Drosophila CaV2 channels harboring human migraine mutations cause synapse hyperexcitability that can be suppressed by inhibition of a Ca2+ store release pathway

Abstract

Gain-of-function mutations in the human CaV2.1 gene CACNA1A cause familial hemiplegic migraine type 1 (FHM1). To characterize cellular problems potentially triggered by CaV2.1 gains of function, we engineered mutations encoding FHM1 amino-acid substitutions S218L (SL) and R192Q (RQ) into transgenes of Drosophila melanogaster CaV2/cacophony. We expressed the transgenes pan-neuronally. Phenotypes were mild for RQ-expressing animals. By contrast, single mutant SL- and complex allele RQ,SL-expressing animals showed overt phenotypes, including sharply decreased viability. By electrophysiology, SL- and RQ,SL-expressing neuromuscular junctions (NMJs) exhibited enhanced evoked discharges, supernumerary discharges, and an increase in the amplitudes and frequencies of spontaneous events. Some spontaneous events were gigantic (10-40 mV), multi-quantal events. Gigantic spontaneous events were eliminated by application of TTX-or by lowered or chelated Ca2+-suggesting that gigantic events were elicited by spontaneous nerve firing. A follow-up genetic approach revealed that some neuronal hyperexcitability phenotypes were reversed after knockdown or mutation of Drosophila homologs of phospholipase Cβ (PLCβ), IP3 receptor, or ryanodine receptor (RyR)-all factors known to mediate Ca2+ release from intracellular stores. Pharmacological inhibitors of intracellular Ca2+ store release produced similar effects. Interestingly, however, the decreased viability phenotype was not reversed by genetic impairment of intracellular Ca2+ release factors. On a cellular level, our data suggest inhibition of signaling that triggers intracellular Ca2+ release could counteract hyperexcitability induced by gains of CaV2.1 function.

Fig 1. SL- and RQ,SL-expressing flies exhibit coarse phenotypes.

(A) Schematic of Ca_V2-type calcium channel α1a subunit, with substitutions to Drosophila Cacophony (Cac) residues indicated (mammalian residues in parentheses) and a CLUSTAL-Omega alignment of Cac, human CACNA1A, and mouse CACNA1A amino acids spanning the relevant region ([*]—fully conserved; [:]—strongly similar; [.]—weakly similar). (B, C) Visible phenotypes resulting from crosses of elaV(C155)-Gal4 females x Balancer/UAS-cac-eGFP^{MUT or WT} males. (B) Premature spiracle protrusion in a larva expressing the UAS-cac-eGFP^SL transgenic line (also observed with UAS-cac-eGFP^RQ,SL expression). The spiracle phenotype did not occur in larvae expressing UAS-cac-eGFP^RQ or UAS-cac-eGFP^WT. (C) Same crosses as in (B) showing diminished UAS-cac-eGFP mutant viability. “UAS-cac Viability Index” = # UAS-cac-eGFP transgenic adult progeny/# Balancer Chromosome siblings, normalized to 100% for WT female progeny counts (Table 1 for raw counts; for all comparisons, n ≥ 115 Balancer sibling progeny counted). *** p < 0.001 by Fisher’s exact test compared to WT sex-specific control. # p = 0.05, ### p < 0.001 by Fisher’s exact test between sexes for the SL or RQ,SL genotypes. (D, E) For both females (D) and males (E), there was starkly diminished longevity for adult flies expressing the RQ,SL transgene. **** p < 0.0001 by Log-rank test.

Fig 2. Localization and expression levels of Cac-GFP transgenes are normal.

(A-D) Images of larval central nervous systems from animals expressing Cac-GFP protein (WT or mutant). Anti-GFP (red), and anti-Bruchpilot (Brp—green) staining are shown. Scale bar 100 μm. (E-H) Wild-type and mutant Cac-GFP successfully localized to NMJ active zones, as indicated by co-staining with anti-Brp (green) and anti-GFP (red). Scale bar 5 μm. (I) Western blots of fruit fly head lysates (10 heads/lane, single sex per Western), from flies expressing either elaV-Gal4 alone or the indicated UAS-cac-eGFP transgene driven by elaV-Gal4. Blots were probed with anti-GFP (top) and anti-Actin (bottom) antibodies. The band corresponding to Cac-GFP is indicated. Other bands are non-specific. (J) Compared to WT, there was no statistically significant change in Cac-GFP expression for any of the transgenic lines utilized in this study (band normalized to actin; p > 0.65, one-way ANOVA with Dunnett’s multiple comparisons vs. WT; GAL4 alone control excluded from analysis). (K) Actin levels were also steady across all transgenic lines (p > 0.71, one-way ANOVA with Dunnett’s multiple comparisons vs. WT).

Fig 3. Hallmarks of NMJ development are normal when Cac-GFP transgenes are expressed.

(A-D) NMJ images of the synapses on Muscle 6/7 of WT- and RQ,SL-expressing third-instar larvae, immunostained with anti-Discs Large (Dlg) and anti-GluRIIA antibodies. Scale bars, 25 μm. (E) For RQ,SL-expressing NMJs, average synaptic bouton numbers were normal, except for a slight undergrowth detected for synapse A2 muscle 6/7 (* p < 0.05, Student’s T-test vs. WT, n ≥ 8 NMJs for all genotypes and segments). (F) The number of glutamate receptor clusters per synapse at RQ,SL-expressing NMJs was not statistically significantly different than WT-expressing NMJs (p > 0.1, Student’s T-test, n ≥ 8 NMJs for all genotypes and segments). (G) For RQ,SL-expressing NMJs, there was a small increase in GluRIIA-containing receptor area coverage. (* p < 0.05 by Student’s T-test vs. WT for both measures, n ≥ 15 NMJs for each genotype).

Fig 4. SL- and RQ,SL-expressing NMJs display hyperexcitability in evoked neurotransmission.

(A) Average EPSP amplitudes at 0.4 mM [Ca²⁺]_e for non-transgenic control (w¹¹¹⁸) or Cac-GFP-expressing lines (** p < 0.01 by one-way ANOVA with Tukey’s post-hoc vs. w¹¹¹⁸; or # p < 0.05 and ### p < 0.001 vs. WT; n ≥ 12 for all genotypes). (B) Average quantal content (QC, estimated as EPSP/mEPSP) at 0.4 mM [Ca²⁺]_e (p > 0.15 by one-way ANOVA with Tukey’s post-hoc for all genotypes, compared to both w¹¹¹⁸ and WT controls). (C) Log-log plots of extracellular calcium concentration vs. QC corrected for non-linear summation (NLS QC). There are no statistically significant differences in calcium cooperativity between genotypes (p = 0.16, linear regression analysis). (D, E) Example electrophysiological traces of (D) normal and (E) abnormal EPSP waveforms. (F) Effect of genotype on EPSP waveforms in response to 30 presynaptic pulses. “RQ only” signifies larvae with a null endogenous cac mutation rescued to viability by the RQ-expressing transgene. (G) Effect of genotype on number of extra discharges observed per 30 presynaptic pulses (* p < 0.05 and *** p < 0.001 vs. WT by one-way Kruskal-Wallis ANOVA with Dunn’s post-hoc). (H) Penetrance and (I) severity of RQ,SL-associated extra discharge waveform dysfunction in low extracellular Mg²⁺ (6 mM). (J) NMJ recordings of 2 min spontaneous neurotransmission with an intact CNS. Measurements assessed: continuous trains of spontaneous activity > 2 sec in duration at any point in the recording; trains with postsynaptic events > 4 mV; trains with postsynaptic events > 10 mV; any observed postsynaptic event (trains or not) > 10 mV; any recording that was continuous trains of throughout (n = 9 for WT, n = 10 for RQ,SL; * p < 0.05, ** p < 0.01 by Fisher’s exact Test). All genotypes abbreviated (WT, RQ, SL, RQ,SL) are elaV(C155)-Gal4/Y; UAS-cac-eGFP^(X)/+ or w¹¹¹⁸ for non-transgenic wild type. Data bars represent the average value and error bars +/- SEM.

Fig 5. SL- and RQ,SL-expressing NMJs have enhanced mEPSPs.

(A) Electrophysiological traces of spontaneous activity at WT- and RQ,SL-expressing NMJs. Example traces with two different scales show variable severity of spontaneous neurotransmission phenotypes, in terms of frequency severity (left) or amplitude severity (right). (B) Effects of genotype on average spontaneous mEPSP amplitude (** p < 0.01 vs. w¹¹¹⁸ by one-way ANOVA with Tukey’s post-hoc; ### p < 0.001 vs. WT by one-way ANOVA with Tukey’s post-hoc. (C) Effects of genotype on spontaneous mEPSP frequency. ## p < 0.01 vs. WT by one-way ANOVA with Tukey’s post-hoc; n ≥ 12 NMJs, all genotypes. (D) Box and whisker plots of mEPSP amplitude range at 0.5 mM extracellular [Ca²⁺]. Box denotes 25^th-75^th percentile; line denotes median; + sign denotes average; whiskers range from 1^st-99^th percentile; individual data points outside the 1^st and 99^th percentiles are plotted; (*** p < 0.001 by Kruskal-Wallis ANOVA with Dunn’s post-hoc vs. either w¹¹¹⁸ or WT; n > 1400 mEPSPs for each genotype). (E) Cumulative probability histogram of the data in (D) showing a marked rightward shift in mEPSP amplitudes for SL- and RQ,SL-expressing NMJs. (F) Box and whisker plot (as in (D)) of mEPSP amplitude at 0.4 mM extracellular [Ca²⁺] with and without the Ca_V2.1 channel modifier BHQ (*** p < 0.001 by Kruskal-Wallis ANOVA with Dunn’s post-hoc vs. identical genotype +/- BHQ; n > 985 mEPSPs for each genotype). (G) Cumulative probability histogram of a subset of data in (F). 5 μM BHQ causes a partial leftward shift in the distribution of events for SL- and RQ,SL-expressing NMJs while not affecting WT-expressing NMJs. (H) Box and whisker plot (as in (D)) of mEPSP amplitude range when expressing the RQ,SL transgene for acute periods of developmental time (*** p < 0.001 by Kruskal-Wallis ANOVA with Dunn’s post-hoc vs. RQ,SL 0 hr., ### p < 0.001 by vs. RQ,SL 24 hr.; n > 1095 mEPSPs for each genotype). (I) Cumulative probability histogram of the data in (H) showing a rightward shift in mEPSP amplitudes for longer periods of RQ,SL expression.

Fig 6. Gigantic spontaneous events vanish in response to diminished Ca²⁺, buffered Ca²⁺, or blocked Na_V.

(A) Box and whisker plot of mEPSP amplitudes at 0.2 mM extracellular Ca²⁺. Plot as in Fig 5 (*** p < 0.001 by Kruskal-Wallis ANOVA with Dunn’s post-hoc vs. either w¹¹¹⁸ or WT; n > 780 mEPSPs for each genotype). (B) Cumulative probability histogram of the data in (A) showing a rightward shift in mEPSP amplitudes for SL- and RQ,SL-expressing NMJs– but less so than for 0.5 mM Ca²⁺, with smaller and fewer gigantic events (compare to Fig 5). (C) Box and whisker plots demonstrating elimination of gigantic spontaneous events by various manipulations. (*** p < 0.001 by Fisher’s exact test examining the incidence of gigantic mEPSPs > 10 mV vs. RQ,SL or SL alone, as appropriate). (D-F) Cumulative probability histograms of mEPSP size separately showing the effects of zero extracellular Ca²⁺ (D); application of BAPTA-AM in 0.5 mM Ca²⁺ (E); application of TTX in 0.5 mM Ca²⁺ (F). In each case, the rightward shift in mEPSP size distribution persists due to RQ,SL expression. However, the gigantic spontaneous events are eliminated (see frequency shift at arrowheads). (G) Box and whisker plot of spontaneous event amplitudes at 0.5 mM extracellular Ca²⁺ + TTX, with an intact central nervous system. (*** p < 0.001 by Mann-Whitney U Test of WT vs. RQ,SL; n = 900 mEPSPs for each genotype). (H) Cumulative probability histogram of the data in (G).

Fig 7. Failure analysis: SL- and RQ,SL-expressing NMJs show elevated release probability at very low extracellular calcium.

(A, B) Frequencies of evoked amplitudes at very low extracellular Ca²⁺ (0.14 mM) for (A) WT-expressing NMJs and (B) RQ,SL-expressing NMJs. For the RQ,SL-expressing NMJs, there is a clear rightward shift in the size distribution of RQ,SL-expressing events, as well as a marked decrease in the frequency of failures (categorized as 0 mV events). (C) For WT-, SL-, and RQ,SL-expressing NMJs, the average EPSP size for successfully evoked events, as well as estimated QC by failure analyses (0.14 mM Ca²⁺) (* p < 0.05; ** p < 0.01 by one-way ANOVA with Tukey’s post-hoc compared to WT). (D) Further lowering extracellular Ca²⁺ (0.1 mM) for RQ,SL reveals a leftward shift in size distribution and an increase in failure percentage compared to (B). (E) Box and whisker data are presented as in Figs 5 and 6 –this time showing the size distributions of spontaneous mEPSP events (WT, RQ,SL, and SL), as well as failure analysis (FA) evoked events for the same genotypes (failures excluded). (F, G) Box and whisker plots for mEPSP rise times (0.5 mM Ca²⁺, see Fig 5D) show a significant increase only for RQ,SL-expressing NMJs (F), as well as a dramatic slowdown for events > 2 mV in size, regardless of genotype (G).

Fig 8. Inhibition of an intracellular Ca²⁺ release pathway dampens gain-of-function phenotypes associated with FHM1-mimicking mutations.

(A) Schematic of an RNA interference (RNAi)-based approach to identify suppressors of gain-of-function electrophysiological phenotypes. The schematic cartoon was adapted from [45]. (B) Knockdown of Plc21C gene function reverses the increase in spontaneous mEPSP amplitude elicited by RQ,SL expression. (C) Box and whisker plots (as before) and (D, E) cumulative probability histograms (as before) demonstrate that heterozygous, loss-of-function point mutations in genes encoding the IP₃ receptor (Itpr^ug3/+) and the Ryanodine receptor (RyR^E4340K/+) significantly diminish the gain-of-function spontaneous mEPSP phenotypes in RQ,SL-expressing NMJs. *** p < 0.001 by Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test vs. RQ,SL alone. (F) The RyR^E4340K/+ condition diminishes evoked EPSP hyperexcitability phenotypes in a RQ,SL-expressing background (# of extra discharges [ED] per muscle–see also Fig 4).

Fig 9. Pharmacological inhibition of intracellular Ca²⁺ release dampens gain-of-function phenotypes associated with FHM1-mimicking mutations.

(A-C) Data displayed and analyzed as before. Box and whisker plots (A) and cumulative probability histograms (B, C) demonstrate that acute application of either LiCl (to block PIP₂ recycling) or Xestospongin C (to block IP₃ receptors) both suppress the gain-of-function spontaneous mEPSP phenotypes in RQ,SL-expressing NMJs. *** p < 0.001 by Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test vs. RQ,SL alone. (D) Cartoon model depicting neuronal components implicated in this study of regulating neurophysiology downstream of migraine-mimicking amino-acid substitutions. Red–Ca_V2 channels; gray–IP₃ receptors; blue–Ryanodine receptors; yellow–Na_V channels.