Of Mice and Math: A Systems Biology Model for Alzheimer's disease

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder in the US, affecting over 1 in 8 people over the age of 65. There are several well-known pathological changes in the brains of AD patients, namely: the presence of diffuse beta amyloid plaques derived from the amyloid precursor protein (APP), hyper-phosphorylated tau protein, neuroinflammation and mitochondrial dysfunction. Recent studies have shown that cholesterol levels in both the plasma and the brain may play a role in disease pathogenesis, however, this exact role is not well understood. Additional proteins of interest have also been identified (ApoE, LRP-1, IL-1) as possible contributors to AD pathogenesis. To help understand these roles better, a systems biology mathematical model was developed. Basic principles from graph theory and control analysis were used to study the effect of altered cholesterol, ApoE, LRP and APP on the system as a whole. Negative feedback regulation and the rate of cholesterol transfer between astrocytes and neurons were identified as key modulators in the level of beta amyloid. Experiments were run concurrently to test whether decreasing plasma and brain cholesterol levels with simvastatin altered the expression levels of beta amyloid, ApoE, and LRP-1, to ascertain the edge directions in the network model and to better understand whether statin treatment served as a viable treatment option for AD patients. The work completed herein represents the first attempt to create a systems-level mathematical model to study AD that looks at intercellular interactions, as well as interactions between metabolic and inflammatory pathways

Modeling Alzheimer\u27s Disease Using CRISPR/CAS9 Gene Editing and Induced Pluripotent Stem Cells Reveals Conserved Cellular Mechanisms

Alzheimer’s disease (AD) is the most common cause of dementia worldwide and a leading cause of death in the United States. Rare cases of autosomal dominant familial AD (fAD) result from genetic mutations in three key genes: amyloid precursor protein (APP), and two APP processing-related genes (presenilin-1 (PSEN1), and presenilin-2 (PSEN2)), supporting the theory that altered APP metabolism is a central cause of AD. However, which product of APP metabolism is causal remains a matter of investigation. A probable source of this lack of understanding stems from the poor disease model systems that have been utilized in the field for many years. Recently, advances in human induced pluripotent stem cell (iPSC) technology has enabled the study of uniquely human diseases, such as AD, in human tissue. However, the inability to precisely and efficiently genetically engineer human iPSCs has limited their use in effectively studying monogenic human diseases like fAD

Thioredoxin-1 in Alzheimer disease

Oxidative stress is one of the earliest signs in Alzheimer Disease (AD) brain. In order to protect themselves against oxidative stress, neurons use antioxidants as a defense mechanism. Such an antioxidant is Thioredoxin-1 (Trx1). Previous studies have shown that the levels of Trx1 are reduced in the brains of AD patients. The aim of this thesis was to further examine the function of Trx1 in AD pathogenesis. In Paper I and III, the role of Trx1 in the mechanisms behind risk-modulating factors is investigated. The incidence of AD is higher in women than in men and one reason for this is thought to be the post-menopausal lack of estrogen. In addition, estrogen was shown to have neuroprotective effects both in vitro and in vivo. In Paper I we studied the protective effect of estrogen against amyloid-beta (Aβ) toxicity in vitro. We found that estrogen is protective via phosphorylation of Protein kinase B (AKT) and inhibition of the Apoptosis signal-regulating kinase 1 (ASK-1) pathway. However, this occurs independently of Trx1 expression. In Paper III we investigated the effect of Apolipoprotein E (ApoE) isoforms on Trx1 in the brain. The ApoE isoform ε4 (ApoE4) is the most important genetic risk factor for sporadic AD and it is also associated with increased oxidative stress in the brain. Furthermore, ApoE4 is suggested to have direct toxic effects via apoptosis. We found that presence of ApoE4 causes a reduction in Trx1 levels, both in vivo, in hippocampus of ApoE Target Replacement Mice, and in vitro, in human primary cortical neurons and neuroblastoma cells. This occurred after leakage of the lysosomal membrane and cytosolic release of Cathepsin D, and it induced apoptotic cell death via activation of the ASK-1 pathway. Thioredoxin-1 can be truncated into an 80 amino acid long peptide called Thioredoxin-80 (Trx80). In Paper II and IV, we demonstrate for the very first time that this peptide is present in the brain, mainly in neurons. The levels were reduced significantly in AD patients and this was also seen in the cerebrospinal fluid (CSF). The reduction in CSF was present already in patients with mild cognitive impairment (MCI). Furthermore, we demonstrate that the peptide is generated by α-secretase cleavage of Trx1 and is secreted from cells in exosomes. The peptide inhibits the aggregation of Aβ and prevents its toxic effects both in vitro and in a Drosophila Melanogaster model of AD. In addition, Trx80 lowers the levels of Aβ, possibly through a mechanism that involves autophagy. These findings give support to the view that oxidative stress in general, and Trx1 in particular, has a key role in AD pathogenesis. It also presents Trx80 as a completely new player to the field that has potential as a specific biomarker for the disease. In addition, therapeutic strategies based on these two peptides could be a possibility in AD that should be further investigation

Yeast as a Model for Studying Aβ Aggregation, Toxicity and Clearance

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the central nervous system, characterised by acute memory loss and behavioural symptoms. The AD brain is characterized by the presence of senile amyloid plaques associated with degenerating neurites and inflammatory processes. The major protein component of these amyloid deposits is the amyloid beta (Aβ) protein. The Aβ protein is a 40 or 42 amino acid cleavage product of APP (Amyloid Precursor Protein) which is produced in low levels in the normal ageing brain. Although senile amyloid plaques is the major pathological hallmark of AD brains, accumulating evidence has been presented to show that increased levels of soluble forms of Aβ42 correlate with the clinical manifestations and severity of the disease. Increased accumulation (both intracellular and extracellular) and toxicity of Aβ42 peptide in the brain play pivotal roles in neurodegeneration and loss of memory functions in the AD brain. Therefore reducing the toxicity of Aβ42 and increasing its clearance from the brain has been considered to be main targets for AD therapeutics. The search for a disease modifying therapy for AD has been very difficult with the majority of agents failing in later stages of clinical trials. The incomplete understanding of drug-target mechanisms and the lack of high-throughput screening systems for identifying selective target based drugs have been some of the main issues expressed for the failure of AD drugs. Yeast offer a simple eukaryotic model for studying pathological mechanisms and compared to other models there is availability of various experimental tools applicable for high throughput analysis of protein-protein, gene-gene and gene-protein interactions and associated cellular functions. It can also offer a versatile model for initial screening in drug development for various human diseases, including AD. Yeast models have been utilised for studying AD related proteins including APP and its processing enzymes (secretases) and tau phosphorylation

The Influence of APOE Genotype on Lipid Droplet Dynamics

Excess lipid droplet (LD) accumulation is associated with several pathological states, including neurodegenerative disorders such as Alzheimer’s disease (AD). However, the mechanism(s) by which changes in LD composition and dynamics may contribute to the pathophysiology of AD remains unclear. Apolipoprotein E (ApoE) is a droplet-related protein with a common variant (ApoE4) that confers the largest increase in genetic risk for late-onset AD. Interestingly, ApoE4 is associated with both increased neuroinflammation and excess LD accumulation. This dissertation work seeks to quantitatively profile the lipid and protein composition of LDs between the ‘neutral’ ApoE3 and ‘risk’ ApoE4 isoforms, in order to gain insight into potential LD-driven contributions to AD pathogenesis. Targeted replacement mice expressing human ApoE3 or ApoE4 were injected with saline (control) or LPS (inflammatory stimulus) and after 24 hours, hepatic lipid droplets were isolated and droplet proteomes and lipidomes were analyzed. Quantitative proteomics showed that LD fractions from E4 mice are enriched for proteins involved in innate immunity, while E3 LDs are enriched for proteins involved in lipid ß-oxidation. Lipidomics revealed a shift in the distribution of glycerophospholipids in E4 LDs with an increase in multiple phosphatidylcholine (PC) species. There was also substantial overlap between LD proteins and AD-proteomes of human whole brain tissue. To translate these findings to the brain, primary microglia from the same strain of mice were exposed to exogenous lipid, inflammatory stimulation, necroptotic N2A cells (nN2A), or a combination of treatments to evaluate lipid droplet accumulation and impact on cell function. Microglia from ApoE4 mice accumulated more LDs at baseline, with exogenous OA, LPS stimulation, and nN2As as a percentage of E3 control across multiple experiments. E4 microglia also secreted significantly more cytokines (TNF, IL-1β, IL-10) than E3 microglia in the control, oleic acid, and nN2A treatment conditions. Interestingly, droplet inhibitors for ACAT and DGAT both decreased droplet accumulation in cells, but did not ameliorate the cytokine response. Finally, we have established a biobank of APOE genotyped peripheral blood mononuclear cells (PBMCs) from research participants. These easily accessible immune cells will serve as a highly translational model to understand LD dynamics as it relates to ApoE and AD risk. In summary, E4 cells accumulate more LDs compared to E3 under all conditions tested, while the proteomic profile of E4 LDs support the hypothesis that E4 expression increases inflammation under basal conditions. This increased LD formation in non-aged, non-diseased E4 cells may suggest preclinical dysfunction associated with the highest risk APOE genotype, and a better understanding of LD dynamics within these cells and their functional implications may provide novel targets to improve E4-related outcomes

Generation of isogenic pluripotent stem cell lines for study of APOE, an Alzheimer’s risk factor

abstract: Alzheimer’s disease (AD), despite over a century of research, does not have a clearly defined pathogenesis for the sporadic form that makes up the majority of disease incidence. A variety of correlative risk factors have been identified, including the three isoforms of apolipoprotein E (ApoE), a cholesterol transport protein in the central nervous system. ApoE ε3 is the wild-type variant with no effect on risk. ApoE ε2, the protective and most rare variant, reduces risk of developing AD by 40%. ApoE ε4, the risk variant, increases risk by 3.2-fold and 14.9-fold for heterozygous and homozygous representation respectively. Study of these isoforms has been historically complex, but the advent of human induced pluripotent stem cells (hiPSC) provides the means for highly controlled, longitudinal in vitro study. The effect of ApoE variants can be further elucidated using this platform by generating isogenic hiPSC lines through precise genetic modification, the objective of this research. As the difference between alleles is determined by two cytosine-thymine polymorphisms, a specialized CRISPR/Cas9 system for direct base conversion was able to be successfully employed. The base conversion method for transitioning from the ε3 to ε2 allele was first verified using the HEK293 cell line as a model with delivery via electroporation. Following this verification, the transfection method was optimized using two hiPSC lines derived from ε4/ε4 patients, with a lipofection technique ultimately resulting in successful base conversion at the same site verified in the HEK293 model. Additional research performed included characterization of the pre-modification genotype with respect to likely off-target sites and methods of isolating clonal variants.Dissertation/ThesisMasters Thesis Bioengineering 201