Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Feb 6;33(6):2605-15.
doi: 10.1523/JNEUROSCI.2898-12.2013.

Mapping the subcellular distribution of α-synuclein in neurons using genetically encoded probes for correlated light and electron microscopy: implications for Parkinson's disease pathogenesis

Daniela Boassa  1 , Monica L Berlanga, Mary Ann Yang, Masako Terada, Junru Hu, Eric A Bushong, Minju Hwang, Eliezer Masliah, Julia M George, Mark H Ellisman
Affiliations

Mapping the subcellular distribution of α-synuclein in neurons using genetically encoded probes for correlated light and electron microscopy: implications for Parkinson's disease pathogenesis

Daniela Boassa et al. J Neurosci. .

Abstract

Modifications to the gene encoding human α-synuclein have been linked to the development of Parkinson's disease. The highly conserved structure of α-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human α-synuclein, including new genetic tags developed for correlated light microscopy and electron microscopy (the tetracysteine-biarsenical labeling system or the new fluorescent protein for electron microscopy, MiniSOG), we determined the distribution of α-synuclein when overexpressed in primary neurons at supramolecular and cellular scales in three dimensions (3D). We observed specific association of α-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, α-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice overexpressing human α-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. Three-dimensional electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulovesicular structures similar to what we observed in vitro. We propose that α-synuclein overexpression is associated with hypertrophy of membrane systems of the presynaptic terminal previously shown to have a role in vesicle recycling. Our data support the conclusion that α-synuclein is involved in processes associated with the sorting, channeling, packaging, and transport of synaptic material destined for degradation.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Recombinant AS-EGFP-4Cys is targeted to presynaptic terminals. A, The combinatorial tag formed by EGFP and the 4Cys motif FLNCCPGCCMEP was fused to the carboxyl terminus of human AS. B, Whole-cell lysates from HEK293 cells expressing AS-EGFP-4Cys (lanes 1 and 3) and EGFP-4Cys (lanes 2 and 4) were Western blotted with a monoclonal antibody specific for GFP (lanes 1 and 2) or AS (lanes 3 and 4). C, The labeling obtained with ReAsH-EDT2 is specific as indicated by the colocalization with the GFP signal (displayed in yellow in the merged image). Inset at higher magnification highlights the strong correspondence between ReAsH labeling and GFP fluorescence (arrowheads). Scale bars, 20 μm. D, Colocalization pattern of AS-EGFP-4Cys with the presynaptic marker synapsin I. Scale bar, 20 μm.
Figure 2.
Figure 2.
FRET-based fluorescent photo-oxidation of ReAsH-labeled AS proteins expressed in neurons specifically labels membranous structures in the presynaptic terminals. A–F, Neurons were transfected with AS-EGFP-4Cys, cultured for 17 days, labeled with ReAsH, and FRET photo-oxidized to avoid nonspecific photo-oxidation of background staining. The EM staining reveals both large tubular membranous structures (arrows) and smaller vesicular structures (arrowheads). C and D are the same terminal, as are E and F. Scale bars, 2 μm (A), 1 μm (B), 200 nm (C–E), 100 nm (F).
Figure 3.
Figure 3.
Recombinant AS-MiniSOG localizes in presynaptic terminals. A, The MiniSOG tag was fused to the carboxyl terminus of human α-synuclein. B, Whole-cell lysates from rat cortical neurons expressing the fusion proteins WT-human AS-MiniSOG (lane 1) and A53T-humanAS-MiniSOG (lane 2) were Western blotted with an antibody specific for AS and then stripped and reprobed against anti-α tubulin as a loading control. C, Rat cortical neurons were transfected with human-AS-MiniSOG, cultured for 21 days, and immunostained with an antibody specific to AS. Images represent single scans by confocal microscopy and are displayed with an inverted color table (black is highest signal, white is no signal, i.e., background). Inset in C is shown at higher magnification. D–G, The MiniSOG fluorescence (D, indicated by black arrows) overlaps with the AS signal (E) and the presynaptic marker Bassoon (F), as observed in the merged image (G, white arrows). MiniSOG is displayed in green, AS labeling in red, and Bassoon in blue. White indicates the overlap of all three channels (displayed at higher magnification in the inset in G, white arrowheads). Scale bars, 10 μm.
Figure 4.
Figure 4.
MiniSOG labeling illuminates the trafficking and degradation of AS. A, The electron micrographs show AS-MiniSOG overexpressing neurons processed for photo-oxidation of DAB into an osmiophilic, electron-dense reaction product resolvable by EM. The darker intensity of the signal reflects the specific AS labeling. The cellular distribution of AS (top row) appears in presynaptic terminals, specifically in synaptic vesicle membranes and in larger tubular membranous structures (A, top row, right), similar to what is observed with the 4Cys labeling in Figure 2. In addition, we observed an accumulation in the membrane of intraluminar vesicles of multivesicular bodies (A, top row, middle, black arrowheads, inset at higher magnification), as well as aggregations of the protein at the cell body of transfected neuronal cells (A, top row, left, black arrows). Controls represent nontransfected rat cortical neurons processed in parallel for photo-oxidation and did not snow this pattern of labeling (bottom row, right, normal presynaptic terminal, arrowheads in the middle panel point at a multivesicular body, with inset at higher magnification and arrows in the left panel pointing at lysosomes in a cell body). Scale bars, 500 nm. B, Zero tilt image from tomogram of cultured neurons expressing AS-MiniSOG processed for photo-oxidation. Extensive stained membranous elements are observed in the presynaptic terminal (highlighted in pink to match the 3D model shown in C). C, Three-dimensional model from EM tomogram of same area shows plasma membrane (pink) and contiguous membranes in three different colors (blue, green, and yellow).
Figure 5.
Figure 5.
Immunoreactivity in transgenic mice overexpressing human α-synuclein. Immunohistochemistry using the human AS antibody reveals strong punctate labeling in the hippocampus, both in CA3 (stratum lucidum, SLu) and CA1 (stratum radiatum, Rad), and in the neocortex of transgenic mice. In nontransgenic controls (littermates), no human AS immunoreactivity was observed.
Figure 6.
Figure 6.
Presynaptic terminals in hippocampus CA3 of AS transgenic mice show convoluted membranous structures. A, EM analysis using TEM in hippocampus CA3 (stratum lucidum) revealed multiple-layered membrane structures in presynaptic terminals of Tg mice overexpressing human AS (top row, white arrows). The non-Tg controls (bottom row) did not show this pattern. B, Analysis of tomographic reconstruction showed that the presynaptic terminal was filled with vesicles and frequent membranous elements around them (left, yellow arrowheads in a tomographic slice). Synapse is indicated by a black arrow. Scale bar, 300 nm. On the right, maximum intensity projection of tomographic reconstruction of the same presynaptic terminal is displayed with an inverted color table (white is highest EM signal, black is no signal). Yellow arrowheads point at membrane convolutions inside the terminal. Two synapses are indicated by the white arrows. C, Using SBEM we observed that the membrane convolutions originate from invaginations of the plasma membrane, as indicated by the white arrow. Three-dimensional volume segmentation of SBEM datasets (image on the right) shows these membrane perturbations inside the complete terminal (plasma membrane is in purple, postsynaptic densities in green, membrane convolutions in magenta, blue, and orange).
Figure 7.
Figure 7.
Presynaptic terminals in hippocampus CA1 of AS transgenic mice show extensive tubulovesicular architecture. Comparison of presynaptic nerve terminals found in the hippocampus CA1 (stratum radiatum) in non-Tg (top left, reconstruction in light blue with synapses in yellow) and AS-Tg mice (top right, reconstruction in green with synapses in red). Enlarged nerve terminals of AS-Tg mice show extensive membranous networks (middle row, white arrowheads) and form synapses (evidenced by postsynaptic densities and accumulation of presynaptic vesicles). Both EM analysis using SBEM (top two rows) and tomography (bottom row) revealed large presynaptic nerve terminals filled with tubulovesicular structures similar to what we observed in vitro. Segmentation of contiguous membrane segments from tomographic reconstruction is shown in three different colors (inset in bottom row, right, corresponding to the area indicated by arrowheads).
Figure 8.
Figure 8.
Atypical presynaptic terminals in neocortex and substantia nigra of AS transgenic mice. A–C, Using SBEM we observed, in the neocortex of AS transgenic mice, enlarged nerve terminals massively filled with an endomembrane network formed of tubulovesicular structures similar to those observed in hippocampus CA1. In C, three-dimensional volume segmentation shows the enlarged terminal (light blue) forming a synapse (postsynaptic density is in yellow, indicated by the white arrow) with a dendritic spine (magenta) compared to a normal terminal (purple) forming a synapse with an adjacent spine on the same dendrite. D, Similarly, TEM analysis of thin sections from substantia nigra of AS transgenic mice revealed endomembrane perturbations in presynaptic terminals characterized by extensive tubulovesicular structures (arrowheads in inset at higher magnification, scale bar, 250 nm). Slim black arrow points at a synapse formed by the enlarged terminal, while the shorter black arrow points at a synapse formed by a normal terminal.
Figure 9.
Figure 9.
Overexpressed AS highlights the intracellular system associated with protein degradation. Soluble AS protein is transported from the cell body to nerve terminals by axonal transport and associates with intracellular membranes in the presynaptic terminal, causing profound membrane perturbations. Consequently, accumulation of AS in components of the protein degradation system such as MVBs and lysosomes might interfere with their normal function to degrade proteins.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Braak H, Del Tredici K, Rüb U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging. 2003;24:197–211. - PubMed
    1. Burré J, Sharma M, Tsetsenis T, Buchman V, Etherton MR, Südhof TC. Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro. Science. 2010;329:1663–1667. - PMC - PubMed
    1. Chartier-Harlin MC, Kachergus J, Roumier C, Mouroux V, Douay X, Lincoln S, Levecque C, Larvor L, Andrieux J, Hulihan M, Waucquier N, Defebvre L, Amouyel P, Farrer M, Destée A. Alpha-synuclein locus duplication as a cause of familial Parkinson's disease. Lancet. 2004;364:1167–1169. - PubMed
    1. Chivet M, Hemming F, Pernet-Gallay K, Fraboulet S, Sadoul R. Emerging role of neuronal exosomes in the central nervous system. Front Physiol. 2012;3:145. doi: 10.3389/fphys.2012.00145. - DOI - PMC - PubMed
    1. Danzer KM, Krebs SK, Wolff M, Birk G, Hengerer B. Seeding induced by alpha-synuclein oligomers provides evidence for spreading of alpha-synuclein pathology. J Neurochem. 2009;111:192–203. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources