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Effect of zinc chelator TPEN on gene expression of retinal ganglion cells during optic nerve injury

ABSTRACT: Retinal ganglion cells (RGCs) convey the major output of information collected from the eye to the brain. RGCs are irreversibly lost when injured in degenerative diseases such as glaucoma; this failure can be partially reversed by eliminating the retinal mobile zinc (Zn2+) and leads to substantial axon regeneration. Here, we conducted an mRNA sequencing of flow cytometry-isolated RGCs 3 days after optic nerve injury (NC) with or without TPEN treatment. We identified differentially expressed genes using a cutoff of at least 1.5-fold change and FDR of at least 0.05. We focused on the intersecting genes that were upregulated after NC and recovered in TPEN (TPEN/NC; fold change < -1.5; P < 0.05), namely, TPEN target genes.

ORGANISM(S): Mus musculus

PROVIDER: GSE208777 | GEO | 2022/11/02

REPOSITORIES: GEO

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Project description:Glaucoma is a group of optic neuropathies characterized by the progressive degeneration of retinal ganglion cells (RGCs) as well as their axons leading to irreversible loss of sight. Medical management of the intraocular pressure (IOP) still represents the gold standard in glaucoma therapy, which only manages a risk factor and does not directly address the neurodegenerative component of this eye disease. Recently, our group showed that antibody derived immunopeptides (encoding complementarity-determining regions, CDRs) provide attractive glaucoma medication candidates and directly interfere its pathogenic mechanisms by different modes of action. In accordance with these findings, the present study showed the synthetic CDR2 peptide (INSDGSSTSYADSVK) significantly increased the RGCs viability in vitro in a concentration-dependent manner (p < 0.05 using a CDR2 concentration of 50 µg/mL). Employing state-of the-art immunoprecipitation experiments, we confirmed that synthetic CDR2 exhibited a high affinity towards the retinal target protein histone H3.1 (HIST1H3A) (p < 0.001 and log2 fold change > 3). Furthermore, virtual docking analyses predicted potential CDR2-specific binding regions of HIST1H3A, which might represent essential PTM sites for epigenetic regulations. Quantitative MS analysis revealed that 39 proteins were significantly changed between CDR2-treated and untreated control retinae (p < 0.05). An up-regulation of proteins involved in the energy production (e.g., ATP5F1B and MT-CO2) as well as the regulatory ubiquitin proteasome system (e.g., PSMC5) was induced by the synthetic CDR2 peptide. On the other hand, metabolic key enzymes (e.g., DDAH1 and MAOB) as well as ER stress-related proteins (e.g., SEC22B and VCP) were found with low abundancy in CDR2-treated retinae and were also partially confirmed by microarray technology. Based on these findings, it can be hypothesized that the specific protein-peptide interaction influences the regulatory epigenetic function of HIST1H3A promoting the neuroprotective mechanism on RGCs in vitro. It also might serve as basis for a synergistic immunotherapy of glaucoma in the future.

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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Effect of zinc chelator TPEN on gene expression of retinal ganglion cells during optic nerve injury

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