Giulia M. Serratto – Erika Pizzi – Luca Murru – Sara Mazzoleni – Silvia Pelucchi – Elena Marcello – Michele Mazzanti – Maria Passafaro – Silvia Bassani
Abstract
PCDH19 encodes for protocadherin-19 (PCDH19), a cell-adhesion molecule of the cadherin superfamily preferentially expressed in the brain. PCDH19 mutations cause a neurodevelopmental syndrome named epileptic encephalopathy, early infantile, 9 (EIEE9) characterized by seizures associated with cognitive and behavioral deficits. We recently reported that PCDH19 binds the alpha subunits of GABAA receptors (GABAARs), modulating their surface availability and miniature inhibitory postsynaptic currents (mIPSCs). Here, we investigated whether PCDH19 regulatory function on GABAARs extends to the extrasynaptic receptor pool that mediates tonic current. In fact, the latter shapes neuronal excitability and network properties at the base of information processing. By combining patch-clamp recordings in whole-cell and cell-attached configurations, we provided a functional characterization of primary hippocampal neurons fromembryonic rats of either sex expressing a specific PCDH19 short hairpin (sh)RNA. We first demonstrated that PCDH19 downregulation reduces GABAAR-mediated tonic current, evaluated by current shift and baseline noise analysis. Next, by single-channel recordings, we showed that PCDH19 regulates GABAARs kinetics without altering their conductance. In particular, GABAARs of shRNA-expressing neurons preferentially exhibit brief openings at the expense of long ones, thus displaying a flickering behavior. Finally, we showed that PCDH19 downregulation reduces the rheobase and increases the frequency of action potential firing, thus indicating neuronal hyperexcitability. These findings establish PCDH19 as a critical determinant of GABAAR-mediated tonic transmission and GABAARs gating, and provide the first mechanistic insights into PCDH19-related hyperexcitability and comorbidities.
Keywords PCDH19 .GABAAR . Hyperexcitability . Intellectual disability . ASD
Introduction
Mutations in the human gene PCDH19 result in an epileptic syndrome known as EIEE9 (epileptic encephalopathy, early infantile, 9; OMIM # 300088), characterized by early-onset seizures, cognitive impairment, and autistic features, in addition to a variety of behavioral problems and sleep dysregulation
[1–3]. PCDH19 is preferentially expressed in the brain, especially the limbic system and cortex [4–6], and encodes for protocadherin-19 (PCDH19), a cell-adhesion molecule of the cadherin superfamily [7, 8]. PCDH19 is supposed to mediate cell-cell recognition within neuronal circuits and their correct
assembly, in accordance with PCDH19 adhesive properties [9] and recent findings that supported PCDH19 involvement in neuronal migration and sorting [10–12]. Despite these advances, most aspects of PCDH19 biological functions remain elusive, and thus the link between PCDH19 neuronal role and EIEE9 pathological phenotype.
We previously reported that PCDH19 binds the alpha subunits of GABAA receptors (GABAARs) [10], ligandgated ion channels that mediate fast inhibitory transmission in the brain [13]. GABAARs are divided between synaptic receptors that mediate phasic inhibition in response to presynaptically released GABA and extrasynaptic receptors that provide tonic inhibition in response to low ambient concentration of GABA [14]. Consistently, extrasynaptic GABAARs generally display higher affinity for GABA, slower desensitization, and lower conductance, with respect to synaptic receptors [15, 16]. GABAARs are composed of the assembly of 5 of 19 available subunits, generally two alpha (alpha 1 to 6), two beta (beta 1 to 3), and either one gamma or delta subunits [17]. As a general rule, the gamma subunit is necessary for the synaptic localization, while deltacontaining receptors are excluded from synapses [18, 19]. However, the composition of extrasynaptic GABAARs that mediate tonic currents appears to be quite heterogeneous. There are evidences supporting the contribution
to tonic transmission of both delta and gamma subunits, and of all of the five different alpha subunits expressed in the hippocampus (alpha 1 to 5) [20–22].
We previously demonstrated that PCDH19 downregulation in hippocampal neurons impaired GABAAR receptor (GABAAR) surface expression and miniature inhibitory postsynaptic currents (mIPSCs) [10]. However, whether PCDH19 might affect GABAAR-mediated tonic current in addition to mIPSCs is unknown. This is a relevant question, since tonic transmission drives the migration and morphological maturation of neurons in the developing brain and is a key determinant of neuronal excitability in the adult brain [23–25]. More in general, tonic inhibition, by shaping neuronal network activity, is involved in epilepsy, sleep, and cognitive processes [22].
Likewise, PCDH19-dependent GABAAR modulatory mechanisms other than receptor surface availability remain unexplored. Indeed, GABAAR-mediated transmission depends both on the number of cell-surface receptors and on their biophysical properties, in primis channel conductanceand kinetics [14, 18, 26–28]. Subunit composition, allosteric modulators, and interacting proteins all contribute to the shaping of GABAAR biophysical properties [26, 29–33].
Here, we provide an expanded view of the GABAAR binding-protein PCDH19 and its modulatory role on GABAergic transmission. Our findings unveil that the regulation of GABAergic transmission by PCDH19 is not restricted to phasic currents but extends to the tonic component. Moreover, PCDH19 emerges as a multitasking GABAAR binding-partner that, besides regulating GABAAR surface expression [10], also regulates channel gating, with crucial implication for neuronal excitability and EIEE9 pathogenesis.
Materials and Methods
Neuronal Cultures and Transfection
For primary neuronal rat cultures, Sprague Dawley timedpregnant adult rats were purchased from Charles River Laboratories (Italy). Hippocampal neurons were prepared from rat embryos of either sex at embryonic day (E)18 as previously described [34]. The neurons were plated on coverslips coated with poly-D-lysine in multiwell culture plates in a 12-well format, at a density of 75,000/well. The neurons were grown in Neurobasal Medium (Life Technologies, Italy) supplemented with homemade B-27, 0.25% L-glutamine, 1% penicillin/streptomycin, and 0.125% glutamate (Sigma Aldrich) and maintained at 37 °C, 5% CO2. The B-27 was prepared as previously described [35], except for a final medium concentration of 2.5 μg/ml of apo-transferrin (Sigma-Aldrich, Italy) instead of 5 μg/ml of holo-transferrin. Hippocampal neurons at days in vitro (DIV) 4 were transfected with standard calcium phosphate method and pyramidal
cells were analyzed at DIV13–15 (Figs. 1 and 5; Supplementary Figure S1 and S2) or DIV11–15 (Figs. 2, 3, and 4). All animal care and experimental procedures were performed in accordance with the CNR licensing and were approved by the Italian Ministry of Health (authorization no. 100/2016 and 2D46A.N.463).
cDNA and shRNA Constructs
All plasmids used to transfect neurons were previously reported in the Bassani et al. paper, in which the validation by western blotting of PCDH19 shRNA and rescue strategy was also provided [10]. Briefly, the PCDH19 open reading frame with a C-terminal V5 tag (human isoform 4, lacking amino acid 892 of the canonical isoform, accession number NM_001184880.1; gift from Prof. J. Gecz, University of Adelaide) was cloned into cFUW (Addgene). PCDH19 short hairpin (sh)RNA (target sequence: 5′-gagcagcatgaccaatacaat-3′) or control shRNA (scramble, target sequence: 5′- gctgagcgaaggagagat-3′) were cloned into the pLVTHMvector (Addgene). The shRNA target sequence has 100% homology with the rat Pcdh19 sequence (NM_001169129.1), while it harbors two mismatches (5′-gagcagcacgaccaatacaac-3′, mismatches in italics) with the human PCDH19 sequence (NM_001184880.1), conferring PCDH19-V5 partial resistance to the shRNA. Hence, PCDH19-V5 and shRNA were used in combination for the rescue experiments.
Immunocytochemistry, Image Acquisition, and Analysis
For the immunocytochemistry (ICC) experiments, hippocampal neurons were fixed atDIV13with 4% paraformaldehyde + 4% sucrose for 8 min at room temperature. The neurons were incubated with primary (rabbit anti-PCDH19, 1:400, Bethyl Laboratories, USA) and secondary antibody (rabbit Alexa fluor
555, 1:300, Thermo Fisher Scientific, Italy) in gelatin detergent buffer (GDB: 30 mM phosphate buffer at pH 7.4 containing 0.2% gelatin, 0.5% Triton X-100, and 0.8 M NaCl) overnight at 4 °C and for 1 h at room temperature, respectively.
Images were acquired with a Zeiss LSM 800 confocal microscope (Carl Zeiss, Italy) by using × 63/1.4 oil objective. The transfected neurons were identified based on the presence of GFP encoded by the pLVTHM vector, and images were obtained from the z-projection (maximum intensity) of 3–4 stacks at 0.60-μm intervals (1024 × 1024 pixel resolution). The images were analyzed with Fiji software: a mask was created on the GFP channel to select neuronal dendrites and
the mean intensity of PCDH19 was measured.
Electrophysiological Recordings
Patch-clamp recordings were collected using an Axopatch 200B amplifier (Molecular Device, CA, USA). Data were digitized at 5 kHz and filtered at 1 kHz with a Digidata 1322 acquisition system. The acquisition software used was
Clampex 9.2 (Axon Instruments, Inc., CA, USA). Patch pipettes (GB150F-8P with filament, Science Products) were pulled from hard borosilicate glass on a Brown-Flaming P-87 puller (Sutter Instruments, Novato, CA, USA) and firepolished to a final electrical resistance of 5–7 MΩ for wholecell
recordings and 8–15MΩ for cell-attached recordings. All the experiments were performed at room temperature on dissociated hippocampal pyramidal neurons maintained in a solution containing (in mM) 138 NaCl, 4 KCl, 2 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 D-glucose, adjusted to pH 7.4 with NaOH. For experiments in whole-cell configuration, series resistance was monitored and, if it was > 20 MΩ, the recording was discarded. Data analysis was performed with Clampfit 10.3 (Molecular Devices, CA, USA).
Tonic Current Measurement and Analysis GABAAR-mediated tonic currents were recorded in whole-cell voltage-clamp mode at a holding potential (Vh) of − 70 mV in the presence of kynurenic acid (3 mM) to block glutamatergic transmission and GABA (0.1 μM) to increase the amplitude of tonic current and reduce variability among cultures. Pipettes were filled with a Cs-based solution containing (in mM) 140 CsCl, 1 MgCl2, 1 CaCl2, 10 EGTA, 10 HEPES, and 0.1 GTP-Na+ (adjusted to pH 7.4 with CsOH) supplemented with the Na+ channel blocker QX 314 bromide (4 mM). The amplitude of GABAAR-mediated tonic current was calculated by the outward shift in the baseline current induced by the GABAAR antagonist bicuculline (40 μM) [16, 28, 36, 37]. After
obtaining a stable recording for at least 1 min (untreated control condition), bicuculline was applied by using a gravity-driven perfusion system (RSC-200, BioLogic, France) through a micropipette positioned close to the soma of the recorded cell. All-points histograms were generated from 60-s epochs relative to untreated and bicuculline conditions. Tonic currents were defined by fitting Gaussian curves to these histograms. The peak of the Gaussian distribution (μ)
represents the mean holding current, while the standard deviation of the curve (σ) represents the root mean square (RMS) of the variance over the 60-s interval. Tonic current was calculated both as the difference between the μ values relative to untreated and bicuculline epochs (current shift, Imean) and difference of RMS noise between the two conditions [28]. Changes in the RMS noise values before and during bicuculline application were also used to predict the singlechannel conductance (γ) of tonic receptors. First, the squared standard deviation of the curve (σ2) was plotted against the Imean to extrapolate the single-channel current (i), based on the parabolic relationship described by the following formula: σ2(Imean) = i(1 − PO)Imean, where PO is the channel open
probability. In our experimental conditions, the PO of the GABAAR mediating tonic current is expected to be small, as the exogenous GABA concentration is low [36]. Therefore, the relationship between σ2 and Imean can be well approximated by the following equation: i = σ2/Imean [16, 36]. Next, single-channel conductance was calculated using the equation γ = i/(Vh − ECl), where Vh is the holding potential and ECl is the chloride electrochemical equilibrium potential (or reversal potential), corresponding to approximately 0 mV in our experimental conditions.
Cell-Attached Recordings GABAAR single-channel currents were recorded in voltage-clamp mode, cell-attached configuration. Patch pipettes were filled with the extracellular solution (composition described above) supplemented with 100 μM 4,4′-diisothiocyanato-2,2′-stilbenedisulfonic acid disodium salt (DIDS) to block ClC type Cl− channel activity, 1 mM 4- aminopyridine (4-AP), and 5 mM tetraethylammonium chloride (TEA-Cl) to block K+ channel activity, and 100 μM GABA to evoke GABAAR activity. Following the formation of a giga seal (> 10 GΩ), the output gain was set to 100 mV/ pA and the headstage switched to the capacitive feedback mode. At the end of each single-channel recording, the patch membrane was ruptured to measure the resting membrane
potential (RMP) of the cell. Although the intrapipette solution was not suitable for whole-cell experiments, RMP values, measured immediately after the rupture of the membrane, were analogous to those obtained in whole-cell experiments using a K-gluconate-based internal solution (values in Fig. 5).
GABAAR single-channel currents were elicited by voltage steps ranging from − 60 to +100mV(corresponding to a patch pipette voltage (Vpip) ranging from − 100 to + 60 mV) in 20-mV increments and 800-ms duration. For each patch, at each voltage step, all the sweeps where the channel opened were concatenated and analyzed. All-points histograms were generated from single-channel openings at each potential and fitted with the Gaussian equation. The peak of the Gaussian distribution represents the mean single-channel current, which was plotted against voltage to obtain the I/V relationship. Voltages were previously adjusted according to the measured RMP for each cell. The point at which the I/V curve crosses the voltage axis represents the reversal potential of the GABAAR-mediated current, which corresponds to the chloride reversal potential (ECl). Single-channel slope conductance for an individual cell was calculated from the slope of the linear regression obtained from the I/V plots. For those cells in which slope conductance could not be obtained, the chord conductance (γchord) at single potential was calculated according to the equation γchord = i/(Vh − ECl) where i is the observed single-channel current, Vh the holding potential, and ECl the chloride reversal potential. Open and close times were analyzed at Vpip = + 60 mV, corresponding to a Vh approximately 60 mV below the ECl, which is the same condition used to measure whole-cell tonic current. Duration histograms were fittedwith amixture of exponential distributions defined by and characterized by a time constant τ and a relative area a. Since concatenated traces were analyzed, we reported only the short close times that likely characterize intra-cluster shutting events, and thus likely define the kinetic of a single channel.
Current-Clamp Recordings The intrinsic excitability of neurons was evaluated by whole-cell recordings. To elicit action potentials (APs), neurons were held at − 60 mV and stimulated with depolarizing current injections of 4 s of duration and amplitude ranging from 0 to + 80 pA (current steps, ΔI = 10 pA). Patch pipettes were filled with a solution containing (in mM) 126 K-gluconate, 4 NaCl, 0.05 CaCl2, 0.1 EGTA, 10 HEPES, 10 D-glucose, 1 MgCl2, 3 ATP-Mg2+, and 0.1 GTPNa+, adjusted to pH 7.2 with KOH. Bath application of bicuculline (40 μM) was used to block GABAAR currents. To assess individual action potential (AP) waveforms, the first spike evoked by the minimum amount of current injected (rheobase) was analyzed. Spike width was measured at 50%
of the peak amplitude, which was calculated from the AP voltage threshold to the peak of APs. To extrapolate the AP voltage threshold values, phase-plane plots were constructed from the first time derivative of voltage (dV/dt) plotted against the membrane voltage [38]. AP threshold was defined as the voltage value at which dV/dt was 4% of its maximal value (dV/dtmax) [39, 40]. Rheobase was calculated by measuring the minimum amount of current injection able to induce a spike from Vh = − 60 mV. Input resistance (R input) was calculated as the slope of the V/I relationship obtained by plotting the steady-state membrane depolarization elicited by a series of subthreshold current steps of 1 pA increment and 4 s of duration.
Statistical Analysis
Three neuronal groups were compared: neurons transfected with scramble, shRNA, and rescue constructs. Statistical significance of the data was assessed by one-way ANOVA, considering transfection condition as the main factor. Two factors were considered for the two-way ANOVA test used to analyze data presented in Fig. 4c, e (transfection and % of τ open or τ close). ANOVA tests were followed by Holm-Sidak’s (Supplementary Figure S1) or Dunnett’s (all other figures) multiple-comparison post hoc test. Two-tailed unpaired Student’s t test was used to assess the statistical significance of the pairwise comparison shown in Fig. 3c. GraphPad Prism (version 6, GraphPad Software) was used for plotting data and for statistical analyses. All data are mean ± SEM. The threshold for statistical significance was p < 0.05. Asterisks indicate a p value < 0.05, and nonsignificance is denoted by “n.s.” Detailed statistical results, including exact p values, F factor, and degrees of freedom (DF), as well as cell number (N), are provided in the figure legends. Neurons were obtained
from the following number of independent preparations: 3 cultures for experiments of Fig. 1 and Supplementary Figure S1; 3–4 cultures for experiments of Figure S2; 3–5 cultures for experiments of Figs. 2, 3, and 4; and 3–7 cultures for experiments of Fig. 5.
Results
PCDH19 Downregulation Reduces the GABAAR Mediated Tonic Current in Hippocampal Neurons
As the first step of our study, we assessed whether PCDH19 downregulation might affect the GABAAR-mediated tonic current in primary hippocampal neurons. The standard method consists in applying a saturating concentration of the GABAAR antagonist bicuculline to block GABAARs and reveal the tonic inhibitory current from the shift in the holding current and the reduction in the current noise, which depend on extrasynaptic receptors [28]. To this end, we transfected hippocampal neurons from embryonic day (E)18 rat embryos at DIV4 with either a shRNA specific for PCDH19 (shRNA), a control shRNA (scramble), or PCDH19 shRNA plus a shRNA-resistant PCDH19 cDNA (rescue condition). We verified by ICC that shRNA neurons displayed a significant downregulation of PCDH19 expression, while rescue neurons showed an overall increase of PCDH19 dendritic signal compared to controls (scramble vs shRNA *p = 0.042, scramble vs rescue *p = 0.031; shRNA vs rescue ***p < 0.001; one-way ANOVA with Holm-Sidak’s post hoc test; Supplementary Figure S1a and S1b). At DIV13–15, we performed wholecell patch-clamp recordings on transfected neurons. GABA (0.1 μM) was added to the extracellular solution in order to avoid fluctuations in the ambient concentration of GABA [36]. Notably, the shRNA expression reduced the current shift upon bicuculline application (40 μM) by more than 50%compared to scramble, and PCDH19 overexpression was able to completely rescue the current shift (scramble vs shRNA, *p =
0.029; scramble vs rescue, n.s.; one-way ANOVA with Dunnett’s post hoc test; Fig. 1a, b). The analysis of the current noise, quantified as the root mean square (RMS) of variance, which is the most accurate method to detect small tonic currents [28, 41], confirmed the reduction of tonic current in shRNA-transfected neurons compared to scramble. In rescue condition, the difference in the RMS noise before and during bicuculline application was fully restored (scramble vs shRNA, *p = 0.037; scramble vs rescue, n.s.; one-way ANOVA with Dunnett’s post hoc test; Fig. 1a, left panel, and c).
Altogether, these results indicate that PCDH19 downregulation leads to a reduction of GABAergic tonic current. Reduced GABAergic tone might be the result of a reduced number of GABAARs on the cell surface, changes in receptor kinetics, or both.
PCDH19 Regulates the Kinetics of GABAARs
Changes in receptor number and kinetics both contribute to set the inhibitory tone. We recently demonstrated that PCDH19 downregulation reduces GABAAR surface expression in hippocampal neurons [10]. Here, we sought to determine whether PCDH19 might also regulate the biophysical properties of GABAARs.
First insights on single-channel conductance were obtained by the analysis of noise variance [16, 28, 36], as described in the methodological section. The predicted unitary current was consistent with the conductance ranges reported for receptors that mediate tonic inhibition [16, 36, 42–44], and was not
statistically different for neurons transfected with either scramble, shRNA, or rescue, suggesting that PCDH19 does not regulate the conductance of GABAARs (Fig. 1d, e).
To confirm these data and study whether PCDH19 might modulate the kinetics of GABAARs, we shifted from wholecell to cell-attached patch-clamp configuration, and recorded GABAAR current from single channels on the somata of neurons. Here, the chance to record currents from extrasynaptic receptors, including those that mediate tonic current, is high, since synaptic receptors are incorporated in clusters that occupy less than 1% (0.72%) of the soma surface [45].
Hippocampal neurons were transfected with shRNA, scramble, and shRNA + PCDH19 (rescue) at DIV4, and single channels were recorded at DIV11–15. We used a high concentration of GABA in the patch pipette (100 μM GABA), which is able to rapidly induce GABAAR desensitization and causes extrasynaptic receptors to enter prolonged closed states interrupted by discrete clusters of openings. Each cluster is supposed to derive from the repeated activation of a
single channel [44].
Irrespective of transfected constructs, recorded GABAARs exhibited three distinct conductance levels, as previously reported in literature [44, 46]. In particular, low-, mid-, and high-conductance states were observed during single-patch recordings, alone or in combination (Fig. 2a–c). The average values of the conductance states were not significantly different between scramble, shRNA, and rescue neurons (Fig. 2d).
The relative percentage of conductance levels did not vary between the different conditions (scramble, shRNA, and rescue) and was characterized by a predominance of a low- followed by a mid-conductance state, while a highconductance state, when displayed, was scarcely represented (Fig. 2e).
In almost all the cells from the three conditions recorded, the GABAARs opened predominately at the low-conductance state (Fig. 2b), which is generally attributed to GABAARs mediating tonic current [16, 36, 42], suggesting that the majority of channels recorded in single-channel experiments were indeed extrasynaptic GABAARs mediating tonic currents.
Irrespective of the transfected constructs, and as expected for GABAAR-mediated currents, the current/voltage (I/V) relationships of the three groups of neurons revealed a chloride reversal potential (ECl) close to − 60 mV, indicating a near completion of ECl shift towards more negative potentials by this time (Fig. 3a, b), consistent with the literature [47, 48]. The well-documented shift of ECl from depolarized to hyperpolarized values in neurons is attributed to the regulated expression of chloride transporters during development [49], and an ECl shift was detected also in our cultures (DIV4 vs DIV18 neurons, *p = 0.033; two-tailed unpaired t test; Fig. 3a, c). These data indicate that shRNA-mediated PCDH19 downregulation between DIV4 and DIV11–15 did not have a major effect on the modulation of ECl.
Next, we compared the single-channel properties of the three groups of neurons transfected at DIV4 and recorded at DIV11–15 (Fig. 4). We focused on the low-conductance state, which was the most represented and typically attributed to tonic current mediating GABAARs, to get insights into channel kinetics following PCDH19 downregulation. In particular, we compared the open and close time properties of GABAARs in neurons expressing scramble,
shRNA, and rescue constructs.
In control neurons (scramble), we observed two open time constants (open τ1 and τ2), characterized by distinct durations, which were not different from those recorded from shRNA and rescue neurons (Fig. 4a, b).
However, the open time distribution, evaluated as a percentage of the open time constant areas, was significantly different following PCDH19 downregulation. In particular, while in scramble and rescue neurons open time was equally distributed between short (τ1) and long (τ2) openings, in shRNA-expressing neurons, the short openings predominated over the long openings (% τ1 open: scramble vs shRNA, **p= 0.005; scramble vs rescue, n.s.; % τ2 open: scramble vs shRNA, **p = 0.005; scramble vs rescue, n.s.; two-way ANOVA with Dunnett’s post hoc test; Fig. 4c).
By contrast, no differences were observed between the three groups of neurons, neither in close time constants (Fig. 4d) nor in close time distribution (Fig. 4e).
The preferential opening of the channel at the shortest time constant in shRNA neurons translates into a flickering behavior of GABAARs, characterized by the rapid transition between open and closed states (Fig. 4a).
In conclusion, these data indicate that PCDH19 downregulation affects GABAAR gating, by increasing the contribution of brief openings at the expense of long ones, which is known to decrease the current flowing through the channel per time unit [50].
PCDH19 Downregulation Increases Neuronal Intrinsic Excitability
The capability of PCDH19 to modulate GABAAR gating provides a mechanism for GABAergic tone regulation, which in turn sets neuronal excitability and hence regulates neuronal network activity [22].
To investigate whether PCDH19 downregulation might affect neuronal intrinsic excitability, hippocampal neurons were transfected as before (at DIV4) with scramble, shRNA, and rescue. At DIV13–15, whole-cell patch-clamp recordings in current-clamp mode were performed to analyze passive and active membrane properties of neurons in all conditions (Fig. 5a–f).
Despite no changes in resting membrane potential (RMP) (Fig. 5b, − BIC), PCDH19 downregulation, contrary to rescue condition, led to a significant increase in the mean input resistance with respect to control neurons (scramble vs shRNA, **p = 0.007; scramble vs rescue, n.s.; one-way ANOVA with Dunnett’s post hoc test; Fig. 5c, − BIC), consistent with the reduction in tonic GABAergic currents described above (Fig. 1a–c). As a consequence, shRNA-expressing neurons required less current injection to trigger an action potential (AP) (rheobase), compared to the control condition, indicating higher intrinsic excitability. The coexpression of shRNA together with PCDH19 (rescue) restored the rheobase to control levels, thus confirming the specificity of shRNA effects (scramble vs shRNA, ***p < 0.0001; scramble vs rescue, n.s.; one-way ANOVA with Dunnett’s post hoc test; Fig. 5d, − BIC).
As further evidence of their hyperexcitability, shRNAexpressing neurons showed a higher firing frequency with respect to controls. Statistical significance was reached between 10 and 40 pA, and a trend was observed for the other current steps (firing frequency at 10 pA, scramble vs shRNA, *p = 0.032; scramble vs rescue, n.s.; 20 pA, scramble vs shRNA, *p = 0.019; scramble vs rescue, n.s.; 30 pA, scramble vs shRNA, *p = 0.014, scramble vs rescue, n.s.; 40 pA, scramble
vs shRNA, *p = 0.024; scramble vs rescue, n.s.; one-way ANOVA with Dunnett’s post hoc test, Fig. 5e and Table S1a).
The analysis of the AP waveform revealed no difference in AP amplitude or width following PCDH19 downregulation (Figure S2a, b, d), and no significant difference in voltage threshold was observed between the three groups of neurons (Figure S2c, d).
To investigate the causal link between reduced GABAARmediated inhibition and increased neuronal excitability in PCDH19 shRNA-expressing neurons, we repeated analogous experiments in the presence of bicuculline (Fig. 5a–d, + BIC; Fig. 5f; and Table S1b). If this hypothesis is true, blocking GABAAR-mediated currents is expected to affect the firing frequency more in controls than in shRNA neurons, thus reducing the firing frequency differences between groups. This
was indeed the case (Fig. 5a, f). As expected, bicuculline bath application (40 μM) increased the firing frequency of scramble and rescue neurons with respect to the basal condition, especially at low values of current injection, as indicated by a firing frequency ratio (+ BIC/− BIC) above 1. By contrast, the effect of bicuculline on shRNA-expressing neurons was negligible, and their firing frequency ratio between bicuculline and basal conditions was significantly lower with respect to the control neurons (firing frequency ratio, + BIC/− BIC, 30 pA: scramble 3.833 ± 1.144, shRNA 0.734 ± 0.292, rescue 2.017 ± 1.127; scramble vs shRNA, *p = 0.026, scramble vs rescue, n.s.; one-way ANOVA with Dunnett’s post hoc test, (Fig. 5a, f and Table S1). No significant differences in RMP
were observed between the three groups of neurons (Fig. 5b, + BIC), and notably, bicuculline erased the differences in input resistance and rheobase between shRNA and scramble neurons (Fig. 5c–d, + BIC).
Altogether, these results indicate that PCDH19 downregulation enhances neuronal intrinsic excitability and suggest a causal link between reduced GABAergic transmission and hyperexcitability, with important implications for the pathogenesis of PCDH19-related epilepsy.
Discussion
This study stemmed from the new recently identified interaction between PCDH19 and GABAARs [10] and sought to provide a more comprehensive understanding of PCDH19’s role in GABAergic transmission and neuronal excitability.
We first demonstrated that, in primary rat hippocampal neurons, PCDH19 downregulation reduces inhibitory tonic currents, and we next investigated both the underlying mechanisms and the consequences on neuronal excitability.
When considering the mechanisms of tonic current reduction, a first explanation might be the reduction of the GABAAR amount on the neuronal surface, as previously demonstrated for alpha1- and alpha2-containing receptors in PCDH19 shRNA-expressing neurons [10]. Besides evidences supporting alpha1 and alpha2 contribution to tonic transmission [21, 22, 45], it is important to note that PCDH19 is also able to associate with typically extrasynaptic GABAAR subunits, most likely because of PCDH19 binding-site conservation among alpha subunits [10].
Notably, we demonstrated that a second non-exclusive mechanism exists and concerns the biophysical properties of GABAARs remaining on the neuronal surface. We first analyzed single-channel conductance that reflects receptor composition in addition to its gating state, i.e., the number of GABA binding sites occupied [16, 36, 44, 51]. We demonstrated that this parameter does not change upon PCDH19 downregulation. In particular, we identified three distinct conductance states within single patches from primary hippocampal neurons, suggesting that we recorded from a heterogeneous extrasynaptic receptor pool. Although we cannot exclude that receptors mediating phasic inhibition but transiently located outside the synapses [52] might have contributed to such heterogeneity, these findings are consistent with the well-known involvement of multiple receptor types (containing both delta and gamma subunits, together with different alpha subunits) in tonic transmission [20–22]. Since PCDH19 downregulation did not affect conductance state values, numbers, or their relative proportions, this suggests the overall retention of extrasynaptic GABAAR pool composition.
Next, we focused on the low-conductance state, which is typical of extrasynaptic receptorsmediating tonic currents [16, 36, 43, 44], and we analyzed channel kinetics. We showed that, following PCDH19 downregulation, GABAARs displayed a significant change in the relative proportion of their opening times, with short openings prevailing over the long ones. The resulting flickering behavior of GABAARs is known to reduce the current flowing through the channel per
time unit [50]. Since PCDH19 directly binds GABAARs, this physical interaction might induce structural rearrangements that stabilize the channel open conformation. In support of this, PCDH19 binds a 10–amino acid sequence within the intracellular loop between the transmembrane domains (TM) 3 and TM4 of alpha subunits [10]. While the regions surrounding the TM2 are important for GABAAR conductance and ion selectivity [53], the TM3–TM4 loop confers gating properties to the channel [54].
Even though conductance analysis argues against major changes in the composition of the extrasynaptic GABAAR pool upon PCDH19 downregulation, as discussed above, we cannot completely exclude some subunit composition changes within the low-conductance receptor pool, able to selectively affect kinetics. For instance, spontaneously opening GABAARs, which do not require GABA binding to activate, display a shorter average open time and lower opening probability but similar conductance with respect to the conventional pool of GABAARs. This likely relies on their subunit composition that, however, remains elusive [55, 56].
Irrespective of their relative contribution, it is reasonable to assume that both altered kinetics and reduced expression of the GABAARs on the neuronal surface
are responsible for tonic current reduction in PCDH19 shRNA-expressing neurons.
Tonic inhibition is a powerful regulator of intrinsic neuronal excitability [22, 23, 57]. Consistently, we observed a decreased rheobase together with an increased spiking frequency following PCDH19 downregulation in neurons. These data fit well with the increased input resistance of shRNA-expressing
neurons. When the RMP is close to ECl, as in our experimental conditions, alterations in Cl− currents are expected to modify the input resistance rather than RMP, thus modulating the responsiveness of neurons to synaptic stimuli. Indeed, although the RMP was not affected by PCDH19 downregulation, small amounts of current injection were more effective in changing the membrane potential, and so the intrinsic excitability, in shRNA-expressing neurons
compared to the control condition.
Input resistance increases as a consequence of reduced GABAergic tone but also in response to reduced cell size [58]. Since a reduced arborization has been observed in hippocampal pyramidal neurons upon PCDH19 downregulation [10], altered neuronal morphology, which might itself arise from the GABAergic signaling impairment [25, 59], could have contributed to increased input resistance and neuronal excitability.
However, acute bath application of bicuculline was able to erase the differences in input resistance between controls and shRNA neurons, thus arguing against a major influence of neuronal arborization. Notably, bicuculline also erased the differences in rheobase between groups and had a negligible effect on the firing frequency of shRNA-expressing neurons, thus supporting the existence of a causal link between reduced GABAAR-mediated inhibition and increased neuronal excitability in PCDH19 shRNA-expressing neurons.
The regulation of neuronal excitability is crucial to maintain the neuronal firing rate within operational ranges to prevent saturation and to limit the number of active neurons, thus allowing a low-noise and sparse information coding system [22, 60, 61]. Indeed, reduced GABAAR-mediated tonic current and neuronal hyperexcitability are associated with epileps [23, 62–64]. Moreover, tonic inhibitory transmission is involved also in the comorbidities of epilepsy, such as cognitive and behavioral processes and sleep [22, 65–68], which are often compromised in EIEE9 patients [1–3].
By describing PCDH19 as a regulator of GABAergic tonic conductance, we provide here a functional link between PCDH19 loss of function and EIEE9 phenotypes. A critical determinant of EIEE9 pathogenesis is thought to be the cell interference: EIEE9 would arise from the coexistence of neurons expressing wild-type PCDH19 and neurons non-expressing or expressing a mutant form of PCDH19 [1, 69]. How would our GABAergic hypothesis fit into this model?
The cell-interference model implies that PCDH19 loss of function and related GABAergic defects involving all brain cells can be compensated for, possibly by homeostatic mechanisms and/or redundant proteins, for instance by other protocadherins that bind GABAARs [70]. By contrast, a mosaic pattern of PCDH19 expression and related heterogeneously distributed GABAergic defects would have detrimental effects for neuronal connectivity. With this regard, pathogenic
variant mosaicism has been reported in genes causing epilepsy-related neurodevelopmental disorders, including gen e s encodi ng for GABAAR subuni t s [71] .
Mechanistically, it is tempting to speculate that a heterogeneous GABAergic tone between PCDH19-positive and PCDH19-negative cells might introduce, together with a scrambled PCDH19 adhesive code, a bias in cell-sorting patterns during brain development, and successively affect memory traces, which rely on the wiring of sparsely distributed neurons sharing the same excitatory level [72–74].
In conclusion, this study expands the knowledge of PCDH19’s role in the modulation of GABAAR-mediated transmission, and sheds light on novel mechanisms in the pathogenesis of EIEE9. The wide influence of PCDH19 on GABAARs is noteworthy. Altogether, the previous [10] and present findings demonstrate that PCDH19 is able to influence both components of GABAergic transmission, i.e., phasic and tonic inhibition. Moreover, PCDH19 demonstrated to act at different levels, regulating not only the amount of GABAARs that reaches the neuronal surface but also the gating of surface receptors. Our findings provide a key to interpret the complex EIEE9 clinical phenotype and to plan future specific therapeutic approaches.
Acknowledgments This work was supported by Fondazione Telethon [grant number GGP17260 to S.B.]; Insieme per la ricerca PCDH19 – ONLUS and The Cute Syndrome Foundation [2014 grant to M.P.]; PCDH19 Alliance [2015 grant to M.P.]; Foundation Jérome Lejeune [2016 grant to M.P.]; Banca Nazionale del Lavoro (BNL) [2016 grant to M.P.]; Italian Ministry of Education, University and Research [2017 PRIN grant, number 2017C9HLW to M.P.], and Fondazione Emilio Trabucchi.
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Conflict of Interest The authors declare that they have no competing interests.
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