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Path from schizophrenia genomics to biology: gene regulation and perturbation in neurons derived from induced pluripotent stem cells and genome editing.

ABSTRACT: Schizophrenia (SZ) is a devastating mental disorder afflicting 1% of the population. Recent genome-wide association studies (GWASs) of SZ have identified >100 risk loci. However, the causal variants/genes and the causal mechanisms remain largely unknown, which hinders the translation of GWAS findings into disease biology and drug targets. Most risk variants are noncoding, thus likely regulate gene expression. A major mechanism of transcriptional regulation is chromatin remodeling, and open chromatin is a versatile predictor of regulatory sequences. MicroRNA-mediated post-transcriptional regulation plays an important role in SZ pathogenesis. Neurons differentiated from patient-specific induced pluripotent stem cells (iPSCs) provide an experimental model to characterize the genetic perturbation of regulatory variants that are often specific to cell type and/or developmental stage. The emerging genome-editing technology enables the creation of isogenic iPSCs and neurons to efficiently characterize the effects of SZ-associated regulatory variants on SZ-relevant molecular and cellular phenotypes involving dopaminergic, glutamatergic, and GABAergic neurotransmissions. SZ GWAS findings equipped with the emerging functional genomics approaches provide an unprecedented opportunity for understanding new disease biology and identifying novel drug targets.

SUBMITTER: Duan J

PROVIDER: S-EPMC4332546 | biostudies-other | 2015 Feb

REPOSITORIES: biostudies-other

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Project description:The gain-of-function mutation of c.G6055A (p.G2019S) in Leucine-rich repeat kinase 2 (LRRK2) gene is the most prevalent genetic cause of Parkinson’s disease (PD). Although clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has been used to generate isogenic control lines, there are risks of creating indels and genomic instability together with a lower efficiency in non-dividing cells. The recent advance of adenine base editors (ABEs) could convert targeted A•T base pairs to G•C base pairs without double-strand DNA breaks or donor DNA templates and function in post-mitotic cells. Here, we demonstrate a complete correction of an induced pluripotent stem cell (iPSC) line derived from a PD patient having LRRK2 p.G2019S mutation using variable genome editing methods, including CRISPR/Cas9-based homology-directed repair (HDR) and ABEs. The corrected isogenic iPSCs derived dopaminergic neurons showed a down-regulated LRRK2 kinase activity, decreased phospho-a-synuclein accumulations, reduced apoptosis and restored neurite shrinkage phenotypes. The mutation correction efficacy, off-target and indels rates between CRISPR/Cas9-HDR and ABE were compared. Among the 47 clones generated by HDR, 3 clones (6.4%) were on-targeted corrected, while ABE showed a much higher correction rate (13 of 53 clones, 24.5%). Whole genome sequencing analysis revealed that there are 27 clones of HDR (57.4%) having deletions but none in clones of ABE, albeit ABE created 14 clones (26.4%) having off-target missense mutations. RNA sequencing and proteomic analysis of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of PD relevant pathways, including calcium ion dependent exocytosis, synaptic transports well as potential novel targets relevant to PD pathophysiology. These results envision that ABE could directly correct the pathogenic PD mutation in iPSCs for exploring the earliest events in PD pathophysiology and providing dopaminergic neurons for future cell therapies.

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Path from schizophrenia genomics to biology: gene regulation and perturbation in neurons derived from induced pluripotent stem cells and genome editing.

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