title: Mitochondria are devoid of amyloid β-protein (Aβ)-producing secretases: Evidence for unlikely occurrence within mitochondria of Aβ generation from amyloid precursor protein.
abstract: Mitochondrial dysfunction is implicated in the pathological mechanism of Alzheimer's disease (AD). Amyloid β-protein (Aβ), which plays a central role in AD pathogenesis, is reported to accumulate within mitochondria. However, a question remains as to whether Aβ is generated locally from amyloid precursor protein (APP) within mitochondria. We investigated this issue by analyzing the expression patterns of APP, APP-processing secretases, and APP metabolites in mitochondria separated from human neuroblastoma SH-SY5Y cells and those expressing Swedish mutant APP. APP, BACE1, and PEN-2 protein levels were significantly lower in crude mitochondria than microsome fractions while those of ADAM10 and the other γ-secretase complex components (presenilin 1, nicastrin, and APH-1) were comparable between fractions. The crude mitochondrial fraction containing substantial levels of cathepsin D, a lysosomal marker, was further separated via iodixanol gradient centrifugation to obtain mitochondria- and lysosome-enriched fractions. Mature APP, BACE1, and all γ-secretase complex components (in particular, presenilin 1 and PEN-2) were scarcely present in the mitochondria-enriched fraction, compared to the lysosome-enriched fraction. Moreover, expression of the β-C-terminal fragment (β-CTF) of APP was markedly low in the mitochondria-enriched fraction. Additionally, immunocytochemical analysis showed very little co-localization between presenilin 1 and Tom20, a marker protein of mitochondria. In view of the particularly low expression levels of BACE1, γ-secretase complex proteins, and β-CTF in mitochondria, we propose that it is unlikely that Aβ generation from APP occurs locally within this organelle.
authors.affiliation: Department of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan; Department of Neurology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. Electronic address: mmd_naomi@yahoo.co.jp.
authors.forename: Naomi
authors.initials: N
authors.lastname: Mamada
authors.affiliation: Department of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan. Electronic address: tanodai@ncgg.go.jp.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Tanokashira
authors.affiliation: Department of Neurology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. Electronic address: kazishii@md.tsukuba.ac.jp.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Ishii
authors.affiliation: Department of Neurology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. Electronic address: atamaoka@md.tsukuba.ac.jp.
authors.forename: Akira
authors.initials: A
authors.lastname: Tamaoka
authors.affiliation: Department of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan. Electronic address: araki@ncnp.go.jp.
authors.forename: Wataru
authors.initials: W
authors.lastname: Araki
mesh_terms: D000072197:ADAM10 Protein / Q000235:genetics / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000235:genetics* / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D010450:Endopeptidases; D005786:Gene Expression Regulation; D006801:Humans; D008247:Lysosomes / Q000737:chemistry* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D053764:Presenilin-1 / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; C508853:PSEN1 protein, human; C469349:PSENEN protein, human; D053764:Presenilin-1; D011956:Receptors, Cell Surface; C084008:TOMM20 protein, human; C413604:nicastrin protein; C475154:APH1A protein, human; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D010447:Peptide Hydrolases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; C533922:CTSD protein, human; D002402:Cathepsin D; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human
keywords: Alzheimer's disease; Amyloid β-protein; BACE1; Mitochondria; Subcellular fractionation; γ-secretase