Cerebral organoid proteomics reveals signatures of dysregulated cortical development associated with human trisomy 21

Abstract

RUNNING TITLE: Proteomics of harmine treated trisomy 21 cerebral organoids 2 ABBREVIATIONS: HSA21, human chromosome 21; DS, Down syndrome; CO, cerebral organoid; EB, embryoid body; iPSC, induced pluripotent stem cell; DMSO, dimethyl sulfoxide; EdU, 5-ethynyl-2'-deoxyuridine; FDR, false discovery rate; T21, the trisomy 21 C2-4 iPSC line; D21, the euploid I50-2 iPSC line; TX100, Triton X-100; NS, neurosphere; DPBS, Dulbecco's phosphate-buffered saline; PBS, phosphate-buffered saline; AGC, Automatic Gain Control; LFQ, label-free quantitation; PCA, principal component analysis; ECM, extracellular matrix; WGCNA, weighted gene co-expression network analysis 3 SUMMARY Human trisomy 21 (Down syndrome) is the most common genetic cause of intellectual disability, and is associated with complex perturbations in protein expression during development. Brain region-specific alterations in neuronal density and composition originate prenatally in trisomy 21 individuals, and are presumed to underlie the intellectual disability and early onset neurodegeneration that characterizes Down syndrome. However, the mechanisms by which chromosome 21 aneuploidy drives alterations in the central nervous system are not well understood, particularly in brain regions that are uniquely human and thus inaccessible to established animal models. Cerebral organoids are pluripotent stem cell derived models of prenatal brain development that have been used to deepen our understanding of the atypical processes associated with human neurobiological disorders, and thus provide a promising avenue to explore the molecular basis for neurodevelopmental alterations in trisomy 21. Here, we employ high-resolution label-free mass spectrometry to map proteomic changes over the course of trisomy 21 cerebral organoid development, and evaluate the proteomic alterations in response to treatment with harmine, a small molecule inhibitor of the chromosome 21 encoded protein kinase DYRK1A. Our results reveal trisomy 21 specific dysregulation of networks associated with neurogenesis, axon guidance and extracellular matrix remodeling. We find significant overlap of these networks with previously identified dysregulated gene expression modules identified in trisomy 21 fetal brain tissue. We show that harmine leads to partial normalization of key regulators of cortical development, including WNT7A and the transcription factors TBR1, BCL11A, and POU3F2, pointing to a causative role for DYRK1A over-expression in neurodevelopmental effects of human trisomy 21. 4

ECE2 regulates neurogenesis and neuronal migration during human cortical development

“…To gain a deeper insight into the mechanisms underlying the observed phenotypes in in vivo and in vitro models, we performed whole‐proteome analysis of 60‐day‐old CTRL and ECE2 KO COs. Mass spectrometry‐based proteomics has recently been used to compare trisomy 21 patient and control‐derived COs and revealed (i) that the neurodevelopmental trajectory shown several times by scRNAseq is also recapitulated on the protein level in COs, (ii) a number of dysregulated proteins in trisomy 21 and (iii) the reversal of dysregulated proteins by drug treatment [preprint: ]. Here, quantitative proteome analysis of whole‐organoid lysate revealed a number of significantly up‐ and downregulated proteins upon ECE2 KO (Fig A and Dataset EV1).…”

Modeling neurodegenerative diseases with cerebral organoids and other three-dimensional culture systems: focus on Alzheimer’s disease

“…The Down Syndrome organoids exhibited dysregulation of neurogenesis, axon guidance and extracellular matrix remodeling. After the treatment with harmine, there was a partial restoration of key regulators of cortical development, transcription factors and normalization of ECM proteins [139].…”

“…In another study, the proteomic changes in Down Syndrome COs was measured before and after treatment with harmine, a β-carboline alkaloid and an inhibitor of chromosome 21 encoded protein kinase (dual specificity tyrosine-phosphorylation-regulated kinase 1A, DYRK1A) [139]. The Down Syndrome organoids exhibited dysregulation of neurogenesis, axon guidance and extracellular matrix remodeling.…”

Integrated transcriptome and proteome analysis reveals posttranscriptional regulation of ribosomal genes in human brain organoids

“…While previous studies have investigated posttranscriptional regulation of specific genes or the impact of loss of key RNA-binding proteins (RBPs) ( Hoye and Silver, 2021 ), proteome-scale analyses of gene regulation during human corticogenesis have been lacking. Currently available proteome data from hiPSC-derived neural progenitors and neurons ( Djuric et al, 2017 ; Varderidou-Minasian et al, 2020 ), cerebral organoids ( McClure-Begley et al, 2018 ; Melliou et al, 2022 ; Nascimento et al, 2019 ), and the fetal brain ( Djuric et al, 2017 ; Kim et al, 2014 ; Melliou et al, 2022 ) are an important step toward establishing a human neurodevelopmental proteomic repertoire. However, these studies suffer from some of the following limitations: (a) due to the inherent diversity of cell types present during corticogenesis, bulk tissue data do not provide cell type-specific information, (b) cell sorting strategies result in datasets that suffer from low number of successfully detected proteins, and (c) importantly, a direct comparison of the transcript and protein expression to understand posttranscriptional gene regulation has been missing.…”