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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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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. Sequence 36–61 with three positively charged residues (at positions 40, 44, and 51), the predicted mitochondrial-targeting sequence, and the mutant construct 3M/APP carrying mutations at these positions are shown. (B) Immunoblot analysis of marker proteins for different subcellular fractions (50 μg protein each) using antibodies to Na+/K+ ATPase, TOM40, calreticulin, βCOP, and p97. The bottom panel represents 200 μg protein from each membrane fraction and was developed with APP Nt Ab. (C) Northern blot analysis of total RNA (25 μg RNA each) from HCN cells treated with PMA for different time intervals. Hybridization with 18S DNA probe served as a loading control. (D) Western blot analysis of mitochondria and PM proteins (200 μg protein each) from HCN cells treated with PMA for different time intervals using APP Nt Ab. (E) Measurement of reduction of MTT dye by freshly isolated mitochondria from HCN-1A cells treated with PMA for various time points was performed as described in the Materials and methods. (F) Immunoblot analysis of glycosidase-treated proteins (200 μg each) from PMA (100 nM)-induced HCN cells using APP Nt Ab. Treatment with glycosidases was performed as described in the Materials and methods. Mito, mitochondria.
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fig1: 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. Sequence 36–61 with three positively charged residues (at positions 40, 44, and 51), the predicted mitochondrial-targeting sequence, and the mutant construct 3M/APP carrying mutations at these positions are shown. (B) Immunoblot analysis of marker proteins for different subcellular fractions (50 μg protein each) using antibodies to Na+/K+ ATPase, TOM40, calreticulin, βCOP, and p97. The bottom panel represents 200 μg protein from each membrane fraction and was developed with APP Nt Ab. (C) Northern blot analysis of total RNA (25 μg RNA each) from HCN cells treated with PMA for different time intervals. Hybridization with 18S DNA probe served as a loading control. (D) Western blot analysis of mitochondria and PM proteins (200 μg protein each) from HCN cells treated with PMA for different time intervals using APP Nt Ab. (E) Measurement of reduction of MTT dye by freshly isolated mitochondria from HCN-1A cells treated with PMA for various time points was performed as described in the Materials and methods. (F) Immunoblot analysis of glycosidase-treated proteins (200 μg each) from PMA (100 nM)-induced HCN cells using APP Nt Ab. Treatment with glycosidases was performed as described in the Materials and methods. Mito, mitochondria.

Mentions: A comparison of NH2-terminal chimeric sequences of APP with those of P4501A1 and P4502B1 is shown in Fig. 1 A. The NH2-terminal 38–amino acid region of APP with a hydrophobic helical structure functions as the ER-targeting domain (Fig. 1 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 (Fig. 1 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).


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)

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. Sequence 36–61 with three positively charged residues (at positions 40, 44, and 51), the predicted mitochondrial-targeting sequence, and the mutant construct 3M/APP carrying mutations at these positions are shown. (B) Immunoblot analysis of marker proteins for different subcellular fractions (50 μg protein each) using antibodies to Na+/K+ ATPase, TOM40, calreticulin, βCOP, and p97. The bottom panel represents 200 μg protein from each membrane fraction and was developed with APP Nt Ab. (C) Northern blot analysis of total RNA (25 μg RNA each) from HCN cells treated with PMA for different time intervals. Hybridization with 18S DNA probe served as a loading control. (D) Western blot analysis of mitochondria and PM proteins (200 μg protein each) from HCN cells treated with PMA for different time intervals using APP Nt Ab. (E) Measurement of reduction of MTT dye by freshly isolated mitochondria from HCN-1A cells treated with PMA for various time points was performed as described in the Materials and methods. (F) Immunoblot analysis of glycosidase-treated proteins (200 μg each) from PMA (100 nM)-induced HCN cells using APP Nt Ab. Treatment with glycosidases was performed as described in the Materials and methods. Mito, mitochondria.
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Related In: Results  -  Collection

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fig1: 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. Sequence 36–61 with three positively charged residues (at positions 40, 44, and 51), the predicted mitochondrial-targeting sequence, and the mutant construct 3M/APP carrying mutations at these positions are shown. (B) Immunoblot analysis of marker proteins for different subcellular fractions (50 μg protein each) using antibodies to Na+/K+ ATPase, TOM40, calreticulin, βCOP, and p97. The bottom panel represents 200 μg protein from each membrane fraction and was developed with APP Nt Ab. (C) Northern blot analysis of total RNA (25 μg RNA each) from HCN cells treated with PMA for different time intervals. Hybridization with 18S DNA probe served as a loading control. (D) Western blot analysis of mitochondria and PM proteins (200 μg protein each) from HCN cells treated with PMA for different time intervals using APP Nt Ab. (E) Measurement of reduction of MTT dye by freshly isolated mitochondria from HCN-1A cells treated with PMA for various time points was performed as described in the Materials and methods. (F) Immunoblot analysis of glycosidase-treated proteins (200 μg each) from PMA (100 nM)-induced HCN cells using APP Nt Ab. Treatment with glycosidases was performed as described in the Materials and methods. Mito, mitochondria.
Mentions: A comparison of NH2-terminal chimeric sequences of APP with those of P4501A1 and P4502B1 is shown in Fig. 1 A. The NH2-terminal 38–amino acid region of APP with a hydrophobic helical structure functions as the ER-targeting domain (Fig. 1 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 (Fig. 1 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).

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