Cheryl F. Dreyfus - US grants

While abundant studies have begun to elucidate ontogeny of the peripheral nervous system, molecular mechanisms underlying brain development remain obscure. This is due in part to the complexity and inaccessibility of the central nervous system (CNS), particularly in developing animals. We, however, are now able to approach questions of developmental regulation in the CNS. We have succeeded in growing brain nuclei in an accessible tissue culture environment and are able to couple study of these nuclei in vitro with sensitive biochemical and morphological techniques. We can thus define molecular events associated with central neuron development. In particular, we have cultured the brain noradrenergic nucleus locus coeruleus. In culture locus neurons express a variety of noradrenergic traits, including endogenous catecholamine (CA), the specific uptake mechanism for norepinephrine (NE) and the CA biosynthetic enzymes, tyrosine hydroxylase and dopamine-Beta-hydroxylase. We thus can use these CA traits to define factors that regulate development of central neurons and determine whether similar rules govern central and peripheral ontogeny. Influences we will study include those of presynaptic depolarizing agents and target tissues. In addition, we will determine the role of glucocorticoids in expression of NE characters. Finally, we will compare regulation of expression of dopamine characters in developing substantia nigra with expression of noradrenergic characters in the locus. These experiments hopefully will provide insight into sites where changes in the normal milieu of a maturing neuron may lead to abnormal brain development. Such an understanding may eventually lead to new therapeutic approaches to congenital birth defects, such as mental retardation and neural tube defects.

A number of psychoactive agents adversely affect brain development. However, underlying molecular mechanisms remain to due, in part, to the complexity and inaccessibility of the brain, particularly in developing animals. We, however, are now able to approach questions of molecular mechanisms governing toxicity in the central nervous system (CNS). We have succeeded in growing the brain nonadrenergic nucleus locus coeruleus in an accessible tissue culture system for prolonged periods. Electrophysiological studies indicate that the locus is responsive to a variety of psychoactive substances, including caffeine, ethanol, amphetamines and tricyclic antidepressants. Moreover, the locus exhibits sensitive and specific phenotypic characters by which ontogeny and toxicity may be monitored at defined molecular loci. In the proposed work, we will evaluate the influence of caffeine, ethanol,locus amphetamines and the tricyclic antidepressant, desmethylimipramine, on ontogeny. Effects of these agents will be assessed by monitoring the noradrenergic traits, tyrosine hydroxylase, dopamine-8-hydroxylase and the specific uptake of norepinephrine, sensitive and specific markers of locus function Specific antagonists will be used to define receptor sites involved in any observed drug action. In addition, agents will be tested on locus cells of varying maturities and critical developmental periods will be defined. Finally, mechanisms underlying ontogeny of drug dependency and tolerance will begin to be evaluated by examining coeruleal traits after prolonged exposure to the psychoactive agents. These experiments promise to identify the molecular loci of action of drugs of abuse in the brain.

This project is designed to test the hypothesis that brain neuronal circuits develop through the complex interaction of specific neurotransmitters and trophic factors. In particular, we hypothesize that glial and neuronal target cells are stimulated by neurotransmitters of specific brain pathways to regulate expression of the very trophic factor(s) and cognate receptor(s) responsible for the development and maintenance of those pathways. Previous work from our laboratory and others indicates that trophic factors regulate normal brain ontogeny. We now will examine the contention that impulse activity regulates trophic factor expression and action through the mediation of specific transmitters. In particular, we will use well-characterized neuronal, glial, and neuronal-glial cultures to 1) define the role of neuronal activity in the regulation of NGF gene expression, 2) define molecular mechanisms underlying the regulation of NGF receptor gene expression, 3) characterize regulation of expression of other members of the NGF gene family in the brain and the actions of these agents. Using this strategy we hope to identify molecular mechanisms governing brain systems formation and function, and define loci where disease processes may intervene, resulting in such abnormalities as cognitive disorders during development.

The applicants propose a core tissue culture facility to provide centralized support for all projects involving substantial culture of primary cells and cell lines.

DESCRIPTION The goal of this proposal is to define the role of oligodendrocytes, the traditional myelinating cells of the central nervous system, as producers of trophic molecules during development. In this capacity, it is hypothesized that oligodendrocytes produce NGF, BDNF, and NT3, molecules that affect survival and function of oligodendrocytes, as well as local neurons. Local neurons, in turn, regulate oligodendrocyte function. It is suggested that critical autocrine and paracrine interactions may exist to optimize survival and function of oligodendrocytes and related neurons in specific brain regions. This issue will be addressed in oligodendrocyte populations associated with the basal forebrain, a region well-known to be sensitive to neurotrophins. Preliminary studies suggest that this oligodendrocyte population expresses and responds to neurotrophins and is regulated by neural signals. To investigate the role of oligodendrocytes as trophin producers the study will 1) examine the maturation of oligodendrocytes as trophin producers and trophin responders during development in culture, 2) investigate the effects of neurotrophin molecules on oligodendrocyte maturation, 3) define regulation of basal forebrain neurons by oligodendrocytes, 4) define the role of neural signaling on oligodendrocyte development, and 5) determine whether effects in culture are relevant in vivo. These studies are designed to explore the role of oligodendrocyte as providers of the trophic molecules, NGF, BDNF, and NT3. It is proposed that these neurotrophins provide trophic support for oligodendrocytes as well as local neuronal populations. The studies will identify factors that support oligodendrocyte function. As such, they promise to provide new insights into deficits that may occur in demyelinating diseases.

[unreadable] DESCRIPTION (provided by applicant): The overall hypothesis of the current work is that oligodendrocytes (OLGs), the myelinating cells of the central nervous system are critically influenced by the well-defined trophic factor, brain-derived neurotrophic factor (BDNF). The effects of BDNF are mediated through trkB and its associated MAP kinase, PI3 kinase-Akt, and PLC-gamma pathways. BDNF supports OLG proliferation and differentiation during brain development and throughout life. Work performed during the initial funding period supports this hypothesis. Using basal forebrain (BF) OLG cultures, we found that BDNF elicits 2-3 fold increases in DNA synthesis and numbers of myelin basic protein + (MBP) cells. Using in vivo studies we found that BDNF knockout animals express 38 percent fewer NG2 oligodendrocyte progenitor cells than wild-type littermates and that BDNF +/_ adult mice exhibit reduced MBP expression. Moreover, coimmunocytochemical analysis revealed that the BDNF receptor, trkB, is expressed on mature APC+ OLGs in adult mice. These observations support the possibility that OLGs are critically regulated by BDNF. To extend these studies we now propose to 1) test in vivo relevance of the culture work and (2) determine signaling mechanisms underlying BDNF actions. In particular, we will: (1) Identify receptors and signaling pathways mediating BDNF effects, (2) Define the in vivo expression pattern of trkB in OLGs and (3) Define the roles of BDNF and BDNF receptors in vivo by using knockout mice. These studies explore the role BDNF may play in the development and maintenance of OLGs in vivo. We suggest that this work may provide significant new insights into deficits in myelination that occur in devastating neural diseases characterized by the loss of oligodendrocytes and an inability to repopulate the lesioned area. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The central objective of this program project is to define integrative principles governing the diverse processes of brain development and plasticity. We hypothesize that reciprocal stem cell differentiation, and neuron-neuron and neuron-Glial-neuron interactions, mediated by a limited set of inter- and intra-cellular signals, coordinate gene expression and seemingly unrelated developmental events. Moreover, specific genes, such as Rab3A, regulate trophin-induced synaptic plasticity. Specifically: a) trophic factors, including the diffusible neurotrophin gene family members, b) growth (mitogenic) factors, including bFGF and IGF-1, mediated by cyclins, CDKs and CKIs, c) membrane-bound cellular labels of the Eph gene family, d) neurotransmitters and the hormone, estradiol and e) the newly discovered intracellular trophin transduction molecule ARMS, working combinatorially, synchronize the developmental sequence. These molecular signals coordinate stem cell commitment and differentiation, neuronal mitosis, selective survival, axonal pathfinding, topographic projection, synaptogenesis and synaptic plasticity. We will employ multidisciplinary molecular genetic, transcriptional, biochemical and morphologic approaches at the population and single cell levels to study development and plasticity in vivo and in culture. We plan to define a) epigenetic regulation of stem cell and precursor mitosis and differentiation, b) the roles of bFGF and IGF-1 in cortical development, c) the role(s) of ARMS in mediating p75 and trk neurotrophin receptor actions in development and plasticity, d) the actions of Eph ligands and receptors in axon defasciculation and synaptogenesis in targets, e) trophic regulation of genes at the single cell level regulating synaptic plasticity and synaptogenesis, and f) the role of astrocyte-neuron interactions in brain development.

CORE ABSTRACT NOT PROVIDED

The Administrative Core will be directed by Dr. Dreyfus and run by Ms. Sohair Ibrahim, Budget Analyst, who has been associated with this Program Project since 1990 when it moved from Cornell Medical College to UMDNJ-RWJMS. Ms. Ibrahim will be responsible for the fiscal management of the Program Project. She will be assisted by Betty Wheeler who will be responsible for purchasing of supplies for the Program Grant, the processing of all receipts and bills and playing a central role in day-to-day expenditures. Ms Wheeler will also support the publication of manuscripts

DESCRIPTION (provided by applicant): The Program Project hypothesizes that the processes of progenitor proliferation, neural differentiation, axon extension and synapse formation are regulated by neuron-neuron and neuron-glial interactions. These cell-cell interactions, in turn, are mediated through the actions of multiple intercellular and intracellular signals that impact seemingly unrelated developmental events. Our studies will elucidate molecules and mechanisms that regulate proliferation, survival, differentiation and synaptogenesis in the developing brain. They focus on the roles of BDNF and Eph family members and lead us to increased understanding of how to effect brain repair and regeneration. Importantly, the studies move from the culture dish, to the developing brain in vivo. Moreover, they extend the work to analysis of a neurotherapeutic molecule used for women of child bearing age and young children. To achieve our goals, Project 1 will examine mechanisms by which the anticonvulsant, valproic acid affects development of neurons and glia, ranging from proliferation control to differentiation and release of BDNF from neurons and glia. Project 2 will explore an emerging field of signal interaction between Eph and Trk family receptors. Project 3 will define mechanisms underlying synaptic plasticity by examining the roles of cytoskeletal structures in transmitter receptor trafficking and spine enlargement/formation and shrinkage and their regulation by intracellular kinases and BDNF. Project 4 will explore mechanisms underlying the processing and release of pro- and mature isoforms of BDNF from neurons. Project 5 will examine mechanisms underlying astrocyte-neuron interaction mediated by the release and synthesis of BDNF. In sum, these interactive projects are designed to explore roles of BDNF in development and disease. Understanding the mechanisms underlying these processes may provide insights into therapeutic interventions that will impact diseases of the developing brain.