Experimental Study On The Gene Regulation Of Alzheimer's Disease (AD) Associated Protein-Amyloid Precursor Protein (APP) Intracellular Domain (AICD)

BackgroundAlzheimer's disease (AD), also named as senile dementia, is the most common degenerative central nervous system disease accompanied by the main clinical manifestations of progressed dysmnesia, overall hypophrenia and abnormal psyche and behavior. The aging problem of the whole world including our country is becoming more and more aggravated due to the constantly elevated living standard and medical technology. In the year 2007, the Alzheimer's disease evaluation report published by American authoritative institution showed that there were 5.1 million people suffering from Alzheimer's disease, among which 96 percent was over 65 years old (late onset) and 4 percent (two hundred thousand) caused by hereditary factor was under 65 years old (early onset). By the year 2050, the AD patients will triple to about sixteen million. In China, there were 3.5 million AD patients, accounting for 5.9 percent of aged over 65 years old people, similar to that in western countries. Therefore, AD has become the hot study spot of degenerative disease all over the world.During the more than 100 years since German Physician firstly described the symptoms of AD in 1906, researchers have carried out lots of studies on its pathologic character, molecular mechanism including the relevant pathopoiesis genes as well as the functions of their encoding proteins, the signal transduction pathways of the molecules and the clinical therapy targets as well as their corresponding medicines. Nevertheless, there is still no effective therapeutical agent which can be clinically utilized. Therefore the molecular mechanisms of AD need to be further investigated.The AD associated proteins include APP (amyloid precursor protein), AP (amyloid P-peptide), PS-1 (presenilin-1), PS-2 (presenilin-2), Tau and ApoE4 (apolipoprotein E4). Up to date, APP is the most widely studied AD associated protein because its proteolytic products play important roles in the pathophysiology of AD. APP is a glucosylated transmembrane protein including three isoforms respectively comprised of 770,751 or 695 amino acids and abundant in brain. After sequential cleavages byβ-andγ-secretase, APP liberates three fragments:(1) sAPPβ, namely secreted APP-N terminal which is a soluble fragment secreted to the ectocyte after the cleavage by P-secretase, accompanied by the APP-C terminal fragment. (2) Aβ, a 42 kDa peptide results from the cleavage of the APP-C terminal byγ-secretase. It is the main toxic substance and can be secreted to brain tissues and aggregates between neurons and synapses to form the amyloid plaque, also known as senile plaque, which was considered to be the main pathological characteristic of brain slice and can induce brain dysfunction including memory and cognitive disorder. (3) AICD (APP intracellular domain):the intracellular functional domain of APP. It is the intracellular C terminal of APP, a peptide with 59 aa which is produced after the cleavage byγ-secretase. It has gene regulation function trough coupling with other protein or transcription factors.The biological functions of a functional molecule can be realized in two ways: The first is the direct way, which is implemented by itself or its molecular fragment binding to its directly interacting molecules such as the interaction between protein and protein by the way of signal transduction or allosteric regulation, or the interaction between protein and nucleic acid. Secondly, the indirect way is activated by the next level molecules mentioned above with multistage action. Therefore the interactions of the functional proteins, including APP and its cleavage products Aβor AICD, are a network and elucidating the biological functions of APP should be a key to explore the molecular mechanism of AD.AICD is the intracellular domain of APP, by which APP exerts its biological functions. Thus, many of the recent studies are focused on whether AICD can act as a transcriptional factor and participate in the gene regulation. In the year 2001, Cao and Sudhof first reported that AICD could act as a transcriptional activator by forming a protein trimer with Fe65 and Tip60, activating the high expression of luciferase report genes in PC 12 and HeLa cells. In this triple complexity, fe65 is a APP binding protein transferring into the nucleus as a transcriptional factor to control the expression of numerous genes. The AICD of APP can confine Fe65 in the cytoplasm prevents its translocation into the nucleus. Tip60 is a 60 kDa histone acetyltransferase capable of remodeling the chromosome mediated by multiple signals. These signals include STAT-3, CREB and c-myc etc. In the year 2004, the study of Rotz showed that AICD could up-regulate its precursor protein through positive feedback mechanism. All of these indicate that AICD plays a signal role of cutting APP, resulting in the continuous supplement of the full length APP. In the subsequent studies, some researchers reported that KA11, glycogen synthetase3, Neprilysin and APP are all regulated by AICD. Most of these reports resulted from investigating the expression of target genes or the indirect action of the functional proteins such as the results after inhibitingγ-secretase, but not the direct interaction between AICD and its target genes. In 2007, Miiller constructed three eukaryotic plasmids:AICD/EGFP, AICD/Fe65, EGFP (as a control) and transfected them into the neuroblastoma cells. Then he studied the changes of gene expression through overexpressing AICD using the Affymetrix Genechip Method. On the basis of screening out eight up-regulated genes, they further identified three highly expressed genes:a2-Actin, Transgelin and Tropomyosin 1. They all associate with actinand exist in the cytoskeleton. The stability of actin is thought to be related with ageing. Because of the above studies were based on RNA level, it is actual that the results of these observations were complicated effects of gene expression influenced by multiple factors in a network, which did not offer the direct gene regulation information of AICD on DNA level. While acquisition of this information can fundamentally clarify the gene targets of APP/AICD and provide the basis for comprehending the molecule mechanism of AD and developping corresponding clinical therapeutic drugs because blocking or altering a gene and its encoding protein function from the root is the most direct and efficient way to treat a disease. Up to now, there is no research about this information of AICD directly related with the mechanisms of AD.Chromatin immunoprecipitation is an experiment Method used to study the interaction between chromatin and DNA. It can be used to detect whether a known intracellular protein can bind to a specific DNA sequence and what type of DNA region it can combine with. In this method, intact cells were fixed using formaldehyde, resulting in cross-linked protein-DNA complexes. The DNA was then sheared into appropriate fragments using enzymatic digestion and then specific protein-DNA complexes were immunoprecipitated using a specific antibody against the DNA binding protein of interest. Following immunoprecipitation, cross-linking was reversed and the DNA is then analyzed by using PCR to determine which DNA fragments or gene sequence was bound by the protein of interest. Thus ChIP can be utilized to study the regulated regions on chromatin DNA as well as genes directly regulated by transcriptional factors.For the fist time, this research studied the gene regulation of APP/AICD on DNA level through finding AICD binding sites on chromatin DNA by using ChIP. Firstly, we cloned several AICD binding fragments on chromatin DNA, which may be DNA regions of gene regulation and whose homologous genes were relevant with the regulation of physiological processes such as cytoskeleton, signal transduction and metabolism of glycogen synthesis. The most important was that, from the AICD dependent binding sites on chromatin DNA obtained in this research, we identified two promoter regions of learning and memory associated genes viz. CaMKIIa and GluR-2 genes. These results in our research have never been reported and may be the experimental evidence for the molecular mechanisms of AD and preparation of clinic therapeutical agents and throw great significance on the genesis, development, prevention and clinical therapy of AD.Research ContentsSection OneExpression of AICD59 Gene and the Purification and Function of the Encoding ProteinPurposeConstructing the MBP-AICD59 fusion protein expression plasmid AICD/pMAL-c2 and then performing prokaryotic expression by transforming it into the E.coli. Preparing the purified MBP-AICD59 fusion protein followed by digestion with thrombin to obtain AICD59 polypeptide. Meanwhile studying the reaction between the MBP-AICD59 fusion protein and the total protein of rat hippocampal neuron cells.MethodsThe gene fragments encoding AICD59 was obtained by using PCR amplification. AICD59/pMAL-c2 plasmid was constructed by ligating this fragment to pMAL-c2 vector followed by being transformed into E.coli DH5a cells. After positive clones were selected and cultured, the DNA fragments were extracted and subjected to sequencing. Then the AICD/pMAL-c2 plasmid was transformed into E.coli BL21 cells. Positive clones were selected and cultured for overnight. Inductive agent was added to induce the expression of MBP-AICD59 fusion protein and then the cells were harvested and lysised to extract the total protein by using the affinity chromatography method. The total cell protein extracts were passed through an amylose resin column and only MBP-AICD59 fusion protein was absorbed by the column. The hybrid proteins were washed with column buffer and the purified MBP-AICD59 fusion protein was eluted from the column with column buffer containing lOmM maltose. The purified MBP-AICD59 fusion protein was concentrated and cut by thrombin to acquire AICD59 polypeptide and MBP. By using Western blot with MBP as control, the reactivity and specificity of MBP-AICD59 fusion protein and AICD59 polypeptide with AICD antibody were detected. At the same time, the total protein of rat hippocampus was extracted and incubated with MBP-AICD59 fusion protein and MBP. SDS-PAGE electrophoresis was used to observe whether AICD could bind some specific protein. Results1. AICD/pMAL-c2 plasmid was acquired and MBP-AICD59 fusion protein was expressed. The MBP-AICD59 fusion protein was purified by using affinity chromatography. AICD59 polypeptide was liberated by cutting MBP-AICD59 fusion protein with thrombin.2. Both MBP-AICD59 fusion protein and AICD polypeptide could be detected by specific antibody against AICD in the Western blot experiment but MBP could not.3. AICD could bind to some hippocampal cell protein with specific molecular weight.Section TwoIdentification of AICD Binding Fragments on Chromatin DNA for Evaluation of the Gene RegulationPurposeTo acquire DNA fragments bound by AICD or AICD contained protein complexity through fixing, digesting and reversing the cross-linked protein-chromatin DNA complexes containing AICD. Then to locate and analyze the genes regulated and targeted by AICD.MethodsHippocampal neurons of one day neonatal rats were prepared for primary culture for about 10 days and then were dealt with 1% formaldehyde to cross-link and fix the protein-chromatin complexes in the nucleus. The nucleus chromatin was extracted by using ChIP-ITTM Express Enzymatic Kit. The ChIP experiment protocol of Active Motif ChIP-ITTM Express Magnetic Immunoprecipitation Kit Manual was strictly followed. The nuclear chromatin DNA extracts were digested into 200-1000bp followed by overnight incubation with G magnetic beads and antibody against AICD or non-specific IgG (negative control) and then the antibody-AICD (or the protein complex contain AICD)-chromatin DNA complexes were separated from magnetic beads. After magnetic beads were precipitated, the antibody-AICD or complexes containing AICD-chromatin complex were segregated. After reversing cross-linking and enzymatic digesting the complexes of antibody-AICD or AICD contained protein, the chromatin DNA fragments bound by AICD or protein complexity containing AICD were obtained.Terminal deoxynucleotidyl transferase was used to add poly dATP to the 3'-OH end of the unknown DNA fragments and PCR amplification was carried out by using poly-dTTP primer with a restriction enzyme cutting site of Pst I (5'-GTCTCTGCAGTTTTTTTTTTTTTTTTTTTTNN-3', NN are random base). The amplified fragments were ligated to pMAL-c2 vector and transformed into E. coli DH5a after being digested with PstⅠ. Positive clones were selected and different plasmids were extracted for sequencing and the insertion sequences were the sequences of AICD dependent protein binding chromatin DNA fragments. The genes with highly similar biological properties to these fragments were found out by using Basic Local Alignment Search Tool (BLAST).On the other hand, specific promoter primers of learning and memory associated genes were used to amplify the chromatin DNA fragments pulled by antibody against AICD and the PCR products were subject to sequencing. After sequencing, the amplified DNA fragments were respectively incubated with the total hippocampus protein extracts of rat to perform the electrophoretic mobility shift assay (EMSA) to observe whether the DNA fragments can be bounded by hippocampus protein(s). Then the incubated mixtures were respctively transferred to polyvinylidene difluoride membrane to identify if the protein-DNA complexes contain AICD by Western blot using antibody against AICD.Results1. The hippocampal neuronal protein-chromatin complexes were isolated and digested into 200-1000bp fragments.2. The chromatin DNA fragments bound by AICD dependent protein was immunoprecipitated by using antibody against AICD followed by being cloned and sequenced. Further local gene sequences comparison discovered the homologous genes of these sequences including Acetyl coenzyme A acyltransferase 2 (ACAA2) gene, Glycogen kinase 2 (GYG2) gene, phosphatidylinositol transfer protein membrane-associated 2 (PITPNM2) gene and SETEX gene.3. The promoter regions of two learning and memory associated genes were amplified and they were proved to be the promoter regions of CaMKⅡand GluR2 genes.Conclusions1. MBP-AICD59 fusion protein was sufficiently expressed by IPTG induction, but the production of AICD59 was very little owing to the low digestion efficiency. Both MBP-AICD59 fusion protein and AICD59 polypeptide could react with antibody against AICD, whereas MBP could not. These indicated that MBP-AICD59 fusion protein and AICD59 polypeptide have good reactivity with antibody against AICD and the antibody has excellent specificity, therefore, all of them could be used in ChIP experiment. 2. MBP-AICD59 fusion protein could bind specific hippocampal cell protein.3. AICD dependent protein binding sites were existed on the chromatin DNA in hippocampal neuron cells. By using BLAST to compare the DNA fragments bound by AICD, the target genes of AICD were found to have high homology with genes that regulate cystoskeleton, signal transduction and glycogenesis metabolism.4. AICD could bind to the promoter regions of learning and memory associated genes and thus it was believed that AICD should be involved in the formation of learning and memory, which was directly related with the dysfuction of learning and memory in AD.