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

et al. (2010) In Vivo Imaging of a-Synuclein in Mouse Cortex Demonstrates Stable Expression and Differential Subcellular Compartment Mobility. PLoS ONE 5(5): e10589. doi:10.1371/journal.pone.0010589 In Vivo Imaging of a-Synuclein in Mouse Cortex Demonstrates Stable Expression and Differential Subcellular Compartment Mobility Vivek K. Unni 0 Tamily A. Weissman 0 Edward Rockenstein 0 Eliezer Masliah 0 Pamela J. McLean 0 Bradley T. Hyman 0 Mark R. Cookson, National Institutes of Health, United States of America 0 1 Alzheimer's Research Unit, MassGeneral Institute for Neurodegenerative Disease, MGH Harvard Medical School , Charlestown , Massachusetts, United States of America, 2 Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University , Cambridge, Massachusetts , United States of America, 3 Department of Neurosciences, University of California San Diego , La Jolla, California , United States of America Background: Regulation of a-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 a-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 a-synuclein (Syn-GFP) transgenic mice and in vivo multiphoton imaging to measure a-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 a-synuclein and related mechanisms of neurodegeneration. Conclusions/Significance: In vivo multiphoton imaging in Syn-GFP transgenic mice demonstrates stable a-synuclein expression and differential subcellular compartment mobility within cortical neurons. This opens new avenues for studying a-synuclein biology in the living brain and testing new therapeutics for PD and related disorders. - Funding: This work was supported by National Institutes of Health Grants NS038372, NS063963, T32AG000222, T32NS048005, AG18440 and AG022074. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Multiple lines of evidence implicate abnormal regulation and aggregation of the synaptic protein a-synuclein in the etiology of Parkinsons Disease (PD) [12]. Because of this there have been significant efforts to better understand the biology of a-synuclein, including mechanisms relating to its synthesis [36], degradation [710], regulation by other proteins [11], and function at synapses [1215]. To date, largely because of technical reasons, these studies have been limited to reduced biochemical preparations, cell culture models and analysis of fixed animal or human tissue. In contrast, the study of other neurodegenerative diseases like Alzheimers Disease (AD) has recently been advanced by development of in vivo multiphoton imaging techniques in mouse models. New insights into the mechanisms of AD involving the formation of extracellular beta-amyloid plaques [1618] and intracellular tau aggregates [1920] have come from these studies that can follow individual plaques and tangles in the mouse brain over time. The study of PD and other related synucleinopathies would benefit from analogous techniques to study the biology of asynuclein in vivo and its role in neurodegeneration. In this study we detail a new experimental paradigm that allows real-time in vivo imaging of fluorescently-tagged human a-synuclein in individual cortical neurons with subcellular resolution over a period of months. We demonstrate that this system is stable and allows for detailed measurements of a-synuclein levels in individual cell bodies and presynaptic terminals. In addition, we use this system to provide the first in vivo evidence that a-synuclein protein is differentially mobile within neurons using the fluorescence recovery after photobleaching (FRAP) technique. To date FRAP measurements have been described in numerous systems [21] and to study a-synuclein in other models [2223], but to our knowledge this is the first in vivo extension of the technique to mammalian neurons, demonstrating its potential feasibility for studying a wide range of neuronal proteins in living brain. Our development of these approaches opens lines of inquiry that are difficult to address otherwise. For instance, chronic imaging of individual Syn-GFP expressing cells and presynaptic terminals allows precise analysis of possible changes in these structures over time. In addition, measuring a-synuclein mobility in different subcellular compartments using FRAP can test how its physical state, ability to bind to partners or other geometrical constraints vary within the cell. Understanding these processes in the living brain is of interest since it may lead to new strategies for developing PD therapies. Materials and Methods Animals Male Syn-GFP transgenic mice were mated with BDF1 female mice by the MGH Center for Comparative Medicine (CCM). Animals were held in a light-dark cycle, temperature and humidity-controlled animal vivarium and maintained under ad libitum food and water diet supplied by the CCM. All experiments were approved by the Subcommittee on Research Animal Care (SRAC) at the MGH and every effort was made to minimize the number of animals used and their suffering. Immunohistochemistry Animals were deeply anesthetized and perfused with a transcardiac approach with ice cold phosphate-buffered saline followed by paraformaldehyde (4%) solution. The brain was quickly removed and placed in paraformaldehyde (4%) at 4 C for a minimum of 24 hr. Next 50200 mm thick floating sections were cut on a freezing microtome (Microm, HM400). Alpha-synuclein immunohistochemistry was performed after blocking tissue with normal goat serum (10%) at room temperature (RT) for 1 hr. Next sections were stained with a human specific a-synuclein antibody LB509 (Zymed, 1:100, 4 C for 24 hr) and a Cy3 anti-mouse secondary antibody (Jackson Immunoresearch, 1:500, RT for 1 hr). All sections were imaged on (...truncated)