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.     

A new method for quantifying mitochondrial axonal transport

Axonal transport of mitochondria is critical for neuronal survival and function. Automatically quantifying and analyzing mitochondrial movement in a large quantity remain challenging. Here, we report an efficient method for imaging and quantifying axonal mitochondrial transport using microfluidic-chamber-cultured neurons together with a newly developed analysis package named “MitoQuant”. This tool-kit consists of an automated program for tracking mitochondrial movement inside live neuronal axons and a transient-velocity analysis program for analyzing dynamic movement patterns of mitochondria. Using this method, we examined axonal mitochondrial movement both in cultured mammalian neurons and in motor neuron axons of Drosophila in vivo. In 3 different paradigms (temperature changes, drug treatment and genetic manipulation) that affect mitochondria, we have shown that this new method is highly efficient and sensitive for detecting changes in mitochondrial movement. The method significantly enhanced our ability to quantitatively analyze axonal mitochondrial movement and allowed us to detect dynamic changes in axonal mitochondrial transport that were not detected by traditional kymographic analyses.     

The genetics of axonal transport and axonal transport disorders

Neurons are specialized cells with a complex architecture that includes elaborate dendritic branches and a long, narrow axon that extends from the cell body to the synaptic terminal. The organized transport of essential biological materials throughout the neuron is required to support its growth, function, and viability. In this review, we focus on insights that have emerged from the genetic analysis of long-distance axonal transport between the cell body and the synaptic terminal. We also discuss recent genetic evidence that supports the hypothesis that disruptions in axonal transport may cause or dramatically contribute to neurodegenerative diseases.     

Motors for fast axonal transport.

In neurons and other animal cells, membrane-bound vesicles course rapidly along cytoskeletal filaments to reach their destinations. Based on a variety of in vivo studies it is becoming clear that the microtubule-based motor, kinesin (and its relatives), drive vesicle movements in axons. Surprisingly, some axonal membranes have the capacity to move on both microtubules and actin filaments.     

A coupled model of fast axonal transport of organelles and slow axonal transport of tau protein

We have developed a model that accounts for the effect of a non-uniform distribution of tau protein along the axon length on fast axonal transport of intracellular organelles. The tau distribution is simulated by using a slow axonal transport model; the numerically predicted tau distributions along the axon length were validated by comparing them with experimentally measured tau distributions reported in the literature. We then developed a fast axonal transport model for organelles that accounts for the reduction of kinesin attachment rate to microtubules by tau. We investigated organelle transport for two situations: (1) a uniform tau distribution and (2) a non-uniform tau distribution predicted by the slow axonal transport model. We found that non-uniform tau distributions observed in healthy axons (an increase in tau concentration towards the axon tip) result in a significant enhancement of organelle transport towards the synapse compared with the uniform tau distribution with the same average amount of tau. This suggests that tau may play the role of being an enhancer of organelle transport.     

Slow axonal transport.

New studies provide further evidence that the neuronal cytoskeleton is the product of a dynamic interplay between axonal transport processes and locally regulated assembly mechanisms. These data confirm that the axonal cytoskeleton in mammalian systems is largely stationary and is maintained by a smaller pool of moving subunits or polymers. Slow axonal transport in certain lower species, however, may exhibit quite different features.     

A fast and robust method for automated analysis of axonal transport

Cargo movement along axons and dendrites is indispensable for the survival and maintenance of neuronal networks. Key parameters of this transport such as particle velocities and pausing times are often studied using kymograph construction, which converts the transport along a line of interest from a time-lapse movie into a position versus time image. Here we present a method for the automatic analysis of such kymographs based on the Hough transform, which is a robust and fast technique to extract lines from images. The applicability of the method was tested on simulated kymograph images and real data from axonal transport of synaptophysin and tetanus toxin as well as the velocity analysis of synaptic vesicle sharing between adjacent synapses in hippocampal neurons. Efficiency analysis revealed that the algorithm is able to detect a wide range of velocities and can be used at low signal-to-noise ratios. The present work enables the quantification of axonal transport parameters with high throughput with no a priori assumptions and minimal human intervention.     

A microfluidic culture platform for CNS axonal injury, regeneration and transport

Investigation of axonal biology in the central nervous system (CNS) is hindered by a lack of an appropriate in vitro method to probe axons independently from cell bodies. Here we describe a microfluidic culture platform that polarizes the growth of CNS axons into a fluidically isolated environment without the use of targeting neurotrophins. In addition to its compatibility with live cell imaging, the platform can be used to (i) isolate CNS axons without somata or dendrites, facilitating biochemical analyses of pure axonal fractions and (ii) localize physical and chemical treatments to axons or somata. We report the first evidence that presynaptic (Syp) but not postsynaptic (Camk2a) mRNA is localized to developing rat cortical and hippocampal axons. The platform also serves as a straightforward, reproducible method to model CNS axonal injury and regeneration. The results presented here demonstrate several experimental paradigms using the microfluidic platform, which can greatly facilitate future studies in axonal biology.     

A model for slow axonal transport and its application to neurofilamentous neuropathies

A model for slow axonal transport is developed in which the essential features are reversible binding of cytoskeletal elements and of soluble cytosolic proteins to each other and to motile elements such as actin microfilaments. Computer simulation of the equations of the model demonstrate that the model can account for many of the features of the SCa and SCb waves observed in pulse experiments. The model also provides a unified explanation for the increase and decrease of neurofilament transport rates observed in various toxicant-induced neuropathies.     

Acrylamide-induced alterations in axonal transport

Alterations in the axonal transport of proteins, glycoproteins, and gangliosides in sensory neurons of the sciatic nerve were examined in adult male rats exposed to acrylamide (40 mg ip/kg body wt/d for nine consecutive days). Twenty-four hours after the last dose, the L5 dorsal root ganglion (DRG) was injected with either [35S]methionine to label proteins or [3H]glucosamine to label glycoproteins and gangliosides. The downflow patterns of radioactivity for [35S]methionine-labeled proteins and [3H]glucosamine-labeled gangliosides were unaltered by acrylamide treatment. In contrast, the outflow pattern of labeled glycoproteins displayed a severely attenuated crest with no alteration in velocity, suggesting a preferential transfer with the unlabeled stationary components in the axolemma. Retrograde accumulation of transported glycoproteins and gangliosides was unaltered for at least 6 h; however, by 24 h, there was a 75% decrease in the amount of accumulated material. The accumulation of [35S]methionine-labeled proteins was not altered. Autoradiographic analysis revealed an acrylamide-induced paucity of transported radiolabeled glycoproteins selectively in myelinated axons with no effect on "nonmyelinated" axons. The pattern of transported proteins was similar in both control and acrylamide-exposed animals. These results suggest a preferential inhibition of glycosylation or axonal transport of glycoproteins in neurons bearing myelinated axons. More importantly, it suggests that interpretations of axonal transport data must be made with the consideration of alterations in selective nerve fibers and not with the tacit assumption that all fibers in the nerve population are equally affected.     

Molecular motors in axonal transport

Neurons require a large amount of intracellular transport. Cytoplasmic polypeptides and membrane-bounded organelles move from the perikaryon, down the length of the axon, and to the synaptic terminals. This movement occurs at distinct rates and is termed axonal transport. Axonal transport is divided into the slow transport of cytoplasmic proteins including glycolytic enzymes and cytoskeletal structures and the fast transport of membrane-bounded organelles along linear arrays of microtubules. The polypeptide compositions of the rate classes of axonal transport have been well characterized, but the underlying molecular mechanisms of this movement are less clear. Progress has been particularly slow toward understanding force-generation in slow transport, but recent developments have provided insight into the molecular motors involved in fast axonal transport. Recent advances in the cellular and molecular biology of one fast axonal transport motor, kinesin, have provided a clearer understanding of organelle movement along microtubules. The availability of cellular and molecular probes for kinesin and other putative axonal transport motors have led to a reevaluation of our understanding of intracellular motility.     

Functions of retrograde axonal transport

Retrograde axonal transport conveys materials from axon to cell body. One function of this process is recycling of materials originally transported from cell body to axon. In motoneurons, 50% of fast-transported protein is returned. Reversal probably occurs mainly at nerve terminals and, for labeled proteins, is nonselective. Proteolysis is not required, although changes in tertiary protein structure may occur with a repackaging of molecules in organelles different from those in which they were anterograde-transported. A second function is transfer of information about axonal status and terminal environment. Premature reversal of transport adjacent to an axon injury may be a component of a signal that initiates cell body chromatolysis. Transport of target cell-derived molecules with trophic effects on the cell body is exemplified by nerve growth factor transport in neurons dependent on it, and is probably a widespread phenomenon in the developing nervous system. Disorders in retrograde transport or reversal occur in some experimental neuropathies, and certain viruses, as well as tetanus toxin, may gain access to the central nervous system by this route.     

Organelles in fast axonal transport

The present minireview describes experiments carried out, in short-term crush-operated rat nerves, using immunofluorescence and cytofluorimetric scanning techniques to study endogenous substances in anterograde and retrograde fast axonal transport. Vesicle membrane components p38 (synaptophysin) and SV2 are accumulating on both sides of a crush, but a larger proportion of p38 (about 3/4) than of SV2 (about 1/2) is recycling toward the cell body, compared to the amount carried with anterograde transport. Matrix peptides, such as CGRP, ChRA, VIP, and DBH are recycling to a minor degree, although only 10-20% of surface-associated molecules, such as synapsins and kinesin, appear to recycle. The described methodological approach to study the composition of organelles in fast axonal transport, anterograde as compared to retrograde, is shown to be useful for investigating neurobiological processes. We make use of the "in vivo chromatography" process that the fast axonal transport system constitutes. Only substances that are in some way either stored in, or associated with, transported organelles can be clearly observed to accumulate relative to the crush region. Emphasis in this paper was given to the synapsins, because of diverging results published concerning the degree of affiliation with various neuronal organelles. Our previously published results have indicated that in the living axons the SYN I is affiliated with mainly anterogradely fast transported organelles. Therefore, some preliminary, previously unpublished results on the accumulations of the four different synapsins (SYN Ia, SYN Ib, SYN IIa, and SYN IIb), using antisera specific for each of the four members of the synapsin family, are described. It was found that SYN Ib clearly has a stronger affiliation to anterogradely transported organelles than SYN Ia, and that both SYN IIa and SYN IIb are bound to some degree to transported organelles.     

Slow axonal transport: the subunit transport model

A central problem concerning slow transport of cytoskeletal proteins along nerve axons is where they are assembled and the form in which they are transported. The polymer and subunit transport models are the two major hypotheses. Recent developments using molecular and cellular biophysics, molecular cell biology and gene technology have enabled visualization of moving forms of cytoskeletal proteins during their transport. Here, we argue that these studies support the subunit transport theory.     

A new methodological approach for studying axonal transport: cytofluorometric scanning of nerves

A new technique for studying axonal transport has been developed. The technique, which is based on histofluorescence techniques, enables the measurement of several different accumulated substances and parameters within a single nerve in relation to a nerve crush or local cooling. Any substance that can be made to fluoresce can be measured. The tissue is treated according to the formaldehyde-induced fluorescence method of Hillarp and Falck for visualization of monoamines, or according to the indirect immunofluorescence method. For immunofluorescence the nerve is cryostat-sectioned and various sections can be incubated with primary antisera against different antigens. After incubation and mounting the sections are placed in a cytofluorimeter (Leitz MPV II). They are passed under a measuring slit at a steady speed by a motor driven cross-table. The fluorescence intensity passing through the measuring slit is continuously registered by a recording unit with an integrator. This recorder produces a graphical nerve accumulation profile, and the area under the profile, relating to the fluorescence, is expressed in arbitrary units. This article presents data on the accumulation of noradrenaline, dopamine beta-hydroxylase, and tyrosine hydroxylase in crush-operated rat sciatic nerve. The time-course accumulations for noradrenaline (visualized by the Falck and Hillarp method) and dopamine beta-hydroxylase (visualized by immunofluorescence) demonstrated a striking similarity, which is to be expected since the two substances are stored in the same organelle. Tyrosine hydroxylase (visualized by immunofluorescence) showed a slower accumulation with time, but faster than would be expected had the enzyme been 100% soluble. Colchicine but not lumi-colchicine blocked the transport of noradrenaline organelles. With the new scanning technique we have the potential to study accumulation profiles of several different substances within a single nerve. Morphometric data, morphological observations, and photograph documentation of the same nerve section are also available.