Temporal Single Cell RNAseq to Identify Genes and Pathways Affected by 15q11.2 Duplication in Autism iPSC-Derived Differentiating Cortical Neurons

点击翻译按钮获取中文摘要

PROJECT SUMMARY We aim to establish a single-cell RNAseq database of differentiating cortical neuronal progenitors (NPCs) and neurons derived from patient-specific induced pluripotent stem cells (iPSCs) for neuro- developmental diseases (NDDs). The database will also include data on differentiation, morphology, formation and functionality of synapses, annotations of analytical findings, and will be supported by the most advanced single-cell RNAseq bioinformatics tools. We will provide all this, together with corresponding NPCs and neurons for research reagents, as tool to support academic research and drug discovery for NDDs in the field. One in 68 children born in 2002 are diagnosed with Autism Spectrum Disorder (ASD), a public health priority in the US. Genetic predispositions in ASD are thought to contribute to the primary pathology by altering neuronal development, evidenced partly by altered gene expressions. Patient-specific induced pluripotent stem cells (iPSCs) have been shown to recapitulate specific disease phenotypes through the neurogenic process and can serve as effective disease models. Hence, single-cell RNAseq along the accessible and controlled process of differentiating ASD-specific iPSCs into neural progenitors and subtypes of neurons can provide insights into the temporal and multi-lineage dimensions of ASD pathogenesis and biomarkers for diagnostics, progression, and therapeutics discovery. Copy-number variants (CNVS) at 15q11.2 is a prominent risk factor for neurological disorders including ASD, epilepsy, and schizophrenia. 15q11-q13 duplication/triplication represent the most common CNVs in patients with ASD (up to 3%). Duplications and microdeletions can both lead to the same disorders, suggesting the importance of this region in normal neurological functions and the necessity to study the impact of both duplications and deletions for a full understanding of the mechanisms. Phase I utilizes four 15q11.2 duplication iPSC lines from ASD patients and two control iPSC lines to: Aim 1: Characterize neural differentiation of ASD and control iPSC-derived neurons. Differentiate iPSCs into cortical NPCs, glutamatergic, and GABAergic subtypes. Identify deficits in ASD lines through morphological studies and structural analyses of synapses. Aim 2: Generate single-cell RNAseq datasets of differentiating NPCs and neuronal subtypes at nine time points during differentiation. Aim 3: Perform bioinformatics analyses. Reconstitute the molecular dynamics underlying neuronal differentiation. Validate experimental conditions including sampling frequency and number of cells. Identify, validate molecular signatures underlying 15q11.2 duplication’s impact on neuronal differentiation and functions. Phase II: utilize the experimental conditions established here to generate single-cell RNAseq datasets from multiple ASD iPSC lines that harbor different genetic mutations to be included in the database; build database structure and user interface, and seek to identify aberrant differentiation and functional development caused by ASD mutations as well as genes and pathways that are commonly and differentially affected across multiple ASD mutations. We will devote resources to annotate the database with our own findings and those that are published by peers to enhance its utility to subscribers.