Investigating impact of schizophrenia-associated non-coding variants on enhancer activity using brain organoids

Project Information

JS_SCZ

Grant agreement ID: 841940

DOI

10.3030/841940

Project closed

EC signature date 3 May 2019

Start date 1 September 2020

End date 31 August 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 186 167,04

EU contribution

€ 186 167,04

Coordinated by

INSTITUT FUER MOLEKULARE BIOTECHNOLOGIE GMBH
Austria

Periodic Reporting for period 1 - JS_SCZ (Investigating impact of schizophrenia-associated non-coding variants on enhancer activity using brain organoids)

Reporting period: 2020-09-01 to 2022-08-31

Summary of the context and overall objectives of the project

Cerebral cortex is the seat of intelligence in the human brain. It develops by a long elaborate process during which multipotent neural progenitors sequentially generate diverse excitatory neurons and glial cells. This process requires complex regulation not only at DNA level to determine which genes are expressed (transcriptional) but also at downstream (post-transcriptional) level to determine the availability of the gene products: RNA and the proteins. Understanding this regulation can help us understand the pathophysiology of many genetic and developmental disorders of the human brain. However, most of our understanding of these phenomena is based on studies using rodent experimental systems. Recent breakthroughs in stem cell-derived brain organoid culture methods have enabled us to recapitulate aspects of human brain development in vitro. Hence, we can now investigate the molecular and cell biological changes that occur during development of the human brain. Taking advantage of the brain organoid system, the following questions about human cerebral cortex development were investigated in this project : a) How does the cell type-specific abundance of RNA and protein change during development? b) Are there distinct RNA regulatory mechanisms that determine the expression trends of RNA and protein during cortex development c) What role do these RNA regulatory mechanisms play in neural progenitor proliferation and subsequent neurogenesis?

Work in this project led to identification of modules of genes showing similar cell type and stage-specific expression patterns of RNA and protein. Investigation of one such module uncovered mTOR pathway-mediated post-transcriptional regulation of ribosomal genes through a common RNA motif present in their mRNAs called the ‘5’TOP motif’. Lastly, it was found that this regulation is crucial to ensure timely progenitor differentiation and neurodevelopment. Thus, this study identified an important gene regulatory mechanism active during early brain development.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In this project, a dual reporter human embryonic stem cell line was generated to sort neural progenitors and neurons from brain organoids. Using this reporter line, a comprehensive dataset of cell type and developmental stage-specific transcriptome and proteome was generated for neural progenitors and neurons in the human brain organoids. These datasets are available for the broad community at NCBI repositories.

By integrating these datasets, modules of gene showing similar cell type and developmental stage-specific expression patterns were identified. Furthermore, RNA regulatory mechanisms enriched for each of these gene expression modules that potentially contribute to the expression pattern were identified. These gene modules are made available through a user-friendly app (https://organoid.multiomics.vbc.ac.at/).

Investigation of one such module through immunohistochemical analyses and reporter assays, uncovered mTOR-mediated translational regulation of ribosomal genes through a common 5’TOP RNA motif present in their mRNA. Further analysis upon mTOR overactivation indicated that partial inhibition of the translation of ribosomal genes in early progenitors is crucial to prevent precocious translation of neuronal and glial markers. This analysis was based on polysome profiling followed by RNA-seq that informed about RNA association with different ribosomal fractions. This dataset is also available for the broad community at NCBI repository.

Overall, the multiomics approach taken in the project revealed novel posttranscriptional regulatory mechanisms crucial for fidelity of cortical development. The findings from this project were disseminated through a preprint which will undergo peer review, dataset deposition to public repositories, presentation at scientific conference and through a custom website dedicated to the project. This study is available as a preprint.
(https://www.biorxiv.org/content/10.1101/2022.10.07.511280v1).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The burden of brain-related psychiatric and developmental disorders on our society is huge. Defects in gene regulation have emerged as a common theme in several neurodevelopmental disorders such as autism spectrum disorder (ASD). Therefore, studies such as the one conducted in this project, that further improve our understanding of human gene regulation are a step towards understanding mechanisms of these diseases. Beyond the gene regulation at DNA-RNA level, this project describes gene expression at RNA-protein level and contributes to holistic understanding of human cortical development. These results not only provide an important dataset for the community, but also reveal a novel RNA regulatory mechanism underlying human cortical development. mTOR-dependency of this regulatory pathway can help us understand cell type and developmental stage specific-mechanisms of many mTOR-related brain disorders such as tuberous sclerosis, focal cortical dysplasia and megalencephaly. Thus, this project provides basic biological insights relevant to understand disease mechanisms and potential therapies.

RNA-protein multiomics revealed gene expression modules active during human neurodevelopment