Mathew M. Blurton-Jones, Ph.D. - US grants

DESCRIPTION (provided by applicant): Alzheimer disease (AD) pathology is characterized by beta-amyloid (A¿) plaques and tau-containing neurofibrillary tangles. Recent evidence suggests that soluble forms of both A¿ and tau can influence cognition and A¿ has been shown to modulate tau pathology. However, the A¿ species that mediates these effects remains largely unresolved. The Arctic A¿ mutation (E22G) has recently been shown to enhance soluble A¿ protofibril formation and cause early-onset familial AD, providing strong genetic evidence that A¿ aggregation state is critical to the development of AD. In this proposal, we will utilize the Arctic mutation to test the hypothesis that soluble A¿ assemblies induce tau pathology and cognitive dysfunction. To facilitate my investigation and greatly enhance my career development, I will receive mentorship from Dr. Frank LaFerla in the design, generation and analysis of transgenic AD models. In addition, Dr. James McGaugh and Dr. Charles Glabe will provide me with training in behavioral analyses and oligomeric protein biochemistry respectively. In Aim 1, we will utilize the Arctic A¿ mutation to develop novel transgenic models of AD with age-dependent and progressive neuropathology to test the hypothesis that soluble A¿ assemblies promote tau pathology from wild type hTau. We anticipate that this model will develop A¿ and tau pathologies without the use of mutant tau, thereby more closely representing the etiology of AD. In Aim 2, we will assess cognitive phenotype and correlations with A¿ and tau pathology. Soluble Arctic A¿ inhibits hippocampal LTP 100-fold more potently than wild-type A¿. Therefore, we hypothesize that the Arctic mutation will rapidly produce cognitive dysfunction via enhancing the generation of soluble A¿ assemblies and modulating the development of tau pathology. Under the guidance of Dr. James McGaugh, I will test this hypothesis by performing a cross-sectional analysis of cognitive function in our novel transgenic models and determine whether cognitive deficits correlate with pathological progression. In Aim 3 we will test the hypothesis that soluble A¿ assemblies induce tau pathology and mediate cognitive decline. In this aim, Dr. Charles Glabe will provide guidance on oligomeric protein biochemistry and his conformational-dependent antibodies will be utilized to test this hypothesis. Combined the proposed aims seek to determine whether soluble A¿ assemblies play a key role in the development of tau pathology and cognitive dysfunction.

For many neurological disorders including Alzheimer Disease (AD), current therapies are largely palliative and based on small molecule designs. However, studies have begun to examine the use of stem cells to both treat and model neurodegenerative disease. Although stem cells have been suggested as a potenfial therapy for AD, to date this approach has not been directly tested in animal models. Consequenfiy, it is critical to obtain pre-clinical evidence to determine whether neural stem cell (NSC) transplantation can offer symptomafic or disease-modifying effects for AD. In preliminary studies, we have found that short-term transplantation of murine NSCs into aged triple transgenic mice (3xTg-AD) improves cognitive function. Interesfingly, NSCs rescue cognifion not by differentiating into neurons or altering levels of AB or tau, but rather by increasing levels of brain-derived neurotrophic factor and enhancing endogenous hippocampal synaptic connectivity. These initial findings suggest that NSC transplantation may provide a promising therapeutic approach. However, AD manifests as a long-term and progressive illness. Thus, it is critical to determine whether NSC transplantation can provide benefits across an extended duration. Here we propose to perform a longitudinal examinafion of the effect of NSC transplantation on AD-related cognitive function in 3xTg-AD mice. We hypothesize that the long-term effectiveness of NSC-based therapies can be improved upon by combining both trophic and disease-modifying approaches. Thus, we will also examine whether NSCs engineered to express an AB-degrading enzyme can provide more substanfial long-term benefit. In addition to their potential therapeutic use, stem cells are being actively studied as a novel and powerful approach to model human disease. To begin to examine the use of stem cells to model AD we therefore propose to generate induced pluripotent stem cells (iPSCs) from AD and control patient fibroblasts Comparisons of AB and tau and their various assembly and phosphorylation states will determine whether genetic factors influence the production, oligomerization, or degradation of these proteins. Likewise analysis of the survival of iPSC-derived neurons in response to AB oligomer treatment will be examined to determine whether AD iPSC-derived neurons are innately more suscepfible to disease-related insults.

DESCRIPTION (provided by applicant): Alzheimer disease (AD) is the leading cause of age-related dementia, affecting over 5 million people in the United States alone. Unfortunately, current therapies are largely palliative and several promising drug candidates have failed in late-stage clinical trials. Hence, there is urgent need to improve our understanding of the basic mechanisms that drive the development of late-onset AD. Recent genetic studies have uncovered a number of genes that influence the risk of developing AD. While many of these gene confer small increases in the risk of AD, one recently discovered gene, triggering receptor expressed on myeloid cells 2 (TREM2), increases AD risk by about 3-fold. Yet the function of TREM2 in the brain and its role in AD pathogenesis remains largely unknown. Here, we will perform a number of studies to test the hypothesis that mutations in TREM2 impair microglial-mediated clearance of beta-amyloid (A?), exacerbate the pro-inflammatory induction of tau pathology, and alter neuronal health. To achieve these goals, we will generate induced pluripotent stem cells (iPSCs) from AD patients carrying the R47H mutation in TREM2. This mutation will then be repaired using TALEN-mediated gene editing technology to create isogenic mutant and control iPSCs. As microglia are the primary cell type within the brain that express TREM2, we will differentiate iPSCs into microglia and examine the effects of TREM2 mutations on microglia function. First, using in vitro approaches, we will quantify the effects of TREM2 R47H mutations on microglia migration and activation state, A? phagocytosis, and neuronal viability. As microglia are inherently plastic cells, their function and activation state s dramatically influenced by other cell types within the brain. We will therefore also study the effects of TREM2 mutations in vivo by transplanting iPSC-derived microglia into two novel xenotransplantation-compatible transgenic mouse models of AD. Examination of these mice will allow us to determine whether TREM2 mutations alter the degradation of A? or modulate the progression of tau pathology in vivo. Using fluorescence activated cell sorting approaches we will also determine the impact of AD pathology on microglia gene expression and activation state. Together, these studies hope to decipher the mechanisms by which TREM2 mutations influence AD and provide a broader understanding of the role of microglia and inflammatory processes in AD pathogenesis.

Core F: iPS Cell Core Project Summary/Abstract Recent studies have uncovered a number of genes that can influence the risk of developing sporadic Alzheimer's disease (AD). However, it remains unknown exactly how these genes impact the function of cells and promote disease. It is also unknown whether drugs can be developed to target these genes in a beneficial way. The UCI Alzheimer's Disease Research Center (ADRC) recently established the first AD induced pluripotent stem (iPS) cell core to provide researchers with a powerful new approach to study the relationship between genetic risk factors and AD pathogenesis in human cells. iPS cells are a specialized cell that can be produced by adding 4 key genes to human skin or blood cells. The resulting cells can then be grown indefinitely and are pluripotent, meaning they can differentiate into any cell type within the body. By producing iPS cells from patients with AD, mild cognitive impairment, and unaffected controls, scientists can study the influence of disease-associated genes on key types of human cells such as neurons, astrocytes, and microglia. One major challenge is that researchers will likely need cells derived from many individual patients to study the effects of genes in a complex disorder such as AD. The UCI ADRC iPS core therefore aims to generate lines from over 140 individual patients. These iPS cell lines will then be made widely available to AD researchers worldwide to accelerate the discovery of novel therapeutics and enhance our understanding of the complex genetic components of AD.

Project Summary The innate immune system is strongly implicated in the pathogenesis of Alzheimer's disease (AD). In contrast, the role of adaptive immunity and peripheral lymphocytes in AD remains largely unexplored. Yet, new studies have begun to implicate these cells in the development and progression of AD in mouse models. For example, we recently found that genetic deletion of T, B, and NK cells in transgenic AD mice leads to dramatic increase in A? pathology. Conversely, replacement of these populations via bone marrow transplantation (BMT) reverses this effect. Thus, mouse studies have begun to implicate a role for the adaptive immune response in modulating the progression of AD. Yet it remains critical to further define the mechanisms by which lymphocytes influence this disease, and whether similar mechanisms are at work in aging humans. For this proposal we have assembled a multidisciplinary team who bring expertise in AD pathogenesis, transgenic modeling, RNA-sequencing, AD neuroimmunology, T-cell biology, and bone marrow transplantation. Together we will examine the role of adaptive immunity in AD pathogenesis and test the hypothesis that: lymphocytes reduce pathology by modulating microglial activation and phagocytosis and that genetic modifiers of peripheral immunity contribute to the development of AD. To achieve these goals we will take advantage of several unique resources including an immune-deficient transgenic AD model and a collection of over 1000 samples of peripheral blood mononuclear cells (PBMCs) derived from well-characterized AD, mild cognitive impairment (MCI), and control patients. In Aim 1, we will determine which peripheral immune cells infiltrate the brain in AD subjects and transgenic models and whether these cells interact directly or indirectly with microglia to modulate A? clearance. In Aim 2, we will determine which specific peripheral immune cell populations most strongly influence beta-amyloid deposition by adoptively transferring specific immune populations and performing bone marrow transplantation studies to determine which cell types are necessary and sufficient to reduce A?. In Aim 3, we will determine whether AD or MCI subjects exhibit changes in peripheral immune cell populations, cytokine production, or antigen receptor repertoires and whether these changes are influenced by AD GWAS risk polymorphisms? Collectively, these studies will greatly enhance our understanding of the immune response in both AD patients and mouse models and determine whether T and B cells play an important role in this disease by promoting the clearance of beta-amyloid.

Project Summary/Abstract Microglia are strongly implicated in the pathogenesis of Alzheimer's disease (AD) and recent genetic studies have identified several microglial-enriched genes that influence AD risk. To study the role of these genes in AD our lab recently developed a fully-defined approach to differentiate induced pluripotent stem cells (iPSCs) into microglia. However, reprogramming erases many of the key signatures of aging, making it difficult to study the interactions between AD genes, pathology, and aging with iPSC-derived cells. One recent study developed an innovative approach to address this challenge by using Progerin, a protein associated with a premature aging disorder. However, the Progerin gene, LMNA, is not normally expressed in human neurons or glia. In contrast, another form of Progeria, Cockayne Syndrome (CS), which is caused by mutations that impair DNA repair, leads to significant neurological defects. Furthermore, two of the genes that are mutated in CS, ERCC1 and ERCC5, are highly expressed in human microglia and their deletion in mice mimics key aspects of microglial aging. Given these findings, we propose to test the hypothesis that deletion or mutations of ERCC1 and ERCC5 will produce changes in iPSC-derived microglia that mimic the effects of chronological aging and impair the response of microglia to AD-associated insults. To achieve these goals, we have assembled a multidisciplinary team who bring expertise in AD iPSC modeling, CRISPR-mediated gene deletion, and microglial differentiation and analysis, RNA-sequencing and bioinformatics, and brain organoid culture systems to test following three specific aims and hypotheses: Aim 1: Examine the impact of ERCC1/5 deletions and mutations on iPSC-derived microglial function. We hypothesize that ERCC knockout and mutant microglia will exhibit an age-associated `primed' activation state with impaired homestatic activity but exacerbated inflammatory responses. Aim 2: Do ERCC1/5 deletions and mutations model the transcriptional effects of aging observed in human brain-derived microglia? We hypothesize that deletion of these genes will produce microglia that exhibit many of the transcriptional changes associated with natural brain aging. Aim 3: Defining the intrinsic versus extrinsic effects of ERCC deletion on microglial aging with brain organoid cultures. We hypothesis that both intrinsic and extrinsic signals influence microglial aging.

Abstract Microglia are the primary immune cells of the CNS and are critical to maintaining neuron health and responding to neuropathology. However, current methods for studying and harnessing the unique biology of human microglia is currently severely limited. Xenotransplantation of human microglia into immunodeficient mice is a promising new approach that provides extensive functional and visual access to human microglia in vitro. Xenotransplanted microglia (XMGs), generated from induced pluripotent stem cells (iPSCs) derived from human patients, can be modified to generate reporter and effector lines for unique microglial active states that activate in response to specific forms of neuropathology. Once transplanted into humanized MITRG mice these XMGs colonize the CNS while maintaining human expression patterns, but it remains to be verified if they exhibit the same response patterns and activity as in human brain tissue. The purpose of this supplement is to contribute to establishing the methodology for using XMGs as a proxy for studying human microglia in vivo and validation of reporter lines for both microglial activity and responses to neuropathology. Functional and morphological XMG responses will be observed in vivo using multiphoton imaging of a reporter for calcium activity, Salsa6f. Calcium signaling patterns will be compared between XMGs and endogenous mouse microglia reacting to laser-induced microlesions in brain tissue to verify that the XMGs retain their human response characteristics. Localized responses of XMGs to ?-amyloid plaques will be evaluated using brain- wide histology of a reporter for CD9, which has implicated as an indicator for the microglial ?-amyloid response state. Using an optical clearing technique, iDISCO+, it is possible to render complete intact mouse brains transparent. These cleared brains can be used to produce highly detailed three-dimensional renders of all XMGs and ?-amyloid plaques throughout the whole brain. From these renders, the distribution of CD9- expressing XMGs will be compared between control and ?-amyloid-expressing brains in order to validate them as a reliable reporter for proximity to ?-amyloid plaques. Validation of these imaging methods and XMG reporter lines may provide for unprecedented access into studying human microglial activity. Exploitation of targeted microglial active states also gives XMGs promising potential as vectors for targeted delivery of effectors localized to neuropathology afflicted regions which may have applications for drug discovery and therapeutics.

Core F: Induced Pluripotent Stem Cell Core Project Summary/Abstract Genome wide association studies have identified almost 30 genes that are associated with altered risk of developing late-onset Alzheimer's disease (AD). Yet, precisely how those genes impact the function of human cells to either promote or protect against the development or progression of AD remains unclear. One promising approach that is increasingly being used to examine such questions involves the use of induced pluripotent stem cells (iPSCs), which can be generated from control and AD subjects and then differentiated into the key brain cells implicated in AD. In 2013, the UCI Alzheimer's disease research center (ADRC) became the first ADRC to establish an induced Pluripotent Stem Cell (iPSC) Core. Since then, the core has generated and provided iPSC lines from AD, MCI and control subjects to researchers around the world who are using these lines to examine the impact of AD genes on human cellular function. In the current application, the iPSC Core will continue to embrace new advances in AD genetics and CRISPR gene editing to generate and distribute additional highly unique iPSC lines from UCI's three ADRC cohorts (UDS, DS, and 90+). Through five specific aims, the iPSC Core will collaborate with AD researchers worldwide (Aims 1-5), employ innovative genetic studies and genome engineering to facilitate the study of genetic AD risk factors in late- onset AD and specialized AD populations (Aims 1, 3, 4), and educate the research and lay communities about the scientific applications and implications of AD iPSCs (Aim 5).