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

Significance

Mutations in prickle genes from flies to humans cause epilepsy, a disorder that affects approximately 1% of the population. Although Prickle has been studied for many years, its molecular function is unknown. We now show that Prickle modulates transport of vesicles in fruit fly neurons. Additionally, we show that prickle mutants have electrophysiological defects consistent with epilepsy, and merely altering the balance of Prickle isoforms causes seizures. Finally, we demonstrate that reducing the level of vesicle transport motor proteins can suppress prickle-mediated seizures, revealing a previously unidentified pathway in the pathophysiology of epilepsy.

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.

Epilepsy, which affects ∼1% of the population, is a disabling and debilitating disease characterized by recurrent seizures. Coupling genetic analysis of human epilepsy cases with functional validation in animal models, we previously showed that mutations in human PRICKLE1 and PRICKLE2, as well as mutations in mouse, zebrafish and fly prickle (pk) orthologs, increase susceptibility to seizures (1–3). We recently showed that both mouse and fly Prickle can associate with Synapsin (4). However, little is known regarding what cellular process connects Prickle with epilepsy. Products of the prickle gene work in concert with a group of cytoplasmic and membrane-associated proteins including Frizzled (Fz) and Dishevelled (Dsh) to establish polarity of structures such as hairs and bristles in the fly epidermis (5–8). The fly pk locus expresses three alternately spliced isoforms, two of which, pk^sple (prickle-spiny-legs) and pk^pk (prickle-prickle), are expressed in postembryonic animals (5, 7). Homozygous pk^pk and pk^sple fly mutants show strong planar cell polarity (PCP) phenotypes in the wing and in the legs, respectively, in addition to other regions of the adult body (6, 7). Prickle (Pk) protein isoforms are localized to the proximal end of fly wing cells, whereas Fz and Dsh are localized distally (8).

Here, we show that vesicle transport dynamics are altered in neurons of Drosophila mutant for either adult isoform of prickle. Seizure-prone pk^sple heterozygotes show enhanced vesicle transport (along with electrophysiological defects similar to other seizure-prone mutant flies), and the seizure phenotypes can be rescued by lowering the dose of vesicle transport motor proteins. However, pk^pk heterozygotes show severely reduced vesicle transport, with loss of both copies of pk^pk showing a marked decrease in viability. These data demonstrate that it is the balance of Pk isoforms that provides normal vesicle transport functions in axons of fly neurons. Additionally, we show that when the Pk^sple isoform predominates, microtubule (MT) polarity can be radically altered in axons. However, when the Pk^pk isoform predominates, MT polarity is normal, whereas MT growth dynamics are altered such that there is an increase in plus-end MT polymerization. Our data establish that Prickle isoforms work in concert to promote the proper transport of vesicles in neurons and demonstrate that Prickle function is necessary for establishment of the correct polarity and growth dynamics of MTs within axons.

Results

To determine whether mutations in pk were solely responsible for the seizure phenotype we previously reported in pk^sple flies (2), we outcrossed both pk^sple1 and pk^pk1 loss-of-function alleles into an Oregon-R (OR) background and then subjected pk^sple/+ and pk^pk/+ flies to a modified bang-sensitivity behavioral assay, used to assess seizures in adult flies (Materials and Methods) (2, 9). Given that homozygous pk^sple and pk^pk loss-of-function alleles result in strong PCP phenotypes on the adult fly body, which might interfere with their climbing ability, we used pk^sple and pk^pk heterozygotes that are devoid of the epidermal PCP phenotypes. As shown in Fig. 1A, pk^sple/+ flies are seizure-prone at all time points (Movie S1, clip 1), whereas pk^pk/+ flies recover as well as (or somewhat better than) the control OR flies (Movie S1, clip 2), and similar results were obtained using additional loss-of-function pk^sple and pk^pk alleles (Fig. S1A). Additionally, pk^sple/+ flies showed a seizure phenotype before outcrossing (2). Collectively, these data demonstrate that genetic background does not significantly influence the seizure phenotype and that the pk^sple mutation is solely responsible for the seizure phenotype. Moreover, considering that both the pk^sple/+ and pk^pk/+ flies are devoid of overt PCP alterations, the epilepsy phenotype can be genetically separated from the PCP phenotypes (5). Generating a pk^pk and pk^sple heteroallelic combination (which restores the balance between the two isoforms) rescues the pk^sple/+ seizure phenotype (Fig. 1A).

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. 9. (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.

To explore whether pk^sple mutants exhibited characteristic seizure-like activity in an intact neuronal circuit similar to what is observed for other seizure-prone fly mutants (9–12), we used an electroconvulsive stimulation (ECS) paradigm (9, 13, 14). High-frequency stimulation across the brain triggers a stereotypic seizure discharge across the whole nervous system. The discharge pattern can be faithfully monitored by recording the spiking activity of the dorsal longitudinal flight muscle (DLM), whose spiking reflects the motor output of its identified motor neuron (Materials and Methods) (9, 13, 14) (Fig. 1 C–E). We found that pk^sple/+flies showed a lower threshold required to trigger seizure discharges compared to WT flies (mean thresholds of 50 ± 4.2 V and 65 ± 3.7 V, respectively, P = 0.01). Furthermore, more spikes were observed during seizure discharges in pk^sple/+ mutants compared to control (Mann–Whitney U, P < 0.05; Fig. 1 D and E, see legend for median spike counts). Although pk^pk/+ flies did not show any significant difference in spiking activity during seizure discharges compared to controls, restoring the balance of the isoforms in the pk^sple/+, +/pk^pk heteroallelic combination suppressed the spiking activity relative to the pk^sple/+ flies (Fig. 1 D and E).

Taken together, both the behavioral and electrophysiological data suggest that the balance of Pk isoforms is critical for producing normal motor function and imply that tipping the balance of isoforms so that Pk^pk predominates might increase susceptibility to seizures. Thus, we used the GAL4/UAS system to overexpress either the Pk^pk isoform (UAS-pk) or the Pk^sple isoform (UAS-sple) in neurons, or neurons plus additional tissues. As shown in Fig. 1B, overexpression of Pk^pk (but not Pk^sple) in brain, motor neurons, and muscles (using the A51-Gal4 driver; Movie S1, clip 3), or all neurons (using the elav-Gal4 driver; Fig. S1B), produced seizure-prone flies that took longer to recover compared to controls. These results are consistent with what is observed for the heterozygous mutant phenotypes whereby tipping the balance such that Pk^pk predominates (as in the pk^sple/+ flies) leads to a seizure phenotype, but tipping the balance such that Pk^sple predominates (as in the pk^pk/+ flies) does not.

A critical determinant of normal bristle and hair polarity in cells of the Drosophila wing is the proper localization of Fz and Dsh to the distal end of the cells, and vesicles have been shown to transport both of these proteins along MTs (15, 16). Because pk^pk homozygous mutants show altered bristle/hair polarity in the wing (5, 7), which would be predicted if Fz/Dsh vesicles were not properly transported distally, we reasoned that Pk^pk might be involved in directly influencing transport of the Fz/Dsh-containing vesicles in the wing cells and that perhaps similar mechanisms might underlie vesicle transport in neurons. We thus used an in vivo system (Materials and Methods) to assess axonal vesicle transport dynamics in Drosophila larvae and expressed a YFP-tagged synaptic vesicle protein (amyloid precursor protein-GFP, or APP-YFP) in third-instar larval segmental nerves in control, pk^pk/+, and pk^sple/+ larvae, followed by fluorescence microscopy of larval fillet preps (Movie S2 and Table S1) (17–19). Removing just one functional copy of the pk^pk isoform was sufficient to severely perturb vesicle transport (Fig. 2B), and significant decreases in both anterograde and retrograde vesicle velocities were observed (Fig. 2 C, i), in addition to an increase in vesicle pause duration (Fig. 2 C, ii) compared to control. Moreover, a greater percentage of vesicles remained stationary, whereas a decreased percentage of vesicles reversed direction, compared to control (Fig. 2D).

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.

Given the severe reduction in vesicle transport (an essential cellular function) when pk^pk isoform levels were reduced (but not when pk^sple isoform levels were reduced, discussed below), we reasoned that loss of pk^pk (but not pk^sple) would likely cause a reduction in viability. We collected 1,000 homozygous mutant embryos for each genotype and monitored their development from embryos to white prepupae and then from pupae to adult flies compared to controls (Table S2). Only the pk^pk mutants showed a significant reduction in the number of embryos that reached the white prepupal stage (P = 0.0172), and we also observed a reduction in the percentage of pk^pk pupae that eclosed to reach adulthood (P = 0.0469). These data are consistent with the hypothesis that there is a high demand for efficient vesicular transport across fly development, most notably during the stages of the greatest growth in size (from embryo to pupae) when embryos undergo substantial cell and tissue differentiation events.

In contrast, removing one functional copy of the pk^sple isoform caused reduced vesicle pause frequencies in both anterograde and retrograde directions compared to control, although only the anterograde reductions were statistically significant (Fig. 2 C, iii). We also observed a decrease in the percentage of vesicles moving in a retrograde direction but found no difference for vesicles moving in the anterograde direction (Fig. 2D). However, all other transport parameters were similar to what was observed in neurons of control animals. Nonetheless, taken as a whole these data demonstrate that overall anterograde vesicle movement is enhanced in the pk^sple/+ background owing to the combination of a net increase in anterograde vesicle distance traveled over time and a greater relative percentage of vesicles moving in the anterograde direction.

To determine whether Pk^pk protein could colocalize with the Kinesin-1 motor protein complex [which is involved in anterograde transport of the APP-YFP vesicles along MTs (17–20)], we performed immunohistochemistry on third-instar larval filet preparations to visualize both a neuronally expressed eGFP-Pk^pk in addition to Kinesin heavy chain (Khc) and observed an overlapping pattern of puncta throughout the axons (Fig. 3 A–C) as well as boutons (Fig. 3 A′–C′). It is important to note that eGFP-Pk^pk, although localized to puncta along the axon, does not move through the axon during live imaging, in contrast to vesicle-associated Kinesins. We speculate that colocalization of eGFP-Pk^pk and Kinesin might be due to traffic jams (axonal blockages) of the Kinesin-associated vesicles that have accumulated at the sites of Pk^pk function (17, 18) owing to the eGFP-Pk^pk overexpression (staining of control larvae for Kinesin does not reveal Kinesin-positive axonal blockages or puncta). It is important to note that axonal blockages are oftentimes seen when genes encoding proteins known to be involved in axonal transport are either reduced in dosage [such as amyloid precursor protein-like (Appl), Khc, Kinesin light chain (Klc), and dynein heavy chain (17, 19, 21)] or overexpressed [such as Appl (17)]. Therefore, our data are consistent with both Pk^pk and Kinesin functioning at the level of the MT to help promote vesicle transport.

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).

Because the pk^sple/+ flies showed a net increase in anterograde vesicle transport, we reasoned that reducing transport of vesicles by reducing the dose of one of the Kinesin-1 anterograde motor subunits (Klc) in the context of the pk^sple mutation might suppress the seizure phenotype. As shown in Fig. 4A, pk^sple/+ flies are strongly seizure-prone, whereas Klc heterozygotes recover similarly to the control. However, removal of one functional copy of Klc in the context of the pk^sple heterozygote (pk^sple/+; +/Klc) fully suppresses the seizure phenotype such that the transheterozygotes now recover as efficiently as controls (Movie S1, clip 4). Additionally, the seizure phenotype of pk^sple /+ flies could be similarly suppressed by removing a functional copy of another subunit of the anterograde motor complex, Khc (Fig. S1C).

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.

We then used the ECS protocol to examine whether reducing the Klc dose in the pk^sple/+ flies would ameliorate their seizure susceptibility (9, 12). Only pk^sple/+, but not Klc/+, flies showed a significant reduction (P = 0.01) in ECS intensity required to trigger a seizure (50 V ± 4.2 and 59 V ± 4.8, respectively) compared to controls (65 V ± 3.7). However, pk^sple/+; +/Klc transheterozygotes showed a normal spiking distribution (Fig. 4 B and C) and no significant difference in seizure threshold (63 V ± 4.5) compared to controls. Comparable results were obtained when we reduced the dose of Khc in the pk^sple/+ flies such that the pk^sple/Khc transheterozygotes showed similar spiking activities (Fig. S2) as well as seizure thresholds compared to controls (62 V ± 6.0 vs. 65 V ± 3.7), as did the Khc/+ flies (69 V ± 6.0). Taken together, these data demonstrate that lowering the dose of either of two Kinesin-1 anterograde motor proteins critical for axonal vesicle transport can suppress the seizure phenotype of pk^sple/+ flies in both seizure behavioral as well as electrophysiological assays.

Having identified a variety of microtubule-mediated vesicle transport defects in the segmental nerves of the pk mutants, we tested whether Pk isoforms might be directly acting at the level of MT organization to influence transport. We imaged motor neurons of larvae overexpressing (i) end binding protein 1 (EB1)-GFP (an MT plus-end tracking protein), (ii) EB1-GFP and the Pk^sple isoform (thus mimicking the isoform imbalance in pk^pk/+ larvae where transport is inhibited), and (iii) EB1-GFP and the Pk^pk isoform (thus mimicking the isoform imbalance in the seizure-prone pk^sple/+ flies where transport is enhanced). In both control neurons and neurons expressing the Pk^pk isoform we observed 98% of the EB1-GFP comets moving toward the nerve terminal (reflecting a unified, canonical polarity of MTs; Fig. 4D, Movie S3, clips 1 and 2, respectively, and Table S3); however, the Pk^pk-expressing larvae showed a significant increase in the length of the EB1-GFP comets compared to control larvae (Mann–Whitney U, P < 0.0001; Fig. 4E), demonstrating that there is an increase in MT plus-end polymerization when the balance is tipped in favor of the Pk^pk isoform (22, 23). In neurons expressing the Pk^sple isoform, a large fraction of the MTs were actually reversed in orientation, with 36% of the comets moving toward the cell body and 64% of the comets moving toward the nerve terminal (Fig. 4D, Movie S3, clip 3, and Table S3). Collectively, these data suggest that Pk^pk promotes and/or stabilizes the proper polarity of axonal MTs in addition to modulating MT growth dynamics, whereas Pk^sple disrupts the unified MT polarity, perhaps by promoting/stabilizing reverse polarity or disrupting a polarizing function of the Pk^pk isoform.

Discussion

Similar to what has been observed regarding the relationship of the two Prickle isoforms while influencing planar cell polarity [with more severe hair and bristle phenotypes observed when one or the other isoform is eliminated and less severe phenotypes observed when both isoforms are eliminated (5)], Pk^pk and Pk^sple must also be in balance in neurons to promote normal vesicle transport and proper neuronal function. Tipping the balance in one direction where Pk^sple predominates creates mixed MT polarity along with severe reductions in transport (and significant reductions in viability but no seizure phenotype), whereas tipping the balance in the other direction where Pk^pk predominates has the opposite effect such that overall anterograde transport is enhanced, with an increase in seizure susceptibility and alteration in MT growth dynamics.

Our data are consistent with a mechanism whereby Prickle promotes cell polarity by influencing MT-dependent vesicle transport, in part by modulating both MT polarity and MT growth dynamics. Shimada and colleagues (15) previously reported that in wing cells Fz is transported in vesicles toward the plus ends of MTs, eventually accumulating at the distal ends of the cells. pk^pk mutants show disruptions in hair and bristle polarity in the wing (5, 7), and recent evidence (16) shows that prickle influences vesicle transport in wing cells in a manner similar to what we have observed in neurons. Given the similar effects on MT polarity in two different cell types, we therefore hypothesize that Prickle plays a more generalized role in affecting vesicle transport in many cell types, with neurons being particularly sensitive to perturbations in vesicle transport given the significant lengths vesicles must traverse in many axons. It is thus not surprising that epilepsy was the first disease to be associated with human PRICKLE mutations.

Our data show that vesicle velocity, directional change, recovery from a stall, and the overall ability to move are severely compromised in pk^pk heterozygotes, thus resulting in an overall decrease in transport. We speculate that when Pk^sple predominates (as in pk^pk/+) the directed MT polarization is lost, creating a network of antiparallel MTs that would significantly impede the sustained transport of a vesicle motor complex along the length of an axon, producing the multitude of vesicle transport defects that we observe. Nonetheless, a comparable percentage of vesicles in the pk^pk heterozygotes compared to controls was transported in both anterograde and retrograde directions (albeit at much slower velocities and increased pause durations; Fig. 2D), and thus there must be sufficient vesicle transport to support life and allow a reasonable level of cellular function in animals when the pk^pk isoform is lost. Although reduction of pk^pk was not associated with a seizure phenotype, we did observe a strong reduction in viability of pk^pk homozygotes compared to pk^sple homozygotes or controls (Table S2), suggesting that there is a developmental cost to compromising efficient cellular vesicle transport.

In contrast to what we observed in pk^pk heterozygotes, vesicles in the seizure-prone pk^sple heterozygotes show a pronounced reduction in pause frequencies, most notably in the anterograde direction, even though vesicle velocities, vesicle pause durations, and vesicle reversals are unaffected. Additionally, although the overall percentage of vesicles that are either stationary or moving in a retrograde direction is roughly the same in OR (∼21%) or pk^sple/+ (∼22%), the relative percentages in each category show an inverse relationship between the genotypes; specifically, in OR, ∼15% of these vesicles move in a retrograde direction whereas ∼6% are stationary. In contrast, in pk^sple/+ animals, ∼14% of the vesicles are stationary whereas only ∼8% move in a retrograde direction. The reason for the reduction in retrograde traffic in the pk^sple heterozygotes is unclear, but these data suggest that a significant proportion of vesicles normally designated for retrograde transport are now stationary in the pk^sple/+ mutant, and that reducing the pk^sple isoform partially compromises retrograde traffic. Taken together, the reduced anterograde pause frequencies as well as the decreased retrograde transport would both serve to enhance the overall anterograde transport relative to retrograde transport, which we believe is the principal cause of the seizure phenotype. Consistent with this model, reducing the level of either of two Kinesin subunits (Klc or Khc), both of which are anterograde motor proteins, fully suppresses the seizure phenotype, perhaps by (i) reestablishing the proper balance of anterograde versus retrograde traffic (such that the reduction in the percentage of vesicles moving in a retrograde fashion in pk^sple/+ animals is balanced by a specific reduction in anterograde transport in pk^sple/+, +/Klc, or pk^sple/Khc) or (ii) suppressing the enhanced anterograde traffic caused by reduced pause frequencies in the pk^sple/+ animals, or a combination of both i and ii. Perhaps Pk^pk biases transport in the anterograde, plus-end direction, which would also be consistent with the requirement of Pk^pk function in wing cells to transport plus-end-directed Fz-containing vesicles to the distal ends of the cells (16). It is intriguing to note that causing Pk^pk to predominate in motor neurons results in altered MT growth dynamics, with MTs showing increased polymerization at their plus ends as evidenced by longer EB1-GFP comets. We speculate that reduced pause frequencies observed in the pk^sple/+ mutants are linked to this change in growth dynamics; perhaps an increase in polymerization results in an overall increase in MT length, thus allowing vesicle motor complexes to move a greater distance before encountering pauses when reaching the end of one MT before transitioning to the next. Further work is required to sort out the precise molecular mechanisms controlled by the Prickle isoforms.

Although this is to our knowledge the first direct genetic evidence demonstrating that mutations in an ortholog of a known human epilepsy gene produce seizures through altered vesicle transport, mutations in genes encoding motor or motor-associated proteins (such as Kinesin-1 family member KIF5A or lissencephaly 1 (LIS1), a gene encoding a factor that clamps dynein to MTs) have been shown to produce seizure activity in mice, worms, and sometimes humans (24–29), suggesting that vesicle transport defects, normally linked to a variety of neurodegenerative diseases, can also be associated with seizures. In the case of KIF5A, it was shown that a conditional knockout of the gene led to impaired GABA_A receptor-mediated synaptic transmission in inhibitory neurons, thus producing epileptic phenotypes, and KIF5A (but neither KIF5B nor KIF5C, the two other Kinesin-1 family members) was shown to directly interact with a GABA_A receptor-associated protein (26). Additionally, rescue experiments showed that expression of KIF5A (but neither KIF5B nor KIF5C) was able to rescue the GABA_A receptor transport defect, demonstrating that only one of the three Kinesin-1 family members was required for this specific transport function. In the case of LIS1, a presynaptic defect was observed such that synaptic vesicles were improperly distributed in GABAergic neurons (25). Although such data point toward motor-associated transport problems as being correlated with seizures, defects in the ability to properly recycle synaptic vesicles via dynamin/shibire in mice/flies, or amphiphysin in mice, can also cause seizure activity, suggesting that overall control of vesicle trafficking in neurons is critical for suppressing seizures (30–32).

How, then, might alteration of vesicle transport dynamics observed in the pk^sple/+ flies be increasing seizure susceptibility? The first possibility is that neuronal and/or axonal migration defects might play a role, because it has been observed that LIS1 mutations are associated with migration defects (33), although this has not been observed in worms (25). While we cannot firmly rule this out, we do not observe obvious defects in neuronal positioning, nor do we see defects in axonal pathfinding to the somatic musculature in pk^sple mutant larval filet preparations. A second possibility is that the enhanced anterograde transport coupled with reduced retrograde transport is perturbing the proper distribution of proteins within the neurons, whether at the nerve terminal or elsewhere. Improper distribution/concentration of voltage-gated ion channels, for example, could have profound effects on neuronal excitability. A corollary to this is that specific classes of neurons might be particularly sensitive to such vesicle transport defects, similar to what is seen for GABAergic neurons in the KIF5A mutants (26). Intriguingly, sodium and potassium channel-containing vesicles have been shown to be transported in axons by Kinesin-1 (34, 35), which is the Kinesin that we have focused on in this study, raising the possibility that transport defects of such channels might underlie the seizure phenotype in the pk^sple/+ mutant. These channels associate directly with Khc (34, 35), yet we are able to suppress the seizure phenotype by reducing the dose of either Klc or Khc; collectively, these data suggest that Klc might be working together with Khc to modulate channel binding or transport, and that lowering the dose of either Kinesin component suppresses the aberrant transport of the channels.

Alternatively, the seizure phenotype might be associated with improper transport of a different set of neuronal components such as those necessary for synaptic maturation and function. The Kinesin-3 motor protein Imac (encoded by immaculate connections) is the Kinesin in flies known to transport many of these components to the nerve terminal, including Synaptotagmin and Vesicular glutamate transporter (36); however, synaptic components have been shown to be transported by Kinesin-1 in rats, including Syntaxin-1 and Bassoon (37), suggesting that altered transport of an as-yet undetermined synaptic component might underlie the seizure phenotype in the pk^sple/+ flies. Experiments are currently underway to identify differentially localized proteins in the pk^sple mutants, as well as to test whether pk^sple-altered vesicle transport in different classes of neurons increases seizure susceptibility. Nonetheless, the full suppression of the seizure phenotype, both behaviorally and electrophysiologically, through reduction of either of two Kinesin motor protein subunits (for which the only known function is MT-mediated transport) firmly demonstrates that altered vesicle transport underlies the seizure phenotype.

It is worth mentioning that Noebels (38) recently presented compelling evidence linking epilepsy and Alzheimer’s disease (AD), and several reports describe connections between genes associated with neurodegeneration and seizures (38–40). We note that two of the AD-associated proteins, amyloid precursor protein and presenilin 1 (APP and PSEN1; Appl and Psn in flies), are components of the same vesicle in mammalian cells, and that Appl and Psn mutations in flies both affect vesicle transport, with reduction in Psn levels linked to increased net vesicle transport, similar to what we observe for the APP-YFP vesicles in the seizure-prone pk^sple/+ flies (17, 19, 38–42). APP binds to a Kinesin subunit (43), and its transport is dependent on Kinesin (44). Thus, alterations in axonal transport may underlie the connection between these disease processes. Additional studies are warranted to investigate the efficacy of drugs developed for AD and other neurodegeneration disorders as potential antiepileptic therapies.

Materials and Methods

Modified Bang-Sensitivity Behavioral Assay.

Flies were collected immediately after eclosion, aged for 7 d, and mechanically stimulated with a vortex mixer for 20 s. After vortex, flies were digitally recorded and their recovery was assessed for up to 25 s; only flies that climbed off the bottom of the vial were considered as recovered flies (2).

Electroconvulsive Stimulation Triggered Seizures.

Action potentials were recorded from flight muscles as previously described (9). ECS, a train of 0.1-ms pulses delivered at 200 Hz for 2 s, was delivered across the brain with voltages ranging from 30–80 V (Supporting Information).

Immunofluorescence.

Third instar larvae were dissected and fixed with 4% (vol/vol) paraformaldehyde for 20 min and stained with rabbit anti-GFP and mouse anti-kinesin SUK4 (Supporting Information).

In Vivo Imaging and Vesicle Analysis.

Larvae expressing YFP-tagged vesicles were dissected and videos were taken at five frames per second at 200 ms for 30 s using MetaMorph software (Supporting Information).

Viability Study.

For each genotype, 1,000 embryos were grown on standard cornmeal media at 25 °C and monitored every 8 h to assess developmental stages (Supporting Information).

EB1-GFP Comet Analysis.

Larvae expressing EB1-GFP were dissected to expose the segmental nerve and imaged using confocal microscopy. EB1-GFP comets were manually counted/tracked and measured using ImageJ software (Supporting Information).