Mol Neurobiol DOI 10.1007/s12035-014-8833-3
Soluble Epoxide Hydrolase Deficiency or Inhibition Attenuates MPTP-Induced Parkinsonism Xiaocui Qin & Qiaoqi Wu & Lifang Lin & Aimin Sun & Shuhu Liu & Xiaowen Li & Xiong Cao & Tianming Gao & Pengcheng Luo & Xinhong Zhu & Xuemin Wang
Received: 15 June 2014 / Accepted: 23 July 2014 # Springer Science+Business Media New York 2014
Abstract Soluble epoxide hydrolase (sEH) inhibition has been demonstrated to have beneficial effects on various diseases, such as hypertension, diabetes, and brain ischemia. However, whether sEH inhibition has therapeutic potential in Parkinson’s disease is still unknown. In this paper, we found that sEH expression is increased in 1-methyl-4-phenyl1,2,3,6-tetrahydro pyridine (MPTP)-treated mice, and sEH deficiency and inhibition significantly attenuated tyrosine hydroxylase (TH)-positive cell loss and improved rotarod performance. The substrate of sEH, 14,15-epoxyeicosatrienoic acid (14,15-EET), protected TH-positive cells and alleviated the rotarod performance deficits of wild-type mice but not sEH-knockout mice. Moreover, the 14,15-EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) abolished the neuronal protective effects of sEH deficiency. In primary cultured cortical neurons, MPP+ induced significant Akt inactivation in neurons from sEH wild-type mice, and this effect was not observed in neurons from knockout mice. Our data indicate that sEH deficiency and inhibition increased 14,15EET in MPTP-treated mice, which activated the Akt-mediated protection of TH-positive neurons and behavioral functioning. We also found that sEH deficiency attenuated TH-positive cell loss in a paraquat-induced mouse model of Parkinson’s. Our Xiaocui Qin and Qiaoqi Wu contributed equally to this work. X. Qin : Q. Wu : L. Lin : S. Liu : X. Li : X. Cao : T. Gao : X. Zhu : X. Wang (*) Department of Neurobiology, Southern Medical University, Guangzhou, Guangdong Province, China e-mail: [email protected]
A. Sun Department of Radiology, Southern Medical University, Guangzhou, Guangdong Province, China P. Luo Huangshi Central Hospital, Hubei Polytechnic University, Hubei Province, China
data suggest that sEH inhibition might be a powerful tool to protect dopaminergic neurons in Parkinson’s disease. Keywords Soluble epoxide hydrolase . EET . Parkinson’s disease . MPTP
Introduction The mammalian soluble epoxide hydrolase (sEH) is a ubiquitously expressed homodimeric enzyme that possesses epoxide hydrolyzing and lipid phosphatase functions . sEH is expressed in multiple human tissues, such as the liver, kidneys, lungs, ovaries, and the brain. In the brain, sEH is highly expressed in the smooth muscles of the arterioles and in neuronal cell bodies, oligodendrocytes, and astrocytes . The enzyme is mainly localized in the cytosol, but in some cell types, sEH exhibits a dual localization in both the cytosol and peroxisomes . Most of the established sEH functions are due to its epoxide hydrolase activity. Mammalian sEH prefers trans- over cis-substituted epoxides. Epoxyeicosatrienoic acids (EETs), including 5,6-EET; 8,9-EET; 11,12-EET; and 14,15-EET, are excellent substrates of sEH, and 14,15-EET is hydrolyzed with the highest Vmax and the lowest Km . EETs have numerous biological activities, such as vasodilation, anti-inflammation, anti-diabetes, and anti-platelet aggregation effects [5, 6]. EETs are metabolized by sEH to the corresponding dihydroxyeicosatrienoic acids (DHETs) . In general, DHETs are much less biologically active than are EETs [8, 9]. Therefore, the inhibition of sEH activity is used to increase the biological activities of EETs. A number of studies have proven that sEH inhibitors (sEHIs) have protective effects in ischemic stroke and cardiovascular, renal, and inflammatory diseases in murine models .
Parkinson’s disease (PD) is the second most common neurodegenerative disease and is characterized by deteriorations in motor functions that include uncontrollable tremor, postural imbalance, bradykinesia, and rigidity. The pathological hallmark of PD is an extensive and progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) . Although the mechanisms underlying this dopaminergic neurodegeneration have not been determined, neuroinflammation, oxidative stress, mitochondrial dysfunction, and impairments of the ubiquitin-proteasome system are considered to be major mechanisms of dopaminergic cell death . Varieties of studies have found that sEH inhibition improves cardiovascular and renal diseases; however, the therapeutic potential of sEH inhibitors in PD remains unknown. Given the beneficial activities of EETs, we hypothesized that sEH might be involved in the pathogenesis of PD via the modulation of the vulnerability of the dopaminergic neurons to environmental insults and that sEHIs would produce protective effects in a PD model. In the present study, we investigated whether sEH deficiency or inhibition would affect the loss of dopamine neurons in 1-methyl-4-phenyl-1,2,3,6tetrahydro pyridine (MPTP)- or paraquat-treated mice. We found that the overexpression of sEH in the in midbrain of MPTP-treated mice and sEH deficiency and inhibition attenuated the loss of dopamine neurons and the deficits in behavioral function. This neuronal protective effect might be mediated by increased EETs, particularly 14,15-EET.
(40 mg/kg) dissolved in 0.5 ml of 0.9 % saline was injected into each mouse intraperitoneally at 16 h intervals as previously reported . Fourteen days later, the mice were killed for immunohistochemistry. To induce paraquat toxicity, 10 mg/kg of paraquat was injected into the mice intraperitoneally once a week for three consecutive weeks as previously reported . The mice were killed at 1 week after the last of three paraquat exposures. Immunohistochemistry The mice were transcardially perfused with 4 % paraformaldehyde (PFA) in phosphate buffer (pH 7.4). The brains were further fixed in the same buffer overnight and then cryoprotected in sucrose solution before cryosectioning. The brains were sectioned in the coronal plane a thickness of 14 μm and collected on slides. For immunohistochemical analysis, the sections were incubated with primary antibody overnight at 4 °C after blocking with 3 % goat serum. Following the primary antibody incubation, the sections were washed in PBS and then incubated in the appropriate biotinylated secondary antibody for 1 h at room temperature. The sections were then washed in PBS and incubated in avidin-biotin peroxidase complex (Vector Laboratories) for 1 h. Immunohistochemical staining
Materials and Methods Chemicals and Antibodies MPTP and 1-methyl-4-phenyl pyridium (MPP+) were purchased from Sigma. 12-(3-Adamantan-1-yl-ureido)dodecanoic acid (AUDA) was from Cayman Chemical. 11,12-EET; 14,15EET; and 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) were from Enzo Life Sciences. Anti-tyrosine hydroxylase (TH) was from Cell Signaling Technology. The sEH antibody was kindly provided by Dr. Bruce D. Hammock. Experimental Animals Male C57BL/6 mice (8–10 weeks old, 22–28 g body weight) were used for all experiments unless otherwise stated. sEHknockout mice were obtained from Dr. Pengcheng Luo at the Hubei Polytechnic University. sEH-knockout mice (bred on a C57BL/6 background) and wild-type littermates were generated by breeding heterozygote sEH mutants. The offspring were analyzed via PCR analysis of tail DNA using primers specific for the sEH sequence (F1, 5′-tgg cac gac cct aat ctt agg ttc-3′; R2, 5′-cac gct ggc att tta aca cca g-3′; R3, 5′-cca atg aca aga cgc tgg gcg-3′) as previously reported . MPTP hydrochloride
Fig. 1 MPTP-induced increase of sEH expression in midbrain of mice. a MPTP induced a significant loss of TH-positive neurons in the wild-type mice. Representative photomicrographs displaying TH-positive cells in the SNc following saline or MPTP treatment in the wild-type mice (left). Quantification of the TH-positive cells in the SNc following saline or MPTP treatment (right). MPTP induced significant TH-positive cell loss. The data are shown as the means±the SDs (n=6). b Western blot analysis revealed increases in sEH expression in the midbrains of the MPTPtreated wild-type mice. At the different days indicated following MPTP treatment, the expressions of sEH in the midbrain tissues were analyzed with anti-sEH antibodies. The membranes were blotted with β-actin as a loading control
was visualized using a 3′3-diaminobenzidine reaction system. Photomicrographs were taken with an Olympus microscope. Quantitative Analysis of TH-Positive Cells in the SNc The loss of neurons in the SNc was determined by serial section analysis of the total number of TH-positive neurons revealed by immunostaining with a specific TH antibody. For each experimental group, six to eight mice were used. For each subject, the numbers of TH-positive neurons with clearly visible processes and nuclei were counted on six sections by an investigator who was blind to the experimental conditions. The serial sections were cut at 14 μm, and the counted sections were spaced 28 μm apart.
Analysis of Cell Viability by MTT Assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added directly to the cells, which were then incubated for 4 h. Next, the medium was removed, and 500 μl of DMSO was added to the cells. One hundred microliters of the supernatant was transferred into a 96-well plate. The optical density (OD) was measured with a spectrophotometer (Victor X3, Perkin Elmer) at 560 nm using 690 nm as the reference readout. A blank with DMSO alone was measured and the resulting value was subtracted from all values. The OD value of the control group was set as 100 % viability, and the cell survival rates of the other groups were calculated as their respective OD values divided by that of the control group.
Primary Neuron Culture Western Blot Analysis Cortical neurons were dissociated from D0 mice as previously described . Briefly, the cortical neurons were dissociated from the D0 mice and cultured in neurobasal medium supplemented with 2 % B27 and 25 μM glutamate on poly-L-lysinecoated plates. Every 3 days, half of the culture medium was replaced with fresh medium lacking glutamate. On the third day in vitro (DIV3), 5 μM cytosine arabinoside was added to the medium.
Fig. 2 sEH is not detectable in knockout mice. a The genotyping of the wild-type, heterozygous, and sEH-knockout mice. b Western blot showing that the expression of sEH in the brain tissues of the sEH-knockout mice is undetectable. c Immunocytofluorescence assay showing that sEH is undetectable in primary cultured cortical neurons from the sEH-knockout mice
The protein concentration in the RIPA buffer was determined using the BCA Protein Assay Kit (Pierce). Protein samples (10– 50 μg) were separated by 10 % SDS-PAGE and transferred to PVDF membranes. The protein levels were assessed using the specific primary antibody. Primary antibody incubation was followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity
was detected by enhanced chemiluminescence reaction (PerkinElmer) and developed on Kodak X-ray films. Rotarod Test Two weeks after the final MPTP injection, the performances of the mice on an accelerating rotarod apparatus (Panlab Harvard Apparatus, LE8500) were evaluated. The speed of the rod was set to accelerate at a constant rate from 4 to 40 rpm over 300 s. The mice were trained for five consecutive days with two trials per day. The latencies to fall from the rod were measured for two trials per day over a 5 day period. Each test trial lasted for a maximum of 6 min. At least 30 min of recovery time was allowed between trials. Statistical Analyses All values are expressed as the mean±the SD. All data were analyzed using one-way ANOVAs followed by least significant difference tests (LSDs) for multiple comparisons or independent Student’s t tests for unpaired groups, unless otherwise stated. Statistical significance was set at P