A Multisystemic Approach Revealed Aminated Polystyrene Nanoparticles-Induced Neurotoxicity.

Exposure to plastic nanoparticles has dramatically increased in the last 50 years, and there is evidence that plastic nanoparticles can be absorbed by organisms and cross the blood-brain-barrier (BBB). However, their toxic effects, especially on the nervous system, have not yet been extensively investigated, and most of the knowledge is based on studies using different conditions and systems, thus hard to compare. In this work, physicochemical properties of non-modified polystyrene (PS) and amine-functionalized PS (PS-NH2 ) nanoparticles are initially characterized. Advantage of a multisystemic approach is then taken to compare plastic nanoparticles effects in vitro, through cytotoxic readouts in mammalian cell culture, and in vivo, through behavioral readouts in the nematode Caenorhabditis elegans (C. elegans), a powerful 3R-complying model organism for toxicology studies. In vitro experiments in neuroblastoma cells indicate a specific cytotoxic effect of PS-NH2 particles, including a decreased neuronal differentiation and an increased Amyloid ß (Aß) secretion, a sensitive readout correlating with Alzheimer's disease pathology. In parallel, only in vivo treatments with PS-NH2 particles affect C. elegans development, decrease lifespan, and reveal higher sensitivity of animals expressing human Aß compared to wild-type animals. In summary, the multisystemic approach discloses a neurotoxic effect induced by aminated polystyrene nanoparticles.

Elegant Nematodes Improve Our Understanding of Human Neuronal Diseases, the Role of Pollutants and Strategies of Resilience.

The prevalence of neurodegenerative disorders such as Alzheimer's and Parkinson's disease are rising globally. The role of environmental pollution in neurodegeneration is largely unknown. Thus, this perspective advocates exposome research in C. elegans models of human diseases. The models express amyloid proteins such as Aß, recapitulate the degeneration of specifically vulnerable neurons and allow for correlated neurobehavioral phenotyping throughout the entire life span of the nematode. Neurobehavioral traits like locomotion gaits, rigidity, or cognitive decline are quantifiable and carefully mimic key aspects of the human diseases. Underlying molecular pathways of neurodegeneration are elucidated in pollutant-exposed C. elegans Alzheimer's or Parkinson's models by transcriptomics (RNA-seq), mass spectrometry-based proteomics and omics addressing other biochemical traits. Validation of the identified disease pathways can be achieved by genome-wide association studies in matching human cohorts. A consistent One Health approach includes isolation of nematodes from contaminated sites and their comparative investigation by imaging, neurobehavioral profiling and single worm proteomics. C. elegans models of neurodegenerative diseases are likewise well-suited for high throughput methods that provide a promising strategy to identify resilience pathways of neurosafety and keep up with the number of pollutants, nonchemical exposome factors, and their interactions.

Exposome, Molecular Pathways and One Health: The Invertebrate Caenorhabditis elegans.

Due to its preferred habitats in the environment, the free-living nematode Caenorhabditis elegans has become a realistic target organism for pollutants, including manufactured nanoparticles. In the laboratory, the invertebrate animal model represents a cost-effective tool to investigate the molecular mechanisms of the biological response to nanomaterials. With an estimated number of 22,000 coding genes and short life span of 2-3 weeks, the small worm is a giant when it comes to characterization of molecular pathways, long-term low dose pollutant effects and vulnerable age-groups. Here, we review (i) flows of manufactured nanomaterials and exposition of C. elegans in the environment, (ii) the track record of C. elegans in biomedical research, and (iii) its potential to contribute to the investigation of the exposome and bridge nanotoxicology between higher organisms, including humans. The role of C. elegans in the one health concept is taken one step further by proposing methods to sample wild nematodes and their molecular characterization by single worm proteomics.

Silica Nanoparticles Disclose a Detailed Neurodegeneration Profile throughout the Life Span of a Model Organism.

The incidence of age-related neurodegenerative diseases is rising globally. However, the temporal sequence of neurodegeneration throughout adult life is poorly understood. To identify the starting points and schedule of neurodegenerative events, serotonergic and dopaminergic neurons were monitored in the model organism C. elegans, which has a life span of 2-3 weeks. Neural morphology was examined from young to old nematodes that were exposed to silica nanoparticles. Young nematodes showed phenotypes such as dendritic beading of serotonergic and dopaminergic neurons that are normally not seen until late life. During aging, neurodegeneration spreads from specifically susceptible ADF and PDE neurons in young C. elegans to other more resilient neurons, such as dopaminergic CEP in middle-aged worms. Investigation of neurodegenerative hallmarks and animal behavior revealed a temporal correlation with the acceleration of neuromuscular defects, such as internal hatch in 2-day-old C. elegans. Transcriptomics and proteomics of young worms exposed to nano silica showed a change in gene expression concerning the gene ontology groups serotonergic and dopaminergic signaling as well as neuropeptide signaling. Consistent with this, reporter strains for nlp-3, nlp-14 and nlp-21 confirmed premature degeneration of the serotonergic neuron HSN and other neurons in young C. elegans. The results identify young nematodes as a vulnerable age group for nano silica-induced neural defects with a significantly reduced health span. Neurodegeneration of specific neurons impairs signaling by classical neurotransmitters as well as neuropeptides and compromises related neuromuscular behaviors in critical phases of life, such as the reproductive phase.

Casein α s1 is expressed by human monocytes and upregulates the production of GM-CSF via p38 MAPK.

Caseins are major constituents of mammalian milks that are thought to be exclusively expressed in mammary glands and to function primarily as a protein source, as well as to ameliorate intestinal calcium uptake. In addition, proinflammatory and immunomodulatory properties have been reported for bovine caseins. Our aim was to investigate whether human casein α s1 (CSN1S1) is expressed outside the mammary gland and possesses immunomodulatory functions in humans as well. For this purpose, CSN1S1 mRNA was detected in primary human monocytes and CD4(+) and CD8(+) T cells, but not in CD19(+) B cells. CSN1S1 protein was traceable in supernatants of cultured primary human CD14(+) monocytes by ELISA. Similarly, CSN1S1 mRNA and protein were detected in the human monocytic cell lines HL60, U937, and THP1 but not in Mono Mac 6 cells. Moreover, permeabilized human monocytes and HL60 cells could be stained by immunofluorescence, indicating intracellular expression. Recombinant human CSN1S1 was bound to the surface of Mono Mac 6 cells and upregulated the expression of GM-CSF mRNA in primary human monocytes and Mono Mac 6 cells in a time- and concentration-dependent manner. A similar increase in GM-CSF protein was found in the culture supernatants. CSN1S1-dependent upregulation of GM-CSF was specifically blocked by the addition of the p38 MAPK inhibitor ML3403. Our results indicated that human CSN1S1 may possess an immunomodulatory role beyond its nutritional function in milk. It is expressed in human monocytes and stimulates the expression of the proinflammatory cytokine GM-CSF.

Distant positioning of proteasomal proteolysis relative to actively transcribed genes.

While it is widely acknowledged that the ubiquitin-proteasome system plays an important role in transcription, little is known concerning the mechanistic basis, in particular the spatial organization of proteasome-dependent proteolysis at the transcription site. Here, we show that proteasomal activity and tetraubiquitinated proteins concentrate to nucleoplasmic microenvironments in the euchromatin. Such proteolytic domains are immobile and distinctly positioned in relation to transcriptional processes. Analysis of gene arrays and early genes in Caenorhabditis elegans embryos reveals that proteasomes and proteasomal activity are distantly located relative to transcriptionally active genes. In contrast, transcriptional inhibition generally induces local overlap of proteolytic microdomains with components of the transcription machinery and degradation of RNA polymerase II. The results establish that spatial organization of proteasomal activity differs with respect to distinct phases of the transcription cycle in at least some genes, and thus might contribute to the plasticity of gene expression in response to environmental stimuli.

Tire components, age and temperature accelerate neurodegeneration in C. elegans models of Alzheimer's and Parkinson's disease.

Increasingly, traffic-related air pollution is linked with Alzheimer's disease, Parkinson's disease and other neurodegenerative conditions. The molecular pathways underlying the epidemiologic observations are unknown. In this study, models of neurodegenerative disorders in the nematode Caenorhabditis elegans were used to investigate effects of the tire wear component nano silica. Life span-resolved exposition of reporter strain GRU102 that expresses the Alzheimer's peptide amyloid beta1-42 with silica nanoparticles significantly reduced locomotory fitness in middle-aged nematodes. A specific vulnerability of 10-day-old nematodes was identified in GRU102 cultivated at ambient temperatures of 15 and 20 °C. Reduction of locomotory fitness was corroborated in the Parkinson's disease model BZ555. Nano silica from different sources, including genuine tire components, accelerated the neurodegeneration of dopaminergic neurons in BZ555 nematodes. Dendritic beading was observed in single PDE neurons along the lateral side of the posterior body. In both, the Alzheimer's disease model GRU102 and the Parkinson's disease model BZ555 increased age and the non-chemical exposome factor temperature aggravated nano silica-induced neurodegeneration. Middle-aged cohorts were defined as the most vulnerable age-group. The results suggest C. elegans disease models as a platform to elucidate the relationships between neurodegeneration, age and the environmental factor ambient temperature after exposition with defined components of non-exhaust emissions or sampled urban aerosols.

How to keep up with the analysis of classic and emerging neurotoxins: Age-resolved fitness tests in the animal model Caenorhabditis elegans - a step-by-step protocol.

The global chemical inventory includes neurotoxins that are mostly interrogated concerning the biological response in developing organisms. Effects of pollutants on adults receive less attention, although vulnerabilities can be expected throughout the entire life span in young, middle-aged and old individuals. We use the animal model Caenorhabditis elegans to systematically quantify neurological outcomes by application of an age-resolved method. Adult hermaphrodite worms were exposed to pollutants or non-chemical stressors such as temperature in liquid culture on microtiter plates and locomotion fitness was analyzed in a whole-life approach. Cultivation at 15, 20 or 25 °C showed that worms held at 15 °C displayed an enhanced level of fitness concerning swimming movements until middle age (11-days-old) and then a decline. In contrast, C. elegans cultivated at ≥ 20 °C continually reduced their swimming movements with increasing age. Here, we provide a step-by-step protocol to investigate the health span of adult C. elegans that may serve as a platform for automation and data collection. Consistent with this, more neurotoxins can be investigated with respect to vulnerable age-groups as well as contributing non-chemical environmental factors such as temperature.

Peripheral neuropathy, protein aggregation and serotonergic neurotransmission: Distinctive bio-interactions of thiacloprid and thiamethoxam in the nematode Caenorhabditis elegans.

Due to worldwide production, sales and application, neonicotinoids dominate the global use of insecticides. While, neonicotinoids are considered as pinpoint neurotoxicants that impair cholinergic neurotransmission in pest insects, the sublethal effects on nontarget organisms and other neurotransmitters remain poorly understood. Thus, we investigated long-term neurological outcomes in the decomposer nematode Caenorhabditis elegans. In the adult roundworm the neonicotinoid thiacloprid impaired serotonergic and dopaminergic neuromuscular behaviors, while respective exposures to thiamethoxam showed no effects. Thiacloprid caused a concentration-dependent delay of the transition between swimming and crawling locomotion that is controlled by dopaminergic and serotonergic neurotransmission. Age-resolved analyses revealed that impairment of locomotion occurred in young as well as middle-aged worms. Treatment with exogenous serotonin rescued thiacloprid-induced swimming deficits in young worms, whereas additional exposure with silica nanoparticles enhanced the reduction of swimming behavior. Delay of forward locomotion was partly caused by a new paralysis pattern that identified thiacloprid as an agent promoting a specific rigidity of posterior body wall muscle cells and peripheral neuropathy in the nematode (lowest-observed-effect-level 10 ng/ml). On the molecular level exposure with thiacloprid accelerated protein aggregation in body wall muscle cells of polyglutamine disease reporter worms indicating proteotoxic stress. The results from the soil nematode Caenorhabditis elegans show that assessment of neurotoxicity by neonicotinoids requires acknowledgment and deeper research into dopaminergic and serotonergic neurochemistry of nontarget organisms. Likewise, it has to be considered more that different neonicotinoids may promote diverse neural end points.

Pollutants corrupt resilience pathways of aging in the nematode C. elegans.

Delaying aging while prolonging health and lifespan is a major goal in aging research. One promising strategy is to focus on reducing negative interventions such as pollution and their accelerating effect on age-related degeneration and disease. Here, we used the short-lived model organism C. elegans to analyze whether two candidate pollutants corrupt general aging pathways. We show that the emergent pollutant silica nanoparticles (NPs) and the classic xenobiotic inorganic mercury reduce lifespan and cause a premature protein aggregation phenotype. Comparative mass spectrometry revealed that increased insolubility of proteins with important functions in proteostasis is a shared phenotype of intrinsic- and pollution-induced aging supporting the hypothesis that proteostasis is a central resilience pathway controlling lifespan and aging. The presented data demonstrate that pollutants corrupt intrinsic aging pathways. Reducing pollution is, therefore, an important step to increasing healthy aging and prolonging life expectancies on a population level in humans and animals.

Effects of Airborne Nanoparticles on the Nervous System: Amyloid Protein Aggregation, Neurodegeneration and Neurodegenerative Diseases.

How the environment contributes to neurodegenerative diseases such as Alzheimer's is not well understood. In recent years, science has found augmenting evidence that nano-sized particles generated by transport (e.g., fuel combustion, tire wear and brake wear) may promote Alzheimer's disease (AD). Individuals residing close to busy roads are at higher risk of developing AD, and nanomaterials that are specifically generated by traffic-related processes have been detected in human brains. Since AD represents a neurodegenerative disease characterized by amyloid protein aggregation, this review summarizes our current knowledge on the amyloid-generating propensity of traffic-related nanomaterials. Certain nanoparticles induce the amyloid aggregation of otherwise soluble proteins in in vitro laboratory settings, cultured neuronal cells and vertebrate or invertebrate animal models. We discuss the challenges for future studies, namely, strategies to connect the wet laboratory with the epidemiological data in order to elucidate the molecular bio-interactions of airborne nanomaterials and their effects on human health.

The Nucleus of Intestinal Cells of the Bacterivore Nematode Caenorhabditis elegans as a Sensitive Sensor of Environmental Pollutants.

Prevalent environmental challenges are climate change, the biodiversity crisis, and the global scale of environmental pollution. We identified the cell nucleus as a sensitive sensor for bio-effects of pollutants such as mercury and nanoparticles. As a major route of pollutant uptake into organisms is ingestion, we have developed a test system that uses single intestinal cells of the nematode roundworm Caenorhabditis elegans. Microscopic observation of the cell nucleus in reporter worms for the methyltransferase fibrillarin (FIB-1::GFP) revealed nuclear staining patterns that are specific for pollutants such as silica nanoparticles, BULK silica particles, silver nanoparticles, ionic AgNO3, and inorganic mercury (HgCl2). While the underlying molecular mechanisms need further investigation, cultivation of the reporter worms in liquid culture on microtiter plates now enables cost-effective screening of more pollutants and samples from the environment, e.g., mesocosm analyses. As C. elegans leads a dual life in the lab and in ecosystems, alteration of nuclear structure and function may likewise explain how environmental pollutants reduce the fitness of wild worms and thus may negatively affect biodiversity.

Nanoparticle-induced cell culture models for degenerative protein aggregation diseases.

Protein aggregates and nuclear inclusions containing components of the ubiquitin-proteasome system, expanded polyglutamine (polyQ) proteins, and transcriptional co-activators characterize cellular responses to stress and are hallmarks of neurodegenerative diseases. For instance, in Huntington's disease, an expansion of a polyQ region causes its aggregation into beta-sheet-containing amyloid fibrils. The biological function of polyQ-containing inclusions is unknown. By means of a silica nanoparticle (NP)-based strategy we induced intranuclear protein inclusions that form amyloid-like structures, recapitulating the protein composition and solubility of polyQ-induced nuclear protein aggregates exactly. We showed that global proteasomal proteolysis increases in silica-NP-treated nuclei and, on the local level, a subpopulation of nuclear inclusions overlaps with focal domains of proteasome-dependent protein degradation. The results suggest that inclusions in the nucleus constitute active proteolysis modules that may serve to concentrate and decompose damaged, mal-folded, or misplaced proteins. While nanoparticle-nucleus interactions turn out to be invaluable tools to study the molecular mechanisms of degenerative protein aggregation diseases, one also has to consider the other side of the coin, namely, emerging environmental risks posed by these very interactions.

Silica nanoparticles disrupt OPT-2/PEP-2-dependent trafficking of nutrient peptides in the intestinal epithelium.

Despite of the increasing application of silica nanoparticles and identification of oral exposure as a major entry portal, we lack understanding of nanosilica effects in the gut. Thus, we investigated biointeractions of nanosilica with single intestinal cells. The invertebrate nematode Caenorhabditis elegans was chosen as model organism with a tractable intestine and realistic target organism of nanomaterials in the environment. We found that nanosilica impairs the intestinal uptake of oligopeptides. Downstream to absorption by the apical OPT-2/PEP-2 transporter dipeptides were trapped in aberrant vesicles that grow over time and reach diameters of ≥6 µm. The peptide vesicles do not correspond to known organelles such as gut granules and form independently of related gene products GLO-1 or GLO-3. Formation of aberrant peptide vesicles also occurred independently of insulin/IGF-I receptor (DAF-2) signaling and daf-2 loss of function mutants showed specific vesicle patterns including distinct localization along the apical membrane of single intestinal cells. As malnutrition of exposed C. elegans manifested in reduced growth and a petite phenotype similar to OPT-2/PEP-2 transporter deficient mutants, we conclude that nanosilica-induced peptide vesicles represent a new compartment of di- and tripeptide trapping which disrupts hydrolysis of nutrient peptides and metabolism.

Life span-resolved nanotoxicology enables identification of age-associated neuromuscular vulnerabilities in the nematode Caenorhabditis elegans.

At present, the majority of investigations concerning nanotoxicology in the nematode C. elegans address short-term effects. While this approach allows for the identification of uptake pathways, exposition and acute toxicity, nanoparticle-organism interactions that manifest later in the adult life of C. elegans are missed. Here we show that a microhabitat composed of liquid S-medium and live bacteria in microtiter wells prolongs C. elegans longevity and is optimally suited to monitor chronic eNP-effects over the entire life span (about 34 days) of the nematode. Silver (Ag) nanoparticles reduced C. elegans life span in concentrations ≥10 µg/mL, whereas nano ZnO and CeO2 (1-160 µg/mL) had no effect on longevity. Monitoring of locomotion behaviors throughout the entire life span of C. elegans showed that Ag NPs accelerate the age-associated decline of swimming and increase of uncoordinated movements at concentrations of ≥10 µg/mL, whereas neuromuscular defects did not occur in response to ZnO and CeO2 NPs. By means of a fluorescing reporter worm expressing tryptophan hydroxylase-1::DsRed Ag NP-induced behavioral defects were correlated to axonal protein aggregation and neurodegeneration in single serotonergic HSN as well as sensory ADF neurons. Notably, serotonergic ADF neurons represented a sensitive target for Ag NPs in comparison to GABAergic neurons that showed no signs of degeneration under the same conditions. We conclude that due to its analogy to the jellylike boom culture of C. elegans on microbe-rich rotting plant material liquid S-medium culture in spatially confined microtiter wells represents a relevant as well as practical tool for comparative identification of age-resolved nanoparticle effects and vulnerabilities in a significant target organism. Consistent with this, specifically middle-aged nematodes showed premature neuromuscular defects after Ag NP-exposure.

Subcellular recruitment of fibrillarin to nucleoplasmic proteasomes: implications for processing of a nucleolar autoantigen.

A prerequisite for proteins to interact in a cell is that they are present in the same intracellular compartment. Although it is generally accepted that proteasomes occur in both, the cytoplasm and the nucleus, research has been focusing on cytoplasmic protein breakdown and antigen processing, respectively. Thus, little is known on the functional organization of the proteasome in the nucleus. Here we report that within the nucleus 20S and 26S proteasomes occur throughout the nucleoplasm and partially colocalize with splicing factor-containing speckles. Because proteasomes are absent from the nucleolus, a recruitment system was used to analyze the molecular fate of nucleolar protein fibrillarin: Subtoxic concentrations of mercuric chloride (HgCl(2)) induce subcellular redistribution of fibrillarin and substantial colocalization (33%) with nucleoplasmic proteasomes in different cell lines and in primary cells isolated from mercury-treated mice. Accumulation of fibrillarin and fibrillarin-ubiquitin conjugates in lactacystin-treated cells suggests that proteasome-dependent processing of this autoantigen occurs upon mercury induction. The latter observation might constitute the cell biological basis of autoimmune responses that specifically target fibrillarin in mercury-mouse models and scleroderma.

Expression and tissue distribution of mouse Hax1.

HAX1 is an ubiquitously expressed human gene. Though a number of cellular and viral proteins are known to interact with HAX1, its function is still not completely understood. On the basis of these identified interaction partners, HAX1 seems to play a role in apoptosis and the organization of the cytoskeleton. The cDNAs for human and mouse Hax1 share 86% identity and 80% identity at the protein level, suggesting a similar functional importance. To date, no conclusive data on the tissue specific expression of the murine Hax1 are available and only one interaction partner has been identified. Here, we show a detailed expression analysis for the murine ortholog by RT-PCR, Northern and Western blot. Furthermore, the distribution of Hax1 within different mouse tissues was studied by immunohistochemistry (IHC). In general, we found a good correlation between the results obtained from different detection techniques. Similar to its human counterpart, mouse Hax1 seems to be ubiquitously expressed. At the RNA level, we found the highest expression of Hax1 in liver, kidney and testis. In sharp contrast to the human HAX1 which is highly expressed in skeletal muscle, the mouse ortholog was detected only at very low levels. Using a specific antibody, we detected Hax1 in the majority of mouse tissues by IHC. Interestingly, the most prominent expression of Hax1 was found in epithelial, endothelial and muscle cells. Surprisingly, thymus, spleen and pancreas did not show detectable immunostaining. Furthermore, we have studied the subcellular localisation of Hax1 in a keratinocyte and a neuronal cell line by immunofluorescence. We found Hax1 to be localised mainly in the cytoplasm and detected a partial colocalisation with mitochondria. The results presented here summarize for the first time the expression of the murine Hax1 in different tissues and two cell lines. Further studies will elucidate the functional importance of this protein in individual cell types with respect to structural aspects, cell mobility and apoptosis.

Anti-amyloid compounds protect from silica nanoparticle-induced neurotoxicity in the nematode C. elegans.

Identifying nanomaterial-bio-interactions are imperative due to the broad introduction of nanoparticle (NP) applications and their distribution. Here, we demonstrate that silica NPs effect widespread protein aggregation in the soil nematode Caenorhabditis elegans ranging from induction of amyloid in nucleoli of intestinal cells to facilitation of protein aggregation in body wall muscles and axons of neural cells. Proteomic screening revealed that exposure of adult C. elegans with silica NPs promotes segregation of proteins belonging to the gene ontology (GO) group of "protein folding, proteolysis and stress response" to an SDS-resistant aggregome network. Candidate proteins in this group include chaperones, heat shock proteins and subunits of the 26S proteasome which are all decisively involved in protein homeostasis. The pathway of protein homeostasis was validated as a major target of silica NPs by behavioral phenotyping, as inhibitors of amyloid formation rescued NP-induced defects of locomotory patterns and egg laying. The analysis of a reporter worm for serotonergic neural cells revealed that silica NP-induced protein aggregation likewise occurs in axons of HSN neurons, where presynaptic accumulation of serotonin, e.g. disturbed axonal transport reduces the capacity for neurotransmission and egg laying. The results suggest that in C. elegans silica NPs promote a cascade of events including disturbance of protein homeostasis, widespread protein aggregation and inhibition of serotonergic neurotransmission which can be interrupted by compounds preventing amyloid fibrillation.

Creatine supplementation normalizes mutagenesis of mitochondrial DNA as well as functional consequences.

Mutations of mitochondrial (mt) DNA play a role in neurodegeneration, normal aging, premature aging of the skin (photoaging), and tumors. We and others could demonstrate that mtDNA mutations can be induced in skin cells in vitro and in normal human skin in vivo by repetitive, sublethal ultraviolet (UV)-A-irradiation. These mutations are mediated by singlet oxygen and persist in human skin as long-term biomarkers of UV exposure. Although mtDNA exclusively encodes for the respiratory chain, involvement of the energy metabolism in mtDNA mutagenesis and a protective role of the energy precursor creatine have thus far not been shown. We assessed the amount of a marker mutation of mtDNA, the so-called common deletion, by real-time PCR. Induction of the common deletion was paralleled by a measurable decrease of oxygen consumption, mitochondrial membrane potential, and ATP content, as well as an increase of matrix metalloproteinase-1. Mitochondrial mutagenesis as well as functional consequences could be normalized by increasing intracellular creatine levels. These data indicate that increase of the energy precursor creatine protects from functionally relevant, aging-associated mutations of mitochondrial DNA.