Axonal transport of membranous and nonmembranous cargoes: a unified perspective

Membranous and nonmembranous cargoes are transported along axons in the fast and slow components of axonal transport, respectively. Recent observations on the movement of cytoskeletal polymers in axons suggest that slow axonal transport is generated by fast motors and that the slow rate is due to rapid movements interrupted by prolonged pauses. This supports a unified perspective for fast and slow axonal transport based on rapid movements of diverse cargo structures that differ in the proportion of the time that they spend moving. A Flash feature (http://www.jcb.org/cgi/content/full/jcb.200212017/DC1) accompanies this Mini-Review.     

Regulation of dynein‐dynactin‐driven vesicular transport

Most of the long‐range intracellular movements of vesicles, organelles and other cargoes are driven by microtubule (MT)‐based molecular motors. Cytoplasmic dynein, a multisubunit protein complex, with the aid of dynactin, drives transport of a wide variety of cargoes towards the minus end of MTs. In this article, I review our current understanding of the mechanisms underlying spatiotemporal regulation of dynein‐dynactin‐driven vesicular transport with a special emphasis on the many steps of directional movement along MT tracks. These include the recruitment of dynein to MT plus ends, the activation and processivity of dynein, and cargo recognition and release by the motor complex at the target membrane. Furthermore, I summarize the most recent findings about the fine control mechanisms for intracellular transport via the interaction between the dynein‐dynactin motor complex and its vesicular cargoes.      

Endogenous GSK‐3/Shaggy Regulates Bidirectional Axonal Transport of the Amyloid Precursor Protein

Neurons rely on microtubule (MT) motor proteins such as kinesin‐1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK‐3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin‐1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, we find that endogenous GSK‐3 is a required negative regulator of both kinesin‐1‐mediated and dynein‐mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK‐3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, we find that GSK‐3 regulates transport by altering the activity of kinesin‐1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK‐3 and APP, and demonstrate that endogenous GSK‐3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK‐3 controls the number of active motors that are moving a cargo .     

Ethanol stimulates the in vivo axonal movement of neuropeptide dense‐core vesicles in Drosophila motor neurons

Proper neuronal function requires essential biological cargoes to be packaged within membranous vesicles and transported, intracellularly, through the extensive outgrowth of axonal and dendritic fibers. The precise spatiotemporal movement of these cargoes is vital for neuronal survival and, thus, is highly regulated. In this study we test how the axonal movement of a neuropeptide‐containing dense‐core vesicle (DCV ) responds to alcohol stressors. We found that ethanol induces a strong anterograde bias in vesicle movement. Low doses of ethanol stimulate the anterograde movement of neuropeptide‐DCV while high doses inhibit bi‐directional movement. This process required the presence of functional kinesin‐1 motors as reduction in kinesin prevented the ethanol‐induced stimulation of the anterograde movement of neuropeptide‐DCV . Furthermore, expression of inactive glycogen synthase kinase 3 (GSK ‐3β) also prevented ethanol‐induced stimulation of neuropeptide‐DCV movement, similar to pharmacological inhibition of GSK ‐3β with lithium. Conversely, inhibition of PI 3K/AKT signaling with wortmannin led to a partial prevention of ethanol‐stimulated transport of neuropeptide‐DCV . Taken together, we conclude that GSK ‐3β signaling mediates the stimulatory effects of ethanol. Therefore, our study provides new insight into the physiological response of the axonal movement of neuropeptide‐DCV to exogenous stressors.

Cover Image for this Issue: doi: 10.1111/jnc.14165 .

     

Primary lysosomal dysfunction causes cargo-specific deficits of axonal transport leading to Alzheimer-like neuritic dystrophy

Abnormally swollen regions of axons and dendrites (neurites) filled mainly with autophagy-related organelles represent the highly characteristic and widespread form of "neuritic dystrophy" in Alzheimer disease (AD), which implies dysfunction of autophagy and axonal transport. In this punctum, we discuss our recent findings that autophagic/lysosomal degradation is critical to proper axonal transport of autophagic vacuoles (AVs) and lysosomes. We showed that lysosomal protease inhibition induces defective axonal transport of specific cargoes, causing these cargoes to accumulate in axonal swellings that biochemically and morphologically resemble the dystrophic neurites in AD. Our findings suggest that a cargo-specific failure of axonal transport promotes neuritic dystrophy in AD, which involves a mechanism distinct from the global axonal transport deficits seen in some other neurodegenerative diseases.     

Cargo crowding at actin‐rich regions along axons causes local traffic jams

Steady axonal cargo flow is central to the functioning of healthy neurons. However, a substantial fraction of cargo in axons remains stationary up to several minutes. We examine the transport of precursors of synaptic vesicles (pre‐SVs), endosomes and mitochondria in Caenorhabditis elegans touch receptor neurons, showing that stationary cargo are predominantly present at actin‐rich regions along the neuronal process. Stationary vesicles at actin‐rich regions increase the propensity of moving vesicles to stall at the same location, resulting in traffic jams arising from physical crowding. Such local traffic jams at actin‐rich regions are likely to be a general feature of axonal transport since they also occur in Drosophila neurons. Repeated touch stimulation of C. elegans reduces the density of stationary pre‐SVs, indicating that these traffic jams can act as both sources and sinks of vesicles. This suggests that vesicles trapped in actin‐rich regions are functional reservoirs that may contribute to maintaining robust cargo flow in the neuron. A video abstract of this article can be found at: Video S1 ; Video S2      

Matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase co‐regulate axonal outgrowth of mouse retinal ganglion cells

Restoration of correct neural activity following central nervous system (CNS) damage requires the replacement of degenerated axons with newly outgrowing, functional axons. Unfortunately, spontaneous regeneration is largely lacking in the adult mammalian CNS. In order to establish successful regenerative therapies, an improved understanding of axonal outgrowth and the various molecules influencing it, is highly needed. Matrix metalloproteinases (MMPs) constitute a family of zinc‐dependent proteases that were sporadically reported to influence axon outgrowth. Using an ex vivo retinal explant model, we were able to show that broad‐spectrum MMP inhibition reduces axon outgrowth of mouse retinal ganglion cells (RGCs), implicating MMPs as beneficial factors in axonal regeneration. Additional studies, using more specific MMP inhibitors and MMP‐deficient mice, disclosed that both MMP‐2 and MT1‐MMP, but not MMP‐9, are involved in this process. Furthermore, administration of a novel antibody to MT1‐MMP that selectively blocks pro‐MMP‐2 activation revealed a functional co‐involvement of these proteinases in determining RGC axon outgrowth. Subsequent immunostainings showed expression of both MMP‐2 and MT1‐MMP in RGC axons and glial cells. Finally, results from combined inhibition of MMP‐2 and β1‐integrin were suggestive for a functional interaction between these molecules. Overall, our data indicate MMP‐2 and MT1‐MMP as promising axonal outgrowth‐promoting molecules.

     

The Translocation Selectivity of the Kinesins that Mediate Neuronal Organelle Transport

Polarized kinesin‐driven transport is crucial for development and maintenance of neuronal polarity. Kinesins are thought to recognize biochemical differences between axonal and dendritic microtubules in order to deliver their cargoes to the appropriate domain. To identify kinesins that mediate polarized transport, we prepared constitutively active versions of all the kinesins implicated in vesicle transport and expressed them in cultured hippocampal neurons. Seven kinesins translocated preferentially to axons and five translocated into both axons and dendrites. None translocated selectively to dendrites. Highly homologous members of the same subfamily displayed distinctly different translocation preferences and were differentially regulated during development. By expressing chimeric kinesins, we identified two microtubule‐binding elements within the motor domain that are important for selective translocation. We also discovered elements in the dimerization domain of kinesin‐2 motors that contribute to their selective translocation. These observations indicate that selective interactions between kinesin motor domains and microtubules can account for polarized transport to the axon, but not for selective dendritic transport.     

Primary lysosomal dysfunction causes cargo-specific deficits of axonal transport leading to Alzheimer-like neuritic dystrophy

Abnormally swollen regions of axons and dendrites (neurites) filled mainly with autophagy-related organelles represent the highly characteristic and widespread form of “neuritic dystrophy” in Alzheimer disease (AD), which implies dysfunction of autophagy and axonal transport. In this punctum, we discuss our recent findings that autophagic/lysosomal degradation is critical to proper axonal transport of autophagic vacuoles (AVs) and lysosomes. We showed that lysosomal protease inhibition induces defective axonal transport of specific cargoes, causing these cargoes to accumulate in axonal swellings that biochemically and morphologically resemble the dystrophic neurites in AD. Our findings suggest that a cargo-specific failure of axonal transport promotes neuritic dystrophy in AD, which involves a mechanism distinct from the global axonal transport deficits seen in some other neurodegenerative diseases.     

Biological Significance of the Importin‐β Family‐Dependent Nucleocytoplasmic Transport Pathways

Importin‐β family proteins (Imp‐βs) are nucleocytoplasmic transport receptors (NTRs) that import and export proteins and RNAs through the nuclear pores. The family consists of 14–20 members depending on the biological species, and each member transports a specific group of cargoes. Thus, the Imp‐βs mediate multiple, parallel transport pathways that can be regulated separately. In fact, the spatiotemporally differential expressions and the functional regulations of Imp‐βs have been reported. Additionally, the biological significance of each pathway has been characterized by linking the function of a member of Imp‐βs to a cellular consequence. Connecting these concepts, the regulation of the transport pathways conceivably induces alterations in the cellular physiological states. However, few studies have linked the regulation of an importin‐β family NTR to an induced cellular response and the corresponding cargoes, despite the significance of this linkage in comprehending the biological relevance of the transport pathways. This review of recent reports on the regulation and biological functions of the Imp‐βs highlights the significance of the transport pathways in physiological contexts and points out the possibility that the identification of yet unknown specific cargoes will reinforce the importance of transport regulation.      

Global and local mechanisms sustain axonal proteostasis of transmembrane proteins

The control of neuronal protein homeostasis or proteostasis is tightly regulated both spatially and temporally, assuring accurate and integrated responses to external or intrinsic stimuli. Local or autonomous responses in dendritic and axonal compartments are crucial to sustain function during development, physiology and in response to damage or disease. Axons are responsible for generating and propagating electrical impulses in neurons, and the establishment and maintenance of their molecular composition are subject to extreme constraints exerted by length and size. Proteins that require the secretory pathway, such as receptors, transporters, ion channels or cell adhesion molecules, are fundamental for axonal function, but whether axons regulate their abundance autonomously and how they achieve this is not clear. Evidence supports the role of three complementary mechanisms to maintain proteostasis of these axonal proteins, namely vesicular transport, local translation and trafficking and transfer from supporting cells. Here, we review these mechanisms, their molecular machineries and contribution to neuronal function. We also examine the signaling pathways involved in local translation and their role during development and nerve injury. We discuss the relative contributions of a transport‐controlled proteome directed by the soma (global regulation) versus a local‐controlled proteome based on local translation or cell transfer (local regulation).      

KIF1A mediates axonal transport of BACE1 and identification of independently moving cargoes in living SCG neurons

Neurons rely heavily on axonal transport to deliver materials from the sites of synthesis to the axon terminals over distances that can be many centimetres long. KIF1A is the neuron‐specific kinesin with the fastest reported anterograde motor activity. Previous studies have shown that KIF1A transports a subset of synaptic proteins, neurofilaments and dense‐core vesicles. Using two‐colour live imaging, we showed that beta‐secretase 1 (BACE1)‐mCherry moves together with KIF1A‐GFP in both the anterograde and retrograde directions in superior cervical ganglions (SCG) neurons. We confirmed that KIF1A is functionally required for BACE1 transport by using KIF1A siRNA and a KIF1A mutant construct (KIF1A‐T312M) to impair its motor activity. We further identified several cargoes that have little or no co‐migration with KIF1A‐GFP and also move independently from BACE1‐mCherry. Together, these findings support a primary role for KIF1A in the anterograde transport of BACE1 and suggest that axonally transported cargoes are sorted into different classes of carrier vesicles in the cell body and are transported by cargo‐specific motor proteins through the axon.      

A Stochastic Multiscale Model That Explains the Segregation of Axonal Microtubules and Neurofilaments in Neurological Diseases

The organization of the axonal cytoskeleton is a key determinant of the normal function of an axon, which is a long thin projection of a neuron. Under normal conditions two axonal cytoskeletal polymers, microtubules and neurofilaments, align longitudinally in axons and are interspersed in axonal cross-sections. However, in many neurotoxic and neurodegenerative disorders, microtubules and neurofilaments segregate apart from each other, with microtubules and membranous organelles clustered centrally and neurofilaments displaced to the periphery. This striking segregation precedes the abnormal and excessive neurofilament accumulation in these diseases, which in turn leads to focal axonal swellings. While neurofilament accumulation suggests an impairment of neurofilament transport along axons, the underlying mechanism of their segregation from microtubules remains poorly understood for over 30 years. To address this question, we developed a stochastic multiscale model for the cross-sectional distribution of microtubules and neurofilaments in axons. The model describes microtubules, neurofilaments and organelles as interacting particles in a 2D cross-section, and is built upon molecular processes that occur on a time scale of seconds or shorter. It incorporates the longitudinal transport of neurofilaments and organelles through this domain by allowing stochastic arrival and departure of these cargoes, and integrates the dynamic interactions of these cargoes with microtubules mediated by molecular motors. Simulations of the model demonstrate that organelles can pull nearby microtubules together, and in the absence of neurofilament transport, this mechanism gradually segregates microtubules from neurofilaments on a time scale of hours, similar to that observed in toxic neuropathies. This suggests that the microtubule-neurofilament segregation can be a consequence of the selective impairment of neurofilament transport. The model generates the experimentally testable prediction that the rate and extent of segregation will be dependent on the sizes of the moving organelles as well as the density of their traffic.     

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (“map”) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. “Cargo mapping” consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to “map” them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.     

Ionizing radiation causes increased tau phosphorylation in primary neurons

Radiotherapy is the major treatment modality for primary and metastatic brain tumors which involves the exposure of brain to ionizing radiation. Ionizing radiation can induce various detrimental pathophysiological effects in the adult brain, and Alzheimer's disease and related neurodegenerative disorders are considered to be late effects of radiation. In this study, we investigated whether ionizing radiation causes changes in tau phosphorylation in cultured primary neurons similar to that in Alzheimer's disease. We demonstrated that exposure to 0.5 or 2 Gy γ rays causes increased phosphorylation of tau protein at several phosphorylation sites in a time‐ and dose‐dependent manner. Consistently, we also found ionizing radiation causes increased activation of GSK3β, c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase before radiation‐induced increase in tau phosphorylation. Specific inhibitors of these kinases almost fully blocked radiation‐induced tau phosphorylation. Our studies further revealed that oxidative stress plays an important role in ionizing radiation‐induced tau phosphorylation, likely through the activation of c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase, but not GSK3β. Overall, our studies suggest that ionizing radiation may cause increased risk for development of Alzheimer's disease by promoting abnormal tau phosphorylation.

     

Tau directs intracellular trafficking by regulating the forces exerted by kinesin and dynein teams

Organelles, proteins, and mRNA are transported bidirectionally along microtubules by plus‐end directed kinesin and minus‐end directed dynein motors. Microtubules are decorated by microtubule‐associated proteins (MAPs) that organize the cytoskeleton, regulate microtubule dynamics and modulate the interaction between motor proteins and microtubules to direct intracellular transport. Tau is a neuronal MAP that stabilizes axonal microtubules and crosslinks them into bundles. Dysregulation of tau leads to a range of neurodegenerative diseases known as tauopathies including Alzheimer's disease (AD). Tau reduces the processivity of kinesin and dynein by acting as an obstacle on the microtubule. Single‐molecule assays indicate that kinesin‐1 is more strongly inhibited than kinesin‐2 or dynein, suggesting tau might act to spatially modulate the activity of specific motors. To investigate the role of tau in regulating bidirectional transport, we isolated phagosomes driven by kinesin‐1, kinesin‐2, and dynein and reconstituted their motility along microtubules. We find that tau biases bidirectional motility towards the microtubule minus‐end in a dose‐dependent manner. Optical trapping measurements show that tau increases the magnitude and frequency of forces exerted by dynein through inhibiting opposing kinesin motors. Mathematical modeling indicates that tau controls the directional bias of intracellular cargoes through differentially tuning the processivity of kinesin‐1, kinesin‐2, and dynein. Taken together, these results demonstrate that tau modulates motility in a motor‐specific manner to direct intracellular transport, and suggests that dysregulation of tau might contribute to neurodegeneration by disrupting the balance of plus‐ and minus‐end directed transport.      

Engineered Tug‐of‐War Between Kinesin and Dynein Controls Direction of Microtubule Based Transport In Vivo

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus‐end directed kinesins and minus‐end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT‐based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug‐of‐war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug‐of‐war between opposing MT motors alone, by attaching a large number of kinesin‐1 motors to organelles transported by dynein to minus‐ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus‐end‐directed dynein‐dependent MT runs, leading to a reversal of the overall direction of dynein‐driven organelles in vivo. Therefore, in the absence of external regulators tug‐of‐war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo.      

Cargo-carrying motor vehicles on the neuronal highway: transport pathways and neurodegenerative disease

Within axons vital cargoes must be transported over great distances along microtubule tracks to maintain neuronal viability. Essential to this system are the molecular motors, kinesin and dynein, which transport a variety of neuronal cargoes. Elucidating the transport pathways, the identity of the cargoes transported, and the regulation of motor-cargo complexes are areas of intense investigation. Evidence suggests that essential components, including signaling proteins, neuroprotective and repair molecules, and vesicular and cytoskeletal components are all transported. In addition newly emerging data indicate that defects in axonal transport pathways may contribute to the initiation or progression of chronic neuronal dysfunction. In this review we concentrate on microtubule-based motor proteins, their linkers, and cargoes and discuss how factors in the axonal transport pathway contribute to disease states. As additional cargo complexes and transport pathways are identified, an understanding of the role these pathways play in the development of human disease will hopefully lead to new diagnostic and treatment strategies.     

Molecular motors and mechanisms of directional transport in neurons

Intracellular transport is fundamental for neuronal morphogenesis, function and survival. Many proteins are selectively transported to either axons or dendrites. In addition, some specific mRNAs are transported to dendrites for local translation. Proteins of the kinesin superfamily participate in selective transport by using adaptor or scaffolding proteins to recognize and bind cargoes. The molecular components of RNA-transporting granules have been identified, and it is becoming clear how cargoes are directed to axons and dendrites by kinesin superfamily proteins. Here we discuss the molecular mechanisms of directional axonal and dendritic transport with specific emphasis on the role of motor proteins and their mechanisms of cargo recognition.