Isolation and Characterization of Rapid Transport Vesicle Subtypes from Rabbit Optic Nerve

Abstract

Subcellular fractionation of rabbit optic nerve resolves three populations of membranes that are rapidly labelled in the axon. The lightest membranes are greater than 200 nm and are relatively immobile. The intermediate density membranes consist of 84 nm vesicles which disappear from the nerve with kinetics identical to those of the rapid component. A third population of membranes, displaying a distinct protein profile, is present in the most dense region of the gradient. Immunological characterization of these membranes suggests the following. (1) The lightest peak contains rapidly transported glucose transporter and most of the total glucose transporters present in the nerve; this peak is therefore enriched in axolemma. (2) The intermediate peak contains rapidly transported glucose transporters and synaptophysin, an integral synaptic vesicle protein, and about half of the total synaptophysin; this peak therefore contains transport vesicles bound for both the axolemma and the nerve terminal, and these subpopulations can be separated by immunoadsorption with specific antibodies against the aforementioned proteins. (3) The heaviest peak contains rapidly transported synaptophysin and tachykinin neuromodulators and about half of the total synaptophysin, and 80% of the total tachykinins present in the nerve; this peak appears to represent a class of synaptic vesicle precursor bound for the nerve terminal exclusively. (4) Synaptophysin is present in the membranes of vesicles carrying tachykinins. (5) Both the intermediate and the heaviest peaks are enriched in kinesin heavy chain, suggesting that both vesicle classes may be transported by the same mechanism.

Apolipoprotein E Is Synthesized in the Retina by Müller Glial Cells, Secreted into the Vitreous, and Rapidly Transported into the Optic Nerve by Retinal Ganglion Cells

“…However, it also could be explained by the delayed export of peptide-containing secretory vesicle precursors that has been previously described in this system (19,21). It is possible that all apoE molecules are taken up into the proximal axons and some are immediately transported in a forward direction, while the other portion is taken back to the Golgi apparatus and sorted into a peptide granule.…”

“…SDS gel electrophoresis was performed on 10 or 10 -20% gels by the method of Laemmli (23). Gels were prepared for autoradiography by immersing them in Resolution (EM Science), followed by drying for 1 h under vacuum as described (19). Films were exposed for appropriate times and developed in an automatic processor.…”

“…Densitometry was carried out using a Pharmacia Biotech laser densitometer. Immunoprecipitations were performed as described previously (19). Briefly, samples were dissolved in 0.1% SDS, 1.0% Triton X-100 in phosphate-buffered saline, and undissolved material was removed by centrifugation at 15,000 ϫ g for 1 min in an Eppendorf centrifuge.…”

“…To characterize the type of transport vesicle that carries apoE into the optic nerve, we fractionated the ON/OT preparations isolated 3 and 6 h after [ 35 S]Met/Cys injection by sucrose density gradient centrifugation as described previously (14,19). We obtained three discrete peaks of radioactivity.…”

The optic nerve head is the site of axonal transport disruption, axonal cytoskeleton damage and putative axonal regeneration failure in a rat model of glaucoma

“…To further characterise the nature of axonal transport disruption during experimental glaucoma, we performed immunostaining for two additional molecules, synaptophysin and brain-derived neurotrophic factor (BDNF), that are synthesised by RGCs and undergo anterograde, fast axonal transport [6, 31]. Synaptophysin displayed broadly similar patterns of accumulation at the ONH as APP, although differences were evident.…”