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The Mechanism of Slow Axonal Transport

Current research on slow axonal transport is focused on addressing four central questions: (1) What are the cargo structures of slow components a and b, (2) what are the tracks along which they move, (3) what are the motors that move them, and (4) how is their movement regulated? The observation of the rapid intermittent movement of neurofilament and microtubule polymers in axons indicates that these cytoskeletal polymers are among the cargo structures of slow axonal transport, although this does not preclude the possibility that cytoskeletal subunit proteins may also move in an unassembled form. The form in which the many other proteins in slow axonal transport move remains unclear. One hypothesis is that they are transported by binding to (and effectively riding piggyback on) moving cytoskeletal polymers. The relatively simple protein composition of slow component a suggests that neurofilaments and microtubules could be the sole carrier structures for this rate component; all the proteins that move in slow component a are either integral parts of these cytoskeletal polymers or are known to associate with these polymers in vivo. The presence of actin in slow component b suggests that microfilaments could function as a carrier structure for this rate component, although it seems unlikely that the several hundred different proteins that move in slow component b all bind to microfilaments. More probably, many of these proteins form supramolecular complexes that, in turn, associate with the moving filaments or that associate with motor proteins independently of the moving filaments. However, this hypothesis is speculative because the movement of microfilaments and most other cytosolic proteins in axons has not yet been demonstrated.

The rapid rate of movement of neurofilaments and microtubules in axons indicates that they are transported by fast motors, perhaps similar to or even the same as the motors that move membranous organelles, but the identity of these motors and the tracks along which they move are not known. By analogy with the movement of membranous organelles, it seems likely that neurofilaments and microtubules and other cargoes of slow axonal transport move along either microtubule or microfilament tracks. For example, there is evidence that dynein may transport axonal microtubules anterogradely along microfilaments and that dynein and kinesin may transport axonal neurofilaments bidirectionally along microtubules. The frequency of neurofilament movement may be regulated by phosphorylation of the neurofilament proteins. Axonal neurofilaments can also interact with myosin Va, but the role of this interaction in neurofilament movement is presently unclear. Although much remains to be learned about the mechanism of movement, it seems likely that the cargo structures of slow axonal transport each interacts with a number of different motor proteins and that these motors act cooperatively to translocate and organize these polymers in both the longitudinal and radial dimensions of the axon.

Slow Axonal Transport Moves Soluble Proteins and Cytoskeletal Structures

The coordinated movement of neurofilament and microtubule proteins provided strong evidence for the “structural hypothesis.” For example, in pulse-labeling experiments labeled neurofilament proteins move as a bell-shaped wave with little or no trailing of neurofilament protein (Baas and Buster, 2004). Neurofilament stability under physiological conditions indicates that soluble neurofilament subunit pools are negligible, so coherent transport of neurofilament triplet proteins implied a transport complex, i.e., neurofilaments. Similarly, coordinate transport of tubulin and MAPs made sense only if microtubules move, because MAPs do not interact with unpolymerized tubulin. The simplest explanation is that neurofilaments and microtubules move as discrete cytological structures, but this idea was controversial for many years.

Development of fluorescently tagged neurofilament or microtubule subunits and methods for visualizing these structures in living cells resolved this issue by documenting movements of individual microtubules and neurofilaments in neurites of cultured neurons (Brown, 2003). Direct observations of individual microtubule or neurofilament segments indicated that they move down axons as assembled polymers. Video images of fluorescently tagged microtubules or neurofilaments reveal discontinuous movements, with long pauses punctuated by brief, rapid translocations at 1–2 μm/sec. Due to long pauses, average rates are 2–3 orders of magnitude slower than instantaneous velocities (Baas and Buster, 2004). Remarkably, dynein plays a major role in slow axonal transport of microtubules and neurofilaments (He et al., 2005).

Studies on transport of neurofilament proteins indicated that little or no degradation occurs until neurofilaments reach nerve terminals, where they are degraded rapidly. Comparable results were obtained with microtubule proteins. Differential metabolism appears to be a key to targeting of cytoplasmic and cytoskeletal proteins. Proteins with slow degradative rates accumulate, reaching higher steady-state levels. Altering degradation rates changes that steady-state concentration, so enrichment of actin in presynaptic terminals is due to slower turnover of actin than neurofilaments and tubulin. As a result, inhibiting calpain causes neurofilament accumulation in terminals. Differential turnover may involve specific proteases or posttranslational modifications that affect susceptibility to degradation. Regardless, cytoplasmic proteins are degraded in the distal axon and do not return in retrograde axonal transport.

Slow Axonal Transport Moves Soluble Components and Cytoskeletal Structures

Slow axonal transport has two major components, both representing the movement of cytoplasmic constituents (Fig. 4.7). The cytoplasmic and cytoskeletal elements of the axon in axonal transport move at rates at least two orders of magnitude more slowly than fast transport. Slow component a is composed largely of cytoskeletal proteins, neurofilaments, and microtubule protein. Slow component b is a complex and heterogeneous rate component, including hundreds of distinct polypeptides ranging from cytoskeletal proteins such as actin (and tubulin in some nerves) to soluble enzymes of intermediary metabolism (such as glycolytic enzymes). Many characteristics of axonal transport have been described, and these characteristics provide the foundation for our understanding of mechanisms.

Neurofilaments and microtubules move as discrete cytological structures (Baas & Buster, 2004). Studies on the transport of neurofilament protein indicate that little degradation or metabolism occurs until neurofilaments reach nerve terminals, where they are degraded rapidly. Comparable results have been obtained in studies labeling microtubule protein by radioactivity or fluorescence. Under favorable conditions, movement of individual microtubules can be detected in neurites or growth cones. Both radiolabeling studies and direct observations of individual microtubules indicate that all microtubules and neurofilaments move down the axon, but the motor protein involved is uncertain. Differential metabolism appears to be a key to the targeting of cytoplasmic and cytoskeletal proteins. Proteins with slow degradative rates accumulate and reach higher steady-state concentrations. Alteration of degradation rates changes the steady-state concentration of a protein. The concentration of actin in presynaptic terminals is explained by a slower turnover in terminals relative to neurofilament proteins and tubulin, and inhibition of calpain causes neurofilament rings to appear in presynaptic terminals. Differential turnover may be accomplished by specific proteases or posttranslational modifications that affect the susceptibility to degradation.

The coherent movement of neurofilaments and microtubule proteins provides strong evidence for the “structural hypothesis.” For example, pulse-labeling experiments show that radiolabeled neurofilament proteins move as a bell-shaped wave with little or no trailing of neurofilament protein. This fits with the observed stability of neurofilaments under physiological conditions. Thus, any soluble pool of neurofilament subunits is negligible. Similarly, the coherent transport of tubulin and MAPs makes sense only if microtubules are moved because MAPs do not interact with unpolymerized tubulin.

A striking demonstration of microtubule movement can be seen with fluorescent analogs of tubulin (Brown, 2003). When local segments of an axon are photoactivated, short fluorescent microtubule segments can be seen to move down the growing neurite. These microtubules move rapidly (1–2 µm/sec) with frequent pauses that produce a net anterograde transport of microtubules at 1–2 mm/day and similar rates are observed for neurofilaments. When studies of axonal transport using radiolabeled amino acids are combined with direct observations of individual microtubules or neurofilaments by video microscopy, there is little doubt that microtubules and neurofilaments can and do move in the axon as intact, individual cytoskeletal elements.

More recently, similar methods have been used to document the transport of classically soluble, cytoplasmic proteins like glycolytic enzymes and synuclein (Roy, Winton, Black, Trojanowski, & Lee, 2007). As with microtubules and neurofilaments, these movements are intermittent and rapid. The extended pauses lead to a net rate of anterograde transport of approximately 2–4 mm/day, consistent with rates obtained in pulse-chase radiolabel studies.

Slow Axonal Transport Moves Soluble Components and Cytoskeletal Structure

Neurofilaments and microtubules move as discrete cytological structures (Brady, 1992). Recent studies on transport of neurofilament protein indicate that little degradation or metabolism occurs until neurofilaments reach nerve terminals, where they are rapidly degraded. Comparable results have been obtained in studies labeling microtubule protein by radioactivity or fluorescence. Under favorable conditions, movement of individual microtubules can be detected in neurites or growth cones. Both radiolabeling studies and direct observations of individual microtubules indicate that all microtubules and neurofilaments move down the axon, but the motor protein involved is uncertain. Differential metabolism appears to be a key to the targeting of cytoplasmic and cytoskeletal proteins. Proteins with slow degradative rates accumulate and reach higher steady-state concentrations. Alteration of degradation rates changes the steady-state concentration of a protein. Concentration of actin in presynaptic terminals is explained by slower turnover in terminals relative to neurofilament proteins and tubulin, and inhibition of calpain causes neurofilament rings to appear in presynaptic terminals. Differential turnover may be accomplished by specific proteases or posttranslational modifications that affect susceptibility to degradation.

The coherent movement of neurofilaments and microtubule proteins provides strong evidence for the “structural hypothesis.” For example, pulse-labeling experiments show that radiolabeled neurofilament proteins move as a bell-shaped wave with little or no trailing of neurofilament protein. This fits with the observed stability of neurofilaments under physiological conditions, which suggests that any soluble pool of neurofilament subunits is negligible. Similarly, the coherent transport of tubulin and MAPs makes sense only if microtubules are moved, because MAPs do not interact with unpolymerized tubulin.

A striking demonstration of microtubule movement can be seen with fluorescent analogs of tubulin (Tanaka and Kirschner, 1991; Reinsch et al, 1991). Tubulin labeled with caged fluorescein can be injected into one cell of a fertilized Xenopus oocyte at the two-cell stage. Such tubulins are fluorescent only after photoactivation. The injected oocyte is then allowed to develop into an embryo. The injected tubulin equilibrates with endogenous tubulin and is incorporated into the microtubules of all cells derived from the original injected cell. Because protein synthesis is minimal in early cell divisions of embryonic development, the labeled tubulin is reused by daughter cells until diluted out by newly synthesized tubulin. When early embryonic neurons are cultured, the caged fluorescent tubulin may be photoactivated and visualized.

When local segments of an axon are photoactivated, patches of fluorescent tubulin can be seen to move down the growing neurite. The fluorescent patches remain discrete during movements in the anterograde direction at slow transport rates. Observations of full microtubules can also be made in embryonic Xenopus neurons by using rhodaminelabeled tubulin. In favorable areas of axons and growth cones, individual fluorescent microtubules can be visualized. Such microtubules can be seen to move down axons and into growth cones. The forces are sufficient to bend these microtubules in conjunction with growth cone movements. When studies of axonal transport using radiolabels are combined with direct observations of individual microtubules by video microscopy, there is little doubt that microtubules and neurofilaments can and do move in the axon as intact, individual cytoskeletal elements.

The Kinetics of Slow Axonal Transport

When considering the rate of slow axonal transport, it is important to make a distinction between measurements made on fast and slow timescales. In radioisotopic pulse-labeling experiments, which are performed on a timescale of days or weeks, each protein conveyed by slow axonal transport moves out along the axon in the form of a unimodal bell-shaped wave that spreads as it moves distally. The rate of slow axonal transport is generally quoted as the rate of movement of the peak of the wave, which represents the modal transport rate, although the width and spreading of the waves clearly indicate that the proteins actually move at a range of rates. The modal rate differs in different neuronal cell types and may also vary spatially (along axons) as well as temporally (during development, maturation, and aging), but it is generally around 0.2–1 mm day^− 1 (0.002–0.02 μm s^− 1) for slow component a and 2–8 mm day^− 1 (0.02–0.09 μm s^− 1) for slow component b (Table 2).

Properties of slow axonal transport suggest molecular mechanisms

Information about molecular mechanisms underlying slow axonal transport is relatively limited. They are energy dependent and require intact MTs. Accordingly, transport of NFs can be pharmacologically uncoupled from MT transport without eliminating slow transport (Griffin et al., 1995). Recent imaging studies in cultured neurons confirmed these findings, further showing that transport of NFs (Francis et al., 2005) and various proteins in SCb does not depend on actin (Roy et al., 2008). In contrast, pharmacological agents that disrupt MTs appear to block slow transport of all components. While this does not rule out a role for the MF cytoskeleton in slow transport movements, MTs appear to be required for the transport of other elements of the cytoskeleton.

The macroscopic transport velocity rates measured by radiolabeling experiments should not be taken to reflect maximum rates of the motors involved. As with mitochondrial transport, the net rate velocity of slow component proteins reflects both the rate of actual movement and the fraction of a time interval that a structure is moving (Brady, 2000). The large size and elongated shape of cytoskeletal structures and their potential for many interactions means that net displacements are discontinuous. If a structure is moving at a speed of 2 µm/sec, but on average only moves at that rate for 1 out of every 100 seconds, then the average rate for the structure will translate to an net rate of only 0.02 µm/sec (Baas & Buster, 2004).

Recently, methods for direct visualization of fluorescently tagged MTs and NFs in cultured neurons have been developed (Brown, 2003). In such studies, relatively short MT or NF segments can be seen to move as rapidly as MBOs moving in fast axonal transport, although they move much less frequently. Integration of these infrequent rapid movements of MTs and NFs over time gives a net rate of approximately 1–2 mm/day instead of the 200–400 mm/day seen for MBOs in mammalian nerve. Other studies permitted visualization of microtubules nucleated at the microtubule organizing center and being translocated toward the cell periphery. The combination of studies of axonal transport using radiolabels and direct observations of individual MTs or NFs with video microscopy provides strong experimental evidence that MTs and NFs can and do move in the axon as intact cytoskeletal structures. As discussed below, there are still questions about the specific motors and mechanisms underlying these movements.

Like membrane proteins, cytoplasmic and cytoskeletal proteins are differentially distributed in neurons and glia. Progress has been made toward identification of targeting mechanisms and some general principles have begun to emerge. Since cytoplasmic constituents move only in the anterograde direction, a key mechanism for targeting of cytoplasmic and cytoskeletal proteins appears to be differential metabolism (Brady, 1993). Concentration of actin and other proteins in presynaptic terminals can be explained by slower turnover in the presynaptic terminal relative to NF proteins and tubulin. Proteins with slow degradative rates in the terminal would accumulate and reach a higher steady-state concentration. Thus, alteration of degradation rates for a protein can change the rate of accumulation for that protein. For example, some protease inhibitors cause the appearance of neurofilament rings in affected presynaptic terminals (Roots, 1983).

Although slow axonal transport of cytoskeletal proteins has received the most attention, all other cytoplasmic proteins must be delivered to specific neuronal compartments of the neuron as well. Many of these have been defined as part of the “cytosol” or soluble fraction that results from biochemical fractionation. These include the enzymes of glycolysis and regulatory proteins like calmodulin and HSC70 (Brady, 1993). However, in pulse-chase radiolabel studies, soluble proteins move down the axon as regularly and systematically as cytoskeletal proteins. Again, this coherent transport of hundreds of different polypeptides appears consistent with the Structural Hypothesis and indicates a higher level of organization of cytoplasmic proteins than has been traditionally assumed (Lasek & Brady, 1982). Such organization is likely necessary to facilitate interactions with motor proteins and targeting mechanisms and to assure a reliable delivery of all required proteins to the axon at appropriate stoichometries.

Kinesins, Liprin, and Presynaptic Vesicle Precursor Transport

There are two major subdivisions of axonal transport: fast and slow. Soluble cytoskeletal proteins such as tau, kinesin, dynein, myosin, and tubulin are transported at a rate of approximately 1 mm day^–1 by slow axonal transport. Membrane-bound proteins, associated with organelles such as presynaptic precursor vesicles, are transported at a rate of approximately 1 μm s^–1 by fast axonal transport. The kinesin family of molecular motors moves soluble proteins and organelles away from the cell body, and dynein moves these materials toward the cell body. When kinesin-1 is disrupted, axonal clogs that contain synaptic components occur along axons and synaptic transmission is disrupted.

While substantial evidence suggests that liprin-α plays a direct role in determining the form and function of synapses, recent data also reveal that liprin-α is involved in the fast axonal transport of presynaptic vesicle precursors (PVPs). PVPs contain the raw materials for the assembly of synapses, and while they are thought to be transported primarily by kinesin-3 (Unc-104, Kif1a), kinesin-1 (conventional kinesin) family members may also play a role in their transport. Since axonal accumulations of endogenous synaptotagmin similar to those found in kinesin heavy chain (Khc) alleles are also observed in mutants lacking Drosophila liprin-α, we speculate that liprin-α is involved in the transport of a subset of synaptic components and that this subset may be transported by more than one family of kinesin.

At one time is was thought that there were molecules whose sole purpose was to link kinesins to cargos, but it now appears that most adaptors are scaffolding proteins that interact with multiple proteins and do not serve as simple linkers. Liprin-α binds to kinesin-1 and kinesin-3. Yet, the balance of the evidence is against liprin-α acting as a simple linker between PVPs and kinesins. When it is disrupted, the transport of PVPs is not halted. Instead, it shifts the probability of transport from anterograde (kinesin-mediated) to retrograde (dynein-mediated) transport. Thus, liprin-α is not required for the movement of PVPs per se. Instead of a simple linker, liprin-α appears as a modulator of axonal transport.

One of the most basic questions in neurobiology is how some types of neuronal components are trafficked to dendrites, and others are selectively transported into axons. Liprin-α is part of a complex that targets postsynaptic components to dendrites and involves GRIP, AMPARs, and kinesin-1. Of note, there are splice variants of liprin-α that lack the GRIP-binding domain. It is tempting to speculate that this differential targeting between axons and dendrites may involve variations in splicing. Yet further work will be required to determine if and how liprin-α could help to selectively guide components into either axonal or dendritic compartments. While several lines of evidence suggest that liprin-α regulates transport, a great deal of work remains to provide a satisfying molecular answer as to how this occurs.

Overview of Toxicant-Induced Neuropathies

In 1969, Pleasure and associates reported marked reduction of slow axonal transport of radiolabeled protein in the central processes of sensory neurons of cats with acrylamide neuropathy.¹⁸⁰ Since that original study, both fast and slow anterograde transport have been measured by various methods in many experimental neuropathies (Table 18-1). Minor reductions in fast-transport velocity have been encountered in the neuropathies induced by zinc pyridinethione (ZPT),¹⁴⁷ acrylamide,²⁶ and vincristine,²⁶ although some studies have reported normal velocities in the same conditions.²¹⁷ More substantial slowing of fast axonal transport, up to 36%, has been seen in hexacarbon neuropathy.¹⁵² Overall, however, one is struck by the extent to which fast anterograde transport seems to persist at full speed despite substantial axonal pathology. Few if any of the toxicant-induced neuropathies involve gross defects of fast anterograde transport. Some more pronounced effects on slow anterograde transport in several different toxic neuropathies are detailed subsequently.

Discovery of Kinesin and Kinesin Superfamily Proteins

The physiological relevance of kinesin-1 in intracellular transport was revealed by genetics studies of fungi, Drosophila (Hurd et al., 1996), and mice (Tanaka et al., 1998). For example, kinesin-1 depletion via gene targeting in mice revealed striking perinuclear clustering of its mitochondrial cargo in extra-embryonic yolk sac cells (Tanaka et al., 1998). This perinuclear clustering was proposed to be due to an imbalance of bidirectional transport in these cells, providing genetic evidence that kinesin can significantly alter the intracellular localization of organelles. Concurrently, positional cloning of yeast and fungal cell division mutants revealed the existence of kinesin-like proteins that shared several amino acid sequences with kinesin-1, such as KAR3 in yeast (Meluh and Rose, 1990). Based on RT-PCR cloning using degenerate primers corresponding to consensus sequences from a mouse brain cDNA library, our group first identified all 45 genes for the mammalian kinesin superfamily proteins (KIFs) (Miki et al., 2001; Nakagawa et al., 1997; Aizawa et al., 1992).

Slow axonal transport

Neurofilaments and other cytoskeletal polymers are transported down the axon at a rate of 0.2–8 mm day⁻¹, in a process known as ‘slow’ axonal transport. This transport is orders of magnitude slower than the transport of vesicular cargos in ‘fast’ axonal transport, at rates of ∼200–400 mm day⁻¹. For a long time, the mechanisms driving the slow anterograde transport of neurofilaments were unclear. However, recent progress has shown that slow transport along the axon involves the same microtubule motor proteins that drive fast transport. The key difference is that for cargo such as neurofilaments that undergo slow transport, rapid excursions in either the anterograde or retrograde direction are punctuated by prolonged pauses. Both the frequent changes in direction and the extended pauses contribute to an overall net slow outward movement.

The reasons why cytoskeletal polymers are transported in such an apparently inefficient mechanism remain to be determined, but one possibility is that the oppositely oriented forces, applied simultaneously by plus and minus end-directed motors, result in a continuous tension on the cargo. In the case of neurofilaments, this tension may keep the long, flexible filaments stretched out along microtubules, and therefore may counteract aggregation. Support for this hypothesis comes from the observation that knockdown of dynein expression or inhibition of dynein/dynactin function leads to the distal accumulation of neurofilaments along neuronal processes.