Publications by authors named "Oliver Kretz"

80 Publications

SRGAP1 Controls Small Rho GTPases To Regulate Podocyte Foot Process Maintenance.

J Am Soc Nephrol 2021 Mar 29;32(3):563-579. Epub 2021 Jan 29.

Institute of Surgical Pathology, Faculty of Medicine, Medical Center - University of Freiburg, Freiburg, Germany

Background: Previous research demonstrated that small Rho GTPases, modulators of the actin cytoskeleton, are drivers of podocyte foot-process effacement in glomerular diseases, such as FSGS. However, a comprehensive understanding of the regulatory networks of small Rho GTPases in podocytes is lacking.

Methods: We conducted an analysis of podocyte transcriptome and proteome datasets for Rho GTPases; mapped , podocyte-specific Rho GTPase affinity networks; and examined conditional knockout mice and murine disease models targeting . To evaluate podocyte foot-process morphology, we used super-resolution microscopy and electron microscopy; proximity ligation assays were used to determine the subcellular localization of the small GTPase-activating protein SRGAP1. We performed functional analysis of CRISPR/Cas9-generated knockout podocytes in two-dimensional and three-dimensional cultures and quantitative interaction proteomics.

Results: We demonstrated SRGAP1 localization to podocyte foot processes and to cellular protrusions . but not knockout mice developed an FSGS-like phenotype at adulthood. Podocyte-specific deletion of by resulted in increased susceptibility to doxorubicin-induced nephropathy. Detailed analysis demonstrated significant effacement of podocyte foot processes. Furthermore, -knockout podocytes showed excessive protrusion formation and disinhibition of the small Rho GTPase machinery . Evaluation of a SRGAP1-dependent interactome revealed the involvement of SRGAP1 with protrusive and contractile actin networks. Analysis of glomerular biopsy specimens translated these findings toward human disease by displaying a pronounced redistribution of SRGAP1 in FSGS.

Conclusions: SRGAP1, a podocyte-specific RhoGAP, controls podocyte foot-process architecture by limiting the activity of protrusive, branched actin networks. Therefore, elucidating the complex regulatory small Rho GTPase affinity network points to novel targets for potentially precise intervention in glomerular diseases.
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http://dx.doi.org/10.1681/ASN.2020081126DOI Listing
March 2021

A muscle growth-promoting treatment based on the attenuation of activin/myostatin signalling results in long-term testicular abnormalities.

Dis Model Mech 2021 Feb 19;14(2). Epub 2021 Feb 19.

School of Biological Sciences, University of Reading, Reading NW1 0TU, UK

Activin/myostatin signalling acts to induce skeletal muscle atrophy in adult mammals by inhibiting protein synthesis as well as promoting protein and organelle turnover. Numerous strategies have been successfully developed to attenuate the signalling properties of these molecules, which result in augmenting muscle growth. However, these molecules, in particular activin, play major roles in tissue homeostasis in numerous organs of the mammalian body. We have recently shown that although the attenuation of activin/myostatin results in robust muscle growth, it also has a detrimental impact on the testis. Here, we aimed to discover the long-term consequences of a brief period of exposure to muscle growth-promoting molecules in the testis. We demonstrate that muscle hypertrophy promoted by a soluble activin type IIB ligand trap (sActRIIB) is a short-lived phenomenon. In stark contrast, short-term treatment with sActRIIB results in immediate impact on the testis, which persists after the sessions of the intervention. Gene array analysis identified an expansion in aberrant gene expression over time in the testis, initiated by a brief exposure to muscle growth-promoting molecules. The impact on the testis results in decreased organ size as well as quantitative and qualitative impact on sperm. Finally, we have used a drug-repurposing strategy to exploit the gene expression data to identify a compound - -methyladenosine - that may protect the testis from the impact of the muscle growth-promoting regime. This work indicates the potential long-term harmful effects of strategies aimed at promoting muscle growth by attenuating activin/myostatin signalling. Furthermore, we have identified a molecule that could, in the future, be used to overcome the detrimental impact of sActRIIB treatment on the testis.
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http://dx.doi.org/10.1242/dmm.047555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903914PMC
February 2021

The proteomic landscape of small urinary extracellular vesicles during kidney transplantation.

J Extracell Vesicles 2020 Oct 19;10(1):e12026. Epub 2020 Nov 19.

Department II of Internal Medicine University of Cologne Faculty of Medicine and University Hospital Cologne Cologne Germany.

Kidney transplantation is the preferred renal replacement therapy available. Yet, long-term transplant survival is unsatisfactory, partially due to insufficient possibilities of longitudinal monitoring and understanding of the biological processes after transplantation. Small urinary extracellular vesicles (suEVs) - as a non-invasive source of information - were collected from 22 living donors and recipients. Unbiased proteomic analysis revealed temporal patterns of suEV protein signature and cellular processes involved in both early response and longer-term graft adaptation. Complement activation was among the most dynamically regulated components. This unique atlas of the suEV proteome is provided through an online repository allowing dynamic interrogation by the user. Additionally, a correlative analysis identified putative prognostic markers of future allograft function. One of these markers - phosphoenol pyruvate carboxykinase (PCK2) - could be confirmed using targeted MS in an independent validation cohort of 22 additional patients. This study sheds light on the impact of kidney transplantation on urinary extracellular vesicle content and allows the first deduction of early molecular processes in transplant biology. Beyond that our data highlight the potential of suEVs as a source of biomarkers in this setting.
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http://dx.doi.org/10.1002/jev2.12026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710132PMC
October 2020

Diminution in sperm quantity and quality in mouse models of Duchenne Muscular Dystrophy induced by a myostatin-based muscle growth-promoting intervention.

Eur J Transl Myol 2020 Jul 22;30(2):8904. Epub 2020 Jun 22.

School of Biological Sciences, University of Reading, UK.

Duchenne Muscular Dystrophy is a devastating disease caused by the absence of a functional rod-shaped cytoplasmic protein called dystrophin. Several avenues are being developed aimed to restore dystrophin expression in boys affected by this X-linked disease. However, its complete cure is likely to need combinational approaches which may include regimes aimed at restoring muscle mass. Augmenting muscle growth through the manipulation of the Myostatin/Activin signalling axis has received much attention. However, we have recently shown that while manipulation of this axis in wild type mice using the sActRIIB ligand trap indeed results in muscle growth, it also had a detrimental impact on the testis. Here we examined the impact of administering a powerful Myostatin/Activin antagonist in two mouse models of Duchenne Muscular Dystrophy. We report that whilst the impact on muscle growth was not always positive, both models showed attenuated testis development. Sperm number, motility and ultrastructure were significantly affected by the sActRIIB treatment. Our report suggests that interventions based on Myostatin/Activin should investigate off-target effects on tissues as well as muscle.
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http://dx.doi.org/10.4081/ejtm.2019.8904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385695PMC
July 2020

Distinct Modes of Balancing Glomerular Cell Proteostasis in Mucolipidosis Type II and III Prevent Proteinuria.

J Am Soc Nephrol 2020 08 8;31(8):1796-1814. Epub 2020 Jul 8.

Institute of Cellular and Integrative Physiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

Background: The mechanisms balancing proteostasis in glomerular cells are unknown. Mucolipidosis (ML) II and III are rare lysosomal storage disorders associated with mutations of the Golgi-resident GlcNAc-1-phosphotransferase, which generates mannose 6-phosphate residues on lysosomal enzymes. Without this modification, lysosomal enzymes are missorted to the extracellular space, which results in lysosomal dysfunction of many cell types. Patients with MLII present with severe skeletal abnormalities, multisystemic symptoms, and early death; the clinical course in MLIII is less progressive. Despite dysfunction of a major degradative pathway, renal and glomerular involvement is rarely reported, suggesting organ-specific compensatory mechanisms.

Methods: MLII mice were generated and compared with an established MLIII model to investigate the balance of protein synthesis and degradation, which reflects glomerular integrity. Proteinuria was assessed in patients. High-resolution confocal microscopy and functional assays identified proteins to deduce compensatory modes of balancing proteostasis.

Results: Patients with MLII but not MLIII exhibited microalbuminuria. MLII mice showed lysosomal enzyme missorting and several skeletal alterations, indicating that they are a useful model. In glomeruli, both MLII and MLIII mice exhibited reduced levels of lysosomal enzymes and enlarged lysosomes with abnormal storage material. Nevertheless, neither model had detectable morphologic or functional glomerular alterations. The models rebalance proteostasis in two ways: MLII mice downregulate protein translation and increase the integrated stress response, whereas MLIII mice upregulate the proteasome system in their glomeruli. Both MLII and MLIII downregulate the protein complex mTORC1 (mammalian target of rapamycin complex 1) signaling, which decreases protein synthesis.

Conclusions: Severe lysosomal dysfunction leads to microalbuminuria in some patients with mucolipidosis. Mouse models indicate distinct compensatory pathways that balance proteostasis in MLII and MLIII.
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http://dx.doi.org/10.1681/ASN.2019090960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460914PMC
August 2020

Neural metabolic imbalance induced by MOF dysfunction triggers pericyte activation and breakdown of vasculature.

Nat Cell Biol 2020 07 15;22(7):828-841. Epub 2020 Jun 15.

Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany.

Mutations in chromatin-modifying complexes and metabolic enzymes commonly underlie complex human developmental syndromes affecting multiple organs. A major challenge is to determine how disease-causing genetic lesions cause deregulation of homeostasis in unique cell types. Here we show that neural-specific depletion of three members of the non-specific lethal (NSL) chromatin complex-Mof, Kansl2 or Kansl3-unexpectedly leads to severe vascular defects and brain haemorrhaging. Deregulation of the epigenetic landscape induced by the loss of the NSL complex in neural cells causes widespread metabolic defects, including an accumulation of free long-chain fatty acids (LCFAs). Free LCFAs induce a Toll-like receptor 4 (TLR4)-NFκB-dependent pro-inflammatory signalling cascade in neighbouring vascular pericytes that is rescued by TLR4 inhibition. Pericytes display functional changes in response to LCFA-induced activation that result in vascular breakdown. Our work establishes that neurovascular function is determined by the neural metabolic environment.
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http://dx.doi.org/10.1038/s41556-020-0526-8DOI Listing
July 2020

Inhibition of Activin/Myostatin signalling induces skeletal muscle hypertrophy but impairs mouse testicular development.

Eur J Transl Myol 2020 Apr 1;30(1):8737. Epub 2020 Apr 1.

School of Biological Sciences, University of Reading, UK.

Numerous approaches are being developed to promote post-natal muscle growth based on attenuating Myostatin/Activin signalling for clinical uses such as the treatment neuromuscular diseases, cancer cachexia and sarcopenia. However there have been concerns about the effects of inhibiting Activin on tissues other than skeletal muscle. We intraperitoneally injected mice with the Activin ligand trap, sActRIIB, in young, adult and a progeric mouse model. Treatment at any stage in the life of the mouse rapidly increased muscle mass. However at all stages of life the treatment decreased the weights of the testis. Not only were the testis smaller, but they contained fewer sperm compared to untreated mice. We found that the hypertrophic muscle phenotype was lost after the cessation of sActRIIB treatment but abnormal testis phenotype persisted. In summary, attenuation of Myostatin/Activin signalling inhibited testis development. Future use of molecules based on a similar mode of action to promote muscle growth should be carefully profiled for adverse side-effects on the testis. However the effectiveness of sActRIIB as a modulator of Activin function provides a possible therapeutic strategy to alleviate testicular seminoma development.
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http://dx.doi.org/10.4081/ejtm.2019.8737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7254437PMC
April 2020

Plasminogen deficiency does not prevent sodium retention in a genetic mouse model of experimental nephrotic syndrome.

Acta Physiol (Oxf) 2021 01 10;231(1):e13512. Epub 2020 Jun 10.

Department of Internal Medicine, Division of Endocrinology, Diabetology and Nephrology, University Hospital Tübingen, Tübingen, Germany.

Aim: Sodium retention is the hallmark of nephrotic syndrome (NS) and mediated by the proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases. Plasmin is highly abundant in nephrotic urine and has been proposed to be the principal serine protease responsible for ENaC activation in NS. However, a proof of the essential role of plasmin in experimental NS is lacking.

Methods: We used a genetic mouse model of NS based on an inducible podocin knockout (Bl6-Nphs2 *Tg(Nphs1-rtTA*3G) *Tg(tetO-cre) or nphs2 ). These mice were crossed with plasminogen deficient mice (Bl6-Plg or plg ) to generate double knockout mice (nphs2 *plg ). NS was induced after oral doxycycline treatment for 14 days and mice were followed for subsequent 14 days.

Results: Uninduced nphs2 *plg mice had normal kidney function and sodium handling. After induction, proteinuria increased similarly in both nphs2 *plg and nphs2 *plg mice. Western blot revealed the urinary excretion of plasminogen and plasmin in nphs2 *plg mice which were absent in nphs2 *plg mice. After the onset of proteinuria, amiloride-sensitive natriuresis was increased compared to the uninduced state in both genotypes. Subsequently, urinary sodium excretion dropped in both genotypes leading to an increase in body weight and development of ascites. Treatment with the serine protease inhibitor aprotinin prevented sodium retention in both genotypes.

Conclusions: This study shows that mice lacking urinary plasminogen are not protected from ENaC-mediated sodium retention in experimental NS. This points to an essential role of other urinary serine proteases in the absence of plasminogen.
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http://dx.doi.org/10.1111/apha.13512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688481PMC
January 2021

Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells.

Cell 2020 05 6;181(5):1080-1096.e19. Epub 2020 May 6.

Institute of Immunology, University Medical Center Mainz, Langenbeckstrasse 1, 55131 Mainz, Germany; Research Centre for Immunotherapy, University Medical Center Mainz, Langenbeckstraße 1, 55131 Mainz, Germany. Electronic address:

Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.
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http://dx.doi.org/10.1016/j.cell.2020.04.022DOI Listing
May 2020

Apoptosis Functions in Defense against Infection of Mammalian Cells with Environmental Chlamydiae.

Infect Immun 2020 05 20;88(6). Epub 2020 May 20.

Institute of Medical Microbiology and Hygiene, Medical Center-University of Freiburg, Faculty of Medicine, Freiburg, Germany

Apoptotic cell death can be an efficient defense reaction of mammalian cells infected with obligate intracellular pathogens; the host cell dies and the pathogen cannot replicate. While this is well established for viruses, there is little experimental support for such a concept in bacterial infections. All are obligate intracellular bacteria, and different species infect vastly different hosts. infects human epithelial cells; replicates in amoebae. We here report that apoptosis impedes growth of in mammalian cells. In HeLa human epithelial cells, infection induced apoptosis, which was inhibited when mitochondrial apoptosis was blocked by codeletion of the mediators of mitochondrial apoptosis, Bax and Bak, by overexpression of Bcl-X or by deletion of the apoptosis initiator Noxa. Deletion of Bax and Bak in mouse macrophages also inhibited apoptosis. Blocking apoptosis permitted growth of in HeLa cells, as measured by fluorescence hybridization, assessment of genome replication and protein synthesis, and the generation of infectious progeny. Coinfection with inhibited -induced apoptosis, suggesting that the known antiapoptotic activity of can also block -induced apoptosis. coinfection could not rescue growth in HeLa; in coinfected cells, even suppressed the growth of independently of apoptosis, while surprisingly enhanced the growth of Our results show that apoptosis can be used in the defense of mammalian cells against obligate intracellular bacteria and suggest that the known antiapoptotic activity of human pathogenic chlamydiae is indeed required to permit their growth in human cells.
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http://dx.doi.org/10.1128/IAI.00851-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240083PMC
May 2020

Renal clearance of polymeric nanoparticles by mimicry of glycan surface of viruses.

Biomaterials 2020 02 23;230:119643. Epub 2019 Nov 23.

Institute for Macromolecular Chemistry, University of Freiburg, 79104, Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104, Freiburg, Germany. Electronic address:

It has been shown that viral particles such as herpes simplex virus-1 and cytomegalovirus show renal clearance despite their large size (155-240 nm). Interestingly, one of the common characteristics of these viruses is their glycoprotein rich viral envelope. Since, glycosaminoglycans (GAGs) share similarities with oligosaccharide chains in the glycoproteins, we hypothesize that modification of nanoparticles (NPs) surface with naturally found GAGs could alter NP clearance characteristics by mimicking physicochemical aspects of viral glycoprotein envelope. We demonstrate that polymeric NP bearing surfaces enriched with dermatan sulfate, chondroitin sulfate, heparin sulfate, and hyaluronic acid undergo rapid renal clearance (74% of injected dose as early as 2 h) while showing reduced liver accumulation. Ultra-structural analyses suggest that the excretion of intact NPs occurs via proximal tubule secretion, but not via glomerular filtration. Finally, we demonstrate that our bioinspired NPs are able to accumulate within the epithelial tumor microenvironment despite their efficient renal clearance. Our system provides a framework to address renal toxicity associated with repeated dosing of NP and a platform to elaborate on plausible mechanism of renal clearance of virus particle.
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http://dx.doi.org/10.1016/j.biomaterials.2019.119643DOI Listing
February 2020

An Early mtUPR: Redistribution of the Nuclear Transcription Factor Rox1 to Mitochondria Protects against Intramitochondrial Proteotoxic Aggregates.

Mol Cell 2020 01 17;77(1):180-188.e9. Epub 2019 Oct 17.

Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany; CIBSS - Centre for Integrative Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany. Electronic address:

The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense.
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http://dx.doi.org/10.1016/j.molcel.2019.09.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941230PMC
January 2020

Management of Tamm-Horsfall Protein for Reliable Urinary Analytics.

Proteomics Clin Appl 2019 11 8;13(6):e1900018. Epub 2019 Sep 8.

Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland.

Purpose: Urinary extracellular vesicles (uEVs) are a novel source of biomarkers. However, urinary Tamm-Horsfall Protein (THP; uromodulin) interferes with all vesicle isolation attempts, precipitates with normal urinary proteins, thus, representing an unwanted "contaminant" in urinary assays. Thus, the aim is to develop a simple method to manage THP efficiently.

Experimental Design: The uEVs are isolated by hydrostatic filtration dialysis (HFD) and treated with a defined solution of urea to optimize release of uEVs from sample. Presence of uEVs is confirmed by transmission electron microscopy, Western blotting, and proteomic profiling in MS.

Results: Using HFD with urea treatment for uEV isolation reduces sample complexity to a great extent. The novel simplified uEV isolation protocol allows comprehensive vesicle proteomics analysis and should be part of any urine analytics to release all sample constituents from THP trap.

Conclusions And Clinical Relevance: The method brings a quick and easy protocol for THP management during uEV isolation, providing major benefits for comprehensive sample analytics.
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http://dx.doi.org/10.1002/prca.201900018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900072PMC
November 2019

The tetraspanin CD9 controls migration and proliferation of parietal epithelial cells and glomerular disease progression.

Nat Commun 2019 07 24;10(1):3303. Epub 2019 Jul 24.

Institut National de la Santé et de la Recherche Médicale (Inserm), Unit 970, Paris Cardiovascular Center - PARCC, 56 rue Leblanc, F-75015, Paris, France.

The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and β1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions.
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http://dx.doi.org/10.1038/s41467-019-11013-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656772PMC
July 2019

Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis.

Kidney Int 2019 08 15;96(2):505-516. Epub 2019 Mar 15.

Department of Nephrology and Clinical Immunology, RWTH Aachen University Clinic, Aachen, Germany; Interdisciplinary Centre for Clinical Research (IZKF Aachen), RWTH Aachen University Hospital, Aachen, Germany; Heisenberg Chair for Preventive and Translational Nephrology, Division of Nephrology, RWTH Aachen University, Aachen, Germany. Electronic address:

Recent developments in optical tissue clearing have been difficult to apply for the morphometric analysis of organs with high cellular content and small functional structures, such as the kidney. Here, we establish combinations of genetic and immuno-labelling for single cell identification, tissue clearing and subsequent de-clarification for histoimmunopathology and transmission electron microscopy. Using advanced light microscopy and computational analyses, we investigated a murine model of crescentic nephritis, an inflammatory kidney disease typified by immune-mediated damage to glomeruli leading to the formation of hypercellular lesions and the rapid loss of kidney function induced by nephrotoxic serum. Results show a graded susceptibility of the glomeruli, significant podocyte loss and capillary injury. These effects are associated with activation of parietal epithelial cells and formation of glomerular lesions that may evolve and obstruct the kidney tubule, thereby explaining the loss of kidney function. Thus, our work provides new high-throughput endpoints for the analysis of complex tissues with single-cell resolution.
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http://dx.doi.org/10.1016/j.kint.2019.02.034DOI Listing
August 2019

Anaerobic Glycolysis Maintains the Glomerular Filtration Barrier Independent of Mitochondrial Metabolism and Dynamics.

Cell Rep 2019 04;27(5):1551-1566.e5

III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address:

The cellular responses induced by mitochondrial dysfunction remain elusive. Intrigued by the lack of almost any glomerular phenotype in patients with profound renal ischemia, we comprehensively investigated the primary sources of energy of glomerular podocytes. Combining functional measurements of oxygen consumption rates, glomerular metabolite analysis, and determination of mitochondrial density of podocytes in vivo, we demonstrate that anaerobic glycolysis and fermentation of glucose to lactate represent the key energy source of podocytes. Under physiological conditions, we could detect neither a developmental nor late-onset pathological phenotype in podocytes with impaired mitochondrial biogenesis machinery, defective mitochondrial fusion-fission apparatus, or reduced mtDNA stability and transcription caused by podocyte-specific deletion of Pgc-1α, Drp1, or Tfam, respectively. Anaerobic glycolysis represents the predominant metabolic pathway of podocytes. These findings offer a strategy to therapeutically interfere with the enhanced podocyte metabolism in various progressive kidney diseases, such as diabetic nephropathy or focal segmental glomerulosclerosis (FSGS).
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http://dx.doi.org/10.1016/j.celrep.2019.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506687PMC
April 2019

Secretome of adipose-derived mesenchymal stem cells promotes skeletal muscle regeneration through synergistic action of extracellular vesicle cargo and soluble proteins.

Stem Cell Res Ther 2019 04 5;10(1):116. Epub 2019 Apr 5.

School of Biological Sciences, University of Reading, Reading, UK.

Background: The mechanisms underpinning the regenerative capabilities of mesenchymal stem cells (MSC) were originally thought to reside in their ability to recognise damaged tissue and to differentiate into specific cell types that would replace defective cells. However, recent work has shown that molecules produced by MSCs (secretome), particularly those packaged in extracellular vesicles (EVs), rather than the cells themselves are responsible for tissue repair.

Methods: Here we have produced a secretome from adipose-derived mesenchymal stem cells (ADSC) that is free of exogenous molecules by incubation within a saline solution. Various in vitro models were used to evaluate the effects of the secretome on cellular processes that promote tissue regeneration. A cardiotoxin-induced skeletal muscle injury model was used to test the regenerative effects of the whole secretome or isolated extracellular vesicle fraction in vivo. This was followed by bioinformatic analysis of the components of the protein and miRNA content of the secretome and finally compared to a secretome generated from a secondary stem cell source.

Results: Here we have demonstrated that the secretome from adipose-derived mesenchymal stem cells shows robust effects on cellular processes that promote tissue regeneration. Furthermore, we show that the whole ADSC secretome is capable of enhancing the rate of skeletal muscle regeneration following acute damage. We assessed the efficacy of the total secretome compared with the extracellular vesicle fraction on a number of assays that inform on tissue regeneration and demonstrate that both fractions affect different aspects of the process in vitro and in vivo. Our in vitro, in vivo, and bioinformatic results show that factors that promote regeneration are distributed both within extracellular vesicles and the soluble fraction of the secretome.

Conclusions: Taken together, our study implies that extracellular vesicles and soluble molecules within ADSC secretome act in a synergistic manner to promote muscle generation.
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http://dx.doi.org/10.1186/s13287-019-1213-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451311PMC
April 2019

Compression of morbidity in a progeroid mouse model through the attenuation of myostatin/activin signalling.

J Cachexia Sarcopenia Muscle 2019 06 27;10(3):662-686. Epub 2019 Mar 27.

School of Biological Sciences, University of Reading, Reading, UK.

Background: One of the principles underpinning our understanding of ageing is that DNA damage induces a stress response that shifts cellular resources from growth towards maintenance. A contrasting and seemingly irreconcilable view is that prompting growth of, for example, skeletal muscle confers systemic benefit.

Methods: To investigate the robustness of these axioms, we induced muscle growth in a murine progeroid model through the use of activin receptor IIB ligand trap that dampens myostatin/activin signalling. Progeric mice were then investigated for neurological and muscle function as well as cellular profiling of the muscle, kidney, liver, and bone.

Results: We show that muscle of Ercc1 progeroid mice undergoes severe wasting (decreases in hind limb muscle mass of 40-60% compared with normal mass), which is largely protected by attenuating myostatin/activin signalling using soluble activin receptor type IIB (sActRIIB) (increase of 30-62% compared with untreated progeric). sActRIIB-treated progeroid mice maintained muscle activity (distance travel per hour: 5.6 m in untreated mice vs. 13.7 m in treated) and increased specific force (19.3 mN/mg in untreated vs. 24.0 mN/mg in treated). sActRIIb treatment of progeroid mice also improved satellite cell function especially their ability to proliferate on their native substrate (2.5 cells per fibre in untreated progeroids vs. 5.4 in sActRIIB-treated progeroids after 72 h in culture). Besides direct protective effects on muscle, we show systemic improvements to other organs including the structure and function of the kidneys; there was a major decrease in the protein content in urine (albumin/creatinine of 4.9 sActRIIB treated vs. 15.7 in untreated), which is likely to be a result in the normalization of podocyte foot processes, which constitute the filtration apparatus (glomerular basement membrane thickness reduced from 224 to 177 nm following sActRIIB treatment). Treatment of the progeric mice with the activin ligand trap protected against the development of liver abnormalities including polyploidy (18.3% untreated vs. 8.1% treated) and osteoporosis (trabecular bone volume; 0.30 mm in treated progeroid mice vs. 0.14 mm in untreated mice, cortical bone volume; 0.30 mm in treated progeroid mice vs. 0.22 mm in untreated mice). The onset of neurological abnormalities was delayed (by ~5 weeks) and their severity reduced, overall sustaining health without affecting lifespan.

Conclusions: This study questions the notion that tissue growth and maintaining tissue function during ageing are incompatible mechanisms. It highlights the need for future investigations to assess the potential of therapies based on myostatin/activin blockade to compress morbidity and promote healthy ageing.
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http://dx.doi.org/10.1002/jcsm.12404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6596402PMC
June 2019

DNA Methyltransferase 1 Controls Nephron Progenitor Cell Renewal and Differentiation.

J Am Soc Nephrol 2019 01 5;30(1):63-78. Epub 2018 Dec 5.

III. Department of Medicine,

Background: Nephron number is a major determinant of long-term renal function and cardiovascular risk. Observational studies suggest that maternal nutritional and metabolic factors during gestation contribute to the high variability of nephron endowment. However, the underlying molecular mechanisms have been unclear.

Methods: We used mouse models, including DNA methyltransferase ( and ) knockout mice, optical projection tomography, three-dimensional reconstructions of the nephrogenic niche, and transcriptome and DNA methylation analysis to characterize the role of DNA methylation for kidney development.

Results: We demonstrate that DNA hypomethylation is a key feature of nutritional kidney growth restriction and and that DNA methyltransferases and are highly enriched in the nephrogenic zone of the developing kidneys. Deletion of in nephron progenitor cells (in contrast to deletion of or ) mimics nutritional models of kidney growth restriction and results in a substantial reduction of nephron number as well as renal hypoplasia at birth. In -deficient mice, optical projection tomography and three-dimensional reconstructions uncovered a significant reduction of stem cell niches and progenitor cells. RNA sequencing analysis revealed that global DNA hypomethylation interferes in the progenitor cell regulatory network, leading to downregulation of genes crucial for initiation of nephrogenesis, and its target Derepression of germline genes, protocadherins, genes, and endogenous retroviral elements resulted in the upregulation of IFN targets and inhibitors of cell cycle progression.

Conclusions: These findings establish DNA methylation as a key regulatory event of prenatal renal programming, which possibly represents a fundamental link between maternal nutritional factors during gestation and reduced nephron number.
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http://dx.doi.org/10.1681/ASN.2018070736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317605PMC
January 2019

The Transcription Factor ETV1 Induces Atrial Remodeling and Arrhythmia.

Circ Res 2018 08;123(5):550-563

From the Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine (C.R., S.R., A.L., M.B., M.S., R.G., T.B., T.S., S.M., L.H.).

Rationale: Structural and electrophysiological remodeling of the atria are recognized consequences of sustained atrial arrhythmias, such as atrial fibrillation. The identification of underlying key molecules and signaling pathways has been challenging because of the changing cell type composition during structural remodeling of the atria.

Objective: Thus, the aims of our study were (1) to search for transcription factors and downstream target genes, which are involved in atrial structural remodeling, (2) to characterize the significance of the transcription factor ETV1 (E twenty-six variant 1) in atrial remodeling and arrhythmia, and (3) to identify ETV1-dependent gene regulatory networks in atrial cardiac myocytes.

Methods And Results: The transcription factor ETV1 was significantly upregulated in atrial tissue from patients with permanent atrial fibrillation. Mice with cardiac myocyte-specific overexpression of ETV1 under control of the myosin heavy chain promoter developed atrial dilatation, fibrosis, thrombosis, and arrhythmia. Cardiac myocyte-specific ablation of ETV1 in mice did not alter cardiac structure and function at baseline. Treatment with Ang II (angiotensin II) for 2 weeks elicited atrial remodeling and fibrosis in control, but not in ETV1-deficient mice. To identify ETV1-regulated genes, cardiac myocytes were isolated and purified from mouse atrial tissue. Active cis-regulatory elements in mouse atrial cardiac myocytes were identified by chromatin accessibility (assay for transposase-accessible chromatin sequencing) and the active chromatin modification H3K27ac (chromatin immunoprecipitation sequencing). One hundred seventy-eight genes regulated by Ang II in an ETV1-dependent manner were associated with active cis-regulatory elements containing ETV1-binding sites. Various genes involved in Ca handling or gap junction formation ( Ryr2, Jph2, Gja5), potassium channels ( Kcnh2, Kcnk3), and genes implicated in atrial fibrillation ( Tbx5) were part of this ETV1-driven gene regulatory network. The atrial ETV1-dependent transcriptome in mice showed a significant overlap with the human atrial proteome of patients with permanent atrial fibrillation.

Conclusions: This study identifies ETV1 as an important component in the pathophysiology of atrial remodeling associated with atrial arrhythmias.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.313036DOI Listing
August 2018

Cilia-localized LKB1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney.

EMBO J 2018 08 19;37(15). Epub 2018 Jun 19.

Renal Department, University Medical Center, Freiburg, Germany

Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2 mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.
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http://dx.doi.org/10.15252/embj.201798615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068446PMC
August 2018

A Multi-layered Quantitative In Vivo Expression Atlas of the Podocyte Unravels Kidney Disease Candidate Genes.

Cell Rep 2018 05;23(8):2495-2508

III. Department of Medicine, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; Department of Medicine IV, Medical Center and Faculty of Medicine, University of Freiburg, 79110 Freiburg, Germany; Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, 79104 Freiburg, Germany; Center for Systems Biology (ZBSA), Albert Ludwigs University, 79104 Freiburg, Germany; BIOSS Centre for Biological Signaling Studies, Albert Ludwigs University Freiburg, 79104 Freiburg, Germany. Electronic address:

Damage to and loss of glomerular podocytes has been identified as the culprit lesion in progressive kidney diseases. Here, we combine mass spectrometry-based proteomics with mRNA sequencing, bioinformatics, and hypothesis-driven studies to provide a comprehensive and quantitative map of mammalian podocytes that identifies unanticipated signaling pathways. Comparison of the in vivo datasets with proteomics data from podocyte cell cultures showed a limited value of available cell culture models. Moreover, in vivo stable isotope labeling by amino acids uncovered surprisingly rapid synthesis of mitochondrial proteins under steady-state conditions that was perturbed under autophagy-deficient, disease-susceptible conditions. Integration of acquired omics dimensions suggested FARP1 as a candidate essential for podocyte function, which could be substantiated by genetic analysis in humans and knockdown experiments in zebrafish. This work exemplifies how the integration of multi-omics datasets can identify a framework of cell-type-specific features relevant for organ health and disease.
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http://dx.doi.org/10.1016/j.celrep.2018.04.059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986710PMC
May 2018

Inhibitor of apoptosis proteins are required for effective fusion of autophagosomes with lysosomes.

Cell Death Dis 2018 05 1;9(5):529. Epub 2018 May 1.

Institute of Medical Microbiology and Hygiene, University Medical Center Freiburg, Freiburg, Germany.

Inhibitor of Apoptosis Proteins act as E3 ubiquitin ligases to regulate NF-κB signalling from multiple pattern recognition receptors including NOD2, as well as TNF Receptor Superfamily members. Loss of XIAP in humans causes X-linked Lymphoproliferative disease type 2 (XLP-2) and is often associated with Crohn's disease. Crohn's disease is also caused by mutations in the gene encoding NOD2 but the mechanisms behind Crohn's disease development in XIAP and NOD2 deficient-patients are still unknown. Numerous other mutations causing Crohn's Disease occur in genes controlling various aspects of autophagy, suggesting a strong involvement of autophagy in preventing Crohn's disease. Here we show that the IAP proteins cIAP2 and XIAP are required for efficient fusion of lysosomes with autophagosomes. IAP inhibition or loss of both cIAP2 and XIAP resulted in a strong blockage in autophagic flux and mitophagy, suggesting that XIAP deficiency may also drive Crohn's Disease due to defects in autophagy.
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http://dx.doi.org/10.1038/s41419-018-0508-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943300PMC
May 2018

Neuronal Growth and Behavioral Alterations in Mice Deficient for the Psychiatric Disease-Associated Gene.

Front Mol Neurosci 2018 9;11:30. Epub 2018 Feb 9.

Department of Anesthesiology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany.

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, -knockout (KO) hippocampus was less permissive for axons grown from EC of β-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of -KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of -KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, -KO mice exhibited impaired social behavior compared to WT littermates. Moreover, -KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders.
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http://dx.doi.org/10.3389/fnmol.2018.00030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811522PMC
February 2018

Mutations in the X-linked cause a glycosylation disorder with autophagic defects.

J Exp Med 2017 Dec 10;214(12):3707-3729. Epub 2017 Nov 10.

Laboratory of Epithelial Biology and Disease, Imagine Institute, Paris, France

The biogenesis of the multi-subunit vacuolar-type H-ATPase (V-ATPase) is initiated in the endoplasmic reticulum with the assembly of the proton pore V0, which is controlled by a group of assembly factors. Here, we identify two hemizygous missense mutations in the extracellular domain of the accessory V-ATPase subunit ATP6AP2 (also known as the [pro]renin receptor) responsible for a glycosylation disorder with liver disease, immunodeficiency, cutis laxa, and psychomotor impairment. We show that deficiency in the mouse liver caused hypoglycosylation of serum proteins and autophagy defects. The introduction of one of the missense mutations into led to reduced survival and altered lipid metabolism. We further demonstrate that in the liver-like fat body, the autophagic dysregulation was associated with defects in lysosomal acidification and mammalian target of rapamycin (mTOR) signaling. Finally, both mutations impaired protein stability and the interaction with ATP6AP1, a member of the V0 assembly complex. Collectively, our data suggest that the missense mutations in lead to impaired V-ATPase assembly and subsequent defects in glycosylation and autophagy.
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http://dx.doi.org/10.1084/jem.20170453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716037PMC
December 2017

Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning.

J Proteome Res 2018 01 20;17(1):86-96. Epub 2017 Nov 20.

Freiburg Institute for Advanced Studies (FRIAS), Albert-Ludwigs University , Freiburg, Germany.

Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally, results of Western blotting confirmed that the membrane protein bands are found in the DP fraction instead of AP. In conclusion, our study validates the use of Triton X-114 phase partitioning protocol on uEVs for a targeted isolation of membrane proteins and to reduce sample complexity. This method successfully facilitates detection of potential biomarkers and druggable targets in uEVs.
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http://dx.doi.org/10.1021/acs.jproteome.7b00386DOI Listing
January 2018

Mitochondrial Priming by CD28.

Cell 2017 Oct 14;171(2):385-397.e11. Epub 2017 Sep 14.

Department of Immunometabolism, Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany. Electronic address:

T cell receptor (TCR) signaling without CD28 can elicit primary effector T cells, but memory T cells generated during this process are anergic, failing to respond to secondary antigen exposure. We show that, upon T cell activation, CD28 transiently promotes expression of carnitine palmitoyltransferase 1a (Cpt1a), an enzyme that facilitates mitochondrial fatty acid oxidation (FAO), before the first cell division, coinciding with mitochondrial elongation and enhanced spare respiratory capacity (SRC). microRNA-33 (miR33), a target of thioredoxin-interacting protein (TXNIP), attenuates Cpt1a expression in the absence of CD28, resulting in cells that thereafter are metabolically compromised during reactivation or periods of increased bioenergetic demand. Early CD28-dependent mitochondrial engagement is needed for T cells to remodel cristae, develop SRC, and rapidly produce cytokines upon restimulation-cardinal features of protective memory T cells. Our data show that initial CD28 signals during T cell activation prime mitochondria with latent metabolic capacity that is essential for future T cell responses.
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http://dx.doi.org/10.1016/j.cell.2017.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5637396PMC
October 2017

Protein and Molecular Characterization of a Clinically Compliant Amniotic Fluid Stem Cell-Derived Extracellular Vesicle Fraction Capable of Accelerating Muscle Regeneration Through Enhancement of Angiogenesis.

Stem Cells Dev 2017 09 22;26(18):1316-1333. Epub 2017 Aug 22.

1 School of Biological Sciences, University of Reading , Reading, United Kingdom .

The secretome of human amniotic fluid stem cells (AFSCs) has great potential as a therapeutic agent in regenerative medicine. However, it must be produced in a clinically compliant manner before it can be used in humans. In this study, we developed a means of producing a biologically active secretome from AFSCs that is free of all exogenous molecules. We demonstrate that the full secretome is capable of promoting stem cell proliferation, migration, and protection of cells against senescence. Furthermore, it has significant anti-inflammatory properties. Most importantly, we show that it promotes tissue regeneration in a model of muscle damage. We then demonstrate that the secretome contains extracellular vesicles (EVs) that harbor much, but not all, of the biological activity of the whole secretome. Proteomic characterization of the EV and free secretome fraction shows the presence of numerous molecules specific to each fraction that could be key regulators of tissue regeneration. Intriguingly, we show that the EVs only contain miRNA and not mRNA. This suggests that tissue regeneration in the host is mediated by the action of EVs modifying existing, rather than imposing new, signaling pathways. The EVs harbor significant anti-inflammatory activity as well as promote angiogenesis, the latter may be the mechanistic explanation for their ability to promote muscle regeneration after cardiotoxin injury.
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http://dx.doi.org/10.1089/scd.2017.0089DOI Listing
September 2017

The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier.

Proc Natl Acad Sci U S A 2017 06 23;114(23):E4621-E4630. Epub 2017 May 23.

Department of Medicine IV, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany;

Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrix-dependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression.
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http://dx.doi.org/10.1073/pnas.1617004114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468651PMC
June 2017

TIR-domain-containing adapter-inducing interferon-β (TRIF) forms filamentous structures, whose pro-apoptotic signalling is terminated by autophagy.

FEBS J 2017 07 19;284(13):1987-2003. Epub 2017 May 19.

Faculty of Medicine, Institute for Medical Microbiology and Hygiene, Medical Center - University of Freiburg, University of Freiburg, Germany.

The formation of amyloid-like protein structures has recently emerged as a feature in signal transduction, particularly in innate immunity. These structures appear to depend on defined domains for their formation but likely also require dedicated ways to terminate signalling. We, here, define the innate immunity protein/Toll-like receptor adaptor TIR-domain-containing adapter-inducing interferon-β (TRIF) as a novel platform of fibril formation and probe signal initiation through TRIF as well as its termination in Toll-like receptor 3 (TLR3)-stimulated melanoma cells. A main signalling pathway triggered by TLR3 caused apoptosis, which was controlled by inhibitor of apoptosis proteins and was dependent on RIPK1 and independent of TNF. Using correlative electron/fluorescence microscopy, we visualised fibrillar structures formed through both Toll/interleukin-1 receptor and RIP homotypic interacting motif regions of TRIF. We provide evidence that these fibrillary structures are active signalling platforms whose activity is terminated by autophagy. TRIF-signalling enhanced autophagy, and fibrillary structures were partly contained within autophagosomes. Inhibition of autophagy increased levels of pro-apoptotic TRIF complexes, leading to the accumulation of active caspase-8 and enhanced apoptosis while stimulation of autophagy reduced TRIF-dependent death. We conclude that pro-death signals through TRIF are regulated by autophagy and propose that pro-apoptotic signalling through TRIF/RIPK1/caspase-8 occurs in fibrillary platforms.
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http://dx.doi.org/10.1111/febs.14091DOI Listing
July 2017