Stuart Feinstein - US grants

Understanding the molecular details of neuronal development and regeneration are fundamental issues for both basic and clinical neurobiology. The goal of this project is to understand the molecular mechanisms regulating the spatial and temporal pattern of cytoskeletal rearrangements and their roles in promoting and maintaining neuronal development. The specific aim is to determine the in vivo molecular functions and regulation of microtubule associated proteins (MAPs) during nerve growth factor (NGF) induced microtubule assembly and neurite outgrowth. Our understanding of the role of MAPs in microtubule assembly is derived almost exclusively from in vitro studies with purified microtubule proteins. In order to ascribe a biological relevance to these in vitro studies, a direct demonstration of MAP function in vivo is required. Unfortunately, no direct in vivo experiments have been possible to date. This application aims to use the novel tools and reagents of molecular biology to overcome existing obstacles and directly assess the in vivo roles of MAPs during microtubule assemble and neurite outgrowth. The experiments will examine the role of MAPs during NGF induced neurite outgrowth of PC12 rat pheochromocytoma cells. They will directly assess the recently proposed model in which the microtubule associated proteins MAP1 and tau are limiting during in vivo microtubule assembly and neurite outgrowth in this system and are therefore key regulatory elements controlling both these events. The experimental program involves isolation of a MAP1 cDNA clone using recombinant DNA technology, which will then become the crucial reagent allowing functional and regulatory studies directly testing the above model.

Understanding the molecular details of neuronal development and regeneration are fundamental issues for both basic and clinical neurobiology. The goal of this project is to understand the molecular mechanisms regulating the spatial and temporal pattern of cytoskeletal rearrangements and their roles in promoting and maintaining neuronal development. The specific aim is to determine the in vivo molecular functions and regulation of microtubule associated proteins (MAPs) during nerve growth factor (NGF) induced microtubule assembly and neurite outgrowth. Our understanding of the role of MAPs in microtubule assembly is derived almost exclusively from in vitro studies with purified microtubule proteins. In order to ascribe a biological relevance to these in vitro studies, a direct demonstration of MAP function in vivo is required. Unfortunately, no direct in vivo experiments have been possible to date. This application aims to use the novel tools and reagents of molecular biology to overcome existing obstacles and directly assess the in vivo roles of MAPs during microtubule assemble and neurite outgrowth. The experiments will examine the role of MAPs during NGF induced neurite outgrowth of PC12 rat pheochromocytoma cells. They will directly assess the recently proposed model in which the microtubule associated proteins MAP1 and tau are limiting during in vivo microtubule assembly and neurite outgrowth in this system and are therefore key regulatory elements controlling both these events. The experimental program involves isolation of a MAP1 cDNA clone using recombinant DNA technology, which will then become the crucial reagent allowing functional and regulatory studies directly testing the above model.

Students of biology must learn the theoretical basis of molecular biology and also acquire practical experience with the methods used in this critically important and rapidly expanding field. This project will establish an instructional unit in molecular biology in the Introductory Biology Laboratory Curriculum at the University of California at Santa Barbara. This unit is composed of two three-hour laboratory exercises, in which students will explore extraction of DNA from intact cells, restriction endonuclease digestion, the electrophoretic separation of DNA, and analysis of DNA fragment size, and a one-hour computer-assisted restriction mapping exercise.

9318142 Christoffersen The goal of this proposal is to purchase a modem computer- controlled fermentor that will be used to express a wide variety of proteins in microbial cells. The various projects that will utilize this facility reflect the breadth of interests present on the UCSB campus. The areas represented include proteins involved in neural growth factor receptor, DNA methylation, interferon regulation of cellular activities, DNA binding domains, plant hormone biosynthesis, plant secondary metabolism, the cellular cytoskeleton, egg sperm receptor and others. The common need to produce large amounts of protein for further biochemical or biophysical studies brings together these diverse investigators on this single proposal. The need for this instrumentation is due to advances in a variety of disciplines but most importantly is the development highly efficient expression vectors for E. coli or yeast cells. These simple, easy to growth organisms are extremely versatile in their ability or produce a variety of proteins under the right experimental conditions. Optimal expression for large scale experiments requires careful control of temperature, dissolved oxygen and agitation. The proposed fermentor would allow complete control of culture conditions and thus facilitate the recovery of milligram amounts of these various proteins.

The purpose of this collaborative proposal is to examine the cellular and molecular mechanisms of action of neurotrophic factors in the mammalian retina. In this proposed set of experiments, we are specifically focusing on brain derived neurotrophic factor (BDNF) and its receptor molecules (trkBs). Photoreceptor outer segment degeneration is one of the main cellular events that occurs when the neural retina is separated from the underlying retinal pigment epithelium (retina detachment). In preliminary experiments, we have determined that the administration of BDNF rescues photoreceptors from certain degeneration following detachment and promotes the regrowth of their outer segments in the absence of reattachment to the retinal pigment epithelium. The overall goals of this proposal are to determine the molecular and cellular mechanisms mediating BDNF's effect. The project will use the tools of molecular and cellular biology to accomplish the following specific aims: l) To identify the repertoire of BDNF receptors (isoforms of trkB) expressed in the retina; 2) To determine the expression profile of these trkB isoforms in normal, detached, BDNF treated non-detached retinas, and BDNF treated detached retinas.; and 3) To begin to assess the functional capabilities of the various trkB isoforms. Because retinal detachment, and other conditions in which photoreceptor outer segments degenerate, are a major cause of visual disability, any advancement in knowledge that may result in either a direct therapy or provide mechanisms for the molecular basis of retinal degeneration are of medical significance. Additionally, this project will add significantly to our understanding of the role of this important family of neurotrophins in the development and maintenance of the normal retina.

DESCRIPTION: (adapted from applicant's abstract) The long term goal of this work is to elucidate the underlying molecular mechanisms leading to the development of neurofibrillary tangles (NFTs), a hallmark pathological feature of Alzheimer's disease. The investigators will focus on the microtubule associated protein, tau, known to be the major molecular component of NFTs. Whereas normal tau is capable of interacting directly with microtubules and regulating their dynamics and stability, the abnormal tau found in NFTs is unable to bind microtubules concomitant with altered structural and functional properties. This project is designed to determine the molecular and structural features of normal tau action, which will be critical information in the quest to understand abnormal tau behavior. The applicants' strategy will be to integrate two laboratories with expertise in two extremely complementary approaches - molecular biology and Xray crystallography. The goals are to identify critical domains of tau and tubulin involved in their binding interaction, to determine the mechanistic capabilities of each microtubule binding domain of tau, and to determine the three dimensional structure of tau functional domains, alone and in association with their cognate tubulin sites. Taken together, achieving these goals will represent a major step forward in gaining a detailed mechanistic understanding of normal and abnormal tau action and a structure based understanding of the biochemistry of NFTs, a critical prerequisite for the development of rational therapies to address the problem of NFT formation.

DESCRIPTION: (adapted from applicant's abstract) The long term goal of this work is to elucidate the underlying molecular mechanisms leading to the development of neurofibrillary tangles (NFTs), a hallmark pathological feature of Alzheimer's disease. The investigators will focus on the microtubule associated protein, tau, known to be the major molecular component of NFTs. Whereas normal tau is capable of interacting directly with microtubules and regulating their dynamics and stability, the abnormal tau found in NFTs is unable to bind microtubules concomitant with altered structural and functional properties. This project is designed to determine the molecular and structural features of normal tau action, which will be critical information in the quest to understand abnormal tau behavior. The applicants' strategy will be to integrate two laboratories with expertise in two extremely complementary approaches - molecular biology and Xray crystallography. The goals are to identify critical domains of tau and tubulin involved in their binding interaction, to determine the mechanistic capabilities of each microtubule binding domain of tau, and to determine the three dimensional structure of tau functional domains, alone and in association with their cognate tubulin sites. Taken together, achieving these goals will represent a major step forward in gaining a detailed mechanistic understanding of normal and abnormal tau action and a structure based understanding of the biochemistry of NFTs, a critical prerequisite for the development of rational therapies to address the problem of NFT formation.

DESCRIPTION (provided by applicant): The microtubule associated protein tau is essential for development and maintenance of the nervous system. On the other hand, tau dysfunction has long been correlated with Alzheimer?s disease pathology. Further, recent genetic evidence demonstrates that mutations affecting either the primary structure of tau or the regulation of tau RNA alternative splicing lead to pathological tau fiber formation similar to that observed in Alzheimer?s disease, neuronal cell death and FTDP-17, a collection of human dementias. Gaining a thorough understanding of tau action is therefore an important goal. Unfortunately, our understanding of tau action is derived primarily from in vitro biochemical studies. It is essential that conclusions drawn from these in vitro studies be assessed and extended in cellular systems. The problem is that all currently available systems to study tau action in cells suffer from major inherent pragmatic or technical limitations. Here, we propose to adapt a cell system with a long and productive history in cell and developmental biology, the echinoderm egg, to overcome many of these limitations. The principle of the assay is that tau, microinjected into eggs prior to fertilization, will inhibit normal fertilization induced microtubule dependent events (such as cell division) in a dose dependent manner, but not microtubule independent events. This assay is rapid, flexible and quantitative - this combination of critical features is not available in any other cellular system to study tau action upon microtubules. For simplicity, we refer to this assay as the Inhibition of Early Development Bioassay, or "IEDB."Using this system, we will (i) test the predictions of in vitro mechanistic studies of tau structure-function and (ii) investigate molecular mechanisms of tau action.

DESCRIPTION (Provided by applicant): The microtubule associated protein tau is essential for development and maintenance of the nervous system. On the other hand, tau dysfunction has long been correlated with Alzheimer's disease pathology. Further, recent genetic evidence demonstrates that mutations affecting either the primary structure of tau or the regulation of tau RNA alternative splicing lead to pathological tau fiber formation similar to that observed in Alzheimer's disease, neuronal cell death and FTDP-17, a collection of human dementias. Gaining a thorough understanding of tau action is therefore an important goal. At a mechanistic level, tau serves its many functions by regulating microtubule dynamics, that is, the growth, shortening and movement of microtubules. This application examines the manner in which tau affects microtubule dynamics under both normal and pathological conditions. These investigations will define the molecular mechanisms by which normal tau regulates microtubule dynamics, and then test the hypothesis that mutations in the tau gene that are linked to neuronal cell death and neurodegenerative disease alter the quantitative and/or qualitative manner by which tau regulates microtubule dynamics. In addition, we will begin to test the hypothesis that modifications in taus ability to regulate microtubule dynamics resulting from the structural and regulatory tau mutations can contribute to neuronal degeneration.

Technical Abstract

The development project is aimed at advancing the design technology for ultra-high-resolution small angle x-ray scattering (SAXS) instrumentation and x-ray optics with broad applications in nanoscale structure characterization. A new optical design concept that can significantly enhance (up to 10-fold) the resolution and signal-to-noise ratio in SAXS measurements will be developed. The superior performance results from a compound optical configuration incorporating both a primary monochromator mirror and a pre-sample secondary focusing mirror to increase resolution and peak intensity. This unique design is expected to supplant the prevalent three-pinhole configuration in current SAXS instruments. The scientific and engineering problems of interest cover a range of multidisciplinary fields including soft condensed matter, biological physics and bioengineering, neuroscience, and bioinorganic chemistry. The instrument will operate out of the x-ray facility of the Materials Research Laboratory, with an established user base of more than 250 researchers from campus, other institutions and local industry. The development should broadly impact the national academic research infrastructure by advancing the optical design and performance of SAXS instrumentation used in diverse research areas in nanoscience and nanotechnology. The investigators of this project will continue their involvement in their numerous outreach programs available at UCSB to improve access to science for diverse groups, including undergraduate and graduate student training, outreach to K-12 students and teachers, and community outreach.

Lay Abstract

Enhancing structure characterization of nanomaterials is critical to the emerging areas of nanoscience, nanotechnology, and biotechnology. We will develop a laboratory-based microbeam x-ray scattering instrument for discovering the structures of novel new materials on the 1 nanometer to 1000 nanometer scale. The performance of this new instrument will exceed the best commercially available x-ray instrument and will provide a significant enhancement to the campus infrastructure in nanoscience and technology. The development of this cutting edge x-ray characterization tool will establish UCSB as one of strongest institutions in the x-ray characterization area, which will attract users not only from multiple campus groups but also from other research institutions as well. The project provides an excellent training opportunity to graduate students and postdoctoral researchers who will not only participate in building the cutting-edge x-ray tool but also be able to utilize it in a series of research projects which will take advantage of the new capability afforded by the instrument. The x-ray development program will be integrated with the ongoing outreach activities of the principle investigators in mentoring undergraduate students and local high school teachers participating in the large number of ongoing summer internship programs at UCSB.

[unreadable] DESCRIPTION (provided by applicant): The microtubule associated protein tau is essential for the development and maintenance of the nervous system. On the other hand, tau hyper-phosphorylation and dysfunction have long been correlated with Alzheimer's disease and a number of related dementias. Recent genetic evidence extended these correlations, demonstrating that mutations affecting either the primary structure of tau or the regulation of tau RNA alternative splicing can cause FTDP-17, a collection of neurodegenerative disorders with many similarities to Alzheimer's disease, including abnormal tau fiber formation, neuronal cell death and dementia. Additional biochemical and genetic data support the conclusion that pathological tau action is likely a key component in Alzheimer's, FTDP-17 and additional "tauopathies". Therefore, gaining a thorough understanding of normal and pathological tau action is of fundamental importance. Mechanistically, tau serves at least many of its functions by regulating the growth and shortening dynamics of microtubules. Since proper regulation of microtubule dynamics is well-established to be essential for cell function and viability, neurons exert tight control over tau action. Indeed, tau activity is regulated by both alternative splicing of tau RNA and phosphorylation mechanisms. In the previous cycle of this project, we performed a detailed mechanistic analysis of the abilities of normal and FTDP-17 mutant tau isoforms to regulate microtubule dynamics, leading us to propose a mis-regulation of microtubule dynamics model in which properly regulated tau maintains MT dynamics within a defined range of tolerable activities whereas errors in tau action lead to inappropriate regulation of MT dynamics, which in turn leads to neuronal cell death and dementia. Here, we propose to superimpose phosphorylation mediated effects upon tau's ability to regulate microtubule dynamics in order to test the hypothesis that biologically relevant but inappropriate phosphorylation of tau compromises its ability to properly regulate microtubule dynamics, thereby promoting cell death. We will also test the hypothesis that inappropriate phosphorylation of tau exacerbates the FTDP- 17 mutation induced inability of tau to properly regulate microtubule dynamics, again promoting neuronal cell death. We will focus our efforts upon CDK/p25, GSK3B and MARK, three kinases with well-established in vivo relevance for tau as well as Pin1, a prolyl isomerase involved in regulating tau phosphorylation and strongly implicated in neurodegeneration. Briefly summarized, this proposal seeks to better understand the precise details underlying neuronal cell death leading to dementia. Achieving this goal will provide excellent potential targets for the development of rationally designed anti-dementia therapeutics. [unreadable] [unreadable]

An award is made to the University of California, Santa Barbara (UCSB) to acquire a high-resolution, fast, laser-scanning confocal microscope. This award will allow critical problems in biophysics, neuroscience, and bioengineering to be addressed, while substantially increasing the research and training capacity of the campus. In particular, this new instrumentation will provide scientists at UCSB with unprecedented abilities to measure the dynamic motions of microscale structures in biological and bioinspired materials. Undergraduate and graduate students will gain expertise in cutting edge technologies, imparting new scientific knowledge, developing new materials, and understanding human health. A partnership with the Wolf Museum of Exploration + Innovation (MOXI) will bring the discoveries enabled by this award to the public.

The fast-scanning, high-resolution confocal microscope with spectral detection capabilities (Leica TCS SP8x) will substantially modernize the Neuroscience Research Institute (NRI)/Molecular Cellular and Developmental Biology Microscopy Facility. The new instrument will transform the institutional research capabilities for collecting quantitative, high spatial and temporal resolution images in order to carry out NSF-funded research addressing diverse and critical research questions in developmental biology, quantitative biophysics, bio-inspired materials research, and neuroscience. These include investigations of: (1) intra- and inter-cellular interactions and the dynamics of morphogenesis and tissue remodeling; (2) human brain development using stem cell-derived cerebral organoids; (3)"smart" materials in which the mechanical properties can be dynamically controlled through responsive molecular architecture; (4) the dynamics of surface diffusion, convection and adhesion; and, (5) the molecular and biophysical mechanisms driving tunable iridescence. In addition to the immediate gains in speed and sensitivity, this project will provide for minor modifications to customize the new microscope and make it compatible with a wide range of customized biophysical tools, including a miniaturized surface forces apparatus (SFA), high-force magnetic tweezers devices, and a scanning probe microscopy system that combines imaging and multielectrode array technologies to map and manipulate neural tissues in culture. Additionally, the proposed instrumentation will be used in a variety of classroom, training, and outreach activities that will significantly enhance the education and mentorship of graduate and undergraduate students, and will bring diverse cohorts of students and experts to UCSB to perform research. A new partnership between the UCSB NRI and the Wolf Museum of Exploration + Innovation (MOXI) will engage the public in active learning about optics, light and imaging, while sharing the important discoveries and applications of the research enabled by this award. These activities will substantially expand the impact of this project beyond our campus, and will further strengthen UCSB's position as a leading institute in quantitative biology, neuroscience, biomaterials and bioengineering research and education.