Publications by authors named "Erich E Wanker"

116 Publications

The ARFRP1-dependent Golgi scaffolding protein GOPC is required for insulin secretion from pancreatic β-cells.

Mol Metab 2020 Dec 23;45:101151. Epub 2020 Dec 23.

German Institute of Human Nutrition (DIfE) Potsdam-Rehbruecke, Germany; German Center for Diabetes Research (DZD) Munich Neuherberg, Germany; University of Potsdam, Institute of Nutritional Sciences, Nuthetal, Germany; Faculty of Health Sciences, Joint Faculty of the Brandenburg University of Technology Cottbus - Senftenberg, the Brandenburg Medical School Theodor Fontane and the University of Potsdam, Germany. Electronic address:

Objective: Hormone secretion from metabolically active tissues, such as pancreatic islets, is governed by specific and highly regulated signaling pathways. Defects in insulin secretion are among the major causes of diabetes. The molecular mechanisms underlying regulated insulin secretion are, however, not yet completely understood. In this work, we studied the role of the GTPase ARFRP1 on insulin secretion from pancreatic β-cells.

Methods: A β-cell-specific Arfrp1 knockout mouse was phenotypically characterized. Pulldown experiments and mass spectrometry analysis were employed to screen for new ARFRP1-interacting proteins. Co-immunoprecipitation assays as well as super-resolution microscopy were applied for validation.

Results: The GTPase ARFRP1 interacts with the Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). Both proteins are co-localized at the trans-Golgi network and regulate the first and second phase of insulin secretion by controlling the plasma membrane localization of the SNARE protein SNAP25. Downregulation of both GOPC and ARFRP1 in Min6 cells interferes with the plasma membrane localization of SNAP25 and enhances its degradation, thereby impairing glucose-stimulated insulin release from β-cells. In turn, overexpression of SNAP25 as well as GOPC restores insulin secretion in islets from β-cell-specific Arfrp1 knockout mice.

Conclusion: Our results identify a hitherto unrecognized pathway required for insulin secretion at the level of trans-Golgi sorting.
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http://dx.doi.org/10.1016/j.molmet.2020.101151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811047PMC
December 2020

RNA Sequencing of Human Peripheral Blood Cells Indicates Upregulation of Immune-Related Genes in Huntington's Disease.

Front Neurol 2020 27;11:573560. Epub 2020 Nov 27.

Department of Neuropsychiatry, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disorder caused by a trinucleotide repeat expansion in the gene. As disease-modifying therapies for HD are being developed, peripheral blood cells may be used to indicate disease progression and to monitor treatment response. In order to investigate whether gene expression changes can be found in the blood of individuals with HD that distinguish them from healthy controls, we performed transcriptome analysis by next-generation sequencing (RNA-seq). We detected a gene expression signature consistent with dysregulation of immune-related functions and inflammatory response in peripheral blood from HD cases vs. controls, including induction of the interferon response genes, and . Our results suggest that it is possible to detect gene expression changes in blood samples from individuals with HD, which may reflect the immune pathology associated with the disease.
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http://dx.doi.org/10.3389/fneur.2020.573560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731869PMC
November 2020

Megadalton-sized Dityrosine Aggregates of α-Synuclein Retain High Degrees of Structural Disorder and Internal Dynamics.

J Mol Biol 2020 12 22;432(24):166689. Epub 2020 Oct 22.

Leibniz Institute of Molecular Pharmacology (FMP-Berlin), Robert-Rössle Strasse 10, 13125 Berlin, Germany. Electronic address:

Heterogeneous aggregates of the human protein α-synuclein (αSyn) are abundantly found in Lewy body inclusions of Parkinson's disease patients. While structural information on classical αSyn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of αSyn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-β amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict αSyn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species, rather than cytochrome c, are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining αSyn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative αSyn aggregation scavenges cytochrome c's activity into the formation of amorphous, high molecular-weight structures that may contribute to the structural diversity of Lewy body deposits.
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http://dx.doi.org/10.1016/j.jmb.2020.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7779668PMC
December 2020

Subcellular Localization And Formation Of Huntingtin Aggregates Correlates With Symptom Onset And Progression In A Huntington'S Disease Model.

Brain Commun 2020 3;2(2):fcaa066. Epub 2020 Aug 3.

Huntington's Disease Centre, Department of Neurodegenerative Disease and UK Dementia Research Institute at UCL, Queen Square Institute of Neurology, UCL, Queen Square, WC1N 3BG, UK.

Huntington's disease is caused by the expansion of a CAG repeat within exon 1 of the gene, which is unstable, leading to further expansion, the extent of which is brain region and peripheral tissue specific. The identification of DNA repair genes as genetic modifiers of Huntington's disease, that were known to abrogate somatic instability in Huntington's disease mouse models, demonstrated that somatic CAG expansion is central to disease pathogenesis, and that the CAG repeat threshold for pathogenesis in specific brain cells might not be known. We have previously shown that the gene is incompletely spliced generating a small transcript that encodes the highly pathogenic exon 1 HTT protein. The longer the CAG repeat, the more of this toxic fragment is generated, providing a pathogenic consequence for somatic expansion. Here, we have used the R6/2 mouse model to investigate the molecular and behavioural consequences of expressing exon 1 with 90 CAGs, a mutation that causes juvenile Huntington's disease, compared to R6/2 mice carrying ∼200 CAGs, a repeat expansion of a size rarely found in Huntington's disease patient's blood, but which has been detected in post-mortem brains as a consequence of somatic CAG repeat expansion. We show that nuclear aggregation occurred earlier in R6/2(CAG) mice and that this correlated with the onset of transcriptional dysregulation. Whereas in R6/2(CAG) mice, cytoplasmic aggregates accumulated rapidly and closely tracked with the progression of behavioural phenotypes and with end-stage disease. We find that aggregate species formed in the R6/2(CAG) brains have different properties to those in the R6/2(CAG) mice. Within the nucleus, they retain a diffuse punctate appearance throughout the course of the disease, can be partially solubilized by detergents and have a greater seeding potential in young mice. In contrast, aggregates from R6/2(CAG) brains polymerize into larger structures that appear as inclusion bodies. These data emphasize that a subcellular analysis, using multiple complementary approaches, must be undertaken in order to draw any conclusions about the relationship between HTT aggregation and the onset and progression of disease phenotypes.
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http://dx.doi.org/10.1093/braincomms/fcaa066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425396PMC
August 2020

Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins and Uncovers Widespread Protein Aggregation in Affected Brains.

Cell Rep 2020 08;32(7):108050

Neuroproteomics, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany. Electronic address:

Interactome maps are valuable resources to elucidate protein function and disease mechanisms. Here, we report on an interactome map that focuses on neurodegenerative disease (ND), connects ∼5,000 human proteins via ∼30,000 candidate interactions and is generated by systematic yeast two-hybrid interaction screening of ∼500 ND-related proteins and integration of literature interactions. This network reveals interconnectivity across diseases and links many known ND-causing proteins, such as α-synuclein, TDP-43, and ATXN1, to a host of proteins previously unrelated to NDs. It facilitates the identification of interacting proteins that significantly influence mutant TDP-43 and HTT toxicity in transgenic flies, as well as of ARF-GEP that controls misfolding and aggregation of multiple ND-causing proteins in experimental model systems. Furthermore, it enables the prediction of ND-specific subnetworks and the identification of proteins, such as ATXN1 and MKL1, that are abnormally aggregated in postmortem brains of Alzheimer's disease patients, suggesting widespread protein aggregation in NDs.
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http://dx.doi.org/10.1016/j.celrep.2020.108050DOI Listing
August 2020

Mixing Aβ(1-40) and Aβ(1-42) peptides generates unique amyloid fibrils.

Chem Commun (Camb) 2020 Aug 6;56(62):8830-8833. Epub 2020 Jul 6.

Magnetic Resonance Center (CERM), University of Florence and Interuniversity Consortium for Magnetic Resonance of Metalloproteins (CIRMMP), Via L. Sacconi 6, 50019, Sesto Fiorentino (FI), Italy.

Recent structural studies show distinct morphologies for the fibrils of Aβ(1-42) and Aβ(1-40), which are believed not to co-fibrillize. We describe here a novel, structurally-uniform 1 : 1 mixed fibrillar species, which differs from both pure fibrils. It forms preferentially even when Aβ(1-42) : Aβ(1-40) peptides are mixed in a non-stoichiometric ratio.
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http://dx.doi.org/10.1039/d0cc02463eDOI Listing
August 2020

Correction: Shedding a new light on Huntington's disease: how blood can both propagate and ameliorate disease pathology.

Mol Psychiatry 2020 Jul 24. Epub 2020 Jul 24.

Centre de Recherche du CHU de Québec-Université Laval, Axe Neurosciences, 2705 boulevard Laurier, Québec, QC, G1V 4G2, Canada.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41380-020-0850-1DOI Listing
July 2020

Shedding a new light on Huntington's disease: how blood can both propagate and ameliorate disease pathology.

Mol Psychiatry 2020 Jun 8. Epub 2020 Jun 8.

Centre de Recherche du CHU de Québec-Université Laval, Axe Neurosciences, 2705 boulevard Laurier, Québec, QC, G1V 4G2, Canada.

Huntington's disease (HD) is a monogenic neurodegenerative disorder resulting from a mutation in the huntingtin gene. This leads to the expression of the mutant huntingtin protein (mHTT) which provokes pathological changes in both the central nervous system (CNS) and periphery. Accumulating evidence suggests that mHTT can spread between cells of the CNS but here, we explored the possibility that mHTT could also propagate and cause pathology via the bloodstream. For this, we used a parabiosis approach to join the circulatory systems of wild-type (WT) and zQ175 mice. After surgery, we observed mHTT in the plasma and circulating blood cells of WT mice and post-mortem analyses revealed the presence of mHTT aggregates in several organs including the liver, kidney, muscle and brain. The presence of mHTT in the brain was accompanied by vascular abnormalities, such as a reduction of Collagen IV signal intensity and altered vessel diameter in the striatum, and changes in expression of Glutamic acid decarboxylase 65/67 (GAD65-67) in the cortex. Conversely, we measured reduced pathology in zQ175 mice by decreased mitochondrial impairments in peripheral organs, restored vessel diameter in the cortex and improved expression of Dopamine- and cAMP-regulated phosphoprotein 32 (DARPP32) in striatal neurons. Collectively, these results demonstrate that circulating mHTT can disseminate disease, but importantly, that healthy blood can dilute pathology. These findings have significant implications for the development of therapies in HD.
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http://dx.doi.org/10.1038/s41380-020-0787-4DOI Listing
June 2020

Sclerotiorin Stabilizes the Assembly of Nonfibrillar Abeta42 Oligomers with Low Toxicity, Seeding Activity, and Beta-sheet Content.

J Mol Biol 2020 03 13;432(7):2080-2098. Epub 2020 Feb 13.

Neuroproteomics, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany. Electronic address:

The self-assembly of the 42-residue amyloid-β peptide, Aβ42, into fibrillar aggregates is associated with neuronal dysfunction and toxicity in Alzheimer's disease (AD) patient brains, suggesting that small molecules acting on this process might interfere with pathogenesis. Here, we present experimental evidence that the small molecule sclerotiorin (SCL), a natural product belonging to the group of azaphilones, potently delays both seeded and nonseeded Aβ42 polymerization in cell-free assays. Mechanistic biochemical studies revealed that the inhibitory effect of SCL on fibrillogenesis is caused by its ability to kinetically stabilize small Aβ42 oligomers. These structures exhibit low β-sheet content and do not possess seeding activity, indicating that SCL acts very early in the amyloid formation cascade before the assembly of seeding-competent, β-sheet-rich fibrillar aggregates. Investigations with NMR WaterLOGSY experiments confirmed the association of Aβ42 assemblies with SCL in solution. Furthermore, using ion mobility-mass spectrometry, we observed that SCL directly interacts with a small fraction of Aβ42 monomers in the gas phase. In comparison to typical amyloid fibrils, small SCL-stabilized Aβ42 assemblies are inefficiently taken up into mammalian cells and have low toxicity in cell-based assays. Overall, these mechanistic studies support a pathological role of stable, β-sheet-rich Aβ42 fibrils in AD, while structures with low β-sheet content may be less relevant.
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http://dx.doi.org/10.1016/j.jmb.2020.01.033DOI Listing
March 2020

Interleukin-12/23 deficiency differentially affects pathology in male and female Alzheimer's disease-like mice.

EMBO Rep 2020 03 30;21(3):e48530. Epub 2020 Jan 30.

Department of Neuropathology, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Pathological aggregation of amyloid-β (Aβ) is a main hallmark of Alzheimer's disease (AD). Recent genetic association studies have linked innate immune system actions to AD development, and current evidence suggests profound gender differences in AD pathogenesis. Here, we characterise gender-specific pathologies in the APP23 AD-like mouse model and find that female mice show stronger amyloidosis and astrogliosis compared with male mice. We tested the gender-specific effect of lack of IL12p40, the shared subunit of interleukin (IL)-12 and IL-23, that we previously reported to ameliorate pathology in APPPS1 mice. IL12p40 deficiency gender specifically reduces Aβ plaque burden in male APP23 mice, while in female mice, a significant reduction in soluble Aβ without changes in Aβ plaque burden is seen. Similarly, plasma and brain cytokine levels are altered differently in female versus male APP23 mice lacking IL12p40, while glial properties are unchanged. These data corroborate the therapeutic potential of targeting IL-12/IL-23 signalling in AD, but also highlight the importance of gender considerations when studying the role of the immune system and AD.
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http://dx.doi.org/10.15252/embr.201948530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054677PMC
March 2020

Common Mode of Remodeling AAA ATPases p97/CDC48 by Their Disassembling Cofactors ASPL/PUX1.

Structure 2019 12 21;27(12):1830-1841.e3. Epub 2019 Oct 21.

Max-Delbrück-Centrum für Molekulare Medizin, 13125 Berlin, Germany; Institut für Chemie und Biochemie, Freie Universität Berlin, 14195 Berlin, Germany. Electronic address:

The hexameric ring structure of the type II AAA+ ATPases is considered as stable and permanent. Recently, the UBX domain-containing cofactors Arabidopsis thaliana PUX1 and human alveolar soft part sarcoma locus (ASPL) were reported to bind and disassemble the cognate AAA+ ATPases AtCDC48 and human p97. Here, we present two crystal structures related to these complexes: a truncated AtCDC48 (AtCDC48-ND1) and a hybrid complex containing human p97-ND1 and the UBX domain of plant PUX1 (p97-ND1:PUX1-UBX). These structures reveal close similarity between the human and plant AAA+ ATPases, but also highlight differences between disassembling and non-disassembling AAA+ ATPase cofactors. Based on an AtCDC48 disassembly assay with PUX1 and known crystal structures of the p97-bound human cofactor ASPL, we propose a general ATPase disassembly model. Thus, our structural and biophysical investigations provide detailed insight into the mechanism of AAA+ ATPase disassembly by UBX domain cofactors and suggest a general mode of regulating the cellular activity of these molecular machines.
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http://dx.doi.org/10.1016/j.str.2019.10.001DOI Listing
December 2019

Maximizing binary interactome mapping with a minimal number of assays.

Nat Commun 2019 08 29;10(1):3907. Epub 2019 Aug 29.

Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute (DFCI), 450 Brookline Avenue, Boston, MA, 02215, USA.

Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes.
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http://dx.doi.org/10.1038/s41467-019-11809-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715725PMC
August 2019

The pathobiology of perturbed mutant huntingtin protein-protein interactions in Huntington's disease.

J Neurochem 2019 11 15;151(4):507-519. Epub 2019 Sep 15.

Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Berlin, Germany.

Mutations are at the root of many human diseases. Still, we largely do not exactly understand how they trigger pathogenesis. One, more recent, hypothesis has been that they comprehensively perturb protein-protein interaction (PPI) networks and significantly alter key biological processes. Under this premise, many rare genetic disorders with Mendelian inheritance, like Huntington's disease and several spinocerebellar ataxias, are likely to be caused by complex genotype-phenotype relationships involving abnormal PPIs. These altered PPI networks and their effects on cellular pathways are poorly understood at the molecular level. In this review, we focus on PPIs that are perturbed by the expanded pathogenic polyglutamine tract in huntingtin (HTT), the protein which, in its mutated form, leads to the autosomal dominant, neurodegenerative Huntington's disease. One aspect of perturbed mutant HTT interactions is the formation of abnormal protein species such as fibrils or large neuronal inclusions as a result of homotypic and heterotypic aberrant molecular interactions. This review focuses on abnormal PPIs that are associated with the assembly of mutant HTT aggregates in cells and their potential relevance in disease. Furthermore, the mechanisms and pathobiological processes that may contribute to phenotype development, neuronal dysfunction and toxicity in Huntington's disease brains are also discussed. This article is part of the Special Issue "Proteomics".
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http://dx.doi.org/10.1111/jnc.14853DOI Listing
November 2019

DCAF8, a novel MuRF1 interaction partner, promotes muscle atrophy.

J Cell Sci 2019 09 6;132(17). Epub 2019 Sep 6.

Intracellular Proteolysis, Max Delbrück Center (MDC) for Molecular Medicine in the Helmholtz Association, Robert-Rössle-Strasse 10, 13125 Berlin-Buch, Germany

The muscle-specific RING-finger protein MuRF1 (also known as TRIM63) constitutes a bona fide ubiquitin ligase that routes proteins like several different myosin heavy chain proteins (MyHC) to proteasomal degradation during muscle atrophy. In two unbiased screens, we identified DCAF8 as a new MuRF1-binding partner. MuRF1 physically interacts with DCAF8 and both proteins localize to overlapping structures in muscle cells. Importantly, similar to what is seen for MuRF1, DCAF8 levels increase during atrophy, and the downregulation of either protein substantially impedes muscle wasting and MyHC degradation in C2C12 myotubes, a model system for muscle differentiation and atrophy. DCAF proteins typically serve as substrate receptors for cullin 4-type (Cul4) ubiquitin ligases (CRL), and we demonstrate that DCAF8 and MuRF1 associate with the subunits of such a protein complex. Because genetic downregulation of DCAF8 and inhibition of cullin activity also impair myotube atrophy in C2C12 cells, our data imply that the DCAF8 promotes muscle wasting by targeting proteins like MyHC as an integral substrate receptor of a Cul4A-containing ring ubiquitin ligase complex (CRL4A).This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.233395DOI Listing
September 2019

The Anti-amyloid Compound DO1 Decreases Plaque Pathology and Neuroinflammation-Related Expression Changes in 5xFAD Transgenic Mice.

Cell Chem Biol 2019 01 21;26(1):109-120.e7. Epub 2018 Nov 21.

Neuroproteomics, Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany. Electronic address:

Self-propagating amyloid-β (Aβ) aggregates or seeds possibly drive pathogenesis of Alzheimer's disease (AD). Small molecules targeting such structures might act therapeutically in vivo. Here, a fluorescence polarization assay was established that enables the detection of compound effects on both seeded and spontaneous Aβ42 aggregation. In a focused screen of anti-amyloid compounds, we identified Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]), a small molecule that potently delays both seeded and non-seeded Aβ42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aβ42 peptides and decreases the seeding activity of Aβ aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aβ plaques and decreased neuroinflammation-related gene expression changes in brains of 5xFAD transgenic mice. Finally, improved nesting behavior was observed upon treatment with the compound. Together, our evidence supports targeting of self-propagating Aβ structures with small molecules as a valid therapeutic strategy.
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http://dx.doi.org/10.1016/j.chembiol.2018.10.013DOI Listing
January 2019

mHTT Seeding Activity: A Marker of Disease Progression and Neurotoxicity in Models of Huntington's Disease.

Mol Cell 2018 09;71(5):675-688.e6

Neuroproteomics, Max Delbrueck Center for Molecular Medicine, 13125 Berlin, Germany; Berlin Institute of Health (BIH), 10178 Berlin, Germany. Electronic address:

Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). Here, we report the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, we assessed the neurotoxicity of mHTT seeds in an inducible Drosophila model transgenic for HTTex1. We found a strong correlation between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo.
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http://dx.doi.org/10.1016/j.molcel.2018.07.032DOI Listing
September 2018

LuTHy: a double-readout bioluminescence-based two-hybrid technology for quantitative mapping of protein-protein interactions in mammalian cells.

Mol Syst Biol 2018 07 11;14(7):e8071. Epub 2018 Jul 11.

Neuroproteomics, Max Delbrück Center for Molecular Medicine and Berlin Institute of Health, Berlin, Germany

Information on protein-protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double-readout bioluminescence-based two-hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence-based co-precipitation (LuC). The double-readout procedure detects interactions with higher sensitivity than traditional single-readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease-causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult-onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease-causing missense mutations L115R and ∆L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease-associated mutations impair protein activity in biological systems.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039870PMC
http://dx.doi.org/10.15252/msb.20178071DOI Listing
July 2018

A Filter Retardation Assay Facilitates the Detection and Quantification of Heat-Stable, Amyloidogenic Mutant Huntingtin Aggregates in Complex Biosamples.

Methods Mol Biol 2018 ;1780:31-40

Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Berlin, Germany.

N-terminal mutant huntingtin (mHTT) fragments with pathogenic polyglutamine (polyQ) tracts spontaneously form stable, amyloidogenic protein aggregates with a fibrillar morphology. Such structures are detectable in brains of Huntington's disease (HD) patients and various model organisms, suggesting that they play a critical role in pathogenesis. Heat-stable, fibrillar mHTT aggregates can be detected and quantified in cells and tissues using a denaturing filter retardation assay (FRA). Here, we describe step-by-step protocols and experimental procedures for the investigation of mHTT aggregates in complex biosamples using FRAs. The methods are illustrated with examples from studies in cellular, transgenic fly, and mouse models of HD, but can be adapted for any disease-relevant protein with amyloidogenic polyQ tracts.
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http://dx.doi.org/10.1007/978-1-4939-7825-0_3DOI Listing
February 2019

Self-assembly of Mutant Huntingtin Exon-1 Fragments into Large Complex Fibrillar Structures Involves Nucleated Branching.

J Mol Biol 2018 06 28;430(12):1725-1744. Epub 2018 Mar 28.

Neuroproteomics, Max Delbrueck Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin, Germany. Electronic address:

Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.
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http://dx.doi.org/10.1016/j.jmb.2018.03.017DOI Listing
June 2018

A Novel RNA Editing Sensor Tool and a Specific Agonist Determine Neuronal Protein Expression of RNA-Edited Glycine Receptors and Identify a Genomic APOBEC1 Dimorphism as a New Genetic Risk Factor of Epilepsy.

Front Mol Neurosci 2017 11;10:439. Epub 2018 Jan 11.

Division Cell Physiology, Zoological Institute, Technische Universität Braunschweig, Braunschweig, Germany.

C-to-U RNA editing of glycine receptors (GlyR) can play an important role in disease progression of temporal lobe epilepsy (TLE) as it may contribute in a neuron type-specific way to neuropsychiatric symptoms of the disease. It is therefore necessary to develop tools that allow identification of neuron types that express RNA-edited GlyR protein. In this study, we identify NH as agonist of C-to-U RNA edited GlyRs. Furthermore, we generated a new molecular C-to-U RNA editing sensor tool that detects Apobec-1- dependent RNA editing in HEPG2 cells and rat primary hippocampal neurons. Using this sensor combined with NH application, we were able to identify C-to-U RNA editing-competent neurons and expression of C-to-U RNA-edited GlyR protein in neurons. Bioinformatic analysis of 1,000 Genome Project Phase 3 allele frequencies coding for human Apobec-1 80M and 80I variants showed differences between populations, and the results revealed a preference of the 80I variant to generate RNA-edited GlyR protein. Finally, we established a new PCR-based restriction fragment length polymorphism (RFLP) approach to profile mRNA expression with regard to the genetic dimorphism of patients with intractable temporal lobe epilepsy (iTLE) and found that the patients fall into two groups. Patients with expression of the Apobec-1 80I variant mostly suffered from simple or complex partial seizures, whereas patients with 80M expression exhibited secondarily generalized seizure activity. Thus, our method allows the characterization of Apobec-1 80M and 80l variants in the brain and provides a new way to epidemiologically and semiologically classify iTLE according to the two different alleles. Together, these results demonstrate Apobec-1-dependent expression of RNA-edited GlyR protein in neurons and identify the 80I/M-coding alleles as new genetic risk factors for iTLE patients.
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http://dx.doi.org/10.3389/fnmol.2017.00439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5768626PMC
January 2018

Epigallocatechin gallate (EGCG) reduces the intensity of pancreatic amyloid fibrils in human islet amyloid polypeptide (hIAPP) transgenic mice.

Sci Rep 2018 01 18;8(1):1116. Epub 2018 Jan 18.

Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany.

The formation of amyloid fibrils by human islet amyloid polypeptide protein (hIAPP) has been implicated in pancreas dysfunction and diabetes. However, efficient treatment options to reduce amyloid fibrils in vivo are still lacking. Therefore, we tested the effect of epigallocatechin gallate (EGCG) on fibril formation in vitro and in vivo. To determine the binding of hIAPP and EGCG, in vitro interaction studies were performed. To inhibit amyloid plaque formation in vivo, homozygous (tg/tg), hemizygous (wt/tg), and control mice (wt/wt) were treated with EGCG. EGCG bound to hIAPP in vitro and induced formation of amorphous aggregates instead of amyloid fibrils. Amyloid fibrils were detected in the pancreatic islets of tg/tg mice, which was associated with disrupted islet structure and diabetes. Although pancreatic amyloid fibrils could be detected in wt/tg mice, these animals were non-diabetic. EGCG application decreased amyloid fibril intensity in wt/tg mice, however it was ineffective in tg/tg animals. Our data indicate that EGCG inhibits amyloid fibril formation in vitro and reduces fibril intensity in non-diabetic wt/tg mice. These results demonstrate a possible in vivo effectiveness of EGCG on amyloid formation and suggest an early therapeutical application.
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http://dx.doi.org/10.1038/s41598-017-18807-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773570PMC
January 2018

Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex.

EMBO J 2018 01 6;37(2):282-299. Epub 2017 Dec 6.

Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene (). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. Using and assays, we have identified a trimeric chaperone complex (Hsc70, Hsp110, and J-protein) that completely suppresses fibrilization of HttExon1Q The composition of this chaperone complex is variable as recruitment of different chaperone family members forms distinct functional complexes. The trimeric chaperone complex is also able to resolubilize Htt fibrils. We confirmed the biological significance of these findings in HD patient-derived neural cells and on an organismal level in Among the proteins in this chaperone complex, the J-protein is the concentration-limiting factor. The single overexpression of DNAJB1 in HEK293T cells is sufficient to profoundly reduce HttExon1Q aggregation and represents a target of future therapeutic avenues for HD.
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http://dx.doi.org/10.15252/embj.201797212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770855PMC
January 2018

Inhibition of Huntingtin Exon-1 Aggregation by the Molecular Tweezer CLR01.

J Am Chem Soc 2017 04 13;139(16):5640-5643. Epub 2017 Apr 13.

Max-Planck-Institut für Kohlenforschung , 45470 Mülheim an der Ruhr, Germany.

Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htt). Above a threshold of 37 glutamine residues, htt starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htt (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htt. Here we identify N17 as a potential target for novel therapeutic intervention using the molecular tweezer CLR01. A combination of biochemical experiments and computer simulations shows that binding of CLR01 induces structural rearrangements within the htt monomer and inhibits htt aggregation, underpinning the key role of N17 in modulating htt toxicity.
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http://dx.doi.org/10.1021/jacs.6b11039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506490PMC
April 2017

Identification of an RNA Polymerase III Regulator Linked to Disease-Associated Protein Aggregation.

Mol Cell 2017 Mar;65(6):1096-1108.e6

European Research Institute for the Biology of Ageing, University of Groningen, University Medical Centre Groningen, 9700 AD Groningen, the Netherlands. Electronic address:

Protein aggregation is associated with age-related neurodegenerative disorders, such as Alzheimer's and polyglutamine diseases. As a causal relationship between protein aggregation and neurodegeneration remains elusive, understanding the cellular mechanisms regulating protein aggregation will help develop future treatments. To identify such mechanisms, we conducted a forward genetic screen in a C. elegans model of polyglutamine aggregation and identified the protein MOAG-2/LIR-3 as a driver of protein aggregation. In the absence of polyglutamine, MOAG-2/LIR-3 regulates the RNA polymerase III-associated transcription of small non-coding RNAs. This regulation is lost in the presence of polyglutamine, which mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol. We then show biochemically that MOAG-2/LIR-3 can also catalyze the aggregation of polyglutamine-expanded huntingtin. These results suggest that polyglutamine can induce an aggregation-promoting activity of MOAG-2/LIR-3 in the cytosol. The concept that certain aggregation-prone proteins can convert other endogenous proteins into drivers of aggregation and toxicity adds to the understanding of how cellular homeostasis can be deteriorated in protein misfolding diseases.
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http://dx.doi.org/10.1016/j.molcel.2017.02.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364375PMC
March 2017

The redox environment triggers conformational changes and aggregation of hIAPP in Type II Diabetes.

Sci Rep 2017 03 13;7:44041. Epub 2017 Mar 13.

Helmholtz Zentrum München, Ingolstädter Landstr. 1, Neuherberg 85764, Germany.

Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in β-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.
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http://dx.doi.org/10.1038/srep44041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347123PMC
March 2017

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders.

Cell Stem Cell 2017 05 26;20(5):659-674.e9. Epub 2017 Jan 26.

Max Delbrueck Center for Molecular Medicine (MDC), 13125 Berlin, Germany. Electronic address:

Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.
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http://dx.doi.org/10.1016/j.stem.2016.12.013DOI Listing
May 2017

Aggregation-induced changes in the chemical exchange saturation transfer (CEST) signals of proteins.

NMR Biomed 2017 01 11;30(1). Epub 2016 Nov 11.

Division of Medical Physics in Radiology, Deutsches Krebsforschungszentrum [German Cancer Research Center] (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Baden-Württemberg, Germany.

Chemical exchange saturation transfer (CEST) is an MRI technique that allows mapping of biomolecules (small metabolites, proteins) with nearly the sensitivity of conventional water proton MRI. In living organisms, several tissue-specific CEST effects have been observed and successfully applied to diagnostic imaging. In these studies, particularly the signals of proteins showed a distinct correlation with pathological changes. However, as CEST effects depend on various properties that determine and affect the chemical exchange processes, the origins of the observed signal changes remain to be understood. In this study, protein aggregation was identified as an additional process that is encoded in the CEST signals of proteins. Investigation of distinct proteins that are involved in pathological disorders, namely amyloid beta and huntingtin, revealed a significant decrease of all protein CEST signals upon controlled aggregation. This finding is of particular interest with regard to diagnostic imaging of patients with neurodegenerative diseases that involve amyloidogenesis, such as Alzheimer's or Huntington's disease. To investigate whether the observed CEST signal decrease also occurs in heterogeneous mixtures of aggregated cellular proteins, and thus prospectively in tissue, heat-shocked yeast cell lysates were employed. Additionally, investigation of different cell compartments verified the assignment of the protein CEST signals to the soluble part of the proteome. The results of in vitro experiments demonstrate that aggregation affects the CEST signals of proteins. This observation can enable hypotheses for CEST imaging as a non-invasive diagnostic tool for monitoring pathological alterations of the proteome in vivo.
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http://dx.doi.org/10.1002/nbm.3665DOI Listing
January 2017

Organ-specific alteration in caspase expression and STK3 proteolysis during the aging process.

Neurobiol Aging 2016 11 15;47:50-62. Epub 2016 Jul 15.

Research Centre on Aging, University of Sherbrooke, Sherbrooke, Faculty of Medicine and Health Sciences, Quebec, Canada; Department of Pharmacology and Physiology, University of Sherbrooke, Sherbrooke, Faculty of Medicine and Health Sciences, Quebec, Canada. Electronic address:

Caspases and their substrates are key mediators of apoptosis and strongly implicated in various physiological processes. As the serine/threonine kinase family is involved in apoptosis and serine/threonine kinase 3 (STK3) is a recently identified caspase-6 substrate, we assessed the expression and cleavage of STK3 in murine peripheral organs and brain regions during the aging process. We also assessed caspase-3, -6, -7, and -8 expression and activity in order to delineate potential mechanism(s) underlying the generation of the STK3 fragments observed and their relation to the apoptotic pathway. We demonstrate for the first time the cleavage of STK3 by caspase-7 and show that STK3 protein levels globally increase throughout the organism with age. In contrast, caspase-3, -6, -7, and -8 expression and activity vary significantly among the different organs analyzed suggesting differential effects of aging on the apoptotic mechanism and/or nonapoptotic functions of caspases throughout the organism. These results further our understanding of the role of caspases and their substrates in the normal aging process and highlight a potential role for STK3 in neurodegeneration.
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http://dx.doi.org/10.1016/j.neurobiolaging.2016.07.003DOI Listing
November 2016

Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers.

Nat Commun 2016 10 20;7:13047. Epub 2016 Oct 20.

Max Delbrück Center for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin, Germany.

Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.
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http://dx.doi.org/10.1038/ncomms13047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5080433PMC
October 2016