Browsing by Author "Nagaraj, Ram"
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Item An Antiapoptotic Peptide for Neuroprotection in Glaucoma(2017-03-14) Krishnamoorthy, Raghu R.; Sampathkumar, Sruthi; Nagaraj, Ram; Stankowska, Dorota L.Purpose: Axonal degeneration and death of retinal ganglion cells (RGC) are primary contributors to vision loss in glaucoma. The purpose of this study was to determine if intraperitoneal administration of the core peptide derived from small heat shock protein αB-crystallin (ABCP) could inhibit RGC death in animal models of glaucoma. Materials and Methods: Brown Norway rats were retrogradely labeled (to detect RGCs) using Fluoro-gold and IOP was elevated (150 mmHg/days) in one eye using the Morrison’s method, while the contralateral eye served as control. The rats were intraperitoneally injected with 10μg of ABCP (n=3 animals per group) three times per week for five weeks. Surviving RGCs were counted in retinal flat mounts. In another model of ischemia reperfusion (I/R) injury, C57BL/6 mice were subjected to IOP elevation of 120 mmHg for 30 min, followed by rapid reperfusion. Intraperitoneal ABCP injections were given 3h before and immediately after the procedure and then once daily post I/R injury for 14 days. RGC apoptosis was assessed using a TUNEL assay (n=2 animals per group). Results: Intraperitoneal injections of ABCP significantly (p Conclusions: Intraperitoneally administered ABCP peptide was able to significantly attenuate RGC death in two animal models of glaucoma. These findings suggest that ABCP has the potential to be developed as a neuroprotective agent in glaucoma.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 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 Novel Peptain for Neuroprotection in Glaucoma(2020) Stankowska, Dorota; Krishnamoorthy, Vignesh; Krishnamoorthy, Raghu; Chaphalkar, Renuka; Nagaraj, Ram; Nahomi, Rooban; Nam, Mi-hyun; Beall, Kallen; Brodrick, Ashley; Kodati, BinduPurpose: To determine if intravitreal administration of the core peptide of [alpha]-B crystallin, peptain-1 (P1) conjugated to a cell permeable peptide (CPP) (named P1-CPP) could inhibit retinal ganglion cell (RGC) death and functional decline in a rodent model of glaucoma. Methods: Primary RGCs were treated with endothelin-3 (ET-3) in the presence of either P1-CPP (12.5 µg/ml) or vehicle, following which RGC survival was assessed. In a different set of experiments, intraocular pressure (IOP) was elevated in one eye of Brown Norway (BN) rats and intravitreally injected with 2 µl of either P1-CPP or vehicle, once in a week for 6 weeks. RGC function was assessed by the pattern electroretinogram (PERG) amplitude. Retinal flat mounts were imaged and surviving RGCs were counted. Results: ET-3 treatment lead to 24% of RGCs loss compared to vehicle treated cells (p< 0.0001). P1-CPP treatment significantly lowered the ET-3-mediated cell loss (7% cell death, p< 0.001). IOP elevation in vehicle injected animals produced 11% and 27% loss of RGCs, in central and peripheral retina respectively, which was significantly lower in P1-CPP treated rats (7% loss in both eccentricities, **p< 0.01). P1-CPP treatment also promoted axonal protection during IOP elevation. IOP elevation caused 63% decline in the PERG amplitude (*p< 0.03) in comparison with naïve rats, which was sustained by P1-CPP treatment. Conclusion:The intravitreally injected P1-CPP provides cellular as well as functional protection of RGCs, which could facilitate neuroprotection against glaucomatous insults.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.