In vivo imaging of α-synuclein in mouse cortex demonstrates stable expression and differential subcellular compartment mobility

Background: Regulation of α-synuclein levels within cells is thought to play a critical role in Parkinson's Disease (PD) pathogenesis and in other related synucleinopathies. These processes have been studied primarily in reduced preparations, including cell culture. We now develop methods to measure α-synuclein levels in the living mammalian brain to study in vivo protein mobility, turnover and degradation with subcellular specificity. Methodology/Principal Findings: We have developed a system using enhanced Green Fluorescent Protein (GFP)-tagged human α-synuclein (Syn-GFP) transgenic mice and in vivo multiphoton imaging to measure α-synuclein levels with subcellular resolution. This new experimental paradigm allows individual Syn-GFP-expressing neurons and presynaptic terminals to be imaged in the living mouse brain over a period of months. We find that Syn-GFP is stably expressed by neurons and presynaptic terminals over this time frame and further find that different presynaptic terminals can express widely differing levels of Syn-GFP. Using the fluorescence recovery after photobleaching (FRAP) technique in vivo we provide evidence that at least two pools of Syn-GFP exist in terminals with lower levels of mobility than measured previously. These results demonstrate that multiphoton imaging in Syn-GFP mice is an excellent new strategy for exploring the biology of α-synuclein and related mechanisms of neurodegeneration. Conclusions/Significance: In vivo multiphoton imaging in Syn-GFP transgenic mice demonstrates stable α-synuclein expression and differential subcellular compartment mobility within cortical neurons. This opens new avenues for studying α-synuclein biology in the living brain and testing new therapeutics for PD and related disorders.