Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Neuroscience

First Advisor

Peter B. Crino

Second Advisor

Steven S. Scherer

Abstract

Pretzel Syndrome (also Polyhydramnios, Megalencephaly, and Symptomatic Epilepsy syndrome; PMSE) is a recently described rare neurodevelopmental disorder occurring in the Old Order Mennonite pediatric population, and characterized by intractable infantile-onset epilepsy, neurocognitive delay, craniofacial dysmorphism, and histopathological evidence of heterotopic neurons in subcortical white matter, suggestive of failed neuronal migration. PMSE is caused by a homozygous deletion of exons 9-13 of LYK5/STRADA, which encodes the pseudokinase STRADA, an upstream inhibitor of mammalian target of rapamycin (mTOR). Therefore, we hypothesize that STRADA plays a critical role in neuronal migration through modulating mTOR (specifically mTOR complex 1, mTORC1) signaling, and that therapeutic mTORC1 inhibition can ameliorate features of the PMSE disease phenotype. To test this hypothesis, we model PMSE in vitro using stable shRNA knockdown of STRADA (STRADA KD) in mouse neural progenitor cells (mNPCs). In vivo, we use in utero electroporation to create focal STRADA KD in the developing mouse brain. We show that STRADA depletion disrupts pathfinding and polarization in migrating mNPCs in vitro, and this effect can be rescued by inhibition of mTORC1 with rapamycin or of its downstream effector p70S6kinase (p70S6K) with PF-4708671 (p70S6Ki), indicating an mTORC1-specific dependence. We then define a pathway for this effect downstream of mTORC1, through insulin receptor substrate 1 (IRS1) signaling to cofilin, and finally modulating actin dynamics. In vivo, we demonstrate that STRADA KD causes a cortical lamination defect in the mouse, which can be rescued with rapamycin treatment, confirming the dependence of STRADA's effect on mTORC1 signaling and suggesting an important target for patient therapy. To correlate our mouse model with PMSE, we demonstrate congruent mTORC1 and downstream signaling and rescue of migration deficit with rapamycin and p70S6Ki in PMSE patient fibroblasts. Finally, we report reduction of seizure frequency with rapamycin treatment in previously intractable PMSE patients. Our findings define a novel role for STRADA in neuronal migration, demonstrate a mechanistic link between STRADA loss and mTORC1 hyperactivity in PMSE, and suggest that mTORC1 inhibition can serve as an effective therapeutic bio-target in PMSE as well as other devastating mTOR-associated neurodevelopmental disorders.

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