The human pathogenic bacterium Clostridioides difficile produces two exotoxins TcdA and TcdB, which inactivate Rho GTPases thereby causing C. difficile‐associated diseases (CDAD) including life‐threatening pseudomembranous colitis. Hypervirulent strains produce additionally the binary actin ADP‐ribosylating toxin CDT. These strains are hallmarked by more severe forms of CDAD and increased frequency and severity. Once in the cytosol, the toxins act as enzymes resulting in the typical clinical symptoms. Therefore, targeting and inactivation of the released toxins are of peculiar interest. Prompted by earlier findings that human α‐defensin‐1 neutralizes TcdB, we investigated the effects of the defensin on all three C. difficile toxins. Inhibition of TcdA, TcdB, and CDT was demonstrated by analyzing toxin‐induced changes in cell morphology, substrate modification, and decrease in transepithelial electrical resistance. Application of α‐defensin‐1 protected cells and human intestinal organoids from the cytotoxic effects of TcdA, TcdB, CDT, and their combination which is attributed to a direct interaction between the toxins and α‐defensin‐1. In mice, the application of α‐defensin‐1 reduced the TcdA‐induced damage of intestinal loops in vivo. In conclusion, human α‐defensin‐1 is a specific and potent inhibitor of the C. difficile toxins and a promising agent to develop novel therapeutic options against C. difficile infections.

Prediction of drug toxicity on the human nervous system still relies mainly on animal experiments. Here, we developed an alternative system allowing assessment of complex signaling in both individual human neurons and on the network level. The LUHMES cultures used for our approach can be cultured in 384-well plates with high reproducibility. We established here high-throughput quantification of free intracellular Ca2+ concentrations [Ca2+]i as broadly applicable surrogate of neuronal activity and verified the main processes by patch clamp recordings. Initially, we characterized the expression pattern of many neuronal signaling components and selected the purinergic receptors to demonstrate the applicability of the [Ca2+]i signals for quantitative characterization of agonist and antagonist responses on classical ionotropic neurotransmitter receptors. This included receptor sub-typing and the characterization of the anti-parasitic drug suramin as modulator of the cellular response to ATP. To exemplify potential studies on ion channels, we characterized voltage-gated sodium channels and their inhibition by tetrodotoxin, saxitoxin and lidocaine, as well as their opening by the plant alkaloid veratridine and the food-relevant marine biotoxin ciguatoxin. Even broader applicability of [Ca2+]i quantification as an end point was demonstrated by measurements of dopamine transporter activity based on the membrane potential-changing activity of this neurotransmitter carrier. The substrates dopamine or amphetamine triggered [Ca2+]i oscillations that were synchronized over the entire culture dish. We identified compounds that modified these oscillations by interfering with various ion channels. Thus, this new test system allows multiple types of neuronal signaling, within and between cells, to be assessed, quantified and characterized for their potential disturbance.

The initial step in the amyloidogenic cascade of amyloid precursor protein (APP) processing is catalyzed by beta-site APP-cleaving enzyme (BACE), and this protease has increased activities in affected areas of Alzheimer's disease brains. We hypothesized that altered APP processing, because of augmented BACE activity, would affect the actions of direct and indirect BACE inhibitors. We therefore compared post-mitotic human neurons (LUHMES) with their BACE-overexpressing counterparts (BLUHMES). Although β-cleavage of APP was strongly increased in BLUHMES, they produced less full-length and truncated amyloid beta (Aβ) than LUHMES. Moreover, low concentrations of BACE inhibitors decreased cellular BACE activity as expected, but increased Aβ levels. Several other approaches to modulate BACE activity led to a similar, apparently paradoxical, behavior. For instance, reduction in intracellular acidification by bepridil increased Aβ production in parallel with decreased BACE activity. In contrast to BLUHMES, the respective control cells (LUHMES or BLUHMES with catalytically inactive BACE) showed conventional pharmacological responses. Other non-canonical neurochemical responses (so-called 'rebound effects') are well-documented for the Aβ pathway, especially for γ-secretase: a partial block of its activity leads to an increased Aβ secretion by some cell types. We therefore compared LUHMES and BLUHMES regarding rebound effects of γ-secretase inhibitors and found an Aβ rise in LUHMES but not in BLUHMES. Thus, different cellular factors are responsible for the γ-secretase- versus BACE-related Aβ rebound. We conclude that increased BACE activity, possibly accompanied by an altered cellular localization pattern, can dramatically influence Aβ generation in human neurons and affect pharmacological responses to secretase inhibitors. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Proteasome inhibition is associated with parkinsonian pathology in vivo and degeneration of dopaminergic neurons in vitro. We explored here the metabolome (386 metabolites) and transcriptome (3257 transcripts) regulations of human LUHMES neurons, following exposure to MG-132 [100 nM]. This proteasome inhibitor killed cells within 24 h but did not reduce viability for 12 h. Overall, 206 metabolites were changed in live neurons. The early (3 h) metabolome changes suggested a compromised energy metabolism. For instance, AMP, NADH and lactate were up-regulated, while glycolytic and citric acid cycle intermediates were down-regulated. At later time points, glutathione-related metabolites were up-regulated, most likely by an early oxidative stress response and activation of NRF2/ATF4 target genes. The transcriptome pattern confirmed proteostatic stress (fast up-regulation of proteasome subunits) and also suggested the progressive activation of additional stress response pathways. The early ones (e.g., HIF-1, NF-kB, HSF-1) can be considered a cytoprotective cellular counter-regulation, which maintained cell viability. For instance, a very strong up-regulation of AIFM2 (=FSP1) may have prevented fast ferroptotic death. For most of the initial period, a definite life–death decision was not taken, as neurons could be rescued for at least 10 h after the start of proteasome inhibition. Late responses involved p53 activation and catabolic processes such as a loss of pyrimidine synthesis intermediates. We interpret this as a phase of co-occurrence of protective and maladaptive cellular changes. Altogether, this combined metabolomics–transcriptomics analysis informs on responses triggered in neurons by proteasome dysfunction that may be targeted by novel therapeutic intervention in Parkinson’s disease.

This Project was performed to provide an in vitro Point of Departure (PoD) for an Adverse Outcome Pathway (AOP)‐informed Integrated Approach to Testing and Assessment (IATA), concerning a potential risk of parkinsonian motor deficits after long‐term exposure to Tebufenpyrad. The AOP considered was AOP:3. Assays were performed for Key Event (KE) 1, KE2 (mitochondrial dysfunction) and KE4 of the AOP, based on the use of human dopaminergic neurons (LUHMES cells). KE1 (inhibition of complex I of the mitochondrial respiratory chain) was considered equivalent to the molecular initiating event (MIE). KE4 (dopaminergic cell degeneration) was considered as alternative AO (target cell degeneration). All necessary and linear steps of the AOP (i.e. KE2,3,4) were investigated by experimental test methods. The three major objectives were: (i) generation of potential PoD data from each assay; (ii) evaluation and optimization of the consistency of these data; (iii) selection of a PoD to be used for further risk assessment. During the optimization phase, assay conditions that reflect brain metabolism (MitoMet conditions) were implemented, and degeneration of neurites was considered the most relevant neuropathological endpoint. With this setting, the potential PoD ranged from 6 nM to 45 nM. The selection of the PoD put emphasis on more chronic effects and on the assay closest to the AO. Thus the KE4 assay, measuring neurite degeneration under MitoMet conditions was chosen, and the PoD was 8 nM. This value has a confidence interval of about one log10‐fold change from the lowest to the highest estimate (range: 3 nM – 30 nM), based on the experimental variations we observed. Some biokinetics measurements were undertaken to help estimating cell concentrations. These data had a high uncertainty concerning the measurements and the model assumptions. Based on the assumptions we consider most realistic and robust, we suggest a cell/tissue (brain) concentration of 8‐40 nM to be associated with a potential onset of toxicity of Tebufenpyrad.