Main Research Findings

1) Epobis is revealed to be a neuroprotective and nonerythropoietic EPO receptor agonist that has the potential to elicit memory enhancing and anti-inflammatory properties.

2) Epobis was found to promote survival of cerebellar and hippocampal neurons and act as a functional agonist of EPO receptors in order to enhance neuroprotection and neurodegeneration.

 

Selected Data

1) The research team of Dmytriyeva et al assessed the mechanism behind the peptide, Epobis and its ability to induce neuritogenesis and promote neuroprotection. The experiment began with isolation of the primary rat motor neurons, followed by dissection of the ventral horns of the lumbar spinal cord from Wistar rat embryos. The motor neurons were plated at a density of 7,000 cells per well and stimulated with diluted Epobis over the course of 24 hours. The neurons were then fixed with 4% formalin and stained with polyclonal rabbit anti-rat growth associated protein-43 antibodies to prepare for analysis via computer-assisted fluorescence microscopy [1].

Secretion of tumor necrosis factor (TNF) from the macrophage cell line was assessed through the macrophage activation assay. The researchers noted that concentrations of TNF can be assessed in this manner considering that fibroblastoid mouse L929 cells are sensitive to TNF when they are exposed to actinomycin D. TNF secretion was induction by treating rat primary microglia cells with lipopolysaccharide, followed by pretreatment either with or without Epobis present. The media was collected 24 hours later and TNF secretion was determined using a rat TNF ELISA assay kit [1].

The rat primary microglia cells used for this portion of the study were prepared from 1-2 day old Wistar rat pups. The cerebral cortices were dissected, chopped and incubated in 0.5% trypsin. After 10 minutes, DMEM, GlutaMAX, 10% fetal calf serum, 100 U/mL of penicillin, and 100 U/mL of streptomycin were added to the simple and the tissue was titrated and centrifuged. The mixed glial culture was grown for 2-3 weeks with the medium changed every 5-6 days. After the growing period the loosely attached microglia cells were shaken off while the medium was collected and centrifuged. The purities of the microglial cultures were stained with rabbit anti-Iba-1 antibody and counterstained with DAPI-containing mounting media [1].

Following the assessment of the mixed glial cell cultures, in vivo experimentation began; all experiments were performed with a license from the Danish Animal experiments Inspectorate. First, in order to detect levels of Epobis in cerebrospinal fluid and plasma, 10 mg/kg of the peptide was subcutaneously injected into anesthetized Wistar rats weighing 200 grams each. The blood samples were collected from the orbital plexus while cerebrospinal fluid samples were collected from the cisterna magna. All samples of serum plasma and cerebrospinal fluid were assessed using a competitive enzyme-linked immunosorbent assay in order to estimate the concentration of the peptide present [1]. This portion of the study was followed by a hematopoiesis assay that estimated the hematopoietic activity of Epobis in female C57BL/6J mice. Each test subject was subcutaneously injected with either 10 mg/kg of Epobis, 10 mg/kg of EPO, or a vehicle, twice weekly over a 5 week treatment period. Blood samples were collected once per week from the orbital plexus in order to evaluate hematocrit values and hemoglobin levels.

Next, the researchers determined the effects of Epobis on experimentally induce autoimmune encephalomyelitis (EAE). EAE was induced in 30 female Lewis rats weighing 200 grams each and 6 healthy, age-matched rats to act as a control group. The test subjects were subcutaneously injected at both sides of the base of the tail with a 0.2 mL emulsion of 1g/L M. tuberculosis and 1 g/L of Guinea pig myelin basic protein. Between days 0 and 21 the weight and symptoms of EAE were recorded daily for each animal. Symptoms were scored on a scale of 0 to 6: 0 was defined as no abnormality; 0.5 was defined as a weak tail; 1 defined as a limp tail; 2 defined as mild palsy of one or both hind legs; 3 defined as severe palsy of one or both legs; 4 defined as complete paralysis of one or both hind legs; 5 defined as paralysis of one or both hind egs and beginning of paralysis of front legs; and 6 defined as moribund [1]. All animals with a score above a 4 were immediately euthanized. After 10 days of induction of EAE the test subjects were administered a once daily treatment of 10 mg/kg of Epobis or 1.0 mL/kg of PBS.

Finally, a social recognition test was performed to evaluate short term and working memory of adult rats considering that these aspects of memory are typically impaired in older rats aged at least 18 months, as well as in animals experiencing early stages of Alzheimer’s disease. In order to assess social recognition in the rats, a habituation session was initially carried out followed by administration of Epobis or PBS, 24 hours later. 1 hour after treatment administration the animals were introduced to a new juvenile male rat for 4 minutes. After 2.5 hours the same rat was reintroduced or another unfamiliar male rat was introduced as a control subject [1].

Long term effects of the peptide were assessed by performing the recognition test 73 hours after treatment administration. Data was expressed as a recognition ratio determined by the time spent observing the juvenile animal during the initial trail (T1) and the test trial (T2). A recognition ratio closer to 0.5 suggests that thes test animals retained no memory of the introduced animal between the two introductions. In comparison, a recognition ratio remarkably lower than 0.5 indicated that the test subjects were able to recognize the introduced animal during the second introduction trial [1].

2) The research team of Pankratova et al looked beyond the hematopoietic activity of EPO in order to evaluate how the Epobis peptide binds to EPO receptors in order to induce neurite growth. Primary cell cultures were collected and isolated from cerebellar granule neurons obtained from 7-day old Wistar rat pups. The cerebella were dissected, cleared from meningeal tissue, and mechanically chopped and trypsinized. This procedure was followed by washing the cells in the presence of DNase and trypsin inhibitors, resuspended and seeded in eight-well slides in a neurobasal medium of 2% B27, 1% glutamax, 100 U/mL penicillin, 100 ug/ml streptomycin, 2% HEPES, and 4% bovine serum albumin [2]. Additionally, hippocampal neurons were dissected and isolated from E19 rats and plated and prepared in the same manner as the cerebellar neurons.

Concentration of Epobis were added to either cerebellar granule neurons or hippocampal neurons as they were plated and the cells were left for 24 hours in order for differentiation to occur. The cerebellar granule neurons were fixed with 4% formaldehyde in PBS and immunostained with polyclonal rabbit anti-rat growth-associated protein (GAP-43 antibodies). The hippocampal cultures were also fixed with 4% formaldehyde and stained. All neuronal samples were incubated for 24 hours with Epobis or recombinant human EPO, followed by fixation and immunostaining for GAP-43 in the same manner [2].

In order to decrease expression of EPO receptors, with the use of electroporation with a nucleofector device and Rat Neuron Nucleofector Kit, neurons were transfected with plasmid DNA encoding shRNA for rat EPO receptors or control shRNA. To the transfection control shRNA a pEGFP-N1 plasmid encoding for a variant of green fluorescent protein was added, and the neurons were immediately seeded on top of L-cells and treated with Epobis for 24 hours. In order to visualize the transfected cells, the neurons were fixed and stained with mouse anti-GFP antibodies and then costained with goat-anti-EPOR and mouse anti-GFP antibodies to confirm the knockdown of the EPO receptors. Prior to the collection of observational images via a confocal laser scanning system, the neurons were incubated with fluorophore conjugated secondary antibodies as a final procedure.

Further images were captured through the use of computer-assisted microscopy in order to evaluate neurite outgrowth. The neurites, defined as the length of neuronal processes, per cell were estimated by using a stereological approach that used software packaging developed at the Protein Laboratory. The research team captured representative images of hippocampal neuron cells stimulated with Epobis; these cells were then further treated and stained for GAP-43 [2].

Neuronal survival was analyzed next, specifically as it relates to potassium withdrawal and kainic acid-induced cytotoxicity. In terms of the potassium withdrawal assay, the CGNs were placed on eight-well slides collated with poly-L-lysine in Neurobasal-A medium containing an elevated concentration of KCl. In order to inhibit glial cell proliferation, cytosine-beta-D-arabinofuranoside was added to the final concentration 24 hours after the samples were plated. After 6 days Basal MEM was used to wash the samples, followed by supplementation with 1% glutamine, 100 U/mL penicillin, 100 ug/mL streptomycin, 1% sodium pyruvate, and 3.5 grams of glucose containing 5 mM of KCl in to induce apoptosis. Epobis, EPO, or human insulin-like growth factor 1 were added to the cells and left to culture for 48 hours; the samples were then fixed, stained, and randomly recorded via computer-controlled fluorescence microscopy [2].

When evaluating kainic acid-induced cytotoxicity, hippocampal neurons we re plated in the same manner as the CGNs, and after 7 days of culturing the neurons were treated with varying concentrations of Epobis, followed by the addition of 300 uM of kainic acid. Neuronal cells were then cultured again for 24 hours, fixed, and stained in order for cell viability to be estimated through the same procedure described for the potassium withdrawal assay. In order to test for the presence of microglial cells in neuronal cultures, both the CGNs and hippocampal neurons were isolated and cultured following the same procedure and double stained with biotinylated tomato lectin and polyclonal rabbit antibodies to GAP-43; this was followed by incubation with streptavidin and secondary anti-rabbit antibodies [2]. In order to create a positive control, primary microglial cells were also obtained for 3 day old rat pups, purified by established procedures, and double stained.

 

Discussion

1) When evaluating the effects on Epobis on TNF release, activation of macrophages and microglia resulting in an increase of proinflammatory cytokine secretion. That being said, the researchers were able to indirectly observe the survival of L-cells in response to conditioned medium in order to conclude that EPO and Epobis reduces the release of TNF. Furthermore, in the rat microglia stimulated for 24 hours with LPS became activated and ended up producing soluble TNF; this was resolved after treatment with 0.9 uM of Epobis was administered. Additionally, when assessing samples of plasma and cerebrospinal fluid after an injection of Epobis, the peptide was identified at the time of the first plasma sample collection and remained detectable for approximately 24 hours with the peak occurring between 2 and 4 hours after administration. Cerebrospinal fluid samples were also observed in order to determine if Epobis has the ability to cross the blood brain barrier; the results revealed that CSF samples contained concentrations of the peptide of 0.2 ug/mL [1].

Figure 1: Concentrations of Epobis found in the plasma samples versus the samples of cerebrospinal fluid.

When evaluating the results of the in vivo portion of the study, the research team was able to determine that twice weekly subcutaneous administration of EPO was able to increase levels of both hematocrit and hemoglobin. It was important to note that twice weekly subcutaneous administration of Epobis resulted in increases in hematocrit and hemoglobin, however, the changes were not deemed significant to the research team [1].

Figure 2: Changes in hematocrit and hemoglobin levels in mice treated with EPO and Epobis.

The following portion of the experiment tested anti-inflammatory properties of the peptide in cases of induced experimental autoimmune encephalomyelitis (EAE). Animals not receiving experimental treatment and experiencing symptoms of EAE typically demonstrated remarkable weight loss, however, there were no significant changes in weight in the animals treated with Epobis. While the peptide did not affect weight changes, Epobis was found to delay the onset and increase of symptom prevalence [1]. Changes in the clinical presentation of signs and symptoms occurred less rapidly in test subjects treated with Epobis in comparison to vehicle-treated animals.

Figure 3: Changes in the presentation of clinical signs and symptoms days after the induction of EAE.

Finally, Epobis was assessed for its potential to enhance short term and working memory through a social recognition test. When performing the test 1 hour after administration of treatment there was a slight increase in recognition ratio in the animals treated with Epobis in comparison to those treated with a vehicle. However, this change was not determined significant by the research team. That being said, when performing the social recognition test 73 hours after administration of treatment, there was a significant increase in the recognition ratio in animals treated with Epobis in comparison to those treated with a vehicle [1].

Figure 4: Changes in recognition ratio and social memory in the control group versus the group treated with Epobis.

2) Results of the study conducted by Pankratova et al initially found that Epobis induces neurite growth based on the evidence that rodent neurons have shown the potential to express EPO receptors, as well as elicit neuritogenic activity. CGNs and hippocampal neurons were grown in the presence of varying concentrations of Epobis for 24 hours. Treatment with the peptide was shown to induce a dose-dependent neuritogenic response in both primary neurons. Additionally, it was indicated that the effects of Epobis were sequence-specific considering that a scrambled version of the peptide was delivered at its optimal effective concentrations and did not result in neurite outgrowth [2].

Evaluation of CGN growth on top of L-cells also allowed the researcher to assess the neuritogenic effects of Epobis. With this experimental setup, the peptide was found to induce neurite outgrowth from CGNs when applied in various concentrations. EPO was added to the CGN and Epobis mixture in order to observe whether the two proteins are synergistic. Cotreatment with Epobis and EPO was not found to have a significant effect on neurite outgrowth or neuritogenesis in comparison to treatment with Epobis alone. Additionally, when testing whether neurite outgrowth induced by Epobis is mediated by EPO receptors, hippocampal neurons were transfected with shRNA for EPO receptors or non-targeting control shRNA [2]. The researchers found that knockdown of EPO receptor expression in hippocampal neurons reduced the neuritogenic effects caused by treatment with Epobis, suggesting that these effects were mediated by the EPO receptors.

When looking at neuronal survival, EPO has been found to have neuroprotective potential, encouraging the research team to assess whether Epobis protects primary neurons in two different survival models related to potassium withdrawal and kainic acid-induced cytotoxicity. In the first survival model, apoptosis of CGNs was induced by applying a low potassium medium to the neurons, this increased neuronal survival when treated with 6.6 nM of insulin-like growth factor, 1 and 1.3 nM of EPO, and Epobis when applied in various concentrations. Overall, it was found that the peptide was able to protect CGNs against induced apoptosis [2].

In the second survival model, 300 uM of kainic acid was applied to hippocampal neurons in order to induce cell death. Treatment with 0.6 nM of EPO was found to protect the neurons from cell death and increase survival. Epobis was also found to have neuroprotective effects that were assessed using TUNEL assay to determine if this protection was due to the inhibition of apoptotic mechanisms. That being said, the amount of TUNEL-positive nuclei in the hippocampal neurons treated with both EPO and Epobis were found to significantly decrease when compared to the control groups receiving no peptide treatment. The research team was able to conclude that neuroprotection induced by Epobis was due to an anti-apoptotic mechanism [2].

Figure 5: Changes in neurite length in CGNs and Hippocampal neurons in response to treatment with varying concentrations of the peptide, Epobis.

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Dmytriyeva O, Pankratova S, Korshunova I, Walmod PS. Epobis is a Nonerythropoietic and Neuroprotective Agonist of the Erythropoietin Receptor with Anti-Inflammatory and Memory Enhancing Effects. Mediators Inflamm. 2016;2016:1346390. doi: 10.1155/2016/1346390. Epub 2016 Nov 21. PMID: 27990061; PMCID: PMC5136666.

[2] Pankratova S, Gu B, Kiryushko D, Korshunova I, Køhler LB, Rathje M, Bock E, Berezin V. A new agonist of the erythropoietin receptor, Epobis, induces neurite outgrowth and promotes neuronal survival. J Neurochem. 2012 Jun;121(6):915-23. doi: 10.1111/j.1471-4159.2012.07751.x. Epub 2012 Apr 24. PMID: 22469063.

 

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