RHOA signaling defects result in impaired axon guidance in iPSC-derived neurons from patients with tuberous sclerosis complex

Abstract

Patients with Tuberous Sclerosis Complex (TSC) show aberrant wiring of neuronal connections formed during development which may contribute to symptoms of TSC, such as intellectual disabilities, autism, and epilepsy. Yet models examining the molecular basis for axonal guidance defects in developing human neurons have not been developed. Here, we generate human induced pluripotent stem cell (hiPSC) lines from a patient with TSC and genetically engineer counterparts and isogenic controls. By differentiating hiPSCs, we show that control neurons respond to canonical guidance cues as predicted. Conversely, neurons with heterozygous loss of TSC2 exhibit reduced responses to several repulsive cues and defective axon guidance. While TSC2 is a known key negative regulator of MTOR-dependent protein synthesis, we find that TSC2 signaled through MTOR-independent RHOA in growth cones. Our results suggest that neural network connectivity defects in patients with TSC may result from defects in RHOA-mediated regulation of cytoskeletal dynamics during neuronal development.

Translating the Role of mTOR- and RAS-Associated Signalopathies in Autism Spectrum Disorder: Models, Mechanisms and Treatment

“…Recent research in a TSC2 knockout iPSC model confirms the altered excitatory/inhibitory imbalance as observed in animal models, which is attributed to an increase in GABAergic signaling at inhibitory synapses [147]. However, neuronal growth cone studies in neurons derived from iPSCs report TSC2 heterozygous knockouts to have normal mTOR signaling but abnormal axon extension and insensitivity to inhibitory axonal guidance cues suggesting mTOR downstream processes to be the culprit and not necessarily mTOR itself [148].…”

Using MS induced pluripotent stem cells to investigate MS aetiology

“…To truly determine whether MS iPSC derived neurons possess intrinsic deficiencies that contribute to MS pathology, more cell lines need to be subjected to differentiation methods that produce specific neuronal subtypes. Intrinsic changes in neuron function have been identified using iPSCs generated from people with other neurological conditions, such as Parkinson's disease (Carola et al, 2021), autism spectrum disorder (Lim et al, 2021) and tuberous sclerosis complex (Catlett et al, 2021). Characterising the earliest drivers of neuron pathology will be critical for developing neuroprotective therapies for MS and this could be achieved by studying MS iPSC-derived neurons.…”

Mechanistic target of rapamycin signaling in human nervous system development and disease

“…2018; Winden et al, 2019;Martin et al, 2020;Catlett et al, 2021;Hisatsune et al, 2021). Hamartomas are potentially caused by "second-hit" somatic mutations in Tsc1 or Tsc2 resulting in loss of heterozygosity (LOH) (Consortium, 1993;Sepp et al, 1996;van Slegtenhorst et al, 1997).…”

“…The inheritance of TSC is autosomal dominant and can be caused by germline mosaicism, although most cases result from de novo mutations during early development ( Rose et al, 1999 ; Orlova and Crino, 2010 ). Interestingly, hiPSC models have shown that neurons with heterozygous Tsc1/2 mutations exhibit atypical morphology, defects in differentiation and axon guidance, and altered excitability and synaptic function ( Li et al, 2017a ; Sundberg et al, 2018 ; Zucco et al, 2018 ; Nadadhur et al, 2019 ; Catlett et al, 2021 ; Hisatsune et al, 2021 ). Homozygous loss of Tsc1/2 in hiPSC-derived neurons generally enhances these phenotypes ( Sundberg et al, 2018 ; Winden et al, 2019 ; Martin et al, 2020 ; Catlett et al, 2021 ; Hisatsune et al, 2021 ).…”

“…Interestingly, hiPSC models have shown that neurons with heterozygous Tsc1/2 mutations exhibit atypical morphology, defects in differentiation and axon guidance, and altered excitability and synaptic function ( Li et al, 2017a ; Sundberg et al, 2018 ; Zucco et al, 2018 ; Nadadhur et al, 2019 ; Catlett et al, 2021 ; Hisatsune et al, 2021 ). Homozygous loss of Tsc1/2 in hiPSC-derived neurons generally enhances these phenotypes ( Sundberg et al, 2018 ; Winden et al, 2019 ; Martin et al, 2020 ; Catlett et al, 2021 ; Hisatsune et al, 2021 ). Hamartomas are potentially caused by “second-hit” somatic mutations in Tsc1 or Tsc2 resulting in loss of heterozygosity (LOH) ( Consortium, 1993 ; Sepp et al, 1996 ; van Slegtenhorst et al, 1997 ).…”