The tau gene encodes a protein (Tau) that is a major neuronal microtubule-associated protein localized mostly in axons. It has microtubule-binding and tubulin-polymerizing activity in vitro and is thought to make short crossbridges between axonal microtubules. Further, tau-transfected non-neuronal cells extend long axon-like processes in which microtubule bundles resembling those in axons are formed. In contrast, tau antisense oligonucleotides selectively suppress axonal elongation in cultured neurons. Thus tau is thought to be essential for neuronal cell morphogenesis, especially axonal elongation and maintenance. To test this hypothesis, we used gene targeting to produce mice lacking the tau gene. We show that the nervous system of tau-deficient mice appears to be normal immunohistologically. Furthermore, axonal elongation is not affected in cultured neurons. But in some small-calibre axons, microtubule stability is decreased and microtubule organization is significantly changed. We observed an increase in microtubule-associated protein 1A which may compensate for the functions of tau in large-calibre axons. Our results argue against the suggested role of tau in axonal elongation but confirm that it is crucial in the stabilization and organization of axonal microtubules in a certain type of axon.

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.

In cells, molecular motors operate in polarized sorting of molecules, although the steering mechanisms of motors remain elusive. In neurons, the kinesin motor conducts vesicular transport such as the transport of synaptic vesicle components to axons and of neurotransmitter receptors to dendrites, indicating that vesicles may have to drive the motor for the direction to be correct. Here we show that an AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor subunit--GluR2-interacting protein (GRIP1)--can directly interact and steer kinesin heavy chains to dendrites as a motor for AMPA receptors. As would be expected if this complex is functional, both gene targeting and dominant negative experiments of heavy chains of mouse kinesin showed abnormal localization of GRIP1. Moreover, expression of the kinesin-binding domain of GRIP1 resulted in accumulation of the endogenous kinesin predominantly in the somatodendritic area. This pattern was different from that generated by the overexpression of the kinesin-binding scaffold protein JSAP1 (JNK/SAPK-associated protein-1, also known as Mapk8ip3), which occurred predominantly in the somatoaxon area. These results indicate that directly binding proteins can determine the traffic direction of a motor protein.

Cytoplasmic dynein, a minus end–directed, microtubule-based motor protein, is thought to drive the movement of membranous organelles and chromosomes. It is a massive complex that consists of multiple polypeptides. Among these polypeptides, the cytoplasmic dynein heavy chain (cDHC) constitutes the major part of this complex. To elucidate the function of cytoplasmic dynein, we have produced mice lacking cDHC by gene targeting. cDHC−/− embryos were indistinguishable from cDHC+/−or cDHC+/+ littermates at the blastocyst stage. However, no cDHC−/− embryos were found at 8.5 d postcoitum. When cDHC−/− blastocysts were cultured in vitro, they showed interesting phenotypes. First, the Golgi complex became highly vesiculated and distributed throughout the cytoplasm. Second, endosomes and lysosomes were not concentrated near the nucleus but were distributed evenly throughout the cytoplasm. Interestingly, the Golgi “fragments” and lysosomes were still found to be attached to microtubules. These results show that cDHC is essential for the formation and positioning of the Golgi complex. Moreover, cDHC is required for cell proliferation and proper distribution of endosomes and lysosomes. However, molecules other than cDHC might mediate attachment of the Golgi complex and endosomes/lysosomes to microtubules.

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.

Microtubule-associated protein 2 (MAP2) is a major component of cross-bridges between microtubules in dendrites, and is known to stabilize microtubules. MAP2 also has a binding domain for the regulatory subunit II of cAMP-dependent protein kinase (PKA). We found that there is reduction in microtubule density in dendrites and a reduction of dendritic length in MAP2-deficient mice. Moreover, there is a significant reduction of various subunits of PKA in dendrites and total amounts of various PKA subunits in hippocampal tissue and cultured neurons. In MAP2-deficient cultured neurons, the induction rate of phosphorylated CREB after forskolin stimulation was much lower than in wild-type neurons. Therefore, MAP2 is an anchoring protein of PKA in dendrites, whose loss leads to reduced amount of dendritic and total PKA and reduced activation of CREB.

Through interactions with microtubules, the kinesin superfamily of proteins (KIFs) could have multiple roles in neuronal function and development. During neuronal development, postmitotic neurons develop primary axons extending toward targets, while other collateral branches remain short. Although the process of collateral branching is important for correct wiring of the brain, the mechanisms involved are not well understood. In this study, we analyzed kif2a(-/-) mice, whose brains showed multiple phenotypes, including aberrant axonal branching due to overextension of collateral branches. In kif2a(-/-) growth cones, microtubule-depolymerizing activity decreased. Moreover, many individual microtubules showed abnormal behavior at the kif2a(-/-) cell edge. Based on these results, we propose that KIF2A regulates microtubule dynamics at the growth cone edge by depolymerizing microtubules and that it plays an important role in the suppression of collateral branch extension.

MAP1B and MAP2 are major members of neuronal microtubule-associated proteins (MAPs). To gain insights into the function of MAP2 in vivo, we generated MAP2-deficient (map2−/−) mice. They developed without any apparent abnormalities, which indicates that MAP2 is dispensable in mouse survival. Because previous reports suggest a functional redundancy among MAPs, we next generated mice lacking both MAP2 and MAP1B to test their possible synergistic functions in vivo. Map2 − /−map1b −/− mice died in their perinatal period. They showed not only fiber tract malformations but also disrupted cortical patterning caused by retarded neuronal migration. In spite of this, their cortical layer maintained an “inside-out” pattern. Detailed observation of primary cultures of hippocampal neurons from map2 −/−map1b −/− mice revealed inhibited microtubule bundling and neurite elongation. In these neurons, synergistic effects caused by the loss of MAP2 and MAP1B were more apparent in dendrites than in axons. The spacing of microtubules was reduced significantly in map2 −/−map1b −/− mice in vitro and in vivo. These results suggest that MAP2 and MAP1B have overlapping functions in neuronal migration and neurite outgrowth by organizing microtubules in developing neurons both for axonal and dendritic morphogenesis but more dominantly for dendritic morphogenesis.