Mechanism Of Fast Axonal Transport Research Articles

Subcellular Localization Determines the Stability and Axon Protective Capacity of Axon Survival Factor Nmnat2

Author SummaryNeurons are polarized cells that rely on bidirectional transport to deliver thousands of cargos between the cell body and the most distal ends of their axons. One cargo that is of particular importance is the NAD-synthesising enzyme Nmnat2. This surprisingly unstable protein is produced in the cell body and its constant supply into axons is required to keep them alive. If this supply is interrupted, Nmnat2 levels in the distal axon drop below a critical threshold, leading to axon degeneration. The rapid turnover of Nmnat2 contributes critically to the time course of axon degeneration. If its half-life could be extended, axons may be able to survive transient interruptions of its supply. In this study, we find that disruption of Nmnat2 localization to axonal transport vesicles increases both its half-life and its capacity to protect injured neurites. Specifically, association of Nmnat2 with transport vesicles reduces it stability by making it vulnerable to ubiquitination and proteasome-mediated degradation. These findings suggest that modulation of the subcellular localization of Nmnat2 on transport vesicles could serve as a potential avenue for therapeutic treatment of axon degeneration.

Neuroaxonal dystrophy in distal symmetric sensory polyneuropathy of the diabetic BB-rat

We and others have previously described neuroaxonal dystrophic changes as one of the hallmarks of structural diabetic autonomic polyneuropathy involving sympathetic nerves. In the present study, a systemic search for similar changes was undertaken in the mainly sensory symmetric polyneuropathy of the spontaneously diabetic BB-rat. Changes identical to those described in sympathetic nerves in this model were found in sensory ganglion cells, in their proximal extramedullary axons, and in proximal and distal myelinated axons of the spinal dorsal columns. The dystrophic substructures consisted of tubulovesicles, tubular rings, layered membranes, electron-dense membranous bodies, and neurofilamentous changes. Neuroaxonal dystrophic abnormalities increased with increasing duration of diabetes, and exhibited a topographic distribution along the sensory neuroaxonal axis, suggesting metabolic abnormalities as well as abnormalities in the turn-around mechanism of fast axonal transport in the pathogenesis of dystrophic changes in diabetic nerves.

A model for fast axonal transport

A model for fast axonal transport is developed in which the essential features are that organelles may interact with mechanochemical cross-bridges that in turn interact with microtubules, forming an organelle-engine-microtubule complex which is transported along the microtubules. Computer analysis of the equations derived to describe such a system show that most of the experimental observations on fast axonal transport can be simulated by the model, indicating that the model is useful for the interpretation and design of experiments aimed at clarifying the mechanism of fast axonal transport.

Bidirectional transport of fluorescently labeled vesicles introduced into extruded axoplasm of squid Loligo pealei.

A reconstituted model was devised to study the mechanisms of fast axonal transport in the squid Loligo pealei. Axonal vesicles were isolated from axoplasm of the giant axon and labeled with rhodamine-conjugated octadecanol, a membrane-specific fluorescent probe. The labeled vesicles were then injected into a fresh preparation of extruded axoplasm in which endogenous vesicle transport was occurring normally. The movement of the fluorescent, exogenous vesicles was observed by epifluorescence microscopy for as long as 5 min without significant photobleaching, and the transport of endogenous, nonfluorescent vesicles was monitored by video-enhanced differential interference-contrast microscopy. The transport of fluorescent, exogenous vesicles was shown to be bidirectional and ATP-dependent and occurred at a mean rate of 6.98 +/- 4.11 micron/s (mean +/- standard deviation, n = 41). In comparison, the mean rate of transport of nonfluorescent, endogenous vesicles in control axoplasm treated with vesicle buffer alone was 4.76 +/- 1.60 micron/s (n = 64). These rates are slightly higher than the mean rate of endogenous vesicle movement in extruded axoplasm (3.56 +/- 1.05 micron/s, n = 40) not subject to vesicles or vesicle buffer. Not all vesicles and organelles, exogenous or endogenous, were observed to move. In experiments in which proteins of the surface of the fluorescent vesicles were digested with trypsin before injection, no movement of the fluorescent vesicles was observed, although the transport of endogenous vesicles and organelles appeared to proceed normally. The results summarized above indicate that isolated vesicles, incorporated into axoplasm, move with the characteristics of fast axonal transport. Because the vesicles are fluorescent, they can be readily distinguished from nonfluorescent, endogenous vesicles. Moreover, this system permits vesicle characteristics to be experimentally manipulated, and therefore may prove valuable for the elucidation of the mechanisms of fast axonal transport.

Gelsolin inhibition of fast axonal transport indicates a requirement for actin microfilaments.

The actions of actin-based microfilaments in cell motility suggest a possible role in the mechanism of fast axonal transport, but the pharmacological data evaluating their role in this process are equivocal. Moreover, microfilaments are difficult to preserve and identify in ultrastructural studies, so the organization and function of axonal actin has remained uncertain. We have now evaluated the role of actin microfilaments in intracellular transport of membranous organelles using video-enhanced contrast microscopy and gelsolin to analyse fast axonal transport directly in isolated axoplasm from the squid giant axon. With this preparation it is possible to perfuse axoplasm with large molecules that do not cross the plasmalemma, while controlling cation levels. The 90,000-molecular weight protein gelsolin depolymerizes actin microfilaments in micromolar Ca2+, but not in the absence of Ca2+. Axonal transport of membranous organelles has previously been shown to be unaffected by levels of Ca2+ up to 10 microM. In the presence of EGTA, gelsolin has no effect on the movement of membranous organelles, but in the presence of 10 microM Ca2+ it completely blocks transport of all membranous organelles. No changes in the organization of the axoplasm were detected. These results and results using other probes for actin are consistent with the hypothesis that actin-based microfilaments are involved in the movement of membranous organelles in the axon.

Fast axonal transport in permeabilized lobster giant axons is inhibited by vanadate

We have developed a method for permeabilizing axons and reactivating the fast transport of microscopically visible organelles. Saltatory movements of organelles in motor axons isolated from lobster walking legs were observed using Nomarski optics and time-lapse video microscopy. In the center of the axon most of the particles and mitochondria moved in the retrograde direction, but immediately below the axolemma the majority moved in the anterograde direction. When axons were permeabilized with 0.02% saponin in an adenosine 5'-triphosphate (ATP)-free "internal" medium, all organelle movement ceased. Saltatory movements resembling those in intact axons immediately reappeared upon the addition of MgATP. Very slight movement could be detected with ATP concentrations as low as 10 microM, and movement appeared to be maximal with 1 to 5 mM ATP. Vanadate, which does not affect axonal transport in intact axons, inhibited the reactivated organelle movements in permeabilized axons. Movement was rapidly and reversibly inhibited by 50 to 100 microM sodium orthovanadate. The effects of vanadate, including the time course of inhibition, its reversibility, and its concentration dependence, are consistent with the hypothesis that a dyneinlike like molecule may play a role in the mechanism of fast axonal transport.

Microinjection into an identified axon to study the mechanism of fast axonal transport.

Microinjection into an axon of an identified invertebrate neuron is shown to be a useful technique for analyzing the mechanisms of fast axonal transport. It permits direct assessment of the effect of agents that cannot permeate the plasma membrane on the translocation of material in the axon. The actin filament depolymerizer DNase I, when injected into the axon of the Aplysia neuron R2, caused a local block of fast transport of [3H]glycoprotein. Two agents that should interfere with the functioning of actin filaments without causing extensive depolymerization, tne N-ethylmaleimide-modified nuclease S1 fragment of myosin (injected) and dihydrocytochalasin B (applied externally). had no effect. Together these results suggest that actin plays a structural role in the axonal cytoskeleton rather than a role in transport force generation, the effect of DNase I being mediated by structural disordering of the axoplasm. Experiments were also done with inhibitors of dynein, the microtubule-associated ATPase. erythro-9-[3-(2-Hydroxynonyl)]adenine blocked transport but vanadate was ineffective.

Analysis of the mechanism of fast axonal transport by intracellular injection of potentially inhibitory macromolecules: evidence for a possible role of actin filaments.

Although actin is thought to participate in several types of cell motility other than muscle contraction, no direct evidence has linked it to the force-generating mechanism for fast axonal transport. We have obtained evidence for the involvement of actin by microinjecting, into the serotonergic giant cerebral neuron of Aplysia, two preparations that have been shown to depolymerize actin filaments. One is a fraction of rabbit serum containing a heat-labile gamma globulin that affects actin polymerization in a manner similar to that of cytochalasin and several proteins that are thought to regulate the length of actin filaments. The other is bovine pancreatic DNase I which binds to actin stoichiometrically. Both preparations substantially decreased the transport of storage vesicles containing [3H]serotonin. Phalloidin, a toxic fungal peptide that binds to actin filaments but stabilizes rather than depolymerizes them, did not inhibit transport. We have not yet determined whether the inhibition od transport occurs during export of [3H]serotonin from the cell body into the axon or during translocation along the axon. Nevertheless, these observations provide a promising experimental indication that actin is involved in fast axonal transport.

The mechanisms of fast axonal transport: a pharmacological approach.

Abstract Eighteen aromatic amines and acids were tested for effects on fast axonal transport of [ 3 H]-leucine-labelled proteins in vitro in the sciatic system of the frog. The methodology used made it possible to resolve effects on synthesis and transport separately. Simple compounds like aniline, benzoic acid, phenylethylamine and phenylpropionic acid, and also more complicated drugs like cphedrine and mescaline at concentrations of 5 mM, were found to reduce the amount of transported material in the nerve without affecting protein synthesis in the dorsal ganglia. The effects of drugs with varying ring structure, indicated a positive correlation between the amphiphilic character and the transport inhibitory effect. No consistent correlation between the length, the structure and the charge of the alkyl chain, and the inhibition of transport was evident. The reversibility of the effects was demonstrated for some of the more potent drugs. Protein synthesis in the ganglia and the transport of labelled material were shown to be inhibited to about the same degree by two inhibitors of oxidative phosphorylation, 2.4-dinitrophenol and sodium cyanide. The effects of the various drugs are discussed in relation to the theories which have been proposed for the mechanism offast axonal transport.