Alzheimer's disease-associated ubiquilin-1 regulates presenilin-1 accumulation and aggresome formation - PubMed (original) (raw)

doi: 10.1111/j.1600-0854.2010.01149.x. Epub 2011 Jan 7.

Annakaisa Haapasalo, Claudia Böttcher, Riitta Miettinen, Kaisa M A Kurkinen, Alice Lu, Anne Thomas, Christa J Maynard, Donna Romano, Bradley T Hyman, Oksana Berezovska, Lars Bertram, Hilkka Soininen, Nico P Dantuma, Rudolph E Tanzi, Mikko Hiltunen

Affiliations

Alzheimer's disease-associated ubiquilin-1 regulates presenilin-1 accumulation and aggresome formation

Jayashree Viswanathan et al. Traffic. 2011 Mar.

Abstract

The Alzheimer's disease (AD)-associated ubiquilin-1 regulates proteasomal degradation of proteins, including presenilin (PS). PS-dependent γ-secretase generates β-amyloid (Aβ) peptides, which excessively accumulate in AD brain. Here, we have characterized the effects of naturally occurring ubiquilin-1 transcript variants (TVs) on the levels and subcellular localization of PS1 and other γ-secretase complex components and subsequent γ-secretase function in human embryonic kidney 293, human neuroblastoma SH-SY5Y and mouse primary cortical cells. Full-length ubiquilin-1 TV1 and TV3 that lacks the proteasome-interaction domain increased full-length PS1 levels as well as induced accumulation of high-molecular-weight PS1 and aggresome formation. Accumulated PS1 colocalized with TV1 or TV3 in the aggresomes. Electron microscopy indicated that aggresomes containing TV1 or TV3 were targeted to autophagosomes. TV1- and TV3-expressing cells did not accumulate other unrelated proteasome substrates, suggesting that the increase in PS1 levels was not because of a general impairment of the ubiquitin-proteasome system. Furthermore, PS1 accumulation and aggresome formation coincided with alterations in Aβ levels, particularly in cells overexpressing TV3. These effects were not related to altered γ-secretase activity or PS1 binding to TV3. Collectively, our results indicate that specific ubiquilin-1 TVs can cause PS1 accumulation and aggresome formation, which may impact AD pathogenesis or susceptibility.

© 2011 John Wiley & Sons A/S.

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Figures

Figure 1

Figure 1. Co-expression of PS1 and ubiquilin-1 TV3 results in the accumulation of HMW-PS1 forms in HEK293-AP-APP cells

A) Schematic representation of the gene structure of ubiquilin-1 transcript variants (TV1–4). Exons encoding the N-terminal ubiquitin-like (UBL) and the C-terminal ubiquitin-associated (UBA) domain are indicated. Black dots represent asparagineand proline-rich repeats that may mediate protein-protein interactions. B) Western blot showing the PS1 C-terminal fragment (PS1-CTF) and full-length PS1 (PS1-FL) levels in HEK293-AP-APP cells transiently co-expressing PS1 and different ubiquilin-1 TVs. Co-expression of PS1 and TV3 results in an increased accumulation of high-molecular weight PS1 (HMW-PS1). P, control plasmid; M, mock-transfection.

Figure 2

Figure 2. Co-expression of PS1 and ubiquilin-1 TVs increase PEN-2 levels and decrease Aβ40 and Aβ42 levels in HEK293-AP-APP cells

A, B) Western blots showing the levels of different γ-secretase complex components in HEK293-AP-APP cells co-expressing PS1 and ubiquilin-1 variants (TV1 and TV2 in A, TV3 and TV4 in B). β-tubulin-normalized PEN-2 levels are significantly increased in cells over-expressing ubiquilin-1 transcript variants, correlating with increased PS1-CTF levels. P, control plasmid; M, mock-transfection. C) Assessment of secreted Aβ40, Aβ42, and total sAPP (sAPPtot) levels from the culture media. Co-expression of PS1 and TV3 significantly reduce total protein-normalized Aβ40, Aβ42, and sAPPtot secretion. D) γ-secretase activity measurements in HEK293-APAPP cells co-transfected with PS1 and TV1, TV3, or control plasmid do not show differences between samples. TV1 and TV3 over-expression status was confirmed from the cytosolic protein fraction using Western blot analysis. Pearson's correlation coefficient test, **p< 0.01, *p<0.05, n ≥ 3, SD.

Figure 3

Figure 3. Over-expression of ubiquilin-1 TV1 or TV3 in HEK293-AP-APP cells affect full-length PS1 half-life differently

A) Western blot of cells co-over-expressing TV1, TV3 or control plasmid with PS1 and treated with cycloheximide (30 μg/ml) for 0, 1, 2, 4 and 8 hours. The HMW-PS1 forms start to accumulate after 2 hours in cells transfected with TV3. Concomitantly, the levels of full-length PS1 (PS1-FL) decrease. B) Quantification of the PS1-FL levels at different times after cycloheximide treatment. TV3 over-expression stabilizes the levels of PS1-FL as compared to TV1-over-expressing or control cells. SEM; M, mock-transfection; t, time (hours after treatment).

Figure 4

Figure 4. N- and C-terminally tagged ubiquilin-1 TV3, but not TV1, increases the accumulation of HMW-PS1 in HEK293-AP-APP cells

A) A schematic representation of the tagged myc-TV1-mRFP and myc-TV3-mRFP cDNA constructs. B) Western blot showing that co-expression of myc-TV3-mRFP with PS1 induced accumulation of HMW-PS1 in a similar manner to the untagged TV3 (shown in Figure 1A). P, control plasmid; mRFP, monomeric red fluorescent protein. Asterisks indicate degradation products of myc-TV1-mRFP and myc-TV3-mRFP.

Figure 5

Figure 5. Over-expression of myc-TV1-mRFP or myc-TV3-mRFP does not result in a general UPS impairment in HEK293T UbG76V-YFP reporter cells

A) Western blot of UbG76V-YFP HEK293T cells over-expressing PS1-FL and myc-TV1-mRFP (*) or myc-TV3-mRFP (arrow head). Middle panel shows HMW-PS1 in myc-TV3-mRFP over-expressing cells. P, control plasmid. B_–_D) Micrographs of UbG76V-YFP HEK293T cells transiently transfected with myc-TV1-mRFP (B), myc-TV3-mRFP (C) or mRFP (control; D). Cells were either left untreated (top panels) or treated for 6 hrs with 10 μM proteasome inhibitor MG132 (bottom panels). Scale bar = 20μm. E) Quantification of YFP fluorescence in UbG76V-YFP HEK293T cells transiently transfected with myc-TV1-mRFP, myc-TV3-mRFP or mRFP. YFP fluorescence was quantified by selecting mRFP-positive objects using Volocity Quantitation software. Error bars represent standard error of mean (SEM) (n > 320 cells). There are no significant differences between YFP values of the mRFP-expressing cells (control) against myc-TV1-mRFP- or myc-TV3-mRFP-expressing cells (Student's _t_-test; untreated cells against MG132-treated cells). YFP, yellow fluorescent protein.

Figure 6

Figure 6. PS1 and ubiquilin-1 TV1 or TV3 co-localize in aggresomes in HEK293-AP-APP cells

A) Localization of PS1 in cells co-transfected with PS1 (green) and mRFP control plasmid (red). B,C) PS1 and TV1 or TV3 co-localize (yellow) in aggresomes in cells co-transfected with PS1 and myc-TV1-mRFP or myc-TV3-mRFP (red). Nuclei are shown in blue. Boxed individual cells containing an aggresome are shown at higher magnification in the insets. Wide-field fluorescence images were taken at 40× magnification. Scale bar = 20 μm for all images. D) Quantification of the number of cells containing TV1- or TV3- and PS1-positive aggresomes. Data are shown as % of cells with aggresomes/total number of cells ± SD, n = 3 (≥ 1000 cells), **p<0.001, One-way ANOVA, Newman-Keuls Multiple Comparison Test.

Figure 7

Figure 7. Vimentin redistributes to the aggresomes in HEK293-AP-APP cells co-transfected with PS1 and ubiquilin-1 TV1 or TV3

A) Normal distribution of vimentin (green) in cells co-transfected with PS1 and mRFP control plasmid (red). B,C) Vimentin redistributes to the aggresomes in cells co-transfected with PS1 and myc-TV1-mRFP or myc-TV3-mRFP (red). Co-localization is indicated by yellow. Nuclei are shown in blue. Boxed individual cells containing an aggresome are shown at higher magnification in the insets. Wide-field fluorescence images were taken at 40× magnification. Scale bar = 20 μm for all images. D) Quantification of the number of cells containing TV1- or TV3- and vimentin-positive aggresomes. Data are shown as % of cells with aggresomes/total number of cells ± SD, n = 3 (≥ 1000 cells), *p<0.01, **p<0.001 as indicated, One-way ANOVA, Newman-Keuls Multiple Comparison Test.

Figure 8

Figure 8. PS1 and ubiquilin-1 TV3 show similar subcellular distribution and co-localize in aggresomes, which are enveloped in a vimentin cage in HEK293-AP-APP cells

A) Subcellular localization of PS1 in cells co-transfected with PS1 (green) and mRFP control plasmid (left panel) or myc-TV3-mRFP (red; right panels). PS1 and TV3 co-localize (yellow) on the plasma membrane and in the aggresomes (arrows) in cells co-transfected with PS1 and myc-TV3-mRFP. B) Vimentin (green) localizes intracellularly and close to the plasma membrane in cells co-transfected with PS1 and mRFP control plasmid (left panel). In cells co-transfected with PS1 and myc-TV3-mRFP, vimentin redistributes to and forms an envelope around the aggresome, as indicated by a green ring surrounding the red TV3-containing aggresome core (right panels). Confocal microscope images show single optical z-sections taken at 60× magnification. Scale bar = 10 μm for all images.

Figure 9

Figure 9. Aggresomes in HEK293-AP-APP cells co-expressing PS1 and ubiquilin-1 TV3 co-localize with γ-tubulin, a marker for the microtubule-organizing center

A) Aggresomes containing myc-TV3-mRFP (red) c o-localize with γ-tubulin (green) at the juxtanuclear microtubule-organizing center (MTOC). Co-localization is indicated by yellow. Nuclei are shown in blue. The boxed individual cell containing an aggresome is shown at higher magnification in the insets. Arrow points to the MTOC. Wide-field fluorescence images were taken at 40× magnification. Scale bar = 20 μm. B) Confocal microscope images of a cell co-transfected with PS1 and myc-TV3-mRFP confirming the co-localization of PS1 and myc-TV3-mRFP with γ-tubulin in the aggresome (cyan blue in merged image). Co-localization of PS1 and TV3 is indicated by yellow. Images show single optical z-sections taken at 60× magnification. Scale bar = 10 μm.

Figure 10

Figure 10. Aggresome formation in primary cortical cells of APP/PS1ΔE9 transgenic mice transfected with ubiquilin-1 TV1 or TV3

A) Western blot showing transient over-expression of myc-TV1 (251 ± 73 % when normalized to endogenous TV1 in control sample) and myc-TV3 (TV3 vs. endogenous TV1 ratio = 0.7 ± 0.1) in E18 cortical cultures from an APP/PS1ΔE9 transgenic mouse. The blot indicates PS1ΔE9 over-expression shown by the strong levels of PS1-FL. Staining with an antibody against PS1 did not demonstrate the presence of HMW-PS1 as in the case of HEK293-AP-APP cells over-expressing myc-TV3 (see Supplement figure 2). P, control plasmid. B) Total protein-normalized Aβ40 and Aβ42 measurements from the culture medium of APP/PS1ΔE9 primary cortical cells show a significant increase in Aβ40 levels in both myc-TV1 and myc-TV3 over-expressing cells. n=4, SD, *p<0.01, **p<0.005. C, D) PS1 co-localizes with TV1 or TV3 in the aggresomes in neurons from APP/PS1ΔE9 transgenic mice transfected with myc-TV1-mRFP or myc-TV3-mRFP. Left panels, control cortical neurons stained with PS1 antibody (green). Right panels, neurons transfected with myc-TV1-mRFP or myc-TV3-mRFP (red) containing an aggresome (arrow) next to the nucleus (blue). Co-localization is indicated by yellow. Wide-field fluorescence images were taken at 40× magnification. Scale bar = 10 μm for all images.

Figure 11

Figure 11. Ubiquilin-1 TV1 and TV3 localize in aggresomes and autophagosomes as revealed by electron microscopy

Electron micrographs of mRFP (A_–_C; scale 500 nm) and myc (D_–_F; scale 200 nm) localization in myc-TV3-mRFP and PS1 (A, B, D, E, F) and myc-TV1-mRFP and PS1 (C) transfected cells. A) Photoconversion of mRFP results in electron dense deposit formation that is located in between the double membranes forming autophagic vacuoles (large arrows). Aggregates are either freely dispersed in the cytoplasm (black star) or inside the forming autophagosome (white star). In general, autophagic vacuoles and lysosomes are numerous in myc-TV3-mRFP transfected cells (thin arrows). B) Large multilamellar-multivesicular inclusions are typically located near the indentation of the nucleus (N). Multiple layers of double membranes (small arrows) wrap around multivesicular bodies and vesicles many of which contain electron dense DAB-deposition (thick arrows). A free cytoplasmic aggresome (black star) is located near the inclusion body. C) As visualized in ultrathin electron microscopic section, short segments of endoplasmic reticulum (small arrows) form a multilamellar inclusion that contains electron dense vesicles. Thin intermediate filaments are seen in the neighbourhood (arrowheads). D) Gold particles are concentrated in the aggregates in a mycimmunostained section. Aggregates in the cytoplasm (black arrows) gather stronger immunoreactivity than that inside the vacuole (white arrow). E) Negative control section contains none or only a few gold particles. F) Higher magnification of an aggresome (black star) that contains multiple gold particles indicating myc-immunoreactivity. This aggresome has close contact with the neighbouring interfilament bundle (dashed line around). The fine structure of the filaments has suffered from etching of the resin that was required for the post-embedding immunostaining.

Figure 12

Figure 12. Potential role of ubiquilin-1 TV1 and TV3 in proteasomal degradation and aggresomal accumulation of PS1

1. TV1 binds poly-ubiquitinated PS1 destined for degradation via its UBA domain. 2. TV1 takes PS1 to the proteasome. The interaction of TV1 with the proteasome is mediated by its UBL domain. 3. PS1 is degraded by the proteasome. 4. TV3 binds poly-ubiquitinated PS1 in the same way as TV1. However, since it lacks most of the UBL domain, TV3 does not efficiently bind to the proteasome, resulting in the accumulation of TV3-PS1 complexes in the cell. 5. Accumulated TV3-PS1 complexes are sequestered to the juxtanuclear MTOC as vimentin-caged aggresomes, rendering the possibly toxic accumulated proteins harmless. 6. The aggresome may be directed for autophagosomal degradation.

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