Yoshiaki Komiya

Two Populations of Axonally Transported Tubulin Differentiated by Their Interactions with Neurofilaments

Abstract: In the sensory fibers of the rat sciatic nerve (fibers of the dorsal root ganglion cells), two components of tubulin transport were observed that differed in the rate of transport, solubility in Triton, and subunit composition. The faster component, migrating ahead of the neurofilament proteins, was soluble in 1% Triton. The slower component, migrating with the neurofilament proteins, was insoluble in 1% Triton and contained a unique polypeptide, “NAP,” in the tubulin region that was not present in the faster component. “NAP” was not a subspecies of tubulin as evidenced by peptide mapping. It seems to be a neurofilament‐associated protein. When a complete separation of the main tubulin wave from the neurofilament wave was achieved in the motor axons of the same nerve (axons of the ventral motoneurons) under the effect of ββ‐iminodipropionitrile, a portion of tubulin was still found associated with the retarded neurofilament wave. The subunit composition of this portion was similar to the slower, neurofilament‐associated component in the sensory fibers under normal conditions, i.e., enriched in “NAP” and the most acidic subtype of (β‐tubulin. It is suggested that two populations of transported tubulin exist that are differentiated by the extent of their interaction with neurofilaments.

Preferential blockade of the tubulin transport by colchicine

L-[35S]Methionine was injected into the dorsal root ganglion (L5) of the adult rat, and migration of the neurofilament polypeptides (the triplet with molecular weights of 200,000, 160,000 and 68,000 daltons), alpha- and beta-tubulins and actin in the sciatic nerve and the dorsal root was quantitatively determined and also examined by fluorography. Colchicine (4 microgram) injected into the ganglion 10 min before methionine preferentially blocked the tubulin transport, with little if any blockade of the triplet and actin. Colchicine at this dose had no effects on the incorporation of L-[14C]leucine into the total protein and also into tubulins. In contrast to colchicine, vinblastine sulphate (4 microgram) injected into the ganglion in a similar way blocked the transport of all the triplet, tubulins and actin. Cytochalasin D (1 microgram) had no effect on the slow axoplasmic transport.

Axonal regeneration in bifurcating axons of rat dorsal root ganglion cells.

Abstract The rate of axonal regeneration in rats was determined by using fast axonal transport, and the following findings are reported. (i) Rate of axonal regeneration in the central branch was lower than that in the peripheral one, and this asymmetry persisted throughout the postnatal life examined. (ii) Rate of axonal regeneration slowed with age in both the central and the peripheral branches. (iii) Initial delay increased with age.

Biochemical Study on Myelin in Adrenoleukodystrophy

Two myelin fractions, heavy and light, were isolated from white matter of a patient with adrenoleukodystrophy. Morphologically, heavy myelin fraction showed compact lamellar structures, and light myelin fraction consisted of loose lamellar structures and rod-like clear space. Analytical studies demonstrated that the chemical composition of heavy myelin fraction was almost the same as that of normal myelin and that light myelin fraction consisted mainly of cholesterol. Long chain saturated fatty acids, C25:0 and C26:0, were increased and C24:1 was decreased in sphingoglycolipids of heavy myelin fraction of the patient.

CMP-sialic acid, the sole sialosyi donor, is intra-axonally transported

N‐Acetyl‐D‐[6‐3H]mannosamine was injected into the 9th dorsal root ganglion of Xenopus laevis and the intra‐axonal transport of chloroform/methanol‐soluble radioactivity was analyzed using thin‐layer chromatography coupled with fluorography. Three radioactive groups were distinct in consecutive segments of the sciatic nerve. The first is due to N‐acetyl‐D‐mannosamine itself which labels the nerve uniformly, but does not seem to migrate within axons. The second group, representing most probably CMP‐sialic acid, migrates at about 8 at 15°C. The third is a species of ganglioside uniquely present in the frog nerve, and this migrates at 1–3 . Our demonstration of the intra‐axonal transport of CMP‐sialic acid affords direct support to the contention that sialosylation of the ganglioside can occur in axon terminals.

A Ganglioside Species (GD1α) Migrates at a Slow Rate and CMP-Sialic Acid Severalfold Faster in Xenopus Sciatic Nerve: Fluorographic Demonstration

Abstract: The ninth dorsal root ganglion of adult Xenopus laevis was labeled with N‐acetyl‐D‐[6‐3H]mannosamine, and intraaxonal migration of gangliosides was examined by analysis of the chloroform/methanol extract of each of 5‐mm consecutive nerve segments by TLC coupled with fluorography. A unique disialoganglioside (GD1α), which amounted to up to 83% of the total ganglioside in this nerve, migrated at 1–2 mm/day at 15°C. This contrasts with the rapid transport of other ganglioside species previously reported in the optic systems of goldfish, rabbits, chickens, and rats. Fluorographic analysis also revealed a trichloro‐acetic acid‐soluble substance migrating at a velocity of 8 mm/day at 15°C. The substance was considered to be CMP‐sialic acid on the basis of observations that it comigrates with authentic CMP‐N‐acetylneuraminic acid in TLC developed with two different solvent systems, it is very labile to weak acid but resistant to neuraminidase from Vibriocholerae, it is converted to N‐acetylmannosamine when treated first with weak acid and subsequently with N‐acetyl‐neuraminic acid aldolase, and it has a β‐sialosyl group in its structure. Because CMP‐sialic acid is believed to be the sole sialosyl donor in the cells, its migration in axons toward terminals, together with the previous demonstration of sialyl‐transferase activity in the synaptosomal plasma membrane, strongly supports the possibility that sialosylation of gangliosides and probably of other sialoglycoproteins is not confined to the Golgi apparatus, but can also occur after the compounds are committed to axonal transport.