ND-13 administration attenuates 6-OHDA toxicity in DJ-1 knockout mice.

<p>(A) IV administration of ND-13 (1.5 mg/Kg dissolved in 200μl saline, 4 hours before 6-OHDA lesioning) significantly reduced the rotational behavior induced by amphetamine injection, 2 and 4 weeks after 6-OHDA striatal lesioning in 6-OHDA hemiparkinsonian transgenic DJ-1 knockout mice. Results are shown as averages ± SD. (# p<0.05, as compared to naïve mice, * p<0.05, as compared to vehicle-treated 6-OHDA mice). (B) IV administration of ND-13 restores dopamine levels, as measured by HPLC in 6-OHDA lesioned DJ-1 knockout mice. Dopamine levels were measured in each cerebral hemisphere of naïve DJ-1 knockout mice (control), or mice lesioned by 6-OHDA treated by ND-13 or vehicle. The dopamine level in the right (6-OHDA lesioned) hemisphere is presented as a percentage of the normal left hemisphere. IV administration of ND-13 (1.5 mg/Kg dissolved in 200 μl, 4 hours before 6-OHDA) statistically restored dopamine levels, as compared to vehicle-treated 6-OHDA mice. Results are shown as averages ± SD. * p<0.05 as compared to vehicle-treated 6-OHDA DJ-1 knockout mice.</p

ND-13 reduced the accumulation of intracellular reactive oxygen species and activated the Nrf2 anti-oxidant system.

<p>(A) Exposure to hydrogen peroxide resulted in intracellular accumulation of reactive oxygen species, as quantified by the DCF assay. Pretreatment with ND-13 resulted in significantly decreased accumulation of reactive oxygen species. The time dependent effect was examined by increasing the time between ND-13 treatment and the oxidative insult. ND-13 showed significant effects when applied up to 6 hours before hydrogen peroxide exposure. Cells were washed before toxin application. (B) ND-13 induced nuclear translocation of Nrf-2, as quantified by cell fractionation and the Nrf2 activity kit (TransAM) on nuclear extracts. Results are shown as averages ± SD. * p<0.05. (C-F) Pretreatment with ND-13 induced early and increased expression of Nrf-2-dependent mRNA of antioxidant genes when cells were exposed to the neurotoxin 6-OHDA. (C) Quantitative real time PCR analysis of the mRNA levels of Nrf2, (D) NAD(P)H quinine oxidoreductase-1, GCLC (E) and HO-1 (F) levels in PC-12 cells, 2 hours after exposure to increasing doses of 6-OHDA (0–50 μM). Real time PCR was done in triplicate, by the ddCT method, with glyceraldehyde 3-phosphate dehydrogenase used as a housekeeping gene. Experiments were repeated 3 times. Results are shown as averages ±SD. * p<0.05. (G, H) Western blots of HO-1 protein levels in cells exposed to increasing doses of 6-OHDA (0–50 μM) for 8 hours. ND-13 pretreated cells showed increased HO-1 protein levels (normalized versus beta-actin levels) after exposure to 6-OHDA.</p

ND-13 increased activation of the Nrf2 after exposure to 6-OHDA.

<p>(A) Quantitative real time PCR analysis of the mRNA levels of Nrf2, (B) HO-1, (C) NQO-1 and (D) GCLC levels of PC-12 cells, 5 hours after exposure to increasing doses of 6-OHDA (0–50 μM). Real time PCR was done in triplicate. GAPDH was used as a housekeeping gene. Real time PCR was done by the ddCT method. Experiments were repeated 3 times. Results are shown as averages ±SD. * p<0.05.</p

ND-13 restores dopamine levels and tyrosine hydroxylase in 6-OHDA hemiparkinsonian mice.

<p>(A) Dopamine levels in brains of naïve (control) mice or mice lesioned by 6-OHDA were measured by HPLC. Dopamine level in the right (6-OHDA lesioned) hemisphere is presented as percentage of the normal left hemisphere. IV injection of the ND-13 (1.5 mg/Kg dissolved in 200 μl, 4 hours before 6-OHDA) or SC injection of ND-13 (3 mg/Kg dissolved in 100 μl saline, 6 hours before and 1 hour after 6-OHDA lesioning) restored dopamine levels as compared to vehicle treated 6-OHDA mice (# p<0.05, as compared to naïve mice, * p<0.05, as compared to vehicle-treated 6-OHDA mice). (B) IV administration of ND-13 (1.5 mg/Kg dissolved in 200μl saline, 4 hours before 6-OHDA lesioning) markedly reduced the loss of tyrosine hydroxylase (TH) staining in the 6-OHDA-lesioned (right) substantia nigra in wild type c57/bl6 mice, as compared to vehicle-treated 6-OHDA mice. TH staining in the left (unlesioned) substantia nigra did not show significant differences.</p

ND-13 attenuates toxicity of trophic factors depletion, oxidative insults and neurotoxins.

<p>SH-SY5Y, human neuroblastoma cells, were exposed to increasing doses of hydrogen peroxide (H₂O₂, 0–60 μM), 6-OHDA (0–25 μM) or dopamine (0–10 μM) for 24 hours. ND-13 (4 μM) or vehicle were applied 1 hour before the toxic insults. (A) ND-13 attenuated H₂O₂-induced cell death, measured by LDH cytotoxicity assay and (B) restored H₂O₂-induced metabolic changes, measured by Alamar blue. As controls we used cells treated with vehicle (phosphate buffered saline) or short peptides (ND-6 and ND-8) with sequences similar to ND-13, attached to the same cell penetrating peptide as ND-13. (C) ND-13 attenuated 6-OHDA and (D) against dopamine toxicity, as measured by the LDH cytotoxicity assay. (E) ND-13 preserved viability of human neuroblastoma cells SH-SY5Y exposed to trophic factors depletion (done by serum deprivation for 48 hours, * p<0.05 as compared to cell culture with serum, # p<0.05 as compared to cell culture without serum or ND-13). (F) ND-13 does not induce cell proliferation. Human neuroblastoma cells (SH-SY5Y) were exposed to 4 μM ND-13 or vehicle for 24 hours. Cell proliferation was quantified by BrdU assay. No significant differences were observed between ND-13 treated cells and vehicle treated cells. Data are presented as means ± S.D. * p<0.05 as compared to cells exposed to vehicle and the same toxic dose. The experiments were repeated 3 times in triplicates.</p

ND-13 blood-brain-barrier penetration.

<p>C57/bl6 mice were injected with FITC-Albumin (FITC-Alb) to visualize intact blood vessels. FITC conjugated TAT peptide (FITC-TAT), FITC conjugated ND-13 lacking the TAT sequence (ND-13C-FITC) or FITC conjugated full length ND-13 (ND-13-FITC) were injected subcutaneous (SC) and monitored by CellVizio system for additional 30 min. Time dependent accumulation of the fluorescent signal in the brain parenchyma around the blood vessels was observed when mice were administrated with either TAT-FITC or ND-13-FITC but not with ND-13C, indicating that the TAT-derived cell penetrating peptide enables ND-13 to penetrate the BBB.</p

In vivo experimental designs.

<p>Experimental designs of the in vivo 6-OHDA hemiparkinsonian mice experiments are shown. (A) Design of the stereotactic intracerebral ND-13 versus vehicle experiment. (B) Design of the intravenous (IV) administration experiment. (C) Design of the subcutaneous (SC) experiment.</p