Impaired axon initial segment structure and function in a model of ARHGEF9 developmental and epileptic encephalopathy

Abstract

Developmental and epileptic encephalopathies (DEE) are rare but devastating and largely intractable childhood epilepsies. Genetic variants in ARHGEF9, encoding a scaffolding protein important for the organization of the postsynaptic density of inhibitory synapses, are associated with DEE accompanied by complex neurological phenotypes. In a mouse model carrying a patient-derived ARHGEF9 variant associated with severe disease, we observed aggregation of postsynaptic proteins and loss of functional inhibitory synapses at the axon initial segment (AIS), altered axo-axonic synaptic inhibition, disrupted action potential generation, and complex seizure phenotypes consistent with clinical observations. These results illustrate diverse roles of ARHGEF9 that converge on regulation of the structure and function of the AIS, thus revealing a pathological mechanism for ARHGEF9-associated DEE. This unique example of a neuropathological condition associated with multiple AIS dysfunctions may inform strategies for treating neurodevelopmental diseases.

Keywords: axon initial segment; epilepsy; hippocampus; mouse model.

Fig. 1.

Generation and seizure phenotyping of the Arhgef9^G55A/Y mouse model. (A) Schematic of CB protein structure with known missense variants mapped to three functional domains. The p.G55A variant (highlighted in red) locates at the N-terminal SH3 domain. (B) Illustration describing the location of the GGA>GCA missense mutation introduced to exon 3 of mouse Arhgef9. (C) Western blot probed for CB and ß-actin using 50 µg of whole-brain lysates from P30 mice and quantification of CB normalized to ß-actin levels (n = 4 to 5 animals per genotype). (D) Schematic of multiday video-EEG recording system and cortical electrode placement. (E) A representative EEG recording showing an episode of generalized tonic-clonic seizure (top record) and SWD events (bottom record) detected in Arhgef9^G55A/Y mice. (F) Quantification of the incidence of GTCS events detected in Arhgef9^G55A/Y and wildtype mice (n = 9 to 10 animals per genotype). (G) Characterization of SWD-like events detected in Arhgef9^G55A/Y mice. EEG trace (top record) and the accompanying spectrogram (bottom record, 0 to 40 Hz). (H) Fundamental frequency analysis of all SWD events detected in Arhgef9^G55A/Y mice (n = 10 animals). (I) Quantification of the incidence of SWD events detected in Arhgef9^G55A/Y and wildtype mice (n = 9 to 10 animals per genotype).

Fig. 2.

Arhgef9^G55A/Y mice exhibit protein aggregates in hippocampal pyramidal neurons. (A) Hippocampal pyramidal layer sections of PND 30 mice stained with antibodies against CB (green) and gephyrin (red). Top: Arhgef9^+/Y brains (n = 5 animals) show CB and gephyrin in microclusters. Bottom: Arhgef9^G55A/Y brains (n = 4 animals) exhibit CB and gephyrin protein aggregates colocalizing with each other. (Scale bar, 10 μm.) (B) Immunostaining of DIV14 Arhgef9^+/Y (Upper panel) and Arhgef9^G55A/Y neurons (Lower panel) probed with antibodies against CB (green, white arrows) and ANK3 (magenta). White boxes indicate AIS and yellow boxes indicate dendrites. (C) Higher magnification images showing CB (green) clustering along the AIS (ANK3, magenta) in Arhgef9^+/Y and Arhgef9^G55A/Y neurons. (D) Immunostaining of DIV14 Arhgef9^+/Y (upper panel) and Arhgef9^G55A/Y neurons (lower panel) probed with antibodies against gephyrin (red, white arrows) and ANK3 (cyan). (E) Quantification of CB and gephyrin puncta density in Arhgef9^+/Y (n = 18 neurons, n = 4 animals) and Arhgef9^G55A/Y neurons (n = 24 neurons, n = 5 animals) at the AIS, somatic, and dendritic compartments. (Scale bar, 5 μm for C and 10 μm for all others.)

Fig. 3.

TEM images show AIS structural changes caused by protein aggregation in Arhgef9^G55A/Y neurons. (A) Longitudinal sections of the AIS (highlighted in pink) in Arhgef9^+/Y and Arhgef9^G55A/Y CA3 pyramidal neurons probed for gephyrin. Asterisks denote gephyrin aggregates associated with microtubules in an Arhgef9^G55A/Y neuron. (B) Cross-sections of the AIS (highlighted in pink) in Arhgef9^+/Y and Arhgef9^G55A/Y neurons probed for gephyrin. A presynaptic terminal in the Arhgef9^+/Y neuron is highlighted in blue. (Scale bars, 500 nm.)

Fig. 4.

Loss of GABAergic synapses at the AIS Arhgef9^G55A/Y primary hippocampal neurons. (A) Immunostaining of DIV14 Arhgef9^+/Y and Arhgef9^G55A/Y neurons probed with antibodies against GABA_AR-α2 (green) and ANK3 (magenta). (B) Immunostaining of DIV14 Arhgef9^+/Y and Arhgef9^G55A/Y neurons probed with antibodies against gephyrin (red), VGAT (green), and ANK3 (magenta). (C) Quantification of GABA_AR-α2 puncta density in Arhgef9^+/Y (n = 21 neurons, n = 4 animals) and Arhgef9^G55A/Y neurons (n = 16 neurons, n = 3 animals). (D) Quantification of VGAT puncta density in Arhgef9^+/Y (n = 8 neurons, n = 3 animals) and Arhgef9^G55A/Y neurons (n = 11 neurons, n = 3 animals). (E) CA3 regions stained with antibodies against VGAT (green) and quantification of normalized intensity in Arhgef9^+/Y and Arhgef9^G55A/Y mice (n = 9 slices, n = 3 animals for both genotypes). Error bars are means ± SEM. Mann–Whitney test. (Scale bar, 10 μm.)

Fig. 5.

Arhgef9^G55A/Y neurons have altered synaptic transmission. (A) Representative traces showing mEPSC (upper traces) and mIPSC (lower traces) and Insets showing illustrative example responses recorded from CA3 pyramidal neurons in Arhgef9^+/Y (black) and Arhgef9^G55A/Y (red) slices. (B) Average peak-aligned mEPSC (dashed lines) and mIPSCs (solid lines) recorded from Arhgef9^+/Y (black) and Arhgef9^G55A/Y (red) slices. (C) Cumulative probability distribution and average (Inset) mEPSC and mIPSC amplitudes. Kolmogorov–Smirnov test. (D) Peak aligned, amplitude normalized, average mEPSC (dashed lines) and mIPSCs (solid lines) recorded from Arhgef9^+/Y (black) and Arhgef9^G55A/Y (red) slices. (E) Cumulative probability distribution and average (Inset) mEPSC and mIPSC decay tau. Kolmogorov–Smirnov test. (F) Average mEPSC and mIPSC total charge transfer recorded from Arhgef9^+/Y and Arhgef9^G55A/Y cells. (G) Event frequency of mEPSCs and mIPSCs recorded from Arhgef9^+/Y and Arhgef9^G55A/Y cells. (B–G) mEPSC and mIPSC analysis (n = 10 to 19 cells from 4 to 5 animals per genotype. Mann–Whitney test. Error bars are means ± SEM). (H) Example two-photon image of a CA3 pyramidal neuron filled with Alexa dye through the patch pipette. RuBi-GABA uncaging ROIs indicated at the dendrite (blue), soma (yellow), and AIS (green) were selected under two-photon microscopy guidance. (I) Representative uncaging-evoked (eIPSC) GABA currents in response to 5 ms laser pulses (red lines) applied at the dendrite, soma, and AIS of Arhgef9^+/Y and Arhgef9^G55A/Y cells. (J) Peak aligned, average eIPSC responses at each cellular compartment recorded from Arhgef9^+/Y and Arhgef9^G55A/Y cells. (K) Quantification of eIPSC responses at each compartment recorded from Arhgef9^+/Y and Arhgef9^G55A/Y cells (n = 6 cells from 3 Arhgef9^+/Y animals and n = 8 cells from 4 Arhgef9^G55A/Y animals. Mann–Whitney test. Error bars are means ± SEM).

Fig. 6.

Impaired AP generation and propagation in Arhgef9^G55A/Y neurons. (A) Representative whole-cell current clamp recordings of APs evoked by increasing levels of current stimuli in Arhgef9^+/Y (black) and Arhgef9^G55A/Y (red) cells. (B) Quantification of the number of APs generation at each current amplitude in Arhgef9^+/Y (black, n = 18 cells from n = 4 animals) and Arhgef9^G55A/Y (red, n = 25 cells from n = 5 animals). (C) Overlay of representative phase-plane plots of the rheobase of an Arhgef9^+/Y neuron (black) and an Arhgef9^G55A/Y neuron (red). (D) Quantification of AP rheobase current. (E) Quantification of average rheobase threshold voltage. (F) Quantification of average AP overshoot voltage. (G) Quantification of average AHP amplitude voltage. (H) Representative voltage record of Arhgef9^+/Y neuron (black) and an Arhgef9^G55A/Y neuron (red) APs and the respective first-order (dV/dt) and second-order (d²V/dt²) derivatives. The Inset shows peaks detected in the d²V/dt² records. (I) Quantification of the number of cells that displayed one versus two peaks in the d²V/dt² record. (J) Quantification of peak d²V/dt² in Arhgef9^+/Y (n = 18 cells from n = 4 animals) and Arhgef9^G55A/Y neurons (n = 25 cells from n = 5 animals). (K) Quantification of minimum d²V/dt² between the AIS and SD peaks within the rising phase of the AP in Arhgef9^+/Y (n = 18 cells from n = 4 animals) and Arhgef9^G55A/Y neurons (n = 25 cells from n = 5 animals). (L) Quantification of the AP interpeak interval of Arhgef9^+/Y (n = 18 cells from n = 4 animals) and Arhgef9^G55A/Y cells (n = 16 cells from n = 5 animals). Arhgef9^G55A/Y cells with only one peak identified are excluded from analysis. Mann–Whitney or Fisher’s exact test. Error bars are means ± SEM.