The ER retention protein RER1 promotes alpha-synuclein degradation via the proteasome

<div><p>Abnormal accumulation of α-synuclein (αSyn) has been linked to endoplasmic-reticulum (ER) stress, defective intracellular protein/vesicle trafficking, and cytotoxicity. Targeting factors involved in ER-related protein processing and trafficking may, therefore, be a key to modulating αSyn levels and associated toxicity. Recently retention in endoplasmic reticulum 1 (RER1) has been identified as an important ER retrieval/retention factor for Alzheimer’s disease proteins and negatively regulates amyloid-β peptide levels. Here, we hypothesized that RER1 might also play an important role in retention/retrieval of αSyn and mediate levels. We expressed RER1 and a C-terminal mutant RER1Δ25, which lacks the ER retention/retrieval function, in HEK293 and H4 neuroglioma cells. RER1 overexpression significantly decreased levels of both wild type and A30P, A53T, and E46K disease causal mutants of αSyn, whereas the RER1Δ25 mutant had a significantly attenuated effect on αSyn. RER1 effects were specific to αSyn and had little to no effect on either βSyn or the Δ71–82 αSyn mutant, which both lack the NAC domain sequence critical for synuclein fibrillization. Tests with proteasomal and macroautophagy inhibitors further demonstrate that RER1 effects on αSyn are primarily mediated through the ubiquitin-proteasome system. RER1 also appears to interact with the ubiquitin ligase NEDD4. RER1 in human diseased brain tissues co-localizes with αSyn-positive Lewy bodies. Together, these findings provide evidence that RER1 is a novel and potential important mediator of elevated αSyn levels. Further investigation of the mechanism of RER1 and downstream effectors on αSyn may yield novel therapeutic targets for modulation in Parkinson disease and related synucleinopathies.</p></div

RER1 effects are specific to αSyn.

<p>(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 (F_2,15 = 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</p

Proteasome inhibition rescues αSyn levels.

<p>(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; F_2,6 = 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.</p

Summary and putative model of RER1 effects on αSyn.

<p>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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184262#pone.0184262.ref048" target="_blank">48</a>]. 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.</p

RER1 localizes in cis-Golgi network.

<p>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.</p

RER1 colocalizes with αSyn in Lewy bodies.

<p>(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).</p

Biochemical analysis of cytosolic antioxidant enzymatic activities in the inner ear tissues of young <i>Gsr</i>^+/+ and <i>Gsr</i>^-/- mice.

<p>The activities of cytosolic SOD (A), catalase (B), COX (C), TXN1 (D), and TXNRD1 (E) were measured in the inner ear tissues from 3–5 month-old <i>Gsr</i>^+/+ and <i>Gsr</i>^-/- males (N = 4–5). Data are shown as means ±SEM. *p<0.05 vs. <i>Gsr</i>^+/+.</p

Body weight of young <i>Gsr</i>^+/+ and <i>Gsr</i>^-/- mice.

<p>Body weights of male and female <i>Gsr</i>^+/+ and <i>Gsr</i>^-/- mice were measured at 3–5 months of age (N = 10–11). Data are shown as means ±SEM.</p

Biochemical analysis of cytosolic glutathione-related enzymatic activities and GSH/GSSG ratios in the inner ear tissues of young <i>Gsr</i>^+/+ and <i>Gsr</i>^-/- mice.

<p>The activities of GSR (A), the levels of GSH (B), GSSG (C), and GSH/GSSG (D), the activities of GPX (E), GCL (F), and GST (G), and the levels of NADPH (H) were measured in the cytosol of the inner ear tissues from 3–5 month-old <i>Gsr</i>^+/+ and <i>Gsr</i>^-/- males (N = 4–5). Data are means ±SEM. *p<0.05 vs. <i>Gsr</i>^+/+. GSR = glutathione reductase, GPX = glutathione peroxidase, GCL = glutamate-cysteine ligase, GST = glutathione transferase.</p

Histological analysis of cochlear spiral ganglion neuron density and stria vascularis thickness in young <i>Gsr</i>^+/+ and <i>Gsr</i>^-/- mice.

<p>(A-G) The densities of spiral ganglion neurons (SGN) in the apical, middle, and basal regions of cochlear tissues from 3–5 month-old male <i>Gsr</i>^+/+ and <i>Gsr</i>^-/- mice (N = 4–5) were counted and quantified (G). (H-N) The thickness of stria vascularis (SV) in the apical, middle, and basal region of cochlear tissues from 3–5 month-old <i>Gsr</i>^+/+ and <i>Gsr</i>^-/- males (N = 4–5) was measured (N). Data are shown as means ±SEM. Scale bar = 25 μm.</p