2021 Fiscal Year Research-status Report
モデルマウスを用いた、正常眼圧緑内障家系で同定された新規原因遺伝子Xの機能解析
| Project/Area Number |
20K18366
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| Research Institution | 独立行政法人国立病院機構(東京医療センター臨床研究センター) |
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Principal Investigator |
潘 洋 独立行政法人国立病院機構(東京医療センター臨床研究センター), 分子細胞生物学研究部, 研究員 (20866389)
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| Project Period (FY) |
2020-04-01 – 2023-03-31
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| Keywords | Normal-tension glaucoma / splicing / causative gene |
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| Outline of Annual Research Achievements |
We identified a novel causative mutation of NTG in gene X by performing WES. Moreover, this novel mutation exhibits the gain of aberrant mRNA splicing that leads to retention of intros or skipping exon and eliminates full-length protein using several different cell culture models, including patient-derived iPSCs. The aberrantly spliced variants affected protein production and altered subcellular localization. Furthermore, we developed and characterized the knock-in and knockout mouse models. Both knock-in and knockout mice had decreased RGCs and retinal thickness. Last, we differentiated iPSCs into RGCs by inhibiting SMAD and Wnt pathways. The mutational effects of the novel gene were analyzed in purified iPSC-RGCs.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
1. We demonstrated this novel mutation exhibits the gain of aberrant mRNA splicing that leads to retention of intros or skipping exon and eliminates full-length protein using several different cell culture models, including patient-derived iPSCs.
2. The aberrantly spliced variants affected protein production and altered sub-cellular localization in three cell lines.
3. To gain further insight into the function of mutation, we analyzed GC complex thickness, disc & cup volume and number of GC in knock-in and knockout mice. B-circular and B-horizontal of OCT scan revealed that the thickness of RGC fiber layer around optic nerve head (ONH) significantly reduced and disc & cup volume increased dramatically in both knock-in and knockout mice compared with controls in 4-month and 6-month of age.
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| Strategy for Future Research Activity |
1. Eases the effects of the novel mutation on RGCs. We have documented decreased ganglion cell complex thickness and increased optic cup volume in knock-in and knockout mice. the effects of this mutation on RGCs will be confirm by robust retinal ganglion cell counts and RGC axon counts within the optic nerve.
robust retinal ganglion cell counts using flat mounts and PPD staining of retrobulbar optic nerve.
2. To strengthen the characterization of the knock-in and knockout mice model, the functional measure of RGC activity, such as pSTR, will be performed.
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| Causes of Carryover |
iPSC-RGCs differentiation proceeded through two phases: PRC induction and differentiation; RGC initiation and differentiation. Because RPCs are multipotent cells, they have the potential to differentiate into neuronal cell types and one glial cell type called the Muller glial cells. Therefore, we need to purify iPSC-RGCs by Thy1 cell surface marker using CD90.2 microbeads.
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[Presentation] The ARMS2 insertion/deletion leads to increased circulating HTRA1 in AMD patients and in vitro2021
Author(s)
Yang Pan, Daisuke Iejima, Mao Nakayama, Akiko Suga, Toru Noda, Inderjeet Kaur, Taraprasad Das, Subhabrata Chakrabarti, Robyn H. Guymer, Margaret M. DeAngelis, Paul N. Baird, Takeshi Iwata
Organizer
第14回RRM
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