Translational investigation of Neurodevelopmental Disorders caused by impairment of the Ubiquitin-Proteasome System – UPS-NDDecipher

The project combined clinical genetics, cell reprogramming, genome editing, neuronal differentiation, advanced imaging and integrative bioinformatics.

Patient blood cells were reprogrammed into induced pluripotent stem cells (hiPSCs), then corrected or engineered by CRISPR-Cas9 to generate mutant and isogenic control lines. A total of 30 validated hiPSC lines were produced, exceeding the initial objective of 24 lines and broadening the genetic scope of the study. These lines were differentiated into neural progenitor cells, cortical glutamatergic neurons and, for selected models, 3D brain organoids to study brain development in a more physiological context.

Phenotypes were assessed using microscopy and functional assays, then integrated with multi-omics datasets including RNA-seq, miRNA-seq, ATAC-seq, ChIP-seq and quantitative mass spectrometry-based proteomics.

The project also relied on the dedicated BioTND-UPS biobank, which includes more than 450 individuals and supports the identification of biomarkers detectable in peripheral blood cells.

The project exceeded its initial objectives by generating 30 hiPSC lines across several UPS genes and establishing robust cellular models for mechanistic studies.

The analyses revealed gene-specific phenotypes, including impaired proliferation and neurite outgrowth in CUL4B models, delayed differentiation and organoid abnormalities in PSMC5 models, and early neurodevelopmental alterations in PSMC3-derived systems.

Multi-omics analyses identified convergent molecular signatures involving neuritogenesis-related genes, defects in proteasome assembly or interaction, and activation of type I interferon signaling. This interferon signature was confirmed in patients’ T lymphocytes across several UPS genes, making it a particularly promising translational biomarker.

The project also led to major publications in top-tier international journals, including Science Translational Medicine, Nature Communications, American Journal of Human Genetics and Genetics in Medicine.

In the short term, ongoing work will complete the multi-omics analyses, deepen the characterization of brain organoids and functionally validate the biomarkers identified so far.

In the medium term, the goal is to develop phenotypic screening assays based on these biomarkers in order to test active compounds targeting the dysregulated pathways, building on the international collaborations already established.

In the longer term, these results could support the development of simple peripheral diagnostic tests based on blood cells and help pave the way toward targeted therapeutic strategies for these rare disorders.

The project has also structured an international network and acted as a lever for major complementary funding, thereby strengthening translational research on UPS-NDD well beyond the initial ANR program.

The ubiquitin-proteasome system (UPS) is one of the major eukaryotic pathways for intracellular degradation of proteins which are misfolded, oxidized, damaged and/or no longer needed. Denatured proteins to be degraded are specifically tagged with ubiquitin chains before being addressed to the 26S proteasome that ensure their hydrolysis. The UPS is essential to neuronal development and function and most of the approximately 1,200 genes which contribute to this protein degradation pathway are highly expressed in brain. Their pathogenic variants are responsible for about 10-15% of neurodevelopmental disorders (NDDs). Yet, since the recognition of UPS-related NDDs (UPS-NDDs) is fairly recent, virtually nothing is known about how UPS genetic variants can lead to abnormal brain development.

The UPS-NDDecipher project was designed to address this question. Its objective is to decipher the physiopathological mechanism of six UPS-NDDs caused by pathogenic genetic variants in USP7, CUL4B, PSMD12, PSMC3, PSMC5 or BAP1. The transdisciplinary approach followed in the project will combine state-of-the-art methods in cell reprogramming and differentiation, neuroimaging and functional genomics applied to patient cells. The implementation of the project is made possible by the contact network with clinical geneticists, scientists and patient organizations that was previously established by members of the UPS-NDDecipher consortium and allowed the initiation of a biocollection dedicated to UPS-NDDs. The occurrence of morphological abnormalities will be monitored during differentiation of patient induced pluripotent stem cells (hiPSC)-derived glutamatergic neurons and culture of mouse hippocampal cell lines. In addition, a specific molecular signature of UPS-NDDS will be sought by comparative integrative multi-omics analysis of patient and isogenic hiPSC-derived neuronal lines. Once a molecular signature specific to the six UPS-NDDs has been identified, selected active compounds will be tested for their ability to restore a normal UPS protein degradation in mutant cells. The ultimate goal of the project is thus to open the way to pre-clinical studies and hopefully to offer therapeutic perspectives to patients.