Publications by authors named "Konstanze F Winklhofer"

83 Publications

Increased ROS-Dependent Fission of Mitochondria Causes Abnormal Morphology of the Cell Powerhouses in a Murine Model of Amyotrophic Lateral Sclerosis.

Oxid Med Cell Longev 2021 14;2021:6924251. Epub 2021 Oct 14.

Department of Cytology, Institute of Anatomy, Medical Faculty, Ruhr University Bochum, D-44801 Bochum, Germany.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in humans and remains to have a fatal prognosis. Recent studies in animal models and human ALS patients indicate that increased reactive oxygen species (ROS) play an important role in the pathogenesis. Considering previous studies revealing the influence of ROS on mitochondrial physiology, our attention was focused on mitochondria in the murine ALS model, wobbler mouse. The aim of this study was to investigate morphological differences between wild-type and wobbler mitochondria with aid of superresolution structured illumination fluorescence microscopy, TEM, and TEM tomography. To get an insight into mitochondrial dynamics, expression studies of corresponding proteins were performed. Here, we found significantly smaller and degenerated mitochondria in wobbler motor neurons at a stable stage of the disease. Our data suggest a ROS-regulated, Ox-CaMKII-dependent Drp1 activation leading to disrupted fission-fusion balance, resulting in fragmented mitochondria. These changes are associated with numerous impairments, resulting in an overall self-reinforcing decline of motor neurons. In summary, our study provides common pathomechanisms with other ALS models and human ALS cases confirming mitochondria and related dysfunctions as a therapeutic target for the treatment of ALS.
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http://dx.doi.org/10.1155/2021/6924251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8531774PMC
October 2021

Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol.

EMBO J 2021 08 30;40(16):e107913. Epub 2021 Jun 30.

Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany.

The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient samples and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but causalities remain unclear. We used Saccharomyces cerevisiae to analyze how mitochondrial processes regulate the behavior of aggregation-prone polyQ protein derived from human huntingtin. Expression of Q97-GFP rapidly led to insoluble cytosolic aggregates and cell death. Although aggregation impaired mitochondrial respiration only slightly, it considerably interfered with the import of mitochondrial precursor proteins. Mutants in the import component Mia40 were hypersensitive to Q97-GFP, whereas Mia40 overexpression strongly suppressed the formation of toxic Q97-GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the post-translational import of mitochondrial precursor proteins into mitochondria competes with aggregation-prone cytosolic proteins for chaperones and proteasome capacity. Mia40 regulates this competition as it has a rate-limiting role in mitochondrial protein import. Therefore, Mia40 is a dynamic regulator in mitochondrial biogenesis that can be exploited to stabilize cytosolic proteostasis.
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http://dx.doi.org/10.15252/embj.2021107913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8365258PMC
August 2021

Remodeling of the Fibrillation Pathway of α-Synuclein by Interaction with Antimicrobial Peptide LL-III.

Chemistry 2021 Aug 22;27(46):11845-11851. Epub 2021 Jul 22.

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227, Dortmund, Germany.

Liquid-liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization, and many recent studies have provided important insights into the role of LLPS in cell biology. There is also evidence that LLPS is associated with a variety of medical conditions, including neurodegenerative disorders. Pathological aggregation of α-synuclein, which is causally linked to Parkinson's disease, can proceed via droplet condensation, which then gradually transitions to the amyloid state. We show that the antimicrobial peptide LL-III is able to interact with both monomers and condensates of α-synuclein, leading to stabilization of the droplet and preventing conversion to the fibrillar state. The anti-aggregation activity of LL-III was also confirmed in a cellular model. We anticipate that studying the interaction of antimicrobial-type peptides with liquid condensates such as α-synuclein will contribute to the understanding of disease mechanisms (that arise in such condensates) and may also open up exciting new avenues for intervention.
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http://dx.doi.org/10.1002/chem.202101592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8457056PMC
August 2021

The N-terminal domain of the prion protein is required and sufficient for liquid-liquid phase separation: A crucial role of the Aβ-binding domain.

J Biol Chem 2021 07 6;297(1):100860. Epub 2021 Jun 6.

Department of Biochemistry of Neurodegenerative Diseases, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany; Cluster of Excellence RESOLV, Ruhr University Bochum, Bochum, Germany. Electronic address:

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrP). PrP is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrP is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aβ oligomers. PrP can undergo LLPS; however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here, we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similar to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aβ-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structures. Through a mutational analysis, we identified three positively charged lysines in the postoctarepeat region as essential drivers of condensate formation, presumably largely via cation-π interactions. These findings provide insights into the molecular basis of LLPS of the mammalian prion protein and reveal a crucial role of the Aβ-binding domain in this process.
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http://dx.doi.org/10.1016/j.jbc.2021.100860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254114PMC
July 2021

Hypochlorous acid-modified human serum albumin suppresses MHC class II - dependent antigen presentation in pro-inflammatory macrophages.

Redox Biol 2021 07 20;43:101981. Epub 2021 Apr 20.

Ruhr University Bochum, Institute of Biochemistry and Pathobiochemistry - Microbial Biochemistry, Universitätsstrasse 150, 44780, Bochum, Germany. Electronic address:

Macrophages are innate immune cells that internalize and present exogenous antigens to T cells via MHC class II proteins. They operate at sites of infection in a highly inflammatory environment, generated in part by reactive oxygen species, in particular the strong oxidant hypochlorous acid (HOCl) produced in the neutrophil respiratory burst. HOCl effectively kills a broad range of pathogens but can also contribute to host tissue damage at sites of inflammation. To prevent tissue injury, HOCl is scavenged by human serum albumin (HSA) and other plasma proteins in interstitial fluids, leading to the formation of variously modified advanced oxidation products (AOPPs) with pro-inflammatory properties. Previously, we showed that HOCl-mediated N-chlorination converts HSA and other plasma proteins into efficient activators of the phagocyte respiratory burst, but the role of these AOPPs in antigen presentation by macrophages remained unclear. Here, we show that physiologically relevant amounts of N-chlorinated HSA can strongly impair the capacity of THP-1-derived macrophages to present antigens to antigen-specific T cells via MHC class II proteins at multiple stages. Initially, N-chlorinated HSA inhibits antigen internalization by converting antigens into scavenger receptor (SR) ligands and competing with the modified antigens for binding to SR CD36. Later steps of antigen presentation, such as intracellular antigen processing and MHC class II expression are negatively affected, as well. We propose that impaired processing of pathogens or exogenous antigens by immune cells at an initial stage of infection prevents antigen presentation in an environment potentially hostile to cells of the adaptive immune response, possibly shifting it towards locations removed from the actual insult, like the lymph nodes. On the flip side, excessive retardation or complete inhibition of antigen presentation by N-chlorinated plasma proteins could contribute to chronic infection and inflammation.
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http://dx.doi.org/10.1016/j.redox.2021.101981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105673PMC
July 2021

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease.

PLoS Genet 2021 04 15;17(4):e1009479. Epub 2021 Apr 15.

Institute of Science and Technology Austria (IST Austria), Klosterneuburg, Austria.

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson's disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.
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http://dx.doi.org/10.1371/journal.pgen.1009479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049241PMC
April 2021

Amyloid precursor protein elevates fusion of promyelocytic leukemia nuclear bodies in human hippocampal areas with high plaque load.

Acta Neuropathol Commun 2021 04 13;9(1):66. Epub 2021 Apr 13.

Department of Molecular Biochemistry, Cell Signalling, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany.

The amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological function but potential impact in neurodegeneration. The current study demonstrates that APP signals to the nucleus causing the generation of aggregates consisting of its adapter protein FE65, the histone acetyltransferase TIP60 and the tumour suppressor proteins p53 and PML. APP C-terminal (APP-CT50) complexes co-localize and co-precipitate with p53 and PML. The PML nuclear body generation is induced and fusion occurs over time depending on APP signalling and STED imaging revealed active gene expression within the complex. We further show that the nuclear aggregates of APP-CT50 fragments together with PML and FE65 are present in the aged human brain but not in cerebral organoids differentiated from iPS cells. Notably, human Alzheimer's disease brains reveal a highly significant reduction of these nuclear aggregates in areas with high plaque load compared to plaque-free areas of the same individual. Based on these results we conclude that APP-CT50 signalling to the nucleus takes place in the aged human brain and is involved in the pathophysiology of AD.
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http://dx.doi.org/10.1186/s40478-021-01174-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042982PMC
April 2021

The key role of solvent in condensation: Mapping water in liquid-liquid phase-separated FUS.

Biophys J 2021 04 28;120(7):1266-1275. Epub 2021 Jan 28.

Department Physical Chemistry, Ruhr-University Bochum, Bochum, Germany. Electronic address:

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has emerged as a pervasive principle in cell biology, allowing compartmentalization and spatiotemporal regulation of dynamic cellular processes. Proteins that form condensates under physiological conditions often contain intrinsically disordered regions with low-complexity domains. Among them, the RNA-binding proteins FUS and TDP-43 have been a focus of intense investigation because aberrant condensation and aggregation of these proteins is linked to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. LLPS occurs when protein-rich condensates form surrounded by a dilute aqueous solution. LLPS is per se entropically unfavorable. Energetically favorable multivalent protein-protein interactions are one important aspect to offset entropic costs. Another proposed aspect is the release of entropically unfavorable preordered hydration water into the bulk. We used attenuated total reflection spectroscopy in the terahertz frequency range to characterize the changes in the hydrogen bonding network accompanying the FUS enrichment in liquid-liquid phase-separated droplets to provide experimental evidence for the key role of the solvent as a thermodynamic driving force. The FUS concentration inside LLPS droplets was determined to be increased to 2.0 mM independent of the initial protein concentration (5 or 10 μM solutions) by fluorescence measurements. With terahertz spectroscopy, we revealed a dewetting of hydrophobic side chains in phase-separated FUS. Thus, the release of entropically unfavorable water populations into the bulk goes hand in hand with enthalpically favorable protein-protein interaction. Both changes are energetically favorable, and our study shows that both contribute to the thermodynamic driving force in phase separation.
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http://dx.doi.org/10.1016/j.bpj.2021.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8059208PMC
April 2021

Protein quality control by the proteasome and autophagy: A regulatory role of ubiquitin and liquid-liquid phase separation.

Matrix Biol 2021 06 28;100-101:9-22. Epub 2020 Nov 28.

Department of Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Universitaetsstrasse 150, 44801 Bochum, Germany. Electronic address:

Degradation of dysfunctional, damaged, or misfolded proteins is a crucial component of the protein quality control network to maintain cellular proteostasis. Dysfunction in proteostasis regulation due to imbalances in protein synthesis, folding, and degradation challenges the integrity of the cellular proteome and favors the accumulation of aggregated proteins that can damage cells by a loss of their functions and/or a gain of adverse functions. Ubiquitination is an essential player in proteostasis regulation but also in orchestrating signaling pathways in response to various stress conditions. Both cellular degradation systems, the proteasome and autophagy, employ ubiquitin for selection and targeting of substrates to the degradative machineries. Here we summarize the manifold functions of ubiquitin in protein degradation and discuss its emerging role in the formation of biomolecular condensates through liquid-liquid phase separation, which allows spatiotemporal regulation of protein quality control.
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http://dx.doi.org/10.1016/j.matbio.2020.11.003DOI Listing
June 2021

SecY-mediated quality control prevents the translocation of non-gated porins.

Sci Rep 2020 10 1;10(1):16347. Epub 2020 Oct 1.

Department Biochemistry of Neurodegenerative Diseases, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Universitätsstr. 150, 44801, Bochum, Germany.

OmpC and OmpF are among the most abundant outer membrane proteins in E. coli and serve as hydrophilic channels to mediate uptake of small molecules including antibiotics. Influx selectivity is controlled by the so-called constriction zone or eyelet of the channel. Mutations in the loop domain forming the eyelet can disrupt transport selectivity and thereby interfere with bacterial viability. In this study we show that a highly conserved motif of five negatively charged amino acids in the eyelet, which is critical to regulate pore selectivity, is also required for SecY-mediated transport of OmpC and OmpF into the periplasm. Variants with a deleted or mutated motif were expressed in the cytosol and translocation was initiated. However, after signal peptide cleavage, import into the periplasm was aborted and the mutated proteins were redirected to the cytosol. Strikingly, reducing the proof-reading capacity of SecY by introducing the PrlA4 substitutions restored transport of OmpC with a mutated channel domain into the periplasm. Our study identified a SecY-mediated quality control pathway to restrict transport of outer membrane porin proteins with a deregulated channel activity into the periplasm.
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http://dx.doi.org/10.1038/s41598-020-73185-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530735PMC
October 2020

PINK1 and Parkin: team players in stress-induced mitophagy.

Biol Chem 2020 05;401(6-7):891-899

Department of Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Universitätsstrasse 150, D-44801, Bochum, Germany.

Mitochondria are highly vulnerable organelles based on their complex biogenesis, entailing dependence on nuclear gene expression and efficient import strategies. They are implicated in a wide spectrum of vital cellular functions, including oxidative phosphorylation, iron-sulfur cluster synthesis, regulation of calcium homeostasis, and apoptosis. Moreover, damaged mitochondria can release mitochondrial components, such as mtDNA or cardiolipin, which are sensed as danger-associated molecular patterns and trigger innate immune signaling. Thus, dysfunctional mitochondria pose a thread not only to the cellular but also to the organismal integrity. The elimination of dysfunctional and damaged mitochondria by selective autophagy, called mitophagy, is a major mechanism of mitochondrial quality control. Certain types of stress-induced mitophagy are regulated by the mitochondrial kinase PINK1 and the E3 ubiquitin ligase Parkin, which are both linked to autosomal recessive Parkinson's disease.
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http://dx.doi.org/10.1515/hsz-2020-0135DOI Listing
May 2020

Propionic Acid Shapes the Multiple Sclerosis Disease Course by an Immunomodulatory Mechanism.

Cell 2020 03 10;180(6):1067-1080.e16. Epub 2020 Mar 10.

Department of Cell Morphology and Molecular Neurobiology, Ruhr University Bochum, Bochum 44801, Germany.

Short-chain fatty acids are processed from indigestible dietary fibers by gut bacteria and have immunomodulatory properties. Here, we investigate propionic acid (PA) in multiple sclerosis (MS), an autoimmune and neurodegenerative disease. Serum and feces of subjects with MS exhibited significantly reduced PA amounts compared with controls, particularly after the first relapse. In a proof-of-concept study, we supplemented PA to therapy-naive MS patients and as an add-on to MS immunotherapy. After 2 weeks of PA intake, we observed a significant and sustained increase of functionally competent regulatory T (Treg) cells, whereas Th1 and Th17 cells decreased significantly. Post-hoc analyses revealed a reduced annual relapse rate, disability stabilization, and reduced brain atrophy after 3 years of PA intake. Functional microbiome analysis revealed increased expression of Treg-cell-inducing genes in the intestine after PA intake. Furthermore, PA normalized Treg cell mitochondrial function and morphology in MS. Our findings suggest that PA can serve as a potent immunomodulatory supplement to MS drugs.
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http://dx.doi.org/10.1016/j.cell.2020.02.035DOI Listing
March 2020

The product PACRG promotes TNF signaling by stabilizing LUBAC.

Sci Signal 2020 02 4;13(617). Epub 2020 Feb 4.

Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, 44801 Bochum, Germany.

The (), which encodes a protein of unknown function, shares a bidirectional promoter with (), which encodes an E3 ubiquitin ligase. Because PRKN is important in mitochondrial quality control and protection against stress, we tested whether PACRG also affected these pathways in various cultured human cell lines and in mouse embryonic fibroblasts. PACRG did not play a role in mitophagy but did play a role in tumor necrosis factor (TNF) signaling. Similarly to Parkin, PACRG promoted nuclear factor κB (NF-κB) activation in response to TNF. TNF-induced nuclear translocation of the NF-κB subunit p65 and NF-κB-dependent transcription were decreased in PACRG-deficient cells. Defective canonical NF-κB activation in the absence of PACRG was accompanied by a decrease in linear ubiquitylation mediated by the linear ubiquitin chain assembly complex (LUBAC), which is composed of the two E3 ubiquitin ligases HOIP and HOIL-1L and the adaptor protein SHARPIN. Upon TNF stimulation, PACRG was recruited to the activated TNF receptor complex and interacted with LUBAC components. PACRG functionally replaced SHARPIN in this context. In SHARPIN-deficient cells, PACRG prevented LUBAC destabilization, restored HOIP-dependent linear ubiquitylation, and protected cells from TNF-induced apoptosis. This function of PACRG in positively regulating TNF signaling may help to explain the association of and polymorphisms with an increased susceptibility to intracellular pathogens.
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http://dx.doi.org/10.1126/scisignal.aav1256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279956PMC
February 2020

Linear Ubiquitin Chains: Cellular Functions and Strategies for Detection and Quantification.

Front Chem 2019 10;7:915. Epub 2020 Jan 10.

Department of Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany.

Ubiquitination of proteins is a sophisticated post-translational modification implicated in the regulation of an ever-growing abundance of cellular processes. Recent insights into different layers of complexity have shaped the concept of the ubiquitin code. Key players in determining this code are the number of ubiquitin moieties attached to a substrate, the architecture of polyubiquitin chains, and post-translational modifications of ubiquitin itself. Ubiquitination can induce conformational changes of substrates and alter their interactive profile, resulting in the formation of signaling complexes. Here we focus on a distinct type of ubiquitination that is characterized by an inter-ubiquitin linkage through the N-terminal methionine, called M1-linked or linear ubiquitination. Formation, recognition, and disassembly of linear ubiquitin chains are highly specific processes that are implicated in immune signaling, cell death regulation and protein quality control. Consistent with their role in influencing signaling events, linear ubiquitin chains are formed in a transient and spatially regulated manner, making their detection and quantification challenging.
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http://dx.doi.org/10.3389/fchem.2019.00915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966713PMC
January 2020

Activation leads to a significant shift in the intracellular redox homeostasis of neutrophil-like cells.

Redox Biol 2020 01 13;28:101344. Epub 2019 Oct 13.

Ruhr University Bochum, Institute of Biochemistry and Pathobiochemistry, Microbial Biochemistry, Bochum, Germany. Electronic address:

Neutrophils produce a cocktail of oxidative species during the so-called oxidative burst to attack phagocytized bacteria. However, little is known about the neutrophils' redox homeostasis during the oxidative burst and there is currently no consensus about the interplay between oxidative species and cellular signaling, e.g. during the initiation of the production of neutrophil extracellular traps (NETs). Using the genetically encoded redox sensor roGFP2, expressed in the cytoplasm of the neutrophil-like cell line PLB-985, we saw that stimulation by both PMA and E. coli resulted in oxidation of the thiol residues in this probe. In contrast to the redox state of phagocytized bacteria, which completely breaks down, the neutrophils' cytoplasmic redox state switched from its intital -318 ± 6 mV to a new, albeit higher oxidized, steady state of -264 ± 5 mV in the presence of bacteria. This highly significant oxidation of the cytosol (p value = 7 × 10) is dependent on NOX2 activity, but independent of the most effective thiol oxidant produced in neutrophils, MPO-derived HOCl. While the shift in the intracellular redox potential is correlated with effective NETosis, it is, by itself not sufficient: Inhibition of MPO, while not affecting the cytosolic oxidation, significantly decreased NETosis. Furthermore, inhibition of PI3K, which abrogates cytosolic oxidation, did not fully prevent NETosis induced by phagocytosis of bacteria. Thus, we conclude that NET-formation is regulated in a multifactorial way, in part by changes of the cytosolic thiol redox homeostasis in neutrophils, depending on the circumstance under which the generation of NETs was initiated.
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http://dx.doi.org/10.1016/j.redox.2019.101344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6807386PMC
January 2020

A new perspective on membrane-embedded Bax oligomers using DEER and bioresistant orthogonal spin labels.

Sci Rep 2019 09 10;9(1):13013. Epub 2019 Sep 10.

Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany.

Bax is a Bcl-2 protein crucial for apoptosis initiation and execution, whose active conformation is only partially understood. Dipolar EPR spectroscopy has proven to be a valuable tool to determine coarse-grained models of membrane-embedded Bcl-2 proteins. Here we show how the combination of spectroscopically distinguishable nitroxide and gadolinium spin labels and Double Electron-Electron Resonance can help to gain new insights into the quaternary structure of active, membrane-embedded Bax oligomers. We show that attaching labels bulkier than the conventional MTSL may affect Bax fold and activity, depending on the protein/label combination. However, we identified a suitable pair of spectroscopically distinguishable labels, which allows to study complex distance networks in the oligomers that could not be disentangled before. Additionally, we compared the stability of the different spin-labeled protein variants in E. coli and HeLa cell extracts. We found that the gem-diethyl nitroxide-labeled Bax variants were reasonably stable in HeLa cell extracts. However, when transferred into human cells, Bax was found to be mislocalized, thus preventing its characterization in a physiological environment. The successful use of spectroscopically distinguishable labels on membrane-embedded Bax-oligomers opens an exciting new path towards structure determination of membrane-embedded homo- or hetero-oligomeric Bcl-2 proteins via EPR.
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http://dx.doi.org/10.1038/s41598-019-49370-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737250PMC
September 2019

α-Synuclein in Parkinson's disease: causal or bystander?

J Neural Transm (Vienna) 2019 07 25;126(7):815-840. Epub 2019 Jun 25.

Department of Neuropathology, Institute of Pathology, University of Würzburg, Würzburg, Germany.

Parkinson's disease (PD) comprises a spectrum of disorders with differing subtypes, the vast majority of which share Lewy bodies (LB) as a characteristic pathological hallmark. The process(es) underlying LB generation and its causal trigger molecules are not yet fully understood. α-Synuclein (α-syn) is a major component of LB and SNCA gene missense mutations or duplications/triplications are causal for rare hereditary forms of PD. As typical sporadic PD is associated with LB pathology, a factor of major importance is the study of the α-syn protein and its pathology. α-Syn pathology is, however, also evident in multiple system atrophy (MSA) and Lewy body disease (LBD), making it non-specific for PD. In addition, there is an overlap of these α-synucleinopathies with other protein-misfolding diseases. It has been proven that α-syn, phosphorylated tau protein (pτ), amyloid beta (Aβ) and other proteins show synergistic effects in the underlying pathogenic mechanisms. Multiple cell death mechanisms can induce pathological protein-cascades, but this can also be a reverse process. This holds true for the early phases of the disease process and especially for the progression of PD. In conclusion, while rare SNCA gene mutations are causal for a minority of familial PD patients, in sporadic PD (where common SNCA polymorphisms are the most consistent genetic risk factor across populations worldwide, accounting for 95% of PD patients) α-syn pathology is an important feature. Conversely, with regard to the etiopathogenesis of α-synucleinopathies PD, MSA and LBD, α-syn is rather a bystander contributing to multiple neurodegenerative processes, which overlap in their composition and individual strength. Therapeutic developments aiming to impact on α-syn pathology should take this fact into consideration.
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http://dx.doi.org/10.1007/s00702-019-02025-9DOI Listing
July 2019

Mitochondria at the interface between neurodegeneration and neuroinflammation.

Semin Cell Dev Biol 2020 03 5;99:163-171. Epub 2019 Jun 5.

Department of Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Germany. Electronic address:

Mitochondria are essential organelles for the maintenance of neuronal integrity, based on their manifold functions in regulating cellular metabolism and coordinating cell death and viability pathways. Accordingly, mitochondrial damage, dysfunction, or ineffective mitochondrial quality control is associated with neurological disorders and can occur as a cause or consequence of neurodegenerative diseases. Recent research revealed that mitochondria play a central role in orchestrating both innate and adaptive immune responses, thereby providing a link between neurodegenerative and neuroinflammatory processes. Here we summarize new insights into the complex interplay between mitochondria, innate immunity and neurodegeneration.
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http://dx.doi.org/10.1016/j.semcdb.2019.05.028DOI Listing
March 2020

A protein quality control pathway regulated by linear ubiquitination.

EMBO J 2019 05 18;38(9). Epub 2019 Mar 18.

Neurobiochemistry, Adolf Butenandt Institute, Ludwig-Maximilians-University Munich, Munich, Germany.

Neurodegenerative diseases are characterized by the accumulation of misfolded proteins in the brain. Insights into protein quality control mechanisms to prevent neuronal dysfunction and cell death are crucial in developing causal therapies. Here, we report that various disease-associated protein aggregates are modified by the linear ubiquitin chain assembly complex (LUBAC). HOIP, the catalytic component of LUBAC, is recruited to misfolded Huntingtin in a p97/VCP-dependent manner, resulting in the assembly of linear polyubiquitin. As a consequence, the interactive surface of misfolded Huntingtin species is shielded from unwanted interactions, for example with the low complexity sequence domain-containing transcription factor Sp1, and proteasomal degradation of misfolded Huntingtin is facilitated. Notably, all three core LUBAC components are transcriptionally regulated by Sp1, linking defective LUBAC expression to Huntington's disease. In support of a protective activity of linear ubiquitination, silencing of OTULIN, a deubiquitinase with unique specificity for linear polyubiquitin, decreases proteotoxicity, whereas silencing of HOIP has the opposite effect. These findings identify linear ubiquitination as a protein quality control mechanism and hence a novel target for disease-modifying strategies in proteinopathies.
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http://dx.doi.org/10.15252/embj.2018100730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484417PMC
May 2019

Author Correction: Laquinimod treatment in the R6/2 mouse model.

Sci Rep 2019 Mar 15;9(1):4960. Epub 2019 Mar 15.

Department of Neurology, St. Josef-Hospital, Ruhr-University Bochum, Bochum, Germany.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-37926-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420624PMC
March 2019

Alterations in the brain interactome of the intrinsically disordered N-terminal domain of the cellular prion protein (PrPC) in Alzheimer's disease.

PLoS One 2018 23;13(5):e0197659. Epub 2018 May 23.

Department Biochemistry of Neurodegenerative Diseases, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Germany.

The cellular prion protein (PrPC) is implicated in neuroprotective signaling and neurotoxic pathways in both prion diseases and Alzheimer's disease (AD). Specifically, the intrinsically disordered N-terminal domain (N-PrP) has been shown to interact with neurotoxic ligands, such as Aβ and Scrapie prion protein (PrPSc), and to be crucial for the neuroprotective activity of PrPC. To gain further insight into cellular pathways tied to PrP, we analyzed the brain interactome of N-PrP. As a novel approach employing recombinantly expressed PrP and intein-mediated protein ligation, we used N-PrP covalently coupled to beads as a bait for affinity purification. N-PrP beads were incubated with human AD or control brain lysates. N-PrP binding partners were then identified by electrospray ionization tandem mass spectrometry (nano ESI-MS/MS). In addition to newly identified proteins we found many previously described PrP interactors, indicating a crucial role of the intrinsically disordered part of PrP in mediating protein interactions. Moreover, some interactors were found only in either non-AD or AD brain, suggesting aberrant PrPC interactions in the pathogenesis of AD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197659PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5965872PMC
November 2018

Dimerization of the cellular prion protein inhibits propagation of scrapie prions.

J Biol Chem 2018 05 10;293(21):8020-8031. Epub 2018 Apr 10.

Department of Biochemistry of Neurodegenerative Diseases, Ruhr University Bochum, D-44801 Bochum, Germany. Electronic address:

A central step in the pathogenesis of prion diseases is the conformational transition of the cellular prion protein (PrP) into the scrapie isoform, denoted PrP Studies in transgenic mice have indicated that this conversion requires a direct interaction between PrP and PrP; however, insights into the underlying mechanisms are still missing. Interestingly, only a subfraction of PrP is converted in scrapie-infected cells, suggesting that not all PrP species are suitable substrates for the conversion. On the basis of the observation that PrP can form homodimers under physiological conditions with the internal hydrophobic domain (HD) serving as a putative dimerization domain, we wondered whether PrP dimerization is involved in the formation of neurotoxic and/or infectious PrP conformers. Here, we analyzed the possible impact on dimerization of pathogenic mutations in the HD that induce a spontaneous neurodegenerative disease in transgenic mice. Similarly to wildtype (WT) PrP, the neurotoxic variant PrP(AV3) formed homodimers as well as heterodimers with WTPrP Notably, forced PrP dimerization via an intermolecular disulfide bond did not interfere with its maturation and intracellular trafficking. Covalently linked PrP dimers were complex glycosylated, GPI-anchored, and sorted to the outer leaflet of the plasma membrane. However, forced PrP dimerization completely blocked its conversion into PrP in chronically scrapie-infected mouse neuroblastoma cells. Moreover, PrP dimers had a dominant-negative inhibition effect on the conversion of monomeric PrP Our findings suggest that PrP monomers are the major substrates for PrP propagation and that it may be possible to halt prion formation by stabilizing PrP dimers.
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http://dx.doi.org/10.1074/jbc.RA117.000990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971439PMC
May 2018

Increased ROS Level in Spinal Cord of Wobbler Mice due to Nmnat2 Downregulation.

Mol Neurobiol 2018 Nov 16;55(11):8414-8424. Epub 2018 Mar 16.

Institute of Anatomy, Department of Cytology, Ruhr University Bochum, 44801, Bochum, Germany.

Amyotrophic lateral sclerosis is a devastating motor neuron disease and to this day not curable. While 5-10% of patients inherit the disease (familiar ALS), up to 95% of patients are diagnosed with the sporadic form (sALS). ALS is characterized by the degeneration of upper motor neurons in the cerebral cortex and of lower motor neurons in the brainstem and spinal cord. The wobbler mouse resembles almost all phenotypical hallmarks of human sALS patients and is therefore an excellent motor neuron disease model. The motor neuron disease of the wobbler mouse develops over a time course of around 40 days and can be divided into three phases: p0, presymptomatic; p20, early clinical; and p40, stable clinical phase. Recent findings suggest an essential implication of the NAD-producing enzyme Nmnat2 in neurodegeneration as well as maintenance of healthy axons. Here, we were able to show a significant downregulation of both gene and protein expression of Nmnat2 in the spinal cord of the wobbler mice at the stable clinical phase. The product of the enzyme NAD is also significantly reduced, and the values of the reactive oxygen species are significantly increased in the spinal cord of the wobbler mouse at p40. Thus, the deregulated expression of Nmnat2 appears to have a great influence on the cellular stress in the spinal cord of wobbler mice.
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http://dx.doi.org/10.1007/s12035-018-0999-7DOI Listing
November 2018

Neutrophil-generated HOCl leads to non-specific thiol oxidation in phagocytized bacteria.

Elife 2018 03 6;7. Epub 2018 Mar 6.

Institute for Biochemistry and Pathobiochemistry - Microbial Biochemistry, Ruhr-Universität Bochum, Bochum, Germany.

Phagocytic immune cells kill pathogens in the phagolysosomal compartment with a cocktail of antimicrobial agents. Chief among them are reactive species produced in the so-called oxidative burst. Here, we show that bacteria exposed to a neutrophil-like cell line experience a rapid and massive oxidation of cytosolic thiols. Using roGFP2-based fusion probes, we could show that this massive breakdown of the thiol redox homeostasis was dependent on phagocytosis, presence of NADPH oxidase and ultimately myeloperoxidase. Interestingly, the redox-mediated fluorescence change in bacteria expressing a glutathione-specific Grx1-roGFP2 fusion protein or an unfused roGFP2 showed highly similar reaction kinetics to the ones observed with roGFP2-Orp1, under all conditions tested. We recently observed such an indiscriminate oxidation of roGFP2-based fusion probes by HOCl with fast kinetics in vitro. In line with these observations, abating HOCl production in immune cells with a myeloperoxidase inhibitor significantly attenuated the oxidation of all three probes in bacteria.
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http://dx.doi.org/10.7554/eLife.32288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839695PMC
March 2018

The Sec61/SecY complex is inherently deficient in translocating intrinsically disordered proteins.

J Biol Chem 2017 12 30;292(52):21383-21396. Epub 2017 Oct 30.

From the Departments of Biochemistry of Neurodegenerative Diseases and

About one-quarter to nearly one-third of the proteins synthesized in the cytosol of eukaryotic cells are integrated into the plasma membrane or are secreted. Translocation of secretory proteins into the lumen of the endoplasmic reticulum or the periplasm of bacteria is mediated by a highly conserved heterotrimeric membrane protein complex denoted Sec61 in eukaryotes and SecYEG in bacteria. To evaluate a possible modulation of the translocation efficiency by secondary structures of the nascent peptide chain, we performed a comparative analysis in bacteria, yeast, and mammalian cells. Strikingly, neither the bacterial SecY nor the eukaryotic Sec61 translocon was able to efficiently transport proteins entirely composed of intrinsically disordered domains (IDDs) or β-strands. However, translocation could be restored by α-helical domains in a position- and organism-dependent manner. In bacteria, we found that the α-helical domains have to precede the IDD or β-strands, whereas in mammalian cells, C-terminally located α-helical domains are sufficient to promote translocation. Our study reveals an evolutionarily conserved deficiency of the Sec61/SecY complex to translocate IDDs and β-strands in the absence of α-helical domains. Moreover, our results may suggest that adaptive pathways co-evolved with the expansion of IDDs in the proteome of eukaryotic cells to increase the transport capacity of the Sec61 translocon.
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http://dx.doi.org/10.1074/jbc.M117.788067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766967PMC
December 2017

Laquinimod treatment in the R6/2 mouse model.

Sci Rep 2017 07 10;7(1):4947. Epub 2017 Jul 10.

Department of Neurology, St. Josef-Hospital, Ruhr-University Bochum, Bochum, Germany.

The transgenic mouse model R6/2 exhibits Huntington's disease (HD)-like deficits and basic pathophysiological similarities. We also used the pheochromocytoma-12 (PC12)-cell-line-model to investigate the effect of laquinimod on metabolic activity. Laquinimod is an orally administered immunomodulatory substance currently under development for the treatment of multiple sclerosis (MS) and HD. As an essential effect, increased levels of BDNF were observed. Therefore, we investigated the therapeutic efficacy of laquinimod in the R6/2 model, focusing on its neuroprotective capacity. Weight course and survival were not influenced by laquinimod. Neither were any metabolic effects seen in an inducible PC12-cell-line model of HD. As a positive effect, motor functions of R6/2 mice at the age of 12 weeks significantly improved. Preservation of morphologically intact neurons was found after treatment in the striatum, as revealed by NeuN, DARPP-32, and ubiquitin. Biochemical analysis showed a significant increase in the brain-derived neurotrophic factor (BDNF) level in striatal but not in cortical neurons. The number of mutant huntingtin (mhtt) and inducible nitric oxide synthase (iNOS) positive cells was reduced in both the striatum and motor cortex following treatment. These findings suggest that laquinimod could provide a mild effect on motor function and striatal histopathology, but not on survival. Besides influences on the immune system, influence on BDNF-dependent pathways in HD are discussed.
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http://dx.doi.org/10.1038/s41598-017-04990-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5504033PMC
July 2017

Alpha-synuclein prevents the formation of spherical mitochondria and apoptosis under oxidative stress.

Sci Rep 2017 02 22;7:42942. Epub 2017 Feb 22.

Department of Molecular Neurology, University Hospital Erlangen, Friedrich-Alexander-University (FAU) Erlangen-Nürnberg, 91054 Erlangen, Germany.

Oxidative stress (OS), mitochondrial dysfunction, and dysregulation of alpha-synuclein (aSyn) homeostasis are key pathogenic factors in Parkinson's disease. Nevertheless, the role of aSyn in mitochondrial physiology remains elusive. Thus, we addressed the impact of aSyn specifically on mitochondrial response to OS in neural cells. We characterize a distinct type of mitochondrial fragmentation, following HO or 6-OHDA-induced OS, defined by spherically-shaped and hyperpolarized mitochondria, termed "mitospheres". Mitosphere formation mechanistically depended on the fission factor Drp1, and was paralleled by reduced mitochondrial fusion. Furthermore, mitospheres were linked to a decrease in mitochondrial activity, and preceded Caspase3 activation. Even though fragmentation of dysfunctional mitochondria is considered to be a prerequisite for mitochondrial degradation, mitospheres were not degraded via Parkin-mediated mitophagy. Importantly, we provide compelling evidence that aSyn prevents mitosphere formation and reduces apoptosis under OS. In contrast, aSyn did not protect against Rotenone, which led to a different, previously described donut-shaped mitochondrial morphology. Our findings reveal a dichotomic role of aSyn in mitochondrial biology, which is linked to distinct types of stress-induced mitochondrial fragmentation. Specifically, aSyn may be part of a cellular defense mechanism preserving neural mitochondrial homeostasis in the presence of increased OS levels, while not protecting against stressors directly affecting mitochondrial function.
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http://dx.doi.org/10.1038/srep42942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320486PMC
February 2017

PERK activation mitigates tau pathology and .

EMBO Mol Med 2017 03;9(3):371-384

Department of Translational Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE), Munich, Germany

The RNA-like endoplasmic reticulum kinase (PERK) is genetically associated with the tauopathy progressive supranuclear palsy (PSP). To elucidate the functional mechanisms underlying this association, we explored PERK activity in brains of PSP patients and its function in three tauopathy models (cultured human neurons overexpressing 4-repeat wild-type tau or treated with the environmental neurotoxin annonacin, and P301S tau transgenic mice). , treatment with a pharmacological PERK activator CCT020312 or PERK overexpression reduced tau phosphorylation, tau conformational change and 4-repeat tau isoforms, and increased cell viability. , the PERK activator significantly improved memory and locomotor function, reduced tau pathology, and prevented dendritic spine and motoneuron loss in P301S tau mice. Importantly, the PERK substrate EIF2A, mediating some detrimental effects of PERK signaling, was downregulated in PSP brains and tauopathy models, suggesting that the alternative PERK-NRF2 pathway accounts for these beneficial effects in the context of tauopathies. In summary, PERK activation may be a novel strategy to treat PSP and eventually other tauopathies.
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http://dx.doi.org/10.15252/emmm.201606664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331260PMC
March 2017

The mitochondrial kinase PINK1: functions beyond mitophagy.

J Neurochem 2016 10 2;139 Suppl 1:232-239. Epub 2016 Jun 2.

Molecular Cell Biology, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Germany.

Mutations in the genes encoding the mitochondrial kinase PINK1 and the E3 ubiquitin ligase Parkin cause autosomal recessive Parkinson's disease (PD). Pioneering work in Drosophila melanogaster revealed that the loss of PINK1 or Parkin function causes similar phenotypes including dysfunctional mitochondria. Further research showed that PINK1 can act upstream of Parkin in a mitochondrial quality control pathway to induce removal of damaged mitochondria in a process called mitophagy. Albeit the PINK1/Parkin-induced mitophagy pathway is well established and has recently been elucidated in great detail, its pathophysiological relevance is being debated. Mounting evidence indicates that PINK1 has additional functions, for example, in regulating complex I activity and maintaining neuronal viability in response to stress. Here, we discuss mitophagy-dependent and -independent functions of PINK1 and their possible role in PD pathogenesis. Mutations in the PINK1 gene, encoding a mitochondrial kinase, are associated with autosomal recessive Parkinson's disease. In this review, we summarize and discuss the functional roles of PINK1 in maintaining mitochondrial integrity, eliminating damaged mitochondria, and promoting cell survival. This article is part of a special issue on Parkinson disease.
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http://dx.doi.org/10.1111/jnc.13655DOI Listing
October 2016
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