␣-Synuclein, a protein implicated in the pathogenesis of Parkinson disease (PD), is thought to affect mitochondrial functions, although the mechanisms of its action remain unclear. In this study we show that the N-terminal 32 amino acids of human ␣-synuclein contain cryptic mitochondrial targeting signal, which is important for mitochondrial targeting of ␣-synuclein. Mitochondrial imported ␣-synuclein is predominantly associated with the inner membrane. Accumulation of wild-type ␣-synuclein in the mitochondria of human dopaminergic neurons caused reduced mitochondrial complex I activity and increased production of reactive oxygen species. However, these defects occurred at an early time point in dopaminergic neurons expressing familial ␣-synuclein with A53T mutation as compared with wild-type ␣-synuclein. Importantly, ␣-synuclein that lacks mitochondrial targeting signal failed to target to the mitochondria and showed no detectable effect on complex I function. The PD relevance of these results was investigated using mitochondria of substantia nigra, striatum, and cerebellum of postmortem late-onset PD and normal human brains. Results showed the constitutive presence of ϳ14-kDa ␣-synuclein in the mitochondria of all three brain regions of normal subjects. Mitochondria of PD-vulnerable substantia nigra and striatum but not cerebellum from PD subjects showed significant accumulation of ␣-synuclein and decreased complex I activity. Analysis of mitochondria from PD brain and ␣-synuclein expressing dopaminergic neuronal cultures using blue native gel electrophoresis and immunocapture technique showed the association of ␣-synuclein with complex I. These results provide evidence that mitochondrial accumulated ␣-synuclein may interact with complex I and interfere with its functions. Parkinson disease (PD)2 is associated with the degeneration of dopaminergic neurons in the substantia nigra pars compacta.

Mitochondrial dysfunction is one of the major intracellular lesions of Alzheimer's disease (AD). However, the causative factors involved in the mitochondrial dysfunction in human AD are not well understood. Here we report that nonglycosylated full-length and C-terminal truncated amyloid precursor protein (APP) accumulates exclusively in the protein import channels of mitochondria of human AD brains but not in age-matched controls. Furthermore, in AD brains, mitochondrially associated APP formed stable ϳ480 kDa complexes with the translocase of the outer mitochondrial membrane 40 (TOM40) import channel and a super complex of ϳ620 kDa with both mitochondrial TOM40 and the translocase of the inner mitochondrial membrane 23 (TIM23) import channel TIM23 in an "N in mitochondria -C out cytoplasm " orientation. Accumulation of APP across mitochondrial import channels, which varied with the severity of AD, inhibited the entry of nuclearencoded cytochrome c oxidase subunits IV and Vb proteins, which was associated with decreased cytochrome c oxidase activity and increased levels of H 2 O 2 . Regional distribution of mitochondrial APP showed higher levels in AD-vulnerable brain regions, such as the frontal cortex, hippocampus, and amygdala. Mitochondrial accumulation of APP was also observed in the cholinergic, dopaminergic, GABAergic, and glutamatergic neuronal types in the category III AD brains. The levels of translocationally arrested mitochondrial APP directly correlated with mitochondrial dysfunction. Moreover, apolipoprotein genotype analysis revealed that AD subjects with the E3/E4 alleles had the highest content of mitochondrial APP. Collectively, these results suggest that abnormal accumulation of APP across mitochondrial import channels, causing mitochondrial dysfunction, is a hallmark of human AD pathology.

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.

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.