In vivo imaging of alpha-synuclein in mouse cortex demonstrates stable expression and differential subcellular compartment mobility.
BACKGROUND
Regulation of alpha-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 alpha-synuclein levels in the living mammalian brain to study in vivo protein mobility, turnover and degradation with subcellular specificity.


RESULTS
We have developed a system using enhanced Green Fluorescent Protein (GFP)-tagged human alpha-synuclein (Syn-GFP) transgenic mice and in vivo multiphoton imaging to measure alpha-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 alpha-synuclein and related mechanisms of neurodegeneration.


CONCLUSIONS
In vivo multiphoton imaging in Syn-GFP transgenic mice demonstrates stable alpha-synuclein expression and differential subcellular compartment mobility within cortical neurons. This opens new avenues for studying alpha-synuclein biology in the living brain and testing new therapeutics for PD and related disorders.
Unni
Vivek K
VK
Alzheimer's Research Unit, MassGeneral Institute for Neurodegenerative Disease, MGH Harvard Medical School, Charlestown, Massachusetts, United States of America.
Weissman
Tamily A
TA
Rockenstein
Edward
E
Masliah
Eliezer
E
McLean
Pamela J
PJ
Hyman
Bradley T
BT
D000818:Animals; D002451:Cell Compartmentation; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D005260:Female; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008297:Male; D051379:Mice; D038761:Photobleaching; D017729:Presynaptic Terminals / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D051844:alpha-Synuclein / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D051844:alpha-Synuclein; D049452:Green Fluorescent Proteins