1998 — 2002 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Rap as a Molecular Chaperone/Escort Protein For Lrp
The LDL receptor-related protein (LRP) is an extremely large (600,000 kDa) single chain protein with four related extracellular domains that are in turn composed of multiple (2, 8, 10, 11 respectively) cysteine-rich ligand binding repeats. LRP has emerged as a unique endocytic receptor due to its ability to bind and endocytose a number of structurally and functionally distinct ligands. These include apolipoprotein E, 2-macroglobulin, tPA, and urokinase-plasminogen activator. A 39kDa receptor-associated protein (RAP) co-purifies with intracellular LRP and blocks ligand binding to LRP in vitro. Preliminary data provided in this proposal demonstrate that the different ligands bind to multiple and distinct regions on LRP and LRP binding sites on RAP. In addition, many cells that synthesize LRP and RAP also synthesize ligands for the receptor, which can prevent the correct folding of LRP if they associate to early. The P.I. hypothesizes that RAP serves a chaperoning function in the folding and proper trafficking of LRP by binding it and preventing premature association with ligand. Experiments are proposed to elucidate the molecular mechanism by which RAP interacts with LRP, explore the structural features of RAP by solving its crystal structure, determine the role of RAP as a folding chaperone using in vivo and in vitro methods, and define the trafficking of RAP and LRP both as individual molecules and interacting complexes.
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1 |
1998 — 2001 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Neurotrophin Regulation of Lrp in Cns Neurons
The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic receptor that is expressed abundantly in neurons of the central nervous system (CNS). Two LRP ligands, apolipoprotein E (apoE)/lipoprotein and beta-amyloid precursor protein (APP), have been shown genetically to play important roles in the pathogenesis of Alzheimer s disease (AD). While mutations in the APP gene cause certain forms of early-onset familial AD, the presence of the xi4 allele of apoE is a risk factor for both familial and sporadic late- onset AD. In addition to its role in the catabolism of its ligands, LRP itself has been identified as a component of senile plaques, a pathological hallmark of AD. We hypothesize that regulation of LRP expression and function in CNS neurons can directly influence the catabolism and functions of apoE/lipoprotein, APP, and beta-amyloid peptide (Abeta), and thereby impact on AD pathogenesis. Over the past few years, we have systematically examined the expression, endocytic function, and the biogenesis of LRP in various cells derived from the CNS. We have shown that normal development of hippocampal neuronal structure in vitro requires functional LRP on the cell surface, and that LRP mediates differential effects of apoE isoforms on neurite outgrowth. Interestingly, our most recent results indicate that cell surface LRP in specific neuronal cell lines can be rapidly up-regulated (over minutes) by nerve growth factor (NGF). To our knowledge, this is the first example in which a neurotrophic factor has been shown to rapidly alter the cellular distribution of an endocytic receptor. The long term goals of this proposal are to elucidate the molecular mechanisms by which LRP is regulated in neuronal systems, the consequences of LRP regulation, and the role of LRP in the pathogenesis of AD. Thus, we propose the following specific aims: 1) to examine the regulation of LRP expression by neurotrophins in primary cultures of CNS neurons; 2) to investigate which intracellular signaling pathway activated by neurotrophins is responsible for LRP up-regulation; 3) to identify the cis-elements within the LRP tail and/or the endosomal component that responds to the neurotrophin signal; and 4) to analyze whether neurotrophin regulation of LRP influences the processing of APP and the catabolism of Abeta. Results from these studies should not only enhance our understanding about the functions of LRP in the CNS, but also provide strategies as to how this receptor and its ligands can be regulated in vivo under pathophysiological conditions.
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1 |
2000 — 2001 |
Bu, Guojun |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Biogenesis of Lipoprotein Receptors
The liver plays a central role in the regulation of lipoprotein metabolism. It has been shown that cellular catabolism of lipoprotein particles is mediated primarily by a family of lipoprotein receptors, which together constitute the low density lipoprotein receptor (LDLR) gene family The LDLR itself and LDLR-related protein (LRP) are the two endocytic receptors that are highly expressed in hepatocytes. Thus, regulation of the cellular expression of the LDLR and LRP is critical for maintaining an appropriate balance of various lipoprotein particles within the circulation. The high cysteine content of the LDLR and LRP and thus the correct linkage of numerous disulfide bonds within these receptors suggest that correct folding of these receptors within the ER requires the participation of molecular chaperones. Previous studies from our laboratory have shown that a receptor-associated protein (RAP) functions a molecular chaperone/escort protein during the biogenesis of LRP. We hypothesis that RAP functions together with other chaperones/factors to assist the folding and trafficking of both the LDLR and LRP during the biogenesis of these lipoprotein receptors, and that their hepatic expression is regulated by molecular chaperones. Thus, we propose the following specific aims: 1) to examine whether RAO serves as an ER chaperone for the LDLR; 2) to identify other chaperones, factors, and protein modifications that are involved in the folding and biogenesis of the LDLR and LRP; 3) to analyze the effects of LDLR mutations on its folding; and 4) to determine whether misfolded LDLR and LRPO are degraded via the ubiquitin-mediated protein degradation system. The mechanisms thus defined for the biogenesis of the LDLR and LRPO should provide us with certain mutations within these receptors result in their misfolding and/or retention within the ER. These studies may also suggest strategies as to how to modulate the expression and folding of these hepatic receptors for individuals with abnormalities in lipoprotein metabolism.
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1 |
2000 — 2002 |
Bu, Guojun |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Role of Lrp &Its Ligand Tpa in Ltp &Aging
low density lipoprotein receptor; long term potentiation; ligands; Alzheimer's disease; plasminogen activator; protein protein interaction; receptor expression; hippocampus; aging; gene expression; neural plasticity; enzyme activity; protein binding; electrophysiology; genetically modified animals; laboratory mouse;
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1 |
2000 — 2001 |
Bu, Guojun |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Morphology Resource
Morphological analysis at the subcellular level is an essential component of modern cell biology and pathobiology research. Experimental approaches proposed in the projects of the current application include extensive use of microscopic analysis on the morphology of subcellular organelles as well as the use of immuno microscopic analysis at the subcellular level on the pattern and localization of specific mutant proteins and chaperones. We will establish a Morphology Core (Core A) consisting of an electron microscopy (EM) services, as well as a central facility equipped with conventional and confocal microscopes The EM service will be established with Dr. Hans Geuze and Janice Griffith at the University of Utrecht, the Netherlands. Dr. Hans Geuze is an internationally recognized expert in both EM techniques and subcellular morphology. His past collaborations and frequent interactions with the Pis in the current proposal has provided the foundation for efficient operation of the EM service. The conventional and confocal microscopes will be equipped with image analysis systems to facilitate data analysis and publication. The usage of the Morphology Core by various projects will include: 1) immuno localization of protein expression within specific subcellular organelles; 2) immunohistochemical and in situ hybridization analysis of protein/mRNA expression in specific tissues; 3) morphological analysis of subcellular organelles under physiologic and pathologic conditions; and 4) defining protein interactions within intact cells using two-fusion fluorescence resonance energy transfer. The establishment of this core facility will facilitate collaboration among the individual investigators and their projects. By using existing expertise and a centralized facility, this core will also increase the quality and efficiency and decrease the cost of the overall program.
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1 |
2001 — 2004 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Biogenesis of Lipoproteins Receptors |
1 |
2004 — 2008 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Pathobiology of Lrp1b Tumor Suppressor
DESCRIPTION (provided by applicant): LRP1 B is a novel member of the low-density lipoprotein receptor (LDLR) family. It was discovered as a putative tumor suppressor and is frequently deleted in lung cancer cells. It shares high sequence and structural homology with another giant member of the LDLR family, LRP. Receptors in the LDLR family mediate endocytosis of a variety of extracellular and membrane ligands and participate in signaling pathways that function in cell migration and differentiation. In addition, several receptors in the family regulate endocytosis and regeneration of urokinase plasminogen activator receptor (uPAR), which plays essential roles in the cell migration and invasion of cancer cells. LRP1 B and LRP have overlapping ligand binding specificities; however, our recent studies demonstrate that LRP1 B exhibits a significantly slower rate of endocytosis when compared to LRP. As a result, expression of LRP1 B prevents the regeneration of uPAR to the cell surface and inhibits cell migration. From these studies, we hypothesize that the tumor suppressive function of LRP1B is related to its ability to retain uPAR in a non-functional state on the cell surface, and that alterations in the expression and endocytic efficiency of LRP1B may impact the level of functional cell surface uPAR, tumor cell migration, invasion, growth, and metastasis. For the current proposal, we have designed experiments to dissect the mechanisms by which LRP1B suppresses uPAR function and to examine how changes in LRP1B expression and endocytosis influence cancer cell migration and tumor growth. We propose the following specific aims: 1) define interactions between LRP1B and the components of the uPAR system, and explore the roles of LRP1B in uPAR/integrin-mediated signaling; 2) dissect the mechanism underlying the slow endocytosis rate of LRP1 B; 3) examine the roles of LRP1B in uPAR-mediated human cancer cell proliferation, migration, and invasion in vitro, and tumorigenesis and metastasis in vivo; and 4) determine whether the LRPIB gene, transcript, and protein levels are altered in human tumor tissues. Results from these proposed studies should allow us to better understand if and how modulation of LRP1B expression in cancer cells influences uPAR-mediated tumor cell behavior. Together, these studies should allow us to vigorously test whether LRP1B is a tumor suppressor, and may provide a framework for the design of therapeutic strategies targeting LRP1B expression and function in human cancers.
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1 |
2004 — 2006 |
Bu, Guojun |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Trafficking of Lrp and Megalin in Polarized Cells
DESCRIPTION (provided by applicant) Megalin and the low-density lipoprotein (LDL) receptor-related protein (LRP) are two large membrane glycoproteins that belong to the LDL receptor gene family. In addition to lipoprotein particles, LRP and megalin also bind and endocytose a variety of structurally and functionally distinct ligands. In polarized cells such as neurons, LRP is localized in the basal lateral/somatodendritic domain, whereas megalin is more restricted to the apical domain of the plasma membrane. Recently, new information has emerged regarding the structural and functional elements within the cytoplasmic tails of the LDL-R family members. Studies from our laboratories have shown that the YATL sequence and the distal di-leucine motif function as the endocytosis signals for LRP. These and other sequence motifs within the LRP tail are similar to the sorting signals present in basolateral proteins expressed in epithelial cells or somatodendritic proteins in neurons. Megalin's cytoplasmic tail is about twice the size of that of LRP, and contains several putative tyrosine-based or di-leucine endocytosis motifs, a proline-rich sequence with homology to a SH3 binding domain, a SEV motif that mediates binding to PDZ proteins, and several putative protein kinase consensus motifs. Because of its role in capturing ligands at the lumenal domain of several epithelia, in addition to internalizing and degrading its ligands, megalin can also transport some of them to the basolateral pole via a transcytosis process. One unexplored aspect in the study of these receptors is the molecular and cellular determinants that dictate their polarized distribution and intracellular trafficking in polarized cells. We hypothesize that LRP is expressed primarily on the basolateral surface of epithelial cells, and its basolateral sorting motif(s) are present in its cytosolic tail. The basolateral expression of LRP is the result of both sorting at the TGN and the early/recycling endosome. Megalin, on the other hand, is expressed predominantly on the apical side of epithelial cells and has different ways to achieve its polarized localization, including targeting, apical retention through tail interactions with the actin cytoskeleton, and association to lipid rafts. The specific aims of the current proposal are 1) to identify and characterize the sorting motif(s) within the cytoplasmic tail of LRP and within the cytoplasmic and/or transmembrane domain of megalin; 2) to study the cellular machinery involved in LRP and megalin sorting; and 3) to analyze the molecular determinants of the endocytic and transcytotic pathways of megalin in polarized epithelial cells. The current proposed research will be performed primarily in Chile as an extension of NIH grant DK61761 (Guojun Bu, PI). Results from these collaborative studies should allow us to elucidate molecular and cellular determinants dictating the differential sorting of megalin and LRP in polarized cells.
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1 |
2006 — 2010 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Apoe Receptor Pathways to Intraneuronal Abeta
DESCRIPTION (provided by applicant): Intraneuronal amyloid-p peptide (AP) accumulation is an early and toxic event in the pathogenesis of Alzheimer's disease (AD). Understanding cellular mechanisms that accelerate or inhibit intraneuronal A[unreadable] accumulation may provide novel therapeutic strategies for AD. A[unreadable] can accumulate inside neurons via receptor-mediated uptake. It can also accumulate via de novo processing of amyloid precursor protein (APR) to A[unreadable] in the endocytic pathway. Our recent studies have shown that apolipoprotein E (apoE) receptors, members of the low-density lipoprotein receptor (LDLR) family, modulate A[unreadable] uptake as well as APR endocytic trafficking and processing to A[unreadable]. In particular, we have demonstrated that LRP overexpression in the brain increases cell-associated A[unreadable]. A[unreadable] can bind to apoE receptors either directly or indirectly via A[unreadable] chaperones such as apoE. This proposal will focus on two apoE receptors, the LDLR-related protein (LRP) and LRP1B. These homologous receptors are both highly expressed in neurons and bind multiple ligands including A[unreadable], apoE, and APP. However, evidence from our lab suggests that LRP and LRP1B play opposing roles in ligand endocytosis. While LRP mediates rapid endocytosis, LRP1B endocytoses very slowly and as a consequence, retains ligands at the cell surface. Our overall hypothesis is that LRP facilitates A[unreadable] uptake, p production, and intraneuronal A[unreadable] accumulation, and that LRP1B blocks these effects, thus inhibiting A[unreadable] toxicity and pathogenesis of Alzheimer's disease. We have designed both in vivo and in vitro approaches to test our hypothesis. In Aim 1, we plan to determine the roles of LRP and LRP1B in intraneuronal accumulation in animal models. Because conventional LRP knockout is early embryonic lethal, our lab has generated conditional LRP forebrain-specific knockout mice. Together with the LRP1B knockout mice, we plan to test the roles of LRP and LRP1B in intraneuronal A[unreadable] accumulation after brain A[unreadable] infusion or after breeding with PDAPP amyloid model mice. In Aim 2, we will define the opposing roles of LRP and LRP1B in apoE-dependent and apoE-independent A[unreadable] uptake in primary neurons. Impacts of altered LRP and/or LRP1B expression in neurons on A[unreadable] uptake and intraneuronal accumulation will be assessed in the absence or presence of apoE. In Aim 3, we plan to dissect the mechanisms underlying the opposing roles of LRP and LRP1B in APP endocytic trafficking and A[unreadable] production. Our proposed studies will take advantage of our experience in studying the cell biology of receptor-mediated endocytosis, APP endocytic trafficking, and A[unreadable] metabolism using both in vitro and in vivo approaches. Because recent studies have established the causal role of intraneuronal A[unreadable] in cognitive deficits prior to A[unreadable] plaques, an understanding of the pathways leading to or protecting against intraneuronal AB accumulation may lead to specific targets for AD therapy.
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1 |
2008 — 2009 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Lrp and App Processing in Neurodegeneration
DESCRIPTION (provided by applicant): Amyloid beta-peptide (AB) accumulation and toxicity in the brain are central events in the pathogenesis of Alzheimer's disease (AD). The low-density lipoprotein receptor-related protein (LRP) interacts with beta-amyloid-precursor protein (APP) and regulates its endocytic trafficking and processing to AB. LRP is also a major receptor in the brain for apolipoprotein E (apoE), which modulates AB clearance, cholesterol metabolism, and cellular signaling. Previous studies have detected abundant functional soluble LRP (sLRP) in peripheral and our preliminary work has detected sLRP in human brain and cerebral spinal fluid (CSF). Molecular and cellular studies have defined Notch/APP-like sequential processing of LRP by matrix metalloprotease (MMP) and by b- and g-secretases. Our long-term goal is to understand how LRP proteolysis is regulated in the brain and dysregulated in AD, and how these proteolytic events and processing products impact its function in apoE metabolism and signaling, as well as APP trafficking and processing to AB. Our preliminary studies have shown that a g-secretase cleavage product, APP intracellular domain (AICD), regulates LRP expression and function by directly binding to LRP promoter. Our studies also identified a novel adaptor protein, sorting nexin 17 (SNX17), that modulates endocytic trafficking and processing of both LRP and APP. Our central hypothesis is that LRP and APP proteolytic processing is dysregulated in AD by pathological ligands and trafficking events, and that this in turn impairs LRP expression and function in apoE metabolism and signaling in the brain. We propose four specific aims to test our hypothesis: 1) to identify LRP shedding enzymes and examine the functional impact of altered LRP shedding on apoE metabolism and signaling;2) to examine how LRP expression, shedding, and proteolysis are altered in AD mouse models and during aging and AD in humans;3) to study how APP processing products and other g-secretase cleavage events regulate LRP expression and function;and 4) to analyze how altered endocytic trafficking of LRP and APP by neuronal adaptor proteins influences their proteolytic processing, apoE metabolism and AB production. Together, these studies should allow us to define the mechanism and regulation of LRP expression, processing and function in the brain during aging and AD. Our proposed studies may also identify novel targets for AD diagnosis and therapy. PUBLIC HEALTH RELEVANCE: The major objective of our proposal is to understand how an apoE receptor LRP undergoes proteolytic processing and how these cellular events are regulated in the brain. Because apoE is a major risk factor for Alzheimer's disease, a leading cause of dementia in elderly, our results may provide knowledge for diagnosis and/or therapy for Alzheimer's disease.
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1 |
2009 — 2013 |
Bu, Guojun |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular and Cell Biology Core (McB Core) @ University of Illinois At Chicago
Apolipoprotein E (apoE) receptors, members of the low-density lipoprotein receptor (LDLR) family, contain common structure modules and share sequence homologue. One of the major challenges in the field of apoE receptor biology is to develop receptor-specific reagents including antibodies and cell lines. In the past, unique and specific reagents for apoE/apoE receptors have been developed by the labs of Drs. Bu, LaDu, Rebeck, Estus, and Weeber, and many of these reagents have been validated during the preliminary stage of this program project. The overall goal of the Molecular Cell Biology (MCB) Core is to facilitate studies in this program project by developing, organizing, and distributing apoE/apoE receptor reagents, and to carry out essential assays pertinent to our proposed studies. The five specific aims of the MCB Core are: 1) to develop and store apoE and apoE receptor-specific antibodies, cDNAs, cell lines, and siRNAs;2) to distribute these reagents and protocols to individual labs as they become available or needed;3) to perform standardized assays for apoE, apoE receptors, amyloid p-peptide (Ap), and Ap plaques;4) to make two new transgenic mouse lines and to assist individual projects to study the in vivo role of apoE receptors in these new and existing transgenic and knockout mice;and 5) to collaborate with individual projects to study apoE receptor proteolysis in Alzheimer's disease brains. The specific plans for the MCB Core include a designated technical personnel under the guidance of Dr. Bu to develop, organize, and supervise this Core facility and an easily accessible database documenting the available reagents and the protocols using them. Dr. Bu and the technical personnel will also provide consultations for the end users for technical issues and trouble shooting. These Core services will allow all individual projects to have access to our unique collection of apoE/apoE receptor reagents and to several standardized assays. It is anticipated that each of the five projects will utilize reagents and services from the MCB Core.
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0.937 |
2009 — 2013 |
Bu, Guojun |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Lrp and App Processing in Neurodegeneraton @ University of Illinois At Chicago
Amyloid p-peptide (A[3) accumulation and toxicity in the brain are central events in the pathogenesis of Alzheimer's disease (AD). The low-density lipoprotein receptor-related protein (LRP) interacts with p- amyloid-precursor protein (APR) and regulates its endocytic trafficking and processing to Ap. LRP is also the major endocytic receptor for apolipoprotein E (apoE), which interacts with Ap and modulates its cellular clearance. Previous studies have detected abundant functional soluble LRP (sLRP) in peripheral and our preliminary work has detected sLRP in human brain and cerebral spinal fluid (CSF). Molecular and cellular studies have defined Notch/APP-like sequential processing of LRP by matrix metalloprotease (MMP) and by P- and y-secretases. Our long-term goal is to understand how LRP proteolysis is regulated in the brain and dysregulated during AD, and how these proteolytic events and processing products impact its function in APP trafficking and processing to Ap. Our preliminary studies have shown that a y-secretase cleavage product, APP intracellular domain (AICD), regulates LRP expression and function by directly binding to LRP promoter. Our studies also identified a novel adaptor protein, sorting nexin 17 (SNX17), that modulates endocytic trafficking and processing of both LRP and APP. Our central hypothesis is that LRP proteolytic processing is dysregulated in AD by altered expression and function of its processing enzymes and intracellular adaptor proteins and this in turn impairs LRP function in apoE metabolism and signaling in the brain. We propose four specific aims to test our hypothesis: 1) to identify LRP shedding enzymes and examine the functional impacts of altered LRP shedding on apoE metabolism and signaling;2) to study how APP and other y-secretase substrates regulate LRP expression, processing and function;3) to analyze how altered endocytic trafficking of LRP and APP by neuronal adaptor proteins influences their proteolytic processing, apoE metabolism and Ap production;and 4) to examine how LRP proteolysis and the expression of its processing enzymes and adaptor proteins are altered in unique AD transgenic mouse models and during aging and AD in human. Through collaborations with the other projects, our goal in this program project is to define the mechanism of apoE receptor processing and effects on their functions. Our proposed studies may identify novel targets for AD diagnosis and therapy.
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0.937 |
2010 — 2014 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Brain Lipid Metabolism, Dendrites and Synapses in Aging and Alzheimer's Disease @ Mayo Clinic Jacksonville
PROJECT SUMMARY/ABSTRACT Brain lipids such as cholesterol play critical roles in neuronal membrane homeostasis and synapse functions. However, the mechanisms that govern their biogenesis and transport to neurons are poorly understood. Apolipoprotein E (apoE) is a major lipid transporter in the brain. Of the three human apoE isoforms (E2, E3 and E4), apoE4 is the predominant risk allele for late-onset AD. Brain apoE/lipoprotein particles, produced primarily by astrocytes, deliver cholesterol and other lipids to neurons via apoE receptors, which belong to the low-density lipoprotein receptor (LDLR) family. To ultimately understand why apoE4 is a risk factor for AD, it is essential to study the differential functions of apoE isoforms in brain lipid transport and synapse functions, and what specific roles apoE receptors play in these processes. We have demonstrated that brain apoE metabolism is mediated by both LDLR and LDLR-related protein 1 (LRP1). However, neuronal deletion of Lrp1, but not Ldlr, impairs cholesterol metabolism in mice. This suggests that LRP1 is the predominant cholesterol transport receptor in neurons. Conditional Lrp1 forebrain knockout (LRP1-KO) mice have decreased brain cholesterol, sulfatide and cerebroside; reduced dendritic spine density and branching; fewer synapses; and diminished synaptic functions. LRP1-KO mice have memory deficits and movement disorders consistent with compromised dendritic spine/synaptic integrity and synaptic functions. Interestingly, LRP1 levels are significantly reduced in human AD brains and in the apoE4-targeted replacement (TR) mice. ApoE4-TR, but not apoE3-TR mice, also exhibit impaired lipid metabolism and synaptic functions, and apoE4 is less stable compared to apoE3. Based on these observations, we hypothesize that apoE4 is inferior to apoE3 in transporting brain lipid and in supporting dendritic spine/synaptic integrity, particularly in aging brains, and that these apoE4 defects can be partially rescued by restoring the expression and function of apoE receptor LRP1. We propose three aims to test our hypothesis. In Aim 1, we will dissect the molecular and cellular mechanisms by which apoE isoforms transport lipids and regulate neuronal functions via LRP1- and LDLR-dependent pathways using astrocytes-secreted apoE/lipoprotein particles and glia-neuron co-culture system. In Aim 2, we will define age-dependent effects of apoE4 on brain lipid metabolism and synaptic functions and examine whether aging brains are more sensitive to the inferior functions of apoE4. In Aim 3, we will determine if overexpressed LRP1 in mouse brains is sufficient to rescue lipid and synaptic impairments in apoE4-TR mice by breeding apoE3-TR and apoE4-TR mice with LRP1 transgenic mice. Together, our proposed studies should generate critical knowledge on how apoE isoforms differentially regulate brain lipid metabolism and synaptic functions via apoE receptors, and why apoE4 is a strong risk factor for AD. Our studies may also define apoE and apoE receptors as critical targets for AD therapy.
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0.903 |
2012 — 2016 |
Bu, Guojun |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Lipoprotein Receptor and Synaptic Regulation of a^ Metabolism
Amyloid-p (AP) peptide accumulation and aggregation are initiating events in the pathogenesis of Alzheimer's disease (AD). AB aggregation is concentration-dependent and can result from its overproduction, inefficient clearance, or both. LRP1 is a multifunctional lipoprotein receptor that modulates both AB production and clearance. While the fast endocytosis of LRP1 modulates the endocytosis and processing of amyloid precursor protein (APP) to influence AB production, the endocytic functions of both LRP1 and cell surface heparan sulfate proteoglycan (HSPG) facilitate cellular uptake and clearance of Ap. A major goal of this project is to examine the in vivo roles of LRP1 and HSPG in brain AP production and clearance in mice with altered gene expression by viral mediated or genetic approaches. Our recent collaborative work with the groups of David Holtzman and John Cirrito has also showed that synaptic activity regulates APP processing to AB, and that this regulation requires the endocytosis of APP. In addition to presynaptic mechanisms, AP producfion can also be regulated by a postsynaptic mechanism that involves NMDARs, which interact with LRP1. Therefore, we plan to determine whether LRP1/HSPGmediated Ap production or clearance is regulated by synaptic activity. Our overall hypothesis is that LRP1 and HSPG play critical roles in synaptic-dependent regulation of both AB production and clearance, and that dysregulation of these pathways leads to AP accumulation and aggregation in AD brains. To test our hypothesis, we will collaborate with Drs. David Holtzman and John Cirrito, who have extensive experience in modulating synaptic activity and measuring AB metabolism in vivo, to pursue three specific aims. In Aim 1, we will dissect the role of LRP1 in synaptic-dependent AB generation in vivo. In Aim 2, we will dissect the role of LRP1 and HSPG in synaptic-dependent AB clearance in vivo. In Aim 3, we will examine the specific roles of LRPI and HSPG in neurons and astrocytes in AB metabolism and amyloid pathology in vivo using conditional knockout mice. Together, these studies should allow us to dissect the molecular mechanisms underlying synaptic regulation of brain AP metabolism and may define novel targets for AD therapy.
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1 |
2012 — 2016 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Apoe Receptor Pathways and Abeta Metabolism @ Mayo Clinic Jacksonville
DESCRIPTION (provided by applicant): Amyloid-? (A?) peptide accumulation and aggregation in the brain lead to synaptic dysfunction and neurotoxicity. Increasing evidence indicates that impaired A? clearance is an early and central pathogenic event in Alzheimer's disease (AD). Our long-term goal is to understand the molecular and cellular pathways by which A? is either cleared from the brain or accumulated to form toxic aggregates. A? clearance and aggregation are regulated by A? chaperones and cell surface receptors. Apolipoprotein E (apoE) is a major A? chaperone and cholesterol carrier in the brain. Of the three apoE isoforms in humans (E2, E3, E4), the ?4 allele of the APOE gene is the strongest genetic risk factor for AD. Mounting evidence indicates that both apoE and apoE receptors, members of the low-density lipoprotein receptor (LDLR) family, play important roles in A? clearance, aggregation and toxicity. During our previous funding period, we have delineated the specific roles of a major apoE receptor, the LDLR-related protein 1 (LRP1), in neuronal A? uptake and trafficking. We found that neuronal LRP1 functions together with cell surface heparan sulfate proteoglycan (HSPG) to mediate A? uptake, trafficking and eventual delivery to lysosomes. Conditional knockout of Lrp1 in neurons in adult mice leads to increase A? accumulation and amyloid plaque deposition. Interestingly, while apoE3 promotes A? degradation, apoE4 facilitates A? accumulation and aggregation in the lysosomes. Finally, we found that the apoE receptor LRP1 and HSPG also play important roles in A? metabolism by astrocytes. Based on these observations, our overall hypothesis is that apoE and apoE receptor LRP1 and HSPG play critical roles in A? uptake and trafficking to lysosomes by neurons and astrocytes, and that the efficiency of A? clearance is differentially regulated by apoE isoforms. Thus, our overall goal for this competitive renewal application is to use both in vitro and in vivo models to systematically dissect how apoE isoforms and apoE receptors regulate A? clearance, aggregation and toxicity. We propose three specific aims to test our hypothesis. In Aim 1, we will use primary cultured neurons and astrocytes to analyze the specific roles of apoE isoforms and apoE receptors in A? endocytic trafficking to lysosomes, its degradation, aggregation and toxicity. In Aim 2, we will use in vivo microdialysis technique to assess the roles of apoE isoforms and apoE receptors in brain A? clearance. In Aim 3, we will use conditional knockout mouse models to examine the roles of apoE receptors in A? aggregation and pathology. Together, our proposed studies will likely provide insight into the mechanisms underlying A? clearance and aggregation pathways in the brain and uncover how apoE isoforms and apoE receptors regulate these events in AD.
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0.903 |
2013 — 2019 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Apoe Isoform-Specific Therapy For Alzheimer Disease @ Mayo Clinic Jacksonville
DESCRIPTION (provided by applicant): The ¿4 allele of the apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer's disease (AD) compared to the more common ¿3 allele. Studies in animal models and humans suggest that apoE4 exhibits both loss-of-function and gain-of-toxic-function compared to apoE3. In regulating amyloid pathology, apoE4 is less efficient than apoE3 in mediating the clearance of amyloid-¿ (A¿) peptides and is more dominant in promoting A¿ aggregation. Outside the A¿ pathway, apoE4 is also less efficient in transporting lipid and supporting synapses. These studies led to an important yet unanswered question as to whether it is better to increase or decrease apoE levels in AD therapy. As there have not been studies addressing the effects of modulating apoE expression in adult mice, we have developed new animal models that allow for inducible and cell-type specific expression of apoE3 or apoE4. To take advantage of these unique animal models, we have established biochemical, pathological, and behavioral analyses that distinguish apoE3- and apoE4-related phenotypes. Thus, the major goal of this proposal is to investigate how an increase or decrease of apoE3 or apoE4 expression with or without amyloid pathology affects apoE isoform-related functions, synapses and behavior. Our overall hypothesis is that decreasing apoE levels in APOE4 carriers and increasing apoE levels in APOE3 carriers respectively represent promising treatment and/or preventive strategies for AD. We propose three specific aims to test our hypothesis. In Aim 1, we will examine how over-expression of apoE3 or apoE4 at different ages and at different stages of amyloid pathology affects A¿ metabolism, plaque deposition, synapses and behavior. These studies will be carried out in the background of apoE3-targeted replacement (TR) mice or apoE4-TR mice, without or with amyloid model APP/PS1 background. In Aim 2, we will investigate how down-regulation of apoE3 or apoE4 expression at different ages and at different stages of amyloid pathology affects A¿ metabolism, plaque deposition, synapses and behavior. These studies will be carried out in the background of apoE knockout mice in the absence or presence of APP/PS1. Finally in Aim 3, we will assess how peripheral expression of apoE3 or apoE4 impacts brain A¿ metabolism, plaque deposition, synapses, behavior and cardiovascular health. These studies will be carried out in the absence of apoE expression in the brain. Together, our studies will for the first time test how up-regulation or down-regulation of apoE isoforms in the adult brain or periphery at different ages and at different stages of amyloid pathology affects AD pathogenesis. Results from these studies will provide mechanistic insights for apoE-based AD prevention and therapy.
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0.903 |
2015 — 2019 |
Bu, Guojun Ertekin-Taner, Nilufer [⬀] |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Integrative Translational Discovery of Vascular Risk Factors in Aging and Dementia @ Mayo Clinic Jacksonville
PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) is the most common cause of dementia characterized by brain accumulation of senile plaques and neurofibrillary tangles. AD risk is likely influenced by a multitude of genetic and environmental risk factors and their complex interplay, which subsequently lead to cascades of downstream pathophysiologic events that include but are not limited to aberrant proteostasis and lipid metabolism, as well as inflammatory, vascular, and oxidative mechanisms. The array of risk factors that lead to AD and their downstream influences are likely to be heterogeneous amongst AD patients, which complicates the search for drug targets, biomarkers and their potential downstream beneficial use in any given AD patient. For this reason, drug target and biomarker discovery efforts in AD have to focus on identification of both molecular mechanisms that are commonly perturbed in AD patients, as well as those mechanisms that may underlie heterogeneity in AD. To overcome this massive challenge, team-science efforts, including the NIH initiatives, Accelerating Medicines Partnership-AD (AMP-AD) and Molecular Mechanisms of the Vascular Etiology of AD (M2OVE-AD) Consortia, have launched large-scale generation and analyses of multi-omics data from well- phenotyped human cohorts and model systems. These consortia aim to integrate multi-omics and clinical endophenotype data to build a model(s) of AD that captures these common and heterogeneous pathomechanisms. Our teams are leading participants of both AMP-AD and M2OVE-AD. The initial findings from these consortia reveal concerted changes in networks of expressed genes and proteins in AD subjects and model systems, with biological significance. Despite this progress and wide and immediate sharing of the data generated by these programs, significant gaps remain in the available ?omics data, and the ability to integrate, harmonize and annotate these datasets. Our proposal is in response to the RFA-AG-17-054, which aims to close these gaps. In this proposal, we maintain the overall objective of our parent funded M2OVE-AD project (RF1 AG51504), which is to determine APOE- and sex-dependent effects, and uncover novel genes and pathways that influence vascular risk in aging, AD and other dementias. Our specific aims are: 1. Integrative functional genomic analysis of human brains to discover novel pathways in AD. 2. Integrative functional genomic analysis in a prospective cohort to validate and discover AD pathways. 3. Investigate the impact of APOE genotype and sex on transcriptional networks and the metabolome in model systems. 4. Perform single-cell profiling to annotate the transcriptome data from AMP-AD and M2OVE-AD. These studies will add key epigenetic data (H3K9Ac and RRBS methylome) to the human and transcriptome and metabolomics data to the mouse cohorts, generate human and mouse single cell transcriptome data, and perform integrative network analyses. We expect this proposal to fill key gaps in knowledge and further enhance the AMP-AD and M2OVE-AD initiatives in their drug and biomarker discovery goals.
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0.903 |
2015 — 2019 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Apoe and Lrp1 in Brain Insulin Signaling and Glucose Metabolism (Competitive Renewal) @ Mayo Clinic Jacksonville
? DESCRIPTION (provided by applicant): APOE4 is the strongest genetic risk factor for Alzheimer's disease (AD); however, how apoE4 predisposes people for the risk for AD is still not clear. Clinical studies have shown that APOE4 carriers, both as healthy adults or with dementia, have lower cerebral glucose metabolism and increased neuroinflammation, conditions that are also common in individuals with diabetes. Interestingly, diabetes and impaired insulin signaling are linked to the pathogenesis of AD. Supporting these, a recent clinical trial with insulin intranasal spray in AD patients has yielded positive results in preventing cognitive decline and this has led to a new national plan for a Phase II/III trial. Thus, there is an urgent need to understand the function and regulation of brain insulin signaling and glucose metabolism in preclinical models. In the previous funding cycle and during the preliminary stage of this project, we found that apoE and its major receptor LRP1 regulate the metabolism of both lipid and glucose in the brain. Specifically, using in vivo microdialysis techniques, we found that brain glucose metabolism is impaired in APOE4-targeted replacement (TR) mice and in Lrp1 neuronal knockout mice. We also found that LRP1 interacts with insulin receptor (IR) and regulates insulin signaling and glucose metabolism in a manner that depends on the function of glucose transporter 4 (GLUT4). Impaired insulin signaling and chronic neuroinflammation were further exacerbated in the APOE4 mice by either amyloid-ß (Aß) pathology or a loss of neuronal LRP1. Thus, our goal for this renewal project is to study the molecular and cellular mechanisms by which apoE4 synergizes with pro-inflammatory cytokines and Aß to impair neuronal insulin signaling and glucose metabolism and test whether a restoration of insulin signaling in APOE4-TR mice allows a rescue of APOE4-related AD phenotypes. We hypothesize that apoE4 impairs neuronal insulin signaling and glucose metabolism in a manner that depends on the functions of apoE receptor LRP1 and glucose transporters, and that neuroinflammation and Aß further exacerbate these events in aging and AD. We propose three Specific Aims to test our hypothesis. In Aim 1, we plan to dissect the mechanisms by which apoE4 impairs neuronal insulin signaling and glucose metabolism in vitro in neurons, in vivo in APOE-TR mice, and in human brains. In Aim 2, we will examine how pro-inflammatory cytokines and Aß synergize with apoE4 in reducing neuronal insulin signaling and glucose metabolism. In Aim 3, we plan to test whether brain administration of insulin or insulin-sensitizing drug metformin, or a restoration of apoE4 expression and lipidation rescues impaired glucose metabolism, reduces apoE4-associated neuroinflammation, and improve synaptic functions and cognition. These studies will not only address the underlying mechanisms of impaired insulin signaling and glucose metabolism in APOE4 carriers and AD patients but will also test therapeutic potentials targeting insulin and apoE pathways.
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0.903 |
2017 |
Bu, Guojun Xu, Huaxi |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Molecular Pathobiology of Soluble Trem2 in Alzheimer's Disease. @ Sanford Burnham Prebys Medical Discovery Institute
PROJECT SUMMARY/ABSTRACT Alzheimer?s disease (AD) is the most common neurodegenerative disorder. Although AD patients feature amyloid plaques and neurofibrillary tangles comprising neurotoxic components such as A? and tau within varying brain regions, components which drive neurodegenerative onset during early stages of AD remain uncertain. Interestingly, genome-wide association studies (GWAS) have uncovered a variety of genes enriched in microglia and myeloid cell types, suggesting that AD may be modulated by microglial activation and function. One particular microglial transmembrane component, TREM2 (Triggering Receptor Expressed on Myeloid cells 2) identified by GWAS presents an interesting case for study as mutations in its ectodomain has been implicated in AD, Nasu-Hakola disease, and frontotemporal dementia. Given that these disorders feature neuroinflammatory etiologies, it seems likely that TREM2 microglial function may modulate neurodegenerative onset. TREM2 is proteolytically processed by ADAM proteases to generate soluble TREM2 (sTREM2) ectodomain fragments. Interestingly, sTREM2 levels have been found to be significantly elevated cerebrospinal fluid (CSF) during early stages of AD onset, and its levels correlate with pathogenic CSF markers such as tau and its hyperphosphorylated forms. Despite the potential use of sTREM2 as an early- stage AD biomarker, virtually nothing is known with regards to its role in microglial or neuronal function. Here we present evidence that sTREM2 can enhance microglial survival and activation to enhance proinflammatory cytokine expression. We also find that sTREM2 expression is induced by oligomerized tau, and exposure to sTREM2 can inhibit A? uptake. With these findings, we hypothesize that sTREM2 can act as a modulator of AD onset, and can thereby influence pathogenic A? plaque deposition/clearance, cognitive behavior, and synaptic function. Since little is known with regards to how sTREM2 is generated, and whether sTREM2 or the sTREM2 R47H risk variant can modulate microglial and neuronal function, we present aims here to define neuropathogenic conditions that may influence sTREM2 induction, and trafficking pathways that may influence sTREM2 generation. We will then determine the effects of sTREM2 on microglial and neuronal function. Using inducible microglial and macrophage sTREM2/sTREM2 R47H overexpression mouse models in AD APP/PS1 or tauopathic P301S Tau-Tg backgrounds, we will then determine whether sTREM2 can modulate cognitive behavior, pathological A?/tau accumulation, and synaptic decline. Together, completion of these aims will derive mechanisms of sTREM2 generation, and consequential effects on microglial and neuronal function. Using inducible sTREM2 mouse models, we will also be able to discern sTREM2 modulatory effects on cognitive, pathological and synaptic decline with AD stress. This will provide evidence that correlative increases in sTREM2 levels observed in early-stage AD will influence AD onset and outcome.
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0.904 |
2017 — 2020 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Apoe Pathway in Cerebrovascular a? Clearance in Alzheimer?S Disease @ Mayo Clinic Jacksonville
PROJECT SUMMARY The major goal of this proposal is to define critical cerebrovasculature pathways mediating the clearance of amyloid-? (A?), the aggregation of which into amyloid plaques represents a pathological hallmark of Alzheimer's disease (AD). In so doing, we will evaluate the specific role of LDL receptor-related protein 1 (LRP1), heparan sulfate proteoglycan (HSPG) and apolipoprotein E (apoE) isoforms in cerebrovascular clearance of A? and formation of cerebral amyloid angiopathy (CAA). The aggregation of A? in the brain is a direct result of its increased brain concentration due to an imbalance of its production and clearance. Although brain A? clearance is mediated by multiple pathways including intracellular degradation and extracellular degradation, much remains unknown about how the cerebrovasculature system clears A? through local cellular, blood-brain barrier (BBB), and perivascular drainage pathways. Given impaired clearance of A? drives late-onset AD (LOAD), we aim to improve understanding of the pathways regulating A? clearance, thereby establishing new targets for AD therapy and prevention that will benefit the vast majority of patients. During the previous funding cycle, we employed several conditional mouse models to demonstrate that deletion of the A? receptor LRP1 leads to slower A? clearance and exacerbated amyloid pathology, while deletion of another A? receptor HSPG in neurons produces the opposite effects. In addition, we and others have shown that apoE, a ligand for LRP1 and HSPG, modulates A? metabolism and pathology in an isoform- dependent manner with apoE4, whose gene allele represents the strongest genetic risk for AD, promoting amyloid deposition and the formation of CAA. Thus, the overall goal of this renewal application is to define the molecular mechanism underlying cerebrovascular clearance of A?. We hypothesize that the A? receptor LRP1 promotes, whereas HSPG inhibits, A? clearance along the cerebrovasculature in an apoE isoform-dependent manner impacting the formation of CAA and the distribution of A? pathology. In Aim 1, we will define the roles of LRP1 and HSPG in cerebrovascular function, clearance of A?, and formation of amyloid plaques and CAA using conditional mouse models inducing vasculature deletion of A? receptors at different ages and at different stages of plaque/CAA pathology. In Aim 2, we will analyze how apoE isoforms affect A? clearance and pathology in cerebrovasculature using cell type-specific and inducible mouse models. In Aim 3, we will define the molecular mechanisms through which LRP1, HSPG and apoE isoforms modulate A? metabolism, BBB integrity and vascular structure using reconstructed model systems from primary mouse cells or induced pluripotent stem cell (iPSC)-derived human cells to improve the likelihood of discoveries translatable to human AD. Finally, we plan to perform unbiased, single cell-type transcriptome analysis to uncover signaling pathways downstream of LRP1/HSPG/apoE. Together, our studies will define the molecular mechanism(s) underlying brain A? clearance and establish new targets for mechanism-based therapy.
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0.903 |
2018 |
Bu, Guojun |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Impact of Apoe2 On Aging-Related Conditions @ Mayo Clinic Jacksonville
Our long-term goal is to understand the molecular and cellular mechanisms by which the ?2 allele of the apolipoprotein E gene (APOE2) protects against Alzheimer's disease (AD), promotes healthy brain aging, and increases longevity, thereby informing the development of therapeutic strategies for AD and other aging-related conditions. Despite intensive effort to understand why APOE4 is a strong risk factor for AD, the reduced risk associated with APOE2 has garnered much less attention. While APOE2 has been shown to protect against amyloid-? (A?) accumulation, a hallmark of AD pathology, emerging evidence, including our recently published work and preliminary data, demonstrates that APOE2 protects against cognitive decline and increases longevity in the absence of AD pathology. Thus, there is an urgent need to understand how A?-dependent and independent pathways collectively support the function of apoE2 protein in protecting against AD and promoting healthy aging. Given apoE2 in the brain forms larger apolipoprotein particles compared to apoE3 and apoE4 suggesting an overall superior function in transporting lipids, we hypothesize that APOE2 protects against aging-related cognitive decline and AD through hyperlipidation leading to more efficient lipid transport, improved synaptic functions, reduced A? aggregation, and longer lifespan. We will test our hypothesis by employing multidisciplinary approaches including animal and cellular models to address the mechanisms and human studies on a healthy aging cohort to address the relevance. In Aim 1, we plan to analyze how astrocytic apoE2 protects against cognitive decline, synaptic functions and amyloid pathology in an age-dependent manner in our novel cell type-specific and inducible mouse models. We will also breed our experimental animals to the Abca1-KO background thus directly assess how alteration of apoE lipidation impacts the protective effects of apoE2. In Aim 2, we plan to dissect the molecular and cellular mechanisms underlying the protective effects of apoE2 using astrocytes and neurons derived from human induced- pluripotent stem cells (iPSCs). ApoE isoform-specific effects will be addressed both in patient background and under isogenic conditions. Multiple experimental systems including neuron-astrocyte co-cultures and 3-D cultures will be employed for these studies. In Aim 3, we plan to determine the impact of APOE2 on clinical outcomes, biomarker status, and pathological/biochemical measures in humans using a longitudinal Mayo Clinic Study of Aging cohort. Specifically, we will measure and correlate apoE and apoE-associated lipids in cerebrospinal fluid and blood and with clinical, imaging/fluid biomarkers, and neuropathological markers. Together, our proposed studies will uncover molecular and cellular pathways associated with APOE2-mediated protection against cognitive decline and increased longevity, thereby elucidating the mechanisms and informing therapeutic strategies against AD and other aging-related conditions.
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0.903 |
2020 — 2021 |
Bu, Guojun Zhao, Na |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Trem2-Mediated Microglial Dynamic Function in Alzheimer Disease @ Mayo Clinic Jacksonville
PROJECT SUMMARY The major goal of this proposal is to address the dynamic role of TREM2-mediated microglial function in brain aging and during different stages of the pathological development of Alzheimer?s disease (AD). TREM2 is a microglial specific gene with several of its rare variants associated with AD risk. Despite some progress, the molecular pathobiology of TREM2 in particular the TREM2-R47H risk variant is still not clear. Studies examining the effects of loss of TREM2 function in mouse models support inconsistent conclusions; with TREM2 deficiency either reduces or enhances amyloid or tau pathology and associated toxicity depending on the stage of the pathological development or the specific mouse models. As such, TREM2-mediated microglial function likely has dynamic effects on amyloid and tau pathologies depending on pathological stages throughout AD progression. Further complicating the challenge of studying the impact of TREM2-R47H variant, a recent study revealed that introducing the R47H mutation into the mouse Trem2 gene locus leads to aberrant splicing and instability of its mRNA. To fill these gaps in knowledge and the lack of appropriate model systems, we have generated novel cell type-specific and inducible mouse models expressing human TREM2 or TREM2- R47H in microglia. To address human relevance and molecular mechanisms, we have also generated human induced pluripotent stem cell (iPSC) lines carrying TREM2 or TREM2-R47H. Thus, the major goal of this proposal is to examine the dynamic effects of human TREM2 and TREM2-R47H on microglial and neuronal functions in aging and AD; while in the process defines the underlying molecular pathways by targeted and non-targeted approaches. We hypothesize that TREM2-mediated microglial function is protective against the development of AD pathologies but can be detrimental when such pathologies are associated with synaptic loss and neurodegeneration. We also hypothesize that TREM2-R47H represents a loss-of-function in particular in microglia-mediated protection against AD-related pathways. We will test our hypothesis through three aims. In Aim 1, we plan to analyze the effects of TREM2 or TREM2-R47H upon injury paradigms and during aging in the absence of AD pathology. In Aim 2, we will examine the effects of TREM2 or TREM2-R47H on the metabolism, deposition, and toxicity of A? and tau at different stages of pathological development. In Aim 3, we plan to identify and validate the molecular pathways associated with TREM2 and TREM2-R47H using iPSC- derived microglia-like cells with or without integration into cerebral organoids. This innovative proposal will take advantage of our existing conditional mouse models and iPSC-derived cellular models combined with state-of- the-art technologies including in vivo microdialysis, two-photon microscopy and molecular profiling by single cell RNA-Seq. These efforts should collectively help to understand how TREM2 modulates microglial dynamic roles in aging and AD pathogenesis and how we can target these pathways to treat AD.
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0.903 |
2021 |
Bu, Guojun |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Impact of Vascular Apoe in Aging and Ad @ Mayo Clinic Jacksonville
PROJECT SUMMARY (APOE U19: Project 4) While the major function of apolipoprotein E (apoE) is to mediate the lipid transport, APOE genotype (APOE2, APOE3 and APOE4) significantly influences cognitive function and Alzheimer?s disease (AD) pathogenesis through multiple pathways. APOE is associated with not only amyloid-? (A?) pathology but also cerebrovascular function including blood-brain barrier integrity and cerebral blood flow. Given that cerebrovascular disturbance substantially contributes to cognitive decline in the elderly and that apoE is abundantly expressed in vascular mural cells (smooth muscle cells and pericytes) as well as astrocytes and microglia in the brain, the overall goal of Project 4 is to define how APOE genotype in vascular mural cells impacts the molecular mechanisms and pathways in the development of cerebrovascular dysregulations and cognitive decline during aging and AD. In Aim 1, we will utilize novel mouse models, in which human APOE2, APOE3, or APOE4 gene is expressed upon an excision of a loxp-flanked STOP cassette by vascular mural cell specific sm22? promoter driven Cre expression to determine the effects of vascular mural cell-specific expression of apoE isoforms on cerebrovascular function and brain cognition during aging. In Aim 2, we will define the impact of apoE isoform deletion in vascular mural cells on age-related cerebrovascular dysfunction and cognitive decline using novel APOE knock-in mice, in which murine Apoe is replaced with floxed APOE2, APOE3, or APOE4 gene. By crossing with sm22?-Cre mice, we will specifically delete individual apoE isoforms in vascular mural cells. In Aim 3, we will examine how vascular-specific expression or deletion of apoE isoforms in conditional mouse models affects AD-related phenotypes by breeding the vascular mural cell-specific apoE isoform expressing or knockout mice with amyloid model APP knockin mice (AppNL-F/NL-F). Using those unique mouse models, we will comprehensively investigate how apoE isoforms in vascular mural cells impact cerebrovascular function, glial phenotypes, neuroinflammation, neurodegeneration, brain cognition and amyloid pathology at different ages. In particular, the apoE properties in the mouse models will be analyzed by the Biochemistry and Structural Modeling Core (Core B). ApoE amounts, oxidation and/or glycation in the mice as well as other AD-rerated fluid biomarkers will be measured through the Biomarker Core (Core D). While neuropathology in the mice will be investigated in Neuropathology Core (Core C), a multi-Omics approach including proteomics, metabolomics, lipidomics and single cell RNA sequencing will be carried out through the Multi-Omics Core (Core F) and Bioinformatics, Biostatistics, and Data Management Core (Core G) to profile molecular phenotypes in these mouse models. Together, our innovative study should fill a critical void in our understanding of how apoE isoforms in cerebrovasculature impact cellular functions and brain homeostasis during aging and AD through synergistic interaction with Core B-G and comparative mouse model studies by Project 2 and Project 3.
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0.903 |
2021 |
Bu, Guojun |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Administrative Core @ Mayo Clinic Jacksonville
PROJECT SUMMARY (APOE U19 Core A: Administrative Core) The Administrative Core will lead this U19 project by providing administrative, fiscal, and scientific oversight and promoting synergy of all Cores and Projects to address the ApoE Cascade Hypothesis. Core A will serve several functions to facilitate the scientific objectives and rigor of this U19. It will also serve the broader scientific community by sharing knowledge, resources, and datasets through a web portal and by promoting collaboration through an annual ApoE Symposium. Core A will cooperate fully with NIA initiatives with a dedicated leadership team and administrative personnel with relevant experience. As the Co-PIs of this U19 project and Co-Leaders of Core A, Drs. Guojun Bu (Mayo Clinic Jacksonville) and David Holtzman (Washington University in St. Louis) are recognized leaders in the apoE field; each has more than 25 years of experience studying apoE and apoE receptors as they relate to the pathogenesis of AD and other aging- related conditions. Drs. Bu and Holtzman have a long history of collaboration and are well positioned to share leadership roles for this U19 program at the two coordinating institutions. They also have strong leadership experience each serving as a Department Chair and an Associate Director of Alzheimer?s Disease Research Center at their respective institution. Together with Dr. Alison Goate, who will serve as the Site-PI at Icahn School of Medicine at Mount Sinai, they will ensure integration and synergy across the U19 by pursuing the following aims: Aim 1: Provide administrative structure, fiscal oversight, and site coordination for the U19 program. Aim 2: Assume responsibility for the quality control of the U19 activities by ensuring responsible conduct of research, rigor, and reproducibility of research practices, and by ensuring compliance with all internal and external regulatory requirements. Promote and track open-access publications and facilitate submission of progress reports to NIH. Aim 3: Promote the scientific direction and integration of the U19 components by organizing meetings of the Internal Steering Committee and External Advisory Committee. Aim 4: Develop and maintain an apoE website what will serve as knowledge portal, and resource and data sharing platform. Aim 5: Organize Annual ApoE Symposium for U19 investigators as well as the broader scientific community to interact and share research findings. These innovative aims will ensure that the robust and rigorous efforts by the U19 team investigators will lead to new discoveries to inform therapeutic strategies.
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0.903 |
2021 |
Bu, Guojun |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Human Ipsc Models Core @ Mayo Clinic Jacksonville
PROJECT SUMMARY (APOE U19 Core E: Human iPSC Models Core) Human somatic and stem cell models have emerged as a powerful system for modeling the complexities of pathological gene expression, particularly in the early phase of disease. Further, human stem cells can be differentiated into cell-types that secrete apoE and that are affected in disease, such as neurons, astrocytes, microglia, and vascular mural cells (VMCs), as well as 3D ?mini-brain? cerebral organoids. The Core E will build upon the existing resources and technology from three institutions at the forefront of stem cell modeling of apoE-related biology and pathobiology in AD: MCJ (Guojun Bu), WUSTL (Celeste Karch), and ISMMS (Julia TCW) to generate a comprehensive collection of well-characterized human iPSC lines with different APOE genotypes from deeply phenotyped patients and through isogenic conversions. The established iPSC lines with different APOE genotypes, sex, ethnicity, and disease status will serve both this U19 and the broader scientific community to address critical gaps in our knowledge of the effects of apoE isoforms in different human brain cell types. In so doing, Core E will support U19 Projects and the broader scientific community by testing a critical component of the ApoE Cascade Hypothesis (ACH): to understand the effects of apoE isoforms on biochemical and cellular events leading to eventual phenotypic outcomes. Thus, Core E will work synergistically with Core A, B, F, G and Projects 1-5 to address the ACH hypothesis and to become an invaluable resource for the broader scientific community.
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0.903 |
2021 |
Bu, Guojun Goate, Alison M (co-PI) [⬀] Holtzman, David M. (co-PI) [⬀] |
U19Activity Code Description: To support a research program of multiple projects directed toward a specific major objective, basic theme or program goal, requiring a broadly based, multidisciplinary and often long-term approach. A cooperative agreement research program generally involves the organized efforts of large groups, members of which are conducting research projects designed to elucidate the various aspects of a specific objective. Substantial Federal programmatic staff involvement is intended to assist investigators during performance of the research activities, as defined in the terms and conditions of award. The investigators have primary authorities and responsibilities to define research objectives and approaches, and to plan, conduct, analyze, and publish results, interpretations and conclusions of their studies. Each research project is usually under the leadership of an established investigator in an area representing his/her special interest and competencies. Each project supported through this mechanism should contribute to or be directly related to the common theme of the total research effort. The award can provide support for certain basic shared resources, including clinical components, which facilitate the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence. |
Biology and Pathobiology of Apoe in Aging and Alzheimer's Disease @ Mayo Clinic Jacksonville
PROJECT SUMMARY (APOE U19: OVERALL) The overarching goal of this U19 project is to comprehensively understand the biology and pathobiology of apolipoprotein E (apoE) in aging and Alzheimer?s disease (AD) to inform therapeutic strategies. The ?4 allele of the APOE gene (APOE4) is the strongest genetic risk factor for AD impacting 50-70% of all AD patients, while the ?2 allele is protective compared to the common ?3 allele. APOE4 is also a strong risk factor for age-related cognitive decline and vascular cognitive impairment. To integrate existing knowledge and address critical gaps, we propose a unified ApoE Cascade Hypothesis that the structural differences and related biochemical properties among the three apoE isoforms initiate their differential effects on a cascade of events at the cellular and systems levels ultimately impacting aging-related pathogenic conditions including AD. Towards this, we have assembled a multi-disciplinary team to synergize expertise and resources across multiple institutions. By integrating five interactive Projects and seven robust Cores, we will create a nexus for apoE-related aging research, sharing the knowledge, expertise and resources with the broader scientific community. Project 1 will work closely with Core B to address the structural and biochemical properties of the three apoE isoforms to generate insights for functional outcomes. Projects 2, 3 and 4 will interactively study how apoE isoforms expressed in astrocytes, microglia, or vascular mural cells impact lipid metabolism, glial and vascular functions, AD-related pathologies, and cellular and molecular pathways using conditional mouse models and systems- based approaches. These studies will generate cell type-specific apoE/lipoprotein particles that will be collected through in vivo microdialysis for structural and biochemical studies. Project 5 will carry out genomic and genetic analyses to identify modifiers of APOE-related age at onset of AD. Studies in Projects 2-5 will be interactively supplemented by neuropathological studies using postmortem brains from healthy aging studies or with AD pathologies (Core C), biomarker studies using both human and mouse biospecimens (Core D), and functional studies using human iPSC-derived cellular and organoid models (Core E). This U19 proposal is supported by a comprehensive Multi-Omics Core (Core F) for centralized proteomics, lipidomics, and metabolomics studies on various animal and iPSC models, as well as human postmortem brains and fluid biospecimens. The Bioinformatics, Biostatistics, and Data Management Core (Core G) will provide critical supports for analyzing large datasets including those from single-cell RNA-seq and biostatistics supports to ensure scientific rigor. Core G will also work closely with the Administrative Core (Core A) to maintain an ApoE Web Portal designated as EPAAD where knowledge, resources, and data will be shared with the scientific community. Core A will also organize annual ApoE Symposium to promote collaboration and engage the ApoE Community. As such, this U19 will drive a team-based effort to generate essential knowledge to guide disease- modifying therapies for AD and other aging-related conditions.
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0.903 |
2021 |
Bu, Guojun |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Apoe Pathway in Cerebrovascular a? Clearance in Alzheimer's Disease @ Mayo Clinic Jacksonville
PROJECT SUMMARY The major goal of this proposal is to define critical cerebrovasculature pathways mediating the clearance of amyloid-? (A?), the aggregation of which into amyloid plaques represents a pathological hallmark of Alzheimer's disease (AD). In so doing, we will evaluate the specific role of LDL receptor-related protein 1 (LRP1), heparan sulfate proteoglycan (HSPG) and apolipoprotein E (apoE) isoforms in cerebrovascular clearance of A? and formation of cerebral amyloid angiopathy (CAA). The aggregation of A? in the brain is a direct result of its increased brain concentration due to an imbalance of its production and clearance. Although brain A? clearance is mediated by multiple pathways including intracellular degradation and extracellular degradation, much remains unknown about how the cerebrovasculature system clears A? through local cellular, blood-brain barrier (BBB), and perivascular drainage pathways. Given impaired clearance of A? drives late-onset AD (LOAD), we aim to improve understanding of the pathways regulating A? clearance, thereby establishing new targets for AD therapy and prevention that will benefit the vast majority of patients. During the previous funding cycle, we employed several conditional mouse models to demonstrate that deletion of the A? receptor LRP1 leads to slower A? clearance and exacerbated amyloid pathology, while deletion of another A? receptor HSPG in neurons produces the opposite effects. In addition, we and others have shown that apoE, a ligand for LRP1 and HSPG, modulates A? metabolism and pathology in an isoform- dependent manner with apoE4, whose gene allele represents the strongest genetic risk for AD, promoting amyloid deposition and the formation of CAA. Thus, the overall goal of this renewal application is to define the molecular mechanism underlying cerebrovascular clearance of A?. We hypothesize that the A? receptor LRP1 promotes, whereas HSPG inhibits, A? clearance along the cerebrovasculature in an apoE isoform-dependent manner impacting the formation of CAA and the distribution of A? pathology. In Aim 1, we will define the roles of LRP1 and HSPG in cerebrovascular function, clearance of A?, and formation of amyloid plaques and CAA using conditional mouse models inducing vasculature deletion of A? receptors at different ages and at different stages of plaque/CAA pathology. In Aim 2, we will analyze how apoE isoforms affect A? clearance and pathology in cerebrovasculature using cell type-specific and inducible mouse models. In Aim 3, we will define the molecular mechanisms through which LRP1, HSPG and apoE isoforms modulate A? metabolism, BBB integrity and vascular structure using reconstructed model systems from primary mouse cells or induced pluripotent stem cell (iPSC)-derived human cells to improve the likelihood of discoveries translatable to human AD. Finally, we plan to perform unbiased, single cell-type transcriptome analysis to uncover signaling pathways downstream of LRP1/HSPG/apoE. Together, our studies will define the molecular mechanism(s) underlying brain A? clearance and establish new targets for mechanism-based therapy.
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0.903 |