Figure 1.
α-synuclein is present in endosomal compartments.
Colocalization experiments of Myc-tagged αSYN (red) with endosome-associated EGFP-tagged Rab proteins (green) in HEK293T cell (A) and SH-SY5Y dopaminergic neuronal cells (B). The subcellular distribution of endogenous αSYN was also examined in SH-SY5Y cells expressing EGFP-tagged Rab proteins (C). Cells were fixed 48 hours post-transfection and were subjected to immunofluorescent analysis. In both cell lines, exogenously expressed αSYN was detected not only throughout the cytosol but also in punctate endosomal structures that were positive for EGFP-Rab5a (early endosome marker), Rab7 (late endosome marker), and Rab11a (recycling endosome marker), respectively. Nuclei were conterstained with TO-PRO3 iodide (pseudocolored blue). The inset picture is a magnified picture of the square area. Immunostaining was performed three times and the experiment three times with the same results. Size bar: 10 µm.
Figure 2.
α-synuclein is detected in supernatant but not in the exosome-containing pellets from neuronal culture medium and CSF by standard immunoblot analysis.
A. wt and A53T mutant αSYN were inducibly expressed in SH-SY5Y cells for 48 hours. Culture medium as well as whole cell lysates (50 µg protein per lane) were subjected to Western blot analysis. The collected media were further separated into the supernatant and exosome-containing pellets before loading onto gels. Alix, Hsp90, and BSA were used as markers for exosome, cytosol, and culture medium, respectively. In the neuronal culture medium, both monomeric/oligomeric wt and mutant αSYN were recovered in the supernatant (dagger) rather than exosome-containing pellets (hash). Asterisk indicates unspecific band. B. The resuspended exosome-containing pellets from the culture medium were further separated by sucrose-density gradient followed by Western blot analysis. Immunoblot probed with synuclein-1 anti-αSYN, anti-PrP Abs and the successful separation of exosome was confirmed by exosomal markers, Flotillin-1 and Alix. As shown in the blot, PrP migrated near the top of the density gradient (fraction #8) with concomitant enrichment of exosome-associated proteins. By contrast, no exosomal enrichment was observed with αSYN. C. CSF (1.5 mL) from 5 PD patients together with age-matched controls was pooled and exosome-containing pellets were isolated by successive centrifugation indicated. Equal concentrations (50 µg per lane) of total CSF samples were loaded alongside CSF-derived exosomes and then probed with anti-αSYN and PrP antibodies. PrP detected in CSF-derived exosomes was enriched compared to neat CSF. αSYN was weakly but specifically detected in neat CSF, whereas no αSYN-positive signal could be detected in CSF-derived exosomes. No significant difference was observed in the expression levels of CSF αSYN between PD patients and normal controls. Representative Western blots from three independent experiments are presented.
Figure 3.
Over-expression of DN VPS4 in HEK293T cells leads to increased extracellular α-synuclein and parallel decrease in lysosome.
A. αSYN-expressing HEK293T cells were co-transfected either with 3XFLAG-tagged wt-VPS4A or DN mutant (E228Q) VPS4A. Forty-eight hours after transfection, HEK293T cells were fractionated into the cytosol (S3), endosome (P4), and lysosome (S4). Fractionated cell lysates as well as protein extracts from the culture medium (50 µg protein per lane) were subjected to Western immunoblot analysis using anti-Myc, anti-ubiquitin, anti-FLAG/M2 Abs. Each fraction was verified by the presence of a specific marker protein: LAMP-1 (late endosome and lysosome), Rab5 (early endosome), Rab11 (recycling endosome), Hsp90 (cytosol), and BSA (culture medium). As shown in the blot, marked increase of extracellular αSYN monomer and multimers (white arrow) concomitant with slightly decreased lysosomal αSYN-immunopositive smear (open triangle) were observed by over-expression of DN VPS4A. Note that endosomal proteins including αSYN seemed to be heavily ubiquitylated (hash). Asterisk indicates endogenous myc band. B. Treatment with lysosomal inhibitor bafilomycin A1 (0–10 nM) for 24 hours induced the buildup of cellular αSYN oligomers (asterisk) in parallel with the increase of extracellular αSYN monomer (closed triangle). C. Subcellular localization of Myc-αSYN (red) in HEK293T cells expressing wt or DN VPS4A (magenta). LAMP-1 (green) was used as a marker for late endosome and lysosome. DN VPS4 distibuted as aberrant cytoplasmic punctate structures, showing a marked contrast to wt-VPS4A with diffuse perinuclear distribution. Representative Western blots from three separate experiments are shown. Scale bar: 10 µm.
Figure 4.
Expression of DN VPS4A increases wt as well as A53T mutant α-synuclein in SH-SY5Y neuronal cells.
A. SH-SY5Y cells inducibly expressing wt or A53T mutant αSYN were further co-transfected with 3XFLAG-tagged wt or DN E228Q mutant VPS4A plasmids. After 48 hours of αSYN induction with doxycycline, whole cell and proteins from culture media (50 µg protein per lane) were subjected to immunoblot analysis using anti- synuclein-1 and anti-FLAG/M2 antibody. Hsp90 and BSA were used as markers for cytosol and culture medium, respectively. Increased extracellular secretion of wt and A53T mutant αSYN were observed by DN VPS4A. Note that the extracellular secretion of monomeric wt-αSYN (black arrow) was higher than that of A53T mutant αSYN (closed triangle) in mock-transfected cells as well as in DN-VPS4A engineered cells. Representative immunoblots from three independent experiments are shown. B. Densitometric measurement of monomeric αSYN secreted into culture media. Values indicate the ratio of αSYN monomer to BSA. Significant increase of wt as well as A53T αSYN in culture media was observed by co-expression of wt and or DN VPS4A (*p<0.05, **p<0.005).
Figure 5.
VPS4 was found to be a component of Lewy body.
Paraffin embedded sections including the substantia nigra and the temporal lobes from four patients with PD with a mean age of 77.5 years and the controls with a mean age of 77.3 years were subjected to immunohistochemical analysis using anti-human VPS4 Ab. Diaminobenzidine were used to visualize the staining and nuclei were counterstained with hematoxylin. In all brain tissues from patients with PD, the core structures of Lewy bodies showed VPS4 immunoreactivity (Fig. 5). Only weak background staining was observed in control brain sections (data not shown). The percentage of VPS4-immunoreactive Lewy bodies in the substantia nigra (A and B) and the temporal lobes (C and D) of PD brains were 90% and 10%, respectively. Scale bar: 20 µm.
Figure 6.
Part of the cellular α-synuclein was trafficked via a recycling endosome pathway for extracellular secretion.
HEK293T cells expressing Myc-αSYN were co-transfected with mock (EGFP), EGFP-wt-Rab11a, EGFP-CA-Rab11a, or EGFP-DN-Rab11a expression plasmids. At 48 hours following transfection, the cells were harvested and fractionated into cytosol, endosome, and lysosome. Fractionated samples as well as total proteins from the culture media (50 µg per lane) were subjected to immunoblot analysis using anti-Myc, anti-EGFP Abs. A successful fraction was verified by the presence of a specific marker proteins. As shown in the blot, secretion of αSYN oligomer in culture medium was partly reduced by the over-expression of GDP-locked DN-Rab11a (asterisk), accompanied by the extensive retention of HMW αSYN species in the endosome (double asterisk). Representative blots from three separate experiments are shown.
Figure 7.
Increased secretion of α-synuclein by DN-VPS4A is restored by DN-Rab11a.
GDP-locked DN-Rab11a strikingly restored the hypersecretion of αSYN triggered by the impaired MVB sorting pathway (closed triangle). HEK293T cells co-expressing Myc-αSYN and 3XFLAG-DN-VPS4A (A) and SH-SY5Y neuronal cells expressing 3XFLAG-DN-VPS4A (B) were further transfected with EGFP-DN-Rab11a. Forty eight hours post transfection, the cells were harvested and solubilized in RIPA buffer. Whole cell lysates as well as total proteins from culture media (50 µg per lane) were then subjected to immunoblot analysis using anti-Myc, anti-synuclein-1, anti-FLAG, and anti-EGFP Abs. Hsp90 and BSA were used as markers for the cytosol and culture medium, respectively. Asterisk indicates unspecific band. Representative blots from three independent experiments are presented.
Figure 8.
Schematic presentation of endosomal pathways and the functional relevance of MVB sorting machinery and Rab11a-mediated recycling pathway in the secretion as well as lysosomal targeting of α-synuclein.
Membrane-associated cargo proteins including αSYN are translocated to early endosomes, which also receive cargoes from the trans-Golgi network. Some cargoes are recycled back to the plasma membrane. Others are sequestered in intraluminal vesicles of MVB. Individual ESCRT proteins and VPS4 contribute to MVB formation through the induction of invagination and final scission of the endosomal membrane. MVB directs either for lysosomal degradation or for secretion as exosomes by exocytic fusion with the plasma membrane. Under the physiolosical condition, αSYN in the early endosome may be transferred to MVB then targeted for lysosomal degradation (A). Alternatively, part of the endosomal αSYN may be cast into the extracellular milieu through the Rab11a-dependent recycling endosome (B) and, to a lesser degree, MVB-exosome pathway (C). If the intracellular αSYN reaches a toxic level or the MVB sorting is dammed up, excessive amounts of endocytic αSYN will flow out mainly through the recycling endosome pathway.