Phenylbutyrate up-regulates the DJ-1 protein and protects neurons in cell culture and in animal models of Parkinson disease

Abstract

Parkinson disease is caused by the death of midbrain dopamine neurons from oxidative stress, abnormal protein aggregation, and genetic predisposition. In 2003, Bonifati et al. (23) found that a single amino acid mutation in the DJ-1 protein was associated with early-onset, autosomal recessive Parkinson disease (PARK7). The mutation L166P prevents dimerization that is essential for the antioxidant and gene regulatory activity of the DJ-1 protein. Because low levels of DJ-1 cause Parkinson, we reasoned that overexpression might stop the disease. We found that overexpression of DJ-1 improved tolerance to oxidative stress by selectively up-regulating the rate-limiting step in glutathione synthesis. When we imposed a different metabolic insult, A53T mutant α-synuclein, we found that DJ-1 turned on production of the chaperone protein Hsp-70 without affecting glutathione synthesis. After screening a number of small molecules, we have found that the histone deacetylase inhibitor phenylbutyrate increases DJ-1 expression by 300% in the N27 dopamine cell line and rescues cells from oxidative stress and mutant α-synuclein toxicity. In mice, phenylbutyrate treatment leads to a 260% increase in brain DJ-1 levels and protects dopamine neurons against 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) toxicity. In a transgenic mouse model of diffuse Lewy body disease, long-term administration of phenylbutyrate reduces α-synuclein aggregation in brain and prevents age-related deterioration in motor and cognitive function. We conclude that drugs that up-regulate DJ-1 gene expression may slow the progression of Parkinson disease by moderating oxidative stress and protein aggregation.

Figures

FIGURE 1.

PB and SB increase DJ-1 expression in N27 and HEK293 cells. A–C, N27 cells were incubated with sodium phenylbutyrate or sodium butyrate at various concentrations for 48 h. The cell lysates were separated in 12% SDS-PAGE and probed with DJ-1 and β-actin antibody. Duplicate treatments in 6-well plates were used, and experiments were repeated three times. A typical Western blot image is shown in A. B and C, quantitative data from Western blot images are shown (*, p < 0.05; **, p < 0.01 compared with control, n = 6). (D and E) HEK293 reporter cells expressing human DJ-1 promoter-Luciferase were treated with PB or SB at indicated doses for 48 h, followed by luciferase assay. Duplicate treatments in 24-well plates were used, and experiments were repeated three times. The average luciferase activity is shown. (*, p < 0.05; **, p < 0.01 compared with control, n = 6.)

FIGURE 2.

Sodium phenylbutyrate protects N27 cells from oxidative stress and mutant α-synuclein toxicity. A–B and E–F, N27 cells were incubated with PB (0.15 mm) for 48 h, followed by 24 h treatment with H2O2 or 6-OHDA at various concentrations. Sample images were shown in A-B with cells identified by GFP adenovirus expression. Cell viability was determined by MTT assays and shown in E–F. C–D and G–H, N27 cells were incubated with various doses of PB for 48 h, followed by 48 h treatment of adenovirus expressing A53T human α-synuclein (200 pfu/cell). N27 cells with α-synuclein aggregates were identified by α-synuclein antibody LB509 staining, with sample images shown in C–D. Three random fields (150–200 cells per field) were examined to determine the percentage of cells with α-synuclein aggregates (arrows in G–H). Triplicate treatments in 24-well plates were used, and experiments were repeated three times. (*, p < 0.05; **, p < 0.01 compared with control, n = 9.)

FIGURE 3.

Knockdown of endogenous DJ-1 abolishes neuroprotection from sodium phenylbutyrate. The N27 cells were transduced with adenovirus expressing shDJ-1 or Ad-GFP at concentration of 200 pfu/cell for 2 days. The cells were then added with or without PB treatment for 48 h, followed by exposure to hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA) for 24 h. A, sample Western blot showed more than 85% reduction of endogenous DJ-1 treated by shDJ-1 adenovirus. B and C, cell viability was measured by MTT assay. Results showed that knockdown of DJ-1 abolished neuroprotective effects of PB in N27 cells (compare vertical and horizontal bars to white or black bars). Triplicate treatments in 24-well plates were used, and experiments were repeated three times. (*, p < 0.05; **, p < 0.01 compared with control, n = 9.)

FIGURE 4.

Sodium phenylbutyrate and sodium butyrate increased DJ-1 expression in mouse brain. A–C, adult C57BL/6 mice (ages 4–6 months old) were treated with PB or SB at different doses in drinking water for 2 weeks. Brain tissues (cortex) were used for Western blotting with DJ-1, α-synuclein, and β-actin antibodies. Four mice were used for each dose, and an equal amount of protein was loaded in each lane after being normalized to β-actin. Typical Western blot images of DJ-1 and α-synuclein are shown in A. B–C, quantitative data of DJ-1 and α-synuclein levels after PB and SB treatment (*, p < 0.–05; **, p < 0.01 compared with control, n = 4.)

FIGURE 5.

Sodium phenylbutyrate prevented mouse dopamine neuron death after MPTP lesion. A–H, adult C57BL/6 mice (4–6 months old) were treated with PB (1000 mg/liter, 5.4 mm) in drinking water for 2 weeks, followed by 4 injections of MPTP at 2-h intervals (20 mg/kg, intraperitoneal). The mice were sacrificed 7 days after MPTP injections. A, dopamine content in mouse striatum tissues were measured by HPLC (6 mice per group, *, p < 0.05 compared with MPTP alone). B–D, striatal TH and DJ-1 protein levels were measured by Western blotting, with sample images shown in B. C–D, quantitative data of TH and DJ-1 levels (n = 6, **, p < 0.01 compared with MPTP alone). E, mouse brains were fixed, and sections were immunostained with TH antibody. The number of dopamine neurons in the substantia nigra was determined using unbiased counting methods (n = 5, *, p < 0.05 compared with MPTP alone). F–H, sample images are shown with TH immunostaining in the substantia nigra in three groups of mice. Bar length, 1 mm for F–H.

FIGURE 6.

Sodium phenylbutyrate improved motor function in aged α-synuclein transgenic mice. The Y39C transgenic mice were divided into two age groups: 6–8 months old (Young Tg, n = 20, A, C) and 10–12 months old (Old Tg, n = 20, B, D). Half of the transgenic mice in each age group (n = 10) received PB (1000 mg/liter, 5.4 mm) in drinking water, while the other mice (n = 10) were treated with water containing sodium chloride in equal molarity (vehicle). At 6 weeks and 12 weeks of drug treatment, all mice were tested for motor function using a rotarod (speed 3–33 rpm). A and C, Young Tg mice had no differences in rotarod performance between PB and vehicle treatments. B and D, PB treatment in Old Tg mice led to significant improvement in rotarod performance compared with vehicle-treated transgenic mice at both 6 week and 12 week tests. (*, p < 0.05; **, p < 0.01, n = 10.)

FIGURE 7.

Sodium phenylbutyrate improved cognitive function in aged α-synuclein transgenic mice. The Y39C transgenic mice were divided into two age groups: 6–8 months old (Young Tg, n = 20, A, C) and 10–12 months old (Old Tg, n = 20, B, D). Half of the transgenic mice in each age group (n = 10) received PB (1000 mg/liter, 5.4 mm) in drinking water, while the other mice (n = 10) were treated with water containing sodium chloride in equal molarity (vehicle). After 6 weeks and 12 weeks of drug treatment, all mice were tested for cognitive function using the Morris water maze. A, C, Young Tg mice had no differences in learning ability between PB and vehicle treatments. B, D, PB treatment in Old Tg mice significantly improved learning ability in the last day of water maze testing compared with vehicle-treated transgenic mice at both 6-week and 12-week tests. (*, p < 0.05; **, p < 0.01, n = 10.)

FIGURE 8.

Sodium phenylbutyrate increased DJ-1 expression and reduced α-synuclein oligomer formation and aggregation in aged transgenic mice. The Y39C transgenic mice were divided into two age groups: 6–8 months old (Young Tg, n = 20) and 10–12 months old (Old Tg, n = 20). Half of the transgenic mice in each age group (n = 10) received PB (1000 mg/liter) in drinking water, while the other mice (n = 10) were given water with equimolar of sodium chloride (vehicle). After 6 weeks and 12 weeks of drug treatment, all mice were tested for motor and cognitive function. After the last behavioral test, half of the mice (n = 5 for each age and treatment group) were sacrificed for biochemical analysis; the remaining half (n = 5 for each age and treatment group) were sacrificed for histology. A, brain tissues (cortex, striatum, and hippocampus) from Young and Old transgenic mice with or without PB treatment were analyzed for α-synuclein aggregation. Western blots showed that PB dramatically reduced α-synuclein oligomer formation in old transgenic mice compared with mice of the same age not receiving PB treatment. A sample blot shows 15 month Tg with (+PB) or without (−PB) treatment), while α-synuclein monomer levels were not changed. B, ratio of α-synuclein oligomer to monomer is shown in Young and Old transgenic mice with or without PB treatment (n = 5, **, p < 0.01). Old transgenic mice had high levels of oligomer (15%). After three months of PB treatment, old mice had much lower levels of oligomer which were similar to the ratio seen in Young transgenic mice. C-D, brain tissues (cortex) from Young and Old transgenic mice with or without PB treatment were analyzed for DJ-1 protein levels using Western blot (C). Results showed that PB treatment significantly increased brain DJ-1 levels in both young and old transgenic mice compared with mice without PB treatment (D; *, p < 0.05, n = 5). E-I, brain sections from Young and Old transgenic mice with or without PB treatment were immunostained with human α-synuclein antibody (LB509). Sections were examined for α-synuclein-positive Lewy body-like inclusions. Sample images from Young and Old transgenic mice with or without PB treatment are shown in E–H. A sample image from 15-month-old non-transgenic mouse is shown in I. J, results showed that the percentage of α-synuclein-positive neurons with LB-like inclusions (arrows) was significantly reduced in Old transgenic mice with PB treatment compared with old mice without PB (*, p < 0.05, n = 5), while there was no significant change in Young transgenic mice after PB treatment. Bar length, 25 μm for E–I.