ROBERDS L.S1, GHAVAMI A2, LEISER S2, KWAN M1, BELTRAN J2, SONG D1, DEVILBISS D1, MAZZELLA M, WONG M2, BRUNNER D3
1PsychoGenics Inc., Tarrytown, NY, USA; 2PsychoGenics Inc., Montvale, NJ, USA; 3Tuberous Sclerosis Alliance, Silver Spring, MD, USA
Individuals with tuberous sclerosis complex (TSC) are impacted by a variety of manifestations caused by mutations in either the TSC1 or TSC2 gene. Of these manifestations, epilepsy is often the most devastating to quality of life and is poorly controlled in many individuals. Preclinical evaluation of new candidate therapeutics requires animal models with robust, translatable endpoints and consistent, rigorous testing procedures. Several mouse models relevant to tuberous sclerosis complex (TSC) develop epilepsy, but Tsc1flox / flox-GFAP-Cre (Tsc1GFAPCKO) mice have a robust, fully penetrant seizure phenotype. In this two-part study, we evaluated incidence of seizures using EEG and changes in mRNA and protein biomarkers in Tsc1GFAPCKO mice.
For measuring seizures, mice were continuously recorded by tethered video-EEG from P35-P49 (6 and 7 weeks of age) and were treated with vehicle between P21-P48 (n=10), vehicle between P21-P34 and rapamycin (3 mg/kg) between P35-P48 (n=8), or rapamycin (3 mg/kg) between P21-P48 (n=10). In a separate cohort, we used quantitative PCR (qPCR) and western blotting to evaluate changes in mRNA, protein, or phospho-protein levels of transcripts and proteins involved in axon formation, synapse function, glutamate transport, mTOR activation, cell adhesion, angiogenesis, cell regulation, inflammation and unfolded protein response activation in brain at 7 weeks of age.
EEG: Tsc1GFAPCKO mice treated only with vehicle exhibited robust electrographic seizures and significantly increased mortality. The vehicle-treated group exhibited on week 6 and 7 an average of 3.5 ± 1.1 and 2.33 ± 1.1 seizures, respectively. Starting rapamycin at P35 caused no clear reduction in seizures during week 6 (1.75 ± 0.65) and a marginally significant reduction in week 7 (0.38 ± 0.26, p=0.06, Tukey-Kramer). However, earlier treatment with rapamycin starting at P21 resulted in complete abolition of seizures and no mortality during the study. Biomarkers: Expression levels (assessed by qPCR) between control (Cre-; Tsc1+/ flox) and heterozygous Cre+; Tsc1+/ flox mice were similar for all transcripts reported in this study. In contrast, Tsc1GFAPCKO mice (homozygous Cre+; Tsc1flox / flox) showed genotype-dependent changes in some transcripts analyzed. Of note were decreased mGluR5 and Tyrosine Kinase c-kit and increased IBA1, CD68, ICAM1, and VEGF-D mRNA expression in Tsc1GFAPCKO mice. Phosphorylation status of several proteins were not affected by genotype. However, phosphorylation levels of AKT, 4EBP1, and Jak1 were decreased, whereas those of mTOR (Ser2448) and pS6 (Ser235/236) were increased. Sacrificing animals by focused microwave irradiation of the brain was necessary to preserve phosphorylation status of about half the protein targets assessed.
We confirmed the protective effects of mTOR inhibition against seizures and premature death in these mice. Our findings confirm clear utility of using these mice to screen potential anti‐epileptic therapeutics. Tsc1GFAPCKO mice showed genotype‐dependent changes in some transcripts involved in synaptic plasticity, inflammation, and the mTOR pathway. Phosphoprotein analysis revealed activation of mTOR pathway as was demonstrated by changes in phosphorylation status of AKT, mTOR, and S6 ribosomal proteins. Overall, our findings suggest a clear utility of using these mice to screen potential anti‐epileptic therapeutics.