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.      

Defect in Synaptic Vesicle Precursor Transport and Neuronal Cell Death in KIF1A Motor Protein–deficient Mice

The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport. Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron– specific microtubule plus end–directed motor and has been proposed as a transporter of synaptic vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769–780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission, resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.     

Motor domain-mediated autoinhibition dictates axonal transport by the kinesin UNC-104/KIF1A

The UNC-104/KIF1A motor is crucial for axonal transport of synaptic vesicles, but how the UNC-104/KIF1A motor is activated in vivo is not fully understood. Here, we identified point mutations located in the motor domain or the inhibitory CC1 domain, which resulted in gain-of-function alleles of unc-104 that exhibit hyperactive axonal transport and abnormal accumulation of synaptic vesicles. In contrast to the cell body localization of wild type motor, the mutant motors accumulate on neuronal processes. Once on the neuronal process, the mutant motors display dynamic movement similarly to wild type motors. The gain-of-function mutation on the motor domain leads to an active dimeric conformation, releasing the inhibitory CC1 region from the motor domain. Genetically engineered mutations in the motor domain or CC1 of UNC-104, which disrupt the autoinhibitory interface, also led to the gain of function and hyperactivation of axonal transport. Thus, the CC1/motor domain-mediated autoinhibition is crucial for UNC-104/KIF1A-mediated axonal transport in vivo.     

KIF1Bbeta- and KIF1A-mediated axonal transport of presynaptic regulator Rab3 occurs in a GTP-dependent manner through DENN/MADD

Synaptic proteins are synthesized in the cell body and transported down the axon by microtubule-dependent motors. We previously reported that KIF1Bbeta and KIF1A motors are essential for transporting synaptic vesicle precursors; however the mechanisms that regulate transport, as well as cargo recognition and control of cargo loading and unloading remain largely unknown. Here, we show that DENN/MADD (Rab3-GEP) is an essential part of the regulation mechanism through direct interaction with the stalk domain of KIF1Bbeta and KIF1A. We also show that DENN/MADD binds preferentially to GTP-Rab3 and acts as a Rab3 effector. These molecular interactions are fundamental as sequential genetic perturbations revealed that KIF1Bbeta and KIF1A are essential for the transport of DENN/MADD and Rab3, whereas DENN/MADD is essential for the transport of Rab3. GTP-Rab3 was more effectively transported than GDP-Rab3, suggesting that the nucleotide state of Rab3 regulates axonal transport of Rab3-carrying vesicles through preferential interaction with DENN/MADD.     

Reconstitution of membrane transport powered by a novel dimeric kinesin motor of the Unc104/KIF1A family purified from Dictyostelium.

Motor-powered movement along microtubule tracks is important for membrane organization and trafficking. However, the molecular basis for membrane transport is poorly understood, in part because of the difficulty in reconstituting this process from purified components. Using video microscopic observation of organelle transport in vitro as an assay, we have purified two polypeptides (245 and 170 kD) from Dictyostelium extracts that independently reconstitute plus-end-directed membrane movement at in vivo velocities. Both polypeptides were found to be kinesin motors, and the 245-kD protein (DdUnc104) is a close relative of Caenorhabditis elegans Unc104 and mouse KIF1A, neuron-specific motors that deliver synaptic vesicle precursors to nerve terminals. A knockout of the DdUnc104 gene produces a pronounced defect in organelle transport in vivo and in the reconstituted assay. Interestingly, DdUnc104 functions as a dimeric motor, in contrast to other members of this kinesin subfamily, which are monomeric.     

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 KIF1Bβ), 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.     

Characterization of the movement of the kinesin motor KIF1A in living cultured neurons

KIF1A is a kinesin motor known to transport synaptic vesicle precursors in neuronal axons, but little is known about whether KIF1A mediates fast and processive axonal transport in vivo. By monitoring movements of EGFP-labeled KIF1A in living cultured hippocampal neurons, we determined the characteristics of KIF1A movements. KIF1A particles moved anterogradely along the neurites with an average velocity of 1.0 microm/s. The movements of KIF1A were highly processive, with an average duration of persistent anterograde movement of 11 s. Some KIF1A particles (17%) exhibited retrograde movements of 0.72 microm/s, although overall particle movement was in the anterograde direction. The anterograde movement of KIF1A, however, did not lead to a detectable accumulation of KIF1A in the periphery of neurons, suggesting that there are mechanisms inhibiting the peripheral accumulation of KIF1A. These results suggest that KIF1A mediates neuronal transport at a high velocity and processivity in vivo.     

The Kinesin-3, Unc-104 Regulates Dendrite Morphogenesis and Synaptic Development in Drosophila

Kinesin-based transport is important for synaptogenesis, neuroplasticity, and maintaining synaptic function. In an anatomical screen of neurodevelopmental mutants, we identified the exchange of a conserved residue (R561H) in the forkhead-associated domain of the kinesin-3 family member Unc-104/KIF1A as the genetic cause for defects in synaptic terminal- and dendrite morphogenesis. Previous structure-based analysis suggested that the corresponding residue in KIF1A might be involved in stabilizing the activated state of kinesin-3 dimers. Herein we provide the first in vivo evidence for the functional importance of R561. The R561H allele (unc-104^bris) is not embryonic lethal, which allowed us to investigate consequences of disturbed Unc-104 function on postembryonic synapse development and larval behavior. We demonstrate that Unc-104 regulates the reliable apposition of active zones and postsynaptic densities, possibly by controlling site-specific delivery of its cargo. Next, we identified a role for Unc-104 in restraining neuromuscular junction growth and coordinating dendrite branch morphogenesis, suggesting that Unc-104 is also involved in dendritic transport. Mutations in KIF1A/unc-104 have been associated with hereditary spastic paraplegia and hereditary sensory and autonomic neuropathy type 2. However, we did not observe synapse retraction or dystonic posterior paralysis. Overall, our study demonstrates the specificity of defects caused by selective impairments of distinct molecular motors and highlights the critical importance of Unc-104 for the maturation of neuronal structures during embryonic development, larval synaptic terminal outgrowth, and dendrite morphogenesis.     

Synaptic organization of a multifunctional interneuron in the central nervous system of Helix pomatia L.

Summary The morphology, axonal arborization and ultrastructure of synaptic connections of the V21 giant neuron in the visceral ganglion of the snail Helix pomatia has been investigated following intracellular labelling with horseradish peroxidase. The V21 neuron establishes several afferent and efferent axo-axonic connections, mainly along the first half of the primary axon. Collaterals of 200–300 m length originate from the primary axon, which shows further arborization, and both afferent and efferent synaptic contacts are formed on these fine axon profiles. Afferent and efferent contacts of the cell occur within very short distances of a few micrometers. On the basis of ultrastructure and vesicle and granule content, several afferent terminals can be distinguished on V21 labelled axon profiles. The majority of these afferent terminals resembles peptidergic-(neurosecretory)-like terminals. This finding supports the possible transmitter role of neuropeptides in the central nervous system of gastropods. Our results are consistent with and provide morphological evidence for recent electrophysiological observations suggesting that, in addition to integrating input, the V21 neuron functions as an interneuron in Helix central nervous system.     

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9‐mediated kif15 mutations accelerate axonal outgrowth during neuronal development and regeneration in zebrafish

KIF15, the vertebrate kinesin‐12, is best known as a mitotic motor protein, but continues to be expressed in neurons. Like KIF11 (the vertebrate kinesin‐5), KIF15 interacts with microtubules in the axon to limit their sliding relative to one another. Unlike KIF11, KIF15 also regulates interactions between microtubules and actin filaments at sites of axonal branch formation and in growth cones. Our original work on these motors was done on cultured rat neurons, but we are now using zebrafish to extend these studies to an in vivo model. We previously studied kif15 in zebrafish by injecting splice‐blocking morpholinos injected into embryos. Consistent with the cell culture work, these studies demonstrated that axons grow faster and longer when KIF15 levels are reduced. In the present study, we applied CRISPR/Cas9‐based knockout technology to create kif15 mutants and labeled neurons with Tg(mnx1:GFP) transgene or transient expression of elavl3:EGFP‐alpha tubulin. We then compared by live imaging the homozygotic, heterozygotic mutants to their wildtype siblings to ascertain the effects of depletion of kif15 during Caudal primary motor neuron and Rohon‐Beard (R‐B) sensory neuron development. The results showed, compared to the kif15 wildtype, the number of branches was reduced while axon outgrowth was accelerated in kif15 homozygotic and heterozygotic mutants. In R‐B sensory neurons, after laser irradiation, injured axons with loss of kif15 displayed significantly greater regenerative velocity. Given these results and the fact that kif15 drugs are currently under development, we posit kif15 as a novel target for therapeutically augmenting regeneration of injured axons.      

Rafting along the axon on Unc104 motors

Neurotransmission depends upon the fast axonal transport of synaptic vesicle precursors by the monomeric kinesin Unc104, a motor whose mechanism of action is a topic of debate. New work suggests that the formation of lipid raft domains triggers the assembly of vesicle-bound Unc104 dimers and the concomitant activation of processive movement, facilitating efficient long-range vesicle transport.     

Distribution of synaptic vesicle proteins within single retinotectal axons of Xenopus tadpoles

In the developing retinotectal projection, retinal axon arbor structure changes rapidly within the target tectal neuropil at stages when the visual system functions to process visual information. In vivo imaging of single retinotectal axon arbors shows that up to 50% of the arbor branch length can be restructured within 8 h and short branchtips have average lifetimes of 10 min. To determine if presynaptic sites are restricted to the relatively stable part of the arbor or if they are also located on the more dynamic portions of the arbor, punctate staining of synaptic vesicle proteins (SVP) synapsin 1 and synaptophysin was mapped within individual retinal axons using double-label confocal immunocytochemistry. We report that SVP puncta were distributed throughout the retinotectal axon arbor. Notably, short branchtips, which are known to be extremely dynamic, contain the presynaptic machinery necessary for synaptic transmission. These data support a model in which activity-dependent mechanisms can influence presynaptic axon arbor morphology by modifying the rate of dynamic rearrangements of axonal branchtips. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 426–434, 1998.     

CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon

Neurons are highly specialized cells with polarized cellular processes and subcellular domains. As vital organelles for neuronal functions, mitochondria are distributed by microtubule-based transport systems. Although the essential components of mitochondrial transport including motors and cargo adaptors are identified, it is less clear how mitochondrial distribution among somato-dendritic and axonal compartment is regulated. Here, we systematically study mitochondrial motors, including four kinesins, KIF5, KIF17, KIF1, KLP-6, and dynein, and transport regulators in C. elegans PVD neurons. Among all these motors, we found that mitochondrial export from soma to neurites is mainly mediated by KIF5/UNC-116. Interestingly, UNC-116 is especially important for axonal mitochondria, while dynein removes mitochondria from all plus-end dendrites and the axon. We surprisingly found one mitochondrial transport regulator for minus-end dendritic compartment, TRAK-1, and two mitochondrial transport regulators for axonal compartment, CRMP/UNC-33 and JIP3/UNC-16. While JIP3/UNC-16 suppresses axonal mitochondria, CRMP/UNC-33 is critical for axonal mitochondria; nearly no axonal mitochondria present in unc-33 mutants. We showed that UNC-33 is essential for organizing the population of UNC-116-associated microtubule bundles, which are tracks for mitochondrial trafficking. Disarrangement of these tracks impedes mitochondrial transport to the axon. In summary, we identified a compartment-specific transport regulation of mitochondria by UNC-33 through organizing microtubule tracks for different kinesin motors other than microtubule polarity.     

Evidence for the involvement of KIF4 in the anterograde transport of L1-containing vesicles

In this study we present evidence about the cellular functions of KIF4. Using subcellular fractionation techniques and immunoisolation, we have now identified a type of vesicle that associates with KIF4, an NH(2)-terminal globular motor domain kinesin-like protein. This vesicle is highly concentrated in growth cones and contains L1, a cell adhesion molecule implicated in axonal elongation. It lacks synaptic vesicle markers, receptors for neurotrophins, and membrane proteins involved in growth cone guidance. In cultured neurons, KIF4 and L1 predominantly localize to the axonal shaft and its growth cone. Suppression of KIF4 with antisense oligonucleotides results in the accumulation of L1 within the cell body and in its complete disappearance from axonal tips. In addition, KIF4 suppression prevents L1-enhanced axonal elongation. Taken collectively, our results suggest an important role for KIF4 during neuronal development, a phenomenon which may be related to the anterograde transport of L1-containing vesicles.     

Characterization of a Kinesin-Related Gene ATSV, within the Tuberous Sclerosis Locus (TSC1) Candidate Region on Chromosome 9Q34

In the search for candidate genes for the tuberous sclerosis (TSC1) disease locus on chromosome 9q34, we have isolated an overlapping series of 22 plasmid and phage cDNA clones covering nearly 7 kb and with an open reading frame of 5070 bp encoding a protein of 1690 amino acids. The putative protein product is a member of the kinesin superfamily and is homologous to the mouse KIF1A and theCaenorhabditas elegansunc-104 genes. Both KIF1A and unc-104 function in the anterograde axonal transport of synaptic vesicles. The human homolog is therefore termed H-ATSV (axonal transporter of synaptic vesicles, HGMW-approved nomenclature ATSV) Screening of DNA from 107 tuberous sclerosis patients and 80 unaffected individuals with H-ATSV cDNA probes by pulsed-field gel electrophoresis/Southern blotting following digestion by rare-cutting methylation-sensitive restriction enzymes showed variant banding patterns in three patients with tuberous sclerosis. However, further analysis indicated that these variant fragments represent a rare polymorphism probably associated with methylation of clustered restriction sites. There is no evidence to support H-ATSV as a candidate gene for TSC1.     

The UNC-104/KIF1 family of kinesins

Most UNC-104/KIF1 kinesins are monomeric motors that transport membrane-bounded organelles toward the plus ends of microtubules. Recent evidence implies that KIF1A, a synaptic vesicle motor, moves processively. This surprising behavior for a monomeric motor depends upon a lysine-rich loop in KIF1A that binds to the negatively charged carboxyl terminus of tubulin and, in the context of motor processivity, compensates for the lack of a second motor domain on the KIF1A holoenzyme.     

A mode control model of a neuron's axon and dendrites

The ability of a neuron network to process information depends upon the ability of the individual neurons to transport impulses and to control the signal transport process in other neurons. The transport process for the action potential seen at the axon depends upon the excitable characteristic of the neural membrane. Propagation of signals in the dendrites, where synaptic imputs are most likely processed, is not clearly understood. Extracellular recordings of dendritic systems indicate that the dendrites are partially excitable and can conduct spikes. Further, electrical stimulation of the reticular formation or specific thalamic nuclei suggest that the conduction process can be modified in the dendrites of cortical cells.A Mode Control model is described which demonstrates many of the observed transport and control properties of dendrite and axon membrane. The model is based upon a simple extension of Fitzhugh's BVP model. Lateral transport over the membrane has been introduced by applying Kirchhoff's laws. Reinterpreting the variables, the influence of membrane potential, pH, and calcium ions can be identified. Modification of the voltage-current characteristic of the membrane model can change the axon model to a dendrite model. The dendrite model possesses a diffusion equation mode, a wave equation mode and a pulse mode. Signals are transferred in the wave and pulse mode and blocked in the diffusion mode. The dendrite's mode is controlled by the resting depolarization level. Experimental evidence tends to confirm these phenomena.The work described in this paper was performed while attending the University of California, Berkeley, under a National Institutes of Health Traineeship.     

Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A

To test the hypothesis that fast anterograde molecular motor proteins power the slow axonal transport of neurofilaments (NFs), we used homologous recombination to generate mice lacking the neuronal-specific conventional kinesin heavy chain, KIF5A. Because null KIF5A mutants die immediately after birth, a synapsin-promoted Cre-recombinase transgene was used to direct inactivation of KIF5A in neurons postnatally. Three fourths of such mutant mice exhibited seizures and death at around 3 wk of age; the remaining animals survived to 3 mo or longer. In young mutant animals, fast axonal transport appeared to be intact, but NF-H, as well as NF-M and NF-L, accumulated in the cell bodies of peripheral sensory neurons accompanied by a reduction in sensory axon caliber. Older animals also developed age-dependent sensory neuron degeneration, an accumulation of NF subunits in cell bodies and a reduction in axons, loss of large caliber axons, and hind limb paralysis. These data support the hypothesis that a conventional kinesin plays a role in the microtubule-dependent slow axonal transport of at least one cargo, the NF proteins.     

Comparison of the dimensions of synaptic vesicles in axon profiles forming synapses on the bodies and dendrites of cat motor cortex neurons]

The synaptic vesicle sizes in the cat motor cortex presynaptic elements was estimated by variational statistics. Populations of axonal profiles synapsing on the pyramidal neurone bodies were found to have significantly smaller synaptic vesicle sizes as compared to the axonal terminals at the dendrite branches and dendrite spines.