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    • To study this complex neuroprotective pathway a

    2018-10-31

    To study this complex neuroprotective pathway, a common endpoint needs to be identified. A potential pathway involves the regulation of the major cellular antioxidant glutathione. Glutathione biosynthesis, which begins with the import of cystine dimers through the cystine glutamate exchanger (System Xc−), is a pivotal step in cellular metabolism, and cysteine availability is the rate limiting to this process. System Xc− is an important transmembrane protein responsible for the intracellular transport of cystine and the export of glutamate. System Xc− is composed of two subunits, the regulatory subunit (4F2hc-CD98) and the catalytic subunit (xCT). The catalytic subunit belongs to a family of transmembrane proteins that have 12 transmembrane spanning regions. CD98 binds to other amino folate analogue transporters while xCT is considered the functional portion of system Xc−. Because of this, xCT and system Xc− will be used interchangeably. There are several studies showing the importance of glutathione and system Xc−. For example, the HIV-1 Tat protein led to a decrease in glutathione and a concomitant increase in system Xc− activity, producing a cell-state more vulnerable to oxidative stresses (Bridges et al., 2004). High extracellular glutamate concentrations inhibited system Xc− and reduced glutathione levels in HT22 cells (Rossler et al., 2004), and diethyl maleate increased the xCT gene at the blood–brain barrier, causing an increase in l-cystine transport (Hosoya et al., 2002). Increased xCT has been demonstrated in retinal ganglion cells exposed to oxidative stress (Dun et al., 2006), and inhibition of system Xc− activity in cultured microglia cells prevented glutamate excitotoxicity and facilitated neuroprotection (Qin et al., 2006). The stress-induced transcription factor, NF-E2 related factor (Nrf2) is considered a defense against oxidative stress and has been shown to upregulate xCT, as well as other glutathione synthesis/release components (Pacchioni et al., 2007).Hypoxic preconditioning protects brains from future insults but its exact mechanism has not been elucidated. We have recently demonstrated that Epo-induced neuroprotection requires system Xc− activity (Sims et al., 2010), and others have shown that overexpression of xCT is neuroprotective against oxidative stress (Shih et al., 2006). Given the possible importance of system Xc− in the neuroprotective mechanism of hypoxic preconditioning, the current study examined the role of system Xc− activity and protein expression during hypoxia. Understanding the regulation of system Xc− activity in vivo could prove to be a novel neuroprotective strategy.
    Results To examine the expression of the cystine glutamate exchanger, postnatal day 7 (PND 7) C57BL/6 mice were exposed to either hypoxia (10% oxygen, treatment group) or normal room air (control group) for 24h. Animals were sacrificed and brain sections were stained for system Xc−. Fig. 1A shows that system Xc− expression increased in animals placed in hypoxia compared to those exposed to normal room air. This increase was most apparent in the hippocampus. Whole brain lysates were collected from both groups of mice for Western Blot analysis of xCT and actin using a standard densitometer (Fig. 1B). The xCT/actin ratio increased by almost 2-fold in the hypoxic mice (0.89±0.1 in control versus 1.62±0.31 in hypoxia animals). To test if the increase in system Xc− expression was due to an increase in translational or post-translational regulation of system Xc− mRNA, total RNA was collected from whole brain of control and hypoxic C57BL/6 mice for real-time PCR analysis. There was an 8-fold increase in xCT mRNA in brains of hypoxic mice compared to those of control mice (Fig. 1D). For system Xc− regulation to be a practical tool for neuroprotection, it must respond in a timely fashion. However, the time course of system Xc− expression during hypoxia is not known. The current experiment was designed to address if system Xc− expression would increase after a short exposure to hypoxia and the time course of this response. PND 7 mice were placed in 10% hypoxia for 45min, and samples were collected from 0 to 24h. Animals were sacrificed, and the right cortex was immediately dissected on ice and placed in lysis buffer at the specified time point. Samples were frozen at −20°C until all time points were collected. Fig. 2A shows a representative Western Blot of both xCT and actin at 0 (1), 4 (2), 8 (3), 12 (4) and 24 (5)h. Fig. 2B is the xCT/actin ratio for all samples. The xCT/actin ratio was 5.64±0.51 (p≤0.01) at 4, 4.61±0.69 (p≤0.01) at 8, 6.49±1.36 (p≤0.01) at 12, and 5.08±1.37 (p≤0.05) at 24h. These data show that there was a persistent elevation in xCT after a short exposure to hypoxia.