A Drosophila Model of Essential Tremor

Abstract

Essential Tremor (ET) is one of the most common neurological diseases, with an estimated 7 million affected individuals in the US; the pathophysiology of the disorder is poorly understood. Recently, we identified a mutation (KCNS2 (Kv9.2), c.1137 T > A, p.(D379E) in an electrically silent voltage-gated K⁺ channel α-subunit, Kv9.2, in a family with ET, that modulates the activity of Kv2 channels. We have produced transgenic Drosophila lines that express either the human wild type Kv9.2 (hKv9.2) or the ET causing mutant Kv9.2 (hKv9.2-D379E) subunit in all neurons. We show that the hKv9.2 subunit modulates activity of endogenous Drosophila K⁺ channel Shab. The mutant hKv9.2-D379E subunit showed significantly higher levels of Shab inactivation and a higher frequency of spontaneous firing rate consistent with neuronal hyperexcitibility. We also observed behavioral manifestations of nervous system dysfunction including effects on night time activity and sleep. This functional data further supports the pathogenicity of the KCNS2 (p.D379E) mutation, consistent with our prior observations including co-segregation with ET in a family, a likely pathogenic change in the channel pore domain and absence from population databases. The Drosophila hKv9.2 transgenic model recapitulates several features of ET and may be employed to advance our understanding of ET disease pathogenesis.

Figure 1

Climbing Response During Lifespan. The climbing index was assessed as the time taken for the first fly to climb 17.5 cm. (A) Expression of hKv9.2 and hKv9.2-D379E channels throughout neuronal development and effects on climbing response during lifespan. The mean climbing index ± SEM as a function of age is shown for Elav/+, Elav > hKv9.2 and Elav > hKv9.2-D379E. Each point represents the mean of 10 flies except for: Week 1, Elav > hKv9.2 (n = 9); Weeks 1–8, Elav > hKv9.2-D379E (n = 11); Week 6, Elav/+ (n = 7); Week 8, Elav/+ (n = 1). Flies expressing either Elav > hKv9.2 and Elav > hKv9.2-D379E displayed significantly faster climbing (p < 0.0003) throughout lifespan. (B) Expression of hKv9.2 and hKv9.2-D379E channels post developmentally in neurons and effects on climbing response during lifespan. The mean climbing index ± SEM as a function of age is shown for Elav-gal4/+ Gal80^ts, Elav-gal4 > Gal80^ts hKv9.2 and Elav-gal4 > Gal80^ts hKv9.2-D379E. Each point represents the mean of 10 or 9 flies. Flies expressing either Elav > hKv9.2 and Elav > hKv9.2-D379E Elav-gal4 > Gal80^ts displayed significantly faster climbing (p = 0.04–0.0001; weeks 3–5) during lifespan.

Figure 2

Lifespan Assays in hKv9.2 Transgenic Lines. (A) Expression of hKv9.2 and hKv9.2-D379E channels throughout neuronal development and effects on lifespan. A total of 200 virgin flies per line were sex-segregated within 4 h of eclosion and maintained in small laboratory vials (n = 20 per vial) containing fresh food in a low-temperature incubator at 25 °C and 40% humidity on a 12/12 h dark/light cycle. The flies were then transferred to fresh food vials every 3-4 days and mortality recorded. Significant differences were not observed between Elav/+ and Elav > hKv9.2 (p = 0.8771) or Elav/+ and Elav > hKv9.2-D379E (p = 0.0656). (B) Expression of hKv9.2 and hKv9.2-D379E channels post developmentally in neurons and effects on lifespan. A total of 100 virgin flies per line were sex-segregated within 4 h of eclosion and age-matched flies were maintained in small laboratory vials (n = 10 per vial) containing fresh food in a high-temperature incubator at 29 °C and 40% humidity on a 12/12 h dark/light cycle. The flies were then transferred to fresh food vials every 2-3 days and mortality recorded. Significant differences were observed between Elav-gal4/+Gal80^ts and Elav-gal4 > Gal80^ts hKv9.2 (p = 0.0012) or Elav-gal4/+Gal80^ts and Elav-gal4 Gal80^ts > hKv9.2-D379E (p = 0.0001).

Figure 3

Expression of hKv9.2 or hKv9.2-D379E subunits causes an alteration in the kinetics of the native Shab (Kv2) channels. (A) Examples of the current evoked from Drosophila Shab when depolarized from −133 mV to between −93 mV and −3 mV in flies expressing only native Shab (left panel), native Shab with the human Kv9.2 (middle panel), or native Shab with hKv9.2-D379E (right panel). The non-inactivating Shab becomes inactivating in the presence of either hKv9.2 subunit. (B) Diagram of the voltage step protocol used. (C) The I-V relationships for Shab determined from the three given genotypes. The peak current (left panel, n = 3) shows that expression of either Kv9.2 subunit causes a shift in activation to more negative voltages. The sustained current (middle panel, n = 3) shows that, at similar voltages, flies expressing either hKv9.2 subunit show reduced Shab current after 200 ms of depolarization. When the sustained current after 200 ms of depolarization to −3 mV is expressed as a percentage of the peak current (right panel, n = 3) the flies expressing only native Shab show high percentages, indicating very low levels of inactivation. When tested by two-way ANOVA, flies expressing either the wild-type hKv9.2 subunit (p < 0.001) or the ET mutant hKv9.2-D379E subunit (p < 0.001) were significantly different to flies expressing only native Shab. The mutant hKv9.2-D379E subunit shows much higher levels of inactivation than the wild-type hKv9.2 subunit (p = 0.0462).

Figure 4

hKv9.2 subunits have significant and different impacts on resulting neuronal activity. (A) Representative example of 5 seconds of spontaneous activity in flies between ZT1 and ZT7 (where ZT0 is lights-on and ZT12 is lights-off) expressing only native Shab (left panel), native Shab with hKv9.2 (middle panel), or native Shab with hKv9.2-D379E (right panel). (B) The spontaneous firing rate of action potentials in flies expressing either hKv9.2 subunit (data are mean ± S.D., n = 3) are significantly different with the mutant subunit having a higher frequency (t(4) = 5.3806, p = 0.0102).

Figure 5

Computational models of Shab ion channel activity describe their differing kinetics. Fitting the electrophysiological data (see Fig. 1) to classical Hodgkin-Huxley equations generated parameters that describe the behaviors (B). These descriptions match well with experimental data acquired (A). The hKv9.2 subunits activate at more negative values (activation Vh) as seen in the experimental data. The extent of inactivation is higher in the mutant hKv9.2-D379E as evidenced by the lower inactivation K. The speed of inactivation is also higher in the mutant hKv9.2-D379E as shown by the higher inactivation σ.

Figure 6

Whole cell computational models using hKv9.2 reflect changes in electrophysiological behavior. (A) Representative example of 5 seconds of spontaneous activity in flies expressing only native Shab (left panel), native Shab with hKv9.2 (middle panel), or native Shab with hKv9.2-D379E (right panel). (B) Model description of 5 seconds of neuronal activity using only native Shab (left panel), native Shab with hKv9.2 (middle panel), or native Shab with hKv9.2-D379E (right panel) at ZT0 (where ZT0 is lights-on/dawn and ZT12 is lights-off/dusk). (C) The model description of the spontaneous firing rate of action potentials in flies expressing either hKv9.2 subunit is not significantly different to the experimental data.

Figure 7

Effects of Expression of hKv9.2 and hKv9.2-D379E channels on Period or Rhythm Statistic. (A) Period of circadian locomotor rhythms in control flies and those expressing hKv9.2 or hKv9.2-D379E under the PDF promoter. (B) Period of circadian locomotor rhythms in control flies and those expressing hKv9.2 or hKv9.2-D379E under the TIM promoter. (C) The corresponding rhythm statistic obtained for flies using the PDF promoter. (D) The corresponding rhythm statistic obtained for flies using the TIM promoter.

Figure 8

Effects of Expression of hKv9.2 and hKv9.2-D379E channels on diurnal/nocturnal index, average night time sleep and total night time activity. (Top row) Flies expressing hKv9.2 or hKv9.2-D379E under the PDF promoter and controls. (Bottom row) Flies expressing hKv9.2 or hKv9.2-D379E under the TIM promoter and controls. (A,B) The diurnal/nocturnal index (D/NI) examining the distribution of day-time and night-time activity. (C,D) The proportion of time flies spent asleep during the night-time, as defined as 5 minutes or more of inactivity. (E,F) The raw activity counts for flies split between day-time (lighter color, left-hand bars) and night-time (darker color, right-hand bars). Significant differences are observed in various comparisons summarized in Table 1.