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, 116 (37), 18429-18434

Disease-associated Mutations Hyperactivate KIF1A Motility and Anterograde Axonal Transport of Synaptic Vesicle Precursors

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Disease-associated Mutations Hyperactivate KIF1A Motility and Anterograde Axonal Transport of Synaptic Vesicle Precursors

Kyoko Chiba et al. Proc Natl Acad Sci U S A.

Abstract

KIF1A is a kinesin family motor involved in the axonal transport of synaptic vesicle precursors (SVPs) along microtubules (MTs). In humans, more than 10 point mutations in KIF1A are associated with the motor neuron disease hereditary spastic paraplegia (SPG). However, not all of these mutations appear to inhibit the motility of the KIF1A motor, and thus a cogent molecular explanation for how KIF1A mutations lead to neuropathy is not available. In this study, we established in vitro motility assays with purified full-length human KIF1A and found that KIF1A mutations associated with the hereditary SPG lead to hyperactivation of KIF1A motility. Introduction of the corresponding mutations into the Caenorhabditis elegans KIF1A homolog unc-104 revealed abnormal accumulation of SVPs at the tips of axons and increased anterograde axonal transport of SVPs. Our data reveal that hyperactivation of kinesin motor activity, rather than its loss of function, is a cause of motor neuron disease in humans.

Keywords: KIF1A; UNC-104; axonal transport; hereditary spastic paraplegia; kinesin.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Complementation of an unc-104 mutant worm by human KIF1A cDNA. (A) Disease-associated mutations analyzed in this study. Mutations shown above and below in the KIF1A schematic indicate de novo and familial mutations, respectively. ID, patients with intellectual disability; no ID, patients without intellectual disability; SPG, patients with SPG. AD and AR indicate autosomal dominant and autosomal recessive mutations, respectively. (See also SI Appendix, Fig. S1.) (B and C) The phenotypes of unc-104(e1265) (B) and unc-104(e1265) expressing human KIF1A cDNA with the unc-104 promoter (C). (Scale bars: 1 mm.) (D) Bar graph showing worm movements. unc-104(e1265) mutant worms without KIF1A expression (control) exhibit severe movement defects compared with the WT N2 strain. Ectopic expression of WT KIF1A, KIF1A(V8M), KIF1A(A255V), and KIF1A(R350G) using the unc-104 promoter in the unc-104(e1265) background results in WT movement. Expression of other KIF1A with disease mutations (S58L, T99M, G199R, E253K, and R307Q) do not fully rescue unc-104(e1265) mutant worms. Asterisks indicate that the velocity is statistically not different from that of WT, indicating complete rescue (Tukey’s multiple comparison test). n = 60 worms for each genotype.
Fig. 2.
Fig. 2.
Single molecule motility assay in solution. (A) Coomassie blue-stained gel showing the purity of recombinant, full-length human KIF1A proteins. The arrow denotes the full-length protein (230 kDa). (B) Representative kymographs showing the motility of purified KIF1A::mScarlet along MTs in vitro. (Scale bars: vertical, 5 s; horizontal, 10 μm.) (C) Representative images showing KIF1A::mScarlet particles (magenta) on MTs (green). Wt, WT KIF1A; V8M, KIF1A(V8M); A255V, KIF1A(A255V); R350G, KIF1A(R350G). (Scale bar: 10 μm.) Note the strong increase in the number of mutant KIF1A molecules bound to MTs compared with the WT motors. (D) Landing rates of purified WT and mutant KIF1A motors: 0.002 ± 0.004/μm/s 1 nM KIF1A, 0.012 ± 0.007/μm/s for 10 nM KIF1A, 0.039 ± 0.012/μm/s for 1 nM KIF1A(V8M), 0.024 ± 0.008/μm/s for 1 nM KIF1A(A255V), and 0.025 ± 0.008/μm/s for 1 nM KIF1A(R350G). Lines show mean ± SD values, and each dot represents 1 counted molecule. n = 27 MTs from at least 3 trials per condition. ****Adjusted P < 0.0001 compared with WT KIF1A, Kruskal–Wallis 1-way ANOVA on ranks and Dunn’s multiple comparisons test. (E) Histograms showing the velocity of KIF1A mutants: 0.77 ± 0.48 μm/s for WT KIF1A, 1.6 ± 0.70 μm/s for KIF1A(V8M), 0.54 ± 0.35 μm/s for KIF1A(A255V), and 2.6 ± 0.85 μm/s for KIF1A(R350G), mean ± SD. n = 143, 260, 203, and 196 molecules, respectively, in >20 trials. ****Adjusted P < 0.0001 compared with WT KIF1A, 1-way ANOVA followed by Dunnett’s multiple comparison test. (See also SI Appendix, Fig. S2.)
Fig. 3.
Fig. 3.
Conservation of disease-associate residues and establishment of disease models. (A) Residues mutated in AD and AR hereditary SPG are conserved in human KIF1A and worm UNC-104. (See also SI Appendix, Fig. S1B.) (B and C) Localization of UNC-104::GFP (UNC-104), UNC-104(V6M)::GFP (V6M), and UNC-104(A252V)::GFP (A252V) in the ALM neuron in vivo. (B) Representative images of proximal and distal regions of the ALM neuron. CB, cell body. (Scale bars: 10 μm.) (C) Mean fluorescent intensity in the cell body and distal axon was measured, and the intensity of cell body:intensity of distal axon ratio was calculated in UNC-104::GFP (wt), UNC-104(V6M)::GFP(V6M), and UNC-104(A252V)::GFP (A252V) expressed in the ALM neuron. n = 20 cells from 20 transgenic animals. Data are mean ± SD. ****Adjusted P < 0.001 compared with WT control, 1-way ANOVA followed by Dunnett’s multiple comparison test. Whole-cell images are shown in SI Appendix, Fig. S3. (D) Body bending assay. The number of body thrashings in M9 buffer in a 1-min observation period were counted in WT N2 (N2), unc-104(ADSPGV6M) (V6M), and unc-104(ARSPGA252V) (A252V) young adult worms (4 d after hatching) and old adult worms (10 d after hatching). Each dot represents an animal. Data are mean ± SD. **Adjusted P < 0.01, ****adjusted P < 0.0001, 1-way ANOVA followed by Tukey’s multiple comparison test. n = 50 worms for each genotype. (E) 4-d-old WT, unc-104(ADSPGV6M), and unc-104(ARSPGA252V) worms were transferred to agarose plates containing 1 mM aldicarb, and worm viability was monitored and plotted. A representative result of 3 independent assays is shown. (See also SI Appendix, Fig. S4.)
Fig. 4.
Fig. 4.
Synaptic phenotypes of SPG disease models. (AG) The localization of SV marker SNB-1::GFP in the ALM neuron. A stably integrated marker, jsIs37, was used. (A) Schema showing the morphology of the ALM neuron. The large and small dotted boxes delineate the areas shown in BD and E, respectively. (B–E) Representative images of the head region (BD) and the tip of primary neurite (E) of WT and disease model worms. (Scale bars: 10 μm.) (F) Quantification of the phenotype in each animal. Young adult worms were scored. n = 40. (See also SI Appendix, Fig. S5.) (G) Quantification of the size of SNB-1::GFP puncta at the tip of axons in WT, unc-104(ADSPGV6M) heterozygote, unc-104(ADSPGV6M) homozygote, unc-104(ARSPGA252V) heterozygote, unc-104(ARSPGA252V) homozygote, dhc-1 homozygote, and dnc-1 homozygote. Only axonal tips showing the aberrant accumulation phenotype in F were compared. All data are plotted along with mean ± SD values. WT and unc-104(ARSPGA252V) heterozygote were not statistically analyzed because of no accumulation. ****Adjusted P < 0.0001. (HJ) Synaptic phenotype of DA9 neurons. Stably integrated marker wyIs85 was used. (H) Line scan images of DA9 neurons. Ten DA9 neurons from independent animals were scanned and aligned. (Scale bar: 5 μm.) Representative synaptic images in the worm body are shown in SI Appendix, Fig. S6 AC. (I) Plots of intersynaptic distances. Each dot represents each intersynaptic distance. n = 60 intersynapses from 3 independent animals. Data are mean ± SD. **Adjusted P < 0.01, Tukey’s multiple comparison test. (J) Mean fluorescent intensity of each dorsal synapse. Data are mean ± SD. *Adjusted P < 0.05, Tukey’s multiple comparison test. n = 102, 102, and 87 synapses from 5 independent animals in WT, unc-104(ADSPGV6M), and unc-104(ARSPGA252V), respectively.
Fig. 5.
Fig. 5.
Electron microscopy analyses of dorsal synapses in WT and unc-104(ADSPGV6M) young adult worms (4 d after hatching). (A and B) Representative images of dorsal synapses in WT (A) and unc-104(ADSPGV6M) (B). AZ, active zone. (Scale bars: 100 nm.) (C) Bar graphs showing the number of SVs in each synaptic bouton reconstituted by serial sections. n = 15 synapses from 2 WT worms and 24 synapses from 3 unc-104(ADSPGV6M) worms reconstituted from 100 serial ultrathin sections. Data are mean ± SEM. *P < 0.05, Welch’s t test. (See also SI Appendix, Fig. S6 DG).
Fig. 6.
Fig. 6.
SPG mutations suppress the arl-8 phenotype. (A) Schematic drawing of the DA9 neuron, showing the dorsal asynaptic region and the commissure that were observed and analyzed. The asterisk indicates the location where the commissure joins the dorsal nerve cord. (BF) Representative images showing the localization of GFP::RAB-3 in WT (wt) (B), arl-8(wy271) (C), arl-8(wy271);unc-104(ADSPGV6M) (D), arl-8(wy271);unc-104(ARSPGA252V) (E), and arl-8(wy271);unc-104(ADSPGV6M)/+ (F). Integrated marker wyIs85 [Pitr-1::GFP::RAB-3] was used. Asterisks indicate the commissure bend shown in A. (Scale bars: 50 μm.) (GI) Statistical analysis of mutant phenotypes. Shown are the length of the asynaptic region (G), the length of synaptic region in the dorsal axon (H), and the number of puncta misaccumulated at the commissure (I). Lines represent the median, and each dot represents 1 animal. Numbers above show actual median values. **Adjusted P < 0.01, ****adjusted P < 0.0001 compared with arl-8, Kruskal–Wallis 1-way ANOVA on ranks and Dunn’s multiple comparisons test. n = 30 animals for each genotype.
Fig. 7.
Fig. 7.
Axonal transport is more active in SPG model worms. (A) Representative kymographs showing the axonal transport of SVPs in WT, unc-104(ADSPGV6M), and unc-104(ARSPGA252V) worms. wyIs251 that stably expresses GFP::RAB-3 under the mig-13 promoter was used. Drawings highlight the locations of processive transport events in the original kymograph. Horizontal and vertical lines represent 5 μm and 10 s, respectively. The left and right sides of the kymographs are the proximal and distal sides, respectively. (B) Histogram showing the velocity of anterogradely transported vesicles. Vesicles that moved >1 μm in 1 min are included. Data are mean ± SD. n = 145, 162 and 150 moving vesicles from more than 20 movies in WT, unc-104(ADSPGV6M), and unc-104(ARSPGA252V), respectively. unc-104(ADSPGV6M), but not unc-104(ARSPGA252V), is statistically faster than WT. P < 0.0001, Tukey’s multiple comparisons test. (C and D) The number of vesicles that were anterogradely (C) or retrogradely (D) moving were plotted and adjusted by axon length and time. Vesicles that moved >1 μm in 1 min are included. *Adjusted P < 0.05, **adjusted P < 0.01, ***adjusted P < 0.001. Kruskal–Wallis 1-way ANOVA on ranks and Dunn’s multiple comparisons test. n = 35 movies from 35 worms. Bars represent mean ± SD. (E and F) Quantification of the dissociation rate (E) and capture probability (F) in the ventral axon in WT, unc-104(ADSPGV6M), and unc-104(ARSPGA252V) worms. The kymographs show a representative dissociation event, capture event, and passing event; 2 μm (horizontal) and 6 s (vertical). Bars represent mean ± SD. *Adjusted P < 0.05, ***adjusted P < 0.001, Kruskal–Wallis 1-way ANOVA on ranks and Dunn’s multiple comparisons test. Actual adjusted P values are 0.0008 (WT vs. ADSPG) and 0.04 (WT vs. ARSPG). n = 42 vesicle pools from 3 movies from 3 worms.

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