Axonal Transport Velocity Research Articles

Using Live-Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons.

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-β, α-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions. Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellulardomain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport. Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

Extreme value analysis of the velocity of axonal transport by kinesin and dynein

Although previous studies have investigated motor differences in in vitro single-molecule experiments, this study aimed to discover motor differences in vivo as well. In particular, we focused on the synaptic cargo transport in DA9 motor neurons of Caenorhabditis elegans. The motion of synaptic cargos was observed by fluorescence microscopy, and the velocity was measured using kymograph analysis. Kinesin and dynein differ in structure, ATP-hydrolysis mechanism, and dynamics. Although the average velocity is sensitive to the cargo size, we consider that the results of extreme value analysis (EVA) are independent of it.

Axonal transport dynamics explain directional bias in tau deposition.

Emerging evidence indicates that defects in axonal transport, an early feature of AD pathology1 , could play a role in how directional biases develop on regional level2 . Specifically, kinesin-1, a motor protein responsible for the anterograde-directed transport of axonal cargoes including tau, is dysregulated by hyperphosphorylated tau species3,4 . Here we demonstrate that a mathematical model of tau-modified axonal transport can reproduce directional biases in tauopathy spread observed in mouse models of disease. We implemented a mathematical model incorporating the effects of aggregation and fragmentation of pathological tau complexes of two types, soluble and insoluble, on axonal transport and diffusion. We simulated the spatiotemporal profiles of each tau species in a multicompartment, two-neuron system using biologically plausible parameters and time scales. Notably, anterograde axonal transport velocity is modeled as being locally enhanced by the presence of soluble pathological tau3 and hindered by insoluble tau4 . We find that changing the balance of tau transport feedback parameters can elicit anterograde and retrograde biases in the distributions of soluble and insoluble tau between somatodendritic compartments in our two-neuron system (Figures 1 & 2). Changing the aggregation and fragmentation parameters perturbs this balance, suggesting complex interplay between these distinct molecular processes (Figures 3 & 4). The model also recreates spread biases comparable to those derived from network spread using AD-like and non-AD-like mouse tauopathy models2 (Figure 5). Our model of tau axonal transport feedback provides a parsimonious, biologically plausible explanation for apparent directionality observed in mouse model tauopathy spread, linking microscopic differences in tau conformational states and variance in clinical presentation.

Multifunctional compounds lithium chloride and methylene Blue attenuate the negative effects of diisopropylfluorophosphate on axonal transport in rat cortical neurons

Organophosphates (OPs) are valuable as pesticides in agriculture and for controlling deadly vector-borne illnesses; however, they are highly toxic and associated with many deleterious health effects in humans including long-term neurological impairments. Antidotal treatment regimens are available to combat the symptoms of acute OP toxicity, which result from the irreversible inhibition of acetylcholinesterase (AChE). However, there are no established treatments for the long-term neurological consequences of OP exposure. In addition to AChE, OPs can negatively affect multiple protein targets as well as biological processes such as axonal transport. Given the fundamental nature of axonal transport to neuronal health, we rationalized that this process might serve as a general focus area for novel therapeutic strategies against OP toxicity. In the studies described here, we employed a multi-target, phenotypic screening, and drug repurposing strategy for the evaluations of potential novel OP-treatments using a primary neuronal culture model and time-lapse live imaging microscopy. Two multi-target compounds, lithium chloride (LiCl) and methylene blue (MB), which are FDA-approved for other indications, were evaluated for their ability to prevent the negative effects of the OP, diisopropylfluorophosphate (DFP) on axonal transport. The results indicated that both LiCl and MB prevented DFP-induced impairments in anterograde and retrograde axonal transport velocities in a concentration dependent manner. While in vivo studies will be required to confirm our in vitro findings, these experiments support the potential of LiCl and MB as repurposed drugs for the treatment of the long-term neurological deficits associated with OP exposure (currently an unmet medical need).

Posters

Background: Neuronal calcium and ATP homeostasis is regulated through alterations in mitochondria fusion/fission events and anterograde and retrograde transport in neurons. Mitochondrial dynamics form an interactome that ultimately controls mitochondrial quality, quantity and metabolism. Our objective is to show that alterations in this interactome lead to point‐of‐no‐return situations characterized as first signs of progressive neurodegeneration. Material and methods: Differentiated human SH‐SY5Y neuroblastoma cells were treated with non‐lethal concentrations of either rotenone (31.25, 62.5, 125 and 250 nM) or 6‐hydroxydopamine (6.25, 12.5, 25 and 50 μmol/L). Cell viability was determined by measuring cell mass, metabolic activity and [ATP] using SRB, resazurin, and CellTiter‐Glo Luminescent Cell Viability assays, respectively, after 96 hours treatment. At earlier time points, before changes on cell viability occurred, cells were labelled with different fluorescent dyes, imaged under a Nikon Ti‐E‐H‐TIRF microscope. Mann‐Whitney's test was used for comparison of two‐mean values of controls and treated cells. Results: In a dose‐dependent manner, rotenone and 6‐hydroxydopamine decreased cell area and number of neuronal processes, leading to the loss of neuronal architecture. Under the same treatment conditions, neuronal mitochondria showed decreased axonal transport (velocities, time and length of movement) along with other dynamic events like fusion/fission cycles that are correlated to mitochondrial shape alterations. Conclusions: Alterations in mitochondrial metabolism and dynamics lead to neuronal cell morphology modifications and neurodegeneration that can ultimately drive cell death. Characterization and prevention of these changes, prior to the escalation to point‐of‐no‐return situations, may be the answer to diagnosis and to design therapeutic interventions to prevent cell death in neurodegenerative diseases.

Slow and fast grouping of cargo velocities in axonal transport due to single versus multi-motor transport

We have recently been exploring the idea that axonal transport velocity is “track and motor limited.” That is, microtubule length as well as microtubule-associated obstructions interact with the number of motors attached to a specific cargo to determine average cargo velocities. We assert that “slow” and “fast” transport as they are commonly referred to in the literature are really single- versus multi-motor transport along interrupted and obstructed track. To this end, we have recently developed a cargo-level motor model that appears to readily reproduce fast and slow transport simply by altering the number of motors. In the work presented here, we explore the ramifications of this model across a wide range of cargo sizes and motor-motor interactions. We find that categorization of cargo transport into “slow” and “fast” might be a natural consequence of track and motor limited transport as cargo load versus average velocity distribution produced by this model are clearly bi-modal with a curved (roughly square-root) relationship between number of motors and cargo load being the best at reproducing experimental data.

MAPT Genetic Variation and Neuronal Maturity Alter Isoform Expression Affecting Axonal Transport in iPSC-Derived Dopamine Neurons.

SummaryThe H1 haplotype of the microtubule-associated protein tau (MAPT) locus is genetically associated with neurodegenerative diseases, including Parkinson's disease (PD), and affects gene expression and splicing. However, the functional impact on neurons of such expression differences has yet to be fully elucidated. Here, we employ extended maturation phases during differentiation of induced pluripotent stem cells (iPSCs) into mature dopaminergic neuronal cultures to obtain cultures expressing all six adult tau protein isoforms. After 6 months of maturation, levels of exon 3+ and exon 10+ transcripts approach those of adult brain. Mature dopaminergic neuronal cultures display haplotype differences in expression, with H1 expressing 22% higher levels of MAPT transcripts than H2 and H2 expressing 2-fold greater exon 3+ transcripts than H1. Furthermore, knocking down adult tau protein variants alters axonal transport velocities in mature iPSC-derived dopaminergic neuronal cultures. This work links haplotype-specific MAPT expression with a biologically functional outcome relevant for PD.

Mitochondrial fission is an acute and adaptive response in injured motor neurons.

Successful recovery from neuronal damage requires a huge energy supply, which is provided by mitochondria. However, the physiological relevance of mitochondrial dynamics in damaged neurons in vivo is poorly understood. To address this issue, we established unique bacterial artificial chromosome transgenic (BAC Tg) mice, which develop and function normally, but in which neuronal injury induces labelling of mitochondria with green fluorescent protein (GFP) and expression of cre recombinase. GFP-labelled mitochondria in BAC Tg mice appear shorter in regenerating motor axons soon after nerve injury compared with mitochondria in non-injured axons, suggesting the importance of increased mitochondrial fission during the early phase of nerve regeneration. Crossing the BAC Tg mice with mice carrying a floxed dynamin-related protein 1 gene (Drp1), which is necessary for mitochondrial fission, ablates mitochondrial fission specifically in injured neurons. Injury-induced Drp1-deficient motor neurons show elongated or abnormally gigantic mitochondria, which have impaired membrane potential and axonal transport velocity during the early phase after injury, and eventually promote neuronal death. Our in vivo data suggest that acute and prominent mitochondrial fission during the early stage after nerve injury is an adaptive response and is involved in the maintenance of mitochondrial and neuronal integrity to prevent neurodegeneration.

Tubulin involvement in Bortezomib peripheral neurotoxicity

Axonal transport of mitochondria (Mt) controlled by specialized motor and docking proteins that distribute Mt throughout the axon where they provide energy for metabolic and synaptic activity is a vulnerable target in neuronal pathology (1). Bortezomib (BZ) is a proteasome inhibitor active in multiple myeloma (2). One of its key toxicities is painful peripheral neuropathy (BIPN), which frequently requires treatment discontinuation (3). BIPN is dose-related and predominantly sensory, resulting from axonal degeneration. Recent results indicate that BZ modifies axonal tubulin dynamic and we hypothesize that BZ alters fast axonal transport. Here we studied using time-lapse imaging the effect of different BZ concentration on axonal Mt transport in isolated dorsal root ganglion (DRG) neurons from adult male mice. We used kymograph to quantify the total number of Mt and to discriminate antero and retrogradely moving Mt from stationary Mt. Twenty-four hours of BZ treatment (0.1 to 15 µM) induced a dose-dependent reduction in Mt trafficking. Moreover, BZ had no impact on MT motion directions, but it induced a progressive reduction of both anterograde and retrograde axonal transport velocities. These events were associated with increase in tubulin polymerization and of MAP2 expression, but they occurred only after 72h of chronic BZ treatment. We have developed an in vitro model of BIPN demonstrating that transport impairment is already present before evident tubulin polymerization, suggesting that transport deficit represents an early stage of axonal dysfunction. Perpetuated transport dysfunction could impair distal organelle supply and play a critical role in advanced stages of BIPN.This work was supported by the University of Milan-Bicocca and University of Michigan research grants

Altered Transport Velocity of Axonal Mitochondria in Retinal Ganglion Cells After Laser-Induced Axonal Injury In Vitro.

Axonal damage precedes retinal ganglion cell (RGC) apoptosis in glaucomatous optic neuropathy. Because mitochondria in RGC axons are damaged before cell death, we examined axonal mitochondrial transport dynamics after axonal injury. Mitochondria in rat-cultivated RGCs were stained with rhodamine 123. After axonal injury induced using a laser microdissection system, axonal transport was evaluated by time-lapse imaging. The RGC apoptosis was detected using ethidium homodimer-1 on day 3 after axonal injury. The rate of stationary mitochondria in RGC axons significantly increased from 37.6% before axonal injury to 70.6% at 5 minutes (P < 0.001) and 63.6% at 18 hours (P < 0.001) after axonal injury. The mean axonal mitochondrial transport velocity in RGCs transiently deteriorated from 0.48 ± 0.01 μm/s before axonal injury to 0.37 ± 0.02 μm/s at 5 minutes after axonal injury (P < 0.001). However, 23.5% of RGCs showed recovered axonal transport velocity at 18 hours after injury. On day 3 after axonal injury, RGCs with the recovery of axonal transport did not undergo apoptosis, whereas 69.2% of RGCs without the recovery of axonal transport underwent apoptosis (P = 0.029). Axonal injury disrupts mitochondrial transport in RGC axons. Irreversible decreased axonal mitochondrial transport velocity may be useful to predict RGC apoptosis after axonal injury.

Axonal transport velocity estimation from kymographs based on curvilinear feature extraction and spline fitting.

Axonal transport velocities are obtained from spatio-temporal maps called kymographs developed from time-lapse confocal microscopy movies of neurons. The kymographs of axonal transport of C.elegans worms are much noisier due to in vivo nature of imaging. Existing methodologies for velocity measurement include laborious manual delineation of axonal movement ridges on the kymographs and thereby determining particle velocities from the slopes of ridges marked. Manual kymograph analysis is not only time consuming but also prone to human errors in marking the ridges. An automated algorithm to extract all the ridges and determine the velocities without significant manual efforts is highly preferred. Not many methods are currently available for such biological studies. We present an almost automated method based on information fusion using LDA classifier, morphological image processing and spline fitting for determining axonal transport velocities. Experimental analysis of 50 kymographs shows considerable reduction of 89% in time taken with manual intervention of 10.83%. Comparitive study with the results of two of the previous literatures shows that our algorithm performs better.

Soluble N-terminal fragment of mutant Huntingtin protein impairs mitochondrial axonal transport in cultured hippocampal neurons

Huntington's disease (HD) is an autosomal dominant, progressive, neurodegenerative disorder caused by an unstable expansion of CAG repeats (>35 repeats) within exon 1 of the interesting transcript 15 (IT15) gene. This gene encodes a protein called Huntingtin (Htt), and mutation of the gene results in a polyglutamine (polyQ) near the N-terminus of Htt. The N-terminal fragments of mutant Htt (mHtt), which tend to aggregate, are sufficient to cause HD. Whether these aggregates are causal or protective for HD remains hotly debated. Dysfunctional mitochondrial axonal transport is associated with HD. It remains unknown whether the soluble or aggregated form of mHtt is the primary cause of the impaired mitochondrial axonal transport in HD pathology. Here, we investigated the impact of soluble and aggregated N-terminal fragments of mHtt on mitochondrial axonal transport in cultured hippocampal neurons. We found that the N-terminal fragment of mHtt formed aggregates in almost half of the transfected neurons. Overexpression of the N-terminal fragment of mHtt decreased the velocity of mitochondrial axonal transport and mitochondrial mobility in neurons regardless of whether aggregates were formed. However, the impairment of mitochondrial axonal transport in neurons expressing the soluble and aggregated N-terminal fragments of mHtt did not differ. Our findings indicate that both the soluble and aggregated N-terminal fragments of mHtt impair mitochondrial axonal transport in cultured hippocampal neurons. We predict that dysfunction of mitochondrial axonal transport is an early-stage event in the progression of HD, even before mHtt aggregates are formed.

The p150Glued CAP-Gly Domain Regulates Initiation of Retrograde Transport at Synaptic Termini

p150(Glued) is the major subunit of dynactin, a complex that functions with dynein in minus-end-directed microtubule transport. Mutations within the p150(Glued) CAP-Gly microtubule-binding domain cause neurodegenerative diseases through an unclear mechanism. A p150(Glued) motor neuron degenerative disease-associated mutation introduced into the Drosophila Glued locus generates a partial loss-of-function allele (Gl(G38S)) with impaired neurotransmitter release and adult-onset locomotor dysfunction. Disruption of the p150(Glued) CAP-Gly domain in neurons causes a specific disruption of vesicle trafficking atterminal boutons (TBs), the distal-most ends of synapses. Gl(G38S) larvae accumulate endosomes along with dynein and kinesin motor proteins within swollen TBs, and genetic analyses show that kinesin and p150(Glued) function cooperatively at TBs to coordinate transport. Therefore, the p150(Glued) CAP-Gly domain regulates dynein-mediated retrograde transport at synaptic termini, and this function of dynactin is disrupted by a mutation that causes motorneuron disease.

Determination of axonal transport velocities via image cross- and autocorrelation

On their way to the synapse and back, neuronal proteins are carried in cargo vesicles along axons and dendrites. Here, we demonstrate that the key parameters of axonal transport, i.e., particle velocities and pausing times can be read out from CCD-camera images automatically. In the present study, this is achieved via cross- and autocorrelation of kymograph columns. The applicability of the method was measured on simulated kymographs and data from axonal transport timeseries of mRFP-labeled synaptophysin. In comparing outcomes of velocity determinations via a performance parameter that is analogous to the signal-to-noise ratio (SNR) definition, we find that outcomes are dependent on sampling, particle numbers and signal to noise of the kymograph. Autocorrelation of individual columns allows exact determination of pausing time populations. In contrast to manual tracking, correlation does not require experience, a priori assumptions or disentangling of individual particle trajectories and can operate at low SNR.

1P-267 神経成長因子による細胞内軸索輸送の促進機構(バイオイメージング,第47回日本生物物理学会年会)

1P-267 神経成長因子による細胞内軸索輸送の促進機構(バイオイメージング,第47回日本生物物理学会年会)

Defective neurofilament transport in mouse models of amyotrophic lateral sclerosis: a review.

Neurofilament proteins synthesized in the cell body of neurons are assembled and transported into axons, where they influence axon radial growth, axonal transport, and nerve conduction velocities. In diseased states, neurofilaments accumulate in cell bodies and proximal axons of affected neurons, and these lesions are characteristic of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), spinal muscular atrophy (SMA), Charcot-Marie-Tooth disease type 2 (CMT2), and hereditary sensory motor neuropathy. Although the molecular mechanisms that contribute to these accumulations are not yet identified, transgenic mouse models are beginning to provide insight into the role of neurofilament transport in disease-related dysfunction of neurons. This review addresses axonal transport in mouse models of ALS and the special significance of neurofilament transport in this disease.

Temporal and spatial variations in slow axonal transport velocity along peripheral motoneuron axons

Neuronal axons are cellular extensions that can reach more than a meter in length. To maintain such a structure, macromolecules synthesized in cell bodies must be transported to the distal axons. Proteins associated with membranous organelles are generally transported in several fast transported groups, while cytoplasmic proteins, mostly composed of cytoskeletal proteins, are transported in slowly transported groups. Neurofilaments are a main component in the slowly transported group. Composed of three polypeptide subunits (NF-H, NF-M and NF-L), they are the most abundant cytoskeletal element in large myelinated axons. In various neurological or neurotoxic disorders, selective accumulation of neurofilaments was observed in different compartments of a neuron (cell bodies, proximal or distal axons). The underlying mechanism for this regional selectivity has been unclear. Using the classical pulse labeling method, we examined the changes in neurofilament transport velocity in transgenic mice that overexpress different neurofilament subunits. We present evidence that at least three velocities of neurofilament transport exist along peripheral motor axons. Each of these velocities was altered differently depending on which neurofilament subunit was overexpressed. We suggest that neurofilament transport in motor axons consists of multiple successive stages and that each of these stages is carried out by different transport mechanisms. These differences provide a basis for the regional deficiencies in axonal transport associated with several neurological disorders.

Overexpression of neurofilament subunit M accelerates axonal transport of neurofilaments

Neurofilaments are composed of three polypeptide subunits (NF-H, NF-M and NF-L). They are the most abundant cytoskeletal element in large myelinated axons and play a central role in development of axonal caliber. To perform this role, neurofilaments are transported from their site of synthesis, the cell bodies, to the distal axons. Previous studies showed that overexpression of NF-M in transgenic mice led to accumulation of neurofilaments in neurons and a reduction in the number of neurofilaments in axons, suggesting that axonal transport of neurofilaments was slowed. To determine whether this was the case, we measured axonal transport velocities in the wild type and transgenic mice overexpressing NF-M by the classical pulse-labeling method using 35S-methionine. We found that neurofilament transport in peripheral motor axons can be described with a model consistent with two linear velocities. Contrary to expectations, both velocities were accelerated by overexpression of NF-M. These results suggest that subunit composition in neurofilaments play a regulatory role in neurofilament transport. In addition, these results show that there are regional differences in neurofilament transport along long axons and these differences may be the basis for selective regional accumulation of neurofilaments in various neurological disorders.

C-terminal phosphorylation of the high molecular weight neurofilament subunit correlates with decreased neurofilament axonal transport velocity

We probed the relationship of NF axonal transport of neurofilaments (NFs) to their phosphorylation state by comparing these parameters in two closely-aged groups of young adult mice — 2 and 5 months of age. This particular time interval was selected since prior studies demonstrate that optic axons have already completed axonal caliber expansion and attained adult NF levels by 2 months but, as shown herein, continue to increase NF-H C-terminal phosphorylation. NF axonal transport was monitored by autoradiographic analysis of the distribution of radiolabeled subunits immunoprecipitated from optic axon segments at intervals following intravitreal injection of 35 S -methionine. Both the peak and front of radiolabeled NFs translocated faster in 2- vs. 5-month-old mice. This developmental decline in NF transport rate was not due to reduced incorporation of NFs into the cytoskeleton, nor to an overall decline in slow axonal transport. By excluding or minimizing other factors, these findings support previous conclusions that C-terminal NF phosphorylation regulates NF axonal transport.

Disruption of type IV intermediate filament network in mice lacking the neurofilament medium and heavy subunits.

To clarify the role of the neurofilament (NF) medium (NF-M) and heavy (NF-H) subunits, we generated mice with targeted disruption of both NF-M and NF-H genes. The absence of the NF-M subunit resulted in a two- to threefold reduction in the caliber of large myelinated axons, whereas the lack of NF-H subunits had little effect on the radial growth of motor axons. In NF-M-/- mice, the velocity of axonal transport of NF light (NF-L) and NF-H proteins was increased by about two-fold, whereas the steady-state levels of assembled NF-L were reduced. Although the NF-M or NF-H subunits are each dispensable for the formation of intermediate filaments, the absence of both subunits in double NF-M; NF-H knockout mice led to a scarcity of intermediate filament structures in axons and to a marked approximately twofold increase in the number of microtubules. Protein analysis indicated that the levels of NF-L and alpha-internexin proteins were reduced dramatically throughout the nervous system. Immunohistochemistry of spinal cord from the NF-M-/-;NF-H-/- mice revealed enhanced NF-L staining in the perikaryon of motor neurons but a weak NF-L staining in axons. In addition, axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed after 30 days very low levels of newly synthesized NF-L proteins in the sciatic nerve of NF-M-/-;NF-H-/- mice. The combined results demonstrate a requirement of the high-molecular-weight subunits for the assembly of type IV intermediate filament proteins and for the efficient translocation of NF-L proteins into the axonal compartment.