Herbert M. Geller - US grants

The long-term objectives of this project are to understand the mechanisms by which histamine can influence the activity of mammalian central neurons, and thereby influence endocrine function. These objectives are to be attained through the use of electrophysiological and pharmacological investigations on neurons of the rat central nervous system. The preparations to be used include long-term organotypic cultures of neonatal rat hypothalamus, slices of adult rat hypothalamus, as well as the intact rat cerebral cortex in situ. Electrophysiological parameters will be recorded both intracellularly and extracellularly, while drugs will be applied systemically or locally by iontophoresis or pressure. A detailed understanding of the cellular effects of histamine should permit a commensurate increase in knowledge about the behavioral and endocrine consequences of the utilization of drugs with affect histaminergic systems. These include conventional antihistamines, which are nonprescription drugs, as well as cimetidine, one of the most widely prescribed therapeutic agents which antagonizes histamine and is known to alter hormone levels in humans.

The objective of this project is to increase our understanding of the role of genetic and epigenetic and epigenetic influences on neuronal growth and development in the mammalian central nervous system. The development of neurons of the hypothalamus, a region of the brain involved in homeostatic regulation, will be followed in tissue culture. Dissociated neurons from the embryonic rat hypothalamus will be cultured in serum- free conditions using a method we have developed, and their growth and development monitored using quantitative morphometric techniques. The expression of specific neuronal phenotypes and of neurotransmitter receptors will be monitored using appropriate monoclonal and monospecific antibodies. Alterations in these parameters as a result of treatment with hormones and growth factors and by different culture substrates will provide evidence for a specific epigenetic influence of these treatments; a stable pattern of gene expression will provide evidence for genetic control. The effect of the culture procedure on neuronal survival and development will also be explored using 3H-thymidine as a marker of neuronal "birthdate". Alterations in neuronal survival or neuronal phenotype as a function of the timing of dissociation will be evaluated. The result of these experiments will be an appreciation of effects of the extracellular environment, including growth factors and hormones, on hypothalamic neuronal development.

This is an application requesting purchase of a laser scanning confocal microscope for use in cell biological and neurobiological research projects at the UMDNJ-Robert Wood Johnson Medical School. Laser scanning confocal microscopy has become an increasingly valuable technique for high-resolution localization and reconstruction of fluorescently-tagged cellular proteins using immunofluorescence methods. Other fluorescent probes are also available for the localization and measurement of cellular calcium, pH and membrane potential. These advances are particularly important to the fields of cell biology and neurobiology because they allow increased understanding of the relationship of cellular constituents to cellular development and function. The equipment requested includes a Bio-Rad MRC-600 laser scanning confocal head and Zeiss Axioplan microscope. The equipment is to be used for the high-resolution localization and three-dimensional reconstruction of the distribution of cellular proteins by a group of highly-qualified and well-funded investigators. Seven major NIH-funded users are identified whose projects concern 1) cell surface proteins on neurons and glia; 2) the molecular basis of lysosomal enzyme targeting; 4) the genetic analysis of the role of tubulin and other proteins in mitosis; 4) role of cell proliferation in nervous system development; 5) the localization of mannose-6-phosphate receptors during neural development; 6) the modulation of cell structure by the adenovirus E1A product; 7) molecular analysis of myosin synthesis and assembly in muscle development. Each of these projects will be advanced significantly by the requested equipment. The research community on the Robert Wood Johnson Medical School/Rutgers University campus consists of over 100 NIH-funded investigators. There is no laser scanning confocal microscope available on this campus. Therefore, in addition to the Major Users, we have projected that approximately 30% of time will be available to these investigators to extend their research through the use of laser scanning confocal microscopy, three dimensional reconstruction and mathematical analysis of the co-localization of cellular proteins. We have included abbreviated descriptions from five of these to indicate the value of the instrument to the research community.

This is a competing continuation grant seeking five years of support. The objectives of the present grant period were to investigate the effects of soluble trophic factors and glial cell surface molecules in regulating the survival and development of hypothalamic neurons. In the next project period we will extend and amplify this work to the role of neural/glial interactions in shaping the development and regeneration of the nervous system. We have identified a population of type 1 astrocytes (named "rocky") which are strongly inhibitory to neuronal adhesion and growth, in contrast to the vast majority of "flat" astrocytes which are permissive to both neuronal adhesion and growth. The rocky astrocyte extracellular matrix (ECM) contains high levels of tenascin and chondroitin sulfate-6- proteoglycan (CS-6-PG), molecules that have been associated with boundary formation in the brain. In response to certain cytokines, flat astrocytes synthesize tenascin and CS-6-PG and become unfavorable for neuronal growth, resembling rocky astrocytes. We propose that, during development and following injury, cytokines have significant actions on astrocytes to alter their expression of cell surface molecules that then alter neuronal migration and neuronal growth. We have three related Specific Aims. Aim 1 will test the hypothesis that cytokines alter permissive flat astrocytes in ways that make them inhibitory to neuronal growth and regeneration. Cultured rat cerebral cortical astrocytes will be exposed to gamma-IFN, TNF-alpha, and TGF-beta1. We will evaluate changes in the composition of the ECM of these astrocyte monolayers using Western blotting and immunocytochemical techniques and also evaluate the adhesion and outgrowth of dissociated embryonic neurons to these cells as well as to ECM preparations of these cells. The dynamic movement of neuronal growth cones on these monolayers will be evaluated using time-lapse video enhanced microscopy and laser scanning confocal microscopy. Specific Aim 2 will test whether the expression of human tenascin in rat astrocytes (from a transgene) leads directly to inhibition of neuronal growth in vitro. Neuronal adhesion and growth will be evaluated on cells with different levels of tenascin expression. We will also test the hypothesis that the balance of expression of permissive vs. non-permissive ECM molecules on cells regulates neuronal adhesion and growth. Finally, we will evaluate the effects of the expression of the alternately spliced variants of human tenascin. Specific Aim 3 will test the effects of blocking tenascin and CS-6-PG with antibodies in order to evaluate the hypothesis that these ECM components have a synergistic, inhibitory effect on neuronal adhesion and neurite outgrowth. We will also evaluate whether the sugar or the protein moieties of the proteoglycans are involved in modulation of neuronal adhesion/growth. We will also test the hypothesis that blocking tenascin and CS-6-PG will reduce or eliminate the ability of growth cones to sense boundaries between permissive and non-permissive astrocytes. The experiments proposed provide a unified approach to a major problem of neural/glial interactions during development and may also apply to the lack of regeneration following injury.

This research to investigate the underlying molecular mechanisms that trigger neuronal apoptosis after DNA damage. While apoptosis is a cardinal feature of programmed cell death during neural development , it has been increasingly recognized that neurons also undergo apoptosis in response to injury. DNA damage produces apoptotic death in cultured mammalian central nervous system (CNS) neurons. Cell death is inhibited by two drugs which inhibit cyclin-dependent kinases. These drugs also protect neurons against apoptotic cell death after trophic factor withdrawal. This suggest that apoptosis in neurons due to both toxic injury and lack of trophic support required enzymes that normally control the cell cycle. Investigating this hypothesis is the major focus of this application. The first two specific aims will determine the role of immediate early genes and cycle-related proteins in apoptosis of cultured rat CNS neurons after DNA damage by irradiation or treatment with camptothecin. We will first determine the changes in immediate early genes (IEGs) and cell cycle-related proteins (cyclin- dependent kinases, Cdks) following DNA damage. We will then determine whether apoptosis is reduced by microinjection of antibodies directed against the IEGs that are increased following DNA damage or by injection of inhibitors or dominant negative mutants the Cdks and also by treatment with antisense oligonucleotides against the cyclins and Cdks that are increased following DNA damage. We will also evaluate the role of the p53 protein in neuronal apoptosis by utilizing neurons from p53 knockout mice. We will also utilize a series of Cdk-inhibitor drugs to determine a structure/activity relationship between Cdk inhibitory activity and reduction of neuronal apoptosis due to DNA damage and also for their ability to produce apoptosis in dividing PC12 cells. Finally, since campotothecin toxicity is reduced by inhibition of transcription, we will evaluate whether inhibition of transcription reduces toxicity of campothecin by reducing 1)DNA damage by camptothecin or 2) the apoptotic process itself or both. These studies should provide significant information about the underlying mechanisms that trigger apoptosis after DNA damage. Moreover, drugs that simultaneously kill dividing cells and protect neurons against toxicity from cancer chemotherapeutic agents or radiation may be exceedingly useful in therapy of brain cancers, in which survivors normally suffer a significant intellectual impairment.

We are expanding the range of assay techniques that will allow us to understand the sulfation code in chondroitin sulfate glycosaminoglycan (CS-GAG) chains. These assay techniques take advantage of specific chromatography techniques (ion exchange, hydrophilicity) to separate the different disaccharides and monosaccharides that comprise the GAG chains. This is the only technique capable of doing this. We have also initiated mass spectrometric analysis of these separated GAG chains to begin to determine the sequence of sulfations on the different parts of the GAG chain. Our results indicate that the sulfation pattern of the non-reducing end of the GAG chain is a major determinant of CS signaling. A patent was awarded based on our research and a publication describing these results is in preparation. We have conducted studies showing that the the LAR family of receptor protein tyrosine phosphatases are are binding partners for CS GAG chains. The binding of the different family members are not all the same. We have identified different regions in the extracellular domains of these molecules that bind GAG chains with different sulfation patterns. In addition our data point to an additional receptor that binds bioactive CSPGs. A publication describing these results is in preparation. We identified the protein plasticity related gene-3 (PRG-3) as one whose phosphorylation changes in response to chondroitin sulfate proteoglycans. PRG-3 is a member of a family of PRGs and our data indicate that these proteins interact to cause physiological changes in cells. A publication describing these results is in preparations. We demonstrated that astrocyte reactivity in response to TGF-β is dependent on the activity of a calcium-activated potassium channel KCa3.1. Pharmacological antagonism or knockout of this channel eliminates glial reactivity in response to TGF-β. Moreover, this is dependent upon the SMAD family of transcription factors.

The objective of this project is to develop potential approaches to the therapy of brain and spinal cord injury. One approach is to limit the production or increase the degradation of chondroitin sulfate proteoglycans (CSPGs). We have demonstrated that the enzyme arylsulfatase B (ARSB), which cleaves the 4-sulfate group from chondroitin sulfate only at the non-reducing end, can dramatically increase neuronal growth on substrates of chondroitin sulfate. We have investigated the role of CSPGS in the injured optic nerve following optic nerve crush. We have demonstrated a significant upregulation of CSPGs in the moue optic nerve. We have also shown that ARSB can significantly improve the regeneration of axons in the mouse optic nerve following optic nerve crush. A manuscript describing these results has been published.