prickle modulates microtubule polarity and axonal transport to ameliorate seizures in flies

Abstract

Recent analyses in flies, mice, zebrafish, and humans showed that mutations in prickle orthologs result in epileptic phenotypes, although the mechanism responsible for generating the seizures was unknown. Here, we show that Prickle organizes microtubule polarity and affects their growth dynamics in axons of Drosophila neurons, which in turn influences both anterograde and retrograde vesicle transport. We also show that enhancement of the anterograde transport mechanism is the cause of the seizure phenotype in flies, which can be suppressed by reducing the level of either of two Kinesin motor proteins responsible for anterograde vesicle transport. Additionally, we show that seizure-prone prickle mutant flies have electrophysiological defects similar to other fly mutants used to study seizures, and that merely altering the balance of the two adult prickle isoforms in neurons can predispose flies to seizures. These data reveal a previously unidentified pathway in the pathophysiology of seizure disorders and provide evidence for a more generalized cellular mechanism whereby Prickle mediates polarity by influencing microtubule-mediated transport.

Keywords: EB1-GFP; epilepsy; neurodegeneration; planar cell polarity; spiny-legs.

Fig. 1.

Tipping the balance of pk isoforms predisposes flies to seizure. (A and B) Modified bang-sensitivity behavioral assay. (A) pk^sple/+ flies (sple/+) are seizure-prone and take longer to recover compared to Oregon-R controls (+/+); all time points (P < 10⁻⁵; Student's t test). Error bars show SEM. (B) Overexpression (in brain, motor neurons, and muscles; A51-Gal4 driver) of pk^pk (UAS-pk) but not pk^sple (UAS-sple) is sufficient to produce seizure-prone flies; all time points (P < 10⁻¹⁵; Student's t test). (C–E) ECS stimulation paradigm. (C) High-frequency ECS applied across the brain triggers a stereotypic seizure discharge; panel adapted from ref. . (D) Representative DLM spiking activity demonstrating that pk^sple/+ flies exhibit pronounced spiking activity at lower voltages. (E) Box blots of spike counts triggered by ECS (60 V) demonstrate that pk^sple/+ flies have a statistically significant increase in total spiking activity (Mann–Whitney U, P < 0.05). Median spike counts: +/+ = 32; pk^sple/+ = 66, pk^pk/+ = 11, and pk^sple/pk^pk = 2.5. n = 10–14 flies per genotype for ECS experiments. n =12 vials per genotype for behavioral experiments, repeated a minimum of two times, with similar results.

Fig. 2.

Heterozygous pk mutations alter axonal transport in larval neurons. (A) Diagram of segmental nerve illustrating vesicle movements. (B) Kymographs of APP-YFP–tagged vesicles show that APP-containing vesicle transport is strongly reduced in pk^pk/+ flies. Rightward and leftward descending lines represent anterograde- and retrograde-moving vesicles, respectively. Vertical lines represent stationary vesicles. (C) Quantification of vesicle movement shows alterations of transport parameters in pk mutant larvae. (i) pk^pk/+ larvae show decreases in duration-weighted segemental velocities of both anterograde and retrograde traveling vesicles. (ii) pk^pk/+ larvae show increases in normalized total pause duration of both anterograde and retrograde traveling vesicles. (iii) pk^sple/+ larvae show a decrease in both anterograde and retrograde vesicle corrected pause frequencies, although only the anterograde pause frequencies were statistically significant. (D) pk^sple/+ larvae show a reduction in vesicles moving in a retrograde direction as well as an increase in number of stationary vesicles. pk^pk/+ larvae show an increase (greater than fourfold) in the number of stationary vesicles and a decrease in the number of reversing vesicles. White bars represent Oregon-R (+/+) larvae, red bars represent pk^sple/+ larvae, and green bars represent pk^pk/+ larvae. n = 5 larvae. Error bars show SEM. See Table S1 for P values.

Fig. 3.

Pk^pk colocalization with a Kinesin motor protein. Confocal images of third-instar larval NMJ preparations show that a neuronally expressed eGFP-Pk^pk (green) colocalizes (arrows) with Kinesin heavy chain (red) in both segmental nerves (A–C) (scale bar, 5 μM) and NMJ terminal boutons (A′–C′) (scale bar, 1 μM).

Fig. 4.

Reducing the dose of a Kinesin motor subunit suppresses the pk^sple/+ seizure phenotype. (A) Modified bang-sensitivity behavioral assay demonstrating that reduction of the dose of Klc in combination with the heterozygous pk^sple/+ mutation suppresses the pk^sple/+ behavioral seizure phenotype. Error bars show SEM. n = 6 vials per genotype, repeated a minimum of two times with similar results. (B) Representative DLM spiking activity demonstrating that reduction of the dose of Klc in combination with the heterozygous pk^sple mutation suppresses the seizure discharge spiking activity of the pk^sple /+ flies. (C) Spike counts after ECS (60 V) demonstrating that the increase in the number of spikes per seizure discharge in the pk^sple /+ flies (Mann–Whitney U, P < 0.05) is suppressed by reducing the dose of Klc. Median spike counts: +/+ = 32, pk^sple/+ = 66, Klc/+ = 31, and pk^sple/+; +/Klc = 18.5. n = 10–14 flies per genotype. (D) Bar graph representation of EB1-GFP comet directionality in the control, UAS-sple, and UAS-pk experimental larval motor neurons demonstrating that a significant fraction of microtubules are reversed in polarity when the Pk^sple isoform predominates. Pink (MT plus ends toward nerve terminal) and blue (MT plus ends toward cell body). n = 8 larvae per genotype. (E) Length of EB1-GFP comets in the control, UAS-sple, and UAS-pk experimental larval motor neurons demonstrating that when Pk^pk predominates the EB1-GFP comets are longer (Mann–Whitney U, P < 0.0001). EB1-GFP median comet length: control, 2.19 µm; UAS-sple, 1.83 µm; and UAS-pk, 4.15 µm. n = 4 larvae per genotype; ∼15 comets were tracked per larva. All UAS constructs were driven by D42-Gal4.