The Role And Underlying Mechanism Of Autophagy In Regulating Dopaminergic Neuron Loss In Substantia Nigra Caused By Systemic Inflammation

PartⅠ Systemic inflammation induced a chronic and progressiveloss of dopaminergic neurons and α-synuclein accumulation inmouse substantia nigraObjective Establish a parkinsonian mouse model by intraperitoneal injection withLPS, and to study the effect of systemic inflammation on dopaminergic neuron loss andα-synuclein accumulation in substantia nigra.Methods Select3,16-month-old C57BL male mice as young and aged mice, asingle intraperitoneal injection of LPS (5mg/kg), motor functions were tested byclimbing pole and step, immunohistochemical method observed substantia nigradopamine neurons and microglia change. Protein expressions of α-synuclein wereassayed by western blot and the levels of α-synuclein transcription were assayed bypolymerase chain reaction (PCR) in ventral midbrain (substantia nigra).Results Compared with the control group,16-month-old LPS treated mice beganto motor dysfunction at4-5months after injection, extend further exacerbated over time,and3-month-old group delay to at7months after injection（*P<0.05）; A large numberof activated microglia and positive expression of IL-1β were seen in substantia nigra ofboth young and aged LPS-treated mice from1day to7months after injection,16-month-old group inflammatory responses were more obvious; dopaminergic neuronsbegan to decrease (27%) in16-month-old LPS treated mice at5months after injection,further decreased (41%) at7months,3-month-old group delay to7months began todecrease (22%). Protein expression of α-synuclein increased in both young and agedLPS-treated mice from1day to7months after injection （**P<0.01）, the levelstranscription of α-synuclein were no significant changes at the same time.Conclusion PD model can be successfully established by systemic inflammation via single intraperitoneal injection with LPS, which induced caused chronic andpersistent neuron inflammation, dopaminergic neurons chronic, progressive loss andα-synuclein accumulation in mouse substantia nigra. Part Ⅱ Systemic inflammation causes a dynamic change ofautophagy and dopaminergic neuron autophagy dysfunction insubstantia nigra.Objective Using LPS PD model, study the changes of autophagy function insubstantia nigra.Methods Select3,16-month-old C57BL male mice as young and aged mice, asingle intraperitoneal injection of LPS (5mg/kg) to establish the PD model. Proteinexpressions of LC3-II, p62, HDAC6were assayed by western blot in ventral midbrain(substantia nigra), neurons ultrastructure were observed by TEM, dopaminergic neuronsand LC3、p62colocalization were observed by immunofluorescence confocal. In vitrostudy, PC12cells were treated by different concentrations of TNF-a, the cell viabilitywas measured by means of MTT methods at24h after treated. Protein expression ofLC3-II, p62and α-synuclein were detected by Western blot, the levels of α-synucleinand p62transcription were assayed by polymerase chain reaction (PCR).Results Compared with the control group, Protein expression of p62increased insubstantia nigra of both young and aged LPS-treated mice from1day to7months afterinjection （**P<0.01）, protein expression of LC3-II and HDAC6dynamic changed,from raised to decline（**P<0.01）. Compared with the3-month-old LPS-treated groups,protein expression of LC3-II and HDAC6decreased earlier in16-month-old groups.Micrographs showed the occurrence of autophagosome with double-membranestructures in neurons at1d after LPS injection, LPS induced positive expression of bothLC3and p62in dopaminergic neurons at the sametime (**P<0.01). The cell viabilitydecreased significantly in a concentration-dependent fashion, TNF-α (50ng/ml) causeda significant up-regulation of LC3-II and p62, as well as α-synuclein proteinaccumulation（*P<0.05）, α-synuclein and p62mRNA levels remained unaltered afterTNF-α(50ng/ml) treatment for24h. Conclusion In this LPS PD model, systemic inflammation causes a dynamicchange of autophagy, from activity to decrease in substantia nigra. Inflammation couldlead to autophagy dysfunction of the dopaminergic neurons, inflammatory cytokinesTNF-α could induce autophagy dysfunction and α-synuclein degradate difficulty indopaminergic cells. Part Ⅲ Minocycline pretreatment alleviate acute inflammationand autophagy dysfunction caused by systemic inflammation insubstantia nigra.Objective Study the effect of minocycline pretreatment on acute inflammation andautophagy in substantia nigra.Methods Minocycline was administered for three consecutive days in3-month-oldC57BL male mice, an hour last injection, intraperitoneal injection of lipopolysaccharide(LPS,5mg/kg). Motor functions were tested by climbing pole and step, the substantianigra microglia activation and positive expression of IL-1β were observed byimmunohistochemical methods, TNF-α levels were detected by ELISA method, proteinexpression of LC3-II, HDAC6, p62and α-synuclein were detected by Western blot，dopaminergic neurons and LC3、 p62colocalization were observed byimmunofluorescence confocal technical.Results Compared with the control group, motor dysfunction were found in mice1d after LPS injection, a large number of activated microglia were seen and the level ofTNF-α and IL-1β expressions increased in substantia nigra of LPS-treated groups,protein expression of LC3-II HDAC6, p62, and α-synuclein appeared simultaneousincrease. Compared with LPS treatment alone group, pre-treatment with minocycline atlow(25mg/kg) and moderate(50mg/kg) doses were observed to improve the motordysfunction, reduced microglia activation, TNF-α generation and IL-1β expressions, atthe same time, protein expression of α-synuclein, LC3-II, p62and HDAC6reducedeither（**P<0.01）. LC3and p62positive expression was significantly decreased indopaminergic neurons with minocycline at moderate (50mg/kg) doses. Pre-treatmentwith minocycline at high(75mg/kg) doses were exacerbated motor dysfunction, and protein expression of p62、α-synuclein、LC3-Ⅱand HDAC6increased.Conclusion In a certain dose range, minocycline could alleviate acuteinflammation, autophagy dysfunction and α-synuclein accumulation caused by systemicinflammation in substantia nigra.