Browsing by Subject "Alzheimer's Disease"
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Item Cortical Astrocyte-Neuron Network Interaction in Health and Disease(2020-09) Lines, JustinCortical activity underlying cognitive function is classically thought to be exclusively mediated by neurons. In contrast, astrocytes are considered to play solely homeostatic roles, without being directly involved in brain function. Yet, astrocytes are emerging as important cells in brain physiology because they interact with neurons establishing Tripartite Synapses, responding to neurotransmitters with rises in internal calcium levels leading to the release of gliotransmitters that regulate synaptic function. While astrocyte calcium and consequent synaptic regulation has been largely documented at the single cell level, astrocyte network activity and its impact on neuronal network function has been minimally explored. Through the dysregulation of this interaction, astrocytes may be involved in brain pathology, contributing to the cognitive deficits in neurodegenerative diseases such as Alzheimer’s disease (AD). AD is the leading cause of dementia in the United States, and yet the mechanisms contributing to cognitive decline are unclear. The disease progression is associated with depositions of senile plaques of beta-amyloid (Aβ) aggregates as well as loss or damage of synapses. Beta-amyloid pathology has been shown to disrupt cortical astrocyte calcium homeostasis and desynchronize neuronal networks, however disturbances caused by astrocyte-neuron dysfunctions on evoked cortical activity in AD remain unknown. The overall goal of this thesis is to identify astrocyte cortical activity, its impact on neuronal network function, and beta-amyloid induced dysregulation of astrocyte – neuron interactions in AD. To assess astrocyte impact on neuronal network function, I monitor astrocyte calcium activity using two-photon microscopy simultaneously with ECoG recordings of neuronal network activity in vivo. While monitoring the somatosensory cortex during hind-paw stimulation, I test the hypothesis that cortical astrocytes respond to sensory stimulation, they impact neuronal network activity, and astrocyte – neuronal interactions are altered in an amyloidosis mouse model of AD. I begin by identifying cortical astrocyte calcium activity in response to sensory stimulation. I then monitor astrocyte calcium simultaneously with neuronal network function during sensory stimulation to identify coordination of neuron – astrocyte network activity. With the use of Designers Receptors Exclusively Activated by Designer Drugs (DREADDs), I show astrocytes alter spontaneous and sensory-evoked neuronal activity. Due to its overexpression of beta-amyloid, I use the well-established APPSwe/PS1dE9 (APP/PS1) mouse model of AD to evaluate the impact of Aβ plaques on cortical astrocyte responsiveness to sensory stimulation. Finally, I monitor astrocyte calcium and neuronal network activity in the APP/PS1 mouse model to show alterations in neuron – astrocyte interactions by beta-amyloid in AD. Astrocyte calcium activity and its relation to neuronal network activity is examined. This project aided in the elucidation of novel cellular and network dynamics that are disrupted in Alzheimer’s disease, and provide new potential therapeutic targets for the treatment of Alzheimer’s disease. During my PhD I received training on a multitude of cutting-edge research techniques that I will be able to use and build upon throughout my career as an independent research scientist.Item Detecting Cognitive Impairment from Language and Speech for Early Screening of Alzheimer's Disease Dementia with Interpretable Transformer-Based Language Models(2024-05) Li, ChangyeAlzheimer’s disease (AD) is a neurodegenerative disorder that affects the use of speech and language and is diffcult to diagnose in its early stages. Neural language models (NLMs) have delivered impressive performance on the task of discriminating between language produced by cognitively healthy individuals, and those with AD. As artificial neural networks (ANNs) grow in complexity, understanding their inner workings becomes increasingly challenging, which is particularly important in healthcare applications. The intrinsic evaluation metrics of autoregressive NLMs (e.g., predicting the next token given the context), such as perplexity (PPL), reflecting a model’s “surprise” at novel input, and have been widely used to understand the behavior of NLMs. As an alternative to fitting model parameters directly, this thesis proposes a novel method by which a pre-trained transformer-based NLM, GPT-2, is paired with an artificially degraded version of itself, GPT-D, to compute the ratio between these two models’ PPLs on language from cognitively healthy and impaired individuals. This technique approaches state-of-the-art (SOTA) performance on text data from a widely used “Cookie Theft” picture description task, and unlike established alternatives also generalizes well to spontaneous conversations, the degraded models generate text with characteristics known to be associated with AD, demonstrating the induction of dementia-related linguistic anomalies. The novel attention head ablation method employed in this thesis exhibits properties attributed to the concepts of cognitive and brain reserve in human brain studies, which postulate that people with more neurons in the brain and more effcient processing are more resilient to neurodegeneration. The results show that larger GPT-2 models require a disproportionately larger share of attention heads to be masked/ablated to display degradation of similar magnitude to masking in smaller models. To realize their benefits for assessment of mental status, transformer-based NLMs require verbatim transcriptions of speech from patients. While such models have shown promise in detecting cognitive impairment from language samples, the feasibility of deploying such automated tools in large-scale clinical settings depends on the ability to reliably capture and transcribe the speech input. Currently available automatic speech recognition ASR solutions have improved dramatically over the last few years but are still not perfect and can have high error rates on challenging speech, such as speech from audio data with sub-optimal recording quality. One of the key questions for successfully applying ASR technology for clinical applications is whether imperfect transcripts generated by ASR provide sucient information for downstream tasks to operate at an acceptable level of accuracy. This thesis examines the relationship between the errors produced by several transformer-based ASR systems and their impact on downstream dementia classification. One of the key findings is that ASR errors may provide important features for this downstream classification task, resulting in better performance compared to using manual transcripts. In summary, this thesis is a step toward a better understanding of the relationships between the inner workings of generative NLMs, the language that they produce, and the deleterious e↵ects of dementia on human speech and language characteristics. The probing methods also suggest that the attention mechanism in transformer models may present an analogue to the notions of cognitive and brain reserve and could potentially be used to model certain aspects of the progression of neurodegenerative disorders and aging. Additionally, the results presented in this thesis suggest that the ASR models and the downstream classification models react to acoustic and linguistic dementia manifestations in systematic and mutually synergistic ways, which would have significant implications for use of ASR technology. This line of research enables the automated analysis of speech collected from patients at least in the dementia screening settings, and it has the potential to expand to a variety of other clinical applications as well in which both language and speech characteristics are affected.Item Determining Cleavage Site Sequences to Characterize the Active Site of BACE1 Aspartic Protease(2015-04) Heink, NicoleProteases play key roles in physiology and disease development due to their active role in the regulation of other proteins. Understanding and characterizing the active site of relevant proteases provides information vital to the production of inhibitors and elucidates potential native substrates that may be affected by inhibitors. Once the preferred sequence of the active site of a target protease, such as β-secretase (BACE1), has been defined, protease inhibitors can be created to treat or manage diseases such as Alzheimer's disease (AD). BACE1 is an aspartic protease that is overexpressed in the AD brain and is believed to initiate the AD disease pathway. As such, it is a strong candidate for drug design. However, chronic administration of BACE1 inhibitors could result in undesirable side effects due to the impairment of its ability to hydrolyze native substrates; therefore, the amino acid peptide sequence preferentially cleaved by BACE1 needs to be characterized. Not only will this indicate potential substrates that may be affected by BACE1 inhibition, it will also aid in the synthesis of viable inhibitors. Recently, a novel method for determing the cleavage sequence of proteases was reported. Proteomic identification of cleavage sites (PICS) is a method designed to accurately identify cleavage sequence preferences and neighbor interactions in the cleavage site that influence protease cleavage. This method gives additional information with less bias than previous methods used to characterize protease active sites. Multiple controls were used to confirm the validity of the procedure. These controls demonstrated our ability to successfully identify the amino acid sequence preferences for well characterized proteases. We were thus able to confidently use PICS to obtain the sequence of amino acids preferentially cleaved by BACE. BACE1A has two noticeable characteristics for the amino acid sequence cleaved: aromatic amino acids are preferred in the P1 site and leucine is strongly preferred in P2'. Other preferred amino acids are observed in the sequence, but not to the extent of P1 and P2'. Neighbor interactions were also investigated. Positive cooperativity resulted with leucine or valine in P3 and with phenylalanine or tyrosine in P1. There were strong interactions between valine in P3 and phenylalanine in P1 and between tyrosine in P1 and valine in P2'. Sequence preferences were also investigated for BACE2A, which exhibited both similarities and differences between BACE1A and BACE2A. The next step in this research will be to use knowledge of the preferred cleavage sites to determine physiological substrates of BACE1A. This will reveal more information about the natural function of BACE1A and identify potential side effects of its inhibition.Item DNA EPIGENETIC MARKS IN THE DEVELOPMENT OF INFLAMMATION ASSOCIATED CANCERS AND ALZHEIMER’S DISEASE(2022-08) Han, QiyuanEpigenetic control of gene expression via DNA methylation, histone modifications, and ncRNAs is critical for ensuring normal cellular development and homeostasis. The best studied DNA epigenetic mark is methylation of cytosine at the C5 position (5mC). This DNA epigenetic mark is introduced by de novo methyltransferases DNMT3a/b and maintained during cell division by maintenance methyltransferase DNMT1. Ten Eleven Translocation (TET) dioxygenases can iteratively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), a process known to induce DNA demethylation and gene reactivation. A precise balance of DNA methylation and demethylation is important for establishing tissue specific gene expression patterns, maintaining cell identity, and guiding normal development. Reprogramming of DNA methylome and hydroxymethylome has been recognized as a critical event in the development of cancers. N6-methyldeoxyadenosine (N6medA) is a potential novel DNA epigenetic mark recently discovered in mammals. Although the exact biological role of N6medA in mammalian genome is still elusive, N6AMT1 and METTL4 are possible methylases that introduce N6medA, while ALKBH1 and ALKBH4 are likely demethylases that remove N6medA. N6medA has been implicated in epigenetic regulation, DNA repair, memory formation, and transposable element suppression, and it has been proposed to play a role in early embryonic development, cancer, and neurological functions. Chapter I of this Thesis provides an overview of the major mechanisms of epigenetic regulation including epigenetic marks of DNA, non-coding RNAs, and histone modifications. A further review of DNA epigenetic marks including the formation, reversibility, abundance, genomic distribution and corresponding biological effects is provided. We then discuss the experimental methodologies for quantifying global levels of epigenetic DNA modifications as well as methods for genome wide mapping of DNA epigenetic marks. Finally, Chapter I covers the role of epigenetics in serving as a molecular link between human disease and its associated risk factors, with a focus on cancer and chronic inflammation, Alzheimer’s Disease, and aging. Chapter II of this Thesis characterizes DNA methylation and hydroxymethylation changes in a mouse model of inflammatory bowel disease/colon cancer. By coupling DNA methylation and hydroxymethylation sequencing data with RNA-seq data for the same tissues, we found that inflammation mediated epigenetic changes altered gene expression levels of key tumorigenesis genes, which could contribute to colon cancer initiation. Chapter III of this thesis examines the timing and the persistence of smoking- mediated epigenetic changes in the lung using a mouse model of smoking induced lung cancer. We characterized DNA methylome, hydroxymethylome, as well as transcriptome changes in the type II alveolar cells of A/J mice exposed to cigarette smoke for 3 weeks, 10 weeks, or 10 weeks followed by a 4 week recovery period. By comparing our data with human lung adenocarcinoma dataset, we identified several cancer related genes that demonstrate early epigenetic changes upon exposure to cigarette smoking. The functional roles of these genes was explored in cell proliferation assay, leading to the identification of OSR2 as a potential protooncogene. In Chapter IV, we explored the dynamics of the novel DNA epigenetic mark N6-methyldeoxyadenosine (N6medA) in the context of human aging and AD. We characterized its global dynamics in human aging and AD via an optimized isotope dilution, high resolution nanoLC-NSI MS/MS method. Global N6medA levels positively correlated with human chronological age. In addition, N6medA was profiled across the human genome using N6medA IP-seq, revealing adenine methylation changes potentially associated with aging and AD. Finally, we identified several specific protein readers of N6medA via mass spectrometry based affinity proteomics. These protein readers have functions in DNA replication, transcription and configuration, implying a regulatory role of N6medA in DNA templated biological processes. Taken together, during the course of the studies described in this Thesis, we have characterized DNA epigenetic changes in two mouse models of inflammation associated cancer, together with gene expression changes. We have also examined the potential oncogenic roles of genes that are differentially methylated upon exposure to cigarette smoking. Finally, we investigated the potential involvement of a novel DNA epigenetic mark, N6-methyl-dA, in human aging and Alzheimer’s Disease. Overall, this work contributes to the current understanding of epigenetic deregulation in inflammation associated cancers and sheds new light on the role of a novel DNA epigenetic marks in human aging and AD.Item Exploring the Dimerization of BACE2-CFP Fusion Protein in HEK293 Cells through Fluorescence Microscopy Techniques(2018-08) Kreitlow, BenjaminAlzheimer’s disease (AD) is a neurodegenerative disorder frequently characterized by the presence of senile amyloid plaques. These plaques are formed through the aggregation of the amyloid β (Aβ) peptide. Aβ is formed through the sequential proteolysis of amyloid precursor protein (APP) by the β-site APP cleaving enzyme-1 (BACE1) and γ-secretase. As the rate-limiting enzyme of Aβ formation, the amyloidogenic properties of BACE1 have been widely examined. Recently, there has been evidence that suggests BACE1 exists as a dimer that is significantly more catalytic than the monomeric form. However, as of yet, there have been no complimentary studies that have explored the dimerization properties of the BACE1 homolog, BACE2. The BACE1 and BACE2 enzymes make up a unique class of membrane-bound aspartyl proteases that share similar extracellular, transmembrane, and cytosolic domains. Interestingly, it is only the BACE1 enzyme that exhibits amyloidogenic properties. Contrarily, BACE2 demonstrates distinct anti-AD characteristics such as the non-amyloidogenic cleavage of APP at α- and θ-sites while also functioning as an efficient Aβ-degrading enzyme. Elucidating the dimerization of BACE2 can help us understand whether dimerization is an inherent characteristic of this unique class of proteases or, alternatively, if dimerization is an attribute required for the amyloidogenic properties of BACE1. In this study, cultured human embryonic kidney 293 (HEK293) cells were transfected with BACE2-CFP fusion protein and imaged using differential interference contrast (DIC) and confocal microscopy to examine sub-cellular localization of the fluorescent construct. Subsequently, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used to examine changes in the fluorescence lifetime distribution of transfected cells. The results of this study suggest that the BACE2 enzyme does not undergo dimerization in contrast to BACE1, as demonstrated in previous studies.Item A Fluorescence Assessment of the Intracellular Trafficking of the Amyloid Precursor Protein within the Secretory Pathway(2020-08) Rodriguez, KassidyAmyloid-β (Aβ) plaque formation in the brain is a major hallmark of Alzheimer’s disease (AD) and has been linked to known symptoms. According to the amyloid cascade hypothesis, Aβ is generated from the cleavage of amyloid-β precursor protein (APP) by BACE1 followed by the regulated intramembrane proteolysis (RIP) by γ-secretase. Aβ plaques are then formed through the extracellular deposition and aggregation of Aβ proteins. Current research suggests that BACE1 dimerizes in cells in a substrate-mediated manner, and that these dimers exhibit a much higher catalytic activity than their monomeric forms within acidic environments, such as endosomal compartments. It was also reported that the interaction between APP and BACE1 increased Aβ production. To date, there has been no research completed using HEK293 cells that compares the degree of co-localization of APP within specific endosomal compartments in the secretory pathway. The co-residence of APP and BACE1 in the same subcellular location would likely favor their interaction and facilitate the proteolysis of APP. Therefore, the study of their intracellular trafficking and localization should indicate where BACE1 is likely to process APP. BACE1 is an attractive therapeutic target for slowing the production of Aβ in the early stages of AD, further highlighting the importance of its study. In this contribution, we describe our efforts to examine where APP-GFP resides along the secretory pathway using fluorescently labeled Rab proteins that target distinct regions of the secretory pathway as points of reference. In addition, we observed an increase in the overall fluorescence intensity in cells co-transfected with APP-GFP and BACE1-YFP. HEK293 cells were transiently transfected with APP-GFP and either Rab5-DsRed, Rab7-DsRed, Rab11-DsRed, or BACE1-YFP fluorescent fusion proteins then imaged using widefield fluorescence and differential interference contrast (DIC) microscopy. The subcellular localization and distribution of each fluorescent fusion protein construct was determined by the colocalization of GFP and DsRed by evaluating Pearson’s correlation coefficients (PCC), Manders’ overlap coefficients (MOC), and Li’s intensity correlation quotients (ICQ). Fluorescence images were analyzed using the JACoP plug-in within FIJI/ImageJ. The results of this study provide evidence that APP-GFP has the highest degree of colocalization with Rab11-DsRed, suggesting that the APP concentration is highest within the recycling endosomes. HEK293 cells co-expressing APP-GFP and BACE1-YFP exhibited an increased fluorescence intensity compared to cells expressing only APP-GFP. These results establish baseline measurements for future cell-based studies of BACE1 with APP-GFP as its substrate.Item HDL-Mimetic Peptides as Potential Therapeutics for Alzheimer's Disease(2018-08) Chernick, DustinAlzheimer’s disease (AD) is the leading cause of dementia worldwide, for which there currently exists no approved disease modifying treatment. A number of large scale human clinical studies have confirmed a robust connection between high density lipoprotein (HDL) – known as the ‘good cholesterol’ levels and AD. Low levels of HDL are associated with increased risk and severity of AD. The role of HDL in the brain is not fully established, however, the anti-inflammatory and anti-oxidative properties of HDL are thought to be critical for its beneficial effects. Apolipoprotein E (apoE) is a key constituent of HDL-like particles in the interstitial fluid (ISF) and cerebral spinal fluid (CSF) in the brain. ApoE exists in 3 common variants in the human population (apoE2, E3, and E4), and the apoE4 isoform is the strongest genetic risk factor for AD, accounting for 40-60% of cases. This risk allele is known to increase neuroinflammation and to promote the aggregation and deposition of amyloid beta (Aβ) in the brain, effects which are influenced by the poor lipidation status of apoE4 (incomplete or improper composition of HDL-like particles) in the brain. Previous studies in the laboratory of Dr. Ling Li have shown that overexpression of human apoA-I, the primary apolipoprotein associated with HDL in the periphery, mitigated amyloid pathology and rescued memory deficits in AD mice. However, a full-length, glycosylated protein is extremely difficult and costly to synthesize and to administer. Therefore, the goal of my research was to test the therapeutic potential of small HDL-mimetic peptides, designed to mimic the beneficial function of their parent apolipoproteins, in AD. My studies focused on 4F, an 18 amino acid HDL-mimetic peptide that has been shown to be safe and well tolerated in human clinical trials for cardiovascular disease. I have demonstrated that the lipidation state of apoE is negatively impacted by the addition of aggregated Aβ to astrocytes from mice and humans, in vitro, an effect that is reversed by the addition of 4F. In addition, I confirmed that apoE4 is less lipidated than apoE2 and E3 at baseline, and demonstrated that apoE4 is more susceptible to the detrimental effects of Aβ on lipidation than apoE2. Intriguingly, 4F was able to completely rescue this effect, bringing apoE4 lipidation levels on par with those of apoE2, even in the presence of Aβ. Preliminary in vivo studies in mice expressing the human apoE isoforms and in a mouse model of AD indicate that 4F reduces soluble amyloid levels in the brain and attenuates memory deficits. As chronic neuroinflammation is a key hallmark of AD pathology, another line of my research focused on a small molecule, called Minnelide. Minnelide is a water soluble, pro-drug of triptolide, which is an anti-inflammatory agent that has been shown in Dr. Li’s lab and in other labs to mitigate AD pathology and rescue memory deficits in animal models. Poor solubility hinders this agent’s prospects in the clinic, and so we sought to test the efficacy of Minnelide in AD. My studies show that Minnelide attenuated age-related cognitive decline in AD mice, independent of Aβ levels in the brains of these animals. These data, taken together, indicate that HDL mimetic peptides, and targeting of inflammatory pathways in the periphery and in the brain are promising avenues for continued efforts to find an effective treatment for AD.Item Integrating summarized imaging and genomic data with GWAS for powerful endophenotype association testing in Alzheimer’s Disease(2021-06) Knutson, KatherineGenome-wide association studies (GWAS) have identified thousands of genetic variants associated with complex traits. However, for most diseases, individual risk variants have small effects which impact disease indirectly through upstream endophenotypes. To improve on the power and interpretability of GWAS, a number of approaches have been developed which aggregate contributions from one or multiple genetic variants to investigate the role of genetically regulated endophenotypes in complex traits. These methods include Mendelian Randomization (MR) and the Transcriptome/Imaging Wide Association Study (TWAS/IWAS, which test for associated gene expression and imaging phenotypes, respectively). In this dissertation, I will compare the performance of these approaches for detecting brain imaging derived phenotypes (IDPs) associated with Alzheimer’s Disease. I will present novel extensions to the TWAS/IWAS framework to account for key biological factors which may impact their performance in practice, namely 1) genetic pleiotropy and 2) population substructure. The first of these factors, genetic pleiotropy, describes the phenomenon in which genetic loci affect multiple intermediate risk phenotypes. The presence of pervasive pleiotropy can result in inconsistent IWAS estimates. I will present a novel extension to the IWAS model (namely, MV-IWAS) which provides consistent causal estimates of endophenotype-trait associations by directly and indirectly accounting for pleiotropic pathways. The second of these factors, population substructure, describes ancestral variation in the underlying genetic architecture of endophenotypes. This variation can lead to ancestry-specific effects of gene expression in TWAS, which go undetected in the standard TWAS framework. Here, I will present a score test to detect heterogeneity in the effects of genetically-regulated gene expression which are correlated with ancestry. By jointly analyzing samples from multiple populations, our multi-ancestry TWAS framework can improve power for detecting genes with shared expression-trait associations across populations through increased sample sizes, as compared to existing stratified TWAS approaches.Item The Societal and Family Lifetime Cost of Dementia(2017-05) Jutkowitz, EricDementia is a complex terminal disease that involves cognitive and functional declines and behavioral/psychological symptoms. Currently >5 million Americans suffer from dementia. The societal economic burden of dementia consists of different types of costs (value of informal care, out-of-pocket expenditures, Medicaid long-term care expenditures, and Medicare expenditures), and several payers (family, Medicaid, and Medicare) shoulder different amounts of the economic responsibility. To facilitate comprehensive planning at the family, state, and federal levels, policymakers must understand who incurs dementia costs over the course of the disease. The objective of this study was to estimate the lifetime and annual cost of dementia care (value of informal care, out-of-pocket expenditures, Medicaid long-term care expenditures, and Medicare expenditures), and the extra cost of caring for someone with dementia compared to someone who did not exhibit dementia clinical features (net cost). To estimate total and net lifetime and annual costs we developed an evidence-based mathematical model to simulate disease progression for newly diagnosed individuals with dementia. Data driven trajectories of three clinical features -cognition, function, and behavioral/psychological symptoms- were used to model disease severity. Personal characteristics, clinical features, place of residence, and dual enrollment status were used to estimate cost. Counterfactual analysis was conducted to compare costs between those who did and did not exhibit clinical features (net cost). From time of diagnosis (mean age of 83 years), discounted total cost of care for a person with dementia was $322,900. Families incurred 72% of the total cost burden ($144,160 for informal caregiving and $88,780 out-of-pocket payments). Medicaid accounted for 12% ($37,390) and Medicare accounted for 16% ($52,540) of total cost, respectively. In counterfactual analysis, net cumulative costs for a person with dementia were $194,890 greater over a lifetime than someone without dementia (85% of net cost incurred by families). Our model extends previous studies by considering costs over the life course of the disease. We found that dementia results in $194,890 additional total care costs over an individual’s lifetime. The extra cost associated with dementia is primarily borne by families (versus Medicare or Medicaid) due to time spent providing informal care and out-of-pocket expenditures.