wts-1 mutants show the premature structural and functional decline of touch receptor neurons (TRNs)

(A, B) Representative images of TRNs ([A] ALM and [B] PLM) in the wild-type and wts-1(ok753) mutant at the L4 stage. In the mutant, morphologically disrupted ALM and PLM were observed to exhibit abnormalities including ectopic swelling/branching on the processes and posterior outgrowth (arrowheads). (C) Newly developed ALM and PLM in the wild-type and the wts-1(ok753) mutant at the early L1 stage. The intact neuronal process is indicated using arrowheads. Intestinal expression of the WTS-1 rescue construct (Popt-2::WTS-1::GFP) is indicated using an arrow. (A–C) TRNs were visualized by expressing GFP under the control of the mec-7 promoter (muIs35[Pmec-7::GFP]) and anterior is to the left unless otherwise noted. Scale bar = 20 μm. (D) Penetration of defective TRNs of the wild-type and the wts-1(ok753) at different developmental stages. Neurons displaying any morphological abnormalities such as swelling, branching, somatic outgrowth and extended distal process were scored as defective neurons. In one experiment, 30 cells were observed for each strain and each stage and the experiments were repeated 3 times. Statistical significance was determined by a two-way ANOVA followed by Bonferroni’s post-test. (E) Quantified touch responses of the wild-type and the wts-1(ok753) mutant at L4 and 1st day of adulthood (1DA). N = 30. Statistical significance was determined by an unpaired t-test. ***, p < 0.001, **, p < 0 .01, *, p < 0.05, n.s, not significant, compared to WT or control, unless otherwise marked on graph. All data are presented as means ± SEM, unless otherwise noted.

yap-1 or egl-44 suppresses premature neuronal decline in wts-1 mutants.

(A, B) Representative images of (A) ALM and (B) PLM in wild-type, wts-1(ok753), wts-1(ok753); yap-1(tm1416) and wts-1(ok753); egl-44(ys41) at the L4 stage. Loss of yap-1 or egl-44 completely restored structural integrity of the wts-1 mutants. Scale bar = 20 μm. (C) Electron microscope images of wild-type, wts-1(ok753) and wts-1(ok753); yap-1(tm1416) at 1DA. TRNs are indicated using boxes. Scale bar = 500 nm. (D, E) Touch responses of wild-type, wts-1(ok753) and wts-1(ok753); yap-1(tm1416) at (D) L4 stage and (G) 1DA stage. For each strain and each stage, 90 animals were tested. Statistical significance was determined using a one-way ANOVA, followed by the Tukey’s multiple comparison test. (E) Survival curve of wild-type, wts-1(ok753) and wts-1(ok753); yap-1(tm1416). Worms were maintained at 20°C and all lines used for the lifespan analysis have muIs35.

wts-1-yap-1 act in a cell-autonomous manner to maintain touch neuronal integrity.

(A) The targeting regions for touch neuronal specific RNAi of wts-1. Sense and antisense (sas) genomic fragments were cloned under the TRNs specific promoter, Pmec-4. (B) Representative images and (C, D) quantified neuronal abnormalities of TRNs in the controls and wts-1-knockdowned animals. Both TRNs-specific-sas1- and sas2-based knock down of wts-1 efficiently induces neuronal abnormalities as seen in wts-1 mutant. (E–F) Touch neuronal rescue of YAP-1 is sufficient to re-induce neuronal dis-integrity in wts-1(ok753); yap-1(tm1416) mutant. (E) Representative images of TRNs of wts-1(ok753); yap-1(tm1416) mutant with the rescue construct or its sibling without transgenes. (F) Quantified results. 60 ALM and PLM of transgenic worms and their siblings were observed. (G, H) Touch neuronal overexpression of YAP-1 is sufficient to induce neuronal abnormalities in wild-type animals. Final concentration of Pmec-4::GFP::YAP-1 in the injection mixture is 100 ng/μl. (I, J) Quantified neuronal defects of (I) ALM and (J) PLM induced by overexpression of Pmec-4::GFP::YAP-1 at each concentration in the injection mixtures. (C, D, I, J) 90 ALM and PLM were observed in each of the three independent lines. Neuronal morphology was examined at L4 stage. Statistical significance was determined by a one-way ANOVA followed by the Dunnett’s multiple comparison test. An unpaired t-test was used for (F). Red: Expression of injection marker, Punc-122::RFP, white arrowhead :neuronal swelling, yellow arrowhead: shortened process. Scale bar = 20 μm.

Abnormal microtubules are responsible for neuronal dis-integrity observed in the wts-1 mutants.

(A–D) Reduced movement resulting from unc-54 knockdown diminishes ectopic swelling or branching in the wts-1(ok753) mutant. (A) unc-54 knockdown leads to uncoordinated movement of worms (yellow arrowhead). (B–C) unc-54 knockdown reduces the number of neuronal lesions on the ALM and PLM. (D) unc-54 knockdown slightly, but significantly, increases the percentages of intact PLM and not ALM. Neurons without any structural defects, including swelling or branching, were considered intact. (E) Quantified motor deficits in animals knocking down various muscle machinery genes. RNAi against unc-15, unc-54 or unc-95 impaired swimming behavior, whereas unc-89 and deb-1 made no differences compared to control. (F) Quantified morphological abnormalities of TRNs in muscle machinery-knockdown animals. Knockdown of unc-89 or deb-1 did not alleviate structural decline of both ALM and PLM. (G–K) Treatment with colchicine reduces ectopic neuronal swelling and increases the percentages of intact neurons in the wts-1(ok753) mutants. F1 progenies grown on the drug-contained plates were scored at (H, I) L4 stage or (J, K) 1DA. (L) Colchicine treatment improved touch responses of wts-1 mutant. The behavior test was done at L4 stage. Statistical significance was determined using an unpaired t-test (C, D, H, L) or a one-way ANOVA, followed by the Dunnett’s multiple comparison test (E, F). The total number of cells or animals analyzed is indicated in each column. Asterisks indicate differences from L4440-fed or drug-untreated-control neurons. Ectopic neuronal swelling and branching were labeled with white arrowheads. Scale bar = 20μm.

Hyper-stabilized microtubules might be responsible for age-associated morphological deformation of touch receptor neurons.

(A, B) Representative images of (A) ALM and (B) PLM of colchicine-treated or untreated wild-type worms. The age of worms is indicated in the image. (C, D) Quantified defects of (C) ALM or (D) PLM in colchicine-treated or untreated worms. Age-synchronized worms were transferred to drug-containing plates on the 1DA and phenotypes were scored on every 2nd, 4th, 6th, 8th, 10th, 15th and 20th day of adulthood. At every time point and in each group, 20 neurons were scored per one experiment and the experiments were repeated 3 times. (E) Colchicine treatment alleviates impaired touch responses of aged animals. Touch responses were scored at 2DA and 10DA. Statistical significance was determined by a two-way ANOVA, followed by the Bonferroni’s post-tests (C, D) or a one-way ANOVA with the Bonferroni’s multiple comparison test (E). (F) Survival curves of colchicine-treated and untreated control animals. Scale bar = 20μm.

Loss of spas-1, a microtubule-severing enzyme, results in premature structural decline.

(A, B) Representative images of defective TRNs of spas-1 mutant on the 2DA. Both deletion mutants, tm683 and ok1608, displayed age-associated morphological alterations of ALM or PLM including ectopic swelling and branching on the neuronal process (white arrowhead), somatic outgrowth and irregular shape of the cell body (yellow arrowhead) precociously. (C, D) Quantified results of structural defects of ALM or PLM in spas-1 mutants. At every time point, 20 neurons were scored and the experiments were repeated 3 times. (E) Results of the rescue experiment of premature neuronal degeneration of spas-1(tm683) with SPAS-1, human SPAST(wt) and human SPAST(K388R). In all cases, the C. elegans spas-1 promoter was used to induce the C-terminally mCherry tagged transgene. (F) Touch neuronal specific expression of SPAS-1 was sufficient to rescue neuronal defects of ALM. (E–F) Neuronal morphology were scored at 2DA. For each rescue construct, three independent transgenic lines were observed that yielded similar results and the results of one line are presented. (G, H) Quantified (G) structural defects of TRNs or (H) touch responses of colchicine-treated and untreated spas-1(tm683) mutant. Analyses were done at 2DA (N =30/one experiment, repeated 3times). Statistical significance was determined using a two-way ANOVA, followed by the Bonferroni’s multiple comparison test (C–D), a one-way ANOVA with the Dunnett’s multiple comparison test (E–F) or with the Turkey’s multiple comparison test (G). Unpaired t-test was used in (H). Scale bar = 20 μm.

wts-1-yap-1 affect neuronal integrity possibly by modulating microtubule stability

(A–C) Loss of dlk-1 or ptl-1 significantly mitigates the structural deformation of wts-1-mutant neurons. (A) Representative images of ALM (upper panels) and PLM (lower panels) of wts-1(ok753), dlk-1(km12), ptl-1(ok621), wts-1(ok753); dlk-1(km12) and wts-1(ok753); ptl-1(ok621) at L4 stage. Ectopic lesions were labeled with arrowhead. (B) Average number of ectopic lesions per neuronal process of each strain. (C) Percentage of intact neurons of each mutant. Loss of dlk-1 or ptl-1 protects TRNs of the wts-1 from premature deformation. (D, E) Loss of yap-1 worsen the neuronal deformation as seen in the ptl-1 mutant. (D) Unlike ptl-1(ok621) single mutant, ptl-1(ok621); yap-1(tm1416) double mutant exhibits severe deformation in ALM and PLM, such as irregular shape of cell body (yellow arrowhead) and ectopic branching (white arrowhead) of the neuronal process. (E) Percentage of undamaged touch neurons in ptl-1(ok621) and ptl-1(ok621); yap-1(tm1416). (A–E) Neurons were analyzed at the L4 stage. The number of scored neurons is indicated in each column. Statistical significance was determined using a one-way ANOVA, followed by the Dunnett’s multiple comparison test. Asterisks indicate differences from the wts-1 or the ptl-1 single mutant. Scale bar = 20 μm.