Abstract
Previously, DJ-1 modulator UCP0054278/comp-B was identified by virtual screening, where comp-B interacts with DJ-1 to produce antioxidant and neuroprotective responses in Parkinson’s disease models. However, the effect of comp-B in an in vivo Alzheimer’s disease (AD) model is yet undetermined. Thus, we examined the effect of comp-B on spatial learning, memory, and amyloid-β (Aβ) clearance in a transgenic mouse model of AD. We found that comp-B resolved the cognitive deficits, reduced insoluble Aβ42 levels, and prevented the degeneration of synaptic functions, thereby suggesting that comp-B may become a major compound for AD treatment.
INTRODUCTION
DJ-1 was first discovered as a novel oncogene product [1] and was later identified as a protein coded by the causative gene of Parkinson’s disease (PD), PARK7 [2]. DJ-1 has multiple functions, including roles in anti-oxidative stress and transcriptional regulation [3, 4]. Previously, we showed that UCP0054278/compound B (comp-B) binds to the SO2H-oxidized C106 regions of DJ-1 using the University Compound Library [5]. Like DJ-1, this compound prevents oxidative stress-induced dopaminergic neuronal death and restores locomotion defects in animal models of PD; it also reduces the infarct size in cerebral ischemia in rats [5–8]. Comp-B itself could not directly scavenge •OH, and the protective functions are lost completely in DJ-1-knockdown cells [7]. Additionally, comp-B can cross the blood–brain barrier [8]. Therefore, comp-B is promising for treating a wide range of neurodegenerative diseases.
The pathological hallmarks of Alzheimer’s disease (AD) include the development of senile plaques via the accumulation of extracellular amyloid-β (Aβ) and neurofibrillary tangles formed by the intraneuronal accumulation of hyperphosphorylated tau. Aβ accumulation in the brain parenchyma is the primary event that influences other AD pathologies, such as the formation of neurofibrillary tangles and neuronal cell death [9]. However, it is not known whether comp-B is effective in treating AD pathologies. Here, we examined the effect of comp-B on spatial learning, memory, and Aβ clearance in a transgenic mouse model of AD.
METHODS
Animals and comp-B administration
All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the Committee for Animal Research at Kyoto Pharmaceutical University. Hemizygous APdE9 mice were purchased from the Jackson Laboratory. Before the drug treatment, we tested APdE9 mice in a Morris water maze (MWM) at 12 months of age. According to the MWM results, the APdE9 mice were balanced into well-matched subgroups. Subsequently, groups of APdE9 mice were intraperitoneally administered 1 mg/kg of comp-B, 3 mg/kg of comp-B, or PBS once daily for 2 months (56 days).
2-[3-(benzyloxy)-4-methoxyphenyl]-N-[2-(7-methoxy-1,3-benzodioxol-5-yl)ethyl] acetamide (Fig. 1A), a DJ-1-binding UCP0054278/comp-B, was synthesized as described previously [5, 8].
Morris water maze
MWM tests were performed at 12 (pre-treatment) and 14 (56 days after treatment) months using an overhead-mounted video camera (Muromachi), as described previously [10].
Sample preparation and analysis
The mice were killed by cervical dislocation on the day after the final drug treatment and the brains were removed. One cerebral hemisphere was prepared for biochemical analysis and the other was post-fixed in 4% paraformaldehyde for histochemical analysis, as described previously [10]. For immunohistochemistry, the brain sections were incubated with anti-Aβ antibody (1 : 3,000; IBL). The amount of Aβ was measured by ELISA, as described previously [10]. The amounts of Aβ in Tris-buffered saline (TBS)-extracted fraction and neutralized formaldehyde (FA)-extracted fraction were tested using a human Aβ42- or Aβ40-specific ELISA kit (IBL).
For the analysis of western blotting, the membrane was incubated with antibody to DJ-1 (1 : 10,000), α-synuclein (1 : 500), synaptophysin (1 : 10,000), or drebrin (1 : 1,000) for 1 h at room temperature [11].
Statistical evaluation
All data were expressed as the mean±S.E. In the MWM tests, the mean sources during acquisition trials were analyzed by two-way repeated measures analysis of variance (ANOVA), with genotype, the test day of acquisition trials or treatment group, and the test day of acquisition trials as sources of variation. For multiple comparisons among APdE9 mice treated with vehicle and comp-B, Fisher’s protected least significant difference test was used to examine post hoc differences. In probe tests, the search bias in the target quadrant was compared between vehicle-treated nontransgenic mice and vehicle-treated APdE9 mice using the Student’s t-test. The search bias among APdE9 mice treated with vehicle and comp-B was evaluated by ANOVA with the Bonferroni/Dunn test. Significant differences among groups for the ELISA results were determined by ANOVA with the Bonferroni/Dunn test.
RESULTS
Before the drug treatment, we subjected APdE9 mice and wild-type mice to the MWM test at 12 months of age. In the acquisition trials, there were no differences in swimming speed (data not shown). A significant effect of genotype was detected on the escape latency in the acquisition trials (Fig. 1B). In the probe test, the APdE9 mice had significantly lower retention times at former platform location (target quadrant) than wild-type mice (Fig. 1C), thereby indicating that APdE9 mice exhibited declines in spatial learning and memory at 12 months of age. From 12 months of age, we intraperitoneally administered groups of APdE9 mice once daily with vehicle or 1 or 3 mg/kg of comp-B for 56 days. Three days before the final drug treatment (at 14 months of age), a second block of acquisition trials and a single probe test were started. In the acquisition trials, there were no differences in swimming speed among any of the groups (data not shown). The comparisons of APdE9 mice detected a significant drug effect on the escape latency in the acquisition trials (Fig. 1B). The post hoc analysis showed that in the acquisition trials, a high dose of comp-B significantly improved theperformance of 14-month-old APdE9 mice. In the probe test, a high dose of comp-B was increased the retention time, and the performance improved significantly with a high dose of comp-B. There was no effect of comp-B on wild-type mice (Fig. 1C). Thus, comp-B treatment improved the spatial learning and memory performance in aged APdE9 mice.
After the MWM experiment, we also examined the Aβ burden in the brains of APdE9 mice. Moderate accumulations of Aβ were detected in APdE9 mice treated with comp-B (Fig. 2A). The amounts of soluble Aβ40 and Aβ42 in the TBS-extracted fractions were almost the same between the drug treatments, whereas those of insoluble Aβ42, but not Aβ40, were decreased significantly in the FA-extracted fractions by the comp-B treatment (Fig. 2B, C). Thus, comp-B promoted Aβ clearance from the brains of APdE9 mice.
We also investigated the effects of comp-B on the expression of DJ-1 and synaptic proteins. In the cytosolic fractions from APdE9 mice, the level of DJ-1 was increased significantly from that of the wild-type mice, but comp-B did not affect the DJ-1 level (Fig. 2D, E). The DJ-1 level was increased significantly in the membrane fractions with 3 mg/kg comp-B (Fig. 2D, F). The α-synuclein levels in both the cytosolic and membrane fractions of APdE9 mice were increased significantly from those of the wild-type mice (Fig. 2D, G, H). The α-synuclein levels were decreased significantly by comp-B. The synaptophysin level in the membrane fraction of APdE9 mice significantly was decreased in comparison to wild-type mice (Fig. 2D, I). The drebrin level in the cytosolic fraction of APdE9 mice tended to decrease, but no significance (Fig. 2D, J). The drebrin level in the membrane fraction did not change (Fig. 2D, K). These protein levels of synaptophysin in the membrane fraction and drebrin in the cytosolic fraction tended to increase by treatment of comp-B. Therefore, comp-B may improve synaptic dysfunctions that are promoted by Aβ accumulation in the brains of APdE9 mice.
DISCUSSION
Oxidative stress can be defined as an imbalance between the generation of reactive oxygen species (ROS) and the antioxidant capacity of a cell. Oxidative stress plays an important role in AD pathogenesis and it is related to an early event in the AD pathology cascade [11]. Significant degeneration of the synaptic functions has been observed consistently in APdE9 mice [12, 13]. In the brains of AD patients, DJ-1 has been reported to co-localize with tau in neurofibrillary tangles, which are major characteristics of the AD pathology [14]. DJ-1 is dramatically increased in the insoluble fraction (including mitochondria) from sporadic PD and dementia with Lewy bodies [15], while abnormal oxidation of DJ-1 is found in PD and AD, which suggests that DJ-1 may play roles in both PD and AD [16]. Low level oxidized DJ-1 can translocate into the mitochondria and scavenge ROS [17]. In the present study, the level of DJ-1 was significantly increased in the membrane fraction (including mitochondria) by comp-B. Therefore, comp-B may maintain and enhance DJ-1-induced anti-oxidative activation, and these responses may help to prevent ROS production. We also found that the dysregulated expression of synaptic proteins was ameliorated by comp-B. Thus, anti-oxidative activation may also prevent degeneration of the synaptic functions caused by Aβ accumulation.
Aβ42 has a greater capacity to form oligomers and thereafter fibrils (the main constituent of amyloid plaques) and therefore has a higher neurotoxicity than its shorter counterpart Aβ40. DJ-1 is a multi-functional protein, having functions a chaperone. DJ-1 regulates its chaperone activity toward α-synuclein aggregate formation [18, 19]. Although it is necessary to further study, comp-B may enhance not only anti-oxidative stress but also the chaperone activity of DJ-1 toward Aβ42 aggregate formation.
The present study suggests that DJ-1 modulators, such as UCP0054278/comp-B, may become lead compounds for treating various oxidative stress-mediated disorders, including PD and AD.
Footnotes
ACKNOWLEDGMENTS
This study was supported partly by grants from JSPS KAKENHI, the Smoking Research Foundation (SRF), and Ritsumeikan University in Japan, and by the Program for Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation (NIBIO) in Japan.
