Fetal Brain mTOR Signaling Activation in Tuberous Sclerosis Complex

Abstract

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.

Normalizing translation through 4E-BP prevents mTOR-driven cortical mislamination and ameliorates aberrant neuron integration

“…That only a subset of our ectopic neurons express Er81 is not surprising, given that Er81 is typically only expressed in 34% of layer 5 callosal projection neurons (41). Analysis of subtype markers Satb2, Ctip2, and Foxp2 also showed that ectopic neurons do not switch projection subtype, which is in agreement with a previous study examining Ctip2 and Tbr1 after in utero electroporation of a Tsc2 shRNA (5).…”

“…Of the many effectors downstream of mTORC1, we identified 4E-BPs as a molecular target that can restore many aspects of normal cortical circuitry. Considering that rapamycin does not fully block mTORC1 signaling through 4E-BPs in vitro (14), our report that normalizing signaling through 4E-BPs, but not S6Ks, prevents Rheb CA -induced mislamination seems to conflict with studies showing that rapamycin can rescue many phenotypes, including mislamination, in TSC models (5,7,49). However, the effects of rapamycin on translation and 4E-BP signaling after chronic administration in vivo are not well studied, particularly in the brain.…”

“…The high incidence of negative outcomes in individuals with such malformations (2), which are often associated with intractable childhood seizures, underscores the need to better understand the molecular etiology of these developmental lesions. Animal models have demonstrated the causative effect of increased mTORC1 signaling on mislamination (3)(4)(5)(6)(7). The pharmacological mTORC1 blocker rapamycin has also been shown to reverse some of the developmental abnormalities and associated seizure activity in several of these mouse models (5,6,(8)(9)(10), further emphasizing the importance of mTORC1 in the disease pathogenesis.…”

mTOR Signaling in Epilepsy: Insights from Malformations of Cortical Development

“…Subsequent studies confirmed seizure relief in several mouse TSC models (for review, see Feliciano et al 2013). Three recent preclinical studies have also shown that in utero prenatal treatment with rapamycin can prevent or significantly diminish the morphological consequences of Tsc1 or Tsc2 loss in the mouse (Anderl et al 2011;Tsai et al 2012b;Way et al 2012). These findings suggest that in utero rapamycin could be used in the setting of a prenatal TSC diagnosis; however, rapamycin alone may have deleterious effects of fetal brain development that may warrant further consideration (Tsai et al 2013).…”

Epilepsy Related to Developmental Tumors and Malformations of Cortical Development

“…1m, n). Interestingly, recent observations indicate that brain malformations in TSC are likely a consequence of increased mTOR activation during fetal brain development [198,209].…”