DJ-1 knockout augments disease severity and shortens survival in a mouse model of ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disorder, characterized by the degeneration of motor neurons. Oxidative stress plays a central role in the disease progression, in concert with an enhanced glutamate excitotoxicity and neuroinflammation. DJ-1 mutations, leading to the loss of functional protein, cause familial Parkinson's disease and motor neuron disease in several patients. DJ-1 responds to oxidative stress and plays an important role in the cellular defense mechanisms. We aimed to investigate whether loss of functional DJ-1 alters the disease course and severity in an ALS mouse model. To this end we used mice that express the human SOD1G93A mutation, the commonly used model of ALS and knockout of DJ-1 mice to generate SOD1 DJ-1 KO mice. We found that knocking out DJ-1in the ALS model led to an accelerated disease course and shortened survival time. DJ-1 deficiency was found to increase neuronal loss in the spinal cord associated with increased gliosis in the spinal cord and reduced antioxidant response that was regulated by the Nrf2 mechanism.The importance of DJ-1 in ALS was also illustrated in a motor neuron cell line that was exposed to glutamate toxicity and oxidative stress. Addition of the DJ-1 derived peptide, ND-13, enhanced the resistance to glutamate and SIN-1 induced toxicity. Thus, our results maintain that DJ-1 plays a role in the disease process and promotes the necessity of further investigation of DJ-1 as a therapeutic target for ALS.

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

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.

Fig 2. Motor function of SOD1 DJ-1 KO mice deteriorated faster than SOD1 mice.

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.

Fig 3. SOD1 DJ-1 KO mice demonstrated an accelerated disease course.

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.

Fig 4. SOD1 DJ-1 KO mice demonstrated shortened survival.

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.

Fig 5. Ventral horn motor neuron survival.

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.

Fig 6. Motor neuron loss and astrogliosis in the spinal cords of SOD1 DJ-1 KO.

Choline acyl transferase (ChAT, A) and glial fibrillary acidic protein (GFAP, B) levels were determined in the spinal cord extracts by Western blot analysis. Proteins levels were normalized to beta actin. Data is presented as averages ± SD. * p<0.05. n = 3 female and 2 males.

Fig 7. Attenuated Nrf2 system in SOD DJ-1 KO mice.

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.

Fig 8. Pre-treatment with DJ-1 derived peptide (ND-13) protected against SIN-1 and glutamate-induced toxicity in vitro.

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.