Daniel R. Marshak

p62dok: A Constitutively Tyrosine-Phosphorylated, GAP-Associated Protein in Chronic Myelogenous Leukemia Progenitor Cells

Characteristic of chronic myelogenous leukemia (CML) is the presence of the chimeric p210(bcr-abl) protein possessing elevated protein tyrosine kinase activity relative to normal c-abl tyrosine kinase. Hematopoietic progenitors isolated from CML patients in the chronic phase contain a constitutively tyrosine-phosphorylated protein that migrates at 62 kDa by SDS-PAGE and associates with the p120 ras GTPase-activating protein (GAP). We have purified p62(dok) from a hematopoietic cell line expressing p210(bcr-abl). p62(dok) is a novel protein with features of a signaling molecule. Association of p62(dok) with GAP correlates with its tyrosine phosphorylation. p62(dok) is rapidly tyrosine-phosphorylated upon activation of the c-Kit receptor, implicating it as a component of a signal transduction pathway downstream of receptor tyrosine kinases.

Increased S100β neurotrophic activity in Alzheimer's disease temporal lobe

The confirming diagnosis of Alzheimers disease includes an assessment of the concentration of neuritic plaques in the temporal lobe of the brain. The presence of abnormal levels of neurotrophic factors in Alzheimers disease is one possible explanation for the increased concentration of aggregates of overgrown neurites in the neuritic plaques of Alzheimers disease. The protein S100 beta, a neurotrophic factor produced by astroglia in the brain, induces neurite outgrowth in cerebral cortical neurons. The generation of specific S100 beta antibodies, the cloning of a full-length cDNA encoding the S100 beta mRNA, and the development of a neurite extension assay system for S100 beta allowed testing of the hypothesis that Alzheimers disease S100 beta expression is elevated in brain temporal lobe where neuritic plaques are concentrated. The levels of S100 beta protein, mRNA, and specific neurotrophic activity were elevated 10-20-fold in extracts of temporal lobe from autopsy samples of Alzheimers disease patients compared to those of aged control patients. The cells containing the increased S100 beta were reactive astrocytes; the neuritic plaques were surrounded by S100 beta-containing astrocytes. The elevated levels of S100 beta provides a link between the prominent reactive gliosis and neuritic plaque formation in this common disease of the elderly and raises the possibility that S100 beta contributes to Alzheimers disease neuropathology.

S100 beta as a neurotrophic factor.

S100 beta is a multifunctional protein that is found in large amounts in astrocytes and a number of other tissues. In the developing nervous system, S100 beta is secreted from proliferating astrocytes during the time of neurite outgrowth from cortical neurons. The secreted form has neurotrophic activity on primary neurons and neuroblastoma cells. The neurotrophic activity is sensitive to reduction of disulfide bonds, and appears to be a disulfide dimer of S100 beta. The accumulation of S100 beta in mature glial cells is associated with microtubule network. The transfection and expression of cDNA for S100 beta in mammalian cells confers neurotrophic activity on extracts of these cells. Based on our observations of a neurotrophic activity for S100 beta, the occurrence of the gene for S100 beta on human chromosome 21 (Allore et al., 1988) and the elevated levels of S100 beta-containing cells in AD and DS brains (Griffin et al., 1989), we suggest that S100 beta plays a role in the abnormal development of the nervous system in DS and the degeneration of central neurons in AD. It is essential at the present stage to demonstrate the action of S100 beta in vivo, and to construct animal models, such as transgenic mice, that overexpress S100 beta. Such models will allow the dissection of the role of S100 beta in the developing and degenerating central nervous system.

Molecular characterization of the Schwann cell myelin protein, SMP: structural similarities within the immunoglobulin superfamily

The Schwann cell myelin protein (SMP), previously defined in quail and chick by a monoclonal antibody, is in vivo exclusively expressed by myelinating and nonmyelinating Schwann cells and oligodendrocytes. The isolation of the complete nucleotide sequence of SMP is reported here. The predicted polypeptide chain reveals that SMP is a transmembrane molecule of the immunoglobulin superfamily showing sequence similarities with several surface glycoproteins expressed in the nervous and immune systems. In spite of a 43.5% overall sequence identity between rat myelin-associated glycoprotein (MAG) and quail SMP, SMP does not seem to be the avian homolog of MAG, since their expression, regulation, and functions are significantly different. Unusual sequence arrangements shared by SMP, MAG, and two lymphoid antigens suggest the existence of a particular subgroup in the immunoglobulin superfamily.

Glial-derived S100b protein selectively inhibits recombinant β protein kinase C (PKC) phosphorylation of neuron-specific protein F1/GAP43

Protein F1/GAP43 is neuron-specific, associated with neurite outgrowth during development and a substrate for PKC. This protein is present in high levels in serotonergic neurons which in culture sprout in response to the glial-derived S100b, the beta-beta homodimer. As an initial step in determining whether S100b acts on F1/GAP43 we studied the regulation by S100b of PKC phosphorylation of F1/GAP43. Either the S100b or a mixture of S100a and S100b, both from a brain glial cell source, inhibited in vitro phosphorylation of purified F1/GAP43 by purified PKC in a dose-dependent manner. Using recombinant PKC subtypes, purified S100b preferentially inhibited the F1/GAP43 phosphorylation by the beta subtype. The IC50 of S100b for beta I and beta II PKC was 8 microM while for alpha and gamma PKC it was 64 microM. S100b inhibition was thus subtype-selective. Histone III-S phosphorylation by the four PKC subtypes was not inhibited by S100b. S100b inhibition was thus substrate-selective. Moreover, the effect of S100b on phosphorylation could not be explained by a direct inhibition of kinase activity. Together with earlier studies implicating a role for S100 in synaptic plasticity and neurite outgrowth, the present results suggest that S100b may regulate such functions through its inhibition of neuron-specific PKC substrate (F1/GAP43) phosphorylation. The regulation of this neuron-specific substrate phosphorylation by glial S100 suggests the potential for a novel neuro-glial interaction. Finally, the location of S100 gene on chromosome 21, trisomic in Downs syndrome, and over-expressed in this disorder, as well as in Alzheimers disease, suggests a link to cognitive impairments in human.

β-Amyloid regulates gene expression of glial trophic substance S100β in C6 glioma and primary astrocyte cultures

S100β, a calcium-binding protein synthesized by CNS astrocytes, has trophic effects in vitro (neurite extension and glial proliferation). In Alzheimers disease and Downs syndrome, severely afflicted brain regions exhibit up to 20-fold higher levels of S100β protein, and astrocytes surrounding neuritic plaques exhibit highly elevated levels of S100β immunostaining. A major constituent of plaques, β-amyloid, has been reported to have neurotoxic and neurotrophic effects in vitro. In our study we examined the responses of CNS glia to β-amyloid. C6 glioma cells and primary rat astrocyte cultures were treated with βA(1–40) peptide at doses up to 1 μM. Weak mitogenic activity, measured by [3H]thymidine incorporation, was observed. Northern blot analysis revealed increases of S100β mRNA within 24 h in a dose-dependent manner. Nuclear run-off transcription assays showed that βA(1–40) specifically induced new synthesis of S100β mRNA in cells maintained in serum, but under serum-freconditions, there was a general elevation of several mRNA species. Corresponding increases of S100β protein synthesis were observed by immunoprecipitation of 35S-labeled cellular proteins. To evaluate whether this effect of β-amyloid was mediated via neurokinin receptors or by calcium fluxes, various agonists and antagonists were tested and found to be ineffective at stimulating S100β synthesis. In sum, these in vitro data suggest that in neuropathological conditions, β-amyloid itself is an agent which may provoke chronic gliosis and the production of trophic substances by astrocytes.

Antipeptide antibodies against the 5-HT1A receptor.

The availability of the primary amino acid sequence for a large number of molecules provides a fruitful opportunity for their cellular localization by utilizing the procedure of antipeptide antibody formation. This procedure permits a synthetic peptide sequence to be attached to a carrier molecule for the purpose of inoculating an animal to raise specific antibodies against the selected protein sequence. In this report we describe a number of steps that can be taken to increase the likelihood that the selected peptide sequence will be specific and antigenic. In addition, we describe how the peptides are synthesized, purified and coupled to keyhold limpet hemocyanin. The preparation of the antibody and its characterization are also presented in this method report. The immunocytochemical staining at both the light and ultrastructural level with serotonin (5-HT1A) receptor antipeptide antibodies is discussed. The advantages and disadvantages of this procedure are summarized.

Chapter 39: S100β and serotonin: a possible astrocytic-neuronal link to neuropathology of Alzheimer's disease

Publisher Summary The chapter discusses a possible role for Sl00β and serotonin (5-HT) as an astrocytic (S100β)/ neuronal (serotonin; 5-HT) link to neuropathologies associated with Alzheimers disease. Sl00β is a protein produced and released from astrocytes in the brain. Extracellular Sl00β can function as a neurotrophic factor to select central and peripheral neurons and a gliotrophic factor, inducing mitosis and morphogenesis of astrocytes. Release of S100β from astrocytes can be stimulated by serotonin acting through 5-HT1, receptors on astrocytes, and S100β functions as a serotonergic neurite extension factor. The abnormal regulation of Sl00β levels or of serotonin-stimulated release of Sl00β in Alzheimers disease contributes to the cascade of events that with aging lead to neuropathologies. It is also suggested that this astrocytic (S100β)/ neuronal (serotonin) link may contribute to neuropathological changes observed not only in Alzheimers disease, but also in Down syndrome.

Umbilical Cord Transforming Growth Factor-β3: Isolation, Comparison with Recombinant TGF-β3 and Cellular Localization

AbstractThe transforming growth factor beta (TGF-β) family of growth modulators play critical roles in tissue development and maintenance. Recent data suggest that individual TGF-β isoforms (TGF-β1, -β2 and -β3) have overlapping yet distinct biological actions and target cell specificities, both in developing and adult tissues. The TGF-β3 isoform was purified to homogeneity from both natural and recombinant sources and characterized by laser desorption mass spectrometry, by protein sequencing, by amino acid analysis and by biological activity. TGF-β3 was the major TGF-β isoform in umbilical cord (230 ng/g), and was physically and biologically indistinguishable from recombinant TGF-β3 and from the tumor growth inhibitory (TGI) protein found in umbilical cord. Immunohistochemistry using antipeptide TGF-β3 specific antibody showed TGF-β3 localization in perivascular smooth muscle.

Cytokines in Human Neurodegenerative Disease

Publisher Summary Cytokines—soluble factors of an information network—are produced by certain cell types and induce specific functions in responder or target cells. Two brain-derived factors, interleukin 1 (IL-1) and S100, are considered as early contributors to a cascade of neurodegenerative events that lead to the neuropathophysiological changes that characterize Alzheimers disease (AD), Down syndrome, and AIDS. This chapter provides an overview of the methods used to study the cellular expression of S100 and IL-1 in samples of temporal lobe of brain from individuals with those neurodegenerative diseases. It describes many functions of IL-1: namely, the macrophage-derived posttranslationally unmodified intracellular α isoform, and the initiation of number of cell-mediated immune responses by cleaved α and β isoforms of IL-1. The chapter also discusses methods to measure the levels and activities of S100 and IL-1 in human brain in health and disease. Prominent proliferation and morphogenesis of astrocytes, overgrowth of neuronal processes, paired helical filaments in neurons, and calcium-induced neuronal cell death are neuropathological changes characteristic of neurodegeneration.