Transcriptome Analysis of iPSC-Derived Neurons from Rubinstein-Taybi Patients Reveals Deficits in Neuronal Differentiation

Luciano Calzari,Cristina Gervasini,Valentina Alari,Silvia Russo,Cristina Barlassina,Daniele Braga,Angel Barco,Rafael Muñoz-Viana,Matteo Barcella,Palma Finelli,Lidia Larizza

doi:10.1007/s12035-020-01983-6

Abstract

Rubinstein-Taybi syndrome (RSTS) is a rare multisystem developmental disorder with moderate to severe intellectual disability caused by heterozygous mutations of either CREBBP or EP300 genes encoding CBP/p300 chromatin regulators. We explored the gene programs and processes underlying the morphological and functional alterations shown by iPSC-derived neurons modeling RSTS to bridge the molecular changes resulting from defective CBP/p300 to cognitive impairment. By global transcriptome analysis, we compared the differentially expressed genes (DEGs) marking the transition from iPSC-derived neural progenitors to cortical neurons (iNeurons) of five RSTS patients carrying private CREBBP/EP300 mutations and manifesting differently graded neurocognitive signs with those of four healthy controls. Our data shows a defective and altered neuroprogenitor to neuron transcriptional program in the cells from RSTS patients. First, transcriptional regulation is weaker in RSTS as less genes than in controls are modulated, including genes of key processes of mature functional neurons, such as those for voltage-gated channels and neurotransmitters and their receptors. Second, regulation is subverted as genes acting at pre-terminal stages of neural differentiation in cell polarity and adhesive functions (members of the cadherin family) and axon extension/guidance (members of the semaphorins and SLIT receptors families) are improperly upregulated. Impairment or delay of RSTS neuronal differentiation program is also evidenced by decreased modulation of the overall number of neural differentiation markers, significantly impacting the initial and final stages of the differentiation cascade. Last, extensive downregulation of genes for RNA/DNA metabolic processes confirms that RSTS is a global transcription disorder, consistent with a syndrome driven by chromatin dysregulation. Interestingly, the morphological and functional alterations we have previously appointed as biomarkers of RSTS iNeurons provide functional support to the herein designed transcriptome profile pointing to key dysregulated neuronal genes as main contributors to patients’ cognitive deficit. The impact of RSTS transcriptome may go beyond RSTS as comparison of dysregulated genes across modeled neurodevelopmental disorders could unveil convergent genes of cognitive impairment.

Highlights

Rubinstein-Taybi syndrome (RSTS1, MIM #180849, RSTS2, MIM #613684) is a multisystem developmental disorder affecting 1:125,000 newborns, characterized by moderate to severe intellectual disability (ID), growth delay, facial dysmorphisms, skeletal abnormalities, mainly of hands and feet, multi-organ malformations, and cancer predisposition [1]
We carried out transcriptome profiling of neural progenitor cells (NPCs) and neurons differentiated using the monolayer protocol [32] after 35 and 70 days, respectively, from the iPSC lines of 5 RSTS patients (4 CREBBP- and 1 EP300-mutated)
The in vitro neuronal iPSC model generated for RubinsteinTaybi syndrome [22,23,24] provided us the platform to search for dysregulated gene pathways which might lead to the morphological and electrophysiological alterations of RSTS neurons appointed as biomarkers of the neurocognitive signs of the patients. This aim has translational relevance because, at difference of CREBBP/EP300 causative gene mutations, the resulting epigenetic modifications are reversible and have been demonstrated to impact both brain development and adult brain function raising the opportunity of postnatal treatment with known and novel compounds to ameliorate the cognitive impairment of the patients [7, 49]

Summary

Introduction

Rubinstein-Taybi syndrome (RSTS1, MIM #180849, RSTS2, MIM #613684) is a multisystem developmental disorder affecting 1:125,000 newborns, characterized by moderate to severe intellectual disability (ID), growth delay, facial dysmorphisms, skeletal abnormalities, mainly of hands and feet, multi-organ malformations, and cancer predisposition [1] It is caused by heterozygous mutations of either CREBBP (cAMP responding element-binding protein (CREB) binding protein) (MIM #600140) (60%) [2] or EP300 (EIA-associated protein p300) (MIM #602700) (8– 10%) [3, 4] genes which encode CBP and p300 homologous transcriptional co-activators with lysine acetyltransferase activity (KAT) acting as epigenetic regulators [5,6,7,8,9]. Further complexity in deciphering molecular pathomechanisms is accounted for by the differential sensitivity of CBP/p300 effectors depending on the cellular context [20] and by the rapid dynamics of the CBP/p300 acetylome [19]

Methods

Results

Conclusion