| 1. The Pathogenesis of Parkinson's diseaseParkinson's disease (PD) is named after James Parkinson, who made a first and detailed description of the disease in the year 1817. PD is a neurodegenerative disorder, usually affects old people at the age of 60, clinically characterized by bradykinesia, rigidity, resting tremor, and a variety of other motor.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. There is growing consensus among parkinsonologists, that PD is probably not a homogenous disease, but a syndrome of different disorders, caused by genetic, environmental, aging, and other etiologies. Although the mechanism of neurodegeneration in PD is not clear, the pathogenesis of PD has been postulated to result from a complex interaction between environmental and genetic factors leading to mitochondrial dysfunction, oxidative stress, inflammation, and excitotoxicity, eventually leading to nigral DAergic neuron degeneration.Impaired degradation of misfolded and aggregated proteins is being increasingly recognized to play an important role in the pathogenesis of PD. Dysfunction of the ubiquitin-proteasome system (UPS) has been already strongly implicated in the pathogenesis of this disease and growing interest has been shown in identifying the role of autophagy-lysosome pathway (ALP) in repair and removal of misfolded proteins.2. The role of dopamine agonist in the treatment of Parkinson's diseaseCurrent drug therapy in Parkinson's disease is symptomatic and primarily aimed at restoring dopaminergic function in the striatum. Levodopa is still the most effective symptomatic treatment. Levodopa is well tolerated, easy to administer, and inexpensive. However, long term use is associated with disabling complications such as fluctuating motor responses and dyskinesias. Besides, it is reported that levodopa is toxic in vitro to dopaminergic neurons and in vivo its use could lead to formation of cytotoxic free radicals when exogenous dopamine is decarboxylated, these would cause damage to surviving dopaminergic neurons and potentially exacerbate the disease . Dopamine agonists were introduced as an adjunct to levodopa treatment and play an important role in antiparkinsonian by acting directly on dopamine receptors and imitating the endogenous neurotransmitter. There are two subclasses of dopamine agonists:ergoline and non-ergoline agonists. The non-ergoline agonists such as pramipexole and ropinirole are more effective than the ergoline agonist, the reason may be that pramipexole is a selective D3 dopamine receptor agonist and with less side effects.3. The animal models of Parkinson's disease:construction and applicationThe successful application of animal models to the study of Parkinson's disease has advanced our knowledge of Parkinson's disease. Neurotoxin-based models have been important in clarifying aspects of the disease, such as selective vulnerability of substantia nigra dopaminergic neurons to the degenerative process. It produces clinical, biochemical, and neuropathological changes of those occurring in idiopathic PD. Several cell death mechanisms have been implicated in MPTP toxicity, including an inhibition of complex I in the mitochondrial electron transport chain, the generation of reactive oxygen species (ROS), inflammation, apoptosis, and autophagia.Exposure of humans to MPTP causes a syndrome that mimics the core neurological symptoms and relatively selective dopaminergic neurodegeneration of PD. Although the monkey MPTP model is the gold standard for the assessment of novel strategies and agents for the treatment of PD symptoms, the monkey MPTP model does not include two characteristic features of PD. First, neurons are not consistently lost from other monaminergic nuclei, a typical feature of PD . Second, although intraneuronal inclusions resembling LBs have been described classical LBs have not been demonstrated convincingly in the brains of MPTP-intoxicated patients or monkeys. Because of practical considerations, MPTP monkeys have not generally been used to explore the molecular mechanisms of dopaminergic neurodegeneration; the MPTP mouse model is typically used for such studies. MPTP toxicity in mice has become the most commonly used animal model of PD for both technical and financial reasons. These studies have focused on three types of dysfunction that may be important in the pathogenesis of PD:oxidative stress, mitochondrial defect, and abnormal protein aggregation.However, several problems need to be emphasized. First, mice are much less sensitive to MPTP than monkeys; thus, much higher doses are required to produce significant SNpc damage in this animal species, presenting a far greater hazardous situation. Second, in contrast to the situation in monkeys, mice treated with MPTP do not develop parkinsonism. Third, the magnitude of nigrostriatal damage depends on the dose and dosing schedule.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 impairment of UPS could lead to the accumulation of toxic proteins and ultimately result in neuronal dysfunction and death. In our lab's previous study, we have showed that direct stereotactic injection with proteasome inhibitor-lactacystin, into the unilateral or bilateral mouse medial forebrain bundle (MFB) 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 and its metabolites, DOPAC and HVA, were significantly reduced, whereas the levels of 5-HT and 5-HIAA were unchanged, indicating that the toxicity of lactacystin might be relatively selective to nigro-striatal dopaminergic pathway.4. ConclusionThe thesis is based on my study during the Joint training of Shandong University and Baylor College of Medicine.Our study on the neuroprotective property of dopamine receptor in two kinds of 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 The neuroprotective property of novel D3 dopamine receptor preferring agonist D-264 in two kinds of Parkinson's disease animal modelsObjectivesThe animal models of Parkinson's disease, induced by MPTP and lactacystin, were constructed. We used a combination study, including a blocking experiment with D3 receptor antagonist U99194, to better understand the mechanism of neuroprotection of selective D3 receptor agonist-D264. We observed the changes of behavioral performances (rotarod, locomotion, pole), the number of dopamine neurons in substantia nigra area, the concentration of dopamine and its metabolites, proteasomal activity, the levels of BDNF and GDNF. After analyzing these changes, we get the conclusions that the mouse models of Parkinson's disease were successfully constructed and the neuroprotective property of D264 was well evaluated.MethodsMale C57BL/6 mice, aged 12 weeks, were randomly assigned into twelve groups:control for MPTP, control for lactacystin, MPTP, lactacystin, D-264 Low Dose (1 mg/kg)+MPTP, D-264 High Dose (5 mg/kg)+MPTP, D-264 Low Dose+lactacystin, D-264 High Dose+lactacystin, D-264 non-lesioned control for MPTP, D-264 non-lesioned control for lactacystin, U99194+D-264 high dose+MPTP, and U99194+D-264 high dose+lactacystin, respectively. The mice were examined 1 day prior to MPTP and lactacystin treatment as a base level and then every week. The mice were sacrificed on day 14 (MPTP) and day 21 (lactacystin), the brains were immediately removed and stored at-80℃until analysis.Results(1) Compared to the control, behavioral performances, the number of TH positive cells in substantia nigra area, the concentration of dopamine and its metabolites, proteasomal activity, the levels of BDNF and GDNF were significantly reduced in MPTP and lactacystin treated mice.(2) Compare to the mice treated with MPTP and lactacystin, pretreatment with D264 at low and high dose significantly attenuated behavioral impairment, showed neuroprotection against both MPTP and lactacystin induced DA neuron loss and depletion of DA and its metabolites in the SN, alleviated lactacystin induced proteasomal inhibition, and increased the BDNF and GDNF levels in MPTP and lactacystin lesioned mice.(3) Pretreatment with U99194 partially but significantly altered the neuroprotective effect of selective D3 agonist-D-264.Conclusion(1) The mouse model induced by MPTP and lactacystin replicated many of the features of PD and were expected to be suitable for our study.(2) D-264 can prevent neurodegeneration induced by the selective neurotoxin MPTP and UPS inhibitor lactacystin, be potentially served as a both symptomatic and neuroprotective treatment agent for PD.(3) Pretreatment with D3 receptor antagonist U99194 significantly altered the effect of neuroprotection conferred by D-264, which showed that the effect of D-264 was mediated partly or completely by D3 receptor. PART TWO Pramipexole in proteasome inhibition induced animal model of Parkinson's disease:Underlying neuroprotective mechanismsObjectivesParkinson's disease (PD) is characterized by the progressive loss of nigral dopamine (DA) neurons in substantia nigra (SN) area and the accumulation of inclusion bodies, known as Lewy bodies (LBs). The cause of the neurodegenerative process in PD remains unclear. Ubiquitin-proteasome system (UPS) impairment has been proposed to play an important role in the pathogenesis of PD. In the present study, we attempt to better evaluate the mechanism of neuroprotection of pramipexole (PPX) in a mouse model of DA neuron degeneration induced by UPS impairment, in addition to test the possible mechanisms of autophagy in prevention of the proteasome inhibition-induced DA neuron degeneration.MethodsMale C57BL/6 mice at the age of 10-12 weeks were were randomly assigned into six groups:vehicle control, lactacystin, PPX alone, PPX Low Dose (01 mg/kg)+lactacystin, PPX High Dose (0.5 mg/kg)+lactacystin, and U99194+PPX High Dose+lactacystin, respectively. Mice treated with PPX (low dose 0.1 mg/kg or high dose 0.5 mg/kg, i.p, twice a day) started 7 days before, and continued after microinjection of proteasome inhibitor lactacystin in the medial forebrain bundle (MFB) for a total 4 weeks. Animal behaviors, pathological and biochemical assays were performed to determine the neuroprotective effect of PPX.ResultsWe found that administration with PPX significantly improved behavioral performance, attenuated DA neuron loss and striatal DA reduction, and alleviated proteasomal inhibition and microglial activation in the SN of lactacystin-lesioned mice. We also documented that PPX can increase the levels of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in the lactacystin-lesioned mice. In addition, we demonstrated an activation of autophagy in PPX treated mice. Furthermore, pretreatment with D3 receptor antagonist U99194 significantly altered the neuroprotection conferred by PPX.ConclusionOur study indicates that, (1) The mouse model microinjected by lactacystin replicated many of the features of PD and were expected to be suitable for our study. (2) Multiple molecular pathways may be attributed to the neuroprotective effects of PPX in the UPS impairment induced animal model of PD. (3) Dysfunction in the autophagy-lysosome pathway (ALP) may cause aggregation of misfolding proteins and attribute to the neurodegenerative diseases, including Parkinson's disease. The results of the study may provide us new insight into the potential novel mechanisms for the treatment of PD. |
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