Brain penetrating IgG-erythropoietin fusion protein is neuroprotective following intravenous treatment in Parkinson's disease in the mouse

Abstract

Parkinson's disease (PD) is caused by oxidative stress, and erythropoietin (EPO) reduces oxidative stress in the brain. However, EPO cannot be developed as a treatment for PD, because EPO does not cross the blood-brain barrier (BBB). A brain penetrating form of human EPO has been developed wherein EPO is fused to a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), which is designated as the cTfRMAb-EPO fusion protein. The TfRMAb acts as a molecular Trojan horse to transport the fused EPO into brain via transport on the BBB TfR. Experimental PD was induced in adult mice by the intra-striatal injection of 6-hydroxydopamine, and PD mice were treated with 1mg/kg of the cTfRMAb-EPO fusion protein intravenously (IV) every other day starting 1 h after toxin injection. Following 3weeks of treatment mice were euthanized for measurement of striatal tyrosine hydroxylase (TH) enzyme activity. Mice treated with the cTfRMAb-EPO fusion protein showed a 306% increase in striatal TH enzyme activity, which correlated with improvement in three assays of neurobehavior. The blood hematocrit increased 10% at 2weeks, with no further changes at 3weeks of treatment. A sandwich ELISA showed the immune reaction against the cTfRMAb-EPO fusion protein was variable and low titer. In conclusion, the present study demonstrates that a brain penetrating form of EPO is neuroprotective in PD following IV administration with minimal effects on erythropoiesis.

Figure 1

Rotation measurements following the administration of apomorphine for PD mice treated with either saline or the cTfRMAb-EPO fusion protein. Data are mean ± S.E. (n=10 mice/group). Statistical differences from the saline treated animals are p<0.005 at weeks 1 and 2 and p<0.001 at week 3 (*).

Figure 2

Rotation measurements following the administration of amphetamine for PD mice treated with either saline or the cTfRMAb-EPO fusion protein. Data are mean ± S.E. (n=10 mice/group). Statistical differences from the saline treated animals are p<0.001 at weeks 2 and 3 (*).

Figure 3

Vibrissae-elicited forelimb placing test scores for right side, which is ipsilateral to the toxin lesion, and the left side, which is contralateral to the toxin lesion, for the saline and cTfRMAb-EPO fusion protein treated mice. All scores were measured at 3 weeks following toxin injection. Data are mean ± S.E. (n=10 mice/group). Statistical differences from the saline treated animals are p<0.001 (*).

Figure 4

Striatal TH enzyme activity on the lesioned side and the non-lesioned side in the striatum and in the frontal cortex in mice treated with either saline or the cTfRMAb-EPO fusion protein. Brain TH activity was measured at 3 weeks after toxin administration. Data are mean ± S.E. (n=10 mice/group). Statistical differences from the saline treated animals in the right striatum are p<0.001(*).

Figure 5

(A) Structure of the 2-site ELISA for detection of antibodies against the cTfRMAb-EPO fusion protein. The cTfRMAb-EPO fusion protein is used as the capture reagent, and the biotinylated cTfRMAb-EPO fusion protein is used as the detector reagent, along with a complex of streptavidin (SA) and horseradish peroxidase (HRP); the biotin moiety is designated, ‘B’. (B) Western blot of cTfRMAb-EPO fusion protein (lane 1) and biotinylated cTfRMAb-EPO fusion protein (lane 2) with a conjugate of avidin and biotinylated peroxidase. (C) Absorbance at 1:50 dilutions of mouse plasma taken at the beginning and the end of the 3-week treatment study of mice with the cTfRMAb-EPO fusion protein treatment groups. (D) Absorbance at dilutions (1:50, 1:300, 1:1000, and 1:3000) of plasma from 4 mice in the cTfRMAb-EPO fusion protein treatment group, including 2 mice that reacted the highest, and 2 mice that reacted the lowest, in the screen at 1:50 dilution (panel B).