Michael L. Shelanski, MD PhD - US grants

This project is divided into four sections all of which share a common focus on the elements of the neuronal cytoskeleton and the molecules that are responsible for the interactions between these elements. The first series of studies focuses on the interactions between neurofilaments and microtubules in vitro with specific attention to the role of microtubule accessory proteins and the P150 and P200 proteins of the neurofilaments in these interactions. The second set uses the techniques of microinjection and video assisted image-intensified microscopy to study the dynamics of DTAF-tubulin in the living cell. Phenomena such as wall exchange, organizing centers and treadmilling will be examined by fluorescence photobleaching recovery. The effecs of microinjected proteins and antibodies on the cytoskeleton will also be assessed. The third section will focus on the separation of the individual MAP components in both the tau and HMW complexes, their comparison by peptide fingerprinting, determination of their function in promoting the assembly and stability of microtubules and the effects of phosphorylation of MAPs on modifying these effects. Antibodies will be raised against each of the MAPs and used for microinjection and localization studies. The final section of the project will address the function of the 94,000 dalton microtubule assembly inhibitory protein in vivo and in vitro. it will be compared to the 95,000 dalton intermediate filament-associated protein and the SV-40 T antigen.

Our aim is to define the role of cell surface glycoproteins (GP) in nervous system development and function. Work will continue on the purification and characterization of the principal antigenic cell surface and released GP of PC12 cells and sympathetic neurons, two homogeneous, well characterized model neuronal systems. A number of antigenic cells surface glycoproteins also present in brain have been identified on PC12 cells and sympathetic neurons including those at apparent Mr = 230, 180, 160, 140, 118 and 25 kd. We have studied the 230 kd component extensively and named it after its properties, the NGF-inducible large external (NILE) GP. Specific antibodies (Ab) have been prepared against the NILE and the 180 kd GP and monoclonal and monospecific polyclonal Ab will be produced against the others. The Ab will be coupled to insoluble matrices and for the immunoaffinity purification of each of these macromolecules. The Ab will also be used to identify cross-reactive proteins in brain which will be purified and compared to their membrane bound and released PC12 and sympathetic neuron counterparts. The polyclonal sera will be used in conjunction with trypsin-treatment of the intact cells and tunicamycin-inhibition of glycosylation to determine the structure of each of these components with respect to the cell membrane. The Ab will also be used in anatomical localization studies in cultured and in fixed CNS, PNS and adrenals of different developmental ages. in an attempt understand the role of these GP, the effects of the antisera and the purified GP will be assessed on a variety of neuronal behaviors. Immunological methods will be used to select mutants of PC12 cells which lack or are altered in specific cell surface GP and these will also be screened for functional changes. Transplacental exposure of embryos to Ab against the individual GP will be studied to understand the developmental role of these molecules. Finally a radioimmunoassay for the released GP will be developed for immunodiagnosis of neural crest tumors in animals.

The role of neuron-astroglial relationships in the development of the normal and injured mammalian brain will be analyzed. The shape and cytoskeletal organization of astroglia in cerebral and cerebellar cortex, regions where astroglia are thought to guide neuronal migration, will be analyzed in the embryonic mouse brain and compared with that of astroglia in the brainstem and spinal cord, regions where an architectonic role for astroglia has not yet emerged, and with optic nerve, an area where immature astroglia might guide axon growth. Neuron-astroglial interactions of cells from various regions will be analyzed, and purified neurons and astroglia from different brain regions and ages will be "mixed and matched" in vitro to assess whether different types of astroglia are present during development and, if so, whether they relate to the region of origin of the astroglia or to their architectonic function. To analyze the formation of neuron-glial associations, the formation, extension and interactions of glial growth cones would be studied using biochemical, immunological and cell biological approaches. The expression of glial filament proteins will be studied using a newly developed cDNA probe for the glial filament protein using both microinjection and in situ hybridization approaches. The interactions of the various components of the astroglial cytoskeleton will be investigated as they relate to celluilar differentiation and function. The insights and tools obtained in the basic biological invetigations of the project will be utilized to understand the cell biology of astroglial reactivity or 'gliosis' after injury to the CNS and to determine the effects of antibodies against astroglial surface macromolecules on the development of the brain, using both the intact embryo and reaggregate cultures of the cerebellum as models. These same tools will be used to study the surfaces of astroglia in neural regeneration.

The early development of nerve-muscle synapses is characterized by three major events: (a) the accumulation of junctional acetylcholine receptors (AChRs), (b) the localization of synaptic acetylcholinesterase (AChE), and (c) the elimination of extrajunctional AChRs. This last event is mechanistically distinct from the accumulation of junctional AChRs and seems to result from the suppression of AChR synthesis in extrajunctional regions of muscle cells caused by muscle contraction. The final result is a muscle cell with a highly elaborated apparatus specialized for efficient synaptic transmission. Experiments in this proposal are designed to understand at the molecular level the way in which neurons and muscle cells communicate to establish this apparatus. The proposal is divided into three major sections. The first will make use of immunocytochemistry and antibody microinjection to demonstrate a functional association between the presence of a newly discovered muscle component (a 37 kilodalton nonmyofibrillar tropomyosin) and the ability to cluster AChRs. This molecule was first identified by its absence from vitally transformed muscle cells which are unable to cluster AChRs at all. The second section of this proposal describes similar techniques to probe other cytoskeletal elements involved in clustering and subsequent structural changes in the muscle cell. These studies are based on the observation from this laboratory that clustering causes a subset of organelles, including myonuclei and the Golgi apparatus, to assume a constant sub-cluster localization. The final section is a study of changes in the levels of AChRs and AChE caused by the increase in muscle cell Ca2+ which occurs during contraction. Also, experiments are designed to test the hypothesis that regional differences in the amount of Ca2+ released during contraction or in the levels of particular Ca2+-binding proteins underly the ability of these cells to specify where particular macromolecules are synthesized. Ca2+ concentration will be measured using the Ca2+-sensitive fluorescent dye fura-2 and optical image processing. Ca2+-binding proteins will be investigated using biochemical and immunological techniques. These experiments should add considerably to our knowledge of how neurons influence properties of their target cells and may contribute to an understanding of a variety of developmental and neurological disorders. In addition, certain results may provide information useful in comprehending cell transformation.

The Center for Alzheimer's Disease Research (CADR) is a multi- disciplinary unit based at the College of Physicians and Surgeons of Columbia University and involving the Schools of Medicine, Public Health, Nursing and Arts and Sciences. It also has major components at the Burke Rehabilitation Hospital, Cornell University School of Medicine, The Harlem Hospital Center and the New York State Psychiatric Institute. The CADR maintains a well-characterize population of patients with Alzheimer's Disease (AD) as well as a large community based population of normal elderly. It provides diagnostic services, serves as a resource for tissue, cells and DNA from AD and control patients, and attempts to educated both health care providers and the community about AD. Its major research avenues are epidemiological, cell biological and molecular biological with developing research in caregiving. Based in Manhattan, the CADR serves a multi-racial, multi-ethnic community with sizable number of minority patients.

The Columbia University M.D./Ph.D. program, a joint endeavor of the Graduate School of Arts and Sciences and the Faculty of Medicine (College of Physicians & Surgeons), was established in 1973 to train physician- scientists for academic careers in biomedical research and teaching. The program admits students who have demonstrated outstanding intellectual achievements, capability in research and strong motivation for an academic career. Students earn the Ph.D. degree by fulfilling the rigorous requirements of the Graduate School of Arts and Sciences and they earn the M.D. degree in a medical curriculum that emphasizes both depth in the basic sciences and a comprehensive grounding in the clinical sciences. Generally, entering trainees first complete the basic science courses of the medical school curriculum. In this period they are helped to develop perspective in science and to explore various training laboratories by basic science research seminars, clinical research orientation seminars, summer research projects, and counseling and guidance by an experienced faculty Advisory Committee. Upon entering a graduate program, the trainee takes additional graduate courses, undertakes an original investigation, and fulfills all requirements for the Ph.D. Degree of the Graduate School of Arts and Sciences. In the graduate training period the student can maintain contact with clinical activities through special exercises (rounds, tutorials, and practical sessions) and the advice and guidance of clinical mentors. The length of the graduate research period is flexible. Thereafter, the trainee completes a clinical clerkship period and electives. Graduates of the double-degree program are highly qualified for careers in academic medicine and biomedical research. The all-University program comprises departments of both the Health Sciences and Morningside campuses, including Anatomy & Cell Biology, Biochemistry & Molecular Biophysics, Biomedical Engineering, Biological Sciences, Chemistry, Epidemiology, Genetics & Development, Medical informatics, Microbiology, Nutrition, Pathology, Pharmacology, and Physiology & Cellular Biophysics.

The Neuropathology Core and Molecular Diagnostics Core is overseen by a professional staff of Michael L. Shelanski, M.D., Ph.D., and Steven S. Chin, M.D., Ph.D. The core provides "state of the art" neuropathologic examination of patients with various neurodegenerative disorders as well as neurologically normal individuals. These patients are clinically evaluated in detail by the Columbia ADRC and other neurology groups. The core provides detailed reports of findings and diagnoses to patient families and their physicians. Diagnoses are rendered according to presently accepted and recommended neuropathological criteria. In particular, the recently developed NIH-Regan Institute Working Group diagnostic criteria for the neuropathological assessment of Alzheimer's disease is utilized. Standard postmortem handling of brain tissues includes hemisection of the fresh brain, quick freezing of coronally sliced slabs of one half of the brain for banking and eventual distribution to investigators within and outside the ADRC, and fixation of the other half in formalin for histopathological examination and diagnosis. Microscopic examination includes application of H&E, thioflavine S, modified Bieschowsky silver, Gallyas silver, anti-ubiquitin, anti-synuclein, anti- tau, and anti-amyloid stains to selected sections from a standardized set of sample neuroanatomic regions. Collections of formalin-fixed and frozen brain tissue, lymphoblastoid cell lines, DNA from study patients, and their corresponding databases are maintained and distributed to investigators by the core. The core actively educates and trains neuropathology fellows, pathology, neurology, and psychiatry residents, and other health professionals in the importance and practical aspects of post-mortem examination and tissue banking. Clinicopathological correlation conferences are held to review discuss interesting and instructive study cases. The core strongly encourages research collaborations, and providing neuropathological and basic research expertise, access to biologic specimens, and assistance and access to specialized instruments, such as fluorescence, electron and laser capture microscopes as well as high speed DNA sequence analysis.

The Alzheimer's Disease Research Center (ADRC) at Columbia University was established to courage and integrate research on the causes of Alzheimer's Disease (AD) and related age-related neurodegenerative diseases and to foster the development of improved diagnosis, prevention and treatment of these conditions. The Clinical Core and Neuropathology Core of the ADRC provide the patient and tissue resources for the examination of new diagnostic and treatment modalities and for biological investigation. The population of the Clinical Core is ethnically diverse with substantial numbers of White, Black and Hispanic patients. The integral research projects of the ADRC are focused on the molecular biology of dementia and on early changes in brain function in AD. These research projects are extended by a large number of independently funded projects on the dementias and their underlying biology which have been nucleated by the Center and draw on ADRC resources. The ADRC actively encourages research in all aspects of AD including caregiving, treatment and biology. The ADRC also serves and a source of education and information on AD. Through the efforts of Education and Information Transfer ore we provide a Web Page with both local and general information; training programs in the biology and psychology of aging; and extensive seminar and works- in-progress series; education for primary care physicians in care of AD patients and educational programs to the lay community. The ADRC offers research services for genotyping, DNA storage, cell line production and DNA sequencing and maintains a bank of appropriate tissues, DNA and cell lines to facilitate research in this and other research centers.

DESCRIPTION (provided by applicant): The demonstration that the drugs rolipram and forskalin reverse the inhibition of LTP induced by alpha beta in hippocampal slices and that exposure of cultured hippocampal neurons to alpha beta causes changes in expression of genes involved in the switch from early to late LTP suggests that the cAMP signaling pathway may be an early target of damage in Alzheimer's disease as well as a therapeutic target, The first portion of work proposed will examine the role of alpha beta on the regulation of cAMP levels and investigate the mechanism of alpha beta Down regulation of PKA activity. Emphasis will be on the regulation of levels of the regulatory subunits of PKA. Experiments of APP Tg mice will examine the mechanism of LTP inhibition in the animals and determine if rolipram and forskalin reverse this inhibition. The second portion of the application examines the role of caspase 2 in cellular responses to alpha beta and will explore the issue of whether caspase 2 is necessary for the synaptic loss in APP Tg mice by crossing these animals with caspase 2 null mice.

DESCRIPTION (provided by applicant): The ADRC at Columbia seeks to advance and disseminate knowledge of the causes, prevention and treatment of Alzheimer's Disease and other age-related neurodegenerative and dementing disorders. Toward this end, we maintain and follow a multi-ethnic and multi-racial patient population of normal elderly and the elderly with cognitive disorders to establish the natural history of the disease as a function of age and of genetic makeup. In this application we propose to use a variety of neuropsychological, neurological and imaging tools to examine the earliest stages of AD and to follow these subjects throughout their lives. At death a comprehensive neuropathologic examination will be performed and the clinical, radiological and pathological views of the disease correlated. Tissues and DNA obtained from these subjects will be available for research on the biology and genetics of the disease. Individual research projects within the ADRC will examine various aspects of the cellular and molecular biology of AD as well as an in-depth analysis of brain function in human subjects using MRI. Patients enrolled in the ADRC will have the opportunity to participate in trials of new drugs and treatments for dementing diseases as they become available. The well-documented cognitive status of these patients makes them highly suitable for inclusion in clinical trials. The Education and Information Core of the Columbia ADRC endeavors to educate both lay and medical communities about AD, about the latest advances in research and about the care of the AD patient. The ADRC serves as a resource for scientists with Columbia as well as outside of it, encouraging new research avenues by the award of pilot grants, by providing tissues and other biological samples, by providing access to a carefully documented patient population and by numerous seminars and Clinical Pathological Correlation Conferences. The Genetics Core serves as the major organizer of family recruitment and identification across the ADRCs and ADCs. HIPAA compliant data organization and statistical consulting services are provided under the ADRC to the research community at Columbia and external to it.

The Administrative Core is the nerve center ofthe Columbia ADRC. It assures communication between the diverse cores and projects. It schedules meetings of both the Internal and External Advisory Committees, developing and implementing action plans based on their recommendations. The Administrative Core is responsible for interactions of the ADRC with the university, with the public and the Alzheimer Association (via the Education Core), with other ADCs, with NACC and with the NIA. It is responsible for administrating, accounting and reporting related to the ADRC grant and related funding; for the appointment of personnel in the university; for assuring the compliance of our cores and projects with regulations on human subjects, animal use, HIPPA and genetic testing. The Administrative Core monitors the mission of the ADRC and modifies it as necessary to meet new knowledge about the disease. The administrative core solicits Pilot Grant proposals on an annual basis, organizes the review of these proposals and their submission to the NIA.

DESCRIPTION (provided by applicant): Parkinson's Disease (PD) is the second most common neurodegenerative disease. As in many other neurodegenerative diseases, conformational alteration of a specific neuronal protein results in the accumulation of fibrillar amyloid inclusions, which in the case of Parkinson's disease, are termed Lewy Bodies (LBs). LBs have a fibrillar core with the fibrils being comprised primarily of a protein of unknown function called alpha-synuclein. Alpha-synuclein mutations cause autosomal dominant Parkinson's disease. Thus both human genetic and histologic evidence link synuclein to Parkinson's disease. Alpha-synuclein is a 140 aa protein which is 'natively unfolded' meaning that it has no identifiable secondary structure. However, in the presence of certain lipid membranes is can fold into a alpha helical conformation, and when incubated alone can fold into a beta-sheet rich conformation which allows it to form amyloid fibrils resembling those seen in Lewy bodies. Consistent with the hypothesis that alteration of synuclein conformation is linked to development of Parkinson's disease, purified mutant synuclein fibrillizes more rapidly than wild-type protein in vitro. Overexpression of synuclein as a transgene results in formation of Lewy body-like pathology in mice and flies. Synuclein expressed at endogenous levels rarely forms amyloid (only in PD patients), is not stably membrane-associated, and remains 'unfolded'. The discrepancy between the in vivo folding parameters and those observed in vitro leads us to hypothesize that synuclein-interacting molecules may regulate synuclein conformation, stabilize it in the 'unfolded' state, or regulate membrane binding. We therefore set up a novel photo-cross linking assay heretofore not used to study synuclein to identify synuclein binding proteins present in brain extracts and present at endogenous levels of expression to begin to determine how synuclein conformation is regulated. We have identified novel synuclein binders. We propose to develop a fluorescence resonance energy transfer assay capable of indicating synuclein conformation both in vivo and in vitro. That will allow for screening of proteins and synthetic agents capable of altering synuclein aggregation. These studies will enable us to define the range of proteins or agents to be further characterized in in vivo models of Parkinson's disease.

DESCRIPTION (provided by applicant): The loss of cognitive function with normal aging is well known and while clearly different from the catastrophic loss of memory seen in Alzheimer's disease and the other age-related dementias, is of concern to an increasing number of Americans. This application seeks support to recruit an outstanding junior faculty member to the Taub Institute at Columbia University to study the normal aging process in either the animal or the human brain. PUBLIC HEALTH RELEVANCE: An understanding of the mechanisms of normal aging in the brain is critical if we are to slow cognitive decline in our aging population. It will enable us to understand the factors that lead to accelerated cognitive decline and dementia.

DESCRIPTION (provided by applicant): The ADRC at Columbia seeks to advance and disseminate knowledge of the causes, prevention and treatment of Alzheimer's Disease and other age-related dementing disorders. To do this, we maintain and follow a multi-ethnic and multi-racial patient population of normal elderly and the elderly with cognitive disorders to establish the natural history of the disease as a function of age and of genetic makeup. In this application we propose to use a variety of neuropsychological, neurological and imaging tools to examine the earliest stages of AD and to follow these subjects throughout their lives and to compare them to other dementing diseases. A comprehensive neuropathologic examination will be performed on the deceased participants correlated with the clinical, radiological and genetic data. Tissues and DNA obtained from these subjects will be available for research on the biology and genetics of the disease. Individual research projects within the ADRC will examine various aspects of the cellular and molecular biology of AD with emphasis on lipids, on retromer function and on the relationship of GBA mutations to dementia. Patients enrolled in the ADRC will have the opportunity to participate in trials of new drugs and treatments for dementing diseases as they become available. The well-documented cognitive status of these patients makes them highly suitable for inclusion in clinical trials. The Education and Information Core of the Columbia ADRC endeavors to educate both lay and medical communities about AD, about the latest advances in research and about the care of the AD patient. The ADRC serves as a resource for scientists with Columbia as well as outside of it, encouraging new research avenues by the award of pilot grants, by providing tissues and other biological samples, by providing access to a carefully documented patient population and by numerous seminars and Clinical Pathological Correlation Conferences. The Genetics Core serves as the major organizer of family recruitment and identification across the ADRCs and ADCs as well as resources for genotyping and gene expression studies. HIPAA compliant data organization and statistical consulting services are provided under the ADRC to the research community at Columbia and external to it.

DESCRIPTION (provided by applicant): Stem cells continuously generate new neurons in restricted regions of the adult mammalian brain. The subventricular zone (SVZ) is the largest germinal region in the adult gives rise to neurons that migrate to the olfactory bulb as well as some oligodendrocytes. The in vivo stem cells are a subset of astrocytes, glial cells classically associated with support functions. These endogenous stem cells potentially represent a pool of cells that can be harnessed for brain repair. A balance between intrinsic and extrinsic signals mediates the activation of quiescent stem cells. However, the ability to distinguish between quiescent and activated stem cells has been hampered by a lack of markers. Here we identify a combination of markers that allow us to identify and isolate four populations of astrocytes from the SVZ directly from the in vivo niche. Based on cell cycle analysis, in vitro stem cell behavior and gene expression profiling, we hypothesize that they correspond to quiescent stem cell astrocytes, activated stem cell astrocytes and niche astrocytes. We propose to: 1) Define the cell cycle kinetics of the purified astrocyte populations, and their ability to act as stem cells in vitro, as well as how their numbers and functional properties change with aging, 2) perform lineage tracing of quiescent and activated stem cells under homeostasis and during regeneration and 3) define the role of PDGFRb in regulating adult neural stem cell quiescence. Together these studies will yield key insights into the regulation of stem cell quiescence in the adult brain.

Project Summary: Research published over the past 15 years has pointed to a critical role of the enzyme Caspase 2 in Alzheimer's disease (AD). Transgenic animals carrying human Amyloid Precursor Protein (APP) genes develop extensive amyloid plaques and show increasing memory impairment and synaptic loss. When Caspase 2 is removed from these animals, the amyloid plaques develop but the synaptic loss and memory impairment do not occur. Cell culture work has demonstrated that Caspase 2 is necessary of A? induced synaptic dysfunction and cell death. These results taken together with the observation that Caspase 2 activity is increased in human AD suggest that Caspase 2 is a potential therapeutic target for AD treatment. Caspase 2 is present at very low levels in the adult brain and increases only in disease or in the AD models suggesting that its inhibition might not affect normal cellular processes. The major obstacle to exploring this avenue of treatment has been the non-specificity of caspase inhibitors. In the work proposed here we will use two highly specific methods of inhibiting Caspase 2. The first uses a cell permeable form of siRNA to diminish the amount of Caspase 2 in the brain while the second uses a newly developed agent, RAIDDpep, which blocks the activation of Caspase 2. These agents will be tested for their ability to block the cognitive and synaptic effects of A? in the J20 APP transgenic mouse and in mice where A? is introduced acutely by stereotaxic injection. The results of this work have the potential to open a new avenue for AD therapy.