High-Content Analysis of Proapoptotic EphA4 Dependence Receptor Functions Using Small-Molecule Libraries

Abstract

Small molecule compounds (SMCs) can provide an inexpensive and selective approach to modifying biological responses. High-content analysis (HCA) of SMC libraries can help identify candidate molecules that inhibit or activate cellular responses. In particular, regulation of cell death has important implications for many pathological conditions. Dependence receptors are a new classification of pro-apoptotic membrane receptors that, unlike classic death receptors, initiate apoptotic signals in the absence of their ligands. EphA4 has recently been identified as a dependence receptor that may have important functions in conditions as disparate as cancer biology and CNS injury and disease. To screen potential candidate SMCs that inhibit or activate EphA4-induced cell death, HCA of a SMC library was performed using stable EphA4-expressing NIH3T3 cells. Our results describe a high-content method for screening dependence receptor-signaling pathways, and demonstrate that several candidate SMCs can inhibit EphA4-mediated cell death.

EphB3 receptors function as dependence receptors to mediate oligodendrocyte cell death following contusive spinal cord injury

“…To examine whether activation of EphB3 following SCI could reverse cell losses, we administered recombinant ephrinB3 33 at a concentration of 50 ng/h through intrathecal infusion into the injury epicenter for a week after the initial insult. Following ephrinB3 infusion, we observed a significantly higher number of GFP-positive OLs (5870±742, n =6) as compared with vehicle controls (3547±510, n =14) ( P <0.05) ( Figure 3c ).…”

The identification of a novel isoform of EphA4 and ITS expression in SOD1 G93A mice

“…EphA4 is distinguished by its ability to bind with both ephrinA and ephrinB ligands (Bowden et al, 2009). Its structure is highly conserved between species; for example, human and mouse EphA4 share about 98.58% amino acid sequence identity (Nelersa et al, 2012).…”

A novel miR17 /protein tyrosine phosphatase-oc/EphA4 regulatory axis of osteoclast activity

“…The mechanistic investigations into this miR17 /PTP-oc/EphA4 regulatory axis have identified two novel potential drug targets for anti-resorptive therapy: 1) EphA4, and 2) miR17 . In this regard, EphA4 is an attractive drug target for an anti-resorptive therapy for the following reasons: a) mature osteoclasts express primarily the EphA4 (27,83,85); b) the forward signaling of EphA4 can effectively be activated by soluble EphA4-binding Efn-fc chimeric proteins (27); c) several Efn fragment-based EphA4 ligands (103) and EphA4-interacting small-molecules (104) have been identified; and d) direct injection of EfnA5-fc (an EphA4 ligand) into the spinal cord of mice with spinal cord injury promoted EphA4-induced axonal regeneration and functional recovery (105,106). The miR17 is also an appealing drug target, as miR17 modulators or mimics could be used to develop novel and effective anti-resorptive therapies.…”