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

Reduced Aβ 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, due to augmented BACE activity, would affect the actions of direct and indirect BACE inhibitors. We therefore compared postmitotic 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 Aβ1-40 levels. Several other approaches to modulate BACE activity led to a similar, apparently paradoxical, behavior. For instance, reduction of 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- vs. 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.publishe

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.publishe

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

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

International audienceBinary 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

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

International audienceWhile 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 mu M 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