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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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Ultrastructural characterization of neuronal defects of the gm379 mutant.(A-C) Transverse EM sections showing the ultrastructure of touch neurons in the wild type (A and C, left panel) and in the mutant (B and C, right panel). (A, B) Microtubule numbers within the touch neuron process were preserved in the mutant. However, a mitochondrion (mt) was located eccentrically, resulting in a marked increase in focal axon diameter (marked by the thick red line). The extracellular matrix (arrows) that normally surrounds the touch neuron process seemed to break at axon swelling sites. Scale bar  = 150 nm. (C) Individual microtubule polymers from the wild type (left) and the mutant (right) both contained 15 protofilaments. The intraluminal electron-dense materials in the microtubule were also preserved in the mutant. Scale bar  = 10 nm. (D) Longitudinal EM section of an ALM neuron in the adult mutant, with anterior to the right. Scale bar  = 5 µm. (D1) Microtubule bundles were compressed (arrow) by some organelles of uncertain identity (asterisk) at the proximal axon segment. (D2) Microtubule polymers assumed straight, more wild-type appearance at certain regions of the axon. (D3) Microtubules were compressed and strangled at focal axon constriction (arrow), which profoundly distorted the axonal cytoskeleton. (D4) Focal mitochondrial accumulation (mt) distorted microtubule bundles (arrows), the latter assuming a curved configuration that was never seen in the wild-type axon. A small protrusion (asterisk) from the axonal membrane contained small membrane-bound profiles of unknown identity. Scale bars  = 0.5 µm. CB, cell body of ALM; Cu, cuticle; Hyp, hypodermal cell; MT, microtubules.
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pgen-1004715-g003: Ultrastructural characterization of neuronal defects of the gm379 mutant.(A-C) Transverse EM sections showing the ultrastructure of touch neurons in the wild type (A and C, left panel) and in the mutant (B and C, right panel). (A, B) Microtubule numbers within the touch neuron process were preserved in the mutant. However, a mitochondrion (mt) was located eccentrically, resulting in a marked increase in focal axon diameter (marked by the thick red line). The extracellular matrix (arrows) that normally surrounds the touch neuron process seemed to break at axon swelling sites. Scale bar  = 150 nm. (C) Individual microtubule polymers from the wild type (left) and the mutant (right) both contained 15 protofilaments. The intraluminal electron-dense materials in the microtubule were also preserved in the mutant. Scale bar  = 10 nm. (D) Longitudinal EM section of an ALM neuron in the adult mutant, with anterior to the right. Scale bar  = 5 µm. (D1) Microtubule bundles were compressed (arrow) by some organelles of uncertain identity (asterisk) at the proximal axon segment. (D2) Microtubule polymers assumed straight, more wild-type appearance at certain regions of the axon. (D3) Microtubules were compressed and strangled at focal axon constriction (arrow), which profoundly distorted the axonal cytoskeleton. (D4) Focal mitochondrial accumulation (mt) distorted microtubule bundles (arrows), the latter assuming a curved configuration that was never seen in the wild-type axon. A small protrusion (asterisk) from the axonal membrane contained small membrane-bound profiles of unknown identity. Scale bars  = 0.5 µm. CB, cell body of ALM; Cu, cuticle; Hyp, hypodermal cell; MT, microtubules.

Mentions: Under serial thin-section electron microscopy, we found that the characteristic 15-protofilament microtubules of C. elegans touch neurons were preserved in the gm379 mutant, including those in the PLM posterior process (Figure 3A-C, S2B-D). Mitochondria could be found where touch neuron processes swelled abnormally (Figure 3B), consistent with our light microscopic observation (Figure 2B). In longitudinal sections, in contrast to the wild type, where neuronal microtubules formed long straight bundles, touch neuron microtubules in the gm379 animals curved focally at sites of organelle accumulation (Figure 3D). We observed bending and splitting of neuronal microtubule bundles at sites of mitochondria accumulation in focal axonal swellings (Figure 3D4). These ultrastructural studies suggest that the microtubule network of the touch neurons is abnormal in the gm379 mutant.


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)

Ultrastructural characterization of neuronal defects of the gm379 mutant.(A-C) Transverse EM sections showing the ultrastructure of touch neurons in the wild type (A and C, left panel) and in the mutant (B and C, right panel). (A, B) Microtubule numbers within the touch neuron process were preserved in the mutant. However, a mitochondrion (mt) was located eccentrically, resulting in a marked increase in focal axon diameter (marked by the thick red line). The extracellular matrix (arrows) that normally surrounds the touch neuron process seemed to break at axon swelling sites. Scale bar  = 150 nm. (C) Individual microtubule polymers from the wild type (left) and the mutant (right) both contained 15 protofilaments. The intraluminal electron-dense materials in the microtubule were also preserved in the mutant. Scale bar  = 10 nm. (D) Longitudinal EM section of an ALM neuron in the adult mutant, with anterior to the right. Scale bar  = 5 µm. (D1) Microtubule bundles were compressed (arrow) by some organelles of uncertain identity (asterisk) at the proximal axon segment. (D2) Microtubule polymers assumed straight, more wild-type appearance at certain regions of the axon. (D3) Microtubules were compressed and strangled at focal axon constriction (arrow), which profoundly distorted the axonal cytoskeleton. (D4) Focal mitochondrial accumulation (mt) distorted microtubule bundles (arrows), the latter assuming a curved configuration that was never seen in the wild-type axon. A small protrusion (asterisk) from the axonal membrane contained small membrane-bound profiles of unknown identity. Scale bars  = 0.5 µm. CB, cell body of ALM; Cu, cuticle; Hyp, hypodermal cell; MT, microtubules.
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pgen-1004715-g003: Ultrastructural characterization of neuronal defects of the gm379 mutant.(A-C) Transverse EM sections showing the ultrastructure of touch neurons in the wild type (A and C, left panel) and in the mutant (B and C, right panel). (A, B) Microtubule numbers within the touch neuron process were preserved in the mutant. However, a mitochondrion (mt) was located eccentrically, resulting in a marked increase in focal axon diameter (marked by the thick red line). The extracellular matrix (arrows) that normally surrounds the touch neuron process seemed to break at axon swelling sites. Scale bar  = 150 nm. (C) Individual microtubule polymers from the wild type (left) and the mutant (right) both contained 15 protofilaments. The intraluminal electron-dense materials in the microtubule were also preserved in the mutant. Scale bar  = 10 nm. (D) Longitudinal EM section of an ALM neuron in the adult mutant, with anterior to the right. Scale bar  = 5 µm. (D1) Microtubule bundles were compressed (arrow) by some organelles of uncertain identity (asterisk) at the proximal axon segment. (D2) Microtubule polymers assumed straight, more wild-type appearance at certain regions of the axon. (D3) Microtubules were compressed and strangled at focal axon constriction (arrow), which profoundly distorted the axonal cytoskeleton. (D4) Focal mitochondrial accumulation (mt) distorted microtubule bundles (arrows), the latter assuming a curved configuration that was never seen in the wild-type axon. A small protrusion (asterisk) from the axonal membrane contained small membrane-bound profiles of unknown identity. Scale bars  = 0.5 µm. CB, cell body of ALM; Cu, cuticle; Hyp, hypodermal cell; MT, microtubules.
Mentions: Under serial thin-section electron microscopy, we found that the characteristic 15-protofilament microtubules of C. elegans touch neurons were preserved in the gm379 mutant, including those in the PLM posterior process (Figure 3A-C, S2B-D). Mitochondria could be found where touch neuron processes swelled abnormally (Figure 3B), consistent with our light microscopic observation (Figure 2B). In longitudinal sections, in contrast to the wild type, where neuronal microtubules formed long straight bundles, touch neuron microtubules in the gm379 animals curved focally at sites of organelle accumulation (Figure 3D). We observed bending and splitting of neuronal microtubule bundles at sites of mitochondria accumulation in focal axonal swellings (Figure 3D4). These ultrastructural studies suggest that the microtubule network of the touch neurons is abnormal in the gm379 mutant.

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