Role of neuronal RNA granules in the etiology of neuropsychiatric disorders

2019 Fiscal Year Research-status Report

• Project/Area Number: 19K16273
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Krzyzanowski Marek 国立研究開発法人理化学研究所, 脳神経科学研究センター, 基礎科学特別研究員 (10837181)
• Project Period (FY): 2019-04-01 – 2023-03-31
• Keywords: RNA granules / translation / autism / brain / neuron

Outline of Annual Research Achievements

In order to explore the potential deregulation of neuronal RNA granules in neuropsychiatric disorders I established a biochemical method to purify RNA granules from mouse brains and cultured neurons. I confirmed the purity of the obtained granules from other cellular components and confirmed the presence of known RNA granule marker proteins (such as FMRP). Furthermore I analyzed the protein composition of the purified granules by quantitative mass spectrometry, confirming again the presence of marker proteins as well as identifying a dozen of other proteins enriched in RNA granules. I have compared the composition of brain RNA granules in wild-type and the neuropsychiatric disease model mice (such as Fmr1 KO). Preliminary results indicate that abundance of several proteins changes in RNA granules of the mutant mice (for example they are no longer enriched in the granules), making them potential targets for further investigation into the etiology of disease phenotypes.

Current Status of Research Progress

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

Reason

In the first year of the project I was able to finalize the development of the method to biochemically purify RNA granules from mouse brains. So far, the experiments with wild-type brains showed satisfactory reproducibility of the mass-spectrometry analysis of protein components. Furthermore I was able to obtain preliminary results of RNA sequencing. Finally I started to apply this methodology to disease model mice and cultured cortical neurons. Therefore the basic methodology needed to study RNA and protein composition of RNA granules has been established at this stage.

Strategy for Future Research Activity

Observed abundance changes of protein components already identified in last year's mass-spectrometry experiments will be tested by western-blotting. Furthermore, in the near future I will focus on obtaining more data on the changes to protein components of RNA granules occurring in neuropsychiatric model mice. In parallel, RNA-sequencing studies will be expanded to identify the disease-related changes to both regulatory RNAs and mRNA cargo.

The big part of next year's effort will be also to test how RNA granules change upon neuronal stimulation (in cortical neuron cultures), to have a full picture of RNA granule components and their mRNA cargo not only in steady state, but also in function of neuronal activity.