Transcriptomic and electrophysiological alterations underlying phenotypic variability in SCN1A-associated febrile seizures

Abstract

Febrile seizures (FS) are a common childhood neurological condition triggered by fever in children without prior neurological disorders. While generally benign, some individuals, particularly those with complex FS or genetic predispositions, may develop epilepsy or other neurological comorbidities. The mechanisms underlying this transition remain unclear. Mutations in SCN1A, encoding the Na_V1.1 sodium channel α-subunit, have been linked to several epilepsy syndromes associated with FS. This study examines phenotypic variability in individuals carrying the same SCN1A c.434T > C mutation, using induced pluripotent stem cell (iPSC)-derived neurons from two siblings with FS. Despite sharing the mutation, only the older sibling developed temporal lobe epilepsy (TLE). Transcriptomic analysis revealed downregulation of GABAergic pathway genes in both siblings' neurons, aligning with SCN1A-associated epilepsy. However, neurons from the sibling with TLE exhibited additional abnormalities, including altered AMPA receptor subunit composition, changes in GABA_A receptor subunits and chloride cotransporters expression, and reduced brain-derived neurotrophic factor (BDNF) levels, indicative of developmental immaturity. Voltage-clamp recordings confirmed impaired GABAergic and AMPA receptor-mediated synaptic activity. These findings suggest that combined GABAergic dysfunction, aberrant AMPA receptor composition, and reduced BDNF signaling contribute to the more severe phenotype and increased epilepsy susceptibility.

Keywords: SCN1A; Febrile seizure; GABAergic dysfunction; Induced pluripotent stem cell-derived neurons; Mesial temporal lobe epilepsy; Voltage-clamp recordings; Voltage-gated sodium channel NaV1.1.

Fig. 1

Generation and characterization of forebrain neurons from iPSC lines. (A–B) Bright-field images of forebrain neurons differentiated from iPSCs at the early (A) and the late (B) stages of differentiation. (C–E) Immunofluorescence staining confirms successful neuronal differentiation, as indicated by the expression of specific neuronal markers: MAP2 and NEFH (C), NEFL (D), and TUBB3 (E). (F) Quantification of TUBB3-positive cells demonstrates a high differentiation efficiency (~ 85–90% at the early stage and ~ 100% at the late stage) across all iPSC lines. Data are presented as mean ± S.E.M., with each data point representing an individual analyzed image.

Fig. 2

Analysis of gene expression profiles and consistency across samples. (A) Box plot illustrating the distribution of gene expression values (FPKM) across replicates for each sample. (B) Density distribution plot depicting the overall gene expression profiles (FPKM values) among all samples. (C) Pearson correlation heatmap showing the correlation between samples based on FPKM values. In the sample names, “E” and “L” denote the early and the late stage, respectively. (D) Principal component analysis (PCA) of mRNA expression data reveals clustering of biological replicates at both early and late stages of differentiation.

Fig. 3

Transcriptomic analysis revealed altered gene expression in FS neurons. (A and B) Volcano plots depicting differentially expressed genes (DEGs) between neurons derived from FS patients and HC at the early (A) and late (B) stages of differentiation. (C) Gene Ontology (GO) enrichment analysis of down-regulated DEGs in FS patient-derived neurons compared to HC at both stages of differentiation. (D and E) Normalized enrichment score (NES) versus significance plot for the early (D) and late (E) stages of differentiation. The red squares represent the FDR q-value, while the black dots denote the nominal p-value for each gene set. Gene sets are considered significant if the FDR < 25% (highlighted in the yellow box). (F–I) Gene set enrichment analysis (GSEA) of GO categories highlights the negative enrichment of GABA_A receptor complex and activity, GABAergic synapse, and GABA signaling pathway in neurons derived from FS patients.

Fig. 4

Impaired glutamatergic signaling and neuronal immaturity in SEV lines. (A) GO enrichment analysis of downregulated DEGs at the early stage of differentiation in SEV versus HC and SEV versus MILD comparisons. (B) GO enrichment analysis of downregulated DEGs at the late stage of differentiation in SEV versus HC and SEV versus MILD comparisons. (C) Gene set enrichment analysis (GSEA) confirms negative enrichment of glutamatergic signaling-related terms in SEV versus HC and SEV versus MILD comparisons. (D and E) Expression of genes encoding for AMPA receptor subunits GRIA1 and GRIA2 across differentiation stages, showing increased GRIA1 and reduced GRIA2 expression in SEV neurons compared to HC and MILD lines. Data are presented as mean ± S.E.M. of 6 biological replicates for controls and 3 biological replicates for patients. (F) GRIA1/GRIA2 ratio in neurons at early and late stages of differentiation.

Fig. 5

Analysis of GABA_AR subunits, chloride channel expression, and BDNF signaling confirm neuronal immaturity in SEV lines. (A) Ratios of GABRA3 to GABRA1 and (B) GABRA3 to GABRB2 are significantly higher in SEV neurons, indicating a more immature neuronal state compared to HC and MILD lines. Data are presented as mean ± S.E.M. of FPKM values, with each biological replicate representing the ratio of the indicated subunits. *p < 0.05, ** p < 0.01, t-test. (C) Expression of chloride channel genes SLC12A2 (NKCC1) and (D) SLC12A5 (KCC2) in neurons during differentiation. Data are presented as mean ± S.E.M., with each dot representing a biological replicate. (E) The NKCC1/KCC2 ratio is elevated in SEV neurons at both differentiation stages compared to HC and MILD lines. **p < 0.01, ***p < 0.001, t-test. (F) Representative immunostaining of neurons at the late stage of differentiation, showing KCC2 (red) and TUBB3 (green). Insets display a higher magnification, highlighting the localization of KCC2 puncta. (G) Quantification of KCC2-positive puncta per cell in HC, SEV, and MILD neurons at the late stage of differentiation. *p < 0.05, t-test. (H) EGR4 expression during differentiation in HC, SEV, and MILD neurons. (I) Negative enrichment of the Neurotrophin Signaling Pathway in SEV versus MILD comparisons at the late stage of differentiation. (J) Expression of the BDNF gene in HC, SEV, and MILD neurons at both early and late stages of differentiation. (K) Expression of NTRK2 gene (coding for the BDNF receptor) in HC, SEV, and MILD neurons at early and late stages of differentiation.

Fig. 6

Analysis of AMPA/GABA current by voltage clamp. (A) AMPA/GABA current ratio (expressed as percentage ± S.E.M.) of 20 μM AMPA- and 500 μM GABA-evoked currents at the early stages of differentiation. The AMPA/GABA current ratio was significantly different between the two phenotypes (blue filled bar: 291.4 ± 29.5%, n = 11; versus green filled bar: 567.6 ± 73. 6%, n = 10; p = 0.002, t-test). The inset on the right shows the GABA- and AMPA-evoked current traces. (B) AMPA-evoked current (percentage ± S.E.M.) blocked by 50 μM IEM 1460 (a selective blocker for GluA2-lacking receptors) in HC and SEV phenotypes (blue filled bar: 63.9 ± 7.2%; n = 10; vs green filled bar: 75.1 ± 2.6%; n = 16; p = 0.128, t-test). The inset on the right shows AMPA-evoked currents recorded from samples with IEM 1460 application (50 μM, black bar) to block GluA2-lacking AMPAR currents. IEM 1460 inhibition was tested using a holding potential of − 80 mV.

Fig. 7

Zn²⁺ selectively inhibits γ-lacking extrasynaptic GABA_ARs. (A) Ratios of GABRA3 to GABRG2 (α3/γ2), showing a lower ratio in SEV neurons compared to HC. Conversely, (B) the ratios of GABRA3 to GABRB3 (α3/β3) and (C) GABRA3 to GABRD (α3/δ) are higher in SEV neurons, indicating a more immature state relative to HC lines. (D) Percentage decrease in GABA current ± S.E.M. following treatment with 40 μM Zn²⁺ (blue filled bar: − 32.2 ± 4.9%; n = 14; vs green filled bar: − 53.1 ± 3.6%; n = 17; p = 0.004, t-test). The inset on the right shows the 200 μM GABA-evoked current before and after treatment with 40 μM Zn^2 + (black traces) for both HC and SEV neurons.