Student Theses and Dissertations
Date of Award
1971
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Bruce McEwen
Keywords
goldfish retina, axonal transport, glycoproteins, 3H-fucose labeling, ganglioside synthesis, retinal ganglion cells
Abstract
The goldfish retino-tectal pathway is a convenient experimental system for studying axonal transport. Radioactive precursors are injected into the eye, and radioactivity which is transported in the axons of the retinal ganglion cells can be studied as it moves in the optic nerve and optic tract to the optic tectum, where the optic fibers terminate. For instance, when labeled amino acids are injected into the goldfish eye, labeled proteins synthesized in the retinal ganglion cells are axonally transported in the optic fibers. Two main waves of protein are transported: most of the labeled protein moves slowly at a rate of about 0.4 mm/day, and much of this slowly transported protein is soluble. A smaller fraction of the transported protein moves much faster, at a rate of about 70-100 mm/day, and most of this rapidly transported protein is particulate. Free amino acids are not transported. The axonal transport of proteins labeled with radioactive amino acids has been studied extensively. However, the axonal transport of glycoproteins labeled with radioactive sugars had not previously been examined. In the experiments reported here, [3H]D-glucosamine and [3H]L-fucose were injected into the goldfish eye. Both sugars are incorporated into macromolecules which are rapidly transported in the optic fibers. The transported glycoproteins move at the same rate as proteins labeled with radioactive amino acids, and this rate is about 70-90 mm/day. Very little of the transported glycoprotein is soluble; most of the transported macromolecules are tightly bound to sedimentable particles. In contrast to the results with amino acids, there is no evidence of a slow component. Inhibition of retinal protein synthesis with a small intraocular dose of acetoxycycloheximide (AXM) prevents the subsequent arrival of labeled transported macromolecules in the tectum, which is evidence that the labeled glycoproteins are synthesized in the retina. Unlike amino acids, the sugar precursors also label a transported TCA-soluble component. Since it is small compared to the macromolecular component, and lags behind the transported macromolecules, the transported TCA-soluble radioactivity does not appear to be a precursor for the local synthesis of the macromolecular component. [3H]Glucosamine also labels transported chloroform-methanol extractable materials. [3H]Fucose is an especially favorable precursor for studying the axonal transport of glycoproteins, because it produces very little "background" labeling, and does not label lipids. More than 90% of the radioactivity which can be released from the transported labeled glycoproteins by acid hydrolysis moves during thin-layer chromatography with the mobility of free fucose. After an intraocular injection, [3H]fucose is incorporated into transported macromolecules for a much longer time than labeled amino acids, and as a result, transported [3H]fucosyl glycoproteins continue to arrive in the tectum after the transport of amino acid-labeled proteins has ceased. N-[3H]acetyl-D-mannosamine, a precursor of the sialic acids, labels rapidly transported TCA-precipitable, TCA-soluble, and chloroform-methanol extractable materials. With N-[3H]acetylmannosamine, unlike [3H]glucosamine, there is more transported radioactivity in the chloroform-methanol extract than in macromolecules. After an intraocular injection of N-[3H]acetylmannosamine, rapidly transported radioactivity is found in highly purified gangliosides isolated from the tecta. Thus, gangliosides as well as glycoproteins are synthesized in the retinal ganglion cell body and then axonally transported. These experiments do not exclude the possibility that some incorporation of carbohydrate into glycoproteins and gangliosides may occur in the axons and nerve endings. It is clear, however, that at least some glycoproteins and gangliosides are first synthesized in the neuron cell body, and then axonally transported.
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Recommended Citation
Forman, David Sholem, "Axonal Transport of Glycoproteins and Glycolipids in the Goldfish Optic System" (1971). Student Theses and Dissertations. 557.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/557
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy