Laboratory project: "Cell Recognition and Synapse Formation"
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5 (561,735 Bytes)
1984-09 (September 1984)
Nirenberg, Marshall W.
Trisler, G. David
National Heart, Lung, and Blood Institute. Laboratory of Biochemical Genetics
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From Neuroblastoma to Homeobox Genes, 1976-1992
Annual Report of the Laboratory of Biochemical Genetics, October 1, 1983 - September 30, 1984 (September 1984)
Project Number: Z01 HL 00009-10 LBG
Period Covered: October 1, 1983 - September 30, 1984
Title of Project: Cell Recognition and Synapse Formation
Marshall Nirenberg, Chief, LBG, NHLBI
David Trisler, Staff Fellow, LBG, NHLBI
Dana Hilt, Staff Fellow, LBG, NHLBI
Maria Giovanni, Staff Fellow, LBG, NHLBI
Hemin Chin, Guest Worker, LBG, NHLBI
Karl Krueger, Staff Fellow, LBG, NHLBI
Patricia Bray, Biologist, LBG, NHLBI
Hsi-Ping Li, Fogarty Fellow, LBG, NHLBI
Gerald Grunwald, Staff Fellow, LBG, NHLBI
Cooperating Units (if any):
William Strauss, Staff Fellow, LDN, NICHD
Victor Ginsberg, Biochemist, LBP, NIADDK
Lab/Branch: Laboratory of Biochemical Genetics
Section: Section of Molecular Biology
Institute and Location: NHLBI, NIH, Bethesda, Maryland 20205
Total Man Years: 12
Summary of Work:
Undifferentiated NG108-15 neuroblastoma-glioma hybrid cells or NS20-Y neuroblastoma cells can be shifted to a differentiated
state by increasing levels of cellular cAMP for several days. cDNA libraries were constructed for species of mRNA that increase
in abundance during differentiation. Elevation of cAMP levels of NG108-15 cells for several days results in the expression
of new glycoproteins, the disappearance of others, changes in the apparent abundance of some glycoproteins and shifts in the
p1 of some glycoproteins. The abundance of a voltage-sensitive calcium channel glycoprotein increases during differentiation.
A large NG108-15 cDNA library was constructed in [lambda]gtll, for use in cloning cDNA for choline acetyltransferase. A monoclonal
antibody that recognizes a plasma membrane protein in chick retina cells that is distributed in a dorsal-ventral gradient
in retina was injected into chick embryos in ovo. A marked reduction in synapses and neurites in the inner synaptic layer
of the retina was observed. cDNA was synthesized frown 14 day chick embryo retina RNA and cloned in [lambda]gtll for use
in cloning cDNA for the gradient protein. Many monoclonal antibodies were obtained that recognize antigens in the developing
nervous system and some of the antigens were characterized. Several antibodies recognize novel gangliosides. A protein was
purified from bovine brain that induces neurite outgrowth at nM concentrations and stimulates the phosphorylation of proteins
in the absence of Ca2+ or cAMP.
To identify and characterize molecules involved in synapse formation and function and to elucidate mechanisms that regulate
gene expression for proteins that are required for synapses.
Populations of relatively undifferentiated NG108-15 neuroblastoma-glioma hybrid cells or NS20-Y neuroblastoma cells can be
shifted a more differentiated state by increasing the levels of cellular cAMP for several days. In previous studies the differentiated
cells were shown to possess functional voltage sensitive channels for Na+, K+, and Ca2+, a Ca2+-dependent K+ channel that
is not voltage-sensitive, long neurites, and small clear vesicles and large dense-core vesicles; whereas, these components
were absent or were reduced in undifferentiated cells. Differentiated cells also exhibit higher specific activities of choline
acetyltransferase and acetylcholinesterase, secrete more acetylcholine when stimulated, and form more synapses with striated
muscle cells than do undifferentiated cells.
During the past year, poly A+ RNA was obtained from differentiated NG108-15 and NS20-Y cells, cDNA was synthesized and then
cloned using plasmid pBR322 as the vector. cDNA corresponding to species of poly A+ RNA that are more abundant in differentiated
cells than in undifferentiated cells were purified by repetitive hybridization with poly A+ RNA from undifferentiated cells;
single-stranded nucleic acids then were separated from double-stranded nucleic acids. The species of cDNA that increase in
abundance as cells differentiate were cloned. Some clones will be used as probes to study the mechanisms of cAMP-dependent
regulation of mRNA in neuroblastoma cells.
Prolonged elevation of cellular cAMP results in an increase in at least one species of protein that is part of the voltage-sensitive
calcium channel complex. Concomitantly, cells acquire functional voltage-sensitive calcium channels. Voltage-sensitive calcium
channel proteins were purified extensively. The cDNA libraries that have been constructed will be screened for clones that
correspond to the channel proteins.
Our previous analysis of glycoproteins synthesized by NG108-15 cells grown in the presence of PGE1, an activator of adenylate
cyclase, or in the absence of PGEl, was extended during the past year. The cells were incubated with [35S]-methionine for
18 hours, solubilized, and the glycoproteins fractionated by wheat germ agglutinin-, ricin-, or lentil lectin-affinity column
chromatography and then by 2-dimensional polyacrylamide gel electrophoresis. 35S-Glycoproteins detected by autoradiography
were compared with those detected by silver staining. Both methods of analysis showed that elevation of intracellular cAMP
levels for several days results in the expression of new glycoproteins, the disappearance of others, changes in the apparent
abundance of some glycoproteins, and shifts in the pI of some glycoproteins. However, silver staining revealed many glycoproteins
that were not detected by autoradiography, including additional glycoproteins that were expressed only by PCE1-treated cells.
These results suggest that elevation of cAMP levels of NG108-15 cells for several days affects the expression of genes for
some glycoproteins and alters the post-translational modification of other glycoproteins. Current studies focus on the purification
of sufficient amounts of some regulated glycoproteins to obtain partial amino acid sequences of the proteins and to obtain
antibodies that recognize the proteins. The cDNA libraries that have been generated will be screened for cDNA that correspond
to some regulated species of glycoproteins.
Whether a peripheral neuroblast will give rise to sympathetic or parasympathetic neurons during differentiation is determined
by mechanisms that regulate the expression of the genes for tyroslne hydroxylase and choline acetyltransferase, respectively.
An extracellular protein and calcium ions are known to be involved in the regulation of these genes, but the mechanisms of
regulation are unknown. Previously, choline acetyltransferase from rat brain was purified to essential homogeneity and 4
monoclonal antibodies that recognize the enzyme were obtained. A large NG108-15 cDNA library was constructed using the bacteriophage.expression
vector, [lambda]gtll. Possible cDNA clones that direct the synthesis of choline acetyltransferase in E. coli have been detected,
but further work is needed to establish the identity of the clones.
A monoclonal antibody was obtained previously that recognizes a large dorsal-ventral concentration gradient of a protein in
plasma membranes of chicken retina cells. The amount of protein detected is a function of the position of the cells in retina
with respect to the dorsal-ventral axis of the retina. The protein is synthesized by proliferating neuroblasts and by nondividing
neurons and the gradient is formed as the retina is formed. The protein was detected on all cells examined in dorsal and
middle retina. Cells that were dissociated from retina and cultured in vitro express the amount of gradient protein that
would be expected of cells in the intact retina depending upon the original position of the cells in the retina. These results
suggest that the gradient is established by an irreversible, clonally inherited mechanism and that once established, the gradient
is perpetuated independently by each cell thereafter.
Monoclonal antibody that recognizes the gradient protein, or hybridoma cells synthesizing the antibody, were injected into
the amniotic cavity of chick embryos in ovo from the second to the fifth day after fertilization and into the vitreal space
of chick embryo eyes to determine whether the antibody affects the development or the spatial organization of the retina.
The retinas of embryos were continuously exposed to antibody throughout development from the second to the twentieth day after
fertilization. Injection of antibody to the gradient protein into the eye resulted in a marked reduction of synapses and
neurites in the inner synaptic layer of the retina; whereas, antibodies synthesized by parental P3X63 Ag8 myeloma cells had
RNA was isolated from 14 day chick embryo retinas and a large cDNA library was constructed in [lambda]gtll that can be used
to direct the synthesis of proteins specified by the cDNA in E. coli. The library currently is being screened for recombinants
that direct the synthesis of the gradient protein. Injection of poly A+ RNA from retina into Xenopus laevis oocytes resulted
in she synthesis of the gradient protein. This assay can be used for the purification of mRNA for the gradient protein.
The cDNA library also is being screened for transducin subunits in collaboration with A. Spiegel.
Seventy-six hybridoma cell lines were generated that synthesize monoclonal antibodies that bind to 8 day chick embryo optic
tectum. Fifteen hundred hybridoma lines were generated from spleen cells of mice immunized with the cervical-thoracic spinal
cord and dorsal root ganglia of 8 day chick embryos. Some of the hybridoma lines synthesize antibodies that recognize antigens
that are restricted to fiber tracts or neuronal cell body regions of the spinal cord.
Additional information was obtained about other antigens that are recognized by monoclonal antibodies. For example, antigen
13H9 was shown to be a protein with an approximate M, of 180,000. The antigen is associated with cell membranes of all chick
retina cells but has not been detected on neurons or glia in other parts of the nervous system. The antigen defines a functional
set of cells in the nervous system.
18B8 antigens are first expressed by ganglion neurons and then by other types of neurons in retina. The antigens are found
on cell soma initially, but later in development antigens disappear from cell soma and can be seen in a highly stratified,
multi-laminar pattern in the inner synaptic layer of the retina and in a circular "organelle" in the outer synaptic
layer. The antigens are expressed by approximately 10% of the cells in retina. In collaboration with Victor Ginsburg and
his colleagues, the antigens were shown to be novel gangliosides of unknown structure that contain disialyl residues whose
abundance and structure change during development; the location of the gangliosides in retina also changes during retinal
development. Most of the antigens are associated with the inner and outer synaptic layers of retina in late embryo and adult
retina. In addition, the antigens for many other monoclonal antibodies were characterized and in some cases were partially
A heat-stable, acidic, soluble, bovine brain protein was found that induces neurite outgrowth from chick embryo cerebral cortical
neurons at nM concentrations in defined medium. The Neurite Extension Factor (NEF) rapidly stimulates the phosphorylatfon
of a protein with an apparent Mr of 90,000 in the absence of calcium ions or cyclic nucleotides. Phosphopeptide mapping results
show that the 90,000 Mr protein is related to an 87,000 Mr protein that is a major substrate for C kinase in brain.
Significance to Biomedical Research
New information was obtained concerning synaptogenesis and synaptic functions.
1. Nirenberg, M., Wilson, S. P., Higashida, H., Rotter, A., Kreuger, K., Busis, N., Ray, R., Kenimer, J., Adler, M., and
Fukui, H.: Synapse Formation by Neuroblastoma Hybrid Cells. In: Molecular Neurobiology. Cold Spring Harbor Symposia on Quantitative
Biology XLVIII: 707-715. (1983).
2. Nirenberg, M., S. Wilson, H. Higashida, A. Rotter, K. Krueger, N. Busis, R. Ray, J. G. Kenimer, and M. Adler.: Modulation
of Synapse Formation by Cyclic Adenosine Monophosphate. Science 222: 794-799. (1983).
3. de Blas, A., Adler, M., Shih, M., Chiang, P. K., Cantoni, G. L., and Nirenberg, M.: Inhibitors of CDP-choline synthesis,
action potential calcium channels, and stimulus-secretion coupling. Proc. Natl. Acad. Sci. 81: 4353-4357. (1984).
5. Trisler, D., Grunwald, G.B., Moskal, J., Darveniza, P., and Nirenberg, M.: Molecules that identify cell types or position
in the retina. In: Neuroimmunology. Behan, P.O. and Spreafico, F. (Eds.). New York, Raven Press, 89-97. (1984).
6. Nirenberg, M., Krueger, K. Rotter, A., Wilson, S., and Higashida, H.: Regulation of Synapse Formation by Cyclic AMP: In:
The Symposium of the International Society for Developmental Neurosciences. In Press.
7. Fredman, P., Magnani, J. L., Nirenberg, M., and Ginsburg, V.: Monoclonal antibody A2B5 reacts with many gangliosides in
neuronal tissue. Archives of Biochemistry and Biophysics. In Press.
8. Strauss, W. L. and Nirenberg, M.: Inhibition of choline acetyltransferase by monoclonal antibodies. Journal of Neuroscience.