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. 2023 Jul:193:107156.
doi: 10.1016/j.eplepsyres.2023.107156. Epub 2023 Apr 27.

Semaphorin 4D induced inhibitory synaptogenesis decreases epileptiform activity and alters progression to Status Epilepticus in mice

Susannah S Adel  1 , Vernon R J Clarke  2 , Aidan Evans-Strong  3 , Jamie Maguire  3 , Suzanne Paradis  4
Affiliations

Semaphorin 4D induced inhibitory synaptogenesis decreases epileptiform activity and alters progression to Status Epilepticus in mice

Susannah S Adel et al. Epilepsy Res. 2023 Jul.

Abstract

Previously we demonstrated that intra-hippocampal infusion of purified, Semaphorin 4D (Sema4D) extracellular domain (ECD) into the mouse hippocampus rapidly promotes formation of GABAergic synapses and decreases seizure susceptibility in mice. Given the relatively fast action of Sema4D treatment revealed by these studies, we sought to determine the time course of Sema4D treatment on hippocampal network activity using an acute hippocampal slice preparation. We performed long-term extracellular recordings from area CA1 encompassing a 2-hour application of Sema4D and found that hippocampal excitation is suppressed for hours following treatment. We also asked if Sema4D treatment could ameliorate seizures in an acute seizure model: the kainic acid (KA) mouse model. We demonstrate that Sema4D treatment delays and suppresses ictal activity, delays the transition to Status Epilepticus (SE), and lessens the severity of SE. Lastly, we sought to explore alternative methods of Sema4D delivery to hippocampus and thus created an Adeno Associated Virus expressing the ECD of Sema4D. Our data reveal that virally delivered, chronically overexpressed Sema4D-ECD promotes GABAergic synapse formation and suppresses ictal activity and progression to SE. These results provide proof of concept that viral delivery of Sema4D is an efficacious and promising delivery method to abate epileptiform activity and progression to SE.

Keywords: GABAergic synapse; Hippocampal inhibition; Refractory epilepsy; Sema4D; Status epilepticus.

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Conflict of interest statement

Declaration of Competing Interest Author Suzanne Paradis holds US Patent US-10626163-B2 entitled "Methods of Modulating GABAergic Inhibitory Synapse Formation and Function Using Sema4D." Co-inventors: Kuzirian, Marissa; Moore, Anna; Paradis, Suzanne. Author Suzanne Paradis is also co-founder and President of Severin Therapeutics, Inc. Author Jamie Maguire is on the Scientific Advisory Board for SAGE Therapeutics. The remaining authors have no conflicts of interest to disclose.

Figures

Figure 1.
Figure 1.. The extracellular domain of Sema4D protein decreases population spike amplitude in acute hippocampal slices
Sema4D-Fc treatment depressed population spike amplitude by ~50% while having no effect on fEPSP slope or paired-pulse facilitation (PPF) ratio. Acute hippocampal slices were isolated from rats of both sexes at 20-25 weeks old. Responses were recorded by low-frequency stimulation of the Schaffer-collateral commissural pathway by stimulating electrodes placed in the stratum radiatum and recording responses at the CA1 pyramidal/stratum oriens border. A stable baseline (1 hour) was obtained before drug application for 2hrs. Comparisons were made 3hrs post washout (i.e., at t=6hrs). A, Time plots represent the pooled effects (both n=8) of control Fc. (black) vs Sema4D-Fc (blue) application to evoked population spikes (Ai), fEPSP slope (Aii) and PPF (measured as the ratio of fEPSP slopes to 2 successive pulses delivered at a 50ms interval; Aiii). Shaded areas are the (bootstrapped) s.e.m. Traces above the time plots represent the raw data comprising averages of time points within each experiment taken at the time points indicated. Fc / Sema4D-Fc application are shown in light gray. For both conditions, vertical and horizontal scale lines indicate 1mV and 5ms, respectively. B, Raw values (points) and summary statistics (mean (bars) and SEM (lines)) of population spike amplitude (Bi), fEPSP slope (Bii) and PPF ratio (Biii) shown at baseline and test time points as in A for control Fc (left, black) vs Sema4D-Fc (right, blue). In the group receiving Sema4D-Fc treatment, the mean spike amplitude at test was significantly lower than its baseline (bootstrapped multiple comparisons test with Holm-Šidák correction); no other conditions differed significantly from one another; * indicates alpha < 0.05. C, Box plot to illustrate Cohen’s d (bootstrap) for population spike amplitude (Ci), fEPSP slope (Cii) and PPF ratio (Ciii). Mean and median effect sizes are represented by point and line, respectively. The interquartile range and 95% confidence interval are illustrated by box and whiskers, respectively. Significant effects from raw data (B) are also illustrated on this plot by asterisk for convenience.
Figure 2.
Figure 2.. Sema4D treatment suppresses seizures and progression to SE in vivo
A, Experimental timeline B, representative electrographic seizure activity from vehicle (black) and Sema4D-Fc-treated mice (red) and C, representative EEG power spectra from vehicle (top) and Sema4D-Fc treated mice (bottom). C57BL/6 male mice aged 10-12 weeks were used for this experiment. Sema4D-Fc was infused unilaterally into the CA1 region of the hippocampus 1hr prior to administration of kainic acid (20mg/kg, i.p.). Electrographic seizure activity was recorded for the entire 4hr period of the experiment. For the two hours following KA injection, the following characteristics were quantified: D, latency to onset of the first seizure; E, cumulative epileptiform activity (% time); F, number of mice that died; G, latency to SE; H, latency to cessation of SE; I, percent of diazepam insensitive mice which was quantified from the one hour following diazepam injection (5mg/kg i.p.). n = 8 mice per experimental group; unpaired Student’s t-test was performed on data in panels D,E,G,H; * denotes p<0.05 unpaired Student’s t-test. N-1 corrected chi-squared test was performed on data in panels F,I; no significant difference was found.
Figure 3.
Figure 3.. Validation of Sema4D-expressing adeno associated viruses in vitro.
A, Schematic representing full-length or extracellular domain of Sema4D or CD4 protein encoded by the AAV viruses. B, Representative images of the CA1 principal cell layer in organotypic slices that were treated with Sema4D-Fc or Fc control proteins or infected with indicated AAV constructs; Sections are stained with an antibody that specifically recognizes GAD65 (red) and DAPI (blue). Scale bar represents 25μm. C, Density of GAD65 puncta per CA1 pyramidal cell soma; error bars are SEM. n = 233-351 neurons per treatment condition (represented by individual points), 16-40 neurons per slice, slices from 6-9 mice per treatment condition, 29 mice total. D, Box plot to illustrate the effect size, Cohen’s d. Mean effect size is represented by single points, interquartile range (IQR) and 95% confidence interval (CI) are illustrated by box and whiskers, respectively for the following comparisons: Fc vs Sema4D-Fc; AAV-CD4-FL vs AAV-Sema4D-FL; AAV-CD4-ECD vs AAV-Sema4D-ECD. A robust mixed effect linear model was fitted to the data with treatment as the fixed effect and animal and slice as random effects in order to control for variability that arises within animals and problems associated with pseudoreplication within slices (modeled as random intercepts) (Lazic et al., 2020). Consequently, data is presented as the estimated marginal means obtained from the model. Effect sizes (Cohen’s d), estimates of s.e.m, IQR, CI and Tukey-corrected multiple comparisons tests are based on these estimates * denotes p<0.05. Significant effects from raw data in C are also indicated by an asterisk in D for convenience.
Figure 4.
Figure 4.. Delivery of AAV-Sema4D-ECD to hippocampus suppresses seizures and decreases severity of SE
A, Experimental timeline B, representative electrographic seizure activity from control AAV-CD4-ECD (black) and AAV-Sema4D-ECD treated mice (red) and C, representative EEG power spectra from control AAV-CD4-ECD (top) and AAV-Sema4D-ECD treated mice (bottom). C57BL/6 male mice aged 10-12 weeks were used for this experiment. AAV-Sema4D-ECD or control virus was injected into the DG region of the hippocampus 1wk prior to administration of KA (20mg/kg, i.p.). Electrographic activity was recorded for 1 hr prior to and 3hrs after KA injection which includes the 1 hour post diazepam administration (5mg/kg, i.p.). For the two hours following KA injection, the following characteristics were quantified: D, latency to onset of the first seizure; E, cumulative epileptiform activity (% time); F, number of mice that died (short red bar indicates 0 for AAV-Sema4D-ECD); G, latency to SE; H, latency to cessation of SE; I, percent of diazepam insensitive mice quantified from the one hour following diazepam injection (5mg/kg i.p.). n = 6-8 mice per experimental group; unpaired Student’s t-test was performed on data in panels D,E,G,H; N-1 corrected chi-squared test was performed on data in panels F,I. * denotes p<0.05 using a Student’s t-test; the chi squared test results were not significant.
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