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.

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.

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.

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.

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.

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.

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.

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.

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.

Amyloid domains in the cell nucleus controlled by nucleoskeletal protein lamin B1 reveal a new pathway of mercury neurotoxicity.

Mercury (Hg) is a bioaccumulating trace metal that globally circulates the atmosphere and waters in its elemental, inorganic and organic chemical forms. While Hg represents a notorious neurotoxicant, the underlying cellular pathways are insufficiently understood. We identify amyloid protein aggregation in the cell nucleus as a novel pathway of Hg-bio-interactions. By mass spectrometry of purified protein aggregates, a subset of spliceosomal components and nucleoskeletal protein lamin B1 were detected as constituent parts of an Hg-induced nuclear aggregome network. The aggregome network was located by confocal imaging of amyloid-specific antibodies and dyes to amyloid cores within splicing-speckles that additionally recruit components of the ubiquitin-proteasome system. Hg significantly enhances global proteasomal activity in the nucleus, suggesting that formation of amyloid speckles plays a role in maintenance of protein homeostasis. RNAi knock down showed that lamin B1 for its part regulates amyloid speckle formation and thus likewise participates in nuclear protein homeostasis. As the Hg-induced cascade of interactions between the nucleoskeleton and protein homeostasis reduces neuronal signalling, amyloid fibrillation in the cell nucleus is introduced as a feature of Hg-neurotoxicity that opens new avenues of future research. Similar to protein aggregation events in the cytoplasm that are controlled by the cytoskeleton, amyloid fibrillation of nuclear proteins may be driven by the nucleoskeleton.

Imaging and quantification of amyloid fibrillation in the cell nucleus.

Xenobiotics, as well as intrinsic processes such as cellular aging, contribute to an environment that constantly challenges nuclear organization and function. While it becomes increasingly clear that proteasome-dependent proteolysis is a major player, the topology and molecular mechanisms of nuclear protein homeostasis remain largely unknown. We have shown previously that (1) proteasome-dependent protein degradation is organized in focal microenvironments throughout the nucleoplasm and (2) heavy metals as well as nanoparticles induce nuclear protein fibrillation with amyloid characteristics. Here, we describe methods to characterize the landscape of intranuclear amyloid on the global and local level in different systems such as cultures of mammalian cells and the soil nematode Caenorhabditis elegans. Application of discrete mathematics to imaging data is introduced as a tool to develop pattern recognition of intracellular protein fibrillation. Since stepwise fibrillation of otherwise soluble proteins to insoluble amyloid-like protein aggregates is a hallmark of neurodegenerative protein-misfolding disorders including Alzheimer's disease, CAG repeat diseases, and the prion encephalopathies, investigation of intracellular amyloid may likewise aid to a better understanding of the pathomechanisms involved. We consider aggregate profiling as an important experimental approach to determine if nuclear amyloid has toxic or protective roles in various disease processes.

Pathology and function of nuclear amyloid. Protein homeostasis matters.

In aging societies increasing cases of neurodegenerative protein deposit diseases urge for the identification of the underlying mechanisms. Expectations are that in 2050 the percentage of population over age 60 is 42% in Japan, 34% in China, and 27% in the US. The cell nucleus is a major target of amyloid-like protein fibrillation in a variety of disorders that are characterized by widespread aggregation of proteins with instable homopolymeric amino acid repeats, ubiquitin, and other proteinaceous components. Additionally, accumulation of insoluble, SDS-resistant proteins has been identified as an intrinsic property of organismal aging. This review collects current knowledge about the composition and function of insoluble, nuclear protein inclusions from the protein homeostasis perspective. It discusses the occurrence and role of nuclear amyloid in the diseased as well as the healthy cell. Features of nuclear inclusions such as protein composition and locally active protein degradation may predict neural fitness and survival in a variety of health or disease settings.

Effect of nanoparticles on the biochemical and behavioral aging phenotype of the nematode Caenorhabditis elegans.

Invertebrate animal models such as the nematode Caenorhabditis elegans (C. elegans) are increasingly used in nanotechnological applications. Research in this area covers a wide range from remote control of worm behavior by nanoparticles (NPs) to evaluation of organismal nanomaterial safety. Despite of the broad spectrum of investigated NP-bio interactions, little is known about the role of nanomaterials with respect to aging processes in C. elegans. We trace NPs in single cells of adult C. elegans and correlate particle distribution with the worm's metabolism and organ function. By confocal microscopy analysis of fluorescently labeled NPs in living worms, we identify two entry portals for the uptake of nanomaterials via the pharynx to the intestinal system and via the vulva to the reproductive system. NPs are localized throughout the cytoplasm and the cell nucleus in single intestinal, and vulval B and D cells. Silica NPs induce an untimely accumulation of insoluble ubiquitinated proteins, nuclear amyloid and reduction of pharyngeal pumping that taken together constitute a premature aging phenotype of C. elegans on the molecular and behavioral level, respectively. Screening of different nanomaterials for their effects on protein solubility shows that polystyrene or silver NPs do not induce accumulation of ubiquitinated proteins suggesting that alteration of protein homeostasis is a unique property of silica NPs. The nematode C. elegans represents an excellent model to investigate the effect of different types of nanomaterials on aging at the molecule, cell, and whole organism level.

Isochronal visualization of transcription and proteasomal proteolysis in cell culture or in the model organism, Caenorhabditis elegans.

Investigation of differential gene regulation by protein degradation requires analysis of the spatial and temporal association between proteolysis and transcription. Here, we describe the isochronal visualization of proteasomal proteolysis and transcription in cell culture or in vivo in the model organism Caenorhabditis elegans. This includes localization of proteasome-dependent proteolysis by fluorescent degradation products of model and endogenous substrates of the proteasome in combination with immunolabelling of RNA polymerase II and transcription in situ run-on assays.

Defining the subcellular interface of nanoparticles by live-cell imaging.

Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP) experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS) indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes.