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Reassessing in vivo conversion of Müller cells to retinal ganglion cells

ABSTRACT: The conversion of Müller cells (MGs) to retinal ganglion cells (RGCs) in vivo is a potential regeneration-based therapeutic strategy for optical nerve injury. It has been reported that Ptbp1 knockdown can efficiently convert MGs into RGCs. However, another study reported that mislabeling could have yielded a possible false positive. No previous studies have examined gene expression at single-cell resolution after Ptbp1 knockdown in MGs. Here, using CasRx-mediated knockdown of Ptbp1 in optic nerve crush model mice, we observed MG-to-RGC conversion in vivo and found that this conversion attenuated the symptoms of visual impairment caused by the loss of RGCs. ScRNA-seq revealed that a cluster of retinal progenitor cells (RPCs) underwent MG-to-RGC conversion after Ptbp1 knockdown. Our study confirmed the presence of intermediate cells during the MG-to-RGC conversion process after Ptbp1 knockdown and discovered transcription regulators including Nfix, Nfia, Crx, Rax, and Neurod1 are functionally involved in regulating this process.

ORGANISM(S): Mus musculus

PROVIDER: GSE208428 | GEO | 2025/07/16

REPOSITORIES: GEO

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Project description:To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested at 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (p<0.05, FDR<1%) were quantified by SWATH-MS. Bioinformatics data analysis showed that there were 35 up-regulated and 24 down-regulated proteins in the temporal quadrant while 19 and 5 proteins were up-regulated and down-regulated, respectively, in the nasal quadrant respectively (n=4, p<0.05; fold change ≥1.4 fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants including CLU, GFAP, GNG5, IRF2BPL, L1CAM and CPLX1. Linear regression analysis indicated a strong association between the data obtained by SWATH-MS and MRM-MS (temporal, R2=0.97; nasal, R2=0.96). Gene ontology analysis reveals statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.

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Reassessing in vivo conversion of Müller cells to retinal ganglion cells

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