investigating the role of oxidative stress in mediating
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Investigating the Role of Oxidative Stress in Mediating Investigating the Role of Oxidative Stress in Mediating
Excitotoxicity Following in vitro Ischemic/Reperfusion Insult Excitotoxicity Following in vitro Ischemic/Reperfusion Insult
Isabella Sturgeon University of Montana, [email protected]
Moira Shea University of Montana, [email protected]
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Sturgeon, Isabella and Shea, Moira, "Investigating the Role of Oxidative Stress in Mediating Excitotoxicity Following in vitro Ischemic/Reperfusion Insult" (2019). University of Montana Conference on Undergraduate Research (UMCUR). 6. https://scholarworks.umt.edu/umcur/2019/pmposters/6
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Methods
Results
Conclusions
IntroductionStroke is responsible for the death of nearly 130,000 Americans annually. Of the
estimated 800,000 strokes that occur in the United States each year, approximately 87% areischemic. Ischemic stroke occurs when a blood vessel is blocked in the brain, hindering thevessel’s ability to provide oxygen and nutrients to brain tissue. Patients who survive ischemicstroke experience delayed neuronal death (DND), which has been linked to an increase in thepresence of Ca2+-permeable AMPA receptors (AMPARs) at the cell surface1.
AMPARs are tetramers composed of GluA1-4 subunits and can be either homomeric orheteromeric2. A majority of AMPARs are impermeable to calcium due to a post-transcriptionalmodification in the channel pore of the GluA2 AMPAR subunit3. GluA2 initially undergoesinternalization and subsequently is trafficked to lysosomal degradation following OGD/R insult4.The subsequent increase in the expression of GluA2-lacking, Ca2+-permeable AMPARs resultsin excitotoxicity, eventually resulting in DND5.
Following OGD/R and the subsequent influx of Ca2+, superoxide generators areactivated6. We hypothesize that the internalization of GluA2-containing AMPARsfollowing oxygen-glucose deprivation/reperfusion (OGD/R), an in vitro model forischemic stroke, is mediated through an oxidative stress signaling cascade.
SNB-19, a human astroglioma cell line, and SH-SY5Y, a human neuroblastoma cell line,were transiently transfected with fluorescently tagged GluA1 and GluA2, as well as differentRab proteins. Rab 5 is localized to early endosomes. Rab 7 is localized to late endosomesdestined for lysosomal degradation. Rab11 is localized to recycling endosomes that undergoexocytosis. We pretreated the transfected cells with MnTMPyP, a superoxide scavenger, andobserved the endocytic trafficking of GluA1 and GluA2 AMPAR subunits following OGD/R .
Investigating the Role of Oxidative Stress in Mediating Excitotoxicity Following in vitro Ischemic/Reperfusion Insult
Isabella Sturgeon*, Moira Shea*, Lindsay Achzet, and Darrell Jackson, PhDUniversity of Montana, Missoula, MT 59812
*Equally contributed to this work.
Literature Cited
• Pre-treatment with MnTMPyP, a superoxide scavenger, ameliorates the ischemic/reperfusioninjury-induced internalization of the GluA1 and GluA2 AMPAR subunits.
• The GluA1 AMPAR subunit colocalizes with Rab11, a marker for recycling endosomesdestined to undergo exocytosis, under both normoxic and ischemic conditions;; indicating thatthere is likely an intracellular pool of GluA1 AMPARs. GluA2 does not colocalize with Rab11under normoxic nor ischemic/reperfusion conditions.
• Following ischemic/reperfusion insult, both the GluA1 and GluA2 AMPAR subunits colocalizewith Rab7, a marker for late endosomes that eventually undergo lysosomal degradation.Treatment with MnTMPyP before ischemic insult ameliorated the degradation of both theGluA1 and GluA2 AMPAR subunits.
• These data indicate that the excitotoxic internalization and degradation of AMPAR subunitsfollowing ischemic/reperfusion injury is oxidative stress mediated.
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Cell CultureSNB-19 cells were generously gifted from Dr. Richard Bridges. SNB-19 cells were maintainedin DMEM/F12, supplemented with fetal bovine serum and penicillin/streptomycin. Once cellsreached 75% confluency, they were passaged.TransfectionSNB-19 cells were transfected with GluA1-tdTomato/GFP or GluA2-tdTomato/GFP, generouslygifted from Dr. Richard Huganir, and either Rab 5-cerulean, Rab7-RFP, and Rab11-RFP. Cellswere transfected using Lipofectamine 2000 in Opti-mem media for 4 hours before the mediawas replaced with phenol red-free DMEM media supplemented with fetal bovine serum andpenicillin/streptomycin. Experiments were performed 48 hours after transfection.AMPA StimulationTransfected SNB-19 cells (75,000 cells/well) were exposed to 100µM AMPA, 100µMcyclothiazide, and 100µM MnTMPyP for 3 minutes before being removed and replaced withphenol-red free DMEM media and fixed with 4% paraformaldehyde at time points: 5 and 10minutes. Cells were then washed 2x with PBS, and mounted on slides.OGD/RTransfected SNB-19 cells were exposed to deoxygenated artificial cerebrospinal fluid withoutglucose in a hypoxic chamber for 20 minutes. Media was then replaced with artificialcerebrospinal fluid containing glucose and cells were reperfused at 0, 5, 15, or 30 minute timepoints. Experiments were also done in the presence of 100µM MnTMPyP, a superoxidedismutase mimetic. Cells were fixed in 4% paraformaldehyde for 20 mins, washed 2x with PBS,and mounted on slides (75,000 cells/well). Normoxic controls were time-matched with thelongest reperfusion time point.Data Analysis/Scientific RigorCells were imaged via confocal microscopy (Fluoview Olympus) and colocalization coefficientswere obtained using FIJI software (NIH) . One-way ANOVA with Tukey post Hoc test wereconducted using GraphPad Prism software to determine significance. All imaging and analysiswas performed blindly.
Stroke is the fifth leading cause of death annually in the United States, resulting in nearly 130,000 deaths per year. Ischemic stroke is the most common form of stroke and occurs when a blood vessel supplying the brain is occluded. This blockage leads to deprivation of oxygen and nutrients to brain tissue, resulting in delayed neuronal death (DND). In the hippocampus, a region susceptible to DND, there is high expression of glutamatergic AMPA receptors (AMPARs). The majority of AMPARs expressed in the hippocampus contain an edited form of the GluA2 (Q607R) subunit, resulting in Ca2+-impermeable AMPARs. Following ischemic/stroke injury, the GluA2 AMPAR subunit is internalized and subsequently degraded, resulting in GluA2-lacking AMPARs that are Ca2+-permeable. Previous studies have indicated an increased production of superoxide radicals following ischemic stroke injury, which have a significant role in mediating oxidative stress-induced neuronal injury. Based upon previous studies, it is suggested that an oxidative stress signaling cascade is responsible for initiating events leading to the internalization and degradation of the GluA2 AMPAR subunit following oxygen-glucose deprivation/reperfusion (OGD/R), an in vitro model for ischemic stroke.
Abstract
1Benjamin, E.J., Virani, S.S., Callaway, C.W., & Chamberlain, A.M. (2018) Circulation, 137 e467-e492.2Blanco-Suarez, E., & Hanley J.G. (2014) J Biol Chem., 298: 4644-4651.3Pellegrini-Giampietry, D.E., Gorter, J.A., Bennett, M.V.L., & Zukinc, R.S. (1997) Trends in Neuroscience, 20: 464-470.4Borges, K., & Dingledine, R. (1998) Prog. Brain Res., 116: 153-1705Sensi, S.L., Yin, H.Z., Carriedo, S.G., Rao, S.S., & Weiss, J.H. (1999) PNAS, 96: 2414-2419.6Beske, P.H., Byrnes, N.M., Astruc-Diaz, D., & Jackson, D.A. (2015) Journal of Neurochemistry, 132: 504-519.