Brain Penetrating Bifunctional Erythropoietin-Transferrin Receptor Antibody Fusion Protein for Alzheimer's Disease

Abstract

Erythropoietin (EPO), a glycoprotein cytokine essential to hematopoiesis, has neuroprotective effects in rodent models of Alzheimer's disease (AD). However, high therapeutic doses or invasive routes of administration of EPO are required to achieve effective brain concentrations due to low blood-brain barrier (BBB) penetrability, and high EPO doses result in hematopoietic side effects. These obstacles can be overcome by engineering a BBB-penetrable analog of EPO, which is rapidly cleared from the blood, by fusing EPO to a chimeric monoclonal antibody targeting the transferrin receptor (cTfRMAb), which acts as a molecular Trojan horse to ferry the EPO into the brain via the transvascular route. In the current study, we investigated the effects of the BBB-penetrable analog of EPO on AD pathology in a double transgenic mouse model of AD. Five and a half month old male APPswe/PSEN1dE9 (APP/PS1) transgenic mice were treated with saline ( n = 10) or the BBB-penetrable EPO ( n = 10) 3 days/week intraperitoneally for 8 weeks, compared to same-aged C57BL/6J wild-type mice treated with saline ( n = 8) with identical regiment. At 9 weeks following treatment initiation, exploration and spatial memory were assessed with the open-field and Y-maze test, mice were sacrificed, and brains were evaluated for Aβ peptide load, synaptic loss, BBB disruption, microglial activation, and microhemorrhages. APP/PS1 mice treated with the BBB-penetrable cTfRMAb-EPO fusion protein had significantly lower cortical and hippocampal Aβ peptide number ( p < 0.05) and immune-positive area ( p < 0.05), a decrease in hippocampal synaptic loss ( p < 0.05) and cortical microglial activation ( p < 0.001), and improved spatial memory ( p < 0.05) compared with APP/PS1 saline controls. BBB-penetrating EPO was not associated with microhemorrhage development. The cTfRMAb-EPO fusion protein offers therapeutic benefits by targeting multiple targets of AD pathogenesis and progression (Aβ load, synaptic loss, microglial activation) and improving spatial memory in the APP/PS1 mouse model of AD.

Keywords: Alzheimer’s disease; blood−brain barrier; erythropoietin; monoclonal antibody; transcytosis; transferrin receptor.

Figure 1:. Effect of the cTfRMAb-EPO fusion protein on Aβ peptide load in the APP/PS1 mice with 6E10 immuno-staining.

Representative images of 6E10 MAb- positive Aβ peptide in the cortex and hippocampus of saline- and cTfRMAb-EPO-treated APP/PS1 and saline treated WT mice (A). Significant reduction in cortical and hippocampal 6E10 MAb-positive Aβ peptide area (B, E) and number (C, F) in the cTfRMAb-EPO treated APP/PS1 mice compared with APP/PS1 treated saline controls. No difference in the 6E10 MAb-positive Aβ peptide size between saline and cTfRMAb- EPO treated APP/PS1 mice (D, G). Scale bar = 100μm. Data are presented as mean ± SD of 8–10 mice per group. One-way ANOVA with Bonferroni’s correction or Kruskal Wallis with Dunn’s correction. *p<0.05, **p<0.01.

Figure 2:. Effect of the cTfRMAb-EPO fusion protein on mature Aβ plaque load in the APP/PS1 mice with thio-S staining.

Representative images of thio-S positive mature Aβ plaque in the cortex of saline- and cTfRMAb-EPO-treated APP/PS1 and saline treated WT mice (A). Significant reduction in cortical thio-S positive mature Aβ plaque area (B) and number (C) in the cTfRMAb-EPO treated APP/PS1 mice compared with saline treated APP/PS1 mice. Cortical thio-S positive mature Aβ plaque size did not differ between saline and cTfRMAb-EPO treated APP/PS1 mice (D). Scale bar = 100μm. Data are presented as mean ± SD of 8–10 mice per group. One-way ANOVA with Bonferroni’s correction for equal variance or Games-Howell for unequal variance, or Kruskal Wallis with Dunn’s correction. *p<0.05, **p<0.01.

Figure 3:. Effect of the cTfRMAb-EPO fusion protein on Iba-1, a marker of microglial activation, in the APP/PS1 mice.

Representative images of cortical and hippocampal Iba-1 immunostaining in the saline treated WT, saline treated APP/PS1 and cTfRMAb-EPO treated APP/PS1 mice (A). WT mice had significantly lower cortical and hippocampal Iba-1 positive immunoreactive area compared with saline treated APP/PS1 mice (B-C). Iba-1 positive immunostaining was found concentrated around plaque like structures in the APP/PS1 mice (A; black arrow heads). Significant reduction in the cortical Iba-1 positive immunoreactive area in the cTfRMAb-EPO treated APP/PS1 mice compared with saline treated APP/PS1 mice (B). A trend towards reduction in the hippocampal Iba-1 positive immunoreactive area in the cTfRMAb-EPO treated APP/PS1 mice compared with saline treated APP/PS1 mice (C). Data are presented as mean ± SD of 5–8 mice per group. One-way ANOVA with Bonferroni’s correction. *p<0.05, ***p<0.001, ****p<0.0001.

Figure 4:. Effect of the cTfRMAb-EPO fusion protein on brain IgG, marker of blood- brain barrier disruption, in the APP/PS1 mice.

Representative images of cortical and hippocampal brain parenchymal IgG immunostaining in the saline treated APP/PS1, cTfRMAb-EPO treated APP/PS1 and saline treated WT mice (A). IgG positive immunostaining was found concentrated around plaque like structures in the APP/PS1 mice (A; black arrow heads). No significant difference in cortical or hippocampal brain IgG between any experimental groups (B-C). Data are presented as mean ± SD of 8–10 mice per group. One-way ANOVA with Bonferroni’s correction for equal variance or Games-Howell for unequal variance, or Kruskal Wallis with Dunn’s correction.

Figure 5:. Effect of the cTfRMAb-EPO fusion protein on cerebral microhemorrhage development.

Prussian blue stained (indicated by arrowheads) representative images showing no significant increase in CMH development with chronic cTfRMAb-EPO treatment. Scale bar = 25μm.

Figure 6.. Effect of the cTfRMAb-EPO fusion protein on synaptophysin, a marker of synaptic loss, in the APP/PS1 mice.

Representative images of hippocampal synaptophysin positive immunostaining (green) in the saline treated WT, saline treated APP/PS1 and cTfRMAb-EPO treated APP/PS1 mice. Primary antibody omitted section represents the control. Blue stain represents DRAQ5 positive nuclei (A). Significantly higher synaptophysin positive MFI in the DG (B), CA3 (C) and total (D) hippocampal region in the cTfRMAb-EPO treated APP/S1 mice compared with the saline treated APP/PS1 mice. Data are presented as mean ± SD of 8–10 mice per group. One-way ANOVA with Bonferroni’s correction for equal variance or Games-Howell for unequal variance, or Kruskal Wallis with Dunn’s correction. *p<0.05.

Figure 7.. Effect of the cTfRMAb-EPO fusion protein on spatial memory and exploration in the APP/PS1 mice.

Representative trajectories of saline treated WT, saline- and cTfRMAb-EPO-treated APP/PS1 mice during the training and testing phase of the Y-maze (A). Six-weeks after treatment initiation, saline treated WT mice showed a preference for the novel arm however, nine-weeks after treatment initiation, both the saline treated WT and cTfRMAb-EPO treated APP/PS1 mice showed a preference for the novel arm (B). Representative trajectories of saline treated WT, saline- and cTfRMAb-EPO-treated APP/PS1 mice during the open-field test (C). Six-weeks after treatment initiation, cTfRMAb-EPO treated APP/PS1 mice had significantly lower distance travelled (D) and mean speed (E) compared with saline treated WT mice. At 9 weeks, no differences in distance travelled or mean speed were observed between any experimental groups (D-E). Data are presented as mean ± SD of 8–10 mice per group. For Y-maze, one-sample t test with a hypothesized value of 33% (indicated by the dotted line). Two-way repeated measures ANOVA for the open-field analysis with Bonferroni’s correction. *p<0.05, **p<0.01.