Browsing by Author "Johnson, Gretchen"
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Item CREB Activation by Mini-Chaperone CPP-P1 Enhances Retinal Ganglion Cell Survival in an Acute Glaucoma Model(2024-03-21) Johnson, Gretchen; Nagaraj, Ram; Stankowska, DorotaPurpose: Alpha-B crystallin is a heat shock protein that has been found to have anti-apoptotic properties and was used to design the novel mini-chaperone called peptain-1 (P1) conjugated with a cell-penetrating peptide (CPP), named CPP-P1. Transcriptomics of primary retinal ganglion cells (RGCs) isolated from adult rats subjected to ocular hypertension and treated with CPP-P1 revealed the activation of CREB signaling as a major pathway activated by the drug. Creb activation by phosphorylation (p-Creb) was previously confirmed in primary RGCs and tested here in a rat model of ocular hypertension. Methods: Adult male Brown Norway rats (N=15, 5 per group) were grouped into naïve, IOP-Vehicle, and IOP-CPP-P1 experimental groups. Silicone oil (20µl) was injected into the anterior chamber, and 2µl of either PBS (vehicle) or CPP-P1 (2µg/µl) was injected intravitreally. On day 7 of elevated IOP, rats were euthanized. Retinal sections obtained were stained with Creb, p-Creb, and DAPI and imaged at 4X for cell counts and at 20X for integrated density measurements. Unpaired t-tests or Mann-Whitney tests in GraphPad Prism were used to calculate statistical significance. Retinal punches from post-mortem human eyes (N=3) were cultured with PBS or CPP-P1 (12.5µg/ml) for 48 hours, and tissue RNA was collected for qPCR. Results: Ganglion cell layer counts obtained from rat retinal sections showed that the silicone oil injury group with no intervention (IOP-Veh) had a 51% decrease in ganglion cells compared to the naïve group (p<0.0001), and the silicone injury with CPP-P1 intervention group (IOP-CPP-P1) showed only a 23% decrease compared to the naïve group (p=0.0016). Integrated density measurements of Creb expression in IOP-Veh was 17% (p=0.615) higher than the naïve group, while in IOP-CPP-P1, it was 136% (p=0.092) higher than the naïve group. Expression of p-Creb showed a decrease in IOP-Veh by 50% (p=0.056) in comparison to the naïve group, while the IOP-CPP-P1 group was significantly higher than the IOP-Veh group (p=0.036). RNA expression of Creb1 in human retinal tissue was increased by 1.7-fold with CPP-P1 treatment. Discussion: IOP-mediated RGC damage was mitigated by CPP-P1 treatment, demonstrating neuroprotective effects when compared to vehicle-treated rats. CREB signaling contributes to CPP-P1-mediated neuroprotection during glaucomatous insults.Item Evaluation of Neuroprotective Effect of hybrid compound SA-2 Nanosuspension in Optic Nerve Crush Mouse Model.(2021) Amankwa, Charles E.; Gondi, Sudershan; Funk, Arlene; Debnath, Biddut; Zhang, Wei; Li, Linya; Ferguson, Jonathan; Johnson, Gretchen; Ellis, Dorette; Stankowska, Dorota; Acharya, SuchismitaThis study examines the cytoprotective effects of hybrid antioxidant-nitric oxide (NO) donating compound SA-2 nanosuspension in normal trabecular meshwork-5 cells (NTM-5) and optic nerve crush (ONC) induced-mouse retinal ganglion cell (RGC) death model. SA-2 was encapsulated in poly-lactic-co-glycolic acid (PLGA) nanoparticles and reported earlier. Total nitrite concentrations of SA-2-NPs was determined using Griess assay in both buffer and NTM-5 cell supernatants. NTM-5 cells were treated with tert-butyl hydrogen peroxide (TBHP, EC50 = 5.5mM) at varying concentrations of SA-2 NPs (1, 0.5, 0.25, 0.125 % w/v) and cell viability was measured. 12-week-old C57BL/6J mice were subjected to ONC injury in the left eye and were administered with either vehicle or 1% SA-2-NPs via intravitreal injections (2uL) or eye drops (5uL). Pattern ERG (PERG) was used to assess retinal function and RGC survival was determined using RBPMS-positive RGCs. Data were presented as mean ± SEM, n=3-8. 1% SA-2-NPs dose dependently increased NTM-5 cell viability and total nitrite concentrations (~50%) lasting over 4 days. There was significant improvement in PERG amplitudes (1.7-1.9 times) following SA-2-NP administration as well as increased RGC numbers (by ~20%) relative to ONC-ed eyes. SA-2 nanosuspension shows strong positive trend in protecting RGC's from oxidative stress-induced apoptosis in ONC rodent model. Further investigation is being done for improved dosing and signaling changes in response to SA-2 NPs therapy.Item Novel Compound SA-21 with Antioxidant Capability - The Prospect for Neuroprotection in Glaucoma.(2021) Ferguson, Jonathan; Johnson, Gretchen; Amankwa, Charles E.; Zhang, Wei; Li, Linya; Gondi, Sudershan; Funk, Arlene; Ellis, Dorette; Acharya, Suchismita; Stankowska, DorotaPurpose: Current treatments for glaucoma do not fully address neurodegeneration of retinal ganglion cells (RGCs). Our objective was to determine if compound SA-21, a hybrid superoxide dismutase and glutathione mimetic, could inhibit death of trabecular meshwork cells (NTM5), RGCs and rescue the functional decline of RGCs in an optic nerve crush (ONC) model. Methods: The structure of SA-21 was confirmed by magnetic resonance (NMR) spectroscopy and mass spectrometry. Reactive oxygen species (ROS) release was performed using pyrogallol assay. NTM5 cells were oxidatively stressed with TBHP (5.5mM) or vehicle in the presence of SA-21 (1mM, 100µM, 10µM, 1µM) for 24h. Cell survival was assessed by MTT assay. C57BL6 male mice (12-weeks old, n=4-5) were anesthetized, underwent ONC surgery, and at day 0 and 3 were intravitreally injected with 1% SA-21 (2µl) or vehicle. On day 7, pattern electroretinogram (PERG) was performed, animals were euthanized, and the number of surviving RBPMS-positive RGCs were counted. Results: SA-21 (1mM) treatment significantly decreased ROS production (~50%) measured by pyrogallol assay and increased NTM5 cell viability (~20%, p< 0.0094) following TBHP treatment compared to cells treated with the vehicle. ONC produced a 48% loss of RGCs, which was decreased in SA-21 treated mice (by ~10%) and demonstrated a trend in increase in PERG amplitude. Conclusions: SA-21 compound has antioxidant capability and protects NTM5 cells from oxidative stress. Intravitreally injected SA-21 at the selected dose in mice demonstrated trend in neuroprotection but further investigation is required.Item P1-CPP promotes Foxo1 and Creb signaling and reduces apoptosis in Neurotrophic Factor-Deprived Primary Retinal Ganglion Cells(2023) Johnson, Gretchen; Pham, Jennifer; Krishnamoorthy, Raghu; Nagaraj, Ram; Stankowska, DorotaPurpose: To elucidate the intracellular mechanisms underlying neuroprotective effects of the core peptide of a-B crystallin, peptain-1 (P1) conjugated to a cell-permeable peptide CPP (P1-CPP) in primary retinal ganglion cells (RGCs). Targets of the investigation were limited to Creb1, Bak1/Bad, and Foxo1, based upon RNA sequencing data obtained from RGCs of IOP-elevated rats treated with P1-CPP in comparison with the vehicle. Methods: Primary RGCs isolated from Sprague Dawley rat pups were deprived of neurotrophic factors (NT) namely, BDNF, CNTF, and Forskolin for 48 hours, either in the presence or absence of P1-CPP (4µM). After the treatments, RNA isolation was carried out using Trizol reagent. Subsequently, cDNA synthesis and qPCR analysis of the target genes expression, including Creb1 (n=2), Foxo1 (n=3), and Bak1 (n=3), was performed. Another set of RGCs subjected to the same treatments was fixed with 4% paraformaldehyde for 20 minutes and used for immunocytochemical analyses of p-CREB (n=3), FOXO1 (n=3), and BAD (n=3) protein expression. Immunostaining with an RBPMS antibody was used as an RGC marker. N indicates experimental repeats. Results: Following NT deprivation, there was an increase in mRNA expression of Creb1 (2-fold) in RGCs treated with P1-CPP, compared to the vehicle-treated RGCs. Moreover, the phosphorylated (active) form, p-CREB, was increased (by 102%; p=0.04) in primary RGCs treated with P1-CPP, compared to the vehicle-treated group. Pro-apoptotic Bak1 mRNA expression was not changed in the P1-CPP-treated RGCs compared to the vehicle-treated group. Primary RGCs stained for BAD protein showed a decrease (by 62%; p=0.08) in the P1-CPP treated group compared to the vehicle-treated RGCs. Foxo1 mRNA levels were increased by more than 2-fold in the P1-CPP treated RGCs, compared to the vehicle-treated RGCs. FOXO1 protein was also elevated in primary RGCs treated with P1-CPP compared to the vehicle group (by 59%). Conclusions: P1-CPP is neuroprotective against neurotrophic factor deprivation through multiple mechanisms, including early changes in the expression of mitochondrial homeostasis regulator Foxo1, activation of the pro-survival CREB pathway, and inhibition of pro-apoptotic members of the BCL-2 family of proteins.