1996 — 2011 |
Lee, Kuo-Fen |
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 Analysis of Erbb2 in Mammalian Development @ Salk Institute For Biological Studies
DESCRIPTION (appended verbatim from investigator's abstract): The long term goal of this research is to elucidate the molecular and cellular mechanisms by which neuregulins and their receptors influence mammalian development. Neuregulins (NRGs) are a family of structurally related signaling molecules that interact with erbB receptor tyrosine kinases (erbB2 erbB3 and erbB4). To understand the physiological role of NRG signaling pathways in mammalian development we established erbB2 null mutant mice that die of cardiac defects before embryonic (E) day 11. To study the role of erbB2 in development after E11 we genetically rescued cardiac defects of erbB2 null mutants that survive until birth. To study the role of erbB2 in development after birth we established erbB2 conditional mutant mice via the Crelox P technology. Preliminary results showed that erbB2 plays an essential role in development of the neuromuscular junction. In addition when crossed with nestin Cre mice erbB2 floxed mice displayed a phenotype resembling human Hirshsprung's disease (HRSD). Together these mice provide an excellent model for genetic anatomical cell and molecular and biochemical studies to further understand the role of erbB2 in neural development. Aim 1 is to determine the role of erbB2 in the development of neuromuscular junction: ErbB2 mutant mice are crossed with agrin and its receptor MuSK or erbB3 mutant mice to determine how their genetic interactions influence pre and postsynaptic development including acetylcholine receptor clustering and gene activation at synaptic sites. Aim 2 is to investigate the regulated protein phosphorylation in erbB2 mutant mice: Signaling mechanisms underlying genetic interactions of erbB2 with agrin/MuSK and erbB3 will be elucidated with an emphasis on protein phosphorylation. Aim 3 is to determine the role of erbB2 in the development of the enteric nervous system. Aim 4 is to characterize the autonomic nervous system in erbB2floxed/nestinCre mutant mice. Results from this research will advance our knowledge on the development of multiple parts of the nervous system and provide insights on several human diseases.
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0.922 |
1997 — 2001 |
Lee, Kuo-Fen |
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. |
Mouse Models of Local Nerve Growth Factor &Acetylcholine Disfunction @ Salk Institute For Biological Studies
The central theme of this Program is to develop animal models both for the understanding of the biology of Alzheimer~s Disease (AD) and for the development of gene therapy of AD. Project 4 focuses on the roles of neurotrophins and acetylcholine within central cholinergic systems in learning and memory. One approach central to this project is to either over-express or abolish expression of the genes encoding nerve growth factor (NGF) or acetylcholine transferase (ChAT), the key enzyme in acetylcholine synthesis, only in specific cholinergic pathways of the adult basal forebrain of the mouse, while expression of these genes is unaffected in the periphery and the remainder of the brain. Over- expression will be achieved by using a tetracycline inducible system. Abolishment of expression will be achieved by employed a conditional mutation system, which is derived from the Cre-loxP recombination system of P1 phage. Recombinase Cre is a 38 kd protein that is able to recognize two recombination sites (termed loxP sites) and catalyze the excision of DNA sequences between two loxP sites. Our preliminary results show that when introduced into hippocampus in an adenoviral vector via stereotaxic injection, the Cre recombinase is indeed able to mediate recombination of a beta-galactosidase target gene construct containing two 1oxP sites. Hence, the strategy is to introduce two loxP sites into the NGF or ChAT gene followed by introduction of the Cre recombinase in specific cholinergic nuclei or target areas of cholinergic neurons. Consequences of these genetic manipulations will be investigated by both neuroanatomical analysis and behavioral tests. The specific aims are: 1. To generate and characterize mice carrying a lost-or-gains-of-function of the NGF or ChAT gene in adult basal forebrain cholinergic pathways. 2. To determine the specificity of the functional (behavioral) consequences of a loss-or gain-of-function of NGF or ChAT in different basal forebrain cholinergic pathways in young and aged mice. 3. To determine the effects of gene therapy in these models by either ex vivo or in vivo gene delivery methods.
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0.922 |
1997 — 2001 |
Lee, Kuo-Fen |
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--Transgenic Mouse @ Salk Institute For Biological Studies
The main objective of Core 9003 is to generated transgenic mice that are instrumental for Projects 4 and 5; to understand the role of neurotrophic factors, cholinergic and glutabminergic pathways in learning and memory, all of which are relevant to the biology of Alzheimer~s Disease. Furthermore these mice will facilitate Project 1 to develop a better gene transfer system in the brain. We will produce transgenic mice via microinjecting DNA into the pronucleus of one-cell embryos and gene targeting in mouse embryonic stem (ES) cells. We will be assisted by Core 9001 in the production of adenoviruses expressing the Cre recombinase that will be used to generate conditional mutant ES cell clones. The specific aims are: 1. To generate transgenic mice by DNA microinjection. 2. To test reagents, maintain ES cell lines, and generate feeder cells for cultivating embryos and ES cells. 3. To generate null and conditional mutant ES cell lines by homologous recombination. 4. To generate chimeras for establishing germ- line mutant mice on an outbred or 129 inbred genetic background.
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0.922 |
1999 — 2002 |
Lee, Kuo-Fen |
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. |
Core--Animal and Transgenic Facility @ Salk Institute For Biological Studies
The main animal facility at the Salk Institute (Cancer Research Animal Facility, or CRAF) contains 24,000 gsf of space and houses 1,000 cages of mice, many transgenic, plus several other species of animals. A separate transgenic mouse facility, contains 8,000 gsf and 3,000 cages of transgenic mice, was completed in 1995. A planned addition to the CRAF, to be completed in the next five years, will comprise 24,000 gsf. The addition will be used exclusively for transgenic animals. A special transgenic core laboratory for microinjection as developed during the previous grant period with core grant support. Services provided by the transgenic core laboratory include microinjection of embryonic stem (ES) cells into blastocysts, microinjection of DNA into one-cell embryos, providing neo-resistant mice for generating feeder cells for ES cell culture, providing inbred strains of mice, re-derivation of mouse lines, and establishment of ES cell lines from wild-type and mutant mice. The core laboratory is operated by a research assistant skilled in these techniques. The core laboratory is operated by a research assistant skilled in these techniques. The core laboratory is equipped with two microinjection microscope, one cooling stage, one needle puller, one needle grinder, one microforge, one incubator, two stereomicroscopes, and surgical instruments, all of which are housed in an isolated procedure room in the newly-constructed transgenic facility. The core microinjection laboratory maintains a mouse colony in a holding room adjacent to the procedure room. The colony houses stud and vasectomized males, and females used as embryo donors or pseudopregnant recipients.
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0.922 |
2002 |
Lee, Kuo-Fen |
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. |
Mouse Models of Local Ngf and Acetylcholine Dysfunction @ Salk Institute For Biological Studies
Alzheimer's Disease (AD) is characterized by the presence of intraneuronal tangles and extracellular amyloid plaques, which are accompanied by neurodegeneration and cognitive impairment. The major goal of this Program Project is to develop animal models that are useful not only for the understanding of the molecular and neural mechanisms of neuronal degeneration and impaired learning and memory in AD, but which are also amenable to the development of effective strategies for gene therapy of AD. Project 2 focuses on the roles of nerve growth factor (NGF), the neurotrophin receptor p75, and acetylcholine (ACh) within central cholinergic systems in learning and memory. NGF, p75m, and choline acetyltransferase (ChAT)(the biosynthetic enzyme) for ACh), conditional mutant mice established by the Cre-LoxP system will be injected with viral vectors expressing Cre into specific regions of adult brain to remove these molecules. Furthermore, mutations in the gene encoding the amyloid precursor protein (APP) have been discovered in familial AD patients, implicating APP in the etiology of AD. Several lines of in vitro evidence suggest that neurotrophins and p75 might modify the expressing, processing, or functions of APP or the aggregation of its processed products, including beta-amyloid. Conversely, APP and its processed products may influence neurotrophin signaling. NGF and p75 mutant mice will e crossed with transgenic mice expressing mutated human APP that display neuroanatomical alterations resembling AD. Time-course of neuroanatomical alterations and behavioral deficits will be determined in these compound mice. The aims are: Aim 1. To determine the role of p75, nerve growth factor (NGF) and acetylcholine (ACh) in the survival and function of adult basal forebrain cholinergic neurons (BFCNs). Aim 2. To determine the role of p75 and NGF in the neurotoxicity of beta-amyloid in adult BFCNs.
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0.922 |
2003 — 2007 |
Lee, Kuo-Fen |
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 Regulation of Neuromuscular Synapse Formation @ Salk Institute For Biological Studies
DESCRIPTION (provided by applicant): The major goal of this project is to elucidate the mechanisms by which synapses are formed. Synapses are the cellular basis of neural circuitry and therefore are fundamental to nervous system function. A cardinal feature of the chemical synapse is the presence of a postsynaptic apparatus containing high concentrations of neurotransmitter receptors closely associated with numerous extracellular, transmembrane, and cytoplasmic scaffolding and signaling components. Perhaps best studied in this respect is the aggregation of acetylcholine receptors (AChRs) at postsynaptic sites of the vertebrate skeletal neuromuscular junction (NMJ) along the central band of the muscle. In the last several decades many studies have led to a neurocentric model in which the specialization of this postsynaptic apparatus is orchestrated by three nerve-derived signals that cluster pre-existing AChR molecules, selectively induce AChR gene expression at synaptic sites and suppress AChR gene expression at extra-synaptic sites. Based on this model, agrin, neuregulin (NRG) and acetylcholine (ACh) were identified as candidate molecules to mediate these three cellular activities, respectively. We have used mouse genetics to determine physiological roles of these three pathways. The results revealed that the muscle intrinsically signals for the initiation of postsynaptic differentiation at the central region of the muscle while the nerve and/or accompanying Schwann cells provide both positive and negative signals that promote differentiation and maintain central location and stability of synapses. In the present proposal, we will focus on understanding the role of ACh and agrin signaling pathways and their interplay in both pre-and post-synaptic development. There are three aims. Aim 1 is to determine the role of target-derived trophic factors in NMJ development in the ChAT mutant mice. Aim 2 is to determine the role of agrin in postsynaptic differentiation in the ChAT mutants. Aim 3 is to determine the role of protein phosphorylation regulated by the interplay of ACh and agrin signaling pathways in NMJ development. The results from this study will advance our knowledge on neuromuscular synapse formation and maintenance and will provide crucial information in designing treatments for neuromuscular diseases and spinal cord injury.
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0.922 |
2004 — 2007 |
Lee, Kuo-Fen |
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. |
Regulation of Synaptic Gene Expression in Muscle @ Salk Institute For Biological Studies
DESCRIPTION (provided by applicant): The goal of this project is to elucidate the mechanisms by which neuregulin (NRG-1), acting through heterodimers or homodimers of erbB receptor tyrosine kinases (erbB2, erbB3 and erbB4), regulates synaptic gene expression at neuromuscular junctions (NMJs). Several lines of evidence suggest that NRG-1 signaling pathways play an important role in Schwann cell development and AChR gene expression. NMJs are comprised of precisely aligned nerve terminals, Schwann cells and muscle cells. A cardinal feature of the NMJ is the presence of a postsynaptic apparatus containing high concentrations of acetylcholine receptors (AChRs) closely associated with numerous extracellular, transmembrane, and cytoplasmic scaffolding and signaling components. The AChR complex in embryonic muscle is organized as a pentamer comprised of four distinct subunits, alpha, beta, gamma, and delta, in the stoichiometry alpha2, beta, gamma, delta. The composition of the AChR undergoes a postnatal switch from alpha2, beta, gamma, delta (embryonic form) to alpha2, beta, epsilon, delta (adult form). The switch from gamma to epsilon subunit occurs during the first two postnatal weeks. Regulation of embryonic and adult AChR subunit gene expression has been an excellent model to understand synapse-specific gene expression. In the present proposal, we will determine the physiological role of erbB receptors in synapse-specific AChR gene expression by generating mice deficient in different erbB receptor heterodimers or homodimers during development or after birth. Furthermore, primary muscle cells will be established from these mutant mice to elucidate signal transduction pathways underlying synapse-specific AChR gene expression. The aims are: Aim 1. To determine the role of the neuregulin signaling pathway in AChR gene expression during embryonic development Aim 2. To determine the role of the neuregulin signaling pathway in regulating expression of adult AchRe subunit Aim 3. To determine the role of protein phosphorylation/dephosphorylation in neuregulin-regulated AchR gene expression Elucidating the mechanisms in the control of neuromuscular synapse formation will further our understanding of general principles governing synapse formation and, hence, the basis of nervous system development and function. These results will undoubtedly provide crucial information in designing strategies to treat neuromuscular diseases and spinal cord injury.
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0.922 |
2005 — 2009 |
Lee, Kuo-Fen |
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--Biology @ Salk Institute For Biological Studies
This Program Project focuses on the role of CRF superfamily peptides and their binding proteins, receptors and modulators in the integration of endocrine, autonomic, behavioral and immune responses to stress. The first aim of this Program is to characterize physical interactions between CRF family ligands, their receptors and binding proteins and to define motifs required to activate downstream signaling events. The second aim is to explore the physiologic and pathophysiologic significance of these molecules at the cellular and system levels and to study the control of protein expression and secretion as well as modes of action. Addressing the questions of a complex ligand/receptor system is facilitated by the availability of key scientific tools. This core application is therefore designed to provide transgenic mice, antibodies, and immunoassays necessary to conduct the integrated studies proposed by the individual Projects. Transgenic mice overexpressing components of the CRF system, or null-mutant mice lacking one or more CRFRs and/or ligands, are essential for elucidating the physiologic roles of these ligands and receptors in normal development and in disease states. Detection and/or neutralization of gene products in vitro and in vivo are dependent on high affinity and high liter antibodies of appropriate specificity against CRF superfamily peptides and their soluble binding proteins and membrane bound receptors. Measurement of factors modulated by the CRF receptor-ligand system, including pituitary hormone ACTH and adrenal steroid corticosterone, are also required. This Core can provide transgenic mice, high quality antibodies and assay services that require highly trained personnel and specialized equipment which would otherwise be unavailable to individual projects due to high cost. The establishment of a Core permits reduced costs, improved quality control, efficiency, specialization of technical personnel, standardization of protocols and the dedication of equipment and space. Core B will be under the overall responsibility of Drs. K.-F. Lee (transgenic mice unit, 5% effort) and W. Vale (antibody production and assay services, 3% effort).
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0.922 |
2008 — 2012 |
Lee, Kuo-Fen |
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 Analysis of the Neurotrophin Receptor P75 in Neural Development @ Salk Institute For Biological Studies
DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the role of the neurotrophin receptor p75 in neural development and regeneration. Although initially identified for its ability to bind neurotrophins, p75 serves as a critical co-receptor to transduce multiple signaling pathways in neural development and function. For example, it is a co-receptor for glycosyl-phosphophatidyl inositol (GPI)-linked NgR and ephrinA5. We have been using the dorsal root ganglion (DRG) sensory neuron system to understand the role of p75 in neural development. DRG neurons are classified by multiple criteria including sizes, neuropeptides, trophic factor-dependence and sensory modalities. Sensory neuron development has embryonic and postnatal phases and is controlled by both extrinsic and intrinsic factors. We previously showed that 50% of DRG neurons are lost in p75 null mutants at birth. Because p75 is expressed in neurons, Schwann and target cells involved in the development of DRG sensory system, we generated p75 conditional mutant mice via the Cre-LoxP system (p75 floxed mice) to understand the role of p75 in these individual cell types. To delete the p75 gene in neurons, we crossed p75 floxed mice with mice expressing Cre under the control of the Islet1 promoter. In contrast to p75 null mutants, there is no DRG neuronal loss in these conditional mutants at birth. However, 20% of total DRG neurons are lost in adult mutants, including a significant number of glial cell-derived neurotrophic factor (GDNF)-dependent c-Ret+IB4+ nociceptive neurons. GDNF-dependent survival of postnatal p75-deficient DRG neurons is markedly decreased, suggesting that p75 is required for GDNF signaling. Preliminary results showed that p75 and GPI-linked GDNF receptor 11/12 (GFR11) and c-Ret are co-localized on DRG neurons and form a GDNF- dependent complex. Furthermore, p75 increases sensitivity of GDNF-induced MAPK Erk1/2 activation in transfected HEK 293 cells. To further understand the cellular and molecular mechanisms of p75-dependent signaling pathways in neural development, three specific aims are proposed. Aim 1 is to determine the role of p75 in neurons, epithelial and Schwann cells during embryonic spinal sensory neuron development. Aim 2 is to determine the role of p75 in nonpeptidergic neuron development. Aim 3 is to determine the role of p75 in GDNF signaling. PUBLIC HEALTH RELEVANCE: P75 may serve as a co-receptor to integrate multiple signaling pathways to regulate diverse cellular activities and is implicated in multiple human diseases in both peripheral and central nervous systems. The results from the present study will provide insights into designing therapeutic strategies to treat neurodegenerative diseases and peripheral neuropathy, including debilitating Alzheimer's Disease and chronic pain.
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0.922 |
2011 — 2015 |
Lee, Kuo-Fen |
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. |
Project 1 - Salk Institute For Biological Studies Ninds Center Core Grant @ Salk Institute For Biological Studies
Individual NINDS grantees have strived to employ and develop cutting-edge genetic tools to manipulate the mouse genome to test specific hypotheses proposed in their NINDS-funded research projects. Producing many of these lines has required multiple steps in generating mouse ES cells and these lines are often laborious or difficult to obtain. The cost and time for generating such ES lines is a major bottleneck for successful animal model development as it prohibits many NINDS funded investigators from generating genetically-engineering mice, even for labs that are versed in the required advanced and very specific technologies. A central genome modification core will provide services to break the barriers to generate genetically modified mouse lines by supporting the production of designer ES-cell lines. A centralized GM Core will increase efficiency and lower the cost of generating such lines. In addition, a central service will provide opportunities for synergistic development of lines that will be of value across all user groups. The Institute has made a commitment to provide space for establishment of the GM core that would lead the way to meet the evolving needs of Neuroscience Center investigators. The major goal of the GM Core is to provide services for manipulation ofthe mouse genome in ES cells that are essential for studies ofthe mechanisms of neural function, structure and development as well as the generation of both in vitro and in vivo mouse models of diseases.
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0.922 |
2012 |
Gage, Fred H (co-PI) [⬀] Lee, Kuo-Fen |
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. |
Exploring Human-Induced Pluripotent Stem Cells For Alzheimer's Disease Research @ Salk Institute For Biological Studies
DESCRIPTION (provided by applicant) Alzheimer's disease (AD) is a neurodegenerative disease characterized by the deposition of amyloid plaque in the extracellular space, formation of neurofibrillary tangles in neurons, and extensive neuronal loss. Growing evidence has shown that increased beta amyloid (A¿) peptide accumulation, aggregation, and deposition in the brain are central to the pathogenesis of AD (1). Recently, the advent of methodologies to reprogram fully differentiated tissues, such as skin, into human-induced pluripotent stem cells (hiPSCs) has made it possible to study AD in a human physiological context. Cell-based human models for AD will be created by directly reprogramming skin fibroblasts from AD patients and controls into hiPSCs. This proposal will focus on reprogrammming skin fibroblasts from sporadic AD patients containing two copies of the ¿4 allele of the apolipoprotein-E (APOE) gene and control subjects containing two copies of the APOE ¿3 allele. Previously established hiPSC lines derived from familial AD patients with mutations in the amyloid precusor proteins (APP) and preselinin (PS) genes will be obtained and differentiated into neuronal cells, including cholinergic neurons. There are three primary aims for this R21 proposal. In Aim 1, fibroblasts from AD patients carrying the APOE ¿4 allele and controls carrying the APOE ¿3allele will be reprogrammed into hiPSCs and then differentiated into neurons. Several parameters associated with AD development such as A¿ expression level, A¿ fibrilgenesis, neuroinflammation, synapse formation and function will be analyzed at different timepoints after neuronal differentiation. Thi will illustrate whether hiPSC-derived neurons can recapitulate the disease progression of AD in vitro. In Aim 2, A¿ 1-42 peptide will be used to treat the hiPSC-derived neurons at low concentrations resembling in vivo A¿ concentration and at high concentrations that induce neuronal toxicity. Various indicators for neuroinflammation, neuronal degeneration and cell death will be measured to examine whether hiPSCs-derived neurons carrying the APOE ¿4 allele react differently to A¿ treatments compared to those carrying the APOE ¿3 allele. In Aim 3, gene expression profiles of hiPSC-derived neurons will be generated using gene expression microarrays. Genes with altered expression in familial AD patients or patients with the APOE ¿4 allele will be cross-referenced with a list of genes modulating A¿ neurotoxicty that we have generated through a genome-wide shRNA library screen. Overlapping genes of interest will be further studied using lentiviral overexpression or shRNA-mediated repression in hiPSC-derived neurons. Studies of hiPSC-derived neurons from AD patients carrying the APOE ¿4 allele have the potential to significantly advance our understanding of the complex mechanisms underlying APOE ¿4-dependent increase of the risk for AD. Results from the proposed studies will likely lead to development of hiPSC-derived neurons-based assays for screening genes or compounds that lead to potential new drug therapies for the treatment of AD. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to create human cell-based models for Alzheimer's disease (AD) by reprogramming skin samples from sporadic AD patients with two copies of the APOE ¿4 allele into human induced pluripotent stem cells (hiPSCs). By differentiating these disease-specific hiPSCs into neurons, we will investigate the underlying mechanisms by which the apoE4 isoform mediates increased risk for AD. Ultimately, we aim to develop genetic or chemical treatments that disrupt molecular pathways that lead to AD.
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0.922 |
2012 — 2014 |
Lee, Kuo-Fen Vale, Wylie W. (co-PI) [⬀] |
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. |
Biology Core @ Salk Institute For Biological Studies
PROJECT SUMMARY (See instructions): This Program Project focuses on the role of CRF superfamily peptides and their binding proteins, receptors and modulators in the integration of endocrine, autonomic, behavioral and immune responses to stress. The first aim of this Program is to characterize physical interacfions between CRF family ligands, their receptors and binding proteins and to define motifs required to activate downstream signaling events. The second aim is to explore the physiologic and pathophysiologic significance of these molecules at the cellular and system levels and to study the control of protein expression and secretion as well as modes of action. Addressing questions of a complex ligand/receptor system is facilitated by the availability of key scientific tools. This core application is therefore designed to provide transgenic mice, antibodies, and immunoassays necessary to conduct the integrated studies proposed by the individual Projects. Transgenic mice over expressing components of the CRF system, or null-mutant mice lacking one or more CRFRs and/or ligands, are essential for elucidating the physiologic roles of these ligand and receptors in normal development and in disease states. Detection and/or neutralization of gene products in vitro and in vivo are dependent on high affinity and high titer antibodies of appropriate specificity against CRF family peptides and their soluble binding proteins and membrane bound receptors. Measurement of factors modulated by the CRF receptor-ligand system, including the pituitary hormone ACTH, the adrenal steroid corticosterone, and the pancreatic hormones insulin and glucagon, are also required. This Core can provide transgenic mice, high quality antibodies and assay services that require highly trained personnel and specialized equipment which would otherwise be unavailable to individual projects due to high cost. The establishment of a Core permits reduced costs, improved quality control, efficiency, specialization of technical personnel, standardization of protocols and the dedication of equipment and space. Core B is jointly directed by Drs. K.-F. Lee (transgenic mice unit, 5% effort) and W. Vale (antibody production and assay services, 3% effort).
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0.922 |
2014 — 2015 |
Jin, Xin (co-PI) [⬀] Lee, Kuo-Fen |
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. |
Optogenetic Dissection of Brain Network Deficits in Alzheimer's Disease @ Salk Institute For Biological Studies
DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive loss of memory and other cognitive functions. Several lines of evidence suggest that neural network impairment leads to cognitive and behavioral deficits in AD, but the underlying cellular and molecular mechanisms are not completely understood. Several neurotransmitter systems are impaired in AD brain, in particular cholinergic neurons. However, drugs, such as acetylcholine esterase inhibitors, that are developed to target individual neurotransmitter systems have met with limited success. As a result, it has been suggested that prolonged and artificially elevated ambient levels of neurotransmitters may interfere with phasic synaptic signaling and lead to aberrant tonic activation of extrasynaptic receptors. Thus, elucidation of systems mechanisms may provide insights into novel strategies to develop more effective treatments for improving cognitive function, including better information on the long-range circuits and local microcircuits underlying cognitive function and better understanding of the roles and molecular basis of phasic and tonic modes of synaptic transmission elicited by individual neurotransmitters and their receptors. The advent of optogenetics coupled with large-scale in vivo recording of freely moving animals performing cognitive tasks provides an excellent opportunity to map functional circuitry and to determine the effect of different synaptic activation patterns to ameliorate cognitive deficits in AD. The prefrontal cortex (PFC) interacts with multiple cortical and subcortical structures and plays an important role in working memory, long-term memory consolidation and execution of actions. As neuronal loss in the nucleus basalis of Meynert (NBM), a component of the basal forebrain that provides long-range projections to the cortex, is a prominent feature in AD brain, we would like to focus on elucidating the cellular and molecular mechanisms of the NBM-PFC network activity underpinning cognitive deficits in AD. Toward this goal, we have provided evidence that APP41 line of AD mice, which express high levels of Amyloid peptide ß (Aß) in the cortex at 3 months of age, displays reduced cholinergic and GABAergic markers, cognitive deficits, altered theta and gamma oscillations and increased epileptic discharge. Furthermore, a neuregulin 1 (NRG1) mutation is associated with late-onset familial AD in patients with psychosis. Our preliminary results show that NRG1 improves cognitive deficits in APP41 mice, forms a complex with muscarinic acetylcholine receptor M2 and is required for ACh-induced neuronal excitability. Neuronal oscillations in the PFC are impaired in mice lacking NRG1 in the cholinergic neurons. To further elucidate the molecular and cellular mechanisms, three aims are proposed in the present application. Aim 1 is to characterize the network activity in the PFC during cognitive tasks in AD mice. Aim 2 is to determine the cholinergic and GABAergic contribution to network pathophysiology and behavior in AD mice. Aim 3 is to determine the role of NRG1 in the development of the NBM-PFC circuit, network activity and cognitive function in AD mice.
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0.922 |
2017 — 2020 |
Lee, Kuo-Fen Reynolds, John H (co-PI) [⬀] |
R24Activity Code Description: Undocumented code - click on the grant title for more information. |
Development of Marmoset Models of Neurodegenerative Disease Using Embryonic Stem Cell-Based Gene-Editing Approaches @ Salk Institute For Biological Studies
PROJECT SUMMARY The long-term goal of this project is to model human neurodegenerative diseases in marmosets via gene- editing in embryonic stem cells (ESCs). The mouse system is a powerful tool for medical research due to the ability to manipulate the mouse genome. However, considerable anatomical, physiological, cognitive, and behavioral differences between mice and humans limit the degree to which insights from mouse models shed light on human diseases. This is reflected in the high number of failed clinical trails for drugs that were effective in treating mouse models of human disease. Several lines of evidence suggest that the marmoset represents an improved animal system for studying a range of human diseases, including stroke and age-associated neurodegenerative diseases such as Alzheimer's disease (AD). Marmosets are the shortest-lived of the anthropoid primates (average lifespan of 5?7 years compared with 25 years for the rhesus macaque) and exhibit age-related changes that are similar to those seen in humans, including ?-amyloid deposition in the cerebral cortex, loss of cholinergic innervation, and reduced neurogenesis, as observed in AD. In addition, marmosets are highly social and communicative and have demonstrated the capacity to learn sophisticated cognitive behaviors. Therefore, marmosets represent an ideal genetic platform for generating models of neurodegenerative diseases that more accurately reflect the human condition and enable the testing of potential autologous (the-same-species) stem cell-based regenerative therapies. Initial efforts will focus on generating a marmoset model of AD. The recent emergence of gene-editing and stem-cell technologies in primates pave the way toward generating marmoset disease models, but improvements in both areas are necessary to make this approach viable. Here, both conventional homologous recombination and CRISPR/Cas9 genome-editing technologies will be employed to modify marmoset ESCs. As genetic evidence demonstrates that mutations in the amyloid precursor protein (APP) gene result in increased ?-amyloid production, the formation of plaques, and cognitive impairment, the marmoset APP will be edited to carry human point mutations. Genetic tools for studying neuronal cell type-specific circuits underlying cognitive impairment and neuropathology in AD will also be generated by inserting a Cre recombinase cassette into 3' end non-translated regions of the parvalbumin and choline acetyltransferase genes. These Cre driver lines will enable the visualization and functional manipulation of these cell types. Successful completion of the proposed Aims will generate a greatly improved animal model of AD, enable testing of stem cell-based regenerative methods for treating AD, and pave the way toward applying these genetic tools for analyzing neuronal circuitry of healthy brains. Establishing gene-editing in marmoset ESCs will also enable the development of additional primate models of human diseases, providing critical experimental resources for research supported by multiple NIH Institutes (e.g., NINDS, NIA, NIMH, NEI, NIAAA, NIDA, NICHG, NIGMS).
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0.922 |
2019 |
Lee, Kuo-Fen Zador, Anthony M |
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. |
High-Throughput Mapping of Selectively Vulnerable Cell Types and Projections in Aging and Alzheimer's Disease @ Salk Institute For Biological Studies
Abstract The long-term goal of this application is to use a cost effective high-throughput approach to identify cell types and map projections that are selectively vulnerable in the progression of aging and Alzheimer's Disease (AD). It is to test the hypothesis that there are concurrent or sequential vulnerabilities in neuroanatomical and protein-interacting network/signaling pathways at critical stages in the progression of aging and AD. A large body of evidence demonstrates that AD is a heterogeneous, multifactorial disease that selectively affects certain regions of the brain, e.g. the entorhinal cortex (EC), while other areas, such as the cerebellum, remain unaffected. Recent studies on the staging of AD neuropathology showed AD-related neuropathology begins in the locus coeruleus (LC) or the EC, followed by the hippocampus (HC) and then the prefrontal cortex (PFC). But cell types, their associated projections and molecular/signaling pathways that are selectively vulnerable at the single neurons level are not well understood. Aging is a major risk factor for AD. Thus, it is important to understand whether there are distinct, similar or overlapping selective vulnerabilities in the progression between aging and AD. Neuronal projections have only been systematically mapped using bulk labeling techniques, e.g., dye tracers, which obscure the diverse projections of intermingled single neurons. This bulk labeling approach to obtain whole brain connectomes requires a large number of animals and is low throughput, labor intensive and expensive. Recently, the MAPseq (Multiplexed Analysis of Projections by Sequencing) approach from one or multiple brain nuclei/sources has been developed to map the projections of thousands or even millions of single neurons by labeling large sets of neurons with random RNA sequences (``barcodes'') in a single brain. Axons are filled with barcode mRNA, each putative projection area is dissected, and the barcode mRNA is extracted and sequenced. Furthermore, each barcoded neuron is also labeled with GFP, enabling fluorescence-activated cell sorting of individual neurons for single cell (nc) RNAseq to obtain transcriptomic information that allows cell type classification and the analysis of biochemical/signaling pathways. A longitudinal study with this MAPseq coupled with scRNAseq will provide unprecedented data informing the cause for the initiation of or contribution to the exacerbation of aging and AD. The multiple source MAPseq coupled with nc-RNAseq will be employed to map the projections and to identify cell types in four brain regions, namely the LC, EC, HC and PFC. The LC provides the major noradrenergic inputs throughout the entire brain. The EC provides key cortical inputs to the HC, which is essential in learning memory. The PFC provides the top-down regulation on various higher order functions, including learning and memory. Both male and female wild-type mice and the APPNLF line of AD mouse, which carries knockin human Swedish and Iberia mutations in the amyloid precursor protein and, importantly, expresses physiological levels of A? and exhibited age-related neuropathology and cognitive impairment, mimicking late onset AD, will be used.
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0.922 |
2019 — 2021 |
Lee, Kuo-Fen |
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. |
Shared Resource-Transgenic Core @ Salk Institute For Biological Studies
Transgenic Core Shared Resource - Project Summary/Abstract The primary goal of the Transgenic Core is to provide Cancer Center members and other investigators at the Salk Institute access to cutting-edge technologies for creating genetically altered mouse models. These transgenic lines of mice represent critical genetic tools that enable researchers to model human diseases, to analyze and functionally manipulate specific genes, and to test potential therapeutic interventions. To achieve this goal, the Transgenic Core provides state-of-the genome editing services, as well as services required to generate and maintain transgenic mice. Specific services include: 1) the microinjection of DNA constructs into early mouse embryos, 2) CRISPR/Cas9 microinjections, 3) lentiviral microinjections, 4) microinjection of gene- targeted mouse embryonic stem (ES) cells into blastocysts, 5) in-vitro fertilization (IVF), 6) embryo and sperm cryopreservation, 7) re-derivation of mouse lines provided to the Institute from non-standard sources, 8) gene- targeting in mouse ES cells, 9) targeting vector design and construction, 10) de novo derivation of ES cell lines, 11) ES cell expansion and preparation for microinjection, and 12) the generation and maintenance of DR4 and CF1 mouse embryonic feeder cells. In addition, the Core offers services for the injection of human embryonic stem cell lines and induced pluripotent stem (iPS) cell lines into immunodeficient mice to assess their ability to form teratomas. Recently the Transgenic Core has expanded services to offer: Southern blot services for confirmation of gene targeting in modified ES cells, PCR-based genotyping of transgenic mice, and mycoplasma testing. Thus, the Transgenic Core seeks to facilitate research of Cancer Center members by generating genetically modified mouse models of cancer, cryopreserving mouse lines that are not currently in use, re-deriving mouse lines provided by non-standard sources, generatimg homologous recombination-based gene targeted mouse ES cell lines, providing free consultations to match current transgenic technologies to investigator needs, and providing free training in all procedures involved in producing transgenic animals.
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0.922 |
2021 |
Lee, Kuo-Fen |
R24Activity Code Description: Undocumented code - click on the grant title for more information. |
Genetic Models of Sporadic Alzheimers Disease in the Marmoset @ Salk Institute For Biological Studies
Abstract The long-term goal of this project is to develop, characterize, and validate genetically modified marmoset models of sporadic Alzheimer's Disease (AD) that will serve as tools for investigating molecular and cellular disease mechanisms, and for identifying therapeutic targets. AD is the most common cause of dementia, with the majority of cases (~95%) appearing to be sporadic, which is caused by complex interactions between multiple gene variants and environmental factors. Numerous models of AD have been developed (e.g., mice); these models are primarily focused on familial forms of AD and have enabled significant progress toward understanding AD, but they fail to recapitulate the full spectrum of molecular, cellular, behavioral, and cognitive pathologies observed in AD and provide poor predictive value when trying to translate findings to human clinical trials. Several lines of evidence suggest that marmosets may effectively bridge the gap between mice and humans for both basic and translational neuroscience research. First, marmosets and humans have very similar brain structures, cognitive/social behavioral repertoires, metabolism, and immune functions. Second, compared to other primates, marmosets have short lifespan, small body size, and high reproductive power. Finally, gene editing tools are now available to generate various types of genetically modified marmosets. Apolipoprotein E (APOE) is the strongest risk factor for late-onset, sporadic AD and also increases the age- dependent risk of monogenic familial AD and incidence of AD in women. There are three APOE alleles in humans with the APOE*?4 allele conferring increased risk and the APOE*?2 allele conferring decreased risk relative to the common APOE*?3 allele. APOE isoforms differentially modulate both amyloid-? (A?)-dependent and A?-independent pathways to affect brain homeostasis, including tau-mediated neurodegeneration, microglial inflammation, lipid transport, synaptic integrity, glucose metabolism and cerebrovascular function. These three APOE alleles differ with regard to cysteine (C) and arginine (R) amino acids at positions 112 and 158 (C112/C158 in APOE2; C112/R158 in APOE3; and R112/R158 in APOE4). Several lines of evidence demonstrate that R61 is critical for APOE4-mediated AD risk. Marmoset APOE (mAPOE) is encoded by a single allele and contains the equivalent of R112 and R158, but lacks the critical R61 equivalent (it contains T instead), suggesting that it behaves like human APOE3. To test this hypothesis, mAPOE T61R mutant protein was generated to test the effect on inflammatory responses induced by lipopolysaccharides in microglial cells. The mAPOE T61R variant resulted in a more robust response compared to wild type mAPOE. Similar results were found when human APOE4 was used (APOE4>APOE3). Taken together, these preliminary results indicate that mAPOE T61R is functionally equivalent to human APOE4. Further, several pairs of CRISPR gRNAs for generating an APOE null mutation have been identified. To investigate the role of APOE in sporadic AD in the marmoset, APOE null and T61R mutant marmosets will be generated and characterized.
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0.922 |
2021 |
Lee, Kuo-Fen Miller, Cory T (co-PI) [⬀] |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Marmoset Bioscience Meeting @ Salk Institute For Biological Studies
Abstract This application is a request for funds to support the 3rd annual Marmoset Bioscience Symposium (MBS) to be held November 11, two days before the start of the 2021 Society for Neuroscience (SFN) meeting in Chicago, IL. This meeting will be registered as an SFN satellite symposium. The common marmoset (Callithrix jacchus) has experienced unprecedented growth in research across the United States and is rapidly emerging as a likely keystone biomedical model system in the next chapter of scientific discovery. The major goal of the meeting is part of a multiple-pronged approach to establish a U.S.-based consortium aimed at highlighting cutting-edge research in marmosets and promoting marmosets as a key model system. This meeting will provide a networking forum for both established investigators and junior scientists from diverse backgrounds to interact and communicate their research findings. Critical to the success of the marmoset model is fostering the development of junior scientists in the field. This meeting is also aimed at attracting investigators who are currently not using marmosets in their research. The principal objectives of the MBS include: (1) to communicate and disseminate new findings from using marmosets as a model organism in diverse fields and development of new genetic, viral, and analytic tools that will advance research in marmosets; (2) to provide an open forum for discussion of new hypotheses and approaches and discrepancies in the literature and to facilitate the exchange of reagents such as antibodies, protocols, and viral toolbox; (3) to increase interactions and collaborations between basic scientists and translational and clinical groups interested in modeling human disease in the marmoset and (4) to provide an atmosphere in which researchers wishing to use marmosets in their research may interact directly with established investigators. These MBSs are highly relevant to the programmatic mission of multiple NIH ICs. The meetings consist of a Keynote Lecture, Young Investigator talks, invited talks from trainees and young investigators that are selected from submitted abstracts, and poster sessions. These meetings offer a unique combination of features, including (1) breadth of research, (2) cutting-edge questions and technologies, (3) mingling of investigators from all ranks and diverse sub-fields and locales, and (4) intimate size and extended discussion time, allowing for sustained interactions. Three tentative themes are planned: (1) Social behavior, physiology, and neural circuits, (2) Aging and neurodegenerative disease, and (3) Genetic, viral, and analytic tools developed for marmoset research. Finally, participation by women and those from underrepresented groups will be emphasized. Half of the oral presentations will be from these groups and half of the travel awards will be given to women. The leadership of the MBS itself is ~43% female.
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0.922 |