Limits...
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.

Show MeSH

Related in: MedlinePlus

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, mitochondria (MITO.), PM, and Golgi fractions (50 μg protein each) and also protein concentrate from 10 ml of cell-free culture medium (CM) were subjected to Western immunoblot analysis using antibodies to APP Nt (A, B, E, F, I, J, M, and N) or Aβ (C, D, G, H, K, L, O, and P). (Q) The level of expression of total APP protein in cells transfected with various cDNA constructs for different time points was monitored by immunoblot analysis of whole cell lysates. The antibody-reactive proteins were quantified using a Bio-Rad Laboratories Fluoro S imaging system. Table I shows the distribution of APP in different membrane fractions at different time points after transfection with WT/APP and SW/APP cDNAs. Values at the bottom of Q show relative APP levels in comparison with the level at 24-h transfection that was considered to be 1. (R) Pulse chase characteristics of mitochondrial- and PM-associated APP. HCN-1A cells transfected with WT/APP cDNA for 24 h were labeled with [35S]Met for 2 h, followed by a chase in normal growth medium containing 3 mM unlabeled Met. Mitochondrial and PM proteins from cells at different points of chase (500 μg each) were immunoprecipitated with APP Nt Ab, analyzed by electrophoresis on 10% SDS-polyacrylamide gels, and imaged through a Bio-Rad Laboratories GS525 molecular imager. The immunoblot at the top of the figure was performed using 100 μg protein from each fraction using APP Nt Ab.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172865&req=5

fig5: 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, mitochondria (MITO.), PM, and Golgi fractions (50 μg protein each) and also protein concentrate from 10 ml of cell-free culture medium (CM) were subjected to Western immunoblot analysis using antibodies to APP Nt (A, B, E, F, I, J, M, and N) or Aβ (C, D, G, H, K, L, O, and P). (Q) The level of expression of total APP protein in cells transfected with various cDNA constructs for different time points was monitored by immunoblot analysis of whole cell lysates. The antibody-reactive proteins were quantified using a Bio-Rad Laboratories Fluoro S imaging system. Table I shows the distribution of APP in different membrane fractions at different time points after transfection with WT/APP and SW/APP cDNAs. Values at the bottom of Q show relative APP levels in comparison with the level at 24-h transfection that was considered to be 1. (R) Pulse chase characteristics of mitochondrial- and PM-associated APP. HCN-1A cells transfected with WT/APP cDNA for 24 h were labeled with [35S]Met for 2 h, followed by a chase in normal growth medium containing 3 mM unlabeled Met. Mitochondrial and PM proteins from cells at different points of chase (500 μg each) were immunoprecipitated with APP Nt Ab, analyzed by electrophoresis on 10% SDS-polyacrylamide gels, and imaged through a Bio-Rad Laboratories GS525 molecular imager. The immunoblot at the top of the figure was performed using 100 μg protein from each fraction using APP Nt Ab.

Mentions: 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 (Fig. 5 ; Table I). Immunoblot in Fig. 5 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 (Fig. 5 B). Notably, the level of secreted Aβ pool (Aβ40, 42, and 43) in the culture fluid (CF) declined steadily (Fig. 5 C), in parallel to declining APP in the PM fraction (Fig. 5 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 (Fig. 5 E), though the level of PM-associated APP and the secreted Aβ peptide (Fig. 5, F and G) 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 (Fig. 5 I) and PM (Fig. 5 J) compartments. This coincided with the increased secretion of total Aβ in the culture fluid (CF) (Fig. 5 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 (Fig. 5 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 (Fig. 5, H and L). We also tested the intracellular distribution of the Swedish mutant of APP (SW/APP), which is implicated in familial AD (Selkoe, 1999). Fig. 5 (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.


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 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, mitochondria (MITO.), PM, and Golgi fractions (50 μg protein each) and also protein concentrate from 10 ml of cell-free culture medium (CM) were subjected to Western immunoblot analysis using antibodies to APP Nt (A, B, E, F, I, J, M, and N) or Aβ (C, D, G, H, K, L, O, and P). (Q) The level of expression of total APP protein in cells transfected with various cDNA constructs for different time points was monitored by immunoblot analysis of whole cell lysates. The antibody-reactive proteins were quantified using a Bio-Rad Laboratories Fluoro S imaging system. Table I shows the distribution of APP in different membrane fractions at different time points after transfection with WT/APP and SW/APP cDNAs. Values at the bottom of Q show relative APP levels in comparison with the level at 24-h transfection that was considered to be 1. (R) Pulse chase characteristics of mitochondrial- and PM-associated APP. HCN-1A cells transfected with WT/APP cDNA for 24 h were labeled with [35S]Met for 2 h, followed by a chase in normal growth medium containing 3 mM unlabeled Met. Mitochondrial and PM proteins from cells at different points of chase (500 μg each) were immunoprecipitated with APP Nt Ab, analyzed by electrophoresis on 10% SDS-polyacrylamide gels, and imaged through a Bio-Rad Laboratories GS525 molecular imager. The immunoblot at the top of the figure was performed using 100 μg protein from each fraction using APP Nt Ab.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172865&req=5

fig5: 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, mitochondria (MITO.), PM, and Golgi fractions (50 μg protein each) and also protein concentrate from 10 ml of cell-free culture medium (CM) were subjected to Western immunoblot analysis using antibodies to APP Nt (A, B, E, F, I, J, M, and N) or Aβ (C, D, G, H, K, L, O, and P). (Q) The level of expression of total APP protein in cells transfected with various cDNA constructs for different time points was monitored by immunoblot analysis of whole cell lysates. The antibody-reactive proteins were quantified using a Bio-Rad Laboratories Fluoro S imaging system. Table I shows the distribution of APP in different membrane fractions at different time points after transfection with WT/APP and SW/APP cDNAs. Values at the bottom of Q show relative APP levels in comparison with the level at 24-h transfection that was considered to be 1. (R) Pulse chase characteristics of mitochondrial- and PM-associated APP. HCN-1A cells transfected with WT/APP cDNA for 24 h were labeled with [35S]Met for 2 h, followed by a chase in normal growth medium containing 3 mM unlabeled Met. Mitochondrial and PM proteins from cells at different points of chase (500 μg each) were immunoprecipitated with APP Nt Ab, analyzed by electrophoresis on 10% SDS-polyacrylamide gels, and imaged through a Bio-Rad Laboratories GS525 molecular imager. The immunoblot at the top of the figure was performed using 100 μg protein from each fraction using APP Nt Ab.
Mentions: 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 (Fig. 5 ; Table I). Immunoblot in Fig. 5 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 (Fig. 5 B). Notably, the level of secreted Aβ pool (Aβ40, 42, and 43) in the culture fluid (CF) declined steadily (Fig. 5 C), in parallel to declining APP in the PM fraction (Fig. 5 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 (Fig. 5 E), though the level of PM-associated APP and the secreted Aβ peptide (Fig. 5, F and G) 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 (Fig. 5 I) and PM (Fig. 5 J) compartments. This coincided with the increased secretion of total Aβ in the culture fluid (CF) (Fig. 5 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 (Fig. 5 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 (Fig. 5, H and L). We also tested the intracellular distribution of the Swedish mutant of APP (SW/APP), which is implicated in familial AD (Selkoe, 1999). Fig. 5 (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.

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