HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-5531fjev1 20/8/1176    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, M. Articles by Kovacs, D. M. Search for Related Content PubMed PubMed Citation Articles by Zhang, M. Articles by Kovacs, D. M.

Presenilin/-secretase activity regulates protein clearance from the endocytic recycling compartment

Neurobiology of Disease Laboratory, Genetics and Aging Research Unit, Department of Neurology/MIND, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA

³Correspondence: Neurobiology of Disease Laboratory, Genetics and Aging Research Unit, Department of Neurology/MIND, Massachusetts General Hospital, Harvard Medical School, 114 16th Street, Charlestown, Massachusetts 02129, USA. E-mail: dora_kovacs{at}hms.harvard.edu

SPECIFIC AIMS

The presenilin (PS)/-secretase complex proteolytically cleaves the amyloid precursor protein (APP), a key protein in the pathogenesis of Alzheimer’s disease (AD). PSs also regulate intracellular trafficking of APP and its C-terminal fragments (CTFs). Our aim was to investigate whether PS/-secretase activity is required for normal endosomal recycling of proteins through a specific endosomal compartment, the endocytic recycling compartment (ERC). We performed live cell imaging experiments with fluorescently labeled transferrin, reported to specifically traffic through the ERC, and green fluorescent protein (GFP)-tagged APP in cells with decreased PS/-secretase function.

PRINCIPAL FINDINGS

1. PS/-secretase activity is required for normal clearance of transferrin from the ERC
To assess the role of PS/-secretase in the recycling of transferrin, we confirmed that transferrin colocalized with Rab11, an ERC marker, and not the late endosomal or lysosomal markers Rab7 or Rab9, indicating that transferrin specifically labeled the ERC in CHO cells (Fig. 1 A, a–e). Next, we labeled CHO cells treated with DAPT, a specific -secretase inhibitor, with transferrin. In control cells, transferrin partially localized to the ERC immediately after labeling (Fig. 1Ab ) and almost completely cleared out from the cells by one hour (Fig. 1Bb ). However, transferrin was still retained in the ERC after one hour in DAPT-treated cells (Fig. 1Bg ). Quantitation revealed that there was about 6 times more transferrin label left in the DAPT-treated cells after 1 hour when compared to the control cells (Fig. 1C ). Similar to the control cells, transferrin colocalized with Rab11, but not with Rab7 or Rab9, at either time point after -secretase inhibition (Fig. 1A and B; h--j ), further implying that transferrin was withheld in the ERC.

View larger version (38K): [in this window] [in a new window]   Figure 1. Transferrin clearance from the endocytic recycling compartment (ERC) is delayed in Chinese hamster ovary (CHO) cells treated with the -secretase inhibitor DAPT. A) Transferrin (Tfn, red; b and g) colocalized with the endocytic recycling compartment marker Rab11 in the ERC (green in a and f) immediately after its uptake (0 h) in both control and DAPT-treated cells. Transferrin did not colocalize with Rab7 or Rab 9, late endosomal and lysosomal markers, in control (d and e) or in DAPT-treated cells (i and j). B) After 1-hour chase, transferrin completely cleared out from the ERC in control cells expressing Rab11 (a–c). In contrast, in DAPT-treated cells, transferrin still colocalized with Rab11 at the ERC (f–h; yellow in h), but not with Rab7 (i) or Rab9 (j). C) Quantitation of transferrin fluorescence in the ERC in CHO cells. Data shown are the average background-corrected fluorescence values in relative units ± SEM in control and DAPT-treated CHO cells immediately (0 h) and 1 h after transferrin labeling. **Statistically significant difference in comparison to control cells (Independent Samples Test, P0.0001). All images are confocal. Scale Bars = 10 µm for a, b, f, g; 5 µm for c–e, h–j.

A similar delay in the clearance of transferrin was observed in CHO cells overexpressing a dominant negative mutant form of PS1 (PS1/D385A), embryonic stem (ES) cells lacking both PS1 and PS2 (PS1/2 dKO) and wild-type ES cells treated with two different -secretase inhibitors, DAPT and L685,458. In all cells, transferrin was present in the ERC immediately after labeling. After chase, however, cells lacking PS/-secretase activity still contained 2–3 times more transferrin in the ERC. Colabeling of the cells with FITC-dextran, a marker for the late endocytic pathway including late endosomes and lysosomes, revealed no colocalization with transferrin, indicating that transferrin was retained in the ERC and not directed to the late endocytic compartments within the -secretase activity-deficient cells.

2. PS/-secretase activity also regulates clearance of APP-CTFs from the ERC
To study the role of PS/-secretase function in the trafficking of APP through the ERC, CHO cells were transiently transfected with yellow fluorescence protein-tagged full-length APP (APP-yellow fluorescent protein), treated with the -secretase inhibitors DAPT or L-685,458, and labeled with transferrin. APP in control cells was mainly localized inside the cells, and it did not colocalize with transferrin to the ERC. However, on treatment of the cells with either of the inhibitors, APP and/or APP-CTFs accumulated on the cell surface and to the ERC, as indicated by its colocalization with transferrin in this compartment. Interestingly, treatment of the cells with -secretase inhibitors resulted in an increase only in APP-CTF, but not full-length APP, levels. This suggests that the newly accumulated CTFs are most likely to accumulate on the cell surface and the ERC subsequent to the -secretase inhibitor treatment.

Finally, we directly assessed the effect of -secretase inhibition of APP-CTF localization by stably transfecting CHO cells with a recombinant APP-C99-GFP construct lacking the whole ectodomain of APP. Similar to CTFs derived from full-length APP, treatment with the -secretase inhibitors DAPT or L-685,458 resulted in the stabilization and accumulation of C99 on the cell surface and in the ERC, as indicated by the colocalization of C99 with transferrin (Fig. 2 A). Western blot analysis further showed that the C99 levels increased in the DAPT- and L685,458-treated cells as compared to the control cells, where C99 levels remained undetectable (Fig. 2B ).

View larger version (50K): [in this window] [in a new window]   Figure 2. C99-GFP accumulates in the ERC after -secretase inhibitor treatment. A) In control cells C99-GFP (green) expression was below detection levels (a), because of constitutive -secretase-mediated processing. Transferrin (Tfn, red) labeling indicated properly formed ERCs in these cells (b and c, arrows). Treatment of the cells with DAPT (d–f) or L-685,458 (g–i) resulted in the stabilization of C99-GFP (d and g), and accumulation in the ERC together with transferrin (wide arrows in f and i, yellow), as well as in the plasma membrane (narrow arrows in f and i). All images are confocal. Scale Bars = 10 µm for a, b, d, e, g, h; 5 µm for c, f, i. B) Western blot analysis showed increased levels of C99-GFP protein in the -secretase inhibitor treated C99-GFP overexpressing cells as compared to those in the control cells. The blot was probed with anti-GFP antibody (top; 1:1000) to visualize C99-GFP levels, and reprobed with antiactin antibody (bottom; 1:1000) to verify equal loading of each lane. Arrows on the right indicate the C99-GFP and actin bands.

CONCLUSIONS AND SIGNIFICANCE

Here we have studied the role of PS/-secretase in protein recycling through a specific endosomal compartment, the ERC. By using fluorescently labeled transferrin, yellow fluorescent protein- and GFP-tagged APP expression constructs, and cells lacking functional PS/-secretase, we showed that PS/-secretase activity is required for normal clearance of soluble transferrin and membrane-bound APP-CTFs from the ERC (Fig. 3 ). This was indicated by the fact that a lack of PS/-secretase activity resulted in a delayed clearance and subsequent accumulation of both transferrin and APP-CTFs in the ERC, whereas in control cells with normal PS/-secretase activity, these proteins were efficiently cleared from the ERC. Presenilins are known to regulate intracellular trafficking of APP and APP-CTFs. PS/-secretase also proteolytically cleaves over 20 different type I membrane proteins in addition to APP, many of which are recycled through the ERC after endocytosis. Therefore, it is possible that PS/-secretase regulates cellular localization, signaling, and proteolysis of many type I membrane proteins, in addition to APP. PS/-secretase-mediated proteolysis of an unidentified membrane protein may account for the observed regulation of endosomal recycling through the ERC in general (Fig. 3) . The connection between these two functions of PS/-secretase, regulation of protein recycling and proteolysis, remains to be clarified. Alternatively, accumulation of CTFs into the ERC in the absence of PS/-secretase activity may delay protein clearance from this compartment.

View larger version (23K): [in this window] [in a new window]   Figure 3. Proposed regulation of protein recycling through the ERC by PS/-secretase. Transferrin (red) is internalized from the plasma membrane into primary endosomes, which fuse with sorting endosomes. These tubular vesicular structures target proteins to late endosomes and lysosomes for degradation or alternatively back to the plasma membrane via the ERC. The ERC is a collection of tubular organelles associated with microtubules. APP (green) and many other type I membrane proteins, some of which are substrates for PS/-secretase, undergo internalization and recycling back to the plasma membrane via the ERC. Our results propose that PS/-secretase activity is essential for correct protein recycling via the ERC, and that in the absence of PS/-secretase activity, proteins are unable to exit the ERC or their exit is slowed down. This results in protein accumulation in the ERC, as indicated by our data. Alternatively, accumulation of CTFs in the absence of PS/-secretase activity may delay protein clearance from the ERC.

The endosomal system is greatly affected in AD, suggesting that disturbances in normal endosomal function may result in neurodegeneration in AD. Endosomal sorting and enhancement of APP endocytosis have been shown to result in increased ßbeta;-amyloid (Aßbeta;) and APP ßbeta;CTF levels. Our imaging studies of -secretase inhibitor-treated C99-GFP expressing cells suggest that the APP-CTFs, in particular, accumulate and colocalize with transferrin to the ERC. Furthermore, Western blotting showed that PS/-secretase inhibitors specifically increased the levels of APP-CTFs, and not full-length APP. Our results are in accordance with previously published data showing that APP-CTFs accumulate on the plasma membrane, endosomes, and other intracellular compartments in PS mutant cells. Most importantly, our results specifically suggest an impaired clearance of APP and/or APP-CTFs from the ERC and trafficking back to the plasma membrane when PS/-secretase activity is inhibited. Thus, our findings imply that -secretase not only cleaves APP-CTFs, but also regulates the delicate balance of APP and/or APP-CTF pools available for Aßbeta; production.

To our knowledge, this is the first report that PS/-secretase activity, perhaps through an unidentified type I membrane protein, is involved in the regulation of protein trafficking through the ERC. This is supported by the notion that trafficking of both transferrin and APP through the ERC is delayed in cells lacking functional PS/-secretase. Our results further indicate that lack of PS/-secretase function results in impaired clearance from a specific endocytic compartment. Even though the precise molecular aspects remain to be elucidated, our results suggest a new mechanism by which impaired PS/-secretase function may eventually result in neurodegeneration.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5531fje

¹ Current address: Synta Pharmaceuticals Corp., 45 Hartwell Ave., Lexington, MA 02421, USA.

² These authors contributed equally to this work.

This article has been cited by other articles:

				R. A. Sharples, L. J. Vella, R. M. Nisbet, R. Naylor, K. Perez, K. J. Barnham, C. L. Masters, and A. F. Hill Inhibition of {gamma}-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes FASEB J, May 1, 2008; 22(5): 1469 - 1478. [Abstract] [Full Text] [PDF]

				E. Repetto, I.-S. Yoon, H. Zheng, and D. E. Kang Presenilin 1 Regulates Epidermal Growth Factor Receptor Turnover and Signaling in the Endosomal-Lysosomal Pathway J. Biol. Chem., October 26, 2007; 282(43): 31504 - 31516. [Abstract] [Full Text] [PDF]

				S. Urra, C. A. Escudero, P. Ramos, F. Lisbona, E. Allende, P. Covarrubias, J. I. Parraguez, N. Zampieri, M. V. Chao, W. Annaert, et al. TrkA Receptor Activation by Nerve Growth Factor Induces Shedding of the p75 Neurotrophin Receptor Followed by Endosomal {gamma}-Secretase-mediated Release of the p75 Intracellular Domain J. Biol. Chem., March 9, 2007; 282(10): 7606 - 7615. [Abstract] [Full Text] [PDF]

				J. Kim, L. Onstead, S. Randle, R. Price, L. Smithson, C. Zwizinski, D. W. Dickson, T. Golde, and E. McGowan A{beta}40 Inhibits Amyloid Deposition In Vivo J. Neurosci., January 17, 2007; 27(3): 627 - 633. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-5531fjev1 20/8/1176    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, M. Articles by Kovacs, D. M. Search for Related Content PubMed PubMed Citation Articles by Zhang, M. Articles by Kovacs, D. M.