Abstracts

Rationale: Pretzel syndrome (PS), a recently described autosomal recessive disorder in Old Order Mennonite children, is characterized by macrocephaly, severe cognitive disability, and intractable epilepsy. All affected children harbor a large homozygous deletion in the STRADα gene (17q23.3), which encodes the pseudokinase STRADα (STE20 Related Adaptor alpha). The underlying pathogenic mechanisms of PS and the functional role of STRADα in cortical development remain unknown. The only characterized function of STRADα is to bind the serine/threonine kinase LKB1, effecting the subcellular localization of LKB1 in the cytoplasm and enhancing its catalytic activity. LKB1 serves as a master upstream kinase to many AMPK-related kinases, including AMPK, which inhibits mTOR signaling by phosphorylating and activating TSC2. In addition, recent reports suggest that LKB1 regulates neuronal migration during corticogenesis. Methods: To define the role of STRADα during corticogenesis, we first analyzed postmortem PS brain tissue by immunohistochemistry and morphometry. Then, we examined the function of STRADα in vitro in mouse neural progenitor cells (mNPCs) using shRNA-mediated STRADα knockdown, and in vivo utilizing in utero electroporation of STRADα shRNA to model PS. Results: Histopathological analysis of postmortem PS brain revealed neuronal cytomegaly and neuronal heterotopia in the subcortical white matter. Immunohistochemical analysis demonstrated absent STRADα expression, enhanced phosphorylation of S6 protein suggestive of mTOR activation, and exclusively nuclear localization of LKB1. shRNA-mediated knockdown of STRADα in mNPCs in vitro demonstrated that STRADα regulates mTOR signaling in an AMPK- and rapamycin-dependent manner. STRADα-depleted mNPCs exhibit nuclear LKB1 localization in contrast to control cells, which express LKB1 in the cytoplasmic compartment. STRADα knockdown in mNPCs resulted in cytomegaly that was prevented by treatment with the mTOR antagonist rapamycin. STRADα knockdown in vivo by in utero electroporation of STRADα shRNA at embryonic day 14 (E14) resulted in aberrant cortical lamination. Specifically, STRADα-depleted cells remain in the ventricular/subventricular zones (VZ/SVZ) at E17 and E19 instead of assuming their appropriate laminar position within the cortical plate (CP). This is of particular interest, since PS cortex contains abundant neuronal heterotopia in the subcortical white matter. Conclusions: These studies, for the first time, delineate a functional role of STRADα during corticogenesis and suggest a potential therapeutic role of rapamycin in PS.