Study On The Mechanisms Of α-synuclein Aggregation In Rotenone Induced Parkinson's Disease

PART I Exp. 1 Subcutaneous rotenone injection destroys dopaminergic neurons and induces Parkinsonism symptoms in ratsObjective To explore a method of reproducing Parkinson's disease models with rotenone in rats. Method Rotenone was injected subcutaneous to the back of rats with the concentration of 2 mg/kg for about 1-8 weeks. The behaviors features of animals, HE pathology in hippocampus, and the immunoreactivity of tyrosine hydroxylase in substantia nigra and corpora striata were observed. Results Rotenone induced Parkinsonism symptoms such as bow-back pose in rats, and it did reduce the tyrosine hydroxylase- immunoreactivity in subtantia nigra and corpora striata, but not affect the neurons in hippocampus during 1-8weeks. Conclusion Rotenone injected subcutaneous in back is an effective method to reproduce the Parkinson's disease model in rats.Exp. 2 Study on protein aggregation and ultramicrostructure in rotenone induced PD ratsObjective: To observe the influence of Rotenone on the distribution ofα-synuclein and ultramicrostructure in rats model of Parkinson's disease. Methods: Wistar rats were randomly divided into two groups and received subcutaneous injections of 2 mg/kg rotenone or sunflower oil for about 4 weeks. Some brain parts as hippocampus, substantia nigra and striatum were observed from global to local.α-Synuclein protein was determined by anti-α-synuclein immunohistochemistry. Electronmicroscope was used to observe the ultramicrostructure of neurons in subtantia nigra. Results: In rotenone treated rats,α-synuclein positive staining enhanced in global brain but not in an even lever. In substantia nigra, ASN positive stuff was found be aggregated in both cytoplasm and nuclear, and some of that formed spherical inclusion. In striatum, ASN positive neuritis end aggregated and agglomerated around neurons. And in hippocampus, there were few dot-like ASN aggregations in cell body and no notable change in nuclear. Several types of cell organ, especially ER, were observed insulted in SN neurons of rotenone induced PD rats. Inclusions:α-Synuclein protein aggregated in the brain of rotenone treated rats, especially in SN. And rotenone could insult endoplasm reticulum in SN neurons.PART II Exp. 1 A Study on Model of Early Apoptosis of Dopaminergic Neurons Induced by Rotenone in vitroObjective: To study the effective time and concentration rule of rotenone on inducing early apoptosis of dopaminergic neurons. Method: Human dopaminergic cells SH-SY5Y were exposed to different concentrations of rotenone for 24 or 48 hours. The cell mortality was measured by MTT, and mitochondrial membrane potential was determined by incubating the cells with rodanmine123, a mitochondria special dye, followed by flow cytometry analysis of fluorescence signals. Results: Lower concentrations (5nmol/L and 20nmol/L) of rotenone affected little on cell mortality within 48 hours, but higher concentrations (>100nmol/L) did much. And the mortality went higher with the concentration going higher. When acting 24 hours, rotenone treatment with concentration of 20nmol/L or higher significantly decreased the mitochondrial membrane potential. Conclusion With the concentration of 20nmol/L, 24 hours rotenone treatment can induce early apoptosis of dopaminergic neurons in vitro.Exp. 2 Rotenone induced endoplasm reticulum stress in dopaminergic neuronsObjective: To explore if rotenone can induce endoplasm reticulum stress in dopaminergic neurons. Methods: Human dopaminergic cells SH-SY5Y were exposed to 30 or 500nmol/L rotenone for 24 to 48 hours. Expressions of GRP78/Bip, XBP-1 and caspase12 in mRNA level were detected by RT-PCR. Results: Rotenone treatment with concentration of 30nmol/L significantly increased the expressions of GRP78/Bip, XBP-1 and caspase12 in mRNA, P<0.05. This phenomenon wasn't seen when treated with 500nmol/L rotenone. Inclusion: Rotenone of low concentrations (less than 30nmol/L) could induce endoplasm reticulum stress in dopaminergic neurons.Exp.3 Effects of Rotenone on expression ofα-synuclein in dopaminergic neurons Objective: To observe the effects of rotenone onα-synuclein expression in dopaminergic neurons. Methods: Human dopaminergic cells SH-SY5Y were exposed to 30 or 500nmol/L rotenone for 24 to 72 hours. Immunocytochemistry was used to observe the disposition ofα-synuclein protein in cells. Expressions ofα-synuclein in mRNA and protein level were separately detected by RT-PCR and Western-blot. Results: Rotenone treatment with concentration of 500nmol/L decreased theα-synuclein protein. When the cultured cells were treated with 30nmol/L rotenone,α-synuclein mRNA increased in 24h, but descended at the point of 48h, and then stepped up in 72h; andα-synuclein protein kept increasing in the period of time. Till 72h, the amount of protein is significant higher than that of control, P<0.05. Inclusion: Rotenone of low concentrations (less than 30nmol/L) could up-regulate the expressions ofα-synuclein in both mRNA and protein level, which might be the base of fact that rotenone could induce endoplasm reticulum stress.