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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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Gross analysis of KIF5A conditional mutant mice (KIF5A/KIF5Aflox; Cresynapsin). (A) Western blot analysis to measure KIF5A protein levels in the brains of KIF5A/KIF5Aflox; Cresynapsin mice. Equal amounts of brain homogenate from 3-wk-old mutant and control (KIF5Aflox/KIF5AWT) littermates were loaded; actin was used as a loading control. Each marked number represents the ratio between the mutant band and control band after normalizing with the actin band. (B and C) KIF5A excision by Cresynapsin transgene in DRG (B) and spinal cord motor neurons (C) of KIF5A/KIF5Aflox; Cresynapsin mutant. Tissue sections from ∼3-wk-old mutant and control littermates were stained with KIF5A-specific antibody. Spinal cord sections were also double stained with an anti-BIP antibody to visualize the motor neuron cell bodies. Note the decreased or lack of KIF5A staining in some neurons (arrowheads). Bar, 100 μm. (D) A comparison of body weight (mean ± SD) among 3-wk-old littermates with different genotypes. Note the obvious low body weight in the KIF5A/KIF5Aflox; Cresynapsin mutant group, n = 4 for each group. *, P < 0.01. (E) Most KIF5A/KIF5Aflox; Cresynapsin mutant mice died around 3 wk of age. Postnatal survival curve of a group of mice (142 total, 113 control and 29 mutant) is shown here. The rate of survival of the different genotypes was plotted against age. (F) Abnormal hind limb posture in an older KIF5A/KIF5Aflox; Cresynapsin mutant mouse. Two 7.5-mo-old littermates (control and KIF5A/KIF5Aflox) are shown.
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fig3: Gross analysis of KIF5A conditional mutant mice (KIF5A/KIF5Aflox; Cresynapsin). (A) Western blot analysis to measure KIF5A protein levels in the brains of KIF5A/KIF5Aflox; Cresynapsin mice. Equal amounts of brain homogenate from 3-wk-old mutant and control (KIF5Aflox/KIF5AWT) littermates were loaded; actin was used as a loading control. Each marked number represents the ratio between the mutant band and control band after normalizing with the actin band. (B and C) KIF5A excision by Cresynapsin transgene in DRG (B) and spinal cord motor neurons (C) of KIF5A/KIF5Aflox; Cresynapsin mutant. Tissue sections from ∼3-wk-old mutant and control littermates were stained with KIF5A-specific antibody. Spinal cord sections were also double stained with an anti-BIP antibody to visualize the motor neuron cell bodies. Note the decreased or lack of KIF5A staining in some neurons (arrowheads). Bar, 100 μm. (D) A comparison of body weight (mean ± SD) among 3-wk-old littermates with different genotypes. Note the obvious low body weight in the KIF5A/KIF5Aflox; Cresynapsin mutant group, n = 4 for each group. *, P < 0.01. (E) Most KIF5A/KIF5Aflox; Cresynapsin mutant mice died around 3 wk of age. Postnatal survival curve of a group of mice (142 total, 113 control and 29 mutant) is shown here. The rate of survival of the different genotypes was plotted against age. (F) Abnormal hind limb posture in an older KIF5A/KIF5Aflox; Cresynapsin mutant mouse. Two 7.5-mo-old littermates (control and KIF5A/KIF5Aflox) are shown.

Mentions: Quantitative immunoblot analysis of a small set of initial animals showed that KIF5A protein levels in mutant brains ranged from 6 to 56% of that of control brains (Fig. 3 A). The heterogeneity in the level of reduction of KIF5A may reflect variable efficiency of Cre-mediated gene excision. To observe directly KIF5A excision at the cellular level, dorsal root ganglion (DRG) and spinal cord from ∼3-wk-old mutants were examined by immunostaining with a KIF5A-specific antibody (Fig. 3, B and C). This staining revealed an absence of KIF5A in some sensory neurons in the mutant. In spinal cord, costaining with a monoclonal BIP antibody (an endoplasmic reticulum marker) indicated that KIF5A staining in the mutant motor neuron cell bodies was much weaker compared with the control; some cells had almost no KIF5A staining. We conclude that KIF5A excision occurred in many DRG sensory neurons and spinal cord motor neurons.


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)

Gross analysis of KIF5A conditional mutant mice (KIF5A/KIF5Aflox; Cresynapsin). (A) Western blot analysis to measure KIF5A protein levels in the brains of KIF5A/KIF5Aflox; Cresynapsin mice. Equal amounts of brain homogenate from 3-wk-old mutant and control (KIF5Aflox/KIF5AWT) littermates were loaded; actin was used as a loading control. Each marked number represents the ratio between the mutant band and control band after normalizing with the actin band. (B and C) KIF5A excision by Cresynapsin transgene in DRG (B) and spinal cord motor neurons (C) of KIF5A/KIF5Aflox; Cresynapsin mutant. Tissue sections from ∼3-wk-old mutant and control littermates were stained with KIF5A-specific antibody. Spinal cord sections were also double stained with an anti-BIP antibody to visualize the motor neuron cell bodies. Note the decreased or lack of KIF5A staining in some neurons (arrowheads). Bar, 100 μm. (D) A comparison of body weight (mean ± SD) among 3-wk-old littermates with different genotypes. Note the obvious low body weight in the KIF5A/KIF5Aflox; Cresynapsin mutant group, n = 4 for each group. *, P < 0.01. (E) Most KIF5A/KIF5Aflox; Cresynapsin mutant mice died around 3 wk of age. Postnatal survival curve of a group of mice (142 total, 113 control and 29 mutant) is shown here. The rate of survival of the different genotypes was plotted against age. (F) Abnormal hind limb posture in an older KIF5A/KIF5Aflox; Cresynapsin mutant mouse. Two 7.5-mo-old littermates (control and KIF5A/KIF5Aflox) are shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Gross analysis of KIF5A conditional mutant mice (KIF5A/KIF5Aflox; Cresynapsin). (A) Western blot analysis to measure KIF5A protein levels in the brains of KIF5A/KIF5Aflox; Cresynapsin mice. Equal amounts of brain homogenate from 3-wk-old mutant and control (KIF5Aflox/KIF5AWT) littermates were loaded; actin was used as a loading control. Each marked number represents the ratio between the mutant band and control band after normalizing with the actin band. (B and C) KIF5A excision by Cresynapsin transgene in DRG (B) and spinal cord motor neurons (C) of KIF5A/KIF5Aflox; Cresynapsin mutant. Tissue sections from ∼3-wk-old mutant and control littermates were stained with KIF5A-specific antibody. Spinal cord sections were also double stained with an anti-BIP antibody to visualize the motor neuron cell bodies. Note the decreased or lack of KIF5A staining in some neurons (arrowheads). Bar, 100 μm. (D) A comparison of body weight (mean ± SD) among 3-wk-old littermates with different genotypes. Note the obvious low body weight in the KIF5A/KIF5Aflox; Cresynapsin mutant group, n = 4 for each group. *, P < 0.01. (E) Most KIF5A/KIF5Aflox; Cresynapsin mutant mice died around 3 wk of age. Postnatal survival curve of a group of mice (142 total, 113 control and 29 mutant) is shown here. The rate of survival of the different genotypes was plotted against age. (F) Abnormal hind limb posture in an older KIF5A/KIF5Aflox; Cresynapsin mutant mouse. Two 7.5-mo-old littermates (control and KIF5A/KIF5Aflox) are shown.
Mentions: Quantitative immunoblot analysis of a small set of initial animals showed that KIF5A protein levels in mutant brains ranged from 6 to 56% of that of control brains (Fig. 3 A). The heterogeneity in the level of reduction of KIF5A may reflect variable efficiency of Cre-mediated gene excision. To observe directly KIF5A excision at the cellular level, dorsal root ganglion (DRG) and spinal cord from ∼3-wk-old mutants were examined by immunostaining with a KIF5A-specific antibody (Fig. 3, B and C). This staining revealed an absence of KIF5A in some sensory neurons in the mutant. In spinal cord, costaining with a monoclonal BIP antibody (an endoplasmic reticulum marker) indicated that KIF5A staining in the mutant motor neuron cell bodies was much weaker compared with the control; some cells had almost no KIF5A staining. We conclude that KIF5A excision occurred in many DRG sensory neurons and spinal cord motor neurons.

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