Author Of 1 Presentation
P0270 - 2-Chlorodeoxyadenosine (Cladribine) preferentially inhibits the biological activity of microglia cells (ID 1011)
Abstract
Background
Background and goals: 2-chlorodeoxyadenosine (CdA, Cladribine) is a compound used in the treatment of MS, which crosses the BBB and is activated by intracellular phosphorylation in specific cell types. We previously demonstrated that CdA inhibits microglial cell proliferation, induces apoptosis and suppress IL-1, IL-6 and TNF-α secretion; effects not observed in the case of astrocytes.
Objectives
To expand previous findings to better explain differences observed between these two cell populations in response to cladribine.
Methods
Primary cultures of microglial cells and astrocytes were prepared from neonatal C57BL/6 mice following the McCarthy and de Vellis protocol. After harvesting, cells were treated with different concentrations of CdA (20 to 200 µM), for periods lasting between 6 and 72 hours. Caspase 3 expression was evaluated by immunocytochemistry. CdA effect on mitochondrial function of microglial cells was measured using an extracellular flux analyzer (Seahorse). Expression of DCK and 5-NT enzymes, as well as of the ABCG2 receptor were measured using RT-PCR. DCK enzyme activity was assessed by ELISA.
Results
Caspase-3 expression was measured in microglial cells, 6-12 hours after exposure to CdA, preceding induction of apoptosis. CdA showed no effect on mitochondrial bioenergetics when cells were treated with a wide range of CdA concentrations (20-200 µM). To investigate differences in response to CdA between microglia and astrocytes, we measured DCK and 5-NT expression as well as expression of the CdA receptor ABCG2 in both cell populations. DCK expression was significantly higher in microglial cells than in astrocytes. By contrast, 5-NT expression was higher in astrocytes than in microglial cells; whereas expression of the ABCG2 receptor was significantly higher in microglia compared to astrocytes. Finally, incubation of microglial cells in the presence of CdA induced significant DCK enzyme activity, which increased further after LPS activation.
Conclusions
Data presented complements previous findings on biological effects induced by CdA on microglial cells, leading to a better understanding of differences in microglia and astrocyte cell responses. We observed that microglia showed: 1) greater expression of the CdA receptor ABCG2, increasing intracellular pro-drug penetration; and 2) higher expression of the phosphorylating enzyme DCK, which transforms CdA into an active drug.