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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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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 showing APP Ab–stained granular structures both overlapping and nonoverlapping with mitochondrial stain, as indicated by arrows. Nonpermeabilized cells (A–C, G–I, and M–O) were double immunostained with APP Nt Ab and monoclonal antibody to Na+/ K+ ATPase. Permeabilized cells (D–F, J–L, and P–R) were double stained with APP Nt Ab and rabbit polyclonal antibodies to TOM40. Staining patterns (A, B, D, E, G, H, J, K, M, N, P, and Q) were developed with appropriate secondary antibodies conjugated to Alexa dyes. (C, F, I, L, O, and R) Respective overlay patterns.
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fig3: 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 showing APP Ab–stained granular structures both overlapping and nonoverlapping with mitochondrial stain, as indicated by arrows. Nonpermeabilized cells (A–C, G–I, and M–O) were double immunostained with APP Nt Ab and monoclonal antibody to Na+/ K+ ATPase. Permeabilized cells (D–F, J–L, and P–R) were double stained with APP Nt Ab and rabbit polyclonal antibodies to TOM40. Staining patterns (A, B, D, E, G, H, J, K, M, N, P, and Q) were developed with appropriate secondary antibodies conjugated to Alexa dyes. (C, F, I, L, O, and R) Respective overlay patterns.

Mentions: 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 (Fig. 3 A), which colocalized with Na+/K+ ATPase (Fig. 3, B and C). In permeabilized cells, the APP Nt Ab stained extranuclear granulate structures (Fig. 3 D), some of which colocalized with mitochondrial-specific marker TOM40 (Fig. 3, E and F). The inset in Fig. 3 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 (Fig. 3, G–I) and also some intracellular staining that did not colocalize with TOM40 stains (Fig. 3, 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.


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)

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 showing APP Ab–stained granular structures both overlapping and nonoverlapping with mitochondrial stain, as indicated by arrows. Nonpermeabilized cells (A–C, G–I, and M–O) were double immunostained with APP Nt Ab and monoclonal antibody to Na+/ K+ ATPase. Permeabilized cells (D–F, J–L, and P–R) were double stained with APP Nt Ab and rabbit polyclonal antibodies to TOM40. Staining patterns (A, B, D, E, G, H, J, K, M, N, P, and Q) were developed with appropriate secondary antibodies conjugated to Alexa dyes. (C, F, I, L, O, and R) Respective overlay patterns.
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Related In: Results  -  Collection

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fig3: 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 showing APP Ab–stained granular structures both overlapping and nonoverlapping with mitochondrial stain, as indicated by arrows. Nonpermeabilized cells (A–C, G–I, and M–O) were double immunostained with APP Nt Ab and monoclonal antibody to Na+/ K+ ATPase. Permeabilized cells (D–F, J–L, and P–R) were double stained with APP Nt Ab and rabbit polyclonal antibodies to TOM40. Staining patterns (A, B, D, E, G, H, J, K, M, N, P, and Q) were developed with appropriate secondary antibodies conjugated to Alexa dyes. (C, F, I, L, O, and R) Respective overlay patterns.
Mentions: 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 (Fig. 3 A), which colocalized with Na+/K+ ATPase (Fig. 3, B and C). In permeabilized cells, the APP Nt Ab stained extranuclear granulate structures (Fig. 3 D), some of which colocalized with mitochondrial-specific marker TOM40 (Fig. 3, E and F). The inset in Fig. 3 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 (Fig. 3, G–I) and also some intracellular staining that did not colocalize with TOM40 stains (Fig. 3, 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.

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