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

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. Electronic supplementary material The online version of this article (10.1007/s12035-020-01983-6) contains supplementary material, which is available to authorized users.

Neural Networks Recapitulation by Cancer Cells Promotes Disease Progression: A Novel Role of p73 Isoforms in Cancer-Neuronal Crosstalk

“…The Affymetrix Human Gene 1.0 ST Array platform was employed. The GEO series GSE135287 [ 60 ] and GSE124308 include genome-wide gene expression analysis by RNA-Seq of human iPSC-derived cortical (i) neural progenitor cells and (ii) mature neurons. The sequencing platforms Illumina NextSeq 500 and Illumina HiSeq 2000 were employed.…”

Rubinstein-Taybi Syndrome: A Model of Epigenetic Disorder

“…In order to understand the phenotypic specificities and elucidate mechanistic insights on cognitive impairment of RSTS patients, different strains of CBP and p300-deficient mice were generated and summarized in this review [ 14 ]. More recently, Alari et al have established iPSC-derived neurons (i-neurons) from peripheral blood samples of three CREBBP - and two EP300 -mutated patients compared to four unaffected controls [ 134 , 135 ].…”

Histone Deacetylase Inhibitors Ameliorate Morphological Defects and Hypoexcitability of iPSC-Neurons from Rubinstein-Taybi Patients

“…One possible explanation for the lack of effect of TSA on neurite outgrowth, one of the critical steps in neuronal differentiation [ 36 ], keeps into account that reduced amount of CBP, the coactivator of CREB (cAMP response element protein) results in impaired calcium-induced activity dependent dendrite morphogenesis [ 37 ]. Impaired and dysregulated neuronal differentiation was revealed by transcriptome analysis of iPSC-derived neurons from a set of RSTS patients, including P149 and P34 [ 19 ]. Hence, one may hypothesize that suppression of HDAC activity by TSA may enhance the differentiation process without correcting the timeline warded off by the genetic defect.…”

“…He also presents agenesis of corpus callosum and hypoplasia of the cerebellar vermis, i.e., cortical malformations which have been linked to cell polarity dysfunction [ 45 ]. We know that several genes acting in cell polarity and adhesion, axonal targeting, and extension, as well as genes involved in key processes of functional neurons, are dysregulated in RSTS neurons [ 19 ], implying that defective CBP primes signaling cascades affecting both neuronal migration and synaptic formation and function. The exact pathway by which TSA altering the local chromatin structure sets in motion genes or cassettes of genes that attenuate the morphological abnormalities of RSTS P149 young neurons and partially rescue the electrophysiological features of differentiated neurons is hard to decode.…”

“…To investigate the effect of TSA on the cells most relevant to the cognitive deficit of the patients, we took advantage of the iPSC-derived neuronal model generated from RSTS patients displaying mental impairment of variable severity [ 18 ]. RNA-Seq analysis of iPSC derived neurons (iNeurons) from a set of RSTS patients showed a defective and altered neuroprogenitor to neuron transcriptional program with respect to healthy iNeurons due to a lower number of regulated genes and a signature compounded by improperly upregulated genes involved in neural migration and axonal targeting and downregulated RNA and DNA metabolic genes [ 19 ]. Out of the modeled patients, we selected the CREBBP -mutated patient 149 (aka P149), who harbors exon 18 c.3474 G>A, p.(Trp1158ter) truncating mutation and manifests the most severe ID [ 7 ] to assess whether short- and long-term exposure of his iPSC-derived neural progenitors to TSA might ameliorate the morphological and electrophysiological alterations exhibited by young and mature neurons from characterized RSTS patients [ 18 ].…”