2021 Fiscal Year Research-status Report
Axonal local translation and its implications in the pathogenesis of amyotrophic lateral sclerosis
| Project/Area Number |
20K07458
|
|---|
| Research Institution | Okinawa Institute of Science and Technology Graduate University |
|---|
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)
|
|---|
| Project Period (FY) |
2020-04-01 – 2023-03-31
|
| Keywords | ALS / biomarkers / IPSC / motor neurons / RNA sequencing / axonal translation / disease onset |
|---|
| Outline of Annual Research Achievements |
We aim to identify biomarkers of and the contribution of local translation in Amyotrophic Lateral Sclerosis (ALS) by deriving motor neurons (MNs) from ALS patient’s human pluripotent stem cells (iPSC).
1) We acquired several commercially available iPSC lines from healthy individuals and ALS patients. We had to maintain each line and improve upon our differentiation protocol, which now provides a very high yield of MNs (over 90%).
2) We characterized the onset of disease for each MN line.
3) We have developed MNs cultures on Boyden chambers to mechanically isolate and separate the axonal compartment from neuronal soma. This allows us to collect specifically axonal RNAs for qPCR and RNAseq. We are currently optimizing these experiments to improve separation of the 2 compartments and increase purity of axonal RNA preparation.
4) We have collected samples from all our ALS patient lines at various stages of maturation in vitro and we are constructing peptide libraries to be able to perform biomarker identification via Mass Spectrometry.
|
| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
We have acquired and characterized 6 iPSC lines from ALS patients and 4 control lines. We started collecting samples for biomarker identification and RNA sequencing. We will perform both screening and validation in 2022 and 2023.
|
| Strategy for Future Research Activity |
1 We have collected samples from conditioned media of human MNs cultures from 6 ALS lines and 4 control lines. We will construct a peptide library and perform proteomic analysis with the aim of identifying secreted proteins in the medium, which could be used as biomarkers for ALS in the clinic.
2. We are collecting RNA from human MNs cultured in Boyden compartmentalized chambers. We will look at the axonal mRNAs in ALS lines vs control lines and candidates of interest emerging from this analysis will then be tested to verify axonal local translation and impact on known physiological hallmark of ALS (i.e. axonal transport, MN survival).
3 We will look at the effect of TDP43 aggregation and phase separation on axonal translation using live imaging and a genetically tagged TDP43 to visualize aggregation. Translation will be detected via puromycin incorporation.
|
| Causes of Carryover |
We have obtained the human IPSCs to use for the proteomic screen. Given the number of different lines in our possession and the length of the differentiation protocol, we have yet not been able to perform the proteomic analysis. Thus, the money carried over to FY2021 will be spent to outsource proteomic analysis for axonally translated proteins and disease progression biomarkers.
|
