Unveiling the Role of Ecto-5′-Nucleotidase/CD73 in Astrocyte Migration by Using Pharmacological Tools

Abstract

CD73 is a bifunctional glycosylphosphatidylinositol (GPI)-anchored membrane protein which functions as ecto-5′-nucleotidase and a membrane receptor for extracellular matrix protein (ECM). A large body of evidence demonstrates a critical involvement of altered purine metabolism and particularly, increased expression of CD73 in a number of human disorders, including cancer and immunodeficiency. Massive up-regulation of CD73 was also found in reactive astrocytes in several experimental models of human neuropathologies. In all the pathological contexts studied so far, the increased expression of CD73 has been associated with the altered ability of cells to adhere and/or migrate. Thus, we hypothesized that increased expression of CD73 in reactive astrocytes has a role in the process of astrocyte adhesion and migration. In the present study, the involvement of CD73 in astrocyte migration was investigated in the scratch wound assay (SW), using primary astrocyte culture prepared from neonatal rat cortex. The cultures were treated with one of the following pharmacological inhibitors which preferentially target individual functions of CD73: (a) α,β-methylene ADP (APCP), which inhibits the catalytic activity of CD73 (b) polyclonal anti-CD73 antibodies, which bind to the internal epitope of CD73 molecule and mask their surface exposure and (c) small interfering CD73-RNA (siCD73), which silences the expression of CD73 gene. It was concluded that approaches that reduce surface expression of CD73 increase migration velocity and promote wound closure in the scratch wound assay, while inhibition of the enzyme activity by APCP induces redistribution of CD73 molecules at the cell surface, thus indirectly affecting cell adhesion and migration. Application of anti-CD73 antibodies induces a decrease in CD73 activity and membrane expression, through CD73 molecules shedding and their release to the culture media. In addition, all applied pharmacological inhibitors differentially affect other aspects of astrocyte function in vitro, including reduced cell proliferation, altered expression of adenosine receptors and increased expression of ERK1/2. Altogether these data imply that CD73 participates in cell adhesion/migration and transmits extracellular signals through interactions with ECM.

Application of Gray Level Co-Occurrence Matrix Analysis as a New Method for Enzyme Histochemistry Quantification

“…CON is a more globally oriented parameter, while inversion second moment gives information of local changes, especially useful when there are specific changes in enzyme activity on a specific cell type or part of a structure. Immunoblot analysis confirmed increase in protein expression of eN, but it should be noted that besides changes in quantity, catalytic activity of enzyme could change, probably resulting in more AMP to adenosine conversion (Grkovic et al, 2014; Adzic & Nedeljkovic, 2018).…”

“…It has been shown that the rate of eN activity does not necessarily correlate with the enzyme protein levels, but rather increases or decreases due to changes in kinetic properties and enzyme efficacy (Stanojevic et al, 2011; Mitrovic et al, 2016, 2017; Grkovic et al, 2018). CD73 also functions as a membrane receptor for extracellular matrix molecules and may participate in a control of cell adhesion and migration, in both normal and neoplastic cells (Sadej et al, 2008; Adzic & Nedeljkovic, 2018). The enzyme has a broad tissue distribution, being expressed in many cell types, including a number of tumor cells (Antonioli et al, 2013 a ).…”

Extracellular vesicles as a platform to study cell-surface membrane proteins

“…enzyme [33] which convert AMP to adenosine and which is over-expressed on tumor cells was also studied as part of our collaboration with Innate Pharma company [34].…”

Purinergic Signaling Participates in a Transition Between Functional States of Reactive Microglia and Controls Astrocyte-Driven Neuroinflammation in the Model of Trimethyltin-Induced Neurodegeneration

“…The activation of the purinoceptors, speci cally the activation of P2Y 6 R in microglial cells promotes the phagocytosis of debris generated by excitotoxic hippocampal damage [51]. Furthermore, NTPDase1/CD39 and eN/CD73 not only that catabolize extracellular ATP and provide adenosine, but function as clusters of differentiation and cell adhesion molecules, which regulate the adhesion and glial cell migration through speci c interactions with extracellular matrix components [52]. Thus, we propose that the up-regulation of NTPDase1/CD39 and eN/CD73 and the change in purinoceptors repertoire induced by TMT are speci c adaptations to the phagocytic activity and amoeboid shape of microglial cells, which migrate along with the wave of spreading neurodegeneration, from hilus towards CA3 and CA1.…”