Ubiquilin-1 regulates amyloid precursor maturation and degradation by stimulating K63-linked polyubiquitination of lysine 688

Amina El Ayadia,1, Emily S. Stierena,1, José M. Barrala,b,c, and Darren Boehninga,b,c,d,2

aDepartment of Neuroscience and Cell Biology, bSealy Center for Structural Biology and Molecular Biophysics, cMitchell Center for Neurodegenerative Diseases, and dInstitute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555

Edited by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved July 10, 2012 (received for review April 22, 2012) The pathogenesis of Alzheimer’s disease (AD) is associated with APP in the Golgi, reducing the amount of APP that reaches the proteolytic processing of the amyloid precursor protein (APP) to an cell surface for processing. SorLA also shuttles APP from the amyloidogenic peptide termed Aβ. Although mutations in APP and early endosomes back to the Golgi, further reducing Aβ pro- the secretase enzymes that mediate its processing are known to duction in late endosomes (3, 12). Clathrin coat and result in familial forms of AD, the mechanisms underlying the adaptor proteins JIP1b, Fe65, retromer, nexin, homer2, homer3, more common sporadic forms of the disease are still unclear. Evi- Numb, and X11 family members also regulate sorting of APP dence suggests that the susceptibility of APP to amyloidogenic through the endocytic pathway by interacting with the intracel- processing is related to its intracellular localization, and that sec- lular tail of APP (3, 6, 9, 13). retase-independent degradation may prevent the formation of Production of Aβ may also be suppressed by factors that target cytotoxic peptide fragments. Recently, single nucleotide polymor- full-length APP or its C-terminal fragments to the or phisms in the UBQLN1 have been linked to late-onset AD, lysosome, resulting in γ-secretase–independent degradation (14– and its protein product, ubiquilin-1, may regulate the maturation 16). Recent studies have suggested a role for ubiquilin-1 in the of full-length APP. Here we show that ubiquilin-1 inhibits the mat- regulation of APP trafficking and processing (17). Specifically, uration of APP by sequestering it in the early secretory pathway, reduction in proteins levels of ubiquilin-1 accelerated the matu- NEUROSCIENCE primarily within the Golgi apparatus. This sequestration signifi- ration of APP through the secretory pathway, which was associ- cantly delayed the proteolytic processing of APP by secretases ated with increased secretion of APPs and increased production and the proteasome. These effects were mediated by ubiquilin- of Aβ (17). Here we show that ubiquilin-1 can sequester APP – 1 stimulated K63-linked polyubiquitination of lysine 688 in the in the Golgi apparatus and delay its access to the cell surface APP intracellular domain. Our results reveal the mechanistic basis and subsequent proteolytic processing. We found that ubiquilin-1 by which ubiquilin-1 regulates APP maturation, with important suppresses APP maturation and proteasomal degradation by consequences for the pathogenesis of late-onset AD. stimulating APP lysine 63 (K63)-linked polyubiquitination. We also found that mutation of lysine 688 arginine (K688R) in the trafficking | | -associated domain | APP intracellular domain abrogated ubiquitination and Golgi ubiquitin-like domain sequestration, suggesting that nondegradative ubiquitination of APP by ubiquilin-1 is a critical sorting signal limiting APP amy- myloid precursor protein (APP) is a type I transmembrane loidogenic processing. Combined with our previous observations Aprotein that matures in the secretory pathway, where it that ubiquilin-1 functions as a molecular and its levels undergoes classical N- and O-linked glycosylation during transit are reduced in Alzheimer’s disease (AD) brains (18), these results through the endoplasmic reticulum (ER) and Golgi, respectively indicate that ubiquilin-1 is a critical quality-control molecule (1, 2). Though most APP is degraded by lysosomes (3), a small regulating APP biosynthesis, trafficking, and degradation. portion reaches the cell surface to be cleaved by the secretases (4). Amyloidogenic processing of APP occurs by sequential Results processing by β- and γ-secretase to release Aβ peptide and the Ubiquilin-1 Sequesters APP in the Early Secretory Pathway. To in- APP intracellular domain (AICD) (5). APP half-life is short, and vestigate the possible effects of exogenous expression of ubiq- thus the amount of APP detected at the cell surface is very low uilin-1 on APP degradation, we monitored the degradation rate (6). The rapid internalization rate of APP is due to the presence of endogenous APP and APP fragments in PC12 cells treated of the highly conserved YENPTY (single amino acid code) se- with the protein synthesis inhibitor cycloheximide (CHX). Traf- quence in the APP cytoplasmic domain, which contains a canon- ficking through the Golgi was monitored by the transition of APP ical NPxY internalization signal for clathrin-mediated endocytosis from an immature form, which migrates faster on SDS/PAGE, (7). Proteolytic processing of APP has been shown to occur in to a mature more slowly migrating form that has undergone O- various sites throughout the secretory and endocytic pathways. linked glycosylation (1, 8). CHX treatment resulted in a time- However, amyloidogenic processing primarily occurs after tran- dependent loss of immature APP and mature APP levels with a sition through the Golgi apparatus (8) and in endosomal com- partments, where acidic conditions promote optimal activity of β the beta-site APP cleaving enzyme (BACE) or -secretase (9). Author contributions: A.E., E.S.S., J.M.B., and D.B. designed research; A.E., E.S.S., and D.B. Deletion or mutation of the YENPTY internalization signal performed research; A.E., E.S.S., J.M.B., and D.B. analyzed data; and A.E. and D.B. wrote leads to an increase in plasma membrane-associated APP and a the paper. significant decrease in Aβ production, underscoring the impor- The authors declare no conflict of interest. tance of endocytic recycling for Aβ generation (5, 7, 10). Various This article is a PNAS Direct Submission. cytoplasmic adaptor proteins have been shown to interact with 1A.E. and E.S.S contributed equally to this work. the YENPTY motif and regulate sorting of APP to different cell 2To whom correspondence should be addressed. E-mail: [email protected]. compartments and thus regulate its proteolytic processing. The This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. protein SorLA or LR11 (11) acts as a sorting receptor that traps 1073/pnas.1206786109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1206786109 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 A C E Control Ubiquilin Vector Ubiquilin CHX(min): 0 15 20 30 40 60 80 100 120 CHX(min): 0 15 20 30 40 60 80 100 120 CHX CHX 10K 100K 10K 100K 10K 100K 10K 100K 201 201 M M 201 116 Im 116 Im M 94 94 116 Im Holo- 94 APP APP 50 APP 50 * 37 37 Myc- 29 29 Ubiquilin 19.4 50 19.4 6.8 Endogenous 6.8 37 c-Myc

116 Myc- 116 Myc- 94 Ubiquilin 94 Ubiquilin F WCL PM Non-PM 50 50 M 116 Holo- Im APP 94 * B D Myc- 1.4 Control Ubiquilin

)lo Ubiquilin

1.2 )lor Ubiquilin: r 1.2 tno p<0.05

1 tn 1 G 1.10 1.10

c

y o

**p<0.05 )dezila t

f 1.05 0.8 is 1.05 c 0.8

o%(PP **p<0.01 f

n 1.00 1.00

0.6 o%(PP Im-APP 0.6 etnI 0.95 p<0.05 0.95 0.4 0.4 **p<0.01 m 0.90

mAPP roN( 0.90 d

0.2 n

A ImAPP 0.2 0.85 0.85 a

A mAPP 0 0 B 0.80 0.80 0 102030406080100120 0 152030406080100120 Ubiquilin: Time post CHX (min) Time post CHX (min) PM Non-PM

Fig. 1. Ubiquilin-1 inhibits APP maturation. (A) Degradation rate of mature (M) and immature (Im) levels of endogenous APP in PC12 cells. Cells were treated with CHX or vehicle and collected at the indicated time points (in minutes). (B) Quantification of three separate experiments as in A represented as the mean ± SEM. (C) Degradation of APP in cells expressing ubiquilin-1 as in A.(D) Quantification from three separate experiments as in B. P values are shown. In A and C, ubiquilin expression was determined by blotting for the myc epitope present on the N terminus of the protein. (E) Subcellular fractionation and endogenous APP distribution in PC12 cells overexpressing ubiquilin-1 or empty-vector transfected cells. Cells were treated with CHX for 2 h, and lysates were prepared and subjected to differential centrifugation. Mature and immature APP bands are identified with an arrow. The asterisk (*) indicates an un- identified reactive band that has faster mobility than immature APP, possibly full-length APP that has not undergone N-linked glycosylation. 10K, 10,000 × g pellet; 100K, 100,000 × g pellet. Equal protein loading was determined by examining endogenous levels of c-Myc (Lower). (F) Biotinylation experiments to examine cell-surface expression of endogenous APP in cells expressing ubiquilin-1 or transfected with empty vector. WCL, whole-cell lysate; PM, plasma membrane; non-PM, nonplasma membrane. (G) Quantification of PM and non-PM APP levels pooled from four experiments.

half-life of ∼40 min (Fig. 1 A and B). In cells overexpressing Sequestration of APP in the secretory pathway would prevent ubiquilin-1, the mature form of APP is degraded at approxi- trafficking of APP to the cell surface. To explore this possibility, mately similar rates; however, the immature form of APP per- cell-surface biotinylation experiments were performed to deter- sists throughout the time course (Fig. 1 C and D). These results mine if ubiquilin-1 overexpression was associated with decreased suggest that ubiquilin-1 may be modulating APP maturation by plasma membrane expression of APP (Fig. 1 F and G). We ob- sequestering immature APP in early secretory compartments. To served that ubiquilin-1 overexpression caused a significant de- explore this possibility, subcellular fractionation experiments were crease in plasma membrane-associated APP, and this decrease performed to examine the compartmentalization of mature and was concomitant with an enrichment of both mature and im- immature APP in the presence and absence of ubiquilin-1 over- mature APP in the nonplasma membrane fraction. expression. PC12 cells were treated with CHX or vehicle for 2 h Ubiquilin-1 Delays the Proteolytic Processing of APP. To study traf- before collection. The lysates were subjected to differential cen- ficking and degradation of APP in real-time, we used live-cell im- trifugation and separated into 10,000 × g pellets (containing heavy aging of PC12 cells expressing an APP–GFP fusion construct membranes such as rough ER, plasma membrane, and high- consisting of a GFP moiety fused to the C terminus of full-length density Golgi) and 100,000 × g pellets (containing light mem- APP (Fig. 2A). Upon APP processing by the secretases, the AICD– branes such as smooth ER, low-density Golgi, and small vesi- fl × GFP fusion is released from the membrane, and the uorescence cles). In vector-only transfected cells, the 10,000 g pellets pattern changes from punctate to diffuse. Before addition of CHX, contained immature, and a small amount of the mature form of, a punctate pattern of GFP fluorescence was observed in cells endogenous APP (Fig. 1E). In control cells, addition of CHX for expressing APP–GFP (Fig. 2B, Upper Left), corresponding to a 2 h resulted in the loss of mature and immature APP in both predominantly vesicular localization of APP–GFP, as expected. fractions. In contrast, in PC12 cells overexpressing ubiquilin-1, After addition of CHX, this punctate fluorescence was gradually there was in increased amount of mature APP in the 10,000 × g replaced by diffuse cytosolic fluorescence over time, reflecting pellet. Furthermore, the immature form of APP persisted in the secretase processing (Fig. 2B, Upper Right and Movie S1). In 10,000 × g pellet after CHX treatment, suggesting that ubiquilin- contrast, in cells coexpressing APP–GFP and ubiquilin-1, the 1 may stabilize APP in light membrane compartments such as the punctate pattern of fluorescence persisted for much longer after Golgi. An additional band with slightly faster mobility (marked the addition of CHX (Fig. 2B, Lower and Movie S2), suggesting by an asterisk) was observed in fractionation experiments but that ubiquilin-1 is able to delay the secretase processing of APP. not in whole-cell lysates, which may correspond to APP before To confirm that the transition from punctate to diffuse fluores- N-linked glycosylation. cence was indeed indicative of APP–GFP processing, expression

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1206786109 El Ayadi et al. Downloaded by guest on September 25, 2021 A B C APP-GFP NGF Differentiated PC12 Cells APP-GFP + Ubiquilin Membrane APP-GFP CHX (min): 15 30 45 15 30 45 Lumen Cytosol 201 mAPP-GFP APP-GFP imAPP-GFP 114

74 APP-GFP + Ubiquilin Secretase 48 AICD- 34 GFP sAPP AICD-GFP 27 0 min 40 min AICD-GFP (OD): 101 111 124 124 115 105 Ratio AICD/m+im: 0.6 0.8 0.9 0.7 0.7 0.7 D E NGF Differentiated PC12 Cells Primary Cortical Neurons CHX CHX 1.2 1.1 1.0 1.0 APP-GFP + Ubiquilin 0.9 APP-GFP + Ubiquilin 0.8 0.8 0.7 0.6 0.6 0.4 APP-GFP 0.5 0.4 APP-GFP 0.2 APP-GFP + Ubiquilin RNAi 0.3 Punctate/Diffuse Index Punctate/Diffuse Index 0102035455 251530 40 0 5 10 15 20 25 30 35 40 45 Time (min) Time (min)

Fig. 2. Ubiquilin-1 inhibits APP–GFP trafficking and secretase processing. (A) Schematic representation of the membrane topology of the APP–GFP construct

used in this study. Secretase processing is predicted to release AICD–GFP from the membrane into the cytosol (resulting in diffuse fluorescence). (B) Fluo- NEUROSCIENCE rescent images showing APP–GFP distribution in NGF-differentiated PC12 cells before and after 40 min of CHX treatment in cells not expressing ubiquilin-1 (Upper) and cells expressing ubiquilin-1 (Lower). See also Movies S1 and S2.(C) Expression of mature APP–GFP, immature APP–GFP, and AICD–GFP in PC12 cells after CHX treatment. Optical-density AICD–GFP expression levels and the ratio of AICD–GFP to full-length APP expression are given below the blot. Note increasing ratio in APP–GFP-expressing cells, which is eliminated in cells overexpressing ubiquilin-1. (D) Quantification of the change in fluorescence from punctate to diffuse over time (punctate/diffuse index) in single PC12 cells. A reduction in this index indicates more homogeneous (i.e., diffuse) distribution of fluorescence. Solid line indicates no change. The ubiquilin-1 RNAi oligonucleotides have been described and validated elsewhere (18). (E) Punctate/diffuse index of APP–GFP in primary rat cortical neurons as in C. Similar results were seen in three independent experiments.

of APP–GFP and AICD–GFP was monitored by Western blot- Left). In cells coexpressing APP–GFP and ubiquilin-1, colocali- ting. As shown in Fig. 2C Left, CHX treatment resulted in a loss zation persists throughout the time course (Fig. 3A, Right and of mature and immature APP–GFP, and a corresponding increase Center Right). To analyze real time-colocalization of APP–GFP in AICD–GFP in cells that were not overexpressing ubiquilin-1. with mCherry–GlcNAc-T1, we performed live-cell imaging of In contrast, ubiquilin-1 overexpression partially suppressed APP– single cells treated with CHX and quantified the colocalization GFP degradation and, more importantly, decreased the forma- by calculating the correlation index (Icorr) (21). In cells expressing tion of AICD–GFP after CHX treatment (Fig. 2C, Right). Thus, APP–GFP and GlcNAc-T1, there was modest colocalization of the transition from punctate to diffuse fluorescence as deter- the two proteins, which was dramatically increased by ubiquilin-1 fi mined in Fig. 2B is indicative of the secretase processing of APP– overexpression (Fig. 3B). Quanti cation of Icorr indicated that – GFP. The transition from punctate to diffuse fluorescence can ubiquilin-1 overexpression increased colocalization of APP GFP ∼ be quantified as the punctate/diffuse index (19). As shown in Fig. and GlcNAc-T1 from 40% to almost 90% after 40 min of CHX 2D, APP–GFP has a reduction in the punctate/diffuse index over treatment. Thus, ubiquilin-1 can function to retain APP in the time after CHX addition, indicating APP processing and libera- Golgi apparatus. tion of GFP–AICD, as expected. In contrast, overexpression of Ubiquilin-1 Inhibits Degradation of APP by Nonlysosomal Pathways. ubiquilin-1 almost completely abrogates APP–GFP processing, – Our results indicate that ubiquilin-1 sequesters APP in the Golgi whereas ubiquilin-1 knockdown accelerated APP GFP process- (Figs. 1 and 3) and delays secretase-dependent processing (Fig. ing (Fig. 2D). Essentially identical results were obtained with 2). Ubiquilin-1 and other members of the ubiquitin-like (UBL)/ primary cortical neurons (Fig. 2E). These results are consistent ubiquitin-associated (UBA) family of proteins have been shown with our previous studies showing that ubiquilin-1 expression to regulate the degradation of various substrates (22–24). To β inhibits the production of A (18). further investigate whether ubiquilin-1 regulates the Because our results suggest that ubiquilin-1 may be regulating of APP, its ability to modulate the half-life of endogenous APP the exit of APP from the Golgi apparatus, we wished to analyze was examined in the presence of the proteasome inhibitor MG132 APP localization within Golgi compartments. We generated a or the lysosomal inhibitor NH4Cl. As shown in Fig. 4A, APP was stable cell line expressing the Golgi-resident enzyme N-acetylglu- rapidly degraded in the absence of MG132, and this degradation cosaminyltransferase 1 (GlcNAc-T1) (20) fused to the mCherry was partially attenuated by proteasome inhibition. Ubiquilin-1 red fluorescent protein, and transiently expressed APP–GFP in overexpression had little effect on APP degradation in the pres- this cell line (Fig. 3A). Cells were treated with CHX or vehicle ence of MG132. When the cells were treated with the lysosomal for 45 min, and subcellular localization was analyzed by confocal inhibitor ammonium chloride (NH4Cl), the degradation rate of microscopy. Treatment with CHX resulted in a considerable APP was significantly delayed (Fig. 4B). These results are con- reduction of colocalization of the two proteins, indicating mat- sistent with previous studies showing that a large proportion of uration and exit of APP from the Golgi (Fig. 3A, Left and Center APP is degraded in acidic compartments, including lysosomes

El Ayadi et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 A APP-GFP APP-GFP APP-GFP APP-GFP + Ubiquilin + Ubiquilin

Fig. 3. Ubiquilin-1 colocalizes with and retains APP–GFP in the Golgi apparatus. (A) Confocal fluo- – 45' CHX 45' CHX rescent images of APP GFP localization in GlcNAc- T1–mCherry-expressing PC12 cells treated with CHX B C for 45 min with or without ubiquilin-1 overex- APP-GFP APP-GFP APP-GFP APP-GFP – – 1.0 pression, as indicated. (B) APP GFP and GlcNAc-T1 mCherry colocalization without (Upper) or with

0.8 corr)

I (Lower) ubiquilin-1 overexpression as determined (noita 0.6 by the normalized mean deviation product (nMDP)

Color Merge Colocalization 1 min Colocalization 20 min Colocalization 40 min (21) in living cells treated with CHX at time 0. (Up- z APP-GFP +Ubiquilin APP-GFP +Ubiquilin APP-GFP +Ubiquilin APP-GFP +Ubiquilin ilacoloC 0.4 per Left and Lower Left) Raw, wide-field fluores- 0.2 cent image at time 0; other panels are nMDP values. nMDP panels are pseudocolored such that low val- 0.0 ues are cool colors, and high values are warmer

APP colors (see scale bar). (C) Quantification of colocal- Color Merge Colocalization 1 min Colocalization 20 min Colocalization 40 min APP + Ubiquilin ization (Icorr) after 40 min CHX treatment. Data are 0.0 nMDP 1.0 presented as the mean ± SEM of 10 separate cells.

(14, 15). Overexpression of ubiquilin-1 almost completely inhib- overexpression potently stimulated K63-linked polyubiquitination ited APP degradation in the presence of the lysosome inhibitor of APP, and to a lesser extent, K48-linked polyubiquitination. (Fig. 4B). This result suggests that nonlysosomal degradation of Ubiquilin-1 is a cytosolic protein and binds to the AICD (18). APP (as determined by half-life in the presence of NH Cl) is The AICD has five lysine residues that could potentially be 4 fi significantly inhibited by ubiquilin-1 overexpression. Presumably ubiquitinated. We mutated all ve lysine residues to arginine in- the nonlysosomal degradation inhibited by ubiquilin-1 overexpres- dividually to determine which residue is targeted for ubiquitina- fi sion is secretase processing (as in shown in Fig. 2) and/or com- tion by ubiquilin-1. One mutant, K688R, had signi cantly reduced – bined secretase and proteasomal degradation. ubiquilin-1 stimulated ubiquitination of APP (Fig. 5C). We rea- soned that if ubiquilin-1 regulates APP trafficking by stimulating Ubiquilin-1 Regulates APP Trafficking and Degradation via Ubiquiti- ubiquitination of K688, then ubiquilin-1 overexpression would nation. We next tested whether ubiquilin-1 regulates APP traffick- have no effect on the subcellular localization of the K688R APP ing and degradation by stimulating ubiquitination. Polyubiquitin mutant. Thus, we examined the ability of ubiquilin-1 to affect the chains conjugated to lysine 29 (K29) or lysine (K48) of ubiquitin subcellular localization of overexpressed wild-type and K688R APP using subcellular fractionation. As shown in Fig. 5D, ubiq- target a substrate protein for proteasomal degradation. However, uilin-1 increases the amount of overexpressed wild-type APP in lysine 63 (K63)-linked polyubiquitination and monoubiquitination both 10,000 and 100,000- × g fractions, which is consistent with are implicated in many nondegradative processes, such as pro- fi the effects of ubiquilin-1 overexpression on endogenous APP tein traf cking, endocytosis, and DNA repair (25, 26). PC12 cells shown in Fig. 1E. In contrast, ubiquilin-1 overexpression had no were transfected with HA-ubiquitin with or without ubiquilin-1 effect on the subcellular distribution of the K688R mutant (Fig. and MG132, and APP ubiquitination was investigated. As shown 5E). Strikingly, there was a complete absence of K688R mutant in in Fig. 5A, ubiquilin-1 stimulated K63-linked polyubiquitination the 100,000 × g pellet fraction, suggesting a dramatically altered of APP as determined by linkage-specific ubiquitin antibodies. subcellular localization. This was examined (as above) using As an alternative approach to confirm linkage specificity, we used APP–GFP and APP–GFP K688R mutant in GlcNAc-T1–mCherry HA-ubiquitin constructs with all lysines mutated to arginine ex- cells treated with CHX. As shown in Fig. 5F, the K688R mutant cept for either K48 or K63. As shown in Fig. 5B, ubiquilin-1 had dramatically reduced Golgi colocalization both before and

A Control Ubiquilin B Control Ubiquilin Control Control APP APP MG132 NH4 Cl Myc- Myc- Ubiqulin Ubiqulin Tubulin Tubulin CHX Time: 0 30 45 60 120 0 30 45 60 120 CHX Time: 0 30 45 60 120 0 30 45 60 120 Fig. 4. Ubiquilin-1 inhibits nonlysosomal degrada-

120 tion of APP. (A) Time course of APP degradation

g 120 ni niniam after addition of cycloheximide in the presence or 100 niam 100 absence of the proteasomal inhibitor MG132 (Up- 80

80 per). (Lower) Quantification of APP band intensities eRn e Ubiquilin + NH4 Cl 60 in an experiment representative of three separate Rnoitca 60 determinations. (B) Time course of APP degrada- 40 Ubiquilin + MG132 oi 40 Control + MG132 tca tion after cycloheximide addition in the absence

20 20 r

r Control Control + NH 4 Cl or presence of lysosomal inhibitor NH4Cl (Upper). Fg F Ubiquilin Control 0 0 Ubiquilin (Lower) Quantification of band intensities in an ex- 0 20 40 60 80 100 120 0 20 40 60 80 100 120 periment representative of three separate determi- Time (min) Time (min) nations. Tubulin is used as a loading control.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1206786109 El Ayadi et al. Downloaded by guest on September 25, 2021 AB C MG132 MG132 HA-Ubq + + + + + Ubiquilin APP K688R Ubiquilin - + - + HA-Ubq WT K48 K63 WT K48 K63 No Ab Ubiquilin - + - +

Anti- Anti- APP Anti-

APP nwod- APP

nw

l

luP

o d-l

nwod-lluPPPA Anti- l

HA uP Anti- PPA Ubiquitin Anti- Fig. 5. Ubiquilin-1 regulates APP trafficking and PP Ubiquitin degradation through K63-linked polyubiquitination A of lysine residue 688. (A) Ubiquitination of endog- Anti- Anti-K63 APP enous immunoprecipitated APP as determined by Ubiquitin linkage-specific antibodies in the presence or ab-

Input Anti- sence of MG132 in cells coexpressing HA-ubiquitin Ubiquilin (HA-Ubq) plus ubiquilin-1 or a control vector as in- dicated at the top. (B) Ubiquitination of endogenous Anti-K48 Ubiquitin DEWT APP K688R APP in the presence of MG132 in cells coexpress- Ubiquilin Mock Ubiquilin Mock ing wild-type or mutant HA-ubiquitin constructs in CHX CHX CHX CHX 10K 100K 10K 100K 10K 100K 10K 100K 10K 100K 10K 100K 10K 100K10K 100K which all lysine residues are mutated to arginine except K63 or K48. (C) Ubiquitination of overex- Anti- Anti- APP 116 pressed wild-type APP or a mutant in which lysine 116 APP 688 is mutated to arginine (K688R). All lanes were Input Anti- Ubiquilin Anti- preincubated with MG132. (D) Subcellular distribu- 250 IP 3 R-1 tion of wild-type overexpressed APP in the presence F or absence of ubiquilin-1 expression and the effects APP-GFP APP-GFP K688R K688R of CHX treatment as indicated. Expression of the ER- resident inositol 1,4,5-trisphosphate receptor type

1(IP3R-1) is used as a loading control. (E) Subcellular

distribution of K688R APP in the presence or ab- NEUROSCIENCE sence of ubiquilin-1 expression and the effects of CHX treatment. (F) Confocal fluorescence images showing the subcellular distribution of wild-type and K688R APP–GFP before and 45 min after CHX 45' CHX 45' CHX treatment.

after CHX treatment, suggesting that ubiquitination of this residue limit amyloidogenic processing of APP and associated neuronal by ubiquilin-1 is a critical regulator of APP retention in the Golgi dysfunction. apparatus. Materials and Methods Discussion Expression Constructs. To generate an APP–GFP fusion construct, APP695 was In the present study, we show that ubiquilin-1 delays the matu- amplified using PCR, incorporating a 5′ HindIII restriction site and a 3′ SacII ration of APP by sequestering it in the Golgi apparatus and pre- restriction site, and was cloned in frame to the HindIII-SacII sites of the venting its transport to the cell surface for subsequent processing. pmaxFP Green-N mammalian expression vector (Amaxa). This vector has been described elsewhere, and the particular GFP variant has been shown to Using a combination of biochemical and cell biological method- have no effect on proteasomal degradation (28). PCR primers were 5′-TTC ologies, our data suggests that ubiquilin-1 inhibits APP matura- AAG CTT CCA TGC TGC CCG GTT TGG CAC TG-3′ and 5′-TCC CCG CGG GTT tion and secretase processing by ubiquitinating lysine residue 688. CTG CAT CTG CTC AAA GAA CTT GTA G-3′. The GlcNAc-T1–mCherry fusion These results indicate that ubiquilin-1 is a potent regulator of construct was kindly provided by Joachim Seemann (University of Texas APP trafficking and secretase processing, and supports a model in Southwestern, Dallas, TX). This construct encodes the cytoplasmic and trans- which age-associated decreases in ubiquilin-1 levels as shown membrane domains of N-acetylglucosaminyltransferase 1, a Golgi-resident previously by our group (18) contribute to increased Aβ produc- enzyme, fused to the mCherry red fluorescent protein, which replaces the tion in late-onset AD. Our results are in agreement with previous C-terminal luminal catalytic domain of the enzyme. Wild-type and mutant studies showing that reduced ubiquilin-1 protein levels correlate HA-ubiquitin constructs were obtained from Addgene. Other expression con- structs have been described previously (18). Ubiquilin-1 RNAi oligonucleotides with accelerated maturation of APP and increased degradation used in Fig. 2D have been described and validated elsewhere (18). rates (17), and provide a mechanistic basis by which ubiquilin-1 fi regulates APP traf cking. Because ubiquilin-1 has no obvious APP Degradation. Cells were transfected with either myc-tagged ubiquilin-1 homology to known ubiquitin ligases, it is likely that ubiquilin-1 or empty vector (pcDNA3.1). At 1 d posttransfection, cells were treated with recruits an E3 ligase to APP while in the Golgi apparatus. Future CHX. CHX was diluted in cell culture medium at a final concentration of 50 μg/ studies will be necessary to identify the putative ligase(s) re- mL before its addition. At the specified time points, one plate from each sponsible for ubiquilin-1–stimulated APP degradation. group was snap-frozen in liquid nitrogen, and plates were stored at −80 °C We have previously shown that ubiquilin-1 functions as a mo- until processing. After completion of the time course, cells lysates were lecular chaperone for APP by binding to the AICD and pre- prepared and run on a 4–20% gradient gel. Control and treated samples venting its aggregation (18). We also demonstrated, regardless were handled under identical conditions, and blots were exposed simulta- neously on the same film. To examine the effects of ubiquilin-1 on APP of ubiquilin-1 genotype, that ubiquilin-1 levels were significantly degradation in the presence of lysosomal/proteasomal inhibitors, cells were reduced in late-onset AD patient brains (18, 27). Combined with treated for 15 min with the proteasome inhibitor MG132 (EMD Biosciences) the results presented here, our results suggest that ubiquilin-1 at a final concentration of 10 μM or the lysosomal inhibitor ammonium is a key regulator of APP biosynthesis, trafficking, and degra- chloride (Sigma) at a final concentration of 25 mM. Both inhibitor treatment dation, and likely functions as a key quality-control molecule to groups were compared with control cells treated with vehicle.

El Ayadi et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 Subcellular Fractionation. PC12 cells were transfected with myc-tagged A-Sepharose beads (Pierce). The beads were then washed with lysis buffer, ubiquilin-1 or a control empty vector. The next day, cells were treated for 2 h boiled with SDS sample buffer, and analyzed by SDS/PAGE. with either CHX or vehicle (DMSO). Cells were then collected and snap-frozen in liquid nitrogen. Differential centrifugation and subsequent analysis was Live-Cell Imaging. PC12 cells were cultured on 25-mm coverslips overnight done as described (29). and then cotransfected with APP–GFP plus ubiquilin-1 or a control vector at 1:1 ratio. At 5 h posttransfection, the media was changed to nerve growth Cell-Surface Biotinylation. Cells were plated on 10-cm plates and transfected factor-containing media to induce cells differentiation for an additional with myc-tagged ubiquilin-1 or a control empty vector. The next day, the media 24 h. The coverslips were transferred to an imaging chamber containing was removed, and cells were washed 3× with 10 mL sterile ice-cold PBS. A cell- imaging media (DMEM without L-glutamine and phenol red supplemented impermeant biotinylation reagent (Sulfo-NHS-SS-Biotin; Pierce) was dissolved with 10 mM Hepes and 1% FBS). For all experiments, transfection effi- fl in ice-cold PBS/MgCl2/CaCl2 at a final concentration of 1 mM, and 6 mL of the ciency, as determined by uorescence microscopy, was greater than 50%. biotin solution was added to each plate. Plates were gently agitated on an All fields were imaged randomly, and all GFP-positive cells in a given field orbital shaker for 3 h at 4 °C to ensure even coverage with the labeling solu- were imaged and quantified. To examine trafficking and degradation, tion. Following incubation, the labeling solution was removed, and cells were cells were treated with CHX to inhibit de novo protein synthesis. The incubated in 8.5 mL quenching solution [PBS (pH 8.0), 10 mM Tris, and 100 mM punctate/diffuse index was determined as described (19), and colocali- glycine] for 10 min at 4 °C. Cells were then lysed in 250 μL lysis buffer containing zation was determined using the Colocalization Colormap plugin in protease inhibitors. Lysates were cleared at 10,000 × g pellet for 20 min at 4 °C, ImageJ (21). and protein content of the supernatant was determined by the Bradford method. A total of 500 μg protein (∼1 mg/mL in lysis buffer) was added to 25 μL Confocal Imaging. For confocal imaging, PC12 cells stably expressing GlcNAc-T1– bed-volume NeutrAvidin agarose resin (Pierce), and mixtures were incubated mCherry were plated on coverslips and the next day transiently transfected overnight at 4 °C with rotation. The resin was washed 10× with the wash buffer with APP–GFP with ubiquilin-1 or a control vector. At 24 h posttransfection, containing protease inhibitors and boiled in 25 μL2× SDS sample buffer, and the cells were treated with CHX or vehicle for specified time points and then eluted proteins were analyzed by SDS/PAGE and Western blotting. washed with PBS and fixed with 4% paraformaldehyde (wt/vol) in PBS solution for 20 min at room temperature. APP–GFP colocalization with GlcNacT1 was Ubiquitination Assays. For ubiquitination experiments, cells were transfected visualized with an Olympus FluoView confocal microscope. with HA-ubiquitin or mutants constructs as indicated in Fig. 5B. The next day, cells were treated with the proteasome inhibitor MG132 for 3 h to induce ACKNOWLEDGMENTS. We thank Dr. Hui Zheng for the APP695 cDNA accumulation of ubiquitinated proteins before harvesting in lysis buffer construct; Drs. Eric J. Brown and Mervyn J. Monteiro for ubiquilin-1 cDNA constructs; and Dr. Joachim Seemann for the GlcNAc-T1–mCherry construct. containing protease inhibitors and the deubiquitinating enzyme inhibitor N- fi μ We also thank Dr. Megan L. Landsverk and Xinmin Wang for assistance ethylmaleimide (Sigma) at 10 mM nal concentration. Lysates (500 g) were with preliminary aspects of this project. This work was supported by Na- precleared by 30-min incubation at 4 °C with protein A from the bacteria tional Institutes of Health Grants R21AG031948 (to J.M.B. and D.B.) and Staphylococcus aureus (Sigma) before incubation with an APP antibody for F30AG030878 (to E.S.S.), as well as funds provided by the Jean C. and William 2 h at 4 °C. Immune complexes were recovered using 50 μL of protein D. Willis Neuroscience Research Endowment (to E.S.S.).

1. Weidemann A, et al. (1989) Identification, biogenesis, and localization of precursors 15. Knops J, Lieberburg I, Sinha S (1992) Evidence for a nonsecretory, acidic degradation of Alzheimer’s disease A4 amyloid protein. Cell 57:115–126. pathway for amyloid precursor protein in 293 cells. Identification of a novel, 22-kDa, 2. Suzuki T, Araki Y, Yamamoto T, Nakaya T (2006) Trafficking of Alzheimer’s disease- beta-peptide-containing intermediate. J Biol Chem 267:16022–16024. related membrane proteins and its participation in disease pathogenesis. J Biochem 16. Nunan J, et al. (2003) Proteasome-mediated degradation of the C-terminus of the 139:949–955. Alzheimer’s disease beta-amyloid protein precursor: Effect of C-terminal truncation 3. Andersen OM, et al. (2005) Neuronal sorting protein-related receptor sorLA/LR11 on production of beta-amyloid protein. J Neurosci Res 74:378–385. regulates processing of the amyloid precursor protein. Proc Natl Acad Sci USA 102: 17. Hiltunen M, et al. (2006) Ubiquilin 1 modulates amyloid precursor protein trafficking – 13461 13466. and Abeta secretion. J Biol Chem 281:32240–32253. fi 4. Selkoe DJ, et al. (1996) The role of APP processing and traf cking pathways in the 18. Stieren ES, et al. (2011) Ubiquilin-1 is a molecular chaperone for the amyloid precursor – formation of amyloid beta-protein. Ann N Y Acad Sci 777:57 64. protein. J Biol Chem 286:35689–35698. 5. De Strooper B, Annaert W (2000) Proteolytic processing and cell biological functions 19. Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR (2000) The coordinate release – of the amyloid precursor protein. J Cell Sci 113:1857 1870. of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell 6. Koo EH, et al. (1990) Precursor of amyloid protein in Alzheimer disease undergoes fast Biol 2:156–162. anterograde axonal transport. Proc Natl Acad Sci USA 87:1561–1565. 20. Kumar R, Yang J, Larsen RD, Stanley P (1990) Cloning and expression of N-acetyl- 7. Perez RG, et al. (1999) Mutagenesis identifies new signals for beta-amyloid precursor glucosaminyltransferase I, the medial Golgi transferase that initiates complex N- protein endocytosis, turnover, and the generation of secreted fragments, including linked carbohydrate formation. Proc Natl Acad Sci USA 87:9948–9952. Abeta42. J Biol Chem 274:18851–18856. 21. Jaskolski F, Mulle C, Manzoni OJ (2005) An automated method to quantify and vi- 8. Tomita S, Kirino Y, Suzuki T (1998) Cleavage of Alzheimer’s amyloid precursor protein sualize colocalized fluorescent signals. J Neurosci Methods 146:42–49. (APP) by secretases occurs after O-glycosylation of APP in the protein secretory 22. Chen L, Madura K (2002) Rad23 promotes the targeting of proteolytic substrates to pathway. Identification of intracellular compartments in which APP cleavage occurs the proteasome. Mol Cell Biol 22:4902–4913. without using toxic agents that interfere with protein metabolism. J Biol Chem 273: 23. Gao L, et al. (2003) Interaction with a ubiquitin-like protein enhances the ubiquiti- 6277–6284. nation and degradation of hepatitis C virus RNA-dependent RNA polymerase. J Virol 9. Small SA, Gandy S (2006) Sorting through the cell biology of Alzheimer’s disease: – Intracellular pathways to pathogenesis. Neuron 52:15–31. 77:4149 4159. 10. Kerr ML, Small DH (2005) Cytoplasmic domain of the beta-amyloid protein precursor 24. Kim I, Xu W, Reed JC (2008) Cell death and endoplasmic reticulum stress: Disease – of Alzheimer’s disease: Function, regulation of proteolysis, and implications for drug relevance and therapeutic opportunities. Nat Rev Drug Discov 7:1013 1030. development. J Neurosci Res 80:151–159. 25. Lim KL, Dawson VL, Dawson TM (2006) Parkin-mediated lysine 63-linked poly- ’ 11. Schmidt V, et al. (2007) SorLA/LR11 regulates processing of amyloid precursor protein ubiquitination: A link to protein inclusions formation in Parkinson s and other con- via interaction with adaptors GGA and PACS-1. J Biol Chem 282:32956–32964. formational diseases? Neurobiol Aging 27:524–529. 12. Spoelgen R, et al. (2006) Interaction of the cytosolic domains of sorLA/LR11 with the 26. Chiu RK, et al. (2006) Lysine 63-polyubiquitination guards against translesion syn- amyloid precursor protein (APP) and beta-secretase beta-site APP-cleaving enzyme. thesis-induced mutations. PLoS Genet 2:e116. J Neurosci 26:418–428. 27. Boehning D, Barral JM (2012) Protein quality control in Alzheimer’s disease: The 13. Traub LM (2005) Common principles in clathrin-mediated sorting at the Golgi and contentious role of ubiquilin-1. Future Neurology 7:5–8. the plasma membrane. Biochim Biophys Acta 1744:415–437. 28. Baens M, et al. (2006) The dark side of EGFP: Defective polyubiquitination. PLoS ONE 14. Caporaso GL, Gandy SE, Buxbaum JD, Greengard P (1992) Chloroquine inhibits in- 1:e54. tracellular degradation but not secretion of Alzheimer beta/A4 amyloid precursor 29. Boehning D, et al. (2003) Cytochrome c binds to inositol (1,4,5) trisphosphate re- protein. Proc Natl Acad Sci USA 89:2252–2256. ceptors, amplifying calcium-dependent apoptosis. Nat Cell Biol 5:1051–1061.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1206786109 El Ayadi et al. Downloaded by guest on September 25, 2021