Production Of Quantum Dot-Labeled?-Synuclein And Study Of Abnormal?-Synuclein Induced Bdnf Retrograde Transport Deficits

Parkinson's disease(PD)is the second most common neurodegenerative disease,characterized by progressive loss of dopaminergic neurons in the substantia nigra and the formation of intracellular aggregates named Lewy Bodies(LB),which are mainly composed of ?-synuclein.It is now well accepted that excessive ?-synuclein easily undergoes aggregation and results in neurotoxcicty,which plays a critical role in the pathogenesis of PD.Brain-derived neurotrophic factor(BDNF)is widely distributed in the brain and found to be necessary for the survival of dopaminergic neurons.Its downstream signal needs to be transported retrogradely from the nerve terminal to the cell body.However,in neurodegenerative diseases,deficit of axonal transport is an early event.Recent studies have showed that overexpressed or aggregated ?-synuclein could impair the retrograde axonal transport.Thus,we investigated the retrograde axonal transport of BDNF in live neurons by using quantum dot-labeled BDNF.We found decreased moving velocities and average speed of BDNF retrograde transport in neurons overexpressing ?-synuclein,accompanied by increased frequency and extended time of pause during the movement.The downstream signals of BDNF such as Erk,Akt were reduced as well.Further study showed that the activation of Rab5 and Rab7 were strengthened due to overexpression of ?-synuclein,which could lead to a rapid maturation and degradation of signaling endosomes.Additionally,the immunoprecipitation results showed that ?-synuclein had an interaction with motor protein dynein.We speculated that dynein might be more prone to be trapped by aggregated ?-synuclein.Our study also found there was no change in the phosphorylation level of tau,indicating that tau might not be involved.So far,many investigations reported a spreading pathology of PD.The underlying molecular mechanism is hypothesized as ?-synuclein could perform intercellular transmission.However,there's no effective model for real time tracking and recording.Therefore,we have designed and produced quantum dot-labeled ?-synuclein.By introducing Avi Tag,?-synuclein could be monobiotinylated and conjugated to one quantum dot.Taking advantage of live imaging,we then observed the internalization of QD-?-synuclein by neurons and microglia.This QD-?-synuclein could be served as a useful tool to investigate the axonal transport and cell-to-cell transmission of ?-synuclein.