cubes

Abstract

Establishment of Tsc1flox / flox -GFAP-Cre (Tsc1GFAPCKO) mice for evaluating potential new antiepileptic therapies for tuberous sclerosis complex

S. C. LEISER1, A GHAVAMI2, M KWAN2, J BELTRAN2, D SONG1, D. M. DEVILBISS1, M WONG3, N RENSING3, S. L. ROBERDS4, D BRUNNER4
1Translational EEG, PsychoGenics Inc., Tarrytown, NY, USA; 2PsychoGenics Inc., Montvale, NJ, USA; 3Dept. of Neurol., Washington Univ. Sch. of Med., St. Louis, MO, USA; 4Tuberous Sclerosis Alliance, Silver Spring, MD, USA

In this two-part study, we evaluated incidence of seizures using EEG and changes in mRNA biomarkers using quantitative PCR (qPCR) in Tsc1GFAPCKO mice. First, we evaluated effects of rapamycin on incidence of seizures and mortality given that previous reports indicated these mice develop spontaneous postnatal epilepsy rescued by treatment with the mTOR inhibitor. EEG from mice were continuously recorded (using Pinnacle Technology’s data acquisition platform) from P35-P49. The vehicle-treated group (n=10) exhibited on week 5 and 6 an average of 3.5 ± 1.1 and 2.33 ± 1.1 seizures, respectively. In contrast, the rapamycin-treated group that started at P35 (n=8) exhibited a reduction in seizures for weeks 5 (1.75 ± 0.65) and 6 (0.38 ± 0.26) nearly reaching significance at week 6 (p=0.06, Tukey-Kramer). Moreover, the rapamycin-treated group that started at P21 (n=10) showed no seizures during week 5 and 6 (p<0.05, Tukey-Kramer). In summary, whereas Tsc1GFAPCKO mice treated with vehicle from P21-48 suffered robust electrographic seizures and a significant mortality over this period, mice treated with 3 mg/kg rapamycin from P21-48 showed zero seizures and zero mortality over the same period. The group receiving rapamycin 3 mg/kg from P35-48 showed reduced seizure frequency and zero mortality. Thus, we confirm the protective effects of mTOR inhibition against seizures and premature death in these mice. Secondly, in a separate cohort, we used qPCR to evaluate changes in mRNA levels of transcripts involved in axon formation, synapse function, glutamate transport, mTOR activation, cell adhesion, angiogenesis, cell regulation, inflammation and unfolded protein response activation in brain at 5 weeks of age. Expression levels between control (Cre-; Tsc1+ /flox) and Cre+; Tsc1+ /flox mice were similar for all transcripts reported in this study. In contrast, Tsc1GFAPCKO mice (Cre+; Tsc1 flox / flox) showed genotype-dependent changes in some transcripts analyzed. Of particular note were decreased mGluR5 and Tyrosine Kinase c-kit and increased IBA1, CD68, ICAM1, VEGF-D, and TLR4 mRNA expression in Tsc1GFAPCKO mice. In some cases affected transcripts in Cre+; Tsc1 flox / flox mice showed significant variation among individuals that cannot simply be explained by differential effectiveness of CRE-mediated knockout since, except one animal, all Cre+; Tsc1 flox / flox mice showed similarly reduced expression levels of Tsc1 mRNA. Further, mRNA levels might covary with seizure frequency or age of seizure onset which needs to be assessed in future studies. Overall, our findings suggest a clear utility of using these mice to screen potential anti-epileptic therapeutics.

 

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