In Vivo Imaging of α-Synuclein in Mouse Cortex Demonstrates Stable Expression and Differential Subcellular Compartment Mobility

National Institutes of Health, United States of America
PLoS ONE (Impact Factor: 3.23). 05/2010; 5(5):e10589. DOI: 10.1371/journal.pone.0010589
Source: PubMed


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.
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.
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.

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Available from: Edward Rockenstein, Oct 07, 2015
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    • "studies , as shown by the recent application of FRET to study signaling ( Stockholm et al . , 2005 ; Rudolf et al . , 2006 ; Breart et al . , 2008 ; Ritsma et al . , 2012a ) , and FRAP , which has been used in the live brain to measure the diffusion of  synuclein , thus opening the door to studying the biophysical properties of proteins in vivo ( Unni et al . , 2010"
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    ABSTRACT: Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology.
    The Journal of Cell Biology 06/2013; 201(7):969-979. DOI:10.1083/jcb.201212130 · 9.83 Impact Factor
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    • "In fact, detection of endogenous emission through two-photon excitation and fluorescence lifetime imaging is becoming a rapidly growing field within IVM research and is providing information about the metabolic state of tissues as well as further contrast signals [149,150]. Tissue contrast can be obtained also by means of expressing a fluorescent protein through viral transduction or direct DNA injection in the whole tissue or organ of interest [23,151–153]. In zebrafish, this has been obtained by injecting second-harmonic generation emitting nanocrystals in the cytoplasm of one-cell stage embryos [154]. "
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    ABSTRACT: Intravital microscopy has become increasingly popular over the past few decades because it provides high-resolution and real-time information about complex biological processes. Technological advances that allow deeper penetration in live tissues, such as the development of confocal and two-photon microscopy, together with the generation of ever-new fluorophores that facilitate bright labelling of cells and tissue components have made imaging of vertebrate model organisms efficient and highly informative. Genetic manipulation leading to expression of fluorescent proteins is undoubtedly the labelling method of choice and has been used to visualize several cell types in vivo. This approach, however, can be technically challenging and time consuming. Over the years, several dyes have been developed to allow rapid, effective and bright ex vivo labelling of cells for subsequent transplantation and imaging. Here, we review and discuss the advantages and limitations of a number of strategies commonly used to label and track cells at high resolution in vivo in mouse and zebrafish, using fluorescence microscopy. While the quest for the perfect label is far from achieved, current reagents are valuable tools enabling the progress of biological discovery, so long as they are selected and used appropriately.
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    • "The uniquely designed PCA presented here has the advantage to detect and visualize α-syn oligomerization in post mortem tissue with minimal tissue processing, but could potentially be utilized to monitor the formation of α-syn oligomers over time in the brain of a living animal using two-photon microscopy. Recent advances in two-photon microscopy have enabled in vivo visualization of protein aggregation and neurodegeneration in the brain of an Alzheimer’s disease mouse model [22,33,34] and protein degradation in the brain of a PD animal model [35-37]. Here, using two-photon microscopy, we demonstrate the ability to image and detect α-syn oligomers in vivo. "
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