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

Xia CH, Roberts EA, Her LS, Liu X, Williams DS, Cleveland DW, Goldstein LS - J. Cell Biol. (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093-0683, USA.

ABSTRACT
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 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.

Show MeSH

Related in: MedlinePlus

Analysis of DRG accumulation and axonal transport in second cohort of 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. (A) Axonal transport of APP, Rab3, synaptophysin, and KIF5C as assessed by Western blotting of proximal and distal segments 6 h after ligation of sciatic nerves from control and mutant animals; unligated controls are from the contralateral unligated nerve. Nerves from each animal were homogenized, and equal amounts of nerve proteins (∼40 μg/lane) were loaded onto SDS-PAGE gels and analyzed by Western blotting. Data from two pairs of mice from different litters are shown. P, proximal side; D, distal side. (B) Analysis of DRG content of mutant and control animals. Approximately 12 μg of DRG lysate from each animal was loaded into each lane. Note increased NF-H and NF-L levels in the mutant DRGs, whereas protein amounts of APP, Rab3, and synaptophysin were not obviously changed. (C) Summary of data from 12 mutant and 12 littermate control animals from five litters. SYP, synaptophysin. As many antibodies as possible were used for reprobing of each filter, but filter lifetime was inconsistent so not all probes could be used on all animals.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172877&req=5

fig7: Analysis of DRG accumulation and axonal transport in second cohort of 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. (A) Axonal transport of APP, Rab3, synaptophysin, and KIF5C as assessed by Western blotting of proximal and distal segments 6 h after ligation of sciatic nerves from control and mutant animals; unligated controls are from the contralateral unligated nerve. Nerves from each animal were homogenized, and equal amounts of nerve proteins (∼40 μg/lane) were loaded onto SDS-PAGE gels and analyzed by Western blotting. Data from two pairs of mice from different litters are shown. P, proximal side; D, distal side. (B) Analysis of DRG content of mutant and control animals. Approximately 12 μg of DRG lysate from each animal was loaded into each lane. Note increased NF-H and NF-L levels in the mutant DRGs, whereas protein amounts of APP, Rab3, and synaptophysin were not obviously changed. (C) Summary of data from 12 mutant and 12 littermate control animals from five litters. SYP, synaptophysin. As many antibodies as possible were used for reprobing of each filter, but filter lifetime was inconsistent so not all probes could be used on all animals.

Mentions: To test further whether KIF5A mutants have a specific defect in slow axonal transport of NFs, we bred a second cohort of 12 3-wk-old KIF5A/KIF5Aflox; Cresynapsin animals and 12 3-wk-old matched littermate control animals from five different litters. We used these animals to assess the integrity of several fast anterograde transport pathways (APP, Rab3, synaptophysin, and KIF5C), using a combination of protein accumulation in sensory neuron cell bodies in DRGs and sciatic nerve ligation experiments (Kamal et al., 2000). Although the small size and fragility of the KIF5A animals and the technical difficulty in performing nerve ligations on them severely limited the sample amounts available, sufficient protein from sciatic nerve ligations of individual animals was obtained to load single lanes on gels and probe these multiple times with different antibodies (some of these filters could not be used for the full antibody set, yielding a reduced n; Fig. 7 C). As before, we observed significantly reduced KIF5A protein levels in all of the mutant DRGs compared with controls (Fig. 7), whereas the axonal transport profile of APP, Rab3, synaptophysin, and KIF5C was indistinguishable from that of their control littermates. 10 out of 12 KIF5A-deficient mice exhibited striking NF protein accumulation in the DRGs, whereas no significant changes in the protein levels of APP, Rab3, and synaptophysin were observed. Interestingly, KIF5C protein amount was slightly decreased in the mutant DRGs, although its abundance in the sciatic nerve and its transport behavior were comparable in the mutant and the control. Although further work will be necessary to understand the origin of KIF5C reductions in the DRG, one obvious possibility is that KIF5C may carry out functions that are redundant with those of KIF5A, such that when less KIF5A is available for transport, more KIF5C is used to compensate for the reduced function of KIF5A. Alternatively, KIF5C may form heterodimers with KIF5A (Kanai et al., 2000), and thus the amount of KIF5C may be decreased when its binding partner is reduced.


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

Xia CH, Roberts EA, Her LS, Liu X, Williams DS, Cleveland DW, Goldstein LS - J. Cell Biol. (2003)

Analysis of DRG accumulation and axonal transport in second cohort of 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. (A) Axonal transport of APP, Rab3, synaptophysin, and KIF5C as assessed by Western blotting of proximal and distal segments 6 h after ligation of sciatic nerves from control and mutant animals; unligated controls are from the contralateral unligated nerve. Nerves from each animal were homogenized, and equal amounts of nerve proteins (∼40 μg/lane) were loaded onto SDS-PAGE gels and analyzed by Western blotting. Data from two pairs of mice from different litters are shown. P, proximal side; D, distal side. (B) Analysis of DRG content of mutant and control animals. Approximately 12 μg of DRG lysate from each animal was loaded into each lane. Note increased NF-H and NF-L levels in the mutant DRGs, whereas protein amounts of APP, Rab3, and synaptophysin were not obviously changed. (C) Summary of data from 12 mutant and 12 littermate control animals from five litters. SYP, synaptophysin. As many antibodies as possible were used for reprobing of each filter, but filter lifetime was inconsistent so not all probes could be used on all animals.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172877&req=5

fig7: Analysis of DRG accumulation and axonal transport in second cohort of 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. (A) Axonal transport of APP, Rab3, synaptophysin, and KIF5C as assessed by Western blotting of proximal and distal segments 6 h after ligation of sciatic nerves from control and mutant animals; unligated controls are from the contralateral unligated nerve. Nerves from each animal were homogenized, and equal amounts of nerve proteins (∼40 μg/lane) were loaded onto SDS-PAGE gels and analyzed by Western blotting. Data from two pairs of mice from different litters are shown. P, proximal side; D, distal side. (B) Analysis of DRG content of mutant and control animals. Approximately 12 μg of DRG lysate from each animal was loaded into each lane. Note increased NF-H and NF-L levels in the mutant DRGs, whereas protein amounts of APP, Rab3, and synaptophysin were not obviously changed. (C) Summary of data from 12 mutant and 12 littermate control animals from five litters. SYP, synaptophysin. As many antibodies as possible were used for reprobing of each filter, but filter lifetime was inconsistent so not all probes could be used on all animals.
Mentions: To test further whether KIF5A mutants have a specific defect in slow axonal transport of NFs, we bred a second cohort of 12 3-wk-old KIF5A/KIF5Aflox; Cresynapsin animals and 12 3-wk-old matched littermate control animals from five different litters. We used these animals to assess the integrity of several fast anterograde transport pathways (APP, Rab3, synaptophysin, and KIF5C), using a combination of protein accumulation in sensory neuron cell bodies in DRGs and sciatic nerve ligation experiments (Kamal et al., 2000). Although the small size and fragility of the KIF5A animals and the technical difficulty in performing nerve ligations on them severely limited the sample amounts available, sufficient protein from sciatic nerve ligations of individual animals was obtained to load single lanes on gels and probe these multiple times with different antibodies (some of these filters could not be used for the full antibody set, yielding a reduced n; Fig. 7 C). As before, we observed significantly reduced KIF5A protein levels in all of the mutant DRGs compared with controls (Fig. 7), whereas the axonal transport profile of APP, Rab3, synaptophysin, and KIF5C was indistinguishable from that of their control littermates. 10 out of 12 KIF5A-deficient mice exhibited striking NF protein accumulation in the DRGs, whereas no significant changes in the protein levels of APP, Rab3, and synaptophysin were observed. Interestingly, KIF5C protein amount was slightly decreased in the mutant DRGs, although its abundance in the sciatic nerve and its transport behavior were comparable in the mutant and the control. Although further work will be necessary to understand the origin of KIF5C reductions in the DRG, one obvious possibility is that KIF5C may carry out functions that are redundant with those of KIF5A, such that when less KIF5A is available for transport, more KIF5C is used to compensate for the reduced function of KIF5A. Alternatively, KIF5C may form heterodimers with KIF5A (Kanai et al., 2000), and thus the amount of KIF5C may be decreased when its binding partner is reduced.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093-0683, USA.

ABSTRACT
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 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.

Show MeSH
Related in: MedlinePlus