Abstract: Objective The abnormality in α-synuclein(α-SYN) expression and the deficiency in mitochondrial function are both implicated in the pathogenesis of Parkinson's disease(PD).And many evidence strongly support a linkage between α-SYN and mitochondria in the pathophysiology of PD,but the molecular basis for the linkage is unknown.Our work is to study whether α-SYN is present in normal rat brain and is able to be transported into mitochondria to help analyzing the asociation of α-SYN with mitochondria in the pathogenesis of PD.Methods Western blot analysis was performed on the isolated brain mitochondria using different anti-α-SYN antibodies(3D5,2E3,EQV1,MDV2,and PQE3),which identify different parts of αSYN.To make clear the precise localization of α-SYN in the mitochondrion,pre-embedding immunoelectron microscopy was performed using 3D5 monoclonal antibody to probe mitochondrial α-SYN and the second antibody-conjugated ultra-small golds to label the 3D5 primary antibody.The total cell α-SYN has been shown to be expressed unevenly in the brain,with its amount being much greater in olfactory bulb,hippocampus,striatum,thalamus and cerebral cortex than in other regions.To answer whether the mitochondrial α-SYN is also richer in these brain regions,mitochondria from different brain regions were isolated,and the relative amount of α-SYN was determined using Western blot analysis.Different concentrations of recombinant human α-SYN was incubated with isolated cerebellar mitochondria where there was no endogenous α-SYN.The reaction mixture was incubated at 37 ℃ for different time.And after thoroughly washing the free α-SYN western blot analysis was used to examine the transportation of α-SYN into mitochondria.Digitonin was used to disrupt the outer mitochondrial membrane to rule out the possibility that the α-SYN transport into mitochondria is because of non-specific binding to the outer mitochondrial membrane.Results Immunoblot analysis identified αthe presence of α-SYN in normal rat brain mitochondria.And shown by immunoelectron microscopy,except sparsely distributed gold particles in the cytoplasm of neurons,densely distributed gold particles are observed in the mitochondria,where the gold particles are localized in the cleft between outer and inner membranes and mitochondrial matrix inside the inner membrane.Mitochondrial α-SYN is differently expressed in different brain regions.In accordance with the total cell α-SYN,the levels ofmitochondrial α-SYN were also higher in olfactory bulb,hippocampus,striatum and thalamus than in cerebral cortex,brain stem and cerebellum.The sequence of the amount of α-SYN in different regions was striatum > hippocampus> olfactory >bulb >thalamus >cortex >brainstem >cerebellum.Incubation of α-SYN protein with isolated mitochondria led to transportation of this protein into the mitochondria in a dose-dependent manner.The import of α-SYN into mitochondria was rapid but not time-dependent manner,with the earliest detection of α-SYN protein in the mitochondria within 5 min.After disrupting the outer membrane with digitonin,most of α-SYN remained in the inner membrane fraction.This suggested that α-SYN is transported into mitochondria rather than bound to the surface of the outer membrane.Conclusion α-SYN is normally present in rat brain mitochondria but varied in expression levels in different brain regions with the amount of α-SYN being much greater in olfactory bulb,hippocampus,striatum and thalamus than in cerebral cortex,brain stem and cerebellum.Except cerebral cortex and cerebellum,the levels of mitochondrial α-SYN is in accordance with the total cell α-SYN,raising a possibility that the αSYN in mitochondria may come from the cytoplasm.Our data provide new evidence for the linkage between α-SYN and mitochondria.Thorough discovery of the role of α-SYN in mitochondria may shed a light on the pathological role of α-SYN in PD.

Key words: α-synuclein, mitochondrion, brain, rat