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Genetic analysis of a novel tubulin mutation that redirects synaptic vesicle targeting and causes neurite degeneration in C. elegans.

Hsu JM, Chen CH, Chen YC, McDonald KL, Gurling M, Lee A, Garriga G, Pan CL - PLoS Genet. (2014)

Bottom Line: This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin.By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal.This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress.

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Axon pathology of the touch neurons in the gm379 mutant.(A-B) Touch neurons in live animals were visualized with zdIs5(Pmec-4::GFP). Asterisks, ALM soma. Scale bar  = 5 µm or 2 µm (insets). (A) ALM processes in the mutant appeared beaded at L2 and developed triangular-shaped swelling at L4 to adult. Anterior is to the left. (B) Mitochondria, labeled with Pmec-7::mito::mCherry, often accumulated at axon swellings. (C) Snapshots of PLM neurite during muscle contraction from a gm379 mutant partially immobilized with microbeads. Arrows and asterisks are buckling and neurite swellings, respectively, that changed their morphology during muscle contraction. Muscles contracted between t  = 2 second and t  = 5 second. (D) ALM neurites of the gm379 or the unc-54; gm379 mutants. (E) Quantification (mean ± S.E.M.) of neurite swellings for single ALM or PLM neurons. Age of the animals was indicated as days in adulthood. *, p≤0.01, **, p<0.0001, Mann-Whitney U test. (F) Longitudinal images of a single ALM neuron from L2 to middle adulthood. Arrows marked the same site over time. The animal died at day 10. Scale bar  = 5 µm. Asterisks, ALM soma. (G) ALM neurites of the wild type, gm379 and the unc-54; gm379 mutants at day 16. Asterisks are ALM soma. Arrows mark neurite breaks and degeneration. (H) Quantification (mean ± S.E.M.) of neurodegeneration. *, p<0.05, **, p<0.0001, Fisher's exact test.
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pgen-1004715-g002: Axon pathology of the touch neurons in the gm379 mutant.(A-B) Touch neurons in live animals were visualized with zdIs5(Pmec-4::GFP). Asterisks, ALM soma. Scale bar  = 5 µm or 2 µm (insets). (A) ALM processes in the mutant appeared beaded at L2 and developed triangular-shaped swelling at L4 to adult. Anterior is to the left. (B) Mitochondria, labeled with Pmec-7::mito::mCherry, often accumulated at axon swellings. (C) Snapshots of PLM neurite during muscle contraction from a gm379 mutant partially immobilized with microbeads. Arrows and asterisks are buckling and neurite swellings, respectively, that changed their morphology during muscle contraction. Muscles contracted between t  = 2 second and t  = 5 second. (D) ALM neurites of the gm379 or the unc-54; gm379 mutants. (E) Quantification (mean ± S.E.M.) of neurite swellings for single ALM or PLM neurons. Age of the animals was indicated as days in adulthood. *, p≤0.01, **, p<0.0001, Mann-Whitney U test. (F) Longitudinal images of a single ALM neuron from L2 to middle adulthood. Arrows marked the same site over time. The animal died at day 10. Scale bar  = 5 µm. Asterisks, ALM soma. (G) ALM neurites of the wild type, gm379 and the unc-54; gm379 mutants at day 16. Asterisks are ALM soma. Arrows mark neurite breaks and degeneration. (H) Quantification (mean ± S.E.M.) of neurodegeneration. *, p<0.05, **, p<0.0001, Fisher's exact test.

Mentions: In addition to SV transport defects, the gm379 mutant touch neurons had progressive neurite swelling and misshapen soma (Figure 2A and S2A). Neurite defects evolved from small beadings at early larval stages into triangular-shaped swellings in L4 and adult animals, and mitochondria were frequently found to be present at the swellings (Figure 2A, 2B and S2A). These swellings were dynamic in morphology, as movements often induced reversible buckling of the neurite and changed the width and height of the swellings (Figure 2C and Video S1), which was similar to what had been described earlier for the tubulin acetyltransferase mutant mec-17[21]. Neurite buckling or swelling were never seen in the wild type even under maximal muscle contraction induced by levamisole. This observation suggests that the gm379 mutation rendered the touch neurite susceptible to deformation under mechanical strain, a phenotype that was also seen when the membrane skeleton protein UNC-70/β-spectrin was lost [22]. We therefore test whether genetic paralysis of the animals suppresses neurite defects of the gm379 mutant. Mutation in the muscle myosin gene unc-54 almost completely paralyzed the animals, and it significantly reduced the number of neurite swellings in the gm379 mutant touch neurons (Figure 2D and 2E). This result implies that the gm379 mutation compromises the ability of the touch neurites to cope with mechanical stress.


Genetic analysis of a novel tubulin mutation that redirects synaptic vesicle targeting and causes neurite degeneration in C. elegans.

Hsu JM, Chen CH, Chen YC, McDonald KL, Gurling M, Lee A, Garriga G, Pan CL - PLoS Genet. (2014)

Axon pathology of the touch neurons in the gm379 mutant.(A-B) Touch neurons in live animals were visualized with zdIs5(Pmec-4::GFP). Asterisks, ALM soma. Scale bar  = 5 µm or 2 µm (insets). (A) ALM processes in the mutant appeared beaded at L2 and developed triangular-shaped swelling at L4 to adult. Anterior is to the left. (B) Mitochondria, labeled with Pmec-7::mito::mCherry, often accumulated at axon swellings. (C) Snapshots of PLM neurite during muscle contraction from a gm379 mutant partially immobilized with microbeads. Arrows and asterisks are buckling and neurite swellings, respectively, that changed their morphology during muscle contraction. Muscles contracted between t  = 2 second and t  = 5 second. (D) ALM neurites of the gm379 or the unc-54; gm379 mutants. (E) Quantification (mean ± S.E.M.) of neurite swellings for single ALM or PLM neurons. Age of the animals was indicated as days in adulthood. *, p≤0.01, **, p<0.0001, Mann-Whitney U test. (F) Longitudinal images of a single ALM neuron from L2 to middle adulthood. Arrows marked the same site over time. The animal died at day 10. Scale bar  = 5 µm. Asterisks, ALM soma. (G) ALM neurites of the wild type, gm379 and the unc-54; gm379 mutants at day 16. Asterisks are ALM soma. Arrows mark neurite breaks and degeneration. (H) Quantification (mean ± S.E.M.) of neurodegeneration. *, p<0.05, **, p<0.0001, Fisher's exact test.
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Related In: Results  -  Collection

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pgen-1004715-g002: Axon pathology of the touch neurons in the gm379 mutant.(A-B) Touch neurons in live animals were visualized with zdIs5(Pmec-4::GFP). Asterisks, ALM soma. Scale bar  = 5 µm or 2 µm (insets). (A) ALM processes in the mutant appeared beaded at L2 and developed triangular-shaped swelling at L4 to adult. Anterior is to the left. (B) Mitochondria, labeled with Pmec-7::mito::mCherry, often accumulated at axon swellings. (C) Snapshots of PLM neurite during muscle contraction from a gm379 mutant partially immobilized with microbeads. Arrows and asterisks are buckling and neurite swellings, respectively, that changed their morphology during muscle contraction. Muscles contracted between t  = 2 second and t  = 5 second. (D) ALM neurites of the gm379 or the unc-54; gm379 mutants. (E) Quantification (mean ± S.E.M.) of neurite swellings for single ALM or PLM neurons. Age of the animals was indicated as days in adulthood. *, p≤0.01, **, p<0.0001, Mann-Whitney U test. (F) Longitudinal images of a single ALM neuron from L2 to middle adulthood. Arrows marked the same site over time. The animal died at day 10. Scale bar  = 5 µm. Asterisks, ALM soma. (G) ALM neurites of the wild type, gm379 and the unc-54; gm379 mutants at day 16. Asterisks are ALM soma. Arrows mark neurite breaks and degeneration. (H) Quantification (mean ± S.E.M.) of neurodegeneration. *, p<0.05, **, p<0.0001, Fisher's exact test.
Mentions: In addition to SV transport defects, the gm379 mutant touch neurons had progressive neurite swelling and misshapen soma (Figure 2A and S2A). Neurite defects evolved from small beadings at early larval stages into triangular-shaped swellings in L4 and adult animals, and mitochondria were frequently found to be present at the swellings (Figure 2A, 2B and S2A). These swellings were dynamic in morphology, as movements often induced reversible buckling of the neurite and changed the width and height of the swellings (Figure 2C and Video S1), which was similar to what had been described earlier for the tubulin acetyltransferase mutant mec-17[21]. Neurite buckling or swelling were never seen in the wild type even under maximal muscle contraction induced by levamisole. This observation suggests that the gm379 mutation rendered the touch neurite susceptible to deformation under mechanical strain, a phenotype that was also seen when the membrane skeleton protein UNC-70/β-spectrin was lost [22]. We therefore test whether genetic paralysis of the animals suppresses neurite defects of the gm379 mutant. Mutation in the muscle myosin gene unc-54 almost completely paralyzed the animals, and it significantly reduced the number of neurite swellings in the gm379 mutant touch neurons (Figure 2D and 2E). This result implies that the gm379 mutation compromises the ability of the touch neurites to cope with mechanical stress.

Bottom Line: This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin.By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal.This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress.

Show MeSH
Related in: MedlinePlus