Asialo-rhuEPO as a Potential Neuroprotectant for Ischemic Stroke Treatment

Abstract

Neuroprotective drugs to protect the brain against cerebral ischemia and reperfusion (I/R) injury are urgently needed. Mammalian cell-produced recombinant human erythropoietin (rhuEPO^M) has been demonstrated to have excellent neuroprotective functions in preclinical studies, but its neuroprotective properties could not be consistently translated in clinical trials. The clinical failure of rhuEPO^M was thought to be mainly due to its erythropoietic activity-associated side effects. To exploit its tissue-protective property, various EPO derivatives with tissue-protective function only have been developed. Among them, asialo-rhuEPO, lacking terminal sialic acid residues, was shown to be neuroprotective but non-erythropoietic. Asialo-rhuEPO can be prepared by enzymatic removal of sialic acid residues from rhuEPO^M (asialo-rhuEPO^E) or by expressing human EPO gene in glycoengineered transgenic plants (asialo-rhuEPO^P). Both types of asialo-rhuEPO, like rhuEPO^M, displayed excellent neuroprotective effects by regulating multiple cellular pathways in cerebral I/R animal models. In this review, we describe the structure and properties of EPO and asialo-rhuEPO, summarize the progress on neuroprotective studies of asialo-rhuEPO and rhuEPO^M, discuss potential reasons for the clinical failure of rhuEPO^M with acute ischemic stroke patients, and advocate future studies needed to develop asialo-rhuEPO as a multimodal neuroprotectant for ischemic stroke treatment.

Keywords: asialo-erythropoietin; cerebral ischemia and reperfusion; clinical trial; erythropoietin; erythropoietin receptor; hematopoietic activity; multimodal neuroprotectant; non-erythropoiesis; preclinical study.

Figure 1

Structural differences between recombinant sialylated rhuEPO^M (A), enzymatically prepared asialo-rhuEPO^E (B) and plant-produced asialo-rhuEPO^P (C). Their protein structures are the same with two separate hematopoietic receptor (EPOR)₂ binding sites (site 1 marked by the green dotted circle at the back side of EPO; site 2 marked by a solid green circle at the front side of EPO). Both binding sites are present distal to the carbohydrate chains. Helix B marked by the red dotted oval is proposed to be involved in EPO-mediated protective functions. All three types of EPO differ with respect to the structure of their N-glycan chains. RhuEPO^M bears sialic acid residues (red diamond) as terminal sugar on bi-, tri- and tetrantennary N-glycans while asialo-rhuEPO^E lacks sialic acid residues and contains β1,4-galactose residues (yellow circles) as terminal sugars. Asialo-rhuEPO^P lacks terminal sialic acid residues like asialo-rhuEPO^E. In addition, asialo-rhuEPO^P bears biantennary N-glycan chains with plant-specific xylose and fucose residues and lacks O-glycan chain.

Figure 2

Mammalian- and plant-produced complex-type N-glycans. Complex N-glycans refer to those in which both the α3- and α6-linked mannose residues are substituted with GlcNAc moieties. In mammals, the N-glycan chains can be bi-, tri-, and tetraantennary, and the GlcNAc residues in each glycan chain are further extended with β1,4-galactose residues and terminal sialic acid residues. The sialic acid in humans is N-acetyl-neuraminic acid (Neu5Ac) while other mammals have both Neu5Ac and N-glycolylneuraminic acids (GlcNGc). In plants, their complex N-glycan chains are biantennary, but do not bear β1,4-galactose and sialic acid residues. The arrow depicts the locations of further expansion of plant complex N-glycans with β1,4-galactose residues by overexpression of human β1,4-galactosyltransferase gene (GalT).

Figure 3

EPO-induced signaling pathways involved in its neuroprotective effects. EPO by binding to either homodimeric (EPOR)₂ receptor or another possible heterodimeric receptor EPOR-βcR activates JAK2 by phosphorylation, followed by activation of downstream STAT5, PI3K/AKT, and MAPK signaling pathways, as well as regulation of voltage-gated Ca²⁺ channel. The activation of these three signaling pathways results in the activation of anti-apoptotic and anti-inflammatory pathways benefiting cell survival while promoting angiogenesis and neurogenesis. The suppression of glutamate release benefits cell survival and inhibits apoptosis through the regulation of voltage-gated calcium ion channels to lower glutamate excitotoxicity.