PathwayNexus: a tool for interactive metabolic data analysis.

MOTIVATION: High-throughput omics methods increasingly result in large datasets including metabolomics data, which are often difficult to analyse. RESULTS: To help researchers to handle and analyse those datasets by mapping and investigating metabolomics data of multiple sampling conditions (e.g. different time points or treatments) in the context of pathways, PathwayNexus has been developed, which presents the mapping results in a matrix format, allowing users to easily observe the relations between the compounds and the pathways. It also offers functionalities like ranking, sorting, clustering, pathway views, and further analytical tools. Its primary objective is to condense large sets of pathways into smaller, more relevant subsets that align with the specific interests of the user. AVAILABILITY AND IMPLEMENTATION: The methodology presented here is implemented in PathwayNexus, an open-source add-on for Vanted available at www.cls.uni-konstanz.de/software/pathway-nexus. CONTACT: falk.schreiber@unikonstanz.de. SUPPLEMENTARY INFORMATION: Website: www.cls.uni-konstanz.de/software/pathway-nexus.

Transcriptional landscape of mitochondrial electron transport chain inhibition in renal cells.

Analysis of the transcriptomic alterations upon chemical challenge, provides in depth mechanistic information on the compound's toxic mode of action, by revealing specific pathway activation and other transcriptional modulations. Mapping changes in cellular behaviour to chemical insult, facilitates the characterisation of chemical hazard. In this study, we assessed the transcriptional landscape of mitochondrial impairment through the inhibition of the electron transport chain (ETC) in a human renal proximal tubular cell line (RPTEC/TERT1). We identified the unfolded protein response pathway (UPR), particularly the PERK/ATF4 branch as a common cellular response across ETC I, II and III inhibitions. This finding and the specific genes elaborated may aid the identification of mitochondrial liabilities of chemicals in both legacy data and prospective transcriptomic studies.

Mapping the cellular response to electron transport chain inhibitors reveals selective signaling networks triggered by mitochondrial perturbation.

Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes.

Neurotoxicity and underlying cellular changes of 21 mitochondrial respiratory chain inhibitors.

Inhibition of complex I of the mitochondrial respiratory chain (cI) by rotenone and methyl-phenylpyridinium (MPP +) leads to the degeneration of dopaminergic neurons in man and rodents. To formally describe this mechanism of toxicity, an adverse outcome pathway (AOP:3) has been developed that implies that any inhibitor of cI, or possibly of other parts of the respiratory chain, would have the potential to trigger parkinsonian motor deficits. We used here 21 pesticides, all of which are described in the literature as mitochondrial inhibitors, to study the general applicability of AOP:3 or of in vitro assays that are assessing its activation. Five cI, three complex II (cII), and five complex III (cIII) inhibitors were characterized in detail in human dopaminergic neuronal cell cultures. The NeuriTox assay, examining neurite damage in LUHMES cells, was used as in vitro proxy of the adverse outcome (AO), i.e., of dopaminergic neurodegeneration. This test provided data on whether test compounds were unspecific cytotoxicants or specifically neurotoxic, and it yielded potency data with respect to neurite degeneration. The pesticide panel was also examined in assays for the sequential key events (KE) leading to the AO, i.e., mitochondrial respiratory chain inhibition, mitochondrial dysfunction, and disturbed proteostasis. Data from KE assays were compared to the NeuriTox data (AO). The cII-inhibitory pesticides tested here did not appear to trigger the AOP:3 at all. Some of the cI/cIII inhibitors showed a consistent AOP activation response in all assays, while others did not. In general, there was a clear hierarchy of assay sensitivity: changes of gene expression (biomarker of neuronal stress) correlated well with NeuriTox data; mitochondrial failure (measured both by a mitochondrial membrane potential-sensitive dye and a respirometric assay) was about 10-260 times more sensitive than neurite damage (AO); cI/cIII activity was sometimes affected at > 1000 times lower concentrations than the neurites. These data suggest that the use of AOP:3 for hazard assessment has a number of caveats: (i) specific parkinsonian neurodegeneration cannot be easily predicted from assays of mitochondrial dysfunction; (ii) deriving a point-of-departure for risk assessment from early KE assays may overestimate toxicant potency.

Correction to: Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

In the original publication of the article.

Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals.

Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.

Development of a neurotoxicity assay that is tuned to detect mitochondrial toxicants.

Many neurotoxicants affect energy metabolism in man, but currently available test methods may still fail to predict mito- and neurotoxicity. We addressed this issue using LUHMES cells, i.e., human neuronal precursors that easily differentiate into mature neurons. Within the NeuriTox assay, they have been used to screen for neurotoxicants. Our new approach is based on culturing the cells in either glucose or galactose (Glc-Gal-NeuriTox) as the main carbohydrate source during toxicity testing. Using this Glc-Gal-NeuriTox assay, 52 mitochondrial and non-mitochondrial toxicants were tested. The panel of chemicals comprised 11 inhibitors of mitochondrial respiratory chain complex I (cI), 4 inhibitors of cII, 8 of cIII, and 2 of cIV; 8 toxicants were included as they are assumed to be mitochondrial uncouplers. In galactose, cells became more dependent on mitochondrial function, which made them 2-3 orders of magnitude more sensitive to various mitotoxicants. Moreover, galactose enhanced the specific neurotoxicity (destruction of neurites) compared to a general cytotoxicity (plasma membrane lysis) of the toxicants. The Glc-Gal-NeuriTox assay worked particularly well for inhibitors of cI and cIII, while the toxicity of uncouplers and non-mitochondrial toxicants did not differ significantly upon glucose ↔ galactose exchange. As a secondary assay, we developed a method to quantify the inhibition of all mitochondrial respiratory chain functions/complexes in LUHMES cells. The combination of the Glc-Gal-NeuriTox neurotoxicity screening assay with the mechanistic follow up of target site identification allowed both, a more sensitive detection of neurotoxicants and a sharper definition of the mode of action of mitochondrial toxicants.

Stage-specific metabolic features of differentiating neurons: Implications for toxicant sensitivity.

Developmental neurotoxicity (DNT) may be induced when chemicals disturb a key neurodevelopmental process, and many tests focus on this type of toxicity. Alternatively, DNT may occur when chemicals are cytotoxic only during a specific neurodevelopmental stage. The toxicant sensitivity is affected by the expression of toxicant targets and by resilience factors. Although cellular metabolism plays an important role, little is known how it changes during human neurogenesis, and how potential alterations affect toxicant sensitivity of mature vs. immature neurons. We used immature (d0) and mature (d6) LUHMES cells (dopaminergic human neurons) to provide initial answers to these questions. Transcriptome profiling and characterization of energy metabolism suggested a switch from predominantly glycolytic energy generation to a more pronounced contribution of the tricarboxylic acid cycle (TCA) during neuronal maturation. Therefore, we used pulsed stable isotope-resolved metabolomics (pSIRM) to determine intracellular metabolite pool sizes (concentrations), and isotopically non-stationary 13C-metabolic flux analysis (INST 13C-MFA) to calculate metabolic fluxes. We found that d0 cells mainly use glutamine to fuel the TCA. Furthermore, they rely on extracellular pyruvate to allow continuous growth. This metabolic situation does not allow for mitochondrial or glycolytic spare capacity, i.e. the ability to adapt energy generation to altered needs. Accordingly, neuronal precursor cells displayed a higher sensitivity to several mitochondrial toxicants than mature neurons differentiated from them. In summary, this study shows that precursor cells lose their glutamine dependency during differentiation while they gain flexibility of energy generation and thereby increase their resistance to low concentrations of mitochondrial toxicants.

Major changes of cell function and toxicant sensitivity in cultured cells undergoing mild, quasi-natural genetic drift.

Genomic drift affects the functional properties of cell lines, and the reproducibility of data from in vitro studies. While chromosomal aberrations and mutations in single pivotal genes are well explored, little is known about effects of minor, possibly pleiotropic, genome changes. We addressed this question for the human dopaminergic neuronal precursor cell line LUHMES by comparing two subpopulations (SP) maintained either at the American-Type-Culture-Collection (ATCC) or by the original provider (UKN). Drastic differences in susceptibility towards the specific dopaminergic toxicant 1-methyl-4-phenylpyridinium (MPP+) were observed. Whole-genome sequencing was performed to identify underlying genetic differences. While both SP had normal chromosome structures, they displayed about 70 differences on the level of amino acid changing events. Some of these differences were confirmed biochemically, but none offered a direct explanation for the altered toxicant sensitivity pattern. As second approach, markers known to be relevant for the intended use of the cells were specifically tested. The "ATCC" cells rapidly down-regulated the dopamine-transporter and tyrosine-hydroxylase after differentiation, while "UKN" cells maintained functional levels. As the respective genes were not altered themselves, we conclude that polygenic complex upstream changes can have drastic effects on biochemical features and toxicological responses of relatively similar SP of cells.

Definition of the Neurotoxicity-Associated Metabolic Signature Triggered by Berberine and Other Respiratory Chain Inhibitors.

To characterize the hits from a phenotypic neurotoxicity screen, we obtained transcriptomics data for valinomycin, diethylstilbestrol, colchicine, rotenone, 1-methyl-4-phenylpyridinium (MPP), carbaryl and berberine (Ber). For all compounds, the concentration triggering neurite degeneration correlated with the onset of gene expression changes. The mechanistically diverse toxicants caused similar patterns of gene regulation: the responses were dominated by cell de-differentiation and a triggering of canonical stress response pathways driven by ATF4 and NRF2. To obtain more detailed and specific information on the modes-of-action, the effects on energy metabolism (respiration and glycolysis) were measured. Ber, rotenone and MPP inhibited the mitochondrial respiratory chain and they shared complex I as the target. This group of toxicants was further evaluated by metabolomics under experimental conditions that did not deplete ATP. Ber (204 changed metabolites) showed similar effects as MPP and rotenone. The overall metabolic situation was characterized by oxidative stress, an over-abundance of NADH (>1000% increase) and a re-routing of metabolism in order to dispose of the nitrogen resulting from increased amino acid turnover. This unique overall pattern led to the accumulation of metabolites known as biomarkers of neurodegeneration (saccharopine, aminoadipate and branched-chain ketoacids). These findings suggest that neurotoxicity of mitochondrial inhibitors may result from an ensemble of metabolic changes rather than from a simple ATP depletion. The combi-omics approach used here provided richer and more specific MoA data than the more common transcriptomics analysis alone. As Ber, a human drug and food supplement, mimicked closely the mode-of-action of known neurotoxicants, its potential hazard requires further investigation.

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.

A quantitative AOP of mitochondrial toxicity based on data from three cell lines.

Adverse Outcome Pathways (AOPs) are increasingly used to support the integration of in vitro data in hazard assessment for chemicals. Quantitative AOPs (qAOPs) use mathematical models to describe the relationship between key events (KEs). In this paper, data obtained in three cell lines, LHUMES, HepG2 and RPTEC/TERT1, using similar experimental protocols, was used to calibrate a qAOP of mitochondrial toxicity for two chemicals, rotenone and deguelin. The objectives were to determine whether the same qAOP could be used for the three cell types, and to test chemical-independence by cross-validation with a dataset obtained on eight other chemicals in LHUMES cells. Repeating the calibration approach for both chemicals in three cell lines highlighted various practical difficulties. Even when the same readouts of KEs are measured, the mathematical functions used to describe the key event relationships may not be the same. Cross-validation in LHUMES cells was attempted by estimating chemical-specific potency at the molecular initiating events and using the rest of the calibrated qAOP to predict downstream KEs: toxicity of azoxystrobin, carboxine, mepronil and thifluzamide was underestimated. Selection of most relevant readouts and accurate characterization of the molecular initiating event for cross-validation are critical when designing in vitro experiments targeted at calibrating qAOPs.

New approach methods (NAMs) supporting read-across: Two neurotoxicity AOP-based IATA case studies

Read-across approaches are considered key in moving away from in vivo animal testing towards addressing data-gaps using new approach methods (NAMs). Ample successful examples are still required to substantiate this strategy. Here we present and discuss the learnings from two OECD IATA endorsed read-across case studies. They involve two classes of pesticides ­ rotenoids and strobilurins ­ each having a defined mode-of-action that is assessed for its neurological hazard by means of an AOP-based testing strategy coupled to toxicokinetic simulations of human tissue concentrations. The endpoint in question is potential mitochondrial respiratory chain mediated neurotoxicity, specifically through inhibition of complex I or III. An AOP linking inhibition of mitochondrial respiratory chain complex I to the degeneration of dopaminergic neurons formed the basis for both cases but was deployed in two different regulatory contexts. The two cases also exemplify several different read-across concepts: analogue versus category approach, consolidated versus putative AOP, positive versus negative prediction (i.e., neurotoxicity versus low potential for neurotoxicity), and structural versus biological similarity. We applied a range of NAMs to explore the toxicodynamic properties of the compounds, e.g., in silico docking as well as in vitro assays and readouts ­ including transcriptomics ­ in various cell systems, all anchored to the relevant AOPs. Interestingly, although some of the data addressing certain elements of the read-across were associated with high uncertainty, their impact on the overall read-across conclusion remained limited. Coupled to the elaborate regulatory review that the two cases underwent, we propose some generic learnings of AOP-based testing strategies supporting read-across.

Shortened derivatives from native antimicrobial peptide LyeTx I: In vitro and in vivo biological activity assessment.

In the continuing search for novel antibiotics, antimicrobial peptides are promising molecules, due to different mechanisms of action compared to classic antibiotics and to their selectivity for interaction with microorganism cells rather than with mammalian cells. Previously, our research group has isolated the antimicrobial peptide LyeTx I from the venom of the spider Lycosa erythrognatha. Here, we proposed to synthesize three novel shortened derivatives from LyeTx I (LyeTx I mn; LyeTx I mnΔK; LyeTx I mnΔKAc) and to evaluate their toxicity and biological activity as potential antimicrobial agents. Peptides were synthetized by Fmoc strategy and circular dichroism analysis was performed, showing that the three novel shortened derivatives may present membranolytic activity, like the original LyeTx I, once they folded as an alpha helix in 2.2.2-trifluorethanol and sodium dodecyl sulfate. In vitro assays revealed that the shortened derivative LyeTx I mnΔK presents the best score between antimicrobial (↓ MIC) and hemolytic (↑ EC50) activities among the synthetized shortened derivatives, and LUHMES cell-based NeuriTox test showed that it is less neurotoxic than the original LyeTx I (EC50 [LyeTx I mnΔK] ⋙ EC50 [LyeTx I]). In vivo data, obtained in a mouse model of septic arthritis induced by Staphylococcus aureus, showed that LyeTx I mnΔK is able to reduce infection, as demonstrated by bacterial recovery assay (∼10-fold reduction) and scintigraphic imaging (less technetium-99m labeled-Ceftizoxime uptake by infectious site). Infection reduction led to inflammatory process and pain decreases, as shown by immune cells recruitment reduction and threshold nociception increment, when compared to positive control group. Therefore, among the three shortened peptide derivatives, LyeTx I mnΔK is the best candidate as antimicrobial agent, due to its smaller amino acid sequence and toxicity, and its greater biological activity.

Pharmacological LRH-1/Nr5a2 inhibition limits pro-inflammatory cytokine production in macrophages and associated experimental hepatitis.

Liver receptor homolog-1 (LRH-1, Nr5a2) is an orphan nuclear receptor mainly expressed in tissues of endodermal origin, where its physiological role has been extensively studied. LRH-1 has been implicated in liver cell differentiation and proliferation, as well as glucose, lipid, and bile acid metabolism. In addition, increasing evidence highlights its role in immunoregulatory processes via glucocorticoid synthesis in the intestinal epithelium. Although the direct function of LRH-1 in immune cells is fairly elucidated, a role of LRH-1 in the regulation of macrophage differentiation has been recently reported. In this study, we aimed to investigate the role of LRH-1 in the regulation of pro-inflammatory cytokine production in macrophages. Our data demonstrate that pharmacological inhibition, along with LRH-1 knockdown, significantly reduced the lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines in the macrophage line RAW 264.7 cells, as well as in primary murine macrophages. This inhibitory effect was found to be independent of defects of LRH-1-regulated cell proliferation or toxic effects of the LRH-1 inhibitors. In contrast, LRH-1 inhibition reduced the mitochondrial ATP production and metabolism of macrophages through downregulation of the LRH-1 targets glucokinase and glutminase-2, and thus impairing the LPS-induced macrophage activation. Interestingly, in vivo pharmacological inhibition of LRH-1 also resulted in reduced tumor necrosis factor (TNF) production and associated decreased liver damage in a macrophage- and TNF-dependent mouse model of hepatitis. Noteworthy, despite hepatocytes expressing high levels of LRH-1, pharmacological inhibition of LRH-1 per se did not cause any obvious liver damage. Therefore, this study proposes LRH-1 as an emerging therapeutic target in the treatment of inflammatory disorders, especially where macrophages and cytokines critically decide the extent of inflammation.

Reductive modification of genetically encoded 3-nitrotyrosine sites in alpha synuclein expressed in E.coli.

Tyrosine nitration is a post-translational protein modification relevant to various pathophysiological processes. Chemical nitration procedures have been used to generate and study nitrated proteins, but these methods regularly lead to modifications at other amino acid residues. A novel strategy employs a genetic code modification that allows incorporation of 3-nitrotyrosine (3-NT) during ribosomal protein synthesis to generate a recombinant protein with defined 3-NT-sites, in the absence of other post-translational modifications. This approach was applied to study the generation and stability of the 3-NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (ASYN) was selected as exemplary protein, relevant in Parkinson's disease (PD). A procedure was established to obtain pure tyrosine-modified ASYN in mg amounts. However, a rapid (t1/2 = 0.4 h) reduction of 3-NT to 3-aminotyrosine (3-AT) was observed. When screening for potential mechanisms, we found that 3-NT can be reduced enzymatically to 3-AT, whilst biologically relevant low molecular weight reductants, such as NADPH or GSH, did not affect 3-NT. A genetic screen for E.coli proteins, involved in the observed 3-NT reduction, revealed the contribution of several, possibly redundant pathways. Green fluorescent protein was studied as an alternative model protein. These data confirm 3-NT reduction as a broadly-relevant pathway in E.coli. In conclusion, incorporation of 3-NT as a genetically-encoded non-natural amino acid allows for generation of recombinant proteins with specific nitration sites. The potential reduction of the 3-NT moiety by E.coli, however, requires attention to the design of the purification strategy for obtaining pure nitrated protein.

Corrigendum to A high-throughput approach to identify specific neurotoxicants / developmental toxicants in human neuronal cell function assays.

In this manuscript, which appeared in ALTEX 35 , 235-253 ( doi:10.14573/altex.1712182 ), the Acknowledgements should read: This work was supported by the Land BW, the Doerenkamp-Zbinden Foundation, the DFG (RTG1331, KoRS-CB), the BMBF (NeuriTox), and it has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 681002 (EU-ToxRisk).

Prevention of neuronal apoptosis by astrocytes through thiol-mediated stress response modulation and accelerated recovery from proteotoxic stress.

The development of drugs directly interfering with neurodegeneration has proven to be astonishingly difficult. Alternative therapeutic approaches could result from a better understanding of the supportive function of glial cells for stressed neurons. Therefore, here, we investigated the mechanisms involved in the endogenous neuro-defensive activity of astrocytes. A well-established model of postmitotic human dopaminergic neurons (LUHMES cells) was used in the absence ('LUHMES' mono-culture) or presence ('co-culture') of astrocytes. Inhibition of the LUHMES proteasome led to proteotoxic (protein aggregates; ATF-4 induction) and oxidative (GSH-depletion; NRF-2 induction) stress, followed by neuronal apoptosis. The presence of astrocytes attenuated the neuronal stress response, and drastically reduced neurodegeneration. A similar difference between LUHMES mono- and co-cultures was observed, when proteotoxic and oxidative stress was triggered indirectly by inhibitors of mitochondrial function (rotenone, MPP+). Human and murine astrocytes continuously released glutathione (GSH) into the medium, and transfer of glia-conditioned medium was sufficient to rescue LUHMES, unless it was depleted for GSH. Also, direct addition of GSH to LUHMES rescued the neurons from inhibition of the proteasome. Both astrocytes and GSH blunted the neuronal ATF-4 response and similarly upregulated NRF-1/NFE2L1, a transcription factor counter-regulating neuronal proteotoxic stress. Astrocyte co-culture also helped to recover the neurons' ability to degrade aggregated poly-ubiquitinated proteins. Overexpression of NRF-1 attenuated the toxicity of proteasome inhibition, while knockdown increased toxicity. Thus, astrocytic thiol supply increased neuronal resilience to various proteotoxic stressors by simultaneously attenuating cell death-related stress responses, and enhancing the recovery from proteotoxic stress through upregulation of NRF-1.

Canagliflozin mediated dual inhibition of mitochondrial glutamate dehydrogenase and complex I: an off-target adverse effect.

Recent FDA Drug Safety Communications report an increased risk for acute kidney injury in patients treated with the gliflozin class of sodium/glucose co-transport inhibitors indicated for treatment of type 2 diabetes mellitus. To identify a potential rationale for the latter, we used an in vitro human renal proximal tubule epithelial cell model system (RPTEC/TERT1), physiologically representing human renal proximal tubule function. A targeted metabolomics approach, contrasting gliflozins to inhibitors of central carbon metabolism and mitochondrial function, revealed a double mode of action for canagliflozin, but not for its analogs dapagliflozin and empagliflozin. Canagliflozin inhibited the glutamate dehydrogenase (GDH) and mitochondrial electron transport chain (ETC) complex I at clinically relevant concentrations. This dual inhibition specifically prevented replenishment of tricarboxylic acid cycle metabolites by glutamine (anaplerosis) and thus altered amino acid pools by increasing compensatory transamination reactions. Consequently, canagliflozin caused a characteristic intracellular accumulation of glutamine, glutamate and alanine in confluent, quiescent RPTEC/TERT1. Canagliflozin, but none of the classical ETC inhibitors, induced cytotoxicity at particularly low concentrations in proliferating RPTEC/TERT1, serving as model for proximal tubule regeneration in situ. This finding is testimony of the strong dependence of proliferating cells on glutamine anaplerosis via GDH. Our discovery of canagliflozin-mediated simultaneous inhibition of GDH and ETC complex I in renal cells at clinically relevant concentrations, and their particular susceptibility to necrotic cell death during proliferation, provides a mechanistic rationale for the adverse effects observed especially in patients with preexisting chronic kidney disease or previous kidney injury characterized by sustained regenerative tubular epithelial cell proliferation.