Alzheimer's amyloid precursor protein 695 (APP) is a plasma membrane protein, which is known to be the source of the toxic amyloid β (Aβ) 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.

We have investigated the mechanism of mitochondrialnuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m-chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Δψ m ) and electron transport-coupled ATP synthesis, which was also accompanied by an elevated steady-state cytosolic Ca 2⍣ level ([Ca 2⍣ ] i ). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor-1 (RyR-1), hence potentiating the Ca 2⍣ release in response to its modulator, caffeine; (ii) enhanced levels of Ca 2⍣ -responsive factors calineurin, calcineurindependent NFATc (cytosolic counterpart of activated T-cell-specific nuclear factor) and c-Jun N-terminal kinase (JNK)-dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF-κB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady-state [Ca 2⍣ ] i were normalized in genetically reverted cells which contain near-normal mtDNA levels. We propose that the mitochondria-to-nucleus stress signaling occurs through cytosolic [Ca 2⍣ ] i changes, which are likely to be due to reduced ATP and Ca 2⍣ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.

Recently we showed that partial depletion of mitochondrial DNA (genetic stress) or treatment with mitochondrial-speci®c inhibitors (metabolic stress) induced a stress signaling that was associated with increased cytoplasmic-free Ca 2+ [Ca 2+ ] c . In the present study we show that the mitochondria-tonucleus stress signaling induces invasive phenotypes in otherwise non-invasive C2C12 myoblasts and human pulmonary carcinoma A549 cells. Tumor-speci®c markers cathepsin L and transforming growth factor b (TGFb) are overexpressed in cells subjected to mitochondrial genetic as well as metabolic stress. C2C12 myoblasts subjected to stress showed 4-to 6-fold higher invasion through reconstituted Matrigel membrane as well as rat tracheal xenotransplants in Scid mice. Activation of Ca 2+ -dependent protein kinase C (PKC) under both genetic and metabolic stress conditions was associated with increased cathepsin L gene expression, which contributes to increased invasive property of cells. Reverted cells with~70% of control cell mtDNA exhibited marker mRNA contents, cell morphology and invasive property closer to control cells. These results provide insights into a new pathway by which mitochondrial DNA and membrane damage can contribute to tumor progression and metastasis.

We have investigated mechanisms of mitochondrial stressinduced phenotypic changes and cell invasion in tumorigenic but poorly invasive human pulmonary carcinoma A549 cells that were partly depleted of mitochondrial DNA (mtDNA). Depletion of mtDNA (genetic stress) caused a markedly lower electron transport-coupled ATP synthesis, loss of mitochondrial membrane potential, elevation of steady state [Ca 2+ ] c , and notably induction of both glycolysis and gluconeogenic pathway enzymes. Markers of tumor invasion, cathepsin L and TGFb1, were overexpressed; calcium-dependent MAP kinases (ERK1 and ERK2) and calcineurin were activated. The levels of anti-apoptotic proteins Bcl2 and Bcl-X L were increased, and the cellular levels of pro-apoptotic proteins Bid and Bax were reduced. Both mtDNA-depleted cells (genetic stress) and control cells treated with carbonyl cyanide m-chlorophenylhydrazone (metabolic stress) exhibited higher invasive behavior than control cells in a Matrigel basement membrane matrix assay system. MtDNA-depleted cells stably expressing anti-sense cathepsin L RNA, TGFb1 RNA, or treated with specific inhibitors showed reduced invasion. Reverted cells with 80% of control cell mtDNA exhibited marker protein levels, cell morphology and invasive property closer to control cells. Our results suggest that the mitochondriato-nucleus signaling pathway operating through increased [Ca 2+ ] c plays an important role in cancer progression and metastasis. Oncogene (2002Oncogene ( ) 21, 7839 -7849. doi:10.1038 Keywords: mitochondria; calcium signaling; cathepsin L; TGFb; tumor invasion; MAP kinases IntroductionThe role of mitochondria in carcinogenesis was initially suggested by Warburg et al. (1926;Warburg, 1956), based on the observation that number of experimentally induced rodent tumors exhibit reduced respiration-coupled oxidative metabolism and increased glycolysis. Since then, altered mitochondrial morphology, as well as changes in mitochondrial enzyme patterns and membrane transport systems, have been described in several tumor types (Zafar et al., 1982). Cell fusion studies (Jonasson et al., 1977;Howell and Sager, 1978;Giguere and Morais, 1981) also suggest that unknown cytoplasmic elements may play roles in carcinogenesis. Fusion between normal cytoplasm and karyoplasts from malignant phenotypes resulted in the ablation of tumorigenicity (Israel and Schaeffer, 1987) and conversely, the cytoplasm of the malignant phenotype could transfer the malignancy to the karyoplasts from normal cells (Israel and Schaeffer, 1988). Studies using mitochondrial DNA (mtDNA)-depleted tumor cells to evaluate the role of mtDNA, and thus mitochondrial function in tumorigenicity have yielded mixed results (Giguere and Morais, 1981;Morais et al., 1994;Cavalli et al., 1997;Cavalli and Liang, 1998;Hofhaus and Gattermann, 1999). Cavalli et al. (1997) found diminished tumor formation by glioblastoma cells following depletion of mtDNA (r 0 cells) with ethidium bromide treatment, while Morais et al. (1994) found increased capacity to p...

Cytochrome P4501A1 is a hepatic, microsomal membrane–bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from β-naphthoflavone–induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30–amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33–44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.