Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/30810
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Browsing Eye / Vision by Author "Johnson, Gretchen A."
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Item Mechanisms of peptain-mediated neuroprotection in retinal ganglion cells(2022) Johnson, Gretchen A.; Pham, Jennifer; Kodati, Bindu; Krishnamoorthy, Raghu; Nagaraj, Ram; Stankowska, DorotaPURPOSE: To determine mechanisms underlying neuroprotective effects of the core peptide of alpha-B crystallin, peptain-1 (P1) conjugated to a cell-permeable peptide CPP (P1-CPP) in retinal ganglion cells (RGCs) in a rodent model of glaucoma. METHODS: Intraocular pressure (IOP) was elevated in Brown Norway (BN) rats and intravitreally injected with 2 µl of either P1-CPP or vehicle, once a week for a period of 2 weeks. Rats were euthanized, primary adult RGCs were isolated by the immunopanning method. Total RNA was isolated using the Trizol/column method. RNA-sequencing was performed using an Illumina platform. The resulting FASTQ files were uploaded into Galaxy for analysis with FASTQC, RNASTAR, feature counts, and finally DESeq2. The results from DESeq2 were then assessed with Qiagen's Ingenuity Pathway Analysis (IPA) to identify significantly upregulated pathways. Relative Creb-1 expression normalized to reference gene GAPDH was determined in IOP-P1-CPP and IOP-vehicle treated rat RGCs. Briefly, quantitative Polymerase Chain Reaction (qPCR) was performed using BioRad's PrimePCR Assay and SsoAdvanced Universal SYBR Green Supermix on the BioRad's CFX96 Real-Time System C1000 Touch Thermal Cycler. RESULTS: RNA-seq analysis from rat RGCs isolated following 2 weeks of IOP-elevation revealed that P1-CPP treated groups had several differentially expressed (DEGs), compared to vehicle-treated groups, including 6343 significantly upregulated and 5960 significantly downregulated. Some significantly upregulated pathways following P1-CPP treatment include phagosome formation, synaptic long-term depression, and CREB signaling in neurons. The IOP and vehicle-treated groups, when compared to the naïve group, demonstrated a decreased expression of members of the CREB signaling pathway (Creb-1, c-RAF, MEK1/2, ERK1/2, and p90RSK). This decline was prevented by P1-CPP treatment. Quantitative PCR further confirmed the RNA-seq findings of the increased expression of Creb-1 in P1-CPP treated rats compared to that of vehicle-treated group. CONCLUSIONS: Mechanism of action of P1-CPP in a rodent model of glaucoma includes the activation of the pro-survival CREB signaling pathway, phagosome formation, and long-term synaptic depression to prevent cell death and vision loss.Item Neuroprotection of human and rodent retinal ganglion cells by a hybrid antioxidant-nitric oxide donor small molecule, SA-2(2022) Pham, Jennifer; Johnson, Gretchen A.; Acharya, Suchismita; Stankowska, DorotaPURPOSE: Current treatments of glaucoma are aimed at lowering intraocular pressure (IOP), which is a key driver of retinal ganglion cell (RGC) death. Another contributing factor to RGC death is exposure to reactive oxygen species (ROS). At present, there is no FDA-approved neuroprotective treatment to prevent glaucomatous optic neuropathy and loss of RGCs. Our novel hybrid molecule, SA-2, contains both a nitric oxide (NO) donating group to lower IOP and a ROS scavenging group to protect RGCs. We hypothesize that SA-2 will inhibit the death of RGCs in an in vitro and an ex vivo neurotrophic factor deprivation model. METHODS: Retinal punches from human explants (n=4 donors/experiments) were isolated and treated with either SA-2 [1 mM] or vehicle and maintained without neurotrophic factors for 7 days ex vivo. In each experiment, 4 baseline retinal explants were collected on day 0. At the end of the experiment, explants were immunostained with RBPMS and Brn-3a (RGC-specific markers) and cell survival was analyzed. In three biological replicates, primary RGCs were isolated from rat pups and treated with either SA-2 (1 mM, 100 µM) or vehicle with or without neurotrophic factors for 48 h. Active caspase 3 and 7 assay was performed and apoptotic cell counts were analyzed. In another set of experiments, rat retinal explants were isolated and incubated with tert-Butyl hydroperoxide (TBHP) along with either SA-2 [1 mM] or vehicle for 2 h (n=2-4 explants/group). Production of superoxide by mitochondria was assessed using MitoSOX reagent according to manufacturer instructions. All cell counts were performed in a masked manner using ImageJ Software. One-way ANOVA or nonparametric Kruskal-Wallis was used for statistical analysis by GraphPad Prism 9 Software. RESULTS: In ex vivo human retinal explants, there was a significant increase in RGC survival by 39% in the SA-2 treated group compared to the vehicle group at day 7 (p< 0.0001). In rodent primary RGCs, SA-2 mediated a significant decrease in apoptotic cells by 30% (p< 0.01) and a 67% (p< 0.05) decrease in dead cell count. In rodent retinal explants, there was a significant decrease (by 59%, p< 0.0001) in the production of superoxide by mitochondria in the TBHP and SA-2 treated group, compared to the TBHP vehicle group. CONCLUSION: SA-2 was shown to be effective at preserving retinal ganglion cell survival in human retinal explants, rat retinal explants and primary rat RGCs by preventing apoptosis and protecting the cells from oxidative stress.