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

Abstract

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.

Methodology/principal findings: 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/significance: 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.

Figure 1. Syn-GFP is present in neuronal cell bodies and presynaptic terminals.

A. Fixed tissue confocal image of Syn-GFP in the cortex shows one neuronal soma and that the vast majority of neuropil puncta staining is present within axons. Endogenous GFP fluorescence is shown without antibody labeling. B. In vivo multiphoton image of Syn-GFP in the cortex shows staining in one neuronal soma and multiple neuropil puncta. Scale bar for A–B 10 µm. C. Fixed tissue confocal image from the cortex of a Syn-GFP animal showing GFP staining in two somata and in the neuropil. D. Staining in the same section for human α-synuclein. E. Merged image shows colocalization between GFP signal and human α-synuclein, as expected for this fusion protein. Scale bar for C–E 20 µm.

Figure 2. Syn-GFP positive cell body and presynaptic terminal density does not vary with age.

A. Group data of average Syn-GFP positive cell body density in layer 2/3 of cortex at different ages demonstrates no difference with age. B. Group data of average Syn-GFP positive presynaptic terminal density in layer 2/3 of cortex at different ages demonstrates no difference with age. C. Representative histograms of mean terminal intensity (in arbitrary units) demonstrate a similar shape at three different ages. n = number of animals, error bars = 1 SD.

Figure 3. Syn-GFP cell body and high intensity presynaptic terminal expression is relatively stable over time.

In vivo multiphoton images of the same region at 2 different time points (A1: day 0, A2: day 49) show that Syn-GFP cell body expression is stable over weeks. Arrows show 4 cell Syn-GFP positive bodies that are present at both time points. Scale bar for A1-2 10 µm. In vivo multiphoton images of two different regions (B1-3 and C1-4) repeatedly imaged at different time points show that high intensity Syn-GFP terminal expression can be followed over months. Arrows show multiple Syn-GFP positive terminals that are present at all the time points and the arrowheads show high intensity terminals that disappeared over time. Scale bars for B 10 µm and C 7.5 µm.

Figure 4. Syn-GFP expression pattern, cell body and terminal density do not vary with time post-window placement.

In vivo multiphoton images of Syn-GFP in the cortex at 3 different times post-window placement (A: day 0, B: 3 months, C: 6 months) shows a similar pattern of staining. Scale bar 10 µm. D. Group data of average Syn-GFP positive cell body density in layer 2/3 of cortex at different times post window placement demonstrates no significant difference. E. Group data of average Syn-GFP positive presynaptic terminal density in layer 2/3 of cortex at different times post-window placement demonstrates no significant difference. F. Representative histograms of mean terminal intensity (in arbitrary units) demonstrate a similar shape at different times post-window placement. n = number of animals, error bars = 1 SD.

Figure 5. Syn-GFP presynaptic terminal signal recovers differently after photobleaching.

A. In vivo multiphoton images of the same region of Syn-GFP positive terminals over time. One normal terminal (marked by the white circle) is photobleached just after t = 0. This sequence shows full recovery of Syn-GFP signal over several minutes. B. Similar time sequence as in panel A but in this case one high intensity terminal (marked by the white circle) is photobleached just after t = 0. This sequence shows partial recovery of Syn-GFP signal. Scale bar A and B 5 µm. C. Group data plotting normalized fluorescence before and after presynaptic terminal photobleaching (marked by yellow arrow; normal terminals n = 15 terminals, high intensity terminals n = 10 terminals; n = 5 animals, error bars = 1 SD; * represents statistically significant difference at t = 8 min, t-test p<0.0001).

Figure 6. Syn-GFP somatic signal recovers slowly after whole-cell photobleaching and is rapidly mobile during half-cell photobleaching.

A. In vivo multiphoton images of the same region of Syn-GFP positive cell bodies over time. One soma (marked by the white circle) is photobleached just after t = 0. This sequence shows partial recovery of Syn-GFP signal over 60 min. B. Similar time sequence as in panel A but in this case half the soma (marked by the white rectangle) is photobleached just after t = 0. Scale bar in A 10 µm and in B 20 µm. C. Group data plotting normalized fluorescence before and after photobleaching (marked by yellow arrow; whole-cell bleach: n = 20 cells, half-cell bleach: n = 3 cells; n = 3 animals, error bars = 1 SD).