Ectopic HCN4 expression drives mTOR-dependent epilepsy in mice

Abstract

The causative link between focal cortical malformations (FCMs) and epilepsy is well accepted, especially among patients with focal cortical dysplasia type II (FCDII) and tuberous sclerosis complex (TSC). However, the mechanisms underlying seizures remain unclear. Using a mouse model of TSC- and FCDII-associated FCM, we showed that FCM neurons were responsible for seizure activity via their unexpected abnormal expression of the hyperpolarization-activated cyclic nucleotide-gated potassium channel isoform 4 (HCN4), which is normally not present in cortical pyramidal neurons after birth. Increasing intracellular cAMP concentrations, which preferentially affects HCN4 gating relative to the other isoforms, drove repetitive firing of FCM neurons but not control pyramidal neurons. Ectopic HCN4 expression was dependent on the mechanistic target of rapamycin (mTOR), preceded the onset of seizures, and was also found in diseased neurons in tissue resected from patients with TSC and FCDII. Last, blocking HCN4 channel activity in FCM neurons prevented epilepsy in the mouse model. These findings suggest that HCN4 play a main role in seizure and identify a cAMP-dependent seizure mechanism in TSC and FCDII. Furthermore, the unique expression of HCN4 exclusively in FCM neurons suggests that gene therapy targeting HCN4 might be effective in reducing seizures in FCDII or TSC.

Figure 1:. Model of mTOR-dependent FCM-associated seizures.

(A) Timeline of the experimental paradigm. (B) Diagram of Rheb^CA effect on mTOR complex 1 (mTORC1). (C) Diagram of a mouse brain with superimposed image of a FCM in the medial prefrontal cortex (mPFC). Linked circles mark the approximate locations of independent pairs of recording electrodes in the ipsilateral and contralateral hemispheres. (D) Confocal images of GFP and tdTomato (pseudo-colored green) fluorescence and phospho-S6 immunostaining (red) in coronal sections from 3 month-old mice expressing GFP (control) or Rheb^CA (+ tdTomato) in the mPFC. Scale bars: 150 μm. (E and F) Bar graphs of the distance from pia surface (E) and soma size (F) of GFP- and Rheb^CA-expressing neurons. (G) B&W phospho-S6 immunostaining from images shown in D. Scale bar: 150 μm. (H) Bar graphs of phospho-S6 (pS6) immunofluorescence in GFP and Rheb^CA-expressing neurons. (I) Image of the video-EEG set-up. (J) Representative examples of an EEG trace and higher temporal resolution traces in the inset. Scale bars: 10 s/200 μV and 2 s/200 μV (inset). Data are mean ± SEM. ***: P<0.001, ****: P<0.0001, ns: not significant. Statistical analysis: MannWhitney U test (E), Student’s t test (F and G). Exact P values can be found in Table S4.

Figure 2:. Silencing cytomegalic neurons in mTOR-driven FCM prevents seizure activity.

(A) Confocal images of GFP and tdTomato fluorescence (pseudo-colored green) and phospho-S6 immunostaining (red) in coronal sections from 4 month-old littermate mice (used in panel J) expressing Rheb^CA + GFP or + Kir2.1 (fused to tdTomato). Scale bar: 100 μm. (B) B&W phospho-S6 immunostaining from images shown in A. (C) Bar graphs of normalized phospho-S6 immunofluorescence intensity and soma size of Rheb^CA neurons co-expressing GFP (green) or Kir2.1 (pink). (D) Bar graphs of cell capacitance, resting membrane potential (RMP), and membrane conductance of Rheb^CA neurons co-expressing GFP or Kir2.1. Patch clamp recordings were obtained in acute slices from P26-P42 mice. (E) Superimposed individual action potentials from Rheb^CA neurons in both conditions. Scale bars: 2 ms/40 mV. (F) Bar graphs of the action potential (AP) half-width and threshold. (G) Representative depolarization and action potentials upon current injection in Rheb^CA co-expressing GFP or Kir2.1. Scales: 200 ms/40 mV. (H) Injected current amplitude against the mean number of action potentials for generating an input-output curve in Rheb^CA neurons. The grey area outlines the SEM for each curve. (I) Heat map of the number of seizures over a 7-day long recording period in mice containing Rheb^CA neurons with GFP or Kir2.1. (J) Bar graphs of the duration and number of seizures per day in the two conditions. Statistical analyses: Student’s t test (C, D, F, and J, seizure frequency), Two-way repeated measure ANOVA, followed by Sidak post-test (H), and Mann Whitney U test (J, seizure duration). Data are mean ± SEM. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, ns: not significant. Exact P values can be found in Table S4.

Figure 3:. Abnormal HCN-mediated currents in FCM neurons are depolarizing and confer cAMP-dependent firing.

(A and B) Bar graphs of the RMP and conductance of control (GFP) and Rheb^CA neurons recorded in littermate P26-P42 mice. (C) Representative depolarization and action potentials upon current injection in Rheb^CA or control (GFP) neurons. Scales: 100 ms/40 mV. (D) Injected current amplitude plotted as a function of the mean number of action potentials for generating an input-output curve in Rheb^CA neurons. The grey area outlines the SEM for each curve. (E) Representative voltage traces in response to a −500 pA step in neurons expressing GFP (black) or Rheb^CA (green). Neurons were recorded in current-clamp at their RMP and voltage traces were superimposed post-recording. The arrow points to a hyperpolarization-induced voltage sag. Scales: 100 ms/10 mV. (F) Voltage traces in response to a −500 pA step from GFP-expressing neurons (black), Rheb^CA-expressing neurons (green), and Rheb^CA-expressing neurons in the presence of zatebradine (40 μM, red). Voltage responses were rescaled and superimposed post-recording. Scale: 100 ms. (G) Plots of the sag amplitude following a −100 pA from GFP or Rheb^CA neurons (left) or a −500 pA step protocol from Rheb^CA neurons under vehicle or zatebradine condition (right). (H) Representative current traces in cortical GFP or Rheb^CA neurons with vehicle or zatebradine. Protocol: conditioning step to −40 mV followed by 10 mV-hyperpolarizing steps from −130 to −40 mV. Scale bars: 200 ms/1 nA. The blue dotted lines illustrate where the difference in current amplitude (ΔI) was measured within each voltage step to generate current-voltage (ΔI-V) curves in K. (I) Current amplitude (I, measured at the end of the trace) versus the voltage in each condition. The grey area indicates the SEM for each curve. (J) Bar graphs of the RMP for each condition. (K) ΔI-V curves. At −90 mV, ΔI corresponds to Ih. Statistics is for Rheb^CA vs Rheb^CA + zatebradine. (L) Scatter plot of Ih (measured at −90 mV) against the RMP. Two-tailed Pearson r with correlation coefficients. (M and N) Voltages traces during bath-application of forskolin on Rheb^CA and control neurons (M), and resulting plots of the voltage changes (N). The red dots indicate the neurons (4/9) that generated repetitive firing upon forskolin application. Scale bars: 5 min/30 mV. Data are mean ± SEM. Statistical analyses: Unpaired Student’s t test (A,B, and J), Mann-Whitney U test (G), paired Student’s t test (N), Two-way repeated measure ANOVA, followed by Sidak post-test (D) and by Tukey’s post-test (I, K). ****:P<0.0001, ***:P<0.001, *:P<0.05, and ns: not significant. Exact P values can be found in Table S4.

Figure 4:. FCM neurons display abnormal mTOR-dependent HCN4 expression.

(A-D) Immunostaining for HCN1 (A), HCN2 (B), and HCN4 (C) in coronal sections containing Rheb^CA neurons co-expressing tdTomato. HCN4 staining was performed using antibodies from Neuromab and Alomone. The bottom images display HCN staining in B&W. Scale bars: 200 μm. The brain section in D was more caudal than the ones in A-C resulting in a larger cortex. In addition, 2.5 instead of 2 μg/μl of Rheb^CA was used. (E) Quantification of HCN1-HCN4 immunostaining in the ipsilateral versus contralateral cortex.. (F) Higher magnification images of HCN4 (green, Neuromab antibody), DAPI, and tdTomato. Scale bar: 60 μm. (G) Quantification of the percentage (%) of tdTomato+ cells expressing HCN4. (H) Images of HCN4 immunostaining, tdTomato fluorescence and DAPI in GFP electroporated mice. Scale bar: 60 μm. (I) Bar graph of integral fluorescence per GFP or Rheb^CA cell. Data are mean ± SEM. Statistical analyses: paired Student t-test (E for HCN1, 3, and 4), unpaired Student t -test (I), and Wilcoxon test (E, for HCN2), P<0.0001, ns: not significant.

Figure 5:. HCN4 expression is mTOR-dependent and precedes seizure onset.

(A and B) HCN4 immunostaining (Neuromab) and tdTomato fluorescence in Rheb^CA mice treated with vehicle (A) or rapamycin (B) and higher magnification images ). Scale bars: 140 and 60 μm, respectively. (C) Integral fluorescence in cells expressing Rheb^CA from mice treated with vehicle or rapamycin. (D) Normalized qRT-PCR values for Hcn1, 2, and 4 as well as Vegf divided by Gapdh from microdissected cortices containing GFP or Rheb^CA. (E and F) Representative current traces in P8-P12 cortical neurons expressing GFP (control) or Rheb^CA. Scale bars: 100 ms/500 pA. The blue dotted lines illustrate where the h current amplitude (Ih) was measured within each voltage step to generate Ih-V curves (F). (G) Plot of the zatebradine block of Ih (measured at −90 mV) over time in a Rheb^CA neuron. Left inset: traces of Zatebradine block at −90 mV in Rheb^CA neurons. Scale bars: 200 ms/100 pA. (H) Bar graphs of -Ih at the different ages under control and Rheb^CA conditions. Statistical analyses: Unpaired Student t-test. (C), Two-way repeated measures ANOVA followed by Tukey’s post-test (F), and One way ANOVA (H). Data are mean ± SEM. ****:P<0.0001, ***:P<0.001, *:P<0.05, and ns: not significant. Exact P values can be found in Table S4.

Figure 6:. Ectopic HCN4 expression in diseased neurons in human FCDII cortices.

(A and B) Staining for HCN4 in patient 1 with FCDII at low (A) and high magnifications (B). Images in B was approximately from the squares in A Scale bars: 250 μm (A) and 30 μm (B). (C) Staining for HCN4 in patient 2 with FCDII at low and high magnifications. The pink arrows point to HCN4-positive cells. Scale bars: 250 μm and 30 μm. (D) Image at a lower magnification for patient 2. Scale bar: 2600 μm. (E and F) Relative soma size (E) and HCN4 intensity (F) in HCN4-positive and HCN4-negative cells averaged per FCDII sample. (G and H) Immunostaining for HCN4 and phospho-S6 (G) or SMI-311 (H) in FCDII tissue from patient 6 co-stained with DAPI. Scale bars: 70 μm (G), 30 μm (H). Statistical analysis: paired Student t-test, ***:P<0.001, **:P<0.01.

Figure 7:. Ectopic HCN4 expression in diseased neurons in human TSC cortices.

(A and C) Immunostaining for HCN4 and phospho-S6 TSC tissue from patient 8 co-stained with DAPI. Scale bars: 70 μm (A) and 40 μm (C). (B) Higher magnification of the cell shown in the white square in A. Scale bar: 35 μm. (D) Quantification of phospho-S6 (pS6) intensity in HCN4+ and HCN- cells. Statistical analysis: paired Student t test. (E) Immunostaining for HCN4 and SMI 311 in TSC tissue from patient 9 co-stained with DAPI. Scale bar: 40 μm. (F) Higher magnification of the cell shown in the white square in E. Scale bar: 20 μm.

Figure 8:. Blocking HCN4 channel activity in FCM neurons prevents epilepsy.

(A and B) Representative voltage-traces of Rheb^CA neurons with and without nonfunctional HCN4 (HCN4^NF) channel expression. Neurons were recorded in acute slices from P21-P35 littermate mice electroporated with Rheb^CA+GFP or Rheb^CA+HCN4^NF (+ tdTomato). (B) Ih-V curve in each condition. (C) Bar graphs of RMP. Recordings were obtained in acute slices from P21-P35 mice. (D) Confocal images of GFP and tdTomato fluorescence (pseudo-colored green) and phospho-S6 immunostaining (red) in coronal sections from 4 month-old mice expressing Rheb^CA+GFP or + HCN4^NF (+ tdTomato). Scale bars: 80 μm. (E) Bar graphs of soma size and normalized (to GFP control cells) phospho-S6 immunofluorescence for neurons expressing Rheb^CA+GFP or Rheb^CA+HCN4^NF. (F) Bar graphs of the action potential (AP) threshold and half-width. (G) Representative depolarization and action potentials upon current injection in Rheb^CA neurons with and without HCN4^NF. Scales: 200 ms/40 mV. (H) Input-output curves of Rheb^CA neurons with and without HCN4^NF. The grey area indicates the SEM for each curve. (I) Representative EEG traces in Rheb^CA mice with and without HCN4^NF. (J) Bar graphs of the number of seizures per day. Data are mean ± SEM. Statistical analyses: Two-way repeated measure ANOVA followed by Sidak post-test (B, H), Student’s t test (C, E, F), and Mann Whitney U test (J). *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, ns: not significant. Exact P values can be found in Table S4.