Noninvasive Brain Delivery and Efficacy of BDNF to Stimulate Neuroregeneration and Suppression of Disease Relapse in EAE Mice

Abstract

The number of FDA-approved protein drugs (biologics), such as antibodies, antibody-drug conjugates, hormones, and enzymes, continues to grow at a rapid rate; most of these drugs are used to treat diseases of the peripheral body. Unfortunately, most of these biologics cannot be used to treat brain diseases such as Alzheimer's disease (AD), multiple sclerosis (MS), and brain tumors in a noninvasive manner due to their inability to permeate the blood-brain barrier (BBB). Therefore, there is a need to develop an effective method to deliver protein drugs into the brain. Here, we report a proof of concept to deliver a recombinant brain-derived neurotrophic factor (BDNF) to the brains of healthy and experimental autoimmune encephalomyelitis (EAE) mice via intravenous (iv) injections by co-administering BDNF with a BBB modulator (BBBM) peptide ADTC5. Western blot evaluations indicated that ADTC5 enhanced the brain delivery of BDNF in healthy SJL/elite mice compared to BDNF alone and triggered the phosphorylation of TrkB receptors in the brain. The EAE mice treated with BDNF + ADTC5 suppressed EAE relapse compared to those treated with BDNF alone, ADTC5 alone, or vehicle. We further demonstrated that brain delivery of BDNF induced neuroregeneration via visible activation of oligodendrocytes, remyelination, and ARC and EGR1 mRNA transcript upregulation. In summary, we have demonstrated that ADTC5 peptide modulates the BBB to permit noninvasive delivery of BDNF to exert its neuroregeneration activity in the brains of EAE mice.

Keywords: ADTC5; BBB modulator (BBBM); BDNF; EAE; blood−brain barrier; cadherin peptide; neuroregeneration.

Figure 1.

Effect of treatment of SJL/elite EAE mice, an animal model for MS, with BDNF (5.71 nmol/kg) + ADTC5 (10 μmol/kg; n = 7), BDNF alone (5.71 nmol/kg; n = 6), ADTC5 alone (10 μmol/kg; n = 5), or vehicle (n = 5) during remission on days 21, 25, 29, 33, 37, 41, 45, and 48. (A) Clinical disease score vs time of mice treated eight times with BDNF + ADTC5, BDNF alone, ADTC5 alone, or vehicle. (B) Comparison of area under the curve (AUC) of the disease scores from days 21 to 55 from EAE mice treated with BDNF + ADTC5, BDNF alone, ADTC5 alone, or vehicle. *p ≤ 0.05; one-way ANOVA (95% confidence).

Figure 2.

Effects of BDNF (5.71 nmol/kg) + ADTC5 (10 μmol/kg), BDNF alone (5.71 nmol/kg), or vehicle treatments on remyelination in the lateral corpus callosum and surrounding cortex of the brains of SJL/elite EAE mice as stained by Luxol fast blue. (A) Grayscale, the binary conversion, and color photomicrograph of myelin images taken under identical exposure of the lateral corpus callosum of EAE mice treated with BDNF + ADTC5, BDNF alone, or vehicle; the red arrows indicate breakages in the myelin. (B) Quantitative myelin densiometric comparison of white spaces (demyelination) in the brain of BDNF + ADTC5, BDNF alone, and vehicle-treated EAE mice; scale bar = 50 μm; **p ≤ 0.01 ***p ≤ 0.001; one-way ANOVA (95% confidence; n = 5).

Figure 3.

Effects of BDNF (5.71 nmol/kg) + ADTC5 (10 μmol/kg), BDNF alone (5.71 nmol/kg), or vehicle treatments in the presence of NG2 receptor in the medial corpus callosum of brains of SJL/elite EAE mice as stained by DAB. (A) Color photomicrograph of anti-NG2 staining (brown) taken under identical conditions from the medial corpus callosum for mice treated with BDNF + ADTC5, BDNF alone, vehicle; the red arrows point to dense regions of activated NG2-glia. (B) Quantitative NG2 density comparison among the EAE mice treated with BDNF + ADTC5, BDNF alone, and vehicle; scale bar = 50 μm; **p ≤ 0.01; one-way ANOVA (95% confidence; n = 5).

Figure 4.

Effects of BDNF (5.71 nmol/kg) + ADTC5 (10 μmol/kg), BDNF alone (5.71 nmol/kg), or vehicle treatments on mRNA expression of EGR1 and ARC in the cortex of the brains of SJL/elite EAE mice. (A, B) Photomicrograph of DAPI (blue), EGR1 (green), ARC (magenta), and composite images taken of the cortex of the midbrain (A) and hindbrain (B) of EAE mice treated with BDNF + ADTC5, BDNF alone, or vehicle. (C) Quantitative comparison of EGR, ARC, and NOS1 mRNA transcript expression, as determined by cell count, for mice treated with BDNF + ADTC5, BDNF alone, or vehicle. (D) Quantitative comparison of DAPI cell count; scale bar = 50 μm; ***p ≤ 0.001; one-way ANOVA (99% confidence; n = 5). Contrast and brightness of images were adjusted only for display purposes.

Figure 5.

Western blot detection of recombinant BDNF and pTrkB from mice treated with either BDNF + ADTC5 or BDNF alone. (A) Western blot probing for recombinant BDNF in the brains of mice that received BDNF (5.71 nmol/kg) + ADTC5 (10 μmol/kg; A1, A2, A3) or BDNF alone (5.71 nmol/kg, B1, B2, B3); “L” represents molecular weight ladder; “+” represents the positive control of recombinant BDNF; the red arrows highlight increased recombinant BDNF detection. (B) Western blot probing for recombinant BDNF after dosage increase in healthy mice that received BDNF (57.1 nmol/kg) + ADTC5 (10 μmol/kg; A1, A2), BDNF (28.6 nmol/kg) + ADTC5 (10 μmol/kg; A3), or BDNF alone (28.6 nmol/kg; B1, B2, B3); the red arrows highlight increased recombinant BDNF detection. (C) Western Blot probing for pTrkB after dosage increase of healthy mice that received BDNF (57.1 nmol/kg) + ADTC5 (10 μmol/kg; A1, A2), BDNF (28.6 nmol/kg) + ADTC5 (10 μmol/kg; A3), or BDNF alone (28.6 nmol/kg; B1, B2, B3); the red arrows highlight increased pTrkB detection. (D) Total protein stain (loading control) for samples treated with BDNF 57.1 nmol/kg or 28.6 nmol/kg in (B) and (C). (E) Graphical representation of recombinant BDNF detection level in mice that received BDNF (57.1 nmol/kg) + ADTC5 (10 μmol/kg; A1, A2), BDNF (28.6 nmol/kg) + ADTC5 (10 μmol/kg; A3), or BDNF alone (28.6 nmol/kg; B1, B2, B3). (F) Graphical representation of pTrkB detection level for mice that received BDNF (57.1 nmol/kg) + ADTC5 (10 μmol/kg; A1, A2), BDNF (28.6 nmol/kg) + ADTC5 (10 μmol/kg; A3), or BDNF alone (28.6 nmol/kg; B1, B2, B3). (G) Graphical representation of total protein loaded among all groups. Contrast and brightness of images were adjusted only for display purposes.