Browsing 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 Modulating mitochondrial calcium channels (TRPM2/MCU/NCX) as a therapeutic strategy for neurodegenerative disorders(Frontiers Media S.A., 2023-11-06) Johnson, Gretchen A.; Krishnamoorthy, Raghu R.; Stankowska, Dorota L.Efficient cellular communication is essential for the brain to regulate diverse functions like muscle contractions, memory formation and recall, decision-making, and task execution. This communication is facilitated by rapid signaling through electrical and chemical messengers, including voltage-gated ion channels and neurotransmitters. These messengers elicit broad responses by propagating action potentials and mediating synaptic transmission. Calcium influx and efflux are essential for releasing neurotransmitters and regulating synaptic transmission. Mitochondria, which are involved in oxidative phosphorylation, and the energy generation process, also interact with the endoplasmic reticulum to store and regulate cytoplasmic calcium levels. The number, morphology, and distribution of mitochondria in different cell types vary based on energy demands. Mitochondrial damage can cause excess reactive oxygen species (ROS) generation. Mitophagy is a selective process that targets and degrades damaged mitochondria via autophagosome-lysosome fusion. Defects in mitophagy can lead to a buildup of ROS and cell death. Numerous studies have attempted to characterize the relationship between mitochondrial dysfunction and calcium dysregulation in neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Amyotrophic lateral sclerosis, spinocerebellar ataxia, and aging. Interventional strategies to reduce mitochondrial damage and accumulation could serve as a therapeutic target, but further research is needed to unravel this potential. This review offers an overview of calcium signaling related to mitochondria in various neuronal cells. It critically examines recent findings, exploring the potential roles that mitochondrial dysfunction might play in multiple neurodegenerative diseases and aging. Furthermore, the review identifies existing gaps in knowledge to guide the direction of future research.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.Item Neuroprotection of Rodent and Human Retinal Ganglion Cells In Vitro/Ex Vivo by the Hybrid Small Molecule SA-2(MDPI, 2022-12-12) Pham, Jennifer H.; Johnson, Gretchen A.; Rangan, Rajiv S.; Amankwa, Charles E.; Acharya, Suchismita; Stankowska, Dorota L.The mechanisms underlying the neuroprotective effects of the hybrid antioxidant-nitric oxide donating compound SA-2 in retinal ganglion cell (RGC) degeneration models were evaluated. The in vitro trophic factor (TF) deprivation model in primary rat RGCs and ex vivo human retinal explants were used to mimic glaucomatous neurodegeneration. Cell survival was assessed after treatment with vehicle or SA-2. In separate experiments, tert-Butyl hydroperoxide (TBHP) and endothelin-3 (ET-3) were used in ex vivo rat retinal explants and primary rat RGCs, respectively, to induce oxidative damage. Mitochondrial and intracellular reactive oxygen species (ROS) were assessed following treatments. In the TF deprivation model, SA-2 treatment produced a significant decrease in apoptotic and dead cell counts in primary RGCs and a significant increase in RGC survival in ex vivo human retinal explants. In the oxidative stress-induced models, a significant decrease in the production of ROS was observed in the SA-2-treated group compared to the vehicle-treated group. Compound SA-2 was neuroprotective against various glaucomatous insults in the rat and human RGCs by reducing apoptosis and decreasing ROS levels. Amelioration of mitochondrial and cellular oxidative stress by SA-2 may be a potential therapeutic strategy for preventing neurodegeneration in glaucomatous RGCs.Item Neuroprotection of Rodent Retinal Ganglion Cells using Hybrid Molecule SA-10(2023) Pham, Jennifer H.; Kodati, Bindu; Johnson, Gretchen A.; Acharya, Suchismita; Stankowska, Dorota L.Purpose: Oxidative stress is the imbalance between the activity of antioxidants and free radical production, which has been shown to be associated with glaucomatous retinal ganglion cell (RGC) degeneration. In this study, we aimed to promote RGC survival by treatment with SA-10, a second-generation hybrid molecule with nitric oxide donating and sulfone reactive oxygen species (ROS) scavenging moieties in vitro and ex vivo following oxidative stress-induced injury. Methods: Endothelin-3, a vasoactive peptide, was used to induce oxidative stress in vitro in rat primary RGCs (n=3 biological replicates) and ex vivo in C57BL/6J mice retinal explants (n=8-9 explants/group). Primary RGCs were isolated from Sprague Dawley rat pups (post-natal days 4-7) and cultured for seven days with neurotrophic factors to allow for neurite outgrowth. The RGCs and retinal explants were pretreated with vehicle (DPBS) or SA-10 [10 µM] for 30 minutes, following which ET-3 treatment [100 nM or 400 nM] was carried out for 1 hour. CellROX™ Green was then used to stain for ROS produced by the cells, and the integrated density was analyzed. Analysis of Variance (ANOVA) or nonparametric Kruskal-Wallis was performed for all experiments. Results: In primary RGCs, ET-3-mediated ROS production decreased by 25.9% (p<0.01) following SA-10 treatment compared to the vehicle. In mice, retinal explants, 400 nM ET-3 induced a 24.4% increase in ROS production compared to the vehicle [0 nM ET-3]. With the SA-10 treatment, the ROS production was decreased by 14.74% (p<0.001) in the ET-3 and SA-10 treated group compared to the ET-3-only treated group. Conclusion: SA-10 effectively protects rodent RGCs in vitro and ex vivo from ET-3-mediated oxidative stress.Item Optimization and Evaluation of qPCR Duplex Assay for mtDNA Copy Number Quantification(2020-05) Johnson, Gretchen A.; Planz, John V.; Phillips, Nicole R.; Zascavage, Roxanne R.Purpose: The mitochondrial genome (mtDNA) encodes thirteen essential proteins in oxidative phosphorylation, the cell's primary energy-generating process. Depending on the cell type and stage of development, each cell contains an average of 103 to 104 copies of mtDNA. Current methods of quantification of mtDNA copy number can be imprecise due to low efficiencies of assays and inherent imbalance of mtDNA copy number with nuclear DNA (nDNA) copy number. Accurate quantification of both mtDNA and nDNA is important when calculating the ratio of mtDNA to nDNA. The goal of this project is to optimize a duplex assay that will give precise and accurate estimates in human samples. Methods: Here we employ synthetic oligomer standards for an absolute real-time qPCR assay. The significance of using absolute qPCR is that the standard curve allows for the direct comparison of unknowns to obtain a copy number. The mitochondrial target is a site in the minor arc (MinArc), and the nuclear target is a single copy locus ([beta]2M). The accuracy of this assay was evaluated using a standard reference material (SRM2372a) and the precision was evaluated via replications. Results: This design resulted in high R2 values for the standards as well as sufficiently high efficiencies. The precision of the assay was analyzed over 6 replicated runs and was deemed effectively reproducible. The accuracy was assessed with the use of a standard reference material (SRM 2372a) and was found to be problematic [Romsos et al., 2018]. This could be from a possible dilution bias of the SRM, effectively changing the copy number ratios in a difficult to predict way [Malik et al., 2011]. An attempt to mathematically correct the data was made but did not provide any solution. Conclusion: The optimization of this assay is ongoing due to the error in accuracy. The assay has proven to be precise and reproducible with sufficient efficiency. Possible future directions include sonication of samples and SRMs to examine if dilution bias could be the cause of inaccurate SRM quantification. Other methods of possibly reducing dilution bias mentioned in Malik et al. [2011] include manual shearing and the use of DNA carriers such as tRNA. Another avenue of future research could include a different method of mathematically correcting the data post run to improve accuracy. This assay has the potential to provide data which can be used to indicate overall mitochondrial health and can be utilized in various research areas such as aging, cancer, forensics and neurodevelopment.Item Optimization and Evaluation of qPCR Duplex Assay for mtDNA Copy Number Quantification(2020) Phillips, Nicole; Planz, John; Johnson, Gretchen A.Purpose: The mitochondrial genome (mtDNA) encodes thirteen essential proteins for oxidative phosphorylation. Depending on the cell type and stage of development, each cell contains an average of 10^3 to 10^4 copies of mtDNA. Current methods of mtDNA copy number quantification can be imprecise due to low efficiencies of assays and inherent imbalance of mtDNA copy number with nuclear DNA (nDNA) copy number. Accurate quantification of both components is important when calculating the ratio of mtDNA to nDNA. The goal of this project is to optimize a duplex assay that will give precise and accurate ratio estimates. Methods: We employ synthetic oligomer standards for an absolute real-time qPCR assay. The significance of using absolute qPCR is that the standard curve allows for the direct comparison of unknowns to obtain a copy number. The mitochondrial target is a site in the minor arc (MinArc), and the nuclear target is a single copy locus (β2M). The accuracy of this assay was evaluated using a standard reference material (SRM2372a) and the precision was evaluated via replications. Results: This assay was optimized so both targets amplify with similar efficiency to give precise and accurate ratio estimates. The precision was demonstrated over repeated runs and shown to be accurate based on SRM quantification. Conclusion: This protocol demonstrates reproducible quantification in both mitochondrial and nuclear targets while reducing time and resources. The data generated can be used to indicate overall mitochondrial health and can be utilized in research areas such as aging, cancer, forensics and neurodevelopment.Item Optimization and Evaluation of qPCR Duplex Assay for mtDNA Copy Number Quantification(2021) Johnson, Gretchen A.; Phillips, NicolePurpose: The mitochondrial genome (mtDNA) encodes thirteen essential proteins in oxidative phosphorylation. Depending on the cell type and stage of development, each cell contains an average of 10^3 to 10^4 copies of mtDNA. Current methods of mtDNA copy number quantification can be imprecise due to low efficiencies of assays and inherent imbalance of mtDNA copy number with nuclear DNA (nDNA) copy number. Accurate quantification of both components is important when calculating the ratio of mtDNA to nDNA. The goal of this project is to optimize a duplex assay that will give precise and accurate ratio estimates. Methods: We employ synthetic oligomer standards for an absolute real-time qPCR assay. The significance of using absolute qPCR is that the standard curve allows for the direct comparison of unknowns to obtain a copy number. The mitochondrial target is a site in the minor arc (MinArc), and the nuclear target is a single copy locus (β2M). The accuracy of this assay was evaluated using a standard reference material (SRM2372a) and the precision was evaluated via replications. Results: This assay was optimized so both targets amplify with similar efficiency to give precise and accurate ratio estimates. The precision was demonstrated over repeated runs and shown to be accurate based on SRM quantification. Conclusion: This protocol demonstrates reproducible quantification in both mitochondrial and nuclear targets while reducing time and resources. The data generated can be used to indicate overall mitochondrial health and can be utilized in research areas such as aging, cancer, forensics and neurodevelopment.