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Browsing Abstracts by Author "Acharya, Suchismita"
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Item Hybrid molecule SA-2 improves both mitochondrial respiration and glycolysis in primary human trabecular meshwork cells(2022) Amankwa, Charles E.; Gondi, Sudershan; Stankowska, Dorota; Acharya, SuchismitaPurpose: Oxidative stress (OS) caused by hypoxia/hyperoxia environment results in progressive loss of trabecular meshwork (TM) cells in primary open angle glaucoma (POAG). Our previous report demonstrated; a hybrid nitric oxide (NO) donor-antioxidant molecule SA-2 protect primary human (h) TM cells against t-butyl hydrogen peroxide (TBHP) -induced cell death and increased superoxide dismutase enzyme level. Here we investigated the effect of SA-2 on mitochondrial energy metabolism by measuring the respiration status, glycolysis rate and energy production. Methods: Primary hTM cells obtained from human donor eyes were seeded in 24-well culture plates (Seahorse XFe 24 Cell Mito Stress test kit, Agilent), and starved for 24h before treatment with SA-2 (1 µM,10µM,100µM, and 1mM). In a separate experiment, the cells were pretreated with TBHP (150µM) for 30 minutes, followed by the addition of SA-2 (10µM,100µM). After 24h, the mitochondrial complex inhibitors and uncoupling reagents (oligomycin, FCCP, rotenone/antimycin A) were added. The plate was analyzed for changes in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer following the manufacturer's instructions. Results: The mean OCR was significantly decreased (>70%) followed by increase in the mean ECAR (~3-fold) after treatment with TBHP compared to oligo/FCCP/rot treated cells, hereafter called as negative control. Treatment with SA-2 at 1 µM,10µM,100µM and 1mM concentrations increased both oligomycin/FCCP induced decrease in ATP production and maximal mitochondrial respiration followed by an increase in the mean ECAR compared to negative control. The mean OCR was higher in SA-2 (100µM) +TBHP treated cells followed by an increase in ECAR in SA-2 (10µM or 100µM) +TBHP treated cells than TBHP and negative control treated cells. N =2-3. Conclusion: Mitochondrial respiration was impaired after TBHP treatment to hTM cells following cell death. While most of the mitochondrial targeting anti-oxidant compounds increase OCR but not ECAR, we found the hybrid NO donor-anti-oxidant compound SA-2 increases ATP production, maximal mitochondrial respiration and increases glycolytic energy production in hTM cells. This finding provides a novel direction for further investigation into the effect of SA-2 and mitochondrial bioenergetics during OS-induced cell death.Item Methionine Synthase: A target for novel small molecules to inhibit cocaine and methamphetamine induced neuronal death(2022) Young, Olivia; Funk, Arlene; Deb, Biddut; Amankwa, Charles E.; Chintagunta, Anila; Gondi, Sudershan; Forster, Michael; Shetty, Ritu; Acharya, SuchismitaPurpose: Oxidative stress-induced cell death is involved in the pathology of psychostimulant addiction neuropathies and ischemic stroke. These conditions potentially cause neuronal and functional changes via different mechanisms - epigenetic alterations (DNA hypomethylation) and reactive oxygen species (ROS) accumulation. Current medications for the treatment of psychostimulants (e.g., cocaine and methamphetamine) induced addiction neuropathies are largely ineffective due to the high rate of relapse and marginal alterations of dependency to these diseases. To circumvent this, our laboratory has synthesized novel hybrid antioxidant small molecules: SA-30 and SA-31, with predictive neuroprotective and broad-spectrum reactive oxygen species (ROS) scavenging abilities in mouse hippocampal HT22 neural cells. Our objective here was to test if the compounds increase cell proliferation, superoxide dismutase (SOD) enzyme, as well as methionine synthase (MS) enzyme, a key enzyme largely responsible for DNA methylation, neuronal growth, and survival using human neuroblastoma cells (SH-SY5Y) expressing dopaminergic neurons. Methods: The synthesis and structure characterization of compounds SA-30 and SA-31 were previously synthesized in Acharya lab using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Human neuroblastoma cells SH-SY5Y expressing dopaminergic neurons were purchased (ATCC), cultured, and treated with different concentrations of cocaine hydrochloride or methamphetamine for 8, 24, and 48h for determining EC50 using MTT assay. In the next experiment, either cocaine hydrochloride (1.5mM) or Methamphetamine (METH, 3.5 mM) was added followed by co-treatment with compounds SA-30 and SA-31 and 4-hydroxy tempol (all at 100µM) for 24 hours. Cell viability was assessed using MTT assay. The level of intracellular MS and SOD enzymes was assessed using ELISA. Results: In SHSY5Y cells, the EC50 of cocaine was 1.5 mM, and for METH was 3.5 mM after 24h of treatment. Both compounds SA-30 and SA-31 were not cytotoxic at varying concentrations (0.01, 0.1, 1, 10, 100µM) and rescued cells from both cocaine and METH-induced oxidative stress/cell death at 100µM concentrations. SA-31 at 100µM significantly increased (~1.5 fold) intracellular MS as compared to control. There was a decrease in MS level after METH treatment and treatment with SA-30 and SA-31 increased the level. SOD levels were significantly higher (~3 fold) in METH+SA-31 treated groups than only METH groups. Conclusion: Both novel hybrid small molecules SA-30 and SA-31 are neuroprotective in SH-SY5Y cells from psychostimulants cocaine and METH-induced oxidative stress/neural cell death. Increasing MS and SOD enzyme activities is one of the mechanisms by which neuroprotection was attained. Future studies will address the potential of both SA-30 and SA-31 to progress further in pre-clinical drug development, with a future application for the treatment of substance abuse disorder.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.