Polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE) is a rare human autosomalrecessive 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.

Tuberous sclerosis complex (TSC) is characterized by developmental malformations of the cerebral cortex known as tubers, comprised of cells that exhibit enhanced mammalian target of rapamycin (mTOR) signaling. To date, there are no reports of mTORC1 and mTORC2 activation in fetal tubers or in neural progenitor cells lacking Tsc2. We demonstrate mTORC1 activation by immunohistochemical detection of substrates phospho-p70S6K1 (T389) and phospho-S6 (S235/236), and mTORC2 activation by substrates phospho-PKCα (S657), phospho-Akt (Ser473), and phospho-SGK1 (S422) in fetal tubers. Then, we show that Tsc2 shRNA knockdown (KD) in mouse neural progenitor cells (mNPCs) in vitro results in enhanced mTORC1 (phospho-S6, phospho-4E-BP1) and mTORC2 (phospho-Akt and phospho-NDRG1) signaling, as well as a doubling of cell size that is rescued by rapamycin, an mTORC1 inhibitor. Tsc2 KD in vivo in the fetal mouse brain by in utero electroporation causes disorganized cortical lamination and increased cell volume that is prevented with rapamycin. We demonstrate for the first time that mTORC1 and mTORC2 signaling is activated in fetal tubers and in mNPCs following Tsc2 KD. These results suggest that inhibition of mTOR pathway signaling during embryogenesis could prevent abnormal brain development in TSC.

Type I and type II focal cortical dysplasias (FCDs) exhibit distinct histopathological features that suggest different pathogenic mechanisms. Type I FCDs are characterized by mild laminar disorganization and hypertrophic neurons whereas type II FCDs exhibit dramatic laminar disorganization and cytomegalic cells (balloon cells). Both FCD types are associated with intractable epilepsy; therefore, identifying cellular or molecular differences between these lesion types that explains the histological differences could provide new diagnostic and therapeutic insights. Type II FCDs express nestin, a neuroglial progenitor protein that is modulated in vitro by the stem cell proteins c-Myc, SOX2, and Oct-4 following activation of mammalian target of rapamycin complex 1 (mTORC1). Since mTORC1 activation has been demonstrated in type II FCDs, we hypothesized that c-Myc, SOX2, and Oct-4 expression would distinguish type II from type I FCDs. In addition, we assayed the expression of progenitor cell proteins FOXG1, KLF4, Nanog, and SOX3. Differential expression of 7 stem cell proteins and aberrant phosphorylation of 2 mTORC1 substrates, S6 and S6 kinase 1 proteins, clearly distinguished type II from type I FCDs (n = 10 each). Our results demonstrate new potential pathogenic pathways in type II FCDs and suggest biomarkers for diagnostic pathology in resected epilepsy specimens.

Our results indicate a new association between HPV16 E6 and FCDIIB and demonstrate for the first time HPV16 E6 in the human brain. We propose a novel etiology for FCDIIB based on HPV16 E6 expression during fetal brain development.

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that results from mutations in the TSC1 or TSC2 genes. TSC is a multisystem hamartoma syndrome with manifestations in the brain, heart, lungs, kidney, skin, and eyes. Neurologically, TSC patients may exhibit severe epilepsy, cognitive disabilities, and autism spectrum disorders. Many TSC patients also present with renal angiomyolipomas, polycystic kidney disease, skin lesions, and lymphangiomyomatosis. TSC1 and TSC2 proteins form a heterodimeric complex that serves to inhibit mammalian target of rapamycin (mTOR) signaling pathway through Ras homolog enriched in brain (Rheb). TSC1 and TSC2 receive activating or inhibitory signals from multiple inputs including growth factors, insulin signaling, energy and amino acid levels, and proinflammatory pathways, which are then integrated to regulate the activity of the mTOR pathway. mTOR signaling plays a critical role in regulating cell growth, transcription, translation, and autophagy. Animal models have shed light on certain features of TSC, but failed to recapitulate the disease completely and currently further research is under way to better understand this devastating disorder. Clinical trials with mTOR inhibitors have shown promising results for some features of TSC, but further research needs to be conducted to establish full indications for therapeutic treatment.