Anterograde axonal transport, transcytosis, and recycling of neurotrophic factors

Traditional views of neurotrophic factor biology held that trophic factors are released from target cells, retrogradely transported along their axons, and rapidly degraded upon arrival in cell bodies. Increasing evidence indicates that several trophic factors such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF-2), glial cell-line derived neurotrophic factor (GDNF), insulin-like growth factor (IGF-I), and neurotrophin-3 (NT-3), can move anterogradely along axons. They can escape the degradative pathway upon internalization and are recycled for future uses. Internalized ligands can move through intermediary cells by transcytosis, presumably by endocytosis via endosomes to the Golgi system, by trafficking of the factor to dendrites or by sorting into anterograde axonal transport with subsequent release from axon terminals and uptake by second- or third-order target neurons. Such data suggest the existence of multiple “trophic currencies,” which may be used over several steps in neural networks to enable nurturing relationships between connected neurons or glial cells, not unlike currency exchanges between trading partners in the world economy. Functions of multistep transfer of trophic material through neural networks may include regulation of neuronal survival, differentiation of phenotypes and dendritic morphology, synapse plasticity, as well as excitatory neurotransmission. The molecular mechanisms of sorting, trafficking, and release of trophic factors from distinct neuronal compartments are important for an understanding of neurotrophism, but they present challenging tasks owing to the low levels of the endogeneous factors.     

The somatodendritic endosomal regulator NEEP21 facilitates axonal targeting of L1/NgCAM

Correct targeting of proteins to axons and dendrites is crucial for neuronal function. We showed previously that axonal accumulation of the cell adhesion molecule L1/neuron-glia cell adhesion molecule (NgCAM) depends on endocytosis (Wisco, D., E.D. Anderson, M.C. Chang, C. Norden, T. Boiko, H. Folsch, and B. Winckler. 2003. J. Cell Biol. 162:1317-1328). Two endocytosis-dependent pathways to the axon have been proposed: transcytosis and selective retrieval/retention. We show here that axonal accumulation of L1/NgCAM occurs via nondegradative somatodendritic endosomes and subsequent anterograde axonal transport, which is consistent with transcytosis. Additionally, we identify the neuronal-specific endosomal protein NEEP21 (neuron-enriched endosomal protein of 21 kD) as a regulator of L1/NgCAM sorting in somatodendritic endosomes. Down-regulation of NEEP21 leads to missorting of L1/NgCAM to the somatodendritic surface as well as to lysosomes. Importantly, the axonal accumulation of endogenous L1 in young neurons is also sensitive to NEEP21 depletion. We propose that small endosomal carriers derived from somatodendritic recycling endosomes can serve to redistribute a distinct set of membrane proteins from dendrites to axons.     

Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons

Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells.     

Endocytosis and endosomes at the crossroads of regulating trafficking of axon outgrowth-modifying receptors

In neurons, many receptors must be localized correctly to axons or dendrites for proper function. During development, receptors for nerve growth and guidance are targeted to axons and localized to growth cones where receptor activation by ligands results in promotion or inhibition of axon growth. Signaling outcomes downstream of ligand binding are determined by the location, levels and residence times of receptors on the neuronal plasma membrane. Therefore, the mechanisms controlling the trafficking of these receptors are crucial to the proper wiring of circuits. Membrane proteins accumulate on the axonal surface by multiple routes, including polarized sorting in the trans Golgi network, sorting in endosomes and removal by endocytosis. Endosomes also play important roles in the signaling pathways for both growth-promoting and -inhibiting molecules: signaling endosomes derived from endocytosis are important for signaling from growth cones to cell bodies. Growth-promoting neurotrophins and growth-inhibiting Nogo-A can use EHD4/Pincher-dependent endocytosis at the growth cone for their respective retrograde signaling. In addition to retrograde transport of endosomes, anterograde transport to axons in endosomes also occurs for several receptors, including the axon outgrowth-promoting cell adhesion molecule L1/NgCAM and TrkA. L1/NgCAM also depends on EHD4/Pincher-dependent endocytosis for its axonal polarization. In this review, we will focus on receptors whose trafficking has been reported to be modulated by the EHD4/Pincher family of endosomal regulators, namely L1/NgCAM, Trk and Nogo-A. We will first summarize the pathways underlying the axonal transport of these proteins and then discuss the potential roles of EHD4/Pincher in mediating their endocytosis.     

Vesicular trafficking of semaphorin 3A is activity-dependent and differs between axons and dendrites

Secreted semaphorins act as guidance cues in the developing nervous system and may have additional functions in mature neurons. How semaphorins are transported and secreted by neurons is poorly understood. We find that endogenous semaphorin 3A (Sema3A) displays a punctate distribution in axons and dendrites of cultured cortical neurons. GFP-Sema3A shows a similar distribution and co-localizes with secretory vesicle cargo proteins. Live-cell imaging reveals highly dynamic trafficking of GFP-Sema3A vesicles with distinct properties in axons and dendrites regarding directionality, velocity, mobility and pausing time. In axons, most GFP-Sema3A vesicles move fast without interruption, almost exclusively in the anterograde direction, while in dendrites many GFP-Sema3A vesicles are stationary and move equally frequent in both directions. Disruption of microtubules, but not of actin filaments, significantly impairs GFP-Sema3A transport. Interestingly, depolarization induces a reversible arrest of axonal transport of GFP-Sema3A vesicles but has little effect on dendritic transport. Conversely, action potential blockade using tetrodotoxin (TTX) accelerates axonal transport, but not dendritic transport. These data indicate that axons and dendrites regulate trafficking of Sema3A and probably other secretory vesicles in distinct ways, with axons specializing in fast, uninterrupted, anterograde transport. Furthermore, neuronal activity regulates secretory vesicle trafficking in axons by a depolarization-evoked trafficking arrest.     

Axonal and dendritic endocytic pathways in cultured neurons

The endocytic pathways from the axonal and dendritic surfaces of cultured polarized hippocampal neurons were examined. The dendrites and cell body contained extensive networks of tubular early endosomes which received endocytosed markers from the somatodendritic domain. In axons early endosomes were confined to presynaptic terminals and to varicosities. The somatodendritic but not the presynaptic early endosomes were labeled by internalized transferrin. In contrast to early endosomes, late endosomes and lysosomes were shown to be predominantly located in the cell body. Video microscopy was used to follow the transport of internalized markers from the periphery of axons and dendrites back to the cell body. Labeled structures in both domains moved unidirectionally by retrograde fast transport. Axonally transported organelles were sectioned for EM after video microscopic observation and shown to be large multivesicular body-like structures. Similar structures accumulated at the distal side of an axonal lesion. Multivesicular bodies therefore appear to be the major structures mediating transport of endocytosed markers between the nerve terminals and the cell body. Late endocytic structures were also shown to be highly mobile and were observed moving within the cell body and proximal dendritic segments. The results show that the organization of the endosomes differs in the axons and dendrites of cultured rat hippocampal neurons and that the different compartments or stages of the endocytic pathways can be resolved spatially.     

NgCAM and VAMP2 reveal that direct delivery and dendritic degradation maintain axonal polarity

Neurons are polarized cells of extreme scale and compartmentalization. To fulfill their role in electrochemical signaling, axons must maintain a specific complement of membrane proteins. Despite being the subject of considerable attention, the trafficking pathway of axonal membrane proteins is not well understood. Two pathways, direct delivery and transcytosis, have been proposed. Previous studies reached contradictory conclusions about which of these mediates delivery of axonal membrane proteins to their destination, in part because they evaluated long-term distribution changes and not vesicle transport. We developed a novel strategy to selectively label vesicles in different trafficking pathways and determined the trafficking of two canonical axonal membrane proteins, neuron–glia cell adhesion molecule and vesicle-associated membrane protein-2. Results from detailed quantitative analyses of transporting vesicles differed substantially from previous studies and found that axonal membrane proteins overwhelmingly undergo direct delivery. Transcytosis plays only a minor role in axonal delivery of these proteins. In addition, we identified a novel pathway by which wayward axonal proteins that reach the dendritic plasma membrane are targeted to lysosomes. These results redefine how axonal proteins achieve their polarized distribution, a crucial requirement for elucidating the underlying molecular mechanisms.     

Botulinum Neurotoxins A and E Undergo Retrograde Axonal Transport in Primary Motor Neurons

The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT) is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs) block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes from nerve terminals to the soma, and represents a general gateway for the delivery of virulence factors and pathogens to the central nervous system.     

Selective binding, uptake, and retrograde transport of tetanus toxin by nerve terminals in the rat iris. An electron microscope study using colloidal gold as a tracer

A series of specific macromolecules (tetanus toxin, cholera toxin, nerve growth factor [NGF], and several lectins) have been shown to be transported retrogradely with high selectivity from terminals to cell bodies in various types of neurons. Under identical experimental conditions (low protein concentrations injected), most other macromolecules, e.g. horseradish peroxidase (HRP), albumin, ferritin, are not transported in detectable amounts. In the present EM study, we demonstrate selective binding of tetanus toxin to the surface membrane of nerve terminals, followed by uptake and subsequent retorgrade axonal transport. Tetanus toxin or albumin was adsorbed to colloidal gold particles (diam 200 A). The complex was shown to be stable and well suited as an EM tracer. 1-4 h after injection into the anterior eye chamber of adult rats, tetanus toxin-gold particles were found to be selectively associated with membranes of nerve terminals and preterminal axons. Inside terminals and axons, the tracer was localized mainly in smooth endoplasmic reticulum (SER)-like membrane compartments. In contrast, association of albumin-gold complexes with nervous structures was never observed, in spite of extensive uptake into fibroblasts. Electron microscope and biochemical experiments showed selective retrograde transport of tetanus toxin-gold complexes to the superior cervical ganglion. Specific binding to membrane components at nerve terminals and subsequent internalization and retrograde transport may represent an important pathway for macromolecules carrying information from target organs to the perikarya of their innervating neurons.     

Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons

To address questions about mechanisms of filament-based organelle transport, a system was developed to image and track mitochondria in an intact Drosophila nervous system. Mutant analyses suggest that the primary motors for mitochondrial movement in larval motor axons are kinesin-1 (anterograde) and cytoplasmic dynein (retrograde), and interestingly that kinesin-1 is critical for retrograde transport by dynein. During transport, there was little evidence that force production by the two opposing motors was competitive, suggesting a mechanism for alternate coordination. Tests of the possible coordination factor P150(Glued) suggested that it indeed influenced both motors on axonal mitochondria, but there was no evidence that its function was critical for the motor coordination mechanism. Observation of organelle-filled axonal swellings ("organelle jams" or "clogs") caused by kinesin and dynein mutations showed that mitochondria could move vigorously within and pass through them, indicating that they were not the simple steric transport blockades suggested previously. We speculate that axonal swellings may instead reflect sites of autophagocytosis of senescent mitochondria that are stranded in axons by retrograde transport failure; a protective process aimed at suppressing cell death signals and neurodegeneration.     

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.     

Mechanistic logic underlying the axonal transport of cytosolic proteins

Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivatable GFP (PAGFP) move with a slow motor-dependent anterograde bias distinct from both vesicular trafficking and diffusion of untagged PAGFP. The overall bias is likely generated by an intricate particle kinetics involving transient assembly and short-range vectorial spurts. In vivo biochemical studies reveal that cytosolic proteins are organized into higher order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supramolecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multiprotein complexes that are directly/indirectly conveyed by motors.     

Axonal transport of proteins. A new view using in vivo covalent labeling

The injection of [2,3-3H]N-succinimidyl propionate ([3H]N-SP) into the rat sciatic nerve was used to covalently label both intra- and extra- axonal proteins. While extra-axonal proteins (e.g., myelin proteins) remained in the injection site, the intra-axonal proteins were transported in both the anterograde and retrograde directions. The mobile labeled proteins appeared to move by normal axonal transport processes because: (a) autoradiographic studies showed that they were localized exclusively within the axon at considerable distances from the injection site, (b) specific and identifiable proteins (by SDS gel electrophoresis) moved at expected rates in the anterograde direction, and (c) an entirely different profile of proteins moved in the anterograde vs. retrograde direction. This novel experimental approach to axonal transport, which is independent of de novo protein synthesis, provided a unique view of slow anterograde transport, and particularly of retrograde transport of endogenous proteins. A large quantity of a 68,000 mol wt proteins, moving at approximately 3-6 mm/day, dominated the retograde transport profile. [3H]N-SP, therefore, represents a new and unique "vital stain" which may find many applications in cell biology.     

Studies on the mechanism of the reversal of rapid organelle transport in myelinated axons of Xenopus laevis

Rapid organelle transport was studied by computer- and video-enhanced microscopy in the region of localized lesions in single myelinated axons of Xenopus laevis. Localized lesions were created that were either impermeable to small ions in the bathing medium or were permeable to agents with molecular weights up to 10,000. Providing the axons were bathed in a suitable "internal" medium, organelle transport continued to within a few micrometers of the lesion whether the lesion was permeable or not. Organelles undergoing anterograde and retrograde transport reversed their direction of transport on reaching the lesion. In preparations with lesions that were permeable, nonhydrolyzable analogs of ATP inhibited normally directed and reversed organelle transport. In permeable preparations, vanadate and EDTA inhibited retrograde and reversed retrograde transport at different intra-axonal concentrations; anterograde and reversed anterograde transport were also differentially inhibited. Anterograde and retrograde organelle transport were also shown to be inhibited at different intraaxonal concentrations of vanadate and EDTA. The results provide evidence for the existence of two different axonal transport mechanisms in myelinated axons. The two mechanisms can account for the normally directed and reversed transport of individual organelles.     

Role of MAP1B in axonal retrograde transport of mitochondria

The MAPs (microtubule-associated proteins) MAP1B and tau are well known for binding to microtubules and stabilizing these structures. An additional role for MAPs has emerged recently where they appear to participate in the regulation of transport of cargos on the microtubules found in axons. In this role, tau has been associated with the regulation of anterograde axonal transport. We now report that MAP1B is associated with the regulation of retrograde axonal transport of mitochondria. This finding potentially provides precise control of axonal transport by MAPs at several levels: controlling the anterograde or retrograde direction of transport depending on the type of MAP involved, controlling the speed of transport and controlling the stability of the microtubule tracks upon which transport occurs.     

Distinct Functions of Nuclear Distribution Proteins LIS1, Ndel1 and NudCL in Regulating Axonal Mitochondrial Transport

Neurons critically depend on the long‐distance transport of mitochondria. Motor proteins kinesin and dynein control anterograde and retrograde mitochondrial transport, respectively in axons. The regulatory molecules that link them to mitochondria need to be better characterized. Nuclear distribution (Nud) family proteins LIS1, Ndel1 and NudCL are critical components of cytoplasmic dynein complex. Roles of these Nud proteins in neuronal mitochondrial transport are unknown. Here we report distinct functions of LIS1, Ndel1 and NudCL on axonal mitochondrial transport in cultured hippocampal neurons. We found that LIS1 interacted with kinsein family protein KIF5b. Depletion of LIS1 enormously suppressed mitochondrial motility in both anterograde and retrograde directions. Inhibition of either Ndel1 or NudCL only partially reduced retrograde mitochondrial motility. However, knocking down both Ndel1 and NudCL almost blocked retrograde mitochondrial transport, suggesting these proteins may work together to regulate retrograde mitochondrial transport through linking dynein‐LIS1 complex. Taken together, our results uncover novel roles of LIS1, Ndel1 and NudCL in the transport of mitochondria in axons.      

Fast axonal transport alterations in amyotrophic lateral sclerosis (ALS) and in parathyroid hormone (PTH)-treated axons

Video-enhanced contrast techniques have been used to study fast axonal transport of organelles in diseased and normal human axons. A broad perspective on the importance of axonal transport in the pathogenesis of human neurological disorders is presented and problems in dealing with human nerve summarized. Results from analysis of organelle traffic in axons from motor nerve in patients with amyotrophic lateral sclerosis (ALS) show: 1) higher mean speed of anterograde organelles, 2) lower mean speed of retrograde organelles, and 3) lower retrograde organelle traffic density. Hyperparathyroidism, another human clinical syndrome, can mimic ALS. The effect of parathyroid hormone (PTH) on axons in vitro is to increase the mean speed of both anterograde and retrograde organelle traffic. The dose response curve and time course of the PTH effect are delineated. Dihydropyridine calcium channel antagonists block the PTH effect, implicating extracellular calcium in the alteration of organelle traffic speed. The results are discussed in relation to neuronal function and the regulation of fast axonal transport.     

Action in the axon: generation and transport of signaling endosomes

Neurons extend axonal processes over long distances, necessitating efficient transport mechanisms to convey target-derived neurotrophic survival signals from remote distal axons to cell bodies. Retrograde transport, powered by dynein motors, supplies cell bodies with survival signals in the form of 'signaling endosomes'. In this review, we will discuss new advances in our understanding of the motor proteins that bind to and move signaling components in a retrograde direction and discuss mechanisms that might specify distinct neuronal responses to spatially restricted neurotrophin signals. Disruption of retrograde transport leads to a variety of neurodegenerative diseases, highlighting the role of retrograde transport of signaling endosomes for axonal maintenance and the importance of efficient transport for neuronal survival and function.     

Pseudorabies Virus Fast Axonal Transport Occurs by a pUS9-Independent Mechanism

Reactivation from latency results in transmission of neurotropic herpesviruses from the nervous system to body surfaces, referred to as anterograde axonal trafficking. The virus-encoded protein pUS9 promotes axonal dissemination by sorting virus particles into axons, but whether it is also an effector of fast axonal transport within axons is unknown. To determine the role of pUS9 in anterograde trafficking, we analyzed the axonal transport of pseudorabies virus in the presence and absence of pUS9.