Multiscale modeling uncovers 7q11.23 copy number variation–dependent changes in ribosomal biogenesis and neuronal maturation and excitability
Marija Mihailovich, … , Michela Matteoli, Giuseppe Testa
Marija Mihailovich, … , Michela Matteoli, Giuseppe Testa
Published July 15, 2024
Citation Information: J Clin Invest. 2024;134(14):e168982. https://doi.org/10.1172/JCI168982.
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Multiscale modeling uncovers 7q11.23 copy number variation–dependent changes in ribosomal biogenesis and neuronal maturation and excitability

Abstract

Copy number variation (CNV) at 7q11.23 causes Williams-Beuren syndrome (WBS) and 7q microduplication syndrome (7Dup), neurodevelopmental disorders (NDDs) featuring intellectual disability accompanied by symmetrically opposite neurocognitive features. Although significant progress has been made in understanding the molecular mechanisms underlying 7q11.23-related pathophysiology, the propagation of CNV dosage across gene expression layers and their interplay remains elusive. Here we uncovered 7q11.23 dosage–dependent symmetrically opposite dynamics in neuronal differentiation and intrinsic excitability. By integrating transcriptomics, translatomics, and proteomics of patient-derived and isogenic induced neurons, we found that genes related to neuronal transmission follow 7q11.23 dosage and are transcriptionally controlled, while translational factors and ribosomal genes are posttranscriptionally buffered. Consistently, we found phosphorylated RPS6 (p-RPS6) downregulated in WBS and upregulated in 7Dup. Surprisingly, p-4EBP was changed in the opposite direction, reflecting dosage-specific changes in total 4EBP levels. This highlights different dosage-sensitive dyregulations of the mTOR pathway as well as distinct roles of p-RPS6 and p-4EBP during neurogenesis. Our work demonstrates the importance of multiscale disease modeling across molecular and functional layers, uncovers the pathophysiological relevance of ribosomal biogenesis in a paradigmatic pair of NDDs, and uncouples the roles of p-RPS6 and p-4EBP as mechanistically actionable relays in NDDs.

Authors

Marija Mihailovich, Pierre-Luc Germain, Reinald Shyti, Davide Pozzi, Roberta Noberini, Yansheng Liu, Davide Aprile, Erika Tenderini, Flavia Troglio, Sebastiano Trattaro, Sonia Fabris, Ummi Ciptasari, Marco Tullio Rigoli, Nicolò Caporale, Giuseppe D’Agostino, Filippo Mirabella, Alessandro Vitriolo, Daniele Capocefalo, Adrianos Skaros, Agnese Virginia Franchini, Sara Ricciardi, Ida Biunno, Antonino Neri, Nael Nadif Kasri, Tiziana Bonaldi, Rudolf Aebersold, Michela Matteoli, Giuseppe Testa

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Figure 1

7q11.23 isogenic iNeurons preserve dosage.

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7q11.23 isogenic iNeurons preserve dosage. (A) Scheme of experimental de...
(A) Scheme of experimental design and generation of 7q11.23 isogenic lines. (B) Two-color FISH analyses using 7 alpha satellite probes (see Supplemental Methods) as a control for the chromosomal number (yellow) and ELN, a WBSCR gene (red). ELN showed 3 signals in 7Dup, 2 in isoCTL, and 1 in isoWBS, corresponding to the 7q11.23 copy number in respective clones. (C–E) WBSCR genes maintain the dosage at the RNA and protein levels. RNA-seq data for WBSCR genes are shown for both patient-derived and isogenic neurons for all 3 genotypes. Although GTF2I transcripts were not downregulated in isoWBS in the RNA-seq analysis, both the translatome and proteome data showed 7q11.23 dosage–dependent expression (Supplemental Figure 1, F and G) that was also confirmed by Western blot (Figure 1D and Supplemental Figure 1H), suggesting that the upregulation observed at the mRNA level is probably an artifact of sequencing of repetitive regions. Western blot results from the same neuronal preparation, run on 2 gels, are shown in D. GAPDH was used as a loading control. Quantification of Western blots (shown in D and Supplemental Figure 1H) is shown as relative expression in E. Non-normalized data are shown as mean ± SEM (n = 4). The statistical comparisons were done with 1-way ANOVA followed by Tukey’s multiple-comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.