A novel strategy to visualize vesicle-bound kinesins reveals the diversity of kinesin-mediated transport.

In mammals, 15 to 20 kinesins are thought to mediate vesicle transport. Little is known about the identity of vesicles moved by each kinesin or the functional significance of such diversity. To characterize the transport mediated by different kinesins, we developed a novel strategy to visualize vesicle-bound kinesins in living cells. We applied this method to cultured neurons and systematically determined the localization and transport parameters of vesicles labeled by different members of the Kinesin-1, -2, and -3 families. We observed vesicle labeling with nearly all kinesins. Only six kinesins bound vesicles that undergo long-range transport in neurons. Of these, three had an axonal bias (KIF5B, KIF5C and KIF13B), two were unbiased (KIF1A and KIF1Bß), and one transported only in dendrites (KIF13A). Overall, the trafficking of vesicle-bound kinesins to axons or dendrites did not correspond to their motor domain preference, suggesting that on-vesicle regulation is crucial for kinesin targeting. Surprisingly, several kinesins were associated with populations of somatodendritic vesicles that underwent little long-range transport. This assay should be broadly applicable for investigating kinesin function in many cell types.

The cellular mechanisms that maintain neuronal polarity.

As polarized cells, neurons maintain different sets of resident plasma membrane proteins in their axons and dendrites, which is consistent with the different roles that these neurites have in electrochemical signalling. Axonal and dendritic proteins are synthesized together within the somatodendritic domain; this raises a fundamental question: what is the nature of the intracellular trafficking machinery that ensures that these proteins reach the correct domain? Recent studies have advanced our understanding of the processes underlying the selective sorting and selective transport of axonal and dendritic proteins and have created potential avenues for future progress.

The Development of Neuronal Polarity: A Retrospective View.

In 1988, Carlos Dotti, Chris Sullivan, and I published a paper on the establishment of polarity by hippocampal neurons in culture, which continues to be frequently cited 30 years later (Dotti et al., 1988). By following individual neurons from the time of plating until they had formed well developed axonal and dendritic arbors, we identified the five stages of development that lead to the mature expression of neuronal polarity. We were surprised to find that, before axon formation, the cells pass through a multipolar phase, in which several, apparently identical short neurites undergo periods of extension and retraction. Then one of these neurites begins a period of prolonged growth, becoming the definitive axon; the remaining neurites subsequently become dendrites. This observation suggested that any of the initial neurites were capable of becoming axons, a hypothesis confirmed by later work. In this Progressions article, I will try to recall the circumstances that led to this work, recapture some of the challenges we faced in conducting these experiments, and consider why some of today's neuroscientists still find this paper relevant.

Selective microtubule-based transport of dendritic membrane proteins arises in concert with axon specification.

The polarized distribution of membrane proteins to axonal or somatodendritic neuronal compartments is fundamental to nearly every aspect of neuronal function. The polarity of dendritic proteins depends on selective microtubule-based transport; the vesicles that carry these proteins are transported into dendrites but do not enter the axon. We used live-cell imaging of fluorescently tagged dendritic and axonal proteins combined with immunostaining for initial segment and cytoskeletal markers to evaluate different models of dendrite-selective transport in cultured rat hippocampal neurons. In mature neurons, dendritic vesicles that entered the base of the axon stopped at the proximal edge of the axon initial segment, defined by immunostaining for ankyrinG, rather than moving into the initial segment itself. In contrast, axonal vesicles passed through the initial segment without impediment. During development, dendrite-selective transport was detected shortly after axons formed, several days before initial segment assembly, before the appearance of a dense actin meshwork in the initial segment, and before dendrites acquire microtubules of mixed polarity orientation. Indeed, some elements of selective transport were detected even before axon specification. These findings are inconsistent with models for selective transport that depend on the presence of an F-actin-based cytoplasmic filter in the initial segment or that posit that transport into dendrites is mediated by dyneins translocating along minus-end out microtubules. Instead our results suggest that selective transport involves the coordinated regulation of the different motor proteins that mediate dendritic vesicle transport and that the selectivity of motor-microtubule interactions is one facet of this process.

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.

Axonal transport plays a crucial role in mediating the axon-protective effects of NmNAT.

Deficits in axonal transport are thought to contribute to the pathology of many neurodegenerative diseases. Expressing the slow Wallerian degeneration protein (Wld(S)) or related nicotinamide mononucleotide adenyltransferases (NmNATs) protects axons against damage from a broad range of insults, but the ability of these proteins to protect against inhibition of axonal transport has received little attention. We set out to determine whether these proteins can protect the axons of cultured hippocampal neurons from damage due to hydrogen peroxide or oxygen-glucose deprivation (OGD) and, in particular, whether they can reduce the damage that these agents cause to the axonal transport machinery. Exposure to these insults inhibited the axonal transport of both mitochondria and of the vesicles that carry axonal membrane proteins; this inhibition occurred hours before the first signs of axonal degeneration. Expressing a cytoplasmically targeted version of NmNAT1 (cytNmNAT1) protected the axons against both insults. It also reduced the inhibition of transport when cells were exposed to hydrogen peroxide and enhanced the recovery of transport following both insults. The protective effects of cytNmNAT1 depend on mitochondrial transport. When mitochondrial transport was inhibited, cytNmNAT1 was unable to protect axons against either insult. The protective effects of mitochondrially targeted NmNAT also were blocked by inhibiting mitochondrial transport. These results establish that NmNAT robustly protects the axonal transport system following exposure to OGD and reactive oxygen species and may offer similar protection in other disease models. Understanding how NmNAT protects the axonal transport system may lead to new strategies for neuroprotection in neurodegenerative diseases.

Kinesin Regulation in the Proximal Axon is Essential for Dendrite-selective Transport.

Neurons are polarized and typically extend multiple dendrites and one axon. To maintain polarity, vesicles carrying dendritic proteins are arrested upon entering the axon. To determine whether kinesin regulation is required for terminating anterograde axonal transport, we overexpressed the dendrite-selective kinesin KIF13A. This caused mistargeting of dendrite-selective vesicles to the axon and a loss of dendritic polarity. Polarity was not disrupted if the kinase MARK2/Par1b was coexpressed. MARK2/Par1b is concentrated in the proximal axon, where it maintains dendritic polarity-likely by phosphorylating S1371 of KIF13A, which lies in a canonical 14-3-3 binding motif. We probed for interactions of KIF13A with 14-3-3 isoforms and found that 14-3-3ß and 14-3-3ζ bound KIF13A. Disruption of MARK2 or 14-3-3 activity by small molecule inhibitors caused a loss of dendritic polarity. These data show that kinesin regulation is integral for dendrite-selective transport. We propose a new model in which KIF13A that moves dendrite-selective vesicles in the proximal axon is phosphorylated by MARK2. Phosphorylated KIF13A is then recognized by 14-3-3, which causes dissociation of KIF13A from the vesicle and termination of transport. These findings define a new paradigm for the regulation of vesicle transport by localized kinesin tail phosphorylation, to restrict dendrite-selective vesicles from entering the axon.

Culture of primary rat hippocampal neurons: design, analysis, and optimization of a microfluidic device for cell seeding, coherent growth, and solute delivery.

We present the design, analysis, construction, and culture results of a microfluidic device for the segregation and chemical stimulation of primary rat hippocampal neurons. Our device is designed to achieve spatio-temporal solute delivery to discrete sections of neurons with mitigated mechanical stress. We implement a geometric guidance technique to direct axonal processes of the neurons into specific areas of the device to achieve solute segregation along routed cells. Using physicochemical modeling, we predict flows, concentration profiles, and mechanical stresses within pertiment sections of the device. We demonstrate cell viability and growth within the closed device over a period of 11 days. Additionally, our modeling methodology may be generalized and applied to other device geometries.

Differential neurite outgrowth is required for axon specification by cultured hippocampal neurons.

Formation of an axon is the first morphological evidence of neuronal polarization, visible as a profound outgrowth of the axon compared with sibling neurites. One unsolved question on the mechanism of axon formation is the role of axon outgrowth in axon specification. This question was difficult to assess, because neurons freely extend their neurites in a conventional culture. Here, we leveraged surface nano/micro-modification techniques to fabricate a template substrate for constraining neurite lengths of cultured neurons. Using the template, we asked (i) Do neurons polarize even if all neurites cannot grow sufficiently long? (ii) Would the neurite be fated to become an axon if only one was allowed to grow long? A pattern with symmetrical short paths (20 µm) was used to address the former question, and an asymmetrical pattern with one path extended to 100 µm for the latter. Axon formation was evaluated by tau-1/MAP2 immunostaining and live-cell imaging of constitutively-active kinesin-1. We found that (1) neurons cannot polarize when extension of all neurites is restricted and that (2) when only a single neurite is permitted to grow long, neurons polarize and the longest neurite becomes the axon. These results provide clear evidence that axon outgrowth is required for its specification.

A change in the selective translocation of the Kinesin-1 motor domain marks the initial specification of the axon.

We used the accumulation of constitutively active kinesin motor domains as a measure of where kinesins translocate in developing neurons. Throughout development, truncated Kinesin-3 accumulates at the tips of all neurites. In contrast, Kinesin-1 selectively accumulates in only a subset of neurites. Before neurons become polarized, truncated Kinesin-1 accumulates transiently in a single neurite. Coincident with axon specification, truncated Kinesin-1 accumulates only in the emerging axon and no longer appears in any other neurite. The translocation of Kinesin-1 along a biochemically distinct track leading to the nascent axon could ensure the selective delivery of Kinesin-1 cargoes to the axon and hence contribute to its molecular specification. Imaging YFP-tagged truncated Kinesin-1 provides the most precise definition to date of when neuronal polarity first emerges and allows visualization of the molecular differentiation of the axon in real time.

Transient receptor potential canonical 5 channels activate Ca2+/calmodulin kinase Igamma to promote axon formation in hippocampal neurons.

Functionality of neurons is dependent on their compartmentalized polarization of dendrites and an axon. The rapid and selective outgrowth of one neurite, relative to the others, to form the axon is critical in initiating neuronal polarity. Axonogenesis is regulated in part by an optimal intracellular calcium concentration. Our investigation of Ca(2+)-signaling pathways involved in axon formation using cultured hippocampal neurons demonstrates a role for Ca(2+)/calmodulin kinase kinase (CaMKK) and its downstream target Ca(2+)/calmodulin kinase I (CaMKI). Expression of constitutively active CaMKI induced formation of multiple axons, whereas blocking CaMKK or CaMKI activity with pharmacological, dominant-negative, or short hairpin RNA (shRNA) methods significantly inhibited axon formation. CaMKK signals via the gamma-isoform of CaMKI as shRNA to CaMKIgamma, but not the other CaMKI isoforms, inhibited axon formation. Furthermore, overexpression of wild-type CaMKIgamma, but not a mutant incapable of membrane association, accelerated the rate of axon formation. Pharmacological or small interfering RNA inhibition of transient receptor potential canonical 5 (TRPC5) channels, which are present in developing axonal growth cones, suppressed CaMKK-mediated activation of CaMKIgamma as well as axon formation. We demonstrate using biochemical fractionation and immunocytochemistry that CaMKIgamma and TRPC5 colocalize to lipid rafts. These results are consistent with a model in which highly localized calcium influx through the TRPC5 channels activates CaMKK and CaMKIgamma, which subsequently promote axon formation.

Axonal degeneration in multiple sclerosis: the mitochondrial hypothesis.

Multiple sclerosis (MS) is a chronic disease of the central nervous system, affecting more than 2 million people worldwide. Traditionally considered an inflammatory demyelinating disease, recent evidence now points to axonal degeneration as crucial to the development of irreversible disability. Studies show that axonal degeneration occurs throughout the entire course of MS. Although the specific mechanisms causing axonal damage may differ at various stages, mitochondrial failure seems to be a common underlying theme. This review addresses the mitochondrial hypothesis for axonal degeneration in MS, highlighting the mechanisms by which mitochondrial dysfunction leads to axonal disruption in acute inflammatory lesions and the chronic axonopathy in progressive MS. Emphasis is placed on Ca(2+), free radical production, and permeability transition pore opening as key players in mitochondrial failure, axonal transport impairment, and subsequent axonal degeneration. In addition, the role of mitochondria as therapeutic targets for neuroprotection in MS is addressed.

Automatic summarization of changes in biological image sequences using algorithmic information theory.

An algorithmic information-theoretic method is presented for object-level summarization of meaningful changes in image sequences. Object extraction and tracking data are represented as an attributed tracking graph (ATG). Time courses of object states are compared using an adaptive information distance measure, aided by a closed-form multidimensional quantization. The notion of meaningful summarization is captured by using the gap statistic to estimate the randomness deficiency from algorithmic statistics. The summary is the clustering result and feature subset that maximize the gap statistic. This approach was validated on four bioimaging applications: 1) It was applied to a synthetic data set containing two populations of cells differing in the rate of growth, for which it correctly identified the two populations and the single feature out of 23 that separated them; 2) it was applied to 59 movies of three types of neuroprosthetic devices being inserted in the brain tissue at three speeds each, for which it correctly identified insertion speed as the primary factor affecting tissue strain; 3) when applied to movies of cultured neural progenitor cells, it correctly distinguished neurons from progenitors without requiring the use of a fixative stain; and 4) when analyzing intracellular molecular transport in cultured neurons undergoing axon specification, it automatically confirmed the role of kinesins in axon specification.

Two distinct mechanisms target membrane proteins to the axonal surface.

We have investigated the trafficking of two endogenous axonal membrane proteins, VAMP2 and NgCAM, in order to elucidate the cellular events that underlie their polarization. We found that VAMP2 is delivered to the surface of both axons and dendrites, but preferentially endocytosed from the dendritic membrane. A mutation in the cytoplasmic domain of VAMP2 that inhibits endocytosis abolished its axonal polarization. In contrast, the targeting of NgCAM depends on sequences in its ectodomain, which mediate its sorting into carriers that preferentially deliver their cargo proteins to the axonal membrane. These observations show that neurons use two distinct mechanisms to polarize proteins to the axonal domain: selective retention in the case of VAMP2, selective delivery in the case of NgCAM.

A WAVE-1 and WRP signaling complex regulates spine density, synaptic plasticity, and memory.

The scaffolding protein WAVE-1 (Wiskott-Aldrich syndrome protein family member 1) directs signals from the GTPase Rac through the Arp2/3 complex to facilitate neuronal actin remodeling. The WAVE-associated GTPase activating protein called WRP is implicated in human mental retardation, and WAVE-1 knock-out mice have altered behavior. Neuronal time-lapse imaging, behavioral analyses, and electrophysiological recordings from genetically modified mice were used to show that WAVE-1 signaling complexes control aspects of neuronal morphogenesis and synaptic plasticity. Gene targeting experiments in mice demonstrate that WRP anchoring to WAVE-1 is a homeostatic mechanism that contributes to neuronal development and the fidelity of synaptic connectivity. This implies that signaling through WAVE-1 complexes is essential for neural plasticity and cognitive behavior.

Putting on the RITz.

Neurons develop two types of processes, axons and dendrites, whose growth must be independently controlled. Recent research has identified the small guanosine triphosphatase Rit as a differential regulator of neurite growth. Activation of Rit enhances axonal growth, whereas inhibition of Rit promotes dendritic growth. These results imply that the reciprocal regulation of a single molecule in the same cell can achieve simultaneous regulation of axonal and dendritic growth.

The role of protein interaction motifs in regulating the polarity and clustering of the metabotropic glutamate receptor mGluR1a.

When expressed in cultured hippocampal neurons, the metabotropic glutamate receptor mGluR1a is polarized to dendrites and concentrated at postsynaptic sites. We used a mutational analysis to determine how previously identified protein interaction motifs in the C terminus of mGluR1a contribute to its localization. Our results show that the polyproline motif that mediates interaction with Homer family proteins is critical for the synaptic clustering of mGluR1a. A single point mutation in this motif, which prevents the binding of Homer with mGluR1a, reduced its colocalization with a postsynaptic marker to near-chance levels but did not affect its dendritic polarity. In contrast, deleting the PDZ (postsynaptic density-95/Discs large/zona occludens-1) binding domain, which interacts with Tamalin and Shank, had no effect on synaptic localization. Neither of these protein interaction motifs is important for trafficking to the plasma membrane or for polarization to dendrites. Although deleting the entire C terminus of mGluR1a only modestly reduced its dendritic polarity, this domain was sufficient to redirect an unpolarized reporter protein to dendrites. These observations suggest that mGluR1a contains redundant dendritic targeting signals. Together, our results indicate that the localization of mGluR1a involves two distinct steps, one that targets the protein to dendrites and a second that sequesters it at postsynaptic sites; different protein interactions motifs mediate each step.

Activated c-Jun N-terminal kinase is required for axon formation.

A critical transition in neuron development is formation of the axon, which establishes the polarized structure of the neuron that underlies its entire input and output capabilities. The morphological events that occur during axonogenesis have long been known, yet the molecular determinants underlying axonogenesis remain poorly understood. We demonstrate here that axonogenesis requires activated c-Jun N-terminal kinase (JNK). JNK is expressed throughout the neuron, but its phosphorylated, activated form is highly enriched in the axon. In young axons, activated JNK forms a proximodistal gradient of increasing intensity, beginning at about the point where the axon exceeds the lengths of the other neurites (minor processes). Treatment with SP600125, a specific inhibitor of JNK, reversibly inhibits axonogenesis but does not prevent the formation of minor processes or their differentiation into dendrites (based on their immunostaining with marker proteins). Expression of a dominant-negative construct against JNK similarly prevents axonogenesis. Investigation of JNK targets revealed that activating transcription factor-2 is phosphorylated under normal conditions in neurons, and its phosphorylation is significantly attenuated after JNK inhibition. These results demonstrate that activated JNK is required for axonogenesis but not formation of minor processes or development of dendrites.

Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila.

Local endosomal recycling at synapses is essential to maintain neurotransmission. Rab4GTPase, found on sorting endosomes, is proposed to balance the flow of vesicles among endocytic, recycling, and degradative pathways in the presynaptic compartment. Here, we report that Rab4-associated vesicles move bidirectionally in Drosophila axons but with an anterograde bias, resulting in their moderate enrichment at the synaptic region of the larval ventral ganglion. Results from FK506 binding protein (FKBP) and FKBP-Rapamycin binding domain (FRB) conjugation assays in rat embryonic fibroblasts together with genetic analyses in Drosophila indicate that an association with Kinesin-2 (mediated by the tail domain of Kinesin-2α/KIF3A/KLP64D subunit) moves Rab4-associated vesicles toward the synapse. Reduction in the anterograde traffic of Rab4 causes an expansion of the volume of the synapse-bearing region in the ventral ganglion and increases the motility of Drosophila larvae. These results suggest that Rab4-dependent vesicular traffic toward the synapse plays a vital role in maintaining synaptic balance in this neuronal network.

Automated semantic analysis of changes in image sequences of neurons in culture.

Quantitative studies of dynamic behaviors of live neurons are currently limited by the slowness, subjectivity, and tedium of manual analysis of changes in time-lapse image sequences. Challenges to automation include the complexity of the changes of interest, the presence of obfuscating and uninteresting changes due to illumination variations and other imaging artifacts, and the sheer volume of recorded data. This paper describes a highly automated approach that not only detects the interesting changes selectively, but also generates quantitative analyses at multiple levels of detail. Detailed quantitative neuronal morphometry is generated for each frame. Frame-to-frame neuronal changes are measured and labeled as growth, shrinkage, merging, or splitting, as would be done by a human expert. Finally, events unfolding over longer durations, such as apoptosis and axonal specification, are automatically inferred from the short-term changes. The proposed method is based on a Bayesian model selection criterion that leverages a set of short-term neurite change models and takes into account additional evidence provided by an illumination-insensitive change mask. An automated neuron tracing algorithm is used to identify the objects of interest in each frame. A novel curve distance measure and weighted bipartite graph matching are used to compare and associate neurites in successive frames. A separate set of multi-image change models drives the identification of longer term events. The method achieved frame-to-frame change labeling accuracies ranging from 85% to 100% when tested on 8 representative recordings performed under varied imaging and culturing conditions, and successfully detected all higher order events of interest. Two sequences were used for training the models and tuning their parameters; the learned parameter settings can be applied to hundreds of similar image sequences, provided imaging and culturing conditions are similar to the training set. The proposed approach is a substantial innovation over manual annotation and change analysis, accomplishing in minutes what it would take an expert hours to complete.