Abstracts

Rationale: Polyhydramnios-megalencephaly and symptomatic epilepsy (PMSE; Pretzel syndrome) is a rare autosomal recessive neurodevelopmental disorder resulting from a homozygous deletion of exons 9-13 in the STRADa (STRADA) gene. Patients suffer from intellectual disability, facial dysmorphism, megalencephaly, and intractable epilepsy. Loss of STRADA results in mTORC1 hyperactivation producing neuronal enlargement and cortical heterotopia. There are no mouse models of PMSE that replicate the clinical phenotype and therefore a model that recapitulates what is observed in patients with PMSE is necessary to define the pathophysiology of PMSE and discover novel treatments. We hypothesize that a murine STRADA knockout model will successfully recapitulate the CNS morphological and pathophysiological characteristics of PMSE. Methods: A 2 KcM deletion of homologous exons 9-13 on the STRADA gene in C57/bl6 mice was generated yielding heterozygous (Het) and knockout (KO) genotypes. Litters were monitored for behavioral abnormalities from P0-P10. Body masses were obtained for live P0 pups. Litters were sacrificed at P0 via ice anesthesia and intracardial perfusion with PBS. Brain specimens were fixed in 4% PFA, paraffin embedded, and sliced on a microtome. Sections were probed with antibodies targeting phosphorylated ribosomal S6 protein (PS6; 235/236), NeuN, and CTIP2. Slides were mounted with DAPI and imaged on a fluorescent microscope. Skin samples were excised prior to fixing to establish primary fibroblasts cultures. Results: The majority of pups did not survive past P10 and all died of yet to be defined causes. KO pups between P5-P10 were observed to have slower ambulation, poor suckling, and repeated tremors compared with WT or Het littermates. KO brain specimens show increased mass, and bilateral ventricular enlargement compared with WT and Het specimens. There was no alteration in cortical laminar structure however CTIP2 staining revealed a large number of heterotopic neurons within the white matter. There were enhanced numbers of PS6 immunoreactive neurons in KO cortex, hippocampus, and thalamus. The heterotopic neurons were PS6 labeled. Quantitative morphometry revealed that PS6+ neurons in KO cortex were larger than from control specimens. In separate experiments, pregnant dams were treated daily with Torin1 (a dual mTORC1/mTORC2 inhibitor) or AICAR from time-of-plug (E0) until delivery to determine if these treatments would enhance perinatal survival.  Litters treated from E0 with Torin1 or AICAR showed a decrease in body and brain mass vs. controls, but did not exhibit enhanced survival. Conclusions: STRADA KO brains display markers of mTOR hyperactivation, ventricular enlargement, neuronal migration deficits, abnormal size, and increased brain weight vs. controls. KO pups have a high rate of perinatal lethality not rescued by pharmacological intervention targeting the mTOR pathway. Therefore, the pathophysiological consequences of STRADA deletion may result from dysfunction in the mTOR pathway as well as other non-mTOR pathways. Funding: R01NS099452