Running head : GENE EXPRESSION AND ALZHEIMER ’ S DISEASE 1 Gene Expression and Alzheimer ’ s Disease Evaluation of Gene Expression Patterns in Brain and Blood for an Alzheimer ’ s Disease Mouse Model

Previous studies have established a causative role for altered gene expression in development of Alzheimer’s disease (AD). These changes can be affected by methylation and miRNA regulation. In this study, expression of miRNA known to change methylation status in AD was assessed by qPCR. Genome-wide expression changes were determined by RNA-sequencing of mRNA from hippocampus and blood of control and AD mice. The qPCR data showed significantly increased expression of Mir 17 in AD, and sequencing data revealed 230 genes in hippocampus, 58 genes in blood, and 8 overlapping genes showing significant differential expression (p value ≤ 0.05). Expression data from this study revealed novel gene expression changes affecting AD and identified changing genes in blood as potential AD biomarkers. GENE EXPRESSION AND ALZHEIMER’S DISEASE 4 Gene Expression and Alzheimer’s Disease: Evaluation of Gene Expression Patterns in Brain and Blood in an Alzheimer’s Disease Mouse Model Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid beta (AB) plaques in the brain and loss of neuronal cells with a corresponding decrease in cognitive function [1]. As one of the foremost health challenges in developed countries, AD affects large numbers of people in a variety of environments [2]. Understanding the biological events involved in AD pathology and determining accurate means of early detection are crucial areas of research. While many researchers have investigated the development of AB plaques and other characteristics of the disease, little is known about the initial causes of AD and the specific biochemical processes of AD development. Analysis of changes in gene expression associated with AD pathology is one important area of current AD research. The development of AD is largely directed by increases and decreases in activity of specific genes related to the disease, and understanding these changes will give better understanding of the process of disease as well as insights into possible treatments. Obtaining a profile of genes that affect AD progression could also be used to develop a diagnostic test for this disease. In pursuance of these goals, my research evaluated changes in gene expression in brain and blood using a mouse model of Alzheimer’s disease. AD and Gene Expression Alzheimer’s Disease Alzheimer’s disease is a progressive neurodegenerative disorder that causes a slow but irreversible loss of cognitive ability. The disease is marked by distinctive neuronal amyloid-beta plaques derived from amyloid precursor protein (APP) as well as GENE EXPRESSION AND ALZHEIMER’S DISEASE 5 by neurofibrillary tangles (NFTs) formed by hyperphosphorylated tau protein [3, 4]. These protein deposits are often accompanied by neuroinflammation and result in eventual neuronal cell death. Many regions of the brain are affected as AB plaques and NFTs form, most notably the hippocampus and neocortex. As the disease develops and neurons die, the patient exhibits the cognitive dysfunction and behavioral changes diagnostic of clinical dementia. Initial memory loss progresses to speech difficulty and problems with the performance of everyday tasks such as driving or making phone calls [4, 5]. This disease affects approximately 15 million people worldwide, and there is no known cure. Diagnosis of the disease is challenging as well, since many of the symptoms of AD (memory loss, difficulty with daily activities, behavioral changes) resemble the signs of normal aging [4]. An autopsy with a histopathological examination of the brain to identify AB plaques and NFTs is required for definitive clinical diagnosis of the disease [5]. In addition, causes of the disease are largely unknown. Mutations in the APP (amyloid precursor protein) and PSEN1 (presenilin 1) genes have been shown to cause AD by increasing deposition of AB plaques, but most AD cases cannot be traced back to a specific genetic mutation [5, 6]. With the large number of people affected by AD, determination of the causes, diagnosis, and treatment of the disease is an active area of current research. Effects of Gene Expression in AD Changes in gene expression are known to influence the progression of AD in the brain. Like other neurodegenerative diseases, AD involves complex interactions between many different factors in the brain over an extended time period. The disease develops when specific genes alter their expression patterns (i.e. increase or decrease transcription GENE EXPRESSION AND ALZHEIMER’S DISEASE 6 of the genes to form mRNA, with a corresponding increase or decrease in the protein products of these genes). For example, in order for AB to deposit as senile plaques on the brain, APP must be processed by secretase enzymes to form toxic AB peptides. This requires an increase in expression of the APP gene to form high amounts of the precursor protein as well as changes in expression of secretase enzymes to form AB plaques [7, 8]. Many other genes have also been implicated in AD including those involved in stress response, inflammation, apoptosis, angiogenesis, and transcriptional regulation [9]. As these changes lead to deposition of AB, the AB plaques themselves further influence gene expression to continue disease development. Toxic AB plaques are known to cause changes in expression of transcription factors, cell-cycle related genes, and genes involved in growth factor regulation in the hippocampus and cortex [10, 11]. Current research focuses on the continuing investigation of genes known to be involved with AD as well as the discovery of relevant genes not yet linked to the disease. The overall goal is to fully understand this disease to determine effective methods of diagnosis and treatment. Epigenetic Control of Gene Expression Gene expression can be controlled by epigenetic modifications (chemical modifications to genes that do not alter the DNA base sequence), and many studies have demonstrated a connection between AD and these epigenetic changes [6, 12, 13]. Previous research has specifically investigated the role of DNA methylation in gene expression and AD development [14-18]. DNA methylation involves the addition of a methyl group to a cytosine base, forming methylated cytosine (5-mC). Methylation decreases gene expression by repressing transcription of the methylated gene. According GENE EXPRESSION AND ALZHEIMER’S DISEASE 7 to studies of the connection between methylation and AD, accumulation of AB and progression of AD pathology involve both increases in methylation (hypermethylation) and decreases in methylation (hypomethylation) in different areas of the genome [19]. Methylation-dependent gene silencing has been directly linked to AD pathology, and several of the specific genes affected have been identified [20]. Since alterations in methylation status and corresponding changes in gene expression are known to affect AD, current research continues to investigate these changes. Gene expression in AD can also be influenced by post-transcriptional regulation via noncoding gene transcripts called microRNA (miRNA). MicroRNAs are small, ~22 nucleotide-long sequences of RNA that are complementary to sections of the 3’ untranslated region (UTR) of mRNA targets [21]. Genes coding for miRNA can be located in introns, exons, or intergenic regions. These genes are transcribed to form long primary miRNAs which are then processed by enzymes including the Drosha complex and Dicer to form the final miRNA. This miRNA joins Argonaute proteins to form the RNA-induced silencing complex (RISC), which functions in translational repression. When miRNA pairs to complementary mRNA targets, the miRNA can either inhibit translation of the gene product or lead to degradation of the mRNA [22]. Posttranscriptional regulation through miRNA is known to play a role in the development of many diseases including AD [22, 23]. In addition, miRNA expression is itself frequently regulated by methylation [21]. Analysis of AD-related changes in miRNA and methylation with the corresponding changes in gene expression is needed for full understanding of the disease. GENE EXPRESSION AND ALZHEIMER’S DISEASE 8 Altered Gene Expression Patterns in Other Tissues While neuronal tissue displays the most distinctive changes in gene expression with AD, other tissues have also been shown to have altered expression in certain genes. These specific expression changes show promise as potential biomarkers for diagnosis of the disease. Definitive AD diagnosis currently requires an autopsy to examine the brain for AB plaques and NFTs [5]. Since clinical diagnosis relies on behavioral signs and can be difficult early in disease progression, tissues other than brain have been investigated in search of diagnostic biomarkers. Some researchers have tested cerebrospinal fluid (CSF) to determine whether levels of mRNA and protein change consistently during AD progression and could be used to diagnose or predict disease development. Promising targets for CSF biomarkers include AB and tau peptides, insulin-like growth factor binding proteins, and various other genes and proteins linked to the disease [10, 24]. AD biomarkers in blood have also been investigated with the goal of developing a simple blood test for AD diagnosis. Gene expression changes in characteristic ways in the blood during AD development, and some of these alterations parallel changes seen in the brain [25]. Ongoing studies focus on identifying the specific changes in blood gene expression in AD, understanding their role in disease development and correlation with brain expression changes, and determining a blood gene expression profile useful for early AD diagnosis. Experimental Design To investigate gene expression, this research utilized a transgenic mouse model of AD. Transgenic animal models have been useful in the study of many disease states due to their ability to mimic human disease development, and several mouse models