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52 Publications visible to you, out of a total of 52
The pseudoprotease iRhom1 controls ectodomain shedding of membrane proteins in the nervous system.

Abstract (Expand)

Proteolytic ectodomain shedding of membrane proteins is a fundamental mechanism to control the communication between cells and their environment. A key protease for membrane protein shedding is ADAM17, … which requires a non-proteolytic subunit, either inactive Rhomboid 1 (iRhom1) or iRhom2 for its activity. While iRhom1 and iRhom2 are co-expressed in most tissues and appear to have largely redundant functions, the brain is an organ with predominant expression of iRhom1. Yet, little is known about the spatio-temporal expression of iRhom1 in mammalian brain and about its function in controlling membrane protein shedding in the nervous system. Here, we demonstrate that iRhom1 is expressed in mouse brain from the prenatal stage to adulthood with a peak in early postnatal development. In the adult mouse brain iRhom1 was widely expressed, including in cortex, hippocampus, olfactory bulb, and cerebellum. Proteomic analysis of the secretome of primary neurons using the hiSPECS method and of cerebrospinal fluid, obtained from iRhom1-deficient and control mice, identified several membrane proteins that require iRhom1 for their shedding in vitro or in vivo. One of these proteins was 'multiple-EGF-like-domains protein 10' (MEGF10), a phagocytic receptor in the brain that is linked to the removal of amyloid beta and apoptotic neurons. MEGF10 was further validated as an ADAM17 substrate using ADAM17-deficient mouse embryonic fibroblasts. Taken together, this study discovers a role for iRhom1 in controlling membrane protein shedding in the mouse brain, establishes MEGF10 as an iRhom1-dependent ADAM17 substrate and demonstrates that iRhom1 is widely expressed in murine brain.

Authors: J. Tushaus, S. A. Muller, J. Shrouder, M. Arends, M. Simons, N. Plesnila, C. P. Blobel, S. F. Lichtenthaler

Date Published: 6th Oct 2021

Publication Type: Journal

Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells.

Abstract (Expand)

The ascorbate peroxidase APEX2 is commonly used to study the neighborhood of a protein of interest by proximity-dependent biotinylation. Here, we describe a protocol for sample processing compatible … with immunoblotting and mass spectrometry, suitable to specifically map the content of autophagosomes and potentially other short-lived endomembrane transport vesicles without the need of subcellular fractionation. By combining live-cell biotinylation with proteinase K digestion of cell homogenates, proteins enriched in membrane-protected compartments can be readily enriched and identified. For complete details on the use and execution of this protocol, please refer to Zellner et al. (2021).

Authors: S. Zellner, K. Nalbach, C. Behrends

Date Published: 18th Jun 2021

Publication Type: Journal

Skin and gut imprinted helper T cell subsets exhibit distinct functional phenotypes in central nervous system autoimmunity.

Abstract (Expand)

Multidimensional single-cell analyses of T cells have fueled the debate about whether there is extensive plasticity or 'mixed' priming of helper T cell subsets in vivo. Here, we developed an … experimental framework to probe the idea that the site of priming in the systemic immune compartment is a determinant of helper T cell-induced immunopathology in remote organs. By site-specific in vivo labeling of antigen-specific T cells in inguinal (i) or gut draining mesenteric (m) lymph nodes, we show that i-T cells and m-T cells isolated from the inflamed central nervous system (CNS) in a model of multiple sclerosis (MS) are distinct. i-T cells were Cxcr6(+), and m-T cells expressed P2rx7. Notably, m-T cells infiltrated white matter, while i-T cells were also recruited to gray matter. Therefore, we propose that the definition of helper T cell subsets by their site of priming may guide an advanced understanding of helper T cell biology in health and disease.

Authors: M. Hiltensperger, E. Beltran, R. Kant, S. Tyystjarvi, G. Lepennetier, H. Dominguez Moreno, I. J. Bauer, S. Grassmann, S. Jarosch, K. Schober, V. R. Buchholz, S. Kenet, C. Gasperi, R. Ollinger, R. Rad, A. Muschaweckh, C. Sie, L. Aly, B. Knier, G. Garg, A. M. Afzali, L. A. Gerdes, T. Kumpfel, S. Franzenburg, N. Kawakami, B. Hemmer, D. H. Busch, T. Misgeld, K. Dornmair, T. Korn

Date Published: 9th Jun 2021

Publication Type: Journal

ADAM10-Mediated Ectodomain Shedding Is an Essential Driver of Podocyte Damage.

Abstract (Expand)

BACKGROUND: Podocytes embrace the glomerular capillaries with foot processes, which are interconnected by a specialized adherens junction to ultimately form the filtration barrier. Altered adhesion and … loss are common features of podocyte injury, which could be mediated by shedding of cell-adhesion molecules through the regulated activity of cell surface-expressed proteases. A Disintegrin and Metalloproteinase 10 (ADAM10) is such a protease known to mediate ectodomain shedding of adhesion molecules, among others. Here we evaluate the involvement of ADAM10 in the process of antibody-induced podocyte injury. METHODS: Membrane proteomics, immunoblotting, high-resolution microscopy, and immunogold electron microscopy were used to analyze human and murine podocyte ADAM10 expression in health and kidney injury. The functionality of ADAM10 ectodomain shedding for podocyte development and injury was analyzed, in vitro and in vivo, in the anti-podocyte nephritis (APN) model in podocyte-specific, ADAM10-deficient mice. RESULTS: ADAM10 is selectively localized at foot processes of murine podocytes and its expression is dispensable for podocyte development. Podocyte ADAM10 expression is induced in the setting of antibody-mediated injury in humans and mice. Podocyte ADAM10 deficiency attenuates the clinical course of APN and preserves the morphologic integrity of podocytes, despite subepithelial immune-deposit formation. Functionally, ADAM10-related ectodomain shedding results in cleavage of the cell-adhesion proteins N- and P-cadherin, thus decreasing their injury-related surface levels. This favors podocyte loss and the activation of downstream signaling events through the Wnt signaling pathway in an ADAM10-dependent manner. CONCLUSIONS: ADAM10-mediated ectodomain shedding of injury-related cadherins drives podocyte injury.

Authors: M. Sachs, S. Wetzel, J. Reichelt, W. Sachs, L. Schebsdat, S. Zielinski, L. Seipold, L. Heintz, S. A. Muller, O. Kretz, M. Lindenmeyer, T. Wiech, T. B. Huber, R. Lullmann-Rauch, S. F. Lichtenthaler, P. Saftig, C. Meyer-Schwesinger

Date Published: 1st Jun 2021

Publication Type: Journal

ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system.

Abstract (Expand)

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome … formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.

Authors: T. N. Nguyen, B. S. Padman, S. Zellner, G. Khuu, L. Uoselis, W. K. Lam, M. Skulsuppaisarn, R. S. J. Lindblom, E. M. Watts, C. Behrends, M. Lazarou

Date Published: 6th May 2021

Publication Type: Journal

White matter aging drives microglial diversity.

Abstract (Expand)

Aging results in gray and white matter degeneration, but the specific microglial responses are unknown. Using single-cell RNA sequencing from white and gray matter separately, we identified white … matter-associated microglia (WAMs), which share parts of the disease-associated microglia (DAM) gene signature and are characterized by activation of genes implicated in phagocytic activity and lipid metabolism. WAMs depend on triggering receptor expressed on myeloid cells 2 (TREM2) signaling and are aging dependent. In the aged brain, WAMs form independent of apolipoprotein E (APOE), in contrast to mouse models of Alzheimer's disease, in which microglia with the WAM gene signature are generated prematurely and in an APOE-dependent pathway similar to DAMs. Within the white matter, microglia frequently cluster in nodules, where they are engaged in clearing degenerated myelin. Thus, WAMs may represent a potentially protective response required to clear degenerated myelin accumulating during white matter aging and disease.

Authors: S. Safaiyan, S. Besson-Girard, T. Kaya, L. Cantuti-Castelvetri, L. Liu, H. Ji, M. Schifferer, G. Gouna, F. Usifo, N. Kannaiyan, D. Fitzner, X. Xiang, M. J. Rossner, M. Brendel, O. Gokce, M. Simons

Date Published: 7th Apr 2021

Publication Type: Journal

Loss of NPC1 enhances phagocytic uptake and impairs lipid trafficking in microglia.

Abstract (Expand)

Niemann-Pick type C disease is a rare neurodegenerative disorder mainly caused by mutations in NPC1, resulting in abnormal late endosomal/lysosomal lipid storage. Although microgliosis is a prominent … pathological feature, direct consequences of NPC1 loss on microglial function remain not fully characterized. We discovered pathological proteomic signatures and phenotypes in NPC1-deficient murine models and demonstrate a cell autonomous function of NPC1 in microglia. Loss of NPC1 triggers enhanced phagocytic uptake and impaired myelin turnover in microglia that precede neuronal death. Npc1(-/-) microglia feature a striking accumulation of multivesicular bodies and impaired trafficking of lipids to lysosomes while lysosomal degradation function remains preserved. Molecular and functional defects were also detected in blood-derived macrophages of NPC patients that provide a potential tool for monitoring disease. Our study underscores an essential cell autonomous role for NPC1 in immune cells and implies microglial therapeutic potential.

Authors: A. Colombo, L. Dinkel, S. A. Muller, L. Sebastian Monasor, M. Schifferer, L. Cantuti-Castelvetri, J. Konig, L. Vidatic, T. Bremova-Ertl, A. P. Lieberman, S. Hecimovic, M. Simons, S. F. Lichtenthaler, M. Strupp, S. A. Schneider, S. Tahirovic

Date Published: 24th Feb 2021

Publication Type: Journal

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