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.     

The phosphorylation of kinesin regulates its binding to synaptic vesicles.

Membrane organella are transported bidirectionally in cells, and the axonal transport system has provided an ideal model system for studying this bidirectional transport. Kinesin and cytoplasmic dynein were identified as candidates for the motor molecules of fast axonal transport, which transport organella along microtubules anterogradely and retrogradely. However, the mechanism that controls this bidirectional transport is unknown. Our previous work revealed that kinesin in axons was associated abundantly with anterogradely transported membranous organella, most of which are believed to be precursors of synaptic vesicles and axonal plasma membranes, while the fractions bound to retrogradely transported ones were very small (Hirokawa, N., Sato-Yoshitake, R., Kobayashi, N., Pfister, K. K., Bloom, G. S., and Brady, S. T. (1991) J. Cell Biol. 114, 295-302). Here we demonstrated in vitro that the binding of kinesin to synaptic vesicles was concentration-dependent and saturable and could be released by high salt concentration. When kinesin was phosphorylated by cAMP-dependent protein kinase, its binding to symaptic vesicles was significantly reduced. By motility assay and by statistical analysis using electron microscopy, we further revealed that synaptic vesicles preincubated with phosphorylated kinesin associated less frequently with microtubules than synaptic vesicles preincubated with unphosphorylated kinesin. The phosphorylation of kinesin should therefore play an essential role in regulating the direction of fast axonal transport by inhibiting its binding to membrane organella, thus releasing it from membrane organella at nerve terminals.     

KIF3A is a new microtubule-based anterograde motor in the nerve axon

Neurons are highly polarized cells composed of dendrites, cell bodies, and long axons. Because of the lack of protein synthesis machinery in axons, materials required in axons and synapses have to be transported down the axons after synthesis in the cell body. Fast anterograde transport conveys different kinds of membranous organelles such as mitochondria and precursors of synaptic vesicles and axonal membranes, while organelles such as endosomes and autophagic prelysosomal organelles are conveyed retrogradely. Although kinesin and dynein have been identified as good candidates for microtubule-based anterograde and retrograde transporters, respectively, the existence of other motors for performing these complex axonal transports seems quite likely. Here we characterized a new member of the kinesin super-family, KIF3A (50-nm rod with globular head and tail), and found that it is localized in neurons, associated with membrane organelle fractions, and accumulates with anterogradely moving membrane organelles after ligation of peripheral nerves. Furthermore, native KIF3A (a complex of 80/85 KIF3A heavy chain and a 95-kD polypeptide) revealed microtubule gliding activity and baculovirus-expressed KIF3A heavy chain demonstrated microtubule plus end-directed (anterograde) motility in vitro. These findings strongly suggest that KIF3A is a new motor protein for the anterograde fast axonal transport.     

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.     

A novel mouse kinesin of the UNC-104/KIF1 subfamily encoded by the Kif1b gene

Kinesin and kinesin-related proteins are microtubule-dependent motor proteins that transport organelles. We have cloned and sequenced a full-length 9924 bp mouse cDNA for a new kinesin of the UNC-104/KIF1 subfamily. Northern blot analysis of mouse RNAs detected high levels of a 10 kb mRNA in brain and eye, but lower levels in other tissues. Human RNA dot-blot analysis detected this mRNA in all tissues examined, although at different levels. The overall structure of the new kinesin (predicted size 204 kDa) was most similar to mouse KIF1A; however, 2.1 kb of the 5' portion of the cDNA were identical to the published sequence for KIF1B (Nangaku, M., Sato-Yoshitake, R., Okada, Y., Noda, Y., Takemura, R., Yamazaki, H., Hirokawa, N., 1994. KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell 79, 1209-1220). We localized the Kif1b gene to the distal end of mouse Chromosome 4 by haplotype analysis of an interspecific backcross from The Jackson Laboratory. We had previously mapped the gene for the novel kinesin to the same location (Gong, T.-W.L., Burmeister, M., Lomax, M.I., 1996b. The novel gene D4Mille maps to mouse Chromosome 4 and human Chromosome 1p36. Mamm. Genome 7, 790-791). We conclude, therefore, that the Kif1b gene generates two major kinesin isoforms by alternative splicing. The shorter 7.8 kb mRNA encodes a 130 kDa kinesin, KIF1Bp130, whereas the 10 kb mRNA encodes a 204 kDa kinesin, KIF1Bp204. In addition, alternative splicing of two exons in the conserved region adjacent to the motor domain generates four different isoforms of each kinesin, leading to eight kinesin isoforms derived from the Kif1b gene.     

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.      

β‐Tubulin mutations that cause severe neuropathies disrupt axonal transport

Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed β‐tubulin mutations that cause neuronal diseases and we have identified mutations that strongly inhibit axonal transport of vesicles and mitochondria. These mutations are in the H12 helix of β‐tubulin and change the negative charge on the surface of the microtubule. This surface is the interface between microtubules and kinesin superfamily motor proteins (KIF). The binding of axonal transport KIFs to microtubules is dominant negatively disrupted by these mutations, which alters the localization of KIFs in neurons and inhibits axon elongation in vivo. In humans, these mutations induce broad neurological symptoms, such as loss of axons in the central nervous system and peripheral neuropathy. Thus, our data identified the critical region of β‐tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations.     

Kinesin superfamily protein 3 (KIF3) motor transports fodrin-associating vesicles important for neurite building

Kinesin superfamily proteins (KIFs) comprise several dozen molecular motor proteins. The KIF3 heterotrimer complex is one of the most abundantly and ubiquitously expressed KIFs in mammalian cells. To unveil the functions of KIF3, microinjection of function-blocking monovalent antibodies against KIF3 into cultured superior cervical ganglion (SCG) neurons was carried out. They significantly blocked fast axonal transport and brought about inhibition of neurite extension. A yeast two-hybrid binding assay revealed the association of fodrin with the KIF3 motor through KAP3. This was further confirmed by using vesicles collected from large bundles of axons (cauda equina), from which membranous vesicles could be prepared in pure preparations. Both immunoprecipitation and immunoelectron microscopy indicated the colocalization of fodrin and KIF3 on the same vesicles, the results reinforcing the evidence that the cargo of the KIF3 motor consists of fodrin-associating vesicles. In addition, pulse-labeling study implied partial comigration of both molecules as fast flow components. Taken together, the KIF3 motor is engaged in fast axonal transport that conveys membranous components important for neurite extension.     

Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta

The kinesin superfamily motor protein KIF1B has been shown to transport mitochondria. Here, we describe an isoform of KIF1B, KIF1Bbeta, that is distinct from KIF1B in its cargo binding domain. KIF1B knockout mice die at birth from apnea due to nervous system defects. Death of knockout neurons in culture can be rescued by expression of the beta isoform. The KIF1B heterozygotes have a defect in transporting synaptic vesicle precursors and suffer from progressive muscle weakness similar to human neuropathies. Charcot-Marie-Tooth disease type 2A was previously mapped to an interval containing KIF1B. We show that CMT2A patients contain a loss-of-function mutation in the motor domain of the KIF1B gene. This is clear indication that defects in axonal transport due to a mutated motor protein can underlie human peripheral neuropathy.     

Effects of ALS-related SOD1 mutants on dynein- and KIF5-mediated retrograde and anterograde axonal transport

Transport of material and signals between extensive neuronal processes and the cell body is essential to neuronal physiology and survival. Slowing of axonal transport has been shown to occur before the onset of symptoms in amyotrophic lateral sclerosis (ALS). We have previously shown that several familial ALS-linked copper–zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interacted and colocalized with the retrograde dynein–dynactin motor complex in cultured cells and affected tissues of ALS mice. We also found that the interaction between mutant SOD1 and the dynein motor played a critical role in the formation of large inclusions containing mutant SOD1. In this study, we showed that, in contrast to the dynein situation, mutant SOD1 did not interact with anterograde transport motors of the kinesin-1 family (KIF5A, B and C). Using dynein and kinesin accumulation at the sciatic nerve ligation sites as a surrogate measurement of axonal transport, we also showed that dynein mediated retrograde transport was slower in G93A than in WT mice at an early presymptomatic stage. While no decrease in KIF5A-mediated anterograde transport was detected, the slowing of anterograde transport of dynein heavy chain as a cargo was observed in the presymptomatic G93A mice. The results from this study along with other recently published work support that mutant SOD1 might only interact with and interfere with some kinesin members, which, in turn, could result in the impairment of a selective subset of cargos. Although it remains to be further investigated how mutant SOD1 affects different axonal transport motor proteins and various cargos, it is evident that mutant SOD1 can induce defects in axonal transport, which, subsequently, contribute to the propagation of toxic effects and ultimately motor neuron death in ALS.     

Nucleotide specificities of anterograde and retrograde organelle transport in Reticulomyxa are indistinguishable

Membrane-bound organelles move bidirectionally along microtubules in the freshwater ameba, Reticulomyxa. We have examined the nucleotide requirements for transport in a lysed cell model and compared them with kinesin and dynein-driven motility in other systems. Both anterograde and retrograde transport in Reticulomyxa show features characteristic of dynein but not of kinesin-powered movements: organelle transport is reactivated only by ATP and no other nucleoside triphosphates; the Km and Vmax of the ATP-driven movements are similar to values obtained for dynein rather than kinesin-driven movement; and of 15 ATP analogues tested for their ability to promote organelle transport, only 4 of them did. This narrow specificity resembles that of dynein-mediated in vitro transport and is dissimilar to the broad specificity of the kinesin motor (Shimizu, T., K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno, F. Malik, and R. D. Vale. 1991. J. Cell Biol. 112: 1189-1197). Remarkably, anterograde and retrograde organelle transport cannot be distinguished at all with respect to nucleotide specificity, kinetics of movement, and the ability to use the ATP analogues. Since the "kinetic fingerprints" of the motors driving transport in opposite directions are indistinguishable, the same type of motor(s) may be involved in the two directions of movement.     

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.     

A novel motor, KIF13A, transports mannose-6-phosphate receptor to plasma membrane through direct interaction with AP-1 complex

Intracellular transport mediated by kinesin superfamily proteins (KIFs) is a highly regulated process. The molecular mechanism of KIFs binding to their respective cargoes remains unclear. We report that KIF13A is a novel plus end-directed microtubule-dependent motor protein and associates with beta 1-adaptin, a subunit of the AP-1 adaptor complex. The cargo vesicles of KIF13A contained AP-1 and mannnose-6-phosphate receptor (M6PR). Overexpression of KIF13A resulted in mislocalization of the AP-1 and the M6PR. Functional blockade of KIF13A reduced cell surface expression of the M6PR. Thus, KIF13A transports M6PR-containing vesicles and targets the M6PR from TGN to the plasma membrane via direct interaction with the AP-1 adaptor complex.     

Disruption of KIF17-Mint1 interaction by CaMKII-dependent phosphorylation: a molecular model of kinesin-cargo release

Establishment and maintenance of cell structures and functions are highly dependent on the efficient regulation of intracellular transport in which proteins of the kinesin superfamily (KIFs) are very important. In this regard, how KIFs regulate the release of their cargo is a critical process that remains to be elucidated. To address this specific question, we have investigated the mechanism behind the regulation of the KIF17-Mint1 interaction. Here we report that the tail region of the molecular motor KIF17 is regulated by phosphorylation. Using direct visualization of protein-protein interaction by FRET and various in vitro and in vivo approaches we have demonstrated that CaMKII-dependent phosphorylation of KIF17 on Ser 1029 disrupts the KIF17-Mint1 association and results in the release of the transported cargo from its microtubule-based transport.     

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.     

Organelle transport along microtubules - the role of KIFs

Organelle transporters are very important for cellular morphogenesis and other cellular functions, conveying and targeting important materials to the correct destination, often at considerable velocities. One of the first proteins to be identified as a motor was kinesin, and recently at least 10 new kinesin superfamily proteins (KIFs) have been described. Characterization of some of them reveals that each member can convey a specific organelle or cargo, although there is some redundancy. It has also become clear that there are distinct subclasses of KIFs that form monomeric, heterodimeric and homodimeric motors. Here, Nobutaka Hirokawa reviews what is known about the kinesin superfamily and discusses how a study of the different types of motors is helping to elucidate the mechanism of mechanical force generation.     

Inhibition of Kinesin Synthesis In Vivo Inhibits the Rapid Transport of Representative Proteins for Three Transport Vesicle Classes into the Axon

Abstract: We have previously demonstrated that the in vivo vitreal injection of an antisense oligonucleotide directed to the kinesin heavy chain inhibits retinal kinesin synthesis by 82% and concomitantly inhibits rapid transport of total protein into the optic nerve by 70%. These results establish a major role for kinesin in rapid axonal transport in vivo. Recently, the cloning of a family of kinesin-like molecules from the mammalian brain has been reported, and some of these proteins are also expressed in neurons. To assign a specific function to the kinesin heavy chain we inhibited the kinesin synthesis with an antisense kinesin oligonucleotide and assessed the axonal transport into the optic nerve of representative proteins from each of three vesicle classes that contain rapidly transported proteins. Marker proteins used were substance P for peptide-containing synaptic vesicles, the amyloid precursor protein for plasma membrane precursor vesicles, and several integral synaptic vesicle proteins. Our results indicate that the major anterograde motor protein for all three vesicle classes utilizes kinesin heavy chain, although we discuss alternative explanations.     

Kinesin Mutations Cause Motor Neuron Disease Phenotypes by Disrupting Fast Axonal Transport in Drosophila

Previous work has shown that mutation of the gene that encodes the microtubule motor subunit kinesin heavy chain (Khc) in Drosophila inhibits neuronal sodium channel activity, action potentials and neurotransmitter secretion. These physiological defects cause progressive distal paralysis in larvae. To identify the cellular defects that cause these phenotypes, larval nerves were studied by light and electron microscopy. The axons of Khc mutants develop dramatic focal swellings along their lengths. The swellings are packed with fast axonal transport cargoes including vesicles, synaptic membrane proteins, mitochondria and prelysosomal organelles, but not with slow axonal transport cargoes such as cytoskeletal elements. Khc mutations also impair the development of larval motor axon terminals, causing dystrophic morphology and marked reductions in synaptic bouton numbers. These observations suggest that as the concentration of maternally provided wild-type KHC decreases, axonal organelles transported by kinesin periodically stall. This causes organelle jams that disrupt retrograde as well as anterograde fast axonal transport, leading to defective action potentials, dystrophic terminals, reduced transmitter secretion and progressive distal paralysis. These phenotypes parallel the pathologies of some vertebrate motor neuron diseases, including some forms of amyotrophic lateral sclerosis (ALS), and suggest that impaired fast axonal transport is a key element in those diseases.     

Kinesin superfamily proteins and their various functions and dynamics

Kinesin superfamily proteins (KIFs) are motor proteins that transport membranous organelles and macromolecules fundamental for cellular functions along microtubules. Their roles in transport in axons and dendrites have been studied extensively, but KIFs are also used in intracellular transport in general. Recent findings have revealed that in many cases, the specific interaction of cargoes and motors is mediated via adaptor/scaffolding proteins. Cargoes are sorted to precise destinations, such as axons or dendrites. KIFs also participate in polarized transport in epithelial cells as shown in the apical transport of annexin XIIIb-containing vesicles by KIFC3. KIFs play important roles in higher order neuronal activity; transgenic mice overexpressing KIF17, which transports N-methyl-d-asp (NMDA) receptors to dendrites, show enhanced memory and learning. KIFs also play significant roles in neuronal development and brain wiring: KIF2A suppresses elongation of axon collaterals by its unique microtubule-depolymerizing activity. X-ray crystallography has revealed the structural uniqueness of KIF2 underlying the microtubule-depolymerizing activity. In addition, single molecule biophysics and optical trapping have shown that the motility of monomeric KIF1A is caused by biased Brownian movement, and X-ray crystallography has shown how the conformational changes occur for KIF1A to move during ATP hydrolysis. These multiple approaches in analyzing KIF functions will illuminate many basic mechanisms underlying intracellular events and will be a very promising and fruitful area for future studies.     

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.