS,TXF and HupA for mRNA and LW,BW and HupA for protein.1.2.STUB1The STIP1 homology and U-box-containing proteinl(STUB1),a cytoplasmic protein also named C terminus of Hsc70-interacting protein(CHIP),is a Hsp70 co-chaperone and an E3 ubiquitin ligase that protects cells from proteotoxic stress,so STUB1 plays the pivotal role in the protein QC system.By its U-box domain,STUB1 acts as an E3 ligase to facilitate the transfer of a polyubiquitin chain to misfolded substrates,while by its TPR domain,STUB1 binds heat shock cognate 70(HSC70)or HSP70,attenuates the HSP40-stimulated ATPase and refolding activities of HSP70, which contributes to the delivery of protein substrates to lysosomes-mediated autophagy. So STUB1 is one of the most important members of UPS and acts as a molecular switch between proteasomal and lysosomal degradation pathways,mediates crosstalk between molecular chaperones and the UPS.This study showed that,with aging process,both mRNA and protein expression of STUB1 in cerebral cortex and hippocampus from SAMR1 were increased after 2-month-old while decreased in the above brain tissues from SAMP8 after 6-month-old;compared with SAMR1,mRNA and protein expression of STUB1 were decreased after 10-month-old SAMP8 respectively and could be up-regulated by the six anti-AD drugs.2.NF-LNeurofilament proteins are specific intermediate filament for neurons and NF-L is the backbone for NF-M and NF-H to polymerize.NF-L plays an important role in maintenance and regulation of the dynamic cystoskeleton of neurons by regulating the growth of neural cells,axis caliber and axonal transport.Consequently,NF-L plays a key role in CNS of the mammal.This study showed that,with aging process,both mRNA and protein expression of NF-L were decreased significantly in cerebral cortex and hippocampus after 10 months of age and which could be up-regulated by HL,DSS, TXF and HupA in cerebral cortex at both mRNA and protein levels while in the hippocampus,all these drugs showed up-regulative effects on both mRNA and protein expression of NF-L.3.NDUFS2NDUFS2 is an important subunit of ironsulfur protein in Complex I for respiratory chain.NDUFS2 has the activity of oxidoreduction and involves in the energy metabolism,which could reflect the level of oxidative phosphorylation.This study showed that mRNA of NDUFS was abnormal expressed in SAMP8 with aging compared with SAMR1,which could be down-regulated by LW,BW,HL,DSS,TXF and HupA in the cerebral cortex of SAMP8 and could be up-regulated by BW,HL,DSS and TXF in the hippocampus of SAMP8.4.Signal transduction related genes CAMK2A,EphB6 and CSTN14.1 CAMK2ACAMK2A is one most abundant subunit of CaMKII in cerebral cortex and hippocampus.The autophosphorylation of the threonine at 286 of CAMK2A is essential for hippocampus long term potentiation(LTP)and spatial learning.CAMK2A may involve in not only phosphorylation at multiple sites of Tau in AD patients,but also phosphorylation of APP in vitro.This study showed that that the levels of both CaMKIIαmRNA and protein decreased significantly in the cerebral cortex of SAMR1 with aging,but increased significantly in the cerebral cortex of SAMP8.Compared with age-matched SAMR1,the expression of mRNA and protein of CaMKIIαsignificantly increased in the cerebral cortex and hippocampus of SAMP8 after 10-months of age. After SAMP8 was treated with the previously mentioned drugs,the abnormally high expression of CaMKIIαwas relatively down-regulated for mRNA in both cerebral cortex and hippocampus,for protein in hippocampus by all the six anti-AD drugs used in this study and was relatively down-regulated for protein in the cerebral cortex by all the six anti-AD drugs with the exception for LW.4.2 EphB6EphB6 is a transmembrane receptor and belongs to protein tyrosine kinase(PTK), which an unique protein that have two-way signal transduction system based on its specific structure.This study showed that EphB6 mRNA has different expression patterns in the hippocampus and cerenral cortex,that is,its expression was increased with aging while showed a highest expression at 6-month-old in hippocampus in SAMR1.While in SAMP8,its expression showed an increase tendency in both the hippocampus and the cerebral cortex.For protein expression of EphB6,it showed an increase at 10-month-old but an decrease at 15-month-old SAMR1 in both the hippocampus and cerebral cortex.However,the protein level was increased after 10-month of age in SAMP8.Compared with SAMR1,the EphB6 protein increased significantly after 10-months of age in SAMP8 which could be down-regulated by LW, HL,TXF and DSS in the cerebral cortex and by LW,BW.TXF and DSS in the hippocampus.4.3 CSTN1CLSTN1 is a specific molecule which could link the proteolysis outside of cytes and the signal transduction of Ca2+within the cytes CLSTN1 locates at the postsynaptic membrane and binds with Ca2+to regulate the signal transduction and cell communication.Study showed that this specific protein could cooperate with APP to increase the secretion of Aβ,thus,this protein may accelerate the AD process.Our study showed that CSTN1 mRNA was highest expressed at 6-month-old SAMR1 in both the cerebral cortex and hippocampus while its expression pattern showed an increase in SAMP8 in both of the above-mentioned brain regions.Compared with SAMR1,this gene increased significantly in SAMP8 and which could be down-regulated by LW,BW,HL and DSS in the cerebral cortex and by BW.HL,DSS and HupA in the hippocampus.5.NRXN1 and NGRNNeurexinâ… could bind withα-latrotoxin as a postsynaptic receptor,thus further activate the release of neurotransmitter(NT).In addition,NRXN1 could play its roles as a cellular adhesion molecule(CAM)and closely linked to synaptogenesis.NGRN has been presumed to play an important role in cell growth,maintenance and neuron differentiation.This paper showed that both NRXN1 and NGRN mRNA were abnormally lower expressed in SAMP8 compared with SAMR1.The lower expression of NRXN1 could be up-regulated by BW,DSS,TXF and HupA in the cerebral cortex in SAMP8 and by all anti-AD drugs with the exception for LW in the hippocampus.And the lower expression of NGRN could be up-regulated by LW,HL,DSS and HupA in the cerebral cortex and by all drugs with the exception for TXF.Part three.Relationship between candidate gene targets for anti-AD drugs and the key pathological proteins in AD1.The distribution of key pathological proteins in ADResults from the study by immunohistochemistry showed that with aging process, APP,Tan and P-Tau showed an apparent increase tendency in the subregions CA3 and dentate gyrus(DG)of hippocampus and the subregion temporal lobe(TL)of cerebral cortex.2.The distribution of AMFR protein in SAM with agingResults from the study by immunohistochemistry showed that with aging process, the positive neural cells decreased apparently in the subregions CA3 and dentate gyrus (DG)of hippocampus and the subregion temporal lobe(TL)of cerebral cortex.While in SAMR1,this phenomenen was not apparent or just showed an increase tendency in the same subregions.3.Co-localization of AMFR,STUB1 and APP in subregion frontal lobe of the cerebral cortexResults from the study by immunofluorescence showed a definite co-localization of AMFR and APP,STUB1 and APP in subregion frontal lobe of the cerebral cortex in 6-month-old SAMP8.Part four.Interaction of candidate gene targets for anti-AD drugs and their roles in AD processFirst.by the molecules interaction analysis software Osprey,the molecules that directly interact with the key pathological protein APP,Tau and the nine candidate molecules were analyzed.The results showed that there were total 49 and 16 molecules directly interacting with APP and Tau,respectively.The result indicated that there are many molecules directly interacting with APP and Tau,which were related to multiple functions.To further study these molecules will be no doubt benefiting the further understanding of the etiopathogenisis for AD and which will help to prevent and cure this disease.There were total 3 and 7 molecules directly interacting with AMFR and STUB1,respectively.The analysis result indicated that involvement in protein metabolism was the major function of AMFR,while for STUB1,besides this function, there were also other important functions,The results showed that there were total 4 molecules directly interacting with NF-L and which indicated that the main function of NF-L is cell organization and cytogenesis.However,the software analysis showed that there were no direct interaction molecules to NDUFS2 and NGRN till now.The direct interaction molecules to CAMK2A,EphB6 and CSTN1 were 14,4 and 3,respectively. Phosphorylation of substrates was the basic function for CAMK2A and CAMK2A is a muir-functional enzyme.EphB6 could involve in phosphorylation of substrates and cell organization and cytogenesis.To further study the functions of CSTN1 will benefit both the understanding and the prevention and cure of AD.The NRXN1 could interaction directly to 20 molecules and the major functions of NRXN1 were transport and cell organization and cytogenesis.There were no direct interacting molecules for NGRN. Then,the possible interaction within the 9 molecules as well as APP,Tau,PS1.PS2 and ApoE were analyzed by osprey and the results are as follows:There were no direct interaction within the 9 molecules and only CSTN1 could directly interact with APP and PS1.Finally,the possible interaction of the 9 molecules and their direct interaction molecules with the key pathological protein of AD was also analyzed by Osprey.The results predicted that nearly 80%of these candidate 9 molecules could indirectly interact with APP and Tan by several pathways.Among of which,AMFR could interact indirectly with APP and Tau through the linkage of EphB6;STUB1 could interact indirectly with APP and Tau;NF-L was much closer to Tau but it could also interact with APP;CaMK2A was much closer to APP but it could also interact with Tau by the linkage of APP;EphB6 could indirectly interact with both APP and Tau;NRXN1 was much closer to APP but it could also interact with Tau.These results indicated that these 9 molecules played their roles in AD onset by the interaction network within them and their interacting molecules instead of their respective sole action.The result supports the hypothesis that the interaction network instead of respective sole action of candidates from "combination targets" for anti-AD drugs involves in the pathology of AD.In sum,in order to preliminary verification of the candidate "combination targets" for anti-AD drugs,from which,this paper selected totally 9 representative genes belonging to five types and used SAMP8 as the animal model for AD.By the methods such as real time Quantitative fluorescence RT-PCR,Western blot, Immunohistochemistry and Immunofluorescence,the auther firstly investigated the age-related expression changes of APP,Tau and these 9 molecules in SAMP8 and SAMR1 with aging.Then,the expression changes of these 9 molecules in 12-month SAMP8 treated with anti-AD drugs LW,BW,HL,DSS,TXF and natural product HupA were also investigated.Finally,the respective networks of these 9 molecules,the interaction of these 9 molecules and their possible roles in AD process had also been studied by bioinformatics.The results showed that these 9 molecules not only closely linked to degeneration in learning and memory ability of SAMP8,but also actively responded to the multiple anti-AD drugs used in this paper.The results also indicated that on the one hand,the same molecule expressed differently at its gene and protein levels,which may be due to the post-transcriptional processing,translation and post-translational modification.The same molecule showed differently distributed patterns in part in different brain regions,which may be due to the distinct functions of its own and different brain regions.On the other hand,the same molecule showed differently responses to the same drug in the different brain regions,which may be caused by the specificities of drugs and specific action region of the molecule.However, the same molecule showed the similar responses to different anti-AD drugs,which indicated that this molecule may be the common target for multiple drugs.And also,the different molecules showed the similar responses to the same drug,which may be resulted from the multiple mechanisms of the same drug.In addition,the 80%of the candidate molecules were predicted to interact with the key pathological protein APP and Tau,and they may involve in the pathology of AD by the interaction network instead of respective sole action of themselves.The results obtained by this paper settled the foundation for designing,screening and further evaluating new drugs for AD, which were based on the bio-regulative network of "combination targets" for anti-AD drugs.In conclusion,this paper supported the hypothesis that "combination targets", based on the bio-regulative network of targets for anti-AD drugs,are of great value to designing,screening and further evaluating new drugs for AD.And the "combination targets" are composed by one group or several groups of molecules,which are closely related to cognitive defects in AD and show complementation and cooperation within them."Combination targets" based on the bio-regulative network of targets for anti-AD drugs,will be of great value not only for designing,screening and further evaluating new drugs for AD,but also for studying and elucidating the pathophysiological processes of AD from the view of pharmacology,and also,will give important cues for discoveries of biomarkers used to predict the risk for the onset of AD,diagnose AD and assess the therapeutic effect by anti-AD drugs.
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