STRADα deficiency results in aberrant mTORC1 signaling during corticogenesis in humans and mice
Ksenia A. Orlova, … , Kevin Strauss, Peter B. Crino
Ksenia A. Orlova, … , Kevin Strauss, Peter B. Crino
Published April 26, 2010
Citation Information: J Clin Invest. 2010;120(5):1591-1602. https://doi.org/10.1172/JCI41592.
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Research Article

STRADα deficiency results in aberrant mTORC1 signaling during corticogenesis in humans and mice

Abstract

Polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE) is a rare human autosomal-recessive disorder characterized by abnormal brain development, cognitive disability, and intractable epilepsy. It is caused by homozygous deletions of STE20-related kinase adaptor α (STRADA). The underlying pathogenic mechanisms of PMSE and the role of STRADA in cortical development remain unknown. Here, we found that a human PMSE brain exhibits cytomegaly, neuronal heterotopia, and aberrant activation of mammalian target of rapamycin complex 1 (mTORC1) signaling. STRADα normally binds and exports the protein kinase LKB1 out of the nucleus, leading to suppression of the mTORC1 pathway. We found that neurons in human PMSE cortex exhibited abnormal nuclear localization of LKB1. To investigate this further, we modeled PMSE in mouse neural progenitor cells (mNPCs) in vitro and in developing mouse cortex in vivo by knocking down STRADα expression. STRADα-deficient mNPCs were cytomegalic and showed aberrant rapamycin-dependent activation of mTORC1 in association with abnormal nuclear localization of LKB1. Consistent with the observations in human PMSE brain, knockdown of STRADα in vivo resulted in cortical malformation, enhanced mTORC1 activation, and abnormal nuclear localization of LKB1. Thus, we suggest that the aberrant nuclear accumulation of LKB1 caused by STRADα deficiency contributes to hyperactivation of mTORC1 signaling and disruption of neuronal lamination during corticogenesis, and thereby the neurological features associated with PMSE.

Authors

Ksenia A. Orlova, Whitney E. Parker, Gregory G. Heuer, Victoria Tsai, Jason Yoon, Marianna Baybis, Robert S. Fenning, Kevin Strauss, Peter B. Crino

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Figure 4

STRADα knockdown in mNPCs.

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STRADα knockdown in mNPCs. (A–G) Transient knockdown of STRADα in mNPCs ...
(A–G) Transient knockdown of STRADα in mNPCs using GFP-shRNA. (A) mNPCs were transfected with vehicle (veh), GFP-shRNA scram, or GFP-shRNA STRADα clones 1–3 and FACS sorted for GFP expression at 5 DPT. Endogenous STRADα expression was assessed by immunoblotting. The blot was reprobed for GAPDH to assess equal loading. Cells were transfected, sorted, and immunoblotted in 3 separate experiments. GFP-shRNA STRADα clone 3 resulted in reproducible knockdown of STRADα. (B–G) mNPCs were transfected with GFP-shRNA STRADα clone 3 (E–G) or control GFP-shRNA scram (B–D) and immunostained for STRADα at 5 DPT. Nuclei were visualized with Hoechst staining (blue, D and G). A GFP-shRNA STRADα clone 3–transfected cell (arrows, E–G) exhibited less immunoreactivity for STRADα than did a GFP-shRNA scram–transfected cell (arrowhead, B–D) or untransfected neighboring cells. (H) Stable knockdown of STRADα using puro-shRNA. mNPCs were transfected with puro-shRNA scram or puro-shRNA STRADα, and transfected cells were selected for in the presence of puromycin. Expression of endogenous STRADα was assessed by immunoblotting approximately 6 weeks post transfection. Untransfected wild-type mNPCs (veh) were used as a control. The blot was reprobed for GAPDH to assess equal loading. Data are representative of 3 separate experiments. Scale bar: 20 μm.