BackgroundDevelopmental neurotoxicity (DNT) of environmental chemicals is a serious threat to human health. Current DNT testing guidelines propose investigations in rodents, which require large numbers of animals. With regard to the “3 Rs” (reduction, replacement, and refinement) of animal testing and the European regulation of chemicals [Registration, Evaluation, and Authorisation of Chemicals (REACH)], alternative testing strategies are needed in order to refine and reduce animal experiments and allow faster and less expensive screening.ObjectivesThe goal of this study was to establish a three-dimensional test system for DNT screening based on human fetal brain cells.MethodsWe established assays suitable for detecting disturbances in basic processes of brain development by employing human neural progenitor cells (hNPCs), which grow as neurospheres. Furthermore, we assessed effects of mercury and oxidative stress on these cells.ResultsWe found that human neurospheres imitate proliferation, differentiation, and migration in vitro. Exposure to the proapoptotic agent staurosporine further suggests that human neurospheres possess functioning apoptosis machinery. The developmental neurotoxicants methylmercury chloride and mercury chloride decreased migration distance and number of neuronal-like cells in differentiated hNPCs. Furthermore, hNPCs undergo caspase-independent apoptosis when exposed toward high amounts of oxidative stress.ConclusionsHuman neurospheres are likely to imitate basic processes of brain development, and these processes can be modulated by developmental neurotoxicants. Thus, this three-dimensional cell system is a promising approach for DNT testing.

The ubiquitin proteasome system plays a fundamental role in the regulation of cellular processes by degradation of endogenous proteins. Proteasomes are localized in both, the cytoplasm and the cell nucleus, however, little is known about nuclear proteolysis. Here, fluorogenic precursor substrates enabled detection of proteasomal activity in nucleoplasmic cell fractions (turnover 0.0541 μM/minute) and nuclei of living cells (turnover 0.0472 μM/minute). By contrast, cell fractions of nucleoli or nuclear envelopes did not contain proteasomal activity. Microinjection of ectopic fluorogenic protein DQ-ovalbumin revealed that proteasomal protein degradation occurs in distinct nucleoplasmic foci, which partially overlap with signature proteins of subnuclear domains, such as splicing speckles or promyelocytic leukemia bodies, ubiquitin, nucleoplasmic proteasomes and RNA polymerase II. Our results establish proteasomal proteolysis as an intrinsic function of the cell nucleus.

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

BackgroundBecause of their lipophilicity, persistent organic pollutants (POPs) cross the human placenta, possibly affecting central nervous system development. Most POPs are known aryl hydrocarbon receptor (AhR) ligands and activators of AhR signaling. Therefore, AhR activation has been suggested to cause developmental neurotoxicity (DNT).ObjectiveWe studied the effects of AhR ligands on basic processes of brain development in two comparative in vitro systems to determine whether AhR-activation is the underlying mechanism for reported DNT of POPs in humans.MethodsWe employed neurosphere cultures based on human neural progenitor cells (hNPCs) and wild-type and AhR-deficient mouse NPCs (mNPCs) and studied the effects of different AhR agonists [3-methylcholanthrene (3-MC), benzo(a)pyrene [B(a)P], and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)] and an antagonist [3′-methoxy-4′-nitroflavone (MNF)] on neurosphere development. Moreover, we analyzed expression of AhR and genes involved in AhR signaling.ResultsIn contrast to wild-type mNPCs, hNPCs and AhR-deficient mNPCs were insensitive to AhR agonism or antagonism. Although AhR modulation attenuated wild-type mNPC proliferation and migration, hNPCs and AhR-deficient mNPCs remained unaffected. Results also suggest that species-specific differences resulted from nonfunctional AhR signaling in hNPCs.ConclusionOur findings suggest that in contrast to wild-type mNPCs, hNPCs were protected against polycyclic aromatic hydrocarbon–induced DNT because of an absence of AhR. This difference may contribute to species-specific differences in sensitivity to POPs.

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. INTRODUCTIONThe bulk of nonlysosomal proteolysis is carried out by the ATP-powered 26S proteasome, which is involved in the regulation of major cellular processes such as progression of the cell cycle, transcription, flux of substrates through metabolic pathways, elimination of abnormal proteins, and antigen processing (Hershko and Ciechanover, 1998;Kloetzel, 2001). In most cultured mammalian cells 80 -90% of the protein breakdown occurs by the proteasome pathway (Lee and Goldberg, 1998). The 26S proteasome is composed of two distinct subcomplexes: the central 20S proteasome, in which proteins are degraded, and two flanking 19S complexes, which provide substrate specificity and regulation. The 20S proteasome forms the core subunit harboring multiple catalytic centers located within the hollow cavity of a cylinder (Finley, 2002). This topology sequesters the catalytic sites from potential substrates (Voges et al., 1999). Most of the substrates of the eukaryotic 26S proteasome must be marked by ubiquitination in order to be destroyed. This involves the covalent attachment of multiple ubiquitin molecules to the target protein (Ciechanover, 1998). However, exceptions to the rule such as ornithine decarboxylase (ODC), which is degraded by the 26S proteasome without ubiquitinylation are known, and more are currently under investigation (Murakami et al., 1992;Verma and Deshaies, 2000).Proteasomes generate oligopeptides, most of which are further degraded by distinct endopeptidases and aminopeptidases into amino acids. However, a fraction of these peptides escape complete destruction and are subjected to antigen presentation. The peptides are transported to the endoplasmic reticulum via the transporter associated with antigen pre...