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

Mitochondrial-associated APP in the brains of transgenic mice overexpressing APP. (A) Mitochondria and PM fractions from different brain regions of control and SWEAPP (2576) transgenic mice (50 μg protein each) were subjected to immunoblot analysis using APP Nt Ab. (B) Mitochondria (50 μg) from different brain regions of transgenic mice overexpressing SW/APP (SWEAPP [2576]) were subjected to trypsin treatment (as described in Fig. 2) followed by immunoblot analysis with APP Nt Ab. (C) CytOX activity in total mitochondrial membrane fraction and (D) total cellular ATP levels were assayed as described in the Materials and methods. MITO., mitochondria; Ctl, control; Tg, transgenic; Ct, cortex; Hp, hippocampus; Ce, cerebellum.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172865&req=5

fig7: Mitochondrial-associated APP in the brains of transgenic mice overexpressing APP. (A) Mitochondria and PM fractions from different brain regions of control and SWEAPP (2576) transgenic mice (50 μg protein each) were subjected to immunoblot analysis using APP Nt Ab. (B) Mitochondria (50 μg) from different brain regions of transgenic mice overexpressing SW/APP (SWEAPP [2576]) were subjected to trypsin treatment (as described in Fig. 2) followed by immunoblot analysis with APP Nt Ab. (C) CytOX activity in total mitochondrial membrane fraction and (D) total cellular ATP levels were assayed as described in the Materials and methods. MITO., mitochondria; Ctl, control; Tg, transgenic; Ct, cortex; Hp, hippocampus; Ce, cerebellum.

Mentions: AD-like plaque pathology was uniformly observed in transgenic mouse models expressing higher levels of human APP protein (for review see Janus and Westaway, 2001). To understand the physiological significance of our results with transfected cells, we analyzed the subcellular distribution of APP in the brains of transgenic mice overexpressing SW/APP (2576). PM and mitochondria from cortex, hippocampus, and cerebellum regions of brains from 12-mo-old amyloid plaque–bearing transgenic mice were isolated and compared with similar regions of age-matched controls. The immunoblot in Fig. 7 A (bottom) shows that the antibody-reactive APP levels in the PM from the cortex, hippocampus, and cerebellar regions of brains from control and transgenic mice were nearly comparable. But the mitochondrial fractions of corresponding mouse brain regions of transgenic mice showed markedly elevated APP compared with counterparts from control brains (Fig. 7 A, top). The cortex and hippocampal regions of transgenic mice contained considerably higher levels of mitochondrial-associated APP compared with the cerebellum. Furthermore, trypsin treatment of mitochondria from different brain regions of transgenic mice yielded a 22-kD protected fragment of APP (Fig. 7 B). These results suggest a transmembrane-arrested topology of mitochondrial-associated APP in transgenic mouse brain similar to that observed in HCN-1A cells transfected with WT/APP cDNA. Increased mitochondrial association of APP in the transgenic mouse model was accompanied by a 20–40% decrease in the CytOX activity (Fig. 7 C) and a 25–50% reduction in total cellular ATP levels (Fig. 7 D) in the cortex and hippocampus. The cerebellar region with a relatively lower level of mitochondrial-associated APP showed a less severe inhibition of CytOX activity (Fig. 7 C) and ATP pool (Fig. 7 D). These results confirm that in both APP-expressing HCN-1A cells and mouse brain, mitochondria are the direct targets of this pathogenic 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)

Mitochondrial-associated APP in the brains of transgenic mice overexpressing APP. (A) Mitochondria and PM fractions from different brain regions of control and SWEAPP (2576) transgenic mice (50 μg protein each) were subjected to immunoblot analysis using APP Nt Ab. (B) Mitochondria (50 μg) from different brain regions of transgenic mice overexpressing SW/APP (SWEAPP [2576]) were subjected to trypsin treatment (as described in Fig. 2) followed by immunoblot analysis with APP Nt Ab. (C) CytOX activity in total mitochondrial membrane fraction and (D) total cellular ATP levels were assayed as described in the Materials and methods. MITO., mitochondria; Ctl, control; Tg, transgenic; Ct, cortex; Hp, hippocampus; Ce, cerebellum.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Mitochondrial-associated APP in the brains of transgenic mice overexpressing APP. (A) Mitochondria and PM fractions from different brain regions of control and SWEAPP (2576) transgenic mice (50 μg protein each) were subjected to immunoblot analysis using APP Nt Ab. (B) Mitochondria (50 μg) from different brain regions of transgenic mice overexpressing SW/APP (SWEAPP [2576]) were subjected to trypsin treatment (as described in Fig. 2) followed by immunoblot analysis with APP Nt Ab. (C) CytOX activity in total mitochondrial membrane fraction and (D) total cellular ATP levels were assayed as described in the Materials and methods. MITO., mitochondria; Ctl, control; Tg, transgenic; Ct, cortex; Hp, hippocampus; Ce, cerebellum.
Mentions: AD-like plaque pathology was uniformly observed in transgenic mouse models expressing higher levels of human APP protein (for review see Janus and Westaway, 2001). To understand the physiological significance of our results with transfected cells, we analyzed the subcellular distribution of APP in the brains of transgenic mice overexpressing SW/APP (2576). PM and mitochondria from cortex, hippocampus, and cerebellum regions of brains from 12-mo-old amyloid plaque–bearing transgenic mice were isolated and compared with similar regions of age-matched controls. The immunoblot in Fig. 7 A (bottom) shows that the antibody-reactive APP levels in the PM from the cortex, hippocampus, and cerebellar regions of brains from control and transgenic mice were nearly comparable. But the mitochondrial fractions of corresponding mouse brain regions of transgenic mice showed markedly elevated APP compared with counterparts from control brains (Fig. 7 A, top). The cortex and hippocampal regions of transgenic mice contained considerably higher levels of mitochondrial-associated APP compared with the cerebellum. Furthermore, trypsin treatment of mitochondria from different brain regions of transgenic mice yielded a 22-kD protected fragment of APP (Fig. 7 B). These results suggest a transmembrane-arrested topology of mitochondrial-associated APP in transgenic mouse brain similar to that observed in HCN-1A cells transfected with WT/APP cDNA. Increased mitochondrial association of APP in the transgenic mouse model was accompanied by a 20–40% decrease in the CytOX activity (Fig. 7 C) and a 25–50% reduction in total cellular ATP levels (Fig. 7 D) in the cortex and hippocampus. The cerebellar region with a relatively lower level of mitochondrial-associated APP showed a less severe inhibition of CytOX activity (Fig. 7 C) and ATP pool (Fig. 7 D). These results confirm that in both APP-expressing HCN-1A cells and mouse brain, mitochondria are the direct targets of this pathogenic 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