TSC patient-derived isogenic neural progenitor cells reveal altered early neurodevelopmental phenotypes and rapamycin-induced MNK-eIF4E signaling

Abstract

Background: Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin. Methods: Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq. Results: Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/ downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs.

RAP-ALO(N)G and Make Me Smart

“…This finding is supported by prior experimental studies in a Tsc1 knockout mice, which has shown that dendritic patterning is modulated in an mTOR-independent manner through mitogen-activated protein kinases (MEK) that regulate phosphorylation of extracellular signal-regulated kinases (ERK). 2,9 The MEK-ERK was shown to be aberrantly activated in SEGAs, 8 and a similar activation and elevation of phosphorylated ERK (pERK) was noticed in the current study. Interestingly, pretreatment with rapamycin, while blocking mTORC1 activation, led to a significant increase in pERK1/2 in TSC-Het and Null and it was abolished by an application of MEK inhibitor trametinib.…”

Pharmacological intervention to restore connectivity deficits of neuronal networks derived from ASD patient iPSC with a TSC2 mutation

“…Previous studies showed that while the involvement of rapamycin in TSC2 patients control tumour growth, its efficacy in managing neuropsychiatric-associated behaviour in TSC2 patients has remained unclear [49]. Rapamycin failed to reverse the enhanced proliferation and altered neurite outgrowth; phenotypes inherent to TSC KO NPCs [50]. As rapamycin only particularly rescues the TSC2 neuronal phenotype, having little effect on the network deficit, we targeted aberrant synchronicity and connectivity deficits with alternative approaches via upstream TSC activation or by manipulating the mTORC1 at the molecular level.…”

Pharmacological intervention to restore connectivity deficits of neuronal networks derived from ASD patient iPSC with a TSC2 mutation

“…Previous studies showed that the effectiveness of rapamycin in control tumour growth for TSC2 patients, its e cacy in managing neuropsychiatric-associated behaviour in TSC2 patients has remained unclear [45]. In a report, rapamycin also failed to reverse the enhanced proliferation and altered neurite outgrowth; phenotypes inherent to TSC KO NPCs [46]. As rapamycin had little effect on the network de cit, we targeted the aberrant synchronicity and connectivity de cits with alternative approaches, examining upstream TSC activation or downstream manipulation the mTORC1 at the molecular level.…”