The movement of membranous organelles in axons. Electron microscopic identification of anterogradely and retrogradely transported organelles

To identify the structures to be rapidly transported through the axons, we developed a new method to permit local cooling of mouse saphenous nerves in situ without exposing them. By this method, both anterograde and retrograde transport were successfully interrupted, while the structural integrity of the nerves was well preserved. Using radioactive tracers, anterogradely transported proteins were shown to accumulate just proximal to the cooled site, and retrogradely transported proteins just distal to the cooled site. Where the anterogradely transported proteins accumulated, the vesiculotubular membranous structures increased in amount inside both myelinated and unmyelinated axons. Such accumulated membranous structures showed a relatively uniform diameter of 50--80 nm, and some of them seemed to be continuous with the axonal smooth endoplasmic reticulum (SER). Thick sections of nerves selectively stained for the axonal membranous structures revealed that the network of the axonal SER was also packed inside axons proximal to the cooled site. In contrast, large membranous bodies of varying sizes accumulated inside axons just distal to the cooled site, where the retrogradely transported proteins accumulated. These bodies were composed mainly of multivesicular bodies and lamellated membranous structures. When horseradish peroxidase was administered in the distal end of the nerve, membranous bodies showing this activity accumulated, together with unstained membranous bodies. Hence, we are led to propose that, besides mitochondria, the membranous components in the axon can be classified into two systems from the viewpoint of axonal transport: "axonal SER and vesiculotubular structures" in the anterograde direction and "large membranous bodies" in the retrograde direction.     

Cytoplasmic dynein and microtubule transport in the axon: The action connection

The neuron uses two families of microtubule-based motors for fast axonal transport, kinesin, and cytoplasmic dynein. Cytoplasmic dynein moves membranous organelles from the distal regions of the axon to the cell body. Because dynein is synthesized in the cell body, it must first be delivered to the axon tip. It has recently been shown that cytoplasmic dynein is moved from the cell body along the axon by two different mechanisms. A small amount is associated with fast anterograde transport, the membranous organelles moved by kinesin. Most of the dynein is transported in slow component b, the actin-based transport compartment. Dynactin, a protein complex that binds dynein, is also transported in slow component b. The dynein in slow component b binds to microtubules in an ATP-dependent manner in vitro, suggesting that this dynein is enzymatically active. The finding that functionally active dynein, and dynactin, are associated with the actin-based transport compartment suggests a mechanism whereby dynein anchored to the actin cytoskeleton via dynactin provides the motive force for microtubule movement in the axon.     

NGF-stimulated retrograde transport of trkA in the mammalian nervous system

The present study was designed to clarify the in vivo function of trkA as an NGF receptor in mammalian neurons. Using the rat sciatic nerve as a model system, we examined whether trkA is retrogradely transported and whether transport is influenced by physiological manipulations. Following nerve ligation, trkA protein accumulates distal to the ligation site as shown by Western blot analysis. The distally accumulating trkA species were tyrosine phosphorylated. The trkA retrograde transport and phosphorylation were enhanced by injecting an excess of NGF in the footpad and were abolished by blocking endogenous NGF with specific antibodies. These results provide evidence that, upon NGF binding, trkA is internalized and retrogradely transported in a phosphorylated state, possibly together with the neurotrophin. Furthermore, our results suggest that trkA is a primary retrograde NGF signal in mammalian neurons in vivo.     

神经退变和再生过程中郎氏结构筑的变化

将夹伤的大鼠坐骨神经分离成单根纤维,在光镜、扫描和透射电镜下观察伤后９８天内郎氏结的构筑变化。发现损伤使细胞器的轴浆转运阻断,积累的退变细胞器使结区的轴突外凸,郎氏结构构筑变形,髓鞘板层失序,轴膜崩解,积累的细胞器逸出,并看到仅残存的郎氏结近心端构筑、由近心端的母体神经和远心端的再生神经共同构筑的新生郎氏结,以及新生郎氏结构的发育过程等特征性图象。再生轴突中转运的微管等细胞器和施旺细胞中富含的线粒     

Endogenous chicken nerve growth factor from sheath cells is transported in regenerating nerve

We report the presence of endogenous nerve growth factor (NGF) in chicken peripheral nerve. The molecule has been detected with antibodies to mouse salivary gland NGF, using immunohistochemical and immunoelectrophoretic techniques. Previous studies have shown that these antibodies inhibit the survival activity of extracts of chicken peripheral nerve. The NGF accumulated distal, but not proximal, to a ligature placed on a peripheral sympathetic nerve demonstrating that it was retrogradely transported. This transport was detected in intact nerve fibers as well as in nerves from which the peripheral target had been ablated 6 hr or 7 days previously. The results indicate that avian NGF is present in adult chicken peripheral nerves and that this molecule shares antigenic determinants with the mouse molecule. The results further demonstrate that regenerating neurons retrogradely transport NGF supplied by cells within the peripheral nerve (presumably Schwann). The possibility that these cells also provide NGF to intact neurons is discussed.     

A neuron-specific cytoplasmic dynein isoform preferentially transports TrkB signaling endosomes

Cytoplasmic dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)-IC incorporates into functional dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP-dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by cytoplasmic dynein containing the neuron-specific IC-1B isoform and not by dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define dynein populations that are selectively recruited to transport distinct cargoes.     

Signalling organelle for retrograde axonal transport of internalized neurotrophins from the nerve terminal

The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. We have been investigating the process of targeting these vesicles for retrograde transport, by examining the transport of [125I]-labelled neurotrophins from the eye to sympathetic and sensory ganglia. With the aid of confocal microscopy, we examined the phenomena further in cultures of dissociated sympathetic ganglia to which rhodamine-labelled nerve growth factor (NGF) was added. We found the label in large vesicles in the growth cone and axons. Light microscopic examination of the sympathetic nerve trunk in vivo also showed the retrogradely transported material to be sporadically located in large structures in the axons. Ultrastructural examination of the sympathetic nerve trunk after the transport of NGF bound to gold particles showed the label to be concentrated in relatively few large organelles that consisted of accumulations of multivesicular bodies. These results suggest that in vivo NGF is transported in specialized organelles that require assembly in the nerve terminal.     

Partial Isolation of Two Classes of Dopamine β-Hydroxylase-Containing Particles Undergoing Rapid Axonal Transport in Rat Sciatic Nerve

The rapid bidirectional transport of dopamine beta-hydroxylase (DBH) in adrenergic axons provides a means of analyzing the life cycle of adrenergic storage vesicles. We compared the physical characteristics of DBH-containing particles traveling to or returning from the terminal varicosities of ligated rat sciatic nerves. Density gradient centrifugation and Sephacryl S1000 gel-permeation chromatography were used to fractionate extracts from nerve segments proximal or distal to the ligatures. A series of experiments indicated the existence of at least two populations of rapidly transported DBH-containing particles, a "light" 85-nm particle and a larger "dense" 120-nm particle. The 85-nm particles were prevalent in unligated nerve, but accounted for only one-third of the total anterogradely transported DBH activity accumulated after 18 h. The 120-nm particles were barely detectable in the unligated nerve, but they accumulated at twice the rate of the 85-nm particles and accounted for the rest of the anterogradely transported particulate DBH activity. These two populations of particles were readily isolated from proximal nerve extracts by sucrose density gradient centrifugation. Similar-appearing dense and light peaks of particulate DBH activity were obtained from distal nerve extracts. Much of the retrogradely transported DBH of the extracts, however, was associated with large particles (greater than 300 nm) not resolved by Sephacryl S1000. Retrogradely transported exogenous NGF was found only in the dense sucrose gradient peak. We propose that the 85-nm DBH-containing particles correspond to "large dense-cored vesicles," and that the 120-nm particles are derived from the dense tubules visualized in adrenergic nerves by the chromaffin reaction.     

Axonal transports of tripeptidyl peptidase II in rat sciatic nerves

Axonal transport of tripeptidyl peptidase II, a putative cholecystokinin inactivating serine peptidase, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. Enzyme activity significantly increased not only in the proximal segment but also in the distal segment 12-72h after ligation, and the maximal enzyme activity was found in the proximal and distal segments at 72h. Western blot analysis of tripeptidyl peptidase II showed that its immunoreactivities in the proximal and distal segments were 3.1- and 1.7-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in immunoreactive tripeptidyl peptidase II level in the proximal and distal segments in comparison with that in the middle segment, indicating that tripeptidyl peptidase II is transported by anterograde and retrograde axonal flow. The results suggest that tripeptidyl peptidase II may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.     

Modelling transport of layered double hydroxide nanoparticles in axons and dendrites of cortical neurons

This paper develops a model of nanoparticle transport in neurons. It is assumed that nanoparticles are transported inside endocytic vesicles by a combined effect of dynein-driven transport and diffusion. It is further assumed that in axons nanoparticles are internalised only at axon terminals, whereas in dendrites nanoparticles can enter through the entire plasma membrane. This causes differences in transport of nanoparticles in axons and dendrites; these differences are investigated in this paper. Another difference is microtubule (MT) orientation in axons and dendrites; in axons, all MTs have their plus-ends oriented towards the axon terminal; in a proximal region of a dendrite, MTs have mixed orientation, whereas in a distal dendritic region the MT orientation is similar to that in an axon. It is shown that if molecular-motor-driven transport were powered by dynein alone, such MT orientation in a dendrite would result in a region of nanoparticle accumulation located at the border between the proximal and distal dendritic regions.     

Modelling transport of layered double hydroxide nanoparticles in axons and dendrites of cortical neurons

This paper develops a model of nanoparticle transport in neurons. It is assumed that nanoparticles are transported inside endocytic vesicles by a combined effect of dynein-driven transport and diffusion. It is further assumed that in axons nanoparticles are internalised only at axon terminals, whereas in dendrites nanoparticles can enter through the entire plasma membrane. This causes differences in transport of nanoparticles in axons and dendrites; these differences are investigated in this paper. Another difference is microtubule (MT) orientation in axons and dendrites; in axons, all MTs have their plus-ends oriented towards the axon terminal; in a proximal region of a dendrite, MTs have mixed orientation, whereas in a distal dendritic region the MT orientation is similar to that in an axon. It is shown that if molecular-motor-driven transport were powered by dynein alone, such MT orientation in a dendrite would result in a region of nanoparticle accumulation located at the border between the proximal and distal dendritic regions.     

Impaired axonal regeneration in acrylamide intoxication

Acrylamide is a neurotoxin known to impair regeneration of axons following nerve crush and to produce structurally abnormal regenerating sprouts. To investigate the mechanism of these abnormalities, protein synthesis and fast axonal transport were studied in acrylamide-intoxicated and control rats 2 weeks after sciatic nerve crush. Using an in vitro preparation of sciatic nerve-dorsal root ganglion, there was no difference in ganglion ³H-leucine incorporation between the two groups. In these preparations of sensory axons, as well as in motor axons studied in vivo, a smaller proportion of rapidly transported radioactivity was carried beyond the crush in the acrylamide-regenerating nerves compared to the control-regenerating nerves. Correlative ultrastructural studies demonstrated that this difference reflected the impaired outgrowth of the acrylamide-regenerating nerves, rather than an abnormality in fast transport. The acrylamide-treated sprouts often developed swellings filled with whorls of neurofilaments; in addition, many sprouts ended in massively enlarged growth cones containing membranous organelles. EM autoradiography showed labeled, rapidly transported organelles accumulated in the neurofilamentous whorls, and therefore suggested that these organelles might be “trapped” or impeded in passage through these regions. However, there was no evidence that the growth cones received insufficient amounts of transported protein; in fact, the distended endings were densely labeled and apparently “ballooned” by transported organelles. These results suggest that acrylamide intoxication does not impair regeneration by diminishing the delivery of rapidly transported materials to the growing tip. Rather, the marked distention of the growth cones is interpreted as the morphological consequence of continued delivery of rapidly transported organelles into sprouts unable to utilize them in outgrowth.     

Anterograde axonal transport of endopeptidase 24.15 in rat sciatic nerves

Axonal transport of endopeptidase 24.15 (EP24.15), a putative neuropeptide degrading-enzyme, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. At 48h after ligation, a significant amount of the axonal transport of EP24.15 activity was found in the proximal segment, while axonal transport of deamidase activity, a lysosomal enzyme, increased in both proximal and distal segments. Western blot analysis of EP24.15 showed that EP24.15 immunoreactivity in the proximal segment was 1.8-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in the immunoreactive EP24.15 in the proximal segment in comparison with that in the middle segment. In the distal segment, no axonal transport of EP24.15 was found in all methods examined, indicating that EP24.15 is mainly transported by an anterograde axonal flow. These observations suggest that EP24.15 may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.     

Fast axonal transport of the vesicular acetylcholine transporter (VAChT) in cholinergic neurons in the rat sciatic nerve

The sciatic nerve, as a part of the peripheral nervous system (PNS), has been used to study axonal transport for decades. It contains motor, sensory as well as autonomic axons. The present study has concentrated on the axonal transport of the synaptic vesicle acetylcholine transporter (VAChT), using the "stop–flow\erve crush” method. After blocking fast axonal transport by means of a crush, distinct accumulations of various synaptic vesicle proteins, including VAChT, and peptides developed during the first hour after crush–operation and marked increases were observed up to 8 h post–operative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immuno–incubated sections, revealed a rapid rate of accumulation proximal to the crush, and that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was about 40%. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. VAChT–immu–noreactive axons were also immunoreactive for choline acetyltransferase (ChAT). Autonomic axons with VAChT also contained VIP–LI.

The results demonstrate (1) that VAChT, as well as other synaptic vesicle proteins, is transported with fast axonal transport in motor axons as well as in autonomic post–ganglionic neurons in this nerve, (2) VAChT colocalized in motor axons with SV1 as well as with synaptophysin, indicating storage in the same axonal particle, (3) in the autonomic postganglionic sympathetic cholinergic fibres, VAChT colocalized with VIP, but VIP–LI was present in rather large granular structures while VAChT–LI was present mostly as small granular elements, (4) in motor as well as in autonomic axons ChAT–LI was present in VAChT–positive axons, and (4) the ratio of recycling (retrogradely accumulated) VAChT–IR was about 40%, in contrast to the recycling fraction of synaptophysin that was about 70%. © 1998 Elsevier Science Ltd. All rights reserved.     

Axonal transport of synaptic vesicle proteins in the rat optic nerve

The optic nerve, as a part of the central nervous system (CNS), has been used to study axonal transport for decades. The present study has concentrated on the axonal transport of synaptic vesicle proteins in the optic nerve, using the “stop-flow/nerve crush” method. After blocking fast axonal transport, distinct accumulations of synaptic vesicle proteins developed during the first hour after crush-operation and marked increases were observed up to 8 h postoperative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immunoincubated sections, revealed that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was much higher (up to 80–90%) for the transmembrane proteins than that for surface adsorbed proteins (only 10–20%). The pattern of axonal transport in the optic nerve was comparable to that in the sciatic nerve. However, clathrin and Rab3a immunoreactivities were accumulated in much lower amounts than that in the sciatic nerve. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. A differential distribution of synaptobrevin I and II, however, was observed in the optic nerve axons; synaptobrevin I was present in large-sized axons, while synaptobrevin II immunoreactivity was present in most axons, including the large ones. The two isoforms were, thus, partially colocalized. The results demonstrate that (1) cytofluorimetric scanning techniques could be successfully used to study axonal transport not only in peripheral nerves, but also in the CNS; (2) synaptic vesicles are transported with fast axonal transport in this nerve; and (3) some differences were noted compared with the sciatic nerve, especially for Rab3a and clathrin. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 237–250, 1997.     

Distinct cytoplasmic dynein complexes are transported by different mechanisms in axons

In neurons, cytoplasmic dynein is synthesized in the cell body, but its function is to move cargo from the axon back to the cell body. Dynein must therefore be delivered to the axon and its motor activity must be regulated during axonal transport. Cytoplasmic dynein is a large protein complex composed of a number of different subunits. The dynein heavy chains contain the motor domains and the intermediate chains are involved in binding the complex to cargo. Five different intermediate chain polypeptides, which are the result of the alternative splicing of the two intermediate chain genes, have been identified. We have characterized two distinct pools of dynein that are transported from the cell body along the axon by different mechanisms. One pool, which contains the ubiquitous intermediate chain, is associated with the membranous organelles transported by kinesin in the fast transport component. The other pool, which contains the other developmentally regulated intermediate chains, is transported in slow component b. The mechanism of dynein regulation will therefore depend on which pool of dynein is recruited to function as the retrograde motor. In addition, the properties of the large pool of dynein associated with actin in slow component b are consistent with the hypothesis that this dynein may be the motor for microtubule transport in the axon.     

High-resolution imaging demonstrates dynein-based vesicular transport of activated Trk receptors

Target-derived neurotrophins signal from nerve endings to the cell body to influence cellular and nuclear responses. The retrograde signal is conveyed by neurotrophin receptors (Trks) themselves. To accomplish this, activated Trks may physically relocalize from nerve endings to the cell bodies. However, alternative signaling mechanisms may also be used. To identify the vehicle wherein the activated Trks are located and transported, and to identify associated motor proteins that would facilitate transport, we use activation-state specific antibodies in concert with immunoelectron microscopy and deconvolution microscopy. We show that the'activated Trks within rat sciatic nerve axons are preferentially localized to coated and uncoated vesicles. These vesicles are moving in a retrograde direction and so accumulate distal to a ligation site. The P-Trk containing vesicles, in turn, colocalize with dynein components, and not with kinesins. Collectively, these results indicate activated Trk within axons travel in vesicles and dynein is the motor that drives these vesicles towards the cell bodies.     

Identification and developmental regulation of a neuron-specific subunit of cytoplasmic dynein.

Cytoplasmic dynein is the microtubule minus-end-directed motor for the retrograde axonal transport of membranous organelles. Because of its similarity to the intermediate chains of flagellar dynein, the 74-kDa intermediate chain (IC74) subunit of dynein is thought to be involved in binding dynein to its membranous organelle cargo. Previously, we identified six isoforms of the IC74 cytoplasmic dynein subunit in the brain. We further demonstrated that cultured glia and neurons expressed different dynein IC74 isoforms and phospho-isoforms. Two isoforms were observed when dynein from glia was analyzed. When dynein from cultured neurons was analyzed, six IC74 isoforms were observed, although the relative amounts of the dynein isoforms from cultured neurons differed from those found in dynein from brain. To better understand the role of the neuronal IC74 isoforms and identify neuron-specific IC74 dynein subunits, the expression of the IC74 protein isoforms and mRNAs of various tissues were compared. As a result of this comparison, the identity of each of the isoform spots observed on two-dimensional gels was correlated with the products of each of the IC74 mRNAs. We also found that between the fifteenth day of gestation (E15) and the fifth day after birth (P5), the relative expression of the IC74 protein isoforms changes, demonstrating that the expression of IC74 isoforms is developmentally regulated in brain. During this time period, there is relatively little change in the abundance of the various IC74 mRNAs. The E15 to P5 time period is one of rapid process extension and initial pattern formation in the rat brain. This result indicates that the changes in neuronal IC74 isoforms coincide with neuronal differentiation, in particular the extension of processes. This suggests a role for the neuronal IC74 isoforms in the establishment or regulation of retrograde axonal transport.     

Transport of Muscarinic Cholinergic Marker Protein Activities in Regenerating Sciatic Nerve of Rat

The transport characteristics of choline acetyltransferase (ChAT; EC 2.3.1.6), acetylcholinesterase (AChE; EC 3.1.1.7), and the muscarinic acetylcholine receptors (mAChR) were studied in perineurally sutured, regenerating rat sciatic nerve. At different times after repair, the sciatic nerve was ligated for 24 h, and the activities of the cholinergic marker proteins, as well as the binding capacity, were measured proximally and distally from the ligature. The number of bidirectionally transported receptors increased linearly up to 5 months postoperatively (6.1-33.6% and 5.6-25.6% of the control level proximal and distal to the ligature, respectively). The quantity of anterogradely transported ChAT reached a plateau 3 months postoperatively (74.9% of the control level), whereas the retrogradely transported enzyme was then only 34.7% of the control value. The activity of AChE increased linearly during nerve regeneration, and exceeded the control level after 4 months (121.0% and 63.7% proximally and distally, respectively). The data indicate that the altered bidirectional transport of cholinergic marker proteins may be monitored quantitatively during nerve regeneration. This method might be suitable for studies of the nerve regeneration process.     

Dynein light chain regulates axonal trafficking and synaptic levels of Bassoon

Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon–DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.