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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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Synaptic vesicle mistargeting and transport defects in the gm379 mutant.(A) A schematic diagram of the C. elegans ALM and PLM neurons and their synapses. The “+” and “−“ signs indicate the dominant microtubule orientation in the anterior ALM and PLM processes. (B-G, B′-G′) Synaptic vesicles in live animals were visualized and quantified with jsIs821(Pmec-7::GFP::RAB-3) (B-E, B′-E′) from the nerve ring synapses (B, B′), ALM soma (C, C′), PLM synapses in the ventral nerve cord (D, D′), PLM soma (E, E′, asterisks) and the PLM posterior process (E, E′). SV mistargeting was independently verified with jsIs37(Pmec-7::SNB-1::GFP) (F, F′) and jsIs219(Psng-1::SNG-1::GFP) (G, G′). Arrows, mistargeted SVs. Scale bar  = 5 µm. (H) SV abundance measured by GFP::RAB-3 quantification (mean ± S.E.M.) in respective locations of the touch neuron circuit. N = 10 animals for each genotype at each time point. (I) Percentage of animals with SV mistargeting. (J) Kymograph of SV in the PLM posterior process of the mutant.
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pgen-1004715-g001: Synaptic vesicle mistargeting and transport defects in the gm379 mutant.(A) A schematic diagram of the C. elegans ALM and PLM neurons and their synapses. The “+” and “−“ signs indicate the dominant microtubule orientation in the anterior ALM and PLM processes. (B-G, B′-G′) Synaptic vesicles in live animals were visualized and quantified with jsIs821(Pmec-7::GFP::RAB-3) (B-E, B′-E′) from the nerve ring synapses (B, B′), ALM soma (C, C′), PLM synapses in the ventral nerve cord (D, D′), PLM soma (E, E′, asterisks) and the PLM posterior process (E, E′). SV mistargeting was independently verified with jsIs37(Pmec-7::SNB-1::GFP) (F, F′) and jsIs219(Psng-1::SNG-1::GFP) (G, G′). Arrows, mistargeted SVs. Scale bar  = 5 µm. (H) SV abundance measured by GFP::RAB-3 quantification (mean ± S.E.M.) in respective locations of the touch neuron circuit. N = 10 animals for each genotype at each time point. (I) Percentage of animals with SV mistargeting. (J) Kymograph of SV in the PLM posterior process of the mutant.

Mentions: In wild-type C. elegans, the bilaterally symmetric touch receptor neurons ALM and PLM develop a single anterior process that forms synapses in the nerve ring and in the ventral nerve cord, respectively (Figure 1A). Touch neuron synapses are enriched in RAB-3-(+) synaptic vesicles (SVs), the active zone protein SYD-2/Liprin-α, and mitochondria (Figure 1B, 1D, and S1) [19], [20]. The PLM neurons also have a short posterior process that does not form synapses. In an EMS mutagenesis screen (see Materials and Methods), we identified gm379, a mutant with prominent SV phenotypes in the touch neurons (Figure 1B′-1G′). gm379 animals lacked RAB-3-(+) SVs at the touch neuron synapses, and instead SVs accumulated in the neuronal soma (Figure 1B′-1E′, 1H). We refer to these as SV transport defects for the rest of the paper. Surprisingly, SVs were also redirected to the PLM posterior process, a phenotype that we call SV mistargeting (Figure 1E′, 1I). These results were confirmed using two other SV reporters, jsIs37(Pmec-7::SNB-1::GFP) that marked the SV membrane protein synaptobrevin/SNB-1, and jsIs219(Psng-1::SNG-1::GFP) labeling another SV protein synaptogyrin/SNG-1, with GFP (Figure 1F-1G′). These ectopic SVs showed very limited motility, and many were stationary (Figure 1J). We followed gm379 mutants through development, and confirmed that SV transport defects and mistargeting were present at early larval stages and progressively worsened (Figure 1H). The transport and targeting of synaptic active zone protein SYD-2 was affected to a much milder degree, and surprisingly, SYD-2 failed to mistarget to the PLM posterior process (Figure S1). The dissociation in the mutant phenotypes of SV and active zone proteins indicates that the gm379 mutation caused relatively specific defects in SV targeting rather than generally impaired axon transport or induced ectopic synapse formation.


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)

Synaptic vesicle mistargeting and transport defects in the gm379 mutant.(A) A schematic diagram of the C. elegans ALM and PLM neurons and their synapses. The “+” and “−“ signs indicate the dominant microtubule orientation in the anterior ALM and PLM processes. (B-G, B′-G′) Synaptic vesicles in live animals were visualized and quantified with jsIs821(Pmec-7::GFP::RAB-3) (B-E, B′-E′) from the nerve ring synapses (B, B′), ALM soma (C, C′), PLM synapses in the ventral nerve cord (D, D′), PLM soma (E, E′, asterisks) and the PLM posterior process (E, E′). SV mistargeting was independently verified with jsIs37(Pmec-7::SNB-1::GFP) (F, F′) and jsIs219(Psng-1::SNG-1::GFP) (G, G′). Arrows, mistargeted SVs. Scale bar  = 5 µm. (H) SV abundance measured by GFP::RAB-3 quantification (mean ± S.E.M.) in respective locations of the touch neuron circuit. N = 10 animals for each genotype at each time point. (I) Percentage of animals with SV mistargeting. (J) Kymograph of SV in the PLM posterior process of the mutant.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230729&req=5

pgen-1004715-g001: Synaptic vesicle mistargeting and transport defects in the gm379 mutant.(A) A schematic diagram of the C. elegans ALM and PLM neurons and their synapses. The “+” and “−“ signs indicate the dominant microtubule orientation in the anterior ALM and PLM processes. (B-G, B′-G′) Synaptic vesicles in live animals were visualized and quantified with jsIs821(Pmec-7::GFP::RAB-3) (B-E, B′-E′) from the nerve ring synapses (B, B′), ALM soma (C, C′), PLM synapses in the ventral nerve cord (D, D′), PLM soma (E, E′, asterisks) and the PLM posterior process (E, E′). SV mistargeting was independently verified with jsIs37(Pmec-7::SNB-1::GFP) (F, F′) and jsIs219(Psng-1::SNG-1::GFP) (G, G′). Arrows, mistargeted SVs. Scale bar  = 5 µm. (H) SV abundance measured by GFP::RAB-3 quantification (mean ± S.E.M.) in respective locations of the touch neuron circuit. N = 10 animals for each genotype at each time point. (I) Percentage of animals with SV mistargeting. (J) Kymograph of SV in the PLM posterior process of the mutant.
Mentions: In wild-type C. elegans, the bilaterally symmetric touch receptor neurons ALM and PLM develop a single anterior process that forms synapses in the nerve ring and in the ventral nerve cord, respectively (Figure 1A). Touch neuron synapses are enriched in RAB-3-(+) synaptic vesicles (SVs), the active zone protein SYD-2/Liprin-α, and mitochondria (Figure 1B, 1D, and S1) [19], [20]. The PLM neurons also have a short posterior process that does not form synapses. In an EMS mutagenesis screen (see Materials and Methods), we identified gm379, a mutant with prominent SV phenotypes in the touch neurons (Figure 1B′-1G′). gm379 animals lacked RAB-3-(+) SVs at the touch neuron synapses, and instead SVs accumulated in the neuronal soma (Figure 1B′-1E′, 1H). We refer to these as SV transport defects for the rest of the paper. Surprisingly, SVs were also redirected to the PLM posterior process, a phenotype that we call SV mistargeting (Figure 1E′, 1I). These results were confirmed using two other SV reporters, jsIs37(Pmec-7::SNB-1::GFP) that marked the SV membrane protein synaptobrevin/SNB-1, and jsIs219(Psng-1::SNG-1::GFP) labeling another SV protein synaptogyrin/SNG-1, with GFP (Figure 1F-1G′). These ectopic SVs showed very limited motility, and many were stationary (Figure 1J). We followed gm379 mutants through development, and confirmed that SV transport defects and mistargeting were present at early larval stages and progressively worsened (Figure 1H). The transport and targeting of synaptic active zone protein SYD-2 was affected to a much milder degree, and surprisingly, SYD-2 failed to mistarget to the PLM posterior process (Figure S1). The dissociation in the mutant phenotypes of SV and active zone proteins indicates that the gm379 mutation caused relatively specific defects in SV targeting rather than generally impaired axon transport or induced ectopic synapse formation.

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