Fig 1.
RER1 localizes in cis-Golgi network.
HEK293 cells were transfected with RER1 or the mutant RER1Δ25 (untagged vs myc-tagged, panels A vs B, respectively) and stained with RER1 and GM130 antibodies (A), myc and GM130 antibodies (B), or RER1 and KDEL antibodies (C). All photos were acquired with an Olympus IX81-DSU Confocal Microscope. The dotted box indicates the area enlarged (in panels iv and viii). (A, B) Perinuclear wild type RER1 immunoreactivity (green) are colocalized with GM130 (red), a cis-Golgi matrix protein. In contrast, perinuclear RER1Δ25 immunoreactivity only partially merges with GM130. Arrows indicate cells with colocalization. (C) Vesicular patterns of both wild type RER1 and RER1Δ25 immunoreactivities (green) do not merge KDEL proteins (red), an ER marker. Scale bar = 10 μm.
Fig 2.
RER1 expression significantly reduces αSyn levels.
(A) αSyn and RER1 were transiently overexpressed in HEK293. Cells were co-stained with αSyn (green) and RER1 (red) specific antibodies. The photos show that αSyn-immunoreactivity was reduced by wild type RER1 and less so by mutant RER1Δ25 expression. (B) Wild type RER1 expression significantly decreased αSyn levels (mean, 87.8 ±7.1%), but expression of RER1Δ25 resulted in significantly smaller effects on levels of αSyn (mean, 24.3 ±10.6%) (*p<0.05, **p<0.01; F2,6 = 17.31, p = 0.0032) (n = 5/control, n = 7/RER1, n = 3/ RER1Δ25) in HEK293. RER1 also decreased αSyn levels in human neuroglioma H4 cells (middle panel). (C) αSyn levels in both 1% Triton X-100 and 2% SDS fractions were decreased by RER1 overexpression (*p<0.05, **p<0.01; F2,9 = 14.02, p = 0.0017) (n = 4/group). C = control; R = RER1 transfected; Δ25 = RER1Δ25. Scale bar is 40 μm.
Fig 3.
RER1 effects are specific to αSyn.
(A) RER1 overexpression decreased the levels of αSyn mutants (A30P, E46K, and A53T). (B) The levels of βSyn did not change with RER1 overexpression (p = 0.725) (n = 4/group). (C) Expression of αSyn Δ71–82 mutant which is unable to aggregate due to the lack of a corresponding middle hydrophobic region, is not significantly decreased by RER1 overexpression (F2,15 = 2.214, p = 0.1438) (n = 6/group). (D) Overexpression of αSyn does not affect the maturation of APP or RER1 retrieval/retention function. C = control; R = RER1 transfected; G = EGFP
Fig 4.
Proteasome inhibition rescues αSyn levels.
(A) 24h post transfection, cells were treated with 10 μM MG132 (MG) or 100 μM chloroquine (Chlor). MG132 treatment partially recovered RER1-mediated reduction of αSyn. In contrast, chloroquine did not rescue αSyn, but increased APP levels consistent with its effects on macroautophagy and the lysosome. (B) Chloroquine treatment blocks autophagy activity. Cells were treated with 10–100 μM Chlorquine for 24 h. Lipidated and sequesterted LC3-II increased by chloroquine treatment (top panel). Cells were transfected with GFP-LC3 and 24 h post transfection, cells were treated with 100 μM Chloroquine for 24 h. Diffused pattern of LC3-I decreased and punctated pattern of LC3-II increased by Chloroquine treatment (bottom panel; scale bar = 10 μm). (C) Cells were co-transfected with αSyn and either EGFP or RER1, and then 24h post transfection treated with DMSO, 100 nM Bafilomycin (Baf), 10 μM MG132 (MG), or 10 μM Eeyarestatin1 (Eer1). In cells co-transfected with EGFP control, MG132 did not increase αSyn levels compared to cells exposed to DMSO. In cells co-transfected with RER1, MG132 showed a similar partial recovery of RER1-mediated αSyn reduction (mean, 69.2%), whereas the macroautophagy and ERAD inhibitors, Bafilomycin and Eeyarestatin1, had no apparent effect (**p<0.01, ***p<0.001; F2,6 = 28, p = 0.0009; n = 3/group). The right 2 lanes (separated by dotted line) show lysates from cells co-transfection of αSyn with RER1Δ25 for comparison. (D) RER1 decreases the levels of αSyn K80R mutant significantly. αSyn wild type or K80R mutant was co-transfected with RER1 or EGFP into HEK293 cells. R = RER1 transfected; G = EGFP. (E) RER1 interacts with NEDD4. RER1 is co-immunoprecipitated with NEDD4 in HEK293 cells co-expressing RER1 and NEDD4. N.C. = negative control using nonreactive serum.
Fig 5.
RER1 colocalizes with αSyn in Lewy bodies.
(A) Photomicrogaphs of tissue from control (left), and LB-positive tissues (right) show that RER1 colocalizes with αSyn in Lewy bodies. RER1 is detected in cell bodies, but appears enriched in round LB-like inclusions (arrows; see also enlargement in inset). (B) Photos show colocalization of both RER1 (green) and phosphorylated αSyn (pSer129: red) immunofluorescence in round, LB-like structures. Below are higher power images and confocal mapping of an inclusion positive for both RER1 and pSer129 immunoreactivity. Images were acquired on TCS SP2 AOBS Spectral Confocal Microscope (Leica) (B).
Fig 6.
Summary and putative model of RER1 effects on αSyn.
1) RER1 expression increases ER retrieval/retention of “immature” proteins in the cis-Golgi compartment which may contribute to ER retention of αSyn. 2) RER1 may indirectly retrieve αSyn back to the ER for degradation via the ERAD and proteasome (unfolded response system). 3) NEDD4 was found to interact with RER1. Although an E3 ligase, NEDD4 has been shown to reduce αSyn though the endosomal-lysosomal pathway [48]. RER1-mediated degradation of αSyn may also occur independent of ubiquitin via the 20S proteasome. 4) RER1 expression may act though maturation of an unknown protein (?) that mediates targeting and disposal of excess cytosolic αSyn via the proteasome (through an ubiquitin independent mechanism). ER, endoplasmic reticulum; ERAD, ER-associated degradation; ERGIC, ER-Golgi intermediate compartment.