| 研究課題/領域番号 |
20K07458
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| 研究種目 |
基盤研究(C)
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| 配分区分 | 基金 |
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| 応募区分 | 一般 |
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| 審査区分 |
小区分49030:実験病理学関連
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| 研究機関 | 沖縄科学技術大学院大学 |
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研究代表者 |
TERENZIO Marco 沖縄科学技術大学院大学, 分子神経科学ユニット, 准教授 (60867513)
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| 研究期間 (年度) |
2020-04-01 – 2024-03-31
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| 研究課題ステータス |
交付 (2022年度)
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| 配分額 *注記 |
4,290千円 (直接経費: 3,300千円、間接経費: 990千円)
2022年度: 1,430千円 (直接経費: 1,100千円、間接経費: 330千円)
2021年度: 1,040千円 (直接経費: 800千円、間接経費: 240千円)
2020年度: 1,820千円 (直接経費: 1,400千円、間接経費: 420千円)
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| キーワード | ALS / biomarkers / IPSC / motor neurons / RNA sequencing / axonal translation / disease onset / protein translation / human IPSC / hIPSC / MNs |
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| 研究開始時の研究の概要 |
To overcome the limitations of murine models, we plan to directly derive motor neurons (MNs) from Amyotrophic Lateral Sclerosis (ALS) patient’s fibroblasts to assess changes in axonal local translation. Axonal degeneration is a primary pathological event determining the demise of MNs in ALS and defects of axonal translation contribute to the axonal pathology. We will investigate differences in axonal translation and test any abnormal protein expression as a possible new biomarker of disease and/or target of new therapies in biological fluids from well characterized cohorts of ALS patients.
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| 研究実績の概要 |
1) We finished the characterization of our motor neuron cultures from a library of 6 ALS and 4 healthy control human iPSC lines from Cedar Sinai Cell Bank for the occurrence of axonal degeneration, protein aggregation and neuronal cell death.
2) We looked at the role of mitochondria and ATP production in correlation with local translation and phase separation for some of the lines.
2) We collected cell pellets and culture medium from pre-symptomatic, early onset and late stage of disease motor neurons in culture.
3) We started to isolate extracellular vesicles, which will be subjected to proteomics and their content will be tested as biomarkers.
4) We started to optimize the proteomic analysis of the collected lysates for cell pellets and media.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
We had delays due to the extensive work required to characterize 10 different hiPSC lines. We extended the previous analysis to mitochondrial activity, ATP production and phase separation. We also started to work on extracellular vesicles isolation as a possible source of biomarkers. We determined the point of insurgence of toxicity for each line and we have produced samples for the planned proteomic analysis for biomarkers. Candidates from this analysis will be validated in 2023.
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| 今後の研究の推進方策 |
1. We will use the collected protein lysates and media from MNs derived by hIPSC to perform proteomic analysis to identify new biomarkers of ALS progression and/or characterize the axonal translatome.
2. MN-muscle co-cultures in MFCs will be used to validate candidates from the proteomic screen and specifically newly axonally synthesized proteins, which will be labeled by the incorporation of puromycin. We will compare healthy MN cultures with cultures of MNs from ALS patients. Candidates will then be tested for their impact on known physiological hallmark of ALS (i.e. axonal transport, MN survival).
3. We will consider testing newly identified biomarkers in spinal fluids of ALS patients.
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