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Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells.

Anandatheerthavarada HK, Biswas G, Robin MA, Avadhani NG - J. Cell Biol. (2003)

Bottom Line: Mutational studies show that the acidic domain, which spans sequence 220-290 of APP, causes the transmembrane arrest with the COOH-terminal 73-kD portion of the protein facing the cytoplasmic side.Accumulation of full-length APP in the mitochondrial compartment in a transmembrane-arrested form, but not lacking the acidic domain, caused mitochondrial dysfunction and impaired energy metabolism.These results show, for the first time, that APP is targeted to neuronal mitochondria under some physiological and pathological conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Alzheimer's amyloid precursor protein 695 (APP) is a plasma membrane protein, which is known to be the source of the toxic amyloid beta (Abeta) peptide associated with the pathogenesis of Alzheimer's disease (AD). Here we demonstrate that by virtue of its chimeric NH2-terminal signal, APP is also targeted to mitochondria of cortical neuronal cells and select regions of the brain of a transgenic mouse model for AD. The positively charged residues at 40, 44, and 51 of APP are critical components of the mitochondrial-targeting signal. Chemical cross-linking together with immunoelectron microscopy show that the mitochondrial APP exists in NH2-terminal inside transmembrane orientation and in contact with mitochondrial translocase proteins. Mutational studies show that the acidic domain, which spans sequence 220-290 of APP, causes the transmembrane arrest with the COOH-terminal 73-kD portion of the protein facing the cytoplasmic side. Accumulation of full-length APP in the mitochondrial compartment in a transmembrane-arrested form, but not lacking the acidic domain, caused mitochondrial dysfunction and impaired energy metabolism. These results show, for the first time, that APP is targeted to neuronal mitochondria under some physiological and pathological conditions.

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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 described in the Materials and methods using 250 μg trypsin/ml of reaction (+). (A, lanes 4–6) Mitochondria were preincubated with or without added inhibitors (50 μM CCCP or 50 μM 2,4 DNP) at 25°C for 10 min before initiating the in vitro transport. (C) Mitochondria were treated with 0.1% Triton X-100 before treatment with trypsin. In each case, 200 μg of mitochondrial protein was used for electrophoresis, and the gels were subjected to fluorography. (D–G) In vivo targeting of WT/APP, 3M/APP, and Δ220–290/APP. HCN-1A cells were transfected with WT/APP (D and G), 3M/APP (E), and Δ220–290/APP (F) cDNA constructs, and PM and mitochondrial fractions were probed with APP Ct Ab (D–F) and APP Nt Ab (G). Trypsin treatment was performed as in A. Treatment with digitonin and extraction with 0.1 M Na2CO3 (D, lanes 9 and 10) were performed as described in the Materials and methods. P, pellet; S, supernatant.
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fig2: 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 described in the Materials and methods using 250 μg trypsin/ml of reaction (+). (A, lanes 4–6) Mitochondria were preincubated with or without added inhibitors (50 μM CCCP or 50 μM 2,4 DNP) at 25°C for 10 min before initiating the in vitro transport. (C) Mitochondria were treated with 0.1% Triton X-100 before treatment with trypsin. In each case, 200 μg of mitochondrial protein was used for electrophoresis, and the gels were subjected to fluorography. (D–G) In vivo targeting of WT/APP, 3M/APP, and Δ220–290/APP. HCN-1A cells were transfected with WT/APP (D and G), 3M/APP (E), and Δ220–290/APP (F) cDNA constructs, and PM and mitochondrial fractions were probed with APP Ct Ab (D–F) and APP Nt Ab (G). Trypsin treatment was performed as in A. Treatment with digitonin and extraction with 0.1 M Na2CO3 (D, lanes 9 and 10) were performed as described in the Materials and methods. P, pellet; S, supernatant.

Mentions: 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. Fig. 2 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 (Fig. 2 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 (Fig. 2 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 (Fig. 2 A, lanes 3–8). Furthermore, 3M/APP with mutated positive residues at +40, +44, and +51, as shown in Fig. 1 A, was not imported significantly (Fig. 2 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 (Fig. 1 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 (Fig. 2 A, lanes 11 and 12). Control experiments in Fig. 2 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.


Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells.

Anandatheerthavarada HK, Biswas G, Robin MA, Avadhani NG - J. Cell Biol. (2003)

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 described in the Materials and methods using 250 μg trypsin/ml of reaction (+). (A, lanes 4–6) Mitochondria were preincubated with or without added inhibitors (50 μM CCCP or 50 μM 2,4 DNP) at 25°C for 10 min before initiating the in vitro transport. (C) Mitochondria were treated with 0.1% Triton X-100 before treatment with trypsin. In each case, 200 μg of mitochondrial protein was used for electrophoresis, and the gels were subjected to fluorography. (D–G) In vivo targeting of WT/APP, 3M/APP, and Δ220–290/APP. HCN-1A cells were transfected with WT/APP (D and G), 3M/APP (E), and Δ220–290/APP (F) cDNA constructs, and PM and mitochondrial fractions were probed with APP Ct Ab (D–F) and APP Nt Ab (G). Trypsin treatment was performed as in A. Treatment with digitonin and extraction with 0.1 M Na2CO3 (D, lanes 9 and 10) were performed as described in the Materials and methods. P, pellet; S, supernatant.
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Related In: Results  -  Collection

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fig2: 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 described in the Materials and methods using 250 μg trypsin/ml of reaction (+). (A, lanes 4–6) Mitochondria were preincubated with or without added inhibitors (50 μM CCCP or 50 μM 2,4 DNP) at 25°C for 10 min before initiating the in vitro transport. (C) Mitochondria were treated with 0.1% Triton X-100 before treatment with trypsin. In each case, 200 μg of mitochondrial protein was used for electrophoresis, and the gels were subjected to fluorography. (D–G) In vivo targeting of WT/APP, 3M/APP, and Δ220–290/APP. HCN-1A cells were transfected with WT/APP (D and G), 3M/APP (E), and Δ220–290/APP (F) cDNA constructs, and PM and mitochondrial fractions were probed with APP Ct Ab (D–F) and APP Nt Ab (G). Trypsin treatment was performed as in A. Treatment with digitonin and extraction with 0.1 M Na2CO3 (D, lanes 9 and 10) were performed as described in the Materials and methods. P, pellet; S, supernatant.
Mentions: 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. Fig. 2 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 (Fig. 2 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 (Fig. 2 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 (Fig. 2 A, lanes 3–8). Furthermore, 3M/APP with mutated positive residues at +40, +44, and +51, as shown in Fig. 1 A, was not imported significantly (Fig. 2 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 (Fig. 1 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 (Fig. 2 A, lanes 11 and 12). Control experiments in Fig. 2 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.

Bottom Line: Mutational studies show that the acidic domain, which spans sequence 220-290 of APP, causes the transmembrane arrest with the COOH-terminal 73-kD portion of the protein facing the cytoplasmic side.Accumulation of full-length APP in the mitochondrial compartment in a transmembrane-arrested form, but not lacking the acidic domain, caused mitochondrial dysfunction and impaired energy metabolism.These results show, for the first time, that APP is targeted to neuronal mitochondria under some physiological and pathological conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Alzheimer's amyloid precursor protein 695 (APP) is a plasma membrane protein, which is known to be the source of the toxic amyloid beta (Abeta) peptide associated with the pathogenesis of Alzheimer's disease (AD). Here we demonstrate that by virtue of its chimeric NH2-terminal signal, APP is also targeted to mitochondria of cortical neuronal cells and select regions of the brain of a transgenic mouse model for AD. The positively charged residues at 40, 44, and 51 of APP are critical components of the mitochondrial-targeting signal. Chemical cross-linking together with immunoelectron microscopy show that the mitochondrial APP exists in NH2-terminal inside transmembrane orientation and in contact with mitochondrial translocase proteins. Mutational studies show that the acidic domain, which spans sequence 220-290 of APP, causes the transmembrane arrest with the COOH-terminal 73-kD portion of the protein facing the cytoplasmic side. Accumulation of full-length APP in the mitochondrial compartment in a transmembrane-arrested form, but not lacking the acidic domain, caused mitochondrial dysfunction and impaired energy metabolism. These results show, for the first time, that APP is targeted to neuronal mitochondria under some physiological and pathological conditions.

Show MeSH
Related in: MedlinePlus