Modeling ferroptosis in human dopaminergic neurons: Pitfalls and opportunities for neurodegeneration research.

The activation of ferroptosis is being pursued in cancer research as a strategy to target apoptosis-resistant cells. By contrast, in various diseases that affect the cardiovascular system, kidneys, liver, and central and peripheral nervous systems, attention is directed toward interventions that prevent ferroptotic cell death. Mechanistic insights into both research areas stem largely from studies using cellular in vitro models. However, intervention strategies that show promise in cellular test systems often fail in clinical trials, which raises concerns regarding the predictive validity of the utilized in vitro models. In this study, the human LUHMES cell line, which serves as a model for human dopaminergic neurons, was used to characterize factors influencing the activation of ferroptosis. Erastin and RSL-3 induced cell death that was distinct from apoptosis. Parameters such as the differentiation state of LUHMES cells, cell density, and the number and timing of medium changes were identified as determinants of sensitivity to ferroptosis activation. In differentiated LUHMES cells, interventions at mechanistically divergent sites (iron chelation, coenzyme Q10, peroxidase mimics, or inhibition of 12/15-lipoxygenase) provide almost complete protection from ferroptosis. LUHMES cells allowed the experimental modulation of intracellular iron concentrations and demonstrated a correlation between intracellular iron levels, the rate of lipid peroxidation, as well as the sensitivity of the cells to ferroptotic cell death. These findings underscore the importance of understanding the various factors that influence ferroptosis activation and highlight the need for well-characterized in vitro models to enhance the reliability and predictive value of observations in ferroptosis research, particularly when translating findings into in vivo contexts.

Non-canonical BIM-regulated energy metabolism determines drug-induced liver necrosis.

Paracetamol (acetaminophen, APAP) overdose severely damages mitochondria and triggers several apoptotic processes in hepatocytes, but the final outcome is fulminant necrotic cell death, resulting in acute liver failure and mortality. Here, we studied this switch of cell death modes and demonstrate a non-canonical role of the apoptosis-regulating BCL-2 homolog BIM/Bcl2l11 in promoting necrosis by regulating cellular bioenergetics. BIM deficiency enhanced total ATP production and shifted the bioenergetic profile towards glycolysis, resulting in persistent protection from APAP-induced liver injury. Modulation of glucose levels and deletion of Mitofusins confirmed that severe APAP toxicity occurs only in cells dependent on oxidative phosphorylation. Glycolytic hepatocytes maintained elevated ATP levels and reduced ROS, which enabled lysosomal recycling of damaged mitochondria by mitophagy. The present study highlights how metabolism and bioenergetics affect drug-induced liver toxicity, and identifies BIM as important regulator of glycolysis, mitochondrial respiration, and oxidative stress signaling.

Identification of the bacterial metabolite aerugine as potential trigger of human dopaminergic neurodegeneration.

The causes of nigrostriatal cell death in idiopathic Parkinson's disease are unknown, but exposure to toxic chemicals may play some role. We followed up here on suggestions that bacterial secondary metabolites might be selectively cytotoxic to dopaminergic neurons. Extracts from Streptomyces venezuelae were found to kill human dopaminergic neurons (LUHMES cells). Utilizing this model system as a bioassay, we identified a bacterial metabolite known as aerugine (C10H11NO2S; 2-[4-(hydroxymethyl)-4,5-dihydro-1,3-thiazol-2-yl]phenol) and confirmed this finding by chemical re-synthesis. This 2-hydroxyphenyl-thiazoline compound was previously shown to be a product of a wide-spread biosynthetic cluster also found in the human microbiome and in several pathogens. Aerugine triggered half-maximal dopaminergic neurotoxicity at 3-4 µM. It was less toxic for other neurons (10-20 µM), and non-toxic (at <100 µM) for common human cell lines. Neurotoxicity was completely prevented by several iron chelators, by distinct anti-oxidants and by a caspase inhibitor. In the Caenorhabditis elegans model organism, general survival was not affected by aerugine concentrations up to 100 µM. When transgenic worms, expressing green fluorescent protein only in their dopamine neurons, were exposed to aerugine, specific neurodegeneration was observed. The toxicant also exerted functional dopaminergic toxicity in nematodes as determined by the "basal slowing response" assay. Thus, our research has unveiled a bacterial metabolite with a remarkably selective toxicity toward human dopaminergic neurons in vitro and for the dopaminergic nervous system of Caenorhabditis elegans in vivo. These findings suggest that microbe-derived environmental chemicals should be further investigated for their role in the pathogenesis of Parkinson's disease.

Dynamic Metabolic and Transcriptional Responses of Proteasome-Inhibited Neurons.

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.

Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models.

Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies.

Human neuronal signaling and communication assays to assess functional neurotoxicity.

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.

Design and evaluation of bi-functional iron chelators for protection of dopaminergic neurons from toxicants.

While the etiology of non-familial Parkinson's disease (PD) remains unclear, there is evidence that increased levels of tissue iron may be a contributing factor. Moreover, exposure to some environmental toxicants is considered an additional risk factor. Therefore, brain-targeted iron chelators are of interest as antidotes for poisoning with dopaminergic toxicants, and as potential treatment of PD. We, therefore, designed a series of small molecules with high affinity for ferric iron and containing structural elements to allow their transport to the brain via the neutral amino acid transporter, LAT1 (SLC7A5). Five candidate molecules were synthesized and initially characterized for protection from ferroptosis in human neurons. The promising hydroxypyridinone SK4 was characterized further. Selective iron chelation within the physiological range of pH values and uptake by LAT1 were confirmed. Concentrations of 10-20 µM blocked neurite loss and cell demise triggered by the parkinsonian neurotoxicants, methyl-phenyl-pyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) in human dopaminergic neuronal cultures (LUHMES cells). Rescue was also observed when chelators were given after the toxicant. SK4 derivatives that either lacked LAT1 affinity or had reduced iron chelation potency showed altered activity in our assay panel, as expected. Thus, an iron chelator was developed that revealed neuroprotective properties, as assessed in several models. The data strongly support the role of iron in dopaminergic neurotoxicity and suggests further exploration of the proposed design strategy for improving brain iron chelation.

Human peptide α-defensin-1 interferes with Clostridioides difficile toxins TcdA, TcdB, and CDT.

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.

Reduced Aß secretion by human neurons under conditions of strongly increased BACE activity.

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 ß-cleavage of APP was strongly increased in BLUHMES, they produced less full-length and truncated amyloid beta (Aß) than LUHMES. Moreover, low concentrations of BACE inhibitors decreased cellular BACE activity as expected, but increased Aß1-40 levels. Several other approaches to modulate BACE activity led to a similar, apparently paradoxical, behavior. For instance, reduction in intracellular acidification by bepridil increased Aß 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 Aß pathway, especially for γ-secretase: a partial block of its activity leads to an increased Aß secretion by some cell types. We therefore compared LUHMES and BLUHMES regarding rebound effects of γ-secretase inhibitors and found an Aß rise in LUHMES but not in BLUHMES. Thus, different cellular factors are responsible for the γ-secretase- versus BACE-related Aß rebound. We conclude that increased BACE activity, possibly accompanied by an altered cellular localization pattern, can dramatically influence Aß 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/.

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.