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57 Publications visible to you, out of a total of 57
Proteomic profiling in cerebral amyloid angiopathy reveals an overlap with CADASIL highlighting accumulation of HTRA1 and its substrates.

Abstract (Expand)

Cerebral amyloid angiopathy (CAA) is an age-related condition and a major cause of intracerebral hemorrhage and cognitive decline that shows close links with Alzheimer's disease (AD). CAA is … characterized by the aggregation of amyloid-beta (Abeta) peptides and formation of Abeta deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Abeta pathology in CAA type 1 and become dysfunctional during disease progression. Here, applying an advanced protocol for the isolation of parenchymal microvessels from post-mortem brain tissue combined with liquid chromatography tandem mass spectrometry (LC-MS/MS), we determined the proteomes of CAA type 1 cases (n = 12) including a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and of AD cases without microvascular amyloid pathology (n = 13) in comparison to neurologically healthy controls (n = 12). ELISA measurements revealed microvascular Abeta(1-40) levels to be exclusively enriched in CAA samples (mean: > 3000-fold compared to controls). The proteomic profile of CAA type 1 was characterized by massive enrichment of multiple predominantly secreted proteins and showed significant overlap with the recently reported brain microvascular proteome of patients with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebral small vessel disease (SVD) characterized by the aggregation of the Notch3 extracellular domain. We found this overlap to be largely attributable to the accumulation of high-temperature requirement protein A1 (HTRA1), a serine protease with an established role in the brain vasculature, and several of its substrates. Notably, this signature was not present in AD cases. We further show that HTRA1 co-localizes with Abeta deposits in brain capillaries from CAA type 1 patients indicating a pathologic recruitment process. Together, these findings suggest a central role of HTRA1-dependent protein homeostasis in the CAA microvasculature and a molecular connection between multiple types of brain microvascular disease.

Authors: A. Zellner, S. A. Muller, B. Lindner, N. Beaufort, A. J. M. Rozemuller, T. Arzberger, N. C. Gassen, S. F. Lichtenthaler, B. Kuster, C. Haffner, M. Dichgans

Date Published: 24th Jan 2022

Publication Type: Journal

Proteomic and lipidomic profiling of demyelinating lesions identifies fatty acids as modulators in lesion recovery.

Abstract (Expand)

After demyelinating injury of the central nervous system, resolution of the mounting acute inflammation is crucial for the initiation of a regenerative response. Here, we aim to identify fatty acids … and lipid mediators that govern the balance of inflammatory reactions within demyelinating lesions. Using lipidomics, we identify bioactive lipids in the resolution phase of inflammation with markedly elevated levels of n-3 polyunsaturated fatty acids. Using fat-1 transgenic mice, which convert n-6 fatty acids to n-3 fatty acids, we find that reduction of the n-6/n-3 ratio decreases the phagocytic infiltrate. In addition, we observe accelerated decline of microglia/macrophages and enhanced generation of oligodendrocytes in aged mice when n-3 fatty acids are shuttled to the brain. Thus, n-3 fatty acids enhance lesion recovery and may, therefore, provide the basis for pro-regenerative medicines of demyelinating diseases in the central nervous system.

Authors: H. Penkert, A. Bertrand, V. Tiwari, S. Breimann, S. A. Muller, P. M. Jordan, M. J. Gerl, C. Klose, L. Cantuti-Castelvetri, M. Bosch-Queralt, I. Levental, S. F. Lichtenthaler, O. Werz, M. Simons

Date Published: 26th Oct 2021

Publication Type: Journal

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

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

Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling.

Abstract (Expand)

Autophagy deficiency in fed conditions leads to the formation of protein inclusions highlighting the contribution of this lysosomal delivery route to cellular proteostasis. Selective autophagy pathways … exist that clear accumulated and aggregated ubiquitinated proteins. Receptors for this type of autophagy (aggrephagy) include p62, NBR1, TOLLIP, and OPTN, which possess LC3-interacting regions and ubiquitin-binding domains (UBDs), thus working as a bridge between LC3/GABARAP proteins and ubiquitinated substrates. However, the identity of aggrephagy substrates and the redundancy of aggrephagy and related UBD-containing receptors remains elusive. Here, we combined proximity labeling and organelle enrichment with quantitative proteomics to systematically map the autophagic degradome targeted by UBD-containing receptors under basal and proteostasis-challenging conditions in human cell lines. We identified various autophagy substrates, some of which were differentially engulfed by autophagosomal and endosomal membranes via p62 and TOLLIP, respectively. Overall, this resource will allow dissection of the proteostasis contribution of autophagy to numerous individual proteins.

Authors: Susanne Zellner, Martina Schifferer, Christian Behrends

Date Published: 18th Mar 2021

Publication Type: Journal

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