Role Of Inflammation Induced By α-synuclein In Etiology Of Parkinson’s Disease

Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder after Alzheimer’s disease (AD) with a prevalence of1%among people older than65years of age. PD is characterized clinically by a classic tetrad of motor symptoms:low-frequency resting tremor, rigidity of the skeletal muscles of the face and hands, reduced motor activity (bradykinesia), and, in later stages of the disorder, postural instability. The cardinal symptoms of PD result mainly from the loss of dopaminergic neurons in the substantial nigra pars compacta and formation of intraneuronal protein aggregates called Lewy bodies. It is widely accepted that the accumulation of a-synuclein is a principal event in the pathogenesis of PD, but the cellular events leading to DA neuronal dysfunction have remained less clear. Studies have suggested that microglia and astrocytes are not mere bystanders but that microglia and astrocytes are central to the initiation and progression of PD. Microglia are activated by and recruited to a-synuclein, phagocytosing it, and secreting cytokines after activation, While astrocytes could support the survival of DA neurons through upregulating the expression of neurotrophic factors. However, overexpression of a-synuclein could block the effect of astrocytes on neurotrophy and inflammation.However, these motor dysfunctions do not become apparent until70%-80%of nigrostriatal nerve terminals have undergone degeneration, suggesting the existence of an impressive compensatory mechanism in the earlier stages of the disease. Although PD has been heavily researched in the last two decades, the precise etiology of the disease is still unknown.In2001, the National Cholesterol Education Program Adult Treatment Panel III guidelines defined a cluster of metabolic factors that increase the risk for atherosclerotic cardiovascular disease. They called this cluster the "metabolic syndrome" and defined afflicted individuals by the presence of three of the following five characteristics:central obesity, hypertriglyceridemia, low high-density lipoprotein (HDL)-cholesterol levels, hypertension, and either impaired fasting glucose or type2diabetes. Type2diabetes (T2D), the most common type of diabetes, characterized by a reduction in the ability of insulin to stimulate glucose utilization, insulin resistance and inadequate pancreatic β-cell insulin secretion in response to hyperglycemia, is related with a faster rate in the decline of cognition in comparison with the general population. Evidence from prospective epidemiological studies has identified T2D as an independent risk factor for multiple complications virtually in all organs, including neurodegenerative diseases such as diabetic neuropathy, stroke, and AD. Recently, some epidemiological researchers focused on the association between T2D and the risk of PD. Gang Hu et al. prospectively followed51552Finnish men and women25-74years of age without a history of PD at baseline, they found that T2D is associated with an increased risk of PD. Qun Xu et al. supported this view with their data that PD risk was40%higher among diabetic patients than among participants without diabetes within a total of1565participants with PD diagnosed after1995were included in the analysis. At about the same time, Eva Schernhammer also suggested that there is the common physiological pathway between PD and T2D. All the epidemiological results above suggested that there is some connection innate existing between PD and T2D.Therefore, the aim of present studies is to investigate the mechanism of a-synuclein involve in PD. In the first part of present work, we used the T2D mice to investigate whether there is the connection between PD and T2D, that could aggravate the progression of PD. Our data revealed that T2D could increase the expression of a-synuclein and reduce the level of BDNF, subsequently, instigate NLRP3inflammasome and ER stress in midbrain. In addition, T2D mice are more sensitive to the neurotoxin MPTP therefore aggravate the injury in midbrain. In the second part, the role of a-synuclein in microglia was investigated on the basis of overexpression of a-synuclein and activation in microglia in midbrain of T2D mice. Finally, it was illustrated that a-synuclein could induce inflammation and reduced the production of neurotrophic factors in astrocytes. Furthermore, a-synuclein had interrupted the effect of D1/2agonists on astrocytes.Part I Relationship between metabolic inflammation and Parkinson’s DiseaseAIM:To investigate the relationship between metabolic inflammation and PD.METHODS:The two-month-old male T2D model mice, db/db and ob/ob mice, were used during this part. The blood glucose and insulin level were measured in the basal condition. The morphologic change of pancreas and liver in T2D mice was observed through HE staining. The level of inflammatory factors in serum of T2D and WT mice, IL-1β, IL-6, IL-18and TNF-a, was tested with ELISA. Immunohistochemistry was taken for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP) and macrophage antigen complex-1(MAC-1) expression to determine the differences between T2D and WT mice of neurons, astrocytes and microglia in midbrain under the basal condition. The inflammatory factors in midbrain of mice, such as IL-1β,IL-4, IL-6, IL-10and TNF-a were analysed by real-time PCR. The western blotting was applied to determine the expression of PD marker protein, a-synuclein, endoplasmic reticulum (ER) stress associated protein, CHOP and GRP78, the neurotrophic factor, BDNF and IL-1β signaling pathway associated NF-κB (p65subunit), NLRP3-caspase-1inflammasome. WT and db/db mice were treated with chronic MPTP intoxication protocol:20mg/kg MPTP in saline was injected subcutaneously, and250mg/kg probenecid in DMSO was injected intraperitoneally every3.5days over a period of5weeks. Immunohistochemistry was taken for tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) in the substantia nigra pars compacts (SNc) and ventral tegmental area (VTA). For acute MPTP neurotoxicity, db/db and WT mice were received four intraperitoneal injections of MPTP20mg/kg in saline at2h intervals on the same day. Analysed the injury in midbrain was going on through immunochemistry of TH, GFAP and Iba-1.RESULTS:1. Inflammation was increased in the liver and pancreas of T2D miceThe morphology of pancreas and liver of ob/ob and db/db had changed significantly compared with WT mice, while the T2D mice exhibiting insulin resistance had a significant high level of inflammatory factors in the serum, such as IL-1β, IL-4, IL-18and TNF-a (P<0.01). The insulin receptor signaling pathway had been blocked in the liver of T2D mice (P<0.05).2. The expression of a-synuclein was upregulated, while the ER stress response was exacerbated in the midbrain of T2D miceThe expression of a-synuclein, PD marker protein, was upregulated not only in pancreas but also in the midbrain of T2D mice (P<0.05). The level of CHOP and GRP78, ER stress associated protein, was significantly higher in the pancreas and midbrain of T2D mice compared with WT mice (P<0.05).3. Microglia in midbrain of T2D mice were activatedUnder the basal condition, the neurons and astrocytes have no apparent injury while the microglia have been activated in the midbrain of T2D mice (P<0.05).4. Inflammtion was aggravated, and expression of neurotrophic factors were downregulated in the midbrain of T2D mice, which also exhibited insulin resistanceThe level of pro-inflammatory factors IL-1β,IL-6and TNF-a was upregulated in the midbrain, while the level of anti-inflammatory factors IL-4and IL-10was down regulated (P<0.05). The IL-1β signaling pathway was activated by increasing the expression of p65and caspase-1inflammasome. Along with the insulin resistance in the periphery tissue, the insulin receptor signaling pathway was also blocked in the midbrain of T2D mice. The expression of neurotrophic factors BDNF and IGF-1, which had the effect to regulate the blood glucose, was decreased in midbrain of T2D mice (P<0.01). 5. T2D mice had more sensitivity to neurotoxin MPTPT2D mice are more susceptible to neurotoxin MPTP, and MPTP exacerbates DA neuron loss and activation of astrocytes and microglia in the midbrain of db/db mice (P<0.01).CONCLUSION:The level of a-synuclein and inflammation factors were increased, and the expression of anti-inflammation factors and neurotrophic factors was decreased, which made T2D mice had more sensitivity to risk factor to had PD.Part Ⅱ The effect of a-synuclein to inflammation in microgliaAIM:To investigate the effect of a-synuclein to microgliaMETHODS:WT and A53Ttg/-mice were subjected to chronic MPTP intoxication. Immunohistochemisty of TH, GFAP and MAC-1was used to measure the injury in midbrain of mice. The murine microglia cell BV-2cells were cultured in vitro. After treating BV-2cells with WT or A53T a-synuclein, we measured the expression of NF-κB (p65), NLRP3and pro-caspase-1/caspase-1. The level of IL-1β in supernatant of BV-2cells stimulated by WT/A53T a-synuclein alone or WT/A53T a-synuclein along with zYVAD was tested through ELISA assay. Immuno-cytofluorescence was used to observe the cytophagy of BV-2cell stimulated by a-synuclein alone, or in present of cytochalasin D. The expression of p-AMPK/AMPK, LC-3Ⅰ/Ⅱ and caspase-1was measure by western blotting. After stimulation of WT or A53T a-synuclein, the morphology of lysosome was observed through labeled by Lyso-Tracker Red, the specific lysosome dye and the expression of cathepsin B was measured by western blotting. The production of ROS from BV-2cells treated with AICAR and cytochalasin D in present of a-synuclein was investigated by cytofluorescence.RESULTS:1. Chronic MPTP induced neurodegeneration in WT and A53Ttg/-mice, however the injury in midbrain of A53Ttg/-mice is more obvious Under the basal condition, the number of DA neurons in WT and A53Ttg/-mice had no difference, but the activated astrocytes and microglia were more than WT mice. Chronic administration of MPTP significantly down-regulated the number of DA neuron, the number of activated astrocytes and microglia was increased significantlly in midbrain of A53Ttg/-mice.2. a-synuclein induced activation of IL-1β signaling pathway in BV-2cellsWT and A53T a-synuclein could upregulate the nuclear translocation of NF-κB and the activation of NLRP3inflammasome, which further induce IL-1βsecretion in BV-2cells. However, zYVAD, the specific inhibitor of caspase-1, could reverse the effect of WT/A53T a-synuclein in BV-2cells.3. a-synuclein activated inflammasome through endocytosis of microgliaa-synuclein was transported into BV-2cells through the cytophagy. The inhibitor of cytophagy, cytochalasin D could inhibit the entering of a-synuclein therefore reduce the activation of inflammasome and the production of IL-1β.4. a-synuclein inhibited autophagy and induced lysosomal damageAfter entering into microglia, WT/A53T a-synuclein could downregulate the expression of p-AMPK and LC-3II, which are autophagy-associated proteins. The lysosome got swollen and distorted, and the cathepsin B released from lysosome was increased after stimulated by synuclein.5. Activation of p-AMPK, inhibition of LC-3Ⅱ and reduced ROS could inhibit a-synuclein induced inflammation in BV-2cellsThe production of ROS was induced by a-synuclein, AICAR and cytochalasin D could reduce the production of ROS and IL-1β through upregulating the expression of p-AMPK and LC-3II.CONCLUSION:A53Ttg/-mice, overexpression of a-synuclein, showed increased DA neuron loss in response to chronic MPTP/p treatment. WT/A53T a-synuclein could enter into microglia by cytophagy. The morphology and function of lysosome was destroyed, therefore the autophagy of cell was inhibited in the present of a-synuclein. The blockage of autophagy made more ROS to activate NF-κB and the inflammasome to produce more IL-1β. Part III The effect of a-synuclein to neurotrophy and inflammation in astrocytesAIM:To investigate the effect and mechanism of a-synuclein in astrocytes on neurotrophic and inflammatory function.METHODS:Mesencephalic primary astrocytes were prepared from the ventral mesencephalic tissue of postnatal (P1-P3) WT and A53Ttg/tg mice. Western blotting was used to analyze the expression of neurotrophic factors, such as BDNF, GDNF and FGF-2, while the NLRP3inflammasome. The WT and A53Ttg/tg astrocytes were exposed to MPP+, then administrated the D1/2agonists SKF38393and quinpirole respectively. The expression of NLRP3, caspase-1, neurotrophic factors, and DA receptor pathway associated proteins were tested with western blotting.RESULTS:1. Overexpression of a-synuclein could downregulate the expression of neurotrophic factors and activate inflammasome in astrocytesThe expression of neurotrophic factor BDNF and GDNF in mesencephalic astrocytes of A53Ttg/tg mice was significantly lower, while the caspase-1inflammasome and IL-1β were much higher compared with WT astrocytes in the basal condition.2. D1/2agonists upregulated expression of neurotrophic factors in WT astrocytes, which could be canceled by overexpression of a-synucleinWT astrocytes exposed to D1/2agonists exhibited high expression of neurotrophic factors, while A53Ttg/ts astrocytes had no significant change.3. Overexpression of a-synuclein inhibited the anti-inflammation and neurotrophic effect of D1/2agonist against MPP+MPP+could induce activation of inflammasome and reduce the expression of neurotrophic factors in WT astrocytes. D1/2agonists could reverse the effect of MPP+. However, the effect of D1/2agonists was canceled in A53Ttg/tg astrocytes.4. Overexpression of a-synuclein inhibited activation of DA signaling pathway by DA agonists in astrocytes.D1/2agonists could activate the DA receptor pathway which was inhibited in present of MPP+, while it had no ameliorate in MPP+reduced activation of DA signaling in A53Ttg/tg astrocytes.CONCLUSION:Overexpression of α-synuclein inhibited the expression of neurotrophic factors and activated inflammasome to promote the production of IL-1β. Under the overexpression of α-synuclein, D1/2agonists could not exhibit the effect of anti-inflammation and neuroprotection.SUMMARY:1. Metabolic inflammation aggravated the inflammation in midbrain of T2D mice, therefore increased the sensitivity to neurotoxin MPTP α-synuclein is expressed much higher T2D mice than WT mice. The inflammation is enhanced in midbrain of T2D mice, reflected in the activation of microglia, the increased level of inflammation. Acute MPTP administration made more serious injury in midbrain of db/db mice compared with WT mice.2. α-synuclein activated microglia through activating NLRP3inflammasome α-synuclein could enter into BV-2cells through cytophagy, inhibit autophagy and breakdown lysosome, therefore promoting the production of ROS to induce the activation of inflammsome. Inhibition the cytophagy and activation of AMPK could reverse the effect of α-synuclein induced in BV-2cells.3. a-synuclein induced inflammation and downregulated expression of neurotrophic factors, and canceled the effect of anti-inflammation and neurotrophy of D1/2agonists in astrocytes Overexpression of α-synuclein promoted the inflammation and inhibited the expression of neurotrophic factors, such as BDNF and GDNF. Overexpression of a-synuclein also could cancel the neuroprotection of D1/2agonists.