Enhanced Delivery of Erythropoietin Across the Blood-Brain Barrier for Neuroprotection against Ischemic Neuronal Injury

Abstract

Due to limited penetration of the BBB, many therapeutic agents in clinical use require higher doses in order to reach effective concentrations in brain. In some instances, these high doses elicit severe side effects. In the case of erythropoietin (EPO), an established neuroprotectant against ischemic brain injury, its low BBB permeability requires such a high therapeutic dose that it can induce dangerous complications such as polycythmia and secondary stroke. The purpose of this study is to generate a modified EPO that has increased facility crossing the BBB without losing its neuroprotective element. We have engineered a fusion protein (EPO-TAT) by tagging a protein transduction domain derived from HIV TAT to the EPO protein. This sequence enhanced the capacity of EPO to cross the BBB in animals at least twofold when IP administered and up to five-fold when IV administered. In vitro experiments showed that this EPO fusion protein retained all its protective properties against neuronal death elicited by oxygen-glucose deprivation and NMDA insults. The needed therapeutic dose of the EPO-TAT was decreased by ~10-fold compared to that of regular EPO to achieve equivalent neuroprotection in terms of reducing volume of infarction induced by middle cerebral artery occlusion in mice. Our results support the approach of using a protein transduction domain coupled to therapeutic agents. In this way, not only can the therapeutic doses be lowered, but agents without BBB permeability may now be available for clinical applications.

Figure 1. Generation of EPO fusion protein containing TAT

(a) A schematic map of plasmid pEPO-TAT-DHFR. TAT tag was PCR-added at the C-terminal of human EPO under the control of CMV promoter. Mouse DHFR was inserted downstream of IRES. A synthetic intron (IVS) was inserted between EPO-TAT and IRES to enhance the stability of the mRNA. (b) Representative graph of Western blot of stable cell lines expressing EPO-TAT. CHO DG44 cells (DHFR ^-/-) were transfected with pEPO-TAT-DHFR or p-EPO-DHFR. Stable cell lines were screened with methotrexate. EPO fusion protein in supernatant was analyzed by Western blot by loading 10 μl of medium from stable cell lines of EPO-TAT (lanes 5-7) and wild-type EPO without TAT (lanes 2-4). Commercial EPO (1 U) was used as control. (c) Western blot analysis of purified EPO-TAT and wild-type EPO recombinant protein.

Figure 2. EPO-TAT fusion protein is neuroprotective against OGD and NMDA toxicity

Cortical neurons at 13 DIV were pretreated for 24 hr with 1 U/ml wild-type EPO or EPO-TAT and then subjected to either OGD (a-g) or NMDA neurotoxicity (h). Representative graphs of Hoechst staining were shown (a-e) after OGD insults. Twenty-four hours of pre-treatment with either wild-type EPO (d) or EPO-TAT (e) reduced neuronal death, while the control protein GFP-TAT had no effect (c). Control neurons and OGD alone treated neuron are shown in panels a and b, respectively. Fragmented or condensed nuclei are indicated by red arrows. (f) Quantitative counting of cell death induced by OGD. (g) Relative LDH release compared with OGD alone. OGD resulted in increased LDH release, which was significantly prevented by EPO or EPO-TAT. (h) Neurons were challenged with 200 μM NMDA and cell death was quantitatively analyzed by Hoechst nucleus staining 24 hours after NMDA treatment. (i) Dose-response curve of EPO-TAT against OGD insults. Neurons were treated with a series concentration of EPO-TAT and EPO, and then subjected to OGD. Cell death was counted 24 hours after OGD. ^** p<0.001 versus OGD control or GFP-TAT group or NMDA alone. Data are mean ± SEM, n=12 from three independent experiments.

Figure 3. TAT fused to EPO enhances the delivery of EPO across the BBB

EPO-TAT or EPO (5000 U/kg BW) was injected either IP (a, b) or IV (c); plasma and CSF were collected 3 hr post-injection. Total EPO protein in the plasma (a) or CSF (b, c) was measured by ELISA. Data are mean ± SEM (n=4 per group). * p<0.05, ** p<0.01 versus PBS controls; ^## p<0.01 versus EPO.

Figure 4. EPO-TAT protects against focal ischemic infarction

Mice were subjected to 60 min of MCAO followed by 72 hr of reperfusion. EPO-TAT or EPO was injected intravenously at the onset of reperfusion. TTC staining was assessed 72 hr after MCAO. (a) Representative photographs of TTC-stained coronal sections of mouse brains subjected to MCAO followed by administration of PBS, EPO-TAT or EPO. (b) Quantitative measurement of infarct volume. Data are presented as mean ± SEM, n=9 per group. * p<0.05 versus PBS; # p<0.05 versus EPO at 1000 U/kg.