Epobis is a Nonerythropoietic and Neuroprotective Agonist of the Erythropoietin Receptor with Anti-Inflammatory and Memory Enhancing Effects

Abstract

The cytokine erythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Moreover, EPO has neuroprotective, anti-inflammatory, and antioxidative effects, but the use of EPO as a neuroprotective agent is hampered by its erythropoietic activity. We have recently designed the synthetic, dendrimeric peptide, Epobis, derived from the sequence of human EPO. This peptide binds the EPO receptor and promotes neuritogenesis and neuronal cell survival. Here we demonstrate that Epobis in vitro promotes neuritogenesis in primary motoneurons and has anti-inflammatory effects as demonstrated by its ability to decrease TNF release from activated AMJ2-C8 macrophages and rat primary microglia. When administered systemically Epobis is detectable in both plasma and cerebrospinal fluid, demonstrating that the peptide crosses the blood-brain barrier. Importantly, Epobis is not erythropoietic, but systemic administration of Epobis in rats delays the clinical signs of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, and the peptide has long-term, but not short-term, effects on working memory, detected as an improved social memory 3 days after administration. These data reveal Epobis to be a nonerythropoietic and neuroprotective EPO receptor agonist with anti-inflammatory and memory enhancing properties.

Figure 1

Effects of Epobis on neurite outgrowth from motor neurons. (a) Representative fluorescence micrographs of rat motoneurons grown for 24 h in the absence (top) or presence (bottom) of 0.33 μM Epobis. Size bar: 10 μm. (b) Neurite outgrowth in response to Epobis. The graph shows mean and SEM from 3 independent experiments. Statistics was performed on nonnormalized data using a one-way ANOVA for repeated measures (F _8,12 = 20.3, p < 0.0001) followed by Tukey's Multiple Comparison Test. ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001.

Figure 2

Effects of Epobis on TNF secretion. (a) Standard curve for the viability of cytokine-sensitive L-cell relative to the concentration of recombinant TNF in the medium. The curve shows mean and SEM of 4 separate experiments. L-cell viability was evaluated using a one-way ANOVA for repeated measures (F _5,18 = 14.96, p < 0.0001) followed by Tukey's Multiple Comparison Test (^∗∗∗ p < 0.001, when compared to 0.00675 ng/mL TNF). (b) L-cell viability in response to exposure to conditioned medium from AMJ2-C8 macrophages. A value of 100% corresponds to the viability of L-cells exposed to conditioned medium from unstimulated AMJ2-C8 cells. EPO was tested at a concentration of 8.4 nM. Data from 4 separate experiments evaluated using one-way ANOVA for repeated measures (F _6,9 = 5.959, p < 0.0160) followed by Tukey's Multiple Comparison Test. ^∗ p < 0.05 when compared to 0 μM Epobis. (c) Representative fluorescence micrograph of the rat microglia cultures utilized for estimates of TNF secretion. The cells are double-stained with DAPI (for visualization of the total number of cells) and an antibody against Iba-1/AIF-1 (for the visualization of microglia). Size bar: 10 μm. (d) Estimate of TNF secretion from primary cultures of rat microglia. The bars show mean and SEM from 3 separate experiments evaluated using Friedman's test (p < 0.0278) followed by Dunn's Multiple Comparison Test. ^∗ p < 0.05, when compared to LPS-stimulated control untreated with Epobis.

Figure 3

Dynamics of Epobis concentrations in plasma. Rats received a single c.s. injection of Epobis (10 mg/kg). Subsequently blood samples were collected at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h; CSF samples were collected only 1 h after administration. The concentrations of Epobis in plasma and CSF (n = 6) were estimated by ELISA.

Figure 4

Effects of Epobis on hematopoiesis in vivo. Mice were injected with Epobis (n = 8), EPO (n = 10), or PBS (n = 6) twice per week for 5 weeks (black arrowheads). Hematocrit (a, b) and hemoglobin levels (c) were estimated from blood samples taken once per week for 6 weeks; first sample day, 0; last sample day, 42. Data are presented as actual values (a) and normalized to vehicle (PBS) from the same day (100%, b and c). Statistics was performed on nonnormalized data using a one-way ANOVA for repeated measures followed by Tukey's Multiple Comparison Test. ^∗ p < 0.05; ^∗∗ p < 0.01; ^∗∗∗ p < 0.001, Epobis versus vehicle.

Figure 5

Effects of Epobis on the clinical signs of EAE. Following induction of EAE the clinical signs were evaluated daily for all animals. Only animals reaching a clinical score ≥1 before day 14 were included in the study. EAE was induced in 30 animals of which 20 developed clinical signs of EAE (10 receiving PBS; 10 receiving Epobis). In the figure, the data have been aligned according to the onset of clinical signs that were scored as follows: 0, no abnormality; 0.5, weak tail; 1, limp tail; 2, mild palsy of one or both hind legs; 3, severe palsy of one or both hind legs; 4, complete paralysis of one or both hind legs; 5, paralysis of one or both hind legs and beginning paralysis of front legs; 6, moribund. Animals with a clinical score ≥4 were sacrificed immediately. The clinical scores of animals sacrificed due to a clinical score ≥4 have been maintained at all subsequent time points. Data are shown as mean and SEM. The data were evaluated using a 2-way ANOVA for repeated measures followed by a Bonferroni posttest. ^∗ p < 0.05 relative to the corresponding control animals.

Figure 6

Effects of Epobis on social memory. Healthy adult rats received a single administration of PBS (open columns, n = 12) or Epobis (solid columns, n = 12), and social recognition tests were initiated 1 h and 73 h later. Data are expressed as a recognition ratio: RR = T2/(T2 + T1), where T1 and T2 are the times spent on investigating the juvenile animal during the initial and the test trial, respectively. Data were evaluated using t-tests. ^∗ p < 0.05 relative to the corresponding control animals.