| Parkinson's disease (PD) which cardinal symptoms are resting tremor, rigidity, bradykinesia and postural abnormality is an age-related neurodegenerative disorder in central nervous system. Its pathological hallmarks are the progressive loss of dopaminergic (DAnergic) neurons in substantia nigra pars compacta (SNpc), depletion of dopatmine (DA) in striatum and formation of inclusion-lewy bodies (LBs) which major component is Synuclein (Syn) in the remaining DAnergic neurons.Syn is a 140 amino acids-containing protein located at presynaptic terminal of DAnergic neurons Aggregation and gene over-expression of a-Syn provoke DAnergic neurodegeneration. Currently, DA replacement therapy is the primary approach to alleviate symptoms of PD. However, symptomatic treatments cannot halt PD progression.In this study, using series of cultures including primary ventral mesencephalic mixed neuron-glia, neuron-enriched, microglia-enriched, astroglia-enrich, mixed neuron-astroglia and reconstituted cell cultures, we explored the role and underlying mechanisms of a-Syn in the continuous microglial activation and DAnergic neurodegeneration. At same time, we investigated the roles and mechanisms of Dextrornethorphan (DM) and its analog 3-hydroxymorphinan (3-HM) in inhibiting the continuous activation of microglia and the production of pro-inflammatory and neurotoxic factors produced by activated microglia and the neurotrophic and neuroprotective effects on DAnergic neurons induced by l-methyl-4-phenyl-l, 2,3,6-tetrahydropyridine (MPTP) and lipopolysaccharide (LPS) PD models.ResultsSection 1: Continuous activation of microglia induced by phagocytosis of a-Syn mediates DAnergic neurodegeneration.Most of the aggregated a-Syn in PBS for up to 14-21 days was soluble or insolublemonomer or polymer confirmed by Western Blot. Syn induced specific reduction of DA uptake capacity and decrease of DAnergic neuronal numbers in ventral mesencephalic neuron-glia cultures after 10 days treatment.a-Syn exerted neurotrophic effect on DAnergic neurons when astroglia were added back to neuron-enriched cultures. However, the DAnergic neurodegeneration induced by a-Syn was significantly enhanced with the increasing amount of microglia added backed to neuron-enriched cultures; a-Syn failed to produce DAnergic neurodegeneration in neuron-astroglia cultures without the existence of microglia, demonstrating the important role of microglia in a-Syn-induced DAnergic neurodegeneration.We observed and found that microglia phagocytized the fluorescently labeled a-Syn under confocal microscope and the phagocytosis of a-Syn by microglia was inhibited by cytochalasin D, a specific inhibitor of phagocytosis, in a dose- and time-dependent manner. Syn-stimulated microglia-mediated the productions of extracellular superoxide and intracellular reactive oxygen species (iROS) were also decreased as microglial phagocytosis of a-Syn was inhibited by cytochalasin D, in a dose-dependent manner, indicating that phagocytosis of a-Syn is essential for continuous microglial activation and the production of reactive oxygen species (ROS).Activated microglia induced by a-Syn presented some changes of functional and morphological plasticity. The levels of pro-inflammatory and neurotoxic factors, such as ROS, including extracellular superoxide and iROS, and prostaglandin (PGE2) were significantly elevated. Activated microglia were larger, irregular and rod- and/or amoeboid-shaped cells, in a dose- and time- dependent manner.a-Syn failed to affect DA uptake capacity and increase the production of extracellular superoxide and iROS significantly in NADPH -null mice compared with wild-type mice, indicating that NADPH oxidase and the production of ROS induced by itself was the important pathway of Syn-elicited DAnergic neurodegeneration.Microglial phagocytosis of a-Syn also damaged mitochondrial function severely as well as activated microglia continuously.Section1: DM protectes MPTP-induced DAnergic neurodegeneration through inhibiting activity of NADPH oxidase.DM is t...
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