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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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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 cell ATP generation (B and C, respectively), and changes in the mitochondrial membrane potential using MitoTracker orange CM-H2TM ROS (D), as described in the Materials and methods. Mitochondrial CytOX activity (2 nmol of cytochrome c oxidized/min/mg mitochondrial protein) from vector alone–transfected cells was used as 100% activity. Values represent mean ± SEM from three separate transfection experiments.
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fig6: 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 cell ATP generation (B and C, respectively), and changes in the mitochondrial membrane potential using MitoTracker orange CM-H2TM ROS (D), as described in the Materials and methods. Mitochondrial CytOX activity (2 nmol of cytochrome c oxidized/min/mg mitochondrial protein) from vector alone–transfected cells was used as 100% activity. Values represent mean ± SEM from three separate transfection experiments.

Mentions: 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 (Fig. 6 A), markedly reduced respiration-coupled (mitochondrially generated) ATP synthesis (Fig. 6 B), a decline in total cellular ATP levels (Fig. 6 C), and disruption of mitochondrial transmembrane potential (Fig. 6 D). These functional parameters were progressively reduced to 50–80% of control cells transfected with vector DNA alone (Fig. 6, 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 (Figs. 6, 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.


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)

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 cell ATP generation (B and C, respectively), and changes in the mitochondrial membrane potential using MitoTracker orange CM-H2TM ROS (D), as described in the Materials and methods. Mitochondrial CytOX activity (2 nmol of cytochrome c oxidized/min/mg mitochondrial protein) from vector alone–transfected cells was used as 100% activity. Values represent mean ± SEM from three separate transfection experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: 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 cell ATP generation (B and C, respectively), and changes in the mitochondrial membrane potential using MitoTracker orange CM-H2TM ROS (D), as described in the Materials and methods. Mitochondrial CytOX activity (2 nmol of cytochrome c oxidized/min/mg mitochondrial protein) from vector alone–transfected cells was used as 100% activity. Values represent mean ± SEM from three separate transfection experiments.
Mentions: 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 (Fig. 6 A), markedly reduced respiration-coupled (mitochondrially generated) ATP synthesis (Fig. 6 B), a decline in total cellular ATP levels (Fig. 6 C), and disruption of mitochondrial transmembrane potential (Fig. 6 D). These functional parameters were progressively reduced to 50–80% of control cells transfected with vector DNA alone (Fig. 6, 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 (Figs. 6, 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.

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