The ALS-associated KIF5A P986L variant is not pathogenic for Drosophila motoneurons

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by the death of motoneurons. Several mutations in the KIF5A gene have been identified in patients with ALS. Some mutations affect the splicing sites of exon 27 leading to its deletion (Δ27 mutation). KIF5A Δ27 is aggregation-prone and pathogenic for motoneurons due to a toxic gain of function. Another mutation found to be enriched in ALS patients is a proline/leucine substitution at position 986 (P986L mutation). Bioinformatic analyses strongly suggest that this variant is benign. Our study aims to conduct functional studies in Drosophila to classify the KIF5A P986L variant. When expressed in motoneurons, KIF5A P986L does not modify the morphology of larval NMJ or the synaptic transmission. In addition, KIF5A P986L is uniformly distributed in axons and does not disturb mitochondria distribution. Locomotion at larval and adult stages is not affected by KIF5A P986L. Finally, both KIF5A WT and P986L expression in adult motoneurons extend median lifespan compared to control flies. Altogether, our data show that the KIF5A P986L variant is not pathogenic for motoneurons and may represent a hypomorphic allele, although it is not causative for ALS.

Fig. 1

Expression level of WT, P986L and Δ27 KIF5A proteins. (a) Representative immunoblots of protein extracts from thoraxes of control, KIF5A WT, KIF5A P986L and KIF5A Δ27-expressing flies using the VGlut-Gal4 motoneuron driver. KIF5A is indicated by a black arrowhead. Note the presence of a non-specific band in all genotypes. Anti-ß-Tubulin was used as a loading control. Western blots were cropped in this figure; full blots are shown in Supplementary Fig. 1. (b) Quantification of gel bands. Data are shown as means ± s.e.m. (a.u. arbitrary units, ns: not significant, ordinary one-way ANOVA with Tukey’s multiple comparisons test, n = 3 gels).

Fig. 2

The morphology of NMJs is not affected by KIF5A P986L. (a) Anti-HRP (red) and anti-Brp (green) labeling were used to visualize the presynaptic motoneuron endings and the active zones, respectively, in control, KIF5A WT and KIF5A P986L-expressing larvae using the VGlut-Gal4 motoneuron driver. Scale bar: 5 µm. (b–e) Quantification of axonal branch length (b), NMJ area (c), number of active zones per NMJ area (d) or per bouton (e). Note that all these parameters are unchanged by KIF5A P986L (white) compared to control (black), while KIF5A WT (grey) induces an increase in the length of axonal branches and NMJ area (ns: not significant, p > 0.05; *p < 0.05; ****p < 0.0001; ordinary one-way ANOVA with Tukey’s multiple comparisons test; n = 10, 13 and 13 for control, KIF5A WT and KIF5A P986L, respectively). (f) Anti-HRP (red) and anti-Dlg (green) antibodies were used to label the presynaptic motoneuron endings and the postsynaptic density, respectively, in control, KIF5A WT and KIF5A P986L-expressing larvae by using the VGlut-Gal4 motoneuron driver. Note that the presynaptic endings and postsynaptic densities are closely apposed for all genotypes. Scale bar: 5 µm.

Fig. 3

The synaptic transmission at NMJs and larval locomotion are not altered by KIF5A P986L. (a) Representative traces of mEJPs (top) and eEJPs (bottom) obtained from control, KIF5A WT and KIF5A P986L-expressing larvae using the VGlut-Gal4 motoneuron driver. (b,c) Amplitude (b) and frequency (c) of mEJPs. (d) Amplitude of eEJPs. (e) Quantal content calculated by dividing the mean of eEJPs by the mean of mEJPs. (f,g) A train of 100 stimulations at a frequency of 20 Hz was applied to NMJs. The A2/A1 (f) and the A100/A1 (g) ratio compare the amplitude of the first response (A1) to the second (A2) or to the 100th (A100), respectively. Note that KIF5A P986L-expressing larvae give rise to similar results as control. Contrary to KIF5A P986L, KIF5A WT-expressing larvae show a reduction of the second (f) or the hundredth (g) response compared to the first one. (b–g) ns: not significant, p > 0.05; *p < 0.05; Kruskal–Wallis with Dunn’s multiple comparisons test (b) or ordinary one-way ANOVA with Tukey’s multiple comparisons test (c–g); n = 11, 12 and 11 for control, KIF5A WT and KIF5A P986L, respectively. (h) Quantification of the distance travelled by control, KIF5A WT and KIF5A P986L-expressing larvae by using the VGlut-Gal4 motoneuron driver. Note that unlike to KIF5A WT, KIF5A P986L does not reduce the distance travelled compared to the control (**p < 0.01; ***p < 0.001; Kruskal–Wallis with Dunn’s multiple comparisons test; n = 30 for each genotype).

Fig. 4

Mitochondria distribution is not affected by KIF5A P986L. (a,b) Anti-HRP (red) and anti-GFP (green) labeling was used to visualize the motoneuron axons (red in (a)) or presynaptic endings (red in (b)) and mitochondria (green in (a,b)), respectively, in control, KIF5A WT and KIF5A P986L-expressing larvae using the VGlut-Gal4 motoneuron driver associated with the UAS-Mito-GFP reporter. (a) Note that mitochondria are enriched in axons of KIF5A WT-expressing larvae compared to control and KIF5A P986L-expressing larvae, which show a similar distribution. Scale bar: 10 µm. (b) Mitochondria are present at NMJs in control and KIF5A P986L-expressing larvae, but at lower levels in KIF5A WT-expressing larvae. Scale bar: 5 µm. (c,d) Quantification of mitochondria fluorescence intensity in the axons (c) and at NMJs (d); a.u. arbitrary units, ns: not significant, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001; Kruskal–Wallis with Dunn’s multiple comparisons test. (c) n = 13, 10 and 8 for control, KIF5A WT and KIF5A P986L, respectively. (d) n = 10, 8 and 11 for control, KIF5A WT and KIF5A P986L, respectively.

Fig. 5

KIF5A WT and P986L are uniformly distributed in motoneuron axons. (a) Anti-HRP (red) and anti-KIF5A (green) antibodies were used to label the motoneuron axons (red) and KIF5A protein (green), respectively, in control, KIF5A WT and KIF5A P986L-expressing larvae using the VGlut-Gal4 motoneuron driver. Note that KIF5A WT and P986L are uniformly distributed along the axons. Scale bar: 10 µm. (b) Quantification of motoneuron axons containing KIF5A-positive aggregates (ns: not significant, p > 0.05; ****p < 0.0001; Kruskal–Wallis with Dunn’s multiple comparisons test. n = 28, 26 and 22 for KIF5A WT, P986L and Δ27, respectively).

Fig. 6

Expression of KIF5A P986L in adult motoneurons does not affect climbing ability and lifespan. (a,b) Crosses were maintained at 18 °C and males were transferred at 29 °C immediately after eclosion to induce KIF5A expression only at the adult stage. Note that the KIF5A Δ27-expressing males were used as a control to confirm their reduced climbing ability and reduced survival. (a) Quantification of the climbing ability of control, KIF5A WT, KIF5A P986L and KIF5A Δ27-expressing male flies using the VGlut-Gal4 motoneuron driver combined with the tub-Gal80^ts transgene. As expected, the climbing ability of all flies decreases with age. Note that at 20 days, all flies expressing KIF5A Δ27 had died. However, the climbing performance was similar between control (black), KIF5A WT (dark grey) and KIF5A P986L (white) expressing flies at 10- and 20 days of age (ns: not significant, p > 0.05; ****p < 0.0001; ordinary one-way ANOVA with Tukey’s multiple comparisons test; at ten days n = 10, 14, 22 and 14 for control, KIF5A WT, P986L and Δ27, respectively. At 20 days n = 40, for all genotypes). (b) Lifespan analysis of control (black), KIF5A WT (green), KIF5A P986L (blue) and KIF5A Δ27 (red) expressing male flies using the VGlut-Gal4 motoneuron driver combined with tub-Gal80^ts transgene. The median survival is 23, 28, 28 and 15 for control, KIF5A WT, P986L and Δ27, respectively [****p < 0.0001; Log-rank (Mantel-cox) test; n = 443, 343, 313 and 444 for control, KIF5A WT, P986L and Δ27, respectively].