Axonal transport of phospholipids in rabbit optic pathway

The uptake of different labeled precursors, their incorporation into lipids, and transport along the rabbit optic pathway [ipsilateral retina and optic nerve (ON), and contralateral optic tract (OT), lateral geniculate body (LGB), and superior colliculus (SC)] were investigated. Albino rabbits were used. The following radioactive precursors, either combined or separately, dissolved in 50 l of saline containing 15% BSA, were injected into vitreous body: [2-³H]glycerol (50 Ci), [1-¹⁴C]palmitate (15 Ci), and [1-¹⁴C]linoleate (7.5 Ci). Animals were killed at different time intervals from 1 hr up to 24 days. The radioactivity of total lipids and of different phospholipid classes from total tissue was measured. One hour after the administration of precursors, the radioactivity into the retina was high and the incorporation of [³H]glycerol and [¹⁴C]palmitate increased until 12 hr and 24 hr, respectively. The incorporation of [¹⁴C]linoleate reached a maximum on the second day. The phospholipids of LGB and SC were intensively labeled after 4–8 hr, and their radioactivity increased up to the 10th day after injection, independent of the precursor employed. The results obtained indicate that the labeled hydrophilic and hydrophobic precursors used were actively incorporated into the retina. The phospholipids were later transported at a rapid rate along the optic pathway.A preliminary report of this study has been presented at the Satellite ISN Meeting, Istanbul, September 8–10, 1979.     

Axonal pathology in myelin disorders

Myelination provides extrinsic trophic signals that influence normal maturation and long-term survival of axons. The extent of axonal involvement in diseases affecting myelin or myelin forming cells has traditionally been underestimated. There are, however, many examples of axon damage as a consequence of dysmyelinating or demyelinating disorders. More than a century ago, Charcot described the pathology of multiple sclerosis (MS) in terms of demyelination and relative sparing of axons. Recent reports demonstrate a strong correlation between inflammatory demyelination in MS lesions and axonal transection, indicating axonal loss at disease onset. Disruption of axons is also observed in experimental allergic encephalomyelitis and in Theiler's murine encephalomyelitis virus disease, two animal models of inflammatory demyelinating CNS disease. A number of dysmyelinating mouse mutants with axonal pathology have provided insights regarding cellular and molecular mechanisms of axon degeneration. For example, the myelin-associated glycoprotein and proteolipid protein have been shown to be essential for mediating myelin-derived trophic signals to axons. Patients with the inherited peripheral neuropathy Charcot-Marie Tooth disease type 1 develop symptomatic progressive axonal loss due to abnormal Schwann cell expression of peripheral myelin protein 22. The data summarized in this review indicate that axonal damage is an integral part of myelin disease, and that loss of axons contributes to the irreversible functional impairment observed in affected individuals. Early neuroprotection should be considered as an additional therapeutic option for these patients.     

Transfer of axonally transported phospholipids into myelin isolated from the rabbit optic pathway

The contribution of the axonal transport to the biosynthesis of myelin phospholipids was investigated in the rabbit optic pathway. A double labeling technique was used. The same animals were injected with one isotope intravitreally and the other intraventricularly. This procedure allows double labeling of the optic nerves, optic tracts, lateral geniculate bodies (LGB), and superior colliculus (SC). The precursors simultaneously injected were: [1-¹⁴C]palmitate (15 Ci intravitreally in both eyes or 50 Ci intraventricularly) and [2-³H]glycerol (50 Ci intravitreally in both eyes or 100 Ci intraventricularly). Twenty four hours and 10 days after the injections, myelin was purified from pooled optic nerves and optic tracts as well as from pooled LGBs or SCs. The phospholipids were extracted and then separated by thin-layer chromatography; the specific radioactivity of the various classes of phospholipids was determined. Using both administration routes of¹⁴C-or³H-precursors, the distribution of label and specific radioactivity of myelin phospholipids in the retina and in all other optic structures were very similar. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine + phosphoinositol were preferentially labeled with both precursors. These results suggest that, in the rabbit optic pathway the phospholipids synthesized in the retinal ganglion cells and transported along the axons, could undergo transaxonal transfer into myelin.     

Axonal composition of the marginal and basal optic tracts in frogs

An electron-microscopic investigation was made of the medial and lateral branches of the marginal and basal optic tracts. The ultrastructure and composition of the branches of the marginal tract are similar. The basal tract has a relatively loose structure and contains a high proportion of myelinated fibers of large diamter. On average these structures contain 3900 (4.7%), 4700 (4.9%), and 700 (28%) of myelinated and 79,800 (95.3%), 91,500 (95.1%), and 1800 (72%) unmyelinated fibers respectively. The myelinated fibers in the branches of the marginal tract have a diameter of 0.4–2.6 (main maximum 1.0, additional maximum 1.6 µ), those in the basal tract from 0.4 to 4.0 µ (main maximum 1.8, small additional maximum at 3.2 µ). The diameters of the unmyelinated fibers in all three tracts are 0.1–0.5 µ; the diameter of 60% of these fibers is approximately 0.2 µ. Degeneration of 99% of myelinated fibers of the optic nerve and 100% of fibers of the basal tract was found 85 days after enucleation and keeping at 18–20°C. Many nondegenerated myelinated fibers were found in the medial and lateral branches of the marginal tract (33 and 13%, ranges of diameters 0.6–1.4 and 0.6–1.0 µ respectively). These fibers perhaps participate in the organization of ipsilateral visual projection of the tectum. The nonmyelinated fibers were unchanged in all the tracts.A. N. Severtsov Institute of Evolutionary Morphology and Ecology of Animals, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 8, No. 1, pp. 54–61, January–February, 1976.     

Axonal transport of lipid in goldfish optic axons

After injection of labeled glycerol, choline, or serine into the eye of goldfish, labeled lipids were axonally transported along the optic nerve to the optic tectum. although the different precursors were presumably incorporated into somewhat different lipid populations, all three were approximately equally effective in labeling the lipids transported to the tectum, but the amount of transported material remaining in the nerve was different, being highest with choline and lowest with serine. The labeled lipids appeared in the tectum within 6 hr of the injection, indicating a fast rate of transport, but continued to accumulate over a period of 1–2 weeks, which presumably reflects the time course of their release from the cell body. Since there was a gradual increase in the proportion of labeled lipid in the tectum during this period, some other process in addition to fast axonal transport may have affected the distribution of the lipids along the optic axons. When [³H]choline was used as precursor, the transported material included a small amount of TCA-soluble material, which was probably mainly phosphorylcholine, with labeled acetylcholine appearing in only insignificant amounts. With serine, which gave rise to a large amount of axonally transported protein in addition to lipid, a late increase in the amount of labeled lipid in the tectum was seen, accompanied by a decrease in labeling of the protein fraction.     

Axonal transport of proteoglycans to the goldfish optic tectum

The study addressed the question of whether³⁵SO₄ labeled molecules that the have been delivered to the goldfish optic nerve terminals by rapid axonal transport include soluble proteoglycans. For analysis, tectal homogenates were subfractionated into a souluble fraction (soluble after centrifugation at 105,000g), a lysis fraction (soluble after treatment with hypotonic buffer followed by centrifugation at 105,000g) and a final 105,000g pellet fraction. The soluble fraction contained 25.7% of incorporated radioactivity and upon DEAE chromatographys was resolved into a fraction of sulfated glycoproteins eluting at 0–0.32 M NaCl and containing 39.5% of total soluble label and a fraction eluting at 0.32–0.60 M NaCl containing 53.9% of soluble label. This latter fraction was included on columns of Sepharose CL-6B with or without 4 M guanidine and after pronase digestion was found to have 51% of its radioactivity contained in the glycosaminoglycans (GAGs) heparan sulfate and chondroitin (4 or 6) sulfate in the ratio of 70% to 30%. Mobility of both intact proteoglycans and constituent GAGs on Sepharose CL-6B indicated a size distribution that is smaller than has been observed for proteoglycans and GAGs from cultured neuronal cell lines. Similar analysis of lysis fraction, containing 11.5% of incorporated³⁵SO₄, showed a mixture of heparan sulfate and chondroitin sulfate containing proteoglycans, apparent free heparan sulfate and few, if any, sulfated glycoproteins. Overall, the result support the hypothesis that soluble proteoglycans are among the molecules axonally transported in the visual system.     

Axonal transport of synaptic vesicle proteins in the rat optic nerve

The optic nerve, as a part of the central nervous system (CNS), has been used to study axonal transport for decades. The present study has concentrated on the axonal transport of synaptic vesicle proteins in the optic nerve, using the “stop-flow/nerve crush” method. After blocking fast axonal transport, distinct accumulations of synaptic vesicle proteins developed during the first hour after crush-operation and marked increases were observed up to 8 h postoperative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immunoincubated sections, revealed that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was much higher (up to 80–90%) for the transmembrane proteins than that for surface adsorbed proteins (only 10–20%). The pattern of axonal transport in the optic nerve was comparable to that in the sciatic nerve. However, clathrin and Rab3a immunoreactivities were accumulated in much lower amounts than that in the sciatic nerve. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. A differential distribution of synaptobrevin I and II, however, was observed in the optic nerve axons; synaptobrevin I was present in large-sized axons, while synaptobrevin II immunoreactivity was present in most axons, including the large ones. The two isoforms were, thus, partially colocalized. The results demonstrate that (1) cytofluorimetric scanning techniques could be successfully used to study axonal transport not only in peripheral nerves, but also in the CNS; (2) synaptic vesicles are transported with fast axonal transport in this nerve; and (3) some differences were noted compared with the sciatic nerve, especially for Rab3a and clathrin. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 237–250, 1997.     

Axonal transport of 4S RNA in the chick optic system

The axonal transport of tRNA has been investigated in the chick optic system. Chicks were injected with [³H]uridine intraocularly or intracranially and the RNA of the retina, nerve complex, and tecta separated by polyacrylamide gel electrophoresis and then counted. The ratio of TRNA to rRNA specific activities increased with time in both the nerve complex and contralateral tectum. The ratio increased more rapidly in the nerve complex than the tectum. However, no increase was observed in the case of intracranially injected animals. This is consistent with the axonal flow of tRNA. When [methyl-³H]methionine was used as precursor, the preferential labeling of 4S RNA to rRNA which resulted more clearly showed a transport of 4S RNA from the retinal cells to the tectum. In conclusion, it was found that about 40% of the radioactive RNA observed within the optic tectum 4 days after an intraocular injection of [³H]uridine was accounted for by 4S RNA which had flowed from the retina. However, the migration of a methylated RNA molecule of size 4S, but unrelated to tRNA, cannot be entirely eliminated.     

D-aspartic acid in purified myelin and myelin basic protein

The presence of the biologically uncommon D-isomer of aspartic acid in the white matter of human brains has been reported previously from this laboratory (1). We now report that the level of D-aspartate in human brains is higher in purified myelin than in white matter and is even higher in the myelin basic protein fraction. There also appears to be a difference in the level of D-aspartate found in human brain as compared to bovine brain, possibly a species or age-related difference.     

Axonal protein synthesis and transport

Recent evidence has challenged our ideas about the nature of axonal protein synthesis and transport. Previous metabolic labeling evidence supported the idea that all axonal proteins were synthesized in the cell body and then transported as formed cytoplasmic structures into the axon. Recent evidence suggests that neither the synthesis nor the transport of axonal proteins is that simple. Though most axonal proteins do appear to be synthesized in the neuronal cell body, a small amount of protein appears to be synthesized intra-axonally in some axons. Though small in amount, intra-axonal protein synthesis may be important functionally in some axons. Recent experiments have also begun to identify the presence of a rich array of transport motors in axons, including many members of the kinesin, dynein and myosin families. Progress is being made in identifying which cargoes are being transported by which of these motors. Finally, recent experiments have addressed an old question about whether axoplasmic proteins are transported as filamentous polymers or as soluble components in axons. The answer is that both mechanism can be used in axons. For example, neurofilament protein can move in its particulate or polymeric state, while tubulin can move in its soluble or unpolymerized state.     

The contribution of axoplasmic flow in optic nerve and protein synthesis within the optic tectum to synaptic membrane proteins of the chick optic tectum

In the 5-day-old chick, radioactive leucine was incorporated into proteins of synaptosomal and subsynaptosomal fractions both by fast axoplasmic flow and synthesis within the optic tectum. The distribution of radioactivity in subsynaptosomal fractions suggested that both pathways contribute to the protein constituents of each fraction. The relative contributions to each fraction were similar except for the supernatant proteins, for which fast axoplasmic flow contributed less than the synthesis within the optic tectum. The qualitative contribution of fast flow and synthesis within the optic tectum to the synaptic membrane fraction was distinctive. Fast axoplasmic flow preferentially labelled the high molecular weight proteins, whereas synthesis within the optic tectum labelled a larger percentage of smaller molecular weight proteins.     

Proximo-distal differences in myelin development in human optic fibers

Summary The observation that the myelin sheaths of human optic fibers seem to form first at the distal end of the tract with a progress of myelination toward the eye was tested by assays of cholesterol and cholesterol esters in segments of human optic nerves and tracts at various phases of development. It was found that the distal to proximal progress of myelination relates mainly to the fact that adult human optic fibers contain in their distal portion about twice the amount of cholesterol that occurs in the proximal portion. This gradient in cholesterol content along the fibers develops gradually in children. If the progress of cholesterol deposition is expressed as a percentage of the adult values, the proximo-distal differences are small, suggesting that myelination develops only slightly faster at the distal end.This investigation was supported by Public Health Grant NB 6239 from the National Institute of Nervous Diseases and Blindness.     

Ganglioside-modulated protein phosphorylation in myelin

Gangliosides have profound effects on the phosphorylation of several proteins in myelin. Addition of polysialogangliosides to purified guinea pig brain myelin enhanced the endogenous phosphorylation of a 62-kDa phosphoprotein, but completely inhibited the phosphorylation of myelin basic protein (MBP) (18.5 kDa). The ganglioside-stimulated phosphorylation of the 62-kDa protein was dose-dependent and -specific. Asialo-GM1, ceramide trihexosides, N-acetylneuraminic acid, or colominic acid alone could not mimic this effect, suggesting that the activation process requires both the hydrophobic head group and the anionic character of the gangliosides. Studies on the time course of this reaction revealed that it was a rapid and reversible process and was affected only very slightly by Ca2+. Thus, the stimulatory effect of gangliosides may not involve Ca2+-gangliosides complexes or proteolysis, but may be mediated through an activation of a ganglioside-dependent protein kinase or due to substrate protein-glycolipid interaction. Modulation of the phosphorylation of MBP by gangliosides varies with the states of phosphorylation of this protein. Prior addition of ganglioside to myelin inhibited the phosphorylation of MBP. However, addition of gangliosides to myelin subsequent to maximal phosphorylation of MBP retarded the dephosphorylation of this protein. Phosphorylation of isolated MBP by protein kinase C was stimulated by gangliosides, provided phosphatidylserine was present. In contrast, the glycolipid inhibited the phosphorylation of a unique site catalyzed by cAMP-dependent protein kinase. This site was distinct from those phosphorylated by protein kinase C and was also sensitive to chymotryptic cleavage. Although the exact physiological significance of protein phosphorylation in myelin has yet to be established, gangliosides may play an important role in the modulation of this reversible post-translational modification mechanism.     

Axonal transport of RNA during regeneration of the optic nerves of goldfish

The distribution of radioactive RNA and RNA precursors in the goldfish optic tecta following intraocular injection of ³H-uridine has been studied during various stages of optic nerve regeneration. ³H-uridine was injected into the posterior chamber of the right eye 17, 30, or 60 days after both optic nerves were crushed. Fish were sacrificed at time intervals ranging from 0.5 to 21 days after injection. One day prior to sacrificing, ¹⁴C-proline was also injected into the right eye as a marker of fast axonal protein transport. Seventeen to 23 days after crushing, the approximate time of nerve reconnection, the amount of radioactive RNA appearing in the left optic tectum was increased by more than ten times control values. Approximately 30 days after crushing the nerve, when the reconnected nerve is maturing, RNA values were still elevated, but significantly decreased from the earlier stage. By 60 days after crushing the optic nerve, the amounts of RNA in the left tectum was close to normal. Evidence suggesting that, at least, some of the radioactive RNA in the tectum originated from RNA transported along optic axons rather than from RNA synthesized locally in the tectum was provided by autoradiographic experiments. Autoradiograms of paraffin sections taken from the goldfish optic tecta after the intraocular injection of ³H-uridine showed a distribution of grains in a linear pattern, suggesting a distribution over the incoming fibers during the reconnection stage of regeneration. Electron microscopic autoradiography of glutaraldehyde fixed epoxy sections confirmed that a significant number of grains (shown to be ³H-RNA) were, in fact, over regenerating optic axons. Intracranial injection of ³H-uridine, during the same stage of regeneration, on the other hand, resulted in a distribution of grains, specifically over cell perikarya. These experiments suggest that during the reconnection phase of nerve regeneration, large amounts of RNA may be carried within regenerating optic axons as they enter the optic tectum.