scholarly journals Validation of a System xc– Functional Assay in Cultured Astrocytes and Nervous Tissue Samples

2022 ◽  
Vol 15 ◽  
Author(s):  
Pauline Beckers ◽  
Olaya Lara ◽  
Ines Belo do Nascimento ◽  
Nathalie Desmet ◽  
Ann Massie ◽  
...  

Disruption of the glutamatergic homeostasis is commonly observed in neurological diseases and has been frequently correlated with the altered expression and/or function of astrocytic high-affinity glutamate transporters. There is, however, a growing interest for the role of the cystine-glutamate exchanger system xc– in controlling glutamate transmission. This exchanger is predominantly expressed in glial cells, especially in microglia and astrocytes, and its dysregulation has been documented in diverse neurological conditions. While most studies have focused on measuring the expression of its specific subunit xCT by RT-qPCR or by Western blotting, the activity of this exchanger in tissue samples remains poorly examined. Indeed, the reported use of sulfur- and carbon-radiolabeled cystine in uptake assays shows several drawbacks related to its short radioactive half-life and its relatively high cost. We here report on the elaborate validation of a method using tritiated glutamate as a substrate for the reversed transport mediated by system xc–. The uptake assay was validated in primary cultured astrocytes, in transfected cells as well as in crude synaptosomes obtained from fresh nervous tissue samples. Working in buffers containing defined concentrations of Na+, allowed us to differentiate the glutamate uptake supported by system xc– or by high-affinity glutamate transporters, as confirmed by using selective pharmacological inhibitors. The specificity was further demonstrated in primary astrocyte cultures from transgenic mice lacking xCT or in cell lines where xCT expression was genetically induced or reduced. As such, this assay appears to be a robust and cost-efficient solution to investigate the activity of this exchanger in physiological and pathological conditions. It also provides a reliable tool for the screening and characterization of new system xc– inhibitors which have been frequently cited as valuable drugs for nervous disorders and cancer.

2021 ◽  
Vol 15 ◽  
Author(s):  
Stefan Passlick ◽  
Christine R. Rose ◽  
Gabor C. Petzold ◽  
Christian Henneberger

High-affinity, Na+-dependent glutamate transporters are the primary means by which synaptically released glutamate is removed from the extracellular space. They restrict the spread of glutamate from the synaptic cleft into the perisynaptic space and reduce its spillover to neighboring synapses. Thereby, glutamate uptake increases the spatial precision of synaptic communication. Its dysfunction and the entailing rise of the extracellular glutamate concentration accompanied by an increased spread of glutamate result in a loss of precision and in enhanced excitation, which can eventually lead to neuronal death via excitotoxicity. Efficient glutamate uptake depends on a negative resting membrane potential as well as on the transmembrane gradients of the co-transported ions (Na+, K+, and H+) and thus on the proper functioning of the Na+/K+-ATPase. Consequently, numerous studies have documented the impact of an energy shortage, as occurring for instance during an ischemic stroke, on glutamate clearance and homeostasis. The observations range from rapid changes in the transport activity to altered expression of glutamate transporters. Notably, while astrocytes account for the majority of glutamate uptake under physiological conditions, they may also become a source of extracellular glutamate elevation during metabolic stress. However, the mechanisms of the latter phenomenon are still under debate. Here, we review the recent literature addressing changes of glutamate uptake and homeostasis triggered by acute metabolic stress, i.e., on a timescale of seconds to minutes.


Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 721
Author(s):  
Srinivasaraghavan Kannan ◽  
Pietro G. A. Aronica ◽  
Thanh Binh Nguyen ◽  
Jianguo Li ◽  
Chandra S. Verma

S100B(ββ) proteins are a family of multifunctional proteins that are present in several tissues and regulate a wide variety of cellular processes. Their altered expression levels have been associated with several human diseases, such as cancer, inflammatory disorders and neurodegenerative conditions, and hence are of interest as a therapeutic target and a biomarker. Small molecule inhibitors of S100B(ββ) have achieved limited success. Guided by the wealth of available experimental structures of S100B(ββ) in complex with diverse peptides from various protein interacting partners, we combine comparative structural analysis and molecular dynamics simulations to design a series of peptides and their analogues (stapled) as S100B(ββ) binders. The stapled peptides were subject to in silico mutagenesis experiments, resulting in optimized analogues that are predicted to bind to S100B(ββ) with high affinity, and were also modified with imaging agents to serve as diagnostic tools. These stapled peptides can serve as theranostics, which can be used to not only diagnose the levels of S100B(ββ) but also to disrupt the interactions of S100B(ββ) with partner proteins which drive disease progression, thus serving as novel therapeutics.


2000 ◽  
Vol 871 (2) ◽  
pp. 175-180 ◽  
Author(s):  
R.D Azbill ◽  
X Mu ◽  
J.E Springer

Talanta ◽  
2015 ◽  
Vol 135 ◽  
pp. 67-74 ◽  
Author(s):  
O. Soldatkin ◽  
A. Nazarova ◽  
N. Krisanova ◽  
A. Borуsov ◽  
D. Kucherenko ◽  
...  

2021 ◽  
Vol 22 (12) ◽  
pp. 6216
Author(s):  
Monika Englert-Golon ◽  
Mirosław Andrusiewicz ◽  
Aleksandra Żbikowska ◽  
Małgorzata Chmielewska ◽  
Stefan Sajdak ◽  
...  

Ovarian cancer remains the leading cause of death due to gynecologic malignancy. Estrogen-related pathways genes, such as estrogen receptors (ESR1 and ESR2) and their coregulators, proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), and proto-oncogene tyrosine-protein kinase c-Src (SRC) are involved in ovarian cancer induction and development, still they require in-depth study. In our study, tissue samples were obtained from 52 females of Caucasian descent (control group without cancerous evidence (n = 27), including noncancerous benign changes (n = 15), and the ovarian carcinoma (n = 25)). Using quantitative analyses, we investigated ESRs, PELP1, and SRC mRNA expression association with ovarian tumorigenesis. Proteins’ presence and their location were determined by Western blot and immunohistochemistry. Results showed that PELP1 and SRC expression levels were found to differ in tissues of different sample types. The expression patterns were complex and differed in the case of ovarian cancer patients compared to controls. The most robust protein immunoreactivity was observed for PELP1 and the weakest for ESR1. The expression patterns of analyzed genes represent a potentially interesting target in ovarian cancer biology, especially PELP1. This study suggests that specific estrogen-mediated functions in the ovary and ovary-derived cancer might result from different local interactions of estrogen with their receptors and coregulators.


1999 ◽  
Vol 56 (6) ◽  
pp. 1095-1104 ◽  
Author(s):  
Hans P. Koch ◽  
Michael P. Kavanaugh ◽  
Christopher S. Esslinger ◽  
Noah Zerangue ◽  
John M. Humphrey ◽  
...  

2004 ◽  
Vol 27 (6) ◽  
pp. 817-820 ◽  
Author(s):  
Atsushi Nishida ◽  
Hiroshi Iwata ◽  
Yukitsuka Kudo ◽  
Tsutomu Kobayashi ◽  
Yuzo Matsuoka ◽  
...  

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