脳のコレステロール代謝を調節する長鎖ノンコーディングRNAの機能解明

2019 Fiscal Year Annual Research Report

• Project/Area Number: 19F19386
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: SHIN JAE・WOO 国立研究開発法人理化学研究所, 生命医科学研究センター, チームリーダー (60553849)
• Co-Investigator(Kenkyū-buntansha): PRABHU ANIKA 国立研究開発法人理化学研究所, 生命医科学研究センター, 外国人特別研究員
• Project Period (FY): 2019-11-08 – 2022-03-31
• Keywords: iPSC to neuron / cholesterol modulation / neuron-astrocyte

Outline of Annual Research Achievements

The aim of this project is to investigate the functionality of non-coding RNAs, with a focus on long non-coding RNAs, in neuronal cholesterol metabolism. Thus far, we have established the model system to be utilized, a highly-scalable iPSC-derived neurons that can be co-cultured with human astrocytes and/or astrocyte-conditioned media. The iPSC differentiation process has been optimized in-house, and we have identified that cholesterol levels increase approximately 10-fold during this process. Cellular cholesterol levels at various stages of this differentiation are ready to be determined, which will allow us to identify the optimal time-points at which to analyze the transcriptomic changes that occur during the dramatic increase in cholesterol synthesis in neurons.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

We plan to use targeted qPCR to finalize the timepoints at which neurons will be analyzed by bulk CAGE sequencing, which will measure total RNA expression and map transcriptional start sites. In addition, small-scale tests with sterol modulators will be used to optimize treatment conditions, prior to sequencing. Genes involved in sterol regulation exhibit a particular transcriptomic pattern in response to sterol modulation. Analysis of the sequencing results will confirm this pattern in known sterol regulatory elements, and help identify novel candidates for further interrogation.

Strategy for Future Research Activity

In FY2020, we will finalize CAGE library production and sequencing. Due to COVID19, the sequencing will be significantly delayed. We ask for kind understanding. Once the data is generated, the top candidates identified will be validated and characterized using CRISPRi/sgRNAs against the lncRNAs of interest and systematically test the effect of lncRNA knockdown on cholesterol metabolism in neurons. This involves performing a longitudinal survival assay to determine the role of the lncRNAs on neuronal health, and measuring the effects of lncRNA knockdown on cholesterol synthesis, uptake, and trafficking. Focusing on a few relevant lncRNAs, we will identify the interactions formed by these lncRNAs through ChIRP-seq which allows the simultaneous profiling of binding proteins and chromatin interacting regions.