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Anti-epileptogenic and Anti-convulsive Effects of Fingolimod in Experimental Temporal Lobe Epilepsy

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Abstract

Temporal lobe epilepsy (TLE) represents a devastating neurological condition, in which approximately 4/5 of patients remain refractory for anti-convulsive drugs. Epilepsy surgery biopsies often reveal the damage pattern of “hippocampal sclerosis” (HS) characterized not only by neuronal loss but also pronounced astrogliosis and inflammatory changes. Since TLE shares distinct pathogenetic aspects with multiple sclerosis (MS), we have here scrutinized therapeutic effects in experimental TLE of the immunmodulator fingolimod, which is established in MS therapy. Fingolimod targets sphingosine-phosphate receptors (S1PRs). mRNAs of fingolimod target S1PRs were augmented in two experimental post status epilepticus (SE) TLE mouse models (suprahippocampal kainate/pilocarpine). SE frequently induces chronic recurrent seizures after an extended latency referred to as epileptogenesis. Transient fingolimod treatment of mice during epileptogenesis after suprahippocampal kainate-induced SE revealed substantial reduction of chronic seizure activity despite lacking acute attenuation of SE itself. Intriguingly, fingolimod exerted robust anti-convulsive activity in kainate-induced SE mice treated in the chronic TLE stage and had neuroprotective and anti-gliotic effects and reduced cytotoxic T cell infiltrates. Finally, the expression profile of fingolimod target-S1PRs in human hippocampal biopsy tissue of pharmacoresistant TLE patients undergoing epilepsy surgery for seizure relief suggests repurposing of fingolimod as novel therapeutic perspective in focal epilepsies.

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Acknowledgements

We thank Lioba Dammer and Vivian Ludwig for excellent technical assistance as well as Thoralf Opitz for critical discussion on the manuscript.

Funding

This work was supported by the Else-Kröner Fresenius Stiftung (JP, JCK, AJB), Novartis Pharma (to AJB), the Deutsche Forschungsgemeinschaft (SFB 1089 to AJB, SS, KMJ; FOR 2715 to AJB), the European Union’s Seventh Framework Program (FP7/2007-2013) under grant agreement n°602102 (EPITARGET; AJB, SS), Bundesministerium für Bildung und Forschung (01GQ0806, SS; the EraNet DeCipher to AJB), Fritz Thyssen Stiftung (Grant Ref 11.15.2.022MN) and the BONFOR program of the University of Bonn Medical Center (AJB, SS), EPICARE grant by Associazione Paolo Zorzi per le Neuroscienze and Ricerca Corrente, and RF151 grants of the Italian ministry of Health (MC).

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Authors and Affiliations

  1. Section for Translational Epilepsy Research, Department of Neuropathology, University of Bonn Medical Center, Sigmund-Freud-Str. 25, 53105, Bonn, Germany

    Julika Pitsch, Julia C. Kuehn, Johannes Alexander Müller, Karen M. J. van Loo, Susanne Schoch & Albert J. Becker

  2. Unit of Epileptology and Experimental Neurophysiology, Fondazione Istituto Neurologico Carlo Besta, 20133, Milan, Italy

    Vadym Gnatkovsky & Marco de Curtis

  3. Clinic for Neurosurgery, University of Bonn Medical Center, 53105, Bonn, Germany

    Hartmut Vatter

  4. Clinic for Epileptology, University of Bonn Medical Center, 53105, Bonn, Germany

    Susanne Schoch & Christian E. Elger

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  1. Julika Pitsch
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Correspondence to Julika Pitsch.

Electronic Supplementary Material

Suppl. Fig. 1

S1PR subunit expression levels in experimental TLE. (A) mRNA expression levels of S1PRs in control animals in CA1 relative to the reference gene β-actin. S1PR2 and S1PR4 showed the lowest expression levels. S1PR5 and S1PR3 were expressed at moderate levels whereas S1PR1 had the highest expression level in CA1 region of naïve mice (n ≥ 5 per group). (B) No significant changes of S1PR4 (n ≥ 5 per group; 2way ANOVA treatment: p = 0.52) and (C) S1PR5 mRNA levels after pilocarpine-induced SE (n ≥ 5 per group; 2way ANOVA treatment: p = 0.88). Error bars indicate mean ± SEM. (PNG 100 kb)

High resolution image (TIF 516 kb)

Suppl. Fig. 2

S1PR expression levels depend on severity of hippocampal sclerosis. (A) The severity of hippocampal sclerosis significantly affects the mRNA expression level of S1PR1 in kainate-induced chronic TLE in CA1 (n ≥ 5; 2way ANOVA: ***p = 0.0002, F(2,58) = 9.921). S1PR1 is strongly increased in severe hippocampal characterized by a strong phenotype depicted in complete granule cell dispersion as well as a severe neuronal cell loss within the hippocampal sclerosis compared to the mild phenotype which resembles a moderate granule cell dispersion in the dentate gyrus and a slight neuronal cell loss in ipsilateral CA1 (Sidak’s post hoc: *p = 0.046). Also in the contralateral CA1 S1PR1 shows an abundance in the severe HS compared to sham-injected control CA1 (Sidak’s post hoc: **p = 0.0039) and mild sclerosis (**p = 0.0026). (B) The expression pattern of S1PR3 mRNA also depends on the severity of hippocampal sclerosis (2way ANOVA: ****p < 0.0001, F(2,58) = 19.61). S1PR3 mRNA shows a strong increased in severe hippocampal sclerosis (n = 5) compared to mild hippocampal sclerosis (n = 11) and sham-injected control animals (n = 11) in the ipsilateral (Sidak’s post hoc: ctrl vs. severe: ***p = 0.0002, mild vs severe: ***p = 0.0003) as well as in the contralateral side (Sidak’s post hoc: ctrl vs. severe: **p = 0.00233, mild vs severe: **p = 0.0021). Error bars indicate mean ± SEM. (PNG 72 kb)

High resolution image (TIF 443 kb)

Suppl. Fig. 3

No changes in gamma frequency during early acute SE. (A–D) EEG spectral analysis demonstrates no significant changes in gamma power increase (AUC, A, B) and duration of acute SE (C, D) between treated and untreated group in both hippocampal CA1. The following low wave delta EEG spectral pattern increase and duration is significantly increased in treated animals. This terminal phase of SE is not correlated with any seizure like behavior and the animal moved normally. (PNG 82 kb)

High resolution image (TIF 530 kb)

Suppl. Fig. 4

No changes in astrogliosis, activated microglia or neuronal cell loss in early treated experimental TLE. (A, B) No significant changes of reactive gliosis between untreated (n = 8) and treated group (n = 9). Mann-Whitney test: ipsilateral: p = 0.6, contralateral: p = 0.8. The staining was rated in relative extent of astrogliosis from 0 to 4 (0 = no reactive gliosis, 4 = strong gliosis). (C, D) No significant changes of activated microglia between untreated (n = 8) and treated group (n = 9). Mann-Whitney test: ipsilateral: p = 0.94, contralateral: p = 0.82. The staining was rated in relative extent of activated microglia from 0 to 4 (0 = no activated microglia, 4 = strong activated microglia) (E, F) No changes with respect to neuronal cell loss (n ≥ 8 per group). 2way ANOVA treatment: CA: p = 0.32; CA3: p = 0.16. Sidak’s post hoc: ipsilateral CA1: p = 0.28, contralateral CA1: 0.98; ipsilateral CA3: p = 0.68, contralateral CA3: p = 0.8. (PNG 138 kb)

High resolution image (TIF 617 kb)

Suppl. Fig. 5

Transient increase of S1PR3 mRNA in CA3 and DG in experimental TLE. (A–D) SE has also an impact on chronic mRNA expression levels of S1PR1 and 3 after pilocarpine-induced SE in CA3 similar to CA1 (n ≥ 5 for all groups; 2way ANOVA: S1PR1: *p = 0.026, F(1,33) = 5.45; S1PR3: ****p < 0.0001, F(1,33) = 127.8) and DG (2way ANOVA: S1PR1: ****p < 0.0001, F(1,33) = 78.65; S1PR3: ****p < 0.0001, F(1,33) = 127.2). (B) S1PR1 is significantly reduced 36 h after SE in CA3 (**p = 0.0073) whereas (C) S1PR3 shows an increase early after SE (12 h: ***p < 0.0001, 36 h: ****p < 0.0001, 72 h: ***p < 0.0001). (D) In DG S1PR1 is significantly reduced early after SE (12 h: ***p = 0.0002, 36 h: ****p < 0.0001, 72 h: **p = 0.008) and (E) S1PR3 is increased (12 h: ***p < 0.0001, 36 h: ****p < 0.0001, 72 h: ***p < 0.0001). Error bars indicate mean ± SEM. (PNG 115 kb)

High resolution image (TIF 578 kb)

Suppl. Fig. 6

S1PR3 protein expression levels 4 days after SE in CA1. Representative protein expression levels of S1PR3 in CA1 4 days after pilocarpine-induced SE with different co-stainings. (A) 4 days after SE S1PR3 expression shows an overlay with GFAP indicating a co-localization of S1PR3 and astrocytes (white arrows). (B) Co-staining with NeuN and (C) DAPI show an increased protein expression not exclusively in the neuronal nuclei but also in the somata (white arrowheads). (D) No co-localization is present with microglia visualized with co-staining using antibodies against Iba-1 (white arrow indicates increased expression on astrocytes). (PNG 5465 kb)

High resolution image (TIF 7092 kb)

Suppl. Fig. 7

S1PR3 protein expression levels early after SE in CA1. (A) Time dependent representative protein expression levels of S1PR3 in CA1 after pilocarpine-induced SE. Strong abundance is present 4 days after. Overlay with GFAP shows a co-localization of S1PR3 in astrocytes (white arrows). Representative staining of S1PR3 in a control animal shows a sparse staining in the neurons of the control animal (upper row). Two days after SE the expression is higher in pyramidal neurons and expression is also present in the somata (white arrowheads). The strongest expression is present at four days after SE with a co-localisation on astrocytes and on neuronal somata. Seven days after SE the protein expression declines but signals are still present on astrocytes and neurons. (PNG 8839 kb)

High resolution image (TIF 14000 kb)

Suppl. Fig. 8

S1PR3 protein expression levels early after SE in CA3. Representative protein expression levels of S1PR3 in CA3 after SE. The expression pattern is similar to CA1 (Fig. 6e). Strong protein increase of S1PR3 is present 4 days after SE. Co-localization analysis with GFAP reveals expression within the astrocytes (see arrows). (PNG 6974 kb)

High resolution image (TIF 8886 kb)

Suppl. Fig. 9

S1PR3 protein expression levels early after SE in DG. Representative protein expression levels of S1PR3 in DG after SE. The expression pattern is similar to CA1 and CA3. Strongest increase of S1PR3 was present 4 days after SE. Co-localization with GFAP again reveals expression by astrocytes (see arrows). (PNG 7613 kb)

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Suppl. Table 1

Sequences of primers that were used for real-time RT-PCR. (PNG 138 kb)

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Suppl. Table 2

Antibodies and dilutions for protein Expression. (PNG 203 kb)

High resolution image (TIF 1496 kb)

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Pitsch, J., Kuehn, J.C., Gnatkovsky, V. et al. Anti-epileptogenic and Anti-convulsive Effects of Fingolimod in Experimental Temporal Lobe Epilepsy. Mol Neurobiol 56, 1825–1840 (2019). https://doi.org/10.1007/s12035-018-1181-y

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