Identification of an axonal kinesin-3 motor for fast anterograde vesicle transport that facilitates retrograde transport of neuropeptides

A screen for genes required in Drosophila eye development identified an UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of green fluorescent protein-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism.     

Mathematical modeling and parameter estimation of axonal cargo transport

This paper is about how cortical recurrent interactions in primary visual cortex (V1) together with feedback from extrastriate cortex can account for spectral peaks in the V1 local field potential (LFP). Recent studies showed that visual stimulation enhances the γ-band (25–90 Hz) of the LFP power spectrum in macaque V1. The height and location of the γ-band peak in the LFP spectrum were correlated with visual stimulus size. Extensive spatial summation, possibly mediated by feedback connections from extrastriate cortex and long-range horizontal connections in V1, must play a crucial role in the size dependence of the LFP. To analyze stimulus-effects on the LFP of V1 cortex, we propose a network model for the visual cortex that includes two populations of V1 neurons, excitatory and inhibitory, and also includes feedback to V1 from extrastriate cortex. The neural network model for V1 was a resonant system. The model’s resonance frequency (ResF) was in the γ-band and varied up or down in frequency depending on cortical feedback. The model’s ResF shifted downward with stimulus size, as in the real cortex, because increased size recruited more activity in extrastriate cortex and V1 thereby causing stronger feedback. The model needed to have strong local recurrent inhibition within V1 to obtain ResFs that agree with cortical data. Network resonance as a consequence of recurrent excitation and inhibition appears to be a likely explanation for γ-band peaks in the LFP power spectrum of the primary visual cortex.     

Multiple layers of spatial regulation coordinate axonal cargo transport

Nerve axons are shaped similar to long electric wires to quickly transmit information from one end of the body to the other. To remain healthy and functional, axons depend on a wide range of cellular cargos to be transported from the neuronal cell body to its distal processes. Because of the extended distance, a sophisticated and well-organized trafficking network is required to move cargos up and down the axon. Besides motor proteins driving cargo transport, recent data revealed that subcellular membrane specializations, including the axon initial segment at the beginning of the axon and the membrane-associated periodic skeleton, which extends throughout the axonal length, are important spatial regulators of cargo traffic. In addition, tubulin modifications and microtubule-associated proteins present along the axonal cytoskeleton have been proposed to bias cargo movements. Here, we discuss the recent advances in understanding these multiple layers of regulatory mechanisms controlling axonal transport.     

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers

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Retrograde axonal transport and motor neuron disease

Transport of material between extensive neuronal processes and the cell body is crucial for neuronal function and survival. Growing evidence shows that deficits in axonal transport contribute to the pathogenesis of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we review recent data indicating that defects in dynein-mediated retrograde axonal transport are involved in ALS etiology. We discuss how mutant copper-zinc superoxide dismutase (SOD1) and an aberrant interaction between mutant SOD1 and dynein could perturb retrograde transport of neurotrophic factors and mitochondria. A possible contribution of axonal transport to the aggregation and degradation processes of mutant SOD1 is also reviewed. We further consider how the interference with axonal transport and protein turnover by mutant SOD1 could influence the function and viability of motor neurons in ALS.     

Microtubule acetylation promotes kinesin-1 binding and transport

Long-distance intracellular delivery is driven by kinesin and dynein motor proteins that ferry cargoes along microtubule tracks . Current models postulate that directional trafficking is governed by known biophysical properties of these motors-kinesins generally move to the plus ends of microtubules in the cell periphery, whereas cytoplasmic dynein moves to the minus ends in the cell center. However, these models are insufficient to explain how polarized protein trafficking to subcellular domains is accomplished. We show that the kinesin-1 cargo protein JNK-interacting protein 1 (JIP1) is localized to only a subset of neurites in cultured neuronal cells. The mechanism of polarized trafficking appears to involve the preferential recognition of microtubules containing specific posttranslational modifications (PTMs) by the kinesin-1 motor domain. Using a genetic approach to eliminate specific PTMs, we show that the loss of a single modification, alpha-tubulin acetylation at Lys-40, influences the binding and motility of kinesin-1 in vitro. In addition, pharmacological treatments that increase microtubule acetylation cause a redirection of kinesin-1 transport of JIP1 to nearly all neurite tips in vivo. These results suggest that microtubule PTMs are important markers of distinct microtubule populations and that they act to control motor-protein trafficking.     

C-terminal kinesin motor KIFC1 participates in acrosome biogenesis and vesicle transport

We have identified a possible role for the KIFC1 motor protein in formation of the acrosome, an organelle unique to spermatogenesis. KIFC1, a C-terminal kinesin motor, first appears on membrane-bounded organelles (MBOs) in the medulla of early spermatids followed by localization to the acrosomal vesicle. KIFC1 continues to be present on the acrosome of elongating spermatids as it flattens on the spermatid nucleus; however, increasing amounts of KIFC1 are found at the caudal aspect of the spermatid head and in distal cytoplasm. The KIFC1 motor is also found in the nucleus of very immature round spermatids just prior to its appearance on the acrosome. In some cases, KIFC1 appears localized just below the nuclear membrane adjacent to the subacrosomal membrane. We demonstrate that KIFC1 is associated with importin beta and colocalizes with this nuclear transport factor on curvilinear structures associated with the spermatid nuclei. These data support a model in which KIFC1, perhaps in association with nuclear factors, assists in the formation and/or elongation of the spermatid acrosome. This article represents the first demonstration of a direct association of a molecular motor with the spermatid acrosome, the formation of which is essential for fertilization.     

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.     

Myelination induces axonal hotspots of synaptic vesicle fusion that promote sheath growth

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Dysfunction of axonal transport in neuropathies and motor neuron diseases

Sensory neurons and motor neurons are particularly vulnerable to axonal transport defects due to the length of their axons. Several mutations have recently been identified in genes coding for cytoskeletal or motor proteins as causative factors in various neurodegenerative diseases. This review synthesizes the main defects found in inherited peripheral neuropathies and in motor neuron diseases as well as in mouse models of these disorders.     

Effect of vesicle traps on traffic jam formation in fast axonal transport

The purpose of this paper is to develop a model for simulation of the formation of organelle traps in fast axonal transport. Such traps may form in the regions of microtubule polar mismatching. Depending on the orientation of microtubules pointing toward the trap region, these traps can accumulate either plus-end or minus-end oriented vesicles. The model predicts that the maximum concentrations of organelles occur at the boundaries of the trap regions; the overall concentration of organelles in the axon with traps is greatly increased compared to that in a healthy axon, which is expected to contribute to mechanical damages of the axon. The organelle traps induce hindrance to organelle transport down the axon; the total organelle flux down the axon with traps is found to be significantly reduced compared to that in a healthy axon.     

The synaptic vesicle membrane: origin, axonal distribution, protein components, exocytosis and recycling

The paper discusses functional and molecular aspects of the synaptic vesicle membrane during its life cycle. The distribution of the synaptic vesicle membrane compartment in an entire cholinergic neuron is monitored using colloidal gold labelling and a monoclonal antibody against the synaptic vesicle membrane protein SV2. This provides new insights concerning vesicle origin and fate in the various compartments of the neuron. A new synaptic vesicle membrane protein (svp25) of Mr 25,000 with properties similar to synaptophysin as well as a synaptic vesicle binding phosphoprotein of the presynaptic membrane (Mr 92,000) likely to be involved in vesicle exocytosis are described. The membrane compartment recycled on induced transmitter release contains synaptic vesicle but not plasma membrane markers and encloses both newly synthesized transmitter and a sample of extracellular medium.     

Slow components of axonal transport: two cytoskeletal networks

We have identified two slowly moving groups of axonally transported proteins in guinea pig retinal ganglion cell axons (4). The slowest group of proteins, designated slow component a (SCa), has a transport rate of 0.25 mm/d and consists of tubulin and neurofilament protein. The other slowly transported group of proteins, designated slow components b (SCb), has a transport rate of 2-3 mm/d and consists of many polypeptides, one of which is actin (4). Our analyses of the transport kinetics of the individual polypeptides of SCa and SCb indicate that (a) the polypeptides of SCa are transported coherently in the optic axons, (b) the polypeptides of SCb are also transported coherently but completely separately from the SCa polypeptides, and (c) the polypeptides of SCa differ completely from those comprising SCb. We relate these results to our general hypothesis that slow axonal transport represents the movements of structural complexes of proteins. Furthermore, it is proposed that SCa corresponds to the microtubule-neurofilament network, and that SCb represents the transport of the microfilament network together with the proteins complexed with microfilaments.     

Application of the fluctuation theorem to motor proteins: from F<Subscript>1</Subscript>-ATPase to axonal cargo transport by kinesin and dynein

The fluctuation theorem is a representative theorem in non-equilibrium statistical physics actively studied in the 1990s. Relating to entropy production in non-equilibrium states, the theorem has been used to estimate the driving power of motor proteins from fluctuation in their motion. In this review, usage of the fluctuation theorem in experiments on motor proteins is illustrated for biologists, especially those who study mechanobiology, in which force measurement is a central issue. We first introduce the application of the fluctuation theorem in measuring the rotary torque of the rotary motor protein F₁-ATPase. Next, as an extension of this application, a recent trial estimating the force generated during cargo transport in vivo by the microtubule motors kinesin and dynein is introduced. Elucidation of the physical mechanism of such transport is important, especially for neurons, in which deficits in cargo transport are deeply related to neuronal diseases. Finally, perspectives on the fluctuation theorem as a new technique in the field of neuroscience are discussed.     

Unc-51/ATG1 controls axonal and dendritic development via kinesin-mediated vesicle transport in the Drosophila brain

Background

Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.

Methodology/Principal Findings

In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II), an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM) and No distributive disjunction (Nod), remains unaltered. Genetic analyses of kinesin light chain (Klc) and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.

Conclusions/Significance

Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of developing neurons.     

Hyperglycemia induces oxidative stress and impairs axonal transport rates in mice

Background

While hyperglycemia-induced oxidative stress damages peripheral neurons, technical limitations have, in part, prevented in vivo studies to determine the effect of hyperglycemia on the neurons in the central nervous system (CNS). While olfactory dysfunction is indicated in diabetes, the effect of hyperglycemia on olfactory receptor neurons (ORNs) remains unknown. In this study, we utilized manganese enhanced MRI (MEMRI) to assess the impact of hyperglycemia on axonal transport rates in ORNs. We hypothesize that (i) hyperglycemia induces oxidative stress and is associated with reduced axonal transport rates in the ORNs and (ii) hyperglycemia-induced oxidative stress activates the p38 MAPK pathway in association with phosphorylation of tau protein leading to the axonal transport deficits.

Research Design and Methods

T₁-weighted MEMRI imaging was used to determine axonal transport rates post-streptozotocin injection in wildtype (WT) and superoxide dismutase 2 (SOD2) overexpressing C57Bl/6 mice. SOD2 overexpression reduces mitochondrial superoxide load. Dihydroethidium staining was used to quantify the reactive oxygen species (ROS), specifically, superoxide (SO). Protein and gene expression levels were determined using western blotting and Q-PCR analysis, respectively.

Results

STZ-treated WT mice exhibited significantly reduced axonal transport rates and significantly higher levels of ROS, phosphorylated p38 MAPK and tau protein as compared to the WT vehicle treated controls and STZ-treated SOD2 mice. The gene expression levels of p38 MAPK and tau remained unchanged.

Conclusion

Increased oxidative stress in STZ-treated WT hyperglycemic mice activates the p38 MAPK pathway in association with phosphorylation of tau and attenuates axonal transport rates in the olfactory system. In STZ-treated SOD-overexpressing hyperglycemic mice in which superoxide levels are reduced, these deficits are reversed.     

The motor protein kinesin-1 links neurofibromin and merlin in a common cellular pathway of neurofibromatosis

Mutations in either of the two tumor suppressor genes NF1 (neurofibromin) and NF2 (merlin) result in Neurofibromatosis, a condition predisposing individuals to developing a variety of benign and malignant tumors of the central and peripheral nervous systems. Here we report the identification of two distinct NF1-containing complexes, one in the soluble and the other in the particulate fraction of HeLa extract. We show that the soluble NF1 complex delineates a large holo-NF1 complex (2 MDa) encompassing the components of a smaller particulate core-NF1 complex (400 kDa). Purification of the core-NF1 complex followed by mass spectrometric analysis revealed the motor protein, kinesin-1 heavy chain (HsuKHC/KIF5B), as a catalytic subunit of both NF-1-containing complexes. Importantly, although NF1 and NF2 are not in a stable association, NF2 is also a component of a distinct kinesin-1-containing complex. These results point to kinesin-1 as a common denominator between NF1 and NF2.