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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.

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Targeted disruption of the mouse KIF5A gene. (A) KIF5A gene targeting strategy. In the targeting vector, the pGK-neo and HSV-tk selection cassette was flanked by two loxP sites (loxP1 and loxP2), and the two exons to be deleted were flanked by loxP2 and loxP3. Two steps of transfection were performed to generate type I deletion ( mutant) and type II deletion ES cells. The first step was done by transfecting linearized targeting vector into ES cells followed by G418 selection; the second step was performed by transfecting a Cre plasmid into recombinant ES cells isolated from the first step, followed by Gancyclovir selection. (B and C) Southern blot analyses of HindIII-digested G418-resistant ES clones after transfecting targeting vector. (B) A 5′ external probe was used to identify the recombinant clones. A 4.7-kb wild-type band was detected, and an additional 8.2 kb (with the addition of a 3.5-kb selection cassette) was also detected in the recombinant clones. (C) The presence of three loxP sites was confirmed by a loxP probe. The correct recombinant clones should have all three loxP sites, both 8.2-kb and 2.5-kb bands should be detected by the probe (clones 2 and 3), whereas clones 1 and 4 only had the first two loxP sites. (D) Southern blot analysis of HindIII-digested Gancyclovir-resistant clones after Cre transfection. With the loxP probe, only a 6-kb band was detected in type I deletion (KIF5A) ES cells, whereas 4.7-kb and 2.5-kb bands were detected in type II (KIF5Aflox) ES cells. (E) No KIF5A protein was detected in KIF5A  mutant mice by Western blot analysis. Mouse brain homogenates were made from two litters of mice, and 100 μg of protein was loaded in each lane. Isoform-specific antibodies were used to probe KIF5A, KIF5B, and KIF5C; α-tubulin was used as a loading control.
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fig1: Targeted disruption of the mouse KIF5A gene. (A) KIF5A gene targeting strategy. In the targeting vector, the pGK-neo and HSV-tk selection cassette was flanked by two loxP sites (loxP1 and loxP2), and the two exons to be deleted were flanked by loxP2 and loxP3. Two steps of transfection were performed to generate type I deletion ( mutant) and type II deletion ES cells. The first step was done by transfecting linearized targeting vector into ES cells followed by G418 selection; the second step was performed by transfecting a Cre plasmid into recombinant ES cells isolated from the first step, followed by Gancyclovir selection. (B and C) Southern blot analyses of HindIII-digested G418-resistant ES clones after transfecting targeting vector. (B) A 5′ external probe was used to identify the recombinant clones. A 4.7-kb wild-type band was detected, and an additional 8.2 kb (with the addition of a 3.5-kb selection cassette) was also detected in the recombinant clones. (C) The presence of three loxP sites was confirmed by a loxP probe. The correct recombinant clones should have all three loxP sites, both 8.2-kb and 2.5-kb bands should be detected by the probe (clones 2 and 3), whereas clones 1 and 4 only had the first two loxP sites. (D) Southern blot analysis of HindIII-digested Gancyclovir-resistant clones after Cre transfection. With the loxP probe, only a 6-kb band was detected in type I deletion (KIF5A) ES cells, whereas 4.7-kb and 2.5-kb bands were detected in type II (KIF5Aflox) ES cells. (E) No KIF5A protein was detected in KIF5A mutant mice by Western blot analysis. Mouse brain homogenates were made from two litters of mice, and 100 μg of protein was loaded in each lane. Isoform-specific antibodies were used to probe KIF5A, KIF5B, and KIF5C; α-tubulin was used as a loading control.

Mentions: We made a KIF5A deletion mutant by homologous recombination. The strategy (Fig. 1 A) was to create a mutant by deleting two critical exons, causing a frame shift of the encoded protein. Mice heterozygous for the KIF5A mutation were mated, and 33 newborn offspring from four litters were analyzed. Initial genotyping revealed that no KIF5A homozygous mutants survived, only three dead homozygous pups were found. Thus, KIF5A mutants are probably neonatal lethal. To directly observe the behavior of KIF5A mutants, KIF5A heterozygous mice were mated, the pregnant female mice were killed by caesarian section (c-section), and the embryonic day 18.5 (E18.5) pups were genotyped. Although live mutant pups were never observed by natural birth, all pups recovered by c-section were alive at E18.5. Whereas control littermates quickly developed a normal breathing pattern, the mutant pups did not (although like control littermates, they gasped just after removal from the uterus). Mutant pups gradually turned blue and usually died within 10 min. In a mixed 129/C57BL background, a total of 45 litters of E18.5 pups obtained by c-section were PCR genotyped. The predicted Mendelian ratio of 1:2:1 was observed: wild type (83/309), heterozygous (153/309), and homozygous mutant (73/309). The KIF5A homozygous mutant pups were indistinguishable from their control littermates by size and appearance. No visible structural defects were observed in any organs.


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)

Targeted disruption of the mouse KIF5A gene. (A) KIF5A gene targeting strategy. In the targeting vector, the pGK-neo and HSV-tk selection cassette was flanked by two loxP sites (loxP1 and loxP2), and the two exons to be deleted were flanked by loxP2 and loxP3. Two steps of transfection were performed to generate type I deletion ( mutant) and type II deletion ES cells. The first step was done by transfecting linearized targeting vector into ES cells followed by G418 selection; the second step was performed by transfecting a Cre plasmid into recombinant ES cells isolated from the first step, followed by Gancyclovir selection. (B and C) Southern blot analyses of HindIII-digested G418-resistant ES clones after transfecting targeting vector. (B) A 5′ external probe was used to identify the recombinant clones. A 4.7-kb wild-type band was detected, and an additional 8.2 kb (with the addition of a 3.5-kb selection cassette) was also detected in the recombinant clones. (C) The presence of three loxP sites was confirmed by a loxP probe. The correct recombinant clones should have all three loxP sites, both 8.2-kb and 2.5-kb bands should be detected by the probe (clones 2 and 3), whereas clones 1 and 4 only had the first two loxP sites. (D) Southern blot analysis of HindIII-digested Gancyclovir-resistant clones after Cre transfection. With the loxP probe, only a 6-kb band was detected in type I deletion (KIF5A) ES cells, whereas 4.7-kb and 2.5-kb bands were detected in type II (KIF5Aflox) ES cells. (E) No KIF5A protein was detected in KIF5A  mutant mice by Western blot analysis. Mouse brain homogenates were made from two litters of mice, and 100 μg of protein was loaded in each lane. Isoform-specific antibodies were used to probe KIF5A, KIF5B, and KIF5C; α-tubulin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Targeted disruption of the mouse KIF5A gene. (A) KIF5A gene targeting strategy. In the targeting vector, the pGK-neo and HSV-tk selection cassette was flanked by two loxP sites (loxP1 and loxP2), and the two exons to be deleted were flanked by loxP2 and loxP3. Two steps of transfection were performed to generate type I deletion ( mutant) and type II deletion ES cells. The first step was done by transfecting linearized targeting vector into ES cells followed by G418 selection; the second step was performed by transfecting a Cre plasmid into recombinant ES cells isolated from the first step, followed by Gancyclovir selection. (B and C) Southern blot analyses of HindIII-digested G418-resistant ES clones after transfecting targeting vector. (B) A 5′ external probe was used to identify the recombinant clones. A 4.7-kb wild-type band was detected, and an additional 8.2 kb (with the addition of a 3.5-kb selection cassette) was also detected in the recombinant clones. (C) The presence of three loxP sites was confirmed by a loxP probe. The correct recombinant clones should have all three loxP sites, both 8.2-kb and 2.5-kb bands should be detected by the probe (clones 2 and 3), whereas clones 1 and 4 only had the first two loxP sites. (D) Southern blot analysis of HindIII-digested Gancyclovir-resistant clones after Cre transfection. With the loxP probe, only a 6-kb band was detected in type I deletion (KIF5A) ES cells, whereas 4.7-kb and 2.5-kb bands were detected in type II (KIF5Aflox) ES cells. (E) No KIF5A protein was detected in KIF5A mutant mice by Western blot analysis. Mouse brain homogenates were made from two litters of mice, and 100 μg of protein was loaded in each lane. Isoform-specific antibodies were used to probe KIF5A, KIF5B, and KIF5C; α-tubulin was used as a loading control.
Mentions: We made a KIF5A deletion mutant by homologous recombination. The strategy (Fig. 1 A) was to create a mutant by deleting two critical exons, causing a frame shift of the encoded protein. Mice heterozygous for the KIF5A mutation were mated, and 33 newborn offspring from four litters were analyzed. Initial genotyping revealed that no KIF5A homozygous mutants survived, only three dead homozygous pups were found. Thus, KIF5A mutants are probably neonatal lethal. To directly observe the behavior of KIF5A mutants, KIF5A heterozygous mice were mated, the pregnant female mice were killed by caesarian section (c-section), and the embryonic day 18.5 (E18.5) pups were genotyped. Although live mutant pups were never observed by natural birth, all pups recovered by c-section were alive at E18.5. Whereas control littermates quickly developed a normal breathing pattern, the mutant pups did not (although like control littermates, they gasped just after removal from the uterus). Mutant pups gradually turned blue and usually died within 10 min. In a mixed 129/C57BL background, a total of 45 litters of E18.5 pups obtained by c-section were PCR genotyped. The predicted Mendelian ratio of 1:2:1 was observed: wild type (83/309), heterozygous (153/309), and homozygous mutant (73/309). The KIF5A homozygous mutant pups were indistinguishable from their control littermates by size and appearance. No visible structural defects were observed in any organs.

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