Multimodal Analysis of STRADA Function in Brain Development

Abstract

mTORopathies are a heterogeneous group of neurological disorders characterized by malformations of cortical development (MCD), enhanced cellular mechanistic target of rapamycin (mTOR) signaling, and epilepsy that results from mutations in mTOR pathway regulatory genes. Homozygous mutations (del exon 9-13) in the pseudokinase STE20related kinase adaptor alpha (STRAD-α; STRADA), an mTOR modulator, are associated with Pretzel Syndrome (PS), a neurodevelopmental disorder within the Old Order Mennonite Community characterized by megalencephaly, intellectual disability, and intractable epilepsy. To study the cellular mechanisms of STRADA loss, we generated CRISPR-edited Strada mouse N2a cells, a germline mouse Strada knockout (KO−/−) strain, and induced pluripotent stem cell (iPSC)-derived neurons from PS individuals harboring the STRADA founder mutation. Strada KO in vitro leads to enhanced mTOR signaling and iPSC-derived neurons from PS individuals exhibit enhanced cell size and mTOR signaling activation, as well as subtle alterations in electrical firing properties e.g., increased input resistance, a more depolarized resting membrane potential, and decreased threshold for action potential (AP) generation. Strada−/− mice exhibit high rates of perinatal mortality and out of more than 100 litters yielding both WT and heterozygous pups, only eight Strada−/− animals survived past P5. Strada−/− mice are hypotonic and tremulous. Histopathological examination (n = 5 mice) revealed normal gross brain organization and lamination but all had ventriculomegaly. Ectopic neurons were seen in all five Strada−/− brains within the subcortical white matter mirroring what is observed in human PS brain tissue. These distinct experimental platforms demonstrate that STRADA modulates mTOR signaling and is a key regulator of cell size, neuronal excitability, and cortical lamination.

Convergent and Divergent Mechanisms of Epileptogenesis in mTORopathies

“…Newer rodent models using IUE to focally express or suppress mTORC1 pathway regulating genes in the cortex (discussed more below), as well as Tsc2 and Depdc5 zebrafish models (Kim S. H. et al, 2011;de Calbiac et al, 2018;Swaminathan et al, 2018), also recapitulate many of the mTORopathy phenotypes. Recent models also include patient-derived induced pluripotent stem cells (IPSCs) and cortical organoids, which provide the advantage of studying mTOR function in human cell populations (Blair et al, 2018;Andrews et al, 2020;Dang et al, 2020;Eichmüller et al, 2020). Both transgenic and IUE-based animal models as well as in vitro models have been vital for mechanistic and preclinical drug studies.…”

STRADA‐mutant human cortical organoids model megalencephaly and exhibit delayed neuronal differentiation

“…To better understand the role of STRADA in human neural development and disease, a human-based model is needed to complement the animal models. Previously, we generated induced pluripotent stem cells (iPSCs) from two individuals with PMSE and differentiated them into cortical-like excitatory neurons in monolayer cultures (Dang et al, 2020). We found loss of STRADA expression, increased pS6 expression, and neuronal cytomegaly, validating that the iPSC neuron model of PMSE demonstrates mTORC1 hyperactivity.…”

“…Fibroblasts were isolated from skin biopsies of two female individuals with PMSE and reprogrammed into iPSCs as previously described (Dang et al, 2020). One iPSC line from each of the two individuals with PMSE was used in this study.…”

“…Cultures were confirmed mycoplasma negative via periodic testing with a PCR-based assay. Each line used for experiments had expression of pluripotency markers (NANOG, OCT3/4, SSEA, SOX2) (Dang et al, 2020;Liu et al, 2013;Tidball et al, 2016). Episomally reprogrammed lines tested negative for episomal reprogramming vector integration.…”

“…These findings suggested an important role for mTOR signaling in cell fate determination or cell migration in neural stem cells. Germline homozygous knockout (KO) of Strada resulted in perinatal lethality, with the recapitulation of various features of PMSE including cerebral ventriculomegaly and enhanced numbers of white matter heterotopic neurons showing exuberant S6 phosphorylation (Dang et al, 2020).…”