1985 — 2006 |
Goldstein, Lawrence S |
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. |
Genetic and Immunologic Analysis of Microtubule Proteins @ University of California San Diego
Our long-term goal is to understand the molecular basis and logic of intracellular transport, which is crucial for normal neuronal and other cellular functions, and may play important roles in neurodegenerative and other disease processes. Our major emphasis is on kinesins, which generate directed movements along microtubules in a variety of cellular contexts including mitosis, vesicle traffic, and axonal transport. Specifically, we want to answer two general questions: What is the logic of kinesin motor utilization? How are kinesin motors regulated and attached to intracellular cargoes? To answer these questions, we propose to focus primarily on the functions of conventional kinesin (kinesin-I), because many of the issues of interest that are playing out in the motor function field can be attacked in studies of this well-studied single motor and its components. Tactically, we will use both Drosophila and mice in our experiments because of their unique and complementary advantages. Thus, Drosophila will be used to identify new genes encoding potential regulatory or attachment proteins and to provide basic information about accessory component functions. Mice will be used for detailed physiological, cell biological, and biochemical analyses of genes first identified or analyzed in Drosophila. To achieve these general goals, we will attack three specific aims in the next project period: 1) To understand the range of functions of conventional kinesin (kinesin-I) by analyzing mutants in the three different kinesin heavy chain subunits in mice (KIF5A, KIF5B, and KIF5C). This work will be carried out by generating systemic and conditional knockout mutants using the lox-cre system. These mutants will be analyzed primarily in six different cell types including cultured embryonic fibroblasts, hepatocytes, photoreceptors, motor neurons, sensory neurons, and cultured hippocampal neurons. 2) To test the hypothesis that kinesin light chain (KLC) is required for the cargo-attachment or regulation of kinesin-I. This aim will be achieved by analyzing the biochemical and cellular phenotype of systemic and conditional mouse mutants lacking each KLC subunit. 3) To identify and analyze new kinesin regulatory and cargo-attachment components. These new components will be identified and cloned in Drosophila and then characterized in depth using genetic, cytological, and biochemical methods.
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0.936 |
1998 — 2002 |
Goldstein, Lawrence S |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Training Program in Basic and Clinical Genetics @ University of California San Diego |
0.936 |
2002 — 2004 |
Goldstein, Lawrence S |
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. |
Genetic Analysis of Dendritic Targeting @ University of California San Diego
DESCRIPTION (provided by applicant) Like polarized epithelial cells, neurons are divided into two functionally distinct compartments and the axon and dendrites of a neuron performs distinctly different functions. Considerable work has established the existence of additional distinct sets of proteins that are selectively partitioned into these compartments. It is obvious that localization of these proteins is critical for proper functioning of the cell and the flow of information in the nervous system. Intuitively such a process should require a sorting system to identify the proteins for a certain intracellular destinations like the dendritic compartment, a transport machine to carry them, and finally, a mechanism that will retain the proteins in their respective place of action. This is an important problem in contemporary Cellular Neurobiology in which mechanistic understanding is quite limited. The goal of this collaborative project is to identify genes needed for preferential sorting of proteins and vesicles to the dendritic compartment of neurons. In order to achieve the stated objective, a live assay for dendritic sorting and transport will be developed in Drosophila using the green fluorescent protein (GFP) tagged tranferrin receptor (TfR) and the Drosophila homologues (ARD, ALS) of the a-subunit of nicotinic acetylcholine receptor (nAChR) proteins. These GFP tagged proteins will be expressed in the embryonic and larval nervous system and their subcellular localization will be characterized in both the live and fixed tissue preparations. An epifluorescence video microscopic set up as well as the confocal microscopic techniques will be used to observe this sorting process. Once established the assay will be used to characterize various known mutants of the fly genome and also to screen for new mutants that will affect the dendritic targeting of TfR/ARD/ALS-GFP proteins. It is expected that, at the end of a three-year period, these experiments will yield a list of candidate proteins involved in the dendritic sorting of TfR, ARD and ALS in Drosophila. In addition, it will also establish a genetically testable cellular assay system for dendritic protein sorting in live animal models. The proposed project will be executed in collaboration with Dr. Krishanu Ray at TIFR, India, and the research will be done primarily in India as an extension of NIH grant # ROl GM 35252.
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0.936 |
2007 — 2011 |
Goldstein, Lawrence S |
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. |
Impairment of Axonal Transport by Amyloid Precursor Protein and Amyloid Beta-Prot @ University of California San Diego
Amyloid precursor protein (APR) is a key player in the development of Alzheimer's Disease (AD) Mutations in humans that alter APR processing or overexpress APP appear to be sufficient to cause AD and to generate the amyloid plaques that are a constent feature of AD neuropathology. Although most work on AD development focuses on the potential toxicity of Abeta proteolytic fragments of APP, numerous observations point to significant neuronal defects caused by other APP proteolytic processing products or overexpression of full length APP itself. A consistent and long-standing set of observations suggest that a highly relevant phenotype caused by excess APP, which may also be found in early and late AD, is poisoning of the axonal transport machinery. This machinery is required for long-range neurotrophic signaling and for the supply of proteins and organelles needed for the maintenance of functional synapses. These observations also provide a way to tie APP behavior to the other major neuropathology found in AD, namely the neurofibrillary tangles, composed of the microtubule binding protein tau, which has also been implicated in controlling the transport of APP and other vesicles and organelles. Because overexpression of mutant forms of human APP in the mouse is one of the major models of AD, and because overexpression of APP may be sufficient to cause some forms of AD, it is crucial to understand the consequences of APP overexpression in neurons, and in particular how excess APP poisons axonal transport. Key issues include resolving whether Abeta plays a role in axonal transport defects and whether the defects generated by APP overexpression and Abeta toxicity are distinct. A related issue that needs to be evaluated further emerges from our recent observation that transport defects may enhance APP processing, potentially causing an autocatalytic spiral of defects. To understand the consequences of APP overexpression in neurons, and in particular how excess APP poisons axonal transport and to resolve whether Abeta plays a role in causing axonal transport defects we propose: 1) To test the hypothesis that APP controls its own transport in "cis". 2) To test the hypothesis that increased APP or its processing products poisons transport in trans and consequently affects synaptic function, and behavior. 3) To test the hypothesis that reduced transport enhances APP processing in neurons.
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0.936 |
2010 |
Goldstein, Lawrence S |
RC1Activity Code Description: NIH Challenge Grants in Health and Science Research |
Human Stem Cell Model of Niemann Pick Type C @ University of California San Diego
DESCRIPTION (provided by applicant): Niemann Pick Type C (NPC1) is a rare but lethal pediatric dementia caused by a mutation in NPC1, a housekeeping protein residing in the late endosomal compartment with a putative role in cholesterol transport. The result is a severe lipidosis characterized by massive accumulation of lysosomal sterols and other lipids that ultimately cause cell death. The disease is of enormous basic science interest as well as a hallmark model of dysfunctional intracellular cholesterol trafficking and because of its similarities with Alzheimer's disease (AD), which suggest shared underlying mechanisms. To date strategies to model NPC have not yet determined how some mutations of NPC1 can cause neuronal failure in humans for two reasons: i) use of animal models that do not replicate all aspects of human pathology, and ii) a focus on accumulation of cholesterol as the cause of neuronal dysfunction in NPC, which may not be the predominant phenotype in neuronal cell populations. We propose to generate the first human neuronal model of NPC1 by genetic engineering of human embryonic stem cells (hESCs) and reprogramming of somatic cells into human induced pluripotency stem cells (hIPSCs). From a broad perspective our approach will have a significant impact in the stem cell field as it cross validates studies in hIPSCs for the study of neurodegenerative diseases. By conducting parallel analysis of hESC and hIPSC lines we will also address the genetic heterogeneity of NPC, and confirm that pathologic phenotypes found in these cells are specifically due to lack of normal NPC1 function. We will use shRNA mediated silencing of NPC1 and insertional methods of viral reprogramming to generate independent sets of NPC1 knockdown hESC lines, and hIPSC lines reprogrammed from NPC fibroblasts respectively. We will follow strict criteria of characterization of these newly generated lines to ensure they maintain stem cell properties, are genetically stable and replicate basic NPC phenotypes described in the mouse literature. We will use protocols we have developed in my lab to generate populations of pure human neurons that we will study in bulk, pure, and compartmented cultures. We will analyze human neurons derived from NPC hESCs and hIPSCs to test specific predictions that have never been probed in a human neuronal model of the disease;i) we will confirm and expand phenotypes typical of NPC that have not been tested in live human neurons, ii) we will evaluate the viability and differentiation capacity of wild type and NPC1 neurons, iii) we will measure the kinetics of cholesterol and lysosomal trafficking and the role of cholesterol in neuronal growth and survival, and iv) we will explore whether NPC is a cell autonomous disease or if neuronal failure in NPC can be affected by the glial environment. Insights generated from our observations have the potential to drastically increase our understanding of how NPC1 causes neuronal failure in humans and guide efforts that may lead to the development of a cure. UCSD is one of the country's leading research institutions. According to data from the U.S. National Science Foundation, UCSD expended nearly $800 million for research and development during the 2007 fiscal year. This project will add to our institution as an economic and academic engine at the state and national level. Our project will contribute to the continued development of the regional and national economy through research, innovation and job creation. Future economic security will be promoted through the continued employment of a senior physician scientist, a senior research technician, as well as the employment of graduate and undergraduate students. The long term impact of this investment will be the creation of knowledge, the preparation of the next generation of academic workforce, the development of new technologies, opportunities for future research and the use of commercial services in the academic sector that have the potential to yield more jobs. PUBLIC HEALTH RELEVANCE: We propose to create the first human neuronal model of Niemann Pick type C1 from human embryonic stem cells and human induced pluripotent stem cells. We will use control and NPC1 human neurons to study the kinetics of cholesterol trafficking in NPC1 and the role of cholesterol in neuronal survival and regeneration. This work will shed important light on an important childhood neurodegenerative disease and on Alzheimer's Disease as well.
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0.936 |
2011 — 2012 |
Goldstein, Lawrence S. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Pluripotent Stem Cell Models of Sporadic Alzheimer's Disease @ University of California San Diego
DESCRIPTION (provided by applicant): How do the genomic variants in individuals with sporadic Alzheimer's Disease (SAD) contribute to the development of Alzheimer's Disease (AD)? To begin addressing this question human induced pluripotent stem cell (hIPSC) technology will be used to begin testing the hypothesis that the genomes of individual SAD patients contain genetic variants that generate biochemically detectable phenotypes in human neurons. Human pluripotent stem cells (hIPSC) allow the genomes of human individuals afflicted with SAD to be captured in a pluripotent stem cell line. Such cells can then be differentiated to human neurons or glia in vitro for evaluating whether the captured genome alters neuronal or glial phenotype in a manner similar to that seen in cells carrying FAD mutations or as predicted by mechanistic models of SAD such as the Ass hypothesis. An hIPSC model may also be useful for addressing human specific effects and avoiding some aspects of the well known limitations of animal models such as high copy number in transgenic models and the absence of a human genetic background. Our underlying hypothesis is that the genome of a patient with SAD contains a set of susceptibility variants that may alter neuronal, glial, or other relevant cellular phenotypes in a detectable manner. Broadly speaking, genomic variants in SAD patients could contribute to disease in a variety of ways ranging from effects on inflammatory pathways, glial turnover of Ass or other potentially toxic molecules generated by neurons and other cells, or by effects in neurons on pathways of APP processing, susceptibility to Ass poisoning, tau hyperphosphorylation, oxidative damage, etc. In this R21 exploratory proposal, we propose to take the first step and use hIPSC technology to test the hypothesis that at least some SAD genomes cause APP expression, APP processing, or tau phosphorylation phenotypes in human neurons that carry the genomes of patients who developed SAD. Identification of such genomes would provide the raw material for further studies using high resolution molecular genetic analyses to define how different genomic variants contribute to neuronal phenotypes and might allow new drug targets and pathways to be identified. Possible predictive measures and diagnostics might also be generated. In this pilot project, we will probe these issues by completing two specific aims: 1) We will generate 3 hIPSC lines each from 5 SAD patients and 5 age-matched normal controls (NC). 2) We will generate purified neurons from each hIPSC line and test whether SAD neurons exhibit APP expression, APP processing, or tau phosphorylation changes typical of FAD.
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0.936 |
2011 — 2013 |
Evans, Sylvia M (co-PI) [⬀] Frazer, Kelly A Goldstein, Lawrence S. Harismendy, Olivier (co-PI) [⬀] Izpisua Belmonte, Juan Carlos (co-PI) [⬀] Yeo, Eugene Wei-Ming |
U01Activity 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. |
Regulatory Genomic Studies in a Cohort of Ips Cell Derived Cardiomyocytes @ University of California San Diego
DESCRIPTION (provided by applicant): The overarching goal of our project is to use IPSC derived cardiomyocytes from genotyped individuals as cellular models to investigate how human genetic variation influences the gene regulatory networks Involved In cardiac biology and disease. Despite current treatment regimens, cardiovascular diseases remain the leading cause of morbidity and mortality in the United States and developed countries. Genome-wide association studies have identified a number of loci associated with cardiovascular disease susceptibility. Our Study will clarify the functional significance of these findings by combining cellular reprogramming Strategies with integrated molecular profiling and cellular assays. We have assembled a team of highly accomplished researchers in stem cell biology, cardiac cell biology, genomics, molecular genetics/epigenetics, biostatistics, and clinical medicine, and are well positioned to achieve the project goals within five years. After collecting fibroblasts and keratinocytes from individuals in the UCSD TSP cohort, the project will be carried out in three phases. In PHASE I, we will establish standardized reagents and procedures for the generation of iPSCs. Additionally, we will take advantage of our ongoing research efforts to increase the efficiency of cardiomyocyte differentiation to 80%, a substantial increase over current protocols (~20%). In PHASE II, we will develop cutting-edge technologies for high throughput generation of IPSCs, which will enable us to generate 600 iPSC lines (3 lines each from 200 individuals) in ~ 24 months. We will also scale our optimized protocols for deriving cardiomyocytes from the IPSC lines. In PHASE III we will initially perform validation experiments to measure the genomic profile variability between isogenic (derived from the same individual) cardiomyocytes. We will then use the derived cardiomyocytes to 1) Identify and characterize the causal DNA variants underlying strong GWAS signals with electrocardiographic traits; and 2) Identify expressed quantitative traits loci (eQTL) in the cohort of IPSC derived cardiomyocytes at baseline (untreated) and after stimulation. (End of Abstract)
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0.936 |
2012 — 2013 |
Goldstein, Lawrence S. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Testing Cell Autonomy of Ad Phenotypes Using Human Ips Cells @ University of California San Diego
DESCRIPTION (provided by applicant): Development of familial and sporadic Alzheimer's Disease (FAD and SAD) therapies requires deeper understanding of disease initiation and progression. A key unanswered question is whether all FAD and SAD neuronal misbehavior is solely the result of processes initiated or enhanced by secreted Amyloid Precursor Protein (APP) fragments such as A¿ and sAPP (and therefore cell non-autonomous), or whether A¿- independent intracellular processes driven by APP proteolytic products, e.g., the C-terminal fragment (CTF) (and therefore potentially cell autonomous) processes are a major contributor. Specifically, the major hypothesis for AD initiation and progression is the amyloid cascade hypothesis, which proposes that Ass peptide fragments of human APP are necessary and sufficient to initiate and to drive all downstream pathologies typical of AD progression including synaptic defects [1]. Alternative ideas include intracellular defects caused by presenilin or APP mutants/fragments that generate defects in endosomal or lysosomal pathways, axonal transport, neurotrophic signaling, transcriptional control, cell cycle reinitiation, oxidative defets, etc. [2-7]. There are also two-hit models in which A¿ is part of an initiating or enhancing insult n combination with intracellular insults [8-11]. These three types of models (autonomous, non-autonomous, two- hit) make distinct experimental predictions for mixed cell culture experiments using neurons derived from human induced pluripotent stem cells (hIPSC). For example, the (non-autonomous) amyloid cascade hypothesis, and related non-autonomous hypotheses based on toxicity of secreted fragments of APP (or other molecules) predict that mixtures of diseased and non-diseased neurons should cause disease phenotypes in the non-diseased neurons owing to secretion of toxic products by diseased neurons. Alternatively, in cell autonomous models of AD that do not posit secretion of toxic products, mixtures of diseased and non-diseased neurons should not lead to disease phenotypes in non-diseased neurons. Finally in two hit models, cell autonomous processes and cell nonautonomous processes might combine such that cell autonomous initiation of phenotypes might be enhanced by A¿ or other secreted toxic mediators. We propose to begin testing these ideas by further developing a new platform hIPSC human neuronal model of AD. These investigations, if successful, can shed new light on the relative contributions of autonomous and non-autonomous processes and two-hit models. We will use neurons made from hIPSC lines derived from a non-demented control patient (NDC), an FAD APPDp patient, and an SAD patient (called SAD2). We have two specific experimental aims: Aim 1) To test the hypothesis that some or all defects observed in purified neurons containing either an FAD APP duplication or a genome from an individual with SAD called SAD2 are cell-autonomous. Aim 2) To test the hypothesis that astrocytes carrying different ApoE alleles enhance or suppress AD phenotypes in purified neurons.
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0.936 |
2014 |
Goldstein, Lawrence S. |
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. |
Elucidating Ad Genotype-Phenotype Relationships Using Genetics of Human Ips Cells @ University of California San Diego
DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is common, devastating, and creates enormous social and financial burdens. At present, no effective disease-modifying AD treatment is available or imminent, in part because we lack a complete understanding of the cellular mechanisms and pathways that fail in human neurons and glial cells during disease, and in part because we don't adequately understand how common genetic variants alter human neuronal and glial phenotypes. Here we propose to test whether APP and PS mutations generated in common genetic backgrounds in human induced pluripotent stem cells (hIPSC) generate the same early neuronal phenotypes and then to investigate the extent to which a candidate set of genes identified by GWAS studies generate comparable phenotypes when reduced, increased, or altered by naturally occurring variants. To tackle both problems, we propose unique applications of hIPSC technology to 1) dissect how FAD mutations alter key pathways and then 2) to test how individual genetic background and identified risk factors predispose to SAD biochemical phenotypes in human neurons and astrocytes. Where possible, we will link the in vitro information to clinical data on individual patients and to post-mortem pathology from the UCSD ADRC. The analysis of hIPSC lines from SAD patients will be crucial to probe how common genetic risk factors act in neurons and astrocytes and will also give an initial estimate of the frequency of genomes in SAD patients and controls that cause relevant SAD phenotypes in neural cells differentiated in vitro. This frequency estimate will help address the important long-term question of whether hIPSC lines can be used to predict the likelihood that a given individual will develop SAD, i.e., to generate a predictive genomic/hIPSC diagnostic for SAD. This proposal capitalizes upon previous work from us and others that analyzed hIPSC lines from patients carrying an APP duplication (APPDp) or trisomy 21. Both situations appear to cause FAD by increasing APP expression by 50% in an otherwise euploid genome. Neurons made from these hIPSC lines exhibit typical AD biochemical alterations including elevated A¿, elevated activation of GSK3, and elevated phosphorylation of tau at a proposed pathological site. We also found that APPV717F but not PS1dE9 mutations cause elevated p-tau levels. Thus, early neuronal phenotypes of APP and presenilin mutations might be different raising the possibility that there may be multiple early pathogenic pathways that can be studied using hIPSC technology. We also found that hIPSC studies can elucidate how one common genetic risk factor, SORL1, acts in human neurons. We thus propose three specific aims: 1) Test the hypothesis that APP, PS1, and ¿-secretase mutations trigger the same early events in human neurons and astrocytes leading to downstream biochemical pathology typical of AD. 2) Test the hypothesis that genes identified as risk factors in GWAS studies generate AD phenotypes and altered endocytosis, trafficking, or transport when over or underexpressed. 3) Test the hypothesis that common genetic variants identified in GWAS studies act by altering gene expression in neurons or astrocytes.
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0.936 |
2014 — 2018 |
Goldstein, Lawrence S. |
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. |
Probing Sorl1 Risk Factors With Human Induced Pluripotent Stem Cell Technology @ University of California San Diego
PROJECT 1: ABSTRACT. A key problem in understanding and eventually treating Alzheimer's disease (AD) is our incomplete understanding of the role of genetic risk factors in late-onset, sporadic AD (SAD). While there is no clear single genetic lesion that causes SAD, the observed high heritability suggests that individual genetic background plays a significant role. Here we propose unique applications of human induced pluripotent stem cell (hIPSC) technology to dissect how individual genetic background and identified risk factors predispose to SAD biochemical phenotypes in human neurons and to link that information to clinical data on individual patients and to post-mortem pathology. We are basing our work on our recent finding using hIPSC technology that tested the hypothesis that R haplotypes cause general reduction of SORL1 expression leading to increased amyloid beta (A?) peptides and consequent risk of developing SAD [3, 7- 10]. Using hIPSC technology we found that R haplotypes impair a signaling input to the SORL1 gene. Specifically, P haplotypes respond to BDNF by inducing SORL1 expression, while R haplotypes do not. Basal expression levels show no correlation with R or P haplotypes. Thus, the SORL1 genetic contribution to SAD may be caused by complex regulatory variation in living human neurons. We now propose to test our working model for how SORL1 haplotypes contribute to SAD neuronal phenotypes and thus SAD risk in humans in vitro, and in vivo in human patients. We propose to test: 1) the hypothesis that BDNF-induced SORL1 expression modulates amyloidogenic processing of APP and downstream SAD-associated biochemical changes; and 2) the hypothesis that effects of SORL1 haplotype on neuronal phenotypes in vitro are mirrored in clinical data on SAD patients, specifically the relative amounts of BDNF and SORL1 proteins in cerebrospinal fluid (CSF) and post-mortem neuropathological phenotypes. In a related goal, we will investigate whether purified neurons made from our collection of patient hIPSC lines correlates with clinical behavior of individual patients. At present, we have too few hIPSC lines to definitively establish the degree of correlation rigorously, but given the existence of the lines, we will begin to collect comparative data as a way of contributing to future studies. Together, these experiments will provide new data about the details of SORL1 variant contributions to SAD phenotypes in human neurons with differing genetic backgrounds and potentially lead to new pathways for drug discovery and stratification of clinical trials based on genetic background. Our specific aims are to: 1. Test the hypothesis that the reduced BDNF induction response of purified human neurons with SORL1 risk variant haplotypes enhances SORL1- dependent downstream biochemical SAD phenotypes. 2. To test the hypothesis that the genetic status of patients at the SORL1 locus has a significant influence on clinical phenotypic markers measured in CSF or by post-mortem pathology.
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0.936 |
2015 — 2019 |
Goldstein, Lawrence S. |
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. |
Scalable Technologies For Genome Engineering in Hipscs @ University of California San Diego
Project Summary Many disorders of mental health, including autism spectrum disorder (ASD) exhibit strong or, in some cases, exclusive, contributions from individual genomic variation. These types of genomic contribution are difficult, if not impossible, to accurately recapitulate in animal in vivo or cellular models. Human pluripotent stem cells (hPSCs) provide the opportunity to use state-of-the-art genetic tools to recreate individual genomic patterns of variation that contribute to, or cause, these disorders. In Project 1, we will develop optimized genome editing methods based on strategies previously developed in non-human models. We will engineer four fluorescent reporter and Cre driver lines (CTIP2-mCherry, GFAP-mCherry, CALB-mCherry-T2A-Cre, and GAD1-mCherry- T2A-Cre), a panel of four monogenic autism models: point mutations in MECP2, SHANK3, TSC2, and FMR1, and a panel of eight CNV autism models: reciprocal duplications and deletions at 7q11.23, 16p11.2, 17p11.2, and 15q11-13. We will also develop new strategies for haplotype exchange of hIPSC models.
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0.936 |
2017 |
Goldstein, Lawrence S. |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Probing the Molecular and Cellular Complexity of App Trafficking and Its Relationship to Ad. @ University of California San Diego
Project Summary Alzheimer's disease (AD) creates substantial human suffering and social financial burdens. At present, the search for effective disease-modifying AD treatments is substantially hampered by our incomplete understanding of the cellular mechanisms and pathways that change in human neurons during AD. Here we propose to substantially increase our understanding of these problems by testing an overarching hypothesis tying variation in Amyloid Precursor Protein (APP) transcytotic trafficking to pathways that lead to AD. Specifically, we propose to test the hypothesis that one major contributor to the molecular and cellular complexity of AD derives from variability in branches of pathways controlling trafficking of APP and its fragments to the neuronal axon. This hypothesis emerges in part from the likely endocytic nature of a number of functions identified by GWAS and in part from our recent work on neuronal trafficking defects in a series of FAD mutations we generated and analyzed using human induced pluripotent stem cell (hIPSC) technology. We also propose to increase our understanding of the uniquely neuronal endocytic and transcytotic pathways mediating APP trafficking to neuronal axons. This trafficking is key to the anatomical location of A? secretion, plaque formation, APP proteolytic processing and the control of axon-specific tau phosphorylation leading to the formation of pathogenic tau aggregates called neurofibrillary tangles (NFT). Thus, we also propose to test a unique model we have developed that describes two different pathways for APP entry into neuronal axons, one of which we suggest controls axonal tau phosphorylation. We will then extend this work to study FAD and AD-protective APP mutations and their interaction with the traffic pathways of APP to the axon. The proposed three specific aims are: 1) Test the hypothesis that there are two somatodendritic pathways that traffic APP to neuronal axons. 2) Test the mechanistic hypothesis that direct interactions of APP and SORLA mediate transcytotic functions that modulate axonal entry and amyloidogenic processing of APP. 3) Test the hypothesis that ESCRT- functions control endo-lysosomal trafficking of APP and subsequent transcytotic amyloidogenic processing and axonal entry. Collectively, the proposed experiments will provide mechanistic insights into the various branches of APP trafficking which are critical for amyloidogenic APP processing and phospho-tau levels, opening up new avenues in AD research and identification of potential targets for therapeutic intervention.
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0.936 |
2020 — 2021 |
Goldstein, Lawrence S. |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Ipsc @ University of California, San Diego
IPSC CORE ? PROJECT SUMMARY/ABSTRACT The Induced Pluripotent Stem Cell (iPSC) Core will provide ADRC researchers access to state of the art human cell-based disease modeling strategies from targeted cohorts of subjects participating within the clinical studies of the ADRC. Additionally, the iPSC Core will provide hands-on training and disseminate protocols and best practices to support researchers within the ADRC, building on NIA Biospecimen Best Practices for iPSCs. Through the Core, the following specific aims will be accomplished: Aim 1 - Isolate and bank subject-specific dermal fibroblast cell lines. In collaboration with the Clinical Core and Neuropathology Cores, the iPSC Core will provide the hands-on expertise needed to isolate primary dermal fibroblasts cells lines from dermal punch biopsies and postmortem dermal explants. Aim 2: Generate and bank of subject-specific iPSC lines. Utilizing state of the art non-integrating reprogramming technologies and a decade of reprogramming experience, the iPSC Core will generate subject-specific iPSC lines from fibroblast cell lines banked in Aim 1. Aim 3: Provide services to confirm cell line identity, genomic analysis, and functional characterization. Utilizing established high-throughput systems in collaboration with the Biomarkers Core, the iPSC Core will validate subject identity and genome integrity for subject-specific fibroblasts cell lines and iPSCs. iPSCs will also undergo functional characterization to validate pluripotency. Aim 4: Implement intelligently designed banking procedures to allow for prolonged resource sharing within and outside of the ADRC. Following best practices for cell line nomenclature and tiered banking structures, the iPSC Core will establish a cell banking strategy that provides clarity and transparency, while also ensuring prolonged access to banked materials. Aim 4 will be closely coordinated with the Indiana University NCRAD center to bank and distribute iPSC cell lines. Aim 5: Provide training and support resources to ADRC researchers utilizing subject-specific cell lines. The iPSC Core will provide the needed resources to ensure that ADRC members are well equipped to utilize cell materials for research purposes. In support of the center?s Research and Education Component, we will provide detailed hands-on training, sharing of research protocols, and advise on best practices for creating subject-specific cell models of Alzheimer?s disease (AD) and AD-related diseases. These same resources will be applied in collaboration with the Latino Core to foster innovative, interdisciplinary research most on genetic AD risk factors and associated disease mechanisms most relevant to this underrepresented community.
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0.936 |