The purpose of this study was to investigate the tissue-protective effects of a nonhematopoietic domain derived from whole-molecule EPO on TBI in a mouse animal model. Attenuated cell death in JM4-treated animals appears to correlate with reduced clinical deficit following TBI. We also showed that despite an initial treatment delay, JM4 boasted a potent therapeutic effect that extended for at least the first 9 h following injury. Finally, the neuroprotective characteristics of whole EPO appear to be contained within the early portion of the intact EPO sequence. The most profound cell death-blocking effects were restricted to the JM4-containing sequence, EP-P#2, and, to a slightly lesser degree, EP-P#3.

JM4 peptide represents a newer class of EPO derivative composed of a small peptide region derived from the AB loop of the whole EPO molecule. We aimed to define a region of the EPO molecule that retained therapeutic efficacy in neuroprotection yet eliminated the undesired hematopoietic interactions that have severely restricted the use of whole EPO treatment in TBI [17]. The JM4 sequence (N28-GCAEHCSLNENITVPDTKV-46C) was chosen for the presence of a disulfide bond capable motif comprised of 2 cysteine residues in the first loop of EPO, allowing for a cyclic structure in solution to enhance stability. Other EPO derivatives based on the same desire to eliminate undesired hematopoiesis have been studied for their use in brain injury, including HBSP and a pHBSP (also known as ARA290) [16]. HBSP and its analog pHBSP have been found to be tissue protective in models of stroke [18], cardiovascular disease [19, 20], experimental autoimmune encephalomyelitis (EAE) [21], and TBI [15]. In our investigation, JM4 also significantly limited the size of the lesion in injured brains following TBI (Fig. 3). We elected to measure cell death with TUNEL staining, which has been used extensively to assess for cell death following TBI [22, 23]. We additionally observed significant attenuation of cell death in brain lesions whether treated with JM4 or EPO, indicating that equivalent levels of neuroprotection can be afforded by both whole-molecule EPO and JM4 (Fig. 2). When the whole EPO molecule was divided into consecutive fragments, our data support the notion that only early segments of the molecule contain the significant neuroprotective effects afforded by EPO (Fig. 6A). This neuroprotective region (AA 28–82) contains the sequences for JM4, HBSP, and pHBSP (Fig. 6B), and, of note, the second fragment (EP-P#2), which contains the JM4 sequence demonstrated to have the most profound cell death-blocking effect. Our data contribute to a small yet steadily growing body of literature demonstrating neuroprotective properties of EPO and the potential for its small peptide derivatives to take on a prominent role in the treatment of brain injury.

Several human trials have revealed that whole-molecule EPO treatment may lead to significantly increased incidence of thrombotic events. More alarmingly, in a phase II/III study on the use of rhEPO for treatment of stroke, the death rate among patients treated with rhEPO was elevated to 16.4 % versus 9.0 % in the placebo group (p = 0.01) [11]. These findings affected a recently published clinical study investigating the use of EPO following TBI to such a degree that the initial 3-dose regimen of EPO was decreased midway through the study to only a single initial dose of EPO after injury [24]. The study failed to report significant benefit in neurological recovery in patients, but it is important to note that the dosage used was about 1/10 of the most effective clinical dose in animal TBI models and that more than half of enrolled patients had a Glasgow Coma Score of 8 or less (severely impaired). In contrast, in our animal model most mice were conscious, alert, and moving within 30–60 min of their TBI procedures and received a complete EPO 3-dose equivalent of JM4. From the mortality data we might gather that the concentration of EPO required in circulating plasma to elicit its neuroprotective effects may risk the activation of erythropoiesis and downstream side effects.

In response to the side effects brought on by EPO treatment, derivatives of the whole EPO protein, including our compound, JM4, have been designed and studied in animal models [25–27]. The goal of the design was to maintain the tissue-protective capabilities of the EPO molecule while eliminating the erythropoietic characteristics that lead to the previously described complications. JM4 has been previously demonstrated to have no effect on hematocrit in animals [17].

We additionally developed a clinical correlate to follow the level of TBI deficit during the disease course following injury. Brain-injured mice treated with JM4 showed fewer deficits than their PBS sham-treated counterparts during the post-TBI course (Fig. 4). Scores for injured mice were significantly higher compared with baseline scores set by sham-operated uninjured mice, allowing us to discount the effects of the craniotomy and other surgical factors unrelated to CCI. From our experience, SNAP provides a useful clinical correlate to track neurologic impairment and recovery [14]. Our results indicate that JM4 remains effective in limiting neurological deficit following injury over a 10-day trial. Interestingly, on day 2, the composite SNAP scores of the JM4- and PBS-treated groups fell dramatically, only to increase again on day 3. TBI has been associated with delayed activation of CCL2 and CCL20, as well as secondary delayed activation of interleukin (IL)-1β and IL-4 in a rat CCI model, suggesting a temporal relationship between the cytokine profile, their targeted cellular infiltrates, and observed clinical deficits [28]. Exploration of EPO as an immunomodulatory agent has revealed that EPO reduces levels of the proinflammatory cytokines tumor necrosis factor (TNF)-α, IL-1β, interferon (IFN)-γ, IL-6, and intracellular adhesion molecule 1 following EPO treatment in TBI and EAE [29–31]. Other studies in EAE and cerebral malaria have demonstrated that whole-molecule EPO treatment can modulate a highly unstable autoimmune environment into an immune system of relatively normal activity, limiting peripheral and CNS infiltration by CD4+ and CD8+ T cells, reducing the number of mononuclear cells and dendritic cells, while suppressing T helper 17 cells, IFN-γ, and TNF mRNA [29–32]. In some studies EPO also induced elevation of IL-10 and FoxP3+ regulatory T cells, factors that contribute toward immune silencing.

An important and highly practical aspect of the design of novel therapeutics is the time frame in which the molecules maintain a high level of efficacy. Whole-molecule EPO reportedly loses protective effectiveness 6 h postinjury [5]. An increase in therapeutic window duration by a few hours could represent a significant advantage over existing therapies. Surprisingly, delaying initial treatment of brain-injured mice with JM4 peptide for 9 h seemed to reduce TUNEL cell death as much as immediate treatment with JM4 (Fig. 5). These findings demonstrate that the therapeutic window for EPO-derived JM4 treatment is open for at least 9 h postinjury and likely extends further toward the 24-h postinjury mark. Studies have observed that TNF-α levels peak between 3 and 8 h following TBI [33, 34]. Though the role of TNF-α in TBI has not been fully elucidated, this response potentially indicates improved JM4 efficacy associated with more optimal inflammatory attenuation by slightly delayed initial therapy.

Though much optimism has surrounded the use of whole EPO and its derivatives in TBI, little is still known about possible mechanisms of action in neuroprotection. In a mouse model of EAE we found that EPO exhibits a strong immunomodulatory effect by inhibiting the proliferation of dendritic cell and antigen-specific T-cell populations while downregulating major histocompatibility complex-II and proinflammatory cytokine (IFN-γ, IL-2, IL-6, TNF-α) expression. Whole-molecule EPO promoted the expansion of T regulatory cells, while also causing a striking decrease in peripheral T helper 17 cell levels [29]. All of these effects occurred in the lymph nodes and spleen (peripheral lymphoid system), as well as within the damaged target organ (spinal cord). This characteristic has also been retained in the JM4 molecule as we have demonstrated that JM4 suppresses IL-2, IL-5, IL-6, TNF-α, and IFN-γ while limiting peripheral mononuclear and dendritic cell populations in EAE-diseased animals [17]. In modulating multiple immune response cell types and their corresponding cytokines, EPO and its JM4 derivative transformed a highly uncontrolled autoimmune process into a more normalized condition while providing significant neuroprotective benefits in both clinical deficit and neuropathology.

Our small peptide EPO derivative JM4 was administered to acutely brain-injured mice. JM4 offers a therapeutic window of at least 9 h following injury. Results indicated remarkable reduction in neural cell death following TBI. JM4 shows promise as an effective tool in treating acute TBI and perhaps a broader range of acute neurological insults.