iPSC-derived neurons of CREBBP - and EP300 -mutated Rubinstein-Taybi syndrome patients show morphological alterations and hypoexcitability

Abstract

Rubinstein-Taybi syndrome (RSTS) is a rare neurodevelopmental disorder characterized by distinctive facial features, growth retardation, broad thumbs and toes and mild to severe intellectual disability, caused by heterozygous mutations in either CREBBP or EP300 genes, encoding the homologous CBP and p300 lysine-acetyltransferases and transcriptional coactivators. No RSTS in vitro induced Pluripotent Stem Cell (iPSC)-neuronal model is available yet to achieve mechanistic insights on cognitive impairment of RSTS patients. We established iPSC-derived neurons (i-neurons) from peripheral blood cells of three CREBBP- and two EP300-mutated patients displaying different levels of intellectual disability, and four unaffected controls. Pan neuronal and cortical-specific markers were expressed by all patients' i-neurons. Altered morphology of patients' differentiating neurons, showing reduced branch length and increased branch number, and hypoexcitability of differentiated neurons emerged as potential disease biomarkers. Anomalous neuronal morphology and reduced excitability varied across different RSTS patients' i-neurons. Further studies are needed to validate these markers and assess whether they reflect cognitive and behavioural impairment of the donor patients.

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

“…Out of RSTS univocal URGs, we underline the most significant top genes, which control phenotypes well recognizable in our in vitro model and hence candidates to account for the morpho-functional alterations detected in neurons from the same patients [ 24 ]. This applies to the semaphorin genes SEMA3F and SEMA5B , encoding members of a family of signaling proteins first described as axon guidance cues and then implicated in multiple aspects of nervous system development [ 41 ], which are sorted under the groups “regulation of axon guidance” (G10) and “axon extension involved in axon guidance” (G8).…”

“…The in vitro neuronal iPSC model generated for Rubinstein-Taybi syndrome [ 22 – 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 ].…”

“…2b ) and DEGs (Fig. 3a ), a finding accounting for the successful differentiation of patients neurons evaluated by qualitative expression of stage-specific immunohistochemical markers in all respective neuronal cultures [ 24 ] (Additional File 1 ). Focus on univocal DEGs lists showed in RSTS improperly upregulated genes of cell polarity and adhesion acting in neuronal migration such as CDH1 , FAT4 , and ENC1 and genes for axonal and dendritic targeting such as SEMA3F , SEMA5B , and SLITRK2/6 (Fig.…”

“…To note that, the genes for semaphorin receptors, neuropilin 1 ( NRP1 ), and plexin A3 ( PLXNA3 ) that mediates cell adhesion via a homophilic binding in the presence of calcium ions were also upregulated in RSTS neurons (Additional File 10 ) highlighting the activation of the semaphorin-plexin axis which usually orchestrates central nervous system connectivity through the differential control of spine morphogenesis and synapse formation [ 54 ]. Incorrect activation of this axis may account for reduced branch length and increased branch number shown by 42 days iNeurons of the same patients [ 24 ]. Furthermore, aberrant upregulation of SEMA3F along with the lack of some genes essential for synaptic signaling and neurotransmission, such as ion voltage-gated activity, neurotransmitter, and neurotransmitter receptors (passive gene modulation), may concur to the hypoexcitability recorded in the 70 days iNeurons [ 24 ].…”

“…iPSCs reprogrammed from blood of RSTS1 and RSTS2 patients successfully generated, likewise iPSCs from healthy individuals, neural progenitor cells (NPCs) which then differentiated into cortical neurons. However, morphological and functional alterations were shown by the young and mature RSTS neurons, respectively [ 24 ], raising the question of exploring the transcriptional dysregulation underlying these defects to understand the molecular basis of RSTS patients cognitive deficits.…”