Study Of Protective Effect Of PARP Inhibitor PJ34 On A Mouse Model Of Parkinson's Disease

PrefaceParkinson's disease(PD) is a neurodegenerative movement disorder usually affecting large numbers of people in middle to old age. The predominant neuropathological feature of PD is characterized by a progressive loss of dopaminergic neurons and a decrease of tyrosine hydroxylase expression in substantia nigra pars compacta(SNc), giving rise to a decrease release of dopamine in nigrostriatal pathway and dopamine-acetylcholine imbalance in striatum. The cardinal physical signs of the disease are resting tremor, rigidity, bradykinesia. Recently with the trends of population aging, the incidence rate of PD is rising every year. Since the pathological mechanism of PD is not well understood, no one has discovered a medicine can slower, prevent or reverse the degeneration process of dopaminergic neurons in clinical research. Exploration of effective and neuroprotective treatment project for PD has become a hotspot at present.Poly(ADP-ribose) polymerase(PARP) is a ribozyme. It has been confirmed in animal experiments that PARP inhibitors exerted protective effects to cardio-cerebral vascular diseases such as focal cerebral ischemia and myocardial ischemia. Recent studies indicated that PARP activation also took part in the pathological process of PD. Apoptosis-inducing factor (AIF) is a mitochondrial intermembrane flavoprotein that is translocated to the nucleus when apoptosis signals stimulate the cell and result in increasing the permeabilization of the mitochondrial outer membrane. AIF plays an important role in inducing nuclear chromatin condensation as well as large-scale DNA fragmentation which can induce cell apoptosis. Animal experiment reports indicate that PARP activation signals AIF release from mitochondria and translocate into nucleus.In the present study 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine(MPTP) -induced mouse model of PD was prepared. The number, ultrastructure, apoptosis of dopaminergic neurons in SNc and behavioral changes of the mice were evaluated. The change of PARP activity as well as translocation and gene expression of AIF was investigated to explore the effect of PARP activity change on the pathological mechanism of PD. PJ34, a novel potent inhibitor of PARP was pretreated to certain PD mice at the first time to study the effect of PJ34 on the results above with the aim to provide theoretical and experimental basis for preventive and therapeutic clues to PD.MethodsMale C57/Black mice (2-3 month old) were used in this study with four intraperitoneal injections. A total of four experimental groups of mice selected with random were divided as below: control group; PJ34 group; MPTP group and PJ34+MPTP group. The symptoms such as tremor, rigidity, bradykinesia were investigated. Pole test and traction test were recorded to evaluate behavioral changes of the mice 10 min after the last injection to prove wether the PD mouse models were successfully made and the effect of PJ34 on PD mouse behavior. Midbrain samples were harvested at 2 h, 24 h and 72 h timepoint. The number of dopaminergic neurons was quantified by tyrosine hydroxylase(TH) immunohistochemical analysis and Nissl staining. The ultrastructural change of dopaminergic neurons was evaluated by transmission electron microscope. PARP activity change was assessed by PAR immunohistochemical and Western blot analysis. Apoptosis in SNc was quantified by TUNEL staining. AIF expression and its localization were examined by using immunohistochemistry. Expression of AIF mRNA was assessed by RT-PCR, and nuclear translocation of AIF was determined by Western blot. Data were expressed as mean±standard deviation (SD) and analyzed using SPSS 13.0. Differences among means were analyzed using one-way ANOVA and t test were used to compare the difference in means between two groups. A P value less than 0.05 was considered statistically significant.Results1. Behavioral observationMPTP group and PJ34+MPTP group mice manifested the abnormal behavior of tremor, tail rigidity, climbing pole slowly and decreasing in locomotor activity 3 min after MPTP injection. This phenomenon could last for 30 min which indicated that the PD model was successfully made. The mice in control group and PJ34 group manifested no abnormal behavior. PJ34 could improve the behavioral impairments of PD mice (P< 0.05).2. Nissl staining and TH immunohistochemical staining assayThe number of Nissl-positive neurons and TH immunoreactive (TH-IR) neurons in SNc of control group and PJ34 group was much more than that of MPTP group at each timepoint (P<0.01). The number of Nissl-positive neurons and TH-IR neurons in SNc of MPTP group and PJ34+MPTP group decreased gradually with the number of the latter was much more than that of the former at each timepoint (P<0.01).3. Transmission electron microscopic assayThe nucleus membrane of dopaminergic neurons appeared shrinking and the chromatin showed agglutination with tendency of border agglutination in the SNc of MPTP group which aggravated gradually. The ultrastructural morphological impairments were markly improved in PJ34+MPTP group compared with MPTP group. The shape of DA neurons in SNc of control and PJ34 group was normal.4. PARP activity assayImmunohistochemical detection of PAR protein showed specific expression of PAR in SNc nuclei of MPTP group which reached its peak at 2h, then decreased with time. PAR expression of PJ34+MPTP group was less than that of MPTP group at each timepoint (P<0.01). In SNc of control and PJ34 group, no PAR was detected.Western blot detection of PAR protein showed specific expression of PAR in nuclear protein of MPTP group which reached its peak at 2h,then decreased with time and was still present at 72h (P<0.01). Specific bands of PAR could only be observed at 2h in nuclear protein of PJ34+MPTP group. PAR expression of PJ34+MPTP group was less than that of MPTP group at each timepoint (P<0.01). In nuclear protein of control and PJ34 group, no PAR specific band was detected.5. TUNEL staining assayIn SNc of control and PJ34 group, no TUNEL positive cell was observed. TUNEL positive nuclei were detected in SNc of MPTP group which reached its peak at 2h, then decreased with time. The number of TUNEL positive nuclei was markly reduced in SNc of PJ34+MPTP group. There was a statistically significant difference between TUNEL positive nuclei number in MPTP and PJ34+MPTP group (P<0.05).6. Nuclear translocation and gene expression of AIF Immunohistochemical detection of AIF protein showed nuclear translocation ofAIF in SNc of MPTP group which reached its peak at 2h,then decreased with time. Nuclear translocation of AIF of PJ34+MPTP group was less than that of MPTP group at each timepoint.In SNc of control and PJ34 group, no nuclear translocation of AIF was detected. There was no statistically significant difference between the protein expression level of AIF in SNc of the four groups (P>0.05).Western blot analysis of AIF nuclear protein showed a specific band in MPTP group which reached its integrated density value peak at 2h, then decreased with time (P<0.01). The specific band could only be observed at 2h in PJ34+MPTP group. There was a statistically significant difference between AIF nuclear translocation in MPTP and PJ34+MPTP group (P<0.01). In control and PJ34 group, no AIF specific band was detected. RT-PCR analysis for AIF mRNA expression showed there was no statistically significant difference in SNc of the four groups (P>0.05).ConclusionPARP is overactivated in SNc of MPTP-induced PD mouse model. PJ34 exerts a protective effect on PD mouse model through inhibiting PARP activity, decreasing nuclear translocation of AIF, reducing cell apoptosis, improving ultrastructural morphological impairments of dopaminergic neurons, increasing viability of dopaminergic neurons in SNc, improving behavioral impairments of the mice. PJ34 has no effect on AIF gene expression in SNc.