Hypothyroidism reduces the rate of slow component A (SCa) axonal transport and the amount of transported tubulin in the hyt/hyt mouse optic nerve.

Thyroid hormone deficiency in the developing brain leads to disorders of neuronal process growth. This is evidenced by reduced axonal and dendritic size and complexity (Garza et al.: Developmental Brain Research 43:287-297, 1988; Ruiz-Marcos: Iodine and the Brain. New York: Plenum Press, pp 91-102, 1989). These findings may be related to alterations in the neuronal cytoskeleton in hypothyroidism, such as reduced or abnormal microtubular number and density (Faivre et al.: Developmental Brain Research 8: 21-30, 1983), and altered assembly, stabilization, and composition of microtubule protein in the hypothyroid brain. Neurofilaments also contribute to axonal caliber and process stability. Similar to microtubules, certain properties of neurofilaments are altered in developing hypothyroid axons (Marc and Rabie: International Journal of Developmental Neuroscience 3: 353-358, 1985; Faivre et al.: Developmental Brain Research 8:21-30, 1983) that may affect axonal caliber and process stability. Normal process growth is predicted on formation of appropriate numbers of microtubules and on the normal synthesis and axonal transport of cytoskeletal components [tubulin, microtubule associated proteins (MAPs), and neurofilament proteins]. Hypothyroidism might alter the neuronal cytoskeleton and neuronal growth either by affecting the developmental programs for expression of specific isoforms of cytoskeletal proteins or by changing the delivery of cytoskeletal proteins via slow axonal transport, particularly slow component a (SCa). Previous studies had demonstrated changes in the amount of specific microtubule protein isoforms and mRNAs (Stein et al.: Iodine and the Brain. New York: Plenum Press, pp 59-78, 1989a). To further elucidate the molecular basis for process growth abnormalities in the hypothyroid brain, we investigated slow axonal transport in the mouse to determine the effects of thyroid hormone deficiency on the rate and composition of SCa. Comparisons of SCa in the optic nerve of hyt/hyt hypothyroid mouse and euthyroid hyt/+ littermates and euthyroid progenitor strain, BALB/cBY +/+ mice, indicated that the velocity of SCa was significantly reduced in hyt/hyt optic nerve relative to hyt/+ and +/+. The axonal transport rate for tubulin, which is carried in SCa, was 0.118 mm/day in the hyt/hyt optic nerves. This rate was significantly different for the tubulin rates for the hyt/+ optic nerves (0.127 mm/day) and for the +/+ optic nerves (0.138 mm/day). Neurofilament proteins, as measured by the 140,000 daltons component, NFM, also appeared to be reduced in velocity in the hyt/hyt versus the hyt/+ and +/+ optic nerves.(ABSTRACT TRUNCATED AT 400 WORDS)     

Axonal transport of a clathrin uncoating ATPase (HSC70): a role for HSC70 in the modulation of coated vesicle assembly in vivo

Clathrin plays an important role in many cellular processes, including endocytosis, secretion, and sorting of membranous organelles. Both the neuronal cell body and presynaptic terminals contain numerous coated vesicles, but few are detectable in the axonal regions that connect these two regions of the neuron. Clathrin heavy chains, light chains, and assembly proteins have all been shown to be axonally transported as part of slow component b (SCb). However, the paucity of coated vesicles present in the axon indicates the existence of a mechanism regulating clathrin coated assembly in vivo. A clathrin uncoating ATPase has been described that binds in stoichiometric amounts to clathrin and dissociates clathrin coats from vesicles in the presence of ATP in vitro. This clathrin uncoating ATPase is a major cytosolic protein of Mr 70 kD in bovine brain, forming 1% of soluble brain protein, and appears to be homologous with a constitutively expressed 70 kd heat shock protein (HSC70). We report here that a major 70 kD protein present in the SCb rate component of axonal transport is identical with HSC70 by both biochemical and immunochemical criteria. The cotransport of HSC70 with clathrin in SCb of axonal transport is consistent with a role for HSC70 in vivo in the regulation of clathrin function during axonal transport.     

Alterations in slow transport kinetics induced by estramustine phosphate, an agent binding to microtubule-associated proteins.

Estramustine phosphate (EP) disassembles microtubules by binding to microtubule-associated proteins (MAPs) rather than tubulin. In this study, EP-induced alterations of MAP integrity caused a unique form of axonal atrophy in rats. Initially, EP-induced axonal atrophy occurred in both proximal and distal axons of the sciatic nerve, characterized by an increase in neurofilament packing density, associated with a decrease in axonal area. In chronic exposure, distal axonal atrophy was associated with decreased numbers of microtubules, while the neurofilament number remained unaltered for the myelin spiral length. Continued exposure caused enlargement of proximal axons associated with an increase in neurofilament content. Correlative slow transport studies done at two different times, 7 and 14 days after [35S] methionine injection showed that EP retards the transport of cytoskeletal proteins migrating with both components of slow transport (SCa and SCb). However, there was a differential effect on SCb which showed progressive slowing along the nerve while the rate of SCa stayed relatively constant. In this model, the early occurring distal axonal atrophy can best be explained by reduced cytoskeletal components, particularly those traveling in SCb. Later in the course of intoxication, a relatively constant rate of SCa permitted continuous transport of neurofilament triplets, accounting for unaltered numbers of neurofilaments in distal axons with increased packing density. This model of axonal atrophy is unique because spacing of neurofilaments, not numbers determined axon size. Furthermore, EP-induced dissociation of the SCa and SCb kinetics suggests that MAPs play a role in the orderly, cohesive migration of slow transport components, essential for the normal organization of cytoskeleton.     

Diversity in the axonal transport of structural proteins: major differences between optic and spinal axons in the rat

Investigations of slow axonal transport reveal variation in both protein composition and the rate of movement. However, these studies involve a variety of nerve preparations in different species, and most lack the resolution needed to determine the kinetics of identified proteins. We have compared the axonal transport of slow-transported proteins in retinal ganglion cells and spinal motor neurons of young rats. Nine proteins that contribute to axonal structures were examined: the neurofilament triplet (NFT), alpha and beta tubulin, actin, fodrin, calmodulin, and clathrin. Axonally transported proteins were pulse-labeled by intraocular or intracord injections of 35S-methionine. After allowing sufficient time for labeled slow-component proteins to enter the spinal or optic nerves, consecutive 2-3 mm nerve segments were subjected to SDS-PAGE. Fluorographs were used as templates for locating the gel regions containing the above polypeptides, and the radioactivity in these regions was measured by liquid-scintillation spectrometry. In retinal ganglion cells, the peak of tubulin labeling advanced at 0.36 mm/d in association with the NFT and fodrin. The cotransport of tubulin and the NFT identified this complex as the slower subcomponent of slow transport, termed slow component a (SCa) and representing the movement of the microtubule-neurofilament network. The peaks of actin and calmodulin labeling were cotransported at 2.3 mm/d in near-register with peaks of fodrin and clathrin labeling. These 4 proteins, moving ahead of the NFT, identified this complex as SCb--the faster subcomponent of slow transport, which represents the movement of the cytoplasmic matrix and microtrabecular lattice. Both subcomponents had the same composition and rate as that reported for the optic axons of guinea pigs and rabbits, establishing a basic mammalian pattern. In spinal motor axons, the SCa tubulin peak advanced at 1.3 mm/d, and the SCb actin and calmodulin peaks were cotransported at 3.1 mm/d. Unlike optic axons, SCa in motor axons was more heavily labeled than SCb, and included labeled peaks of actin, clathrin, and calmodulin moving in register with the SCa tubulin peak. Actin was the most heavily labeled of these SCb proteins moving with SCa, and it left a higher plateau of radioactivity behind the advancing SCa peak. The SDS-PAGE labeling pattern for SCb did not differ from that seen in optic axons, except that some tubulin was found to form a peak that advanced in register with the actin and calmodulin peaks.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assembly of microfilaments and microtubules from axonally transported actin and tubulin after axotomy

The slow component (SC) of axonal transport conveys structural proteins, regulatory proteins, and glycolytic enzymes toward the axon tip at 1–6 mm/day. Following axon interruption (axotomy), the rate of outgrowth corresponds to the rate of SCb—the fastest subcomponent of SC. Both axonal outgrowth and SCb accelerate 20–25% after axotomy. Tubulin and actin are the major proteins being carried by SCb. To further characterize the acceleration of SCb, we measured the equilibrium between subunits and polymers for both actin and tubulin. We radiolabeled newly synthesized proteins in rat motor neurons by microinjecting [³⁵S]methionine into the spinal cord 7 days after crushing the sciatic nerve (85 mm from the spinal cord). Nerves were removed 7 days later for homogenization in polymer-stabilizing buffer (PSB) and centrifugation, followed by SDS-PAGE of supernatants (S) and pellets (P). We removed β-tubulin, actin, and the medium-weight neurofilament protein (NF-M) from each gel by using the fluorogram as a template. After solubilizing gel segments for liquid scintillation spectrometry, we expressed counts as a polymerization ratio: P/[S + P]. In the nerve segments that contained radiolabeled SCb proteins, located 24–36 mm from the spinal cord, axotomy increased the polymerization ratio of SCb actin from 0.23 to 0.36 (P < 0.05) but had no effect on SCb β-tubulin. In a separate experiment, we added 12 μM taxol to PSB to stabilize newly assembled microtubules. Adding taxol did not alter the polymerization ratio for SCb β-tubulin in sham-axotomized nerves but did increase the ratio in axotomized nerves, from 0.44 to 0.63 (P < 0.05); polymerization ratios for SCb actin were unaffected. We conclude that the assembly of microfilaments and microtubules increases to provide cytoskeletal elements for axon sprouts. The resulting loss of actin and tubulin subunits may play a role in the acceleration of SCb. © 1996 Wiley-Liss, Inc.     

Altered slow axonal transport and regeneration in a myelin-deficient mutant mouse: the trembler as an in vivo model for Schwann cell-axon interactions

The thickness of the myelin sheath in normal myelinated nerve is proportional to the diameter of the axon. In the demyelinating mutant mouse, Trembler, not only is the thickness of the myelin sheath reduced, but the caliber of associated axons is smaller. This correlation suggests that the interaction between axons and Schwann cells may affect the shape and function of axons as well as properties of myelin. Since axonal diameter depends in part on the cytoskeleton and its movement with slow axonal transport, we have compared the properties of slow transport in the sciatic nerve of control and Trembler mice. Studies of the sciatic nerve of normal mice showed that the rates for proteins moving in slow component a (SCa) and slow component b (SCb) are similar to those previously measured in rat. In Trembler mice, tubulin was transported significantly faster than in control mice, with a rate of 1.73 mm/d for Trembler compared to 1.56 mm/d in the control. In contrast, the rate for neurofilament proteins was significantly slower in the Trembler (1.15 mm/d compared to 1.38 mm/d in the control). The majority of proteins in SCb were also transported slower in Trembler than control: actin and calmodulin were transported at 2.29 mm/d as compared to 2.73 mm/d in control, while spectrin and clathrin were transported at 2.01 and 2.43 mm/d, respectively, as compared to 2.54 mm/d in control. The importance of slow axonal transport in regeneration has been suggested by the clear correlation between the rates of regeneration and the rates of SCb. Therefore, we evaluated regeneration of motor axons in Trembler mice to determine whether the regenerative response was affected by deficient Schwann cells. A slower regeneration rate was found in the Trembler (1.7 mm/d) motor axon when compared to the control (2.29 mm/d), but elongation of fibers in regeneration began after a shorter delay in the Trembler (1.6 d) than in control (2.5 d). Thus, deficient Schwann cells and poor myelination appear to affect both quantitative and qualitative properties of slow axonal transport. These changes lead to alterations in the morphological and physiological properties of affected axons.     

Conditioning nerve crush accelerates cytoskeletal protein transport in sprouts that form after a subsequent crush.

To examine the relationship between axonal outgrowth and the delivery of cytoskeletal proteins to the growing axon tip, outgrowth was accelerated by using a conditioning nerve crush. Because slow component b (SCb) of axonal transport is the most rapid vehicle for carrying cytoskeletal proteins to the axon tip, the rate of SCb was measured in conditioned vs. sham-conditioned sprouts. In young Sprague-Dawley rats, the conditioning crush was made to sciatic nerve branches at the knee; 14 days later, the test crush was made where the L4 and L5 spinal nerves join to form the sciatic nerve in the flank. Newly synthesized proteins were labeled in motor neurons by injecting 35S-methionine into the lumbar spinal cord 7 days before the test crush. The wave of pulse-labeled SCb proteins reached the crush by the time it was made and subsequently entered sprouts. The nerve was removed and sectioned for SDS-PAGE and fluorography 4-12 days after the crush. Tubulins, neurofilament proteins, and representative "cytomatrix" proteins (actin, calmodulin, and putative microtubule-associated proteins) were removed from gels for liquid scintillation counting. Labeled SCb proteins entered sprouts without first accumulating in parent axon stumps, presumably because sprouts begin to grow within hours after axotomy. The peak of SCb moved 11% faster in conditioned than in sham-conditioned sprouts: 3.0 vs. 2.7 mm/d (p less than 0.05). To confirm that sprouts elongate more rapidly when a test crush is preceded by a conditioning crush, outgrowth distances were measured in a separate group of rats by labeling fast axonal transport with 3H-proline 24 hours before nerve retrieval.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport of cytoskeletal elements from parent axons into regenerating daughter axons

The kinetics of slow axonal transport in newly regenerating axonal sprouts were compared with those in nonelongating axons. The slowly transported cytoskeletal proteins of ventral motor axons were prelabeled by microinjection of 35S-methionine into the spinal cord. Pulse-labeled slow transport "waves" were observed as they progressed from the surviving "parent" axon stumps (located proximal to a crush lesion) into regenerating "daughter" axon sprouts (located distal to the lesion). Prelabeled cytoskeletal elements of the parent axons were transported into daughter axons, to become distributed into 2 transport waves, "a" and "b." The rate and composition of these waves corresponded to the slow transport subcomponents, SCa and SCb. The shapes of the "a" and "b" waves suggested that the cytoskeletal elements had been reorganized at the junction between the parent and daughter axons. This hypothesis was supported by quantitative analyses of the transport distribution for individual radiolabeled cytoskeletal proteins (actin, spectrin, a 58-67 kDa group that includes microtubule-associated proteins, calmodulin, and tubulin). Specifically, during the first week of outgrowth, the amounts of radiolabeled calmodulin and 58-67 kDa proteins were greater in daughter axons than in nonregenerating control axons. These results support Paul Weiss's "conservative" model of axonal regeneration, which holds that the preexisting transported cytoskeletal elements that continually maintain axonal structure can also provide the cytoskeletal elements required for axonal regeneration. In addition, the results elucidate some of the reorganizational changes in cytoskeletal elements that occur when these are recruited from the parent axon to form daughter axons.     

Synapsin I: A regulated synaptic vesicle organizing protein

Synapsin is a protein initially discovered and characterized as a target for cyclic AMP and Ca/calmodulin-regulated protein kinases that is concentrated in nerve endings and is localized on the surface of small synaptic vesicles. Synapsin shares antigenic sites and some local regions of homology with erythrocyte protein 4.1, although these proteins in general are quite different in sequence. Protein 4.1 and synapsin share several local regions of homology with erythrocyte spectrin alpha subunit. Protein 4.1 and synapsin may be related to each other through a common relationship with spectrin. Synapsin binds to synaptic vesicles and membrane sites with a Kd of 0.01-0.02 microM and associates with a Kd of 0.5-4 microM to spectrin, microtubules and neurofilaments in in vitro assays. Synapsin interconnects synaptic vesicles to membranes, and this activity is inhibited by phosphorylation with Ca/calmodulin-dependent protein kinase. Synapsin may have a role in neurons as a structural protein capable of interconnecting small synaptic vesicles with a number of proteins, including spectrin, microtubules, neurofilaments, and membrane sites. A physiological function of synapsin could be as a vesicle-organizing protein that mediates calcium-regulated association of vesicles with cytoskeletal proteins during axonal transport and attaches vesicles to active zones in nerve endings.     

Abnormal growth of the corticospinal axons into the lumbar spinal cord of the hyt/hyt mouse with congenital hypothyroidism

Thyroid hormone deficiency may cause severe neurological disorders resulting from developmental deficits of the central nervous system. The mutant hyt/hyt mouse, characterized by fetal-onset, life-long hypothyroidism resulting from a point mutation of the thyroid-stimulating hormone receptor of the thyroid gland, displays a variety of abnormalities in motor behavior that are likely associated with dysfunctions of specific brain regions and a defective corticospinal tract (CST). To test the hypothesis that fetal and neonatal hypothyroidism cause abnormal CST development, the growth of the CST was investigated in hypothyroid hyt/hyt mice and their euthyroid progenitors, the BALB/cByJ mice. Anterograde labeling with biotinylated dextran amine demonstrated a decrease in the number of CST axons in the hyt/hyt mouse at the first lumbar level at postnatal day (P) 10. After retrograde tracing with fast blue (FB), fewer FB-labeled neurons were found in the motor cortex, the red nucleus, and the lateral vestibular nucleus of the hyt/hyt mouse. At the fourth lumbar level, the hyt/hyt mouse also showed smaller CST cross-sectional areas and significantly lower numbers of unmyelinated axons, myelinated axons, and growth cones within the CST during postnatal development. At P10, the hyt/hyt mouse demonstrated significantly lower immunoreactivity of embryonic neural cell adhesion molecule in the CST at the seventh cervical level, whereas the expression of growth-associated protein 43 remained unchanged. Our study demonstrated an abnormal development of the CST in the hyt/hyt mouse, manifested by reduced axon quantity and retarded growth pattern at the lumbar spinal cord.     

Decreased axon caliber and neurofilaments in hereditary motor and sensory neuropathy, type I

The axons of large- and intermediate-diameter myelinated fibers of sural nerves of patients with hereditary motor and sensory neuropathy, type I (HMSN-I), were previously found to be attenuated relative to their myelin spiral length. We inferred that axonal atrophy might account for secondary segmental demyelination and remyelination. To assess whether the observed axonal atrophy could be explained by a decrease in neurofilaments, we have evaluated the number of neurofilaments, microtubules, and other axon organelles in sural nerves of patients with HMSN-I. Whereas the density per square micrometer of neurofilaments or microtubules in diseased nerves was not significantly different from that in control specimens, the number of neurofilaments per axon as related to myelin spiral length was significantly less for intermediate and large myelinated fibers in HMSN-I nerves. The regression lines for the number of microtubules per axon on myelin spiral lengths were also less steep in HMSN-I, but the difference did not reach statistical significance. These results indicate that the number of neurofilaments is proportional to axon diameter but significantly below that expected considering myelin spiral length. Decreased neurofilament synthesis, assembly, or transport may underlie the axonal atrophy in HMSN-I.     

Slow component B protein kinetics in optic nerve and tract windows

The transport kinetics of 3 radiolabeled slow component b (SCb) proteins (a 30 kDa protein, clathrin, and actin) were examined in the axons of mouse retinal ganglion cells. To view the transit of these proteins through the entire optic pathway between the eye and the target cells, we used two different windows: (1) a 2 mm segment from the optic nerve located 3-5 mm from the eye, and (2) a 2 mm segment from the optic tract located past the chiasm 6-8 mm from the eye. The radiolabeled proteins from these windows were separated by 1- and 2-dimensional SDS-PAGE, and the individual radiolabeled bands were quantified. Radiolabeled proteins entered and cleared the optic axons between 1 and 119 days post-labeling. All these proteins had broader transport waves in the more distal optic tract window than in the more proximal optic nerve window. The spreading of transport waves as they advance along the axon appears to be produced by a playing out of the natural heterogeneity of axonal transport rates within each population of labeled proteins. Our results confirm the proposals that clathrin and the 30 kDa protein are transported principally with SCb and that actin is transported both with SCb and with SCa. Although these proteins can be generally classified with SCb, their detailed kinetics differed (for example, their median transit times differed) and, in summary, their characteristic rates of movement can be ordered as: clathrin greater than 30 kDa protein greater than actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modifications in the cytoskeleton transported by slow component A during axonal regeneration

We studied the modifications occurring in the parent cytoskeleton carried by SCa (the slower of the two slow axonal transport subcomponents) after peripheral nerve crush. The proteins transported in rat sciatic motor axons were radiolabelled by injecting [35S]methionine into the ventral horn of the spinal cord, and the nerve was crushed so as to entrap only the proteins transported by SCa along the parent axon. Two weeks after the crush, the regenerating nerve was removed and the distributions of the polymerized and unpolymerized radiolabelled cytoskeletal proteins were compared with those in normal, non-regenerating nerves. We found that in the parent axons, most of the radioactive neurofilaments were arrested by the crush, but microtubules, soluble tubulin, insoluble and soluble actin were normally transported. Thus, when the resulting cytoskeleton transported by SCa entered the daughter axon, it was enriched in microtubules and actin, and partially depleted of neurofilaments. This cytoskeleton contained larger proportions of soluble tubulin and insoluble actin than the parent cytoskeleton, but retained its coordinated progression and transport velocity, suggesting that after axotomy, the main destiny of the parent cytoskeleton carried by SCa is to become the equivalent cytoskeleton in the daughter axons.     

Effect of changes in neurofilament content on caliber of small axons: the beta,beta'-iminodipropionitrile model

The structural role of neurofilaments in the normal axon and the consequences of altered axonal transport of neurofilaments have been extensively studied in large axons. These studies suggest that neurofilament numbers and interneurofilament spacing are major determinants of axonal cross-sectional area. In contrast, in small axons and dendrites, microtubules and membranous organelles appear to be the most closely correlated with size and shape of the cell process. In this study we have examined the effect of impairment in neurofilament transport on small axons, typical of most CNS pathways. Neurofilament transport was impaired by administration of beta,beta'-iminodipropionitrile (IDPN), resulting in proximal accumulation and distal depletion of neurofilaments. The evolution of these changes was studied in the optic nerves of guinea pigs treated with IDPN, 1-35 weeks following intoxication. The effect of this redistribution of neurofilaments on cross-sectional area of small axons was evaluated using quantitative ultrastructural methods. Our results show that with the alteration in neurofilament transport seen with IDPN intoxication, there is a wide spectrum of neurofilament densities, ranging from a 5-fold increase above normal in the proximal axon, to a 5-fold decrease below normal in the distal axon. Although the optic nerve fibers enlarge with the increase in neurofilament content, they do not atrophy significantly with the continued loss of neurofilaments. We conclude that factors other than neurofilament content are capable of maintaining size and shape of these small axons. Candidate organelles include microtubules and membranous organelles and possibly other axonal elements.     

Axonal transport of the cytoskeleton in regenerating motor neurons: constancy and change

We have examined slow axonal transport in regenerating motor neurons of the rat sciatic nerve. Using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) we previously found that the slow component is the vehicle for the axonal cytoskeletal proteins, i.e. the neurofilament triplet proteins, tubulin and actin. When these proteins are pulse-labeled by injecting [3H]- or [35S]-amino acids into the spinal cord, they are transported distally in the nerve as two distinguishable waves of radioactivity, SCa and SCb. In normal motor neurons, the neurofilament triplet proteins and the tubulin are transported in SCa at an average velocity of 1.7 mm/day; the less heavily labeled SCb which moves at 2-5 mm/day is the primary vehicle for actin. We now find that during regeneration the velocity of SCa is unchanged in the region of the axon between the cell body and the lesion, but the amount of labeled neurofilament triplet and associated tubulin transported in the axon is decreased in neurons which had been labeled 20 days post-lesion. In contrast, the labeling of the slowly transported proteins moving ahead of the neurofilament triplet is greater in regenerating nerves than in controls. On the basis of our findings, we propose that in motor axons the normal supply of cytoskeletal protein, which is continuously transported in the slow component, is sufficient to support regeneration. Nevertheless, the neuron cell body can alter the supply of these cytoskeletal proteins so as to enhance its regenerative capacity.     

Selective impairment of slow axonal transport after optic nerve injury in adult rats.

To investigate cellular responses of injured mammalian CNS neurons, we examined the slow transport of cytoskeletal proteins in rat retinal ganglion cell (RGC) axons within the ocular stump of optic nerves that were crushed intracranially. RGC proteins were labeled by an intravitreal injection of 35S-methionine, and optic nerves were examined by SDS PAGE at different times after injury. In one group of rats, the RGC proteins were labeled 1 week after crushing. From 14 to 67 d after axotomy, the labeling of tubulin and neurofilaments was reduced in relation to other labeled proteins and to the labeling of tubulin and neurofilaments in the intact optic nerve of controls. To determine whether this reduction in labeling was due to an alteration in axonal transport after axotomy, we prelabeled RGC proteins 1 week before crushing. In such experiments, the rate of slow axonal transport of tubulin and neurofilaments decreased approximately 10-fold from 6 to 60 d after injury. Our results cannot be due only to the retrograde degeneration of RGCs and injured axons caused by axotomy in the optic nerve, because fast axonal protein transport and the fluorescent labeling of many axons were preserved in the ocular stumps of these optic nerves. This selective failure of the slow axonal transport of tubulin and neurofilaments may affect the renewal of the cytoskeleton and contribute to the gradual degeneration of RGCs that is observed after axotomy. The alterations in slow transport we document here differ from the enhanced rates we previously reported when injured RGC axons regenerated along peripheral nerve segments grafted to the ocular stump of transected optic nerves (McKerracher et al., 1990).     

Axonal transport in neurological disease

The axonal transport systems have a wide variety of primary roles and secondary responses in neurological disease processes. Recent advances in understanding these roles have built on the increasingly detailed insights into the cell biology of the axon and its supporting cells. Fast transport is a microtubule-based system of bidirectional movement of membranous organelles; the mechanism of translocation of these organelles involves novel proteins, including the recently described protein of fast anterograde transport, kinesin. Slow transport conveys the major cytoskeletal elements, microtubules, and neurofilaments. Several types of structural changes in diseased nerve fibers are understood in terms of underlying transport abnormalities. Altered slow transport of neurofilaments produces changes in axonal caliber (swelling or atrophy) and is involved in some types of perikaryal neurofibrillary abnormality. Secondary changes in slow axonal transport--for example, the reordered synthesis and delivery of cytoskeletal proteins after axotomy--also can produce changes in axonal caliber. Secondary demyelination can be a prominent late consequence of a sustained alteration of neurofilament transport. Impaired fast transport is found in experimental models of distal axonal degeneration (dying back). Retrograde axonal transport provides access to the central nervous system for agents such as polio virus and tetanus toxin, as well as access for known and hypothetical trophic factors. Correlative studies of axonal transport, axonal morphometry, cytoskeletal ultrastructure, and molecular biology of cytoskeletal proteins are providing extremely detailed reconstructions of the pathogenesis of experimental models of neurological disorders. A major challenge lies in the extension of these approaches to clinical studies.     

Correlation of axonal regeneration and slow component B in two branches of a single axon

We investigated the relationship between slow axonal transport and axonal regeneration in the rat dorsal root ganglion (DRG) cell. The DRG cell sends out a single axon which bifurcates within the ganglion; one axon proceeds centrally into the spinal cord and the other proceeds peripherally. The rate of axonal regeneration is approximately 2 times faster for the peripheral processes (4.6 +/- 0.9 mm/day) than for the central processes (2.1 +/- 0.5 mm/day). The peripheral and central processes regenerate through dissimilar environments (sciatic nerve and dorsal root, respectively); thus, environmental factors may account for the differences in regeneration rates. We tested this possibility by measuring the regeneration of motoneuron axons within the ventral root (histologically similar to the dorsal root). The motoneuron regeneration rate within the ventral root is similar to the motoneuron regeneration rate within the sciatic nerve, suggesting that factors within the DRG cell produce the differences in regeneration rate. Slow axonal transport is classified into two distinct components: slow component a (SCa), corresponding to the microtubule/neurofilament network of the axonal cytoskeleton, and slow component b (SCb), corresponding to the microfilament complex/axoplasmic matrix. The transport rate of SCa and SCb in the peripheral sensory axons is approximately 2 times faster than their counterparts in the central sensory axons. SCa moves at 1.0 to 3.0 mm/day in the peripheral processes and 0.5 to 1.0 mm/day in the central processes; SCb moves at 3.5 to 6.5 mm/day in the peripheral processes and 2.0 to 3.5 mm/day in the central processes. In each branch of the DRG cell, the rate of axonal regeneration is similar to the rate of SCb transport. These results support the hypothesis that SCb is a rate-limiting factor in axonal regeneration because of its role in providing the cytoskeletal elements which are directly involved in the motility of the growth cone and elongation of the axon.     

Stable clathrin: uncoating protein (hsc70) complexes in intact neurons and their axonal transport.

We have studied the organization of clathrin during its transport in axons. Using immunoprecipitation techniques we have confirmed earlier findings that clathrin is transported as part of slow component b, but we also detect small amounts of clathrin in fast component. As fast component is known to correspond to the transport of membraneous material, including coated vesicle membrane components, our findings suggest that some clathrin in axons undergoes transport in the form of coated membranes and that a portion of the clathrin delivered to axons and axon terminals arrives by way of fast component. The organizational form of clathrin in slow component b (SCb) was examined in more detail, as it is thought to represent a non-membrane-associated species, is relatively long-lived, and at any instant represents the major transport species in axons. We used nondenaturing immunoprecipitation methods with stringent wash procedures to identify other SCb proteins that interact with clathrin. The immunoprecipitates contained major labeled bands that corresponded to clathrin heavy and light chains, along with a prominent 70-kDa band and several minor bands that ranged in apparent Mr from 70,000 to 150,000; the 70-kDa band was shown to be the ATP-dependent uncoating protein by two-dimensional gel electrophoresis. A very similar profile of polypeptides was also immunoprecipitated from extracts of cultured neurons. The results from a variety of control immunoprecipitations, including the use of antisera preadsorbed with purified clathrin trimers or clathrin light chains, indicate that coprecipitation of clathrin and uncoating protein with the other 70,000-150,000-Da polypeptides from SCb reflects specific interactions. Including exogenous uncoating protein in the lysis buffer had no detectable effect on the levels of endogenous uncoating protein recovered in the immunoprecipitates, indicating that complexes of clathrin, uncoating protein, and the other coimmunoprecipitating SCb protein existed in the intact neurons prior to lysis. Finally, a specific and functional association is further supported by the release of uncoating protein, but not the other 70,000-150,000-Da polypeptides, from the immunoprecipitated complexes on the addition of ATP. Collectively, these observations provide the first direct evidence of interaction between clathrin and uncoating protein in intact cells, lend strong support to the concept that uncoating protein plays an intimate role in clathrin dynamics within cells, and reveal a family of 70,000-150,000-Da polypeptides that form a stable nonmembranous association with clathrin in intact cells.     

Neurofilaments are transported rapidly but intermittently in axons: implications for slow axonal transport.

Slow axonal transport conveys cytoskeletal proteins from cell body to axon tip. This transport provides the axon with the architectural elements that are required to generate and maintain its elongate shape and also generates forces within the axon that are necessary for axon growth and navigation. The mechanisms of cytoskeletal transport in axons are unknown. One hypothesis states that cytoskeletal proteins are transported within the axon as polymers. We tested this hypothesis by visualizing individual cytoskeletal polymers in living axons and determining whether they undergo vectorial movement. We focused on neurofilaments in axons of cultured sympathetic neurons because individual neurofilaments in these axons can be visualized by optical microscopy. Cultured sympathetic neurons were infected with recombinant adenovirus containing a construct encoding a fusion protein combining green fluorescent protein (GFP) with the heavy neurofilament protein subunit (NFH). The chimeric GFP-NFH coassembled with endogenous neurofilaments. Time lapse imaging revealed that individual GFP-NFH-labeled neurofilaments undergo vigorous vectorial transport in the axon in both anterograde and retrograde directions but with a strong anterograde bias. NF transport in both directions exhibited a broad spectrum of rates with averages of approximately 0.6-0.7 microm/sec. However, movement was intermittent, with individual neurofilaments pausing during their transit within the axon. Some NFs either moved or paused for the most of the time they were observed, whereas others were intermediate in behavior. On average, neurofilaments spend at most 20% of the time moving and rest of the time paused. These results establish that the slow axonal transport machinery conveys neurofilaments.