Neuroprotective effects evaluation of a recombinant human low-glycosylation erythropoietin isoform.

Abstract

Erythropoietin (Epo) is a glycoprotein hormone that mainly regulates hematopoiesis, has a molecular weight of approximately 34 KDa and is made up of 165 amino acids, it has potential N- and Oglycosylation sites at residues Asn 24, 38, 83 and Ser 126 respectively. Its tetra-antennary and tetra-sialylated glycosylation pattern is specifically related to its affinity for its receptor and directly related to its half-life in circulation. Epo exerts its effect by binding to its receptor (EpoR), which belongs to the type I cytokine receptor superfamily, weighs approximately 66 KDa, and binds to its ligand forming homodimers on the cell surface. This interaction amplifies a signaling cascade, primarily through STAT5, PI3K, MAPK, and PKC, resulting in positive regulation of anti-apoptotic Bcl-2 family proteins, such as Bcl-xL. Furthermore, the presence of the Epo receptor in non-erythroid tissues, such as the central nervous system (CNS), suggests a production of the hormone in these tissues, with a relevant role in the protection against cellular stress, preventing apoptosis through the activation of its receptor and the previously mentioned pathways. The clinical use of Epo depends on its recombinant production, which is developed in mammalian cell culture expression systems as bioreactors, being commonly used Chinese hamster ovary cells, The main disadvantage of this system is its low efficiency and high production cost. As an alternative to this expression system, the Biotechnology and Biopharmaceuticals Laboratory has developed a new method for the production of recombinant human erythropoietin, which consists of a system of direct transduction of goat mammary gland with adenoviral vectors containing the human erythropoietin gene information. This system allows the efficient production of the desired recombinant protein, generating a variant of Epo with a bi-antennary glycosylation pattern and lacking sialic acid, which has a direct effect on the decrease of its hematopoietic activity (EpoL). Therefore, the present project focused on studying the EpoL/EpoR interaction in the CNS and its neuroprotective effects, mediated by a recombinant Epo variant with a bi-antennary and asialylated glycosylation pattern, against chronic oxidative stress models. Specifically, the aim of the project was to evaluate and molecularly characterize the neuroprotective capacity of EpoL, comparing it with native Epo. From the results we can point out that this variant, has a neuroprotective effect mediated by EpoR activation, increasing intracellular signaling of anti-apoptotic pathways, decreasing local oxidative stress and improving the synaptic parameters evaluated. All these effects were obtained at a concentration ten times lower than with native Epo and without altering hematocrit, which leads us to suggest the potential use of EpoL as a biopharmaceutical for the study and treatment of CNS-related diseases with an inflammatory and oxidative stress component such as cerebrovascular infarction or neurodegenerative diseases.