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65 Publications visible to you, out of a total of 65
Nonvesicular lipid transfer drives myelin growth in the central nervous system.

Abstract (Expand)

Oligodendrocytes extend numerous cellular processes that wrap multiple times around axons to generate lipid-rich myelin sheaths. Myelin biogenesis requires an enormously productive biosynthetic machinery … for generating and delivering these large amounts of newly synthesized lipids. Yet, a complete understanding of this process remains elusive. Utilizing volume electron microscopy, we demonstrate that the oligodendroglial endoplasmic reticulum (ER) is enriched in developing myelin, extending into and making contact with the innermost myelin layer where growth occurs. We explore the possibility of transfer of lipids from the ER to myelin, and find that the glycolipid transfer protein (GLTP), implicated in nonvesicular lipid transport, is highly enriched in the growing myelin sheath. Mice with a specific knockout of Gltp in oligodendrocytes exhibit ER pathology, hypomyelination and a decrease in myelin glycolipid content. In summary, our results demonstrate a role for nonvesicular lipid transport in CNS myelin growth, revealing a cellular pathway in developmental myelination.

Authors: Jianping Wu, Georg Kislinger, Jerome Duschek, Ayşe Damla Durmaz, Benedikt Wefers, Ruoqing Feng, Karsten Nalbach, Wolfgang Wurst, Christian Behrends, Martina Schifferer, Mikael Simons

Date Published: 11th Nov 2024

Publication Type: Journal

Loss of CLN3 in microglia leads to impaired lipid metabolism and myelin turnover.

Abstract (Expand)

Loss-of-function mutations in CLN3 cause juvenile Batten disease, featuring neurodegeneration and early-stage neuroinflammation. How loss of CLN3 function leads to early neuroinflammation is not yet … understood. Here, we have comprehensively studied microglia from Cln3<sup>∆ex7/8</sup> mice, a genetically accurate disease model. Loss of CLN3 function in microglia leads to lysosomal storage material accumulation and abnormal morphology of subcellular organelles. Moreover, pathological proteomic signatures are indicative of defects in lysosomal function and abnormal lipid metabolism. Consistent with these findings, CLN3-deficient microglia are unable to efficiently turnover myelin and metabolize the associated lipids, showing defects in lipid droplet formation and cholesterol accumulation. Accordingly, we also observe impaired myelin integrity in aged Cln3<sup>∆ex7/8</sup> mouse brain. Autophagy inducers and cholesterol-lowering drugs correct the observed microglial phenotypes. Taken together, these data implicate a cell-autonomous defect in CLN3-deficient microglia that impacts their ability to support neuronal cell health, suggesting microglial targeted therapies should be considered for CLN3 disease.

Authors: Seda Yasa, Elisabeth S Butz, Alessio Colombo, Uma Chandrachud, Luca Montore, Sarah Tschirner, Matthias Prestel, Steven D Sheridan, Stephan A Müller, Janos Groh, Stefan F Lichtenthaler, Sabina Tahirovic, Susan L Cotman

Date Published: 22nd Oct 2024

Publication Type: Journal

Proteomic Characterization of Ubiquitin Carboxyl-Terminal Hydrolase 19 Deficient Cells Reveals a Role for USP19 in the Secretion of Lysosomal Proteins.

Abstract (Expand)

Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase, characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain … that anchors them to the endoplasmic reticulum. Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from endoplasmic reticulum-associated protein degradation in a catalytic-independent manner, promote autophagy, and address proteins to lysosomal degradation via endosomal microautophagy. USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson's disease-associated protein α-synuclein. Despite mounting evidence that USP19 plays crucial roles in several biological processes, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by the loss of USP19 to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired the release of several lysosomal proteins, including legumain (LGMN) and several cathepsins. In order to understand the underlaying mechanism, we dissected the USP19-regulated secretion of LGMN in several cell types. We found that LGMN was not a deubiquitinase substrate of USP19 and that its USP19-dependent release did not require their direct interaction. LGMN secretion occurred by a mechanism that involved the Golgi apparatus, autophagosome formation, and lysosome function. This mechanism resembled the recently described "lysosomal exocytosis," by which lysosomal hydrolases are secreted, when ubiquitination of p62 is increased in cells lacking deubiquitinases such as USP15 and USP17. In conclusion, our proteomic characterization of USP19 has identified a collection of proteins in the secretome and within the cell that are regulated by USP19, which link USP19 to the secretion of lysosomal proteins, including LGMN.

Authors: Simone Bonelli, Margot Lo Pinto, Yihong Ye, Stephan A Müller, Stefan F Lichtenthaler, Simone Dario Scilabra

Date Published: 9th Oct 2024

Publication Type: Journal

Development of a Proteomic Workflow for the Identification of Heparan Sulphate Proteoglycan-Binding Substrates of ADAM17.

Abstract (Expand)

Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase … ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signalling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g., HB-EGF) are known to bind to heparan sulphate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-treated secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e., HB-EGF, CX3CL1) and 14 novel HSPG-binding putative ADAM17 substrates. Two of these, MHC-I and IL1RL1, were validated as ADAM17 substrates by immunoblotting.

Authors: Matteo Calligaris, Donatella Pia Spanò, Maria Chiara Puccio, Stephan A Müller, Simone Bonelli, Margot Lo Pinto, Giovanni Zito, Carl P Blobel, Stefan F Lichtenthaler, Linda Troeberg, Simone Dario Scilabra

Date Published: 24th Sep 2024

Publication Type: Journal

A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates.

Abstract (Expand)

Over 600 E3 ligases in humans execute ubiquitination of specific target proteins in a spatiotemporal manner to elicit desired signaling effects. Here, we developed a ubiquitin-specific proximity-based … labeling method to selectively biotinylate substrates of a given ubiquitin ligase. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich bona fide substrates of a ligase using a one-step streptavidin pulldown under denaturing conditions. We applied our method, which we named Ub-POD, to the really interesting new gene (RING) E3 ligase RAD18 and identified proliferating cell nuclear antigen and several other critical players in the DNA damage repair pathway. Furthermore, we successfully applied Ub-POD to the RING ubiquitin ligase tumor necrosis factor receptor-associated factor 6 and a U-box-type E3 ubiquitin ligase carboxyl terminus of Hsc70-interacting protein. We anticipate that our method could be widely adapted to all classes of ubiquitin ligases to identify substrates.

Authors: Urbi Mukhopadhyay, Sophie Levantovsky, Teresa Maria Carusone, Sarah Gharbi, Frank Stein, Christian Behrends, Sagar Bhogaraju

Date Published: 9th Aug 2024

Publication Type: Journal

Rational correction of pathogenic conformational defects in HTRA1.

Abstract (Expand)

Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we establish independent approaches to achieve the functional correction of trimer assembly … defects. Focusing on the prototypical R274Q mutation, we identify an HTRA1 variant that promotes trimer formation thus restoring enzymatic activity in vitro. Genetic experiments in Htra1<sup>R274Q</sup> mice further demonstrate that expression of this protein-based corrector in trans is sufficient to stabilize HtrA1-R274Q and restore the proteomic signature of the brain vasculature. An alternative approach employs supramolecular chemical ligands that shift the monomer-trimer equilibrium towards proteolytically active trimers. Moreover, we identify a peptidic ligand that activates HTRA1 monomers. Our findings open perspectives for tailored protein repair strategies.

Authors: Nathalie Beaufort, Linda Ingendahl, Melisa Merdanovic, Andree Schmidt, David Podlesainski, Tim Richter, Thorben Neumann, Michael Kuszner, Ingrid R Vetter, Patricia Stege, Steven G Burston, Anto Filipovic, Yasser B Ruiz-Blanco, Kenny Bravo-Rodriguez, Joel Mieres-Perez, Christine Beuck, Stephan Uebel, Monika Zobawa, Jasmin Schillinger, Rainer Malik, Katalin Todorov-Völgyi, Juliana Rey, Annabell Roberti, Birte Hagemeier, Benedikt Wefers, Stephan A Müller, Wolfgang Wurst, Elsa Sanchez-Garcia, Alexander Zimmermann, Xiao-Yu Hu, Tim Clausen, Robert Huber, Stefan F Lichtenthaler, Carsten Schmuck, Michael Giese, Markus Kaiser, Michael Ehrmann, Martin Dichgans

Date Published: 16th Jul 2024

Publication Type: Journal

Multiomic ALS signatures highlight subclusters and sex differences suggesting the MAPK pathway as therapeutic target.

Abstract (Expand)

Amyotrophic lateral sclerosis (ALS) is a debilitating motor neuron disease and lacks effective disease-modifying treatments. This study utilizes a comprehensive multiomic approach to investigate the … early and sex-specific molecular mechanisms underlying ALS. By analyzing the prefrontal cortex of 51 patients with sporadic ALS and 50 control subjects, alongside four transgenic mouse models (C9orf72-, SOD1-, TDP-43-, and FUS-ALS), we have uncovered significant molecular alterations associated with the disease. Here, we show that males exhibit more pronounced changes in molecular pathways compared to females. Our integrated analysis of transcriptomes, (phospho)proteomes, and miRNAomes also identified distinct ALS subclusters in humans, characterized by variations in immune response, extracellular matrix composition, mitochondrial function, and RNA processing. The molecular signatures of human subclusters were reflected in specific mouse models. Our study highlighted the mitogen-activated protein kinase (MAPK) pathway as an early disease mechanism. We further demonstrate that trametinib, a MAPK inhibitor, has potential therapeutic benefits in vitro and in vivo, particularly in females, suggesting a direction for developing targeted ALS treatments.

Authors: Lucas Caldi Gomes, Sonja Hänzelmann, Fabian Hausmann, Robin Khatri, Sergio Oller, Mojan Parvaz, Laura Tzeplaeff, Laura Pasetto, Marie Gebelin, Melanie Ebbing, Constantin Holzapfel, Stefano Fabrizio Columbro, Serena Scozzari, Johanna Knöferle, Isabell Cordts, Antonia F Demleitner, Marcus Deschauer, Claudia Dufke, Marc Sturm, Qihui Zhou, Pavol Zelina, Emma Sudria-Lopez, Tobias B Haack, Sebastian Streb, Magdalena Kuzma-Kozakiewicz, Dieter Edbauer, R Jeroen Pasterkamp, Endre Laczko, Hubert Rehrauer, Ralph Schlapbach, Christine Carapito, Valentina Bonetto, Stefan Bonn, Paul Lingor

Date Published: 1st Jul 2024

Publication Type: Journal

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