Authors :
Presenting Author: Mi Jiang, MD – University of Michigan
Tao Yang, Ph.D – PostDoc, Department of Neurology, University of Michigan; Rajat Banerjee, PhD – Research Specialist, Neurology, University of Michigan Medical School; Yu Wang, MD. PhD. – Assistant professor, Department of Neurology, University of Michigan Medical School
Rationale:
Low-frequency somatic mutations in key genes within the mechanistic target of rapamycin (mTOR) pathway have been frequently identified in patients with malformation of cortical development, such as focal cortical dysplasia type II (FCDII) and hemimegalencephaly, that are collectively referred as mTORopathies, the most common underlying pathologies in children with severe refractory epilepsies. However, systemic administration of mTOR inhibitor only has modest efficacy and causes global effects at the whole brain level, not just a few low-frequency mutant cells in a focal cortical region. Therefore, a new strategy to specifically inhibit mTOR hyperactivity in mutant cells needs to be developed.
Methods:
We constructed a mutant RHEB expression plasmid based on the patient-specific gain-of-function (GoF) mutation (RHEB, c.110C >T, p.P37L) and applied
in utero electroporation-mediated somatic mutagenesis to overexpress RHEBP37L in the mouse cortical dorsal progenitors that give rise to cortical excitatory neuron. We constructed the SPAR (Small regulatory Polypeptide of Amino Acid Response) plasmid to inhibit mTORC1 recruitment to the lysosome membrane and negatively regulate its activation. To investigate whether turning on SPAR expression at chronic disease stages is effective, we constructed a conditional SPAR expression system in which the 4-OHT–activatable Cre recombinase is expressed under the control of CAG promoter (CAG-ER
T2CreER
T2) and SPAR expression vector contains a neomycin resistance gene (
neo) with a stop codon flanked by
loxP sites (CALNL-SPAR). 24/7 video-EEG monitoring was performed to characterize seizure phenotype and assess therapeutic responses.
Results:
Our preliminary data showed that neurons expressing RHEBP37L were cytomegalic and had highly increased pS6 immunoreactivity, suggesting mTOR hyperactivation. At P45, all animals with IUE-RHEBP37L developed frequent seizures. SPAR significantly inhibits mTOR activity in mutant neurons. When co-expressed with Depdc5 CRISPR or RHEBP37L IUE at E15, SPAR successfully rescued cytomegaly, reduced pS6 immunostaining, and prevented animals from developing seizures, suggesting mTOR-dependent disease initiation. In vitro and in vivo experiments showed that the conditional expression system is efficient and is under tight control.
Conclusions:
Our preliminary suggested SPAR could be a novel therapeutic peptide for mTORopathies.
Funding: R01 NS113824 (Wang) NIH (NINDS) 9/2019~9/2024 Cortical development and pathogenesis in DEPDC5-related epilepsies.