Age-related dysfunction of the autophago-lysosomal pathway in human endothelial cells.

Senescent cells, which are cells in a post-proliferative state, show an increased number of dysfunctional mitochondria and oxidatively damaged and aggregated proteins. The mitochondrial-lysosomal axis theory of aging proposes that the autophago-lysosomal system is unable to cope with the rising amount of damaged organelles and proteins. We used human umbilical vein endothelial cells (HUVEC) as in vitro model system to determine which part/s of the autophago-lysosomal pathway become deficient by aging. Senescent HUVEC contained a much larger population of autophagosomes and lysosomes compared to young cells. Transcriptome analysis comparing young and old cells demonstrated several age-related changes of autophagy gene expression. One reason for the observed increase of autophagosomes was an impairment of the autophagic flux in senescent cells due to reduced V-ATPase activity required for acidification of the lysosomes and thus functionality of lysosomal hydrolases. The hypothesis that reduced mitochondrial ATP production underlies low V-ATPase activity was supported by addition of exogenous ATP. This procedure rescued the lysosomal acidification and restored the autophagic flux. Thus, we propose impaired lysosomal acidification due to ATP shortage which may result from mitochondrial dysfunction as a mechanism underlying the accumulation of dysfunctional cellular constituents during aging.

Use of HuH6 and other human-derived hepatoma lines for the detection of genotoxins: a new hope for laboratory animals?

Cell lines which are currently used in genotoxicity tests lack enzymes which activate/detoxify mutagens. Therefore, rodent-derived liver preparations are used which reflect their metabolism in humans only partly; as a consequence misleading results are often obtained. Previous findings suggest that certain liver cell lines express phase I/II enzymes and detect promutagens without activation; however, their use is hampered by different shortcomings. The aim of this study was the identification of a suitable cell line. The sensitivity of twelve hepatic cell lines was investigated in single cell gel electrophoresis assays. Furthermore, characteristics of these lines were studied which are relevant for their use in genotoxicity assays (mitotic activity, p53 status, chromosome number, and stability). Three lines (HuH6, HCC1.2, and HepG2) detected representatives of five classes of promutagens, namely, IQ and PhIP (HAAs), B(a)P (PAH), NDMA (nitrosamine), and AFB1 (aflatoxin), and were sensitive towards reactive oxygen species (ROS). In contrast, the commercially available line HepaRG, postulated to be a surrogate for hepatocytes and an ideal tool for mutagenicity tests, did not detect IQ and was relatively insensitive towards ROS. All other lines failed to detect two or more compounds. HCC1.2 cells have a high and unstable chromosome number and mutated p53, these features distract from its use in routine screening. HepG2 was frequently employed in earlier studies, but pronounced inter-laboratory variations were observed. HuH6 was never used in genotoxicity experiments and is highly promising, it has a stable karyotype and we demonstrated that the results of genotoxicity experiments are reproducible.

Decreased expression of Drp1 and Fis1 mediates mitochondrial elongation in senescent cells and enhances resistance to oxidative stress through PINK1.

Mitochondria display different morphologies, depending on cell type and physiological situation. In many senescent cell types, an extensive elongation of mitochondria occurs, implying that the increase of mitochondrial length in senescence could have a functional role. To test this hypothesis, human endothelial cells (HUVECs) were aged in vitro. Young HUVECs had tubular mitochondria, whereas senescent cells were characterized by long interconnected mitochondria. The change in mitochondrial morphology was caused by downregulation of the expression of Fis1 and Drp1, two proteins regulating mitochondrial fission. Targeted photodamage of mitochondria induced the formation of reactive oxygen species (ROS), which triggered mitochondrial fragmentation and loss of membrane potential in young cells, whereas senescent cells proved to be resistant. Alterations of the Fis1 and Drp1 expression levels also influenced the expression of the putative serine-threonine kinase PINK1, which is associated with the PARK6 variant of Parkinson's disease. Downregulation of PINK1 or overexpression of a PINK1 mutant (G309D) increased the sensitivity against ROS in young cells. These results indicate that there is a Drp1- and Fis1-induced, and PINK1-mediated protection mechanism in senescent cells, which, when compromised, could contribute to the age-related progression of Parkinson's disease and arteriosclerosis.

Short- and long-term alterations of mitochondrial morphology, dynamics and mtDNA after transient oxidative stress.

Cells are exposed during their life span to fluctuating levels of reactive oxygen species (ROS). To investigate the effects of a single ROS boost in vitro, human endothelial cells (HUVEC) were treated with one short-term dose of hydrogen peroxide. This treatment resulted in a short, dose-dependent ROS peak that caused transient changes in the mitochondrial morphology and fine structure, in the frequency of mitochondrial fission and fusion and in the mRNA levels of distinct fission and fusion factors. Treatment with a higher dose induced prolonged mtDNA damage; these cells exhibited a significantly shortened replicative lifespan, indicating dose-dependent effects of oxidative stress on mitochondria.

Loss of PINK1 impairs stress-induced autophagy and cell survival.

The mitochondrial kinase PINK1 and the ubiquitin ligase Parkin are participating in quality control after CCCP- or ROS-induced mitochondrial damage, and their dysfunction is associated with the development and progression of Parkinson's disease. Furthermore, PINK1 expression is also induced by starvation indicating an additional role for PINK1 in stress response. Therefore, the effects of PINK1 deficiency on the autophago-lysosomal pathway during stress were investigated. Under trophic deprivation SH-SY5Y cells with stable PINK1 knockdown showed downregulation of key autophagic genes, including Beclin, LC3 and LAMP-2. In good agreement, protein levels of LC3-II and LAMP-2 but not of LAMP-1 were reduced in different cell model systems with PINK1 knockdown or knockout after addition of different stressors. This downregulation of autophagic factors caused increased apoptosis, which could be rescued by overexpression of LC3 or PINK1. Taken together, the PINK1-mediated reduction of autophagic key factors during stress resulted in increased cell death, thus defining an additional pathway that could contribute to the progression of Parkinson's disease in patients with PINK1 mutations.

Autophagy proteins LC3B, ATG5 and ATG12 participate in quality control after mitochondrial damage and influence lifespan.

Mitochondrial health is maintained by the quality control mechanisms of mitochondrial dynamics (fission and fusion) and mitophagy. Decline of these processes is thought to contribute to aging and neurodegenerative diseases. To investigate the role of mitochondrial quality control in aging on the cellular level, human umbilical vein endothelial cells (HUVEC) were subjected to mitochondria-targeted damage by combining staining of mitochondria and irradiation. This treatment induced a short boost of reactive oxygen species, which resulted in transient fragmentation of mitochondria followed by mitophagy, while mitochondrial dynamics were impaired. Furthermore, targeted mitochondrial damage upregulated autophagy factors LC3B, ATG5 and ATG12. Consequently these proteins were overexpressed in HUVEC as an in vitro aging model, which significantly enhanced the replicative life span up to 150% and the number of population doublings up to 200%, whereas overexpression of LAMP-1 did not alter the life span. Overexpression of LC3B, ATG5 and ATG12 resulted in an improved mitochondrial membrane potential, enhanced ATP production and generated anti-apoptotic effects, while ROS levels remained unchanged and the amount of oxidized proteins increased. Taken together, these data relate LC3B, ATG5 and ATG12 to mitochondrial quality control after oxidative damage, and to cellular longevity.

A new link to mitochondrial impairment in tauopathies.

Tauopathies like the "frontotemporal dementia with Parkinsonism linked to chromosome 17" (FTDP-17) are characterized by an aberrant accumulation of intracellular neurofibrillary tangles composed of hyperphosphorylated tau. For FTDP-17, a pathogenic tau mutation P301L was identified. Impaired mitochondrial function including disturbed dynamics such as fission and fusion are most likely major pathomechanisms of most neurodegenerative diseases. However, very little is known if tau itself affects mitochondrial function and dynamics. We addressed this question using SY5Y cells stably overexpressing wild-type (wt) and P301L mutant tau. P301L overexpression resulted in a substantial complex I deficit accompanied by decreased ATP levels and increased susceptibility to oxidative stress. This was paralleled by pronounced changes in mitochondrial morphology, decreased fusion and fission rates accompanied by reduced expression of several fission and fusion factors like OPA-1 or DRP-1. In contrast, overexpression of wt tau exhibits protective effects on mitochondrial function and dynamics including enhanced complex I activity. Our findings clearly link tau bidirectional to mitochondrial function and dynamics, identifying a novel aspect of the physiological role of tau and the pathomechanism of tauopathies.

Mitochondrion-derived reactive oxygen species lead to enhanced amyloid beta formation.

AIMS: Intracellular amyloid beta (Aß) oligomers and extracellular Aß plaques are key players in the progression of sporadic Alzheimer's disease (AD). Still, the molecular signals triggering Aß production are largely unclear. We asked whether mitochondrion-derived reactive oxygen species (ROS) are sufficient to increase Aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. RESULTS: Complex I and III dysfunction was induced in a cell model using the respiratory inhibitors rotenone and antimycin, resulting in mitochondrial dysfunction and enhanced ROS levels. Both treatments lead to elevated levels of Aß. Presence of an antioxidant rescued mitochondrial function and reduced formation of Aß, demonstrating that the observed effects depended on ROS. Conversely, cells overproducing Aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. Again, the capability of these cells to generate Aß was partly reduced by an antioxidant, indicating that Aß formation was also ROS dependent. Moreover, mice with a genetic defect in complex I, or AD mice treated with a complex I inhibitor, showed enhanced Aß levels in vivo. INNOVATION: We show for the first time that mitochondrion-derived ROS are sufficient to trigger Aß production in vitro and in vivo. CONCLUSION: Several lines of evidence show that mitochondrion-derived ROS result in enhanced amyloidogenic amyloid precursor protein processing, and that Aß itself leads to mitochondrial dysfunction and increased ROS levels. We propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic AD.

Aging of different avian cultured cells: lack of ROS-induced damage and quality control mechanisms.

Elevated reactive oxygen species (ROS) levels have been observed in mammals during aging, implying an important role of ROS in the aging process. Most bird species are known to live longer and to contain lower ROS levels than mammals of the same body weight. The influence of ROS on the aging process of birds has been investigated using pigeon embryonic fibroblasts (PEF) and chicken embryonic fibroblasts (CEF). ROS levels in young avian cells were much lower than in human cells. When cultivated till replicative senescence, PEF proliferated about one-third longer compared to CEF. However, both senescent avian cell populations showed no increased ROS levels or accumulation of ROS-induced damage on the mtDNA or protein level. The investigation for quality control (QC) mechanisms revealed that the autophagosomal/lysosomal pathway was not downregulated in old avian cells and stable overexpression of the autophagy protein ATG5 improved mitochondrial fitness, enhanced the resistance against oxidative stress and prolonged the life span of CEF. Oxidative stress-mediated apoptosis induced a dose-dependent cell proliferation in CEF as well as in PEF. Taken together, our data indicate that autophagy and compensatory proliferation act as QC mechanisms, while ROS did not influence the aging process in avian cells.

Structural implications of mitochondrial dynamics.

Mitochondrial components are continuously distributed throughout the whole chondriome of a cell by fusion and fission. Thus, a single mitochondrion represents a transient fraction of the chondriome. Mitochondrial dynamics are responsible for intracellular distribution and reaction of mitochondria to functional requirements. Dynamics occur on different levels: overall morphology, inner membrane-matrix compartment, turnover and rearrangements of mitochondrial proteins and DNA. Electron micrographs of serial sections of human umbilical vein endothelial cells reveal perinuclear mitochondria of extreme length and with branches in those cells that also have short peripheral mitochondria. Interactions of mitochondria with cytoskeletal elements are revealed in cells treated with cytochalasin D to destroy actin fibrillar structures or after disassembling microtubule by nocodazole. In the latter case mitochondria not only become immobilized, they also acquire a multiple ring structure. In F-actin-disturbed cells, motility (shape changes in particular) is increased and the mitochondria become elongated. Mechanisms of how F-actin might render mitochondria immobile may involve dynamin-related protein 1 (DRP1) or interaction with anion channels. This may be responsible for the lack of mitochondrial motility in senescent cells. Fusion between mitochondria revealed local fluctuations of mitochondrial red fluorescent protein (mtRFP), indicating novel fast inner membrane reorganizations. Mitochondrial dynamics result from a complex interplay between the molecular organization of the inner membrane-matrix complex and cytoskeletal elements outside.