Axonal Transport In Neurons Research Articles

A novel algorithm to generate kymographs from dynamic axons for the quantitative analysis of axonal transport

The biological and clinical relevance of axonal transport has driven the development of a variety of new approaches to its study, including the generation of fluorescence or brightfield movies of moving cargoes within axons. Kymograph analysis is a simple and effective tool used to analyze axonal transport in neurons. Typically, kymographs are built by having a user trace the path of the axon in one frame of a time-lapse movie and extracting intensity profiles from subsequent frames along that path. This method cannot accommodate movies in which translation of the axon, or changes in axonal orientation or geometry, occur. Both are frequently observed in long-term movies of neurons, both in vitro and in vivo. To solve this problem and automate the creation of kymographs from these movies, we developed a two step algorithm. The first step implemented a simple image registration algorithm that aligned axons based on identification of a reference point on the axon in each image. The second step used a Hough transformation (HT) to automatically detect the axonal contour in each frame. Intensity profiles along this contour were then used to construct a kymograph. This algorithm was able to build an accurate kymograph of mitochondrial and actin transport in dynamic cultured sensory neurons, which were not amenable to previously used analytical methods. Although developed as a tool for analyzing transport, this algorithm is easily modified to analyze movies for the directionality and speed of axonal outgrowth, another metric of interest to neuroscientists.

Comparison of active transport in neuronal axons and dendrites

This paper presents a theoretical study, based on modified Smith–Simmons equations, that compares transport of intracellular organelles in two different neurite outgrowths, dendrites and axons. It is demonstrated that the difference in microtubule polarity orientations in dendrites and axons has significant implications on motor-assisted transport in these neurite outgrowths. The developed approach presents a qualitative theoretical basis for understanding important questions such as why axons exhibit almost an unlimited grows potential in vitro while dendrites remain relatively short. It is shown that the difference in a microtubule polarity arrangement between axons and dendrites may be a regulatory mechanism for limiting dendritic growth. Other biological implications of the developed theory as well as other possible reasons for the difference in microtubule structure between axons and dendrites are discussed.

Modeling Bidirectional Transport of New and Used Organelles in Fast Axonal Transport in Neurons

This paper develops a model for simulating transport of newly synthesized material from the neuron body toward the synapse of the axon as well as transport of misfolded and aggregated proteins back to the neuron body for recycling. The model demonstrates that motor-assisted transport, much similar to diffusion, can occur due to a simple concentration difference between the cell body and the synapse; organelles heading to the synapse do not need to attach preferably to plus-end-directed molecular motors, same as organelles heading to the neuron body for recycling do not need to attach preferably to minus-end-directed molecular motors. The underlying mechanics of molecular-motor-assisted transport is such that organelles would be transported to the right place even if new and used organelles had the same probability of attachment to plus-end-directed (and minus-end-directed) motors. It is also demonstrated that the axon with organelle traps and a region with a reversed microtubule polarity would support much smaller organelle fluxes of both new and used organelles than a healthy axon. The flux of organelles is shown to decrease as the width of organelle traps increases.

High Precision Tracking of Intracellular Transport with Fluorescent Nanoparticles

To grow and maintain eukaryotic cilia and flagella, axonemal precursors and membrane proteins are moved continuously along the unipolar array of outer microtubules by intraflagellar transport (IFT). To understand the role of dynein and kinesin motors in this process, we have used paralyzed flagella mutants of Chlamydomonas as a model organism. Flagella was rigidly immobilized to a glass coverslip and fluorescent nanoparticles were attached to transmembrane component of moving cargoes. Fluorescent signals of nanoparticles were tracked by ultrahigh spatial (∼1 nm error) and temporal (600 microsecond) resolution. Cargoes were moved by 8 nm steps towards both directions, in agreement with kinesin and dynein step sizes. Movement was highly directional (no backward steps) which is different from other bidirectional transport systems, i.e. axonal transport in neurons. The bead usually changes the direction of movement at the tip of the flagellum and change in direction in the middle of the flagella was less common. Remarkably, just before the change in direction we observed high fluctuations in position for ∼50 msec. The cargo then abruptly starts moving on the opposite direction without showing any forward-backward stepping. The results imply that the motors responsible of the transport can be switched on and off quickly in turnaround zones by the cell so that they do not compete against each other to determine the direction of cargo transport.

Phosphorylated Tau Interacts with c-Jun N-terminal Kinase-interacting Protein 1 (JIP1) in Alzheimer Disease

In Alzheimer disease (AD) and frontotemporal dementia the microtubule-associated protein Tau becomes progressively hyperphosphorylated, eventually forming aggregates. However, how Tau dysfunction is associated with functional impairment is only partly understood, especially at early stages when Tau is mislocalized but has not yet formed aggregates. Impaired axonal transport has been proposed as a potential pathomechanism, based on cellular Tau models and Tau transgenic mice. We recently reported K369I mutant Tau transgenic K3 mice with axonal transport defects that suggested a cargo-selective impairment of kinesin-driven anterograde transport by Tau. Here, we show that kinesin motor complex formation is disturbed in the K3 mice. We show that under pathological conditions hyperphosphorylated Tau interacts with c-Jun N-terminal kinase- interacting protein 1 (JIP1), which is associated with the kinesin motor protein complex. As a result, transport of JIP1 into the axon is impaired, causing JIP1 to accumulate in the cell body. Because we found trapping of JIP1 and a pathological Tau/JIP1 interaction also in AD brain, this may have pathomechanistic implications in diseases with a Tau pathology. This is supported by JIP1 sequestration in the cell body of Tau-transfected primary neuronal cultures. The pathological Tau/JIP1 interaction requires phosphorylation of Tau, and Tau competes with the physiological binding of JIP1 to kinesin light chain. Because JIP1 is involved in regulating cargo binding to kinesin motors, our findings may, at least in part, explain how hyperphosphorylated Tau mediates impaired axonal transport in AD and frontotemporal dementia.

Tau and Microtubule Motors

The microtubule-associated protein tau is implicated in neurodegenerative diseases, including Alzheimer’s disease, but neither its normal nor its pathological role are well understood. Microtubules in cells are densely coated with microtubule-associated proteins, including tau, and Dixit et al . wanted to learn how motor proteins navigate on coated microtubules. The authors incorporated several isoforms and truncations of tau in a cell-free assay to build a complex system mimicking the cell environment. Encounters between individual motor proteins and tau on microtubules were observed directly, which revealed dramatic differences between the effects of tau on the motility of two microtubule-based motors, dynein versus kinesin. It appears likely that tau can spatially regulate the balance of microtubule-dependent axonal transport in neurons. R. Dixit, J. L. Ross, Y. E. Goldman, E. L. F. Holzbaur, Differential regulation of dynein and kinesin motor proteins by tau. Science 319 , 1086-1089 (2008). [Abstract] [Full Text]

Altered Cellular Distribution of Phospho‐Tau Proteins Coincides with Impaired Retrograde Axonal Transport in Neurons of Aged Rats

We hypothesize that the age-related degeneration of cytoskeleton in basal forebrain cholinergic neurons renders the NGF-TrkA signaling system non-functional and thereby impairs trophic support. Comparing young (4 months) and aged (28 months) rat brain, we examined immunohistochemically the compartmentalization of phosphorylated Tau protein using antibodies phospho-Tau404 and phospho-Tau231 of the GSK3beta kinase, known to phosphorylate Tau, the neurotrophin NGF, and its receptor P-TrkA. Retrograde labeling of basal forebrain cholinergic cells after injection of fluorogold into multiple sites in cortex and hippocampus revealed a significantly lower number of fluorogold-positive cells in aged brain. Despite a lower density of P-TrkA immunoreactivity in cortex and hippocampus of aged rats, there was no difference in NGF expression. In young animals phospho-Tau404, phospho-Tau231, and GSK3 immunoreactivity was observed mainly in neuronal fibers with lower staining in somata both in cortex and hippocampus. By contrast, Tau and GSK3 labeling were confined to the cell bodies in aged rats. This is confirmation that aging leads to a redistribution of cytoskeletal proteins. Since a somatic localization of phospho-Tau is indicative of cytoskeletal breakdown, we suggest that failure of axonal trafficking may be responsible for the lack of trophic support in aged cholinergic neurons of the basal forebrain.

HSV trafficking and development of gene therapy vectors with applications in the nervous system

Herpes simplex virus type 1 (HSV-1) is a neurotropic double-stranded DNA virus that causes cold sores, keratitis, and rarely encephalitis in humans. Nonpathogenic HSV-1 gene transfer vectors have been generated by elimination of viral functions necessary for replication. The life cycle of the native virus includes replication in epithelial cells at the site of initial inoculation followed by retrograde axonal transport to the nuclei of sensory neurons innervating the area of cutaneous primary infection. In this review, we summarize the current understanding of the molecular basis for HSV cell entry, nuclear transport of the genome, virion egress following replication, and retrograde and anterograde axonal transport in neurons. We discuss how each of these properties has been exploited or modified to allow the generation of gene transfer vectors with particular utility for neurological applications. Recent advances in engineering virus entry have provided proof of principle that vector targeting is possible. Furthermore, significant and potentially therapeutic modifications to the pathological responses to various noxious insults have been demonstrated in models of peripheral nerve disease. These applications exploit the natural axonal transport mechanism of HSV, allowing transgene expression in the cell nucleus within the inaccessible trigeminal ganglion or dorsal root ganglion, following the noninvasive procedure of subcutaneous vector inoculation. These findings demonstrate the importance of understanding basic virology in the design of vector systems and the powerful approach of exploiting favorable properties of the parent virus in the generation of gene transfer vectors.

Mutant superoxide dismutase disrupts cytoplasmic dynein in motor neurons

Cytoplasmic dynein and dynactin drive retrograde axonal transport in neurons, and mutations in dynein/dynactin cause motor neuron degeneration. To test whether defects in dynein/dynactin function are involved in the neurodegenerative disease amyotrophic lateral sclerosis, we examined neurotracer transport from muscle to motor neuron in a transgenic mouse model of amyotrophic lateral sclerosis. Significant inhibition was observed, which was temporally correlated with declines in muscle strength. No decrease in dynein/dynactin expression was observed, but immunohistochemistry suggests that dynein associates with aggregates of mutant Cu/Zn superoxide dismutase 1. Expression of mutant Cu/Zn superoxide dismutase 1 in primary motor neurons altered the cellular localization of dynein, suggesting an inhibition of dynein/dynactin function. Thus, inhibition of dynein/dynactin function may have a role in motor neuron degeneration in amyotrophic lateral sclerosis.

Myosin-dependent transport in neurons.

Axonal transport in neurons has been shown to be microtubule dependent, driven by the molecular motor proteins kinesin and dynein. However, organelles undergoing fast transport can often pause or rapidly change directions without apparent dissociation from their transport tracks. Cytoskeletal polymers such as neurofilaments and microtubules have also been shown to make infrequent but rapid movements in axons indicating that their transport is likely to involve molecular motors. In addition, neurons have multiple compartments that are devoid of microtubules where transport of organelles is still seen to occur. These areas are rich in other cytoskeletal polymers such as actin filaments. Transported organelles have been shown to associate with multiple motor proteins including myosins. This suggests that nonmicrotubule-based transport may be myosin driven. In this review we will focus our attention on myosin motors known to be present in neurons and evaluate the evidence that they contribute to transport or other functions in the different compartments of the neuron.

Impaired fast axonal transport in neurons of the sciatic nerves from dystonia musculorum mice.

Dystonia musculorum (dt) mice suffer from a severe sensory neuropathy caused by mutations in the gene encoding the cytoskeletal cross-linker protein dystonin/bullous pemphigoid antigen 1 (Bpag1). Loss of function of dystonin/Bpag1 within neurons leads to a loss in the maintenance of cytoskeletal organization and to the development of focal axonal swellings prior to death of the neuron. In the present study, we demonstrate that neurons within the sciatic nerves of dt27J mice undergo axonal degeneration as has been previously reported for the dorsal roots. Furthermore, ultrastructural studies reveal a perturbed organization of the neurofilament and microtubule networks within the axons of sciatic nerves in dt27J mice. The disrupted cytoskeletal organization suggested that axonal transport is affected in dt mice. To address this, we assessed fast axonal transport by measuring the rate of accumulation of acetylcholinesterase (AChE) proximal and distal to a surgically introduced ligature on the sciatic nerves of normal and dt27J mice. Our findings demonstrate that axonal transport of AChE in both orthograde and retrograde directions is markedly affected, and allow us to conclude that axonal transport defects do exist in the sciatic nerves of dt27J mice.

Clonal tests of conventional kinesin function during cell proliferation and differentiation.

Null mutations in the Drosophila Kinesin heavy chain gene (Khc), which are lethal during the second larval instar, have shown that conventional kinesin is critical for fast axonal transport in neurons, but its functions elsewhere are uncertain. To test other tissues, single imaginal cells in young larvae were rendered null for Khc by mitotic recombination. Surprisingly, the null cells produced large clones of adult tissue. The rates of cell proliferation were not reduced, indicating that conventional kinesin is not essential for cell growth or division. This suggests that in undifferentiated cells vesicle transport from the Golgi to either the endoplasmic reticulum or the plasma membrane can proceed at normal rates without conventional kinesin. In adult eye clones produced by null founder cells, there were some defects in differentiation that caused mild ultrastructural changes, but they were not consistent with serious problems in the positioning or transport of endoplasmic reticulum, mitochondria, or vesicles. In contrast, defective cuticle deposition by highly elongated Khc null bristle shafts suggests that conventional kinesin is critical for proper secretory vesicle transport in some cell types, particularly ones that must build and maintain long cytoplasmic extensions. The ubiquity and evolutionary conservation of kinesin heavy chain argue for functions in all cells. We suggest interphase organelle movements away from the cell center are driven by multilayered transport mechanisms; that is, individual organelles can use kinesin-related proteins and myosins, as well as conventional kinesin, to move toward the cell periphery. In this case, other motors can compensate for the loss of conventional kinesin except in cells that have extremely long transport tracks.

Mouse p150Glued(Dynactin 1) cDNA Sequence and Evaluation as a Candidate for the Neuromuscular Disease Mutationmnd2

p150Glued(Dynactin 1) is a component of the dynactin complex that is essential for retrograde axonal transport in neurons. The mouse p150GluedcDNA was isolated from mouse brain by RT-PCR. The complete sequence of the full length mouse cDNA was determined, including 19 bp of 5′ UTR, 3843 bp of coding sequence, and 162 bp of 3′ UTR. The predicted protein has a molecular mass of 142 kDa and exhibits 95% amino acid sequence identity to the predicted amino acid sequence of human p150Glued(DCTN1). The mouseDctn1gene was previously mapped in the central region of mouse chromosome 6, close to the neuromuscular disease genemnd2.Northern blot analysis, complete sequencing of the cDNA, Western blot, and functional tests of the protein did not detect any abnormalities of p150Gluedinmnd2mice.

The cytoskeleton and disease: genetic disorders of intermediate filaments.

Specialized cytoskeletons play many fascinating roles, including mechanical integrity and wound-healing in epidermal cells, cell polarity in simple epithelia, contraction in muscle cells, hearing and balance in the inner ear cells, axonal transport in neurons, and neuromuscular junction formation between muscle cells and motor neurons. These varied functions are dependent upon cytoplasmic networks of actin microfilaments (6 nm), intermediate filaments (10 nm) and microtubules (23 nm), and their many associated proteins. In this chapter, I review what is known about the cytoskeletons of intermediate filaments and their associated proteins. I focus largely on epidermal cells, which devote most of their protein-synthesizing machinery to producing an extensive intermediate filament network composed of keratin. Recent studies have shown that many of the devastating human disorders that arise from degeneration of this cell type have as their underlying basis either defects in the genes encoding keratins or abnormalities in keratin IF networks. I discuss what we know about the functions of IFs, and how the link to genetic disease has enhanced this understanding.

Cytoplasmic polarity and endoplasmic microtubules associated with the nucleus and organelles are ubiquitous features of food‐conducting cells in bryoid mosses (Bryophyta)

summaryAn ultrastructural survey of food conducting cells in the gametophytes and sporophytes of bryoid mosses, including the highly specialized leptoids of Polytrichales, has revealed that, without exception, these cells have a polarized organization and contain an axial system of endoplasmic microtubules (MTs). The orientation of polarity is highly consistent with the putative direction of the source to sink gradient, with distal cellular ends, i.e. those toward the sink, containing denser cytoplasm than proximal ends. In most cases the nucleus is spindle‐shaped and also lies at the distal end. Longitudinal arrays of MTs are associated with the nuclear envelope. These MTs extend into the cytoplasm for long distances and may come into contact with the plasma membrane lining the terminal walls at the distal ends. Cortical MTs are absent. The endoplasmic MTs are closely associated with a variety of organelles including mitochondria, plastids. endoplasmic reticulum and membrane‐bounded tubules and vesicles. The organelles aligned along the MT bundles lie within longitudinal cytoplasmic strands clearly visible with light microscopy. The discovery of cytoplasmic polarity and endoplasmic MTs as distinctive features of food conducting cells of mosses sets these cells apart from the sieve elements of vascular plants. The MT‐organelle associations, as observed in the food conducting cells of mosses, are hitherto undescribed in plants but are reminiscent of MT arrays functioning in organelle transport in neuronal axons and other animal and fungal systems.

A paradigm for examining toxicant effects on viability, structure, and axonal transport of neurons in culture

N1E.115 murine neuroblastoma cells differentiating in serum-free medium were used to develop a paradigm for testing neurotoxicity in vitro. The paradigm was designed to test the effects of toxicants on four different aspects of cell function or structure: 1. Viability as shown by the retention of cellular radiolabel (51Cr); 2. Growth and maintenance of neurites as reflected by the incidence and average length of these processes; 3. Gross structure of neurites; and 4. Velocity and flux of rapid anterograde and retrograde axonal transport as judged by video-enhanced differential interference contrast microscopy. To evaluate this paradigm, colchicine and vinblastine were used as neurotoxicants with a well-understood mechanism of action. These agents were only weakly cytotoxic according to the Cr-release assay, but were able to interfere with neurite outgrowth at nanomolar concentrations. Neurites that were elaborated in the presence of vinblastine and colchicine were often disfigured by numerous swellings packed with organelles. In established neurites, micromolar concentrations of vinblastine inhibited organellar motility with great rapidity, blocking all signs of transport within 20 min. The effect of colchicine was slower and less complete, but still impressive. We suggest that this four-part analysis represents a highly sensitive in vitro test for neurotoxicity, and a means of analyzing the relation between abnormalities of transport and structural damage of nerve cells.

Translocations of fodrin and its binding proteins

Fodrin, a protein related to erythrocyte spectrin, redistributes within the cell in certain situations. We compare such movements of fodrin and several fodrin binding proteins during the processes of axonal transport in neurons, and capping of surface proteins in lymphocytes. In neurons, three different populations of newly synthesized fodrin appear to be transported down the axons at different velocities corresponding to those of groups of transported proteins designated II, IV, and V. Actin, which can interact with fodrin, is transported at the velocity of group IV. Synapsin, a component of synaptic vesicles, is also reported to bind to fodrin. One population of synapsin is transported more rapidly than fodrin, at the velocity of group I; two additional populations of transported synapsin may overlap fodrin in groups II and IV. We consider possible functional associations of these different populations of fodrin and fodrin binding proteins. We note that the transport of group IV proteins resembles in certain respects the process of capping in lymphocytes, suggesting the possibility of a common mechanism. We outline one of several possible mechanisms.