Motor Neuron Synapse and Axon Defects in a C. elegans Alpha-Tubulin Mutant

Regulation of microtubule dynamics underlies many fundamental cellular mechanisms including cell division, cell motility, and transport. In neurons, microtubules play key roles in cell migration, axon outgrowth, control of axon and synapse growth, and the regulated transport of vesicles and structural components of synapses. Loss of synapse and axon integrity and disruption of axon transport characterize many neurodegenerative diseases. Recently, mutations that specifically alter the assembly or stability of microtubules have been found to directly cause neurodevelopmental defects or neurodegeneration in vertebrates. We report here the characterization of a missense mutation in the C-terminal domain of C. elegans alpha-tubulin, tba-1(ju89), that disrupts motor neuron synapse and axon development. Mutant ju89 animals exhibit reduction in the number and size of neuromuscular synapses, altered locomotion, and defects in axon extension. Although null mutations of tba-1 show a nearly wild-type pattern, similar axon outgrowth defects were observed in animals lacking the beta-tubulin TBB-2. Genetic analysis reveals that tba-1(ju89) affects synapse development independent of its role in axon outgrowth. tba-1(ju89) is an altered function allele that most likely perturbs interactions between TBA-1 and specific microtubule-associated proteins that control microtubule dynamics and transport of components needed for synapse and axon growth

Increased serum TSH response to TRH in cystic fibrosis.

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The doublecortin-related gene zyg-8 is a microtubule organizer in Caenorhabditis elegans neurons.

International audienceDoublecortin-domain containing (DCDC) genes play key roles in the normal and pathological development of the human brain cortex. The origin of the cellular specialization and the functional redundancy of these microtubule (MT)-associated proteins (MAPs), especially those of Doublecortin (DCX) and Doublecortin-like kinase (DCLKs) genes, is still unclear. The DCX domain displays the ability to control MT architecture and bundling. However, the physiological significance of such properties is not fully understood. To address these issues, we sought post-mitotic roles of zyg-8, the sole representative of the DCX-DCLK subfamily of genes in C. elegans. Previously, zyg-8 has been shown to control anaphase-spindle positioning in one-cell stage embryos, but functions of the gene later in development have not been investigated.Here we show that wild-type zyg-8 is required beyond early embryonic divisions for proper development, spontaneous locomotion and touch sensitivity of adult worms. Consistently, we find zyg-8 expression in the six touch receptor neurons (TRNs), as well as in a subset of other neuronal and non neuronal cells. In TRNs and motoneurons, zyg-8 controls cell body shape/polarity and process outgrowth and morphology. Ultrastructural analysis of mutant animals reveals that zyg-8 promotes structural integrity, length and number of individual MTs, as well as their bundled organization in TRNs, with no impact on MT architecture

GABAergic Motor Neuron Synapses are Defective in <i>tba-1(ju89</i>) Mutants.

<p>(A) Diagram of <i>en passant</i> neuromuscular synapses in <i>C. elegans</i>. Dots represent synapses; dorsal D neurons (DDs) form synapses onto dorsal body-wall muscles, and ventral D neurons (VDs) synapse with ventral muscles. Presynaptic termini of the inhibitory GABAergic type D motor neurons are visualized with the synaptic vesicle marker P<i>_unc-25</i>SNB-1::GFP. Each GFP puncta visible by fluorescence microscopy corresponds to the cumulative signal from all GFP-tagged vesicles at an individual synapse. (B) Morphology of the six DD motor neurons. Cell bodies are located in the ventral nerve cord. Each D neuron extends a ventral process, branches to form a commissure, and bifurcates at the dorsal nerve cord to form a dorsal process along the dorsal nerve cord. At the distal tip of each process, gap junctions are formed with adjacent DD neurons <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009655#pone.0009655-White1" target="_blank">[17]</a>. Expression pattern of SNB-1::GFP in wild-type (C) and mutant (D) dorsal nerve cord. Irregular size puncta and reduced numbers of puncta are evident in <i>ju89</i> mutants. An arrow designates the position of a DD commissure. (E) SNT-1 (synaptotagmin) expression in the dorsal nerve cord of wild-type and (F) <i>ju89</i> mutant animals. UNC-10::GFP expression along the dorsal nerve cord of (G) wild-type and (H) <i>ju89</i> worms. (I) Expression of the GABA-B receptor subunit, UNC-49::GFP, along dorsal muscles of wild-type animals and (J) <i>ju89</i> mutant worms. Gaps in UNC-49::GFP expression demonstrate the lack of post-synaptic structures in these regions. Wild-type <i>C. elegans</i> (K) have a larger body size than <i>tba-1(ju89)</i> (L). <i>C. elegans</i> move in a wavelike pattern. <i>ju89</i> mutants are uncoordinated, and the amplitude of the wave pattern is severely reduced compared to wild-type animals.</p

Ultrastructure of <i>tba-1(ju89)</i> Motor Neuron Synapses.

<p>The ultrastructure of one adult wild-type and two <i>ju89</i> adult mutant worm were examined by EM (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009655#s4" target="_blank">Methods</a>). 1500 continuous sections of 50 nm each were collected from 1 wild-type adult animal and 400 sections from each of two <i>ju89</i> adult mutant worms. Sections were collected in the anterior of each animal between the nerve ring and the vulva. Inhibitory GABAergic motor neurons form synapses directly onto dorsal and ventral muscles; excitatory cholinergic motor neurons form dyadic synapses with muscles and a GABAergic motor neuron dendrite. Representative GABAergic motor neuron synapses with body wall muscle arms in (A) wild-type and (C) mutant <i>tba-1(ju89)</i> animals, and cholinergic motor neuron synapses in (B) wild-type and (D) <i>ju89</i> worms. Fewer synaptic vesicles are visible in both types of synapses in the mutants. (E) The number of synaptic vesicles per active zone (p<.05), (F) length of the active zone in nm (p<.05), and (G) number of synaptic vesicles/active zone length for the cholinergic motor neuron synapses in one animal are shown. Cholinergic synapse size is reduced in the <i>ju89</i> mutant. Error bars: S.E.M.</p

<i>ju89</i> is a Novel Allele of <i>C. elegans</i> Alpha-Tubulin <i>tba-1</i>.

<p>(A) location of <i>ju89</i> on <i>C. elegans</i> chromosome I based on genetic map data, (B) Transformation rescue of <i>ju89</i> by F24E4.8 (<i>tba-1)</i>. The minimal rescuing activity for <i>ju89</i> was narrowed to an overlapping region of cosmid F26E4 and fosmid HO1I17 containing the <i>tba-1-drsh-1</i> operon. <i>pCZ485</i>, a 4.5 kb subclone containing upstream sequence and only the <i>tba-1</i> coding region (F26E4.8) is sufficient to rescue the SNB-1:GFP and locomotion defects of <i>ju89</i> mutants. (C) <i>ju89</i> is a missense mutation that converts a conserved glycine to arginine in the H11–H12 loop of the TBA-1 C-terminus. C<i>-</i>terminal domain structure based on Lowe et al., 2001. Sequences used for the alignment are <i>C. elegans</i> TBA-1 (CAB03001), TBA-2 (CAB16856), human TUBA3/TUBA1A (NP006000), <i>Mus musculus</i> TUBA1A (AAH83344), and <i>Danio rerio</i> (NP919369). (D) Crystal structure of alpha-beta tubulin dimer generated by Polyview based on Nogales et al, 1998 and Lowe et al., 2001 (PDB#1JFF). Red highlights the residue altered in <i>ju89</i> mutants (G414 in TBA-1 and G416 in TUBA1A) located at the beginning of helix 12. Blue highlights the R402 residue mutated in human lissencephaly (R400 in TBA-1) in the H11–H12 loop adjacent to H11.</p

Synapse Loss in DD Axons of <i>tba-1(ju89)</i> Animals.

<p>The DD neuron axon marker P<i>_flp-13</i>::GFP was coexpressed with a presynaptic marker P<i>_unc-25</i> mCherry::RAB-3 in wild-type (A, C, E) and mutant (B, D, F) <i>tba-1(ju89)</i> animals (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009655#s4" target="_blank">Methods</a>). Axon extension was similar in both the wild-type (A) and mutant (B) axons depicted. mCherry::RAB-3 puncta were reduced or missing in the <i>tba-1(ju89)</i> mutant axon (D, F) compared to the wild-type worm (C, E). The bracket in (F) delineates a region in which mCherry::RAB-3 puncta are missing or greatly reduced in size in the mutant. Scale bar 20 µM.</p

SNB-1::GFP Expression by D Motor Neurons.

<p>Genetic analysis of tubulin mutant synaptic defects based on expression pattern of the synaptic vesicle marker Synaptobrevin::GFP (SNB-1::GFP). Young adult worms were scored for the number of SNB-1::GFP puncta in the ventral and dorsal nerve cords. Animals carrying the <i>ju89</i> mutant chromosome in trans to a wild-type (<i>+/ju89)</i> or deficiency (<i>Df9/ju89)</i> chromosome or the <i>tba-1</i> deletion allele (<i>ok1135/ju89</i>) exhibit partial defects. Deficiency/+ and <i>ok1135/+</i> heterozygotes appear wild-type. Animals that express an extrachromosomal array containing <i>tba-1</i> amplified from <i>ju89</i> animals, <i>Extba-1(R414)</i> resemble <i>ju89</i> mutants. Animals homozygous for the null <i>tbb-2</i> allele <i>(gk207)</i> have a similar decrease in total SNB-1::GFP as single mutants and in combination with <i>tba-1(ju89)</i>. Mean ± sd.</p><p>*p<.001, two-tailed T test.</p><p>**Synapse loss is strongly enhanced in <i>tba-1(ju89); tbb-1 (gk207)</i> animals due to loss of axons in the dorsal and ventral nerve cord.</p

DD Neuron Outgrowth and Stability.

<p>The axon morphology of the six dorsal D (DD) motor neurons was visualized with P<i>_flp-13</i>GFP <i>(juIs145)</i> and P<i>_unc-25</i>GFP (<i>juIs76)</i> transgenes in L1 larvae and with P<i>_flp-13</i>GFP in young adults (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009655#s4" target="_blank">Methods</a>). All 6 DD axons were evaluated in animals expressing <i>juIs76</i>, and DD1-DD5 axons were examined in worms that expressed the <i>juIs145</i> reporter. Animals were scored for gaps along the dorsal nerve cord, commissures that stopped short of the dorsal nerve cord, and abnormal branching morphology where commissures join the dorsal nerve cord (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009655#pone-0009655-g003" target="_blank">Figure 3</a>). <i>tbb-2(gk130)</i> mutants and <i>tba-1(ju89);tbb-2(gk130)</i> animals exhibit similar DD axon defects, whereas axon defects are increased in <i>tba-1(ju89);tbb-1(gk207)</i> double mutants.</p><p>*Two-tailed Z-test, p<.005.</p><p>**p<.05.</p