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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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Accumulation of NF proteins in DRG sensory neuron cell bodies of KIF5A/KIF5Aflox; Cresynapsin mutant mice. (A) Western blot analyses of DRGs from the first cohort of 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. In the litter shown, control DRGs (C) were pooled from one II/+ and one Cre/+ littermates; mutant DRGs (M) were pooled from two mutant littermates. Note the obvious increase in dephosphorylated NF-H (revealed by the lower band labeled by NF-H, a polyclonal antibody against the COOH terminus of NF-H, and by antibody SMI-32) as well as the increase in NF-M, NF-L, and peripherin. (B) Western blot of brain and sciatic nerve from 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. 20 μg of proteins of mutant and control (II/+) littermates was loaded in each lane. Note the clear reduction of KIF5A protein in the mutant. The levels of NFs and peripherin were not significantly changed in the mutant brain and sciatic nerve. (C and D) Immunostaining of DRG sensory neurons of 7.5-mo-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. Double staining with KIF5A (green) and SMI-32 (recognizes dephosphorylated NF-H, red) was performed on a mutant and a control littermate. (C) Control DRG staining. Bottom panel, higher magnification. (D) Mutant DRG staining, higher magnification in the lower panel. Note the apparent intense SMI-32 staining in some DRG neurons. Bars, 200 μm.
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fig6: Accumulation of NF proteins in DRG sensory neuron cell bodies of KIF5A/KIF5Aflox; Cresynapsin mutant mice. (A) Western blot analyses of DRGs from the first cohort of 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. In the litter shown, control DRGs (C) were pooled from one II/+ and one Cre/+ littermates; mutant DRGs (M) were pooled from two mutant littermates. Note the obvious increase in dephosphorylated NF-H (revealed by the lower band labeled by NF-H, a polyclonal antibody against the COOH terminus of NF-H, and by antibody SMI-32) as well as the increase in NF-M, NF-L, and peripherin. (B) Western blot of brain and sciatic nerve from 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. 20 μg of proteins of mutant and control (II/+) littermates was loaded in each lane. Note the clear reduction of KIF5A protein in the mutant. The levels of NFs and peripherin were not significantly changed in the mutant brain and sciatic nerve. (C and D) Immunostaining of DRG sensory neurons of 7.5-mo-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. Double staining with KIF5A (green) and SMI-32 (recognizes dephosphorylated NF-H, red) was performed on a mutant and a control littermate. (C) Control DRG staining. Bottom panel, higher magnification. (D) Mutant DRG staining, higher magnification in the lower panel. Note the apparent intense SMI-32 staining in some DRG neurons. Bars, 200 μm.

Mentions: To test whether diminished axonal caliber was caused by reduced NF transport into, and accumulation within, sensory axons, we used immunoblotting of extracts of DRGs (comprised primarily of the cell bodies of sensory neurons). As expected if KIF5A was needed for efficient NF transport, NF subunits accumulated in KIF5A-depleted DRGs, while KIF5A levels were reduced to 20% of that in the controls (three litters were examined; one representative litter is shown in Fig. 6 A). A polyclonal antibody against the COOH terminus of NF-H, which recognizes NF-H independent of its phosphorylation state, revealed a significant (∼150%) increase in total NF-H protein in mutant DRGs. The amount of NF-L was increased in mutant DRGs by an equivalent amount (∼150%), while NF-M amounts were increased but to a lower extent (by 20–50%). The increase in NF-H was confirmed with the monoclonal antibody SMI-32, which is specific for the dephosphorylated isoform of NF-H, whereas a monoclonal antibody specific for phosphorylated NF-H (SMI-31) revealed a slight (10–15%) decrease. The type III intermediate filament protein peripherin was also increased in the mutant DRGs, whereas the neuron-specific βIII-tubulin isoform was not. Increases in NF-H, NF-M, NF-L, and peripherin were selective for the DRGs. No obvious changes in the amounts of NF-H, NF-M, NF-L, or peripherin were found in the brain and sciatic nerve (Fig. 6 B).


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)

Accumulation of NF proteins in DRG sensory neuron cell bodies of KIF5A/KIF5Aflox; Cresynapsin mutant mice. (A) Western blot analyses of DRGs from the first cohort of 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. In the litter shown, control DRGs (C) were pooled from one II/+ and one Cre/+ littermates; mutant DRGs (M) were pooled from two mutant littermates. Note the obvious increase in dephosphorylated NF-H (revealed by the lower band labeled by NF-H, a polyclonal antibody against the COOH terminus of NF-H, and by antibody SMI-32) as well as the increase in NF-M, NF-L, and peripherin. (B) Western blot of brain and sciatic nerve from 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. 20 μg of proteins of mutant and control (II/+) littermates was loaded in each lane. Note the clear reduction of KIF5A protein in the mutant. The levels of NFs and peripherin were not significantly changed in the mutant brain and sciatic nerve. (C and D) Immunostaining of DRG sensory neurons of 7.5-mo-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. Double staining with KIF5A (green) and SMI-32 (recognizes dephosphorylated NF-H, red) was performed on a mutant and a control littermate. (C) Control DRG staining. Bottom panel, higher magnification. (D) Mutant DRG staining, higher magnification in the lower panel. Note the apparent intense SMI-32 staining in some DRG neurons. Bars, 200 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172877&req=5

fig6: Accumulation of NF proteins in DRG sensory neuron cell bodies of KIF5A/KIF5Aflox; Cresynapsin mutant mice. (A) Western blot analyses of DRGs from the first cohort of 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. In the litter shown, control DRGs (C) were pooled from one II/+ and one Cre/+ littermates; mutant DRGs (M) were pooled from two mutant littermates. Note the obvious increase in dephosphorylated NF-H (revealed by the lower band labeled by NF-H, a polyclonal antibody against the COOH terminus of NF-H, and by antibody SMI-32) as well as the increase in NF-M, NF-L, and peripherin. (B) Western blot of brain and sciatic nerve from 3-wk-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. 20 μg of proteins of mutant and control (II/+) littermates was loaded in each lane. Note the clear reduction of KIF5A protein in the mutant. The levels of NFs and peripherin were not significantly changed in the mutant brain and sciatic nerve. (C and D) Immunostaining of DRG sensory neurons of 7.5-mo-old KIF5A/KIF5Aflox; Cresynapsin mutant mice. Double staining with KIF5A (green) and SMI-32 (recognizes dephosphorylated NF-H, red) was performed on a mutant and a control littermate. (C) Control DRG staining. Bottom panel, higher magnification. (D) Mutant DRG staining, higher magnification in the lower panel. Note the apparent intense SMI-32 staining in some DRG neurons. Bars, 200 μm.
Mentions: To test whether diminished axonal caliber was caused by reduced NF transport into, and accumulation within, sensory axons, we used immunoblotting of extracts of DRGs (comprised primarily of the cell bodies of sensory neurons). As expected if KIF5A was needed for efficient NF transport, NF subunits accumulated in KIF5A-depleted DRGs, while KIF5A levels were reduced to 20% of that in the controls (three litters were examined; one representative litter is shown in Fig. 6 A). A polyclonal antibody against the COOH terminus of NF-H, which recognizes NF-H independent of its phosphorylation state, revealed a significant (∼150%) increase in total NF-H protein in mutant DRGs. The amount of NF-L was increased in mutant DRGs by an equivalent amount (∼150%), while NF-M amounts were increased but to a lower extent (by 20–50%). The increase in NF-H was confirmed with the monoclonal antibody SMI-32, which is specific for the dephosphorylated isoform of NF-H, whereas a monoclonal antibody specific for phosphorylated NF-H (SMI-31) revealed a slight (10–15%) decrease. The type III intermediate filament protein peripherin was also increased in the mutant DRGs, whereas the neuron-specific βIII-tubulin isoform was not. Increases in NF-H, NF-M, NF-L, and peripherin were selective for the DRGs. No obvious changes in the amounts of NF-H, NF-M, NF-L, or peripherin were found in the brain and sciatic nerve (Fig. 6 B).

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