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Alzheimers disease is a progressive neurodegenerative disease where it slowly takes away a persons ability to functionally compose thoughts, retain memories, and carry out simple tasks such as self maintenance. In the United States alone, approximately 5.3 million people over the age of 65 suffer from AD, making it the 7th leading cause of death. There are several forms of AD, Late-Onset Alzheimer's Disease (LOAD) is the most common form typically affecting individuals over the age of 65 and approximately 90% of AD patients have LOAD. The sporadic and non-genetic nature of LOAD separates it from its counterparts Early-Onset and Familial AD, where they cover the remaining 10% of AD patients (Alzheimer's Association). It is unclear whether aging is the cause or a part of AD since most AD patients are over the age of 65 (Swerdlow, 2006). As people age, it is not unusual that their ability to recollect specific events dwindles. Or that it takes them longer to perform certain task given that the neuronal synapses in their brain are not as efficient as before in connecting and relaying messages. Occasional episode of forgetfulness is a common aspect of aging. However, repeated poor judgment or reasoning as well as cognitive impairments are not simply due to poor synaptic connections but the deterioration of brain cells. Symptoms of AD are often misdiagnosed as depression, Huntington's disease, brain tumors, or natural signs of aging (CureResearch.com). Therefore, definite confirmation of AD is typically not until after death when the brain is thoroughly analyzed for presence of Î²-amyloid plaques and neurofibrillary tau tangles. The accumulations of Î²-amyloid plaques and neurofibrillary tau tangles found in the cerebral cortex and hippocampus regions of the brain have been shown to be strong neuropathological indicators of AD (Alzheimer's Association).
The cerebral cortex, consisting of frontal, parietal, temporal and occipital lobes of the brain is in charge of many important and vital functions of the human body. Respectively, these four lobes are responsible for functions like movement control, memory, intellect, body awareness, and but not limited to speech/visual comprehension. The hippocampus plays a vital role in learning and processing spatial memory and navigation (Healthcommunities.com). Therefore, the aggregation of plaques and tangles in any of these areas is detrimental which will result in death as brain tissues shrink and fail to function properly (National Institute on Aging).
In healthy young people, the CpG-rich promoter region of genes is highly regulated by DNA methylation. When a gene promoter is heavily methylated the gene is silenced. However, when hypomethylation is observed in a gene's promoter it leads to the expression of genes in AD (Zawia et al, 2009). In AD, it is the loss of methylation in the promoters that leads to the expression of APP, BACE, and MAPT genes (Zawia et al, 2009). Not only does the misregulation of DNA methylation play a role in AD but histone posttranslational modifications specifically increasing histone acetylation allow neurons to undergo apoptosis (Mattson, 2003).
The first of the two structures that have been identified in LOAD leading to the destruction of neurons is Î²- amyloid plaques. Î²-amyloid plaques are insoluble protein fibrils of Î² pleated sheets that accumulate in between neurons (Haass & Selkoe, 2007). Amyloid Precursor Protein gene (APP) is found on chromosome 21. It is the proteolysis of Î² -amyloid plaque protein (APP) that leads to amyloid beta formation (Haass & Selkoe, 2007). In LOAD patients, the APP gene has decreased methylation therefore leading to the increased expression of APP which can lead to the increase amyloid beta accumulation (Mattson). The aggregation of Amyloid beta is responsible for the disruption of neurons which leads to cell death in AD by causing oxidative stress and disturbing the calcium homeostasis (Mattson). Î² site APP-cleaving enzyme gene (BACE) is found on chromosome 11, it is the loss of methylation of the BACE genes that leads to the over expression of BACE and the production of the Î² -site aspartyl cleaving enzyme which cleaves APP and leads to the accumulation of Amyloid beta (Mastronei et al , 2008). In studies by Kobayashi et al, 2008 showed that when BACE was knocked out in mice that it did indeed abolish the accumulation of amyloid beta however more severe phenotypes and neurological impairments were observed.
There are two pathways (benign, AD) for how APP is cleaved and depending on the specific enzyme that cleaves APP, which is embedded in the neuronal cell membrane, depends on which pathway will occur. The normal benign pathway is when the enzyme Î± secretase cleaves the APP molecule in the site where amyloid Î² could form and therefore no accumulation of this plaque (National Institute of Aging). Instead of AÎ² being cleaved from the neuron, sAPPÎ± is released which promotes cell survival and growth. Then the gamma secretase cleaves at the end of the APP fragment which is attached to the neuron releasing a small fragment outside of the neuron and the larger fragment stays within in neuron interacting within the nucleus of the cell (National Institute of Aging). Whereas the AD cleavage pathways involves the Î²-secretase cleaving APP at one end of the Î²- amyloid peptide releasing sAPPÎ² (National Institute of Aging). Gamma-secretase cleaves at the other end and Î²-amyloid is then released outside of the cell where it can bind with other Î²-amyloid peptides and accumulate in between neurons. Once Î²-amyloid accumulation of oligomers form they can interact and disrupt the receptors on surrounding cells and interfere with synapses which lead to memory loss and eventually cell death (National Institute of Aging). Since the promoter's of APP and BACE genes are unmethylated and through Histone PTM specifically acetylation that increase expression of these genes which contribute to the production and formation of amyloid beta plaques that are responsible for neuronal cell death. However, it is not only plaques that lead AD but also neurofibrillary tangles formed by protein tau. In order for the amyloid beta accumulation which leads to neurodegeneration in AD both amyloid plaques and tangles must be present.
Neurofibrillary tau tangles are primarily composed of hyperphosphorylated microtubule-associated protein tau (MAPT) encoded by the MAPT gene. In normal brain cells, these insoluble tau proteins form part of the microtubules that stabilize the structure of neurons. However, in AD patients, aggregation of tau proteins are found abnormally phosphorylated and twisted within neurons preventing proper signaling between neurons which could be one of the factors inducing cell death and therefore shrinkage of the brain. Neurons that are depleted of abnormal phosphorylated tau are observed to have rapid microtubules turnover to maintain cell structure whereas in AD patients, neurodegeneration occurs when microtubules turnover cease (ScienceNews). The specific mechanism of how these proteins are made and regulated is unknown. However, it is postulated that the demethylation of protein phosphatase 2A (PP2A) which is an enzyme produced from PPP2CA gene disrupts the binding of PP2A catalytic and regulatory subunits thereby preventing PP2A from dephosphorylating tau. The reduced expression of PP2A in AD patients is believed to be the cause of decreased tau dephosphorylation. When PP2A expression decreases, it induces abnormal hyperphosphorylation of MAPT which stimulates the accumulation of neurofibrillary tau tangles observed within neurons of AD patients (Zhang C.-E, 2007). In addition, it is postulated that the demethylation properties of JMJD and Histone PTM by H3S10 that contribute to the self assembly of phosphorylated tau when tau is hyperphosphorylated.
The goal of this research is to determine the mechanisms that cause this loss of methylation in gene promoters associated with AD by looking at how activating epigenetic histone modifications play a role in the progression of Late-Onset AD. Therefore by identifying the specific histone posttranslational modifications that occur at these gene promoters, they will determine solutions for how to target the misregulation of gene promoters that leads to LOAD. Our research will provide the answer to the gap of information so that better approaches can be made on how to prevent or slow down the progression of neurodegeneration in AD to improve the quality of life.
Research Design and Methods:
AIM 1: Produce Alzheimer's disease transgenic mice with strains expressing APP, MAPT, and BACE gene and isolate tissues from the cerebral cortex and hippocampus regions of brain known to be associated with AD.
Experimental Design: The first gene to be successfully transferred into mice was in 1980, since then further advancements have increased the likelihood of generating an ideal human pathology model for research. This technique is an excellent way to allow researchers to observe how genes function in development, physiology and disease pathology. Transgenic animals are generated in two different ways. The first technique is to microinject the desire gene into the pronucleus of zygotes and the other technique is to inject embryonic stem cells into blastocysts of experiment animals (Kimball's Biology Pages). Due to the nature of our research we believe injecting APP, MAPT, and BACE genes into the pronucleus of mouse zygotes will minimize the potential errors and cost of the embryonic stem cells technique. Using the recombinant DNA methods, APP, MAPT, and BACE genes will be inserted into vector DNAs which will allow our desired molecules to be inserted into the mice DNA molecules. Newly fertilized eggs will be collected before sperm head form a pronucleus. The desired DNAs are then injected into the male pronucleus and pronuclei will then fuse forming the diploid zygote nucleus. Once zygote divide to form 2-cell embryo, the embryo will be implanted into a pseudopregnant (mating of female with vesectomized male) female mouse. Pseudopregnancy will bring about hormonal changes that will allow the female uterus to be more receptive of implanted embryos. After birth, DNA (tissue from tail) of healthy pups is examined for desired genes. Pups carrying the genes are heterozygous for the genes; however the mating of two heterozygous mice will produce (1:4) offspring that will be homozygous for the transgene. Offspring will be screened for transgene (Kimball's Biology Pages). Once mice are shown to be expressing the desired genes, tissues from the cerebral cortex and hippocampus are collected to be further analyzed. AD transgenic mice will be made by the University of Missouri-Columbia Transgenic Core if needed.
Transgenic mouse will be sacrificed; the brain will be removed and rinsed in ice cold DEPC treated Milli Q water to remove any surface blood. The right and left hemisphere are then divided on a cold metal plate. The desired regions, cerebral cortex consisting of frontal, temporal, parietal, and occipital lobes as well as the hippocampus are collected and flash freeze in liquid nitrogen and store at -80° C (Chiu, 2007).
Possible Limitations: Even though it has been shown that generating transgenic mice with desired genes is very successful, however, in order to prevent a complete stop in our research due to the inability of generating AD transgenic mice if it occurs, we will resort to injecting mice with chemicals that will induce AD symptoms in mice. For example, homocysteine (Hcy) can be injected via vena caudalis to our treatment group and saline to our control group. Hcy will cause significant tau hyperphosphorylation as seen in AD patients (Zhang, Chang-E, 2007). Also, generating AD transgenic mice and collecting tissue samples could pose to be very time consuming procedures. In order to prevent the cost of procedural errors when collecting tissues from AD transgenic mice, tissues collection will be practiced on normal mice.
AIM 2: To verify and quantify the presence of APP and MAPT proteins in tissues from AD transgenic mice versus normal mice.
Experimental Design: Previous studies have shown that in the cerebral cortex and hippocampus areas of AD patients, there is an elevated level of Î²-amyloid protein plaques and neurofibrillary tangles formed from the cleavage of Î²-amyloid precursor protein (APP) and hyperphosphorylated microtubule-associated protein tau (MAPT) respectively when compared to normal healthy brain. Therefore, in order to confirm that the homogenized tissue extracts isolated from AD transgenic mice exhibit similar pathology as AD patients, Western blot will be used to analyze the presence of APP and MAPT proteins in treatment and control mice samples. The bands result from the Western blot will be quantitatively analyzed for elevated presence of APP and MAPT proteins in AD mice versus normal control mice. Tissue extracts from treatment and control groups are sonicated into smaller fragments and are then combined with detergents, salts, and buffer to solubilize the proteins. Proteins are then separated by gel electrophoresis, SDS polyacrylamide gel. A marker with defined protein mixture is also loaded onto gel to be used as a comparison and location identification of APP and MAPT proteins. Once separated, proteins are then transferred onto nitrocellulose membrane retaining same pattern of separation as on gel. Special antibodies specific for APP and MAPT proteins are applied to the membrane to bind to desired proteins. The membrane will be rinsed of any unbound proteins. When incubated, the enzymes in the antibodies will convert the colorless substrate to a colored product revealing the antibodies' location (Davidson College). The various bands revealed from Western blot will be qualitatively analyzed for enrichment of APP and MAPT proteins in treatment and control groups.
Possible limitations: If the Western blot fails to clearly separate and reveal the location of desired proteins, as an alternative an ELISA assay could also be done to determine the presence and quantity of the specific APP and MAPT antibodies.
AIM 3: To verify that APP, BACE, and MAPT genes have loss of methylation in their promoters in the transgenic AD versus control mice.
Experimental Design: It has been proved that there is loss of methylation observed in gene promoters in AD resulting in the increased expression of certain genes, APP, BACE, and MAPT (Mastroeni et al, 2008). First, PCR amplification assays will be developed using the human genome that is publicly available (NCBI) because our transgenic mice have the human genes of the abovementioned genes. PCR primers will be designed for the promoter regions of APP, BACE, and MAPT genes to amplify regions (~500-600 bp) within theses regions with more > 50 CpG dinucleotides. APP gene is located on chromosome 21, and is 290, 272 bp. The protein is 770 amnio acids in length. BACE gene is located on chromosome 11 and is 30,559 bp. The BACE protein is 501 amino acids long. MAPT gene is located on chromosome 17 and is 133,924 bp with a protein of 758 amino acids in length. Then sequencing will be done to verify that we are amplifying the correct genes using the University of Missouri's DNA Core. Once it is verified we are in the correct loci then PCR bisulfite primers will be designed for the previously mentioned promoter regions (unmethylated promoters of AD mice) and for the control (methylated promoter of normal mice) to determine the methylation status. To verify that our transgenic mice are indeed expressing the human genes associated in AD we will perform Combined Bisulfite Restriction Analysis (COBRA) on tissues collected from the brain in the abovementioned procedure for both the AD and control mice. The tissue samples will be put into DNA lysis buffer (Tris-Lysis Buffer, Proteinase K). The following day the DNA will be extracted using Phenol-Cholorform. Bisulfite mutagenesis will be performed using a commercially available kit (Sigma, Imprint DNA Modification Kit). This will allow all the unmethylated cytosines to be converted to uracils and then to thymines while the methylated cytosines remain unchanged during PCR amplification. A polyacrylamide gel electrophoresis (PAGE) will be done to verify the assay works before restriction enzyme digestion. Then using restriction enzymes that are methylation sensitive to only cut unmethylated promoter regions (AD mice) using PAGE to determine the methylation status. Next, each gene digestion for both the control and AD mice will be analyzed using commercially available quantification software to determine methylation percentages.
Possible Limitations: In previous studies that used both human and mice models showed that there is loss of methylation observed in LOAD vs control models(Mastroeni, et al 2008, Kobayashi et al, 2007). In order to prove that our transgenic mice with human APP, BACE, and MAPT genes are functioning properly we need to prove that there is methylation differences between our treatment and control groups before we can further progress with our project. Alternative methods have been addressed in the first part of how if the transgenic mice don't work how we will overcome this issue and retest the mice using this procedure.
AIM 4: To prove that in the AD genes that there are activating histones marks that allow for the increased expression observed in the APP, BACE, and MAPT genes.
Experimental Design: Chromatin Immunoprecipitation (chIP) assay will be performed. ChIP will allow us to determine protein-DNA interactions on a specific gene locus of interest, APP, BACE, and MAPT. Using brain tissue collected in the above mentioned protocol. ChIP will allow us to determine the epigenetic marks specifically activating histone acetylation and phosphorylation that are present at each of the loci of interest, which will determine the mechanisms of gene expression in AD. Brain tissue will be collected and used from treatment and control groups. Using commercially available kits e.i. (Active Motif- ChIP IT) and already established protocols in the lab. The first step of ChIP is the cross-linking of protein-DNA using 1% final Concentration of formaldehyde. Next, after cross-linking, the cells are lysed to release chromatin and crude extracts are sonicated which allows the DNA to be sheared into fragment of ~600bp. Proteins together with cross-linked DNA are subsequently immunoprecipitated using 5 ug of Antibodies (for each specific loci of interest APP- H3K9ac, K14ac, H4K5ac, K16 ac; BACE- H3K9ac, K18ac, H4K5ac, H2BK6ac, MAPT- H3S10 phos., JMJD2A and C) antibody. JMJD2 A and C demethylate repressive histone trimethylation marks of H3K9 and H3K36, therefore increasing the expression of MAPT. Last the Protein-DNA crosslink's are reversed followed by PCR amplification. Using primers to amplify the promoter region of APP, BACE, and MAPT using assays designed in AIM 3. Then an agarose gel will be made to verify if the histone mark of interest was present for not in the specific promoter region.
Possible Limitations: It is possible that the specific histone acetylation and phosphorylation, demethylation marks aren't the ones that are present in these specific promoters and that other histone marks are present. If this is the case will be ChIP with other histone antibodies.
Timeline of proposal:
Transgenic mice: up to 6-7 months to generate AD transgenic mice. From 8-16 months, tissue will be collected from AD mice allotted time to observe phenotype and behavior similarities in AD. During this time control mice will be analyzed and PCR, western, ChIP assays will be developed (18 months). Once the transgenic mice are of age then the previously developed assays will be used (10 months).