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.

No abstract

Abstract. In mature neurons, tau is abundant in axons, whereas microtubule-associated protein 2 (MAP2) and MAP2C are specifically localized in dendrites. Known mechanisms involved in the compartmentalization of these cytoskeletal proteins include the differential localization of mRNA (MAP2 mRNA in dendrites, MAP2C mRNA in cell body, and Tau mRNA in proximal axon revealed by in situ hybridization) (Garner, C.C., R.P. Tucker, and A. Matus. 1988. Nature (Lond.). 336: 674--677; Litman, P., J. Barg, L. Rindzooski, and I. Ginzburg. 1993. Neuron. 10:627-638), suppressed transit of MAP2 into axons (revealed by cDNA transfection into neurons) (Kanai, Y., and N. Hirokawa. 1995. Neuron. 14:421-432), and differential turnover of MAP2 in axons vs dendrites (Okabe, S., and N. Hirokawa. 1989. Proc. Natl. Acad. Sci. USA. 86:4127-4131). To investigate whether differential turnover of MAPs contributes to localization of other major MAPs in general, we microinjected biotinylated tau, MAP2C, or MAP2 into mature spinal cord neurons in culture (~3 wk) and then analyzed their fates by antibiotin immunocytochemistry. Initially, each was detected in axons and dendrites, although tau persisted only in axons, whereas MAP2C and MAP2 were restricted to cell bodies and dendrites. Injected MAP2C and MAP2 bound to dendritic microtubules more firmly than to microtubules in axons, while injected tau bound to axonal microtubules more firmly than to microtubules in dendrites. Thus, beyond contributions from mRNA localization and selective axonal transport, compartmentalization of each of the three major MAPs occurs through local differential turnover.

Abstract. Neurofilaments (NFs) are composed of triplet proteins, NF-H, NF-M, and NF-L. To understand the dynamics of NFs in vivo, we studied the dynamics of NF-H and compared them to those of NF-L, using the combination of microinjection technique and fluorescence recovery after photobleaching. In the case of NF-L protein, the bleached zone gradually restored its fluorescence intensity with a recovery half time of ,'~ 35 min. On the other hand, recovery of the bleached zone of NF-H was considerably faster, taking place in ,,o19 min. However, in both cases the bleached zone was stationary. Thus, it was suggested that NF-H is the dynamic component of the NF array and is interchangeable, but that it assembles with the other neurofilament triplet proteins in a more exchangeable way, implying that the location of NF-H is in the periphery of the core NF array mainly composed of NF-L subunits. Immunoelectron microscopy investigations of the incorporation sites of NF-H labeled with biotin compounds also revealed the lateral insertion of NF-H subunits into the preexisting NF array, taking after the pattern seen in the case of NF-L. In summary, our results demonstrate that the dynamics of the L and H subunit proteins in situ are quite different from each other, suggesting different and separated mechanisms or structural specialization underlying the behavior of the two proteins.

Abbreviations: 3MA, 3-methyladenine; CPS1, carbamoyl-phosphate synthase 1, mitochondrial; CASP3, caspase 3, apoptosis-related cysteine peptidase; CTSD, cathepsin D; COX4I1, cytochrome c oxidase subunit IV isoform 1; GalN, D-galactosamine; IL1B, interleukin1, b; LPS, lipopolysaccharide; LAMP, lysosomal-associated membrane protein; MT-mKO1, mitochondrially tagged monomeric Kusabira Orange1; mGFP, monomeric green fluorescent protein; mRFP, monomeric red fluorescent protein; MEF, mouse embryonic fibroblast; PMN, polymorphonuclear leukocyte; SQSTM1/p62, sequestosome 1; tfLC3, tandem fluorescent-tagged LC3; TFEB, transcription factor EB; TNF, tumor necrosis factor; VDAC1, voltage-dependent anion channel 1.Sepsis/endotoxemia is elicited by the circulatory distribution of pathogens/endotoxins into whole bodies, and causes profound effects on human health by causing inflammation in multiple organs. Mitochondrial damage is one of the characteristics of the cellular degeneration observed during sepsis/endotoxemia. Elimination of damaged mitochondria through the autophagy-lysosome system has been reported in the liver, indicating that autophagy should play an important role in liver homeostasis during sepsis/endotoxemia. An increased appearance of mitochondrial DNA and proteins in the plasma is another feature of sepsis/endotoxemia, suggesting that damaged mitochondria are not only eliminated within the cells, but also extruded through currently unknown mechanisms. Here we provide evidence for the secretion of mitochondrial proteins and DNA from lipopolysaccharide (LPS)-stimulated rat hepatocytes as well as mouse embryonic fibroblasts (MEFs). The secretion of mitochondrial contents is accompanied by the secretion of proteins that reside in the lumenal space of autolysosomes (LC3-II and CTSD/cathepsin D), but not by a lysosomal membrane protein (LAMP1). The pharmacological inhibition of autophagy by 3MA blocks the secretion of mitochondrial constituents from LPS-stimulated hepatocytes. LPS also stimulates the secretion of mitochondrial as well as autolysosomal lumenal proteins from wild-type (Atg5 C/C ) MEFs, but not from atg5 ¡/¡ MEFs. Furthermore, we show that direct exposure of purified mitochondria activates polymorphonuclear leukocytes (PMNs), as evident by the induction of IL1B/interlekin-1b, a pro-inflammatory cytokine. Taken together, the data suggest the active extrusion of mitochondrial contents, which provoke an inflammatory response of immune cells, through the exocytosis of autolysosomes by cells stimulated with LPS.