Upregulated function of mitochondria-associated ER membranes in Alzheimer disease
2012; Springer Nature; Volume: 31; Issue: 21 Linguagem: Inglês
10.1038/emboj.2012.202
ISSN1460-2075
AutoresEstela Área-Gómez, Maria del Carmen Lara Castillo, Marc D. Tambini, Cristina Guardia‐Laguarta, Ad J.C. de Groof, Moneek Madra, Junichi Ikenouchi, Masato Umeda, Thomas D. Bird, Stephen L. Sturley, Eric A. Schon,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoArticle14 August 2012Open Access Upregulated function of mitochondria-associated ER membranes in Alzheimer disease Estela Area-Gomez Estela Area-Gomez Department of Neurology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Maria del Carmen Lara Castillo Maria del Carmen Lara Castillo Department of Neurology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Marc D Tambini Marc D Tambini Department of Cellular, Molecular and Biophysical Studies, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Cristina Guardia-Laguarta Cristina Guardia-Laguarta Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Ad J C de Groof Ad J C de Groof Department of Neurology, Columbia University Medical Center, New York, NY, USA Department of Cell Biology, NCMLS, Radboud University, Nijmegen, The NetherlandsPresent address: Merck/Intervet International bv, Wim de Körverstraat 35, PO Box 31, 5830 AA Boxmeer, The Netherlands Search for more papers by this author Moneek Madra Moneek Madra Department of Pediatrics, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Junichi Ikenouchi Junichi Ikenouchi Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto, Japan Search for more papers by this author Masato Umeda Masato Umeda Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto, Japan Search for more papers by this author Thomas D Bird Thomas D Bird Division of Neurogenetics, University of Washington and Geriatrics Research Center, VA Medical Center, Seattle, WA, USA Search for more papers by this author Stephen L Sturley Stephen L Sturley Department of Pediatrics, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Eric A Schon Corresponding Author Eric A Schon Department of Neurology, Columbia University Medical Center, New York, NY, USA Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Estela Area-Gomez Estela Area-Gomez Department of Neurology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Maria del Carmen Lara Castillo Maria del Carmen Lara Castillo Department of Neurology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Marc D Tambini Marc D Tambini Department of Cellular, Molecular and Biophysical Studies, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Cristina Guardia-Laguarta Cristina Guardia-Laguarta Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Ad J C de Groof Ad J C de Groof Department of Neurology, Columbia University Medical Center, New York, NY, USA Department of Cell Biology, NCMLS, Radboud University, Nijmegen, The NetherlandsPresent address: Merck/Intervet International bv, Wim de Körverstraat 35, PO Box 31, 5830 AA Boxmeer, The Netherlands Search for more papers by this author Moneek Madra Moneek Madra Department of Pediatrics, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Junichi Ikenouchi Junichi Ikenouchi Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto, Japan Search for more papers by this author Masato Umeda Masato Umeda Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto, Japan Search for more papers by this author Thomas D Bird Thomas D Bird Division of Neurogenetics, University of Washington and Geriatrics Research Center, VA Medical Center, Seattle, WA, USA Search for more papers by this author Stephen L Sturley Stephen L Sturley Department of Pediatrics, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Eric A Schon Corresponding Author Eric A Schon Department of Neurology, Columbia University Medical Center, New York, NY, USA Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA Search for more papers by this author Author Information Estela Area-Gomez1, Maria del Carmen Lara Castillo1, Marc D Tambini2, Cristina Guardia-Laguarta3, Ad J C de Groof1,4, Moneek Madra5, Junichi Ikenouchi6, Masato Umeda6, Thomas D Bird7, Stephen L Sturley5 and Eric A Schon 1,8 1Department of Neurology, Columbia University Medical Center, New York, NY, USA 2Department of Cellular, Molecular and Biophysical Studies, Columbia University Medical Center, New York, NY, USA 3Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA 4Department of Cell Biology, NCMLS, Radboud University, Nijmegen, The Netherlands 5Department of Pediatrics, Columbia University Medical Center, New York, NY, USA 6Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Kyoto, Japan 7Division of Neurogenetics, University of Washington and Geriatrics Research Center, VA Medical Center, Seattle, WA, USA 8Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA *Corresponding author. Department of Neurology, Columbia University Medical Center, Room P&S 4-449, 630 West 168th Street, New York, NY 10032, USA. Tel.:+1 212 305 1665; Fax:+1 212 305 3986; E-mail: [email protected] The EMBO Journal (2012)31:4106-4123https://doi.org/10.1038/emboj.2012.202 There is a Have you seen? (November 2012) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Alzheimer disease (AD) is associated with aberrant processing of the amyloid precursor protein (APP) by γ-secretase, via an unknown mechanism. We recently showed that presenilin-1 and -2, the catalytic components of γ-secretase, and γ-secretase activity itself, are highly enriched in a subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAMs). We now show that MAM function and ER–mitochondrial communication—as measured by cholesteryl ester and phospholipid synthesis, respectively—are increased significantly in presenilin-mutant cells and in fibroblasts from patients with both the familial and sporadic forms of AD. We also show that MAM is an intracellular detergent-resistant lipid raft (LR)-like domain, consistent with the known presence of presenilins and γ-secretase activity in rafts. These findings may help explain not only the aberrant APP processing but also a number of other biochemical features of AD, including altered lipid metabolism and calcium homeostasis. We propose that upregulated MAM function at the ER–mitochondrial interface, and increased cross-talk between these two organelles, may play a hitherto unrecognized role in the pathogenesis of AD. Introduction Alzheimer disease (AD) is a late onset neurodegenerative disorder characterized by progressive neuronal loss, especially in the cortex and the hippocampus (Goedert and Spillantini, 2006). The vast majority of AD is sporadic (SAD), but mutations in the amyloid precursor protein (APP) and in presenilin-1 (PS1) and -2 (PS2), which are components of the γ-secretase complex that processes APP to produce amyloid-β (Aβ), have been identified in the familial form (FAD), which is similar to SAD but has an earlier age of onset. To date, there is no unifying hypothesis that can explain the diverse and apparently unrelated morphological and biochemical abnormalities—extracellular plaques containing Aβ fibrils (Pimplikar, 2009), intracellular tangles containing hyperphosphorylated forms of the microtubule-associated protein tau (Pimplikar, 2009), elevated serum cholesterol (Stefani and Liguri, 2009), altered phospholipid metabolism (Wells et al, 1995; Pettegrew et al, 2001), aberrant calcium homeostasis (Bezprozvanny and Mattson, 2008), and mitochondrial dysfunction (Wang et al, 2009)—detected in tissues and in cultured cells from AD patients (Pimplikar, 2009), with no apparent direct link among them. In the case of FAD, investigations into a common source of these apparently unrelated features have been complicated by uncertainty regarding the subcellular distribution of the presenilins. PS1 and PS2 have been located to numerous subcellular compartments, including endoplasmic reticulum (ER) (Annaert et al, 1999), Golgi (Annaert et al, 1999), plasma membrane (PM) (Marambaud et al, 2002), nuclear envelope (Kimura et al, 2001), endosomes (Vetrivel et al, 2004), lysosomes (Pasternak et al, 2003), and mitochondria (Ankarcrona and Hultenby, 2002). On the other hand, it is generally accepted that APP (Kosicek et al, 2010), presenilins, Aβ, and γ-secretase activity are enriched in lipid rafts (LRs) (Urano et al, 2005; Vetrivel et al, 2005), which are specialized domains rich in cholesterol and sphingolipids that form detergent-insoluble aggregates in cell membranes (i.e., detergent-resistant membranes, or DRMs) (Simons and Vaz, 2004). These regions have a liquid-ordered structure with unique biophysical characteristics that differs from the rest of the cell's liquid-disordered membranes (Simons and Vaz, 2004). Traditionally, LRs have been considered to be present only in the PM (Simons and Vaz, 2004). However, γ-secretase activity is negligible in this compartment, forming the basis of what has been called ‘the spatial paradox’ (Cupers et al, 2001). Recent evidence, however, has in fact indicated the existence of intracellular LRs/DRMs that are different in protein composition from those in the PM (Browman et al, 2006). We recently found that PS1, PS2, APP, and γ-secretase activity are not homogeneously distributed in the ER, but rather are enriched in mitochondria-associated ER membranes (ER-MAMs or MAMs) (Area-Gomez et al, 2009). MAM is a dynamic subcompartment of the ER connected physically and biochemically to mitochondria that is involved in a number of key metabolic functions (Hayashi et al, 2009), including cholesterol metabolism (Rusinol et al, 1994), the synthesis and transfer of phospholipids between the ER and mitochondria (Vance, 2003), and calcium homeostasis (Csordas et al, 2010). We now report that MAM is a complex elaboration of the ER with the characteristics of an LR. Moreover, using a number of relevant tissues and cell lines (Supplementary Table S1), we show that mutations in presenilins perturb MAM function significantly, and that these perturbations are also present in cells from FAD patients with mutations in PS1, PS2, and APP, and in SAD patients with no known genetic aetiology. These findings may shed new light on the biology of presenilins and on our understanding of some of the features associated with the pathogenesis of AD. Results MAM displays the characteristics of an intracellular LR MAM is a dynamic domain of the ER responsible for the integration of several cellular functions, including Ca2+ signalling, lipid transport, energy metabolism, and cellular survival. For this reason, we speculated that MAM might have the characteristics of an LR/DRM in order for it to recruit and orientate the different signalling proteins needed for cellular homeostasis and for the effective cross-talk between mitochondria and ER (Hayashi and Fujimoto, 2010; Williamson et al, 2011; Fujimoto et al, 2012). In addition, the fact that γ-secretase activity is present in LRs (Vetrivel et al, 2005) and is also enriched in MAM (Area-Gomez et al, 2009) provided indirect support for the idea that MAM could be an LR/DRM. We therefore incubated purified MAM from mouse tissues (Area-Gomez et al, 2009; Supplementary Figure S1) with and without Triton X-100 (TX100), and loaded both samples onto a Percoll gradient under the same conditions used for its initial isolation. The TX100-treated MAM sample was fundamentally intact and migrated to the identical position in the gradient as did the untreated sample, consistent with the behaviour of a DRM (Figure 1A). To separate LR from other cell contents, we loaded TX100-treated and untreated control MAM from mouse brain onto a sucrose gradient, and analysed fractions for the known MAM markers Pemt (phosphatidylethanolamine N-methyltransferase; Vance, 1990), Vdac1 (voltage-dependent anion channel 1; Hayashi et al, 2009), and Ps1 (Area-Gomez et al, 2009; Figure 1B). The proteins migrated at similar positions in the lower density fractions, and were unaffected by detergent treatment (Figure 1B), consistent with the behaviour of MAM as a DRM. By contrast, purified mitochondria and bulk ER from the bottom of the gradient behaved like detergent-soluble fractions (Supplementary Figure S2), indicating the absence of DRMs in these organelles, as expected (Zheng et al, 2009). MAM was not contaminated with PM rafts, as Src, a marker for PM rafts (Morrow and Parton, 2005), was observable in sucrose gradient fractions from purified PM, but not from the crude mitochondrial fraction from which the MAM fraction was derived (Figure 1C). Moreover, the cholesterol content of MAM was higher than that found in the cytoplasm, mitochondria, bulk ER, and total PM, and was comparable to that of LR from PM (Simons and Vaz, 2004; Figure 2A). These results are consistent with the numerous reports showing that presenilins and γ-secretase activity reside in LRs and suggest that MAM is an intracellular LR-like domain that may recruit and orient various signalling proteins needed to regulate cross-talk between ER and mitochondria. Figure 1.MAM displays the features of a lipid raft. (A) Mouse liver Percoll-purified MAM treated with or without TX100 prior to centrifugation through a second Percoll gradient. The low density fraction (arrow) is detergent resistant but solubilizable by methanol (MeOH), implying that it is a DRM. (B) Western blot of fractions from a 5–30% sucrose gradient (triangle; lower density at left) of MAM isolated from a Percoll gradient (as in A). The pellet (P) denotes TX100-soluble material. (C) Western blot of gradient fractions of mouse liver PM and crude mitochondrial extract (CM) to detect Src (PM marker) and Pemt (MAM marker). Download figure Download PowerPoint Figure 2.Cholesterol metabolism in PS-mutant and AD cells. (A) Total cholesterol in the indicated mouse brain fractions (n=3, except PM rafts; n=2). (B) ACAT activity in mouse brain fractions (n=4). Inset: western blot to detect ACAT protein; 20 μg protein loaded in each lane. Asterisk denotes significant difference versus ER and mito fractions (P<0.05). (C) Content of cholesterol species in PS-mutant MEFs relative to that in WT MEFs (numbers denote average amounts of the indicated cholesterol species, in ng/mg protein). (D) ACAT activity (i.e., conversion of 3H-cholesterol to 3H-cholesteryl esters) in MEFs after 6 h (n=6). (E) Kinetics of CE synthesis (performed as in D) in Ps1-KD cells (note increased slope (line of best fit, in cpm/μg/h) versus control). (F) Quantitation of 3H-CE synthesis after 6 h in fibroblasts from FAD (n=5; 4 PS1 (circles), 1 PS2 (triangle)) and SAD (n=9) patients versus paired controls. For cell lines that were evaluated multiple times (see Supplementary Table S1), the data point represents an average of the assays. Boxes with centred lines denote averages±s.d.; asterisks denote significant difference versus WT (P 3-fold in the DKO MEFs as compared to WT (Figure 4A), suggesting an upregulation of MAM function and of ER–mitochondrial cross-talk in these cells. In a control experiment, we determined that this upregulation was not due to an increase in the expression of Ptdss1, Ptdss2, or Pisd, three key genes involved in transport of phospholipids between ER and mitochondria (Supplementary Figure S3). Figure 4.Phospholipid synthesis in PS-mutant and AD cells. (A) Synthesis of 3H-PtdSer and 3H-PtdEtn after labelling DKO MEFs with 3H-Ser for the indicated times (h) (n=3). (B) Pulse-chase. MEFs were labelled for 1 h with 3 H-Ser and chased with cold Ser for the indicated times (n=3). Note the steeper slopes (i.e., rates of 3 H-Ser incorporation) for both PtdSer (negative slopes) and PtdEtn (positive slopes), indicative of a more rapid conversion of PtdSer to PtdEtn, especially in the DKO cells. (C) Phospholipid synthesis in crude mitochondria from Ps-KO MEFs (n=3 or 4, as indicated; error bars, s.e.). (D) Kinetics of PtdSer and PtdEtn synthesis (as in A) in Ps1-KD cells. (E) Phospholipid synthesis after 6 h (as in A) in fibroblasts from FAD (n=6) and SAD (n=9) patients. Note: three of the SAD PtdEtn values were unusually high (∼600% of control) and were omitted from the statistical analyses. Other notation as in Figure 2. Download figure Download PowerPoint To determine the kinetics of this upregulation, we performed pulse-chase analysis by incubating the MEFs with 3H-Ser for 1 h, followed by a chase with cold serine for various time periods (Figure 4B). The incorporation of label into 3H-PtdSer during the pulse (time 0 in Figure 4B) was significantly higher in the Ps1-KO and DKO MEFs than in control. During the chase, the amount of 3H-PtdSer decreased and that of 3H-PtdEtn increased, consistent with the conversion of the former into the latter, with higher rates in the Ps1-KO and DKO MEFs (up to three-fold higher). While the increase in lipid synthesis in the pulse-chase was not altered significantly in the Ps2-KO MEFs, Ps2 clearly contributes to phospholipid metabolism and MAM function, as lipid synthesis in the Ps1+Ps2 double knockout was much more pronounced than in the Ps1-knockout alone (Figure 4B). These results were confirmed in isolated MEF crude mitochondrial fractions (containing essentially only ER, MAM, and mitochondria; Area-Gomez et al, 2009) (Figure 4C). The rate of phospholipid synthesis was also increased in Ps1-KD cells (Figure 4D) and, importantly, in FAD and SAD fibroblasts (by ∼1.5- to 2-fold over controls) (Figure 4E). Since some of the PtdEtn synthesized is exported to the inner leaflet of the PM (Vance, 2008), we hypothesized that it would be elevated in the PM of mutant cells. Accordingly, we treated cells with two highly related antibiotics, cinnamycin (Cin; also called Ro09-0198) (Choung et al, 1988) and duramycin (Dura) (Marki et al, 1991), both of which are 19-aa cyclic peptides that form a complex specifically with PtdEtn to induce pore formation in the PM in a PtdEtn concentration-dependent manner, followed by rapid cell death (Makino et al, 2003) (see example in Figure 5A, left and middle panels). Ps1-KO and DKO MEFs were ∼3.5-fold more Cin sensitive than were controls (Figure 5A, right panel). Similarly, Ps1-KD cells were ∼3-fold more Cin sensitive than were controls; as before, this sensitivity could be rescued by overexpression of human WT, but not A246E mutant, PS1 (Figure 5B, left panel). Notably, FAD and SAD cells were significantly (∼3- to 5-fold) more Cin sensitive than were controls (Figure 5B, right panel). We also were able to visualize the presence of PtdEtn on the cell surface by staining cells with FL-SA-Ro (Figure 5C), a fluorescent-conjugated form of cinnamycin that binds to PtdEtn on the PM but does not initiate cell death (Emoto et al, 1996). In agreement with the Cin-sensitivity results, ∼4 times as many FAD and SAD cells were stained with FL-SA-Ro as compared to controls (Figure 5C; Supplementary Figure S7). Figure 5.Cinnamycin sensitivity in PS-mutant and AD cells. (A) Left: Example of live/dead assays (1 μM cinnamycin for 10 min at 37°C). Middle: Example of cinnamycin-sensitivity curves in PS-mutant MEFs. Right: Summary of cinnamycin sensitivity assays (1 μM Cin for 10 min) in PS-mutant MEFs. (B) Left: Example of cinnamycin sensitivity in Ps1-KD cells versus mismatch control (C). Note that overexpression of human WT PS1, but not A246E mutant PS1, could ‘rescue’ Cin sensitivity. Right: Cin/Dura sensitivity in fibroblasts from FAD (n=7) and SAD (n=8) patients versus controls (n=7). (C) Left: Example of staining of control and AD patient cells with fluorescent cinnamycin (FL-SA-Ro; orange); cells were counterstained with calcein (green) to visualize overall cell morphology. Right: Quantitation of FL-SA-Ro staining in fibroblasts from FAD (n=3) and SAD (n=3) patients compared to controls (n=3). See other examples in Supplementary Figure S7. Other notations as in Figures 2 and 3. Download figure Download PowerPoint Together with the CE data, these results point to an upregulation of MAM function in AD, either by mutations in presenilins or APP or, in the case of SAD, by unknown causes. Increased ER–mitochondrial contacts in PS-mutant cells and AD patient fibroblasts The increased biochemical activity of MAM in PS-mutant cells prompted us to see if ER–mitochondrial contacts were physically altered. We therefore transfected cells with DsRed-Mito to detect mitochondria (in red) and with GFP-Sec61β to detect ER (in green), and used confocal microscopy and Image J analysis to detect and quantitate regions where the two signals were in close apposition (see example in Figure 6A). Using this method, we found that the degree of ER–mitochondrial apposition was significantly higher in Ps1-KO (∼34±10% of the total signal), Ps2-KO (34±6%), and DKO (56±6%) MEFS than in WT MEFS (12±4%) (Figure 6B, left). Moreover, the degree of apposition was significantly higher in fibroblasts from both FAD (26±4%) and SAD (24±6%) patients than in those from controls (11±2%) (Figure 6B, right). Figure 6.ER–mitochondrial colocalization in Ps-mutant and AD cells. (A) Example of confocal images of cells stained with Mito DS Red (red) and GFP-Sec61β (green). In the insets, note the large number of discrete red and green signals in the WT as compared to the Ps-mutant MEFs, which have more overlap (orange and yellow signals). (B) Quantitation of colocalization (as in A) by Image J in WT (average of 10 images ±s.d.), Ps1-KO (n=11), Ps2-KO (n=13), and DKO (n=7) MEFs (left), and in fibroblasts from FAD (n=2) and SAD (n=5) patients compared to controls (n=3) (right). Other notations as in Figures 2 and 3. Download figure Download PowerPoint In order to observe ER–mitochondrial apposition at higher resolution, we imaged MEFs and patient cells by electron microscopy (EM). We observed a significant increase in the length of mitochondrial-ER contacts (i.e., MAM) in DKO as compared to WT MEFs (Figure 7). Specifically, there were significantly more numerous ‘long’ (50–200 nm; Figure 7C) and ‘very long’ (>200 nm; Figure 7E) contacts in DKO MEFs than in WT MEFs (∼5-fold and >10-fold, respectively; Figure 7F, left), whereas connections in WT MEFs were much shorter and more ‘punctate’ (<50 nm; Figure 7B). We found a similar increase in ‘long’ and ‘very long’ contacts in AD patients (Figure 7F, right; Supplementary Figure S8). Thus, the increased biochemical activity of MAM in PS-mutant and in AD cells correlated with an increased area of physical association between the two organelles. Figure 7.Electron microscopy of Ps-mutant and AD cells. (A, C, E) DKO MEFs. (B, D) WT MEFs. Note increased length of regions of contact between ER and mitochondria (M) (arrowheads) in DKO MEFs, and, in (E), a region of ER ‘sandwiched’ between two mitochondria. (F) Quantitation of ER–mitochondrial co
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