Spastic Paraplegia Mutation N256S in the Neuronal Microtubule Motor KIF5A Disrupts Axonal Transport in a <em>Drosophila</em> HSP Model

<div><p>Hereditary spastic paraplegias (HSPs) comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a <em>Drosophila</em> model for HSP type 10 (SPG10), caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc) or the affected kinesin-1 complexes. Ectopic expression of <em>Drosophila</em> Khc carrying a human SPG10-associated mutation (N256S) is sufficient to disturb axonal transport and to induce motoneuron disease in <em>Drosophila</em>. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP–like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality.</p> </div

Establishment of a <i>Drosophila</i> SPG 10 model.

<p>(A) Khc protein alignment across species (<i>Danio rerio</i> (Dr), <i>Tribolium castaneum</i> (Tc), <i>Xenopus tropicalis</i> (Xt), <i>Drosophila melanogaster</i> (Dm), <i>Homo sapiens</i> (Hs), <i>Rattus norvegicus</i> (Rn)) shows unique conservation of the amino acids spanning from the β-sheet 7 (β7) to the α-helix 4 (α4). Pathological mutations (red dots) are highly enriched in the latter half of loop 11 (L11) that links β7 to α4. The human mutation at position 256 (red star) was selected for further analysis. Accession numbers for the proteins used in the alignment are: Dr: GenBank_CAQ15489.1; Tc: EFA10675.1; Xt: NCBI_NP_001096215.1; Dm: GenBank_AAF58029.1, Hs: NCBI_NP_004975.2; Rn: EDM 16486.1 (B) 3D protein structure of rat Khc (3KIN) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066-Kozielski1" target="_blank">[8]</a>. The microtubule binding site is shown in yellow and green. L11 (brown cylinder), which was not resolved in the crystal structure, was added between β7 (yellow) and α4 (pink). (C) Analysis of larval locomotion. Larvae overexpressing D42>Khc^N262S (25°C) are almost completely (excluding the head) paralyzed (red arrow), whereas D42>Khc^wt+N262S larvae are still crawling, but display a tail-flipping phenotype (green arrowhead). (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s007" target="_blank">Videos S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s008" target="_blank">S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s009" target="_blank">S3</a>) (D) Kaplan-Meier survival curve recorded at 18°C (Genotypes: black: D42>w¹¹¹⁸; Green: D42>Khc^wt; Blue: D42>Khc^N262S; Red: D42>Khc^wt+N262S) (E) Summary of the data derived from the survival analysis shown in (D). Statistical significance of data was determined by a series of Mantel-Cox tests. *** p<0.001.</p

Characterization of adult <i>Drosophila</i> SPG 10 model flies.

<p>(A) Analysis of adult wing posture. While control flies have a normal wing posture (green arrowheads), Khc^N262S expressing flies frequently display abnormal wing postures such as holding their wings up (red arrow), suggesting either the degeneration or functional impairment of the indirect flight muscles. 2-day old male flies raised at 18°C are shown. All flies contained a copy of the motoneuron specific driver D42-Gal4. (B) Analysis of thorax muscle integrity provided no evidence for the degeneration of the indirect flight muscles. Scale bar: 100 µm. (C) Western blot of whole fly head extracts after adult-onset pan-neuronal (elav^C155-Gal4/tub-gal80ts) expression of Khc^N262S and Khc^wt at 29°C for 13 days. Levels of endogenous Khc from 10 heads of control flies (w¹¹¹⁸) are comparable to Khc levels in 2.5 heads from flies overexpressing Khc (Khc^N262S and Khc^wt). The number of heads loaded per lane is indicated. β-Tubulin was used as loading control. (D) Climbing assay of adult flies 16 days after adult-onset pan-neuronal expression of Khc (white bars). Although Khc^wt expression caused no adverse effects on climbing scores compared with control (grey bars), Khc^N262S expression significantly lowered the climbing score. Statistical significance was tested using an unpaired, two-tailed student's t-test (*** p<0.001). The standard error of the mean (s.e.m) is shown as a box, standard deviation (s.d.) as a black line.</p

Expression of Khc^N262S causes length-dependent synaptic defects at the neuromuscular junction.

<p>Confocal images and quantification of immunofluorescent staining of synaptic marker proteins. NMJs 6/7, segment A5 of mid-third-instar <i>Drosophila</i> larva, were selected for analysis. D42>w¹¹¹⁸ and D42>khc^wt larvae were used as controls for the mutant phenotype D42>khc^N262S. All three selected synaptic marker proteins were reduced in abundance at the NMJs of khc^N262S-expressing larvae. Larvae were raised at 29°C. (A,B) The active zone protein Brp, as well as the synaptic vesicle proteins (C,D) DV-Glut and (E,F) CSP, were selected as synaptic marker proteins. (A,C,E) Confocal images revealed that DV-Glut and CSP abundance is increased in dystrophic boutons (C,E, arrows) and reduced elsewhere (C,E, arrowheads) at NMJs of D42>khc^N262S larvae. Scale bar: 5 µm; inset 2.5 µm. (B,D,F) For quantification, n = 8–10 NMJs were analyzed per genotype. Statistical significance was determined using a Kruskal-Wallis H-test followed by a Dunn's test for comparisons between multiple groups. The standard error of the mean (s.e.m.) is shown as a box, the standard deviation (s.d.) as a black line. * p<0.05; ** p<0.01.</p

Light microscopic analysis of NMJ degeneration.

<p>Degeneration was revealed and scored by immunofluorescent staining. All larvae carried one copy of the motoneuron-specific driver D42-Gal4 and were raised at 29°C. (A–D) Confocal images of immunofluorescent staining showing NMJs 6/7, segment A5 of mid-third-instar <i>Drosophila</i> larvae. To visualize the subsynaptic reticulum, we used a GFP insertion in the discs-large locus. Neuronal membranes were visualized with an antibody against horseradish peroxidase (HRP). Synaptic vesicles were stained using m-α-Synapsin (Syn) antibody. For Khc^N262S- and Khc^wt+N262S-expressing larvae, which showed a strong reduction in Syn intensity, an additional, false-colored panel (high exposure) is shown. In this panel, the brightness was adjusted for better visibility of weak signals. Scale bar: 10 µm; right panels 5 µm. Genotypes: (A) D42>w¹¹¹⁸; (B) D42>Khc^wt; (C) D42>Khc^N262S; (D) D42>Khc^wt+N262S. (E) To integrate the frequency of retractions, we used a neurodegenerative scoring system to combine the occurrence of dystrophic boutons and minor pathological alterations at the NMJs into a single measure for the degree of pathological alterations (for details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s001" target="_blank">Figure S1A</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s001" target="_blank">S1F</a>). Using this scoring system, we detected a significant degree of neurodegenerative alterations in larvae expressing Khc^N262S either alone or in combination with Khc^wt. Statistical significance was determined using a Kruskal-Wallis H-test followed by a Dunn's test for comparisons between multiple groups. The standard error of the mean (s.e.m.) is shown as a box, the standard deviation (s.d.) as a black line. ** p<0.01.</p

<i>In vivo</i> analysis of axonal transport.

<p>(A–D) Confocal analysis of mitochondrial transport in living larvae after bleaching a segment of the nerve. (A–D) Movement of mitochondria is recorded in anesthetized larvae. A representative frame of a confocal movie is shown. (A^I–D^I) Kymographs of mitochondrial transport in larvae. Mitochondria moving in anterograde and retrograde directions appear as oblique lines. Scale bar: 5 µm. (E,F) Neither anterograde nor retrograde velocity of mitochondria is significantly different between any of the examined genotypes. Both anterograde and retrograde flux of mitochondria is reduced in mutant larvae (D42>khc^N262S and D42>khc^wt+N262S) compared with controls (D42>w¹¹¹⁸ and D42>khc^wt). Statistical significance was determined using a Kruskal-Wallis H-test followed by a Dunn's test for comparisons between multiple groups. The standard error of the mean (s.e.m) is shown as a box, the standard deviation (s.d.) as a black line. * p<0.05; ** p<0.01. (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s010" target="_blank">Videos S4</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s011" target="_blank">S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s012" target="_blank">S6</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003066#pgen.1003066.s013" target="_blank">S7</a>).</p

Neuromuscular function.

<p>Evoked junctional potentials (EJPs), the half width of EJPs and mini excitatory junctional potentials (mEJPs) were determined in D42>w¹¹¹⁸, D42>khc^N262S and D42>khc^wt+N262S larvae. (A) Averaged traces of EJPs. Stimulation artifacts were removed. (B) Quantification of averaged EJP size. (C) Half width of EJPs. (D) Averaged amplitudes of mEJPs from control and mutant larvae. (E) Quantification of averaged mEJP size. (F) Quantification of the number of vesicles released per action potential. Statistics n = 8–9 larvae per genotype for B,C,E,F. One-way ANOVA followed by Tukey-Kramer post-test ** p<0.01; *** p<0.001. The standard error of the mean (s.e.m) is shown as a box, the standard deviation (s.d.) as a black line.</p

Analysis of larval locomotion and morphometric analysis of segmental nerves.

<p>Larvae used to assay locomotion were raised at 25°C. Larvae used for analysis of axonal cargo accumulations were raised at 29°C. All larvae (except khc^−/− and khc ^+/−) carried one copy of the motoneuron-specific driver D42-Gal4. (A) Analysis of larval locomotion velocities. (B–I) Analysis of axonal cargo accumulation was performed by staining segmental nerves of third instar larva for the membrane marker anti-HRP and for various cargos. Axonal cargo accumulations (arrowheads in B,D,F,H) were defined as segments of the nerve characterized by a bright anti-HRP staining and the simultaneous accumulation of cargo. Both the area fraction of the nerve filled with cargo accumulations and the number of cargo accumulations per 1000 µm² of the nerve were significantly increased in larvae expressing khc^N262S, either alone, or in combination with Khc^wt. (B,C) Cysteine-string protein (CSP) and (D,E) the vesicular glutamate transporter (DV-Glut) were selected as markers for synaptic vesicles. The kinesin-3 cargos (F,G) ANF-GFP and (H,I) Brp were used as markers for dense core vesicles and active zone precursor vesicles, respectively. Scale bars in B–I: 10 µm. For all quantifications, n = 7–10 axons per genotype were used. Statistical significance (A,C,E,G,I) was determined by using a Kruskal-Wallis H-test followed by a Dunn's test for comparisons between multiple groups. The standard error of the mean (s.e.m.) is shown as a box, the standard deviation (s.d.) as a black line. * p<0.05; ** p<0.01, *** p<0.001.</p

Mitochondrial content.

<p>Mitochondrial content was scored by immunofluorescent staining of larvae expressing mito-GFP in motoneurons. D42>w¹¹¹⁸ or D42>khc^wt larvae were used as controls for the two mutant phenotypes D42>khc^N262S and D42>khc^wt+N262S. (A) Confocal images of immunofluorescent staining of the ventral nerve cord (VNC) of mid-third-instar larvae. Staining for Even-skipped (eve, mangenta) was used to visualize the RP2 and aCC motoneuron (white arrow heads). The medially located interneurons (cyan arrowheads) are negative for mito-GFP (green). No reduction on mitochondrial content was observed in the two mutant phenotypes. Scale bar: 20 µm, and 5 µm in right panels. The star indicates the segment in the left panel that was used for the right panels. (B) Confocal images of immunofluorescent staining showing NMJ 6/7, segment A2 of mid-third-instar <i>Drosophila</i> larvae. In mutant larvae the mitochondrial content at the NMJ was lowered compared to the control larvae. An anti-HRP immunofluorescent staining was used to visualize neuronal membranes. Scale bar: 10 µm. The arrowhead indicates the section in the upper panel that was used for the enlargement shown in the lower panel. (C–E) Quantification of the mitochondrial number (C), size (D) and density (E) at NMJ 6/7, segment A2 of mid-third-instar <i>Drosophila</i> larvae. Mitochondrial Density is the mitochondrial Area Fraction relative to the NMJ size as quantified by the HRP staining. Statistical significance was determined using a Kruskal-Wallis H-test followed by a Dunn's test for comparisons between multiple groups. ** p<0.01, *** p<0.001. The standard error of the mean (s.e.m.) is shown as a box, the standard deviation (s.d.) as a black line.</p

Characterization of axonal swellings.

<p>(A) Electron micrographs of segmental nerves of mid-L2 wild-type (D42>w¹¹¹⁸) and mutant (D42>Khc^N262S) larvae. Nerves of wild-type larvae contain mitochondria (green arrowhead) and microtubules (cyan arrowhead). Nerves of larvae expressing Khc^N262S are frequently swollen. These swollen axons are filled with mitochondria (green arrowheads), prelysosomal vacuoles (red arrowheads), autophagosomes (dark blue arrowheads), and multivesicular bodies (purple arrowheads). Scale bars: 100 nm. (B) Box-plot of axon diameters in mid-L2 wild-type (D42>w¹¹¹⁸) and mutant (D42>Khc^N262S) larvae as determined by electron microscopy. The box displays median, upper, and lower quartile. The whiskers represent the 1^st to 99^th percentile. (C,D) Confocal images of immunofluorescent staining showing segmental nerves of mid-third-instar <i>Drosophila</i> larvae. Larvae were stained for the membrane marker anti-HRP as well as for the lysosome marker LAMP-GFP (C) and the autophagosome marker ATG8-mRFP (D). Axonal swellings are positive for autophagosomes and lysosomal organelles. Scale bars in C and D: 10 µm.</p