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Slow Axonal Transport 1 Author's personal copy Slow Axonal Transport 1 Slow Axonal Transport A Brown , Ohio State University, Columbus, OH, USA The different rates of fast and slow axonal trans- ã 2009 Elsevier Ltd. All rights reserved. port have long been assumed to reflect fundamentally distinct mechanisms of movement, but direct obser- vations of membranous organelles and cytoskeletal polymers in living cells now suggest that all axonal Overview cargoes are transported by fast motors. Thus, the Axons can be 1 m or more in length in large animals, principal difference between fast and slow transport yet they are dependent on the cell body for the syn- is not thought to be the rate of movement per se but, thesis of most of their components. Materials des- rather, the frequency of movement and the nature of the cargo structures. The terms fast and slow tined for the axon are transported anterogradely, toward the axon tip, and materials destined to return refer to the average rate of movement on a timescale are transported retrogradely, toward the cell body. of days or weeks and do not reflect the actual velocity of the transport motors. This bidirectional transport process, known as axo- nal transport, is not fundamentally different from the pathways of macromolecular and membrane traffic Methods for Studying Slow Axonal found in other parts of the neuron, or indeed in any Transport eukaryotic cell, but it is unique for the volume and scale of the traffic. There are essentially three different kinds of experi- mental techniques for studying axonal transport: Kinetic studies on axonal transport in vivo using radioisotopic pulse labeling have defined two distinct accumulation techniques, pulse-labeling techniques, patterns of movement, termed fast and slow. Fast axo- and imaging techniques (Table 1). Although these approaches can all be applied to both fast and slow nal transport represents the movement of membranous organelles, including Golgi-derived vesicles, endocytic axonal transport, their use for studies on slow axonal organelles, mitochondria, peroxisomes, lysosomes, transport is emphasized here. Each technique has and autophagosomes. For the most part, these orga- strengths and limitations, and each reveals different aspects of the motility, so they should be regarded as nelles move rapidly and frequently at rates of up to several hundred millimeters per day, which corre- complementary approaches, not as alternatives. sponds to several micrometers per second. Some of Accumulation Techniques these organelles move anterogradely, some retro- gradely, and some (such as mitochondria) are capable The oldest and simplest technique for studying axo- of frequent reversals and move in both directions. nal transport is to locally impede movement along an axon or nerve and then observe what cargo structures Ribosomes and ribonucleoprotein particles, which are transported along developing and regenerating axons, accumulate and the rate at which they do so. Retro- probably also move by fast axonal transport. gradely moving materials accumulate on the distal side of the blockade and become depleted on the In contrast to fast axonal transport, slow axonal transport represents the movement of proteins that proximal side, whereas anterogradely moving materi- make upthe cytoskeleton and cytosol of axons. These als accumulate on the proximal side and become proteins, which are not associated with membra- depleted on the distal side. The most commonly nous organelles, move in an intermittent manner at used approaches are to surgically ligate or crush a average rates of approximately millimeters or tenths nerve in vivo. A variant on these approaches is the of millimeters per day, which corresponds to several cold block, in which local cooling is applied to a nerve in vivo. This method is technically more challenging, nanometers or tens of nanometers per second, much slower than fast axonal transport. The cargo struc- but eliminates the tissue damage that is associated tures of slow axonal transport are largely unknown, with the ligation and crush approaches. Another approach, which has been applied to isolated axons but they are believed to include cytoskeletal polymers and cytosolic macromolecular complexes. For many teased apart from peripheral nerves and to single years, it was assumed that slow axonal transport axons of cultured neurons, is to locally compress is an exclusively anterograde movement, but it is axons with fine nylon or glass fibers (Figure 1). now clear that at least some of the cargoes of slow Pulse-Labeling Techniques axonal transport can move in both anterograde and retrograde directions, although the net direction of The most widely used pulse-labeling technique is radio- movement may still be anterograde. isotopic pulse labeling, which was first introduced Encyclopedia of Neuroscience (2009), vol. 9, pp. 1-9 Author's personal copy 2 Slow Axonal Transport Table 1 Three experimental methods for studying slow axonal transport Accumulation techniques Pulse-labeling techniques Imaging techniques Example Ligate or constrict single axons Inject radioactive amino acids into the Inject or transfect neurons with or entire nerves and observe vicinity of nerve cell bodies in vivo and fluorescent proteins and observe the the accumulation of then sacrifice the animal and analyze moving structures by fluorescence transported materials the distribution of the radiolabeled microscopy proteins along the axons Strengths Simple; can be performed Permits the kinetics and composition of High temporal resolution (seconds or in vivo or in cell culture axonal transport to be studied in vivo milliseconds); high spatial resolution (micrometers); permits individual cargoes to be tracked Limitations Poor spatial resolution; poor Poor spatial resolution (1 mm at best); Not well suited to long-timescale temporal resolution poor temporal resolution (hours or days studies; cannot presently be at best); each animal yields only one performed in vivo time point, so multiple animals are required for each analysis in the late 1950s and 1960s. Most of what we know 0 min about the composition and kinetics of slow axonal transport has come from studies using this approach, a and it remains to this day the preferred method for 1 min studying the composition and kinetics of slow axonal transport in vivo. Essentially, this technique involves b the injection of radiolabeled precursors of macromole- 30 min cules (amino acids, sugars, or nucleotides) into the vicinity of neuronal cell bodies in an animal. Most published studies have used radiolabeled amino acids, c which permit the movement of proteins to be investi- 120 min gated. The radiolabeled amino acids are taken up by the cell bodies, creating a pulse of radiolabeled pro- d teins. Those proteins destined for the axon move into Fixed and along the axon in association with distinct cargo structures. By injecting numerous animals and sacrifi- e cing them at various time intervals, the kinetics of transport can be analyzed (each animal yields a single NF protein time point). The most powerful approach is to dissect out the nerves containing the radiolabeled proteins f and then cut them into contiguous segments, permit- Figure 1 Constriction of an axon of a cultured neuron growing ting biochemical analysis of the radiolabeled proteins on a glass coverslip: (a) axon prior to constriction; (b) axon after by subcellular fractionation, immunoprecipitation, 1 min constriction with a flexible glass fiber (1 mm in diameter); (c) axon after 30 min; (d) axon after 120 min; (e) constricted axon and/or electrophoresis (Figure 2). after fixation and removal of the glass fiber; (f) distribution of Radioisotopic pulse-labeling studies of slow axonal neurofilament protein in the fixed axon, visualized by immunofluo- transport can be performed on a variety of different rescence microscopy. Note that the glass fiber compresses the nerve cell types. Those that are best suited are ones axon without severing it. Anterogradely and retrogradely moving whose cell bodies are located in an anatomically dis- membranous organelles, which are cargoes of fast axonal trans- port, accumulate rapidly, causing the axon to swell on both sides crete region, facilitating reproducible injection of the radioisotope, and ones whose axons extend in a of the constriction (proximal is left, distal is right). As shown in (f), the net anterograde movement of neurofilaments in these axons nerve that can be dissected readily and that is long results in an accumulation of neurofilament protein proximal to with minimal branching. Most radioisotopic pulse- the constriction, which is evident from the bright fluorescence labeling studies have been performed on rats or in the proximal swelling. Scale bar ¼ 5 mm. Reproduced from Koehnle TJ and Brown A (1999) Slow axonal transport of neuro- mice, and the most commonly used axons are the filament proteins in cultured neurons. Journal of Cell Biology 144: retinal ganglion cell axons of the optic nerve (radio- 447–458. labeled precursor injected into the eye) and the motor Encyclopedia of Neuroscience (2009), vol. 9, pp. 1-9 Author's personal copy Slow Axonal Transport 3 Nerve segments Nerve segments Inject radiolabeled 1 234 1234 amino acid Dorsal ganglion Dorsal root Axons of sciatic nerve Time 1 Time 2 Spinal cord 1234 1234 Time 3 Time 4 Nerve segments 1234 a b1 234 1234 Assay for various radiolabeled proteins Figure 2 The radioisotopic pulse-labeling technique. (a) Radiolabeled amino acids are injected into the vicinity of nerve
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