James E. Goldman, MD PhD - US grants

Astrocytes comprise a large proportion of CNS cells. They are thought to originate among the neuroectodermal cells of germinal zones. While immature cells form few cellular contacts, astrocytes form many close interactions with other cells and with extracellular elements. Astrocytic differentiation thus must involve major changes in the cell surface. Little is known of the molecular characteristics of astrocyte membranes, however. Astrocytes also comprise the major cellular component of glial scars, a pathological result common to a great many neurological disorders, including demyelinating diseases, trauma, stroke and neurodegenerative diseases. In scarring, astrocytes accumulate large amounts of the intermediate filament protein, glial fibrillary acidic protein (GFAP). We propose a series of studies to examine several morphological and biochemical characteristics of astrocytes during glial differentiation and during gliosis. We have recently found that GD3 ganglioside is a major and characteristic cell surface component of immature CNS cells, and of astrocytes in glial scars. Because of the restricted localization of this molecule to immature cells, we will use an anti-GD3 antibody to select for immature cells from newborn rat CNS. We are developing a cell culture system in chemically-defined, serum-free medium to allow proliferation of these cells. Our preliminary studies show this is possible and, furthermore, that we will be able to induce astrocytic differentiation by alterations in growth medium. We propose to study glial differentiation in detail, examining possible stimuli that induce and maintain the process. We will concentrate on intermediate filament expression and characterization of cell surface molecules. We will also continue our current studies on cytoskeletal organization and metabolism in astrocytes, in which we have found experimental means of modulating GFAP expression. One of these means appears to be cAMP-dependent. Finally, we will examine molecular changes in astrocytes during gliosis, in particular changes in GFAP expression and surface molecules. We will use in vivo systems of traumatic lesions, and murine mutants which display gliosis.

The PI is continuing studies on the regulation and function of aB- crystallin, a major component of the vertebrate lens and a member of the small "heat shock" protein family. AB-crystallin accumulates in CNS glia and neurons in a variety of neurological disorders, in some cases actually forming inclusions in glial cells, such as the Rosenthal fibers of Alexander disease. The gene is upregulated in response to physiological stresse and the protein serves to protect cells from the adverse consequences of those stresses, but the modes of regulation and the mechanisms of protection are not fully understood. Over the previous grant period, the laboratory has begun to characterize two different transcription controlpathways through which aB-crystallin expression is regulated in astrocytes subjected to physiological stresses, one through a heat shock factor pathway and one through a heat shock-independent pathway. The PI has also begun to explore how this protein confers protection upon cells. He proposes a set of interrelated specific aims for the next grant period to examine further the transcriptional regulation of the aB-crystalling gene and the function of the protein in protecting CNS cells from physiological stress. Specifically, he plans to: 1. continue our studies todefine the transcriptional regulation of the aB-crystallin gene during phsiological stress. 2. further define how aB-crystallinprotects cells from hypertonic and other stresses, 3. explore mechanisms of Rosenthal Fiber production.

Over the past several years, our laboratory has been studying the genesis of astrocytes and oligodendrocytes during early CNS development. We have been and will continue to follow developmental fates and migration of progenitors from germinative zones of the postnatal rat CNS in vivo using replication deficient retroviral vectors and to chart the lineage and migration of immature cells and their differentiation into glia. We propose a series of integrated approaches to begin to answer the following questions. 1. What are the early stages of glial migration and differentiation? We will study early events in antigen acquisition and morphological changes during migration of forebrain subventricular zone (SVZ) cells into the cortex. We suggest that contact with blood vessels or pia is an early event in astrocyte development. We are developing an in vitro system in which SVZ cells aggregate in clusters, and migrate from clusters along glial "cables". We will also visualize the migration and differentiation of immature cells in brain slices to study modes and directions of migration by infecting progenitors with a retrovirus encoding an endogenously fluorescent protein (GFP), using a beta-gal substrate in living cells, or marking SVZ cells with fluorescent dyes. We will begin to ask what are the signals that determine cell migration and fate during gliogenesis, using "cable" cultures to give migrating SVZ cells choices between cables and either blood vessels or neurons (axons) to ask if such exposure will activate astrocyte or oligodendrocyte differentiation, respectively. We will introduce genes that may play roles in progenitor proliferation, migration, and differentiation into SVZ cells in vivo using retroviruses to see if expression will alter migration or developmental fate. 2. What is the extent of clonal dispersion and phenotypic heterogeneity of the progeny of postnatal SVZ cells? We will use a library of 100 different retroviruses coupled with polymerase chain reaction to recover and identify the specific virus in each cell in a single brain. 3. What are the patterns of gliogenesis in the cerebellum? We will examine the origins and migratory paths of glial progenitors in the cerebellum by retroviral labeling in vivo and after labeling in living, long-term cerebellar slices. 4. What is the nature of immature, proliferating cells in the adult CNS subcortical white matter and residual SVZ? What is (are) their normal fates? Do they continue to migrate through the CNS? If they do not normally differentiate into mature cells, do they have the potential to develop into neurons or glia? We will inject retroviruses into the SVZ and white matter of adult rats and characterizing the labeled cells in vivo and in vitro.

histopathology; postmortem; brain disorder diagnosis; biomedical facility; Alzheimer's disease; tissue resource /registry; training; meeting /conference /symposium; human genetic material tag; human tissue; cryopreservation;

The Pathology-Molecular Core for this project will continue to provide postmortem diagnosis and will now provide apolipoprotein-E genotypes and storage capacity for serum and plasma for the entire cohort. The primary purpose of this core has been to support the clinical projects by providing state of the art neuropathological diagnosis. Because the program project was, from its inception, designed to be a large-scale epidemiologic project, routine or mandatory brain autopsy was not a realistic criterion for inclusion into the studies. However, we now intend to provide educational seminars in the community with the hope of enhancing autopsy acquisition. Our banked tissue has also been used for several studies examining the relationship between Alzheimer's disease (AD) and APOE and in a collaborative study of APOE in the diagnosis of AD. The importance of having autopsy confirmed diagnoses is obvious, and now, we will make a concerted effort to ascertain autopsy in patients at any stge of disease and controls. Because this project has made great strides in community relations regarding autopsy we are confident that the rate of autopsy confirmation will improve. Preliminary data indicates with efforts focused on the families of patients with late-stage disease, the rate of acceptance of autopsy can approach 50%. In addition to providing a neuropathological examination of participation who die in the program project, we will complete data entry forms for entry into program project database, collect and store fresh blood samples as serum and plasma for planned investigations, extract and store DNA for APOE genotypes and other planned genetic studies, distribute additional serum, plasma or DNA as needed to study investigators, provide relevant tissues to complete aims in projects related to APOE expression and interact with Columbia University's Taub Alzheimer's Disease Center Research Center.

DESCRIPTION(Verbatim from the Applicant's Abstract): Our laboratory has been examining the genesis of astrocytes and oligodendrocytes during early CNS development and the nature and differentiation potential of immature glial cells in the adult CNS. We propose to continue a series of studies that include: 1. Further examination of early gliogenesis. a. We will continue to examine early stages of astrocyte development in vivo by looking for the concurrent expression of astrocyte genes and the contact between progenitors and blood vessels in the developing neocortex. We will also continue our real-time video imaging of migrating progenitors to examine the early stages of astrocyte development. b. We will ask if interactions between progenitors from the SVZ and endothelial cells induce astrocyte phenotype, co-culturing progenitors from the neonatal SVZ with endothelial cells. c. We will continue to study pathways of progenitor migration. Since our previous data suggests that radial glia represent one of the major pathways along which progenitors from the SVZ migrate into gray matter and white matter, we will try to visualize that interaction directly using retroviral gene transfer into the SVZ of the transgenic mouse that expresses green fluorescent protein driven by the GFAP promoter. d. We will further characterize cycling cells in the neonatal SVZ. 2. We will continue to characterize gliogenesis and neurogenesis in the postnatal cerebellum. a. Are the dividing progenitors in cerebellar white matter multipotent cells or are there separate progenitors for neurons and glia? We will approach this question by performing a series of in vitro and in vivo clonally analyses and by looking for markers that might distinguish neuronal from glial progenitors. b. What are the patterns of migration and cell development in the postnatal cerebellum? We hope to be able to perform video imaging of glial development in cerebellar slices for several days, since such slices can be kept alive for prolonged periods. 3. Further characterization of cycling cells in the adult CNS. We have begun to characterize a population of dividing progenitors in the adult rat subcortical white matter and neocortex, and have shown that some of these cells will differentiate into myelinating oligodendrocytes in response to a local demyelination. We will continue to characterize these cells in several respects to ask what growth factor receptors do these cells express and how do they respond to specific growth factors. b. How do these cycling cells respond to pathological conditions? We are particularly interested in understanding whether such pathologies as demyelination and trauma will cause these progenitors to accumulate, through increased proliferation and/or decreased cell death, as well as how pathologies induce glial differentiation.

We propose to examine several aspects of intermediate filament (IF) organization and cellular stress reactions that may be germane to an understanding of the pathophysiology of Alexander disease. Thus, the over-expression of IF proteins may lead to an abnormal organization of filaments, which subsequently leads to up-regulating the transcription and translation of the small hsps, alphabeta-crystallin and hsp27. We must also consider that Alexander disease is a disorder in which a mutant gene expressed on one cell type (astrocyte) results in degeneration and/or abnormal development of another cell type (oligodendrocyte). We propose three specific aims: 1) How are mutant GFAPs expressed in cells and do they result in an abnormal organization of IFs? Is there an accumulation of IFs? Does the expression of mutant GFAPs alter IF turnover. 2. Does the accumulation of IFs lead to a cellular stress response, part of which leads to the up-regulation of small hsps? If so, what are the mechanism(s) that produce such a stress response? If IF accumulation induces small hsps, we will investigate intracellular stress signal pathways that may underlie this effect, focusing on HSF1 activation, MAP kinase activation (ERK1/2, JNK, p38 kinase), NF- kappaB activation, and protein kinase-N activation. 3. How does the expression of a mutant protein in one cell type in the CNS (astrocytes) produce deleterious effects on another cell type (oligodendrocytes)? Is the cellular stress response an important part of this link? Do the pathological changes in astrocytes interfere with oligodendrocyte differentiation and/or myelination? We will examine possible further consequences of IF aggregation, focusing on the possibility that "stressed" astrocytes regulate cytokines. These experiments will focus on how the accumulation of a protein in one cell type (Astrocytes) appears to produce deleterious effects on another cell type (oligodendrocytes), the rationale taken from experiments in which cytokines are toxic to oligodendrocytes. In addition, we will examine other potentially toxic substances, including reactive oxygen species. If the neuropathology of the transgenic mice that is constructing suggest a defect in oligodendrocyte development and/or myelination, then we will examine directly the interactions between astrocytes expressing GFAP mutations and oligodendrocyte progenitors using a cell culture system.

[unreadable] DESCRIPTION (provided by applicant): We are requesting $500,000 to purchase a massively parallel DNA sequencer for use by the Columbia University community and collaborators at other institutions. There is no comparable instrument available on any of the campuses of Columbia University. A single sequencing run on this instrument will achieve the equivalent of over 100 sequencing runs on each of Columbia University's currently available 2 instruments, thus dramatically expanding the scope of research programs. [unreadable] This instrument will be used by a broad range of NIH-funded investigators. Projects range from comparative genomics of microorganisms to identify genes and genome regions associated with distinct forms of pathogenicity, a metagenomics study of the effect of influenza virus infection on bacterial flora, global basic research studies of motifs involved in splicing, transcription, and of tissue specific miRNA species, and populations of sequences in directed enzyme evolution; much of the projected use will involve identification of genetic changes involved in a wide range of diseases from glomerulonephritis to diabetes, neuropsychiatric disorders to alopecia, lymphoma to arrhythmias, with results anticipated to provide insight into the mechanism of disease, diagnosis, prognosis and treatment. [unreadable] The instrument, housed in the Genome Center, will be run by staff well experienced with DNA sequencing and methods development, and supported by all required ancillary instruments and computational resources. Columbia University has demonstrated its commitment to the maintenance and operation of this instrument by pledging support for warranty extension and a technician covering the first year and a half of operation, following which the instrument will be supported by user fees. Given the large number of biomedical investigators on the campuses of Columbia University, the presence of such an instrument would have a significant impact on NIH-funded research. [unreadable] [unreadable] [unreadable]

Address; Agonist; Alexander Disease; Alexander syndrome; Ammon Horn; Apoptosis; Apoptosis Pathway; Assay; Astrocytes; Astrocytus; Astroglia; Astroprotein; Autophagocytosis; Bioassay; Biologic Assays; Biological Assay; Brain; Buffers; Bundling; CDK6 Inhibitor p18; CDK6-associated protein p18; CDKN2C; CDKN2C Protein; CDKN6; CSBP1; CSBP2; CSPB1; Cell Communication and Signaling; Cell Death, Programmed; Cell Signaling; Cell Survival; Cell Viability; Cells; Cellular Stress; Chronic; Communicating Junction; Connexin 43; Connexin43; Cornu Ammonis; Coupling; Crystallins; Cx43; Cyclin-Dependent Inhibitor; Cyclin-Dependent Kinase 4 Inhibitor C; Cyclin-Dependent Kinase 6 Inhibitor; Cyclin-Dependent Kinase 6 Inhibitor p18; Cyclin-Dependent Kinase Inhibitor 2C; Cyclin-Dependent Kinase Inhibitor 2C (p18, Inhibits CDK4); Data; Dependence; Dysfunction; EC 2.7; EXIP; Encephalon; Encephalons; Enzymes; Exhibits; Exposure to; Extracellular Signal-Regulated Kinase Gene; FK506 Binding Protein 12-Rapamycin Associated Protein 1; FK506 Binding Protein 12-Rapamycin Associated Protein 2; FK506 binding protein 12-rapamycin associated protein 1, human; FKBP-Rapamycin Associated Protein; FKBP-rapamycin associated protein, human; FKBP12 Rapamycin Complex Associated Protein 1; FRAP1 protein, human; Filament; Frequencies (time pattern); Frequency; Fugu Toxin; Functional disorder; GFA-Protein; GFAP; Gap Junctions; Genetic Alteration; Genetic Change; Genetic defect; Glial Fibrillary Acid Protein; Glial Fibrillary Acidic Protein; Glial Intermediate Filament Protein; Glutamate Ammonia Ligase (ADP); Glutamate Dehydrogenase; Glutamate Translocase; Glutamate Transport Glycoprotein; Glutamate Transporter; Glutamate-Ammonia Ligase; Glutamates; Glutamine Synthetase; HSP; Heat shock proteins; Hippocampus; Hippocampus (Brain); INK4C; INK4C protein; Intracellular Communication and Signaling; JUN Family Gene; JUN Proto-oncogene Family; JUN gene; Kinases; Knock-in; Knock-in Mouse; L-Glutamate; L-Glutamate[{..}]NAD+ oxidoreductase (deaminating); L-Glutamate[{..}]ammonia ligase (ADP-forming); Lens Proteins; Link; Low-resistance Junction; MAP Kinase Gene; MAPK; MAPK14; MAPK14 gene; Mammals, Mice; Measures; Mediating; Metabolic; Mice; Mice, Mutant Strains; Mice, Transgenic; Mitogen-Activated Protein Kinase Gene; Murine; Mus; Mutant Strains Mice; Mutation; Mxi2; Nerve Cells; Nerve Unit; Nervous System, Brain; Neural Cell; Neurocyte; Neurons; Nexus; Nexus Junction; Numbers; Oligodendrocytes; Oligodendrocytus; Oligodendroglia; Oligodendroglia Cell; Overexpression; P01 Mechanism; P01 Program; PRKM14; PRKM15; Pathway interactions; Phosphotransferases; Physiopathology; Predisposition; Process; Program Project Grant; Program Research Project Grants; Proteasome Inhibition; Protein Overexpression; Proteins; Pyramidal neuron; RAFT1 protein, human; RAPT1 protein, human; Rapamycin Target Protein; Research Program Projects; SAPK2A; Seizures; Signal Transduction; Signal Transduction Systems; Signaling; Slice; Stimulus; Stress; Stress Proteins; Study of Disease Mechanisms; Sum; Susceptibility; Synapses; Synaptic; System; System, LOINC Axis 4; TSC1/2; TSC1/2 gene; TSC1/TSC2; TSC2/TSC1; TTX; Tarichatoxin; Testing; Tetradotoxin; Tetrodotoxin; Transgenic Mice; Transgenic Organisms; Transphosphorylases; Whole-Cell Recordings; Work; autophagy; biological adaptation to stress; biological signal transduction; c jun; c-jun Gene; cell type; cellular pathology; cyclin-dependent kinase inhibitor p18; demyelinogenic leukodystrophy; disease mechanisms study; dysmyelinogenic leukodystrophy; established cell line; fibrinoid degeneration of astrocytes; fibrinoid leukodystrophy; gene product; genome mutation; glutamate synthetase; glutamic dehydrogenase; glutamine synthase; hippocampal; hippocampal pyramidal neuron; human FRAP1 protein; in vivo; mTOR; mTOR inhibition; macrocephaly with feeblemindedness and encephalopathy with peculiar deposits; megalencephaly with hyaline panneuropathy; mouse mutant; mutant; neuron toxicity; neuronal; neuronal excitability; neuronal toxicity; neurotoxicity; overexpress; p18; p18 protein; p18-INK4C; p18-INK6; p18INK4c protein; p38; p38 MAPK Gene; p38Alpha; pathophysiology; pathway; puffer fish toxin; rapamycin and FKBP12 target 1 protein, human; reaction; crisis; response; stress response; stress; reaction; transgenic