Axonal Transport Mechanisms and Target Site of Toxicity

G. Jean Harry
Head, Neurotoxicology Group
National Institute of Environmental Health Sciences

Objective:

The purpose of this presentation is to describe the basic process by which the neuron of the nervous system transports newly synthesized material from the cell body to the various distant cell processes. This process of axonal transport requires transport of materials over long distances and represents a process critical to the functioning and maintenance of the neuronal processes. Target sites that are vulnerable to interruption by pharmacological agents and toxic chemicals will be briefly discussed.

Introduction:

All types of cells are required to transport proteins and other molecular components from their site of synthesis near the nucleus to the various other sites of usage in the cell. In the nervous system, the neuron has three well-defined regions 1) the cell body, the site of the synthesis of materials, 2) the axon and dendrites in which materials are moved by axoplasmic transport, and 3) the nerve terminal. In the cell body, proteins and polypeptides are synthesized under the direction of the nucleus and may be either quickly transported in the axons or stored in a cell body compartment for later export into the processes. The egress of various components into the neurites is controlled by a "gate" which has been identified as the Golgi apparatus. The cell body of the neuron must maintain the functions normally associated with its own support, as well as, provide continuous support of membraneous organelles to its various processes.

Axonal Transport

Axonal transport is the process by which the neuron replenishes components of the axon and the nerve terminal. Axonal membrane is constantly being replaced by new components arriving from the cell body as are the macromolecular components of the nerve terminal needed for synaptic transmission.

Classification of Axonal Transport:

Anterograde Transport:
Transport of proteins and other molecular components from their site of synthesis near the nucleus to the various other sites of usage in the cell. Once material enters the rapid transport vector, the continuation of rapid transport is independent of the cell body and the electrical activity of the axon.

Fast Anterograde Transport:
Materials move at a rate of 200-400 mm/day and are predominantly membrane associated. It depends on one or more of the filaments that make up the neuron's cytoskeleton and on microtubules that provide a stationary track on which specific organelles move. Fast anterograde is driven by a microtubule associated ATP-ase, kinesin.

Slow Anterograde Transport:
Includes proteins that are part of the cytoskeletal structures, including microtubules and neurofilaments, or are linked to these structures.

Slow component a (SCa)- Travels at a rate of 0.2-1.0 mm/day and carries proteins used to make up the fibrillary elements of the cytoskeleton - subunits of neurofilaments and microtubules. Slow component b (Scb)-Travels at a rate of 2.0-5.0 mm/day and contains a more complex protein content including actin, myosin, clathrin, enzymes of intermediary metabolism, and calmodulin.

Retrograde Transport:
The process by which material returns from the terminals to the cell body either for degradation or recycle. Transport rate is approximately 200mm/day. Particles are driven along microtubules by a microtubule-associated ATPase - dynein. The composition of the material is similar to that of the anterograde fast component and is packaged in large membrane-bound organelles. Extracellular components can be taken up at the terminals by endocytosis and transported to the cell body by retrograde transport.

Table 1. Major rate components of axonal transport

Rate Component (mm/day) Structure and composition FAST TRANSPORT Anterograde 200-400 Small vesiculotubular structures, neurotransmitters,membrane proteins and lipids Mitochondria 50-100 Mitochondria Retrograde 200-300 Lysosomal vesicles and enzymes SLOW TRANSPORT SCb (slow component b) 2-8 Microfilaments, metabolic enzymes, clathrin complex SCa (slow component a! 0.2-1 Neurofilaments and microtubules

Procedures to Examine Anterograde Transport

A labeled precursor is injected into the neuronal cell body, allowed to incorporate into various compounds, and given sufficient time for the labeled synthesized materials to be carried out into the axon. The nerve is then removed to determine the proximo-distal pattern of radioactivity distribution with respect to time. The downflow curve is determined by taking a series of small, equal, transverse segments of the nerve successively along it's length and plotting the radioactivity content against the distance from the cell body. Such downflow curves show a pool of radioactivity in the segment containing the cell bodies, followed by a lower plateau region which rises to a crest before abruptly falling to baseline levels forming the front (foot) of the crest. The rate of transport (mm/hr) is estimated from a linear regression of distance down the axon relative to time.

Lag time is identified by the intercept of the regression line and is often taken to represent the intracellular processing and commitment to transport in the Golgi apparatus.

Nerve Constriction (Ligatures): Transported material can be examined as material accumulated at either the proximal side of a ligature (anterograde transport) or the distal side of a ligature (retrograde transport). incorporation of labeled precursor into the cell bodies and for the initiation of transport, the constriction can be placed on the nerve either prior to the arrival of transported material to the site of blockage or it can be placed on the nerve once the radiolabeled material has been transported past the site. If placed on the nerve before transported material has reached the site and the material is allowed to accumulate at the block information can be obtained with regard to rate and amount of transported material. This procedure has also been used to detect selected material, radiolabeled or unlabelled, by the accumulation at the block site. A constriction placed on the axon after the passage of labeled material will allow for the detection of material accumulated not only in the anterograde direction but also in the retrograde direction.

Alterations In Axonal Transport:

  1. Interference with a specific step in the mechanism of axonal transport
  2. Altered movement of a specific transported component
  3. Perturbation of a general biochemical process such as, energy metabolism
  4. Structural alterations in the axon
  5. Alterations in the cell body -morphological or physical
  6. Temperature of the axon

Although axonal transport is altered by various different environmental agents and pharmaceuticals it is still unclear if an axonopathy can be produced by a direct effect on axonal transport. Various target sites and processes have been proposed in chemical induced peripheral neuropathies.

Neurofilaments.
Structural components intrinsic to the axonal transport system are a point of vulnerability and may be directly altered. Neurofilaments are presumed to be the primary target site for a number of neurotoxic substances. Focal accumulations of neurofilaments within neurons and their processes have been observed following exposure to certain chemicals. Aluminum salts and vinca alkaloids produce an accumulation of neurofilaments in the cell body which blocks the export of transported materials. IDPN causes an accumulation in the proximal axon while carbon disulfide and acrylamide produce a similar pattern in the distal axon. An alteration in neurofilament transport would be the result of the slow component of transport and often in these cases fast transport is not altered until a physical disruption and blockage of the axon occurs.

Smooth membranes.
Distal axonal swellings comprised of accumulations of vesiculotubular membrane structures are produced by several neurotoxic chemicals including pBromophenylacetylurea, zinc pyridine thione, and certain organophosphorous compounds. These structures are derived from the smooth endoplasmic reticulum. However, it is unknown whether they reflect a direct effect of the toxicant on SER or a nonspecific reactive response of the axon. Chemicals which produce accumulations of membraneous structures appear to have little or no effect on slow axonal transport until the axonopathy is severe and the axon shows signs of degeneration.

Cell body. Alterations in axonal transport can be seen as the result of alterations in the complex processes of transport initiation in the cell body. Methyl mercury is a specific toxicant that changes reported in axonal transport are likely to be reflective of changes in somal synthesis and processing rather than changes in axonal transport per se.

Points for Consideration:

  1. Non-specific labeling of axon - sampling from contralateral axon
  2. Specific radiolabeled precursor injected into the cell body.
  3. Cell body processing and commitment of material to transport.
  4. Synergistic effects with body temperature.
  5. Experimental approach: downflow, ligature, double-ligature,
  6. Retrograde transport: delivery to, turn-around at, or uptake of labeled material in the nerve ending,
  7. Nerve fiber population affected: myelinated, unmyelinated, sensory, or motor,
  8. Morphology of the axon.

All of these factors contribute to the apparent conflicts in the literature regarding both the morphological and biochemical alterations produced by various toxicants. Many neurotoxicants and disease processes appear to have a direct pathological effect on axons however, the question remains as to whether any of these agents produce a direct effect upon axonal transport prior to the appearance of morphological alterations or clinical signs. The disruption of the transport process remains a critical part of the cellular pathology determining the subsequent course of the neuropathy. Whether alterations in axonal transport are a cause or an effect of neuropathy the information obtained about the process is an important factor in the further understanding of the pathogenesis and progression of toxicant-induced neuropathies.