The genetics of axonal transport and axonal transport disorders

Neurons are specialized cells with a complex architecture that includes elaborate dendritic branches and a long, narrow axon that extends from the cell body to the synaptic terminal. The organized transport of essential biological materials throughout the neuron is required to support its growth, function, and viability. In this review, we focus on insights that have emerged from the genetic analysis of long-distance axonal transport between the cell body and the synaptic terminal. We also discuss recent genetic evidence that supports the hypothesis that disruptions in axonal transport may cause or dramatically contribute to neurodegenerative diseases.     

Changes in axonal transport of phospholipids in the regenerating goldfish optic system

Changes in axonally transported phospholipids of regenerating goldfish optic nerve were studied by intraocular injection of [2-³H]glycerol 9 days and 16 days after nerve crush at 30°C. The four major glycerophospholipids all showed substantial increases in transported radioactivity above non-regenerating controls at both time points, these being maximal (15- to 35-fold) in the optic nerve-tract at 9 days and about half as great at 16 days. In the contralateral optic tectum transported label increased 6- to 13-fold at 9 days and 10- to 25-fold at 16 days in the various glycerophospholipids. While all glycerophospholipids showed absolute increases in both tissues, PS and PI increased relatively more, especially in the tectum. The regeneration-associated increases in transported label of all glycerophospholipids were larger than those previously demonstrated for gangliosides and glycoproteins in the same system. Special Issue dedicated to Dr. Eugene Kreps.     

Control of axonal caliber by neurofilament transport

The role of neurofilaments, the intermediate filaments of nerve cells, has been conjectural. Previous morphological studies have suggested a close relationship between neurofilament content and axonal caliber. In this study, the regenerating neuron was used as a model system for testing the hypotheses that neurofilaments are intrinsic determinants of axonal caliber, and that neurofilament content is controlled by the axonal transport of neurofilaments. This system was chosen because previous studies had shown that, after axotomy, axonal caliber was reduced within the proximal stump of the regenerating nerve and, because the relative amount of neurofilament protein undergoing axonal transport in regenerating axons was selectively reduced. The relationship between axonal caliber and neurofilament number was examined in a systematic fashion in both regenerating and control motor axons in rat L5 ventral root. Reconstruction of the spatial and temporal sequences of axonal atrophy in the proximal stump after axotomy showed that reductions in axonal caliber were first detected in the most proximal region of the root and subsequently progressed in a proximal-to-distal direction at a rate of 1.7 mm/day, which is identical to the rate of neurofilament transport in these neurons. Quantitative ultrastructural studies showed that these reductions in caliber correlated with a proportional decrease in the number of axonal neurofilaments but not microtubules. These results support the hypotheses that neurofilament content is a major intrinsic determinant of axonal caliber and that neurofilament content is controlled by the axonal transport of neurofilaments. On this basis, we suggest a role for neurofilaments in the control of axonal volume.     

Acrylamide-induced alterations in axonal transport

Alterations in the axonal transport of proteins, glycoproteins, and gangliosides in sensory neurons of the sciatic nerve were examined in adult male rats exposed to acrylamide (40 mg ip/kg body wt/d for nine consecutive days). Twenty-four hours after the last dose, the L5 dorsal root ganglion (DRG) was injected with either [35S]methionine to label proteins or [3H]glucosamine to label glycoproteins and gangliosides. The downflow patterns of radioactivity for [35S]methionine-labeled proteins and [3H]glucosamine-labeled gangliosides were unaltered by acrylamide treatment. In contrast, the outflow pattern of labeled glycoproteins displayed a severely attenuated crest with no alteration in velocity, suggesting a preferential transfer with the unlabeled stationary components in the axolemma. Retrograde accumulation of transported glycoproteins and gangliosides was unaltered for at least 6 h; however, by 24 h, there was a 75% decrease in the amount of accumulated material. The accumulation of [35S]methionine-labeled proteins was not altered. Autoradiographic analysis revealed an acrylamide-induced paucity of transported radiolabeled glycoproteins selectively in myelinated axons with no effect on "nonmyelinated" axons. The pattern of transported proteins was similar in both control and acrylamide-exposed animals. These results suggest a preferential inhibition of glycosylation or axonal transport of glycoproteins in neurons bearing myelinated axons. More importantly, it suggests that interpretations of axonal transport data must be made with the consideration of alterations in selective nerve fibers and not with the tacit assumption that all fibers in the nerve population are equally affected.     

Molecular motors in axonal transport

Neurons require a large amount of intracellular transport. Cytoplasmic polypeptides and membrane-bounded organelles move from the perikaryon, down the length of the axon, and to the synaptic terminals. This movement occurs at distinct rates and is termed axonal transport. Axonal transport is divided into the slow transport of cytoplasmic proteins including glycolytic enzymes and cytoskeletal structures and the fast transport of membrane-bounded organelles along linear arrays of microtubules. The polypeptide compositions of the rate classes of axonal transport have been well characterized, but the underlying molecular mechanisms of this movement are less clear. Progress has been particularly slow toward understanding force-generation in slow transport, but recent developments have provided insight into the molecular motors involved in fast axonal transport. Recent advances in the cellular and molecular biology of one fast axonal transport motor, kinesin, have provided a clearer understanding of organelle movement along microtubules. The availability of cellular and molecular probes for kinesin and other putative axonal transport motors have led to a reevaluation of our understanding of intracellular motility.     

Organelles in fast axonal transport

The present minireview describes experiments carried out, in short-term crush-operated rat nerves, using immunofluorescence and cytofluorimetric scanning techniques to study endogenous substances in anterograde and retrograde fast axonal transport. Vesicle membrane components p38 (synaptophysin) and SV2 are accumulating on both sides of a crush, but a larger proportion of p38 (about 3/4) than of SV2 (about 1/2) is recycling toward the cell body, compared to the amount carried with anterograde transport. Matrix peptides, such as CGRP, ChRA, VIP, and DBH are recycling to a minor degree, although only 10-20% of surface-associated molecules, such as synapsins and kinesin, appear to recycle. The described methodological approach to study the composition of organelles in fast axonal transport, anterograde as compared to retrograde, is shown to be useful for investigating neurobiological processes. We make use of the "in vivo chromatography" process that the fast axonal transport system constitutes. Only substances that are in some way either stored in, or associated with, transported organelles can be clearly observed to accumulate relative to the crush region. Emphasis in this paper was given to the synapsins, because of diverging results published concerning the degree of affiliation with various neuronal organelles. Our previously published results have indicated that in the living axons the SYN I is affiliated with mainly anterogradely fast transported organelles. Therefore, some preliminary, previously unpublished results on the accumulations of the four different synapsins (SYN Ia, SYN Ib, SYN IIa, and SYN IIb), using antisera specific for each of the four members of the synapsin family, are described. It was found that SYN Ib clearly has a stronger affiliation to anterogradely transported organelles than SYN Ia, and that both SYN IIa and SYN IIb are bound to some degree to transported organelles.     

Changes in axonal transport in neurones of Asterias vulgaris and Asterias forbesei produced by colchicine and dimethyl sulfoxide

Summary Gas flow analysis, autoradiography, and electron microscopy were performed on radial nerve cords of sea stars injected intragastrically with [¹⁴C] leucine tracer, a precursor of axonal proteins. Neurotubules, neurofilaments, and axonal transport were demonstrated for the first time in sea stars. This transport occurred at two rates (fast phase: 240–480 mm/da, slow phase: 20–40 mm/da) moving in both the cellulifugal and centripetal directions. The rate in the centripetal direction was slightly slower. Displacement of peaks of radioactivity appeared cyclic.Transneuronal transport apparently occurred between several groups of neurones suggesting the possibility of metabolic communication, perhaps across the synapse, between groups of neurones or even areas of the nervous system. Regenerating nerve cords demonstrated an increase in the rate (400–600 mm/da) and quantity (6–11 times as much) of label transported to cord termini.The relationship between colchicine and the disruption of neurotubular integrity, resulting in disturbances in fast phase transport, was confirmed. More significantly, the association between nerve injury and site or concentration of treatment is emphasized. Treatment of nerve cords with dimethyl sulfoxide (DMSO) produced more subtle effects than did colchicine, reversible 4–6 hours following the local treatment of the involved nerves. Axon bundles swelled and the organization of axons within the bundles was distorted. Stereochemical structure was suggested from this work as playing a key role in effecting fast phase transport.The authors are indebted to Dr. B. Kiefer, Department of Biology, Wesleyan University, Dr. L. Ross, Department of Anatomy, Medical College of Pennsylvania, and Dr. M. Shelanski, Department of Molecular Biology, Harvard University for their help and constructive criticism.     

Slow axonal transport.

New studies provide further evidence that the neuronal cytoskeleton is the product of a dynamic interplay between axonal transport processes and locally regulated assembly mechanisms. These data confirm that the axonal cytoskeleton in mammalian systems is largely stationary and is maintained by a smaller pool of moving subunits or polymers. Slow axonal transport in certain lower species, however, may exhibit quite different features.     

Depression of fast axonal transport produced by tullidora

The fast axoplasmic transport of labeled proteins was studied in cats showing hindlimb paralysis 4-7 weeks after a single oral dose of tullidora (Karwinskia humboldtiana) toxins. The isotope (3H-leucine) was injected into the spinal ganglion and the contralateral spinal cord of the seventh lumbar segment in order to study transport in sensory and motor fibers. The axoplasmic transport in motor fibers of the sciatic nerve was clearly altered in tullidora-treated cats. The majority of these animals showed a gradual decline of radioactivity from the cord to the periphery instead of the clear-cut wave front always seen in normal cats. An apparent wave was seen in three treated cats but the wave peak was behind the normal position and the slope of the wave front was reduced. While the rate of transport indicated by the farthest extent of the foot of the slope was not in all cases significantly changed, the results all indicated a hindered transport by the reduced slope front in the distal segments of the motor axons. In contrast, the axoplasmic transport appeared normal in the sensory fibers of all but one tullidora-treated cat. Light and electron microscopy of medial gastrocnemius and sural (cutaneous) nerves revealed axonal constrictions and axolemal irregularities associated with organelle retention after tullidora treatment. Also, some mitochondria appeared swollen. These changes were more frequent and intense in the motor nerve fibers than in the cutaneous nerve fibers.     

Changes in the axonal protein transport under conditions of elevated functional activity of the neurons

The prolonged nonintensive physical activity by swimming without load (12 +/- 2 h.) has no effect on the overall amount of fast and slow transported proteins of transport velocity in rat central and peripheral sensory fibres of the sciatic nerve. However, the rate of fast axonal transport in the motor fibres decreases by 18% and the amount of proteins by a factor of 2 as compared with control. The rate of slow axonal transport does not change, but the mean level of transported labeled proteins decreases by 1.9 times. The relatively short-term but more intensive activity (swimming with the load during 60 +/- 10 min.) provokes an increase of the rate by 10% and the overall amount of fast transported proteins by 2 times. The rest of the animals during 6 h. returns the above parameters to control values. A suggestion is made that the rate and the amount of transported proteins depend on the variations in functional state of the neurons and their axons.     

A paradigm for axonal transport

Axonal transport has been extensively studied for a period of 20–30 years, but there is still no general consensus concerning the mechanism by which this transport process operates. An important development in this regard is the recent studies in the physical biochemistry group in the Department of Biochemistry at Monash University where it has been demonstrated that ordered flows may be generated spontaneously in polymer systems under non-equilibeium conditions. The new phenomenon exhibits many novel features, particularly with respect to polymer transport, which bear marked similarity to the behaviour of components in axonal transport. This article sets out to essentiallybring to the attention of those in the neurosciences some of the properties of ordered structured flows in polymer solutions. These properties may generate a different view in the understanding of the mechanism of axonal transport.     

Functions of retrograde axonal transport

Retrograde axonal transport conveys materials from axon to cell body. One function of this process is recycling of materials originally transported from cell body to axon. In motoneurons, 50% of fast-transported protein is returned. Reversal probably occurs mainly at nerve terminals and, for labeled proteins, is nonselective. Proteolysis is not required, although changes in tertiary protein structure may occur with a repackaging of molecules in organelles different from those in which they were anterograde-transported. A second function is transfer of information about axonal status and terminal environment. Premature reversal of transport adjacent to an axon injury may be a component of a signal that initiates cell body chromatolysis. Transport of target cell-derived molecules with trophic effects on the cell body is exemplified by nerve growth factor transport in neurons dependent on it, and is probably a widespread phenomenon in the developing nervous system. Disorders in retrograde transport or reversal occur in some experimental neuropathies, and certain viruses, as well as tetanus toxin, may gain access to the central nervous system by this route.     

Slow axonal transport: the subunit transport model

A central problem concerning slow transport of cytoskeletal proteins along nerve axons is where they are assembled and the form in which they are transported. The polymer and subunit transport models are the two major hypotheses. Recent developments using molecular and cellular biophysics, molecular cell biology and gene technology have enabled visualization of moving forms of cytoskeletal proteins during their transport. Here, we argue that these studies support the subunit transport theory.