Dual localization of APP in human neuronal HCN-1A cells
A comparison of NH2
-terminal chimeric sequences of APP with those of P4501A1 and P4502B1 is shown in
A. The NH2
-terminal 38–amino acid region of APP with a hydrophobic helical structure functions as the ER-targeting domain ( A). Immediately COOH terminal to this region contains positively charged residues at positions 40, 44, and 51 that mimic the cryptic mitochondrial-targeting signals of P4501A1 and P4502B1 ( A). The positively charged residues within sequence 20–30 of P4502B1 and sequence 32–44 of P4501A1 have been shown to be critical for mitochondrial targeting (Addya et al., 1997
; Anandatheerthavarada et al., 1999
Figure 1. Chimeric signal properties of APP and its bimodal targeting to mitochondria and PMs. (A) Chimeric signals of APP and comparison with the signal domains of P4501A1 and 2B1. The ER-targeting sequence (1–36) of APP is indicated as a dark shaded area. (more ...)
The purity of subcellular fractions was established by immunoblot analysis of various membrane fractions from HCN-1A cells using antibodies to Na+
ATPase (PM specific), TOM40 (mitochondria specific), calreticulin (ER specific), βCOP (Golgi specific), and p97 (nuclear specific) as markers ( B). Immunoblot analysis using APP Nt Ab (antibody specific for the NH2
-terminal amino acids 42–66) in the bottom panel of B shows the endogenous levels of 95-kD APP in mitochondria and ~110-kD APP in the PM, ER, as well as Golgi fractions of HCN cells. PMA, a known inducer of APP expression (Ringheim et al., 1996
), was used to test the effects of elevated APP on mitochondrial targeting. Northern blot analysis ( C) shows that ~2.1-kb APP695 mRNA was induced at 12 and 24 h of PMA treatment ( C). Although not shown, mRNAs for APP770, APP751, and APLP2 were not detectable by the Northern blot using isoform-specific probes (Shepherd et al., 2000
). These results suggest that HCN-1A cells express predominantly the APP695 species. Consistent with induced mRNA levels, the antibody-reactive protein in both PM and mitochondria increased by 10–15-fold by PMA treatment ( D). At 24 h of treatment, the mitochondrial level was nearly 60% of that detected in the PM. Coincident with increased mitochondrial accumulation of APP, the ability of mitochondria to reduce methylthiazoletetrazolium (MTT), which is widely used as a measure of mitochondrial function, was also progressively diminished ( E).
Immunoblot in F shows that the 110-kD protein associated with the PM is converted to a 95-kD species after treatment with glycosidases (PNGase F and O-glycanase). APP associated with the mitochondrial fraction, on the other hand, did not show any difference in size by these treatments. These results show that a substantial amount (~25–30%) of induced APP695 protein exists in association with the mitochondrial fraction in a nonglycosylated form.
The chimeric NH2-terminal signal of APP and its incomplete translocation through mitochondrial membranes
The nature of mitochondrial targeting of APP695 was further studied using an in vitro mitochondrial import assay, in which protection against limited proteolytic digestion was used as a criterion for the import of 35S-labeled proteins into mitochondria.
A (lane 1) shows that a full-length APP of 95 kD is recovered in a reisolated mitochondrial fraction after in vitro incubation, suggesting that APP indeed associates with mitochondrial membrane. Trypsin treatment of in vitro–incubated mitochondria resulted in the protection of a 22-kD fragment of APP ( A, lane 2). These results suggest that the 22-kD protected region of APP is located inside the mitochondrial membrane, whereas the remaining ~73-kD portion might be exposed outside. Under similar import conditions, however, the full-length P4501A1 and 2B1 proteins were protected, indicating their complete translocation ( B). Both APP binding to mitochondria and partial internalization were markedly inhibited by uncouplers of mitochondrial membrane potential, CCCP and 2,4 DNP, and also by mitochondrial swelling ( A, lanes 3–8). Furthermore, 3M/APP with mutated positive residues at +40, +44, and +51, as shown in A, was not imported significantly ( A, lanes 9 and 10), suggesting the importance of these residues for mitochondrial targeting. The possibility of the acidic domain spanning amino acids 220–290 imposing a barrier for complete translocation was verified by using a deletion mutant of APP lacking this domain ( A, Δ220–290/APP). The Δ220–290/APP protein was completely internalized by mitochondria, as indicated by the protection of nearly the full-length of input protein by trypsin ( A, lanes 11 and 12). Control experiments in C show that the 22-kD protease-protected fragment is located inside the mitochondrial membranes because disruption of membrane by Triton X-100 (0.1%) rendered the protein sensitive to protease. Furthermore, labeled APP protein in reactions without added mitochondria was completely sensitive to trypsin, dispelling the possibility that resistance to protease was due to some unusual structural features of the protein. These results together show that APP is targeted to mitochondria, although the acidic domain containing 70 negatively charged residues likely imposes a structural barrier for the complete translocation of APP into the mitochondrial compartment.
Figure 2. Mitochondrial targeting of APP under in vitro and in vivo conditions. (A, B and C) WT/APP, 3M/APP, Δ220–290/APP, P450 MT2 (+33/1A1), and P450 MT4 (2B1) proteins were used for the in vitro transport in isolated rat brain mitochondria as (more ...)
The bimodal targeting of APP695 in vivo was studied by transfection of HCN-1A neuronal cells with WT/APP, 3M/APP, and Δ220–290/APP cDNAs. PM and mitochondrial proteins from transfected cells were subjected to immunoblot analysis. Both intact and digitonin-treated mitochondria from transfected cells contained significant putative nonglycosylated 95-kD APP (25–30% of the total pool; D, lanes 3 and 5–7). Mitochondria treated with trypsin showed a 22-kD antibody-reactive fragment (lane 4). Digitonin treatment, which strips off the outer membrane, did not reduce the level of 95-kD protein ( D, lanes 5–7), suggesting its association with the inner membrane matrix compartment. Trypsin treatment of digitonin-treated mitochondria resulted in a slight reduction in the size of protected fragment by ~1–2 kD ( D, lane 8), which may account for the region spanning the intermembrane space of translocation-arrested protein. Resistance to alkaline Na2CO3 extraction ( D, lanes 9 and 10) of mitochondrial-associated APP further supports its transmembrane topology. Mutations targeted to the three positively charged residues (3M/APP) completely abolished mitochondrial targeting, though the mutant protein was efficiently targeted to the PM ( E). A deletion construct lacking the acidic domain (Δ220–290/APP), however, was targeted to both the PM and mitochondria ( F). In agreement with the results of in vitro mitochondrial import in A, the entire length of the Δ220–290/APP protein was protected by protease treatment, suggesting that it is completely internalized by mitochondria ( F, last lane). Finally, the Cout orientation of the mitochondrial transmembrane-arrested APP was verified using an antibody specific for the COOH-terminal end of APP (APP Ct Ab). As shown in G, APP Ct Ab cross-reacted with intact 95-kD mitochondrially associated APP but failed to interact with the protease-protected 22-kD fragment, confirming the Nin orientation. These results further demonstrate that mitochondrially targeted APP occurs in a transmembrane-arrested orientation with the NH2 terminus buried inside mitochondria.
The dual localization of APP695 in mitochondria and the PM was further investigated by immuno-colocalization of the protein in HCN-1A cells transfected with WT/APP and 3M/APP cDNA constructs for 24 h. Triton-permeabilized cells were subjected to double immunostaining with APP Nt Ab and antibody to mitochondrial outer membrane receptor TOM40. Nonpermeabilized cells were immunostained with APP Nt Ab and antibody to the PM-specific marker Na+/K+ ATPase. In nonpermeabilized cells transfected with WT/APP, a robust staining around the PM by APP antibody was observed (
A), which colocalized with Na+/K+ ATPase (). In permeabilized cells, the APP Nt Ab stained extranuclear granulate structures ( D), some of which colocalized with mitochondrial-specific marker TOM40 (). The inset in F shows a region of the cell with high mitochondrial content, which shows APP-stained structures both overlapping and nonoverlapping with mitochondrial-specific stain. These results show that ectopically expressed APP is targeted to both the PM (through the ER route) and mitochondria. Predictably, transfection with 3M/APP cDNA yielded predominantly PM-specific staining (, G–I) and also some intracellular staining that did not colocalize with TOM40 stains (, J–L). The level of accumulation of APP in the Golgi apparatus after 48 h of transfection was generally higher in HCN-1A cells overexpressing WT/APP than in cells overexpressing 3M/APP (unpublished data). Reasons for this difference currently remain unclear.
Figure 3. Subcellular localization of WT/APP and 3M/APP by immunofluorescence microscopy. HCN-1A cells (A–L) and COS cells (M–R) were transfected with WT/APP (A–F) or 3M/APP (G–L). The inset in F is a twofold enlargement of a region (more ...)
The generality of observations with HCN-1A cells were tested in COS cells transfected with WT/APP cDNA for 24 h. As expected, in intact cells, the antibody stained the PM fraction similar to that in HCN-1A cells (, M–O). In permeabilized cells, the APP Nt Ab stained extranuclear punctuate structures, which colocalized with mitochondrial-specific marker TOM40 (, P–R). Although not shown, immunoblot analysis showed the presence of a 95-kD antibody-reactive protein in mitochondria from transfected COS cells. A more punctate mitochondrial staining was observed in COS cells versus more granular structures in HCN-1A cells, probably reflecting cell-specific differences.
Association of transmembrane-arrested APP with mitochondrial translocases
Interaction of nascent proteins with mitochondrial outer and inner membrane translocase complexes (TOMs and TIMs, respectively) is a critical requirement for mitochondrial import of proteins. Because of the dynamic nature of the transport process, the association of nascent proteins with TOMs and TIMs is detectable only by generating translocation intermediates using fusion proteins with dihydrofolate reductase (DHFR) in the presence of added methatrexate (MTX), a ligand of DHFR (Eilers and Schatz, 1986
). To test the association of APP with translocase proteins, we generated a fusion construct consisting of the 1–220 amino acid region of APP fused to DHFR (
A). As shown in A, the 20-kD DHFR is not imported significantly into mitochondria. However, the 1–220/APP–DHFR fusion protein (42 kD) is imported into mitochondria and rendered resistant to trypsin. In a reaction mixture with added MTX, a 22-kD fragment of the fusion protein is protected, suggesting that only part of the fusion protein enters due to the ligand-mediated translocation arrest.
Figure 4. Transmembrane-arrested APP exists in contact with mitochondrial translocase proteins. 35S-labeled APP (1–220) fused to DHFR (APP–DHFR), WT/APP, or Δ220–290/APP proteins were used for in vitro transport with isolated yeast (more ...)
B shows the extent of interaction of the 1–220/APP–DHFR fusion protein with various translocase proteins, as determined by chemical cross-linking followed by immunoprecipitation. The fusion protein from an in vitro reaction mixture without added cross-linker is immunoprecipitated by APP Nt Ab but not by pre-immune Ab. The APP Nt Ab also yielded a 42-kD immunoprecipitate with no detectable cross-linked product from a reaction mixture without added MTX, probably reflecting the dynamic nature of the import process. A reaction mixture with added MTX, however, yielded two major cross-linked products (82 and 62 kD) in addition to the 42-kD input protein with Nt Ab. The 82- and 62-kD products were not seen in reactions without added cross-linker. Immunoprecipitation with antibodies specific for different translocase proteins shows that the 82-kD band represents cross-linked products with TOM40 and TIM44, both of which have nearly comparable molecular mass. The 62-kD component represents cross-linked product with TIM23. These results show that the NH2-terminal 220–amino acid sequence of APP can indeed direct the mitochondrial targeting of a nonmitochondrial reporter protein, DHFR, and that it interacts with mitochondrial translocase proteins.
C shows the cross-linked products of translocation-arrested APP with mitochondrial translocase complexes. It is seen that reaction with Δ220–290/APP, which is transported into mitochondria without any translocation arrest, predominantly yielded the input protein without any detectable higher molecular forms. Reactions with WT/APP, on the other hand, yielded three higher molecular weight components of ~110, 120, and 140 kD with APP Nt Ab. Immunoprecipitation with antibodies against specific translocase proteins indicated that the 140-kD species consists of APP cross-linked to TOM40 or TIM44, whereas the 120-kD species represents cross-linked product with TIM23. The nature of the 110-kD species remains unclear, though it may represent cross-linked products with some of the smaller TOM or TIM proteins. These results provide confirmatory evidence that the mitochondrial-associated WT/APP exists in transmembrane-arrested form in association with various translocase proteins.
A direct evidence for the transmembrane orientation of mitochondrial-targeted APP was sought by immunoelectron microscopy of HCN-1A cells transfected with WT and mutant APP cDNAs for 32 h. We used primary antibodies specific for the NH2 and COOH termini of APP and antibody for TOM40 simultaneously for probing the sections, and costained with colloidal gold–conjugated secondary antibodies, each specific for a given antibody. The anti–Nt APP Ab was conjugated to 5-nm gold particles, anti–Aβ APP Ab to 10-nm gold, and anti-TOM40 Ab to 20-nm gold particles. The electron micrograph in D shows that APP-specific (5 and 10 nm) electron-dense particles are associated with mitochondria, the ER, the Golgi apparatus, and secretory vesicles. Interestingly, the mitochondrial-associated APP is organized in an Nin–Cout orientation invariably in close proximity to TOM40 protein (20-nm particle). The positions of the NH2- and COOH-terminal–specific stains suggest that the APP protein is in direct contact with, and possibly traversing, the channel-forming TOM40 protein. As expected, APP associated with the Golgi apparatus, the ER, and secretory vesicles showed only APP NH2- ( D, arrow 3) and COOH-terminal Aβ ( D, arrow 1)–specific stains, but not TOM40-specific stain. In keeping with the results of immunoblot analysis and immunofluorescence microscopy ( E and , J–L), cells transfected with 3M/APP cDNA showed vastly reduced mitochondrial localization but significant localization in the Golgi apparatus and transport vesicles ( E). Cells transfected with Δ220–290/APP, on the other hand, showed the distribution of COOH- ( E, arrow 1) and NH2-terminal ( E, arrow 3)–specific stains in different subcellular compartments, including mitochondria ( F). Interestingly, both the COOH- and NH2-terminal–specific stains for Δ220–290/APP protein were localized well inside the mitochondrial inner membrane, confirming that it is completely internalized. These results show that mitochondrial-associated APP exists in a translocation-arrested orientation through the mitochondrial membrane. Results also provide further confirmation that the negatively charged domain is essential for causing the translocation arrest of APP.
Inverse patterns of APP accumulation in the mitochondrial and PM fractions at longer time intervals
We next determined the time course of accumulation of APP695 in the mitochondrial and PM fractions of HCN cells transfected with WT/APP, 3M/APP, and Δ220–290/APP cDNA constructs and also the accumulation of the intra- as well as the extracellular Aβ peptide at these time points (
; ). Immunoblot in A shows a steady increase in the level of WT/APP in the mitochondrial fraction from 0 to 96 h of transfection, whereas the level in the PM declined steadily during this time ( B). Notably, the level of secreted Aβ pool (Aβ40, 42, and 43) in the culture fluid (CF) declined steadily ( C), in parallel to declining APP in the PM fraction ( B). Use of peptide-specific antibodies to Aβ40 and Aβ42 indicated that cells transfected with 3M/APP cDNA excreted mostly Aβ40 peptide, whereas cells transfected with WT/APP cDNA excreted a mixture of Aβ40 and 42 peptides after 24 h (unpublished data). In cells transfected with 3M/APP, however, no significant mitochondrial accumulation of APP was observed ( E), though the level of PM-associated APP and the secreted Aβ peptide () increased with time. Furthermore, cells transfected with Δ220–290/APP showed a time-dependent increase in the accumulation of mutant protein in both the mitochondrial ( I) and PM ( J) compartments. This coincided with the increased secretion of total Aβ in the culture fluid (CF) ( K). As the Golgi network has been shown to be an important cellular site of Aβ production (Greenfield et al., 1999
), we examined the level of the Aβ peptide in the purified Golgi fraction of transfected cells. It is seen that by 48 h of transfection, there was an increased accumulation of the peptide in the Golgi apparatus of cells transfected with WT/APP ( D). Surprisingly, accumulation of processed 4-kD Aβ peptide in the Golgi apparatus did not occur in cells transfected with 3M/APP and Δ220–290/APP constructs (). We also tested the intracellular distribution of the Swedish mutant of APP (SW/APP), which is implicated in familial AD (Selkoe, 1999
). (M–P) shows that the pattern of accumulation of APP in mitochondria and the PM and the level of secreted Aβ in the extracellular compartment are nearly similar to those with WT/APP. The only difference is the time frame of accumulation of 4-kD Aβ peptide in the Golgi apparatus, which occurs at 24 h in the case of SW/APP and 48 h in the case of WT/APP. These results show that the levels of mitochondrial targeting of WT/APP and SW/APP are nearly similar.
Figure 5. Subcellular distribution of ectopically expressed APPs in HCN-1A cells. Cells were transfected with WT/APP (A–D), 3M/APP (E–H), Δ220–290/APP (I–L), and SW/APP (M–P). At indicated times after transfection, (more ...)
Subcellular distribution of APP in cells transfected with WT/APP and SW/APP constructs
Total cell extracts from companion cells, as in (A, E, I, and M), were subjected to immunoblot analysis to determine the level of expression of WT and mutant forms of APP at various time points of transfection. Results in Q show that the levels of expression of WT, 3M/APP, and Δ220–290/APP at all time points were nearly comparable with an ~10–20% increase by 48 and 96 h as compared with the 24 h time point. These results suggest that the observed differences in the distribution of APP and Aβ peptide levels in different cell compartments were not due to vastly variable levels of transfection or expression.
The reason for the contrasting levels of APP in the mitochondrial and PM fractions at longer time intervals was verified by pulse chase experiments. After 24 h of transfection with WT/APP cDNA, cells were labeled for 2 h with [35S]Met and chased for various time periods up to 16 h ( R) by growing in a normal medium. APP protein from the mitochondrial and PM fractions at each time point was immunoprecipitated and resolved on an SDS gel, and the radioactivity was quantified. R shows that the radioactivity in the mitochondrial-associated APP increased steadily up to 16 h of chase, whereas the radioactivity in the PM fraction declined steadily. The steady-state APP levels in the mitochondrial and PM fractions, as indicated in the immunoblot at the top, essentially concur with the pulse chase pattern. These results show that the pools of APP from the mitochondria and PM are turned over quite differently and provide a rational basis for the observed differences in the steady-state levels of APP in the two compartments at 48 and 96 h after transfection.
Accumulation of transmembrane-arrested APP disrupts mitochondrial functions
To understand the patho-physiological relevance of the mitochondrial accumulation of APP in a transmembrane-arrested orientation, we assessed mitochondrial functional parameters. A time-dependent accumulation of APP in the mitochondria of cells transfected with WT/APP was accompanied by a decline in the CytOX activity (
A), markedly reduced respiration-coupled (mitochondrially generated) ATP synthesis ( B), a decline in total cellular ATP levels ( C), and disruption of mitochondrial transmembrane potential ( D). These functional parameters were progressively reduced to 50–80% of control cells transfected with vector DNA alone (, A–D). Transfection with 3M/APP (which showed no detectable mitochondrial accumulation) and Δ220–290/APP (which did not cause translocational arrest), however, showed no effect on the cellular or mitochondrial ATP pools, CytOX activity, or transmembrane potential (, A–D). Transfection with SW/APP with intact NH2-terminal signal sequence and the acidic domain affected mitochondrial functional parameters at levels similar to that with WT/APP. These results show, for the first time, the progressive nature of mitochondrial accumulation of transmembrane-arrested APP and its adverse effects on energy production in cells overexpressing the protein.
Figure 6. Effects of transmembrane-arrested APP on mitochondrial functions. Total cell extracts or mitochondria from HCN-1A cells transfected with WT/APP, 3M/APP, Δ220–290/APP, and SW/APP were analyzed for CytOX activity (A), mitochondria and total (more ...)
Transmembrane-arrested APP and impaired energy metabolism in brain mitochondria from a mouse model for AD
AD-like plaque pathology was uniformly observed in transgenic mouse models expressing higher levels of human APP protein (for review see Janus and Westaway, 2001
). To understand the physiological significance of our results with transfected cells, we analyzed the subcellular distribution of APP in the brains of transgenic mice overexpressing SW/APP (2576). PM and mitochondria from cortex, hippocampus, and cerebellum regions of brains from 12-mo-old amyloid plaque–bearing transgenic mice were isolated and compared with similar regions of age-matched controls. The immunoblot in
A (bottom) shows that the antibody-reactive APP levels in the PM from the cortex, hippocampus, and cerebellar regions of brains from control and transgenic mice were nearly comparable. But the mitochondrial fractions of corresponding mouse brain regions of transgenic mice showed markedly elevated APP compared with counterparts from control brains ( A, top). The cortex and hippocampal regions of transgenic mice contained considerably higher levels of mitochondrial-associated APP compared with the cerebellum. Furthermore, trypsin treatment of mitochondria from different brain regions of transgenic mice yielded a 22-kD protected fragment of APP ( B). These results suggest a transmembrane-arrested topology of mitochondrial-associated APP in transgenic mouse brain similar to that observed in HCN-1A cells transfected with WT/APP cDNA. Increased mitochondrial association of APP in the transgenic mouse model was accompanied by a 20–40% decrease in the CytOX activity ( C) and a 25–50% reduction in total cellular ATP levels ( D) in the cortex and hippocampus. The cerebellar region with a relatively lower level of mitochondrial-associated APP showed a less severe inhibition of CytOX activity ( C) and ATP pool ( D). These results confirm that in both APP-expressing HCN-1A cells and mouse brain, mitochondria are the direct targets of this pathogenic protein.
Figure 7. Mitochondrial-associated APP in the brains of transgenic mice overexpressing APP. (A) Mitochondria and PM fractions from different brain regions of control and SWEAPP (2576) transgenic mice (50 μg protein each) were subjected to immunoblot analysis (more ...)