High-throughput screening identifies histone deacetylase inhibitors that modulate GTF2I expression in 7q11.23 microduplication autism spectrum disorder patient-derived cortical neurons

Abstract

Background Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental condition affecting almost 1% of children, and represents a major unmet medical need with no effective drug treatment available. Duplication at 7q11.23 (7Dup), encompassing 26–28 genes, is one of the best characterized ASD-causing copy number variations and offers unique translational opportunities, because the hemideletion of the same interval causes Williams–Beuren syndrome (WBS), a condition defined by hypersociability and language strengths, thereby providing a unique reference to validate treatments for the ASD symptoms. In the above-indicated interval at 7q11.23, defined as WBS critical region, several genes, such as GTF2I, BAZ1B, CLIP2 and EIF4H, emerged as critical for their role in the pathogenesis of WBS and 7Dup both from mouse models and human studies. Methods We performed a high-throughput screening of 1478 compounds, including central nervous system agents, epigenetic modulators and experimental substances, on patient-derived cortical glutamatergic neurons differentiated from our cohort of induced pluripotent stem cell lines (iPSCs), monitoring the transcriptional modulation of WBS interval genes, with a special focus on GTF2I, in light of its overriding pathogenic role. The hits identified were validated by measuring gene expression by qRT-PCR and the results were confirmed by western blotting. Results We identified and selected three histone deacetylase inhibitors (HDACi) that decreased the abnormal expression level of GTF2I in 7Dup cortical glutamatergic neurons differentiated from four genetically different iPSC lines. We confirmed this effect also at the protein level. Limitations In this study, we did not address the molecular mechanisms whereby HDAC inhibitors act on GTF2I. The lead compounds identified will now need to be advanced to further testing in additional models, including patient-derived brain organoids and mouse models recapitulating the gene imbalances of the 7q11.23 microduplication, in order to validate their efficacy in rescuing phenotypes across multiple functional layers within a translational pipeline towards clinical use. Conclusions These results represent a unique opportunity for the development of a specific class of compounds for treating 7Dup and other forms of intellectual disability and autism.

KMT2D haploinsufficiency in Kabuki syndrome disrupts neuronal function through transcriptional and chromatin rewiring independent of H3K4-monomethylation

“…Intellectual disability is a hallmark of KS and one of its greatest burdens 28 . In order to elucidate its molecular underpinnings, we hypothesized that cortical neurons could reveal pathogenic vulnerabilities and differentiated iPSCs into excitatory cortical neurons through the transposon-based inducible Ngn2 overexpression 29 system that we recently validated 30 (hereafter iNeurons). We performed morphological reconstruction on control and KS1 patient-derived iNeurons 23 days after Ngn2 induction.…”

“…As bona fide pathogenic endophenotype, such robust KS1-specific neuronal network properties are ideally suited for middle-to-high-throughput drug screenings. Given the major neuronal defect in H3K27ac deposition, these could be productively oriented towards histone deacetylase inhibitors, which have already proven valuable for a condition with a similar degree of intellectual disability (7q11.23 duplication syndrome, OMIM 609757) 30 .…”

“…Pluripotent cells were engineered using an all-in-one ePiggyBac (ePB) transposon. We cloned the sequence of Ngn2 – P2A – EGFP – T2A – PuroR 29 sequence into an ePB backbone containing a blasticidin resistance cassette and doxycycline responsive element under the control of hUbC promoter 30 . iPSCs were electroporated with the Neon system (ThermoFisher) with the following parameters: 1200V, 30 ms, 1 pulse using 0,5 µg of a helper plasmid expressing a transposase and 4,5 µg of donor plasmid with a transposable element.…”

Contribution of Human Pluripotent Stem Cell-Based Models to Drug Discovery for Neurological Disorders

“…The first advantage is to be able to explore genes and pathways expressed at physiological levels without the need to introduce exogenous gene expression systems. This was exemplified in studies that screened compounds for genetic forms of ASD induced by the loss of function of FRMP [ 104 , 105 , 106 ] and duplication of a segment of chromosome 7 [ 107 ]. FXS was the first neurodevelopmental disorder to be modeled with this aim using iPSCs.…”

Autism Spectrum Disorders: Analysis of Mobile Elements at 7q11.23 Williams–Beuren Region by Comparative Genomics

“…GTF2I, is part of a complex that exerts epigenetic effects that are crucial for neurodevelopment [1]. In a recent study, three compounds were able to epigenetically inhibit the abnormal mRNA and protein level of GTF2I in 7DupASD patient-derived cell models thus representing promising therapeutic results [19].…”