Neuroprotection Of Nurr1Activator In UPS Impairment Induced Animal Model Of Parkinson's Disease

1. Pathogenesis of Parkinson's diseaseParkinson's disease (PD) is one of the most common neurodegenerative disease, clinically characterized by resting tremors, bradykinesia, rigidity, postural instability, and a variety of other motor and non-motor symptoms [1,2,3]. The pathological hallmark of PD is progressive loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNPc), and the presence of cytoplasmic inclusions, termed Lewy bodies [4].Recently, numerous researchers concentrated on the pathogenesis, clinical features and the treatment strategy of PD. There is growing consensus among parkinsonologists that PD is probably not a homogenous disease, but a syndrome of different disorders, caused by genetic abnormity(such as abnormal expression of Pitx3, Nurr1, α-synuclein, parkin, PINK1, LRRK2), environmental effect, aging, and other etiologies[4,5,6,7,8,9]. Recently, a lot of studies demonstrated that Ubiquitin-Proteasome System (UPS) may also involve in the pathogenesis of PD [10].2. Nurr1gene and Parkinson's diseaseNuclear receptor superfamily includes a variety of transcription factors, such as receptors for steroid and thyroid hormones, and Vitamins A and D. Nurr1(also called NR4A2/NOT/TINUR/RNR-1/HZF-3) is a member of this nuclear receptor superfamily of transcription factors, first identified from mouse brain cDNA library in1992[11] and localized to human chromosome2q22-q23in1994[12]. Nurr1gene includes eight exons. The open reading frame initiates in the third exon and terminates at the upstream region of the eighth exon that encode for598amino acids. Nurr1is expressed predominantly in the central nervous system, especially in substantia nigra (SN), ventral tegmental area (VTA), and limbic areas. In addition, it is expressed highly in olfactory bulb, hippocampus, temporal cortex, subiculum, cerebellum, posterior hypothalamus and habenuclear nuclei [13,14,15]. Several lines of evidence have indicated that Nurrl is essential for the development, migration and survival of dopaminergic neurons [16,17]. Zetterstrom et al found that mice with a targeted deletion of the Nurrl gene died1day after birth. They have also observed that mice with deletion of the Nurrl gene (Nurr-/-) cannot suck milk and have poor motor function. The histopathological examination has found a lack of dopaminergic neurons in SN and VTA in the midbrain [16]. Previous studies of Nurr1+/-mice demonstrated that the gene deletion significantly increased the susceptibility of SN impairment induced by lactacystin [18]. Motor function and numbers of DA neurons in SN in older Nurr1+/-mice significantly reduced compared to the wild type [19,20]. Quantitatively measuring the level of Nurrl mRNA in human peripheral blood lymphocytes has revealed a significant decrease in individuals with PD and parkinsonian syndromes [21,22,23].To sum up, Nurrl gene plays an important role in DA neurons development and functional maintenance, which make Nurrl gene a potential target in PD new treatment strategy. The first part of our experiment is to evaluate the effect of Nurrl gene activator-Compound1in lactacystin induced PD model. The results of behavior test, DA neurons in the SN and the biochemistry experiment may show us the bioactivity and the neuroprotective effect of Nurrl gene activator Compound1.3. Construction of Parkinson's disease modelThe successful application of animal models to the study of Parkinson's disease has advanced our knowledge of Parkinson's disease. Lactacystin is a selective inhibitor of Ubiquitin Proteasome system (UPS), which is responsible for the degradation of normal processed cellular proteins as well as misfolded proteins, it is believed that inhibition or impaired of UPS could lead to the accumulation of toxic proteins and ultimately result in neuronal dysfunction and death [24].In our previous studies, we have showed that direct stereotactic injection with proteasome inhibitor-lactacystin into the medial forebrain bundle (MFB) of mice can cause severe nigral cell degeneration and inclusion body-like protein aggregate formation, which resemble some neuropathological features of PD. In addition, we have found that in the lactacystin-injected mice, the striatal levels of dopamine(DA), DOPAC and HVA were significantly reduced, whereas the levels of5-HT and5-HIAA were unchanged, indicating that the toxicity of lactacystin might be relatively selective to nigro-striatal dopaminergic pathway.4. Pitx3and Parkinson's diseasePitx3(paired-like homeodomain transcription factor3or pituitary homeobox3), which belongs to a pituitary homeobox subfamily, is a homeodomain-containing transcription factor [25].Pitx3was found to be transiently expressed in skeleton muscle and eye lens during embryogenesis. After birth, Pitx3has highly restricted and constitutive expression in the SNc and VTA of the midbrain [26,27]. Studies in the Pitx3-deficiency mice showed that, TH positive cells in the ventral midbrain are significantly decreased compared to the wild-type mice. In addition, the behavior test of Pitx3-deficiency mice was also appeared significantly decrease compared to the wild-type mice [28].Our recent studies also demonstrated that Pitx3may play an important role in the expression of BDNF and GDNF in the DA [29].Although most studies considered that Pitx3and Nurr1may influence development and functional maintenance of DA neurons through different pathways, Frank et al indicted that Pitx3may potentiate Nurr1in dopamine neuron terminal differentiation through release of SMRT-mediated repression [30]. Therefore, we propose that Pitx3and Nurrl deficiency may synergetically affect dopamine neuron development and functional maintenance.In the second part of our experiment, immunohistochemistry, behavior test, biochemistry test and Real-time PCR were performed in mice with three different genotypes (Pitx3-/-, Nurr1+/-and Nurr1+/-Pitx3-/-).The results showed us different effect by Pitx3and Nurrl in DA neuron development and functional maintenance.5. Brief summaryThe thesis is based on my study during the Joint PhD program of Shandong University and Baylor College of Medicine.Our study on the neuroprotective property of Nurrl gene activator in Parkinson's disease animal models has not only improved the knowledge on neurodegeneration disease, but also focus on novel therapeutic strategies. It may provide us further communication between neurology and neurosurgery in different fields of central nervous system diseases. Part One Anti-parkinsonian effects of Nurrl activator in UPS impairment induced animal model of Parkinson's diseaseObjectivesThe animal model of Parkinson' disease induced by lactacystin was constructed to observe the effect of Nurrl gene activator. We observed the changes of behavioral performances (rotarod and locomor test), the numbers of dopamine neurons in substantia nigra area, the concentration of dopamine and its metabolites. After analyzing these changes, we get the conclusions that the mice model of Parkinson's disease was successfully constructed and the neuroprotective property of Nurrl gene activator was well evaluated.MethodsMale C57BL/6mice, aged8weeks, were randomly assigned into4groups: vehicle control, lactacystin, lactacystin plus Compound1, lactacystin plus Compound2(drug control).The mice were examined1day prior to lactacystin treatment as a base level and then every7days. The mice were sacrificed on day28, and the brains were immediately removed and stored at-80℃until analysis.Results(1) Compared to the vehicle control, behavioral performances, the number of TH positive cells in substantia nigra area, the concentration of dopamine and its metabolites were significantly reduced in lactacystin treated mice.(2)Compared to the lactacystin treated mice, behavioral performances, the number of TH positive cells in substantia nigra area, the concentration of dopamine and its metabolites, mRNA levels of Nurrl, DAT and Vmat2were significantly improved in Compound1treated mice.(3) Compared to the lactacystin treated mice, there is no significant change in Compound2treated mice.Conclusions (1)Mice models induced by lactacystin replicated many of the features of PD and were expected to be suitable for our study.(2)Nurrl gene activator prevent neurodegeneration induced by the UPS inhibitor lactacystin, be potentially served as a neuroprotective treatment agent for PD. Part twoPitx3and Nurrl deficiency synergetically affect dopamine neuron development and functional maintenanceObjectivesBoth Pitx3gene and Nurrl gene play important roles in the DA neurons development and functional maintenance. Recent studies indicted that Pitx3potentiates Nurrl in dopamine neuron terminal differentiation through release of SMRT-mediated repression and they may affect dopamine neuron development and functional maintenance synergetically. Immunohistochemistry, behavior test, biochemistry test and Real-time PCR were performed in mice with three different genotypes (Pitx3-/-, Nurr1+/-and Nurrl+/-Pitx3-/-).The results showed us different effect by Pitx3and Nurrl in DA neuron development and functional maintenance.MethodsMice with different genotypes were assigned into4groups:wild type, Pitx3-/-, Nurr1+/-, Nurr1+/-Pitx3-/-. Behavior test was performed in day30and60after they were born and the mice were sacrificed on day60.The brains were immediately removed and stored at-80℃until analysis.Results(1) Compared to the wild type, behavioral performances, the number of TH positive cells in substantia nigra area, mRNA levels of TH and DAT, the concentration of dopamine and its metabolites were significantly reduced in the Pitx3-/-mice.(2)Compared to the Pitx3-/-mice and the Nurr1+/-mice, behavioral performances, the number of TH positive cells in substantia nigra area, mRNA levels of TH and DAT, the concentration of dopamine and its metabolites were significantly reduced in the Nurr1+/-Pitx3-/-mice(3)Compared to the Nurr1+/-mice, mRNA levels of TH and DAT was significantly reduced in the Nurr1+/-Pitx3-/-mice. Conclusions(1) Pitx3gene and Nurrl gene play important roles in the DA neurons development and functional maintenance.(2) Pitx3and Nurrl may affect dopamine neuron development and functional maintenance synergetically.