2022 Fiscal Year Research-status Report
Axonal local translation and its implications in the pathogenesis of amyotrophic lateral sclerosis
| Project/Area Number |
20K07458
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
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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| Project Period (FY) |
2020-04-01 – 2024-03-31
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| Keywords | ALS / biomarkers / IPSC / motor neurons / RNA sequencing / axonal translation / disease onset |
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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
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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| Strategy for Future Research Activity |
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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| Causes of Carryover |
The money will be used for the planned proteomic analysis for axonally translated proteins and disease progression biomarkers. In addition, the money will be spent in consumables necessary for the candidate validation of the proteomic screen.
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