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Synuclein Family Members Prevent Membrane Damage by Counteracting α-Synuclein Aggregation

May 9, 2022 | 2021, Biomolecules, Journal publications

Abstract

The 140 amino acid protein α-synuclein (αS) is an intrinsically disordered protein (IDP) with various roles and locations in healthy neurons that plays a key role in Parkinson’s disease (PD). Contact with biomembranes can lead to α-helical conformations, but can also act as s seeding event for aggregation and a predominant β-sheet conformation. In PD patients, αS is found to aggregate in various fibrillary structures, and the shift in aggregation and localization is associated with disease progression. Besides full-length αS, several related polypeptides are present in neurons. The role of many αS-related proteins in the aggregation of αS itself is not fully understood Two of these potential aggregation modifiers are the αS splicing variant αS Δexon3 (Δ3) and the paralog β-synuclein (βS). Here, polarized ATR-FTIR spectroscopy was used to study the membrane interaction of these proteins individually and in various combinations. The method allowed a continuous monitoring of both the lipid structure of biomimetic membranes and the aggregation state of αS and related proteins. The use of polarized light also revealed the orientation of secondary structure elements. While αS led to a destruction of the lipid membrane upon membrane-catalyzed aggregation, βS and Δ3 aggregated significantly less, and they did not harm the membrane. Moreover, the latter proteins reduced the membrane damage triggered by αS. There were no major differences in the membrane interaction for the different synuclein variants. In combination, these observations suggest that the formation of particular protein aggregates is the major driving force for αS-driven membrane damage. The misbalance of αS, βS, and Δ3 might therefore play a crucial role in neurodegenerative disease.

Specific Attenuation of Purinergic Signaling during Bortezomib-Induced Peripheral Neuropathy In Vitro

May 9, 2022 | 2022, International Journal of Molecular Sciences, Journal publications

Abstract

Human peripheral neuropathies are poorly understood, and the availability of experimental models limits further research. The PeriTox test uses immature dorsal root ganglia (DRG)-like neurons, derived from induced pluripotent stem cells (iPSC), to assess cell death and neurite damage. Here, we explored the suitability of matured peripheral neuron cultures for the detection of sub-cytotoxic endpoints, such as altered responses of pain-related P2X receptors. A two-step differentiation protocol, involving the transient expression of ectopic neurogenin-1 (NGN1) allowed for the generation of homogeneous cultures of sensory neurons. After >38 days of differentiation, they showed a robust response (Ca2+-signaling) to the P2X3 ligand α,β-methylene ATP. The clinical proteasome inhibitor bortezomib abolished the P2X3 signal at ≥5 nM, while 50−200 nM was required in the PeriTox test to identify neurite damage and cell death. A 24 h treatment with low nM concentrations of bortezomib led to moderate increases in resting cell intracellular Ca2+ concentration but signaling through transient receptor potential V1 (TRPV1) receptors or depolarization-triggered Ca2+ influx remained unaffected. We interpreted the specific attenuation of purinergic signaling as a functional cell stress response. A reorganization of tubulin to form dense structures around the cell somata confirmed a mild, non-cytotoxic stress triggered by low concentrations of bortezomib. The proteasome inhibitors carfilzomib, delanzomib, epoxomicin, and MG-132 showed similar stress responses. Thus, the model presented here may be used for the profiling of new proteasome inhibitors in regard to their side effect (neuropathy) potential, or for pharmacological studies on the attenuation of their neurotoxicity. P2X3 signaling proved useful as endpoint to assess potential neurotoxicants in peripheral neurons.

Next-generation risk assessment of chemicals – Rolling out a human-centric testing strategy to drive 3R implementation: The RISK-HUNT3R project perspective

May 9, 2022 | 2022, Altex, Journal publications

Abstract

In many industrial sectors, there is a need for reliable ways to evaluate the safety of chemicals with methods anchored to human biology and pathology. For this purpose, many animal-free new approach methods (NAMs) have been developed and implemented in various stages of risk assessment. Now it is time to assemble individual NAMs into a comprehensive next-generation risk assessment (NGRA) strategy. The European Horizon 2020 RISK-HUNT3R project (Risk assessment of chemicals integrating human-centric next-generation testing strategies promoting the 3Rs) has been designed to promote a combination of computational toxicology, in vitro toxicology, and systems biology. It is anticipated that this approach will lead to faster and more accurate risk assessment procedures. The RISK-HUNT3R NGRA strategy will be developed to address the implementation of a comprehensive NAM toolbox into the regulatory framework. Critical conceptual approaches of the project include i) the integration of human-relevant data on biotransformation and elimination, ii) the translation of high-content mode-of-action datasets into predictions of adverse outcomes, iii) development of quantitative adverse outcome pathways (qAOPs), and iv) quantification of uncertainties associated with the predictions based on NGRA strategies. Many of the project steps will be used iteratively to generate datasets with sufficient quality and certainty for NGRA. Scientists and regulators will work together on case studies to evaluate practical applicability of NAMs and strategies to combine information therefrom. Here we delineate how the strategy will be deployed to establish an overall NGRA framework for chemicals, pesticides, food additives, and drugs.

A framework for chemical safety assessment incorporating new approach methodologies within REACH

Mar 15, 2022 | 2022, Archives of Toxicology, Journal publications

Abstract

The long-term investment in new approach methodologies (NAMs) within the EU and other parts of the world is beginning to result in an emerging consensus of how to use information from in silico, in vitro and targeted in vivo sources to assess the safety of chemicals. However, this methodology is being adopted very slowly for regulatory purposes. Here, we have developed a framework incorporating in silico, in vitro and in vivo methods designed to meet the requirements of REACH in which both hazard and exposure can be assessed using a tiered approach. The outputs from each tier are classification categories, safe doses, and risk assessments, and progress through the tiers depends on the output from previous tiers. We have exemplified the use of the framework with three examples. The outputs were the same or more conservative than parallel assessments based on conventional studies. The framework allows a transparent and phased introduction of NAMs in chemical safety assessment and enables science-based safety decisions which provide the same level of public health protection using fewer animals, taking less time, and using less financial and expert resource. Furthermore, it would also allow new methods to be incorporated as they develop through continuous selective evolution rather than periodic revolution.

Circulating (Poly)phenol Metabolites: Neuroprotection in a 3D Cell Model of Parkinson’s Disease

Mar 15, 2022 | 2022, Journal publications, Molecular nutrition & Food Research

Abstract

Scope: Diets rich in (poly)phenols have been associated with positive effects on neurodegenerative disorders, such as Parkinson’s disease (PD). Several low-molecular weight (poly)phenol metabolites (LMWPM) are found in the plasma after consumption of (poly)phenol-rich food. It is expected that LMWPM, upon reaching the brain, may have beneficial effects against both oxidative stress and neuroinflammation, and possibly attenuate cell death mechanisms relate to the loss of dopaminergic neurons in PD.

Methods and results: This study investigates the neuroprotective potential of two blood-brain barrier permeant LMWPM, catechol-O-sulfate (cat-sulf), and pyrogallol-O-sulfate (pyr-sulf), in a human 3D cell model of PD. Neurospheroids were generated from LUHMES neuronal precursor cells and challenged by 1-methyl-4-phenylpyridinium (MPP+ ) to induce neuronal stress. LMWPM pretreatments were differently neuroprotective towards MPP+ insult, presenting distinct effects on the neuronal transcriptome. Particularly, cat-sulf pretreatment appeared to boost counter-regulatory defense mechanisms (preconditioning). When MPP+ is applied, both LMWPM positively modulated glutathione metabolism and heat-shock response, as also favorably shifting the balance of pro/anti-apoptotic proteins.

Conclusions: Our findings point to the potential of LMWPM to trigger molecular mechanisms that help dopaminergic neurons to cope with a subsequent toxic insult. They are promising molecules to be further explored in the context of preventing and attenuating parkinsonian neurodegeneration.

Keywords: dopaminergic neurons; gene expression; neurodegeneration; preconditioning; transcriptomics.

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

Mar 15, 2022 | 2021, Archives of Toxicology, News

Abstract

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.