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

Abstract

Background Tuberous sclerosis complex (TSC) is a rare genetic multisystemic disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes. It is characterised by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway and has severe neurodevelopmental and neurological components including autism, intellectual disability and epilepsy. In human and rodent models, loss of the TSC proteins causes neuronal hyperexcitability and synaptic dysfunction, although the consequences of these changes for the developing central nervous system are currently unclear. Methods Here we apply multi-electrode array-based assays to study the effects of TSC2 loss on neuronal network activity using autism spectrum disorder (ASD) patient-derived iPSCs. We examine both temporal synchronisation of neuronal bursting and spatial connectivity between electrodes across the network. Results We find that ASD patient-derived neurons with a functional loss of TSC2, in addition to possessing neuronal hyperactivity, develop a dysfunctional neuronal network with reduced synchronisation of neuronal bursting and lower spatial connectivity. These deficits of network function are associated with elevated expression of genes for inhibitory GABA signalling and glutamate signalling, indicating a potential abnormality of synaptic inhibitory–excitatory signalling. mTORC1 activity functions within a homeostatic triad of protein kinases, mTOR, AMP-dependent protein Kinase 1 (AMPK) and Unc-51 like Autophagy Activating Kinase 1 (ULK1) that orchestrate the interplay of anabolic cell growth and catabolic autophagy while balancing energy and nutrient homeostasis. The mTOR inhibitor rapamycin suppresses neuronal hyperactivity, but does not increase synchronised network activity, whereas activation of AMPK restores some aspects of network activity. In contrast, the ULK1 activator, LYN-1604, increases the network behaviour, shortens the network burst lengths and reduces the number of uncorrelated spikes. Limitations Although a robust and consistent phenotype is observed across multiple independent iPSC cultures, the results are based on one patient. There may be more subtle differences between patients with different TSC2 mutations or differences of polygenic background within their genomes. This may affect the severity of the network deficit or the pharmacological response between TSC2 patients. Conclusions Our observations suggest that there is a reduction in the network connectivity of the in vitro neuronal network associated with ASD patients with TSC2 mutation, which may arise via an excitatory/inhibitory imbalance due to increased GABA-signalling at inhibitory synapses. This abnormality can be effectively suppressed via activation of ULK1.

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].…”

Human In Vitro Models of Epilepsy Using Embryonic and Induced Pluripotent Stem Cells

“…The first group is the studies that described the generation of patient-specific iPSC lines [ 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 ] that carry genetic variants in genes associated with epilepsy, such as SCN1A , GNB5, LGI1, GRIN2A, KCNC1, or KCNA2 ( Figure 3 , Table S1 ) [ 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 ]. The second group is the studies that assessed the effects of convulsant and anticonvulsant drugs on hiPSC-derived neurons and astrocytes ( Figure 4 , Table S1 ) [ 57 , 58 , 59 ]. Some of these studies used healthy human stem cells to either generate an in vitro disease model by genetically manipulating a gene of interest; or by assessing the effects of different compounds on chemically induced “epilepsy-like” phenotype on otherwise healthy neurons [ 57 , 58 , 59 , 60 ].…”

“…Some of these studies used healthy human stem cells to either generate an in vitro disease model by genetically manipulating a gene of interest; or by assessing the effects of different compounds on chemically induced “epilepsy-like” phenotype on otherwise healthy neurons [ 57 , 58 , 59 , 60 ]. Others used the iPSCs derived from patients carrying a specific gene variance of interest ( Figure 4 ) [ 27 , 36 , 38 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 ,…”

The trilateral interactions between mammalian target of rapamycin (mTOR) signaling, the circadian clock, and psychiatric disorders: an emerging model

“…This reduced inhibition further causes the mTOR hyperactivity, altered protein synthesis, enhancement of proliferation. Also, a study by Alsaqati et al demonstrated the TSC disorder which they characterized by hyperactivation of the mTORC1 pathway using the iPSC from ASD patients [ 150 ]. Secondly, the mutations in the Fmr1 gene leads to FXS and associated to abnormalities in the mTOR-dependent protein synthesis.…”