Titanium dioxide and carbon black nanoparticles disrupt neuronal homeostasis via excessive activation of cellular prion protein signaling.

BACKGROUND: Epidemiological emerging evidence shows that human exposure to some nanosized materials present in the environment would contribute to the onset and/or progression of Alzheimer's disease (AD). The cellular and molecular mechanisms whereby nanoparticles would exert some adverse effects towards neurons and take part in AD pathology are nevertheless unknown. RESULTS: Here, we provide the prime evidence that titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) bind the cellular form of the prion protein (PrPC), a plasma membrane protein well known for its implication in prion diseases and prion-like diseases, such as AD. The interaction between TiO2- or CB-NPs and PrPC at the surface of neuronal cells grown in culture corrupts PrPC signaling function. This triggers PrPC-dependent activation of NADPH oxidase and subsequent production of reactive oxygen species (ROS) that alters redox equilibrium. Through PrPC interaction, NPs also promote the activation of 3-phosphoinositide-dependent kinase 1 (PDK1), which in turn provokes the internalization of the neuroprotective TACE α-secretase. This diverts TACE cleavage activity away from (i) TNFα receptors (TNFR), whose accumulation at the plasma membrane augments the vulnerability of NP-exposed neuronal cells to TNFα -associated inflammation, and (ii) the amyloid precursor protein APP, leading to overproduction of neurotoxic amyloid Aß40/42 peptides. The silencing of PrPC or the pharmacological inhibition of PDK1 protects neuronal cells from TiO2- and CB-NPs effects regarding ROS production, TNFα hypersensitivity, and Aß rise. Finally, we show that dysregulation of the PrPC-PDK1-TACE pathway likely occurs in the brain of mice injected with TiO2-NPs by the intra-cerebro-ventricular route as we monitor a rise of TNFR at the cell surface of several groups of neurons located in distinct brain areas. CONCLUSION: Our in vitro and in vivo study thus posits for the first time normal cellular prion protein PrPC as being a neuronal receptor of TiO2- and CB-NPs and identifies PrPC-coupled signaling pathways by which those nanoparticles alter redox equilibrium, augment the intrinsic sensitivity of neurons to neuroinflammation, and provoke a rise of Aß peptides. By identifying signaling cascades dysregulated by TiO2- and CB-NPs in neurons, our data shed light on how human exposure to some NPs might be related to AD.

Roadbumps at the Crossroads of Integrating Behavioral and In Vitro Approaches for Neurotoxicity Assessment.

With the appreciation that behavior represents the integration and complexity of the nervous system, neurobehavioral phenotyping and assessment has seen a renaissance over the last couple of decades, resulting in a robust database on rodent performance within various testing paradigms, possible associations with human disorders, and therapeutic interventions. The interchange of data across behavior and other test modalities and multiple model systems has advanced our understanding of fundamental biology and mechanisms associated with normal functions and alterations in the nervous system. While there is a demonstrated value and power of neurobehavioral assessments for examining alterations due to genetic manipulations, maternal factors, early development environment, the applied use of behavior to assess environmental neurotoxicity continues to come under question as to whether behavior represents a sensitive endpoint for assessment. Why is rodent behavior a sensitive tool to the neuroscientist and yet, not when used in pre-clinical or chemical neurotoxicity studies? Applying new paradigms and evidence on the biological basis of behavior to neurobehavioral testing requires expertise and refinement of how such experiments are conducted to minimize variability and maximize information. This review presents relevant issues of methods used to conduct such test, sources of variability, experimental design, data analysis, interpretation, and reporting. It presents beneficial and critical limitations as they translate to the in vivo environment and considers the need to integrate across disciplines for the best value. It proposes that a refinement of behavioral assessments and understanding of subtle pronounced differences will facilitate the integration of data obtained across multiple approaches and to address issues of translation.

Prenatal exposure to paraquat and nanoscaled TiO2 aerosols alters the gene expression of the developing brain.

Nanopesticides are innovative pesticides involving engineered nanomaterials in their formulation to increase the efficiency of plant protection products, while mitigating their environmental impact. Despite the predicted growth of the nanopesticide use, no data is available on their inhalation toxicity and the potential cocktail effects between their components. In particular, the neurodevelopmental toxicity caused by prenatal exposures might have long lasting consequences. In the present study, we repeatedly exposed gestating mice in a whole-body exposure chamber to three aerosols, involving the paraquat herbicide, nanoscaled titanium dioxide particles (nTiO2), or a mixture of both. Particle number concentrations and total mass concentrations were followed to enable a metrological follow-up of the exposure sessions. Based on the aerosols characteristics, the alveolar deposited dose in mice was then estimated. RNA-seq was used to highlight dysregulations in the striatum of pups in response to the in utero exposure. Modifications in gene expression were identified at post-natal day 14, which might reflect neurodevelopmental alterations in this key brain area. The data suggest an alteration in the mitochondrial function following paraquat exposure, which is reminiscent of the pathological process leading to Parkinson disease. Markers of different cell lineages were dysregulated, showing effects, which were not limited to dopaminergic neurons. Exposure to the nTiO2 aerosol modulated the regulation of cytokines and neurotransmitters pathways, perhaps reflecting a minor neuroinflammation. No synergy was found between paraquat and nTiO2. Instead, the neurodevelopmental effects were surprisingly lower than the one measured for each substance separately.

The Challenges of 21st Century Neurotoxicology: The Case of Neurotoxicology Applied to Nanomaterials.

After a short background discussing engineered nanomaterials (ENMs) and their physicochemical properties and applications, the present perspective paper highlights the main specific points that need to be considered when examining the question of neurotoxicity of nanomaterials. It underlines the necessity to integrate parameters, specific tools, and tests from multiple sources that make neurotoxicology when applied to nanomaterials particularly complex. Bringing together the knowledge of multiple disciplines e.g., nanotoxicology to neurotoxicology, is necessary to build integrated neurotoxicology for the third decade of the 21st Century. This article focuses on the greatest challenges and opportunities offered by this specific field. It highlights the scientific, methodological, political, regulatory, and educational issues. Scientific and methodological challenges include the determination of ENMs physicochemical parameters, the lack of information about protein corona modes of action, target organs, and cells and dose- response functions of ENMs. The need of standardization of data collection and harmonization of dedicated neurotoxicological protocols are also addressed. This article highlights how to address those challenges through innovative methods and tools, and our work also ventures to sketch the first list of substances that should be urgently prioritized for human modern neurotoxicology. Finally, political support with dedicated funding at the national and international levels must also be used to engage the communities concerned to set up dedicated educational program on this novel field.

Chronic Oral Exposure to Synthetic Amorphous Silica (NM-200) Results in Renal and Liver Lesions in Mice.

INTRODUCTION: Silicon dioxide, produced as synthetic amorphous silica (SAS), is made of nanoparticles (NPs), either present as such or as agglomerates and aggregates, and is widely used in many types of food processes and products as an additive. To assess whether repeated, long-term exposure to SAS NPs may result in adverse effects, mice were exposed for 18 months via drinking water to NM-200, one of the reference nanostructured silica used for applications related to food, at 4.8 mg NM-200/kg body weight per day, a dose relevant to the estimated dietary exposure to SAS in humans. METHODS: The experiment focused on the kidney and liver as target organs and was carried out in parallel using 3 mouse lines (wild type and transgenic) differing for the expression of α-synuclein, that is, murine and human mutated (A53T). Sensitive determination of silicon revealed higher contents in liver and kidneys of NM-200-exposed mice compared with unexposed aged-matched controls. RESULTS: Histological abnormalities, such as vacuolization of tubular epithelial cells, were detected in all kidneys, as well as inflammatory responses that were also detected in livers of exposed animals. Less frequent but more deleterious, amyloidosis lesions were observed in glomeruli, associated with perivascular amyloid accumulation in liver. CONCLUSION: These histological findings, in conjunction with the observation of detectable deposition of silica, highlight that chronic oral intake of SAS may pose a health risk to humans and need to be examined further.

Nano- and neurotoxicology: An emerging discipline.

The present critical review analyzes the question of how nanoparticles from continuously growing industrial production and use of nanomaterials may impact human brain health. Available evidence suggests incomplete effectiveness of protective barriers of the brain against nanoparticles translocation to the brain. This raises concerns of potential effects of manufactured nanoparticles on brain functions, given that nanoparticle's potential to induce oxidative stress, inflammation, death by apoptosis, or changes in the level of expression of certain neurotransmitters. Most concerns have not been studied sufficiently and many questions are still open: Are the findings in animals transposable to humans? What happens when exposure is chronic or protracted? What happens to the developing brain when exposure occurs in utero? Are some nanoparticles more deleterious, given their ability to alter protein conformations and aggregation? Aside from developments in nanomedicine, the evidence already available fully justifies the need to specifically evaluate the interactions between nanoparticles and the nervous system. The available data clearly indicates the need for original dedicated experimental models and tools for neurotoxicological research on the one hand, and the need for epidemiological studies of neurodegenerative diseases in manufactured nanoparticle-exposed populations, on the other. A combination of nanotoxicology with neurology in a novel discipline, with its specific tools and methods of investigation, should enable answering still unresolved questions.

Oral Exposure to Paraquat Triggers Earlier Expression of Phosphorylated α-Synuclein in the Enteric Nervous System of A53T Mutant Human α-Synuclein Transgenic Mice.

The misfolded α-synuclein protein, phosphorylated at serine 129 (pSer129 α-syn), is the hallmark of Parkinson disease (PD). Detected also in the enteric nervous system (ENS), it supports the recent theory that PD could start in the gut, rather than the brain. In a previous study, using a transgenic mouse model of human synucleinopathies expressing the A53T mutant α-synuclein (TgM83), in which a neurodegenerative process associated with α-synuclein occurs spontaneously in the brain, we have shown earlier onset of pSer129 α-syn in the ENS. Here, we used this model to study the impact of paraquat (PQ) a neurotoxic herbicide incriminated in PD in agricultural workers) on the enteric pSer129 α-syn expression in young mice. Orally delivered in the drinking water at 10 mg/kg/day for 6-8 weeks, the impact of PQ was measured in a time-dependent manner on weight, locomotor abilities, pSer129 α-syn, and glial fibrillary acidic protein (GFAP) expression levels in the ENS. Remarkably, pSer129 α-syn was detected in ENS earlier under PQ oral exposure and enteric GFAP expression was also increased. These findings bring additional support to the theory that neurotoxic agents such as PQ initiate idiopathic PD after oral delivery.

Concerns regarding nanosized titanium dioxide nasal exposure and neurotoxicity study by Ze et al.

Scientific articles dealing with nanotoxicology are particularly awaited and important, as in the field of nanotechnology, a relatively new domain; they likely have a significant impact not only on our perception of the risk and danger potentially associated with nanomaterials but also on regulatory decisions with regard to their use in consumer products. Because nanotoxicology refers to the work of researchers from different domains of expertise, it appears also more difficult to secure accurate review and thus accurate conclusions. In that context, particular attention must be drawn on these studies. Still for the readers of such articles, of which most of are not experts in several aspects of nanotoxicology it would be tempting to take the messages for granted, thinking the studies are well conducted and reported with accuracy. In the case of the article by Ze and colleagues, we have noticed a certain number of critical comments that should be brought to the attention of the readers of J. Biomed Mater res A, because this study presents several problems.

Early and persistent expression of phosphorylated α-synuclein in the enteric nervous system of A53T mutant human α-synuclein transgenic mice.

Alpha-synuclein is a key protein in Parkinson disease (PD) and dementia with Lewy bodies. It is found in Lewy bodies in the brains of PD patients and has been reported in the peripheral nervous system in postmortem tissues from PD patients and in biopsies from patients in the preclinical phase of PD. Here, we used a transgenic mouse model of human synucleinopathies expressing the A53T mutant α-synuclein (TgM83) in which a neurodegenerative process associated with α-synuclein occurs spontaneously and increases with age. In particular, α-synuclein protein phosphorylated at serine 129 (pSer129 α-synuclein) naturally and progressively increases in diseased brains. We examined the time course of pSer129 α-synuclein presence in the gut of these mice between 1.5 and 22 months of age using immunohistochemistry and paraffin-embedded tissue blots. The pSer129 α-synuclein accumulated early (before the onset of motor signs) and persistently in the enteric nervous system and was concomitantly found in the brain. These results suggest that the accumulation of phosphorylated α-synuclein in the enteric and central nervous systems may result from parallel pathologic processes when the disease is linked to a mutation of α-synuclein.

[Is the brain protected from the impact of nanomaterial exposure?]. / Le cerveau est-il à l'abri d'un impact d'une exposition à des nanomatériaux ?

Beside natural and entropic nanoparticules (NPs), the engineered nanoparticules are now more and more present in many different industrial and medical applications. Notably, despite this fast development of the nanotechnologies, little is known about a possible impact on health and environment. Above all, the impact on human body and especially on the brain is not known. Among the possible ways of exposure to NPs, inhalation and ingestion are those probably the most effective to reach the brain. Until recently the scientific literature on the subject was very poor, but looking back to the last 10 years scientific productions, it is now possible to identify critical elements that should help to evaluate how well the brain is spared from a potential NP impact. First we recall some properties that characterize the nervous system as compared to other peripheral organs. Then we review the possible ways of exposure that lead to efficient direct and indirect translocation to the brain, we describe some significant data that allow to show which cells and in which sub-cellular compartment NPs are detected. We propose to review the NPs parameters that could favor translocation to the brain. Then with several examples we report the different types of neurotoxicity that have been described until now. Finally we raise several questions that need to be seriously evaluated through new experiments in order to complete our knowledge on the precise impact of NPs on brain function.

L-type bovine spongiform encephalopathy in genetically susceptible and resistant sheep: changes in prion strain or phenotypic plasticity of the disease-associated prion protein?

BACKGROUND: Sheep with prion protein (PrP) gene polymorphisms QQ171 and RQ171 were shown to be susceptible to the prion causing L-type bovine spongiform encephalopathy (L-BSE), although RQ171 sheep specifically propagated a distinctive prion molecular phenotype in their brains, characterized by a high molecular mass protease-resistant PrP fragment (HMM PrPres), distinct from L-BSE in QQ171 sheep. METHODS: The resulting infectious and biological properties of QQ171 and RQ171 ovine L-BSE prions were investigated in transgenic mice expressing either bovine or ovine PrP. RESULTS: In both mouse lines, ovine L-BSE transmitted similarly to cattle-derived L-BSE, with respect to survival periods, histopathology, and biochemical features of PrPres in the brain, as well as splenotropism, clearly differing from ovine classic BSE or from scrapie strain CH1641. Nevertheless and unexpectedly, HMM PrPres was found in the spleen of ovine PrP transgenic mice infected with L-BSE from RQ171 sheep at first passage, reminiscent, in lymphoid tissues only, of the distinct PrPres features found in RQ171 sheep brains. CONCLUSIONS: The L-BSE agent differs from both ovine classic BSE or CH1641 scrapie maintaining its specific strain properties after passage in sheep, although striking PrPres molecular changes could be found in RQ171 sheep and in the spleen of ovine PrP transgenic mice.

Unique properties of the classical bovine spongiform encephalopathy strain and its emergence from H-type bovine spongiform encephalopathy substantiated by VM transmission studies.

In addition to classical bovine spongiform encephalopathy (C-BSE), which is recognized as being at the origin of the human variant form of Creutzfeldt-Jakob disease, 2 rare phenotypes of BSE (H-type BSE [H-BSE] and L-type BSE [L-BSE]) were identified in 2004. H-type BSE and L-BSE are considered to be sporadic forms of prion disease in cattle because they differ from C-BSE with respect to incubation period, vacuolar pathology in the brain, and biochemical properties of the protease-resistant prion protein (PrP) in natural hosts and in some mouse models that have been tested. Recently, we showed that H-BSE transmitted to C57Bl/6 mice resulted in a dissociation of the phenotypic features, that is, some mice showed an H-BSE phenotype, whereas others had a C-BSE phenotype. Here, these 2 phenotypes were further studied in VM mice and compared with cattle C-BSE, H-BSE, and L-BSE. Serial passages from the C-BSE-like phenotype on VM mice retained similarities with C-BSE. Moreover, our results indicate that strains 301V and 301C derived from C-BSE transmitted to VM and C57Bl/6 mice, respectively, are fundamentally the same strain. These VM transmission studies confirm the unique properties of the C-BSE strain and support the emergence of a strain that resembles C-BSE from H-BSE.

Differentiation of prions from L-type BSE versus sporadic Creutzfeldt-Jakob disease.

We compared transmission characteristics for prions from L-type bovine spongiform encephalopathy and MM2-cortical sporadic Creutzfeldt-Jakob disease in the Syrian golden hamster and an ovine prion protein-transgenic mouse line and isolated distinct prion strains. Our findings suggest the absence of a causal relationship between these diseases, but further investigation is warranted.

Selection of distinct strain phenotypes in mice infected by ovine natural scrapie isolates similar to CH1641 experimental scrapie.

A few cases of transmissible spongiform encephalopathies in sheep have been described in France in which the protease-resistant prion protein (PrP(res)) exhibited some features in Western blot of experimental bovine spongiform encephalopathy in sheep. Their molecular characteristics were indistinguishable from those produced in the CH1641 experimental scrapie isolate. Four of these CH1641-like isolates were inoculated intracerebrally into wild-type C57Bl/6 mice. In striking contrast to previous results in ovine transgenic mice, CH1641 transmission in wild-type mice was efficient. Several components of the strain signature, that is, PrP(res) profile, brain distribution, and morphology of the deposits of the disease-associated prion protein, had some similarities with "classical" scrapie and clearly differed from both bovine spongiform encephalopathy in sheep and CH1641 transmission in ovine transgenic mice. These results on CH1641-like isolates in wild-type mice may be consistent with the presence in these isolates of mixed conformers with different abilities to propagate and mediate specific disease phenotypes in different species.

Prion-like acceleration of a synucleinopathy in a transgenic mouse model.

Our aim in this study was to investigate experimentally the possible in vivo transmission of a synucleinopathy, using a transgenic mouse model (TgM83) expressing the human A53T mutated α-synuclein. Brain homogenates from old TgM83 mice showing motor clinical signs due to the synucleinopathy and containing insoluble and phosphorylated (pSer129) α-synuclein were intracerebrally inoculated in young TgM83 mice. This triggered an early onset of characteristic motor clinical signs, compared with uninoculated TgM83 mice or to mice inoculated with a brain homogenate from a young, healthy TgM83 mouse. This early disease was associated with insoluble α-synuclein phosphorylated on Ser129, as already identified in old and sick uninoculated TgM83 transgenic mice. Although the molecular mechanisms remain to be determined, acceleration of the pathology following inoculation of mice expressing human mutated α-synuclein with tissues from mice affected by the synucleinopathy, could be consistent with "prion-like" propagation of the disease.

Stem cell therapy extends incubation and survival time in prion-infected mice in a time window-dependant manner.

Prion diseases, which are mostly represented in humans by Creutzfeldt-Jakob disease, are transmissible neurodegenerative disorders characterized by vacuolization and neuronal loss, as well as by the accumulation of an abnormal form of the prion protein. These disorders have yet no effective treatment, and drugs that block prion replication in vitro do not significantly slow down the progression of the disease when used in vivo at late stages. Cell therapy that has been already tested in other neurodegenerative disorders therefore represents an interesting alternative approach. In this study, we showed for the first time in prion diseases that intracerebral transplantation of fetal neural stem cells significantly extended both incubation and survival time. This result was dependant on the time window chosen for the engraftment and was obtained with both genetically modified and wild-type stem cells, therefore forging a path toward efficient stem cell therapy for human prion diseases.

Histopathological studies of "CH1641-like" scrapie sources versus classical scrapie and BSE transmitted to ovine transgenic mice (TgOvPrP4).

The possibility of the agent causing bovine spongiform encephalopathy (BSE) infecting small ruminants is of serious concern for human health. Among scrapie cases, the CH1641 source in particular appears to have certain biochemical properties similar to the BSE strain. In France, several natural scrapie cases were identified as "CH1641-like" natural scrapie isolates in sheep and goats. The Tg(OvPrP4) mouse line expressing the ovine prion protein is a sensitive model for studying and identifying strains of agents responsible for scrapie and BSE. This model is also very useful when studying specific scrapie source CH1641, known to be not transmissible to wild-type mice despite the similarity of some of its biochemical properties to those of the BSE strain. As it is important to be able to fully distinguish CH1641 from BSE, we herein report the histopathological data from CH1641 scrapie transmission experiments compared to specific cases of "CH1641-like" natural scrapie isolates in sheep, murine scrapie strains and BSE. In addition to the conventional vacuolar lesion profile approach and PrP(d) brain mappings, an innovative differential PET-blot analysis was introduced to classify the different strains of agent and revealed the first direct concordance between ways of grouping strains on the basis of PrP(d) biochemical characteristics.

Transmission of prion strains in a transgenic mouse model overexpressing human A53T mutated α-synuclein.

There is a growing interest in the potential roles of misfolded protein interactions in neurodegeneration. To investigate this issue, we inoculated 3 prion strains intracerebrally into transgenic (TgM83) mice that overexpress human A53T α-synuclein. In comparison to nontransgenic controls, there was a striking decrease in the incubation periods of scrapie, classic and H-type bovine spongiform encephalopathies(C-BSE and H-BSE), with conservation of the histopathologic and biochemical features characterizing these 3 prion strains. TgM83 mice died of scrapie or C-BSE prion diseases before accumulating the insoluble and phosphorylated forms of α-synuclein specific to late stages of synucleinopathy. In contrast, the median incubation time for TgM83 mice inoculated with H-BSE was comparable to that observed when these mice were uninfected, thereby allowing the development of molecular alterations of α-synuclein. The last 4 mice of this cohort exhibited early accumulations of H-BSE prion protein along with α-synuclein pathology. The results indicate that a prion disease was triggered concomitantly with an overt synucleinopathy in some transgenic mice overexpressing human A53T α-synuclein after intracerebral inoculation with an H-BSE prion strain.