Biallelic Mutations inTSC2Lead to Abnormalities Associated with Cortical Tubers in Human iPSC-Derived Neurons

Abstract

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in TSC1 or TSC2. Patients frequently have epilepsy, autism spectrum disorder, and/or intellectual disability, as well as other systemic manifestations. In this study, we differentiated human induced pluripotent stem cells (iPSCs) from a female patient with TSC with one or two mutations in TSC2 into neurons using induced expression of NGN2 to examine neuronal dysregulation associated with the neurological symptoms in TSC. Using this method, neuronal differentiation was comparable between the three genotypes of iPSCs. We observed that TSC2 ϩ/Ϫ neurons show mTOR complex 1 (mTORC1) hyperactivation and associated increased cell body size and process outgrowth, as well as exacerbation of the abnormalities by loss of the second allele of TSC2 in TSC2 Ϫ/Ϫ neurons. Interestingly, iPSC-derived neurons with either a single or biallelic mutation in TSC2 demonstrated hypersynchrony and downregulation of FMRP targets. However, only neurons with biallelic mutations of TSC2 demonstrated hyperactivity and transcriptional dysregulation observed in cortical tubers. These data demonstrate that loss of one allele of TSC2 is sufficient to cause some morphological and physiological changes in human neurons but that biallelic mutations in TSC2 are necessary to induce gene expression dysregulation present in cortical tubers. Finally, we found that treatment of iPSC-derived neurons with rapamycin reduced neuronal activity and partially reversed gene expression abnormalities, demonstrating that mTOR dysregulation contributes to both phenotypes. Therefore, biallelic mutations in TSC2 and associated molecular dysfunction, including mTOR hyperactivation, may play a role in the development of cortical tubers.

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

“…This can explain the patterns of network disorganisation that we observe, manifest as a high number of random and uncorrelated spikes. Furthermore a large reduction in the number of SBs detected in the TSC2 model was associated with an increase in the burst length, consistent with previous reports in both animal and human models [16,40]. Our pharmacological pro ling demonstrated that consistent with that already shown in control human iPSC-derived neurons and in rodent neurons, glutamate and GABA signalling are the primary drivers for the network activity in TSC2 neurons [27].…”

“…We showed that TSC2-derived neurons have common neuronal defects caused by both autosomal dominant TSC1 and TSC2 mutations and that neuronal hyperexcitability of TSC patient-derived cells was reversed by rapamycin [17]. A subsequent study used induced expression NGN2 to generate excitatory neurons from TSC2 +/-, TSC2 -/and TSC2 +/+ cells and replicated the observations on TSC neurons hyperexcitability [16].…”

“…In that study, as NGN-2 directed neuronal differentiation produces only excitatory neurons it was not possible to monitor aberrant network behaviour arising from an excitatory/inhibitory imbalance [39]. Our current data demonstrates that TSC2 neurons possess aberrant synchrony and connectivity, in addition to neuronal hyperexcitability described previously [16,17]. Sundberg et al found that TSC2 patient neurons exhibited reduced synaptic activity, measured by a decrease in the number of functional glutamatergic synapses in relation to controls with no effect on the glutamate receptor properties [11].…”

“…In animal models, treatment with mTORC1 inhibitor, rapamycin recovered the behavioural de cits including learning, memory, and autistic-like features and the neuronal hyperexcitability [13][14][15]. In human models, rapamycin reversed neuronal hyperexcitability and improved recall memory in patients with angiomyolipomas associated with TSC [16][17][18]. However, other neuropsychological measures including executive functions and recognition memory showed reduction in some participants [18].…”

Progress of Induced Pluripotent Stem Cell Technologies to Understand Genetic Epilepsy

“…More recently, Winden et al confirmed the increased activity levels of human TSC2−/− neurons by MEA, showing also a difference between control and heterozygous networks. Hypersynchronous neuronal discharges were also found in both TSC2+/− and TSC2−/− neurons [48].…”