SOD1 DJ-1 KO mice demonstrated shortened survival.

<p>Kaplan-Meier survival curves of (A) male and female SOD1 DJ-1 KO mice and SOD1 mice.(B) The average survival (in days) of SOD1 DJ-1 KO mice and SOD1 mice is presented as averages ± SD. ** P<0.01. n = SOD1 mice 9F and 9M, SOD1 DJ-1 KO mice 14F and 22M.</p

SOD1 DJ-1 KO mice demonstrated an accelerated disease course.

<p>Accelerated deterioration of gait was observed in SOD1 DJ-1 KO mice as compared to SOD1 mice. The score for gait of (A) female and (B) male mice was evaluated on a clinical scale of 5 (normal) to 1 (severely pathologic). Hind limb splay reflex of female (C) and male (D) mice p<0.05, SPSS repeated measures. n = SOD1 mice 9F and 9M, SOD1 DJ-1 KO mice 14F and 22M.</p

Pre-treatment with DJ-1 derived peptide (ND-13) protected against SIN-1 and glutamate-induced toxicity <i>in vitro</i>.

<p>NSC-34 cells were exposed to peroxynitrite ion generator 3-morpholinosydnonimine (SIN-1), and cell survival was evaluated by Alamar blue assay (A). The experiment was reapeted 3 times and the results are presented as averages ± SD. * p<0.05. Glutamate-induced apoptosis was measured by Annexin V-FITC, PI staining using FACS analysis (B). Different stages of apoptosis were differentiated as follows: early apoptosis (Annexin V+/PI−); late apoptosis/necrosis cells (Annexin V+/PI+); and viable cells (Annexin V−/PI−). Histograms represent the proportion of apoptotic cells relative to total cells. Plots were calculated from the histogram distributions (avarages ± SD). # p<0.05; ## p<0.01; ***, ### p<0.001; * vs. control; and # vs. glutamate-treated group.</p

Attenuated Nrf2 system in SOD DJ-1 KO mice.

<p>Mutated SOD1 increased the expression of Nrf2 and HO-1 while SOD1 DJ-1 KO demonstrated reduction. Nuclear factor erythroid-2 related factor 2 (Nrf2, A) and hemeoxygenase 1(HO-1, B) protein levels were determined in the spinal cord extracts by Western blot anlysis. Proteins levels were normalized to beta actin. Data is presented as averages ± SD. * p<0.05. n = 3 female and 2 males.</p

Motor function of SOD1 DJ-1 KO mice deteriorated faster than SOD1 mice.

<p>The motor function and deterioration was evaluated by Rotarod (A, female mice; B, male mice). The age at which the performance of the animals fell below 150 seconds was taken as an index ofdeteriorationonset. (C, females; D, males).Data is presented as averages ± SE. ** p<0.01. n = SOD1 mice 9F and 9M, SOD1 DJ-1 KO mice 14F and 22M.</p

SOD1 DJ-1 KO-mice lost weight earlier and faster than SOD1 mice.

<p>A, Weight of female mice. B, Weight of male mice. Disease onset was calculated retrospectively as the day the mouse reached peak body weight. Disease onset was significantly earlier in both male and female SOD1 DJ-1 KO mice as compared to SOD1 mice (C, female mice; D, male mice). Data is presented as averages ± SE. * p<0.05, ** p<0.01. n = SOD1 mice 9F and 9M, SOD1 DJ-1 KO mice 14F and 22M.</p

Ventral horn motor neuron survival.

<p>Lumbar spinal cord sections were stained by Nissl stain. Motor neurons were identified according to the criteria detailed in the methods section. At symptomatic disease stage (15 weeks), motor neurons loss was augmented in SOD1 DJ-1 KO as compared to SOD1 mice. Nissl Stain of lumbar spinal cord sections of SOD1 mice are presented in A (low magnification) and B (high magnification). Nissl Stain of lumbar spinal cord sections of SOD1 DJ-1 KO mice are presented in C (low magnification) and D (high magnification). The red circles signify the ventral horns, from which the high magnification pictures were taken. Quantification of motor neurons in the ventral horn of the lumbar spinal cord in the different groups is shown graphically (E). The values are presented as averages ± SD. ** p< 0.001 vs. WT mice; # p< 0.01 SOD1 DJ-1 KO mice vs. SOD1 mice.</p

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 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